Plant Physiology

Schaller, G. Eric; Voesenek, Laurentius A.C.J.

doi: 10.1104/pp.15.01242pmid: 26342109

First discovered as a plant growth regulator over a century ago, ethylene continues to be a focus for research worldwide due to the many roles it plays in growth, development, and adaptive responses to biotic and abiotic factors. Ethylene is frequently manipulated for agricultural purposes, with both the inhibition and induction of ethylene responses being of commercial value. Ethylene is removed during the storage of fruit and vegetables by scrubbers and absorbants, its biosynthesis inhibited by aminovinylglycine, which targets the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, and its perception blocked at the receptor level by silver or cyclopropenes. Conversely, ethylene treatment, often applied in the form of ethephon, is used to regulate ripening, flowering, abscission, and even the flow of latex from rubber trees. Ethylene was previously featured in a 2004 Plant Physiology Focus issue edited by Caren Chang and Anthony Bleecker. We contributed research articles to the 2004 Focus issue, and there is a symmetry to our now taking on the editorial role for this 2015 Focus issue on the same subject. There have been substantial advances in our understanding of how ethylene functions in the decade since the previous Focus issue, and a comparison of the research articles found in the two issues provides a snapshot of which aspects of the field have changed and which have remained the same. For example, in the 2004 Focus issue, four studies employed Arabidopsis (Arabidopsis thaliana), two studies employed tobacco (Nicotiana tabacum), and the remaining three studies employed petunia (Petunia spp.), oak (Quercus ilex), and marsh dock (Rumex palustris). By comparison, in this issue, 10 studies employed Arabidopsis, two studies each employed rice (Oryza sativa) and maize (Zea mays), and the other studies employed pea (Pisum sativum), tobacco, apple (Malus domestica), the legume Medicago truncatula, and the moss Physcomitrella patens. Although both issues feature a diversity of experimental subjects, Arabidopsis is the most prominent of these, emphasizing the continued utility of Arabidopsis for mechanistic studies. Perhaps the most significant change in terms of experimental subjects is the presence of four articles in this issue that explore ethylene function in monocots, no such studies being present in the earlier issue. There has been a change in the overall research focus of the articles between the two issues. In the 2004 issue, five articles, representing over half the issue, explored molecular mechanisms of the ethylene signal transduction pathway, analyzing the roles of receptors, the transmembrane protein ETHYLENE INSENSITIVE2 (EIN2), and the EIN3 family of transcription factors. In this issue, there are three articles that emphasize the study of primary pathway elements. Bakshi et al. (2015b) explore a diverging role of the receptor receiver domain in transmitting the ethylene signal; Yasumura et al. (2015)take an evolutionary approach to characterize the role of CONSTITUTIVE TRIPLE RESPONSE1 in ethylene signaling; and Yang et al. (2015) characterize the function of the EIN3 transcription factor family in rice. There are also two articles that focus on novel signaling elements, which interact with and regulate the response from the primary signaling pathway. Shi et al. (2015) provide evidence that members of the ARGOS gene family, which encode short transmembrane proteins, regulate ethylene signaling, whereas Tao et al. (2015) identify a protein that interacts with and stabilizes the receptor against degradation. Interestingly, three of these articles address, although in different ways, cross talk between the ethylene and abscisic acid hormonal responses (Bakshi et al., 2015b; Shi et al., 2015; Yasumura et al., 2015). A major area of interest in this Focus issue is how ethylene regulates specific aspects of growth and development. In the 2004 issue, two articles focused on specific downstream ethylene responses of shade avoidance and hyponastic growth. This issue features studies on aerenchyma formation (Yamauchi et al., 2015), root growth (Street et al., 2015), hypocotyl growth (Sun et al., 2015), hyponastic growth (Polko et al., 2015), abscission (Eccher et al., 2015), leaf senescence (Ueda and Kusaba, 2015), and the interaction of ethylene with light to regulate development (Weller et al., 2015). Of note, three articles in this issue emphasize a previously unheralded role for ethylene as an inhibitor of cell proliferation in both the shoot and root of Arabidopsis (Polko et al., 2015; Street et al., 2015; Tao et al., 2015). Another major area of interest in this Focus issue is in the roles that ethylene plays in mediating plant responses to their environment. In the 2004 issue, abiotic interactions of shade, flooding, heat, and drought stress were represented, but biotic interactions were conspicuously absent. In this issue, four articles analyze the role of ethylene in nodulation (Larrainzar et al., 2015) and in defense responses against Pseudomonas syringae (Zhang, 2015) and aphids (Casteel et al., 2015; Louis et al., 2015). Three other articles explore abiotic interactions, analyzing the role of ethylene in response to drought (Shi et al., 2015), salt (Yang et al., 2015), and osmotic (Dubois et al., 2015) stresses. Although the focus has shifted away from the mechanistic analysis of pathway elements, mutants of these pathway elements serve prominently as genetic tools in the Arabidopsis articles as a means to decipher the role(s) of ethylene in its diversity of downstream regulation. Commensurate with the diversity of roles for ethylene in plant physiology, this Focus issue features Update articles that illuminate how ethylene is synthesized (Booker and DeLong, 2015), its signal transduced (Ju and Chang, 2015), and the roles it plays in development (Van de Poel et al., 2015), microbial interactions (Gamalero and Glick, 2015), and abiotic stress responses (García et al., 2015; Gibbs et al., 2015; Müller and Munné-Bosch, 2015; Sasidharan and Voesenek, 2015; Thao et al., 2015). Just months after the 2004 Focus issue, Tony Bleecker passed away from cancer. His significance to the field was commemorated in a 2006 piece by Edgar Spalding (Spalding, 2006) and more recently in a broad historical perspective on the history of ethylene studies written by Brad Binder, Caren Chang, and members of their laboratories (Bakshi et al., 2015a). Tony’s presence is strongly felt in this Focus issue through the contributions of Chang, Binder, and Schaller, whose studies on ethylene were launched as a result of their interactions with Tony. Chang collaborated with Tony on the cloning of the ethylene receptor gene ETHYLENE RESPONSE1 of Arabidopsis while a postdoc in the laboratory of Elliot Meyerowitz, and Binder and Schaller studied ethylene signaling as postdocs in the Bleecker laboratory. In addition to establishing a family of ethylene researchers, one of Tony’s enduring legacies is the infectious intellectual joy he brought to scientific study. For these reasons, we dedicate this Focus issue to the memory and legacy of Tony Bleecker. LITERATURE CITED Bakshi A , Shemansky JM, Chang C, Binder BM ( 2015 a ) History of research on the plant hormone ethylene . J Plant Growth Regul http://dx.doi.org/10.1007/s00344-015-9522-9 Google Scholar OpenURL Placeholder Text WorldCat Bakshi A, Wilson RL, Lacey RF, Kim H, Wupalapapati SK, Binder BM (2015b) Identification of regions in the receiver domain of the ETHYLENE RESPONSE1 ethylene receptor of Arabidopsis important for functional divergence. Plant Physiol 169: 219 – 232 Booker MA , DeLong A ( 2015 ) Producing the ethylene signal: regulation and diversification of ethylene biosynthetic enzymes . Plant Physiol 169 : 61 – 69 Google Scholar Crossref Search ADS PubMed WorldCat Casteel CL , De Alwis M, Bak A, Dong H, Whitham SA, Jander G ( 2015 ) Disruption of ethylene responses by Turnip mosaic virus mediates suppression of plant defense against the green peach aphid vector . Plant Physiol 169 : 209 – 218 Google Scholar Crossref Search ADS PubMed WorldCat Dubois M , Van den Broeck L, Claeys H, Van Vlierberghe K, Matsui M, Inzé D ( 2015 ) The ETHYLENE RESPONSE FACTORs ERF6 and ERF11 antagonistically regulate mannitol-induced growth inhibition in Arabidopsis . Plant Physiol 169 : 166 – 179 Google Scholar Crossref Search ADS PubMed WorldCat Eccher G , Begheldo M, Boschetti A, Ruperti B, Botton A ( 2015 ) Roles of ethylene production and ethylene receptor expression in regulating apple fruitlet abscission . Plant Physiol 169 : 125 – 137 Google Scholar Crossref Search ADS PubMed WorldCat Gamalero E , Glick BR ( 2015 ) Bacterial modulation of plant ethylene levels . Plant Physiol 169 : 13 – 22 Google Scholar Crossref Search ADS PubMed WorldCat García MJ , Romera FJ, Lucena C, Alcántara E, Pérez-Vicente R ( 2015 ) Ethylene and the regulation of physiological and morphological responses to nutrient deficiencies . Plant Physiol 169 : 51 – 60 Google Scholar Crossref Search ADS PubMed WorldCat Gibbs DJ , Vicente Conde J, Berckhan S, Prasad G, Mendiondo GM, Holdsworth MJ ( 2015 ) Group VII ethylene response factors coordinate oxygen and nitric oxide signal transduction and stress responses in plants . Plant Physiol 169 : 23 – 31 Google Scholar Crossref Search ADS PubMed WorldCat Guan R , Su J, Meng X, Li S, Liu Y, Xu J, Zhang S ( 2015 ) Multilayered regulation of ethylene induction plays a positive role in Arabidopsis resistance against Pseudomonas syringae . Plant Physiol 169 : 299 – 312 Google Scholar Crossref Search ADS PubMed WorldCat Ju C , Chang C ( 2015 ) Mechanistic insights in ethylene perception and signal transduction . Plant Physiol 169 : 85 – 95 Google Scholar Crossref Search ADS PubMed WorldCat Larrainzar E , Riely BK, Kim SC, Carrasquilla-Garcia N, Yu HJ, Hwang HJ, Oh M, Kim GB, Surendrarao AK, Chasman D, et al. ( 2015 ) Deep sequencing of the Medicago truncatula root transcriptome reveals a massive and early interaction between Nodulation factor and ethylene signals . Plant Physiol 169 : 233 – 265 Google Scholar Crossref Search ADS PubMed WorldCat Louis J, Basu S, Varsani S, Castano-Duque L, Jiang V, Williams WP, Felton GW, Luthe DS (2015) Ethylene contributes to maize insect resistance1-mediated maize defense against the phloem sap-sucking corn leaf aphid. Plant Physiol 169: 313 – 324 Müller M , Munné-Bosch S ( 2015 ) Ethylene response factors: a key regulatory hub in hormone and stress signaling . Plant Physiol 169 : 32 – 41 Google Scholar Crossref Search ADS PubMed WorldCat Polko JK, van Rooij JA, Vanneste S, Pierik R, Ammerlaan AMH, Vergeer-van Eijk MH, McLoughlin F, Guhl K, Van Isterdael G, Voesenek LACJ, et al (2015) Ethylene-mediated regulation of A2-type CYCLINs modulates hyponastic growth in Arabidopsis. Plant Physiol 169: 194 – 208 Sasidharan R , Voesenek LACJ ( 2015 ) Ethylene-mediated acclimations to flooding stress . Plant Physiol 169 : 3 – 12 Google Scholar Crossref Search ADS PubMed WorldCat Shi J , Habben JE, Archibald RL, Drummond BJ, Chamberlin MA, Williams RW, Lafitte HR, Weers BP ( 2015 ) Overexpression of ARGOS genes modifies plant sensitivity to ethylene, leading to improved drought tolerance in both Arabidopsis and maize . Plant Physiol 169 : 266 – 282 Google Scholar Crossref Search ADS PubMed WorldCat Spalding EP ( 2006 ) The contributions of Anthony B. Bleecker to ethylene signaling and beyond . Plant Cell 18 : 3347 – 3349 Google Scholar Crossref Search ADS WorldCat Street IH , Aman S, Zubo Y, Ramzan A, Wang X, Shakeel SN, Kieber JJ, Schaller GE ( 2015 ) Ethylene inhibits cell proliferation of the Arabidopsis root meristem . Plant Physiol 169 : 338 – 350 Google Scholar Crossref Search ADS PubMed WorldCat Sun J , Ma Q, Mao T ( 2015 ) Ethylene regulates Arabidopsis microtubule-associated protein WAVE-DAMPENED2-LIKE5 in etiolated hypocotyl elongation . Plant Physiol 169 : 325 – 337 Google Scholar Crossref Search ADS PubMed WorldCat Tao JJ , Cao YR, Chen HW, Wei W, Li QT, Ma B, Zhang WK, Chen SY, Zhang JS ( 2015 ) Tobacco translationally controlled tumor protein interacts with ethylene receptor tobacco Histidine Kinase1 and enhances plant growth through promotion of cell proliferation . Plant Physiol 169 : 96 – 114 Google Scholar Crossref Search ADS PubMed WorldCat Thao NP , Khan MIR, Thu NBA, Hoang XLT, Asgher M, Khan NA, Tran LSP ( 2015 ) Role of ethylene and its cross talk with other signaling molecules in plant responses to heavy metal stress . Plant Physiol 169 : 73 – 84 Google Scholar Crossref Search ADS PubMed WorldCat Ueda H , Kusaba M ( 2015 ) Strigolactone regulates leaf senescence in concert with ethylene in Arabidopsis . Plant Physiol 169 : 138 – 147 Google Scholar Crossref Search ADS PubMed WorldCat Van de Poel B , Smet D, Van Der Straeten D ( 2015 ) Ethylene and hormonal cross talk in vegetative growth and development . Plant Physiol 169 : 61 – 72 Google Scholar Crossref Search ADS PubMed WorldCat Weller JL , Foo EM, Hecht V, Ridge S, Vander Schoor JK, Reid JB ( 2015 ) Ethylene signaling influences light-regulated development in pea . Plant Physiol 169 : 115 – 124 Google Scholar Crossref Search ADS PubMed WorldCat Yamauchi T , Shiono K, Nagano M, Fukazawa A, Ando M, Takamure I, Mori H, Nishizawa NK, Kawai-Yamada M, Tsutsumi N, et al. ( 2015 ) Ethylene biosynthesis is promoted by very-long-chain fatty acids during lysigenous aerenchyma formation in rice roots . Plant Physiol 169 : 180 – 193 Google Scholar Crossref Search ADS PubMed WorldCat Yang C, Ma B, He SJ, Xiong Q, Duan KX, Yin CC, Chen H, Lu X, Chen SY, Zhang JS (2015) MAOHUZI6/ETHYLENE INSENSITIVE3-LIKE1 and ETHYLENE INSENSITIVE3-LIKE2 regulate ethylene response of roots and coleoptiles and negatively affect salt tolerance in rice. Plant Physiol 169: 148 – 165 Yasumura Y, Pierik R, Kelly S, Sakuta M, Voesenek LACJ, Harberd NP (2015) An ancestral role for CONSTITUTIVE TRIPLE RESPONSE1 proteins in both ethylene and abscisic acid signaling. Plant Physiol 169: 283 – 298 Author notes www.plantphysiol.org/cgi/doi/10.1104/pp.15.01242 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Sasidharan, Rashmi; Voesenek, Laurentius A.C.J.

doi: 10.1104/pp.15.00387pmid: 25897003

Abstract Flooding is detrimental for plants, primarily because of restricted gas exchange underwater, which leads to an energy and carbohydrate deficit. Impeded gas exchange also causes rapid accumulation of the volatile ethylene in all flooded plant cells. Although several internal changes in the plant can signal the flooded status, it is the pervasive and rapid accumulation of ethylene that makes it an early and reliable flooding signal. Not surprisingly, it is a major regulator of several flood-adaptive plant traits. Here, we discuss these major ethylene-mediated traits, their functional relevance, and the recent progress in identifying the molecular and signaling events underlying these traits downstream of ethylene. We also speculate on the role of ethylene in postsubmergence recovery and identify several questions for future investigations. Come gather round people wherever you roam And admit that the waters around you have grown And accept it that soon you’ll be drenched to the bone If your time to you is worth saving And you’d better start swimming or you’ll sink like a stone For the times they are a-changing… THE TIMES THEY ARE A-CHANGIN' Words and Music by Bob Dylan Copyright © 1963, 1964 Warner Bros. Inc. Copyright Renewed 1991, 1992 Special Rider Music International Copyright Secured. All Rights Reserved. Reprinted by Permission. Fifty years later, these prophetic words from Dylan’s legendary song strangely ring true. We live in an increasingly wetter world. Flooding events have become more frequent, severe, and unpredictable, a trend that is linked to climate change (Arnell and Liu, 2001; Hirabayashi et al., 2013). Not only do they destroy human lives, but also, they affect plants, on which we depend so much. Flooding negatively affects plant biodiversity, natural species distribution, and global food production because of crop losses (Silvertown et al., 1999; Normile, 2008), because most terrestrial plants, including major crops, are extremely sensitive to wet conditions. It might seem counterintuitive that a molecule so biologically benign and indispensable for plant growth and function is harmful when present in excess. However, this is attributed to the fact that water is an extremely poor medium for gas diffusion. Hampered gas exchange with flooded organs leads to restriction of two vital plant processes: photosynthesis and respiration. The problem is compounded by stagnant and/or turbid floodwaters, because this further restricts the availability of light and oxygen. An energy crisis quickly results owing to an imbalance between energy production and consumption, ultimately causing plant mortality. Flooding survival tactics in the plant kingdom vary widely and include several morphological, anatomical, physiological, and molecular changes that can prolong survival and even facilitate permanent habitation in wet environments (Voesenek and Bailey-Serres, 2015). Initiation of these changes requires accurate and timely perception of water inundation to initiate adaptive responses. The term flooding encompasses both waterlogging and submergence. Waterlogging implies soil flooding, where only roots are exposed to wet conditions. Submergence also immerses the shoot (partially or wholly). Unless otherwise specified, this nomenclature will be used throughout this article. ETHYLENE: AN EARLY AND RELIABLE SIGNAL Other than O2 and CO2, restricted gas diffusion in flooded plant organs also affects the dynamics of another volatile: ethylene. Internal changes in oxygen and ethylene are considered primary signals triggering plant-adaptive responses to flooding. However, the temporal and spatial dynamics of these two gases during the course of a flooding event can be very distinct (Voesenek and Sasidharan, 2013). Even in nonflooded plants, steep oxygen gradients occur in organs, such as seeds and fruits, because of their density and high metabolic demand (Van Dongen and Licausi, 2015). Flooding causes a decrease in oxygen availability to all plant cells. However, this drop in oxygen levels is not uniform and can vary, especially between the shoot and root (Fig. 1). Different oxygen dynamics between these two organs are caused by their direct environments. Flooded roots in waterlogged soils are rapidly depleted of oxygen because of microbial and root respiration (Vashisht et al., 2011). Root oxygen content is consequently strongly dependent on photosynthetically derived oxygen from the shoot when the plant is completely submerged or oxygen that diffuses into an emerged shoot. The flow of oxygen from the shoot to the root is, in turn, influenced by source to sink strength, tissue porosity, and root respiratory demand. Submerged shoots are surrounded by water that is relatively more oxygen replete (Vashisht et al., 2011), especially upper layers that are in contact with the atmosphere (Setter et al., 1987). Internal oxygen levels in the shoot are determined by light availability, presence of leaf gas films, and leaf traits that facilitate underwater photosynthesis and inward diffusion of oxygen, even under restricted conditions of the aquatic environment (Mommer et al., 2004; Pedersen et al., 2009). In fact, in planta oxygen measurements show that, in the presence of sufficient illumination, internal oxygen content can stay at normoxic values in submerged shoots (Vashisht et al., 2011; van Veen et al., 2013). Figure 1. Open in new tabDownload slide Oxygen dynamics during flooding. The root and shoot of a flooded plant have very different oxygen dynamics. Depicted are the general trends in endogenous oxygen levels in the shoot and root of plants when submerged in the light or in darkness. Also shown are typical oxygen concentrations in the surrounding floodwater and soil. Generalized trend lines shown are based on measurements on submerged Arabidopsis plants over a 24-h period (Vashisht et al., 2011). Figure 1. Open in new tabDownload slide Oxygen dynamics during flooding. The root and shoot of a flooded plant have very different oxygen dynamics. Depicted are the general trends in endogenous oxygen levels in the shoot and root of plants when submerged in the light or in darkness. Also shown are typical oxygen concentrations in the surrounding floodwater and soil. Generalized trend lines shown are based on measurements on submerged Arabidopsis plants over a 24-h period (Vashisht et al., 2011). All cells of a plant are capable of synthesizing ethylene, and endogenous levels are largely determined by biosynthesis rates and amounts lost by diffusion to the external environment. Measurements of endogenous ethylene concentrations in flooded plant organs all consistently report rapid (within 1 h) elevation of ethylene to physiologically saturating (1 μL L−1) concentrations after the onset of flooding (Voesenek and Sasidharan, 2013). Flooding-induced increases in the expression and activity of the ethylene biosynthetic enzymes 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC synthase have been reported in several species (Van Der Straeten et al., 2001; Lee et al., 2011; van Veen et al., 2013). However, it is ethylene’s sluggish outward diffusion in water that causes its fast physical entrapment and accumulation to saturating levels in flooded tissues. This rapid buildup upon flooding, independent of most environmental conditions, makes ethylene a reliable and timely signal conveying flooding stress ahead of the onset of hypoxic and anoxic conditions. It is, therefore, not surprising that ethylene is a key regulator of several flood-adaptive traits. FLOODING Waterlogging Although soil flooding directly exposes only plant roots to the stress, whole-plant functioning is affected in the absence of timely stress perception and initiation of appropriate adaptive responses (Sauter, 2013). As soil microbes and roots rapidly consume the remaining oxygen in the waterlogged soil, roots switch to inefficient anaerobic fermentation to generate ATP needed for proper functioning. Ultimately, available carbohydrate reserves are used, and as anoxia sets in, starvation, impaired membrane integrity, and entry of phytotoxic compounds from the waterlogged soil can all combine to severely compromise root growth and function. The resulting inability to transport water and nutrients also affects shoot function, resulting in symptoms such as wilting, senescence, and death. Adaptive traits that improve aeration, thereby preventing root anoxia, are, therefore, critical to maintain root function and whole-plant survival of waterlogging. These traits include the formation of a suberin/lignin barrier in the root that prevents radial loss of oxygen to enhance its delivery to the root tip (Shiono et al., 2011), increased formation of air spaces (aerenchyma) that increases organ porosity and root aeration (Takahashi et al., 2014), and formation of aerenchyma-rich adventitious roots (ARs; Sauter, 2013). Submergence Complete submergence is even more detrimental, completely cutting off plant access to the aerial environment and seriously compromising photosynthesis (Voesenek and Bailey-Serres, 2015). As with waterlogging, strategies to cope with submergence are directed toward improving aeration. An escape strategy involving directed shoot growth out of floodwaters restores atmospheric contact (Hattori et al., 2011; Sasidharan et al., 2013; van Veen et al., 2013). After oxygen entry into the shoot, oxygenation of the rest of the plant is facilitated by aerenchymatous tissue (Pierik et al., 2009). Some species improve underwater photosynthesis rates aided by specialized leaf traits and gas films (Mommer and Visser, 2005). If the floods are deep, the energy-depleting escape strategy is not beneficial, because plant growth will not result in emergence. In this case, restricted growth is an alternative strategy that economizes on reserves for postsubmergence growth reestablishment (Fukao et al., 2006; Sasidharan et al., 2013; van Veen et al., 2013). Ethylene is an important regulator of several of the aforementioned traits, including adaptive systemic responses of the shoot upon waterlogging. During waterlogging, the ethylene precursor ACC produced in the flooded root is transported through the xylem stream to the shoot (Jackson, 2002). Here, oxygen-mediated conversion of ACC to ethylene triggers the adaptive changes typically observed in shoots of waterlogged plants, such as shoot aerenchyma formation and leaf nastic movements (Jackson, 2002). Ethylene-Mediated Flooding-Adaptive Traits Aerenchyma: Airing the Plant Aerenchymatous tissue can develop in both roots and shoots depending on the species and environmental conditions (Colmer and Pedersen, 2008; Parlanti et al., 2011; Steffens et al., 2011). These air-filled breaches extending throughout the inner plant connect flooded parts with those still in aerial contact and greatly improve internal aeration. It also reduces the number of oxygen-consuming cells, which is obviously advantageous in low-oxygen environments. Even during complete submergence, aerenchymatous tissue could facilitate access to available oxygen sources, such as the relatively oxygen-rich floodwater, gas films on submerged leaves, and oxygen produced by underwater photosynthesis. Two main types of aerenchymatous tissue are lysigenous (formed by regulated cortical cell death) and schizogenous (involving cell separation during tissue development; Takahashi et al., 2014). Both types can be either constitutive or inducible upon flooding. Constitutive aerenchyma is found in both wetland and nonwetland plants and can be further enhanced by flooding (Drew et al., 2000; Yamauchi et al., 2014). In other species, waterlogging induces aerenchyma formation (Rajhi et al., 2011; Yamauchi et al., 2014). Most research on aerenchyma formation has focused on lysigenous aerenchyma, which will be discussed here. Ethylene is an important hormonal signal triggering flooding-induced lysigenous aerenchyma formation in several species studied, including rice (Oryza sativa; Fukao and Bailey-Serres, 2008a; Sauter, 2013), maize (Zea mays; Rajhi et al., 2011), and wheat (Triticum aestivum; Yamauchi et al., 2014). Aerenchyma formation can be induced in maize roots by ethylene application, and conversely, inhibitors of ethylene biosynthesis or perception can block the process in flooded or hypoxic roots (Rajhi et al., 2011). In rice, constitutive aerenchyma forms during normal development but gets further augmented in response to flooding and hypoxia in a process that is ethylene dependent (Takahashi et al., 2014; Yukiyoshi and Karahara, 2014). Ethylene was so far not considered relevant to the formation of constitutive aerenchyma, but a recent report suggests otherwise. In a so-called sandwich method, Japonica rice caryopses were germinated between two agar slabs. This allowed application of different chemicals to different sides of the same emergent root. ACC treatment only on one side resulted in a higher percentage of aerenchyma in the ACC-exposed developing root, whereas 1-methylcyclopropane (an ethylene perception inhibitor) elicited a reverse trend (Yukiyoshi and Karahara, 2014). The ethylene dependency and inducibility of aerenchyma formation can strongly vary between genotypes (Parlanti et al., 2011; Yin et al., 2013). For example, the rice varieties FR13A and Arborio Precoce both show constitutive aerenchyma in leaf sheaths that is further enhanced by submergence. However, ethylene signaling was implicated in aerenchyma formation only in the Arborio Precoce variety (Parlanti et al., 2011). Downstream of the ethylene-signaling module, several signaling components have been identified using pharmacological approaches in maize roots (Fig. 2). In these experiments, manipulation of Ca2+ levels and use of chemicals interfering with phosphorylation and phosphoinositide signaling helped identify a signaling pathway leading from ethylene and involving heterotrimeric G proteins, protein phosphorylation, and Ca2+ as essential signaling components (Drew et al., 2000). Reactive oxygen species (ROS) are another important component in the ethylene-mediated signaling network. In rice internodes, ethylene-induced formation of stem aerenchyma involved increased superoxide radicals and hydrogen peroxide in preaerenchyma cells. Genetic manipulation and exogenous application of hydrogen peroxide showed that ROS could induce aerenchyma in a dose-dependent manner (Steffens et al., 2011). In wheat roots as well, ethylene-mediated aerenchyma formation was dependent on controlled ROS production by NADPH oxidases (Yamauchi et al., 2014). ROS are important components in cell death signaling, and their regulated generation might be important in triggering physiological cell death to form aerenchyma. The demise of specific cells in the root cortex by genetically programmed cell death is the terminal step in aerenchyma formation. In maize cells, the process has been followed in detail using light and electron microscopy and revealed the distinct stages of controlled cell death during aerenchyma formation (Gunawardena et al., 2001). These start with plasma membrane invagination and vesicle formation followed by nuclear events, such as chromatin condensation and DNA fragmentation, and ultimately, cell wall breakdown and collapse of the entire cell, leaving behind empty air spaces. Cell wall degradation involves ethylene-mediated increases in activities of enzymes, such as cellulases, pectinases, and xylanases (Bragina et al., 2003; Xu et al., 2013). Figure 2. Open in new tabDownload slide Ethylene-mediated flood-adaptive traits. An overview of the ethylene signaling networks regulating flooding-induced shoot elongation (A), hyponasty (B), aerenchyma (C), and AR growth (D). Depicted are generalized schemes based on studies in one or more species. Interactions and hierarchy of signaling components can vary depending on species. Images shown are R. palustris (A), Arabidopsis (B), barley (Hordeum vulgare) root cross sections (C), and rice stem nodes (D). Photographs courtesy of Shiono Katsuhiro (B) and Bianka Steffens (D). COP1, CONSTITUTIVE PHOTOMORPHOGENIC1; MT2B, METALLOTHIONEIN 2B; PIF, PHYTOCHROME INTERACTING FACTOR; RBOH, RESPIRATORY BURST OXIDASE HOMOLOG; SLR1, SLENDER RICE1; SLRL1, SLENDER RICE-LIKE1; XTH, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE. Figure 2. Open in new tabDownload slide Ethylene-mediated flood-adaptive traits. An overview of the ethylene signaling networks regulating flooding-induced shoot elongation (A), hyponasty (B), aerenchyma (C), and AR growth (D). Depicted are generalized schemes based on studies in one or more species. Interactions and hierarchy of signaling components can vary depending on species. Images shown are R. palustris (A), Arabidopsis (B), barley (Hordeum vulgare) root cross sections (C), and rice stem nodes (D). Photographs courtesy of Shiono Katsuhiro (B) and Bianka Steffens (D). COP1, CONSTITUTIVE PHOTOMORPHOGENIC1; MT2B, METALLOTHIONEIN 2B; PIF, PHYTOCHROME INTERACTING FACTOR; RBOH, RESPIRATORY BURST OXIDASE HOMOLOG; SLR1, SLENDER RICE1; SLRL1, SLENDER RICE-LIKE1; XTH, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE. Recent microarray studies using microdissected cortical cells from maize root tips and 1-methylcyclopropane have identified ethylene-mediated transcriptomic changes occurring in preaerenchymatous cortical cells (Rajhi et al., 2011). Major changes were observed in functional gene categories associated with ethylene signaling, ROS metabolism, cell wall degradation, and calcium signaling, providing support to the signaling network constructed from the more physiological studies mentioned above. What determines the lysis of only specific cortical cells is still unclear. Preaerenchymatous cells in rice have distinct characteristics, including low starch, thinner cell walls, less chlorophyll, and higher amounts of ROS (Steffens et al., 2011). However, details of how and when this cellular identity is established and what marks these cells for their final fate need to be determined. One possibility is the differential ethylene sensitivity of preaerenchyma cells, but this remains to be verified. Advantageous: Adventitious Roots These postembryonic roots originating from shoots and the upper parts of the original roots are observed in several species, including rice, Solanum dulcamara, Rumex spp., and tomato (Solanum lycopersicum) upon flooding (Visser et al., 1996; Steffens et al., 2006; Vidoz et al., 2010; Dawood et al., 2014). Aerenchyma-rich ARs improve shoot-root gas diffusion and can completely replace flood-damaged soil-borne roots. Ethylene is important for AR formation, although its role can differ depending on the species (McDonald and Visser, 2003; Steffens et al., 2006; Vidoz et al., 2010). For example, in waterlogged Rumex palustris, AR formation is mediated by ethylene-induced increase in the auxin sensitivity of root-forming tissue (Visser et al., 1996). In tomato, waterlogging-induced AR formation requires ethylene perception by the Never Ripe receptor. Elevated ethylene levels stimulate auxin transport to the shoot, where an induction of ACC synthase genes results in increased stem ethylene. This then directs auxin flow toward the submerged stem to initiate AR growth. Accordingly, an inhibition of auxin transport hampers normal adventitious rooting (Vidoz et al., 2010). Flooding-induced AR growth from preexisting root primordia, such as in rice, requires penetration of overlying cell layers. Studies in deep-water rice internodes have unraveled how AR growth from root primordia is coordinated with the death of overlying epidermal cells to facilitate AR emergence. Physiological experiments have convincingly shown the primary role of ethylene in triggering AR growth and epidermal cell death, and both processes are synergistically enhanced by GA and inhibited by abscisic acid (ABA; Steffens and Sauter, 2005; Steffens et al., 2006). Although microarray studies revealed that the epidermal cells overlaying root primordia have a very distinct cellular identity (Steffens and Sauter, 2009), the precise signal that caused only these specific cells to die remained unknown. It is now clear that this trigger is the mechanical stimulus provided by the underlying root primordia when they start growing (Steffens et al., 2012). The growing ARs exert a mechanical force on the epidermal cells overlying them in a process that also requires ethylene-mediated ROS formation (Fig. 2). Interestingly, ROS or ethylene alone could only induce ectopic cell death when a dummy force was also present. These studies provide a clear example of how a mechanical force can provide the spatial signal required to localize ethylene action to a targeted cluster of cells during flooding when ethylene accumulates equally in all flooded cells. Rising to the Occasion: Shoot Hyponasty Upward leaf movement or hyponasty is considered an important acclimation to flooding and results from an unequal growth rate of the cells on the abaxial and adaxial sides of the affected organ (Cox et al., 2004; Polko et al., 2012). Flooding-induced shoot hyponasty has been observed in species, such as Rumex spp. and Arabidopsis (Arabidopsis thaliana), in response to flooding (Cox et al., 2003; Lee et al., 2011; Rauf et al., 2013). During the course of a flooding event, as floodwaters rise, hyponastic growth, especially in such rosette species, would elevate the leaves above the water. When submerged, the almost vertical reorientation of Rumex spp. leaves also directs subsequent shoot elongation on the shortest path out of the water. In R. palustris, early hyponastic growth upon submergence is a prerequisite for subsequent shoot elongation to outgrow floodwaters (Cox et al., 2003). In Arabidopsis, such flooding-induced petiole elongation is absent, but hyponasty is observed upon both waterlogging and complete submergence (Lee et al., 2011; Rauf et al., 2013). In both R. palustris and Arabidopsis, hyponastic growth is driven by ethylene, and exogenous application of ethylene mimics this trait, even in the absence of flooding (Millenaar et al., 2005; Heydarian et al., 2010). Increased ethylene biosynthesis in waterlogged Arabidopsis is linked to transcript accumulation of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE5 (ACO5). ACO5 is a direct target of the NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC2]) transcription factor SPEEDY HYPONASTIC GROWTH (SHYG) that is induced in shoots upon waterlogging. SHYG itself is ethylene inducible, although it is unclear what causes its early induction in the shoots upon root flooding. Accordingly, waterlogging fails to induce a wild-type hyponastic response in shyg and aco5 mutants (Rauf et al., 2013). In R. palustris, ethylene regulates rapid hyponastic growth by interaction with auxin and ABA (Cox et al., 2004). Within the first 1 h of submergence, a dramatic ethylene-mediated depletion of ABA is required to prevent the inhibitory effect of this hormone on the initiation, speed, and maintenance of hyponastic growth. Ethylene also promotes a lateral redistribution of auxin to the outer cell layers in the petiole (Cox et al., 2004), likely causing differential growth because of expansion of specific cells. Indeed, in Arabidopsis petioles, ethylene causes expansion of cells in a proximal 3- to 4-mm zone in the abaxial epidermal cell layer coinciding with the selective expression of cell wall-modifying expansins and a transverse growth-promoting orientation of cortical microtubules (Polko et al., 2012; Rauf et al., 2013). In R. palustris, a third hormone, GA, positively regulates the speed of hyponasty. However, this is attributed to GA already present in the petioles, because submergence-induced GA levels increase only after the onset of hyponasty (Benschop et al., 2006). Studies in Arabidopsis have also added brassinosteroids (BRs) downstream of ethylene. In the ROTUNDIFOLIA3 mutant, affected in a cytochrome P450 involved in BR biosynthesis, ethylene was unable to induce differential cell expansion and therefore, leaf hyponasty. Similar observations were made upon chemical perturbation of BR biosynthesis, supporting the involvement of BR action in ethylene-mediated hyponasty (Polko et al., 2013; Fig. 2). The Right Time to Grow: Ethylene-Regulated Shoot Elongation Depending upon the flooding regime, plants in such hydrological niches show distinct growth responses classified into two antithetical strategies: escape and quiescence (Voesenek and Bailey-Serres, 2015). Entrapped ethylene is the primary regulator for both of these strategies, invoking species-dependent stimulated or restricted shoot growth. Escape A robust escape response is observed in many species inhabiting niches with prolonged and shallow flooding. This brisk growth of the youngest leaves keeps them ahead of the rising floodwaters and in atmospheric contact. In the well-studied models of rice and Rumex spp., the hormonal trinity of ethylene, GA, and ABA forms the conserved regulatory core of this response (Fig. 2). In R. palustris, the escape response can be replicated by ethylene application and conversely, strongly dampened by inhibition of ethylene perception (Cox et al., 2004; Heydarian et al., 2010). Detailed growth kinetics, physiological analyses, and endogenous hormone measurements coupled with genome-wide transcriptome profiling have facilitated a detailed reconstruction of the timeline of molecular events underlying this impressive growth response (van Veen et al., 2013; Voesenek and Bailey-Serres, 2015). Ethylene rapidly accumulates to saturating levels (>1 μL L−1) in submerged R. palustris. Although measurements indicate that this takes up to 1 h, ethylene-mediated effects are detected much earlier. Ethylene-induced petiole cell wall acidification occurs within 20 min after submergence (Vreeburg et al., 2005). This sets the optimal milieu for the activities of cell wall-modifying proteins, like expansins and xyloglucan endotransglucosylase/hydrolases, that mediate cellular expansion (Sasidharan et al., 2011). Within the first 1 h, ethylene also causes a massive (up to 80%) depletion of endogenous ABA, which is critical to evince shoot elongation. This is mediated by a down-regulation of the anabolic enzyme 9-cis-epoxycarotenoid dioxygenase and an up-regulation of the catabolic enzyme ABA-8-hydroxylase (Benschop et al., 2005; van Veen et al., 2013). ABA does not curb early ethylene-induced apoplastic acidification or the up-regulation of specific expansin genes (Vreeburg et al., 2005). However, the maintained block on ABA by ethylene is essential to permit GA-mediated growth stimulation, which occurs after 4 to 5 h of submergence (Benschop et al., 2006; van Veen et al., 2013). In this later phase, flooded petioles also show increased expression of genes associated with shade avoidance and photomorphogenesis. These genes include orthologs of the Arabidopsis E3-ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1, the basic helix-loop-helix protein KIDARI, and PHYTOCHROME INTERACTING FACTORS, which have established roles in promoting growth-related events linked to light signaling. However, in submerged R. palustris, the accumulation of these transcripts is not associated with changes in the light environment but instead, requires ethylene-mediated ABA reduction (van Veen et al., 2013). Interestingly, ABA depletion mediated by ethylene was found to be key factor regulating natural variation in flooding-induced shoot elongation in natural accessions of R. palustris (Chen et al., 2010). Deep-water rice escapes from submergence with a spectacular growth response (20–25 cm d−1), which allows the hollow rice stem to stay above water and aerate the rest of the plant (Hattori et al., 2009). Flooding-induced intralacunar accumulation of ethylene is the primary regulator of this internodal elongation (Hattori et al., 2009). As in R. palustris, this positive effect of ethylene is the result of modulation of the contrasting effects of ABA and GA on shoot elongation (Fukao and Bailey-Serres, 2008a). Ethylene’s primary effects in this respect are 2-fold. First, it causes depletion of ABA by regulation of ABA metabolism; second, it promotes GA-mediated internodal elongation by increasing tissue sensitivity to and biosynthesis of GA (Hoffmann-Benning and Kende, 1992; Saika et al., 2007). GA is essential for internodal elongation, and flooded rice has increased endogenous levels of bioactive GA1 (Hoffmann-Benning and Kende, 1992). Exogenous ABA application restricts elongation because of reduced tissue responsiveness to GA. Farther downstream, induction of the expression and activity of expansins facilitates cellular expansion and growth (Choi et al., 2003). Shoot elongation is a quantitative trait attributed primarily to the SNORKEL (SK) locus on chromosome 12, which together with two other loci on chromosomes 1 and 3, can account for the full escape response (Hattori et al., 2008, 2009, 2011). The SK locus encodes two ethylene-inducible group VII ETHYLENE RESPONSE FACTOR (ERF) transcription factors: SK1 and SK2. Expression of these genes in nondeep-water rice triggers internodal elongation, even in nonflooded plants (Hattori et al., 2009). How signaling from the SKs leads to downstream events culminating in shoot elongation is unclear. Quantitative trait loci analyses on GA-controlled responses of deep-water rice internodes indicate that signaling downstream of the SKs and the other uncharacterized quantitative trait loci regions coordinately affects GA biosynthesis (Ayano et al., 2014; Nagai et al., 2014). Quiescence The escape strategy is only beneficial if leaves emerge, thereby restoring gas exchange with the atmosphere. Additionally, this response must bring the leaves above the water before depletion of existing carbohydrate reserves, and the shoot must be porous enough to act as a snorkel for the rest of the submerged plant (Pierik et al., 2009; Akman et al., 2012). When floods are transient or too deep to outgrow, the quiescence strategy is favored (Fukao et al., 2006; Akman et al., 2012; van Veen et al., 2013). The Rumex sp. Rumex acetosa and lowland rice are well-studied examples of quiescent behavior, where restriction of growth and other energetically expensive processes allow conservation of resources until the floodwaters recede. In R. acetosa, submergence causes an active restriction of petiole growth (van Veen et al., 2013). Although ethylene accumulates to saturating levels, there is no ABA down-regulation or GA increase (Benschop et al., 2005). Instead, R. acetosa displays metabolic adjustments, consistent with energy conservation (van Veen et al., 2013). The quiescent characteristics of lowland rice are attributed to the SUBMERGENCE1 (SUB1) locus originally identified in the submergence tolerant FR13A landrace. This locus encodes two to three (SUB1A, SUB1B, or SUB1C) transcription factors also belonging to the group VII ERF family. Rice varieties possessing the SUB1A gene are tolerant to complete submergence and survive such conditions for up to 2 weeks (Fukao et al., 2006; Xu et al., 2006; Perata and Voesenek, 2007). Comparison of near-isogenic lines differing only in the possession of the SUB1 locus revealed the mechanism by which SUB1A confers submergence tolerance (Fukao et al., 2006, 2011). Submergence causes massive accumulation of SUB1A transcripts, much higher than can be replicated with ethylene application alone. It is presumed that SUB1A is also positively regulated by other flooding-associated signals, such as low oxygen or starvation. Interestingly, SUB1A induction feeds back on ethylene biosynthesis, and it dampens both ethylene production and responsiveness (Fukao et al., 2006), thereby curbing ethylene-mediated shoot elongation in SUB1A-containing lines. SUB1A enhances transcript abundance of Slender Rice1 and Slender Rice-Like1, which negatively regulate GA responses (Fukao and Bailey-Serres, 2008b). The presence of SUB1A, therefore, restricts growth by blocking GA-mediated activation of growth-promoting genes. Submergence-induced or ectopic expression of SUB1A has revealed that it is responsible for the lower expression of genes associated with cell elongation, starch metabolism, and induction of fermentation genes (Fukao et al., 2006). SUB1A-mediated tolerance, therefore, stems from curbing carbohydrate use and preventing an energy crisis during submergence. ETHYLENE AND PLANT OXYGEN SENSING Arabidopsis group VII ERFs have also been intensively studied in the context of their regulatory role in acclimative responses to flooding and hypoxic stress (Sasidharan and Mustroph, 2011; Bailey-Serres et al., 2012; Van Dongen and Licausi, 2015). Arabidopsis has five group VII ERFs: Related to APETALA2 12 (RAP2.12), RAP2.2, RAP2.3, Hypoxia responsive1 (HRE1), and HRE2 (Bailey-Serres et al., 2012). At least four (except RAP2.3) members have been shown to be redundantly involved in the regulation of hypoxia-responsive gene expression and survival (Papdi et al., 2008; Hinz et al., 2010; Licausi et al., 2010; Hess et al., 2011). All five Arabidopsis group VII ERFs possess a signature N-terminal motif that makes them susceptible to oxygen-dependent degradation through the N-end rule pathway (NERP) of targeted proteolysis (Gibbs et al., 2011; Licausi et al. 2011; Bailey-Serres et al., 2012). This conserved protein degradation pathway links the fate of a protein to its N-end terminus. Recent studies in Arabidopsis have shown that group VII ERF abundance and consequently, hypoxia responses are regulated by the oxygen-dependent degradation of group VII ERFs through the NERP (Gibbs et al., 2011; Licausi et al., 2011; Sasidharan and Mustroph, 2011). In these proteins possessing the characteristic N-terminal sequence starting with Met-Cys, the cleavage of the terminal Met exposes the Cys residue. During normoxic conditions, the oxidation of this Cys commits the protein to degradation through the NERP. A drop in oxygen levels limits the degradation of these proteins, allowing them to move to the nucleus and switch on target gene expression, including anaerobic metabolism and other survival-related genes (Gibbs et al., 2011; Licausi et al., 2011). Experiments have also established that, during normoxia, RAP2.12 can escape degradation because of its association with plasma membrane proteins Acyl CoA Binding Protein1 (ACBP1) and ACBP2. Hypoxia triggers RAP2.12 dissociation and translocation to the nucleus to initiate target gene expression (Licausi et al., 2011). Interestingly, the N termini of the rice SKs deviate from the conserved N-terminal degrons associated with the NERP, and experiments have established that SUB1A is not an N-end rule substrate (Gibbs et al., 2011). It is speculated that this N-end rule independence of SUB1A coupled with its ethylene inducibility would initiate quiescence-related energy management before the onset of hypoxia and result in higher submergence tolerance. A detailed update on the understanding of group VII ERFs and the N-end rule-mediated mechanism of oxygen sensing can be found elsewhere (Gibbs et al., 2015). AFTER THE FLOODS: ETHYLENE AND POSTSUBMERGENCE ACCLIMATION When floodwaters subside, energy-depleted plant tissues acclimated to low-oxygen and low-light conditions are abruptly reexposed to the terrestrial environment. Reaeration is typically associated with an increased formation of reactive ROS molecules and harmful metabolites. Normally, ROS production occurs as part of normal cellular metabolism and is kept in check with an active scavenging system (Blokhina and Fagerstedt, 2010). However, flooding stress disrupts this carefully maintained homeostasis. This is evidenced by the high levels of ROS-related lipid peroxidation and cellular damage observed during reoxygenation (Fukao et al., 2011). Root cell membrane damage resulting from this lipid peroxidation is likely the cause of another frequently observed symptom of reaeration: dehydration stress. Despite excessive water in the soil, some plants display symptoms of water deficit after reoxygenation, such as wilted leaves, and up-regulate dehydration-responsive genes (Setter et al., 2010; Fukao et al., 2011; Tamang et al., 2014; Tsai et al., 2014). These symptoms could be caused by reduced hydraulic conductance of flooded roots (Rodríguez-Gamir et al., 2011). Flooding tolerance, therefore, involves surviving not just submergence but also, thereafter, limiting dehydration and oxidative stress and recovering growth and photosynthesis. The molecular responses and signaling events occurring in plants postflooding have received scant attention, and also, the possible role of ethylene after desubmergence has received little attention. Although ethylene trapped by floodwaters would escape from submerged plant organs upon desubmergence, studies report that ethylene biosynthesis increases during reoxygenation (Voesenek et al., 2003; Tsai et al., 2014). An assessment of ethylene production after desubmergence in several species revealed that the strongest response was in flooding-escape species, assigning it a functional significance (Voesenek et al., 2003). This ethylene production would allow shoot elongation to continue, even after the leaf tips have reemerged. However, postsubmergence ethylene signaling might also be of relevance in nonescape species, albeit for different functional reasons, such as improving postsubmergence recovery. In Arabidopsis, reoxygenation is associated with increased expression of ethylene biosynthetic enzymes (Tsai et al., 2014). Furthermore, the ethylene-insensitive (ein) mutants ein2-5 and ein3eil1 showed increased sensitivity to postanoxic stress. This could be linked to the impaired regulation of many functional gene clusters associated with ABA biosynthesis, dehydration, and heat shock proteins (Tsai et al., 2014). The submergence tolerance of SUB1 rice also extends to better postsubmergence recovery. The ethylene-inducible SUB1A gene mediates improved tolerance to dehydration and oxidative stress by inducing genes associated with ROS amelioration and acclimation to dehydration (Fukao et al., 2011). Although it is apparent that ethylene modulates plant responses postsubmergence, there is plenty to be investigated. The role of ethylene in regulating postsubmergence-mediated drought responses, the interaction with ROS, and ethylene’s possibly distinct roles in postsubmergence recovery of quiescent/escape species are some interesting aspects for future research. FUTURE OUTLOOK Studies so far have linked ethylene primarily to the regulation of morphological and anatomical traits that improve aeration in a flooded plant. These traits are triggered early upon flooding before the onset of severe oxygen deprivation. Because of the oxygen dependency of ethylene biosynthesis, ethylene is not considered an important regulator of anoxia tolerance. However, recent studies showing improved anoxia survival in ethylene-pretreated plants (van Veen et al., 2013) challenge this perception. Indeed, ethylene signaling may be of little relevance during anoxia, but its presence in the early stages of flooding could already prime for forthcoming oxygen deprivation. Ethylene, therefore, seems to mediate plant responses to all stages of a flooding event. The challenge of future studies will be to further unravel the distinct molecular events occurring in each of these flooding-related phases. The molecular basis of ethylene priming, the downstream events that ethylene mediates, especially through various ERFs, its interaction with other flooding-associated signals, such as ROS, sugars, and nitric oxide, and its regulation of postsubmergence recovery are just some of the pertinent research areas pending additional investigation. ACKNOWLEDGMENTS We thank Katsuhiro Shiono, Bianka Steffens, and Joanna Polko for image inputs and useful comments for the figures in this article. Glossary AR adventitious root BR brassinosteroid NERP N-end rule pathway ROS reactive oxygen species LITERATURE CITED Akman M , Bhikharie AV, McLean EH, Boonman A, Visser EJW, Schranz ME, van Tienderen PH ( 2012 ) Wait or escape? Contrasting submergence tolerance strategies of Rorippa amphibia, Rorippa sylvestris and their hybrid . Ann Bot (Lond) 109 : 1263 – 1276 Google Scholar Crossref Search ADS WorldCat Arnell N , Liu C ( 2001 ) Hydrology and water resources. In JJ McCarthy, OF Canziani, NA Leary, DJ Dokken, KS White, eds, Climate Change 2001: Impacts, Adaptation and Vulnerability. Cambridge University Press, Cambridge, UK, pp 191–234 Ayano M , Kani T, Kojima M, Sakakibara H, Kitaoka T, Kuroha T, Angeles-Shim RB, Kitano H, Nagai K, Ashikari M ( 2014 ) Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice . Plant Cell Environ 37 : 2313 – 2324 Google Scholar Crossref Search ADS PubMed WorldCat Bailey-Serres J , Fukao T, Gibbs DJ, Holdsworth MJ, Lee SC, Licausi F, Perata P, Voesenek LACJ, van Dongen JT ( 2012 ) Making sense of low oxygen sensing . Trends Plant Sci 17 : 129 – 138 Google Scholar Crossref Search ADS PubMed WorldCat Benschop JJ , Bou J, Peeters AJ, Wagemaker N, Gühl K, Ward D, Hedden P, Moritz T, Voesenek LA ( 2006 ) Long-term submergence-induced elongation in Rumex palustris requires abscisic acid-dependent biosynthesis of Gibberellin1 . Plant Physiol 141 : 1644 – 1652 Google Scholar Crossref Search ADS PubMed WorldCat Benschop JJ , Jackson MB, Gühl K, Vreeburg RAM, Croker SJ, Peeters AJM, Voesenek LACJ ( 2005 ) Contrasting interactions between ethylene and abscisic acid in Rumex species differing in submergence tolerance . Plant J 44 : 756 – 768 Google Scholar Crossref Search ADS PubMed WorldCat Blokhina O , Fagerstedt KV ( 2010 ) Oxidative metabolism, ROS and NO under oxygen deprivation . Plant Physiol Biochem 48 : 359 – 373 Google Scholar Crossref Search ADS PubMed WorldCat Bragina TV , Rodionova NA, Grinieva GM ( 2003 ) Ethylene production and activation of hydrolytic enzymes during acclimation of maize seedlings to partial flooding . Russ J Plant Physiol 50 : 794 – 798 Google Scholar Crossref Search ADS WorldCat Chen X , Pierik R, Peeters AJM, Poorter H, Visser EJW, Huber H, de Kroon H, Voesenek LACJ ( 2010 ) Endogenous abscisic acid as a key switch for natural variation in flooding-induced shoot elongation . Plant Physiol 154 : 969 – 977 Google Scholar Crossref Search ADS PubMed WorldCat Choi D , Lee Y, Cho HT, Kende H ( 2003 ) Regulation of expansin gene expression affects growth and development in transgenic rice plants . Plant Cell 15 : 1386 – 1398 Google Scholar Crossref Search ADS PubMed WorldCat Colmer TD , Pedersen O ( 2008 ) Oxygen dynamics in submerged rice (Oryza sativa) . New Phytol 178 : 326 – 334 Google Scholar Crossref Search ADS PubMed WorldCat Cox MCH , Benschop JJ, Vreeburg RAM, Wagemaker CA, Moritz T, Peeters AJM, Voesenek LACJ ( 2004 ) The roles of ethylene, auxin, abscisic acid, and gibberellin in the hyponastic growth of submerged Rumex palustris petioles . Plant Physiol 136 : 2948 – 2960 Google Scholar Crossref Search ADS PubMed WorldCat Cox MCH , Millenaar FF, Van Berkel YE, Peeters AJ, Voesenek LA ( 2003 ) Plant movement. Submergence-induced petiole elongation in Rumex palustris depends on hyponastic growth . Plant Physiol 132 : 282 – 291 Google Scholar Crossref Search ADS PubMed WorldCat Dawood T , Rieu I, Wolters-Arts M, Derksen EB, Mariani C, Visser EJW ( 2014 ) Rapid flooding-induced adventitious root development from preformed primordia in Solanum dulcamara . AoB Plants 6 : plt058 Google Scholar Crossref Search ADS PubMed WorldCat Drew MC , He CJ, Morgan PW ( 2000 ) Programmed cell death and aerenchyma formation in roots . Trends Plant Sci 5 : 123 – 127 Google Scholar Crossref Search ADS PubMed WorldCat Fukao T , Bailey-Serres J ( 2008 a ) Ethylene - A key regulator of submergence responses in rice . Plant Sci 175 : 43 – 51 Google Scholar Crossref Search ADS WorldCat Fukao T , Bailey-Serres J ( 2008 b ) Submergence tolerance conferred by Sub1A is mediated by SLR1 and SLRL1 restriction of gibberellin responses in rice . Proc Natl Acad Sci USA 105 : 16814 – 16819 Google Scholar Crossref Search ADS PubMed WorldCat Fukao T , Xu K, Ronald PC, Bailey-Serres J ( 2006 ) A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice . Plant Cell 18 : 2021 – 2034 Google Scholar Crossref Search ADS PubMed WorldCat Fukao T , Yeung E, Bailey-Serres J ( 2011 ) The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice . Plant Cell 23 : 412 – 427 Google Scholar Crossref Search ADS PubMed WorldCat Gibbs DJ , Conde JV, Berckhan S, Prasad G, Mendiondo GM, Holdsworth MJ ( 2015 ) Group VII ethylene response factors coordinate oxygen and nitric oxide signal transduction and stress responses in plants . Plant Physiol 169 : 23 – 31 Google Scholar Crossref Search ADS PubMed WorldCat Gibbs DJ , Lee SC, Isa NM, Gramuglia S, Fukao T, Bassel GW, Correia CS, Corbineau F, Theodoulou FL, Bailey-Serres J, et al. ( 2011 ) Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants . Nature 479 : 415 – 418 Google Scholar Crossref Search ADS PubMed WorldCat Gunawardena AHLAN , Pearce DM, Jackson MB, Hawes CR, Evans DE ( 2001 ) Characterisation of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize (Zea mays L.) . Planta 212 : 205 – 214 Google Scholar Crossref Search ADS PubMed WorldCat Hattori Y , Nagai K, Ashikari M ( 2011 ) Rice growth adapting to deepwater . Curr Opin Plant Biol 14 : 100 – 105 Google Scholar Crossref Search ADS PubMed WorldCat Hattori Y , Nagai K, Furukawa S, Song XJ, Kawano R, Sakakibara H, Wu J, Matsumoto T, Yoshimura A, Kitano H, et al. ( 2009 ) The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water . Nature 460 : 1026 – 1030 Google Scholar Crossref Search ADS PubMed WorldCat Hattori Y , Nagai K, Mori H, Kitano H, Matsuoka M, Ashikari M ( 2008 ) Mapping of three QTLs that regulate internode elongation in deepwater rice . Breed Sci 58 : 39 – 46 Google Scholar Crossref Search ADS WorldCat Hess N , Klode M, Anders M, Sauter M ( 2011 ) The hypoxia responsive transcription factor genes ERF71/HRE2 and ERF73/HRE1 of Arabidopsis are differentially regulated by ethylene . Physiol Plant 143 : 41 – 49 Google Scholar Crossref Search ADS PubMed WorldCat Heydarian Z , Sasidharan R, Cox MCH, Pierik R, Voesenek LACJ, Peeters AJM ( 2010 ) A kinetic analysis of hyponastic growth and petiole elongation upon ethylene exposure in Rumex palustris . Ann Bot (Lond) 106 : 429 – 435 Google Scholar Crossref Search ADS WorldCat Hinz M , Wilson IW, Yang J, Buerstenbinder K, Llewellyn D, Dennis ES, Sauter M, Dolferus R ( 2010 ) Arabidopsis RAP2.2: an ethylene response transcription factor that is important for hypoxia survival . Plant Physiol 153 : 757 – 772 Google Scholar Crossref Search ADS PubMed WorldCat Hirabayashi Y , Mahendran R, Koirala S, Konoshima L, Yamazaki D, Watanabe S, Kim H, Kanae S ( 2013 ) Global flood risk under climate change . Nat Clim Chang 3 : 816 – 821 Google Scholar Crossref Search ADS WorldCat Hoffmann-Benning S , Kende H ( 1992 ) On the role of abscisic Acid and gibberellin in the regulation of growth in rice . Plant Physiol 99 : 1156 – 1161 Google Scholar Crossref Search ADS PubMed WorldCat Jackson MB ( 2002 ) Long-distance signalling from roots to shoots assessed: the flooding story . J Exp Bot 53 : 175 – 181 Google Scholar Crossref Search ADS PubMed WorldCat Lee SC , Mustroph A, Sasidharan R, Vashisht D, Pedersen O, Oosumi T, Voesenek LACJ, Bailey-Serres J ( 2011 ) Molecular characterization of the submergence response of the Arabidopsis thaliana ecotype Columbia . New Phytol 190 : 457 – 471 Google Scholar Crossref Search ADS PubMed WorldCat Licausi F , Kosmacz M, Weits DA, Giuntoli B, Giorgi FM, Voesenek LACJ, Perata P, van Dongen JT ( 2011 ) Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization . Nature 479 : 419 – 422 Google Scholar Crossref Search ADS PubMed WorldCat Licausi F , van Dongen JT, Giuntoli B, Novi G, Santaniello A, Geigenberger P, Perata P ( 2010 ) HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana . Plant J 62 : 302 – 315 Google Scholar Crossref Search ADS PubMed WorldCat McDonald MP , Visser EJW ( 2003 ) A study of the interaction between auxin and ethylene in wild type and transgenic ethylene-insensitive tobacco during adventitious root formation induced by stagnant root zone conditions . Plant Biol 5 : 550 – 556 Google Scholar Crossref Search ADS WorldCat Millenaar FF , Cox MCH, van Berkel YE, Welschen RAM, Pierik R, Voesenek LACJ, Peeters AJM ( 2005 ) Ethylene-induced differential growth of petioles in Arabidopsis. Analyzing natural variation, response kinetics, and regulation . Plant Physiol 137 : 998 – 1008 Google Scholar Crossref Search ADS PubMed WorldCat Mommer L , Pedersen O, Visser EJW ( 2004 ) Acclimation of a terrestrial plant to submergence facilitates gas exchange under water . Plant Cell Environ 27 : 1281 – 1287 Google Scholar Crossref Search ADS WorldCat Mommer L , Visser EJW ( 2005 ) Underwater photosynthesis in flooded terrestrial plants: a matter of leaf plasticity . Ann Bot (Lond) 96 : 581 – 589 Google Scholar Crossref Search ADS WorldCat Nagai K , Kondo Y, Kitaoka T, Noda T, Kuroha T, Angeles-Shim RB, Yasui H, Yoshimura A, Ashikari M ( 2014 ) QTL analysis of internode elongation in response to gibberellin in deepwater rice . AoB Plants 6 : plu028 Normile D ( 2008 ) Agricultural research. Reinventing rice to feed the world . Science 321 : 330 – 333 Google Scholar Crossref Search ADS PubMed WorldCat Papdi C , Abrahám E, Joseph MP, Popescu C, Koncz C, Szabados L ( 2008 ) Functional identification of Arabidopsis stress regulatory genes using the controlled cDNA overexpression system . Plant Physiol 147 : 528 – 542 Google Scholar Crossref Search ADS PubMed WorldCat Parlanti S , Kudahettige NP, Lombardi L, Mensuali-Sodi A, Alpi A, Perata P, Pucciariello C ( 2011 ) Distinct mechanisms for aerenchyma formation in leaf sheaths of rice genotypes displaying a quiescence or escape strategy for flooding tolerance . Ann Bot (Lond) 107 : 1335 – 1343 Google Scholar Crossref Search ADS WorldCat Pedersen O , Rich SM, Colmer TD ( 2009 ) Surviving floods: leaf gas films improve O₂ and CO₂ exchange, root aeration, and growth of completely submerged rice . Plant J 58 : 147 – 156 Google Scholar Crossref Search ADS PubMed WorldCat Perata P , Voesenek LACJ ( 2007 ) Submergence tolerance in rice requires Sub1A, an ethylene-response-factor-like gene . Trends Plant Sci 12 : 43 – 46 Google Scholar Crossref Search ADS PubMed WorldCat Pierik R , van Aken JM, Voesenek LACJ ( 2009 ) Is elongation-induced leaf emergence beneficial for submerged Rumex species? Ann Bot (Lond) 103 : 353 – 357 Google Scholar Crossref Search ADS WorldCat Polko JK , Pierik R, van Zanten M, Tarkowská D, Strnad M, Voesenek LACJ, Peeters AJM ( 2013 ) Ethylene promotes hyponastic growth through interaction with ROTUNDIFOLIA3/CYP90C1 in Arabidopsis . J Exp Bot 64 : 613 – 624 Google Scholar Crossref Search ADS PubMed WorldCat Polko JK , van Zanten M, van Rooij JA, Marée AFM, Voesenek LACJ, Peeters AJM, Pierik R ( 2012 ) Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion . New Phytol 193 : 339 – 348 Google Scholar Crossref Search ADS PubMed WorldCat Rajhi I , Yamauchi T, Takahashi H, Nishiuchi S, Shiono K, Watanabe R, Mliki A, Nagamura Y, Tsutsumi N, Nishizawa NK, et al. ( 2011 ) Identification of genes expressed in maize root cortical cells during lysigenous aerenchyma formation using laser microdissection and microarray analyses . New Phytol 190 : 351 – 368 Google Scholar Crossref Search ADS PubMed WorldCat Rauf M , Arif M, Fisahn J, Xue GP, Balazadeh S, Mueller-Roeber B ( 2013 ) NAC transcription factor speedy hyponastic growth regulates flooding-induced leaf movement in Arabidopsis . Plant Cell 25 : 4941 – 4955 Google Scholar Crossref Search ADS PubMed WorldCat Rodríguez-Gamir J , Ancillo G, González-Mas MC, Primo-Millo E, Iglesias DJ, Forner-Giner MA ( 2011 ) Root signalling and modulation of stomatal closure in flooded citrus seedlings . Plant Physiol Biochem 49 : 636 – 645 Google Scholar Crossref Search ADS PubMed WorldCat Saika H , Okamoto M, Miyoshi K, Kushiro T, Shinoda S, Jikumaru Y, Fujimoto M, Arikawa T, Takahashi H, Ando M, et al. ( 2007 ) Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8′-hydroxylase in rice . Plant Cell Physiol 48 : 287 – 298 Google Scholar Crossref Search ADS PubMed WorldCat Sasidharan R , Mustroph A ( 2011 ) Plant oxygen sensing is mediated by the N-end rule pathway: a milestone in plant anaerobiosis . Plant Cell 23 : 4173 – 4183 Google Scholar Crossref Search ADS PubMed WorldCat Sasidharan R , Mustroph A, Boonman A, Akman M, Ammerlaan AMH, Breit T, Schranz ME, Voesenek LACJ, van Tienderen PH ( 2013 ) Root transcript profiling of two Rorippa species reveals gene clusters associated with extreme submergence tolerance . Plant Physiol 163 : 1277 – 1292 Google Scholar Crossref Search ADS PubMed WorldCat Sasidharan R , Voesenek LACJ, Pierik R ( 2011 ) Cell wall modifying proteins mediate plant acclimatization to biotic and abiotic stresses . Crit Rev Plant Sci 30 : 548 – 562 Google Scholar Crossref Search ADS WorldCat Sauter M ( 2013 ) Root responses to flooding . Curr Opin Plant Biol 16 : 282 – 286 Google Scholar Crossref Search ADS PubMed WorldCat Setter TL , Bhekasut P, Greenway H ( 2010 ) Desiccation of leaves after de-submergence is one cause for intolerance to complete submergence of the rice cultivar IR 42 . Funct Plant Biol 37 : 1096 – 1104 Google Scholar Crossref Search ADS WorldCat Setter TL , Kupkanchanakul T, Kupkanchanakul K, Bhekasut P, Wiengweera A, Greenway H ( 1987 ) Concentrations of CO2 and O2 in floodwater and in internodal lacunae of floating rice growing at 1-2 metre water depths . Plant Cell Environ 10 : 767 – 776 Google Scholar OpenURL Placeholder Text WorldCat Shiono K , Ogawa S, Yamazaki S, Isoda H, Fujimura T, Nakazono M, Colmer TD ( 2011 ) Contrasting dynamics of radial O2-loss barrier induction and aerenchyma formation in rice roots of two lengths . Ann Bot (Lond) 107 : 89 – 99 Google Scholar Crossref Search ADS WorldCat Silvertown J , Dodd ME, Gowing DJG, Mountford JO ( 1999 ) Hydrologically defined niches reveal a basis for species richness in plant communities . Nature 400 : 61 – 63 Google Scholar Crossref Search ADS WorldCat Steffens B , Geske T, Sauter M ( 2011 ) Aerenchyma formation in the rice stem and its promotion by H2O2 . New Phytol 190 : 369 – 378 Google Scholar Crossref Search ADS PubMed WorldCat Steffens B , Kovalev A, Gorb SN, Sauter M ( 2012 ) Emerging roots alter epidermal cell fate through mechanical and reactive oxygen species signaling . Plant Cell 24 : 3296 – 3306 Google Scholar Crossref Search ADS PubMed WorldCat Steffens B , Sauter M ( 2005 ) Epidermal cell death in rice is regulated by ethylene, gibberellin, and abscisic acid . Plant Physiol 139 : 713 – 721 Google Scholar Crossref Search ADS PubMed WorldCat Steffens B , Sauter M ( 2009 ) Epidermal cell death in rice is confined to cells with a distinct molecular identity and is mediated by ethylene and H2O2 through an autoamplified signal pathway . Plant Cell 21 : 184 – 196 Google Scholar Crossref Search ADS PubMed WorldCat Steffens B , Wang J, Sauter M ( 2006 ) Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice . Planta 223 : 604 – 612 Google Scholar Crossref Search ADS PubMed WorldCat Takahashi H , Yamauchi T, Colmer T, Nakazono M ( 2014 ) In JT van Dongen , F Licausi , eds, Aerenchyma Formation in Plants , Vol 21,. Springer , Vienna , pp 247 – 265 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Tamang BG , Magliozzi JO, Maroof MAS, Fukao T ( 2014 ) Physiological and transcriptomic characterization of submergence and reoxygenation responses in soybean seedlings . Plant Cell Environ 37 : 2350 – 2365 Google Scholar PubMed OpenURL Placeholder Text WorldCat Tsai KJ , Chou SJ, Shih MC ( 2014 ) Ethylene plays an essential role in the recovery of Arabidopsis during post-anaerobiosis reoxygenation . Plant Cell Environ 37 : 2391 – 2405 Google Scholar PubMed OpenURL Placeholder Text WorldCat Van Der Straeten D , Zhou Z, Prinsen E, Van Onckelen HA, Van Montagu MC ( 2001 ) A comparative molecular-physiological study of submergence response in lowland and deepwater rice . Plant Physiol 125 : 955 – 968 Google Scholar Crossref Search ADS PubMed WorldCat Van Dongen JT , Licausi F ( January 12, 2015 ) Oxygen sensing and signaling . Annu Rev Plant Biol 10.1146/annurev-arplant-043014-114813 van Veen H , Mustroph A, Barding GA, Vergeer-van Eijk M, Welschen-Evertman RAM, Pedersen O, Visser EJW, Larive CK, Pierik R, Bailey-Serres J, et al. ( 2013 ) Two Rumex species from contrasting hydrological niches regulate flooding tolerance through distinct mechanisms . Plant Cell 25 : 4691 – 4707 Google Scholar Crossref Search ADS PubMed WorldCat Vashisht D , Hesselink A, Pierik R, Ammerlaan JMH, Bailey-Serres J, Visser EJW, Pedersen O, van Zanten M, Vreugdenhil D, Jamar DCL, et al. ( 2011 ) Natural variation of submergence tolerance among Arabidopsis thaliana accessions . New Phytol 190 : 299 – 310 Google Scholar Crossref Search ADS PubMed WorldCat Vidoz ML , Loreti E, Mensuali A, Alpi A, Perata P ( 2010 ) Hormonal interplay during adventitious root formation in flooded tomato plants . Plant J 63 : 551 – 562 Google Scholar Crossref Search ADS PubMed WorldCat Visser E , Cohen JD, Barendse G, Blom C, Voesenek L ( 1996 ) An ethylene-mediated increase in sensitivity to auxin induces adventitious root formation in flooded Rumex palustris Sm . Plant Physiol 112 : 1687 – 1692 Google Scholar Crossref Search ADS PubMed WorldCat Voesenek LACJ , Bailey-Serres J ( 2015 ) Flood adaptive traits and processes: an overview . New Phytol 206 : 57 – 73 Google Scholar Crossref Search ADS PubMed WorldCat Voesenek LACJ , Jackson MB, Toebes AHW, Huibers W, Vriezen WH, Colmer TD ( 2003 ) De-submergence-induced ethylene production in Rumex palustris: regulation and ecophysiological significance . Plant J 33 : 341 – 352 Google Scholar Crossref Search ADS PubMed WorldCat Voesenek LACJ , Sasidharan R ( 2013 ) Ethylene—and oxygen signalling—drive plant survival during flooding . Plant Biol (Stuttg) 15 : 426 – 435 Google Scholar Crossref Search ADS PubMed WorldCat Vreeburg RAM , Benschop JJ, Peeters AJM, Colmer TD, Ammerlaan AH, Staal M, Elzenga TM, Staals RH, Darley CP, McQueen-Mason SJ, et al. ( 2005 ) Ethylene regulates fast apoplastic acidification and expansin A transcription during submergence-induced petiole elongation in Rumex palustris . Plant J 43 : 597 – 610 Google Scholar Crossref Search ADS PubMed WorldCat Xu K , Xu X, Fukao T, Canlas P, Maghirang-Rodriguez R, Heuer S, Ismail AM, Bailey-Serres J, Ronald PC, Mackill DJ ( 2006 ) Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice . Nature 442 : 705 – 708 Google Scholar Crossref Search ADS PubMed WorldCat Xu QT , Fan HY, Jiang Z, Zhou ZQ, Yang L, Mei FZ, Qu LH ( 2013 ) Cell wall degradation and the dynamic changes of Ca2+ and related enzymes in the developing aerenchyma of wheat (Triticum aestivum L.) under waterlogging . Acta Biol Hung 64 : 328 – 340 Google Scholar Crossref Search ADS PubMed WorldCat Yamauchi T , Watanabe K, Fukazawa A, Mori H, Abe F, Kawaguchi K, Oyanagi A, Nakazono M ( 2014 ) Ethylene and reactive oxygen species are involved in root aerenchyma formation and adaptation of wheat seedlings to oxygen-deficient conditions . J Exp Bot 65 : 261 – 273 Google Scholar Crossref Search ADS PubMed WorldCat Yin D , Chen S, Chen F, Jiang J ( 2013 ) Ethylene promotes induction of aerenchyma formation and ethanolic fermentation in waterlogged roots of Dendranthema spp . Mol Biol Rep 40 : 4581 – 4590 Google Scholar Crossref Search ADS PubMed WorldCat Yukiyoshi K , Karahara I ( 2014 ) Role of ethylene signalling in the formation of constitutive aerenchyma in primary roots of rice . AoB Plants 6 : plu043 Author notes 1 This work was supported by the Netherlands Organization for Scientific Research (De Nederlandse Organisatie voor Wetenschappelijk Onderzoek/Aarden Levenswetenschappen grant no. 822.01.007 to R.S. and De Nederlandse Organisatie voor Wetenschappelijk Onderzoek-Veni grant no. 863.12.013 to R.S.). * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00387 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Gamalero, Elisa; Glick, Bernard R.

doi: 10.1104/pp.15.00284pmid: 25897004

Abstract A focus on the mechanisms by which ACC deaminase-containing bacteria facilitate plant growth.Bacteria that produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, when present either on the surface of plant roots (rhizospheric) or within plant tissues (endophytic), play an active role in modulating ethylene levels in plants. This enzyme activity facilitates plant growth especially in the presence of various environmental stresses. Thus, plant growth-promoting bacteria that express ACC deaminase activity protect plants from growth inhibition by flooding and anoxia, drought, high salt, the presence of fungal and bacterial pathogens, nematodes, and the presence of metals and organic contaminants. Bacteria that express ACC deaminase activity also decrease the rate of flower wilting, promote the rooting of cuttings, and facilitate the nodulation of legumes. Here, the mechanisms behind bacterial ACC deaminase facilitation of plant growth and development are discussed, and numerous examples of the use of bacteria with this activity are summarized. Agricultural development policies and practices in the past sixty years have largely been based on external inputs (pesticides and fertilizers) to control soil-borne diseases and increase crop yields. Recently, stimulated by the awareness of potentially serious environmental and human health damage caused by the over use of agricultural chemicals (Alavanja et al., 2004; Leach and Mumford, 2008; Damalas and Eleftherohorinos, 2011), the controversy regarding the use of pesticides and fertilizers has gained prominence. Therefore, worldwide agricultural practice is moving toward a more sustainable and environmentally friendly approach. In 2002, in the European Union, 5.7 million ha were designated as being cultivated organically, and by 2011, this number had increased to 9.6 million ha (http://ec.europa.eu/agriculture/markets-and-prices/more-reports/pdf/organic-2013_en.pdf). In other words, in 10 years, the area devoted to organic agriculture in the European Union increased by approximately 400,000 ha per year. This growth in organic agriculture notwithstanding, the total amount of organically cultivated land represents only 5.4% of the total agricultural land in Europe. In this context, the use of microbial inoculants instead of traditional chemicals is gaining popularity, and a number of new products have been formulated, marketed, and applied successfully. The soil surrounding plant roots (the rhizosphere) is one of the main sources of bacteria expressing plant-beneficial activities ( i.e. plant growth-promoting bacteria [PGPB]; Bashan and Holguin, 1998). Stimulation of growth and protection of different crops from pathogens and abiotic stressors by PGPB is well documented under both controlled conditions and in the field, and a large number of papers on this topic are available (Reed and Glick, 2005, 2013; Thakore, 2006). The positive effects induced by PGPB on plant growth are based on: (1) the improvement of mineral nutrition (nitrogen fixation, phosphate solubilization, and iron sequestration), (2) the enhancement of plant tolerance to biotic and abiotic stress (largely mediated by 1-aminocyclopropane-1-carboxylate [ACC] deaminase), (3) the modification of root development (via phytohormone synthesis), and (4) the suppression of phytopathogens (by antibiotics, competition, lytic enzymes, systemic resistance, etc.; Fig. 1). The current knowledge of microorganisms living in the rhizosphere, their role, and their biotechnological and environmental applications has been summarized in several reviews (Glick, 2012; Hirsch and Mauchline, 2012; Bakker et al., 2013; Mendes et al., 2013; Reed and Glick, 2013). This review focuses on the role of bacterial ACC deaminase in supporting the growth of plants exposed to environmental stress. In addition, the issues of the distribution and phylogeny of ACC deaminase, and the possible role of ACC as a signaling molecule, are addressed. Figure 1. Open in new tabDownload slide Schematic overview of the main mechanisms used by PGPB. Following the release of root exudates, a variety of soil microorganisms are attracted to the root. Some of them can efficiently colonize the root surface while others (endophytes) can penetrate the root tissue and spread inside the plant. Plant growth promotion by beneficial microorganisms may occur by either direct or indirect mechanisms. Direct promotion of plant growth involves the improvement of mineral nutrition via nitrogen fixation, phosphate solubilization, and iron chelation, as well as the modulation of phytohormones levels (auxins, cytokinins, GAs, and ethylene). In addition to the increase of biomass, PGPB can positively affect the nutritional value of fruits and edible seeds. The indirect mechanisms are based on the improvement of plant health via suppression of soil-borne diseases by antibiotics, lytic enzymes, siderophore production, induced systemic resistance involving jasmonate and ethylene signaling within the plant, and other molecules (the O-antigenic side chain of the bacterial outer membrane protein lipopolysaccharide, flagellar fractions, pyoverdine, 2,4-diacetylphloroglucinol, cyclic lipopeptide, surfactants, and salicylic acid) that stimulate the host plant’s resistance to pathogens. Figure 1. Open in new tabDownload slide Schematic overview of the main mechanisms used by PGPB. Following the release of root exudates, a variety of soil microorganisms are attracted to the root. Some of them can efficiently colonize the root surface while others (endophytes) can penetrate the root tissue and spread inside the plant. Plant growth promotion by beneficial microorganisms may occur by either direct or indirect mechanisms. Direct promotion of plant growth involves the improvement of mineral nutrition via nitrogen fixation, phosphate solubilization, and iron chelation, as well as the modulation of phytohormones levels (auxins, cytokinins, GAs, and ethylene). In addition to the increase of biomass, PGPB can positively affect the nutritional value of fruits and edible seeds. The indirect mechanisms are based on the improvement of plant health via suppression of soil-borne diseases by antibiotics, lytic enzymes, siderophore production, induced systemic resistance involving jasmonate and ethylene signaling within the plant, and other molecules (the O-antigenic side chain of the bacterial outer membrane protein lipopolysaccharide, flagellar fractions, pyoverdine, 2,4-diacetylphloroglucinol, cyclic lipopeptide, surfactants, and salicylic acid) that stimulate the host plant’s resistance to pathogens. RHIZOSPHERIC BACTERIA VERSUS ENDOPHYTES VERSUS RHIZOBIA Thanks to carbon-rich exudates released from plant roots, bacteria in the rhizosphere establish themselves and proliferate along the roots, giving rise to a biofilm surrounding the roots’ surface (Danhorn and Fuqua, 2007). Following rhizosphere colonization, some of these microorganisms can penetrate the root tissue, therefore shifting their habitus from rhizospheric to endophytic. Endophytic bacteria include: (1) facultative endophytes living inside the plants as well as in other habitats, (2) obligate endophytes that can only live inside plant tissues, and (3) opportunistic endophytes that can occasionally enter plants and live inside their tissues. However, the fact that scientists can isolate and culture specific endophytic strains means that they are likely dealing exclusively with facultative endophytes that may be isolated from rhizosphere soil samples as well as from inside the plant. According to Wilson (1995), endophytes are those microorganisms living inside plant tissues without harming the plant. Internal colonization typically starts in the zone of lateral root emergence or in root wounds and cracks; from there, endophytic bacteria proliferate, spread through xylematic vessels, and reach different plant compartments (Compant et al., 2008). Bacterial endophytes have been detected inside the endorhiza in stems, leaves, and flowers (Compant et al., 2010; Reinhold-Hurek and Hurek, 2011) of a number of plant species. Inside plant tissues, endophytic bacteria express their physiological activities, synthesize secondary metabolites, and may, both directly and indirectly, facilitate plant development through phytopathogen suppression, mineral nutrition improvement, and enhancement of plant tolerance to stress. Consequently, a number of studies have focused on the application of bacterial endophytes as biofertilizers for phytostimulation and as biological control agents (Kuklinsky-Sobral et al., 2004; Gaiero et al., 2013). Recently, based on genome sequences of 304 Proteobacteria, Bruto et al. (2014) analyzed the distribution of 23 genes that may contribute to the ability of these bacteria to promote plant growth. These authors suggest that gene transfers, predominantly ancient, resulted in characteristic gene combinations according to taxonomic subgroups of PGPB strains. In other words, genes associated with plant growth, such as the ACC deaminase structural gene (acdS), are found in rhizospheric bacteria as a consequence of ancient horizontal gene transfer, and are also present in endophytic bacteria. Thus, understanding the mechanisms utilized by rhizospheric bacteria also provides insight into the mechanisms used by endophytic bacteria. Rhizobia represent a particular group of endophytic microorganisms able to improve plant mineral nutrition, primarily through nitrogen fixation. They colonize plant roots and establish a mutualistic symbiosis with compatible legume plants. The strict and highly specific relationship between these bacteria and the plant host induces physiological, genetic, and morphological changes in the plant. This includes the formation of root nodules containing bacteria (bacteroids), where nitrogen fixation occurs, under limited oxygen concentration via the action of the enzyme nitrogenase. However, rhizobia, moving from the root toward the shoot (Chi et al., 2005) can, to some extent, colonize internal root tissues of cereal crop plants, such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), and wheat (Triticum aestivum), increasing plant biomass and grain yield independently of root nodule formation and nitrogen fixation (Biswas et al., 2000; Gutiérrez-Zamora and Martínez-Romero, 2001; Lupway et al., 2004; García-Fraile et al., 2012). Open-field application of rhizobia as biofertilizers for legume or cereal crop plants of agricultural importance facilitates plant development and high productivity when cultivated under low fertilization regimes. In this regard, rhizobia have been used to promote plant growth in the field for more than 100 years. ACC DEAMINASE Biochemistry of ACC Deaminase The (largely bacterial) enzyme ACC deaminase (3.5.99.7) cleaves ACC, the immediate precursor of ethylene in plants, producing ammonia and α-ketobutyrate (Honma and Shimomura, 1978), reducing the amount of ethylene that the plant can synthesize (Glick et al., 1998). Ethylene is a gaseous hormone displaying a wide range of biological effects in plants at concentrations as low as 0.05 μL L–1 (Abeles et al., 1992). Ethylene is involved in seed germination, tissue differentiation, formation of root and shoot primordia, root branching and elongation, lateral bud development, flowering, flower senescence, fruit ripening and abscission, anthocyanin production, synthesis of volatile organic compounds responsible for aroma formation in fruits, storage product hydrolysis, leaf senescence, and abscission (Abeles et al., 1992; Glick, 2014). Local increases in the concentration of this hormone also occur during the establishment of symbioses between plants and microorganisms, including rhizobia and mycorrhizal fungi. In these cases, by locally lowering ethylene levels, ACC deaminase-producing bacteria can facilitate symbiosis development (Ma et al., 2003; Gamalero et al., 2008). In all higher plants, ethylene is produced from S-adenosyl-Met via the action of the enzyme ACC synthase, both during normal plant development and when the plant is exposed to various environmental stresses (Abeles et al., 1992). By modulating ethylene levels, ACC deaminase represents one of the key bacterial physiological activities supporting plant growth under stressed conditions, where the ethylene concentration inside the plant might otherwise reach levels inhibitory to plant growth (Glick et al., 1998, 2007; Glick, 2014). As a consequence of the wide range of potential applications of bacteria that produce ACC deaminase, there has been considerable interest in the biochemistry and functioning of this enzyme. Thus, a number of different ACC deaminases have now been characterized. ACC deaminase is a multimeric enzyme, cytoplasmically localized, that utilizes the coenzyme pyridoxal phosphate as a tightly bound cofactor. Its subunit mass is approximately 35 to 42 kD, while its native size is estimated to be approximately 100 to 112 kD (Sheehy et al., 1991; Jacobson et al., 1994; Hontzeas et al., 2004). Based on its protein fold, ACC deaminase has been classified as belonging to the Trp synthase β superfamily of pyridoxal phosphate-binding proteins (Glick et al., 2007). The affinity of this enzyme for the substrate is not particularly high (K m = 1.5–6.0 mm). Most organisms with ACC deaminase contain a basal level of enzyme activity. However, ACC deaminase synthesis is induced by ACC, at levels as low as 100 nm (Jacobson et al., 1994), with full induction requiring up to 10 h. The amino acids l-Ala, dl-Ala, and dl-Val can also induce enzyme activity to a small extent, and γ-aminoisobutyric acid can induce activity to almost the same level as ACC (Honma, 1983). Maximal enzyme activity typically occurs at 30°C and pH 8.5. The affinity for the substrate ACC and the competitive inhibitors l-Ala and l-Ser is also highest at pH 8.5 (Hontzeas et al., 2006). Yoon and Kieber (2013) have recently posited a model in which, in addition to acting as the immediate precursor to ethylene, ACC may also act as a signaling molecule in several plant processes, including root-to-shoot communication. With this scenario, the interaction of plants with ACC deaminase-producing PGPB might be expected to decrease the extent of ACC signaling of specific plant functions such as the regulation of cell wall function. Unlike experiments that utilize chemical inhibitors of ethylene biosynthesis or ethylene perception, ACC deaminase specifically decreases ACC levels. Thus, to test the ability of ACC to act directly as a signaling molecule, one might repeat some of the experiments cited by Yoon and Kieber (2013) in the presence of ACC deaminase. In this regard, while ACC deaminase may not completely breakdown all of the available ACC, the resultant low levels of ACC may be readily quantified (Penrose et al., 2001). Distribution and Phylogeny of ACC Deaminase The bacterium Pseudomonas sp. ACP and the yeast Cyberlindnera saturnus (previously Hansenula saturnus) were the two first microorganisms reported to synthesize ACC deaminase (Honma and Shimomura, 1978; Minami et al., 1998). Subsequently, ACC deaminase activity has been found in numerous bacteria, both gram positive and negative with a variety of different types of metabolism (for review, see Gamalero and Glick, 2012; Glick, 2014). ACC deaminase genes (including both the structural gene acdS and the regulatory gene acdR) have been found in many different rhizobacteria (rhizospheric, endophytic, and rhizobia), including Azospirillum spp., Rhizobium spp., Agrobacterium spp., Achromobacter spp., Burkholderia spp., Ralstonia spp., Pseudomonas spp., and Enterobacter spp. (Blaha et al., 2006). More importantly, even if some strains of a particular genus and species have an acdS gene, not all strains do. The frequency of ACC deaminase activity in various soil bacteria has been estimated, especially in rhizobia. Of 13 rhizobial strains tested, five (38%) isolates were able to synthesize ACC deaminase, while seven out of 13 (54%) possessed the acdS gene. This discrepancy was related to the fact that two strains, belonging to the genus Mesorhizobium are only able to produce the enzyme during the symbiotic phase, when localized inside a root nodule (Ma et al., 2003). It subsequently was shown that ACC deaminase genes in Mesorhizobium spp. were, unlike all other known ACC deaminases genes, under the transcriptional control of the nitrogen fixation positive regulatory gene nifA2 promoter and expressed only within root nodules (Nukui et al., 2006). In this regard, it has been suggested that the expression of ACC deaminase genes within nitrogen-fixing nodules may decrease the rate of nodule senescence, as nitrogen fixation with its high-energy demand could activate stress ethylene synthesis (Murset et al., 2012). Another study, including a much larger number of rhizobial isolates (233; Duan et al., 2009), revealed that 27 strains (12%) expressed ACC deaminase. These 27 strains were characterized for the presence of the acdS gene; while 17 of them had genes that were 99% identical to the previously characterized ACC deaminase structural gene (acdS) from Rhizobium leguminosarum bv viciae 128C53K, the other 10 strains were found to be 84% identical compared with the acdS gene from strain 128C53K (Duan et al., 2009). The observation that rhizobia strains with ACC deaminase activity from a wide geographic area showed very little diversity was somewhat surprising. It was then argued that given the harsh winters and lack of diverse vegetation in southern Saskatchewan (where these strains were isolated), there might be intrinsic limits to the diversity of these microorganisms (Duan et al., 2009). Bacterial ACC deaminase activity is relatively common in rhizosphere bacteria, especially in soils that are often subjected to stressful conditions (Timmusk et al., 2011). Thus, rhizosphere bacteria that contain ACC deaminase may endow some plants with the ability to better withstand, and therefore survive in, harsh environmental conditions. When analyzing the sequences of acdS genes, Blaha et al. (2006) found a high level of polymorphism. Consequently, they defined three acdS groups: groups I and II included sequences originating from the β- and γ-Proteobacteria, while group III was composed of α-Proteobacteria. Looking at their geographical origin and habitat, strains from a given acdS group originated from different plant hosts. Moreover, by comparing the sequences of 45 different acdS genes, from seven α-Proteobacteria, 35 β-Proteobacteria, and three γ-Proteobacteria, Prigent-Combaret et al. (2008) found a high similarity (62.1%–89.4%) with the acdS gene of the model strain Pseudomonas putida UW4 and 53.9% to 93.5% with the gene from Azospirillum lipoferum 4B. A complete description of the phylogeny and evolution of the genes encoding acdS and its major regulatory gene, acdR, has been recently elaborated (Bruto et al., 2014; Nascimento et al., 2014). Information regarding acdS/acdR sequences must be considered together with the habitat, the origin, and the enzymatic activity of completely sequenced bacterial strains to obtain a comprehensive view. Overall, the data show that ACC deaminase activity is prevalent in some bacteria, fungi, and members of stramenopiles. Stramenopiles are a monophyletic eukaryotic group of organisms bearing an immature flagellum with tripartite hairs comprising more than 100,000 species and including a variety of life forms (single cells, large plasmodia, and complex multicellular thalli). The best known members of the group are the colorless oomycetes (aquatic fungi, including plant pathogens for cultivated crops), diatoms, chrysophyte algae, and giant kelp seaweeds. Stramenopiles able to perform photosynthesis are the predominant eukaryotes in most aquatic environments, where they are major primary producers (Yoon et al., 2009). In parallel, through multiple searches of the National Center for Biotechnology Information database, acdS genes have been found in Actinobacteria, members from the Deinococcus-Thermus phylum (Meiothermus spp.), α-, β- and, γ-Proteobacteria, various fungi (Ascomycota and Basidiomycota), and some stramenopiles (Nascimento et al., 2014). Although ACC deaminase genes are mainly transmitted vertically in various microorganisms, occasional horizontal gene transfer, including interkingdom transfer events, occur. It is possible that acdS genes had an ancient origin in a eukaryote and bacterial common ancestor. Then, during vertical transmission, different constraints, such as adaptation to specific niches, induced acdS divergence or gene loss. The advantages conferred by ACC deaminase activity have been positively selected by evolution, leading to intragenomic transfers of acdS genes from primary chromosomes to plasmids and increased divergence of acdS genes. In fact, acdS genes in most Burkholderia and Cupriavidus spp. strains are located on a second smaller chromosome, while in other β-Proteobacteria (e.g. Ralstonia solanacearum), it is located on the primary chromosome or on megaplasmids (Nascimento et al., 2014). Here, it should be noted that some strains of Burkholderia spp. are exclusively rhizospheric, while others are facultative endophytes. Because plasmids are transmissible between bacteria via conjugation, it’s possible that some dispersion of acdS genes occurred. This is in agreement with work that previously reported the occurrence of horizontal acdS/acdR genes transfer in Proteobacteria and in many Mesorhizobium species (Hontzeas et al., 2005; Blaha et al., 2006; Nascimento et al., 2012). Moreover, due to intragenomic transfer events, many microorganisms may have lost acdS genes. Consistently, it has been reported that, during phenotypic variation events, A. lipoferum strain 4B readily loses the plasmid containing an acdS gene (Prigent-Combaret et al., 2008). Model Including IAA Feedback Inhibition of Ethylene Action In addition to being rich in sugars, root exudates contain high amounts of amino acids. Among them, Trp is released by the roots and may be taken up by bacterial cells in the rhizosphere. Bacteria use Trp to synthesize the phytohormone indole-3-acetic acid (IAA), some of which is then taken up by the plant. Production of IAA is widespread among soil bacteria; it has been estimated that approximately 80% of rhizosphere bacteria and a significant fraction of bacterial endophytes produce IAA (Patten and Glick, 1996). The bacterial IAA, together with endogenous plant IAA, can regulate several phases of plant development, such as seed and tuber germination, xylem formation, plant cell proliferation and elongation, vegetative growth, emergence of lateral and adventitious roots, plant responses to light and gravity, and florescence and fructification (Tsakelova et al., 2006). IAA can also affect the synthesis of ACC deaminase by activating the transcription of the plant enzyme ACC synthase (that catalyzes the conversion of ACC from S-adenosyl-Met). As a consequence of an increased amount of ACC, the ethylene level inside a plant is increased inducing a plant stress response. Bacteria that produce high levels of IAA often inhibit plant growth. However, this does not necessarily occur because as plant ethylene levels increase, the transcription of auxin response factors is inhibited (Pierik et al., 2006; Prayitno et al., 2006; Czarny et al., 2007; Glick et al., 2007; Stearns et al., 2012), thereby limiting the extent that IAA can activate ACC synthase transcription. Moreover, some ACC is released by the roots (Bayliss et al., 1997; Penrose and Glick, 2001), taken up by the bacteria, and through the action of ACC deaminase, converted to ammonia and α-ketobutyrate. As a result, the amount of ethylene produced by the plant is reduced. Therefore, root colonization by bacteria that synthesize ACC deaminase prevents a rise in ethylene levels that might otherwise become growth inhibitory (Glick, 1995). In plants inoculated with bacteria that produce both IAA and ACC deaminase, ethylene levels do not become elevated to the same extent as when plants interact with bacteria that synthesize IAA but not ACC deaminase. When bacterial ACC deaminase is induced and expressed, ethylene is synthesized at a relatively low level, and the bacterial IAA can continue to both stimulate plant growth and enhance the transcription of ACC synthase. However, a large portion of the ACC synthesized is released by the root, taken up by the bacterial cells and finally cleaved by ACC deaminase (Fig. 2). Consequently, the cross talk between IAA and ethylene enables ACC deaminase to effectively facilitate the stimulation of plant growth by IAA. Bacteria that synthesize both ACC deaminase and IAA may facilitate plant growth in the presence of several ethylene-producing environmental stresses (Gamalero and Glick, 2010). Figure 2. Open in new tabDownload slide Schematic representation of how PGPB that produce both ACC deaminase and IAA facilitate plant growth. A detailed explanation is given in the text. SAM, S-Adenosyl Met. Figure 2. Open in new tabDownload slide Schematic representation of how PGPB that produce both ACC deaminase and IAA facilitate plant growth. A detailed explanation is given in the text. SAM, S-Adenosyl Met. Galland et al. (2012) reported that treatment of Arabidopsis (Arabidopsis thaliana) seedlings with the rhizospheric plant growth-promoting bacterium Phyllobacterium brassicacearum STM196 caused a significant increase in plant root hair elongation. Following this bacterial treatment, these workers were unable to detect any significant increase in ethylene biosynthesis. Moreover, this signaling pathway activation does not depend on local plant auxin biosynthesis. However, this bacterium also produces and secretes IAA so plant IAA biosynthesis is not needed to activate ACC synthase transcription. By using ethylene-insensitive mutants of Arabidopsis, Zamioudis et al. (2013) clarified which plant growth parameter is affected by the ethylene pathway. They concluded that the main impact of a PGPB strain, able to directly affect auxin signaling in plants, is on the length of the primary root; moreover, they demonstrated that other plant parameters such as lateral root and the root hair formation are affected by the strain independently by the ethylene pathways. AMELIORATING PLANT STRESS VIA ACC DEAMINASE In the past, stress ethylene has been suggested to both alleviate and exacerbate some of the effects of pathogen infection (Abeles et al., 1992). However, a simple model (originally developed to explain the effects of stress ethylene following biotic stress and later extended to include abiotic stress as well) was proposed to explain these seemingly contradictory results (Glick et al., 2007). That is, a short time following the onset of the stress, a small peak of ethylene is produced. This small peak of ethylene is thought to consume the existing pool of ACC within plant tissues and likely activates the synthesis of defensive genes within the plant (Stearns et al., 2012). Subsequently, following the synthesis of additional ACC within the plant, a second much larger peak of ethylene is typically observed. The second peak of ethylene occurs as a consequence of increased transcription of ACC synthase genes, mostly triggered by environmental cues, and acts as a signal to initiate processes such as senescence, chlorosis, and abscission, all of which are inhibitory to plant growth and survival. Thus, a significant fraction of the damage that occurs to a plant following a biotic or abiotic stress is due to the second (large) peak of ethylene that is synthesized by the plant rather than to the direct effects of the stress itself. Based on this model, it was predicted that bacteria, which produce an amount of ACC deaminase that can reduce the magnitude of the second ethylene peak, should decrease the damage to plants that occurs as a consequence of a wide range of biotic and abiotic stresses. Flooding and Anoxia Plant roots typically respond to flooding by synthesizing a high level of ACC, and as a consequence of a lack of oxygen, the ACC is translocated to shoots, where it becomes a substrate for ACC oxidase and is converted to ethylene (Bradford and Yang, 1980; Else and Jackson, 1998). Ethylene synthesis in flooded plants induces the expression of various symptoms such as epinasty, leaf chlorosis, and necrosis (Li et al., 2013). Bacteria able to limit the increase of ethylene through the action of ACC deaminase can be useful in supporting plant growth in such adverse conditions (Grichko and Glick, 2001; Barnawal et al., 2012; Li et al., 2013) The protein profile of cucumber (Cucumis sativus) roots, inoculated or not with P. putida UW4 and able to synthesize ACC deaminase, in normoxic (no oxygen limitation) and hypoxic conditions has been characterized (Li et al., 2013). In normoxic conditions, no significant change in protein expression occurred in cucumber seedling roots treated with P. putida UW4. However, expression of several root proteins changed following the plant’s inoculation with P. putida UW4 under hypoxic stress, including those involved in carbohydrate and nitrogen metabolism, defense stress, antioxidant activity, and binding to host plants (Li et al., 2013). Drought The first report of ACC deaminase-producing bacteria facilitating the growth of plants under drought stress was by Mayak et al. (2004a), who used Achromobacter piechaudii ARV8, from the rhizosphere of Lycium shawii from the Arava region of the Negev desert, to inoculate tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants exposed to drought stress. Plants inoculated with the bacterial strain had 4 times the biomass compared with noninoculated controls, concomitant with a significant reduction of the ethylene level. Similar experiments (in the laboratory and in the field) with several plants (pea [Pisum sativum], maize, wheat, mung bean [Vigna radiata], and Trigonella foenum-graecum) and different ACC deaminase-producing bacteria have since demonstrated the efficacy of using bacteria able to synthesize ACC deaminase in protecting plants against yield loss induced by drought stress (Arshad et al., 2008; Belimov et al., 2009; Shakir et al., 2012; Barnawal et al., 2013; Sarma and Saikia, 2014; Zafarul-Hye et al., 2014). Salt Worldwide, the total area of salt-affected soil is about one billion ha, mainly in the arid-semiarid regions of Asia, Australia, and South America. In addition, salinity affects about 1 million ha in the European Union and is a major cause of desertification. In Spain, for example, 3% of the 3.5 million ha of irrigated land is severely affected, while another 15% of this land is considered to be under serious risk (Soil Atlas of Europe, European Soil Bureau Network European Commission 2005, http://eusoils.jrc.ec.europa.eu/projects/soil_atlas/pages/117.html). Salt stress inhibits plant growth, inducing osmotic stress, Na+ and Cl– toxicity, ethylene production, plasmolysis, nutrient imbalance, production of reactive oxygen species, and interference with photosynthesis. Inhibition of seed germination, seedling growth and vigor, flowering, and fruit set occur as a consequence of these physiological changes (Sairam and Tyagi, 2004). The initial responses of most plants to drought and salinity are very similar; both are attributed to water stress. When plants are exposed to high salt, a decrease in the growth rate followed by a slow recovery to a new reduced growth rate is the plant’s first response to the decrease in water potential caused by salt, rather than to any salt-specific toxicity. Subsequently, metabolic toxicity in plants caused by sodium ions is attributed to these ions competing with potassium ions for binding sites essential for cellular functioning (Gamalero et al., 2009a). Mayak et al. (2004b) first reported on the ability of A. piechaudii ARV8 to promote tomato plant growth in the presence of up to 172 mm NaCl salt. This work has served as a model for other researchers employing similar bacterial strains to facilitate the growth of plants in the presence of inhibitory salt levels (Gamalero et al., 2010; Nadeem et al., 2010; Ahmad et al., 2011; Siddikee et al., 2011; Chookietwattana and Maneewan, 2012; Karthikeyan et al., 2012; Bal et al., 2013; Ramadoss et al., 2013; Akhgar et al., 2014; Ali et al., 2014; Barnawal et al., 2014; Chang et al., 2014). Fungal and Bacterial Pathogens Ethylene levels inside plants increase following pathogen infection, and this induces the appearance of specific symptoms (van Loon et al., 2006). In this context, seedling inoculation with bacteria expressing ACC deaminase may reduce pathogen-induced ethylene, e.g. for soil-borne disease caused by pathogenic bacteria such as Pseudomonas syringae pv tomato (Indiragandhi et al., 2008), Agrobacterium tumefaciens (Toklikishvili et al., 2010; Hao et al., 2011), Erwinia spp. (Wang et al., 2000), and fungi, including Pythium ultimum (Wang et al., 2000), Pythium aphanidermatum (El-Tarabily, 2013), and Pyricularia oryzae (Amutharaj et al., 2012). Nematodes Recently, bacterial ACC deaminase has been identified as a key trait in suppression of the pathogenic nematode Bursaphelenchus xylophilus causing pine wilt disease (Nascimento et al., 2013). Thus, seedling inoculation with bacteria able to synthesize ACC deaminase may lead to plant resistance to nematode-induced diseases. Metals and Organic Contaminants Phytoremediation is the use of plants, able to tolerate/accumulate/degrade organic or inorganic chemicals and/or producing high biomass, to clean up polluted soils (Pilon-Smits, 2005). However, plants tolerant to xenobiotics do not develop high biomass, often limiting the practical application of this technology (Khan et al., 2000). PGPB can often facilitate phytoremediation (Glick, 2010) by promoting plant growth, improving their health, enhancing root development, or increasing plant tolerance to the stress imposed by environmental toxicants (Burd et al., 1998; Huang et al., 2004; Reed and Glick, 2005; Gamalero et al., 2009b; Gurska et al., 2009; Glick, 2012). Flower Wilting To extend the shelf life of cut flowers, treatments with, potentially environmentally harmful, chemical ethylene inhibitors are routinely performed (Reid and Wu, 1991). The application of bacteria that produce ACC deaminase to lower the amount of ethylene in cut flowers represents a safer alternative. To prolong the lifetime of cut flowers, bacterial cells must be taken up by the cut flowers. In this context, the use of ACC deaminase-expressing endophytes, which are adapted to live inside plant tissues, may assure the efficacy of this treatment. Consistent with this hypothesis, Ali et al. (2012) demonstrated that two endophytic bacterial strains, Pseudomonas fluorescens YsS6 and Pseudomonas migulae 8R6, both of which internally colonize the stems of the cut flowers, lower the flower ethylene levels and delay flower senescence by 2 to 3 d. Rooting of Cuttings The impact of inoculating plant cuttings with bacteria that are able to produce ACC deaminase was described by Mayak et al. (1999), who treated mung bean cuttings with P. putida GR12-2 or with its mutant lacking ACC deaminase activity. While the number of adventitious roots was similar in the two treatments, the length of the newly generated roots was significantly greater in mung bean cuttings inoculated with the wild-type strain. Similarly, carnation cuttings treated with a strain of Azospirillum brasilense engineered to synthesize ACC deaminase produced significantly more and longer roots than untreated cuttings (Li et al., 2005). Montero-Calasanz (2013) measured the rooting efficiency of olive (Olea europaea) cuttings following inoculation with five bacterial strains with different physiological traits: Pantoea spp. AG9, the only strain able to express ACC deaminase, was the most efficient strain in enhancing the rooting of these cuttings. SUMMARY AND CONCLUSION Plants that are grown in the field are subject to more or less continuous exposure to one stress after another, all of which can potentially inhibit plant growth and development. These stresses may be caused by biotic factors such as viruses, nematodes, insects, bacteria, or fungi or by abiotic factors such as extremes of temperature, high light, flooding, drought, the presence of toxic metals, and organic contaminants. While various plants may respond somewhat differently to stresses, nearly all plants respond to stress by producing ethylene. Lowering the amount of ethylene that is synthesized in response to stress through the application of ACC deaminase-producing bacteria can significantly decrease the extent of plant growth inhibition that accrues from the stress. From a practical perspective, as a consequence of the fundamental knowledge of plant growth-promoting bacterial modes of action that has been gained over the past 10 to 20 years, specifically emphasizing an understanding of the key role of ACC deaminase, this technology is currently accessible for use in agriculture, horticulture, and environmental cleanup technologies in both the developed and the developing world. Glossary ACC 1-aminocyclopropane-1-carboxylate IAA indole-3-acetic acid PGPB plant growth-promoting bacteria LITERATURE CITED Abeles FB , Morgan PW, Saltveit ME Jr ( 1992 ) Ethylene in Plant Biology , Ed 2. Academic Press , New York Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Ahmad M , Zahir ZA, Asghar HN, Asghar M ( 2011 ) Inducing salt tolerance in mung bean through coinoculation with rhizobia and plant-growth-promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase . Can J Microbiol 57 : 578 – 589 Google Scholar Crossref Search ADS PubMed WorldCat Akhgar AR , Arzanlou M, Bakker PAHM, Hamidpour M ( 2014 ) Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing Pseudomonas spp. in the rhizosphere of salt-stressed canola . Pedosphere 24 : 461 – 468 Google Scholar Crossref Search ADS WorldCat Alavanja MCR , Hoppin JA, Kamel F ( 2004 ) Health effects of chronic pesticide exposure: cancer and neurotoxicity . Annu Rev Public Health 25 : 155 – 197 Google Scholar Crossref Search ADS PubMed WorldCat Ali S , Charles TC, Glick BR ( 2012 ) Delay of carnation flower senescence by bacterial endophytes expressing ACC deaminase . J Appl Microbiol 113 : 1139 – 1144 Google Scholar Crossref Search ADS PubMed WorldCat Ali S , Charles TC, Glick BR ( 2014 ) Amelioration of high salinity stress damage by plant growth-promoting bacterial endophytes that contain ACC deaminase . Plant Physiol Biochem 80 : 160 – 167 Google Scholar Crossref Search ADS PubMed WorldCat Amutharaj P , Sekar C, Natheer SE ( 2012 ) Intergeneric microbial coaggregates: bioinoculation effect of ACC deaminase positive wild type strains of Pseuodomonas and Paenibacillus, as coaggregates, on the maximization of ISR against Pyricularia oryzae in upland rice cv. ASD-19 . CIBTech J Microbiol 1 : 57 – 66 Google Scholar OpenURL Placeholder Text WorldCat Arshad M , Shaharoona B, Mahmood T ( 2008 ) Inoculation with Pseudomonas spp. containing ACC deaminase partially eliminates the effects of drought stress on growth, yield, and ripening of pea (Pisum sativum L.) . Pedosphere 18 : 611 – 620 Google Scholar Crossref Search ADS WorldCat Bakker PA , Berendsen RL, Doornbos RF, Wintermans PC, Pieterse CM ( 2013 ) The rhizosphere revisited: root microbiomics . Front Plant Sci 4 : 165 Google Scholar Crossref Search ADS PubMed WorldCat Bal HB , Nayak L, Das S, Adhya TK ( 2013 ) Isolation of ACC deaminase PGPR from rice rhizosphere and evaluating their plant growth promoting activity under salt stress . Plant Soil 366 : 93 – 105 Google Scholar Crossref Search ADS WorldCat Barnawal D , Bharti N, Maji D, Chanotiya CS, Kalra A ( 2012 ) 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation . Plant Physiol Biochem 58 : 227 – 235 Google Scholar Crossref Search ADS PubMed WorldCat Barnawal D , Bharti N, Maji D, Chanotiya CS, Kalra A ( 2014 ) ACC deaminase-containing Arthrobacter protophormiae induces NaCl stress tolerance through reduced ACC oxidase activity and ethylene production resulting in improved nodulation and mycorrhization in Pisum sativum . J Plant Physiol 171 : 884 – 894 Google Scholar Crossref Search ADS PubMed WorldCat Barnawal D , Maji D, Bharti N, Chanotiya CS, Kalra A ( 2013 ) ACC deaminase-containing Bacillus subtilis reduces stress ethylene-induced damage and improves mycorrhizal colonization and rhizobial nodulation in Trigonella foenum-graecum under drought stress . J Plant Growth Regul 32 : 809 – 822 Google Scholar Crossref Search ADS WorldCat Bashan Y , Holguin G ( 1998 ) Proposal for the division of plant growth-promoting rhizobacteria into two classifications: biocontrol-PGPB (Plant Growth-Promoting Bacteria) and PGPB . Soil Biol Biochem 30 : 1225 – 1228 Google Scholar Crossref Search ADS WorldCat Bayliss C , Bent E, Culham DE, MacLellan S, Clarke AJ, Brown GL, Wood JM ( 1997 ) Bacterial genetic loci implicated in the Pseudomonas putida GR12-2R3-anola mutualism: identification of an exudate-inducible sugar transporter . Can J Microbiol 43 : 809 – 818 Google Scholar Crossref Search ADS PubMed WorldCat Belimov AA , Dodd IC, Hontzeas N, Theobald JC, Safronova VI, Davies WJ ( 2009 ) Rhizosphere bacteria containing 1-aminocyclopropane-1-carboxylate deaminase increase yield of plants grown in drying soil via both local and systemic hormone signalling . New Phytol 181 : 413 – 423 Google Scholar Crossref Search ADS PubMed WorldCat Biswas JC , Ladha JK, Dazzo FB ( 2000 ) Rhizobia inoculation improves nutrient uptake and growth in lowland rice . Soil Sci Soc Am J 64 : 1644 – 1650 Google Scholar Crossref Search ADS WorldCat Blaha D , Prigent-Combaret C, Mirza MS, Moënne-Loccoz Y ( 2006 ) Phylogeny of the 1-aminocyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography . FEMS Microbiol Ecol 56 : 455 – 470 Google Scholar Crossref Search ADS PubMed WorldCat Bradford KJ , Yang SF ( 1980 ) Xylem transport of 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor, in waterlogged tomato plants . Plant Physiol 65 : 322 – 326 Google Scholar Crossref Search ADS PubMed WorldCat Bruto M , Prigen-Combaret C, Muller , D, Moënne-Loccoz , Y ( 2014 ) Analysis of genes contributing to plant-beneficial functions in plant growth-promoting rhizobacteria and related Proteobacteria . Sci Rpts 4 : 6261 Burd GI , Dixon DG, Glick BR ( 1998 ) A plant growth-promoting bacterium that decreases nickel toxicity in seedlings . Appl Environ Microbiol 64 : 3663 – 3668 Google Scholar Crossref Search ADS PubMed WorldCat Chang P , Gerhardt KE, Huang XD, Yu XM, Glick BR, Gerwing PD, Greenberg BM ( 2014 ) Plant growth-promoting bacteria facilitate the growth of barley and oats in salt-impacted soil: implications for phytoremediation of saline soils . Int J Phytoremediation 16 : 1133 – 1147 Google Scholar Crossref Search ADS PubMed WorldCat Chi F , Shen SH, Cheng HP, Jing YX, Yanni YG, Dazzo FB ( 2005 ) Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology . Appl Environ Microbiol 71 : 7271 – 7278 Google Scholar Crossref Search ADS PubMed WorldCat Chookietwattana K , Maneewan K ( 2012 ) Selection of efficient salt-tolerant bacteria containing ACC deaminase for promotion of tomato growth under salinity stress . Soil Environ 31 : 30 – 36 Google Scholar OpenURL Placeholder Text WorldCat Compant S , Clement C, Sessitsch A ( 2010 ) Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization . Soil Biol Biochem 42 : 669 – 678 Google Scholar Crossref Search ADS WorldCat Compant S , Kaplan H, Sessitsch A, Nowak J, Ait Barka E, Clément C ( 2008 ) Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues . FEMS Microbiol Ecol 63 : 84 – 93 Google Scholar Crossref Search ADS PubMed WorldCat Czarny JC , Shah S, Glick BR ( 2007 ) Response of canola plants at the transcriptional level to expression of a bacterial ACC deaminase in the roots . In A Ramina , C Chang , J Giovannoni , H Klee , P Perata , E Woltering , eds, Advances in Plant Ethylene Research. Springer , Dordrecht, Netherlands , pp 377 – 382 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Damalas CA , Eleftherohorinos IG ( 2011 ) Pesticide exposure, safety issues, and risk assessment indicators . Int J Environ Res Public Health 8 : 1402 – 1419 Google Scholar Crossref Search ADS PubMed WorldCat Danhorn T , Fuqua C ( 2007 ) Biofilm formation by plant-associated bacteria . Annu Rev Microbiol 61 : 401 – 422 Google Scholar Crossref Search ADS PubMed WorldCat Duan J , Müller KM, Charles TC, Vesely S, Glick BR ( 2009 ) 1-Aminocyclopropane-1-carboxylate (ACC) deaminase genes in rhizobia from southern Saskatchewan . Microb Ecol 57 : 423 – 436 Google Scholar Crossref Search ADS PubMed WorldCat Else MA , Jackson MB ( 1998 ) Transport of 1-aminocyclopropane-1-carboxylic acid (ACC) in the transpiration stream of tomato (Lycopersicon esculentum) in relation to foliar ethylene production and petiole epinasty . Aust J Plant Physiol 25 : 453 – 458 Google Scholar OpenURL Placeholder Text WorldCat El-Tarabily KA ( 2013 ) Biocontrol of damping-off and root and crown rots of cucumber caused by Pythium aphanidermatum by ACC-deaminase producing endophytic actinomycetes . Phytopathology 103 : 40 Google Scholar OpenURL Placeholder Text WorldCat Gaiero JR , McCall CA, Thompson KA, Day NJ, Best AS, Dunfield KE ( 2013 ) Inside the root microbiome: bacterial root endophytes and plant growth promotion . Am J Bot 100 : 1738 – 1750 Google Scholar Crossref Search ADS PubMed WorldCat Galland M , Gamet L, Varoquaux F, Touraine B, Touraine B, Desbrosses G ( 2012 ) The ethylene pathway contributes to root hair elongation induced by the beneficial bacteria Phyllobacterium brassicacearum STM196 . Plant Sci 190 : 74 – 81 Google Scholar Crossref Search ADS PubMed WorldCat Gamalero E, Berta G, Glick BR (2009a) The use of microorganisms to facilitate the growth of plants in saline soils. In MS Khan, A Zaidi, J Musarrat, eds, Microbial Strategies for Crop Improvement. Springer-Verlag, Berlin, pp 1–22 Gamalero E , Berta G, Lingua G, Glick BR ( 2009 b ) Effects of plant growth-promoting bacteria and AM fungi on the response of plants to heavy metal stress . Can J Microbiol 55 : 501 – 514 Google Scholar Crossref Search ADS PubMed WorldCat Gamalero E , Berta G, Massa N, Glick BR, Lingua G ( 2008 ) Synergistic interactions between the ACC deaminase-producing bacterium Pseudomonas putida UW4 and the AM fungus Gigaspora rosea positively affect cucumber plant growth . FEMS Microbiol Ecol 64 : 459 – 467 Google Scholar Crossref Search ADS PubMed WorldCat Gamalero E , Berta G, Massa N, Glick BR, Lingua G ( 2010 ) Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences for the growth of cucumber under salt-stress conditions . J Appl Microbiol 108 : 236 – 245 Google Scholar Crossref Search ADS PubMed WorldCat Gamalero E , Glick BR ( 2010 ) Bacterial ACC deaminase and IAA: interactions and consequences for plant growth in polluted environments . In IA Golubev , ed, Handbook of Phytoremediation. Nova Science Publishers , New York , pp 763 – 774 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Gamalero E, Glick BR (2012) Ethylene and abiotic stress tolerance in plants. In P Ahmad, MNV Prasad, eds, Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change. Springer-Verlag, Berlin, pp 395–412 García-Fraile P , Carro L, Robledo M, Ramírez-Bahena MH, Flores-Félix JD, Fernández MT, Mateos PF, Rivas R, Igual JM, Martínez-Molina E, et al. ( 2012 ) Rhizobium promotes non-legumes growth and quality in several production steps: towards a biofertilization of edible raw vegetables healthy for humans . PLoS ONE 7 : e38122 Google Scholar Crossref Search ADS PubMed WorldCat Glick BR ( 1995 ) The enhancement of plant growth by free-living bacteria . Can J Microbiol 41 : 109 – 117 Google Scholar Crossref Search ADS WorldCat Glick BR ( 2010 ) Using soil bacteria to facilitate phytoremediation . Biotechnol Adv 28 : 367 – 374 Google Scholar Crossref Search ADS PubMed WorldCat Glick BR (September 13, 2012) Plant growth-promoting bacteria: mechanisms and applications. Scientifica http://dx.doi.org/10.6064/2012/963401 Glick BR ( 2014 ) Bacteria with ACC deaminase can promote plant growth and help to feed the world . Microbiol Res 169 : 30 – 39 Google Scholar Crossref Search ADS PubMed WorldCat Glick BR , Cheng Z, Czarny J, Duan J ( 2007 ) Promotion of plant growth by ACC deaminase-containing soil bacteria . Eur J Plant Pathol 119 : 329 – 339 Google Scholar Crossref Search ADS WorldCat Glick BR , Penrose DM, Li J ( 1998 ) A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria . J Theor Biol 190 : 63 – 68 Google Scholar Crossref Search ADS PubMed WorldCat Grichko VP , Glick BR ( 2001 ) Amelioration of flooding stress by ACC deaminase-containing plant growth-promoting bacteria . Plant Physiol Biochem 39 : 11 – 17 Google Scholar Crossref Search ADS WorldCat Gurska J , Wang W, Gerhardt KE, Khalid AM, Isherwood DM, Huang XD, Glick BR, Greenberg BM ( 2009 ) Field test of a multi-process phytoremediation system at a petroleum sludge contaminated land farm . Environ Sci Technol 43 : 4472 – 4479 Google Scholar Crossref Search ADS PubMed WorldCat Gutiérrez-Zamora ML , Martínez-Romero E ( 2001 ) Natural endophytic association between Rhizobium etli and maize (Zea mays L.) . J Biotechnol 91 : 117 – 126 Google Scholar Crossref Search ADS PubMed WorldCat Hao Y , Charles TC, Glick BR ( 2011 ) An ACC deaminase containing A. tumefaciens strain D3 shows biocontrol activity to crown gall disease . Can J Microbiol 57 : 278 – 286 Google Scholar Crossref Search ADS PubMed WorldCat Hirsch PR , Mauchline TH ( 2012 ) Who’s who in the plant root microbiome? Nat Biotechnol 30 : 961 – 962 Google Scholar Crossref Search ADS PubMed WorldCat Honma M ( 1983 ) Enzymatic determination of 1-aminocyclopropane-1-carboxylate deam-inase . Agric Biol Chem 47 : 617 – 618 Google Scholar OpenURL Placeholder Text WorldCat Honma M , Shimomura T ( 1978 ) Metabolism of 1-aminocyclopropane-1-carboxylic acid . Agric Biol Chem 42 : 1825 – 1831 Google Scholar OpenURL Placeholder Text WorldCat Hontzeas N , Hontzeas CE, Glick BR ( 2006 ) Reaction mechanisms of the bacterial enzyme 1-aminocyclopropane-1-carboxylate deaminase . Biotechnol Adv 24 : 420 – 426 Google Scholar Crossref Search ADS PubMed WorldCat Hontzeas N , Richardson AO, Belimov AA, Safranova VI, Abu-Omar MM, Glick BR ( 2005 ) Evidence for horizontal gene transfer (HGT) of ACC deaminase genes . Appl Environ Microbiol 71 : 7556 – 7558 Google Scholar Crossref Search ADS PubMed WorldCat Hontzeas N , Zoidakis J, Glick BR, Abu-Omar MM ( 2004 ) Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the rhizobacterium Pseudomonas putida UW4: a key enzyme in bacterial plant growth promotion . Biochim Biophys Acta 1703 : 11 – 19 Google Scholar Crossref Search ADS PubMed WorldCat Huang XD , El-Alawi Y, Penrose DM, Glick BR, Greenberg BM ( 2004 ) A multi-process phytoremediation system for removal of polycyclic aromatic hydrocarbons from contaminated soils . Environ Pollut 130 : 465 – 476 Google Scholar Crossref Search ADS PubMed WorldCat Indiragandhi P , Anandham R, Kim K, Yim WJ, Madhaiyan M, Sa TM ( 2008 ) Induction of defense responses in tomato against Pseudomonas syringae pv. tomato by regulating the stress ethylene level with Methylobacterium oryzae CBMB20 containing 1-aminocyclopropane-1-carboxylate deaminase . World J Microbiol Biotechnol 24 : 1037 – 1045 Google Scholar Crossref Search ADS WorldCat Jacobson CB , Pasternak JJ, Glick BR ( 1994 ) Partial purification and characterization of ACC deaminase from the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 . Can J Microbiol 40 : 1019 – 1025 Google Scholar Crossref Search ADS WorldCat Karthikeyan B , Joe MM, Islam MR, Sa T ( 2012 ) ACC deaminase containing diazotrophic endophytic bacteria ameliorate salt stress in Catharanthus roseus through reduced ethylene levels and induction of antioxidative defense systems . Symbiosis 56 : 77 – 86 Google Scholar Crossref Search ADS WorldCat Khan AG , Kuek C, Chaudhry TM, Khoo CS, Hayes WJ ( 2000 ) Role of plants, mycorrhizae and phytochelators in heavy metal contaminated land remediation . Chemosphere 41 : 197 – 207 Google Scholar Crossref Search ADS PubMed WorldCat Kuklinsky-Sobral J , Araújo WL, Mendes R, Geraldi IO, Pizzirani-Kleiner AA, Azevedo JL ( 2004 ) Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion . Environ Microbiol 6 : 1244 – 1251 Google Scholar Crossref Search ADS PubMed WorldCat Leach AW , Mumford JD ( 2008 ) Pesticide Environmental Accounting: a method for assessing the external costs of individual pesticide applications . Environ Pollut 151 : 139 – 147 Google Scholar Crossref Search ADS PubMed WorldCat Li J , McConkey BJ, Cheng Z, Guo S, Glick BR ( 2013 ) Identification of plant growth-promoting bacteria-responsive proteins in cucumber roots under hypoxic stress using a proteomic approach . J Proteomics 84 : 119 – 131 Google Scholar Crossref Search ADS PubMed WorldCat Li Q , Saleh-Lakha S, Glick BR ( 2005 ) The effect of native and ACC deaminase-containing Azospirillum brasilense Cd1843 on the rooting of carnation cuttings . Can J Microbiol 51 : 511 – 514 Google Scholar Crossref Search ADS PubMed WorldCat Lupway N , Clayton G, Hanson K, Rice W, Bierderbeck V ( 2004 ) Endophytic rhizobia in barley, wheat, and canola roots . Can J Plant Sci 84 : 37 – 45 Google Scholar Crossref Search ADS WorldCat Ma W , Guinel FC, Glick BR ( 2003 ) Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants . Appl Environ Microbiol 69 : 4396 – 4402 Google Scholar Crossref Search ADS PubMed WorldCat Mayak S , Tirosh T, Glick BR ( 1999 ) Effect of wild-type and mutant plant growth-promoting rhizobacteria on the rooting of mung bean cuttings . J Plant Growth Regul 18 : 49 – 53 Google Scholar Crossref Search ADS PubMed WorldCat Mayak S , Tirosh T, Glick BR ( 2004 a ) Plant growth-promoting bacteria that confer resistance to water stress in tomato and pepper . Plant Sci 166 : 525 – 530 Google Scholar Crossref Search ADS WorldCat Mayak S , Tirosh T, Glick BR ( 2004 b ) Plant growth-promoting bacteria confer resistance in tomato plants to salt stress . Plant Physiol Biochem 42 : 565 – 572 Google Scholar Crossref Search ADS PubMed WorldCat Mendes R , Garbeva P, Raaijmakers JM ( 2013 ) The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms . FEMS Microbiol Rev 37 : 634 – 663 Google Scholar Crossref Search ADS PubMed WorldCat Minami R , Uchiyama K, Murakami T, Kawai J, Mikami K, Yamada T, Yokoi D, Ito H, Matsui H, Honma M ( 1998 ) Properties, sequence, and synthesis in Escherichia coli of 1-aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus . J Biochem 123 : 1112 – 1118 Google Scholar Crossref Search ADS PubMed WorldCat Montero-Calasanz MC , Santamaría C, Albareda M, Daza A, Duan J, Glick BR, Camacho M ( 2013 ) Alternative rooting induction of semi-hardwood olive cuttings by several auxin-producing bacteria for organic agriculture systems . Span J Agric Res 11 : 146 – 154 Google Scholar Crossref Search ADS WorldCat Murset V , Hennecke H, Pessi G ( 2012 ) Disparate role of rhizobial ACC deaminase in root-nodule symbioses . Symbiosis 57 : 43 – 50 Google Scholar Crossref Search ADS WorldCat Nadeem SM , Zahair ZA, Naveed M, Asghar HN, Asghar M ( 2010 ) Rhizobacteria capable of producing ACC-deaminase may mitigate salt stress in wheat . Soil Sci Soc Am J 74 : 533 – 542 Google Scholar Crossref Search ADS WorldCat Nascimento FX , Brígido C, Glick BR, Oliveira S ( 2012 ) ACC deaminase genes are conserved among Mesorhizobium species able to nodulate the same host plant . FEMS Microbiol Lett 336 : 26 – 37 Google Scholar Crossref Search ADS PubMed WorldCat Nascimento FX , Rossi MJ, Soares CRFS, McConkey BJ, Glick BR ( 2014 ) New insights into 1-aminocyclopropane-1-carboxylate (ACC) deaminase phylogeny, evolution and ecological significance . PLoS ONE 9 : e99168 Google Scholar Crossref Search ADS PubMed WorldCat Nascimento FX , Vicente CSL, Barbosa P, Espada M, Glick BR, Oliveira S, Mota M ( 2013 ) The use of the ACC deaminase producing bacterium Pseudomonas putida UW4 as a biocontrol agent for pine wilt disease . BioControl 58 : 427 – 433 Google Scholar Crossref Search ADS WorldCat Nukui N , Minamisawa K, Ayabe S, Aoki T ( 2006 ) Expression of the 1-aminocyclopropane-1-carboxylic acid deaminase gene requires symbiotic nitrogen-fixing regulator gene nifA2 in Mesorhizobium loti MAFF303099 . Appl Environ Microbiol 72 : 4964 – 4969 Google Scholar Crossref Search ADS PubMed WorldCat Patten CL , Glick BR ( 1996 ) Bacterial biosynthesis of indole-3-acetic acid . Can J Microbiol 42 : 207 – 220 Google Scholar Crossref Search ADS PubMed WorldCat Penrose DM , Glick BR ( 2001 ) Levels of ACC and related compounds in exudate and extracts of canola seeds treated with ACC deaminase-containing plant growth-promoting bacteria . Can J Microbiol 47 : 368 – 372 Google Scholar Crossref Search ADS PubMed WorldCat Penrose DM , Moffatt BA, Glick BR ( 2001 ) Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings . Can J Microbiol 47 : 77 – 80 Google Scholar Crossref Search ADS PubMed WorldCat Pierik R , Tholen D, Poorter H, Visser EJW, Voesenek LACJ ( 2006 ) The Janus face of ethylene: growth inhibition and stimulation . Trends Plant Sci 11 : 176 – 183 Google Scholar Crossref Search ADS PubMed WorldCat Pilon-Smits E ( 2005 ) Phytoremediation . Annu Rev Plant Biol 56 : 15 – 39 Google Scholar Crossref Search ADS PubMed WorldCat Prayitno J , Rolfe BG, Mathesius U ( 2006 ) The ethylene-insensitive sickle mutant of Medicago truncatula shows altered auxin transport regulation during nodulation . Plant Physiol 142 : 168 – 180 Google Scholar Crossref Search ADS PubMed WorldCat Prigent-Combaret C , Blaha D, Pothier JF, Vial L, Poirier MA, Wisniewski-Dyé F, Moënne-Loccoz Y ( 2008 ) Physical organization and phylogenetic analysis of acdR as leucine-responsive regulator of the 1-aminocyclopropane-1-carboxylate deaminase gene acdS in phytobeneficial Azospirillum lipoferum 4B and other Proteobacteria . FEMS Microbiol Ecol 65 : 202 – 219 Google Scholar Crossref Search ADS PubMed WorldCat Ramadoss D , Lakkineni VK, Bose P, Ali S, Annapurna K ( 2013 ) Mitigation of salt stress in wheat seedlings by halotolerant bacteria isolated from saline habitats . Springerplus 2 : 6 Google Scholar Crossref Search ADS PubMed WorldCat Reed MLE , Glick BR ( 2005 ) Growth of canola (Brassica napus) in the presence of plant growth-promoting bacteria and either copper or polycyclic aromatic hydrocarbons . Can J Microbiol 51 : 1061 – 1069 Google Scholar Crossref Search ADS PubMed WorldCat Reed MLE , Glick BR ( 2013 ) Applications of plant growth-promoting bacteria for plant and soil systems . In VK Gupta , M Schmoll , M Maki , M Tuohy , MA Mazutti , eds, Applications of Microbial Engineering. Taylor and Francis , Enfield, CT , pp 181 – 229 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Reid MS , Wu MJ ( 1991 ) Ethylene in flower development and senescence . In AK Mattoo , JC Suttle , eds The Plant Hormone Ethylene. CRC Press , Boca Raton, FL , pp 215 – 234 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Reinhold-Hurek B , Hurek T ( 2011 ) Living inside plants: bacterial endophytes . Curr Opin Plant Biol 14 : 435 – 443 Google Scholar Crossref Search ADS PubMed WorldCat Sairam RK , Tyagi A ( 2004 ) Physiology and molecular biology of salinity stress tolerance in plants . Curr Sci 86 : 407 – 421 Google Scholar OpenURL Placeholder Text WorldCat Sarma RK , Saikia R ( 2014 ) Alleviation of drought stress in mung bean by strain Pseudomonas aeruginosa GGRJ21 . Plant Soil 377 : 111 – 126 Google Scholar Crossref Search ADS WorldCat Shakir MA , Asghari B, Arshad M ( 2012 ) Rhizosphere bacteria containing ACC deaminase conferred drought tolerance in wheat grown under semi-arid climate . Soil Environ 31 : 108 – 112 Google Scholar OpenURL Placeholder Text WorldCat Sheehy RE , Honma M, Yamada M, Sasaki T, Martineau B, Hiatt WR ( 1991 ) Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase . J Bacteriol 173 : 5260 – 5265 Google Scholar Crossref Search ADS PubMed WorldCat Siddikee MA , Glick BR, Chauhan PS, Yim WJ, Sa T ( 2011 ) Enhancement of growth and salt tolerance of red pepper seedlings (Capsicum annuum L.) by regulating stress ethylene synthesis with halotolerant bacteria containing ACC deaminase activity . Plant Physiol Biochem 49 : 427 – 434 Google Scholar Crossref Search ADS PubMed WorldCat Stearns JC , Woody OZ, McConkey BJ, Glick BR ( 2012 ) Effects of bacterial ACC deaminase on Brassica napus gene expression . Mol Plant Microbe Interact 25 : 668 – 676 Google Scholar Crossref Search ADS PubMed WorldCat Thakore Y ( 2006 ) The biopesticide market for global agricultural use . Ind Biotechnol (New Rochelle NY) 2 : 194 – 208 Google Scholar Crossref Search ADS WorldCat Timmusk S , Paalme V, Pavlicek T, Bergquist J, Vangala A, Danilas T, Nevo E ( 2011 ) Bacterial distribution in the rhizosphere of wild barley under contrasting microclimates . PLoS ONE 6 : e17968 Google Scholar Crossref Search ADS PubMed WorldCat Toklikishvili N , Dandurishvili N, Vainstein A, Tediashvili M, Giorgobiani N, Lurie S, Szegedi E, Glick BR, Chernin L ( 2010 ) Inhibitory effect of ACC deaminase-producing bacteria on crown gall formation in tomato plants infected by Agrobacterium tumefaciens or A. vitis . Plant Pathol 59 : 1023 – 1030 Google Scholar Crossref Search ADS WorldCat Tsakelova EA , Klimova SY, Cherdyntseva TA, Netrusov AI ( 2006 ) Microbial producers of plant growth stimulators and their practical use: a review. Appl Biochem Microbiol 42 : 117 – 126 Google Scholar Crossref Search ADS WorldCat van Loon LC , Geraats BPJ, Linthorst HJM ( 2006 ) Ethylene as a modulator of disease resistance in plants . Trends Plant Sci 11 : 184 – 191 Google Scholar Crossref Search ADS PubMed WorldCat Wang C , Knill E, Glick BR, Défago G ( 2000 ) Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities . Can J Microbiol 46 : 898 – 907 Google Scholar Crossref Search ADS PubMed WorldCat Wilson D ( 1995 ) Endophyte: the evolution of a term and clarification of its use and definition . Oikos 72 : 274 – 276 Google Scholar Crossref Search ADS WorldCat Yoon GM, Kieber JK (March 11, 2013) 1-Aminocyclopropane-1-carboxylic acid as a signalling molecule in plants. AoB Plants http://dx.doi.org/10.1093/aobpla/plt017 Yoon HS, Andersen RA, Boo SM, Bhattacharya D (2009) Stramenopiles. In M Schaechter, ed, Encyclopedia of Microbiology, Ed 3. Academic Press, New York, pp 721–731 Zafarul Hye M , Farooq HM, Zahir ZA, Hussain M, Hussain A ( 2014 ) Application of ACC-deaminase containing rhizobacteria with fertilizer improves maize production under drought and salinity stress . Int J Agric Biol 16 : 591 – 596 Google Scholar OpenURL Placeholder Text WorldCat Zamioudis C , Mastranesti P, Dhonukshe P, Blilou I, Pieterse CMJ ( 2013 ) Unraveling root developmental programs initiated by beneficial Pseudomonas spp. bacteria . Plant Physiol 162 : 304 – 318 Google Scholar Crossref Search ADS PubMed WorldCat Author notes * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00284 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Gibbs, Daniel J.; Conde, Jorge Vicente; Berckhan, Sophie; Prasad, Geeta; Mendiondo, Guillermina M.; Holdsworth, Michael J.

doi: 10.1104/pp.15.00338pmid: 25944828

Abstract The group VII ethylene response factors (ERFVIIs) are plant-specific transcription factors that have emerged as important regulators of abiotic and biotic stress responses, in particular, low-oxygen stress. A defining feature of ERFVIIs is their conserved N-terminal domain, which renders them oxygen- and nitric oxide (NO)-dependent substrates of the N-end rule pathway of targeted proteolysis. In the presence of these gases, ERFVIIs are destabilized, whereas an absence of either permits their accumulation; ERFVIIs therefore coordinate plant homeostatic responses to oxygen availability and control a wide range of NO-mediated processes. ERFVIIs have a variety of context-specific protein and gene interaction partners, and also modulate gibberellin and abscisic acid signaling to regulate diverse developmental processes and stress responses. This update discusses recent advances in our understanding of ERFVII regulation and function, highlighting their role as central regulators of gaseous signal transduction at the interface of ethylene, oxygen, and NO signaling. The ethylene response factor (ERF) transcription factors are plant-specific proteins characterized by a single DNA-binding APETALA2 (AP2)/ethylene-responsive element-binding protein domain (Nakano et al., 2006; Licausi et al., 2013). This domain was first discovered in ethylene-responsive element-binding proteins in tobacco (Nicotiana tabacum) and shown to bind to the GCC element of ethylene-responsive elements in promoters (Ohme-Takagi and Shinshi, 1995). ERFs constitute one of the largest transcription factor families in plants: a genome-wide analysis found that rice (Oryza sativa) has 139 and Arabidopsis (Arabidopsis thaliana) 122, clustered into 15 and 12 subgroups, respectively, based on the presence of conserved features (Nakano et al., 2006). One of these subgroups, the group VII ethylene response factors (ERFVIIs), has been heavily studied in recent years due to the roles its members play in orchestrating a wide range of plant growth, development, and stress responses. In addition to the AP2 domain, ERFVIIs are characterized by several other motifs, including, most importantly, N-terminal (Nt)-MCGGAII/L (Nakano et al., 2006; van Veen et al., 2014), which is a conserved feature of ERFVIIs in all flowering plant species (Fig. 1, A and B). A recent study investigated the phylogenetic origin of ERFVIIs using an analysis of synteny in 16 angiosperm species, revealing that ERFVIIs originated from two ancestral genes, and that subsequent gene duplication resulted in the appearance of five members before mono- and dicotyledonous plants divided (Fig. 1A). Of the five Arabidopsis ERFVIIs, HYPOXIA RESPONSIVE1 (HRE1), RELATED TO APETALA2.2 (RAP2.2), and RAP2.12 were shown to share syntenic origins, whereas HRE2 and RAP2.3 evolved from a separate ancestral protein (van Veen et al., 2014). Figure 1. Open in new tabDownload slide Phylogeny and features of ERFVII transcription factors. A, Phylogenetic relationship of the five ERFVIIs in Arabidopsis, showing their groupings into two syntenic blocks (SBI and SBII; van Veen et al., 2014). A schematic representation of each family member is shown, including the Nt (conserved motif [CM]VII-1) and AP2 domains, which are defining features of the ERFVIIs found in all family members, and several other amino acid motifs that are conserved in some but not all ERFVIIs (Nakano et al., 2006). B, Sequence logo showing the first 10 amino acids of ERFVII protein sequences from Zea mays, Brachypodium distachyon, rice, Arabidopsis lyrata, Arabidopsis, Prunus persica, Medicago truncatula, and Populus trichocarpa. Logo was generated from 89 sequences using Geneious (Blosum 62 matrix, gap open penalty 15, gap extension penalty 3; Kearse et al., 2012). The observed frequency of amino acids at each position is measured in bits. The individual Arabidopsis ERFVII Nts are shown underneath. Figure 1. Open in new tabDownload slide Phylogeny and features of ERFVII transcription factors. A, Phylogenetic relationship of the five ERFVIIs in Arabidopsis, showing their groupings into two syntenic blocks (SBI and SBII; van Veen et al., 2014). A schematic representation of each family member is shown, including the Nt (conserved motif [CM]VII-1) and AP2 domains, which are defining features of the ERFVIIs found in all family members, and several other amino acid motifs that are conserved in some but not all ERFVIIs (Nakano et al., 2006). B, Sequence logo showing the first 10 amino acids of ERFVII protein sequences from Zea mays, Brachypodium distachyon, rice, Arabidopsis lyrata, Arabidopsis, Prunus persica, Medicago truncatula, and Populus trichocarpa. Logo was generated from 89 sequences using Geneious (Blosum 62 matrix, gap open penalty 15, gap extension penalty 3; Kearse et al., 2012). The observed frequency of amino acids at each position is measured in bits. The individual Arabidopsis ERFVII Nts are shown underneath. ERFVIIs CONTROL FLOODING RESPONSES A major role for ERFVIIs in controlling flooding and low oxygen (hypoxia) tolerance in plants has been discovered in recent years. For example, in Arabidopsis, hre1hre2 mutant seedlings showed drastically reduced survival in anoxia (0% oxygen), whereas ectopic overexpression of HRE1 enhanced survival by increasing the expression of core hypoxia-responsive genes, including ALCOHOL DEHYDROGENASE1 (ADH1; Licausi et al., 2010; Hess et al., 2011). A similar function was observed for RAP2.2, where ectopic expression increased ADH1 transcripts in hypoxia (5% oxygen), whereas rap2.2 mutants had reduced survival under this stress (Hinz et al., 2010). Other studies in Arabidopsis have also pointed to key roles for these proteins in controlling hypoxia and ethylene-regulated submergence responses (Papdi et al., 2008; Yang et al., 2011). Flood-tolerant plants typically cope with submergence through two opposite survival strategies: quiescence during flash floods and escape during deep-water floods (Voesenek and Bailey-Serres, 2015). The quiescence strategy is characterized by reduced growth and respiration, metabolic changes that enhance use of carbohydrate reserves, induced responses against oxidative damage, and inhibition of floral initiation (Peña-Castro et al., 2011; Bailey-Serres et al., 2012). In contrast, the deep-water escape strategy requires rapid growth of petioles and stems, and vascular changes to facilitate gas diffusion (Bailey-Serres et al., 2012; Voesenek and Bailey-Serres, 2015). In rice, three different members of the ERFVII family control these antithetical survival strategies: SUBMERGENCE1A (SUB1A), one of three ERFVIIs located at the Sub1 locus, promotes quiescence, whereas SNORKEL1 and SNORKEL2 regulate escape (Xu et al., 2006; Hattori et al., 2009). These ERFVIIs are transcriptionally induced by ethylene, which rapidly accumulates in flooded tissues, but regulate their respective growth strategies by modifying GA signaling in opposite directions. Ethylene induction of SUB1A increases the accumulation of SLENDER RICE1 (SLR1) and SLENDER RICE1 LIKE1, two GA-labile signaling repressors that inhibit the transcription of GA-inducible genes, arresting elongation and promoting catabolism of carbohydrates (Fukao et al., 2006; Fukao and Bailey-Serres, 2008; Hirano et al., 2012). Furthermore, there is a strong up-regulation of GA-deactivating GA 2-oxidase in SUB1A-containing lines, which would reduce the levels of active GA and enhance SLR1 stability (Jung et al., 2010). In contrast, during flooding in deep-water rice varieties, the induction of SNORKEL1 and SNORKEL2 by ethylene triggers internode elongation by up-regulating GA 20-oxidase, which increases the accumulation of active GA (Raskin and Kende, 1984; Hattori et al., 2009; Ayano et al., 2014). Similar quiescent and escape-like flooding responses have also recently been identified in two related Rumex spp. species from contrasting hydrological niches, which may also be regulated by ERFVIIs (van Veen et al., 2013). Together with ethylene and GA, abscisic acid (ABA) is also an important hormone involved in the signaling network regulated by SUB1A in rice (Hoffmann-Benning and Kende, 1992; Fukao and Bailey-Serres, 2008; Chen et al., 2010; Fukao et al., 2011). When flood waters subside, desubmergence can lead to rapid dehydration of the leaves (Setter et al., 2010; Fukao et al., 2011). ABA levels decline upon submergence (Fukao and Bailey-Serres, 2008), and SUB1A prevents leaf desiccation by increasing ABA responsiveness, which promotes the expression of several drought-associated DEHYDRATION RESPONSE ELEMENT-BINDING PROTEIN1 and LATE EMBRYOGENESIS ABUNDANT genes, limits the spread of reactive oxygen species (ROS), and induces enzymes (e.g. superoxide dismutases and catalases) that neutralize the oxidative damage associated with dehydration (Fukao et al., 2011). The role of SUB1A in fine tuning ROS levels seems to be also essential for flooding tolerance, specifically regulating the role of hydrogen peroxide in aerenchyma formation, but at the same time limiting its toxicity during the stress (Jung et al., 2010; Parlanti et al., 2011). Furthermore, there is evidence supporting a role for SUB1A in maintaining the levels of chlorophyll and carbohydrates during leaf senescence promoted by ethylene, a process associated with several stresses, including drought (Fukao et al., 2012). It has also been reported that GA 2-oxidase may inhibit leaf growth and promote root growth during drought (Wang et al., 2011). Intriguingly, recent data show a positive role in drought of GA-INSENSITIVE DWARF1, the GA receptor that counteracts SUB1A action by degrading SLR1 (Du et al., 2014); future studies will be required to unravel the interaction between these factors during dehydration. ERFVIIs ARE SUBSTRATES OF THE N-END RULE PATHWAY OF PROTEOLYSIS In addition to ethylene accumulation, another key signal associated with flooding is reduced oxygen availability (van Dongen and Licausi, 2015). It has been shown that ERFVIIs function as homeostatic sensors of hypoxia via the N-end rule pathway of targeted proteolysis (Gibbs et al., 2011; Licausi et al., 2011), an ancient and conserved branch of the ubiquitin proteasome system that relates the stability of a protein to the nature of its N terminus (Fig. 2; Bachmair et al., 1986; Varshavsky, 2011; Gibbs et al., 2014a). Moreover, this ERFVII-based system has also emerged as a critical mechanism for NO sensing (Gibbs et al., 2014b), highlighting a central role for ERFVIIs and their proteolytic control by the N-end rule pathway in gaseous signal transduction. Figure 2. Open in new tabDownload slide Oxygen (O2) and nitric oxide (NO) signal transduction via N-end rule regulation of ERFVII stability. Arabidopsis ERFVII transcription factors are transcribed either constitutively and/or in response to several upstream signals, including ethylene. MET AMINOPEPTIDASE (MetAP) enzymes cleave Nt-Met cotranslationally to reveal Nt-Cys (C). In the presence of oxygen and NO (which may be derived from a variety of cellular sources: NR, nitrate reductase; NOA1, nitric oxide associated protein1), Nt-Cys is oxidized to Cys-sulfinic or Cys-sulfonic acid (C*). This oxidation is facilitated by PLANT CYS OXIDASE (PCO) enzymes (Weits et al., 2014). Oxidized Nt-Cys is then arginylated (R) by ARGINYL tRNA TRANSFERASES (ATE1/2). Nt-Arg-ERFVII is recognized by the N-end rule E3 ligase (N-recognin) PROTEOLYSIS6 (PRT6), which targets the protein for proteasomal degradation via polyubiquitination (Ubi; Gibbs et al., 2011; Licausi et al., 2011). ERFVII degradation initiates germination, inhibits hypocotyl growth, stimulates stomatal closure, represses the hypoxia transcriptional response, and promotes photomorphogenesis. In conditions in which either oxygen or NO (or both) become limiting, Nt-Cys oxidation is inhibited, and ERFVIIs are stabilized (Gibbs et al., 2014b). Stable ERFVIIs promote anaerobic gene expression, maintain seed dormancy and open stomata, promote hypocotyl growth, and repress photomorphogenesis. Regulation of ERFVII stability by the N-end rule pathway therefore controls the homeostatic response to oxygen and NO availability. Figure 2. Open in new tabDownload slide Oxygen (O2) and nitric oxide (NO) signal transduction via N-end rule regulation of ERFVII stability. Arabidopsis ERFVII transcription factors are transcribed either constitutively and/or in response to several upstream signals, including ethylene. MET AMINOPEPTIDASE (MetAP) enzymes cleave Nt-Met cotranslationally to reveal Nt-Cys (C). In the presence of oxygen and NO (which may be derived from a variety of cellular sources: NR, nitrate reductase; NOA1, nitric oxide associated protein1), Nt-Cys is oxidized to Cys-sulfinic or Cys-sulfonic acid (C*). This oxidation is facilitated by PLANT CYS OXIDASE (PCO) enzymes (Weits et al., 2014). Oxidized Nt-Cys is then arginylated (R) by ARGINYL tRNA TRANSFERASES (ATE1/2). Nt-Arg-ERFVII is recognized by the N-end rule E3 ligase (N-recognin) PROTEOLYSIS6 (PRT6), which targets the protein for proteasomal degradation via polyubiquitination (Ubi; Gibbs et al., 2011; Licausi et al., 2011). ERFVII degradation initiates germination, inhibits hypocotyl growth, stimulates stomatal closure, represses the hypoxia transcriptional response, and promotes photomorphogenesis. In conditions in which either oxygen or NO (or both) become limiting, Nt-Cys oxidation is inhibited, and ERFVIIs are stabilized (Gibbs et al., 2014b). Stable ERFVIIs promote anaerobic gene expression, maintain seed dormancy and open stomata, promote hypocotyl growth, and repress photomorphogenesis. Regulation of ERFVII stability by the N-end rule pathway therefore controls the homeostatic response to oxygen and NO availability. ERFVIIs and Oxygen Sensing A defining feature of the ERFVIIs is the presence of an Nt motif that initiates with the residues Met-Cys (Fig. 1, A and B; Gibbs et al., 2011; Licausi et al., 2011). This motif is highly conserved across ERFVIIs in flowering plants and functions as an N-degron, rendering these proteins substrates of the N-end rule pathway. The Nt-Met of ERFVIIs is cotranslationally cleaved by cytosolic MET AMINOPEPTIDASEs to reveal a tertiary destabilizing Nt-Cys residue that is susceptible to oxidative modifications to produce an oxidized Cys (Cys sulfinic or sulfonic acid; Hu et al., 2005), which is then arginylated by ATEs. Nt-Arg-Cys is then recognized by the N-end rule pathway E3 ligase (N-recognin) PRT6, which targets the ERFVII for destruction via polyubiquitination (Fig. 2). In the presence of oxygen (and NO; see next section), oxidation of Nt-Cys therefore catalyzes protein degradation, whereas under hypoxia, ERFVIIs accumulate to coordinate the transcriptional response to oxygen limitation (Gibbs et al., 2011; Licausi et al., 2011). In Arabidopsis, RAP2.12, RAP2.2, and RAP2.3 are all constitutively expressed, whereas HRE1 and HRE2 are hypoxia inducible, suggesting that there is a cascade of transcription and stabilization in response to declining oxygen levels, and that individual ERFVIIs have different contributions to the response (Licausi et al., 2010; Bui et al., 2015). It was hypothesized that reliance upon spontaneous Nt-Cys oxidation alone would not allow plants to fine tune their response to hypoxia, and would instead expose ERFVIIs to unregulated fluctuations in cell redox status. The stability of the mammalian hypoxia sensor protein HYPOXIA INDUCIBLE FACTOR1α is regulated in an oxygen-dependent manner by prolyl hydroxylases (Kaelin and Ratcliffe, 2008). A survey of hypoxia-responsive genes in Arabidopsis identified several PCO enzymes, which were shown to oxidize Nt-Cys using oxygen as a cosubstrate (Weits et al., 2014). These enzymes promote degradation of RAP2.12 in the presence of oxygen, and therefore play a similar regulatory role to mammalian prolyl hydroxylases (Weits et al., 2014). PCOs were shown to counteract RAP2.12-mediated induction of hypoxia-responsive reporter genes, and hypoxic induction of PCO1 and PCO2 indicates that they are important for dampening anaerobic gene transcription through negative regulation of RAP2.12, and likely the other ERFVIIs (Weits et al., 2014). Interestingly, analysis of rice SUB1A-1 demonstrated that it is not an N-end rule substrate in vitro, in contrast to all five of the Arabidopsis ERFVIIs and at least one barley (Hordeum vulgare) ERFVII (Gibbs et al., 2011; Mendiondo et al., 2015). Enhanced tolerance of rice varieties carrying the SUB1A-1 gene might therefore be due to the increased stability of the protein, although more research is needed to fully support this hypothesis. Perhaps the semiaquatic nature of rice has placed evolutionary pressure on ERFVII dynamics to enhance survival in fluctuating water schemes; it will be interesting to see whether ERFVIIs from other wetland species, for example, those from Rumex spp. (van Veen et al., 2014), are also uncoupled from N-end rule regulation. Arabidopsis N-end rule pathway mutants have altered responses to hypoxia or flooding, either enhancing or negatively impacting survival rates, depending upon the context of the stress and recovery conditions (Gibbs et al., 2011; Licausi et al., 2011; Riber et al., 2015). This indicates that the N-end rule pathway is a promising target for manipulating flooding tolerance in crops. In barley, the ERFVII BARLEY ERF1 was shown to be a putative N-end rule substrate in vitro, and posttranscriptional accumulation of an artificial N-end rule reporter protein consisting of the ERFVII Nt domain fused to GUS (MCGGAIL-GUS) was observed under waterlogged conditions, indicating that ERFVIIs are also stabilized by low oxygen in monocots (Mendiondo et al., 2015). Barley PRT6 (HvPRT6) RNA interference lines with reduced HvPRT6 expression had increased levels of anaerobic response gene transcripts (including ADHs), similar to what is observed in Arabidopsis N-end rule mutants (Gibbs et al., 2011; Licausi et al., 2011). Furthermore, RNA interference lines performed better than null controls under waterlogging stress, as evidenced by retention of chlorophyll, increased biomass, and sustained yield poststress (Mendiondo et al., 2015). This translational study highlights the value of targeting ERFVIIs and the N-end rule pathway for engineering flooding tolerance in agronomically important species. A recent study has shown that hypoxia can also act as an important environmental positional cue (Abbas et al., 2015). It was found that low oxygen levels repress photomorphogenesis in dicot species, promoting the maintenance of a skotomorphogenic developmental program. This response was linked to stabilized ERFVIIs, which actively maintained a closed apical hook, and repressed chlorophyll biosynthesis and cotyledon greening. Counterintuitively, hypoxic conditions were beneficial to seedlings, helping to protect them from photooxidative damage following extended darkness and dramatically enhancing survival rates once light was perceived (Abbas et al., 2015). Remarkably, hypocotyl elongation still occurred under hypoxia, demonstrating an active role for oxygen sensing by the ERFVIIs in protecting the stem cell niche, as opposed to inducing a quiescent-like state, as can occur during flooding stress. This study indicates that oxygen availability may have a wider role in regulating general plant growth and development than has been previously considered. NO Signal Transduction via Proteolytic Control of ERFVIIs An important signal associated with flood-induced hypoxia is the accumulation of NO, which alongside the activity of class-1 nonsymbiotic hemoglobins plays a role in balancing the antioxidant status of the cell (van Dongen and Licausi, 2015). NO is a highly reactive gaseous signaling molecule that is known to regulate a diverse range of processes in plants (Yu et al., 2014). In contrast to animals, which produce NO through the action of NO synthases, the origins of NO in plants are less well defined, and production can occur through several different reductive and oxidative pathways (Yu et al., 2014). NO typically induces effects through covalent modification of proteins, thereby altering function, such as via S-nitrosylation, Tyr-nitration, and metal nitrosylation (Besson-Bard et al., 2008). Cys S-nitrosylation has been shown to control a number of key regulatory proteins during plant stress responses. For example, in Arabidopsis, S-nitrosylation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D regulates the salicylic acid-induced hypersensitive response (Yun et al., 2011), whereas S-nitrosylation of SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2.6/OPEN STOMATA1 negatively regulates ABA signaling in guard cells (Wang et al., 2015). However, in these instances, the downstream effect of this modification is process or stress specific, and no unifying sensing mechanism for coordinating multiple transcriptional, developmental, and physiological responses to NO had been identified until recently. Destabilization of Nt-Cys-initiating REGULATOR OF G PROTEIN SIGNALING (RGS) substrates during cardiovasculature development in mammals requires NO in addition to oxygen (Hu et al., 2005; Jaba et al., 2013). It was hypothesized that Nt-Cys of RGS proteins is first S-nitrosylated, and then subsequently further oxidized to permit arginylation and degradation (Hu et al., 2005). An analysis of ERFVII stability revealed that ERFVII degradation is also dependent on NO, indicating that a similar mechanism occurs in plants (Fig. 2; Gibbs et al., 2014b). Under NO-limited conditions, such as in the nitrate reductase1 (nia1)nia2 double mutant or using pharmacological NO scavengers, ERFVII proteins are stabilized. Remarkably, prt6 and ate1ate2 N-end rule mutants, in which ERFVIIs constitutively accumulate, were completely insensitive to exogenous NO for a wide range of responses, including induction of germination, inhibition of hypocotyl elongation in the dark, and stomatal closure, suggesting that N-end rule-mediated proteolysis is essential for NO signal transduction. Furthermore, the NO insensitivity of N-end rule mutants was genetically linked to ERFVIIs for each of the processes investigated, revealing that ERFVIIs play a key role in regulating plant NO responses (Gibbs et al., 2014b). It will be important to elucidate the exact mechanism by which NO controls the stability of these transcription factors and the relationship between NO and oxygen during this process; for example, it is not yet known if NO spontaneously modifies ERFVIIs, or whether the effect of NO on their stability is dependent on enzymatic activity or occurs indirectly. REGULATION OF PLANT RESPONSES TO OTHER ENVIRONMENTAL STRESSES BY ERFVIIs In addition to hypoxia, ERFVIIs from a range of flowering plant species enhance tolerance to other abiotic and biotic stresses. For example, a recent study of Arabidopsis ERFVIIs found that the constitutively expressed group members also regulate responses to oxidative and osmotic stresses, which are both also associated with submergence (Papdi et al., 2015). ERFVII genes are up-regulated in response to phytohormones and stresses, including ethylene, ABA, sodium chloride, salicylic acid, cold and heat, drought, and osmotic stress (Yi et al., 2004; Jung et al., 2007; Xu et al., 2007; Zhang et al., 2009, 2010; Park et al., 2011; Chen et al., 2012; Zhu et al., 2013; Yang et al., 2014). Pathogen infection was shown to increase expression of RAP2.2 in Arabidopsis in response to Botrytis cinerea (Zhao et al., 2012), BENZOTHIADIAZOLE (bth)-INDUCED ERF1 (OsBIERF1) and OsBIERF4 in rice in response to Magnaporthe grisea (Cao et al., 2006), PATHOGEN FREEZING TOLERANCE PROTEIN1 (CaPF1) in Capsicum annuum in response to Xanthomonas axonopodis (Yi et al., 2004), GmERF3 in soybean (Glycine max) in response to soybean mosaic virus (Zhang et al., 2009), and wheat (Triticum aestivum) TaERF1 in response to infection with Blumeria graminis (Xu et al., 2007). Ectopic expression of ERFVIIs in transgenic plants increased cross tolerance to multiple stresses. Arabidopsis HRE2 overexpression increased tolerance to salt and mannitol, whereas the hre2 mutant showed higher sensitivity to these stresses (Park et al., 2011). CaPF1 overexpressed in Arabidopsis and tobacco increased tolerance to freezing and Pseudomonas syringae (Yi et al., 2004), and in Pinus virginiana (Virginia pine), to the heavy metals cadmium, copper, and zinc; to heat; and to the pathogens Bacillus thuringiensis and Staphylococcus epidermidis (Tang et al., 2005). Ectopic expression of GmERF3 in transgenic tobacco enhanced resistance to Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus, and improved tolerance to salt and dehydration (Zhang et al., 2009). Expression of tomato (Solanum lycopersicum) JASMONATE AND ETHYLENE RESPONSE FACTOR1 in tobacco and rice led to increased tolerance to salt and drought (Zhang et al., 2004, 2010), and in rice, resistance to Rhizoctonia solani (Pan et al., 2014). Ectopic expression of TaERF1 and barley ROOT ABUNDANT FACTOR in Arabidopsis led to increased tolerance, respectively, to salt, drought, cold, B. cinerea, and R. solanacearum (Jung et al., 2007; Xu et al., 2007). Transgenic tobacco expressing the Jatropha curcas ERFVII JcERF1 showed increased salt tolerance (Yang et al., 2014). In many cases, similar alterations associated with ectopic expression hint at the downstream molecular and biochemical mechanisms that are enhanced by overexpressing ERFVIIs. There is certainly evidence of repression of ROS production by ERFVIIs. Tobacco BRIGHT YELLOW-2 cells expressing Arabidopsis RAP2.3 were more resistant to hydrogen peroxide (H2O2), and showed increased expression of the H2O2-induced GLUTATHIONE S-TRANSFERASE6 gene (Ogawa et al., 2005), whereas transgenic Arabidopsis expressing HRE2 showed increased tolerance to methyl viologen (MV)-induced oxidative stress and lower levels of ROS in response to high salt (Park et al., 2011). Levels of several antioxidant enzymes were increased in Virginia pine overexpressing CaPF1 (Tang et al., 2005). It was also previously shown that the Sub1 locus increases seedling tolerance to MV and H2O2 due to enhanced transcript levels of genes encoding ascorbate peroxidase, superoxide dismutase, and catalase (Fukao et al., 2011). In fact, transcripts for SUB1A and several other ERFVIIs increase in response to MV treatment (Jung et al., 2007; Fukao et al., 2011; Park et al., 2011). In addition, in several cases, overexpression of ERFVIIs from a variety of species resulted in increased expression of pathogenesis-related genes, many of which contain GCC boxes in their promoters, suggesting the mechanism through which ERFVIIs may increase tolerance to pathogens (Yi et al., 2004; Ogawa et al., 2005; Jung et al., 2007; Xu et al., 2007; Zhang et al., 2009). To understand how ectopic expression of ERFVIIs regulates plant responses to diverse stresses, it will be essential to determine the mechanism controlling N-end rule (and potentially other)-mediated protein stabilization and destabilization. None of the published studies of ectopically expressed ERFVIIs from non-Arabidopsis species have analyzed levels of transgenic ERFVII protein, but as similar phenotypes are invariably observed, it is very likely that their overexpression overrides the destabilizing function of the N-end rule pathway. As it has been shown (at present, only in vitro) that SUB1A is not a substrate of the N-end rule pathway (Gibbs et al., 2011), it is likely that this protein is constitutively stable, thus mimicking the phenotypes of ectopically expressed ERFVIIs. It is unlikely that oxygen levels vary under the nonhypoxic abiotic and biotic stresses analyzed, suggesting that either NO (Gibbs et al., 2014b) or protection of the N terminus (Shemorry et al., 2013) may be involved in modulating stability. KEY INTERACTIONS MEDIATING ERFVII FUNCTION How do the ERFVII transcription factors control such a diverse range of plant developmental and stress responses, and how do they distinguish between oxygen and NO signals to appropriately regulate gene expression? It is probable that several factors are involved, including diversity in gene targets, differences in temporal and tissue-specific expression or subcellular localization, and context-specific protein-protein interactions. Variation in gene targets and interaction partners is conceivable, since comparative analysis of ERFVIIs reveals that they are highly variable in sequence length and identity outside of the N-terminal and AP2 domains (Fig. 1A; Nakano et al., 2006). For example, Arabidopsis RAP2.3 was shown to associate with OCTOPINE SYNTHASE GENE ELEMENT BINDING FACTOR4 (Büttner and Singh, 1997), and RAP2.2 with SEVEN IN ABSENTIA OF ARABIDOPSIS2 (SINAT2; Welsch et al., 2007). SINAT2 is a REALLY INTERESTING NEW GENE E3 ligase, and a recent study showed that GFP-RAP2.12, in which the N-degron is removed, was stabilized in SINAT1/2-silenced Arabidopsis lines (Papdi et al., 2015). Furthermore, an N-terminal YELLOW FLUORESCENT PROTEIN-RAP2.3 fusion was also shown to be degraded in response to light independently of the Cys-2 N-degron (Abbas et al., 2015). Both of these findings indicate that ERFVII stability is regulated by more than one proteolytic mechanism, suggesting that complex posttranslational control of ERFVIIs occurs in plants. ERFVIIs have previously been shown to associate with a range of promoter DNA motifs, including GCC boxes (Büttner and Singh, 1997; Zhang et al., 2004; Gibbs et al., 2014b) and the ATCTA sequence (Welsch et al., 2007), suggesting they have diverse gene targets. Furthermore, there is evidence that posttranslational phosphorylation by kinases might affect ERFVII activity (Cheong et al., 2003; Xu et al., 2007). Combined with these previous reports, recent studies indicate that ERFVIIs function as promiscuous transcription factors, providing mechanistic insight into how they regulate diverse signal- and context-specific responses. ERFVII-Protein Interactions during the Low-Oxygen Response Licausi et al. (2011) discovered that, under normoxic conditions, RAP2.12 is localized to the plasma membrane (PM) via interaction with acyl-CoA binding protein1 (ACBP1) and ACBP2, two PM-associated members of the six-member ACBP family (Fig. 3A; Xiao and Chye, 2009; Licausi et al., 2011). RAP2.3 had also previously been identified as a direct interaction partner of ACBP2 (Li and Chye, 2004). In normoxia, this protein association seems to protect a pool of ERFVIIs from degradation, whereas hypoxia induces a localization shift from the PM to the nucleus (Licausi et al., 2011). This relocalization is triggered once oxygen levels decrease to approximately one-half that of normal air, and accumulation in the nucleus at this oxygen tension coincides with the first induction of hypoxia-responsive gene expression (Kosmacz et al., 2015). In addition, reduction in oxygen availability would also permit stabilization of de novo synthesized ERFVIIs; therefore, the pool of active ERFVIIs in the nucleus likely comprises factors of these two different origins. This is supported by the finding that RAP2.12 transcript levels in polysomal complexes are not dramatically altered by hypoxia (Mustroph et al., 2009). Once normal oxygen levels return, RAP2.12 degradation occurs within 3 h, coinciding with down-regulation of hypoxia-adaptive gene expression (Kosmacz et al., 2015). The PM localization of RAP2.12 is speculated to be a key component of the hypoxia-sensing mechanism, and it will be interesting to see whether other members of the ERFVII family behave similarly, whether interactions with soluble ACBP proteins also occur, and whether this mechanism is conserved across species. A key focus should be placed on understanding how RAP2.12 relocalization occurs in response to low oxygen, and whether changes in NO availability play a role. Furthermore, exactly how a small pool of RAP2.12 evades degradation to make it to the PM remains unanswered. Figure 3. Open in new tabDownload slide Functional ERFVII protein-protein and protein-gene interactions in Arabidopsis. A, ERFVIIs interact with PM-associated ACBP1 and ACBP2 under normoxic conditions. When oxygen levels decline, this association is reduced and ERFVIIs accumulate in the nucleus (Licausi et al., 2011; Kosmacz et al., 2015). B, The ERFVII RAP2.12 induces expression of HYPOXIA RESPONSE ATTENUATOR1 (HRA1), which physically associates with RAP2.12 and attenuates anaerobic gene expression (for example, PYRUVATE DECARBOXYLASE1 [PDC1]) as part of a negative feedback module (Giuntoli et al., 2014). C, Stabilized ERFVIIs in the mature seed endosperm promote ABI5 expression, enhancing ABA responsiveness and maintaining seed dormancy. NO-mediated abolishment of ERFVIIs down-regulates ABI5, reducing ABA sensitivity and initiating germination (Gibbs et al., 2014b). D, DELLA proteins interact with ERFVIIs via the AP2 domain, which inhibits the ability of ERFVIIs to bind to target genes (X). This suggests that when GA levels are low, accumulated DELLA proteins inhibit ERFVII-mediated gene expression (Marín-de la Rosa et al., 2014). For each interaction, the validated Arabidopsis ERFVII family members are listed. Figure 3. Open in new tabDownload slide Functional ERFVII protein-protein and protein-gene interactions in Arabidopsis. A, ERFVIIs interact with PM-associated ACBP1 and ACBP2 under normoxic conditions. When oxygen levels decline, this association is reduced and ERFVIIs accumulate in the nucleus (Licausi et al., 2011; Kosmacz et al., 2015). B, The ERFVII RAP2.12 induces expression of HYPOXIA RESPONSE ATTENUATOR1 (HRA1), which physically associates with RAP2.12 and attenuates anaerobic gene expression (for example, PYRUVATE DECARBOXYLASE1 [PDC1]) as part of a negative feedback module (Giuntoli et al., 2014). C, Stabilized ERFVIIs in the mature seed endosperm promote ABI5 expression, enhancing ABA responsiveness and maintaining seed dormancy. NO-mediated abolishment of ERFVIIs down-regulates ABI5, reducing ABA sensitivity and initiating germination (Gibbs et al., 2014b). D, DELLA proteins interact with ERFVIIs via the AP2 domain, which inhibits the ability of ERFVIIs to bind to target genes (X). This suggests that when GA levels are low, accumulated DELLA proteins inhibit ERFVII-mediated gene expression (Marín-de la Rosa et al., 2014). For each interaction, the validated Arabidopsis ERFVII family members are listed. Once ERFVIIs accumulate in the nucleus, a number of essential anaerobic response genes are switched on, including many of the “core 49” hypoxia response mRNAs previously identified (Mustroph et al., 2009). Sustained expression of many of these genes can be detrimental, and so counterbalancing mechanisms must be in place. In addition to the PCO enzymes discussed earlier, the trihelix transcription factor HRA1 plays such a role (Giuntoli et al., 2014; Fig. 3B). HRA1 is both induced by RAP2.12 and counteracts anaerobic gene induction by physically interacting with RAP2.12. HRA1 was shown to be particularly important for attenuating hypoxia responses in young tissues and meristematic regions, and may play a role in dampening low-oxygen responses under aerobic conditions in regions of the plant that are experiencing physiological hypoxia. In addition to antagonizing RAP2.12-mediated gene expression, HRA1 also negatively regulates its own transcription, providing a second feedback loop. Interestingly, HRA1 did not interact with any of the other four Arabidopsis ERFVIIs, further indicating that each family member may have specific interaction partners (Giuntoli et al., 2014). ERFVII Cross Talk with Hormone Signaling Pathways In addition to the discovery of functionally relevant protein-protein interactions, ERFVII proteins are known to interact with diverse hormone signaling pathways at the transcriptional and protein level. Gibbs et al. (2014b) discovered that ERFVIIs mediate cross talk between NO availability and ABA signaling during the regulation of seed dormancy through their positive control of ABSICISIC ACID INSENSITIVE5 (ABI5) expression, a major negative regulator of germination (Fig. 3C; Holdsworth et al., 2008). Constitutively expressed RAP2.2, RAP2.12, and RAP2.3 all induced ABI5 expression in protoplasts, and a physical interaction between RAP2.3 and a GCC box DNA motif in the ABI5 promoter was confirmed (Gibbs et al., 2014b). This functional interaction retrospectively explained why both prt6 and ate1ate2 mutants, which constitutively accumulate ERFVIIs, have heightened levels of seed dormancy and ABA hypersensitivity relative to the wild type (Holman et al., 2009). It has long been known that NO acts as a potent disrupter of seed dormancy, able to trigger germination (Bethke et al., 2007), and that following seed imbibition, there is a well-documented burst of NO (Liu et al., 2009). Rapid increases in cellular NO as well as oxygenation of the seed during imbibition would stimulate ERFVII degradation, leading to a down-regulation of ABI5 expression in the endosperm, reducing sensitivity to ABA, and promoting germination (Gibbs et al., 2014b). Future analyses should focus on whether other key ABA-regulated responses are also regulated by ERFVIIs. A recent large-scale yeast two-hybrid screen of Arabidopsis proteins identified RAP2.3 as an interaction partner of the DELLA protein GA INSENSITIVE (GAI; Marín-de la Rosa et al., 2014). Interactions between RAP2.3 and the DELLA protein REPRESSOR OF THE ga1-3 MUTANT, and between GAI and RAP2.12 were also identified. These DELLAs associate with the ERFVII DNA-binding domain, and disrupt their ability to interact with target genes (Fig. 3D; Marín-de la Rosa et al., 2014). This suggests that, in the absence of GA, accumulated DELLA proteins act as negative regulators of ERFVII-mediated gene expression, whereas GA-induced destruction of DELLA proteins would relieve this repression. This hypothesis was investigated within the context of seedling apical hook development. The apical hook is a hallmark feature of etiolated seedlings; its formation is promoted by both ethylene and GA, which have additive effects enhancing hook angle (Abbas et al., 2013). In the pentuple erfVII mutant, which lacks all five ERFVIIs, hooks were able to respond similarly to the wild type for ethylene, but the single and additive effects of exogenous GA were significantly reduced compared with the wild type, indicating a role for ERFVIIs in GA regulation of early seedling development. As discussed earlier, there is a well-documented link between ERFVIIs and GA signaling in rice: SNORKELs and SUB1A-1 promote and inhibit downstream GA responses, respectively (Fukao and Bailey-Serres, 2008; Hattori et al., 2009). Furthermore, Arabidopsis ERFVIIs regulate processes dependent on cell elongation (e.g. germination and hypocotyl elongation; Holman et al., 2009; Gibbs et al., 2014b), suggesting that ERFVII interactions with GA signaling pathways may be more significant than is currently understood. CONCLUSION Recent data indicate that the long-known distinctive Nt structure of the ERFVIIs, conferring oxygen- and NO-dependant stability, provides these proteins with a conditional activity that has diverse consequences during growth and development, and in response to environmental stress. Many aspects of ERFVII biology remain to be determined to enable an understanding of how changed stability is related to distinct responses to different conditions. Future studies aimed at defining the specificity of ERFVII protein-protein and protein-gene interactions, as well as the contribution of non-N-end rule-mediated ubiquitination to their stability, will shed light onto the complex nature of ERFVII signal transduction, and may help understand how this small family of transcriptional regulators controls such an array of plant responses. In addition, determining the overlapping and unique roles of individual members of the group will help in understanding adaptive molecular responses to evolutionary pressure related to specific stresses. Further study of these factors and mechanisms influencing the changes in their stability should also provide a deeper understanding of how plants integrate development and stress responses. The observation that ERFVIIs from diverse species positively influence tolerance to multiple stresses in similar ways indicates that they are good targets for breeding and biotechnological approaches to increase stability of plant growth and yield in response to environmental stress. Glossary Nt N-terminal ROS reactive oxygen species NO nitric oxide H2O2 hydrogen peroxide MV methyl viologen PM plasma membrane LITERATURE CITED Abbas M , Alabadí D, Blázquez MA ( 2013 ) Differential growth at the apical hook: all roads lead to auxin . Front Plant Sci 4 : 441 Google Scholar Crossref Search ADS PubMed WorldCat Abbas M , Berckhan S, Rooney DJ, Gibbs DJ, Conde JV, Correia CS, Bassel GW, Marin-de la Rosa N, Leon J, Alabadi D , et al ( 2015 ) Oxygen sensing coordinates photomorphogenesis to facilitate seedling survival . Curr Biol 25 : 1483 – 1488 Google Scholar Crossref Search ADS PubMed WorldCat Ayano M , Kani T, Kojima M, Sakakibara H, Kitaoka T, Kuroha T, Angeles-Shim RB, Kitano H, Nagai K, Ashikari M ( 2014 ) Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice . Plant Cell Environ 37 : 2313 – 2324 Google Scholar Crossref Search ADS PubMed WorldCat Bachmair A , Finley D, Varshavsky A ( 1986 ) In vivo half-life of a protein is a function of its amino-terminal residue . Science 234 : 179 – 186 Google Scholar Crossref Search ADS PubMed WorldCat Bailey-Serres J , Fukao T, Gibbs DJ, Holdsworth MJ, Lee SC, Licausi F, Perata P, Voesenek LA, van Dongen JT ( 2012 ) Making sense of low oxygen sensing . Trends Plant Sci 17 : 129 – 138 Google Scholar Crossref Search ADS PubMed WorldCat Besson-Bard A , Pugin A, Wendehenne D ( 2008 ) New insights into nitric oxide signaling in plants . Annu Rev Plant Biol 59 : 21 – 39 Google Scholar Crossref Search ADS PubMed WorldCat Bethke PC , Libourel IG, Aoyama N, Chung YY, Still DW, Jones RL ( 2007 ) The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy . Plant Physiol 143 : 1173 – 1188 Google Scholar Crossref Search ADS PubMed WorldCat Bui LT , Giuntoli B, Kosmacz M, Parlanti S, Licausi F ( 2015 ) Constitutively expressed ERF-VII transcription factors redundantly activate the core anaerobic response in Arabidopsis thaliana. Plant Sci 236 : 37 – 43 Google Scholar Crossref Search ADS PubMed WorldCat Büttner M , Singh KB ( 1997 ) Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein . Proc Natl Acad Sci USA 94 : 5961 – 5966 Google Scholar Crossref Search ADS PubMed WorldCat Cao Y , Song F, Goodman RM, Zheng Z ( 2006 ) Molecular characterization of four rice genes encoding ethylene-responsive transcriptional factors and their expressions in response to biotic and abiotic stress . J Plant Physiol 163 : 1167 – 1178 Google Scholar Crossref Search ADS PubMed WorldCat Chen N , Yang QL, Su MW, Pan LJ, Chi XY, Chen MN, He YA, Yang Z, Wang T, Wang MA , et al. ( 2012 ) Cloning of six ERF family transcription factor genes from peanut and analysis of their expression during abiotic stress . Plant Mol Biol Rep 30 : 1415 – 1425 Google Scholar Crossref Search ADS WorldCat Chen X , Pierik R, Peeters AJM, Poorter H, Visser EJW, Huber H, de Kroon H, Voesenek LACJ ( 2010 ) Endogenous abscisic acid as a key switch for natural variation in flooding-induced shoot elongation . Plant Physiol 154 : 969 – 977 Google Scholar Crossref Search ADS PubMed WorldCat Cheong YH , Moon BC, Kim JK, Kim CY, Kim MC, Kim IH, Park CY, Kim JC, Park BO, Koo SC, et al. ( 2003 ) BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor . Plant Physiol 132 : 1961 – 1972 Google Scholar Crossref Search ADS PubMed WorldCat Du H , Chang Y, Huang F, Xiong L ( 2014 ) GID1 modulates stomatal response and submergence tolerance involving abscisic acid and gibberellic acid signaling in rice . J Integr Plant Biol Google Scholar OpenURL Placeholder Text WorldCat Fukao T , Bailey-Serres J ( 2008 ) Submergence tolerance conferred by Sub1A is mediated by SLR1 and SLRL1 restriction of gibberellin responses in rice . Proc Natl Acad Sci USA 105 : 16814 – 16819 Google Scholar Crossref Search ADS PubMed WorldCat Fukao T , Xu K, Ronald PC, Bailey-Serres J ( 2006 ) A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice . Plant Cell 18 : 2021 – 2034 Google Scholar Crossref Search ADS PubMed WorldCat Fukao T , Yeung E, Bailey-Serres J ( 2011 ) The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice . Plant Cell 23 : 412 – 427 Google Scholar Crossref Search ADS PubMed WorldCat Fukao T , Yeung E, Bailey-Serres J ( 2012 ) The submergence tolerance gene SUB1A delays leaf senescence under prolonged darkness through hormonal regulation in rice . Plant Physiol 160 : 1795 – 1807 Google Scholar Crossref Search ADS PubMed WorldCat Gibbs DJ , Bacardit J, Bachmair A, Holdsworth MJ ( 2014 a ) The eukaryotic N-end rule pathway: conserved mechanisms and diverse functions . Trends Cell Biol 24 : 603 – 611 Google Scholar Crossref Search ADS PubMed WorldCat Gibbs DJ , Lee SC, Isa NM, Gramuglia S, Fukao T, Bassel GW, Correia CS, Corbineau F, Theodoulou FL, Bailey-Serres J , et al. ( 2011 ) Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants . Nature 479 : 415 – 418 Google Scholar Crossref Search ADS PubMed WorldCat Gibbs DJ , Md Isa N, Movahedi M, Lozano-Juste J, Mendiondo GM, Berckhan S, Marín-de la Rosa N, Vicente Conde J, Sousa Correia C, Pearce SP, et al. ( 2014 b ) Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors . Mol Cell 53 : 369 – 379 Google Scholar Crossref Search ADS PubMed WorldCat Giuntoli B , Lee SC, Licausi F, Kosmacz M, Oosumi T, van Dongen JT, Bailey-Serres J, Perata P ( 2014 ) A trihelix DNA binding protein counterbalances hypoxia-responsive transcriptional activation in Arabidopsis . PLoS Biol 12 : e1001950 Google Scholar Crossref Search ADS PubMed WorldCat Hattori Y , Nagai K, Furukawa S, Song XJ, Kawano R, Sakakibara H, Wu J, Matsumoto T, Yoshimura A, Kitano H, et al. ( 2009 ) The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water . Nature 460 : 1026 – 1030 Google Scholar Crossref Search ADS PubMed WorldCat Hess N , Klode M, Anders M, Sauter M ( 2011 ) The hypoxia responsive transcription factor genes ERF71/HRE2 and ERF73/HRE1 of Arabidopsis are differentially regulated by ethylene . Physiol Plant 143 : 41 – 49 Google Scholar Crossref Search ADS PubMed WorldCat Hinz M , Wilson IW, Yang J, Buerstenbinder K, Llewellyn D, Dennis ES, Sauter M, Dolferus R ( 2010 ) Arabidopsis RAP2.2: an ethylene response transcription factor that is important for hypoxia survival . Plant Physiol 153 : 757 – 772 Google Scholar Crossref Search ADS PubMed WorldCat Hirano K , Kouketu E, Katoh H, Aya K, Ueguchi-Tanaka M, Matsuoka M ( 2012 ) The suppressive function of the rice DELLA protein SLR1 is dependent on its transcriptional activation activity . Plant J 71 : 443 – 453 Google Scholar PubMed OpenURL Placeholder Text WorldCat Hoffmann-Benning S , Kende H ( 1992 ) On the role of abscisic acid and gibberellin in the regulation of growth in rice . Plant Physiol 99 : 1156 – 1161 Google Scholar Crossref Search ADS PubMed WorldCat Holdsworth MJ , Bentsink L, Soppe WJ ( 2008 ) Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination . New Phytol 179 : 33 – 54 Google Scholar Crossref Search ADS PubMed WorldCat Holman TJ , Jones PD, Russell L, Medhurst A, Ubeda Tomás S, Talloji P, Marquez J, Schmuths H, Tung SA, Taylor I, et al. ( 2009 ) The N-end rule pathway promotes seed germination and establishment through removal of ABA sensitivity in Arabidopsis . Proc Natl Acad Sci USA 106 : 4549 – 4554 Google Scholar Crossref Search ADS PubMed WorldCat Hu RG , Sheng J, Qi X, Xu Z, Takahashi TT, Varshavsky A ( 2005 ) The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators . Nature 437 : 981 – 986 Google Scholar Crossref Search ADS PubMed WorldCat Jaba IM , Zhuang ZW, Li N, Jiang Y, Martin KA, Sinusas AJ, Papademetris X, Simons M, Sessa WC, Young LH , et al. ( 2013 ) NO triggers RGS4 degradation to coordinate angiogenesis and cardiomyocyte growth . J Clin Invest 123 : 1718 – 1731 Google Scholar Crossref Search ADS PubMed WorldCat Jung J , Won SY, Suh SC, Kim H, Wing R, Jeong Y, Hwang I, Kim M ( 2007 ) The barley ERF-type transcription factor HvRAF confers enhanced pathogen resistance and salt tolerance in Arabidopsis . Planta 225 : 575 – 588 Google Scholar Crossref Search ADS PubMed WorldCat Jung KH , Seo YS, Walia H, Cao P, Fukao T, Canlas PE, Amonpant F, Bailey-Serres J, Ronald PC ( 2010 ) The submergence tolerance regulator Sub1A mediates stress-responsive expression of AP2/ERF transcription factors . Plant Physiol 152 : 1674 – 1692 Google Scholar Crossref Search ADS PubMed WorldCat Kaelin WG Jr , Ratcliffe PJ ( 2008 ) Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway . Mol Cell 30 : 393 – 402 Google Scholar Crossref Search ADS PubMed WorldCat Kearse M , Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, et al. ( 2012 ) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data . Bioinformatics 28 : 1647 – 1649 Google Scholar Crossref Search ADS PubMed WorldCat Kosmacz M , Parlanti S, Schwarzlander M, Kragler F, Licausi F, Van Dongen JT ( 2015 ) The stability and nuclear localization of the transcription factor RAP2.12 are dynamically regulated by oxygen concentration . Plant Cell Environ 38 : 1094 – 1103 Google Scholar Crossref Search ADS PubMed WorldCat Li HY , Chye ML ( 2004 ) Arabidopsis Acyl-CoA-binding protein ACBP2 interacts with an ethylene-responsive element-binding protein, AtEBP, via its ankyrin repeats . Plant Mol Biol 54 : 233 – 243 Google Scholar Crossref Search ADS PubMed WorldCat Licausi F , Kosmacz M, Weits DA, Giuntoli B, Giorgi FM, Voesenek LA, Perata P, van Dongen JT ( 2011 ) Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization . Nature 479 : 419 – 422 Google Scholar Crossref Search ADS PubMed WorldCat Licausi F , Ohme-Takagi M, Perata P ( 2013 ) APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs . New Phytol 199 : 639 – 649 Google Scholar Crossref Search ADS PubMed WorldCat Licausi F , van Dongen JT, Giuntoli B, Novi G, Santaniello A, Geigenberger P, Perata P ( 2010 ) HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana . Plant J 62 : 302 – 315 Google Scholar Crossref Search ADS PubMed WorldCat Liu Y , Shi L, Ye N, Liu R, Jia W, Zhang J ( 2009 ) Nitric oxide-induced rapid decrease of abscisic acid concentration is required in breaking seed dormancy in Arabidopsis . New Phytol 183 : 1030 – 1042 Google Scholar Crossref Search ADS PubMed WorldCat Marín-de la Rosa N , Sotillo B, Miskolczi P, Gibbs DJ, Vicente J, Carbonero P, Oñate-Sánchez L, Holdsworth MJ, Bhalerao R, Alabadí D , et al. ( 2014 ) Large-scale identification of gibberellin-related transcription factors defines group VII ETHYLENE RESPONSE FACTORS as functional DELLA partners . Plant Physiol 166 : 1022 – 1032 Google Scholar Crossref Search ADS PubMed WorldCat Mendiondo GM, Gibbs DJ, Szurman-Zubrzycka M, Korn A, Marquez J, Szarejko I, Maluszynski M, King J, Axcell B, Smart K, et al. (2015) Enhanced waterlogging tolerance in barley by manipulation of expression of the N-end rule pathway E3 ligase PROTEOLYSIS6. Plant Biotechnol J DOI: 10.1111/pbi.12334 Mustroph A , Zanetti ME, Jang CJ, Holtan HE, Repetti PP, Galbraith DW, Girke T, Bailey-Serres J ( 2009 ) Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis . Proc Natl Acad Sci USA 106 : 18843 – 18848 Google Scholar Crossref Search ADS PubMed WorldCat Nakano T , Suzuki K, Fujimura T, Shinshi H ( 2006 ) Genome-wide analysis of the ERF gene family in Arabidopsis and rice . Plant Physiol 140 : 411 – 432 Google Scholar Crossref Search ADS PubMed WorldCat Ogawa T , Pan L, Kawai-Yamada M, Yu LH, Yamamura S, Koyama T, Kitajima S, Ohme-Takagi M, Sato F, Uchimiya H ( 2005 ) Functional analysis of Arabidopsis ethylene-responsive element binding protein conferring resistance to Bax and abiotic stress-induced plant cell death . Plant Physiol 138 : 1436 – 1445 Google Scholar Crossref Search ADS PubMed WorldCat Ohme-Takagi M , Shinshi H ( 1995 ) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element . Plant Cell 7 : 173 – 182 Google Scholar PubMed OpenURL Placeholder Text WorldCat Pan X , Li Y, Zhang H, Huang R, Liu W, Ming J, Liu S, Li X ( 2014 ) Expression of signalling and defence-related genes mediated by over-expression of JERF1, and increased resistance to sheath blight in rice . Plant Pathol 63 : 109 – 116 Google Scholar Crossref Search ADS WorldCat Papdi C , Abrahám E, Joseph MP, Popescu C, Koncz C, Szabados L ( 2008 ) Functional identification of Arabidopsis stress regulatory genes using the controlled cDNA overexpression system . Plant Physiol 147 : 528 – 542 Google Scholar Crossref Search ADS PubMed WorldCat Papdi C, Pérez-Salamó I, Joseph MP, Giuntoli B, Bögre L, Koncz C, Szabados L (2015) The low oxygen, oxidative and osmotic stress responses synergistically act through the ethylene response factor VII genes RAP2.12, RAP2.2 and RAP2.3. Plant J doi: 10.1111/tpj.12848 Park HY , Seok HY, Woo DH, Lee SY, Tarte VN, Lee EH, Lee CH, Moon YH ( 2011 ) AtERF71/HRE2 transcription factor mediates osmotic stress response as well as hypoxia response in Arabidopsis . Biochem Biophys Res Commun 414 : 135 – 141 Google Scholar Crossref Search ADS PubMed WorldCat Parlanti S , Kudahettige NP, Lombardi L, Mensuali-Sodi A, Alpi A, Perata P, Pucciariello C ( 2011 ) Distinct mechanisms for aerenchyma formation in leaf sheaths of rice genotypes displaying a quiescence or escape strategy for flooding tolerance . Ann Bot (Lond) 107 : 1335 – 1343 Google Scholar Crossref Search ADS WorldCat Peña-Castro JM , van Zanten M, Lee SC, Patel MR, Voesenek LA, Fukao T, Bailey-Serres J ( 2011 ) Expression of rice SUB1A and SUB1C transcription factors in Arabidopsis uncovers flowering inhibition as a submergence tolerance mechanism . Plant J 67 : 434 – 446 Google Scholar Crossref Search ADS PubMed WorldCat Raskin I , Kende H ( 1984 ) Role of gibberellin in the growth response of submerged deep water rice . Plant Physiol 76 : 947 – 950 Google Scholar Crossref Search ADS PubMed WorldCat Riber W , Müller JT, Visser EJ, Sasidharan R, Voesenek LA, Mustroph A ( 2015 ) The greening after extended darkness1 is an N-end rule pathway mutant with high tolerance to submergence and starvation . Plant Physiol 167 : 1616 – 1629 Google Scholar Crossref Search ADS PubMed WorldCat Setter TL , Bhekasut P, Greenway H ( 2010 ) Desiccation of leaves after de-submergence is one cause for intolerance to complete submergence of the rice cultivar IR 42 . Funct Plant Biol 37 : 1096 – 1104 Google Scholar Crossref Search ADS WorldCat Shemorry A , Hwang CS, Varshavsky A ( 2013 ) Control of protein quality and stoichiometries by N-terminal acetylation and the N-end rule pathway . Mol Cell 50 : 540 – 551 Google Scholar Crossref Search ADS PubMed WorldCat Tang W , Charles TM, Newton RJ ( 2005 ) Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine (Pinus Virginiana Mill.) confers multiple stress tolerance and enhances organ growth . Plant Mol Biol 59 : 603 – 617 Google Scholar Crossref Search ADS PubMed WorldCat van Dongen JT , Licausi F ( 2015 ) Oxygen sensing and signaling . Annu Rev Plant Biol 66 : 345 – 367 Google Scholar Crossref Search ADS PubMed WorldCat van Veen H , Akman M, Jamar DC, Vreugdenhil D, Kooiker M, van Tienderen P, Voesenek LA, Schranz ME, Sasidharan R ( 2014 ) Group VII ethylene response factor diversification and regulation in four species from flood-prone environments . Plant Cell Environ 37 : 2421 – 2432 Google Scholar PubMed OpenURL Placeholder Text WorldCat van Veen H , Mustroph A, Barding GA, Vergeer-van Eijk M, Welschen-Evertman RA, Pedersen O, Visser EJ, Larive CK, Pierik R, Bailey-Serres J , et al. ( 2013 ) Two Rumex species from contrasting hydrological niches regulate flooding tolerance through distinct mechanisms . Plant Cell 25 : 4691 – 4707 Google Scholar Crossref Search ADS PubMed WorldCat Varshavsky A ( 2011 ) The N-end rule pathway and regulation by proteolysis . Protein Sci 20 : 1298 – 1345 Google Scholar Crossref Search ADS PubMed WorldCat Voesenek LA , Bailey-Serres J ( 2015 ) Flood adaptive traits and processes: an overview . New Phytol 206 : 57 – 73 Google Scholar Crossref Search ADS PubMed WorldCat Wang D , Pan Y, Zhao X, Zhu L, Fu B, Li Z ( 2011 ) Genome-wide temporal-spatial gene expression profiling of drought responsiveness in rice . BMC Genomics 12 : 149 Google Scholar Crossref Search ADS PubMed WorldCat Wang P , Du Y, Hou YJ, Zhao Y, Hsu CC, Yuan F, Zhu X, Tao WA, Song CP, Zhu JK ( 2015 ) Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1 . Proc Natl Acad Sci USA 112 : 613 – 618 Google Scholar Crossref Search ADS PubMed WorldCat Weits DA , Giuntoli B, Kosmacz M, Parlanti S, Hubberten HM, Riegler H, Hoefgen R, Perata P, van Dongen JT, Licausi F ( 2014 ) Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway . Nat Commun 5 : 3425 Google Scholar Crossref Search ADS PubMed WorldCat Welsch R , Maass D, Voegel T, Dellapenna D, Beyer P ( 2007 ) Transcription factor RAP2.2 and its interacting partner SINAT2: stable elements in the carotenogenesis of Arabidopsis leaves . Plant Physiol 145 : 1073 – 1085 Google Scholar Crossref Search ADS PubMed WorldCat Xiao S , Chye ML ( 2009 ) An Arabidopsis family of six acyl-CoA-binding proteins has three cytosolic members . Plant Physiol Biochem 47 : 479 – 484 Google Scholar Crossref Search ADS PubMed WorldCat Xu K , Xu X, Fukao T, Canlas P, Maghirang-Rodriguez R, Heuer S, Ismail AM, Bailey-Serres J, Ronald PC, Mackill DJ ( 2006 ) Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice . Nature 442 : 705 – 708 Google Scholar Crossref Search ADS PubMed WorldCat Xu ZS , Xia LQ, Chen M, Cheng XG, Zhang RY, Li LC, Zhao YX, Lu Y, Ni ZY, Liu L , et al. ( 2007 ) Isolation and molecular characterization of the Triticum aestivum L. ethylene-responsive factor 1 (TaERF1) that increases multiple stress tolerance . Plant Mol Biol 65 : 719 – 732 Google Scholar Crossref Search ADS PubMed WorldCat Yang CY , Hsu FC, Li JP, Wang NN, Shih MC ( 2011 ) The AP2/ERF transcription factor AtERF73/HRE1 modulates ethylene responses during hypoxia in Arabidopsis . Plant Physiol 156 : 202 – 212 Google Scholar Crossref Search ADS PubMed WorldCat Yang H , Yu C, Yan J, Wang X, Chen F, Zhao Y, Wei W ( 2014 ) Overexpression of the Jatropha curcas JcERF1 gene coding an AP2/ERF-type transcription factor increases tolerance to salt in transgenic tobacco . Biochemistry (Mosc) 79 : 1226 – 1236 Google Scholar Crossref Search ADS PubMed WorldCat Yi SY , Kim JH, Joung YH, Lee S, Kim WT, Yu SH, Choi D ( 2004 ) The pepper transcription factor CaPF1 confers pathogen and freezing tolerance in Arabidopsis . Plant Physiol 136 : 2862 – 2874 Google Scholar Crossref Search ADS PubMed WorldCat Yu M , Lamattina L, Spoel SH, Loake GJ ( 2014 ) Nitric oxide function in plant biology: a redox cue in deconvolution . New Phytol 202 : 1142 – 1156 Google Scholar Crossref Search ADS PubMed WorldCat Yun BW , Feechan A, Yin M, Saidi NB, Le Bihan T, Yu M, Moore JW, Kang JG, Kwon E, Spoel SH , et al. ( 2011 ) S-nitrosylation of NADPH oxidase regulates cell death in plant immunity . Nature 478 : 264 – 268 Google Scholar Crossref Search ADS PubMed WorldCat Zhang G , Chen M, Li L, Xu Z, Chen X, Guo J, Ma Y ( 2009 ) Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco . J Exp Bot 60 : 3781 – 3796 Google Scholar Crossref Search ADS PubMed WorldCat Zhang H , Huang Z, Xie B, Chen Q, Tian X, Zhang X, Zhang H, Lu X, Huang D, Huang R ( 2004 ) The ethylene-, jasmonate-, abscisic acid- and NaCl-responsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco . Planta 220 : 262 – 270 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Z , Li F, Li D, Zhang H, Huang R ( 2010 ) Expression of ethylene response factor JERF1 in rice improves tolerance to drought . Planta 232 : 765 – 774 Google Scholar Crossref Search ADS PubMed WorldCat Zhao Y , Wei T, Yin KQ, Chen Z, Gu H, Qu LJ, Qin G ( 2012 ) Arabidopsis RAP2.2 plays an important role in plant resistance to Botrytis cinerea and ethylene responses . New Phytol 195 : 450 – 460 Google Scholar Crossref Search ADS PubMed WorldCat Zhu Z , Shi J, Xu W, Li H, He M, Xu Y, Xu T, Yang Y, Cao J, Wang Y ( 2013 ) Three ERF transcription factors from Chinese wild grapevine Vitis pseudoreticulata participate in different biotic and abiotic stress-responsive pathways . J Plant Physiol 170 : 923 – 933 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the Biotechnology and Biological Sciences Research Council (grant nos. BB/M002268/1 and BB/M007820/1 to M.J.H. and grant no. BB/K000144/1 to J.V.C. and M.J.H., including financial support from SABMiller), the University of Birmingham (Fellowship to D.J.G.), Government of India National Overseas Scholarship for higher studies (PhD studentship to G.P.), the University of Nottingham (PhD studentship to S.B.), and a Barry Axcell postdoctoral fellowship (to G.M.M.). * Address correspondence to [email protected] and [email protected] www.plantphysiol.org/cgi/doi/10.1104/pp.15.00338 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Müller, Maren; Munné-Bosch, Sergi

doi: 10.1104/pp.15.00677pmid: 26103991

Abstract Ethylene is essential for many developmental processes and a key mediator of biotic and abiotic stress responses in plants. The ethylene signaling and response pathway includes Ethylene Response Factors (ERFs), which belong to the transcription factor family APETALA2/ERF. It is well known that ERFs regulate molecular response to pathogen attack by binding to sequences containing AGCCGCC motifs (the GCC box), a cis-acting element. However, recent studies suggest that several ERFs also bind to dehydration-responsive elements and act as a key regulatory hub in plant responses to abiotic stresses. Here, we review some of the recent advances in our understanding of the ethylene signaling and response pathway, with emphasis on ERFs and their role in hormone cross talk and redox signaling under abiotic stresses. We conclude that ERFs act as a key regulatory hub, integrating ethylene, abscisic acid, jasmonate, and redox signaling in the plant response to a number of abiotic stresses. Environmental stresses, including drought, salinity, high light, and extreme temperatures, influence plant growth and productivity. These abiotic stresses result in reductions in growth, stomatal and nonstomatal limitations on photosynthesis, and alterations in both hormonal balance and reduction/oxidation (redox) processes, potentially leading to enhanced lipid peroxidation, protein oxidation, and DNA damage (Munns and Tester, 2008; Mittler et al., 2011; Munné-Bosch et al., 2013). Plant responses and adaptation to abiotic stresses are controlled by molecular signal transduction cascades. In these cascades, plant hormones as a part of the signal network function as central integrators that link and reprogram the complex stress-adaptive signaling cascades (Ma et al., 2006; Golldack et al., 2014). Several plant hormones, such as ethylene (Zhao and Schaller, 2004; Cheng et al., 2013), abscisic acid (ABA; Wu et al., 2007), jasmonates (Cela et al., 2011; Cheng et al., 2013), salicylic acid (Jayakannan et al., 2015), GAs (Magome et al., 2008), cytokinins (Wu et al., 2014b), auxin (He et al., 2005), and brassinosteroids (Divi et al., 2010), have been reported to be involved in stress signaling. Despite its simple two-carbon structure, the plant hormone ethylene serves as a key mediator of biotic and abiotic stress factors. Transcription factors (TFs) control the majority of stress response genes, and of more than 1,800 TFs identified in Arabidopsis (Arabidopsis thaliana; Arabidopsis Genome Initiative, 2000; Riechmann et al., 2000; Gong et al., 2004), the APETALA2 (AP2)/Ethylene Response Factor (ERF) superfamily plays a pivotal role in adaptation to biotic and abiotic stresses, such as those caused by pathogens, wounding, cold and heat stress, UV light, drought, and salinity (Mizoi et al., 2012). A genome-wide analysis of plant species has identified, for instance, the following numbers of members of the AP2/ERF superfamily: Arabidopsis, 147 (Nakano et al., 2006); Populus spp., 200 (Zhuang et al., 2008); Brassica spp., 291 (Song et al., 2013); Citrus spp., 108 (Ito et al., 2014); Vitis spp., 149 (Licausi et al., 2010); Solanum spp., 155 (Charfeddine et al., 2015); and Oryza spp., 180 (Nakano et al., 2006; Sharoni et al., 2011). Sakuma et al. (2002) classified AP2/ERF TFs into five subfamilies: AP2, related to ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1), dehydration-responsive element (DRE) binding protein, ERF, and others according to the number and similarity of the DNA binding domains. ERFs have been extensively reported to be involved in the response to pathogen attack by binding to sequences containing AGCCGCC motifs (the GCC box), a cis-acting ethylene response element (Solano et al., 1998; Berrocal-Lobo et al., 2002; Lorenzo et al., 2002). However, recent research has shown that several ERFs also bind to DREs and play a regulatory role in plant responses to abiotic stresses (Cheng et al., 2013). Although the role of AP2/ERF TFs as mediators of stress responses and development programs has been reviewed recently (Xu et al., 2011; Mizoi et al., 2012; Licausi et al., 2013), little emphasis has been put on the role of ERFs in abiotic stress tolerance. Here, we focus on the role of ERFs in plant tolerance to abiotic stresses, with an emphasis on hormone cross talk, redox regulation, and abiotic stress signaling. ERFs IN ETHYLENE RESPONSE The ethylene signaling and response pathway to the cell nucleus, where the transcription of hundreds of genes is altered, was revealed as a result of the analysis of the model system Arabidopsis (Fig. 1). Ethylene is sensed by five receptors localized at the endoplasmic reticulum membrane that are divided into subfamily I (Ethylene Response1 [ETR1] and Ethylene Response Sensor1 [ERS1]) and subfamily II (ETR2, ERS2, and Ethylene Insensitive4 [EIN4]; Chen et al., 2005; Lacey and Binder, 2014). The ethylene signaling and response pathway also includes the downstream components Constitutive Triple Response1 (CTR1), EIN2, EIN3/Ethylene Insensitive-Like Protein1 (EIL1), and ERFs (Kendrick and Chang, 2008; Stepanova and Alonso, 2009). CTR1 is a negative regulator of ethylene signaling. In the absence of ethylene, the receptors promote CTR1 kinase activity, which phosphorylates the C-terminal domain of EIN2 and thereby, prevents its nucleus localization. However, in the presence of ethylene, the receptors and CTR1 are inactive (Kieber et al., 1993; Gao et al., 2003; Huang et al., 2003; Ju et al., 2012). In contrast, EIN2, which is localized at the ER membrane along with the ethylene receptors and CTR1, positively regulates ethylene signaling (Bisson et al., 2009; Bisson and Groth, 2010). In the absence of ethylene, EIN2 protein levels are reduced because of the interaction with two F-box proteins: Ethylene Insensitive2-Targeting Protein1 (ETP1) and ETP2 (Qiao et al., 2009). In the presence of ethylene, the inactivation of the receptors and CTR1 results in the dephosphorylation and cleavage of the EIN2 C terminus and translocation to the nucleus, where it directly or indirectly regulates EIN3/EIL1 activation (Ju et al., 2012). An MA3 domain-containing protein (ECIP1) interacts with the EIN2 C terminus, leading to enhanced ethylene response (Lei et al., 2011). In the absence of ethylene, the EIN3/EIL1 protein levels are extremely low because of the protein turnover involving the F-box proteins Ethylene Insensitive3-Binding F-Box Protein1 (EBF1) and EBF2. The presence of ethylene down-regulates EBF1 and EBF2 and leads to the accumulation of EIN3/EIL1 proteins, which initiates a transcriptional cascade that results in the activation and repression of hundreds of genes (An et al., 2010). In Arabidopsis, one of the direct targets of EIN3 is the ERF genes (Solano et al., 1998). AtERF and ERF will be used in this review interchangeably. Figure 1. Open in new tabDownload slide Model of the ethylene (ET) signaling pathway to ERFs. In the absence of ethylene (left), the ethylene receptors promote CTR1 kinase activity, resulting in the phosphorylation of the C-terminal domain of EIN2. Because of the protein turnover involving the F-box proteins ETP1/2 and EBF1/2, the protein levels of both EIN2 and EIN3/EIL1 are extremely low. In the presence of ethylene (right), the inactivation of the ethylene receptors and CTR1 results in the dephosphorylation and cleavage of the EIN2 C terminus and translocation to the nucleus, where they regulate EIN3/EIL1 activation directly or indirectly. The direct targets of EIN3 are the TF genes ERFs, such as ERF1, which activates, depending on the stress conditions (either biotic [pathogen infection] or abiotic [e.g. dehydration, salinity, or heat shock] stress), a specific set of stress response genes by binding to the specific cis-acting GCC box and DRE elements. ER, Endoplasmic reticulum; ECIP1, EIN2 C-TERMINUS INTERACTING PROTEIN1. Figure 1. Open in new tabDownload slide Model of the ethylene (ET) signaling pathway to ERFs. In the absence of ethylene (left), the ethylene receptors promote CTR1 kinase activity, resulting in the phosphorylation of the C-terminal domain of EIN2. Because of the protein turnover involving the F-box proteins ETP1/2 and EBF1/2, the protein levels of both EIN2 and EIN3/EIL1 are extremely low. In the presence of ethylene (right), the inactivation of the ethylene receptors and CTR1 results in the dephosphorylation and cleavage of the EIN2 C terminus and translocation to the nucleus, where they regulate EIN3/EIL1 activation directly or indirectly. The direct targets of EIN3 are the TF genes ERFs, such as ERF1, which activates, depending on the stress conditions (either biotic [pathogen infection] or abiotic [e.g. dehydration, salinity, or heat shock] stress), a specific set of stress response genes by binding to the specific cis-acting GCC box and DRE elements. ER, Endoplasmic reticulum; ECIP1, EIN2 C-TERMINUS INTERACTING PROTEIN1. Recent studies revealed that ERFs are key regulators in abiotic stress tolerance in several species. Enhanced ERF expression has been reported after drought, salinity, light stress, and cold and heat treatments among other abiotic stresses (Table I). Several ERF genes have been reported to be induced by salt stress (38 study cases), drought (27 study cases), low temperature (18 study cases), heat stress (3 study cases), and changes in light availability (14 study cases). It should be noted that ERF gene expression is common to various abiotic stresses, including salt, drought, and cold stress treatment (12 study cases), salt and drought (8 study cases), and others. The effects of overexpressing ERF genes in plant response to abiotic stress have been studied in various plant systems (Table II). Expression of genes from the ERF subfamily under abiotic stress Table I. Expression of genes from the ERF subfamily under abiotic stress —, Not studied; JERF1, Jasmonate and Ethylene Response Factor1; OPBP1, Osmotin Promoter Binding Protein1; BIERF3, BTH-Induced ERF Transcriptional Factor3; TSRF1, Tomato Stress-Responsive Factor1; TERF, Tomato Ethylene Response Factor; SlERF, Tomato Ethylene Response Factor; SHN1, ethylene-responsive transcription factor WIN1. Species . ERFs . Up-Regulation under Stress . References . Salt . Drought . Cold . Heat . Light . Arabidopsis AtERF1 a Yes Yes — Yes — Cheng et al., 2013 Arabidopsis AtERF4 Yes — — — — Seo et al., 2010 Arabidopsis AtERF5 No Yes — — — Dubois et al. (2013) Arabidopsis AtERF6 Yes Yes Yes No Yes Dubois et al. (2013); Sewelam et al. (2013); Wang et al. (2013a); Vogel et al. (2014) Arabidopsis AtERF98 Yes — — — — Zhang et al. (2012) Arabidopsis AtERF104 — — — — Yes Vogel et al. (2014) Arabidopsis AtERF105 — — — — Yes Vogel et al. (2014) Brassica rapa BrERF4 No — — — — Seo et al. (2010) Pepper (Capsicum annuum) CaERFLP1 a Yes No No — — Lee et al. (2004) Pepper CaPF1 a Yes Yes Yes — — Yi et al. (2004) Chickpea (Cicer arietinum) CarERF116 Yes Yes Yes Yes — Deokar et al. (2015) Chrysanthemum nankingense CnERF1 No — Yes — — Gao et al. (2015) Camellia reticulata CitERF Yes Yes Yes — — Yang et al. (2011) Citrus sinensis CsERF Yes Yes Yes — — Ma et al. (2014) Gossypium hirsutum GhERF1 Yes Yes Yes — — Qiao et al. (2008) G. hirsutum GhERF2 Yes Yes Yes — — Jin et al. (2010) G. hirsutum GhERF3 Yes Yes Yes — — Jin et al. (2009) G. hirsutum GhERF5 Yes Yes Yes — — Jin et al. (2010) G. hirsutum GhERF6 Yes Yes Yes — — Jin et al. (2010) Soybean (Glycine max) GmERF3 a Yes Yes No — — Zhang et al. (2009) Jatropha curcas JcERF1 Yes Yes — — — Yang et al. (2014) Chinese boxthorn (Lycium chinense) LchERF Yes Yes — — — Wu et al. (2014a, 2014b) Common bird's-foot trefoil Lotus corniculatus LcERF054 Yes No — — — Sun et al. (2014a) L. corniculatus LcERF080 Yes — — — — Sun et al. (2014b) Alfala (Medicago sativa) MsERF8 Yes Yes — — — Chen et al. (2012) Tobacco (Nicotiana tabacum) JERF1 a Yes Yes Yes — — Wu et al. (2007) Tobacco JERF3 a Yes Yes Yes — — Wang et al. (2004) Tobacco OPBP1 Yes — — — — Guo et al. (2004) Tobacco OsBIERF3 Yes — — — — Cao et al. (2006) Tobacco Tsi1 a Yes — — — — Park et al. (2001) Tomato (Lycopersicon esculentum) LeERF3b Yes Yes Yes No Yes Chen et al. (2008); Severo et al. (2015) Tomato TSRF1 a Yes Yes — — — Zhang et al. (2007) Tomato TERF1 a Yes Yes — — — Gao et al. (2008) Tomato LeERF1 Yes — — — — Hu et al. (2014) Tomato LeERF2 Yes — — — Yes Hu et al. (2014); Severo et al. (2015) Tomato SlERF5 Yes Yes Yes Yes — Pan et al. (2012) Tomato ERF A.1 — — — — Yes Severo et al. (2015) Tomato ERF A.3 (Pti4) — — — — Yes Severo et al. (2015) Tomato ERF B.1 — — — — Yes Severo et al. (2015) Tomato ERF B.2 — — — — Yes Severo et al. (2015) Tomato ERF B.3 (LeERF4) — — — — Yes Severo et al. (2015) Tomato ERF C.6 (ERF5) — — — — Yes Severo et al. (2015) Tomato ERF D.1 — — — — Yes Severo et al. (2015) Tomato ERF D.3 — — — — Yes Severo et al. (2015) Tomato ERF G.2 (Pti6) — — — — Yes Severo et al. (201) Tomato TERF2/LeERF2 — No Yes — — Zhang and Huang (2010) Sugarcane (Saccharum officinarum) SodERF3 Yes — — — — Trujillo et al. (2008) Wheat (Triticum aestivum) TaERF1 a Yes Yes Yes — — Xu et al. (2007) T. aestivum TaERF3 Yes Yes — — — Rong et al. (2014) Tamarix hispida ThERF1 a Yes Yes — — — Wang et al. (2014); Liu et al. (2014) T. hispida ThERF5 Yes Yes — — — Liu et al. (2014) T. hispida ThERF12 Yes No — — — Liu et al. (2014) Triticum turgidum TdSHN1 a Yes Yes Yes — — Djemal and Khoudi (2015) Species . ERFs . Up-Regulation under Stress . References . Salt . Drought . Cold . Heat . Light . Arabidopsis AtERF1 a Yes Yes — Yes — Cheng et al., 2013 Arabidopsis AtERF4 Yes — — — — Seo et al., 2010 Arabidopsis AtERF5 No Yes — — — Dubois et al. (2013) Arabidopsis AtERF6 Yes Yes Yes No Yes Dubois et al. (2013); Sewelam et al. (2013); Wang et al. (2013a); Vogel et al. (2014) Arabidopsis AtERF98 Yes — — — — Zhang et al. (2012) Arabidopsis AtERF104 — — — — Yes Vogel et al. (2014) Arabidopsis AtERF105 — — — — Yes Vogel et al. (2014) Brassica rapa BrERF4 No — — — — Seo et al. (2010) Pepper (Capsicum annuum) CaERFLP1 a Yes No No — — Lee et al. (2004) Pepper CaPF1 a Yes Yes Yes — — Yi et al. (2004) Chickpea (Cicer arietinum) CarERF116 Yes Yes Yes Yes — Deokar et al. (2015) Chrysanthemum nankingense CnERF1 No — Yes — — Gao et al. (2015) Camellia reticulata CitERF Yes Yes Yes — — Yang et al. (2011) Citrus sinensis CsERF Yes Yes Yes — — Ma et al. (2014) Gossypium hirsutum GhERF1 Yes Yes Yes — — Qiao et al. (2008) G. hirsutum GhERF2 Yes Yes Yes — — Jin et al. (2010) G. hirsutum GhERF3 Yes Yes Yes — — Jin et al. (2009) G. hirsutum GhERF5 Yes Yes Yes — — Jin et al. (2010) G. hirsutum GhERF6 Yes Yes Yes — — Jin et al. (2010) Soybean (Glycine max) GmERF3 a Yes Yes No — — Zhang et al. (2009) Jatropha curcas JcERF1 Yes Yes — — — Yang et al. (2014) Chinese boxthorn (Lycium chinense) LchERF Yes Yes — — — Wu et al. (2014a, 2014b) Common bird's-foot trefoil Lotus corniculatus LcERF054 Yes No — — — Sun et al. (2014a) L. corniculatus LcERF080 Yes — — — — Sun et al. (2014b) Alfala (Medicago sativa) MsERF8 Yes Yes — — — Chen et al. (2012) Tobacco (Nicotiana tabacum) JERF1 a Yes Yes Yes — — Wu et al. (2007) Tobacco JERF3 a Yes Yes Yes — — Wang et al. (2004) Tobacco OPBP1 Yes — — — — Guo et al. (2004) Tobacco OsBIERF3 Yes — — — — Cao et al. (2006) Tobacco Tsi1 a Yes — — — — Park et al. (2001) Tomato (Lycopersicon esculentum) LeERF3b Yes Yes Yes No Yes Chen et al. (2008); Severo et al. (2015) Tomato TSRF1 a Yes Yes — — — Zhang et al. (2007) Tomato TERF1 a Yes Yes — — — Gao et al. (2008) Tomato LeERF1 Yes — — — — Hu et al. (2014) Tomato LeERF2 Yes — — — Yes Hu et al. (2014); Severo et al. (2015) Tomato SlERF5 Yes Yes Yes Yes — Pan et al. (2012) Tomato ERF A.1 — — — — Yes Severo et al. (2015) Tomato ERF A.3 (Pti4) — — — — Yes Severo et al. (2015) Tomato ERF B.1 — — — — Yes Severo et al. (2015) Tomato ERF B.2 — — — — Yes Severo et al. (2015) Tomato ERF B.3 (LeERF4) — — — — Yes Severo et al. (2015) Tomato ERF C.6 (ERF5) — — — — Yes Severo et al. (2015) Tomato ERF D.1 — — — — Yes Severo et al. (2015) Tomato ERF D.3 — — — — Yes Severo et al. (2015) Tomato ERF G.2 (Pti6) — — — — Yes Severo et al. (201) Tomato TERF2/LeERF2 — No Yes — — Zhang and Huang (2010) Sugarcane (Saccharum officinarum) SodERF3 Yes — — — — Trujillo et al. (2008) Wheat (Triticum aestivum) TaERF1 a Yes Yes Yes — — Xu et al. (2007) T. aestivum TaERF3 Yes Yes — — — Rong et al. (2014) Tamarix hispida ThERF1 a Yes Yes — — — Wang et al. (2014); Liu et al. (2014) T. hispida ThERF5 Yes Yes — — — Liu et al. (2014) T. hispida ThERF12 Yes No — — — Liu et al. (2014) Triticum turgidum TdSHN1 a Yes Yes Yes — — Djemal and Khoudi (2015) a ERFs described as capable of binding to GCC box and DRE elements. Open in new tab Table I. Expression of genes from the ERF subfamily under abiotic stress —, Not studied; JERF1, Jasmonate and Ethylene Response Factor1; OPBP1, Osmotin Promoter Binding Protein1; BIERF3, BTH-Induced ERF Transcriptional Factor3; TSRF1, Tomato Stress-Responsive Factor1; TERF, Tomato Ethylene Response Factor; SlERF, Tomato Ethylene Response Factor; SHN1, ethylene-responsive transcription factor WIN1. Species . ERFs . Up-Regulation under Stress . References . Salt . Drought . Cold . Heat . Light . Arabidopsis AtERF1 a Yes Yes — Yes — Cheng et al., 2013 Arabidopsis AtERF4 Yes — — — — Seo et al., 2010 Arabidopsis AtERF5 No Yes — — — Dubois et al. (2013) Arabidopsis AtERF6 Yes Yes Yes No Yes Dubois et al. (2013); Sewelam et al. (2013); Wang et al. (2013a); Vogel et al. (2014) Arabidopsis AtERF98 Yes — — — — Zhang et al. (2012) Arabidopsis AtERF104 — — — — Yes Vogel et al. (2014) Arabidopsis AtERF105 — — — — Yes Vogel et al. (2014) Brassica rapa BrERF4 No — — — — Seo et al. (2010) Pepper (Capsicum annuum) CaERFLP1 a Yes No No — — Lee et al. (2004) Pepper CaPF1 a Yes Yes Yes — — Yi et al. (2004) Chickpea (Cicer arietinum) CarERF116 Yes Yes Yes Yes — Deokar et al. (2015) Chrysanthemum nankingense CnERF1 No — Yes — — Gao et al. (2015) Camellia reticulata CitERF Yes Yes Yes — — Yang et al. (2011) Citrus sinensis CsERF Yes Yes Yes — — Ma et al. (2014) Gossypium hirsutum GhERF1 Yes Yes Yes — — Qiao et al. (2008) G. hirsutum GhERF2 Yes Yes Yes — — Jin et al. (2010) G. hirsutum GhERF3 Yes Yes Yes — — Jin et al. (2009) G. hirsutum GhERF5 Yes Yes Yes — — Jin et al. (2010) G. hirsutum GhERF6 Yes Yes Yes — — Jin et al. (2010) Soybean (Glycine max) GmERF3 a Yes Yes No — — Zhang et al. (2009) Jatropha curcas JcERF1 Yes Yes — — — Yang et al. (2014) Chinese boxthorn (Lycium chinense) LchERF Yes Yes — — — Wu et al. (2014a, 2014b) Common bird's-foot trefoil Lotus corniculatus LcERF054 Yes No — — — Sun et al. (2014a) L. corniculatus LcERF080 Yes — — — — Sun et al. (2014b) Alfala (Medicago sativa) MsERF8 Yes Yes — — — Chen et al. (2012) Tobacco (Nicotiana tabacum) JERF1 a Yes Yes Yes — — Wu et al. (2007) Tobacco JERF3 a Yes Yes Yes — — Wang et al. (2004) Tobacco OPBP1 Yes — — — — Guo et al. (2004) Tobacco OsBIERF3 Yes — — — — Cao et al. (2006) Tobacco Tsi1 a Yes — — — — Park et al. (2001) Tomato (Lycopersicon esculentum) LeERF3b Yes Yes Yes No Yes Chen et al. (2008); Severo et al. (2015) Tomato TSRF1 a Yes Yes — — — Zhang et al. (2007) Tomato TERF1 a Yes Yes — — — Gao et al. (2008) Tomato LeERF1 Yes — — — — Hu et al. (2014) Tomato LeERF2 Yes — — — Yes Hu et al. (2014); Severo et al. (2015) Tomato SlERF5 Yes Yes Yes Yes — Pan et al. (2012) Tomato ERF A.1 — — — — Yes Severo et al. (2015) Tomato ERF A.3 (Pti4) — — — — Yes Severo et al. (2015) Tomato ERF B.1 — — — — Yes Severo et al. (2015) Tomato ERF B.2 — — — — Yes Severo et al. (2015) Tomato ERF B.3 (LeERF4) — — — — Yes Severo et al. (2015) Tomato ERF C.6 (ERF5) — — — — Yes Severo et al. (2015) Tomato ERF D.1 — — — — Yes Severo et al. (2015) Tomato ERF D.3 — — — — Yes Severo et al. (2015) Tomato ERF G.2 (Pti6) — — — — Yes Severo et al. (201) Tomato TERF2/LeERF2 — No Yes — — Zhang and Huang (2010) Sugarcane (Saccharum officinarum) SodERF3 Yes — — — — Trujillo et al. (2008) Wheat (Triticum aestivum) TaERF1 a Yes Yes Yes — — Xu et al. (2007) T. aestivum TaERF3 Yes Yes — — — Rong et al. (2014) Tamarix hispida ThERF1 a Yes Yes — — — Wang et al. (2014); Liu et al. (2014) T. hispida ThERF5 Yes Yes — — — Liu et al. (2014) T. hispida ThERF12 Yes No — — — Liu et al. (2014) Triticum turgidum TdSHN1 a Yes Yes Yes — — Djemal and Khoudi (2015) Species . ERFs . Up-Regulation under Stress . References . Salt . Drought . Cold . Heat . Light . Arabidopsis AtERF1 a Yes Yes — Yes — Cheng et al., 2013 Arabidopsis AtERF4 Yes — — — — Seo et al., 2010 Arabidopsis AtERF5 No Yes — — — Dubois et al. (2013) Arabidopsis AtERF6 Yes Yes Yes No Yes Dubois et al. (2013); Sewelam et al. (2013); Wang et al. (2013a); Vogel et al. (2014) Arabidopsis AtERF98 Yes — — — — Zhang et al. (2012) Arabidopsis AtERF104 — — — — Yes Vogel et al. (2014) Arabidopsis AtERF105 — — — — Yes Vogel et al. (2014) Brassica rapa BrERF4 No — — — — Seo et al. (2010) Pepper (Capsicum annuum) CaERFLP1 a Yes No No — — Lee et al. (2004) Pepper CaPF1 a Yes Yes Yes — — Yi et al. (2004) Chickpea (Cicer arietinum) CarERF116 Yes Yes Yes Yes — Deokar et al. (2015) Chrysanthemum nankingense CnERF1 No — Yes — — Gao et al. (2015) Camellia reticulata CitERF Yes Yes Yes — — Yang et al. (2011) Citrus sinensis CsERF Yes Yes Yes — — Ma et al. (2014) Gossypium hirsutum GhERF1 Yes Yes Yes — — Qiao et al. (2008) G. hirsutum GhERF2 Yes Yes Yes — — Jin et al. (2010) G. hirsutum GhERF3 Yes Yes Yes — — Jin et al. (2009) G. hirsutum GhERF5 Yes Yes Yes — — Jin et al. (2010) G. hirsutum GhERF6 Yes Yes Yes — — Jin et al. (2010) Soybean (Glycine max) GmERF3 a Yes Yes No — — Zhang et al. (2009) Jatropha curcas JcERF1 Yes Yes — — — Yang et al. (2014) Chinese boxthorn (Lycium chinense) LchERF Yes Yes — — — Wu et al. (2014a, 2014b) Common bird's-foot trefoil Lotus corniculatus LcERF054 Yes No — — — Sun et al. (2014a) L. corniculatus LcERF080 Yes — — — — Sun et al. (2014b) Alfala (Medicago sativa) MsERF8 Yes Yes — — — Chen et al. (2012) Tobacco (Nicotiana tabacum) JERF1 a Yes Yes Yes — — Wu et al. (2007) Tobacco JERF3 a Yes Yes Yes — — Wang et al. (2004) Tobacco OPBP1 Yes — — — — Guo et al. (2004) Tobacco OsBIERF3 Yes — — — — Cao et al. (2006) Tobacco Tsi1 a Yes — — — — Park et al. (2001) Tomato (Lycopersicon esculentum) LeERF3b Yes Yes Yes No Yes Chen et al. (2008); Severo et al. (2015) Tomato TSRF1 a Yes Yes — — — Zhang et al. (2007) Tomato TERF1 a Yes Yes — — — Gao et al. (2008) Tomato LeERF1 Yes — — — — Hu et al. (2014) Tomato LeERF2 Yes — — — Yes Hu et al. (2014); Severo et al. (2015) Tomato SlERF5 Yes Yes Yes Yes — Pan et al. (2012) Tomato ERF A.1 — — — — Yes Severo et al. (2015) Tomato ERF A.3 (Pti4) — — — — Yes Severo et al. (2015) Tomato ERF B.1 — — — — Yes Severo et al. (2015) Tomato ERF B.2 — — — — Yes Severo et al. (2015) Tomato ERF B.3 (LeERF4) — — — — Yes Severo et al. (2015) Tomato ERF C.6 (ERF5) — — — — Yes Severo et al. (2015) Tomato ERF D.1 — — — — Yes Severo et al. (2015) Tomato ERF D.3 — — — — Yes Severo et al. (2015) Tomato ERF G.2 (Pti6) — — — — Yes Severo et al. (201) Tomato TERF2/LeERF2 — No Yes — — Zhang and Huang (2010) Sugarcane (Saccharum officinarum) SodERF3 Yes — — — — Trujillo et al. (2008) Wheat (Triticum aestivum) TaERF1 a Yes Yes Yes — — Xu et al. (2007) T. aestivum TaERF3 Yes Yes — — — Rong et al. (2014) Tamarix hispida ThERF1 a Yes Yes — — — Wang et al. (2014); Liu et al. (2014) T. hispida ThERF5 Yes Yes — — — Liu et al. (2014) T. hispida ThERF12 Yes No — — — Liu et al. (2014) Triticum turgidum TdSHN1 a Yes Yes Yes — — Djemal and Khoudi (2015) a ERFs described as capable of binding to GCC box and DRE elements. Open in new tab Abiotic stress response studied in transgenic plants overexpressing ERFs Table II. Abiotic stress response studied in transgenic plants overexpressing ERFs BIERF3, BTH-induced ERF transcriptional Factor3; TERF, Tomato Ethylene Response Factor; SlERF, Tomato Ethylene Response Factor. Gene . Transgenic Plants . Effect . References . AtERF1 Arabidopsis Salt, drought, and heat stress tolerance Cheng et al. (2013) AtERF4 Arabidopsis Hypersensitive to salt stress Yang et al. (2005) AtERF5 Arabidopsis Hypersensitive to osmotic stress Dubois et al. (2013) AtERF6 Arabidopsis Hypersensitive to osmotic stress Dubois et al. (2013) AtERF98 Arabidopsis Salt tolerance Zhang et al. (2012) BrERF4 Arabidopsis Salt and drought tolerance Seo et al. (2010) CarERF116 Arabidopsis Osmotic and freezing tolerance Deokar et al. (2015) LcERF054 Arabidopsis Salt tolerance Sun et al. (2014a) CaPF1 Arabidopsis Freezing tolerance Yi et al. (2004) CaPF1 Virginia pine (Pinus virginiana) Heat and heavy metal tolerance Tang et al. (2005) CaPF1 Potato (Solanum tuberosum) Drought, freezing, heat, and heavy metal tolerance Youm et al. (2008) CaERFLP1 Tobacco Salt tolerance Lee et al. (2004) CsERF Tobacco Cold tolerance Ma et al. (2014) JERF1 Rice (Oryza sativa) Drought tolerance Zhang et al. (2010) JERF3 Tobacco Salt, drought, and freezing tolerance Wang et al. (2004); Wu et al. (2008) Tsi1 Tobacco Salt tolerance Park et al. (2001) OPBP1 Tobacco Salt tolerance Guo et al. (2004) TaERF1 Arabidopsis Salt, drought, and freezing tolerance Xu et al. (2007) OsBIERF3 Tobacco Salt tolerance Cao et al. (2006) SodERF3 Tobacco Salt and drought tolerance Trujillo et al. (2008) TERF1 Rice Salt and drought tolerance Gao et al. (2008) TERF1 Tobacco Salt tolerance Huang et al. (2004) MsERF8 Tobacco Salt tolerance Chen et al. (2012) GmERF8 Tobacco Salt and drought tolerance Zhang et al. (2009) JcERF1 Tobacco Salt tolerance Yang et al. (2014) LchERF Tobacco Salt tolerance Wu et al. (2014a) TSRF1 Tobacco Negative regulator of salt stress Zhang et al. (2007) TSRF1 Zea mays Salt tolerance Wang et al. (2013a, 2013b) TSRF1 Rice Drought tolerance Quan et al. (2010) LeERF1 Tomato Salt tolerance Hu et al. (2014) LeERF2 Tomato Salt tolerance Hu et al. (2014) SlERF5 Tomato Salt and drought tolerance Pan et al. (2012) ThERF1 Arabidopsis Negative regulator of salt and drought stress Wang et al. (2014) TERF2/LeERF2 Tobacco Freezing tolerance Zhang and Huang (2010) Gene . Transgenic Plants . Effect . References . AtERF1 Arabidopsis Salt, drought, and heat stress tolerance Cheng et al. (2013) AtERF4 Arabidopsis Hypersensitive to salt stress Yang et al. (2005) AtERF5 Arabidopsis Hypersensitive to osmotic stress Dubois et al. (2013) AtERF6 Arabidopsis Hypersensitive to osmotic stress Dubois et al. (2013) AtERF98 Arabidopsis Salt tolerance Zhang et al. (2012) BrERF4 Arabidopsis Salt and drought tolerance Seo et al. (2010) CarERF116 Arabidopsis Osmotic and freezing tolerance Deokar et al. (2015) LcERF054 Arabidopsis Salt tolerance Sun et al. (2014a) CaPF1 Arabidopsis Freezing tolerance Yi et al. (2004) CaPF1 Virginia pine (Pinus virginiana) Heat and heavy metal tolerance Tang et al. (2005) CaPF1 Potato (Solanum tuberosum) Drought, freezing, heat, and heavy metal tolerance Youm et al. (2008) CaERFLP1 Tobacco Salt tolerance Lee et al. (2004) CsERF Tobacco Cold tolerance Ma et al. (2014) JERF1 Rice (Oryza sativa) Drought tolerance Zhang et al. (2010) JERF3 Tobacco Salt, drought, and freezing tolerance Wang et al. (2004); Wu et al. (2008) Tsi1 Tobacco Salt tolerance Park et al. (2001) OPBP1 Tobacco Salt tolerance Guo et al. (2004) TaERF1 Arabidopsis Salt, drought, and freezing tolerance Xu et al. (2007) OsBIERF3 Tobacco Salt tolerance Cao et al. (2006) SodERF3 Tobacco Salt and drought tolerance Trujillo et al. (2008) TERF1 Rice Salt and drought tolerance Gao et al. (2008) TERF1 Tobacco Salt tolerance Huang et al. (2004) MsERF8 Tobacco Salt tolerance Chen et al. (2012) GmERF8 Tobacco Salt and drought tolerance Zhang et al. (2009) JcERF1 Tobacco Salt tolerance Yang et al. (2014) LchERF Tobacco Salt tolerance Wu et al. (2014a) TSRF1 Tobacco Negative regulator of salt stress Zhang et al. (2007) TSRF1 Zea mays Salt tolerance Wang et al. (2013a, 2013b) TSRF1 Rice Drought tolerance Quan et al. (2010) LeERF1 Tomato Salt tolerance Hu et al. (2014) LeERF2 Tomato Salt tolerance Hu et al. (2014) SlERF5 Tomato Salt and drought tolerance Pan et al. (2012) ThERF1 Arabidopsis Negative regulator of salt and drought stress Wang et al. (2014) TERF2/LeERF2 Tobacco Freezing tolerance Zhang and Huang (2010) Open in new tab Table II. Abiotic stress response studied in transgenic plants overexpressing ERFs BIERF3, BTH-induced ERF transcriptional Factor3; TERF, Tomato Ethylene Response Factor; SlERF, Tomato Ethylene Response Factor. Gene . Transgenic Plants . Effect . References . AtERF1 Arabidopsis Salt, drought, and heat stress tolerance Cheng et al. (2013) AtERF4 Arabidopsis Hypersensitive to salt stress Yang et al. (2005) AtERF5 Arabidopsis Hypersensitive to osmotic stress Dubois et al. (2013) AtERF6 Arabidopsis Hypersensitive to osmotic stress Dubois et al. (2013) AtERF98 Arabidopsis Salt tolerance Zhang et al. (2012) BrERF4 Arabidopsis Salt and drought tolerance Seo et al. (2010) CarERF116 Arabidopsis Osmotic and freezing tolerance Deokar et al. (2015) LcERF054 Arabidopsis Salt tolerance Sun et al. (2014a) CaPF1 Arabidopsis Freezing tolerance Yi et al. (2004) CaPF1 Virginia pine (Pinus virginiana) Heat and heavy metal tolerance Tang et al. (2005) CaPF1 Potato (Solanum tuberosum) Drought, freezing, heat, and heavy metal tolerance Youm et al. (2008) CaERFLP1 Tobacco Salt tolerance Lee et al. (2004) CsERF Tobacco Cold tolerance Ma et al. (2014) JERF1 Rice (Oryza sativa) Drought tolerance Zhang et al. (2010) JERF3 Tobacco Salt, drought, and freezing tolerance Wang et al. (2004); Wu et al. (2008) Tsi1 Tobacco Salt tolerance Park et al. (2001) OPBP1 Tobacco Salt tolerance Guo et al. (2004) TaERF1 Arabidopsis Salt, drought, and freezing tolerance Xu et al. (2007) OsBIERF3 Tobacco Salt tolerance Cao et al. (2006) SodERF3 Tobacco Salt and drought tolerance Trujillo et al. (2008) TERF1 Rice Salt and drought tolerance Gao et al. (2008) TERF1 Tobacco Salt tolerance Huang et al. (2004) MsERF8 Tobacco Salt tolerance Chen et al. (2012) GmERF8 Tobacco Salt and drought tolerance Zhang et al. (2009) JcERF1 Tobacco Salt tolerance Yang et al. (2014) LchERF Tobacco Salt tolerance Wu et al. (2014a) TSRF1 Tobacco Negative regulator of salt stress Zhang et al. (2007) TSRF1 Zea mays Salt tolerance Wang et al. (2013a, 2013b) TSRF1 Rice Drought tolerance Quan et al. (2010) LeERF1 Tomato Salt tolerance Hu et al. (2014) LeERF2 Tomato Salt tolerance Hu et al. (2014) SlERF5 Tomato Salt and drought tolerance Pan et al. (2012) ThERF1 Arabidopsis Negative regulator of salt and drought stress Wang et al. (2014) TERF2/LeERF2 Tobacco Freezing tolerance Zhang and Huang (2010) Gene . Transgenic Plants . Effect . References . AtERF1 Arabidopsis Salt, drought, and heat stress tolerance Cheng et al. (2013) AtERF4 Arabidopsis Hypersensitive to salt stress Yang et al. (2005) AtERF5 Arabidopsis Hypersensitive to osmotic stress Dubois et al. (2013) AtERF6 Arabidopsis Hypersensitive to osmotic stress Dubois et al. (2013) AtERF98 Arabidopsis Salt tolerance Zhang et al. (2012) BrERF4 Arabidopsis Salt and drought tolerance Seo et al. (2010) CarERF116 Arabidopsis Osmotic and freezing tolerance Deokar et al. (2015) LcERF054 Arabidopsis Salt tolerance Sun et al. (2014a) CaPF1 Arabidopsis Freezing tolerance Yi et al. (2004) CaPF1 Virginia pine (Pinus virginiana) Heat and heavy metal tolerance Tang et al. (2005) CaPF1 Potato (Solanum tuberosum) Drought, freezing, heat, and heavy metal tolerance Youm et al. (2008) CaERFLP1 Tobacco Salt tolerance Lee et al. (2004) CsERF Tobacco Cold tolerance Ma et al. (2014) JERF1 Rice (Oryza sativa) Drought tolerance Zhang et al. (2010) JERF3 Tobacco Salt, drought, and freezing tolerance Wang et al. (2004); Wu et al. (2008) Tsi1 Tobacco Salt tolerance Park et al. (2001) OPBP1 Tobacco Salt tolerance Guo et al. (2004) TaERF1 Arabidopsis Salt, drought, and freezing tolerance Xu et al. (2007) OsBIERF3 Tobacco Salt tolerance Cao et al. (2006) SodERF3 Tobacco Salt and drought tolerance Trujillo et al. (2008) TERF1 Rice Salt and drought tolerance Gao et al. (2008) TERF1 Tobacco Salt tolerance Huang et al. (2004) MsERF8 Tobacco Salt tolerance Chen et al. (2012) GmERF8 Tobacco Salt and drought tolerance Zhang et al. (2009) JcERF1 Tobacco Salt tolerance Yang et al. (2014) LchERF Tobacco Salt tolerance Wu et al. (2014a) TSRF1 Tobacco Negative regulator of salt stress Zhang et al. (2007) TSRF1 Zea mays Salt tolerance Wang et al. (2013a, 2013b) TSRF1 Rice Drought tolerance Quan et al. (2010) LeERF1 Tomato Salt tolerance Hu et al. (2014) LeERF2 Tomato Salt tolerance Hu et al. (2014) SlERF5 Tomato Salt and drought tolerance Pan et al. (2012) ThERF1 Arabidopsis Negative regulator of salt and drought stress Wang et al. (2014) TERF2/LeERF2 Tobacco Freezing tolerance Zhang and Huang (2010) Open in new tab Several TFs from Arabidopsis and other plant species that belong to the ERF subfamily have been reported to be capable of binding to both GCC box and DRE elements (Table I). For instance, AtERF1 binds specifically to GCC boxes in the promoter regions of the ethylene- and jasmonate-responsive plant defensin (PDF1.2) and basic-chitinase (b-CHI) genes (Solano et al., 1998) and DRE elements in the promoters of the Ɗ1-Pyrroline-5-Carboxylate Synthetase1 (P5CS1), Germin-Like Protein9 (GLP9), osmotin34 (OSM34), similar to RCD one5 (SRO5), responsive to desiccation29B (RD29B), Early Response to Dehydration7 (ERD7), and RD20 genes, thus conferring not only resistance to pathogen attack but also, tolerance to several abiotic stresses, including drought, salt, and heat stress (Cheng et al., 2013). Interestingly, the affinity of ERF1 for the DRE elements in the promoter of the P5CS1 was much higher than it was for the GCC box in the promoters of b-CHI and PDF1.2 (Cheng et al., 2013). Other ERFs also specifically bind to both GCC box and DRE elements (Table I). The pepper ethylene-responsive factor-like protein1 (ERFLP1) gene was identified from Xanthomonas spp.-infected plants and encodes the CaERFLP1 protein. CaERFLP1 showed enhanced expression under salt stress but not cold or drought stress. Overexpression of CaERFLP1 led to increased salt stress tolerance in tobacco plants (Lee et al., 2004; Table II). Expression of pathogen and freezing tolerance-related protein1 (CaPF1) was induced in plant response to various abiotic stresses, including cold, salt, and drought stress in Arabidopsis. Overexpression of CaPF1 resulted in enhanced resistance to freezing in Arabidopsis (Yi et al., 2004), and in potato, it resulted in enhanced resistance to freezing, heat, heavy metal, and oxidative stress (Youm et al., 2008). Other examples of ERFs binding to both GCC box and DRE elements include ERF3 from soybean (Zhang et al., 2009); JERF1, JERF3, and tobacco stress-induced protein1 (Tsi1) from tobacco plants (Park et al., 2001; Wang et al., 2004; Wu et al., 2007); TSRF1 from tomato plants (Zhang et al., 2007); and SHN1 from wheat (Triticum durum; Djemal and Khoudi, 2015), which are all induced by drought, salt, and/or cold stress. A complete list of the TF genes that encode proteins from the ERF subfamily, including those that bind to both GCC box and DRE elements, and are involved in abiotic stress tolerance is given in Table I. ERFs AND HORMONE CROSS TALK Plant response and adaptation to environmental stresses require the coordinated interaction of hormone signaling pathways to regulate the expression of TF genes that allows the plant to fine tune specific stress responses. Expression of ERFs as downstream components of the ethylene signaling and response pathway can be induced by ethylene as well as biotic and abiotic stresses. Jasmonic acid and ABA have also been reported to be involved in the regulation of ERFs under abiotic stresses. Moreover, ethylene signaling interacts with other plant hormone pathways, such as those regulated by salicylic acid, gibberellins, and brassinosteroids, during plant adaptation to abiotic stresses. Indeed, exogenous application of these phytohormones has led to induced expression of a number of ERF genes (Table III). However, the molecular transduction mechanisms underlying pathway cross talk are still only partly understood. Hormonal effects on the transcription of ERF genes Table III. Hormonal effects on the transcription of ERF genes BRs, Brassinosteroids; GAs, gibberellins; —, not studied; TERF, Tomato Ethylene Response Factor; SlERF, Tomato Ethylene Response Factor. Gene . Induced Expression . References . Ethylene . ABA . Jasmonates . Salicylic Acid . GAs . Auxin . BRs . AtERF1 a Yes Yes Yes — — — — Cheng et al. (2013) AtERF4 Yes Yes Yes — — — — Yang et al. (2005) BrERF4 Yes No Yes — — — — Seo et al. (2010) CaPF1 a Yes Yes Yes — — — — Yi et al. (2004) CarERF116 Yes Yes — Yes Yes — — Deokar et al. (2015) CsERF Yes Yes Yes Yes No No No Ma et al. (2014) GhERF1 Yes Yes — — — — — Qiao et al. (2008) GhERF2 Yes Yes — — — — — Jin et al. (2010) GhERF3 Yes Yes — — — — — Jin et al. (2009) GhERF5 Yes Yes — — — — — Jin et al. (2010) GhERF6 Yes Yes — — — — — Jin et al. (2010) GmERF3 a Yes Yes Yes Yes — — — Zhang et al. (2009) JcERF1 Yes Yes — — — — — Yang et al. (2014) LchERF Yes — — — — — — Wu et al. (2014a) LcERF054 Yes Yes Yes No — — — Sun et al. (2014a) TSRF1 Yes — — Yes — — — Zhang et al. (2007) MsERF8 Yes Yes Yes Yes Yes Yes — Chen et al. (2012) JERF1 a Yes Yes Yes — — — — Wu et al. (2007) JERF3 a Yes Yes Yes — — — — Wang et al. (2004) OPBP1 Yes — Yes No — — — Guo et al. (2004) Tsi1 a Yes No Yes Yes — — — Park et al. (2001) TERF1 a Yes — — — — — — Gao et al. (2008) SlERF5 Yes Yes No No — — — Pan et al. (2012) SodERF3 Yes Yes Yes No — — — Trujillo et al. (2008) TaERF1 a Yes Yes — Yes — — — Xu et al. (2007) Gene . Induced Expression . References . Ethylene . ABA . Jasmonates . Salicylic Acid . GAs . Auxin . BRs . AtERF1 a Yes Yes Yes — — — — Cheng et al. (2013) AtERF4 Yes Yes Yes — — — — Yang et al. (2005) BrERF4 Yes No Yes — — — — Seo et al. (2010) CaPF1 a Yes Yes Yes — — — — Yi et al. (2004) CarERF116 Yes Yes — Yes Yes — — Deokar et al. (2015) CsERF Yes Yes Yes Yes No No No Ma et al. (2014) GhERF1 Yes Yes — — — — — Qiao et al. (2008) GhERF2 Yes Yes — — — — — Jin et al. (2010) GhERF3 Yes Yes — — — — — Jin et al. (2009) GhERF5 Yes Yes — — — — — Jin et al. (2010) GhERF6 Yes Yes — — — — — Jin et al. (2010) GmERF3 a Yes Yes Yes Yes — — — Zhang et al. (2009) JcERF1 Yes Yes — — — — — Yang et al. (2014) LchERF Yes — — — — — — Wu et al. (2014a) LcERF054 Yes Yes Yes No — — — Sun et al. (2014a) TSRF1 Yes — — Yes — — — Zhang et al. (2007) MsERF8 Yes Yes Yes Yes Yes Yes — Chen et al. (2012) JERF1 a Yes Yes Yes — — — — Wu et al. (2007) JERF3 a Yes Yes Yes — — — — Wang et al. (2004) OPBP1 Yes — Yes No — — — Guo et al. (2004) Tsi1 a Yes No Yes Yes — — — Park et al. (2001) TERF1 a Yes — — — — — — Gao et al. (2008) SlERF5 Yes Yes No No — — — Pan et al. (2012) SodERF3 Yes Yes Yes No — — — Trujillo et al. (2008) TaERF1 a Yes Yes — Yes — — — Xu et al. (2007) a ERFs described as capable of binding to GCC box and DRE elements. Open in new tab Table III. Hormonal effects on the transcription of ERF genes BRs, Brassinosteroids; GAs, gibberellins; —, not studied; TERF, Tomato Ethylene Response Factor; SlERF, Tomato Ethylene Response Factor. Gene . Induced Expression . References . Ethylene . ABA . Jasmonates . Salicylic Acid . GAs . Auxin . BRs . AtERF1 a Yes Yes Yes — — — — Cheng et al. (2013) AtERF4 Yes Yes Yes — — — — Yang et al. (2005) BrERF4 Yes No Yes — — — — Seo et al. (2010) CaPF1 a Yes Yes Yes — — — — Yi et al. (2004) CarERF116 Yes Yes — Yes Yes — — Deokar et al. (2015) CsERF Yes Yes Yes Yes No No No Ma et al. (2014) GhERF1 Yes Yes — — — — — Qiao et al. (2008) GhERF2 Yes Yes — — — — — Jin et al. (2010) GhERF3 Yes Yes — — — — — Jin et al. (2009) GhERF5 Yes Yes — — — — — Jin et al. (2010) GhERF6 Yes Yes — — — — — Jin et al. (2010) GmERF3 a Yes Yes Yes Yes — — — Zhang et al. (2009) JcERF1 Yes Yes — — — — — Yang et al. (2014) LchERF Yes — — — — — — Wu et al. (2014a) LcERF054 Yes Yes Yes No — — — Sun et al. (2014a) TSRF1 Yes — — Yes — — — Zhang et al. (2007) MsERF8 Yes Yes Yes Yes Yes Yes — Chen et al. (2012) JERF1 a Yes Yes Yes — — — — Wu et al. (2007) JERF3 a Yes Yes Yes — — — — Wang et al. (2004) OPBP1 Yes — Yes No — — — Guo et al. (2004) Tsi1 a Yes No Yes Yes — — — Park et al. (2001) TERF1 a Yes — — — — — — Gao et al. (2008) SlERF5 Yes Yes No No — — — Pan et al. (2012) SodERF3 Yes Yes Yes No — — — Trujillo et al. (2008) TaERF1 a Yes Yes — Yes — — — Xu et al. (2007) Gene . Induced Expression . References . Ethylene . ABA . Jasmonates . Salicylic Acid . GAs . Auxin . BRs . AtERF1 a Yes Yes Yes — — — — Cheng et al. (2013) AtERF4 Yes Yes Yes — — — — Yang et al. (2005) BrERF4 Yes No Yes — — — — Seo et al. (2010) CaPF1 a Yes Yes Yes — — — — Yi et al. (2004) CarERF116 Yes Yes — Yes Yes — — Deokar et al. (2015) CsERF Yes Yes Yes Yes No No No Ma et al. (2014) GhERF1 Yes Yes — — — — — Qiao et al. (2008) GhERF2 Yes Yes — — — — — Jin et al. (2010) GhERF3 Yes Yes — — — — — Jin et al. (2009) GhERF5 Yes Yes — — — — — Jin et al. (2010) GhERF6 Yes Yes — — — — — Jin et al. (2010) GmERF3 a Yes Yes Yes Yes — — — Zhang et al. (2009) JcERF1 Yes Yes — — — — — Yang et al. (2014) LchERF Yes — — — — — — Wu et al. (2014a) LcERF054 Yes Yes Yes No — — — Sun et al. (2014a) TSRF1 Yes — — Yes — — — Zhang et al. (2007) MsERF8 Yes Yes Yes Yes Yes Yes — Chen et al. (2012) JERF1 a Yes Yes Yes — — — — Wu et al. (2007) JERF3 a Yes Yes Yes — — — — Wang et al. (2004) OPBP1 Yes — Yes No — — — Guo et al. (2004) Tsi1 a Yes No Yes Yes — — — Park et al. (2001) TERF1 a Yes — — — — — — Gao et al. (2008) SlERF5 Yes Yes No No — — — Pan et al. (2012) SodERF3 Yes Yes Yes No — — — Trujillo et al. (2008) TaERF1 a Yes Yes — Yes — — — Xu et al. (2007) a ERFs described as capable of binding to GCC box and DRE elements. Open in new tab Jasmonates Lorenzo et al. (2002) have reported that ERF1 is a downstream component of not only the ethylene but also, the jasmonate signaling pathway in Arabidopsis. Lorenzo et al. (2002) observed that ERF1 expression can be induced rapidly by ethylene and jasmonic acid as well as synergistically by both hormones, and they suggested that ERF1 acts as a key element in the regulation of ethylene/jasmonic acid-dependent defense response genes (Lorenzo et al., 2002). Recent studies using the jasmonic acid-insensitive mutant jasmonic acid amido-synthetase1-1 exposed to drought, salt, and heat stress revealed blocked ERF1 expression, indicating that ERF1 induction requires jasmonic acid as well as ethylene signaling under a number of abiotic stresses (Cheng et al., 2013). Ethylene/jasmonic acid signaling is also required in the induction of other ERFs to a number of abiotic stresses; examples of these ERFs include ERF6 (Sewelam et al., 2013), JERF1 (Wu et al., 2007, 2008), JERF3 (Wang et al., 2004), Tsi1 (Park et al., 2001), OPBP1 (Guo et al., 2004) and GmERF3 (Zhang et al., 2009). ABA ABA plays an important role in the response of plants to abiotic stresses, such as drought, salinity, and extreme temperatures. ERF1 overexpression has been observed to enhance drought, salt, and heat stress resistance in Arabidopsis plants accompanied with increased levels of ABA and Pro (Cheng et al., 2013). As an osmolite, Pro contributes to stress tolerance because its accumulation may prevent water loss. ABA has been reported to partially modulate Pro accumulation (Sharma et al., 2011). However, ABA negatively regulates ERF1 induction as shown in the ABA-hypersensitive abi1 and abi2 knockout mutants. Nevertheless, in the constitutive ethylene signaling mutant ctr1, ERF1 expression was even higher than in wild-type plants after ABA treatment. This indicates that ethylene/jasmonic acid signaling could not be blocked by the negative effect of ABA (Cheng et al., 2013). ABA treatment also repressed the expression of ERF6 in Arabidopsis (Sewelam et al., 2013). In contrast to ERF1 and ERF6, other ERF genes have been reported to be induced by ABA, including CsERF (Ma et al., 2014), GmERF3 (Zhang et al., 2009), LchERF (Wu et al., 2014a), JERF3 (Wu et al., 2008), TSRF1 (Quan et al., 2010), TaERF1 (Xu et al., 2007), and JERF1 (Wu et al., 2007). In addition, plants overexpressing TaERF1 were found to be highly sensitive to exogenous ABA treatment, resulting in rapid stomatal closure (Xu et al., 2007). Interestingly, JERF1 overexpression also increased leaf and root growth of tobacco significantly under salinity and low temperature accompanied by increased ABA levels (Wu et al., 2007). JERF1 was found to interact with multiple cis-acting elements and may activate both stress-responsive and ABA biosynthesis-related genes (such as tobacco short-chain dehydrogenase/reductase [NtSDR]), resulting in enhanced tolerance to salinity and cold stress in tobacco (Wu et al., 2007). Also, TSRF1 overexpression in tobacco enhanced expression of the ABA biosynthesis-related gene NtSDR, resulting in increased ABA contents. Moreover, overexpression of TSRF1 in tobacco plants resulted in enhanced drought tolerance and increased Pro contents (Quan et al., 2010; Cheng et al., 2013). Complex interaction signaling between ABA, ethylene, and ERF proteins, such as ERF1, JERF1, and TSRF1, seems to regulate ABA biosynthesis; however, ABA also acts as a negative regulator of ERF1 gene induction (Cheng et al., 2013). Interestingly, all three ERF proteins can bind to both GCC box and DRE elements and induce salt, heat, drought, and cold tolerance (Table I). A model for ethylene, jasmonic acid, and ABA cross talk through ERF1 under abiotic stress is shown in Figure 2. Figure 2. Open in new tabDownload slide Proposed model for ethylene (ET), jasmonic acid (JA), and ABA cross talk through ERFs under abiotic stress. ERF1 induces expression of genes involved in abiotic stress tolerance. It has been postulated that, through the activation of JERF1 and TSRF1 (ERFs from the same ERF subfamily), ERF1 activates expression of NtSDR, an ABA biosynthesis-related gene. In turn, ABA might down-regulate ERF1 expression under abiotic stress. However, the negative effect of ABA does not seem to block ET/JA signaling. LEA4-5, Late-Embryogenesis Abundant Protein4-5; HSFA3, Heat-Shock Transcription Factor A3; HSP101, Heat-Shock Protein101. Figure 2. Open in new tabDownload slide Proposed model for ethylene (ET), jasmonic acid (JA), and ABA cross talk through ERFs under abiotic stress. ERF1 induces expression of genes involved in abiotic stress tolerance. It has been postulated that, through the activation of JERF1 and TSRF1 (ERFs from the same ERF subfamily), ERF1 activates expression of NtSDR, an ABA biosynthesis-related gene. In turn, ABA might down-regulate ERF1 expression under abiotic stress. However, the negative effect of ABA does not seem to block ET/JA signaling. LEA4-5, Late-Embryogenesis Abundant Protein4-5; HSFA3, Heat-Shock Transcription Factor A3; HSP101, Heat-Shock Protein101. Some proteins from the ERF subfamily can also act as negative regulators, such as AtERF4 and AtERF7, which are localized in the nuclear bodies and modulate ABA responses. Induced AtERF4 expression has been reported to make plants less sensitive to ABA, inhibit the expression of genes that are responsive to ABA, and confer hypersensitivity to salt stress in Arabidopsis (Yang et al., 2005). It has been suggested that AtERF7 activation inhibits the expression of genes induced by ABA, thereby decreasing tolerance to drought stress (Song et al., 2005). Other Hormones Salicylic acid has long been known to play a role in the induction of defense mechanisms in plants; however, recent studies revealed that it participates in abiotic stress signaling (Stevens et al., 2006; Horváth et al., 2007). It has been revealed that salicylic acid signaling enhances salt and oxidative stress tolerance in Arabidopsis by the induction of the NONEXPRESSER OF PATHOGENESIS RELATED1 (NPR1) gene (Jayakannan et al., 2015). Upon pathogen infection, ethylene is known to enhance salicylic acid/NPR1-dependent defenses through the ethylene signaling and response pathway (Leon-Reyes et al., 2009). Salicylic acid treatment induced expression of the ethylene TF genes AtERF6 (Sewelam et al., 2013), TaERF1 (Xu et al., 2007), TSRF1 (Huang et al., 2004), MsERF8 (Chen et al., 2012), GmERF3 (Zhang et al., 2009), and CarERF116 (Deokar et al., 2015), whereas expression of SodERF3 (Trujillo et al., 2008) and CsERF (Ma et al., 2014) was reduced by salicylic acid. Recently, ERF6 has been reported to be involved in cross talk between the ethylene and gibberellin/DELLA pathway. ERF6 expression inhibits leaf growth by activating the transcription of the GIBBERELN2-OXIDASE6 gene, resulting in inactivation of gibberellins by DELLA stabilization under osmotic stress conditions in Arabidopsis (Dubois et al., 2013). However, the rapid ERF6 activation was found to be independent of EIN3/EIL1. It has been shown that ERF6 is activated by a mitogen-activated protein kinase cascade, including MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3)/MPK6. Phosphorylation of ERF6 by MPK3/MPK6 in gain-of-function transgenic plants increases ERF6 protein stability in vivo (Dubois et al., 2013; Meng et al., 2013). Brassinosteroids have been found to mediate thermotolerance and salt tolerance and induce expression of several hormone-responsive genes, such as PDF1.2, suggesting cross talk between brassinosteroids and the ethylene, jasmonic acid, ABA, and salicylic acid signaling pathways (Divi et al., 2010). In Citrus spp. plants, expression of the CsERF gene was neither induced nor reduced after treatment with brassinosteroids, auxin, and gibberellin 3 (Ma et al., 2014). In contrast, gibberellin 3 treatment induced expression of both the CaERF116 and MsERF8 genes (Chen et al., 2012; Deokar et al., 2015). ERFs AND REDOX SIGNALING The reactive oxygen species (ROS) signaling network controls a broad range of biological processes, including biotic and abiotic stress responses, by activating defense genes (Mittler et al., 2011). ROS, such as 1O2, hydrogen peroxide, O2 −, and •HO, are molecules that are considered to be both signaling and potentially damaging molecules (Iqbal et al., 2014). Salinity, drought, and cold stresses enhance ROS production, which results in an imbalance between ROS production and ROS scavenging (Miller et al., 2010). Antioxidants, such as ascorbic acid, glutathione, carotenoids, and tocopherols, as well as enzymes, such as superoxide dismutase, ascorbate peroxidase, catalase, and glutathione peroxidase, play an essential role in ROS scavenging mechanisms (Apel and Hirt, 2004; Munné-Bosch et al., 2013). Moreover, Pro plays a potential role in ROS detoxification because it is typically accumulated in response to osmotic stress (Sharma et al., 2011). Extracellular ROS, which are produced by peroxidases and NADPH oxidases, can transmit intracellular signals rapidly to the nucleus and/or amplify signals passing from the chloroplast to the cell nucleus through the action of secondary messengers, such as mitogen-activated protein kinases and plant hormones. Therefore, the TFs activated by ROS result in the transcription of a large number of genes (Miller et al., 2010; Munné-Bosch et al., 2013). Oxidative stress treatment induced ERF1 expression (Sewelam et al., 2013), and overexpression of ERF1 leads to Pro accumulation and induces expression of P5CS1 (Cheng et al., 2013). This, in turn, catalyzes the first step of Pro synthesis, resulting in enhanced drought tolerance in Arabidopsis (Cheng et al., 2013). This result suggests, on the one hand, that ERF1 might regulate ROS signaling and on the other hand, that Pro accumulation seems to be a common response of ERFs to stress. This is, indeed, documented for the majority of TFs from the ERF subfamily that enhance abiotic stress tolerance, such as JERF1 (Zhang et al., 2010), TSRF1 (Quan et al., 2010), GmERF3 (Zhang et al., 2009), Tomato Ethylene-Response Factor5 (Pan et al., 2012), CsERF (Ma et al., 2014), JcERF1 (Yang et al., 2014), LeERF1, LeERF2 (Hu et al., 2014), MsERF8 (Chen et al., 2012), LcERF054, LcERF080 (Sun et al., 2014a, 2014b), LchERF (Wu et al., 2014a), and TaERF3 (Rong et al., 2014). Overexpression of LeERF1, LeERF2, and MsERF8 has been reported to elevate Pro accumulation and reduce malondialdehyde levels, an indicator of lipid peroxidation, in tomato and tobacco plants under salt stress (Cheng et al., 2013; Hu et al., 2014). Tang et al. (2005) have reported that overexpression of CaPF1 enhances biotic and abiotic stress tolerance in transgenic Virginia pine by regulating antioxidant enzyme activities. This result coincides with that for transgenic potato plants that overexpress CaPF1 and were more tolerant after oxidative stress treatment than control plants (Youm et al., 2008). AtERF98 has been reported to enhance salt tolerance by regulating the expression of ascorbate biosynthesis genes, resulting in reduced ROS levels in Arabidopsis (Zhang et al., 2012). JERF3 regulates the expression of ROS-related genes, such as SUPEROXIDE DISMUTASE, ASCORBATE PEROXIDASE1 (APX1), NtAPX2, and GLUTATHIONE PEROXIDASE in tobacco plants, resulting in decreased accumulation of ROS and enhancing tolerance to drought, salt, and freezing (Wu et al., 2008). ERF6 shows highly induced expression under oxidative stress treatment, such as hydrogen peroxide, and high light stress. Up-regulation of ROS-responsive gene expression analyzed in erf6 mutants revealed that ERF6 seems to be a negative regulator of ROS-responsive gene expression. In contrast, several antioxidant enzymes, such as MONODEHYDROASCORBATE REDUCTASE3, CATALASE3, and VITAMIN C DEFECTIVE2, showed down-regulation in erf6 mutants, indicating that ERF6 is a positive antioxidant regulator under biotic and abiotic stresses (Sewelam et al., 2013). Wang et al. (2013a) reported that ERF6 can bind specifically to the ROS-responsive cis-acting element7 (ROSE7)/GCC box, and enhances high light tolerance given that ROSE7-type genes showed no activation in erf6-1 mutants under high light stress. So far, little is known about the involvement of ROS signaling in the activation of ERFs. The MAPK cascade is well known to play an important role in ROS signaling. Ethylene synthesis is known to be positively regulated by the MPK6-mediated phosphorylation of ACC SYNTHASE2 (ACS2) and ACS6 (Liu and Zhang, 2004). Recently, several studies revealed that ERF6 is activated independently of EIN3/EIL1 by MPK6 phosphorylation, resulting in ROS-responsive gene expression in Arabidopsis (Dubois et al., 2013; Meng et al., 2013; Sewelam et al., 2013; Wang et al., 2013b). It is hypothesized that inactive ERF6 is kept at a basal level and can be rapidly phosphorylated, thereby reducing the time lag in transcriptional activation (Dubois et al., 2013; Fig. 3). Figure 3. Open in new tabDownload slide Proposed model for ROS signaling to ERFs. Biotic and abiotic stresses enhance ROS production, resulting in the activation of MPK6, which activates ethylene biosynthesis by phosphorylation of ACS6. Then, EIN2, EIN3/EIL1, and finally, ERF1 are activated, which could result in the activation of ROS gene expression that enhances stress tolerance. Recently, it has been suggested that ERF6 is activated by MPK6 phosphorylation independently of EIN3/EIL1 under oxidative stress. Figure 3. Open in new tabDownload slide Proposed model for ROS signaling to ERFs. Biotic and abiotic stresses enhance ROS production, resulting in the activation of MPK6, which activates ethylene biosynthesis by phosphorylation of ACS6. Then, EIN2, EIN3/EIL1, and finally, ERF1 are activated, which could result in the activation of ROS gene expression that enhances stress tolerance. Recently, it has been suggested that ERF6 is activated by MPK6 phosphorylation independently of EIN3/EIL1 under oxidative stress. ERFs may also play a role in linking redox and hormonal regulation in plant responses to abiotic stresses. Tocopherols, which belong to the vitamin E group of compounds, are lipid-soluble antioxidants found in the chloroplasts. Photosynthetic tissues accumulate α-tocopherol in chloroplasts and to a lesser extent, its immediate precursor γ-tocopherol. ERF1 expression in the γ-tocopherol methyltransferase (vte4) mutant, which is deficient in α-tocopherol but accumulates γ-tocopherol, was reduced in parallel with lower jasmonic acid levels in the vte4 mutant compared with the wild type (Cela et al., 2011). These results indicate that γ-tocopherol represses jasmonic acid and ethylene signaling and response pathways in salt-stressed vte4 plants, thus suggesting a link between redox and hormonal signaling in the regulation of ERF1 expression in Arabidopsis. In another example of retrograde signaling from the chloroplast to the nucleus, Vogel et al. (2014) found that ERF6, ERF104, and ERF105 expression was rapidly (within 1 min) up-regulated upon exposure to high light in Arabidopsis. This response was deregulated in triose phosphate translocator (tpt) mutants. Similarly, activation of MPK6 was up-regulated after 1 min in the wild type but not in the tpt mutant (Vogel et al., 2014). Vogel et al. (2014) propose that metabolite export through the tpt in the chloroplast leads to subsequent MPK6 activation and ERF gene expression in the nucleus, therefore representing an additional mechanism of chloroplast to nucleus retrograde signaling. CONCLUSION AND PROSPECTS Molecular genetic studies have been pivotal in dissecting the ethylene signaling and response pathway. New insights from recent years have revealed that ERFs regulate not only biotic but also, abiotic stress responses. Thus, overexpression of a number of ERFs enhances salt, drought, light stress, and cold and heat tolerance as well as pathogen resistance in Arabidopsis plants. Ethylene, jasmonic acid, and ABA have been reported to be involved in the regulation of ERFs under abiotic stresses. For instance, ERF1 is involved in both ethylene and jasmonic acid signaling pathways. Moreover, plants that overexpress ERF1 enhance ABA levels under drought stress, indicating that ERF1 may regulate ABA biosynthesis. However, ABA negatively regulates ERF1 induction. Furthermore, induced ERF expression under oxidative stress suggests that ERFs might regulate ROS-responsive gene expression, thereby conferring stress tolerance. Because of the fact that many stresses act hand in hand with each other and not in isolation, it is clear that there is cross talk between biotic and abiotic stress responses. The specific binding activity of several ERFs to both GCC box and DRE elements depending on the stress conditions supports this hypothesis. There are still, however, many gaps in our knowledge on ERFs and hormonal cross talk, and the answers to remaining questions are important to increase our understanding of stress adaptation. Alongside the cross talk with jasmonic acid and ABA, the cross talk between ERFs and auxin, cytokinin, gibberellin, salicylic acid, and brassinosteroid responses should be studied in more detail. ERFs seem to regulate ROS-responsive gene expression, but more evidence of synergistic ERFs- and ROS-responsive genes is needed. Glossary ABA abscisic acid ROS reactive oxygen species TF transcription factor LITERATURE CITED An F , Zhao Q, Ji Y, Li W, Jiang Z, Yu X, Zhang C, Han Y, He W, Liu Y, et al. ( 2010 ) Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis . Plant Cell 22 : 2384 – 2401 Google Scholar Crossref Search ADS PubMed WorldCat Apel K , Hirt H ( 2004 ) Reactive oxygen species: metabolism, oxidative stress, and signal transduction . Annu Rev Plant Biol 55 : 373 – 399 Google Scholar Crossref Search ADS PubMed WorldCat Arabidopsis Genome Initiative ( 2000 ) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana . Nature 408 : 796 – 815 Crossref Search ADS PubMed WorldCat Berrocal-Lobo M , Molina A, Solano R ( 2002 ) Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi . Plant J 29 : 23 – 32 Google Scholar Crossref Search ADS PubMed WorldCat Bisson MM , Bleckmann A, Allekotte S, Groth G ( 2009 ) EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1 . Biochem J 424 : 1 – 6 Google Scholar Crossref Search ADS PubMed WorldCat Bisson MM , Groth G ( 2010 ) New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2 . Mol Plant 3 : 882 – 889 Google Scholar Crossref Search ADS PubMed WorldCat Cao Y , Wu Y, Zheng Z, Song F ( 2006 ) Overexpression of the rice EREBP-like gene OsBIERF3 enhances disease resistance and salt tolerance in transgenic tobacco . Physiol Mol Plant Pathol 67 : 202 – 211 Google Scholar Crossref Search ADS WorldCat Cela J , Chang C, Munné-Bosch S ( 2011 ) Accumulation of γ- rather than α-tocopherol alters ethylene signaling gene expression in the vte4 mutant of Arabidopsis thaliana . Plant Cell Physiol 52 : 1389 – 1400 Google Scholar Crossref Search ADS PubMed WorldCat Charfeddine M , Saïdi MN, Charfeddine F, Hammami A, Bouzid RG ( 2015 ) Genome-wide analysis and expression profiling of the ERF transcription factor family in potato (Solanum tuberosum L.) . Mol Biotechnol 57 : 348 – 358 Google Scholar Crossref Search ADS PubMed WorldCat Chen G , Hu Z, Grierson D ( 2008 ) Differential regulation of tomato ethylene responsive factor LeERF3b, a putative repressor, and the activator Pti4 in ripening mutants and in response to environmental stresses . J Plant Physiol 165 : 662 – 670 Google Scholar Crossref Search ADS PubMed WorldCat Chen T , Yang Q, Gruber M, Kang J, Sun Y, Ding W, Zhang T, Zhang X ( 2012 ) Expression of an alfalfa (Medicago sativa L.) ethylene response factor gene MsERF8 in tobacco plants enhances resistance to salinity . Mol Biol Rep 39 : 6067 – 6075 Google Scholar Crossref Search ADS PubMed WorldCat Chen YF , Etheridge N, Schaller GE ( 2005 ) Ethylene signal transduction . Ann Bot (Lond) 95 : 901 – 915 Google Scholar Crossref Search ADS WorldCat Cheng MC , Liao PM, Kuo WW, Lin TP ( 2013 ) The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals . Plant Physiol 162 : 1566 – 1582 Google Scholar Crossref Search ADS PubMed WorldCat Deokar AA , Kondawar V, Kohli D, Aslam M, Jain PK, Karuppayil SM, Varshney RK, Srinivasan R ( 2015 ) The CarERF genes in chickpea (Cicer arietinum L.) and the identification of CarERF116 as abiotic stress responsive transcription factor . Funct Integr Genomics 15 : 27 – 46 Google Scholar Crossref Search ADS PubMed WorldCat Divi UK , Rahman T, Krishna P ( 2010 ) Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid, ethylene and salicylic acid pathways . BMC Plant Biol 10 : 151 – 165 Google Scholar Crossref Search ADS PubMed WorldCat Djemal R , Khoudi H ( February 17, 2015 ) Isolation and molecular characterization of a novel WIN1/SHN1 ethylene-responsive transcription factor TdSHN1 from durum wheat (Triticum turgidum. L. subsp. durum) . Protoplasma 10.1007/s00709-015-0775-8 OpenURL Placeholder Text WorldCat Dubois M , Skirycz A, Claeys H, Maleux K, Dhondt S, De Bodt S, Vanden Bossche R, De Milde L, Yoshizumi T, Matsui M, et al. ( 2013 ) ETHYLENE RESPONSE FACTOR6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis . Plant Physiol 162 : 319 – 332 Google Scholar Crossref Search ADS PubMed WorldCat Gao C , Li P, Song A, Wang H, Wang Y, Ren L, Qi X, Chen F, Jiang J, Chen S ( 2015 ) Isolation and characterization of six AP2/ERF transcription factor genes in Chrysanthemum nankingense . Int J Mol Sci 16 : 2052 – 2065 Google Scholar Crossref Search ADS PubMed WorldCat Gao S , Zhang H, Tian Y, Li F, Zhang Z, Lu X, Chen X, Huang R ( 2008 ) Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity . Plant Cell Rep 27 : 1787 – 1795 Google Scholar Crossref Search ADS PubMed WorldCat Gao Z , Chen YF, Randlett MD, Zhao XC, Findell JL, Kieber JJ, Schaller GE ( 2003 ) Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes . J Biol Chem 278 : 34725 – 34732 Google Scholar Crossref Search ADS PubMed WorldCat Golldack D , Li C, Mohan H, Probst N ( 2014 ) Tolerance to drought and salt stress in plants: unraveling the signaling networks . Front Plant Sci 5 : 151 Google Scholar Crossref Search ADS PubMed WorldCat Gong W , Shen YP, Ma LG, Pan Y, Du YL, Wang DH, Yang JY, Hu LD, Liu XF, Dong CX, et al. ( 2004 ) Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes . Plant Physiol 135 : 773 – 782 Google Scholar Crossref Search ADS PubMed WorldCat Guo ZJ , Chen XJ, Wu XL, Ling JQ, Xu P ( 2004 ) Overexpression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance in tobacco . Plant Mol Biol 55 : 607 – 618 Google Scholar Crossref Search ADS PubMed WorldCat He XJ , Mu RL, Cao WH, Zhang ZG, Zhang JS, Chen SY ( 2005 ) AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development . Plant J 44 : 903 – 916 Google Scholar Crossref Search ADS PubMed WorldCat Horváth E , Szalai G, Janda T ( 2007 ) Induction of abiotic stress tolerance by salicylic acid signaling . J Plant Growth Regul 26 : 290 – 300 Google Scholar Crossref Search ADS WorldCat Hu N , Tang N, Yan F, Bouzayen M, Li Z ( 2014 ) Effect of LeERF1 and LeERF2 overexpression in the response to salinity of young tomato (Solanum lycopersicum cv. Micro-Tom) seedlings . Acta Physiol Plant 36 : 1703 – 1712 Google Scholar Crossref Search ADS WorldCat Huang Y , Li H, Hutchison CE, Laskey J, Kieber JJ ( 2003 ) Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis . Plant J 33 : 221 – 233 Google Scholar Crossref Search ADS PubMed WorldCat Huang Z , Zhang Z, Zhang X, Zhang H, Huang D, Huang R ( 2004 ) Tomato TERF1 modulates ethylene response and enhances osmotic stress tolerance by activating expression of downstream genes . FEBS Lett 573 : 110 – 116 Google Scholar Crossref Search ADS PubMed WorldCat Iqbal N , Umar S, Khan NA, Khan MIR ( 2014 ) A new perspective of phytohormones in salinity tolerance: regulation of proline metabolism . Environ Exp Bot 100 : 34 – 42 Google Scholar Crossref Search ADS WorldCat Ito TM , Polido PB, Rampim MC, Kaschuk G, Souza SGH ( 2014 ) Genome-wide identification and phylogenetic analysis of the AP2/ERF gene superfamily in sweet orange (Citrus sinensis) . Genet Mol Res 13 : 7839 – 7851 Google Scholar Crossref Search ADS PubMed WorldCat Jayakannan M , Bose J, Babourina O, Shabala S, Massart A, Poschenrieder C, Rengel Z ( 2015 ) The NPR1-dependent salicylic acid signalling pathway is pivotal for enhanced salt and oxidative stress tolerance in Arabidopsis . J Exp Bot 66 : 1865 – 1875 Google Scholar Crossref Search ADS PubMed WorldCat Jin LG , Huang B, Li H, Liu JY ( 2009 ) Expression profile and transactivation analysis of a novel ethylene-responsive transcription factor gene GhERF5 from cotton . Prog Nat Sci 19 : 563 – 572 Google Scholar Crossref Search ADS WorldCat Jin LG , Li H, Liu JY ( 2010 ) Molecular characterization of three ethylene responsive element binding factor genes from cotton . J Integr Plant Biol 52 : 485 – 495 Google Scholar PubMed OpenURL Placeholder Text WorldCat Ju C , Yoon GM, Shemansky JM, Lin DY, Ying ZI, Chang J, Garrett WM, Kessenbrock M, Groth G, Tucker ML, et al. ( 2012 ) CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis . Proc Natl Acad Sci USA 109 : 19486 – 19491 Google Scholar Crossref Search ADS PubMed WorldCat Kendrick MD , Chang C ( 2008 ) Ethylene signaling: new levels of complexity and regulation . Curr Opin Plant Biol 11 : 479 – 485 Google Scholar Crossref Search ADS PubMed WorldCat Kieber JJ , Rothenberg M, Roman G, Feldmann KA, Ecker JR ( 1993 ) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases . Cell 72 : 427 – 441 Google Scholar Crossref Search ADS PubMed WorldCat Lacey RF , Binder BM ( 2014 ) How plants sense ethylene gas—the ethylene receptors . J Inorg Biochem 133 : 58 – 62 Google Scholar Crossref Search ADS PubMed WorldCat Lee JH , Hong JP, Oh SK, Lee S, Choi D, Kim WT ( 2004 ) The ethylene-responsive factor like protein 1 (CaERFLP1) of hot pepper (Capsicum annuum L.) interacts in vitro with both GCC and DRE/CRT sequences with different binding affinities: possible biological roles of CaERFLP1 in response to pathogen infection and high salinity conditions in transgenic tobacco plants . Plant Mol Biol 55 : 61 – 81 Google Scholar Crossref Search ADS PubMed WorldCat Lei G , Shen M, Li ZG, Zhang B, Duan KX, Wang N, Cao YR, Zhang WK, Ma B, Ling HQ, et al. ( 2011 ) EIN2 regulates salt stress response and interacts with a MA3 domain-containing protein ECIP1 in Arabidopsis . Plant Cell Environ 34 : 1678 – 1692 Google Scholar Crossref Search ADS PubMed WorldCat Leon-Reyes A , Spoel SH, De Lange ES, Abe H, Kobayashi M, Tsuda S, Millenaar FF, Welschen RAM, Ritsema T, Pieterse CMJ ( 2009 ) Ethylene modulates the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 in cross talk between salicylate and jasmonate signaling . Plant Physiol 149 : 1797 – 1809 Google Scholar Crossref Search ADS PubMed WorldCat Licausi F , Giorgi FM, Zenoni S, Osti F, Pezzotti M, Perata P ( 2010 ) Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera . BMC Genomics 11 : 719 Google Scholar Crossref Search ADS PubMed WorldCat Licausi F , Ohme-Takagi M, Perata P ( 2013 ) APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs . New Phytol 199 : 639 – 649 Google Scholar Crossref Search ADS PubMed WorldCat Liu W , Wang Y, Gao C ( 2014 ) The ethylene response factor (ERF) genes from Tamarix hispida respond to salt, drought and ABA treatment . Trees 28 : 317 – 327 Google Scholar Crossref Search ADS WorldCat Liu Y , Zhang S ( 2004 ) Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis . Plant Cell 16 : 3386 – 3399 Google Scholar Crossref Search ADS PubMed WorldCat Lorenzo O , Piqueras R, Sánchez-Serrano JJ, Solano R ( 2002 ) ETHYLENE RESPONSE FACTOR1 integrates signal from ethylene and jasmonate pathway in plant defense . Plant Cell 15 : 165 – 178 Google Scholar Crossref Search ADS WorldCat Ma S , Gong Q, Bohnert HJ ( 2006 ) Dissecting salt stress pathways . J Exp Bot 57 : 1097 – 1107 Google Scholar Crossref Search ADS PubMed WorldCat Ma Y , Zhang L, Zhang J, Chen J, Wu T, Zhu S, Yan S, Zhao X, Zhong G ( 2014 ) Expressing a Citrus ortholog of Arabidopsis ERF1 enhanced cold-tolerance in tobacco . Sci Hortic 174 : 64 – 76 Google Scholar Crossref Search ADS WorldCat Magome H , Yamaguchi S, Hanada A, Kamiya Y, Oda K ( 2008 ) The DDF1 transcriptional activator upregulates expression of a gibberellin-deactivating gene, GA2ox7, under high-salinity stress in Arabidopsis . Plant J 56 : 613 – 626 Google Scholar Crossref Search ADS PubMed WorldCat Meng X , Xu J, He Y, Yang KY, Mordorski B, Liu Y, Zhang S ( 2013 ) Phosphorylation of an ERF transcription factor by Arabidopsis MPK3/MPK6 regulates plant defense gene induction and fungal resistance . Plant Cell 25 : 1126 – 1142 Google Scholar Crossref Search ADS PubMed WorldCat Miller G , Suzuki N, Ciftci-Yilmaz S, Mittler R ( 2010 ) Reactive oxygen species homeostasis and signalling during drought and salinity stresses . Plant Cell Environ 33 : 453 – 467 Google Scholar Crossref Search ADS PubMed WorldCat Mittler R , Vanderauwera S, Suzuki N, Miller G, Tognetti VB, Vandepoele K, Gollery M, Shulaev V, Van Breusegem F ( 2011 ) ROS signaling: the new wave? Trends Plant Sci 16 : 300 – 309 Google Scholar Crossref Search ADS PubMed WorldCat Mizoi J , Shinozaki K, Yamaguchi-Shinozaki K ( 2012 ) AP2/ERF family transcription factors in plant abiotic stress responses . Biochim Biophys Acta 1819 : 86 – 96 Google Scholar Crossref Search ADS PubMed WorldCat Munné-Bosch S , Queval G, Foyer CH ( 2013 ) The impact of global change factors on redox signaling underpinning stress tolerance . Plant Physiol 161 : 5 – 19 Google Scholar Crossref Search ADS PubMed WorldCat Munns R , Tester M ( 2008 ) Mechanisms of salinity tolerance . Annu Rev Plant Biol 59 : 651 – 681 Google Scholar Crossref Search ADS PubMed WorldCat Nakano T , Suzuki K, Fujimura T, Shinshi H ( 2006 ) Genome-wide analysis of the ERF gene family in Arabidopsis and rice . Plant Physiol 140 : 411 – 432 Google Scholar Crossref Search ADS PubMed WorldCat Pan Y , Seymour GB, Lu C, Hu Z, Chen X, Chen G ( 2012 ) An ethylene response factor (ERF5) promoting adaptation to drought and salt tolerance in tomato . Plant Cell Rep 31 : 349 – 360 Google Scholar Crossref Search ADS PubMed WorldCat Park JM , Park CJ, Lee SB, Ham BK, Shin R, Paek KH ( 2001 ) Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco . Plant Cell 13 : 1035 – 1046 Google Scholar PubMed OpenURL Placeholder Text WorldCat Qiao H , Chang KN, Yazaki J, Ecker JR ( 2009 ) Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis . Genes Dev 23 : 512 – 521 Google Scholar Crossref Search ADS PubMed WorldCat Qiao ZX , Huang B, Liu JY ( 2008 ) Molecular cloning and functional analysis of an ERF gene from cotton (Gossypium hirsutum) . Biochim Biophys Acta 1779 : 122 – 127 Google Scholar Crossref Search ADS PubMed WorldCat Quan R , Hu S, Zhang Z, Zhang H, Zhang Z, Huang R ( 2010 ) Overexpression of an ERF transcription factor TSRF1 improves rice drought tolerance . Plant Biotechnol J 8 : 476 – 488 Google Scholar Crossref Search ADS PubMed WorldCat Riechmann JL , Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, et al. ( 2000 ) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes . Science 290 : 2105 – 2110 Google Scholar Crossref Search ADS PubMed WorldCat Rong W , Qi L, Wang A, Ye X, Du L, Liang H, Xin Z, Zhang Z ( 2014 ) The ERF transcription factor TaERF3 promotes tolerance to salt and drought stresses in wheat . Plant Biotechnol J 12 : 468 – 479 Google Scholar Crossref Search ADS PubMed WorldCat Sakuma Y , Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K ( 2002 ) DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression . Biochem Biophys Res Commun 290 : 998 – 1009 Google Scholar Crossref Search ADS PubMed WorldCat Seo YJ , Park JB, Cho YJ, Jung C, Seo HS, Park SK, Nahm BH, Song JT ( 2010 ) Overexpression of the ethylene-responsive factor gene BrERF4 from Brassica rapa increases tolerance to salt and drought in Arabidopsis plants . Mol Cells 30 : 271 – 277 Google Scholar Crossref Search ADS PubMed WorldCat Severo J , Tiecher A, Pirrello J, Regad F, Latché , Pech JC, Bouzayen M, Rombaldi CV ( 2015 ) UV-C radiation modifies the ripening and accumulation of ethylene response factor (ERF) transcripts in tomato fruits . Postharvest Biol Technol 102 : 9 – 16 Google Scholar Crossref Search ADS WorldCat Sewelam N , Kazan K, Thomas-Hall SR, Kidd BN, Manners JM, Schenk PM ( 2013 ) Ethylene response factor 6 is a regulator of reactive oxygen species signaling in Arabidopsis . PLoS One 8 : e70289 Google Scholar Crossref Search ADS PubMed WorldCat Sharma S , Villamor JG, Verslues PE ( 2011 ) Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential . Plant Physiol 157 : 292 – 304 Google Scholar Crossref Search ADS PubMed WorldCat Sharoni AM , Nuruzzaman M, Satoh K, Shimizu T, Kondoh H, Sasaya T, Choi IR, Omura T, Kikuchi S ( 2011 ) Gene structures, classification and expression models of the AP2/EREBP transcription factor family in rice . Plant Cell Physiol 52 : 344 – 360 Google Scholar Crossref Search ADS PubMed WorldCat Solano R , Stepanova A, Chao Q, Ecker JR ( 1998 ) Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1 . Genes Dev 12 : 3703 – 3714 Google Scholar Crossref Search ADS PubMed WorldCat Song CP , Agarwal M, Ohta M, Guo Y, Halfter U, Wang P, Zhu JK ( 2005 ) Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses . Plant Cell 17 : 2384 – 2396 Google Scholar Crossref Search ADS PubMed WorldCat Song X , Li Y, Hou X ( 2013 ) Genome-wide analysis of the AP2/ERF transcription factor superfamily in Chinese cabbage (Brassica rapa ssp. pekinensis) . BMC Genomics 14 : 573 – 588 Google Scholar Crossref Search ADS PubMed WorldCat Stepanova AN , Alonso JM ( 2009 ) Ethylene signaling and response: where different regulatory modules meet . Curr Opin Plant Biol 12 : 548 – 555 Google Scholar Crossref Search ADS PubMed WorldCat Stevens J , Senaratna T, Sivasithamparam K ( 2006 ) Salicylic acid induces salinity tolerance in tomato (Lycopersico nesculentum cv. Roma): associated changes in gas exchange, water relations and membrane stabilization . Plant Growth Regul 49 : 77 – 83 Google Scholar OpenURL Placeholder Text WorldCat Sun ZM , Zhou ML, Xiao XG, Tang YX, Wu YM ( 2014 a ) Genome-wide analysis of AP2/ERF family genes from Lotus corniculatus shows LcERF054 enhances salt tolerance . Funct Integr Genomics 14 : 453 – 466 Google Scholar Crossref Search ADS PubMed WorldCat Sun ZM , Zhou ML, Xiao XG, Tang YX, Wu YM ( 2014 b ) Overexpression of a Lotus corniculatusAP2/ERF transcription factor gene, LcERF080, enhances tolerance to salt stress in transgenic Arabidopsis . Plant Biotechnol Rep 8 : 315 – 324 Google Scholar Crossref Search ADS WorldCat Tang W , Charles TM, Newton RJ ( 2005 ) Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine (Pinus Virginiana Mill.) confers multiple stress tolerance and enhances organ growth . Plant Mol Biol 59 : 603 – 617 Google Scholar Crossref Search ADS PubMed WorldCat Trujillo LE , Sotolongo M, Menéndez C, Ochogavía ME, Coll Y, Hernández I, Borrás-Hidalgo O, Thomma BP, Vera P, Hernández L ( 2008 ) SodERF3, a novel sugarcane ethylene responsive factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants . Plant Cell Physiol 49 : 512 – 525 Google Scholar Crossref Search ADS PubMed WorldCat Vogel MO , Moore M, König K, Pecher P, Alsharafa K, Lee J, Dietz KJ ( 2014 ) Fast retrograde signaling in response to high light involves metabolite export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF transcription factors in Arabidopsis . Plant Cell 26 : 1151 – 1165 Google Scholar Crossref Search ADS PubMed WorldCat Wang H , Huang Z, Chen Q, Zhang Z, Zhang H, Wu Y, Huang D, Huang R ( 2004 ) Ectopic overexpression of tomato JERF3 in tobacco activates downstream gene expression and enhances salt tolerance . Plant Mol Biol 55 : 183 – 192 Google Scholar Crossref Search ADS PubMed WorldCat Wang L , Qin L, Liu W, Zhang D, Wang Y ( 2014 ) A novel ethylene-responsive factor from Tamarix hispida, ThERF1, is a GCC-box- and DRE-motif binding protein that negatively modulates abiotic stress tolerance in Arabidopsis . Physiol Plant 152 : 84 – 97 Google Scholar Crossref Search ADS PubMed WorldCat Wang M , Liu C, Li S, Zhu D, Zhao Q, Yu J ( 2013 a ) Improved nutritive quality and salt resistance in transgenic maize by simultaneously overexpression of a natural lysine-rich protein gene, SBgLR, and an ERF transcription factor gene, TSRF1 . Int J Mol Sci 14 : 9459 – 9474 Google Scholar Crossref Search ADS PubMed WorldCat Wang P , Du Y, Zhao X, Miao Y, Song CP ( 2013 b ) The MPK6-ERF6-ROS-responsive cis-acting Element7/GCC box complex modulates oxidative gene transcription and the oxidative response in Arabidopsis . Plant Physiol 161 : 1392 – 1408 Google Scholar Crossref Search ADS PubMed WorldCat Wu D , Ji J, Wang G, Guan C, Jin C ( 2014 a ) LchERF, a novel ethylene-responsive transcription factor from Lycium chinense, confers salt tolerance in transgenic tobacco . Plant Cell Rep 33 : 2033 – 2045 Google Scholar Crossref Search ADS PubMed WorldCat Wu L , Chen X, Ren H, Zhang Z, Zhang H, Wang J, Wang XC, Huang R ( 2007 ) ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesis-related gene enhances the tolerance under salinity and cold in tobacco . Planta 226 : 815 – 825 Google Scholar Crossref Search ADS PubMed WorldCat Wu L , Zhang Z, Zhang H, Wang XC, Huang R ( 2008 ) Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt, drought, and freezing . Plant Physiol 148 : 1953 – 1963 Google Scholar Crossref Search ADS PubMed WorldCat Wu X , He J, Chen J, Yang S, Zha D ( 2014 b ) Alleviation of exogenous 6-benzyladenine on two genotypes of eggplant (Solanum melongena Mill.) growth under salt stress . Protoplasma 251 : 169 – 176 Google Scholar Crossref Search ADS PubMed WorldCat Xu ZS , Chen M, Li LC, Ma YZ ( 2011 ) Functions and application of the AP2/ERF transcription factor family in crop improvement . J Integr Plant Biol 53 : 570 – 585 Google Scholar Crossref Search ADS PubMed WorldCat Xu ZS , Xia LQ, Chen M, Cheng XG, Zhang RY, Li LC, Zhao YX, Lu Y, Ni ZY, Liu L, et al. ( 2007 ) Isolation and molecular characterization of the Triticum aestivum L. ethylene-responsive factor 1 (TaERF1) that increases multiple stress tolerance . Plant Mol Biol 65 : 719 – 732 Google Scholar Crossref Search ADS PubMed WorldCat Yang H , Yu C, Yan J, Wang X, Chen F, Zhao Y, Wei W ( 2014 ) Overexpression of the Jatropha curcas JcERF1 gene coding an AP2/ERF-type transcription factor increase tolerance to salt in transgenic tobacco . Biochemistry 79 : 1226 – 1236 Google Scholar PubMed OpenURL Placeholder Text WorldCat Yang XY , Xie JX, Lu XP, Liu YZ, Peng SA ( 2011 ) Isolation of a citrus ethylene-responsive element binding factor gene and its expression in response to abiotic stress, girdling and shading . Sci Hortic 127 : 275 – 281 Google Scholar Crossref Search ADS WorldCat Yang Z , Tian L, Latoszek-Green M, Brown D, Wu K ( 2005 ) Arabidopsis ERF4 is a transcriptional repressor capable of modulating ethylene and abscisic acid responses . Plant Mol Biol 58 : 585 – 596 Google Scholar Crossref Search ADS PubMed WorldCat Yi SY , Kim JH, Joung YH, Lee S, Kim WT, Yu SH, Choi D ( 2004 ) The pepper transcription factor CaPF1 confers pathogen and freezing tolerance in Arabidopsis . Plant Physiol 136 : 2862 – 2874 Google Scholar Crossref Search ADS PubMed WorldCat Youm JW , Jeon JH, Choi D, Yi SY, Joung H, Kim HS ( 2008 ) Ectopic expression of pepper CaPF1 in potato enhances multiple stresses tolerance and delays initiation of in vitro tuberization . Planta 228 : 701 – 708 Google Scholar Crossref Search ADS PubMed WorldCat Zhang G , Chen M, Li L, Xu Z, Chen X, Guo J, Ma Y ( 2009 ) Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco . J Exp Bot 60 : 3781 – 3796 Google Scholar Crossref Search ADS PubMed WorldCat Zhang H , Li W, Chen J, Yang Y, Zhang Z, Zhang H, Wang XC, Huang R ( 2007 ) Transcriptional activator TSRF1 reversely regulates pathogen resistance and osmotic stress tolerance in tobacco . Plant Mol Biol 63 : 63 – 71 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Z , Huang R ( 2010 ) Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis . Plant Mol Biol 73 : 241 – 249 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Z , Li F, Li D, Zhang H, Huang R ( 2010 ) Expression of ethylene response factor JERF1 in rice improves tolerance to drought . Planta 232 : 765 – 774 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Z , Wang J, Zhang R, Huang R ( 2012 ) The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis . Plant J 71 : 273 – 287 Google Scholar Crossref Search ADS PubMed WorldCat Zhao XC , Schaller GE ( 2004 ) Effect of salt and osmotic stress upon expression of the ethylene receptor ETR1 in Arabidopsis thaliana . FEBS Lett 562 : 189 – 192 Google Scholar Crossref Search ADS PubMed WorldCat Zhuang J , Cai B, Peng RH, Zhu B, Jin XF, Xue Y, Gao F, Fu XY, Tian YS, Zhao W, et al. ( 2008 ) Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa . Biochem Biophys Res Commun 371 : 468 – 474 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the Spanish Government (grant no. BFU2012–32057) and the Generalitat de Catalunya (Catalan Institution for Research and Advanced Studies Academia Award to S.M.-B.). * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00677 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Booker, Matthew A.; DeLong, Alison

doi: 10.1104/pp.15.00672pmid: 26134162

Abstract Strictly controlled production of ethylene gas lies upstream of the signaling activities of this crucial regulator throughout the plant life cycle. Although the biosynthetic pathway is enzymatically simple, the regulatory circuits that modulate signal production are fine tuned to allow integration of responses to environmental and intrinsic cues. Recently identified posttranslational mechanisms that control ethylene production converge on one family of biosynthetic enzymes and overlay several independent reversible phosphorylation events and distinct mediators of ubiquitin-dependent protein degradation. Although the core pathway is conserved throughout seed plants, these posttranslational regulatory mechanisms may represent evolutionarily recent innovations. The evolutionary origins of the pathway and its regulators are not yet clear; outside the seed plants, numerous biochemical and phylogenetic questions remain to be addressed. Ethylene gas is a crucial regulator of numerous aspects of plant development and physiology, including germination, seedling growth and morphology, fruit ripening, organ senescence, and stress and defense responses (Abeles et al., 1992). Biosynthetic capacity for ethylene production may be nearly ubiquitous throughout the plant body, but biosynthesis is generally maintained at low levels by regulatory circuitry that confers tight control while allowing rapid and dramatic increases under conditions such as wounding or fruit ripening (Vandenbussche et al., 2012). Ethylene biosynthesis levels change in response to endogenous developmental cues as well as exogenous signals including light, abiotic stress, and pathogens (for review, see De Paepe and Van der Straeten, 2005; Argueso et al., 2007; Lin et al., 2009; Rodrigues et al., 2014), and it is clear that both transcriptional and posttranslational mechanisms provide strict control of biosynthetic enzyme activity levels. Because of ethylene's many roles during vegetative and reproductive development, stress and defense responses, and the postharvest phase, biosynthesis of this simple molecule is of considerable agricultural significance. During vegetative development (and during ripening of most fruits), ethylene perception typically down-regulates the biosynthetic pathway. However, ethylene metabolism is of particular interest in the biology of climacteric fruits, which achieve ripening through a coordinated burst of respiration and ethylene production (for review, see Alexander and Grierson, 2002; Klee and Giovannoni, 2011). Although the enzymology of climacteric ethylene production is unaltered, the regulation is changed profoundly, as the biosynthetic pathway becomes part of a positive feedback loop (autocatalytic). Here, we focus on recent advances in our understanding of the biosynthetic pathway, with particular emphasis on posttranslational modifications that control the stability of biosynthetic enzymes and on phylogenetic analysis of biosynthetic capacity in land plants. HARDWARE OF THE BIOSYNTHETIC PATHWAY The biosynthetic pathway for ethylene in flowering plants was defined over 30 years ago (for review, see Yang and Hoffman, 1984) and includes only two committed enzymatic steps (Fig. 1). The first, conversion of S-adenosyl-Met (SAM) to 1-aminocyclopropane-1-carboxylate (ACC) and methylthioadenosine, is performed by the enzyme ACC synthase (ACS) in an elimination reaction that requires a pyridoxal phosphate (PLP) cofactor. In the second step, ACC is converted to ethylene, CO2, and cyanide by 1-aminocyclopropane-1-carboxylate oxidase (ACO). The toxicity of cyanide production is ameliorated at the cellular level by the detoxifying enzyme β-cyano-Ala synthase (Yip and Yang, 1988). Recent work has shown that the iron center in the ACO active site is also protected from cyanide poisoning; in the presence of oxygen, bicarbonate ion, and ascorbate cofactors, ACO breaks the ACC ring and releases the unstable cyanoformate ion, sequestering cyanide until the reaction products have diffused away (Murphy et al., 2014). ACC synthesis is generally rate limiting for ethylene production during vegetative growth, although ACO activity may be limiting under some conditions (e.g. in fruit and flowers; Yang and Hoffman, 1984). Both ACS and ACO are encoded by multigene families in seed plants. Figure 1. Open in new tabDownload slide Biosynthesis of ethylene and metabolism of ethylene precursors. The core pathway is shown in blue, with additional enzyme activities (italics) and reaction products indicated. Dashed lines indicate additional enzymatic steps. Figure 1. Open in new tabDownload slide Biosynthesis of ethylene and metabolism of ethylene precursors. The core pathway is shown in blue, with additional enzyme activities (italics) and reaction products indicated. Dashed lines indicate additional enzymatic steps. This apparent simplicity overlies a dynamic interplay of metabolic branch pathways that modulate the availability precursors as well as the production of conjugated ACC derivatives (Fig. 1). First, reactions of the Yang cycle salvage the methyl-thio group of methylthioadenosine into Met, stabilizing Met pools for essential primary metabolic functions even in periods of rapid ethylene synthesis (for review, see Sauter et al., 2013). Similarly, SAM is not only the methyl group donor for many methylation reactions, but also the precursor for polyamine and nicotianamine synthesis, so its levels must be maintained by adequate SAM synthetase activity. Free ACC levels also are modulated by conjugation with malonyl-CoA (yielding 1-malonyl-ACC) and with glutathione (yielding γ-glutamyl-ACC), as well as conjugation to jasmonic acid (JA; yielding JA-ACC; for review, see Van de Poel and Van Der Straeten, 2014). This last conjugate suggests a mechanism for coregulating JA and ACC availability (Staswick and Tiryaki, 2004). The relative abundance of ACC conjugates varies significantly among species (Wang et al., 2007), and it is unclear whether ACC is recovered for ethylene synthesis from these derivatives, although exogenous JA-ACC apparently is cleaved to release ACC (Staswick and Tiryaki, 2004). Another route of possible hormone cross talk is suggested by the characterization of the REVERSAL OF SAV3 PHENOTYPE1 (VAS1) aminotransferase activity, a reaction that reduces pools of both auxin and ACC by converting the auxin biosynthetic intermediate indole-3-pyruvate to Trp using an amino acid (preferentially Met) as the amino donor (Zheng et al., 2013). Finally, ACC deaminase, an enzyme produced by numerous bacteria, can affect ACC pools and alter growth characteristics of plants that interact with these bacterial strains (for review, see Update by Gamalero and Glick [2015], in this issue). Down-regulation of either ACO or ACS expression via gene silencing approaches can reduce ethylene production levels dramatically (for review, see Lin et al., 2009). Ethylene production can also be blocked or reduced by compounds that inhibit the enzymatic activities of ACO (e.g. cobalt or α-aminoisobutyric acid) or ACS (e.g. aminoethoxyvinyl-Gly and aminooxyacetic acid, which compete with the required PLP cofactor). New inhibitors that may show improved specificity for ACS were recently identified (Lin et al., 2010); these compounds do not compete with PLP, reducing side effects on other PLP-dependent enzymes. Nonetheless, severe effects on seedling root growth should be expected for all of these inhibitors of ethylene synthesis, because reduced ethylene synthesis appears to be a primary defect in PLP-deficient roots, leading to impairments in auxin synthesis and distribution and in apical meristem function (Boycheva et al., 2015). Intriguingly, genetic analysis revealed an essential embryonic requirement for ACS function (but not ethylene signaling) in Arabidopsis (Arabidopsis thaliana; Tsuchisaka et al., 2009), and additional work has suggested that ACC may act in an ethylene-independent mode to signal loss of cell wall integrity (Xu et al., 2008; Tsang et al., 2011). Biosynthesis of ethylene frequently involves spatial or temporal separation of the two committed reaction steps. A recent study characterized the distribution of ethylene metabolic enzymes and intermediate metabolites in ripening tomato (Solanum lycopersicum) fruits and found the locular gel and central tissues to be sites where intermediates including free and conjugated ACC accumulate, whereas ACC synthesis is concentrated in central tissues and peak ethylene production maps to the fruit’s outer (pericarp) layer (Van de Poel et al., 2014). These data suggest that intermediates may be stored in tissue that has limited biosynthetic capacity. Recent work indicates that some cells actively export ACC (Pesquet and Tuominen, 2011), and that at least one amino acid transporter, the LYS HIS TRANSPORTER1 of Arabidopsis (Shin et al., 2015), promotes uptake of extracellular ACC. Additionally, the importance of long-range transport of ACC for optimal ethylene synthesis in specific tissues has been characterized in a number of contexts, particularly flooding and hypoxia (Bradford and Yang, 1980; for review, see Van de Poel and Van Der Straeten, 2014). KEEPING SYNTHESIS ON A SHORT LEASH Accumulation of both ACS and ACO mRNA is responsive to endogenous and exogenous cues. Several ACS genes are subject to negative feedback loops that reduce mRNA levels, whereas expression of several ACO or ACO-like genes is increased by ethylene treatment (for review, see Lin et al., 2009; Van de Poel and Van Der Straeten, 2014). Other hormones, circadian regulation, and light-signaling pathways show complex interactions with ACS and ACO mRNA accumulation, with auxin having a particularly strong positive effect on expression of ACS mRNAs (Tsuchisaka and Theologis, 2004b; Lin et al., 2009; Rodrigues et al., 2014). Furthermore, rapid effects mediated by posttranslational modification can be reinforced via coordinate transcriptional regulation (Li et al., 2012). ACS activity levels are regulated by proteasome-mediated degradation and by reversible protein phosphorylation (Fig. 2). ACS isozymes belong to three different subclades or types with distinct regulatory features (Figs. 2 and 3; Chae and Kieber, 2005). The three isozyme types were originally defined by the presence or absence of regulatory motifs in the noncatalytic C-terminal domains (Fig. 2, A and B). Type 1 ACS isozymes carry target sites for mitogen-activated protein kinase (MAPK) phosphorylation; this MAPK motif lies immediately downstream from a putative CDPK phosphorylation site (Hernández Sebastià et al., 2004; Liu and Zhang, 2004; Kamiyoshihara et al., 2010). Type 2 isozymes carry only the putative CDPK target motif (Fig. 2, B and C), and type 3 isozymes have a short C-terminal extension that contains neither motif (Chae and Kieber, 2005). All three isozyme types are degraded via proteasome-dependent mechanisms (for review, see Lyzenga and Stone, 2012). Figure 2. Open in new tabDownload slide Determinants of ACS stability regulate ethylene production. A, Model summarizing regulation of ubiquitin-dependent ACS turnover for representative Arabidopsis ACS isozymes. Sequences that regulate ACS stability (cyan) occur both N and C terminally, relative to conserved catalytic domains (green). Specific E3 ubiquitin ligases (orange ovals) acting on type 2 and type 3 isozymes, but not type 1 isozymes, have been identified. Kinase/phosphatase factors that positively regulate ACS stability are shown in blue, whereas negative regulators are shown in red. C-terminal motifs containing three Ser-Pro motifs phosphorylated by MAPKs (M) or a Target of ETO1 (TOE) interaction motif regulate turnover of type 1 and type 2 isozymes, respectively. The PP2C isoform AP2C1 negatively regulates MPK6 (Schweighofer et al., 2007). B, ACS isozyme types from Arabidopsis (ACS) and tomato (SlACS) share posttranslational stability determinants. Type 1 (light-blue), 2 (light-orange), and 3 (light-pink) ACS isoforms interact with regulatory factors that stabilize (S), destabilize (D), or have no effect on turnover of the proteins (U); some physical interaction assay results (+ and −) have not been tested for correlating effects on ACS stability. Predicted target sites for regulators are present (*) or absent (gray shading) but untested in several ACS isozymes. Protein half-lives have been measured for ACS6, SlACS2, ACS7, and ACS5 (Chae et al., 2003; Joo et al., 2008; Kamiyoshihara et al., 2010; Lyzenga et al., 2012; Xiong et al., 2014); for epitope-tagged reporters, the tag is indicated (myc, HA, or Flag). C, A complex overlay of motifs surrounds the TOE sequence in type 2 isozymes. Sequences from the corresponding region of several type 1 and type 2 isozymes are aligned, with the consensus motifs or target residue for calcium-dependent protein kinase (CDPK) phosphorylation (Hernández Sebastià et al., 2004), ETO1 interaction (TOE; Yoshida et al., 2005, 2006), and casein kinase 1.8 (CK1.8) phosphorylation (Tan and Xue, 2014) indicated in red. Isoforms highlighted in orange are known to interact with ETO1, whereas gray highlighting indicates failure to interact (Yoshida et al., 2005; Christians et al., 2009). Nonconservative substitutions at conserved positions in the TOE motif are shown in lowercase in type 2 isozyme sequences. Residues highlighted in black have been shown to undergo regulatory phosphorylation (Tatsuki and Mori, 2001; Tan and Xue, 2014); residues highlighted in yellow are phosphorylatable residues at the position of CK1.8 phosphorylation in ACS5. Sequences matching the CDPK target motif are underlined. D, The N-terminal extensions of ACS7 and other type 3 isozymes show weak sequence conservation. N-terminal sequences of Arabidopsis ACS7 (AtACS7) and type 3 isoforms from several other species were aligned manually; the alignment shown ends at the first residue of the conserved Box1 motif (Yamagami et al., 2003), and the consensus sequence (ConS) shows invariant (underlined), strongly conserved (≥9/11, uppercase), and weakly conserved (≥6/11, lowercase) residues. The first 14 amino acids (bold face) in ACS7 constitute an N-terminal stability determinant (ACS7-N14; Xiong et al., 2014). Sequences in the N-terminal extension (pink shading) are not conserved, and similarity to the ACS7-N14 motif is found only in sequences from cruciferous species. Similar to ACS7, turnover of AtACS4 is regulated by XBAT32, but the N-terminal sequence of ACS4 (blue) lacks any similarity to ACS7-N14 and differs from the ACS7 consensus at several conserved positions (gray letters). Cr, Capsella rubella; Cs, Camelina sativa; Es, Eutrema salsugineum; Ch, Cleome hassleriana; Mn, Morus notabilis; Ns, Nicotiana sylvestris; Sl, Solanum lycopersicon; Mt, Medicago truncatula; Ob, Oryza brachyantha; Sb, Sorghum bicolor; Si, Setaria italica; Bd, Brachypodium distacyon; Os, O. sativa. Figure 2. Open in new tabDownload slide Determinants of ACS stability regulate ethylene production. A, Model summarizing regulation of ubiquitin-dependent ACS turnover for representative Arabidopsis ACS isozymes. Sequences that regulate ACS stability (cyan) occur both N and C terminally, relative to conserved catalytic domains (green). Specific E3 ubiquitin ligases (orange ovals) acting on type 2 and type 3 isozymes, but not type 1 isozymes, have been identified. Kinase/phosphatase factors that positively regulate ACS stability are shown in blue, whereas negative regulators are shown in red. C-terminal motifs containing three Ser-Pro motifs phosphorylated by MAPKs (M) or a Target of ETO1 (TOE) interaction motif regulate turnover of type 1 and type 2 isozymes, respectively. The PP2C isoform AP2C1 negatively regulates MPK6 (Schweighofer et al., 2007). B, ACS isozyme types from Arabidopsis (ACS) and tomato (SlACS) share posttranslational stability determinants. Type 1 (light-blue), 2 (light-orange), and 3 (light-pink) ACS isoforms interact with regulatory factors that stabilize (S), destabilize (D), or have no effect on turnover of the proteins (U); some physical interaction assay results (+ and −) have not been tested for correlating effects on ACS stability. Predicted target sites for regulators are present (*) or absent (gray shading) but untested in several ACS isozymes. Protein half-lives have been measured for ACS6, SlACS2, ACS7, and ACS5 (Chae et al., 2003; Joo et al., 2008; Kamiyoshihara et al., 2010; Lyzenga et al., 2012; Xiong et al., 2014); for epitope-tagged reporters, the tag is indicated (myc, HA, or Flag). C, A complex overlay of motifs surrounds the TOE sequence in type 2 isozymes. Sequences from the corresponding region of several type 1 and type 2 isozymes are aligned, with the consensus motifs or target residue for calcium-dependent protein kinase (CDPK) phosphorylation (Hernández Sebastià et al., 2004), ETO1 interaction (TOE; Yoshida et al., 2005, 2006), and casein kinase 1.8 (CK1.8) phosphorylation (Tan and Xue, 2014) indicated in red. Isoforms highlighted in orange are known to interact with ETO1, whereas gray highlighting indicates failure to interact (Yoshida et al., 2005; Christians et al., 2009). Nonconservative substitutions at conserved positions in the TOE motif are shown in lowercase in type 2 isozyme sequences. Residues highlighted in black have been shown to undergo regulatory phosphorylation (Tatsuki and Mori, 2001; Tan and Xue, 2014); residues highlighted in yellow are phosphorylatable residues at the position of CK1.8 phosphorylation in ACS5. Sequences matching the CDPK target motif are underlined. D, The N-terminal extensions of ACS7 and other type 3 isozymes show weak sequence conservation. N-terminal sequences of Arabidopsis ACS7 (AtACS7) and type 3 isoforms from several other species were aligned manually; the alignment shown ends at the first residue of the conserved Box1 motif (Yamagami et al., 2003), and the consensus sequence (ConS) shows invariant (underlined), strongly conserved (≥9/11, uppercase), and weakly conserved (≥6/11, lowercase) residues. The first 14 amino acids (bold face) in ACS7 constitute an N-terminal stability determinant (ACS7-N14; Xiong et al., 2014). Sequences in the N-terminal extension (pink shading) are not conserved, and similarity to the ACS7-N14 motif is found only in sequences from cruciferous species. Similar to ACS7, turnover of AtACS4 is regulated by XBAT32, but the N-terminal sequence of ACS4 (blue) lacks any similarity to ACS7-N14 and differs from the ACS7 consensus at several conserved positions (gray letters). Cr, Capsella rubella; Cs, Camelina sativa; Es, Eutrema salsugineum; Ch, Cleome hassleriana; Mn, Morus notabilis; Ns, Nicotiana sylvestris; Sl, Solanum lycopersicon; Mt, Medicago truncatula; Ob, Oryza brachyantha; Sb, Sorghum bicolor; Si, Setaria italica; Bd, Brachypodium distacyon; Os, O. sativa. Figure 3. Open in new tabDownload slide Dedicated ACS activity is specific to the seed plant lineage. ACS and ACS-like isoforms were identified via BLAST search from Arabidopsis, tomato, rice, Amborella trichopoda, conifers, the lycophyte Selaginella moellendorffii, the moss Physcomitrella patens., humans, the cnidarian Nematostella vectensis, and the green algae Ostreococcus lucimarinus Chlamydomonas reinhardtii, and Volvox carteri. The amino acid sequences of the catalytic domain (residues 9–438 in Arabidopsis ACS2 or 9–436 in SlACS2) were aligned using muscle, and a phylogenetic tree was constructed using the Bayesian software package MrBayes (Ronquist et al., 2012). Clade credibility values are indicated at nodes. The tree was rooted using Ala aminotransferase sequences from plants and animals. Highlighted clades identified include Ala aminotransferases (purple), eukaryotic ACS-like deaminases (yellow), ACS-like aminotransferases (green), type 1 (blue), type 2 (orange), and type 3 (pink) ACS. Accession numbers for sequences used are provided in Supplemental Table S1. Figure 3. Open in new tabDownload slide Dedicated ACS activity is specific to the seed plant lineage. ACS and ACS-like isoforms were identified via BLAST search from Arabidopsis, tomato, rice, Amborella trichopoda, conifers, the lycophyte Selaginella moellendorffii, the moss Physcomitrella patens., humans, the cnidarian Nematostella vectensis, and the green algae Ostreococcus lucimarinus Chlamydomonas reinhardtii, and Volvox carteri. The amino acid sequences of the catalytic domain (residues 9–438 in Arabidopsis ACS2 or 9–436 in SlACS2) were aligned using muscle, and a phylogenetic tree was constructed using the Bayesian software package MrBayes (Ronquist et al., 2012). Clade credibility values are indicated at nodes. The tree was rooted using Ala aminotransferase sequences from plants and animals. Highlighted clades identified include Ala aminotransferases (purple), eukaryotic ACS-like deaminases (yellow), ACS-like aminotransferases (green), type 1 (blue), type 2 (orange), and type 3 (pink) ACS. Accession numbers for sequences used are provided in Supplemental Table S1. As discussed below, recent work has bolstered our understanding of ACS turnover. Regulatory circuits that govern ACS stability often affect multiple isozymes of a common type (e.g. Joo et al., 2008; Christians et al., 2009), suggesting that members of these subclades diversified within a common regulatory context. However, several newly identified regulatory factors target ACSs of different types (e.g. Lyzenga et al., 2012; Yoon and Kieber, 2013a), highlighting the layering and integration of mechanisms that control ACS turnover. Overall, the stability control mechanisms clearly link ethylene production to exogenous (e.g. light, wounding, pathogen infection, and abiotic stress) or endogenous signals (e.g. hormone levels, developmental transitions). Light affects ACS stability both positively and negatively; for instance, turnover of ACS5 is decreased (Yoon and Kieber, 2013a), whereas ACS7 turnover is increased (Xiong et al., 2014) by light. Exogenous cytokinin and brassinosteroid treatments increase ethylene production via posttranslational stabilization (Woeste et al., 1999; Hansen et al., 2009), and pathogen infection increases ACS abundance at both transcriptional and posttranslational levels (Han et al., 2010; Li et al., 2012). One challenging question for future analysis is how the homo- and heterodimerization of ACS isozymes affect the interplay of these turnover mechanisms. Analysis of ACO gene regulation has been hampered by an incomplete definition of the gene family members. ACO belongs to a large family of dioxygenases with high sequence similarity (Zhang et al., 2004), and numerous ACO-like family members have been defined by their amino acid sequence similarity with a group of tomato gene products known to convert ACC to ethylene (for review, see Lin et al., 2009). However, only a subset of ACO-like gene products group closely with bona fide ACOs in phylogenetic analyses (see below), and only a few of these have been assayed for their enzymatic functions (Hamilton et al., 1991; Dong et al., 1992; Gómez-Lim et al., 1993; Bidonde et al., 1998). Interestingly, the ACO-like protein E8 may participate in a negative feedback loop that limits ethylene production in fruit, but this antagonistic relationship does not appear to involve direct interaction between ACO and E8 proteins (Van de Poel et al., 2014). Phosphorylation and Proteasome-Mediated Degradation: Leitmotifs of ACS Regulation Phosphorylation of the Arabidopsis type 1 isozymes ACS2 and ACS6 by stress-responsive MAPKs (MAPK3 [MPK3] and MPK6) results in increased ethylene synthesis through protein stabilization (Liu and Zhang, 2004; Joo et al., 2008; Han et al., 2010). MAPK activity on ACS6 is counteracted by protein phosphatase2A (PP2A)-mediated dephosphorylation (Skottke et al., 2011); moreover, okadaic acid and calyculin (which inhibit both PP2A and PP1) have been shown to cause the accumulation of stabilized MAPK- and CDPK-phosphorylated SlACS2 in tomato fruit (Kamiyoshihara et al., 2010). Unphosphorylated type 1 isozymes are rapidly turned over via a 26S proteasome-dependent pathway, and the noncatalytic C-terminal region containing the CDPK and MAPK phosphorylation motifs is sufficient to confer instability on reporter protein fusions (Joo et al., 2008). In rice (Oryza sativa), salt stress activates an analogous MAPK cascade downstream of the Salt Intolerance1 receptor-like kinase, promoting ethylene synthesis (Li et al., 2014), although target ACS isozymes were not identified in that work. CDPK-mediated phosphorylation of a tomato type 1 ACS (SlACS2) stabilizes the enzyme and leads to increased ACS activity in wounded tomato tissue (Tatsuki and Mori, 2001; Kamiyoshihara et al., 2010). However, the corresponding site in Arabidopsis type 1 isozymes (ACS1, ACS2, and ACS6) conforms poorly to the consensus site utilized by several CDPKs (Fig. 2C; Hernández Sebastià et al., 2004) and has not been shown to be phosphorylated in vivo or in vitro. Type 2 ACS isozymes are recruited for ubiquitin-dependent proteolysis by ETHYLENE-OVER-PRODUCING1 (ETO1) and the ETHYLENE-OVER-PRODUCING1-LIKE (EOL1 and EOL2) proteins, which are subunits of Cul3a/3b-based E3 ubiquitin ligases (Chae et al., 2003; Wang et al., 2004; Gingerich et al., 2005; Yoshida et al., 2005; Christians et al., 2009). Ethylene overproduction in the Arabidopsis eto1 mutant is caused by decreased proteolytic turnover of type 2 ACS isozymes. ETO/EOL-mediated regulation requires recognition of a C-terminal TOE motif that is conserved in numerous species; in Arabidopsis, this motif is altered by the dominant acs5eto2 and acs9eto3 mutations (Fig. 2C). Each of these mutations stabilizes the ACSeto protein product by preventing its interaction with ETO1 (Chae et al., 2003; Wang et al., 2004; Yoshida et al., 2006; Christians et al., 2009). Ethylene overproduction due to loss of ETO/EOL-mediated regulation is dramatic in etiolated seedlings but is also observed in light-grown seedlings and adult plants (Christians et al., 2009; Yoon and Kieber, 2013a). Phosphorylation also regulates type 2 ACS turnover. A CK1.8 isoform was recently shown to phosphorylate a Ser residue in the TOE motif of ACS5 and thereby promote ETO1-mediated ACS degradation (Fig. 2, A–C; Tan and Xue, 2014), providing an example of a destabilizing phosphorylation signal. PP2A-mediated dephosphorylation stabilizes ACS5 (Skottke et al., 2011), but it has not yet been determined whether ACS5 is the direct target for phosphatase action, or whether this effect is ETO/EOL or CK1.8 dependent. The putative CDPK target motif of type 2 ACS proteins overlaps with the TOE sequence (Fig. 2C). This motif can be phosphorylated in vitro (Hernández Sebastià et al., 2004), but the role of phosphorylation at this site is uncertain. Substitution of either a phosphomimicking or a nonphosphorylatable residue for the putative target Ser does not affect the interaction of ACS5 with ETO/EOL proteins in a yeast two-hybrid assay (Christians et al., 2009). Regulation of ACS7 levels is of particular interest because it is the sole representative of the type 3 class in Arabidopsis, and may be capable of forming active heterodimers with both type 1 (ACS6) and type 2 (ACS4, 8, and 9) isozymes in vivo (Tsuchisaka and Theologis, 2004a). Type 3 isozymes are encoded by small multiisoform gene families in some other species (e.g. rice; Iwai et al., 2006), and Arabidopsis ACS7 appears to make a significant contribution to baseline ethylene production in light-grown seedlings (Tsuchisaka et al., 2009; Li et al., 2012) as well as in etiolated seedlings (Dong et al., 2011), indicating that regulation of type 3 function may be equally important to that of type 1 and 2 isozymes. Analysis of ACS7 turnover raises the interesting and experimentally challenging question of whether ACS proteins may carry both N- and C-terminal destabilizing sequences. To allow specific detection of individual ACS isozymes, in vivo turnover experiments have usually relied on ACS alleles in which an epitope tag is fused at one end of the protein (most frequently the N terminus), potentially masking some determinants of protein stability. In the absence of isozyme-specific anti-ACS antibodies, these constructs have been invaluable tools; however, the weak phenotypes of acs single mutants have typically precluded rigorous complementation testing to confirm that the tagged alleles show native function. ACS7 lacks the C-terminal regulatory motifs recognized in type 1 and type 2 isozymes but is destabilized by the ubiquitin ligase XB3 ORTHOLOG2 IN ARABIDOPSIS (XBAT32; Fig. 2A; Lyzenga et al., 2012). Similar to the recognition of TOE motifs by ETO/EOL proteins, XBAT recognition of ACS7 can be reconstituted in a yeast two-hybrid assay and in vitro, and has been assayed using N-terminally tagged ACS7 constructs (Prasad et al., 2010; Lyzenga et al., 2012). Interestingly, though, turnover of a C-terminally epitope-tagged ACS7 requires a motif contained within the 14 N-terminal amino acids (ACS7-N14; Xiong et al., 2014). Neither the XBAT32 dependence of ACS7-N14-mediated turnover nor the ACS7 dependence of ethylene overproduction in xbat32 mutants has been directly tested, thus it is unclear whether these constitute a single regulatory mechanism or two different turnover systems for ACS7. The N-terminal sequence of type 3 isozymes is very poorly conserved (Xiong et al., 2014), and sequences resembling the ACS7-N14 motif are found only in type 3 isozymes from species within the Brassicaceae (Fig. 2D). Newly Identified Posttranslational Mechanisms Regulate Multiple ACS Types A network of regulatory inputs has been superimposed on the ACS subclades to provide very finely modulated control of ethylene synthesis. Although the type 1, 2, and 3 designations accurately represent ACS subclades (Fig. 3), several recently elucidated regulatory factors target ACSs of different types (Fig. 2B), suggesting rapid evolution of regulatory mechanisms. For instance, the putative target site for destabilizing phosphorylation by CK1.8 (T463) is present in type 2 isozymes ACS5 and ACS9 plus several type 1 isozymes, but absent from type 2 isozymes ACS4 and ACS8 (Fig. 2B). Similarly, interaction with 14-3-3 proteins stabilizes at least one isozyme of each type (ACS2, ACS5, and ACS7; Yoon and Kieber, 2013a, 2013b). An abundant family of small dimeric proteins that typically bind phospho-Ser, 14-3-3 proteins interact with a wide array of client phosphoproteins; the effects of 14-3-3 binding are variable (Denison et al., 2011). The mechanism for 14-3-3 stabilization of ACS2 and ACS7 is not known. In the case of ACS5, 14-3-3 interaction antagonizes ETO/EOL-mediated turnover both by stabilizing the ACS protein and by destabilizing ETO/EOL proteins. Interaction of 14-3-3 with ACS is not dependent on the TOE sequence of ACS5 (Yoon and Kieber, 2013a). Because 14-3-3 binding is generally phosphorylation dependent, the discovery of 14-3-3-mediated stabilization suggests that these isozymes may undergo stabilizing phosphorylation in the catalytic domain, the only domain that is common to all three types. Interestingly, cell-free degradation assays show that XBAT32 not only regulates ACS7 (type 3) stability, but also promotes turnover of ACS4 (Prasad et al., 2010; Lyzenga et al., 2012), a type 2 isozyme that is a predicted ETO1 target (Chae et al., 2003; Wang et al., 2004; Gingerich et al., 2005; Yoshida et al., 2005; Christians et al., 2009). In addition to its C-terminal TOE motif (Yoshida et al., 2006; Christians et al., 2009), ACS4 also carries a destabilizing N-terminal sequence (Schlögelhofer and Bachmair, 2002). This N-terminal stability determinant has not been mapped precisely; the 94-amino acid region shown to destabilize a reporter protein includes two of the conserved structural elements of the catalytic domain (Yamagami et al., 2003) but lacks any similarity to the ACS7-N14 motif (Fig. 2D). Thus, both ACS4 and ACS7 carry N-terminal stability determinants, and both are regulated by XBAT32; the signals recognized by XBAT32 remain to be identified. EVOLUTION OF THE BIOSYNTHETIC PATHWAY The three ACS isozyme types, with their characteristic C-terminal motifs, are represented in monocots as well as dicots (Fig. 3; Zhang et al., 2012), but these motifs are not conserved outside the flowering plants. Nonetheless, conifer ACS isozymes are clearly related to isoforms in the type 1, type 2, and type 3 subclades (Fig. 3; Ralph et al., 2007; Lyzenga et al., 2012; Zhang et al., 2012). This is consistent with the diversification of ACS isozymes into three types after the split of seed plants from other land plants (Fig. 3) and the innovation of posttranslational regulatory switches controlling ACS stability very early in flowering plant evolution. Conifers express ACO isoforms that group phylogenetically with the angiosperm ACOs known to synthesize ethylene as well as additional ACO-like isoforms, indicating that diversification of ACO isoforms occurred prior to the split of flowering plants and gymnosperms (Hudgins et al., 2006; Ruduś et al., 2013). The conservation of ACS and ACO isozymes in conifers suggests that the flowering plant ethylene biosynthesis pathway is conserved throughout seed plants, although the posttranslational regulation of specific isoforms likely differs between gymnosperms and angiosperms, because the gymnosperm ACS isoforms exhibit none of the known regulatory sequence motifs. Because of the conservation of ethylene responses, the evolutionary origin of the biosynthetic pathway is of considerable interest, and the ACS and ACO gene families are clearly present in seed plants. However, physiological studies of ethylene biosynthesis outside the seed plants do not provide a consistent mechanistic picture of a conserved biosynthesis pathway. In flowering plants and in all four gymnosperm groups (Osborne et al., 1996), feeding the biosynthetic intermediate ACC to whole plants or live tissue results in the production of ethylene, consistent with an ACO activity conserved throughout seed plants. Similarly, ACC treatment increases ethylene production in the charophyte Spirogyra pratensis, the chlorophytes Haematococcus pluvialis and Ulva intestinalis, and the red alga Pterocladiella capillacea, consistent with conservation throughout the plant kingdom (Maillard et al., 1993; Plettner et al., 2005; Garcia-Jimenez et al., 2013; Ju et al., 2015). However, treatment of liverworts, moss, and ferns with exogenous ACC does not yield increased ethylene production, despite detectable uptake and metabolism of labeled ACC (Osborne et al., 1996). These results are perplexing since they suggest that seed plants and red and green algae convert ACC to ethylene, whereas land plants other than seed plants use an alternate pathway. Additional work will be required to clarify the biosynthetic pathway in nonseed plants. ACS and ACS-Like Gene Families Aminotransferase activity likely represents the ancestral function of the ACS-like enzyme family. Phylogenetic analyses indicate a complex evolutionary history for the subclade of the PLP-dependent aminotransferase gene family that contains ACS, and suggest that the role of ACS in ethylene biosynthesis is an evolutionary novelty that arose specifically in plants. The ACS-like subfamily of aminotransferases includes both true ACS isozymes and a group of related proteins that lack ACS enzymatic activity (Fig. 3). Members of the ACS-like protein family are conserved in vertebrates, and biochemical characterization of a mammalian isoform shows that it does not convert SAM to ACC at physiologically relevant rates, but does deaminate the ACS inhibitor 1-aminovinyl-Gly (Koch et al., 2001). Thus, animal ACS-like proteins may catalyze a similar but distinct deaminase or aminotransferase reaction (putative deaminase clade; Fig. 3). Similarly, the Arabidopsis ACS-like proteins ACS10 and ACS12 are Asp, Phe, and Tyr aminotransferases, not ACSs (Yamagami et al., 2003). ACS10 and ACS12 are members of an ACS-like clade specific to seed plants (putative aminotransferase clade; Fig. 3), whereas type 1, 2, and 3 ACSs form a separate clade shared throughout seed plants. These relationships suggest that true ACS enzymes and a separate group of ACS-like amino transferases diverged after the split of seed plants from mosses (Fig. 3; Ralph et al., 2007; Zhang et al., 2012). Furthermore, the ethylene-related clade is under positive selection (Zhang et al., 2012), consistent with diversification of an ancestral ACS-like aminotransferase clade and functional specialization of true ACS isoforms after the divergence of seed plants from other plants. Under this model, the ACS-like sequences recently identified in green algae, red algae, glaucophytes, cyanobacteria (Ju et al., 2015), mosses (Rensing et al., 2008), and lycophytes (Banks, 2009) might catalyze aminotransferase reactions rather than synthesize ACC. The recently identified putative ACS from S. pratensis (SpACS1 in Fig. 3; Ju et al., 2015) groups outside of the clade that contains both true ACS enzymes and ACS-like aminotransferases, leaving its function ambiguous. However, a simple amino acid sequence comparison (using BLAST) reveals that the S. pratensis ACS-like protein sequence (Ju et al., 2015) is more similar to that of ACS8 and other ACC-producing isoforms than it is to ACS-like amino acid aminotransferase proteins (e.g. ACS10 and ACS12). This suggests that ACS/ACS-like isoforms outside the seed plants may be dual-function ACC-producing aminotransferases, with subsequent subfunctionalization of ACC synthase and amino acid aminotransferase activities occurring in seed plants. Further characterization of nonseed plant ACS isoforms is needed to clearly define the enzymatic activities and metabolic roles of these proteins. ACO Genes: Disambiguation Desired The ACO gene family is less well studied, and the available data on evolution of ACO function are not clear cut. ACO isozymes are members of a large Fe(II)-requiring dioxygenase/oxidase superfamily encompassing roughly 100 Arabidopsis proteins (Zhang et al., 2004), most of which do not exhibit known ACC oxidase activity. Only a few proteins (e.g. tomato ACO1–3 [Hamilton et al., 1991; Bidonde et al., 1998], MdACO1 from apple [Malus domestica; Dong et al., 1992], and Arabidopsis ACO4 [Gómez-Lim et al., 1993]) are known to exhibit ACC oxidase activity in vitro. Arabidopsis ACO2 and ACO3 and tomato ACO4 group with these active ACOs in phylogenetic analyses along with some conifer enzymes, whereas tomato ACO5 and Arabidopsis ACO1 and ACO5 cluster together in a sister clade that is separate from the group of enzymatically defined ACO isoforms (Hudgins et al., 2006; Ruduś et al., 2013). Furthermore, a set of Arabidopsis dioxygenase gene products with a more distant relationship to the known ACOs (Ruduś et al., 2013) have been designated as ACO6 to ACO13 on the basis of sequence similarity alone (Clouse and Carraro, 2014). Genes encoding isoforms of the dioxygenase superfamily have been identified in mosses, lycophytes, and green algae, but it is currently unknown whether these enzymes catalyze an ACO reaction (Clouse and Carraro, 2014). This uncertainty about which flowering plant dioxygenase genes encode bona fide ACOs has hindered analysis of ACO-like enzymes from other plants. PROSPECTS AND QUESTIONS IN ETHYLENE BIOSYNTHESIS The preceding summary clearly points to a number of questions for future research on the ethylene biosynthetic pathway and its regulation. Are the ACC conjugates that have been identified in several systems simple sinks that reduce the ACC pools available for ethylene production, or are they nodes of hormonal cross talk and/or physiologically significant reservoirs for ACC that can be mobilized by conjugate cleavage? What are the constituents of the circuits that provide feedback regulation on ACS and ACO mRNA levels? How does ACS dimerization affect turnover? What are the mechanisms that regulate and integrate the protein kinase and phosphatase activities that modulate turnover? How widely conserved are the posttranslational regulatory mechanisms that control ACS stability? Can the conclusions derived from analysis in Arabidopsis and tomato be extrapolated to more distantly related, agriculturally important plants such as banana (Musa spp.), kiwi (Actinidia deliciosa), and avocado (Persea americana)? Did ancestral ACS enzymes contain noncatalytic regulatory domains? The evolutionary history of the biosynthetic pathway presents additional puzzles. Although components of the ethylene response pathway are distinctive and provide a clear phylogenetic signature, ACS and ACO are members of multiisoform gene families that catalyze distinct but chemically similar enzymatic reactions; gene family members are difficult to distinguish from each other on the basis of simple sequence similarity. Thus, the pathway for ethylene biosynthesis outside of the seed plants is somewhat unclear, because ACS-like enzymes in moss and algae may convert SAM to ACC, or they may perform a different but chemically similar reaction. The functions of ACO-like dioxygenases are even harder to predict in the absence of biochemical data. The phylogenetic position of true ACOs within the dioxygenase family will provide interesting insight into the evolution of this function and thus the configuration of the pathway in evolutionary history. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers listed in Supplemental Table S1. Supplemental Data The following supplemental materials are available. Supplemental Table S1. Names and accession numbers of protein sequences represented in Figure 3 phylogeny. ACKNOWLEDGMENTS We thank Caren Chang, Alexander Leydon, Gloria Muday, and Gyeong Mee Yoon for critical comments on the manuscript. Glossary SAM S-adenosyl-Met ACC 1-aminocyclopropane-1-carboxylate PLP pyridoxal phosphate CDPK calcium-dependent protein kinase JA jasmonic acid LITERATURE CITED Abeles FB , Morgan PW, Saltveit MEJ ( 1992 ) Ethylene in Plant Biology , Ed 2. Academic Press , San Diego, CA Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Alexander L , Grierson D ( 2002 ) Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening . J Exp Bot 53 : 2039 – 2055 Google Scholar Crossref Search ADS PubMed WorldCat Argueso C , Hansen M, Kieber JJ ( 2007 ) Regulation of ethylene biosynthesis . J Plant Growth Regul 26 : 92 – 105 Google Scholar Crossref Search ADS WorldCat Banks JA ( 2009 ) Selaginella and 400 million years of separation . Annu Rev Plant Biol 60 : 223 – 238 Google Scholar Crossref Search ADS PubMed WorldCat Bidonde S , Ferrer MA, Zegzouti H, Ramassamy S, Latché A, Pech JC, Hamilton AJ, Grierson D, Bouzayen M ( 1998 ) Expression and characterization of three tomato 1-aminocyclopropane-1-carboxylate oxidase cDNAs in yeast . Eur J Biochem 253 : 20 – 26 Google Scholar Crossref Search ADS PubMed WorldCat Boycheva S , Dominguez A, Rolcik J, Boller T, Fitzpatrick TB ( 2015 ) Consequences of a deficit in vitamin B6 biosynthesis de novo for hormone homeostasis and root development in Arabidopsis . Plant Physiol 167 : 102 – 117 Google Scholar Crossref Search ADS PubMed WorldCat Bradford KJ , Yang SF ( 1980 ) Xylem transport of 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor, in waterlogged tomato plants . Plant Physiol 65 : 322 – 326 Google Scholar Crossref Search ADS PubMed WorldCat Chae HS , Faure F, Kieber JJ ( 2003 ) The eto1, eto2, and eto3 mutations and cytokinin treatment increase ethylene biosynthesis in Arabidopsis by increasing the stability of ACS protein . Plant Cell 15 : 545 – 559 Google Scholar Crossref Search ADS PubMed WorldCat Chae HS , Kieber JJ ( 2005 ) Eto Brute? Role of ACS turnover in regulating ethylene biosynthesis . Trends Plant Sci 10 : 291 – 296 Google Scholar Crossref Search ADS PubMed WorldCat Christians MJ , Gingerich DJ, Hansen M, Binder BM, Kieber JJ, Vierstra RD ( 2009 ) The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively to regulate ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels . Plant J 57 : 332 – 345 Google Scholar Crossref Search ADS PubMed WorldCat Clouse RM , Carraro N ( 2014 ) A novel phylogeny and morphological reconstruction of the PIN genes and first phylogeny of the ACC-oxidases (ACOs) . Front Plant Sci 5 : 296 Google Scholar Crossref Search ADS PubMed WorldCat Denison FC , Paul A-L, Zupanska AK, Ferl RJ ( 2011 ) 14-3-3 proteins in plant physiology . Semin Cell Dev Biol 22 : 720 – 727 Google Scholar Crossref Search ADS PubMed WorldCat De Paepe A , Van der Straeten D ( 2005 ) Ethylene biosynthesis and signaling: an overview . Vitam Horm 72 : 399 – 430 Google Scholar Crossref Search ADS PubMed WorldCat Dong H , Zhen Z, Peng J, Chang L, Gong Q, Wang NN ( 2011 ) Loss of ACS7 confers abiotic stress tolerance by modulating ABA sensitivity and accumulation in Arabidopsis . J Exp Bot 62 : 4875 – 4887 Google Scholar Crossref Search ADS PubMed WorldCat Dong JG , Fernández-Maculet JC, Yang SF ( 1992 ) Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit . Proc Natl Acad Sci USA 89 : 9789 – 9793 Google Scholar Crossref Search ADS PubMed WorldCat Gamalero E , Glick BR ( 2015 ) Bacterial modulation of plant ethylene levels . Plant Physiol 169 : 13 – 22 Google Scholar Crossref Search ADS PubMed WorldCat Garcia-Jimenez P , Brito-Romano O, Robaina RR ( 2013 ) Production of volatiles by the red seaweed Gelidium arbuscula (Rhodophyta): emission of ethylene and dimethyl sulfide . J Phycol 49 : 661 – 669 Google Scholar Crossref Search ADS PubMed WorldCat Gingerich DJ , Gagne JM, Salter DW, Hellmann H, Estelle M, Ma L, Vierstra RD ( 2005 ) Cullins 3a and 3b assemble with members of the broad complex/tramtrack/bric-a-brac (BTB) protein family to form essential ubiquitin-protein ligases (E3s) in Arabidopsis . J Biol Chem 280 : 18810 – 18821 Google Scholar Crossref Search ADS PubMed WorldCat Gómez-Lim MA , Valdés-López V, Cruz-Hernandez A, Saucedo-Arias LJ ( 1993 ) Isolation and characterization of a gene involved in ethylene biosynthesis from Arabidopsis thaliana . Gene 134 : 217 – 221 Google Scholar Crossref Search ADS PubMed WorldCat Hamilton AJ , Bouzayen M, Grierson D ( 1991 ) Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast . Proc Natl Acad Sci USA 88 : 7434 – 7437 Google Scholar Crossref Search ADS PubMed WorldCat Han L , Li GJ, Yang KY, Mao G, Wang R, Liu Y, Zhang S ( 2010 ) Mitogen-activated protein kinase 3 and 6 regulate Botrytis cinerea-induced ethylene production in Arabidopsis . Plant J 64 : 114 – 127 Google Scholar PubMed OpenURL Placeholder Text WorldCat Hansen M , Chae HS, Kieber JJ ( 2009 ) Regulation of ACS protein stability by cytokinin and brassinosteroid . Plant J 57 : 606 – 614 Google Scholar Crossref Search ADS PubMed WorldCat Hernández Sebastià C , Hardin SC, Clouse SD, Kieber JJ, Huber SC ( 2004 ) Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate . Arch Biochem Biophys 428 : 81 – 91 Google Scholar Crossref Search ADS PubMed WorldCat Hudgins JW , Ralph SG, Franceschi VR, Bohlmann J ( 2006 ) Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells . Planta 224 : 865 – 877 Google Scholar Crossref Search ADS PubMed WorldCat Iwai T , Miyasaka A, Seo S, Ohashi Y ( 2006 ) Contribution of ethylene biosynthesis for resistance to blast fungus infection in young rice plants . Plant Physiol 142 : 1202 – 1215 Google Scholar Crossref Search ADS PubMed WorldCat Joo S , Liu Y, Lueth A, Zhang S ( 2008 ) MAPK phosphorylation-induced stabilization of ACS6 protein is mediated by the non-catalytic C-terminal domain, which also contains the cis-determinant for rapid degradation by the 26S proteasome pathway . Plant J 54 : 129 – 140 Google Scholar Crossref Search ADS PubMed WorldCat Ju C , Van de Poel B, Cooper ED, Thierer JH, Gibbons TR, Delwiche CF, Chang C ( 2015 ) Conservation of ethylene as a plant hormone over . Nature Plants 1 : 14004 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kamiyoshihara Y , Iwata M, Fukaya T, Tatsuki M, Mori H ( 2010 ) Turnover of LeACS2, a wound-inducible 1-aminocyclopropane-1-carboxylic acid synthase in tomato, is regulated by phosphorylation/dephosphorylation . Plant J 64 : 140 – 150 Google Scholar PubMed OpenURL Placeholder Text WorldCat Klee HJ , Giovannoni JJ ( 2011 ) Genetics and control of tomato fruit ripening and quality attributes . Annu Rev Genet 45 : 41 – 59 Google Scholar Crossref Search ADS PubMed WorldCat Koch KA , Capitani G, Gruetter MG, Kirsch JF ( 2001 ) The human cDNA for a homologue of the plant enzyme 1-aminocyclopropane-1-carboxylate synthase encodes a protein lacking that activity . Gene 272 : 75 – 84 Google Scholar Crossref Search ADS PubMed WorldCat Li CH , Wang G, Zhao JL, Zhang LQ, Ai LF, Han YF, Sun DY, Zhang SW, Sun Y ( 2014 ) The receptor-like kinase SIT1 mediates salt sensitivity by activating MAPK3/6 and regulating ethylene homeostasis in rice . Plant Cell 26 : 2538 – 2553 Google Scholar Crossref Search ADS PubMed WorldCat Li G , Meng X, Wang R, Mao G, Han L, Liu Y, Zhang S ( 2012 ) Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis . PLoS Genet 8 : e1002767 Google Scholar Crossref Search ADS PubMed WorldCat Lin LC , Hsu JH, Wang LC ( 2010 ) Identification of novel inhibitors of 1-aminocyclopropane-1-carboxylic acid synthase by chemical screening in Arabidopsis thaliana . J Biol Chem 285 : 33445 – 33456 Google Scholar Crossref Search ADS PubMed WorldCat Lin Z , Zhong S, Grierson D ( 2009 ) Recent advances in ethylene research . J Exp Bot 60 : 3311 – 3336 Google Scholar Crossref Search ADS PubMed WorldCat Liu Y , Zhang S ( 2004 ) Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis . Plant Cell 16 : 3386 – 3399 Google Scholar Crossref Search ADS PubMed WorldCat Lyzenga WJ , Booth JK, Stone SL ( 2012 ) The Arabidopsis RING-type E3 ligase XBAT32 mediates the proteasomal degradation of the ethylene biosynthetic enzyme, 1-aminocyclopropane-1-carboxylate synthase 7 . Plant J 71 : 23 – 34 Google Scholar Crossref Search ADS PubMed WorldCat Lyzenga WJ , Stone SL ( 2012 ) Regulation of ethylene biosynthesis through protein degradation . Plant Signal Behav 7 : 1438 – 1442 Google Scholar Crossref Search ADS PubMed WorldCat Maillard P , Thepenier C, Gudin C ( 1993 ) Determination of an ethylene biosynthesis pathway in the unicellular green alga, Haematococcus pluvialis: relationship between growth and ethylene production . J Appl Phycol 5 : 93 – 98 Google Scholar Crossref Search ADS WorldCat Murphy LJ , Robertson KN, Harroun SG, Brosseau CL, Werner-Zwanziger U, Moilanen J, Tuononen HM, Clyburne JA ( 2014 ) A simple complex on the verge of breakdown: isolation of the elusive cyanoformate ion . Science 344 : 75 – 78 Google Scholar Crossref Search ADS PubMed WorldCat Osborne DJ , Walters J, Milborrow BV, Norville A, Stange LMC ( 1996 ) Special publication evidence for a non-ACC ethylene biosynthesis pathway in lower plants . Phytochemistry 42 : 51 – 60 Google Scholar Crossref Search ADS WorldCat Pesquet E , Tuominen H ( 2011 ) Ethylene stimulates tracheary element differentiation in Zinnia elegans cell cultures . New Phytol 190 : 138 – 149 Google Scholar Crossref Search ADS PubMed WorldCat Plettner I , Steinke M, Malin G ( 2005 ) Ethene (ethylene) production in the marine macroalga Ulva (Enteromorpha) intestinalis L. (Chlorophyta, Ulvophyceae): effect of light-stress and co-production with dimethyl sulphide . Plant Cell Environ 28 : 1136 – 1145 Google Scholar Crossref Search ADS WorldCat Prasad ME , Schofield A, Lyzenga W, Liu H, Stone SL ( 2010 ) Arabidopsis RING E3 ligase XBAT32 regulates lateral root production through its role in ethylene biosynthesis . Plant Physiol 153 : 1587 – 1596 Google Scholar Crossref Search ADS PubMed WorldCat Ralph SG , Hudgins JW, Jancsik S, Franceschi VR, Bohlmann J ( 2007 ) Aminocyclopropane carboxylic acid synthase is a regulated step in ethylene-dependent induced conifer defense. Full-length cDNA cloning of a multigene family, differential constitutive, and wound- and insect-induced expression, and cellular and subcellular localization in spruce and Douglas fir . Plant Physiol 143 : 410 – 424 Google Scholar Crossref Search ADS PubMed WorldCat Rensing SA , Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H, Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y, et al. ( 2008 ) The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants . Science 319 : 64 – 69 Google Scholar Crossref Search ADS PubMed WorldCat Rodrigues MA , Bianchetti RE, Freschi L ( 2014 ) Shedding light on ethylene metabolism in higher plants . Front Plant Sci 5 : 665 Google Scholar Crossref Search ADS PubMed WorldCat Ronquist F , Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP ( 2012 ). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space . Syst Biol 61 : 539 – 542 Google Scholar Crossref Search ADS PubMed WorldCat Ruduś I , Sasiak M, Kępczyński J ( 2013 ) Regulation of ethylene biosynthesis at the level of 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene . Acta Physiol Plant 35 : 295 – 307 Google Scholar Crossref Search ADS WorldCat Sauter M , Moffatt B, Saechao MC, Hell R, Wirtz M ( 2013 ) Methionine salvage and S-adenosylmethionine: essential links between sulfur, ethylene and polyamine biosynthesis . Biochem J 451 : 145 – 154 Google Scholar Crossref Search ADS PubMed WorldCat Schlögelhofer P , Bachmair A ( 2002 ) A test of fusion protein stability in the plant Arabidopsis thaliana reveals degradation signals from ACC synthase and from the plant N-end rule pathway . Plant Cell Rep 21 : 174 – 179 Google Scholar OpenURL Placeholder Text WorldCat Schweighofer A , Kazanaviciute V, Scheikl E, Teige M, Doczi R, Hirt H, Schwanninger M, Kant M, Schuurink R, Mauch F, et al. ( 2007 ) The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis . Plant Cell 19 : 2213 – 2224 Google Scholar Crossref Search ADS PubMed WorldCat Shin K , Lee S, Song WY, Lee RA, Lee I, Ha K, Koo JC, Park SK, Nam HG, Lee Y, Soh MS ( 2015 ) Genetic identification of ACC-RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana . Plant Cell Physiol 56 : 572 – 582 Google Scholar Crossref Search ADS PubMed WorldCat Skottke KR , Yoon GM, Kieber JJ, DeLong A ( 2011 ) Protein phosphatase 2A controls ethylene biosynthesis by differentially regulating the turnover of ACC synthase isoforms . PLoS Genet 7 : e1001370 Google Scholar Crossref Search ADS PubMed WorldCat Staswick PE , Tiryaki I ( 2004 ) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis . Plant Cell 16 : 2117 – 2127 Google Scholar Crossref Search ADS PubMed WorldCat Tan ST , Xue HW ( 2014 ) Casein kinase 1 regulates ethylene synthesis by phosphorylating and promoting the turnover of ACS5 . Cell Reports 9 : 1692 – 1702 Google Scholar Crossref Search ADS PubMed WorldCat Tatsuki M , Mori H ( 2001 ) Phosphorylation of tomato 1-aminocyclopropane-1-carboxylic acid synthase, LE-ACS2, at the C-terminal region . J Biol Chem 276 : 28051 – 28057 Google Scholar Crossref Search ADS PubMed WorldCat Tsang DL , Edmond C, Harrington JL, Nühse TS ( 2011 ) Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway . Plant Physiol 156 : 596 – 604 Google Scholar Crossref Search ADS PubMed WorldCat Tsuchisaka A , Theologis A ( 2004 a ) Heterodimeric interactions among the 1-amino-cyclopropane-1-carboxylate synthase polypeptides encoded by the Arabidopsis gene family . Proc Natl Acad Sci USA 101 : 2275 – 2280 Google Scholar Crossref Search ADS PubMed WorldCat Tsuchisaka A , Theologis A ( 2004 b ) Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate synthase gene family members . Plant Physiol 136 : 2982 – 3000 Google Scholar Crossref Search ADS PubMed WorldCat Tsuchisaka A , Yu G, Jin H, Alonso JM, Ecker JR, Zhang X, Gao S, Theologis A ( 2009 ) A combinatorial interplay among the 1-aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana . Genetics 183 : 979 – 1003 Google Scholar Crossref Search ADS PubMed WorldCat Vandenbussche F , Vaseva I, Vissenberg K, Van Der Straeten D ( 2012 ) Ethylene in vegetative development: a tale with a riddle . New Phytol 194 : 895 – 909 Google Scholar Crossref Search ADS PubMed WorldCat Van de Poel B , Vandenzavel N, Smet C, Nicolay T, Bulens I, Mellidou I, Vandoninck S, Hertog ML, Derua R, Spaepen S, et al. ( 2014 ) Tissue specific analysis reveals a differential organization and regulation of both ethylene biosynthesis and E8 during climacteric ripening of tomato . BMC Plant Biol 14 : 11 Google Scholar Crossref Search ADS PubMed WorldCat Van de Poel B , Van Der Straeten D ( 2014 ) 1-aminocyclopropane-1-carboxylic acid (ACC) in plants: more than just the precursor of ethylene! Front Plant Sci 5 : 640 Google Scholar Crossref Search ADS PubMed WorldCat Wang KL , Yoshida H, Lurin C, Ecker JR ( 2004 ) Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein . Nature 428 : 945 – 950 Google Scholar Crossref Search ADS PubMed WorldCat Wang L , Halitschke R, Kang JH, Berg A, Harnisch F, Baldwin IT ( 2007 ) Independently silencing two JAR family members impairs levels of trypsin proteinase inhibitors but not nicotine . Planta 226 : 159 – 167 Google Scholar Crossref Search ADS PubMed WorldCat Woeste KE , Ye C, Kieber JJ ( 1999 ) Two Arabidopsis mutants that overproduce ethylene are affected in the posttranscriptional regulation of 1-aminocyclopropane-1-carboxylic acid synthase . Plant Physiol 119 : 521 – 530 Google Scholar Crossref Search ADS PubMed WorldCat Xiong L , Xiao D, Xu X, Guo Z, Wang NN ( 2014 ) The non-catalytic N-terminal domain of ACS7 is involved in the post-translational regulation of this gene in Arabidopsis . J Exp Bot 65 : 4397 – 4408 Google Scholar Crossref Search ADS PubMed WorldCat Xu SL , Rahman A, Baskin TI, Kieber JJ ( 2008 ) Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis . Plant Cell 20 : 3065 – 3079 Google Scholar Crossref Search ADS PubMed WorldCat Yamagami T , Tsuchisaka A, Yamada K, Haddon WF, Harden LA, Theologis A ( 2003 ) Biochemical diversity among the 1-amino-cyclopropane-1-carboxylate synthase isozymes encoded by the Arabidopsis gene family . J Biol Chem 278 : 49102 – 49112 Google Scholar Crossref Search ADS PubMed WorldCat Yang SF , Hoffman NE ( 1984 ) Ethylene biosynthesis and its regulation in higher plants . Annu Rev Plant Physiol 35 : 155 – 189 Google Scholar Crossref Search ADS WorldCat Yip W-K , Yang SF ( 1988 ) Cyanide metabolism in relation to ethylene production in plant tissues . Plant Physiol 88 : 473 – 476 Google Scholar Crossref Search ADS PubMed WorldCat Yoon GM , Kieber JJ ( 2013 a ) 14-3-3 regulates 1-aminocyclopropane-1-carboxylate synthase protein turnover in Arabidopsis . Plant Cell 25 : 1016 – 1028 Google Scholar Crossref Search ADS PubMed WorldCat Yoon GM , Kieber JJ ( 2013 b ) ACC synthase and its cognate E3 ligase are inversely regulated by light . Plant Signal Behav 8 : e26478 Google Scholar Crossref Search ADS PubMed WorldCat Yoshida H , Nagata M, Saito K, Wang KL, Ecker JR ( 2005 ) Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases . BMC Plant Biol 5 : 14 Google Scholar Crossref Search ADS PubMed WorldCat Yoshida H , Wang KL, Chang CM, Mori K, Uchida E, Ecker JR ( 2006 ) The ACC synthase TOE sequence is required for interaction with ETO1 family proteins and destabilization of target proteins . Plant Mol Biol 62 : 427 – 437 Google Scholar Crossref Search ADS PubMed WorldCat Zhang TC , Qiao Q, Zhong Y ( 2012 ) Detecting adaptive evolution and functional divergence in aminocyclopropane-1-carboxylate synthase (ACS) gene family . Comput Biol Chem 38 : 10 – 16 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Z , Ren J-S, Clifton IJ, Schofield CJ ( 2004 ) Crystal structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase--the ethylene-forming enzyme . Chem Biol 11 : 1383 – 1394 Google Scholar Crossref Search ADS PubMed WorldCat Zheng Z , Guo Y, Novák O, Dai X, Zhao Y, Ljung K, Noel JP, Chory J ( 2013 ) Coordination of auxin and ethylene biosynthesis by the aminotransferase VAS1 . Nat Chem Biol 9 : 244 – 246 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the National Science Foundation (grant no. IOS–1145585) and partially supported by the National Institutes of Health (predoctoral training program grant no. GM007601 to M.A.B.). * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00672 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

García, María José; Romera, Francisco Javier; Lucena, Carlos; Alcántara, Esteban; Pérez-Vicente, Rafael

doi: 10.1104/pp.15.00708pmid: 26175512

Abstract To cope with nutrient deficiencies, plants develop both morphological and physiological responses. The regulation of these responses is not totally understood, but some hormones and signaling substances have been implicated. It was suggested several years ago that ethylene participates in the regulation of responses to iron and phosphorous deficiency. More recently, its role has been extended to other deficiencies, such as potassium, sulfur, and others. The role of ethylene in so many deficiencies suggests that, to confer specificity to the different responses, it should act through different transduction pathways and/or in conjunction with other signals. In this update, the data supporting a role for ethylene in the regulation of responses to different nutrient deficiencies will be reviewed. In addition, the results suggesting the action of ethylene through different transduction pathways and its interaction with other hormones and signaling substances will be discussed. When plants suffer from a mineral nutrient deficiency, they develop morphological and physiological responses (mainly in their roots) aimed to facilitate the uptake and mobilization of the limiting nutrient. After the nutrient has been acquired in enough quantity, these responses need to be switched off to avoid toxicity and conserve energy. In recent years, different plant hormones (e.g. ethylene, auxin, cytokinins, jasmonic acid, abscisic acid, brassinosteroids, GAs, and strigolactones) have been implicated in the regulation of these responses (Romera et al., 2007, 2011, 2015; Liu et al., 2009; Rubio et al., 2009; Kapulnik et al., 2011; Kiba et al., 2011; Iqbal et al., 2013; Zhang et al., 2014). Before the 1990s, there were several publications relating ethylene and nutrient deficiencies (cited in Lynch and Brown [1997] and Romera et al. [1999]) without establishing a direct implication of ethylene in the regulation of nutrient deficiency responses. In 1994, Romera and Alcántara (1994) published an article in Plant Physiology suggesting a role for ethylene in the regulation of Fe deficiency responses. In 1999, Borch et al. (1999) showed the participation of ethylene in the regulation of P deficiency responses. Since then, evidence has been accumulating in support of a role for ethylene in the regulation of both Fe (Romera et al., 1999, 2015; Waters and Blevins, 2000; Lucena et al., 2006; Waters et al., 2007; García et al., 2010, 2011, 2013, 2014; Yang et al., 2014) and P deficiency responses (Kim et al., 2008; Lei et al., 2011; Li et al., 2011; Nagarajan and Smith, 2012; Wang et al., 2012, 2014c). Both Fe and P may be poorly available in most soils, and plants develop similar responses under their deficiencies (Romera and Alcántara, 2004; Zhang et al., 2014). More recently, a role for ethylene has been extended to other deficiencies, such as K (Shin and Schachtman, 2004; Jung et al., 2009; Kim et al., 2012), S (Maruyama-Nakashita et al., 2006; Wawrzyńska et al., 2010; Moniuszko et al., 2013), and B (Martín-Rejano et al., 2011). Ethylene has also been implicated in both N deficiency and excess (Tian et al., 2009; Mohd-Radzman et al., 2013; Zheng et al., 2013), and its participation in Mg deficiency has been suggested (Hermans et al., 2010). In this update, we will review the information supporting a role for ethylene in the regulation of different nutrient deficiency responses. For information relating ethylene to other aspects of plant mineral nutrition, such as N2 fixation and responses to excess of nitrate or essential heavy metals, the reader is referred to other reviews (for review, see Maksymiec, 2007; Mohd-Radzman et al., 2013; Steffens, 2014). ETHYLENE SYNTHESIS AND SIGNALING UNDER NUTRIENT DEFICIENCIES Nutrient deficiencies can influence both ethylene synthesis and signaling. In general, ethylene production increases under different nutrient deficiencies. Additionally, ethylene production can increase upon excess of some nutrients, like nitrate (Tian et al., 2009; Mohd-Radzman et al., 2013) or essential heavy metals (Maksymiec, 2007). In 1999, Romera et al. (1999) showed that Fe-deficient roots of several dicots produced more ethylene than the Fe-sufficient ones, even before the plants showed any other symptom of deficiency (which could lead to tissue necrosis and thereby, stimulation of wound ethylene; Lynch and Brown, 1997). At the same time, Borch et al. (1999) and Gilbert et al. (2000) showed that P-deficient roots produced more ethylene than the P-sufficient ones. After these reports, the higher ethylene production by Fe-deficient roots has been confirmed by other authors (cited in García et al. [2010] and Romera et al. [2015]). In relation to P, there has been research confirming higher ethylene production by P-deficient roots (Li et al., 2009) and showing higher ethylene production by P-deficient shoots (Kim et al., 2008). In the last 10 years, increased ethylene production by roots and/or shoots has been described for other nutrient deficiencies, such as K (Shin and Schachtman, 2004; Benlloch-González et al., 2010), S (Zuchi et al., 2009; Moniuszko et al., 2013), N (Zheng et al., 2013), and Mg (Hermans et al., 2010). The higher ethylene production described for nutrient deficiencies has been further supported by results showing up-regulation of genes implicated in ethylene synthesis. Ethylene is synthesized from Met through a pathway that requires the enzymes S-adenosyl methionine synthetases (SAMS), 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and 1-aminocyclopropane-1-carboxylic acid oxidade (ACO; Sauter et al., 2013). SAMS, ACS, and ACO genes (Table I shows gene names and functions) were up-regulated under Fe deficiency (for review, see Romera et al., 2015) and also, P deficiency (Hernández et al., 2007; Lei et al., 2011; O’Rourke et al., 2013; Wang et al., 2014b). Shin and Schachtman (2004) have found up-regulation of two Arabidopsis (Arabidopsis thaliana) ACOs under K deficiency, Nikiforova et al. (2003) have found up-regulation of AtSAMS under S deficiency, Zhao et al. (2015) have found up-regulation of an AtACO under N deficiency, and Hermans et al. (2010) have found up-regulation of several AtACS under Mg deficiency. Genes related to nutrient deficiency responses used in this update Table I. Genes related to nutrient deficiency responses used in this update ER, endoplasmic reticulum; TF, transcription factor. Name . Function . Ethylene synthesis genes  SAMS SAMS  ACS ACS  ACO ACO Ethylene signaling genes  ETRs and ERSs Ethylene receptors  CTR1 a Kinase  EIN2 b Protein acting downstream of CTR1 (localized to ER membrane)  EIN3 c TF acting downstream of EIN2  EILs c TFs acting downstream of EIN2  SLIM1 (EIL3) SLIM1 is an allele of EIL3  ERFs TFs acting downstream of EIN3  RAP2.11 Is an ERF Fe-related genes  FIT (FER homolog) TF (master regulator of most Fe acquisition genes)  MED16 Mediator (interacts with EIN3/EIL1 for FIT transcription) P-related genes  PT1, PT2, and PT5 Phosphate transporters  ACP5 and PAP1 Acid phosphatases  PHO2 E2 conjugase (negative regulator of P deficiency responses) S-related genes  SULTRs Sulfate transporters  APR Adenosine 5′-phosphosulfate reductase  APS4 ATP sulfurylase (negative regulator of S deficiency responses) K-related genes  HAK5 K+ transporter N-related genes  NRTs Nitrate transporters Name . Function . Ethylene synthesis genes  SAMS SAMS  ACS ACS  ACO ACO Ethylene signaling genes  ETRs and ERSs Ethylene receptors  CTR1 a Kinase  EIN2 b Protein acting downstream of CTR1 (localized to ER membrane)  EIN3 c TF acting downstream of EIN2  EILs c TFs acting downstream of EIN2  SLIM1 (EIL3) SLIM1 is an allele of EIL3  ERFs TFs acting downstream of EIN3  RAP2.11 Is an ERF Fe-related genes  FIT (FER homolog) TF (master regulator of most Fe acquisition genes)  MED16 Mediator (interacts with EIN3/EIL1 for FIT transcription) P-related genes  PT1, PT2, and PT5 Phosphate transporters  ACP5 and PAP1 Acid phosphatases  PHO2 E2 conjugase (negative regulator of P deficiency responses) S-related genes  SULTRs Sulfate transporters  APR Adenosine 5′-phosphosulfate reductase  APS4 ATP sulfurylase (negative regulator of S deficiency responses) K-related genes  HAK5 K+ transporter N-related genes  NRTs Nitrate transporters a Its mutation leads to a constitutive triple-response phenotype, similar to the one of wild-type plants treated with ethylene. b Its mutation leads to an ethylene-insensitive phenotype. c Their mutations lead to ethylene-insensitive phenotypes. Open in new tab Table I. Genes related to nutrient deficiency responses used in this update ER, endoplasmic reticulum; TF, transcription factor. Name . Function . Ethylene synthesis genes  SAMS SAMS  ACS ACS  ACO ACO Ethylene signaling genes  ETRs and ERSs Ethylene receptors  CTR1 a Kinase  EIN2 b Protein acting downstream of CTR1 (localized to ER membrane)  EIN3 c TF acting downstream of EIN2  EILs c TFs acting downstream of EIN2  SLIM1 (EIL3) SLIM1 is an allele of EIL3  ERFs TFs acting downstream of EIN3  RAP2.11 Is an ERF Fe-related genes  FIT (FER homolog) TF (master regulator of most Fe acquisition genes)  MED16 Mediator (interacts with EIN3/EIL1 for FIT transcription) P-related genes  PT1, PT2, and PT5 Phosphate transporters  ACP5 and PAP1 Acid phosphatases  PHO2 E2 conjugase (negative regulator of P deficiency responses) S-related genes  SULTRs Sulfate transporters  APR Adenosine 5′-phosphosulfate reductase  APS4 ATP sulfurylase (negative regulator of S deficiency responses) K-related genes  HAK5 K+ transporter N-related genes  NRTs Nitrate transporters Name . Function . Ethylene synthesis genes  SAMS SAMS  ACS ACS  ACO ACO Ethylene signaling genes  ETRs and ERSs Ethylene receptors  CTR1 a Kinase  EIN2 b Protein acting downstream of CTR1 (localized to ER membrane)  EIN3 c TF acting downstream of EIN2  EILs c TFs acting downstream of EIN2  SLIM1 (EIL3) SLIM1 is an allele of EIL3  ERFs TFs acting downstream of EIN3  RAP2.11 Is an ERF Fe-related genes  FIT (FER homolog) TF (master regulator of most Fe acquisition genes)  MED16 Mediator (interacts with EIN3/EIL1 for FIT transcription) P-related genes  PT1, PT2, and PT5 Phosphate transporters  ACP5 and PAP1 Acid phosphatases  PHO2 E2 conjugase (negative regulator of P deficiency responses) S-related genes  SULTRs Sulfate transporters  APR Adenosine 5′-phosphosulfate reductase  APS4 ATP sulfurylase (negative regulator of S deficiency responses) K-related genes  HAK5 K+ transporter N-related genes  NRTs Nitrate transporters a Its mutation leads to a constitutive triple-response phenotype, similar to the one of wild-type plants treated with ethylene. b Its mutation leads to an ethylene-insensitive phenotype. c Their mutations lead to ethylene-insensitive phenotypes. Open in new tab Other than ethylene synthesis, nutrient deficiencies can also affect ethylene responsiveness. He et al. (1992) showed increased sensitivity to ethylene in N- and P-deficient roots, which has been further supported in more recent publications (Ma et al., 2003; Kim et al., 2008). Although ethylene’s mode of action is not fully understood, a linear signaling pathway has been proposed in Arabidopsis (Shakeel et al., 2013; Wang et al., 2013): where ET indicates ethylene, ─╢ indicates negative effect, → indicates positive effect, CTR1 indicates Constitutive Triple Response1, EIN2 indicates Ethylene Insensitive2, EIL indicates Ethylene Insensitive-Like, and ERF indicates Ethylene Response Factor. In the absence of ethylene, the kinase CTR1 phosphorylates EIN2 (which is localized to the endoplasmic reticulum membrane), preventing the cleavage and translocation of the EIN2 C-terminal fragment into the nucleus. In the presence of ethylene, this is bound to its receptors, and CTR1 is inactivated, resulting in dephosphorylation of EIN2 and its cleavage. The EIN2 C-terminal fragment is then translocated into the nucleus, where it participates in stabilization of the transcription factor EIN3 and downstream gene activation (Shakeel et al., 2013; Wang et al., 2013). EIN3 belongs to a small family of transcription factors that also includes various EIL proteins: EIL1, EIL2, and EIL3 (Wang et al., 2013). Mutants of CTR1 present constitutive activation of ethylene signaling, whereas mutants of EIN2 and EIN3/EILs display reduced sensitivity to ethylene (Shakeel et al., 2013; Wang et al., 2013). The ERF transcription factors act downstream of EIN3 to activate or repress ethylene-responsive genes, although some ERFs can be activated by ethylene-independent transcription factors not related to EIN3 (Wang et al., 2013; Thirugnanasambantham et al., 2015). Ethylene responsiveness could be related to changes in the expression of genes implicated in ethylene signaling. Several of these genes were up-regulated under Fe deficiency, like Ethylene Triple Responses (ETRs; coding for ethylene receptors), Ethylene Response Sensors (ERSs; coding for ethylene receptors), EIN2, EIN3, EILs, and ERFs (O’Rourke et al., 2007; García et al., 2010, 2014; Wang et al., 2014a). Similarly, Shin and Schachtman (2004) found increased expression of AtETR2 (encoding an ethylene receptor) and Kim et al. (2012) found increased expression of Arabidopsis Rhoptry-Associated Protein2.11 (AtRAP2.11; encoding an ERF) under K deficiency. In relation to N, Zheng et al. (2013) have shown increased expression of AtEIN3 and AtEIL1, and Zhao et al. (2015) have shown increased expression of several AtERFs under this deficiency. Very recently, Ramaiah et al. (2014) have described the up-regulation of AtERF070 (encoding an ERF) in P-deprived roots and shoots. Whether the expression of these genes enhances or decreases the sensitivity to ethylene deserves a deeper investigation. Because ethylene receptors act as negative regulators of ethylene signaling, their increase would decrease sensitivity to ethylene (Wang et al., 2013). Possibly, the induction of ethylene receptor genes may function as a dampening mechanism, slowing down an ethylene response after it has been initiated. ETHYLENE PARTICIPATION IN NUTRIENT DEFICIENCY RESPONSES In addition to the higher ethylene production of nutrient deficient plants (see above), other results also support a role for ethylene in the regulation of nutrient deficiency responses. These other results are mainly based on the use of ethylene inhibitors, like cobalt or silver thiosulfate (STS), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene-releasing substance ethephon, ethylene itself, ethylene mutants (ethylene insensitive, ethylene constitutive, or ethylene overproducers), and molecular biology techniques, such as transgenic lines, transcriptomics, fluorescence imaging, luciferase imaging, GUS assay, and yeast (Saccharomyces cerevisiae) two-hybrid assay (Romera and Alcántara, 2004; Maruyama-Nakashita et al., 2006; Jung et al., 2009; García et al., 2010; Lei et al., 2011; Kim et al., 2012; Yang et al., 2014). In most cases, ethylene, with production that increases under the nutrient deficiency, acts as activator of the responses. Consequently, ethylene inhibitors block the responses (Fig. 1), whereas ethylene itself or ethylene precursors (ACC and ethephon) promote them (Romera and Alcántara, 1994, 2004; Jung et al., 2009; Tian et al., 2009; Lei et al., 2011; Li et al., 2011; Wang et al., 2012). Figure 1. Open in new tabDownload slide Ethylene generally acts as an activator of responses to nutrient deficiencies. Consequently, ethylene inhibitors block these responses. As an example, ferric reductase activity is enhanced under Fe deficiency (denoted by the purple color of the assay solution) but blocked upon application of the ethylene inhibitor STS. Tomato plants grown in complete nutrient solution (+Fe) were transferred for the last 3 d to nutrient solution either without Fe (−Fe) or without Fe plus 400 μm STS (−Fe + STS). Ferric reductase activity was determined as described by Romera and Alcántara (1994). Figure 1. Open in new tabDownload slide Ethylene generally acts as an activator of responses to nutrient deficiencies. Consequently, ethylene inhibitors block these responses. As an example, ferric reductase activity is enhanced under Fe deficiency (denoted by the purple color of the assay solution) but blocked upon application of the ethylene inhibitor STS. Tomato plants grown in complete nutrient solution (+Fe) were transferred for the last 3 d to nutrient solution either without Fe (−Fe) or without Fe plus 400 μm STS (−Fe + STS). Ferric reductase activity was determined as described by Romera and Alcántara (1994). Ethylene has been implicated in the regulation of both morphological and physiological responses to nutrient deficiencies (Romera and Alcántara, 1994, 2004; Jung et al., 2009; Lei et al., 2011; Wang et al., 2012, 2014c). Morphological responses include responses like changes in root system architecture (RSA), development of root hairs, development of cluster or proteoid roots (clusters of closely spaced short lateral rootlets formed in some plant species adapted to poor soils; Wang et al., 2014b), and development of root transfer cells (cells with increased surface area because of invaginations of the plasma membrane; Kramer et al., 1980). Most of these root modifications enhance nutrient uptake by increasing the surface of contact of roots with soil and chemically modifying the soil environment (Wang et al., 2014b). Physiological responses are changes in processes aimed to facilitate the mobilization and uptake of nutrients. Some physiological responses are the acidification of the rhizosphere, the release of chelating agents into the medium, the increased amount of specific transporters in root epidermal cells, the increase of internal transporters and chelating agents (to improve mobilization of nutrients inside the plant), the enhancement of root ferric reductase activity, and the enhancement of root acid phosphatase activity. Morphological Responses Ethylene has been implicated in the development of subapical root hairs, root transfer cells, and cluster roots induced under Fe or P deficiency (Kramer et al., 1980; Romera and Alcántara, 1994, 2004; Schmidt et al., 2000a; Waters and Blevins, 2000; Schmidt and Schikora, 2001; Schikora and Schmidt, 2002; Zaid et al., 2003; Zhang et al., 2003, 2014; Wang et al., 2014b). Other than these deficiencies, ethylene has also been implicated in the development of root hairs caused by K (Jung et al., 2009) or B deficiency (Martín-Rejano et al., 2011). Nutrient deficiencies can also change RSA by altering the number, length, and diameter of roots (Gruber et al., 2013). The RSA modifications depend on each specific nutrient and the extent of the deficiency (mild, moderate, or severe). Generally, nutrient-deficient plants exhibit a shallower architecture that results from inhibition of primary root elongation (Kramer et al., 1980; López-Bucio et al., 2003; Ma et al., 2003; Jung et al., 2009; Martín-Rejano et al., 2011; Nagarajan and Smith, 2012, Wang et al., 2012; Gruber et al., 2013; Zhang et al., 2014). Some exceptions are S deficiency, with relatively little influence on the morphology of roots, and N deficiency, which stimulates primary root elongation and particularly, lateral root elongation (Gruber et al., 2013). Several deficiencies, like B, Fe, or P deficiency, can cause an increase in lateral root density (Kramer et al., 1980; López-Bucio et al., 2003; Miura et al., 2011; Gruber et al., 2013; Zhang et al., 2014). Ethylene has been implicated in most of these RSA changes (Borch et al., 1999; Gilbert et al., 2000; Ma et al., 2003; Zhang et al., 2003, 2014; Jung et al., 2009; Martín-Rejano et al., 2011; Miura et al., 2011; Chérel et al., 2014) along with auxin and other hormones and signaling substances (“Ethylene Interacts with Other Signals for the Regulation of Nutrient Deficiency Responses”). Very recently, Ramaiah et al. (2014) have found that AtERF070 (encoding an ERF) was greatly induced under P deprivation and can affect RSA. This indicates that ethylene, through an ERF transcription factor that participates in ethylene signaling (see above), can modify the architecture of roots. The participation of ethylene in the regulation of the morphological responses described above is supported by results showing that ethylene inhibitors negatively affect these changes in nutrient-deficient plants, whereas ethylene precursors promote them in nutrient-sufficient plants (Romera and Alcántara, 1994; Borch et al., 1999; Schmidt et al., 2000a; Ma et al., 2003; Zaid et al., 2003; Zhang et al., 2003, 2014; Romera et al., 2007; Wang et al., 2012, 2014b). As an example, ethylene inhibitors almost totally block the development of subapical root hairs in Fe-, K-, or P-deficient plants (Zhang et al., 2003; Romera and Alcántara, 2004; Jung et al., 2009). Additionally, results obtained with ethylene mutants support this participation (Jung et al., 2009; Wang et al., 2012; see below). Physiological Responses In addition to a role for ethylene in the regulation of morphological responses, many data support its role in the regulation of physiological responses. In relation to Fe nutrition, ethylene participates in the up-regulation of many genes implicated in Fe acquisition and homeostasis (Lucena et al., 2006; Waters et al., 2007; García et al., 2010; Romera et al., 2015). Some of the genes up-regulated by ethylene encode transcription factors that are key regulators of most of the responses to Fe deficiency, such as Arabidopsis Fer-like Fe Deficiency Transcription Factor (AtFIT; Colangelo and Guerinot, 2004) or its tomato (Solanum lycopersicum) homolog tomato Fe Efficiency Response (SlFER; Brumbarova and Bauer, 2005). Very recently, it has been shown that AtEIN3 and AtEIL1, both related to ethylene signaling (see above), interact with Arabidopsis Mediator16 (AtMED16) to form a complex implicated in the transcription of AtFIT (Yang et al., 2014). In the last 10 years, the role of ethylene in the regulation of physiological responses has been extended to other nutrient deficiencies. In K nutrition, ethylene has been shown to be implicated in the up-regulation of the K+ transporter Arabidopsis High Affinity K+ Transporter5 (AtHAK5; Jung et al., 2009), possibly through RAP2.11 (an ERF; Kim et al., 2012). In S nutrition, the up-regulation of several sulfate transporter genes (Arabidopsis Sulfate Transporter1;1 [AtSULTR1;1], AtSULTR1;2, AtSULTR3;4, and AtSULTR4;2) as well as other S-responsive genes is greatly diminished in sulfur limitation1 (slim1; eil3) mutants (Maruyama-Nakashita et al., 2006). The similarity of AtEIL3 with AtEIN3 suggests that it could be a positive regulator of ethylene signaling (see above). The participation of ethylene in S deficiency responses has also been supported by other experimental results (Koprivova et al., 2008; Wawrzyńska et al., 2010; Iqbal et al., 2012; Moniuszko et al., 2013). Koprivova et al. (2008) found up-regulation of several Arabidopsis Adenosine 5′-Phosphosulfate Reductases (AtAPRs; encoding adenosine 5′-phosphosulfate reductase, a key enzyme of sulfate assimilation) upon ACC treatment, and Iqbal et al. (2012) found increased ATP-sulfurylase activity (also implicated in sulfate assimilation) upon ethephon application. However, Wawrzyńska et al. (2010) showed that tobacco (Nicotiana tabacum) Upregulated by Sulfur Deficit 9C, a tobacco gene strongly induced by S deficiency, is activated by the NtEIL2 transcription factor related to ethylene signaling (see above). In P nutrition, Lei et al. (2011) in Arabidopsis and Li et al. (2011) in Medicago falcata have implicated ethylene in up-regulation of phosphate transporter genes (Arabidopsis Phosphate Transporter1 [AtPT1], AtPT2, MfPT1, and MfPT5) and enhanced phosphatase activity (through higher expression of Arabidopsis Acid Phosphatase5 (AtACP5) and M. falcata Purple Acid Phosphatase1 (MfPAP1) encoding phosphatases) of P-deficient roots. In N nutrition, ethylene has been shown to up-regulate Arabidopsis Nitrate Transporter1.1 (AtNRT1.1), whereas it down-regulates AtNRT2.1 (both encoding nitrate transporters; Tian et al., 2009; Zheng et al., 2013). The negative effect of ethylene on the regulation of some nutrient deficiency responses has also been shown in P starvation-induced anthocyanin (Lei et al., 2011; Wang et al., 2012) and N starvation-induced anthocyanin (Wang et al., 2015), both of them inhibited by ethylene. ARE THE DIFFERENT NUTRIENT DEFICIENCY RESPONSES REGULATED SIMILARLY BY ETHYLENE? Because ethylene has been implicated in the regulation of many nutrient deficiency responses, the question arises as to whether ethylene regulates all of them through the same transduction pathway. The answer to this question is clearly that different responses can be regulated by ethylene through different transduction pathways based on results with ethylene mutants. However, many results suggest that ethylene does not act alone but acts in conjunction with other hormones and signaling substances to regulate the responses. Results from ethylene-insensitive mutants suggest that, even within a deficiency, different responses can be regulated through different transduction pathways. The tomato ethylene-insensitive mutant Never ripe does not increase adventitious root formation under P deficiency but is normal in other morphological responses (Kim et al., 2008). Similarly, the development of subapical root hairs is impaired in the Arabidopsis ethylene-insensitive mutant ein2 under Fe deficiency (Schmidt and Schikora, 2001), whereas the enhanced ferric reductase activity (Fig. 2) and the expression of Fe acquisition genes are not impaired (García et al., 2010). Figure 2. Open in new tabDownload slide Effect of P deficiency on AtPT2 gene expression (A) and Fe deficiency on ferric reductase activity (B) in Arabidopsis wild-type (WT) Columbia-0 plants and ethylene mutants (ctr1, ethylene-constitutive mutant; ein2, ethylene-insensitive mutant; eto1, ethylene overproducer mutant). Fold changes were normalized to transcript levels of the wild type on P sufficiency (A) and ferric reductase activity of the wild type on Fe sufficiency (B). Data for P treatments were redrawn from Lei et al. (2011) with permission, and data for Fe were from García et al. (2010, 2014) and M.J. García, F.J. Romera, C. Lucena, E. Alcántara, and R. Pérez-Vicente (unpublished data). Figure 2. Open in new tabDownload slide Effect of P deficiency on AtPT2 gene expression (A) and Fe deficiency on ferric reductase activity (B) in Arabidopsis wild-type (WT) Columbia-0 plants and ethylene mutants (ctr1, ethylene-constitutive mutant; ein2, ethylene-insensitive mutant; eto1, ethylene overproducer mutant). Fold changes were normalized to transcript levels of the wild type on P sufficiency (A) and ferric reductase activity of the wild type on Fe sufficiency (B). Data for P treatments were redrawn from Lei et al. (2011) with permission, and data for Fe were from García et al. (2010, 2014) and M.J. García, F.J. Romera, C. Lucena, E. Alcántara, and R. Pérez-Vicente (unpublished data). The idea of multiple transduction pathways for regulating nutrient deficiency responses by ethylene is further reinforced by comparing different deficiencies and looking at the results from ethylene overproducer and ethylene-constitutive mutants. The up-regulation of the AtPT2 gene (induced under P deficiency; Fig. 2) and the AtHAK5 gene (induced under K deficiency) is impaired in the Arabidopsis ein2 mutant (Jung et al., 2009; Lei et al., 2011), whereas increased ferric reductase activity under Fe deficiency is not impaired (Fig. 2). Both the Arabidopsis ethylene-constitutive mutant ctr1 and the Arabidopsis ethylene overproducer mutant eto have constitutive subapical root hairs (a nutrient deficiency symptom) in complete nutrient solution; however, neither of these mutants has full constitutive activation of P, Fe, or K physiological responses (Fig. 2; Schmidt et al., 2000b; Romera and Alcántara, 2004; García et al., 2007, 2014; Jung et al., 2009; Lei et al., 2011; Wang et al., 2012). In the same way, root hairs, transfer cells, and cluster roots are almost fully induced by ACC or ethephon in plants grown with high levels of P, Fe, or K, whereas physiological responses are activated to a lesser degree than when applied to plants grown with low levels or in absence of these nutrients (Romera and Alcántara, 1994; Schmidt et al., 2000a; Zaid et al., 2003; Zhang et al., 2003; Jung et al., 2009; Lucena et al., 2006; Lei et al., 2011; Li et al., 2011; García et al., 2013). From all of the results above, several conclusions can be drawn. First, different nutrient deficiency responses can be regulated by ethylene through distinct transduction pathways. Second, for some responses, like Fe physiological responses, ethylene could act through a pathway where EIN2 and possibly, CTR1 are not strictly required (Fig. 3). Third, morphological and physiological responses can be differently regulated by ethylene. Fourth, for the regulation of physiological responses, ethylene could act in conjunction with nutrient-related repressive signals. The existence of an alternate route for ethylene signaling, other than the conventional one including CTR1 and EIN2 (Fig. 3; Shakeel et al., 2013), is further supported by results showing that the Arabidopsis ctr1 and ein2 mutants respond to both ACC (García et al., 2010, 2014) and ethylene inhibitors (García et al., 2007; Jung et al., 2009) for some physiological responses. In relation to the last two conclusions, we can speculate that, because physiological responses are not fully activated in the ctr1 and eto mutants (Fig. 2), whereas morphological responses (at least in root hairs) are, some nutrient-related signals act negatively to block physiological responses. These signals probably act downstream of CTR1 in the ethylene signaling pathway. This does not preclude that these signals can also affect ethylene synthesis. Figure 3. Open in new tabDownload slide Ethylene (ET) could regulate different nutrient deficiency responses through two distinct signal transduction pathways. One pathway is CTR1-EIN2 dependent, and the other is CTR1-EIN2 independent (instead, using Arabidopsis His Phosphotransfer [AHP] proteins and Arabidopsis Response Regulators [ARRs]; Shakeel et al., 2013). This model is supported by data showing that ctr1 and ein2 mutants respond to both ACC and ET inhibitors for some physiological responses (García et al., 2007, 2010, 2014; Jung et al., 2009). It is possible that both pathways can act independently (A) or that both can interact and converge downstream through EIN3/EILs (B) depending on the responses (Table I). ─╢, Inhibition; →, promotion. Figure 3. Open in new tabDownload slide Ethylene (ET) could regulate different nutrient deficiency responses through two distinct signal transduction pathways. One pathway is CTR1-EIN2 dependent, and the other is CTR1-EIN2 independent (instead, using Arabidopsis His Phosphotransfer [AHP] proteins and Arabidopsis Response Regulators [ARRs]; Shakeel et al., 2013). This model is supported by data showing that ctr1 and ein2 mutants respond to both ACC and ET inhibitors for some physiological responses (García et al., 2007, 2010, 2014; Jung et al., 2009). It is possible that both pathways can act independently (A) or that both can interact and converge downstream through EIN3/EILs (B) depending on the responses (Table I). ─╢, Inhibition; →, promotion. ETHYLENE INTERACTS WITH OTHER SIGNALS FOR THE REGULATION OF NUTRIENT DEFICIENCY RESPONSES Morphological and physiological responses work together to effectively increase nutrient uptake (Lucena et al., 2006; Jung et al., 2009; Wang et al., 2014b). Consequently, their regulation is coordinated through the participation of similar signals for both kind of responses, like hormones (e.g. auxin, ethylene, cytokinins, jasmonic acid, brassinosteroids, gibberellins, abscisic acid, and strigolactones) and other signaling substances, such as nitric oxide (NO), reactive oxygen species (ROS), and sugars (Romera et al., 2007, 2011, 2015; Rubio et al., 2009; Hammond and White, 2011; Kapulnik et al., 2011; Lei and Liu, 2011; Iqbal et al., 2013; Zhang et al., 2014). Other than these common signals, there are other more nutrient-specific signals, such as mineral ions, microRNAs, reduced glutathione (GSH), and peptides (Lappartient et al., 1999; Liu et al., 2009; Buhtz et al., 2010; García et al., 2013; Zeng et al., 2014; Zhang et al., 2014), that could confer specificity (at least a certain degree of specificity) to the different nutrient deficiency responses. Despite this, cross talk in the activation of physiological responses under different nutrient deficiencies (e.g. a K physiological response can be activated under P deficiency) has been described (Shin et al., 2005; Waters et al., 2012; Wang et al., 2014b), probably because of the common implication of ethylene and other signals in their regulation. Different results suggest that some nutrient-related repressive signals (e.g. mineral ions, peptides, and GSH) can move from shoots to roots through the phloem (Dong et al., 1998; Lappartient et al., 1999; García et al., 2013; Zhang et al., 2014). Additionally, other signals, like auxin, sugars, and microRNAs, can also move through the phloem (Romera et al., 2007, 2011; Buhtz et al., 2010; Lei and Liu, 2011). This would provide a way for shoots to inform roots of their nutrient status and could serve to integrate the role of both shoots and roots in the regulation of nutrient deficiency responses. Auxin, sugars, NO, and ROS generally accumulate in roots under nutrient deficiencies (Shin et al., 2005; Tewari et al., 2006; Graziano and Lamattina, 2007; Romera et al., 2007; Jung et al., 2009; Wang et al., 2010, 2014b; Kiba et al., 2011; Iqbal et al., 2013) and can positively interact with ethylene to regulate nutrient deficiency responses. Auxin can stimulate ethylene production, and ethylene can affect auxin transport and accumulation (Romera et al., 2007, 2011; Muday et al., 2012). The exact relationship of sugars and ethylene is not totally known, but hexokinases are a critical node in mediating plant Glc and ethylene responses (Karve et al., 2012). Ethylene can stimulate both ROS and NO accumulation (Shin et al., 2005; Jung et al., 2009; García et al., 2011; Steffens, 2014), whereas both ROS and NO can stimulate ethylene production (Ahlfors et al., 2009; García et al., 2011; Iqbal et al., 2013). Furthermore, ROS can stimulate ethylene production through NO accumulation (Ahlfors et al., 2009). Probably, ROS and NO influence the production of ethylene in a positive feedback loop, leading to enhancement of the nutrient deficiency signal as described for ethylene and NO (Fig. 4; García et al., 2011). Figure 4. Open in new tabDownload slide Working model to explain the role of ethylene (ET) on the regulation of responses to different nutrient deficiencies in plants. Nutrient deficiencies can enhance ET production by up-regulating SAMS, ACS, and ACO genes (Shin and Schachtman, 2004; García et al., 2010; Hermans et al., 2010), although the steps leading to this up-regulation are not yet clear. A possibility is that, at first, nutrient deficiencies cause oxidative stress and consequently, ROS accumulation and possibly, NO accumulation (Shin et al., 2005; Tewari et al., 2006; Graziano and Lamattina, 2007; Ahlfors et al., 2009; Jung et al., 2009; García et al., 2011; Iqbal et al., 2013; Steffens, 2014). This ROS-NO accumulation would stimulate ET production, which in turn, could increase ROS-NO production (Ahlfors et al., 2009; Jung et al., 2009; García et al., 2011; Iqbal et al., 2013) in a positive feedback loop, leading to enhancement of the nutrient deficiency signal (García et al., 2011). When ET is perceived by the receptors, it could act through a CTR1-EIN2-dependent pathway for the regulation of some responses or a CTR1-EIN2-independent pathway for the regulation of other responses (Fig. 3). At the end of these transduction pathways, different ET-related transcription factors, such as RAP2.11, ERF070, EIL3, EIN3/EIL1, or others, would activate different nutrient responses (Maruyama-Nakashita et al., 2006; Kim et al., 2012; Ramaiah et al., 2014; Yang et al., 2014). Under nutrient sufficiency, several signals that can move through the phloem (mineral ions, peptides, GSH, etc.; Lappartient et al., 1999; García et al., 2013; Zhang et al., 2014) could negatively interact with ET to inactivate nutrient responses. However, under nutrient deficiency, other signals that can move through the phloem (microRNAs, auxin, sugars, etc.; Kasajima et al., 2007; Lejay et al., 2008; Hammond and White, 2011; Lei and Liu, 2011; Hu et al., 2015) could positively interact with ET to activate the responses. Additionally, ET can influence auxin accumulation and distribution and the expression of some microRNAs (see text for details). Yellow background indicates signals that activate responses, orange background indicates signals that repress responses. AHP, Arabidopsis His Phosphotransfer; ARR, Arabidopsis Response Regulators; ─╢, inhibition; →, promotion. Figure 4. Open in new tabDownload slide Working model to explain the role of ethylene (ET) on the regulation of responses to different nutrient deficiencies in plants. Nutrient deficiencies can enhance ET production by up-regulating SAMS, ACS, and ACO genes (Shin and Schachtman, 2004; García et al., 2010; Hermans et al., 2010), although the steps leading to this up-regulation are not yet clear. A possibility is that, at first, nutrient deficiencies cause oxidative stress and consequently, ROS accumulation and possibly, NO accumulation (Shin et al., 2005; Tewari et al., 2006; Graziano and Lamattina, 2007; Ahlfors et al., 2009; Jung et al., 2009; García et al., 2011; Iqbal et al., 2013; Steffens, 2014). This ROS-NO accumulation would stimulate ET production, which in turn, could increase ROS-NO production (Ahlfors et al., 2009; Jung et al., 2009; García et al., 2011; Iqbal et al., 2013) in a positive feedback loop, leading to enhancement of the nutrient deficiency signal (García et al., 2011). When ET is perceived by the receptors, it could act through a CTR1-EIN2-dependent pathway for the regulation of some responses or a CTR1-EIN2-independent pathway for the regulation of other responses (Fig. 3). At the end of these transduction pathways, different ET-related transcription factors, such as RAP2.11, ERF070, EIL3, EIN3/EIL1, or others, would activate different nutrient responses (Maruyama-Nakashita et al., 2006; Kim et al., 2012; Ramaiah et al., 2014; Yang et al., 2014). Under nutrient sufficiency, several signals that can move through the phloem (mineral ions, peptides, GSH, etc.; Lappartient et al., 1999; García et al., 2013; Zhang et al., 2014) could negatively interact with ET to inactivate nutrient responses. However, under nutrient deficiency, other signals that can move through the phloem (microRNAs, auxin, sugars, etc.; Kasajima et al., 2007; Lejay et al., 2008; Hammond and White, 2011; Lei and Liu, 2011; Hu et al., 2015) could positively interact with ET to activate the responses. Additionally, ET can influence auxin accumulation and distribution and the expression of some microRNAs (see text for details). Yellow background indicates signals that activate responses, orange background indicates signals that repress responses. AHP, Arabidopsis His Phosphotransfer; ARR, Arabidopsis Response Regulators; ─╢, inhibition; →, promotion. Signals Interacting with Ethylene for the Regulation of Morphological Responses One of the signals most closely related to ethylene for the regulation of morphological responses is auxin. In supporting this view, it should be noted that subapical root hairs (Schmidt and Schikora, 2001; Zhang et al., 2003; Romera et al., 2007; Martín-Rejano et al., 2011), transfer cells (Schmidt et al., 2000a; Schikora and Schmidt, 2002), and cluster roots (Zaid et al., 2003; Wang et al., 2014b) are similarly affected by either ethylene or auxin treatments. In the same way, lateral root formation and inhibition of root elongation under different deficiencies (“Ethylene Participation in Nutrient Deficiency Responses”) have been associated with auxin and ethylene (Zhang et al., 2003; López-Bucio et al., 2003; Miura et al., 2011; Muday et al., 2012; Chérel et al., 2014). Both hormones synergistically inhibit root elongation and play an antagonistic role on lateral root formation, where auxin stimulates while ethylene inhibits it (López-Bucio et al., 2003; Miura et al., 2011; Muday et al., 2012; Chérel et al., 2014; Wang et al., 2014b). Ethylene increases rootward auxin transport by up-regulating PIN-FORMED3 (PIN3) and PIN7 (auxin efflux carriers) in the central cylinder, which may deplete the lateral root-forming zone of auxin while increasing auxin accumulation in the root apex. This effect may be responsible for the negative regulation of lateral root formation by ethylene (Muday et al., 2012; Chérel et al., 2014). At the root tip, up-regulation of AUXIN RESISTANT1 (AUX1; auxin influx carrier) and PIN2 (auxin efflux carrier) enhances shootward auxin transport into the elongation zone, thereby reducing primary root elongation and promoting the development of subapical root hairs by up-regulating root hair-specific genes (Muday et al., 2012; Lee and Cho, 2013). Strigolactones are other hormones that participate in the regulation of root hair elongation, and their role has been related to interactions with auxin and ethylene (Kapulnik et al., 2011). NO has also been implicated in the development of subapical root hairs (Graziano and Lamattina, 2007) and cluster roots (Wang et al., 2010) under Fe or P deficiency. Similarly, ROS has also been implicated in the development of subapical root hairs under K, N, and P deficiency (Shin et al., 2005; Jung et al., 2009). As previously described, both NO and ROS can influence the production of ethylene and vice versa. Moreover, ACC and ethephon induce NO and ROS accumulation in the subapical region of the roots, where subapical root hairs develop (Jung et al., 2009; García et al., 2011). Signals Interacting with Ethylene for the Regulation of Physiological Responses Different signals could interact with ethylene to activate or suppress physiological responses depending on the nutrient status of the plants. Under nutrient sufficiency, several nutrient-related repressive signals, some of them moving through the phloem (e.g. mineral ions, peptides, and GSH), could negatively interact with ethylene to inactivate nutrient responses (Fig. 4; Lappartient et al., 1999; García et al., 2013; Zhang et al., 2014). It has been proposed that some Fe compound moves through the phloem (probably an Fe peptide) and could negatively interact with ethylene signaling to regulate Fe physiological responses (García et al., 2013; Romera et al., 2015). Similarly, GSH moving in the phloem has been described as a suppressor of some S physiological responses (Lappartient et al., 1999), and GSH could negatively interact with ethylene signaling (Chen et al., 2013). Cytokinins have also been described as negative regulators of several physiological responses to nutrient deficiencies (Rubio et al., 2009; Hammond and White, 2011), although their exact relationship with ethylene will require additional research. Under nutrient deficiency, some signals, such as auxin, NO, ROS, and sugars, have been described as activators of physiological responses to different deficiencies (Graziano and Lamattina, 2007; Kasajima et al., 2007; Lejay et al., 2008; Jung et al., 2009; García et al., 2010, 2011; Wang et al., 2010; Hammond and White, 2011; Kiba et al., 2011; Lei and Liu, 2011; Zhang et al., 2014). Because these signals can interact with ethylene in several ways (see above), it is possible that the roles of all of them on the activation of the responses could be tightly interrelated (Fig. 4; Jung et al., 2009; Romera et al., 2011, 2015). Under nutrient deficiency, some nutrient-related signals that can move through the phloem, like microRNAs (Buhtz et al., 2010), have also been described as activators of many physiological responses (Zeng et al., 2014; Hu et al., 2015). Many microRNAs increase their expression under nutrient deficiencies: as examples, microRNA399 (miRNA399) is strongly induced under P, Fe, or K deficiency; miR827 is strongly induced under P deficiency; miR395 is strongly induced under S deficiency; miR158 is strongly induced under Fe deficiency; and miR397, miR398, and miR857 are strongly induced under Cu deficiency (Buhtz et al., 2010; Kawashima et al., 2011; Waters et al., 2012; Lin et al., 2013; Zeng et al., 2014; Hu et al., 2015). They usually down-regulate the expression of target genes by posttranscriptional cleavage (Kawashima et al., 2011; Zeng et al., 2014; Hu et al., 2015). The down-regulation of some target genes, such as Arabidopsis Phosphate2 (AtPHO2; encoding an E2 ubiquitin conjugase) or Arabidopsis ATP Sulfurylase4 (AtAPS4; encoding an ATP sulfurylase), caused by miR399 or miR395, respectively, leads to the accumulation of P or S in shoots and the prevention of their transport in the phloem from shoots to roots (Dong et al., 1998; Liang et al., 2010; Kawashima et al., 2011; Zhang et al., 2014). This suggests that some microRNAs could restrict the movement of nutrient-related repressive signals from shoots to roots, thereby positively interacting with ethylene in the activation of the responses (Fig. 4). Moreover, ethylene could potentiate the effects of some microRNAs. As an example, miR395 is induced by S deficiency in a SLIM1(EIL3)-ethylene-dependent manner (Fig. 4; Kawashima et al., 2011). CONCLUSION In addition to the participation of ethylene in the regulation of Fe and P deficiency responses, its role has been extended to other deficiencies. This implies that it acts as a general coordinator of many nutrient deficiency responses. Its participation in so many deficiencies suggests that, to confer specificity to the different responses, ethylene should act through different transduction pathways and/or in conjunction with other signals. Its interaction with some of these signals, such as auxin and NO, is partly known. However, its interaction with other signals, such as peptides, microRNAs, and GSH-related compounds moving in the phloem, is practically unknown. Deeper research is required in the near future to clarify the shared steps related to ethylene signaling for the different responses and the signaling steps specific for each response. In addition, it would be necessary to extend research on the role of ethylene to other nutrient deficiency responses not studied yet. ACKNOWLEDGMENTS We thank Dong Liu (School of Life Sciences, Tsinghua University) for permission to elaborate figure 2 and Brian M. Waters (University of Nebraska) and Jon Shaff (Robert Holley Center for Agriculture and Health) for English correction of the article. Glossary ACC 1-aminocyclopropane-1-carboxylic acid GSH reduced glutathione NO nitric oxide ROS reactive oxygen species RSA root system architecture STS silver thiosulfate LITERATURE CITED Ahlfors R , Brosché M, Kangasjärvi J ( 2009 ) Ozone and nitric oxide interaction in Arabidopsis thaliana: a role for ethylene? Plant Signal Behav 4 : 878 – 879 Google Scholar Crossref Search ADS PubMed WorldCat Benlloch-González M , Romera J, Cristescu S, Harren F, Fournier JM, Benlloch M ( 2010 ) K+ starvation inhibits water-stress-induced stomatal closure via ethylene synthesis in sunflower plants . J Exp Bot 61 : 1139 – 1145 Google Scholar Crossref Search ADS PubMed WorldCat Borch K , Bouma TJ, Lynch JP, Brown KM ( 1999 ) Ethylene: a regulator of root architectural responses to soil phosphorus availability . Plant Cell Environ 22 : 425 – 431 Google Scholar Crossref Search ADS WorldCat Brumbarova T , Bauer P ( 2005 ) Iron-mediated control of the basic helix-loop-helix protein FER, a regulator of iron uptake in tomato . Plant Physiol 137 : 1018 – 1026 Google Scholar Crossref Search ADS PubMed WorldCat Buhtz A , Pieritz J, Springer F, Kehr J ( 2010 ) Phloem small RNAs, nutrient stress responses, and systemic mobility . BMC Plant Biol 10 : 64 Google Scholar Crossref Search ADS PubMed WorldCat Chen HJ , Huang CS, Huang GJ, Chow TJ, Lin YH ( 2013 ) NADPH oxidase inhibitor diphenyleneiodonium and reduced glutathione mitigate ethephon-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato (Ipomoea batatas) . J Plant Physiol 170 : 1471 – 1483 Google Scholar Crossref Search ADS PubMed WorldCat Chérel I , Lefoulon C, Boeglin M, Sentenac H ( 2014 ) Molecular mechanisms involved in plant adaptation to low K(+) availability . J Exp Bot 65 : 833 – 848 Google Scholar Crossref Search ADS PubMed WorldCat Colangelo EP , Guerinot ML ( 2004 ) The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response . Plant Cell 16 : 3400 – 3412 Google Scholar Crossref Search ADS PubMed WorldCat Dong B , Rengel Z, Delhaize E ( 1998 ) Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of Arabidopsis thaliana . Planta 205 : 251 – 256 Google Scholar Crossref Search ADS PubMed WorldCat García MJ , García-Mateo MJ, Lucena C, Romera FJ, Rojas CL, Alcántara E, Pérez-Vicente R ( 2014 ) Hypoxia and bicarbonate could limit the expression of iron acquisition genes in Strategy I plants by affecting ethylene synthesis and signaling in different ways . Physiol Plant 150 : 95 – 106 Google Scholar Crossref Search ADS PubMed WorldCat García MJ , Lucena C, Romera FJ, Alcántara E, Pérez-Vicente R ( 2010 ) Ethylene and nitric oxide involvement in the up-regulation of key genes related to iron acquisition and homeostasis in Arabidopsis . J Exp Bot 61 : 3885 – 3899 Google Scholar Crossref Search ADS PubMed WorldCat García MJ , Romera FJ, Pérez-Vicente R, Lucena C, Alcántara E ( 2007 ) Ferric reductase and iron transporter gene expression in different Arabidopsis ethylene mutants . In A Ramina , C Chang , J Giovannoni , H Klee , P Perata , E Woltering , eds, Advances in Plant Ethylene Research. Springer , Dordrecht, The Netherlands , pp 401 – 403 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC García MJ , Romera FJ, Stacey MG, Stacey G, Villar E, Alcántara E, Pérez-Vicente R ( 2013 ) Shoot to root communication is necessary to control the expression of iron-acquisition genes in Strategy I plants . Planta 237 : 65 – 75 Google Scholar Crossref Search ADS PubMed WorldCat García MJ , Suárez V, Romera FJ, Alcántara E, Pérez-Vicente R ( 2011 ) A new model involving ethylene, nitric oxide and Fe to explain the regulation of Fe-acquisition genes in Strategy I plants . Plant Physiol Biochem 49 : 537 – 544 Google Scholar Crossref Search ADS PubMed WorldCat Gilbert GA , Knight JD, Vance CP, Allan DL ( 2000 ) Proteoid root development of phosphorus deficient lupin in mimicked by auxin and phosphonate . Ann Bot 85 : 921 – 928 Google Scholar Crossref Search ADS WorldCat Graziano M , Lamattina L ( 2007 ) Nitric oxide accumulation is required for molecular and physiological responses to iron deficiency in tomato roots . Plant J 52 : 949 – 960 Google Scholar Crossref Search ADS PubMed WorldCat Gruber BD , Giehl RFH, Friedel S, von Wirén N ( 2013 ) Plasticity of the Arabidopsis root system under nutrient deficiencies . Plant Physiol 163 : 161 – 179 Google Scholar Crossref Search ADS PubMed WorldCat Hammond JP , White PJ ( 2011 ) Sugar signaling in root responses to low phosphorus availability . Plant Physiol 156 : 1033 – 1040 Google Scholar Crossref Search ADS PubMed WorldCat He CJ , Morgan PW, Drew MC ( 1992 ) Enhanced sensitivity to ethylene in nitrogen- or phosphate-starved roots of Zea mays L. during aerenchyma formation . Plant Physiol 98 : 137 – 142 Google Scholar Crossref Search ADS PubMed WorldCat Hermans C , Vuylsteke M, Coppens F, Cristescu SM, Harren FJ, Inzé D, Verbruggen N ( 2010 ) Systems analysis of the responses to long-term magnesium deficiency and restoration in Arabidopsis thaliana . New Phytol 187 : 132 – 144 Google Scholar Crossref Search ADS PubMed WorldCat Hernández G , Ramírez M, Valdés-López O, Tesfaye M, Graham MA, Czechowski T, Schlereth A, Wandrey M, Erban A, Cheung F, et al. ( 2007 ) Phosphorus stress in common bean: root transcript and metabolic responses . Plant Physiol 144 : 752 – 767 Google Scholar Crossref Search ADS PubMed WorldCat Hu B , Wang W, Deng K, Li H, Zhang Z, Zhang L, Chu C ( 2015 ) MicroRNA399 is involved in multiple nutrient starvation responses in rice . Front Plant Sci 6 : 188 Google Scholar PubMed OpenURL Placeholder Text WorldCat Iqbal N , Khan NA, Nazar R, Teixeira da Silva JA ( 2012 ) Ethylene-stimulated photosynthesis results from increased nitrogen and sulfur assimilation in mustard types that differ in photosynthetic capacity . Environ Exp Bot 78 : 84 – 90 Google Scholar Crossref Search ADS WorldCat Iqbal N , Trivellini A, Masood A, Ferrante A, Khan NA ( 2013 ) Current understanding on ethylene signaling in plants: the influence of nutrient availability . Plant Physiol Biochem 73 : 128 – 138 Google Scholar Crossref Search ADS PubMed WorldCat Jung JY , Shin R, Schachtman DP ( 2009 ) Ethylene mediates response and tolerance to potassium deprivation in Arabidopsis . Plant Cell 21 : 607 – 621 Google Scholar Crossref Search ADS PubMed WorldCat Kapulnik Y , Resnick N, Mayzlish-Gati E, Kaplan Y, Wininger S, Hershenhorn J, Koltai H ( 2011 ) Strigolactones interact with ethylene and auxin in regulating root-hair elongation in Arabidopsis . J Exp Bot 62 : 2915 – 2924 Google Scholar Crossref Search ADS PubMed WorldCat Karve A , Xia X, Moore Bd ( 2012 ) Arabidopsis Hexokinase-Like1 and Hexokinase1 form a critical node in mediating plant glucose and ethylene responses . Plant Physiol 158 : 1965 – 1975 Google Scholar Crossref Search ADS PubMed WorldCat Kasajima I , Ohkama-Ohtsu N, Ide Y, Hayashi H, Yoneyama T, Suzuki Y, Naito S, Fujiwara T ( 2007 ) The BIG gene is involved in regulation of sulfur deficiency-responsive genes in Arabidopsis thaliana . Physiol Plant 129 : 351 – 363 Google Scholar Crossref Search ADS WorldCat Kawashima CG , Matthewman CA, Huang S, Lee BR, Yoshimoto N, Koprivova A, Rubio-Somoza I, Todesco M, Rathjen T, Saito K, et al. ( 2011 ) Interplay of SLIM1 and miR395 in the regulation of sulfate assimilation in Arabidopsis . Plant J 66 : 863 – 876 Google Scholar Crossref Search ADS PubMed WorldCat Kiba T , Kudo T, Kojima M, Sakakibara H ( 2011 ) Hormonal control of nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin . J Exp Bot 62 : 1399 – 1409 Google Scholar Crossref Search ADS PubMed WorldCat Kim HJ , Lynch JP, Brown KM ( 2008 ) Ethylene insensitivity impedes a subset of responses to phosphorus deficiency in tomato and petunia . Plant Cell Environ 31 : 1744 – 1755 Google Scholar Crossref Search ADS PubMed WorldCat Kim MJ , Ruzicka D, Shin R, Schachtman DP ( 2012 ) The Arabidopsis AP2/ERF transcription factor RAP2.11 modulates plant response to low-potassium conditions . Mol Plant 5 : 1042 – 1057 Google Scholar Crossref Search ADS PubMed WorldCat Koprivova A , North KA, Kopriva S ( 2008 ) Complex signaling network in regulation of adenosine 5′-phosphosulfate reductase by salt stress in Arabidopsis roots . Plant Physiol 146 : 1408 – 1420 Google Scholar Crossref Search ADS PubMed WorldCat Kramer D , Römheld V, Landsberg E, Marschner H ( 1980 ) Induction of transfer-cell formation by iron deficiency in the root epidermis of Helianthus annuus L . Planta 147 : 335 – 339 Google Scholar Crossref Search ADS PubMed WorldCat Lappartient AG , Vidmar JJ, Leustek T, Glass ADM, Touraine B ( 1999 ) Inter-organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloem-translocated compound . Plant J 18 : 89 – 95 Google Scholar Crossref Search ADS PubMed WorldCat Lee RDW , Cho HT ( 2013 ) Auxin, the organizer of the hormonal/environmental signals for root hair growth . Front Plant Sci 4 : 448 Google Scholar PubMed OpenURL Placeholder Text WorldCat Lei M , Liu D ( 2011 ) Sucrose regulates plant responses to deficiencies in multiple nutrients . Plant Signal Behav 6 : 1247 – 1249 Google Scholar Crossref Search ADS PubMed WorldCat Lei M , Zhu C, Liu Y, Karthikeyan AS, Bressan RA, Raghothama KG, Liu D ( 2011 ) Ethylene signalling is involved in regulation of phosphate starvation-induced gene expression and production of acid phosphatases and anthocyanin in Arabidopsis . New Phytol 189 : 1084 – 1095 Google Scholar Crossref Search ADS PubMed WorldCat Lejay L , Wirth J, Pervent M, Cross JMF, Tillard P, Gojon A ( 2008 ) Oxidative pentose phosphate pathway-dependent sugar sensing as a mechanism for regulation of root ion transporters by photosynthesis . Plant Physiol 146 : 2036 – 2053 Google Scholar Crossref Search ADS PubMed WorldCat Li YS , Gao Y, Tian QY, Shi FL, Li LH, Zhang WH ( 2011 ) Stimulation of root acid phosphatase by phosphorus deficiency is regulated by ethylene in Medicago falcata . Environ Exp Bot 71 : 114 – 120 Google Scholar Crossref Search ADS WorldCat Li YS , Mao XT, Tian QY, Li LH, Zhang WH ( 2009 ) Phosphorus deficiency-induced reduction in root hydraulic conductivity in Medicago falcata is associated with ethylene production . Environ Exp Bot 67 : 172 – 177 Google Scholar Crossref Search ADS WorldCat Liang G , Yang F, Yu D ( 2010 ) MicroRNA395 mediates regulation of sulfate accumulation and allocation in Arabidopsis thaliana . Plant J 62 : 1046 – 1057 Google Scholar PubMed OpenURL Placeholder Text WorldCat Lin WY , Huang TK, Chiou TJ ( 2013 ) Nitrogen limitation adaptation, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis . Plant Cell 25 : 4061 – 4074 Google Scholar Crossref Search ADS PubMed WorldCat Liu TY , Chang CY, Chiou TJ ( 2009 ) The long-distance signaling of mineral macronutrients . Curr Opin Plant Biol 12 : 312 – 319 Google Scholar Crossref Search ADS PubMed WorldCat López-Bucio J , Cruz-Ramírez A, Herrera-Estrella L ( 2003 ) The role of nutrient availability in regulating root architecture . Curr Opin Plant Biol 6 : 280 – 287 Google Scholar Crossref Search ADS PubMed WorldCat Lucena C , Waters BM, Romera FJ, García MJ, Morales M, Alcántara E, Pérez-Vicente R ( 2006 ) Ethylene could influence ferric reductase, iron transporter, and H+-ATPase gene expression by affecting FER (or FER-like) gene activity . J Exp Bot 57 : 4145 – 4154 Google Scholar Crossref Search ADS PubMed WorldCat Lynch J , Brown KM ( 1997 ) Ethylene and plant responses to nutritional stress . Physiol Plant 100 : 613 – 619 Google Scholar Crossref Search ADS WorldCat Ma Z , Baskin TI, Brown KM, Lynch JP ( 2003 ) Regulation of root elongation under phosphorus stress involves changes in ethylene responsiveness . Plant Physiol 131 : 1381 – 1390 Google Scholar Crossref Search ADS PubMed WorldCat Maksymiec W ( 2007 ) Signaling responses in plants to heavy metal stress . Acta Physiol Plant 29 : 177 – 187 Google Scholar Crossref Search ADS WorldCat Martín-Rejano EM , Camacho-Cristóbal JJ, Herrera-Rodríguez MB, Rexach J, Navarro-Gochicoa MT, González-Fontes A ( 2011 ) Auxin and ethylene are involved in the responses of root system architecture to low boron supply in Arabidopsis seedlings . Physiol Plant 142 : 170 – 178 Google Scholar Crossref Search ADS PubMed WorldCat Maruyama-Nakashita A , Nakamura Y, Tohge T, Saito K, Takahashi H ( 2006 ) Arabidopsis SLIM1 is a central transcriptional regulator of plant sulfur response and metabolism . Plant Cell 18 : 3235 – 3251 Google Scholar Crossref Search ADS PubMed WorldCat Miura K , Lee J, Gong Q, Ma S, Jin JB, Yoo CY, Miura T, Sato A, Bohnert HJ, Hasegawa PM ( 2011 ) SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation . Plant Physiol 155 : 1000 – 1012 Google Scholar Crossref Search ADS PubMed WorldCat Mohd-Radzman NA , Djordjevic MA, Imin N ( 2013 ) Nitrogen modulation of legume root architecture signaling pathways involves phytohormones and small regulatory molecules . Front Plant Sci 4 : 385 Google Scholar Crossref Search ADS PubMed WorldCat Moniuszko G , Skoneczny M, Zientara-Rytter K, Wawrzyńska A, Głów D, Cristescu SM, Harren FJM, Sirko A ( 2013 ) Tobacco LSU-like protein couples sulphur-deficiency response with ethylene signalling pathway . J Exp Bot 64 : 5173 – 5182 Google Scholar Crossref Search ADS PubMed WorldCat Muday GK , Rahman A, Binder BM ( 2012 ) Auxin and ethylene: collaborators or competitors? Trends Plant Sci 17 : 181 – 195 Google Scholar Crossref Search ADS PubMed WorldCat Nagarajan VK , Smith AP ( 2012 ) Ethylene’s role in phosphate starvation signaling: more than just a root growth regulator . Plant Cell Physiol 53 : 277 – 286 Google Scholar Crossref Search ADS PubMed WorldCat Nikiforova V , Freitag J, Kempa S, Adamik M, Hesse H, Hoefgen R ( 2003 ) Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: interlacing of biosynthetic pathways provides response specificity . Plant J 33 : 633 – 650 Google Scholar Crossref Search ADS PubMed WorldCat O’Rourke JA , Charlson DV, Gonzalez DO, Vodkin LO, Graham MA, Cianzio SR, Grusak MA, Shoemaker RC ( 2007 ) Microarray analysis of iron deficiency chlorosis in near-isogenic soybean lines . BMC Genomics 8 : 476 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC O’Rourke JA , Yang SS, Miller SS, Bucciarelli B, Liu J, Rydeen A, Bozsoki Z, Uhde-Stone C, Tu ZJ, Allan D, et al. ( 2013 ) An RNA-Seq transcriptome analysis of orthophosphate-deficient white lupin reveals novel insights into phosphorus acclimation in plants . Plant Physiol 161 : 705 – 724 Google Scholar Crossref Search ADS PubMed WorldCat Ramaiah M , Jain A, Raghothama KG ( 2014 ) ETHYLENE RESPONSE FACTOR070 regulates root development and phosphate starvation-mediated responses . Plant Physiol 164 : 1484 – 1498 Google Scholar Crossref Search ADS PubMed WorldCat Romera FJ , Alcántara E ( 2004 ) Ethylene involvement in the regulation of Fe-deficiency stress responses by Strategy I plants . Funct Plant Biol 31 : 315 – 328 Google Scholar Crossref Search ADS PubMed WorldCat Romera FJ , Alcántara E ( 1994 ) Iron-deficiency stress responses in cucumber (Cucumis sativus L.) roots (a possible role for ethylene?) . Plant Physiol 105 : 1133 – 1138 Google Scholar Crossref Search ADS PubMed WorldCat Romera FJ , Alcántara E, de la Guardia MD ( 1999 ) Ethylene production by Fe-deficient roots and its involvement in the regulation of Fe-deficiency stress responses by Strategy I plants . Ann Bot 83 : 51 – 55 Google Scholar Crossref Search ADS WorldCat Romera FJ , García MJ, Alcántara E, Pérez-Vicente R ( 2011 ) Latest findings about the interplay of auxin, ethylene and nitric oxide in the regulation of Fe deficiency responses by Strategy I plants . Plant Signal Behav 6 : 167 – 170 Google Scholar Crossref Search ADS PubMed WorldCat Romera FJ , Lucena C, Alcántara E ( 2007 ) Plant hormones influencing iron uptake in plants . In LL Barton , J Abadía , eds, Iron Nutrition in Plants and Rhizospheric Microorganisms. Springer , Dordrecht, The Netherlands , pp 251 – 278 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Romera FJ , Lucena C, García MJ, Alcántara E, Pérez-Vicente R ( 2015 ) The role of ethylene and other signals in the regulation of Fe deficiency responses by dicot plants . In M Sarwat , ed, Stress Signaling in Plants: Genomics and Proteomics Perspectives , Vol 2. Springer , Dordrecht, The Netherlands Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Rubio V , Bustos R, Irigoyen ML, Cardona-López X, Rojas-Triana M, Paz-Ares J ( 2009 ) Plant hormones and nutrient signaling . Plant Mol Biol 69 : 361 – 373 Google Scholar Crossref Search ADS PubMed WorldCat Sauter M , Moffatt B, Saechao MC, Hell R, Wirtz M ( 2013 ) Methionine salvage and S-adenosylmethionine: essential links between sulfur, ethylene and polyamine biosynthesis . Biochem J 451 : 145 – 154 Google Scholar Crossref Search ADS PubMed WorldCat Schikora A , Schmidt W ( 2002 ) Formation of transfer cells and H(+)-ATPase expression in tomato roots under P and Fe deficiency . Planta 215 : 304 – 311 Google Scholar Crossref Search ADS PubMed WorldCat Schmidt W , Schikora A ( 2001 ) Different pathways are involved in phosphate and iron stress-induced alterations of root epidermal cell development . Plant Physiol 125 : 2078 – 2084 Google Scholar Crossref Search ADS PubMed WorldCat Schmidt W , Schikora A, Pich A, Bartels M ( 2000 a ) Hormones induce an Fe-deficiency-like root epidermal cell pattern in the Fe-inefficient tomato mutant fer . Protoplasma 213 : 67 – 73 Google Scholar Crossref Search ADS WorldCat Schmidt W , Tittel J, Schikora A ( 2000 b ) Role of hormones in the induction of iron deficiency responses in Arabidopsis roots . Plant Physiol 122 : 1109 – 1118 Google Scholar Crossref Search ADS PubMed WorldCat Shakeel SN , Wang X, Binder BM, Schaller GE ( 2013 ) Mechanisms of signal transduction by ethylene: overlapping and non-overlapping signalling roles in a receptor family . AoB Plants 5 : plt010 Google Scholar Crossref Search ADS PubMed WorldCat Shin R , Berg RH, Schachtman DP ( 2005 ) Reactive oxygen species and root hairs in Arabidopsis root response to nitrogen, phosphorus and potassium deficiency . Plant Cell Physiol 46 : 1350 – 1357 Google Scholar Crossref Search ADS PubMed WorldCat Shin R , Schachtman DP ( 2004 ) Hydrogen peroxide mediates plant root cell response to nutrient deprivation . Proc Natl Acad Sci USA 101 : 8827 – 8832 Google Scholar Crossref Search ADS PubMed WorldCat Steffens B ( 2014 ) The role of ethylene and ROS in salinity, heavy metal, and flooding responses in rice . Front Plant Sci 5 : 685 Google Scholar Crossref Search ADS PubMed WorldCat Tewari RK , Kumar P, Sharma PN ( 2006 ) Magnesium deficiency induced oxidative stress and antioxidant responses in mulberry plants . Sci Hortic (Amsterdam) 108 : 7 – 14 Google Scholar Crossref Search ADS WorldCat Thirugnanasambantham K , Durairaj S, Saravanan S, Karikalan K, Muralidaran S, Islam VIH ( 2015 ) Role of Ethylene Response Transcription Factor (ERF) and its regulation in response to stress encountered by plants . Plant Mol Biol Rep 33 : 347 – 357 Google Scholar Crossref Search ADS WorldCat Tian QY , Sun P, Zhang WH ( 2009 ) Ethylene is involved in nitrate-dependent root growth and branching in Arabidopsis thaliana . New Phytol 184 : 918 – 931 Google Scholar Crossref Search ADS PubMed WorldCat Wang BL , Tang XY, Cheng LY, Zhang AZ, Zhang WH, Zhang FS, Liu JQ, Cao Y, Allan DL, Vance CP, et al. ( 2010 ) Nitric oxide is involved in phosphorus deficiency-induced cluster-root development and citrate exudation in white lupin . New Phytol 187 : 1112 – 1123 Google Scholar Crossref Search ADS PubMed WorldCat Wang F , Cui X, Sun Y, Dong CH ( 2013 ) Ethylene signaling and regulation in plant growth and stress responses . Plant Cell Rep 32 : 1099 – 1109 Google Scholar Crossref Search ADS PubMed WorldCat Wang J , Wang Y, Yang J, Ma C, Zhang Y, Ge T, Qi Z, Kang Y ( 2015 ) Arabidopsis ROOT HAIR DEFECTIVE3 is involved in nitrogen starvation-induced anthocyanin accumulation . J Integr Plant Biol 57 : 708 – 721 Google Scholar Crossref Search ADS PubMed WorldCat Wang L , Dong J, Gao Z, Liu D ( 2012 ) The Arabidopsis gene hypersensitive to phosphate starvation 3 encodes ethylene overproduction 1 . Plant Cell Physiol 53 : 1093 – 1105 Google Scholar Crossref Search ADS PubMed WorldCat Wang S , Lu B, Wu T, Zhang X, Xu X, Han Z, Wang Y ( 2014 a ) Transcriptomic analysis demonstrates the early responses of local ethylene and redox signaling to low iron stress in Malus xiaojinensis . Tree Genet Genomes 10 : 573 – 584 Google Scholar Crossref Search ADS WorldCat Wang Z , Straub D, Yang H, Kania A, Shen J, Ludewig U, Neumann G ( 2014 b ) The regulatory network of cluster-root function and development in phosphate-deficient white lupin (Lupinus albus) identified by transcriptome sequencing . Physiol Plant 151 : 323 – 338 Google Scholar Crossref Search ADS PubMed WorldCat Waters BM , Blevins DG ( 2000 ) Ethylene production, cluster root formation, and localization of iron(III) reducing capacity in Fe deficient squash roots . Plant Soil 225 : 21 – 31 Google Scholar Crossref Search ADS WorldCat Waters BM , Lucena C, Romera FJ, Jester GG, Wynn AN, Rojas CL, Alcántara E, Pérez-Vicente R ( 2007 ) Ethylene involvement in the regulation of the H(+)-ATPase CsHA1 gene and of the new isolated ferric reductase CsFRO1 and iron transporter CsIRT1 genes in cucumber plants . Plant Physiol Biochem 45 : 293 – 301 Google Scholar Crossref Search ADS PubMed WorldCat Waters BM , McInturf SA, Stein RJ ( 2012 ) Rosette iron deficiency transcript and microRNA profiling reveals links between copper and iron homeostasis in Arabidopsis thaliana . J Exp Bot 63 : 5903 – 5918 Google Scholar Crossref Search ADS PubMed WorldCat Wawrzyńska A , Lewandowska M, Sirko A ( 2010 ) Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene induced by sulphur starvation . J Exp Bot 61 : 889 – 900 Google Scholar Crossref Search ADS PubMed WorldCat Yang Y , Ou B, Zhang J, Si W, Gu H, Qin G, Qu LJ ( 2014 ) The Arabidopsis Mediator subunit MED16 regulates iron homeostasis by associating with EIN3/EIL1 through subunit MED25 . Plant J 77 : 838 – 851 Google Scholar Crossref Search ADS PubMed WorldCat Zaid H , El Morabet R, Diem HG, Arahou M ( 2003 ) Does ethylene mediate cluster root formation under iron deficiency? Ann Bot (Lond) 92 : 673 – 677 Google Scholar Crossref Search ADS WorldCat Zeng H , Wang G, Hu X, Wang H, Du L, Zhu Y ( 2014 ) Role of microRNAs in plant responses to nutrient stress . Plant Soil 374 : 1005 – 1021 Google Scholar Crossref Search ADS WorldCat Zhang YJ , Lynch JP, Brown KM ( 2003 ) Ethylene and phosphorus availability have interacting yet distinct effects on root hair development . J Exp Bot 54 : 2351 – 2361 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Z , Liao H, Lucas WJ ( 2014 ) Molecular mechanisms underlying phosphate sensing, signaling, and adaptation in plants . J Integr Plant Biol 56 : 192 – 220 Google Scholar Crossref Search ADS PubMed WorldCat Zhao W , Yang X, Yu H, Jiang W, Sun N, Liu X, Liu X, Zhang X, Wang Y, Gu X ( 2015 ) RNA-Seq-based transcriptome profiling of early nitrogen deficiency response in cucumber seedlings provides new insight into the putative nitrogen regulatory network . Plant Cell Physiol 56 : 455 – 467 Google Scholar Crossref Search ADS PubMed WorldCat Zheng D , Han X, An YI, Guo H, Xia X, Yin W ( 2013 ) The nitrate transporter NRT2.1 functions in the ethylene response to nitrate deficiency in Arabidopsis . Plant Cell Environ 36 : 1328 – 1337 Google Scholar Crossref Search ADS PubMed WorldCat Zuchi S , Cesco S, Varanini Z, Pinton R, Astolfi S ( 2009 ) Sulphur deprivation limits Fe-deficiency responses in tomato plants . Planta 230 : 85 – 94 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the European Union (European Regional Development Fund), the Ministerio de Economía y Competitividad (project no. AGL2013–40822–R), and the Junta de Andalucía (Research Groups AGR115 and BIO159). * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00708 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Van de Poel, Bram; Smet, Dajo; Van Der Straeten, Dominique

doi: 10.1104/pp.15.00724pmid: 26232489

Abstract Ethylene is a gaseous plant hormone that most likely became a functional hormone during the evolution of charophyte green algae, prior to land colonization. From this ancient origin, ethylene evolved into an important growth regulator that is essential for myriad plant developmental processes. In vegetative growth, ethylene appears to have a dual role, stimulating and inhibiting growth, depending on the species, tissue, and cell type, developmental stage, hormonal status, and environmental conditions. Moreover, ethylene signaling and response are part of an intricate network in cross talk with internal and external cues. Besides being a crucial factor in the growth control of roots and shoots, ethylene can promote flowering, fruit ripening and abscission, as well as leaf and petal senescence and abscission and, hence, plays a role in virtually every phase of plant life. Last but not least, together with jasmonates, salicylate, and abscisic acid, ethylene is important in steering stress responses. This Update provides recent insights into the role of ethylene on vegetative growth, both at the cellular and the whole-plant levels, with special attention to hormonal cross talk. Due to space restrictions, this Update is mainly focused on Arabidopsis (Arabidopsis thaliana). CELLULAR RESPONSES: ETHYLENE IN THE CONTROL OF CELL DIVISION, CELL ELONGATION, AND CELL DEATH Ethylene and Cell Division Few reports have addressed the role of ethylene on cell division, revealing opposite actions on the cell cycle, depending on tissue type and internal and external cues. Ethylene stimulates cell division in the subepidermal layers during apical hook development, probably acting in cooperation with auxins (Raz and Koornneef, 2001). The role of ethylene on cell division in roots is somewhat conflicting. Ethylene does not change the expression pattern of the CYCLIN-DEPENDENT PROTEIN KINASE B1;1 (CYCB1;1)-GUS reporter, indicating that it does not affect mitotic activity in the root (Růzicka et al., 2007). Yet, it was shown that ethylene modulates cell division in the stem cell niche of the quiescent center (QC), resulting in supernumerary QC cells that will develop in extra columella layers in the root cap (Ortega-Martínez et al., 2007). Furthermore, root apical meristem (RAM) size and cell number were shown to be controlled by CULLIN3-type E3 ligases in an ethylene-dependent manner (Thomann et al., 2009). Recently, it was confirmed genetically and pharmacologically that ethylene inhibits cell proliferation of the RAM, resulting in smaller meristems (Street et al., 2015). The action of ethylene on RAM size was shown to be mediated by the ethylene receptors signaling primarily via the canonical ethylene signaling pathway (over CONSTITUTIVE TRIPLE RESPONSE1 [CTR1]-ETHYLENE INSENSITIVE2 [EIN2]-EIN3/ETHYLENE INSENSITIVE3-LIKE [EIL]) and in part via an alternative two-component signaling cascade involving ARABIDOPSIS RESPONSE REGULATOR1 (ARR; a B-type ARR transcription factor; Street et al., 2015). Furthermore, in the control of RAM proliferation, ethylene was found to interact with both cytokinins (CKs) and auxin, the latter likely mediated by the auxin repressor SHORT HYPOCOTYL2 (INDOLE ACETIC ACID3; Street et al., 2015). Ethylene also plays a role during the differentiation of vascular tissue, where it controls cell division rate (Etchells et al., 2012). It was shown that ETHYLENE OVERPRODUCER eto1 and eto2 mutations stimulate vascular cell division and ethylene signaling was required for vascular cell differentiation (Etchells et al., 2012). In leaves, ethylene was shown to cause cell cycle arrest upon mild osmotic stress, through the inhibition of CYCLIN-DEPENDENT KINASE A activity and independently from EIN3 transcriptional control (Skirycz et al., 2011). Ethylene also inhibits the mitotic cell cycle in the abaxial cells of the leaf petiole through the down-regulation of CYCLIN2A;1 expression, partially contributing to the hyponastic response (Polko et al., 2015). Ethylene and Cell Elongation Although ethylene is best known for its inhibition of cell elongation both in dark-grown seedlings (as part of the triple response; Bleecker et al., 1988; Guzmán and Ecker, 1990) and in light-grown plants (Rodrigues-Pousada et al., 1993), there are several observations of ethylene-stimulated cell elongation. For example, ethylene induces cell elongation in the hypocotyl of seedlings grown in the light (Smalle et al., 1997) as well as root hair elongation (Pitts et al., 1998) and petiole elongation in certain ecotypes (Millenaar et al., 2005). Cellular elongation is the net result of several processes, including but not limited to the rearrangement of the cytoskeleton, the relaxation of the cell wall, and the uptake of water to establish sufficient turgor pressure. Some of these processes are (at least in part) regulated by ethylene. Modification of the interior cytoskeleton, mainly via the rearrangement of cortical microtubules (CMT), controls the direction of elongation (Bashline et al., 2014). Cell elongation is facilitated by changing the orientation of the CMT perpendicular to the growth axis (Bashline et al., 2014). Ethylene has been shown to rapidly (within 10 min) affect microtubule orientation in Arabidopsis roots and hypocotyls, likely preventing cell elongation and facilitating radial swelling (Le et al., 2004, 2005). The opposite process has been observed in upper hypocotyl cells when grown in the light (Le et al., 2005). Ethylene can also induce petiole elongation as part of a hyponastic growth response by rearranging the CMT from a longitudinal toward a transverse orientation in the abaxial cells (but not the adaxial cells; Polko et al., 2012). Recently, it was shown that auxins, like ethylene, can also rapidly induce the reorientation of CMT to prevent root and hypocotyl elongation (Chen et al., 2014). However, this was challenged by Baskin (2015), who demonstrated that, upon removal of nearly all CMT by an oryzalin treatment, seedlings still showed a strong inhibition of root growth rate by auxin, suggesting that the auxin-induced inhibition of cell expansion is independent from CMT. It is also very likely that auxin and ethylene signal via cross talk to regulate CMT orientation, although this remains to be investigated. Cellular elongation also requires cell wall loosening, which is achieved by rearranging the cell wall matrix polymers with the aid of cell wall-remodeling enzymes (Cosgrove, 2000). Four major groups of cell wall-remodeling enzymes control elongation: expansins (EXP), xyloglucan endotransglycolases/hydrolases (XTH), endo-1,4-β-glucanases, and pectin methylesterases (Sasidharan et al., 2011; Bashline et al., 2014). Of these four, mainly EXP and XTH are involved in ethylene-regulated cellular elongation. In Arabidopsis, expansins have been shown to be ethylene regulated during root hair formation (Cho and Cosgrove, 2002) and the submergence-escape elongation response (Polko et al., 2012; Rauf et al., 2013). During these processes, ethylene induces EXP expression and triggers a local, often tissue-specific, elongation response. It has also been shown that ethylene can regulate XTH expression during root hair initiation (Vissenberg et al., 2001) and the submergence-induced hyponastic response of Arabidopsis (Rauf et al., 2013). Ethylene and Cell Death At given stages in the life cycle of plants, vegetative development requires programmed cell death (PCD). PCD facilitates organ growth and/or abscission as well as defense reactions against biotic and abiotic stresses (Van Hautegem et al., 2015). One of the best known examples of PCD is the formation of xylem cells, called xylogenesis (Bollhöner et al., 2012). It was shown that, during the active phase of xylogenesis, the expression of ethylene biosynthesis genes (both 1-aminocyclopropane-1-carboxylic acid [ACC] synthases [ACS] and ACC oxidases [ACO]) and, consequently, ethylene production were increased, stimulating xylem formation (Andersson-Gunnerås et al., 2003; Love et al., 2009; Pesquet and Tuominen, 2011). Ethylene production and ACO protein abundance were also up-regulated in xylem cells during gravitational bending of woody species (Andersson-Gunnerås et al., 2003), resulting in the modification of xylem cell morphology (Love et al., 2009; Ramos and Herrera, 2013), ultimately leading to the formation of tension wood. Another example of PCD is the formation of aerenchyma, which stimulates gas exchange during hypoxic conditions, typical for semiaquatic species (Drew et al., 2000). Aerenchyma formation was shown to be ethylene regulated in many species, including Arabidopsis (Mühlenbock et al., 2007). ETHYLENE AND GERMINATION A complex balance between plant hormones regulates seed dormancy and germination in response to environmental signals. Ethylene biosynthesis, relying on the activities of ACS and ACO, which convert the precursor ACC to ethylene (Yang and Hoffman, 1984), increases during germination, with a peak production upon radicle protrusion (Corbineau et al., 2014). Although ACS is usually the rate-limiting enzyme, ACO activity plays a crucial role in seed germination. ACO1 and ACO2 are the most important ACOs for ethylene biosynthesis in the Arabidopsis seed (Linkies et al., 2009). Ethylene promotes the germination of primary and secondary dormant seeds and even of nondormant seeds under unfavorable environmental conditions (Corbineau et al., 2014). Consistently, gain-of-function mutations in ETHYLENE RESPONSE1 (ETR1) and loss-of-function mutations of EIN2, which decrease the ethylene response, increased dormancy and postponed germination, whereas mutations in ETO1 and CTR1, which increase ethylene production and response, respectively, reduced dormancy and accelerated germination (Beaudoin et al., 2000; Ghassemian et al., 2000; Chiwocha et al., 2005; Subbiah and Reddy, 2010). A recent study by Wilson et al. (2014b) indicated that, under salt stress, the ethylene receptors ETR1, ETR2, and EIN4 have contrasting functions during Arabidopsis seed germination. Loss of ETR2 delayed germination, while loss of ETR1 and EIN4 advanced germination. However, the involvement of ethylene in the regulation of germination under salt stress by ETR1 and ETR2 appeared to be minor. A difference in ethylene production or sensitivity was not accountable for the contrasting effect on germination upon the loss of ETR1 and ETR2. Furthermore, another study by Wilson et al. (2014a) demonstrated that light, an important trigger of germination, affects the function of ETR1 in germination. In etr1 loss-of-function alleles, germination was promoted in darkness and under far-red light, although not under other light conditions. The latter result and the genetic interaction of ETR1 with PHYTOCHROME A (PHYA) and PHYB suggest that the regulation of germination by ETR1 is mediated by PHYA and PHYB under far-red light. Nevertheless, under white light and in darkness, ETR1 may affect germination independent of PHY signaling. Consistent with Wilson et al. (2014b), ETR1 and ETR2 also had contrasting functions under far-red light, and the involvement of ethylene in the regulation of seed germination by ETR1 again appeared to be minor (Wilson et al., 2014a). Apart from plant hormones, a role for reactive oxygen species (ROS) in the regulation of seed germination emerged. Recently, the accumulation ROS concomitant with radicle protrusion was shown to be required for seed germination (Liu et al., 2010; Leymarie et al., 2012). Ethylene and ROS interplay is involved in the regulation of cell elongation (El-Maarouf-Bouteau et al., 2014), possibly through cell wall remodeling (Linkies and Leubner-Metzger, 2012); however, this needs to be confirmed for Arabidopsis. Increasing evidence in various plant species, including Arabidopsis, suggests that protein modifications represent a putative mechanism for the cross talk between ethylene and the ROS nitric oxide in the regulation of germination (Arc et al., 2013). Ethylene Cross Talk during Germination Seed dormancy and germination are regulated by a complex cross talk of multiple plant hormones. The importance of abscisic acid (ABA) and GAs in the regulation of seed germination has long been established. ABA initiates and maintains seed dormancy, whereas GAs release dormancy and initiate seed germination (Arc et al., 2013; Corbineau et al., 2014; Miransari and Smith, 2014). However, brassinosteroids (BRs), auxins, CKs, and jasmonates (JAs) are also involved in the regulation of seed germination (Linkies and Leubner-Metzger, 2012; Miransari and Smith, 2014). Nevertheless, a possible cross talk with ethylene has not yet been established. Ethylene works antagonistically with ABA but synergistically with GAs in the regulation of Arabidopsis seed germination. The activity of ethylene in the regulation of seed dormancy and germination is based on reciprocal effects on both ABA and GA biosynthesis and signaling (Arc et al., 2013; Corbineau et al., 2014). Upon ABA and GA treatment during germination, ethylene biosynthesis is affected through changes in ACO rather than ACS expression. Furthermore, by means of the ethylene biosynthesis mutant aco2, ethylene production by ACO2 has been demonstrated to hinder the ABA-controlled inhibition of endosperm rupture (Linkies et al., 2009; Linkies and Leubner-Metzger, 2012). Apart from ABA biosynthesis, ethylene also affects the sensitivity to ABA in the regulation of germination (Beaudoin et al., 2000; Ghassemian et al., 2000; Cheng et al., 2009). In the eto3 and ctr1 mutants, ABA sensitivity was significantly reduced, while it was significantly enhanced in ethylene-insensitive alleles of etr1, ein2, and ein6 (Subbiah and Reddy, 2010). Wilson et al., (2014b) demonstrated that loss-of-function etr1 and etr2 mutants have reduced and enhanced ABA sensitivity during salt stress, consistent with their faster and delayed germination, respectively. The function of ETR1 and ETR2 during salt stress thus seems to be mediated by ABA rather than ethylene signaling. Moreover, under far-red light, loss of ETR1 was demonstrated to affect ABA and GA biosynthesis and sensitivity during germination (Wilson et al., 2014a). ETHYLENE AND APICAL HOOK DEVELOPMENT To protect the delicate shoot apical meristem and cotyledons from damage upon soil protrusion after underground germination, the hypocotyl of dicotyledonous seedlings forms a hook-like structure (Darwin and Darwin, 1881; Guzmán and Ecker, 1990). The formation of the apical hook initiates shortly after germination. When the hook is completely closed, it is maintained until exposure to light after emergence from the soil, which heralds its unfolding (Raz and Ecker, 1999). The gradual bending of the hook rests predominantly on differential growth mediated by an auxin gradient, regulated by a complex hormonal cross talk (Fig. 1; Abbas et al., 2013; Mazzella et al., 2014; Žádníková et al., 2015). Figure 1. Open in new tabDownload slide Simplified cross talk diagram on the regulation of the dynamics of apical hook development. Apical hook development proceeds in three distinct phases: formation (F), maintenance (M), and opening (O). Hook formation initiates shortly after germination. After complete formation, the hook is maintained for several hours before opening (the apical hook dynamics shown here are for complete darkness). The formation of the apical hook depends on differential elongation driven by an auxin gradient, which is formed, maintained, and lost concomitant with the three phases of apical hook development. Ethylene plays a central role upstream of auxin. BRs, GAs, and JAs interact with ethylene in the regulation of the three phases of apical hook development and the intermediary transitions (T). Red arrows indicate inhibitory effects, whereas green arrows indicate stimulatory effects. Figure 1. Open in new tabDownload slide Simplified cross talk diagram on the regulation of the dynamics of apical hook development. Apical hook development proceeds in three distinct phases: formation (F), maintenance (M), and opening (O). Hook formation initiates shortly after germination. After complete formation, the hook is maintained for several hours before opening (the apical hook dynamics shown here are for complete darkness). The formation of the apical hook depends on differential elongation driven by an auxin gradient, which is formed, maintained, and lost concomitant with the three phases of apical hook development. Ethylene plays a central role upstream of auxin. BRs, GAs, and JAs interact with ethylene in the regulation of the three phases of apical hook development and the intermediary transitions (T). Red arrows indicate inhibitory effects, whereas green arrows indicate stimulatory effects. In dark-grown Arabidopsis seedlings, ethylene elicits an exaggeration of the apical hook, the inhibition of both hypocotyl and root elongation, and radial swelling of the seedling stem. The exaggeration of the hook curvature as part of the triple response (Bleecker et al., 1988; Guzmán and Ecker, 1990) stresses an important function of ethylene in apical hook development and results from a delay in the transition between hook formation and maintenance (Fig. 1; Vandenbussche et al., 2010; Žádníková et al., 2010). Ethylene also regulates the transition between hook maintenance and opening by preventing hook opening (Fig. 1; Vandenbussche et al., 2010; Gallego-Bartolomé et al., 2011; Smet et al., 2014). Ethylene biosynthesis, perception, and signaling are indispensable for a flawless development of the apical hook. Ethylene-insensitive gain-of-function receptor mutants have a defective apical hook, whereas ethylene-hypersensitive alleles exhibit an exaggerated apical hook curvature in the presence of ethylene. The constitutive ethylene response mutant ctr1 exhibits an exaggerated hook in the absence of ethylene (Abbas et al., 2013; Mazzella et al., 2014). Apical hook development is deficient in the ethylene-insensitive mutant ein2 (Guzmán and Ecker, 1990), resulting from a severe inhibition of apical hook formation (Smet et al., 2014). Similarly, loss of function of the transcription factor EIN3 results in a reduced curvature, whereas EIN3 overexpression induces an enhanced apical hook curvature (An et al., 2012a). The ethylene-responsive gene HOOKLESS1 (HLS1), which encodes an N-acetyltransferase, is indispensable for apical hook formation (Guzmán and Ecker, 1990; Lehman et al., 1996). Recently, An et al. (2012a) showed that ethylene activates the transcription of HLS1 through direct binding of the EIN3/EIL1 transcription factors to its promoter. Ethylene Cross Talk during Apical Hook Development The differential elongation of the hypocotyl, underlying apical hook development, is driven by an auxin gradient (Friml et al., 2002; Li et al., 2004; Vandenbussche et al., 2010; Žádníková et al., 2010; Gallego-Bartolomé et al., 2011). Auxin is differentially distributed in the apical part, with an accumulation at the concave side exceeding that of the convex side, concomitant with hook formation and maintenance. Hook opening is associated with the fading of the asymmetrical auxin distribution (Fig. 1). The auxin gradient is established by polar auxin transport regulated by a complex hormonal network (Abbas et al., 2013; Mazzella et al., 2014; Žádníková et al., 2015). Ethylene acts directly upstream of auxin in the regulation of apical hook development, with HLS1 functioning as a key factor between both plant hormones (Lehman et al., 1996; Stowe-Evans et al., 1998; Li et al., 2004). Auxin is able to restore the defective hook formation in the ethylene-insensitive mutant ein2, indicating the importance of ethylene-mediated auxin biosynthesis in the regulation of hook development. In support, ethylene was shown to promote auxin production at the concave side of the hook through up-regulation of the auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE1 and TRYPTOPHAN AMINOTRANSFERASE-RELATED2 (Vandenbussche et al., 2010). Furthermore, the regulation of hook formation by ethylene depends on auxin transport. Inhibition of auxin influx as well as efflux hindered hook formation and exaggeration by ethylene. Moreover, ethylene affects apical hook development by regulating auxin transport. The expression of the auxin influx carriers AUXIN RESISTANT1 (AUX1)/LIKE AUXIN RESISTANCE3 and the auxin efflux carrier PIN-FORMED3 (PIN3) was enhanced, while that of PIN1 and PIN4 was reduced, by ethylene (Vandenbussche et al., 2010; Žádníková et al., 2010). BR, GA, and JA also impinge on auxin biosynthesis, transport, and signaling in the regulation of auxin distribution in the apical hook, whether or not through ethylene (De Grauwe et al., 2005; Gallego-Bartolomé et al., 2011; An et al., 2012a; Smet et al., 2014; Zhang et al., 2014b). Upstream of auxin, GAs interact with ethylene in the regulation of apical hook development. Both hormones promote hook formation and prevent hook opening (Fig. 1; Vandenbussche et al., 2010; Žádníková et al., 2010; Gallego-Bartolomé et al., 2011; Smet et al., 2014). GA biosynthesis is indispensable for apical hook formation and the ethylene-induced apical hook exaggeration (Gallego-Bartolomé et al., 2011; An et al., 2012a), and ethylene promotes GA biosynthesis in the apical hook (Mazzella et al., 2014). Moreover, ethylene biosynthesis is also targeted by GAs during hook development. GAs enhanced the expression of ACS5/ETO2 and ACS8 in etiolated Arabidopsis seedlings (Gallego-Bartolomé et al., 2011). della quintuple mutants exhibited an exaggerated apical hook curvature, which suggested that GAs promote the ethylene-mediated hook formation through the promotion of DELLA protein degradation. Ethylene insensitivity inhibited hook exaggeration in the della quintuple mutant, supporting an ethylene-dependent regulation of apical hook formation by GAs. Also, HLS1 proved indispensable for the constitutive apical hook exaggeration in the della quintuple mutant (An et al., 2012a). Both ethylene and GAs promoted HLS1 expression (Gallego-Bartolomé et al., 2011; An et al., 2012a). EIN3/EIL1 emerged as DELLA-associated transcription factors, leading An et al. (2012a) to postulate a GA-nullified DELLA inhibition of EIN3/EIL1 as the mechanism for the ethylene-GA cross talk in the regulation of apical hook formation. However, GA partially restored apical hook curvature of the ethylene-insensitive mutant ein2, also indicating an ethylene-independent GA regulation of hook formation (Gallego-Bartolomé et al., 2011). Apart from apical hook formation, GAs and ethylene are also involved in hook opening. Both in the ethylene-insensitive mutant ein2 and in the GA-insensitive mutant gai-1, hook maintenance is inhibited while hook opening is advanced, suggesting that both plant hormones postpone apical hook opening (Vandenbussche et al., 2010; Gallego-Bartolomé et al., 2011; Smet et al., 2014). In support, apical hook opening is even prevented in the della quintuple mutant upon the administration of ACC (Gallego-Bartolomé et al., 2011). Recently, a cross talk between BRs and ethylene in the regulation of apical hook development emerged. Etiolated BR-deficient mutants are hookless (Chory et al., 1991; Kauschmann et al., 1996; Li et al., 1996; Szekeres et al., 1996). Furthermore, the auxin response in the hook changes when the biosynthesis of BRs is affected (De Grauwe et al., 2005). Ethylene failed to exaggerate the hook curvature upon the inhibition of BR biosynthesis, suggesting that BRs are indispensable for the ethylene regulation of hook formation (Fig. 1). Nevertheless, in the absence of ethylene, the hook is not exaggerated by BRs. The reduced exaggeration of apical hook curvature by ethylene in the presence of BRs suggests, however, that BRs down-regulate the ethylene activity in apical hook formation (De Grauwe et al., 2005; Smet et al., 2014). Apart from hook formation, BRs and ethylene also interact in the regulation of hook maintenance and opening. BRs postponed the transition between hook maintenance and opening and, therefore, similar to ethylene and GAs, hamper apical hook opening (Fig. 1; Vandenbussche et al., 2010; Gallego-Bartolomé et al., 2011; Smet et al., 2014). However, ethylene postponed hook opening in the absence of BR biosynthesis, suggesting that, similar to hook formation, BRs also down-regulate the ethylene activity in apical hook opening (Smet et al., 2014). In contrast with ethylene, BRs, and GAs, JAs recently emerged as negative regulators of hook formation (Fig. 1). The JA regulation of apical hook development is also mediated by ethylene. JAs inhibited the exaggeration of apical hook curvature induced by enhanced ethylene signaling in etiolated seedlings. However, upon JA insensitivity, apical hook development and ethylene-induced hook exaggeration remained unaffected, indicating that JAs are dispensable for apical hook development and the ethylene activity therein (Ellis and Turner, 2002; Turner et al., 2002). The JA insensitivity of hls1 seedlings again suggested a cross talk between ethylene and JAs at the level of HLS1. Consistently, JAs were shown to affect HLS1 expression in an EIN3/EIL1-dependent manner. MYC2 was subsequently shown to impede EIN3/EIL1 transcriptional activity through direct binding with the promoter of EIN3. Hence, JAs are assumed to counteract ethylene in the regulation of apical hook development by inhibiting the EIN3/EIL1-mediated HLS1 expression through the binding of MYC2 to EIN3 (Song et al., 2014; Zhang et al., 2014b). ETHYLENE AND HYPOCOTYL GROWTH Ethylene regulates hypocotyl growth both in light and in darkness. In the dark, ethylene inhibits, whereas in the light, it promotes, the growth of Arabidopsis hypocotyls (Fig. 2). Light-grown eto2, ctr1, and EIN3-overexpressing seedlings had longer hypocotyls, whereas the hypocotyls of ethylene-insensitive etr1, ein2, ein3, and ein3 eil1 seedlings were shorter (Smalle et al., 1997; Alonso et al., 1999; Zhong et al., 2012; Yu et al., 2013). During skotomorphogenesis, hypocotyl growth is promoted, whereas during photomorphogenesis, it is inhibited, which is opposite to the effect of ethylene during the respective developmental programs. Consequently, a light-dependent regulation of hypocotyl growth by ethylene is expected. Figure 2. Open in new tabDownload slide Simplified cross talk diagram showing the effects of and interactions between plant hormones in the regulation of hypocotyl development. The effect of the plant hormones on elongation and their interactions often differ between light and darkness. The hormonal cross talk is less well established in darkness compared with light. Ethylene plays an important role in the regulation of hypocotyl development, in the light and presumably also in darkness, interacting with other plant hormones. Red arrows indicate inhibitory effects, whereas green arrows indicate stimulatory effects. Figure 2. Open in new tabDownload slide Simplified cross talk diagram showing the effects of and interactions between plant hormones in the regulation of hypocotyl development. The effect of the plant hormones on elongation and their interactions often differ between light and darkness. The hormonal cross talk is less well established in darkness compared with light. Ethylene plays an important role in the regulation of hypocotyl development, in the light and presumably also in darkness, interacting with other plant hormones. Red arrows indicate inhibitory effects, whereas green arrows indicate stimulatory effects. In the dark, photomorphogenesis is repressed by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1; Leivar and Quail, 2011; Lau and Deng, 2012). COP1 proved to function downstream of EIN3 in the light. Conversely, in darkness, COP1 acts upstream of EIN3. Both ethylene and overexpression of EIN3 reduced the expression of ETHYLENE RESPONSE FACTOR1 (ERF1) in etiolated wild-type seedlings, whereas in dark-grown cop1 seedlings, ERF1 expression was enhanced, suggesting that COP1 regulates the transcriptional activity of EIN3 (Liang et al., 2012). Apart from COP1, photomorphogenesis is also repressed in the dark by PHYTOCROME INTERACTING FACTORS (PIFs; Leivar and Quail, 2011; Lau and Deng, 2012). PIF3 proved to be indispensable for the regulation of hypocotyl extension by ethylene in the light, with EIN3 promoting PIF3 expression by directly binding to its promoter. In darkness, the ethylene-induced inhibition of hypocotyl elongation is also mediated by EIN3/EIL1, although independently of PIF3. Both in light and in darkness, ethylene enhanced ERF1 expression. Nevertheless, overexpression of ERF1 substantially inhibited hypocotyl elongation in the dark, suggesting that ethylene depends on ERF1 for the inhibition of hypocotyl growth in the dark (Zhong et al., 2012). As opposed to COP1 and PIFs, LONG HYPOCOTYL5 (HY5) transcription factors promote photomorphogenesis (Leivar and Quail, 2011; Lau and Deng, 2012), which suggests their involvement in the regulation of hypocotyl elongation. In support, ethylene was shown to depend on HY5 in the regulation of hypocotyl growth in the light but not in darkness (Liang et al., 2012; Yu et al., 2013). HY5 emerged as an inhibitor of ethylene during hypocotyl growth in the light. As for PIF3 (Zhong et al., 2012), HY5 appeared to act downstream of EIN3 in the regulation of hypocotyl growth by ethylene in the light. Moreover, ethylene suppressed the accumulation but not the expression of HY5. Ethylene promoted the nuclear localization of COP1 in the light, driving the degradation of HY5 (Yu et al., 2013). Hence, COP1, PIF3, and HY5 implement light and ethylene in the regulation of hypocotyl growth. Vandenbussche et al. (2007) demonstrated that the regulation of hypocotyl elongation by ethylene is blue light dependent and mediated by cryptochrome signaling. Ethylene Cross Talk during Hypocotyl Growth Hypocotyl growth is regulated by a complex hormone cross talk involving, apart from ethylene, also auxins, BRs, GAs, ABA, and CKs. Some of these plant hormones cooperate with ethylene therein. However, mutual interactions independent of ethylene were also demonstrated. As for ethylene, auxin is able to both inhibit and promote the elongation of Arabidopsis hypocotyls (Smalle et al., 1997; Collett et al., 2000). Inhibition of auxin efflux hinders hypocotyl elongation in the light, whereas in darkness, the effect is negligible (Garbers et al., 1996; Lehman et al., 1996; Jensen et al., 1998). However, hypocotyl elongation was significantly inhibited in dark-grown auxin-resistant mutants (Liang et al., 2012). Auxin biosynthesis, transport, and signaling proved to be important for ethylene-induced hypocotyl growth in the light (Fig. 2). Auxin accumulation was enhanced by ethylene, concomitant with an increased expression of the auxin biosynthesis genes YUCCA1 and YUCCA5. The inhibition of auxin efflux suppressed ethylene-induced hypocotyl elongation in the light. Ethylene also up-regulated the expression of the auxin transport genes AUX1, PIN3, and PIN7 in light-grown seedlings. Furthermore, the elongation of the hypocotyl in response to ethylene was inhibited in auxin-insensitive mutants in the light (Liang et al., 2012). Hence, the cross talk between ethylene and auxin in hypocotyl growth seems to be mediated by light, presumably via COP1 (Liang et al., 2012; Yu et al., 2013). Furthermore, HY5 has been shown to regulate the expression of genes involved in auxin signaling (Jing et al., 2013). GAs stimulate hypocotyl elongation mainly by targeting the degradation of DELLA proteins, which negatively affect growth (Schwechheimer, 2008; Hauvermale et al., 2012). In light-grown Arabidopsis seedlings, GAs promote hypocotyl elongation (Fig. 2), whereas in etiolated seedlings, hypocotyl lengths remain unaltered. However, GA levels are significantly higher in etiolated seedlings, promoting hypocotyl elongation, which explains the absence of additional stimulation of hypocotyl elongation by exogenous GAs (Cowling and Harberd, 1999; Gendreau et al., 1999; Saibo et al., 2003). Consistently, upon inhibition of GA biosynthesis, GA significantly enhanced hypocotyl elongation in dark-grown Arabidopsis seedlings (Zhang et al., 2010; Fig. 2). The importance of GAs for hypocotyl elongation in the dark is also supported by the deetiolated growth of GA biosynthesis and signaling mutants in darkness and the wild type treated with the GA biosynthesis inhibitor paclobutrazol (Alabadí et al., 2004; Vriezen et al., 2004). As for ethylene, GAs negatively and positively regulate HY5 and PIFs, respectively, through COP1 or DELLAs (Jing et al., 2013; Zhang et al., 2014a). Hence, a cross talk between ethylene and GAs in the light-mediated regulation of hypocotyl elongation is expected. Consistently, in light-grown Arabidopsis seedlings, hypocotyl elongation is even more enhanced when ethylene and GA are administered together (Saibo et al., 2003). In blue light, the inhibition of GA biosynthesis completely abolished the ethylene-induced hypocotyl elongation, suggesting a GA-dependent regulation (Vandenbussche et al., 2007). Like GAs, BRs are growth-promoting hormones. Arabidopsis BR biosynthesis and perception mutants have a dwarfed stature and are constitutively photomorphogenic in the dark (Chory et al., 1991; Kauschmann et al., 1996; Li et al., 1996; Szekeres et al., 1996). BRs promote hypocotyl elongation in the light, whereas in darkness, depending on the dose, hypocotyl lengths are unaffected or even reduced (Fig. 2). In the dark, the ethylene-induced inhibition of hypocotyl elongation was not promoted by BRs (Chen et al., 2013). Furthermore, ethylene insensitivity, but not deficiency, abolished the BR-induced inhibition of hypocotyl elongation (Deslauriers and Larsen, 2010). Hence, in the dark, an enhanced ethylene response mediates the BR-induced inhibition of hypocotyl elongation (Fig. 2). In the light, BRs further enhanced hypocotyl elongation induced by ethylene, and BR deficiency reduced the hypocotyl length in response to ethylene (Chen et al., 2013). Furthermore, the BR-induced hypocotyl elongation in the light was suppressed upon inhibition of ethylene perception. Ethylene and BRs thus appear to work antagonistically and interdependently in the regulation of hypocotyl elongation in the light (Fig. 2). The receptor-like kinase FERONIA was suggested to be a key modulator of the BR and ethylene responses in hypocotyl growth (Deslauriers and Larsen, 2010). ETHYLENE AND PRIMARY ROOT GROWTH Since the discovery of the triple response, it has become well established that ethylene plays an important role during primary root growth (Smalle and Van Der Straeten, 1997). More recent work with an octuple acs mutant revealed a slightly smaller root phenotype and an increased sensitivity toward externally applied ACC (Tsuchisaka et al., 2009), suggesting that a small amount of ethylene production is essential for normal root development. Ethylene affects primary root growth at two different levels. First, ethylene induces stem cell division in the RAM (Ortega-Martínez et al., 2007). Second, ethylene inhibits cell expansion in the root elongation zone (Le et al., 2001). The action of ethylene on primary root growth is the net result of a complex cross talk with other hormones. Currently, the ethylene-auxin cross talk is best established, but more recent studies also highlight that ethylene interacts with other hormones during root development. Ethylene Cross Talk during Primary Root Growth The auxin-ethylene cross talk during root growth has been primarily characterized at the molecular level by Stepanova et al. (2007), Swarup et al. (2007), and Růzicka et al. (2007). Ultimately, this led to a hormone interaction model that has been reviewed by several authors (Benková and Hejátko, 2009; Vanstraelen and Benková, 2012; Takatsuka and Umeda, 2014). An important aspect of primary root development is the establishment of an auxin gradient along the longitudinal root axis. Polar transport drives the auxin flux through the inner stele from the hypocotyl toward the RAM in the QC, where a local auxin maximum drives the maintenance of the meristem and pattern formation. Subsequently, auxin is transported back upward, predominantly through the epidermis cells by means of auxin carriers, creating an auxin gradient and determining cell fate along the root axis. This auxin gradient is strictly regulated, and ethylene is one of the important players that regulates the auxin flow (Fig. 3). Over the years, an ethylene-auxin interaction model has been proposed for primary root growth. Ethylene can stimulate local auxin biosynthesis in the root apex and stimulate shootward auxin transport. Ethylene also enhances the auxin sensitivity of cells in the elongation zone. Altogether, ethylene is required for the establishment of a normal auxin gradient. Abnormal levels of ethylene will result in an imbalance in the auxin gradient, which in turn will lead to a higher auxin content in the elongation zone, resulting in the inhibition of cell elongation, a typical ethylene feature. Figure 3. Open in new tabDownload slide Simplified cross talk diagram showing the interaction model between ethylene and other plant hormones that regulate primary root development through the modulation of cell elongation. Ethylene has a central hub position acting downstream of ABA, CKs, GAs, and BRs. Ethylene, in its turn, regulates primarily auxin biosynthesis, transport, and signaling, which is crucial for the establishment of the auxin gradient in the root, which drives the regulation of cell elongation. Two intermediate signaling molecules that affect root development and that are positioned between ethylene and auxins are flavonols and PLS. Red arrows indicate inhibitory effects, whereas green arrows indicate stimulatory effects. Figure 3. Open in new tabDownload slide Simplified cross talk diagram showing the interaction model between ethylene and other plant hormones that regulate primary root development through the modulation of cell elongation. Ethylene has a central hub position acting downstream of ABA, CKs, GAs, and BRs. Ethylene, in its turn, regulates primarily auxin biosynthesis, transport, and signaling, which is crucial for the establishment of the auxin gradient in the root, which drives the regulation of cell elongation. Two intermediate signaling molecules that affect root development and that are positioned between ethylene and auxins are flavonols and PLS. Red arrows indicate inhibitory effects, whereas green arrows indicate stimulatory effects. It is important to note that two intermediate signaling components can intervene with the ethylene-auxin cross talk. The first group of secondary messengers is flavonols, which inhibit shootward auxin transport and cause the inhibition of root elongation (Muday et al., 2012; Fig. 3). Both ethylene and auxin can induce flavonol biosynthesis via two independent signaling pathways that converge through the MYB12 transcription factor (Lewis et al., 2011b). A second important messenger is POLARIS (PLS), a peptide acting as a negative regulator of the ethylene-induced inhibition of root growth (Casson et al., 2002; Chilley et al., 2006; Fig. 3). Ethylene down-regulates PLS expression, while auxins up-regulate PLS expression (Chilley et al., 2006). The pls mutant shows a short-root phenotype and increased levels of the auxin efflux carriers PIN1 and PIN2, while a PLS overexpression line shows a decrease in PIN1 and PIN2 levels (Liu et al., 2013). The double mutant pls etr1-1 did not show altered PIN levels, suggesting that PLS-mediated changes in PIN levels are regulated by ethylene signaling, which in turn alters the auxin gradient and, therefore, root growth (Liu et al., 2013). GAs also play an important role in root growth. GAs promote the degradation of DELLA proteins, which were shown to accumulate in the endodermal cells of the elongation zone, where they inhibit root growth (Davière and Achard, 2013). Recently, it was shown that fluorescein-coupled GA also accumulates in the endodermal cells of the elongation zone, where it likely regulates DELLA stability. Interestingly, ACC treatment inhibited fluorescein-coupled GA accumulation, while having no effect on the fluorescein-coupled GA levels in ein2-5, suggesting that GA transport in the root epidermal cells is regulated by ethylene (Shani et al., 2013). CKs also regulate root growth via cross talk with ethylene (Fig. 3). Originally, it was shown that CKs inhibit root growth and that this inhibition was mediated via the ethylene signaling pathway (Cary et al., 1995). Furthermore, CKs can induce ethylene biosynthesis by stabilizing ACS5 protein levels (Chae et al., 2003), resulting in a higher endogenous ACC content (Žd’árská et al., 2013). CKs also up-regulate the levels of S-adenosyl methionine synthetase and ACO in the root (Žd’árská et al., 2013), most likely leading to a local increase in ethylene production and, consequently, to the inhibition of root elongation. Contradictorily, Kushwah et al. (2011) showed that CKs can induce root elongation instead of inhibition and that this response is also mediated by the ethylene signaling pathway. More research will be needed to unravel these aspects of CK-ethylene cross talk on root development. It was originally shown that ABA inhibits root growth and that this ABA response is mediated by the ethylene signaling pathway but not by ethylene biosynthesis (Beaudoin et al., 2000; Ghassemian et al., 2000; Thole et al., 2014). Recent reports have shown that ABA can stabilize the C-terminal part of ACS6, leading to higher ethylene production and, consequently, inhibition of root elongation (Luo et al., 2014). On the other hand, the ABA signaling protein PHOSPHATASE2C ABSCISIC ACID-INSENSITIVE2 can dephosphorylate ACS6, directing it for degradation, thus lowering ethylene production (Ludwików et al., 2014; Fig. 3). Besides the dual role of ABA on ethylene biosynthesis, it was shown that plants with a lower ethylene production capacity (aminoethoxyvinylglycine-treated plants and acs multiple knockout lines) all have decreased ABA sensitivity with relation to inhibition of root growth, while the ethylene-overproducing mutant eto shows increased ABA sensitivity (Luo et al., 2014). Thus, ABA can mediate ethylene biosynthesis, and it inhibits root elongation through the downstream action of ethylene. BRs also play a dual role in root growth. Low concentrations of BRs can induce root growth, while high concentrations inhibit root growth, a response that is independent of ethylene (Clouse et al., 1996; Müssig et al., 2003). On the other hand, BRs can induce ethylene production in roots, suggesting that BRs can direct the ethylene-regulated inhibition of root growth (Müssig et al., 2003; Fig. 3). Recent work by Fridman et al. (2014) reported a cell type-specific cross talk interaction between ethylene and BRs. A cell type-specific expression of the BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) in root hair cells induced cell elongation in all root cells, while a specific expression of BRI1 in nonhair cells inhibited root cell elongation. Moreover, nonhair cell expression of BRI1 appeared to induce ACS expression, and EIN2 was indispensable for the BRI1-induced root cell inhibition, suggesting that BR acts upstream of ethylene to inhibit root elongation. Altogether, it is clear that ethylene interacts with other hormones to regulate primary root growth. A general interaction model where ethylene plays a central role is presented (Fig. 3). GAs, CKs, ABA, and BRs act (in part) upstream of ethylene, directing ethylene biosynthesis and/or requiring ethylene signaling in order to regulate the inhibition of root elongation, while auxin seems to act downstream of ethylene. ETHYLENE AND LATERAL ROOT DEVELOPMENT Besides primary root development, ethylene also plays an important role during the initiation and growth of lateral roots. It was initially shown that enhanced ethylene synthesis (upon ACC treatment or in eto1 mutants) or signaling (in ctr1 mutants) reduced lateral root formation. Ethylene-insensitive mutants (etr1 and ein2), on the other hand, enhance lateral root formation (Negi et al., 2008). Low doses of ACC promote the formation of lateral root primordia, as also evidenced by ACS1 expression (Rodrigues-Pousada et al., 1993), but the outgrowth of these primordia through the pericycle is prevented by ethylene (Ivanchenko et al., 2008). Similar to other vegetative developmental stages, ethylene does not act alone on lateral root development but interacts with other hormones. Ethylene Cross Talk during Lateral Root Development A major player in lateral root development is auxin, which promotes lateral root development. Auxin will prime pericycle cells, initiate cell cycle progression, stimulate asymmetric division, and ensure the emergence and elongation of the lateral root (for review, see Lavenus et al., 2013). Auxin and ethylene have antagonistic effects in lateral root development. Ethylene reduces DR5rev:GFP expression in the regions where lateral roots emerge, suggesting a locally reduced auxin responsiveness (Lewis et al., 2011a). Arabidopsis seedlings also show a local depletion of PIN3 and PIN7 abundance in the region just below developing lateral root primordia, matching the DR5rev:GFP expression pattern, suggesting a local depletion of auxin. This, in turn, results in a local auxin accumulation just above the lateral root primordia, giving rise to the formation of lateral roots (Lewis et al., 2011a; Aloni, 2013). Ethylene will prevent this local auxin depletion, predominantly by increasing PIN3 and PIN7 expression and abundance, thus increasing rootward auxin transport, which suppresses the local auxin maxima at lateral root primordia, inhibiting their outgrowth (Lewis et al., 2011a; Muday et al., 2012; Aloni, 2013). ETHYLENE AND ADVENTITIOUS ROOT DEVELOPMENT Adventitious roots are lateral roots initiated aboveground from the hypocotyl, instead of belowground from the primary root. In Arabidopsis, ethylene inhibits the formation of adventitious roots (Sukumar, 2010). Root-excised seedlings treated with ACC, as well as the eto1 and ctr1 mutants, showed fewer adventitious roots, while ethylene-insensitive mutants showed an increase in adventitious roots. On the other hand, ethylene inhibits auxin-stimulated adventitious root formation. It must be noted that, in other species, ethylene can stimulate adventitious root formation and that other hormones are also involved in the formation of adventitious roots (for review, see Bellini et al., 2014; Verstraeten et al., 2014). ETHYLENE AND ROOT HAIR DEVELOPMENT The development of root hairs is stimulated by ethylene (Tanimoto et al., 1995). Ethylene-insensitive etr1 and ein2 mutant alleles show shorter root hairs, while ethylene-overproducing eto1 mutants show longer root hairs, supporting that ethylene stimulates root hair elongation (Pitts et al., 1998; Rahman et al., 2002). Ethylene also regulates the differentiation of root epidermal cells, since the ctr1-1 mutant bears ectopic root hairs, as do wild-type roots treated with the ethylene precursor ACC (Tanimoto et al., 1995; Cao et al., 1999). The higher order acs mutants (hextuple and octuple mutants) exhibit an increased sensitivity toward ACC, reflected by an increase of root hair formation when treated with ACC (Tsuchisaka et al., 2009). The position of root hair emergence is also ethylene regulated, because the ethylene-overproducing mutant eto1 forms root hairs closer to the apical end of the root hair cell, while a dominant ethylene-insensitive etr1 mutant forms root hairs closer to the basal end of the root hair cell (Masucci and Schiefelbein, 1996). The exact location of root hair emergence is determined by the local auxin gradient, which, in turn, is partially regulated by ethylene (Ikeda et al., 2009). The action of ethylene (and other hormones like CKs) is at least in part mediated via the transcription factor C2H2 ZINC FINGER PROTEIN, which is a key regulator of root hair initiation and development in Arabidopsis (An et al., 2012b). Auxin can also stimulate root hair formation independent from as well as in collaboration with ethylene (Grierson et al., 2014). For example, both auxin and ethylene can independently restore the root hair-deficient (rhd) phenotype of the rhd6 mutant (Masucci and Schiefelbein, 1994). On the other hand, auxin is able to rescue root hair defects in the ein2-1 mutant (Rahman et al., 2002). Auxin transport, and more precisely auxin influx via AUX1, is another point of cross talk that regulates root hair elongation, as evidenced by the suppression of the long-root-hair phenotype of the eto1 mutant in the eto1 aux1 double mutant (Strader et al., 2010). Ethylene also interacts with JA to regulate root hair development (Zhu et al., 2006). JA biosynthesis inhibitors (ibuprofen and salicylhydroxamic acid) block the ACC-induced root hair formation, while ethylene inhibitors (Ag+ and aminoethoxyvinylglycine) block the JA-promoted formation of root hairs (Zhu et al., 2006). CONCLUSION AND FUTURE PERSPECTIVES Ethylene is a developmental regulator that operates in manifold physiological processes in all tissues throughout the plant life cycle. Compared with animals, plants have relatively few hormones, suggesting the necessity for hormonal cross talk. Over the years, hormonal biosynthesis and signaling pathways have been elucidated, allowing scientists to identify crucial players in hormonal interactions. This Update summarized key findings on ethylene and its cross talk with other hormones in the model species Arabidopsis, focusing on major developmental and growth phases of the vegetative life cycle and highlighting the different modes of action of ethylene in diverse tissues. Future challenges in the field of hormone research are grand, because it has become evident over the years that hormonal cross talk is not linear and should be tackled in a multidimensional space. This means that scientists should try to understand the three-dimensional spatial but also temporal relationships between two hormones, as well as the real-time concentration dependence, and their interdependent relationships with other hormones. The latter complex network of hormonal cross talk is not static but highly dynamic, again affected by time (development)- and space (cell and tissue specificity)-related factors. This Update highlighted the first studies where ethylene sensitivity and cross talk is cell type specific within a certain tissue and developmental stage. Future endeavors should provide more insight in these single-cell regulatory mechanisms. Last but not least, the Gordian knot of hormonal cross talk dynamics is also affected by external clues such as, for example, light or temperature. Furthermore, it remains critical to precisely link hormone dynamics with a well-characterized phenotype. Major progress in the field of hormone biology will most likely be backed up by advancements in computer-aided phenotyping. The latter will enable plant scientists to further comprehend and visualize the vast hormonal cross talk dynamics. Glossary QC quiescent center RAM root apical meristem CMT cortical microtubules PCD programmed cell death ACC 1-aminocyclopropane-1-carboxylic acid ROS reactive oxygen species ABA abscisic acid BR brassinosteroid CK cytokinin JA jasmonate LITERATURE CITED Abbas M , Alabadí D, Blázquez MA ( 2013 ) Differential growth at the apical hook: all roads lead to auxin . Front Plant Sci 4 : 441 Google Scholar Crossref Search ADS PubMed WorldCat Alabadí D , Gil J, Blázquez MA, García-Martínez JL ( 2004 ) Gibberellins repress photomorphogenesis in darkness . Plant Physiol 134 : 1050 – 1057 Google Scholar Crossref Search ADS PubMed WorldCat Aloni R ( 2013 ) Role of hormones in controlling vascular differentiation and the mechanism of lateral root initiation . Planta 238 : 819 – 830 Google Scholar Crossref Search ADS PubMed WorldCat Alonso JM , Hirayama T, Roman G, Nourizadeh S, Ecker JR ( 1999 ) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis . Science 284 : 2148 – 2152 Google Scholar Crossref Search ADS PubMed WorldCat An F , Zhang X, Zhu Z, Ji Y, He W, Jiang Z, Li M, Guo H ( 2012 a ) Coordinated regulation of apical hook development by gibberellins and ethylene in etiolated Arabidopsis seedlings . Cell Res 22 : 915 – 927 Google Scholar Crossref Search ADS PubMed WorldCat An L , Zhou Z, Sun L, Yan A, Xi W, Yu N, Cai W, Chen X, Yu H, Schiefelbein J, et al. ( 2012 b ) A zinc finger protein gene ZFP5 integrates phytohormone signaling to control root hair development in Arabidopsis . Plant J 72 : 474 – 490 Google Scholar Crossref Search ADS PubMed WorldCat Andersson-Gunnerås S , Hellgren JM, Björklund S, Regan S, Moritz T, Sundberg B ( 2003 ) Asymmetric expression of a poplar ACC oxidase controls ethylene production during gravitational induction of tension wood . Plant J 34 : 339 – 349 Google Scholar Crossref Search ADS PubMed WorldCat Arc E , Sechet J, Corbineau F, Rajjou L, Marion-Poll A ( 2013 ) ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination . Front Plant Sci 4 : 63 Google Scholar PubMed OpenURL Placeholder Text WorldCat Bashline L , Lei L, Li S, Gu Y ( 2014 ) Cell wall, cytoskeleton, and cell expansion in higher plants . Mol Plant 7 : 586 – 600 Google Scholar Crossref Search ADS PubMed WorldCat Baskin TI ( 2015 ) Auxin inhibits expansion rate independently of cortical microtubules . Trends Plant Sci 20 : 471 – 472 Google Scholar Crossref Search ADS PubMed WorldCat Beaudoin N , Serizet C, Gosti F, Giraudat J ( 2000 ) Interactions between abscisic acid and ethylene signaling cascades . Plant Cell 12 : 1103 – 1115 Google Scholar Crossref Search ADS PubMed WorldCat Bellini C , Pacurar DI, Perrone I ( 2014 ) Adventitious roots and lateral roots: similarities and differences . Annu Rev Plant Biol 65 : 639 – 666 Google Scholar Crossref Search ADS PubMed WorldCat Benková E , Hejátko J ( 2009 ) Hormone interactions at the root apical meristem . Plant Mol Biol 69 : 383 – 396 Google Scholar Crossref Search ADS PubMed WorldCat Bleecker AB , Estelle M, Sommerville C, Kende H ( 1988 ) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana . Science 240 : 1086 – 1089 Google Scholar PubMed OpenURL Placeholder Text WorldCat Bollhöner B , Prestele J, Tuominen H ( 2012 ) Xylem cell death: emerging understanding of regulation and function . J Exp Bot 63 : 1081 – 1094 Google Scholar Crossref Search ADS PubMed WorldCat Cao XF , Linstead P, Berger F, Kieber J, Dolan L ( 1999 ) Differential ethylene sensitivity of epidermal cells is involved in the establishment of cell pattern in the Arabidopsis root . Physiol Plant 106 : 311 – 317 Google Scholar Crossref Search ADS PubMed WorldCat Cary AJ , Liu W, Howell SH ( 1995 ) Cytokinin action is coupled to ethylene in its effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings . Plant Physiol 107 : 1075 – 1082 Google Scholar Crossref Search ADS PubMed WorldCat Casson SA , Chilley PM, Topping JF, Evans IM, Souter MA, Lindsey K ( 2002 ) The POLARIS gene of Arabidopsis encodes a predicted peptide required for correct root growth and leaf vascular patterning . Plant Cell 14 : 1705 – 1721 Google Scholar Crossref Search ADS PubMed WorldCat Chae HS , Faure F, Kieber JJ ( 2003 ) The eto1, eto2, and eto3 mutations and cytokinin treatment increase ethylene biosynthesis in Arabidopsis by increasing the stability of ACS protein . Plant Cell 15 : 545 – 559 Google Scholar Crossref Search ADS PubMed WorldCat Chen IJ , Lo WS, Chuang JY, Cheuh CM, Fan YS, Lin LC, Wu SJ, Wang LC ( 2013 ) A chemical genetics approach reveals a role of brassinolide and cellulose synthase in hypocotyl elongation of etiolated Arabidopsis seedlings . Plant Sci 209 : 46 – 57 Google Scholar Crossref Search ADS PubMed WorldCat Chen X , Grandont L, Li H, Hauschild R, Paque S, Abuzeineh A, Rakusová H, Benkova E, Perrot-Rechenmann C, Friml J ( 2014 ) Inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules . Nature 516 : 90 – 93 Google Scholar Crossref Search ADS PubMed WorldCat Cheng WH , Chiang MH, Hwang SG, Lin PC ( 2009 ) Antagonism between abscisic acid and ethylene in Arabidopsis acts in parallel with the reciprocal regulation of their metabolism and signaling pathways . Plant Mol Biol 71 : 61 – 80 Google Scholar Crossref Search ADS PubMed WorldCat Chilley PM , Casson SA, Tarkowski P, Hawkins N, Wang KL, Hussey PJ, Beale M, Ecker JR, Sandberg GK, Lindsey K ( 2006 ) The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling . Plant Cell 18 : 3058 – 3072 Google Scholar Crossref Search ADS PubMed WorldCat Chiwocha SDS , Cutler AJ, Abrams SR, Ambrose SJ, Yang J, Ross ARS, Kermode AR ( 2005 ) The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination . Plant J 42 : 35 – 48 Google Scholar Crossref Search ADS PubMed WorldCat Cho HT , Cosgrove DJ ( 2002 ) Regulation of root hair initiation and expansin gene expression in Arabidopsis . Plant Cell 14 : 3237 – 3253 Google Scholar Crossref Search ADS PubMed WorldCat Chory J , Nagpal P, Peto CA ( 1991 ) Phenotypic and genetic analysis of det2, a new mutant that affects light-regulated seedling development in Arabidopsis . Plant Cell 3 : 445 – 459 Google Scholar Crossref Search ADS PubMed WorldCat Clouse SD , Langford M, McMorris TC ( 1996 ) A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development . Plant Physiol 111 : 671 – 678 Google Scholar Crossref Search ADS PubMed WorldCat Collett CE , Harberd NP, Leyser O ( 2000 ) Hormonal interactions in the control of Arabidopsis hypocotyl elongation . Plant Physiol 124 : 553 – 562 Google Scholar Crossref Search ADS PubMed WorldCat Corbineau F , Xia Q, Bailly C, El-Maarouf-Bouteau H ( 2014 ) Ethylene, a key factor in the regulation of seed dormancy . Front Plant Sci 5 : 539 Google Scholar Crossref Search ADS PubMed WorldCat Cosgrove DJ ( 2000 ) Loosening of plant cell walls by expansins . Nature 407 : 321 – 326 Google Scholar Crossref Search ADS PubMed WorldCat Cowling RJ , Harberd NP ( 1999 ) Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation . J Exp Bot 50 : 1351 – 1357 Google Scholar Crossref Search ADS WorldCat Darwin C , Darwin F ( 1881 ). The Power of Movement in Plants. D Appleton , New York Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Davière JM , Achard P ( 2013 ) Gibberellin signaling in plants . Development 140 : 1147 – 1151 Google Scholar Crossref Search ADS PubMed WorldCat De Grauwe L , Vandenbussche F, Tietz O, Palme K, Van Der Straeten D ( 2005 ) Auxin, ethylene and brassinosteroids: tripartite control of growth in the Arabidopsis hypocotyl . Plant Cell Physiol 46 : 827 – 836 Google Scholar Crossref Search ADS PubMed WorldCat Deslauriers SD , Larsen PB ( 2010 ) FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in Arabidopsis hypocotyls . Mol Plant 3 : 626 – 640 Google Scholar Crossref Search ADS PubMed WorldCat Drew MC , He CJ, Morgan PW ( 2000 ) Programmed cell death and aerenchyma formation in roots . Trends Plant Sci 5 : 123 – 127 Google Scholar Crossref Search ADS PubMed WorldCat Ellis C , Turner JG ( 2002 ) A conditionally fertile coi1 allele indicates cross-talk between plant hormone signalling pathways in Arabidopsis thaliana seeds and young seedlings . Planta 215 : 549 – 556 Google Scholar Crossref Search ADS PubMed WorldCat El-Maarouf-Bouteau H , Sajjad Y, Bazin J, Langlade N, Cristescu SM, Balzergue S, Baudouin E, Bailly C ( 2014 ) Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination . Plant Cell Environ 38 : 364 – 374 Google Scholar Crossref Search ADS PubMed WorldCat Etchells JP , Provost CM, Turner SR ( 2012 ) Plant vascular cell division is maintained by an interaction between PXY and ethylene signalling . PLoS Genet 8 : e1002997 Google Scholar Crossref Search ADS PubMed WorldCat Fridman Y , Elkouby L, Holland N, Vragović K, Elbaum R, Savaldi-Goldstein S ( 2014 ) Root growth is modulated by differential hormonal sensitivity in neighboring cells . Genes Dev 28 : 912 – 920 Google Scholar Crossref Search ADS PubMed WorldCat Friml J , Wiśniewska J, Benková E, Mendgen K, Palme K ( 2002 ) Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis . Nature 415 : 806 – 809 Google Scholar Crossref Search ADS PubMed WorldCat Gallego-Bartolomé J , Arana MV, Vandenbussche F, Zádníková P, Minguet EG, Guardiola V, Van Der Straeten D, Benková E, Alabadí D, Blázquez MA ( 2011 ) Hierarchy of hormone action controlling apical hook development in Arabidopsis . Plant J 67 : 622 – 634 Google Scholar Crossref Search ADS PubMed WorldCat Garbers C , DeLong A, Deruére J, Bernasconi P, Söll D ( 1996 ) A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis . EMBO J 15 : 2115 – 2124 Google Scholar Crossref Search ADS PubMed WorldCat Gendreau E , Orbovic V, Höfte H, Traas J ( 1999 ) Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl . Planta 209 : 513 – 516 Google Scholar PubMed OpenURL Placeholder Text WorldCat Ghassemian M , Nambara E, Cutler S, Kawaide H, Kamiya Y, McCourt P ( 2000 ) Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis . Plant Cell 12 : 1117 – 1126 Google Scholar Crossref Search ADS PubMed WorldCat Grierson C , Nielsen E, Ketelaarc T, Schiefelbein J ( 2014 ) Root hairs . The Arabidopsis Book 12 : e0172 , doi/10.1199/tab.0172 Google Scholar Crossref Search ADS PubMed WorldCat Guzmán P , Ecker JR ( 1990 ) Exploiting the triple response of Arabidopsis to identify ethylene-related mutants . Plant Cell 2 : 513 – 523 Google Scholar PubMed OpenURL Placeholder Text WorldCat Ikeda Y , Men S, Fischer U, Stepanova AN, Alonso JM, Ljung K, Grebe M ( 2009 ) Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis . Nat Cell Biol 11 : 731 – 738 Google Scholar Crossref Search ADS PubMed WorldCat Hauvermale AL , Ariizumi T, Steber CM ( 2012 ) Gibberellin signaling: a theme and variations on DELLA repression . Plant Physiol 160 : 83 – 92 Google Scholar Crossref Search ADS PubMed WorldCat Ivanchenko MG , Muday GK, Dubrovsky JG ( 2008 ) Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana . Plant J 55 : 335 – 347 Google Scholar Crossref Search ADS PubMed WorldCat Jensen PJ , Hangarter RP, Estelle M ( 1998 ) Auxin transport is required for hypocotyl elongation in light-grown but not dark-grown Arabidopsis . Plant Physiol 116 : 455 – 462 Google Scholar Crossref Search ADS PubMed WorldCat Jing Y , Zhang D, Wang X, Tang W, Wang W, Huai J, Xu G, Chen D, Li Y, Lin R ( 2013 ) Arabidopsis chromatin remodeling factor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation . Plant Cell 25 : 242 – 256 Google Scholar Crossref Search ADS PubMed WorldCat Kauschmann A , Jessop A, Koncz C, Szekeres M, Willmitzer L, Altmann T ( 1996 ) Genetic evidence for an essential role of brassinosteroids in plant development . Plant J 9 : 701 – 713 Google Scholar Crossref Search ADS WorldCat Kushwah S , Jones AM, Laxmi A ( 2011 ) Cytokinin interplay with ethylene, auxin, and glucose signaling controls Arabidopsis seedling root directional growth . Plant Physiol 156 : 1851 – 1866 Google Scholar Crossref Search ADS PubMed WorldCat Lau OS , Deng XW ( 2012 ) The photomorphogenic repressors COP1 and DET1: 20 years later . Trends Plant Sci 17 : 584 – 593 Google Scholar Crossref Search ADS PubMed WorldCat Lavenus J , Goh T, Roberts I, Guyomarc’h S, Lucas M, De Smet I, Fukaki H, Beeckman T, Bennett M, Laplaze L ( 2013 ) Lateral root development in Arabidopsis: fifty shades of auxin . Trends Plant Sci 18 : 450 – 458 Google Scholar Crossref Search ADS PubMed WorldCat Le J , Vandenbussche F, De Cnodder T, Van Der Straeten D, Verbelen JP ( 2005 ) Cell elongation and microtubule behavior in the Arabidopsis hypocotyl: responses to ethylene and auxin . J Plant Growth Regul 24 : 166 – 178 Google Scholar Crossref Search ADS WorldCat Le J , Vandenbussche F, Van Der Straeten D, Verbelen JP ( 2001 ) In the early response of Arabidopsis roots to ethylene, cell elongation is up- and down-regulated and uncoupled from differentiation . Plant Physiol 125 : 519 – 522 Google Scholar Crossref Search ADS PubMed WorldCat Le J , Vandenbussche F, Van Der Straeten D, Verbelen JP ( 2004 ) Position and cell type-dependent microtubule reorientation characterizes the early response of the Arabidopsis root epidermis to ethylene . Physiol Plant 121 : 513 – 519 Google Scholar Crossref Search ADS WorldCat Lehman A , Black R, Ecker JR ( 1996 ) HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl . Cell 85 : 183 – 194 Google Scholar Crossref Search ADS PubMed WorldCat Leivar P , Quail PH ( 2011 ) PIFs: pivotal components in a cellular signaling hub . Trends Plant Sci 16 : 19 – 28 Google Scholar Crossref Search ADS PubMed WorldCat Lewis DR , Negi S, Sukumar P, Muday GK ( 2011 a ) Ethylene inhibits lateral root development, increases IAA transport and expression of PIN3 and PIN7 auxin efflux carriers . Development 138 : 3485 – 3495 Google Scholar Crossref Search ADS PubMed WorldCat Lewis DR , Ramirez MV, Miller ND, Vallabhaneni P, Ray WK, Helm RF, Winkel BSJ, Muday GK ( 2011 b ) Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks . Plant Physiol 156 : 144 – 164 Google Scholar Crossref Search ADS PubMed WorldCat Leymarie J , Vitkauskaité G, Hoang HH, Gendreau E, Chazoule V, Meimoun P, Corbineau F, El-Maarouf-Bouteau H, Bailly C ( 2012 ) Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy . Plant Cell Physiol 53 : 96 – 106 Google Scholar Crossref Search ADS PubMed WorldCat Li H , Johnson P, Stepanova A, Alonso JM, Ecker JR ( 2004 ) Convergence of signaling pathways in the control of differential cell growth in Arabidopsis . Dev Cell 7 : 193 – 204 Google Scholar Crossref Search ADS PubMed WorldCat Li J , Nagpal P, Vitart V, McMorris TC, Chory J ( 1996 ) A role for brassinosteroids in light-dependent development of Arabidopsis . Science 272 : 398 – 401 Google Scholar Crossref Search ADS PubMed WorldCat Liang X , Wang H, Mao L, Hu Y, Dong T, Zhang Y, Wang X, Bi Y ( 2012 ) Involvement of COP1 in ethylene- and light-regulated hypocotyl elongation . Planta 236 : 1791 – 1802 Google Scholar Crossref Search ADS PubMed WorldCat Linkies A , Leubner-Metzger G ( 2012 ) Beyond gibberellins and abscisic acid: how ethylene and jasmonates control seed germination . Plant Cell Rep 31 : 253 – 270 Google Scholar Crossref Search ADS PubMed WorldCat Linkies A , Müller K, Morris K, Turecková V, Wenk M, Cadman CSC, Corbineau F, Strnad M, Lynn JR, Finch-Savage WE, et al. ( 2009 ) Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana . Plant Cell 21 : 3803 – 3822 Google Scholar Crossref Search ADS PubMed WorldCat Liu J , Mehdi S, Topping J, Friml J, Lindsey K ( 2013 ) Interaction of PLS and PIN and hormonal crosstalk in Arabidopsis root development . Front Plant Sci 4 : 75 Google Scholar PubMed OpenURL Placeholder Text WorldCat Liu Y , Ye N, Liu R, Chen M, Zhang J ( 2010 ) H2O2 mediates the regulation of ABA catabolism and GA biosynthesis in Arabidopsis seed dormancy and germination . J Exp Bot 61 : 2979 – 2990 Google Scholar Crossref Search ADS PubMed WorldCat Love J , Björklund S, Vahala J, Hertzberg M, Kangasjärvi J, Sundberg B ( 2009 ) Ethylene is an endogenous stimulator of cell division in the cambial meristem of Populus . Proc Natl Acad Sci USA 106 : 5984 – 5989 Google Scholar Crossref Search ADS PubMed WorldCat Ludwików A , Cieśla A, Kasprowicz-Maluśki A, Mituła F, Tajdel M, Gałgański Ł, Ziółkowski PA, Kubiak P, Małecka A, Piechalak A, et al. ( 2014 ) Arabidopsis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis . Mol Plant 7 : 960 – 976 Google Scholar Crossref Search ADS PubMed WorldCat Luo X , Chen Z, Gao J, Gong Z ( 2014 ) Abscisic acid inhibits root growth in Arabidopsis through ethylene biosynthesis . Plant J 79 : 44 – 55 Masucci JD , Schiefelbein JW ( 1994 ) The rhd6 mutation of Arabidopsis thaliana alters root-hair initiation through an auxin- and ethylene-associated process . Plant Physiol 106 : 1335 – 1346 Google Scholar Crossref Search ADS PubMed WorldCat Masucci JD , Schiefelbein JW ( 1996 ) Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root . Plant Cell 8 : 1505 – 1517 Google Scholar PubMed OpenURL Placeholder Text WorldCat Mazzella MA , Casal JJ, Muschietti JP, Fox AR ( 2014 ) Hormonal networks involved in apical hook development in darkness and their response to light . Front Plant Sci 5 : 52 Google Scholar Crossref Search ADS PubMed WorldCat Millenaar FF , Cox MCH, van Berkel YE, Welschen RAM, Pierik R, Voesenek LAJC, Peeters AJM ( 2005 ) Ethylene-induced differential growth of petioles in Arabidopsis: analyzing natural variation, response kinetics, and regulation . Plant Physiol 137 : 998 – 1008 Google Scholar Crossref Search ADS PubMed WorldCat Miransari M , Smith DL ( 2014 ) Plant hormones and seed germination . Environ Exp Bot 99 : 110 – 121 Google Scholar Crossref Search ADS WorldCat Muday GK , Rahman A, Binder BM ( 2012 ) Auxin and ethylene: collaborators or competitors? Trends Plant Sci 17 : 181 – 195 Google Scholar Crossref Search ADS PubMed WorldCat Mühlenbock P , Plaszczyca M, Plaszczyca M, Mellerowicz E, Karpinski S ( 2007 ) Lysigenous aerenchyma formation in Arabidopsis is controlled by LESION SIMULATING DISEASE1 . Plant Cell 19 : 3819 – 3830 Google Scholar Crossref Search ADS PubMed WorldCat Müssig C , Shin GH, Altmann T ( 2003 ) Brassinosteroids promote root growth in Arabidopsis . Plant Physiol 133 : 1261 – 1271 Google Scholar Crossref Search ADS PubMed WorldCat Negi S , Ivanchenko MG, Muday GK ( 2008 ) Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana . Plant J 55 : 175 – 187 Google Scholar Crossref Search ADS PubMed WorldCat Ortega-Martínez O , Pernas M, Carol RJ, Dolan L ( 2007 ) Ethylene modulates stem cell division in the Arabidopsis thaliana root . Science 317 : 507 – 510 Google Scholar Crossref Search ADS PubMed WorldCat Pesquet E , Tuominen H ( 2011 ) Ethylene stimulates tracheary element differentiation in Zinnia elegans cell cultures . New Phytol 190 : 138 – 149 Google Scholar Crossref Search ADS PubMed WorldCat Pitts RJ , Cernac A, Estelle M ( 1998 ) Auxin and ethylene promote root hair elongation in Arabidopsis . Plant J 16 : 553 – 560 Google Scholar Crossref Search ADS PubMed WorldCat Polko JK , van Rooij JA, Vanneste S, Pierik R, Ammerlaan AMH, Vergeer-van Eijk MH, McLoughlin F, Gühl K, Van Isterdael G, Voesenek LACJ, et al. ( 2015 ) Ethylene-mediated regulation of A2-type CYCLINs modulates hyponastic growth in Arabidopsis . Plant Physiol 169: 194 – 208 Google Scholar OpenURL Placeholder Text WorldCat Polko JK , van Zanten M, van Rooij JA, Marée AF, Voesenek LA, Peeters AJ, Pierik R ( 2012 ) Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion . New Phytol 193 : 339 – 348 Google Scholar Crossref Search ADS PubMed WorldCat Rahman A , Hosokawa S, Oono Y, Amakawa T, Goto N, Tsurumi S ( 2002 ) Auxin and ethylene response interactions during Arabidopsis root hair development dissected by auxin influx modulators . Plant Physiol 130 : 1908 – 1917 Google Scholar Crossref Search ADS PubMed WorldCat Ramos P , Herrera R ( 2013 ) Anatomical changes of xylem cells in stem of Pinus radiata seedlings exposed to inclination and ethylene . Biol Plant 57 : 525 – 530 Google Scholar Crossref Search ADS WorldCat Rauf M , Arif M, Fisahn J, Xue GP, Balazadeh S, Mueller-Roeber B ( 2013 ) NAC transcription factor speedy hyponastic growth regulates flooding-induced leaf movement in Arabidopsis . Plant Cell 25 : 4941 – 4955 Google Scholar Crossref Search ADS PubMed WorldCat Raz V , Ecker JR ( 1999 ) Regulation of differential growth in the apical hook of Arabidopsis . Development 126 : 3661 – 3668 Google Scholar Crossref Search ADS PubMed WorldCat Raz V , Koornneef M ( 2001 ) Cell division activity during apical hook development . Plant Physiol 125 : 219 – 226 Google Scholar Crossref Search ADS PubMed WorldCat Rodrigues-Pousada RA , De Rycke R, Dedonder A, Van Caeneghem W, Engler G, Van Montagu M, Van Der Straeten D ( 1993 ) The Arabidopsis 1-aminocyclopropane-1-carboxylate synthase gene 1 is expressed during early development . Plant Cell 5 : 897 – 911 Google Scholar Crossref Search ADS PubMed WorldCat Růzicka K , Ljung K, Vanneste S, Podhorská R, Beeckman T, Friml J, Benková E ( 2007 ) Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution . Plant Cell 19 : 2197 – 2212 Google Scholar Crossref Search ADS PubMed WorldCat Saibo NJM , Vriezen WH, Beemster GTS, Van Der Straeten D ( 2003 ) Growth and stomata development of Arabidopsis hypocotyls are controlled by gibberellins and modulated by ethylene and auxins . Plant J 33 : 989 – 1000 Google Scholar Crossref Search ADS PubMed WorldCat Sasidharan R , Voesenek LA, Pierik R ( 2011 ) Cell wall modifying proteins mediate plant acclimatization to biotic and abiotic stresses . CRC Crit Rev Plant Sci 30 : 548 – 562 Google Scholar Crossref Search ADS WorldCat Schwechheimer C ( 2008 ) Understanding gibberellic acid signaling: are we there yet? Curr Opin Plant Biol 11 : 9 – 15 Google Scholar Crossref Search ADS PubMed WorldCat Shani E , Weinstain R, Zhang Y, Castillejo C, Kaiserli E, Chory J, Tsien RY, Estelle M ( 2013 ) Gibberellins accumulate in the elongating endodermal cells of Arabidopsis root . Proc Natl Acad Sci USA 110 : 4834 – 4839 Google Scholar Crossref Search ADS PubMed WorldCat Skirycz A , Claeys H, De Bodt S, Oikawa A, Shinoda S, Andriankaja M, Maleux K, Eloy NB, Coppens F, Yoo SD, et al. ( 2011 ) Pause-and-stop: the effects of osmotic stress on cell proliferation during early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest . Plant Cell 23 : 1876 – 1888 Google Scholar Crossref Search ADS PubMed WorldCat Smalle J , Haegman M, Kurepa J, Van Montagu M, Van Der Straeten D ( 1997 ) Ethylene can stimulate Arabidopsis hypocotyl elongation in the light . Proc Natl Acad Sci USA 94 : 2756 – 2761 Google Scholar Crossref Search ADS PubMed WorldCat Smalle J , Van Der Straeten D ( 1997 ) Ethylene and vegetative development . Physiol Plant 100 : 593 – 605 Google Scholar Crossref Search ADS WorldCat Smet D , Žádníková P, Vandenbussche F, Benková E, Van Der Straeten D ( 2014 ) Dynamic infrared imaging analysis of apical hook development in Arabidopsis: the case of brassinosteroids . New Phytol 202 : 1398 – 1411 Google Scholar Crossref Search ADS PubMed WorldCat Song SS , Huang H, Gao H, Wang JJ, Wu DW, Liu XL, Yang SH, Zhai QZ, Li CY, Qi TC, et al. ( 2014 ) Interaction between MYC2 and ETHYLENE INSENSITIVE3 modulates antagonism between jasmonate and ethylene signaling in Arabidopsis . Plant Cell 26 : 263 – 279 Google Scholar Crossref Search ADS PubMed WorldCat Stepanova AN , Yun J, Likhacheva AV, Alonso JM ( 2007 ) Multilevel interactions between ethylene and auxin in Arabidopsis roots . Plant Cell 19 : 2169 – 2185 Google Scholar Crossref Search ADS PubMed WorldCat Stowe-Evans EL , Harper RM, Motchoulski AV, Liscum E ( 1998 ) NPH4, a conditional modulator of auxin-dependent differential growth responses in Arabidopsis . Plant Physiol 118 : 1265 – 1275 Google Scholar Crossref Search ADS PubMed WorldCat Strader LC , Chen GL, Bartel B ( 2010 ) Ethylene directs auxin to control root cell expansion . Plant J 64 : 874 – 884 Google Scholar Crossref Search ADS PubMed WorldCat Street IH , Aman S, Zubo Y, Ramzan A, Wang X, Shakeel SN, Kieber JJ, Schaller GE ( 2015 ) Ethylene inhibits cell proliferation of the Arabidopsis root meristem . Plant Physiol 169 : 338 – 350 Google Scholar Crossref Search ADS PubMed WorldCat Subbiah V , Reddy KJ ( 2010 ) Interactions between ethylene, abscisic acid and cytokinin during germination and seedling establishment in Arabidopsis . J Biosci 35 : 451 – 458 Google Scholar Crossref Search ADS PubMed WorldCat Sukumar P (2010) The role of auxin and ethylene in adventitious root formation in Arabidopsis and tomato. PhD thesis. Wake Forest University, Winston-Salem, NC Swarup R , Perry P, Hagenbeek D, Van Der Straeten D, Beemster GTS, Sandberg G, Bhalerao R, Ljung K, Bennett MJ ( 2007 ) Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation . Plant Cell 19 : 2186 – 2196 Google Scholar Crossref Search ADS PubMed WorldCat Szekeres M , Németh K, Koncz-Kálmán Z, Mathur J, Kauschmann A, Altmann T, Rédei GP, Nagy F, Schell J, Koncz C ( 1996 ) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis . Cell 85 : 171 – 182 Google Scholar Crossref Search ADS PubMed WorldCat Takatsuka H , Umeda M ( 2014 ) Hormonal control of cell division and elongation along differentiation trajectories in roots . J Exp Bot 65 : 2633 – 2643 Google Scholar Crossref Search ADS PubMed WorldCat Tanimoto M , Roberts K, Dolan L ( 1995 ) Ethylene is a positive regulator of root hair development in Arabidopsis thaliana . Plant J 8 : 943 – 948 Google Scholar Crossref Search ADS PubMed WorldCat Thole JM , Beisner ER, Liu J, Venkova SV, Strader LC ( 2014 ) Abscisic acid regulates root elongation through the activities of auxin and ethylene in Arabidopsis thaliana . G3 Genes Genomes Genet 4 : 1259 – 1274 Thomann A , Lechner E, Hansen M, Dumbliauskas E, Parmentier Y, Kieber J, Scheres B, Genschik P ( 2009 ) Arabidopsis CULLIN3 genes regulate primary root growth and patterning by ethylene-dependent and -independent mechanisms . PLoS Genet 5 : e1000328 Google Scholar Crossref Search ADS PubMed WorldCat Tsuchisaka A , Yu G, Jin H, Alonso JM, Ecker JR, Zhang X, Gao S, Theologis A ( 2009 ) A combinatorial interplay among the 1-aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana . Genetics 183 : 979 – 1003 Google Scholar Crossref Search ADS PubMed WorldCat Turner JG , Ellis C, Devoto A ( 2002 ) The jasmonate signal pathway . Plant Cell ( Suppl ) 14 : S153 – S164 Google Scholar Crossref Search ADS PubMed WorldCat Vandenbussche F , Petrásek J, Zádníková P, Hoyerová K, Pesek B, Raz V, Swarup R, Bennett M, Zazímalová E, Benková E, et al. ( 2010 ) The auxin influx carriers AUX1 and LAX3 are involved in auxin-ethylene interactions during apical hook development in Arabidopsis thaliana seedlings . Development 137 : 597 – 606 Google Scholar Crossref Search ADS PubMed WorldCat Vandenbussche F , Vancompernolle B, Rieu I, Ahmad M, Phillips A, Moritz T, Hedden P, Van Der Straeten D ( 2007 ) Ethylene-induced Arabidopsis hypocotyl elongation is dependent on but not mediated by gibberellins . J Exp Bot 58 : 4269 – 4281 Google Scholar Crossref Search ADS PubMed WorldCat Van Hautegem T , Waters AJ, Goodrich J, Nowack MK ( 2015 ) Only in dying, life: programmed cell death during plant development . Trends Plant Sci 20 : 102 – 113 Google Scholar Crossref Search ADS PubMed WorldCat Vanstraelen M , Benková E ( 2012 ) Hormonal interactions in the regulation of plant development . Annu Rev Cell Dev Biol 28 : 463 – 487 Google Scholar Crossref Search ADS PubMed WorldCat Verstraeten I , Schotte S, Geelen D ( 2014 ) Hypocotyl adventitious root organogenesis differs from lateral root development . Front Plant Sci 5 : 495 Google Scholar Crossref Search ADS PubMed WorldCat Vissenberg K , Fry SC, Verbelen JP ( 2001 ) Root hair initiation is coupled to a highly localized increase of xyloglucan endotransglycosylase action in Arabidopsis roots . Plant Physiol 127 : 1125 – 1135 Google Scholar Crossref Search ADS PubMed WorldCat Vriezen W , Achard P, Harberd N, Van Der Straeten D ( 2004 ) Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent . Plant J 37 : 505 – 516 Google Scholar Crossref Search ADS PubMed WorldCat Wilson RL , Bakshi A, Binder BM ( 2014 a ) Loss of the ETR1 ethylene receptor reduces the inhibitory effect of far-red light and darkness on seed germination of Arabidopsis thaliana . Front Plant Sci 5 : 433 Google Scholar Crossref Search ADS PubMed WorldCat Wilson RL , Kim H, Bakshi A, Binder BM ( 2014 b ) The ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE2 have contrasting roles in seed germination of Arabidopsis during salt stress . Plant Physiol 165 : 1353 – 1366 Google Scholar Crossref Search ADS PubMed WorldCat Yang SF , Hoffman NE ( 1984 ) Ethylene biosynthesis and its regulation in higher plants . Annu Rev Plant Physiol 35 : 155 – 189 Google Scholar Crossref Search ADS WorldCat Yu Y , Wang J, Zhang Z, Quan R, Zhang H, Deng XW, Ma L, Huang R ( 2013 ) Ethylene promotes hypocotyl growth and HY5 degradation by enhancing the movement of COP1 to the nucleus in the light . PLoS Genet 9 : e1004025 Google Scholar Crossref Search ADS PubMed WorldCat Zádníková P , Petrásek J, Marhavy P, Raz V, Vandenbussche F, Ding Z, Schwarzerová K, Morita MT, Tasaka M, Hejátko J, et al. ( 2010 ) Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana . Development 137 : 607 – 617 Google Scholar Crossref Search ADS PubMed WorldCat Žádníková P , Smet D, Zhu Q, Van Der Straeten D, Benková E ( 2015 ) Strategies of seedlings to overcome their sessile nature: auxin in mobility control . Front Plant Sci 6 : 218 Google Scholar Crossref Search ADS PubMed WorldCat Žd’árská M , Zatloukalová P, Benítez M, Šedo O, Potě¡il D, Novák O, Svačinová J, Pe¡ek B, Malbeck J, Va¡íčková J, et al. ( 2013 ) Proteome analysis in Arabidopsis reveals shoot- and root-specific targets of cytokinin action and differential regulation of hormonal homeostasis . Plant Physiol 161 : 918 – 930 Google Scholar Crossref Search ADS PubMed WorldCat Zhang D , Jing Y, Jiang Z, Lin R ( 2014 a ) The chromatin-remodeling factor PICKLE integrates brassinosteroid and gibberellin signaling during skotomorphogenic growth in Arabidopsis . Plant Cell 26 : 2472 – 2485 Google Scholar Crossref Search ADS PubMed WorldCat Zhang X , Zhu Z, An F, Hao D, Li P, Song J, Yi C, Guo H ( 2014 b ) Jasmonate-activated MYC2 represses ETHYLENE INSENSITIVE3 activity to antagonize ethylene-promoted apical hook formation in Arabidopsis . Plant Cell 26 : 1105 – 1117 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Y , Liu Z, Wang L, Zheng S, Xie J, Bi Y ( 2010 ) Sucrose-induced hypocotyl elongation of Arabidopsis seedlings in darkness depends on the presence of gibberellins . J Plant Physiol 167 : 1130 – 1136 Google Scholar Crossref Search ADS PubMed WorldCat Zhong S , Shi H, Xue C, Wang L, Xi Y, Li J, Quail PH, Deng XW, Guo H ( 2012 ) A molecular framework of light-controlled phytohormone action in Arabidopsis . Curr Biol 22 : 1530 – 1535 Google Scholar Crossref Search ADS PubMed WorldCat Zhu C , Gan L, Shen Z, Xia K ( 2006 ) Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis . J Exp Bot 57 : 1299 – 1308 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the Research Foundation Flanders (grant nos. G.0306.12N and G.0656.13N to D.V.D.S.) and Ghent University. B.V.d.P. is a postdoctoral associate of Ghent University (Bijzonder Onderzoeksfonds). D.S. is a research assistant of the Research Foundation Flanders. 2 These authors contributed equally to the article. * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00724 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Thao, Nguyen Phuong; Khan, M. Iqbal R.; Thu, Nguyen Binh Anh; Hoang, Xuan Lan Thi; Asgher, Mohd; Khan, Nafees A.; Tran, Lam-Son Phan

doi: 10.1104/pp.15.00663pmid: 26246451

Abstract Excessive heavy metals (HMs) in agricultural lands cause toxicities to plants, resulting in declines in crop productivity. Recent advances in ethylene biology research have established that ethylene is not only responsible for many important physiological activities in plants but also plays a pivotal role in HM stress tolerance. The manipulation of ethylene in plants to cope with HM stress through various approaches targeting either ethylene biosynthesis or the ethylene signaling pathway has brought promising outcomes. This review covers ethylene production and signal transduction in plant responses to HM stress, cross talk between ethylene and other signaling molecules under adverse HM stress conditions, and approaches to modify ethylene action to improve HM tolerance. From our current understanding about ethylene and its regulatory activities, it is believed that the optimization of endogenous ethylene levels in plants under HM stress would pave the way for developing transgenic crops with improved HM tolerance. In addition to common abiotic stresses seen in agricultural production, such as drought, submerging, and extreme temperatures (Thao and Tran, 2012; Xia et al., 2015), heavy metal (HM) stress has arisen as a new pervasive threat (Srivastava et al., 2014; Ahmad et al., 2015). This is mainly due to the unrestricted industrialization and urbanization carried out during the past few decades, which have led to the increase of HMs in soils. Plants naturally require more than 15 different types of HM as nutrients serving for biological activities in cells (Sharma and Chakraverty, 2013). However, when the nutritional/nonnutritional HMs are present in excess, plants have to either suffer or take these up from the soil in an unwilling manner (Nies, 1999; Sharma and Chakraverty, 2013). Upon HM stress exposure, plants induce oxidative stress due to the excessive production of reactive oxygen species (ROS) and methylglyoxal (Sharma and Chakraverty, 2013). High levels of these compounds have been shown to negatively affect cellular structure maintenance (e.g. induction of lipid peroxidation in the membrane, biological macromolecule deterioration, ion leakage, and DNA strand cleavage; Gill and Tuteja, 2010; Nagajyoti et al., 2010) as well as many other biochemical and physiological processes (Dugardeyn and Van Der Straeten, 2008). As a result, plant growth is retarded and, ultimately, economic yield is decreased (Yadav, 2010; Anjum et al., 2012; Hossain et al., 2012; Asgher et al., 2015). Moreover, the accumulation of metal residues in the major food chain has been shown to cause serious ecological and health problems (Malik, 2004; Verstraeten et al., 2008). Plants employ different strategies to detoxify the unwanted HMs. Among the common responses of plants to HM stress are increases in ethylene production due to the enhanced expression of ethylene-related biosynthetic genes (Asgher et al., 2014; Khan and Khan, 2014; Khan et al., 2015b) and/or changes in the expression of ethylene-responsive genes (Maksymiec, 2007). Conventionally, this hormone has been established to modulate a number of important plant physiological activities, including seed germination, root hair and root nodule formation, and maturation (fruit ripening in particular; Dugardeyn and Van Der Straeten, 2008). On the other hand, although ethylene has also been suggested to be a stress-related hormone responding to a number of biotic and abiotic triggers, little is known about the exact role of elevated HM stress-related ethylene in plants (Zapata et al., 2003). Enhanced production of ethylene in plants subjected to toxic levels of cadmium (Cd), copper (Cu), iron (Fe), nickel (Ni), and zinc (Zn) has been shown (Maksymiec, 2007). As an example, Cd- and Cu-mediated stimulation of ethylene synthesis has been reported as a result of the increase of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity, one of the enzymes involved in the ethylene synthesis pathway (Schlagnhaufer and Arteca, 1997; Khan et al., 2015b). Plants tend to adjust or induce adaptation or tolerance mechanisms to overcome stress conditions. To develop stress tolerance, plants trigger a network of hormonal cross talk and signaling, among which ethylene production and signaling are prominently involved in stress-induced symptoms in acclimation processes (Gazzarrini and McCourt, 2003). Therefore, the necessity of controlling ethylene homeostasis and signal transduction using biochemical and molecular tools remains open to combat stress situations. Stress-induced ethylene acts to trigger stress-related effects on plants because of the autocatalytic ethylene synthesis. Autocatalytic stress-related ethylene production is controlled by mitogen-activated protein kinase (MAPK) phosphorylation cascades (Takahashi et al., 2007) and through stabilizing ACS2/6 (Li et al., 2012). Strong lines of evidence have shown the multiple facets of ethylene in plant responses to different abiotic stresses, including excessive HM, depending upon endogenous ethylene concentration and ethylene sensitivities that differ in developmental stage, plant species, and culture systems (Pierik et al., 2006; Kim et al., 2008; Khan and Khan, 2014). Under HM stress conditions, plants show a rapid increase in ethylene production and reduced plant growth and development, suggesting a negative regulatory role of ethylene in plant responses to HM stress (Schellingen et al., 2014; Khan et al., 2015b). On the other hand, a potential involvement of ETHYLENE INSENSITIVE2 (EIN2), a central component of the ethylene signaling pathway, as a positive regulator in lead (Pb) resistance in Arabidopsis (Arabidopsis thaliana) has also been demonstrated (Cao et al., 2009). More recently, Khan and Khan (2014) showed that ethylene-regulated antioxidant metabolism maintained a higher level of reduced glutathione (GSH) and alleviated photosynthetic inhibition in mustard (Brassica juncea) plants exposed to Ni, Zn, or Cd through the optimization of ethylene homeostasis (Masood et al., 2012). Taken together, the purpose of this review is to update the research community with our current understanding of the roles of ethylene and its signaling in plant responses to HM stress. Moreover, the cross talk of ethylene with other phytohormones and signaling molecules upon HM stress will also be discussed. ETHYLENE AND PLANT RESPONSES TO HM STRESS The role of ethylene in plant responses to HMs has been a concern of many plant molecular biologists, biochemists, and physiologists, but in-depth and convincing research on how ethylene regulates different HM tolerance mechanisms is still a matter of task. Under unstressed conditions, ethylene is synthesized from an activated form of Met in plants (Xu and Zhang, 2015). ACS converts S-adenosyl-methionine (SAM) to ACC, and the oxidization of ACC is then executed by ACC oxidase (ACO) to form ethylene (Fig. 1). ACS and ACO, the two major enzymes in ethylene biosynthesis, are encoded by multigene families, which are also the primary regulation points in the ethylene biosynthetic pathway (Xu and Zhang, 2015). HM stress increases the activity of these two enzymes, resulting in increased ethylene production (Schellingen et al., 2014; Khan et al., 2015b). The Cu-inducible expression of the ACS genes in potato (Solanum tuberosum) and the accumulation of the ACS transcripts in different varieties of tobacco (Nicotiana tabacum) have been reported (Schlagnhaufer et al., 1997). Recently, transcriptome analysis of chromium-treated rice (Oryza sativa) roots also indicated enhanced expression of four ethylene biosynthesis-related genes (ACS1, ACS2, ACO4, and ACO5), suggesting the participation of ethylene in chromium signaling in rice (Steffens, 2014; Trinh et al., 2014). These findings together demonstrated that ethylene is enhanced in response to various excessive metals in a wide range of plant species (Maksymiec, 2007; Peñarrubia et al., 2015). Figure 1. Open in new tabDownload slide Ethylene biosynthesis under normal conditions and HM stress. Ethylene biosynthesis under normal conditions starts from the conversion of Met into SAM catalyzed by SAM synthetase. Furthermore, SAM is catalyzed by ACS to form ACC, an immediate precursor of ethylene. At the last step, ACC is oxidized by ACO to form ethylene. At this step, CO2 and cyanide (HCN) are produced as by-products. Under HM stress, ethylene biosynthesis rapidly increased due to the excessive ROS production, resulting in oxidative burst of the cell and activation of the MAPK3 and MAPK6 cascade. The activated MAPK cascade phosphorylates ACS2 and ACS6 enzymes. Both native and phosphorylated ACS enzymes are functional; however, phosphorylated ACS is more stable and active compared with native ACS. Phosphorylated ACS induces stress ethylene. However, HM-induced stress ethylene can be controlled either by the manipulation of ethylene biosynthetic genes using biotechnological tools or by pharmacological tools, such as the ethylene biosynthesis inhibitors aminoethoxyvinylglycine (AVG) and cobalt (Co) that inhibit ACS and ACO activities, respectively. Additionally, stress ethylene action can be blocked by using ethylene receptor inhibitor norbornadiene (NBD), silver nitrate (AgNO3), 1-methylcyclopropene (1-MCP), or silver thiosulfate (STS). The dashed line indicates possible regulation under HM stress. Arrows and T-bars represent positive and negative regulation, respectively, upon HM stress. Pi, Inorganic phosphate. Figure 1. Open in new tabDownload slide Ethylene biosynthesis under normal conditions and HM stress. Ethylene biosynthesis under normal conditions starts from the conversion of Met into SAM catalyzed by SAM synthetase. Furthermore, SAM is catalyzed by ACS to form ACC, an immediate precursor of ethylene. At the last step, ACC is oxidized by ACO to form ethylene. At this step, CO2 and cyanide (HCN) are produced as by-products. Under HM stress, ethylene biosynthesis rapidly increased due to the excessive ROS production, resulting in oxidative burst of the cell and activation of the MAPK3 and MAPK6 cascade. The activated MAPK cascade phosphorylates ACS2 and ACS6 enzymes. Both native and phosphorylated ACS enzymes are functional; however, phosphorylated ACS is more stable and active compared with native ACS. Phosphorylated ACS induces stress ethylene. However, HM-induced stress ethylene can be controlled either by the manipulation of ethylene biosynthetic genes using biotechnological tools or by pharmacological tools, such as the ethylene biosynthesis inhibitors aminoethoxyvinylglycine (AVG) and cobalt (Co) that inhibit ACS and ACO activities, respectively. Additionally, stress ethylene action can be blocked by using ethylene receptor inhibitor norbornadiene (NBD), silver nitrate (AgNO3), 1-methylcyclopropene (1-MCP), or silver thiosulfate (STS). The dashed line indicates possible regulation under HM stress. Arrows and T-bars represent positive and negative regulation, respectively, upon HM stress. Pi, Inorganic phosphate. A classic example illustrating the involvement of ethylene in plant responses to HM stress was the study of Sandmann and Böger (1980), which demonstrated that the synthesis of ethylene and the inhibition of photosynthetic electron transport in isolated spinach (Spinacia oleracea) chloroplasts were induced by Cu stress. It is possible that the high content of ethylene led to the inhibition of the photosystems, which might also trigger senescence processes at the late phase of growth or after a longer exposure to the excessive Cu in runner bean (Phaseolus coccineus; Maksymiec and Baszyński, 1996). Moreover, Arteca and Arteca (2007) showed that the application of Cu or Cd induced various levels of ethylene production in different plant parts, among which the highest amount was recorded in inflorescences. This group affirmed that Cu and Cd induced similar levels of ethylene production in both inflorescence stalks and leaves. This observation was different from earlier results that demonstrated that Cd promoted a greater increase in ethylene production in bean leaves than Cu or other HMs tested (Rodecap et al., 1981; Fuhrer, 1982). Interestingly, it was reported that ethylene biosynthesis was diminished in the Arabidopsis copper transporter5 (copt5) mutant, which is defective in Cu transport, resulting in the hypersensitivity of copt5 to Cd stress (Carrió-Seguí et al., 2015). This finding suggests that an optimal endogenous Cu level might help plants better tolerate HM stress. Another independent study noticed that Ni and Zn did not stimulate ethylene production in Arabidopsis (Arteca and Arteca, 2007). However, these two HMs increased ethylene levels in mustard plants by enhancing ACS activity (Khan and Khan, 2014). In other recent studies, Jakubowicz et al. (2010) reported that 2.5 mm Cu induced ethylene biosynthesis in broccoli (Brassica oleracea) seedlings, and Franchin et al. (2007) noted significantly enhanced ethylene production with Cu concentration within a range of 5 to 500 μm, causing leaf toxicity and impairing root formation in poplar (Populus alba). In contrast, Cu at 25 and 50 μm did not significantly induce ethylene production in Arabidopsis seedlings (Lequeux et al., 2010). Collectively, these data might suggest that the HM-induced ethylene production is plant specific and/or dose dependent. Ethylene was shown to be involved in the regulation of P. coccineus responses to Cd stress (Maksymiec, 2011). The Cd-induced ROS decreased in roots, and Cd-induced inhibition of leaf growth was completely ameliorated by the ethylene action inhibitor STS (Maksymiec, 2011). More recently, Schellingen et al. (2014) reported that the expression of ethylene-responsive genes, such as ACO2, ETHYLENE RESPONSE2 (ETR2), and ETHYLENE RESPONSE FACTOR1 (ERF1), was up-regulated by Cd treatment, while ethylene elevation during stress resulted in negative effects on leaf biomass in Arabidopsis plants. Together, these data suggest that the induction of ethylene by HMs may cause unbeneficial symptoms in plants that were exposed to HMs. However, although it was also reported that HM stress-induced ethylene had negative effects on mustard plants, an optimized level of ethylene, which was lower than the HM stress-induced ethylene level but still higher than the ethylene level of control plants under unstressed conditions, could lead to beneficial plant responses, such as increased photosynthesis under Cd stress (Masood et al., 2012). These findings together suggest the complex and biphasic regulatory function of ethylene under stressful environments, which depends on its endogenous level. EFFECTS OF ETHYLENE MODULATORS ON ETHYLENE BIOSYNTHESIS UNDER HM STRESS It has been evident that the ethylene biosynthesis pathway is well regulated under HM stress in plants. The increase of endogenous ethylene levels under HM stress caused negative effects on plant growth and developmental processes (Maksymiec, 2011; Schellingen et al., 2014). By contrast, reducing HM-induced ethylene production to keep ethylene at an optimized level shows the positive regulatory role of ethylene in plant responses to various HMs (Maksymiec, 2011). Understanding these important issues, scientists have been able to control plant growth and development under HM stress conditions, including Cd, Ni, and Zn stresses, using ethylene action or ethylene biosynthetic inhibitors at low concentrations (Maksymiec and Krupa, 2007; Khan et al., 2015b). More interestingly, the inhibitors of ethylene production do not protect the commodity from exogenous ethylene (Zhang and Wen, 2010; Iqbal et al., 2012). They disrupt the ethylene biosynthesis pathway by targeting either ACS or ACO, whereas ethylene action inhibitors occupy ethylene receptors and block ethylene action (Serek et al., 2006). Co, a beneficial metal for plant development at moderate levels, is known as an inhibitor of ethylene production (Palit et al., 1994; Yıldız et al., 2009; Chmielowska-Bąk et al., 2014). Although many studies showed that Cd, Cu, Fe, and Zn induce ethylene production in plants (Wise and Naylor, 1988; Maksymiec, 2007), excessive Co treatment of HM-stressed plants does not lead to enhanced ethylene levels, since Co inhibits the ACO enzymatic activity in the ethylene synthetic pathway. Thus, Co has been widely used as an ethylene biosynthesis inhibitor to study the effects of ethylene on plant responses to HM stress (Sun et al., 2010; Chmielowska-Bąk et al., 2014). However, in soybean (Glycine max) seedlings, coapplication of Co and Cd negatively affected cell viability as well as the expression of Cd-induced genes encoding MAPK KINASE2, DNA BINDING WITH ONE FINGER1 (DOF1), and BASIC LEUCINE ZIPPER2 (bZIP2) transcription factors, suggesting that Co increased Cd toxicity to soybean plants and that this happened independently from ethylene action (Chmielowska-Bąk et al., 2014). Moreover, excessive Co also increased oxidative stress and photosynthesis inhibition as well as caused alterations in germination, sex ratio, photoperiodism, and uptake of other elements (Yıldız et al., 2009; Hasan et al., 2011). Therefore, the use of Co as an ethylene biosynthesis inhibitor in research should be interpreted with caution. AVG, another inhibitor of ethylene synthesis, has been shown to decrease ethylene production by inhibiting ACS activity (Masood et al., 2012). Iakimova et al. (2008) reported that the combination of ethylene and Cd treatments to tomato (Solanum lycopersicum) suspension cells resulted in increased cell death, which could be rescued by adding AVG (Fig. 1). Besides the application of ethylene biosynthesis inhibitors, ethephon, an exogenous ethylene-releasing compound, has also been widely used to control endogenous ethylene production and function under Cd stress (Masood et al., 2012) and Ni or Zn stress (Khan and Khan, 2014). Although under nonstressed conditions, ethephon treatment has been shown to increase the level of endogenous ethylene in plants (Cooke and Randall, 1968; Khan, 2004), interestingly, the level of HM-induced ethylene was shown to be decreased by ethephon treatment, which led to the induction of an antioxidant system and increased photosynthesis. As a result, ethephon-treated plants were found to be more tolerant to HM stress (Masood et al., 2012; Khan and Khan, 2014). More investigations should be carried out to better clarify the role of ethephon in the regulation of ethylene homeostasis and sensitivity under HM stress. ETHYLENE SIGNALING AND PLANT RESPONSES TO HM STRESS Ethylene receptors are similar to bacterial two-component receiver domains. Ethylene in Arabidopsis is perceived by a five-member family of ethylene receptors, including products encoded by the ETR1 and ETR2, ETHYLENE RESPONSE SENSOR1 (ERS1) and ERS2, and EIN4 genes (Clark et al., 1998; Yoo et al., 2009). In Arabidopsis, in the absence of ethylene, CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a Raf-like MAPK KINASE KINASE, interacts with the ethylene receptors to suppress the downstream component EIN2 by directly phosphorylating its cytosolic C-terminal domain, leading to the inactivation of EIN3 and ETHYLENE-INSENSITIVE3-LIKE1 (EIL1; Guo and Ecker, 2004; Ju et al., 2012; Shan et al., 2012). Upon the binding of ethylene to the receptors with the help of the Cu ions delivered by the Cu transporter RESPONSIVE TO ANTAGONIST1 (RAN1), CTR1 becomes inactivated, consequently resulting in the cleavage of CARBOXYL END OF EIN2 from the endoplasmic reticulum-located EIN2. As a result, the moving of EIN2 to the nucleus is facilitated, which leads to the stabilization of EIN3 protein that initiates the signaling cascade (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012). The MAPK cascade has been shown to be involved in ethylene signaling and/or ethylene biosynthetic pathways by targeting at least ACS2 and ACS6 (Liu and Zhang, 2004; Hahn and Harter, 2009; Yoo et al., 2009; Opdenakker et al., 2012). Under HM stress, such as Cd stress, ethylene production has also been found to be induced mainly through the accumulation of ACS2 and ACS6 transcripts (Schellingen et al., 2014). The Arabidopsis acs2-1 acs6-1 double knockout mutant exposed to Cd showed a decreased ethylene level, leading to a positive effect on leaf biomass (Schellingen et al., 2014), suggesting the negative regulation of HM stress-induced ethylene in plant development. As the number of studies on ethylene signaling under HM stress has been limited, more effort should be taken in this important research area. Since blockers of the ethylene receptor protect the tissues from both endogenous and exogenous ethylenes, ethylene action inhibitors are considered very potent for agricultural use (Sisler and Serek, 1997; Feng et al., 2000). They are more specific than ethylene biosynthetic inhibitors because they bind to a specific receptor (Sisler and Serek, 1997; Hua and Meyerowitz, 1998; Klee, 2004). The use of 1-MCP, a blocker of ethylene action in plants, has been reviewed extensively (Sisler and Serek, 1997; Blankenship and Dole, 2003), and numerous applications of 1-MCP in the amelioration of stress responses in plants have been reported (Grimmig et al., 2003; Huang and Lin, 2003; Yokotani et al., 2004). Recently, Montero-Palmero et al. (2014b) reported the involvement of ethylene as a negative regulator of mercury (Hg)-induced responses in alfalfa (Medicago sativa) using 1-MCP. Similarly, the application of STS, an inhibitor of ethylene reception, is another efficient means of controlling ethylene action and thus is being used for both agronomic and research purposes (Ichimura and Niki, 2014; Pacifici et al., 2014). Silver is thought to occupy the Cu-binding site of ethylene receptors and to interact with ethylene to inhibit the ethylene response (Rodríguez et al., 1999; Zhao et al., 2002; Binder et al., 2007). NBD, the third ethylene action inhibitor compound, is also a very common tool used to reduce ethylene-induced stress effects under Ni and Zn treatment (Sisler and Serek, 1997; Khan and Khan, 2014). Using NBD, which was expected to inhibit ethylene action by blocking receptors, Khan and Khan (2014) have verified the involvement of ethylene in the reversal of photosynthetic inhibition by Ni and Zn stress, which was caused by changes in PSII activity, and the enhancement of photosynthetic nitrogen use efficiency and antioxidant capacity. These findings together suggest that appropriate control of ethylene action using ethylene action inhibitors could lead to the positive regulation role of this hormone in plant responses to HM stress. ETHYLENE AND ITS CROSS TALK WITH OTHER HORMONES AND SIGNALING MOLECULES IN THE REGULATION OF PLANT TOLERANCE TO HM STRESS The molecular mechanism of how plants can cope with different HM stresses varies from plant to plant, but in general, ethylene and its cross talk with other phytohormones or with signaling molecules are important for plant adaptation to HM-induced oxidative stress (Thapa et al., 2012; Montero-Palmero et al., 2014a, 2014b). It has been found that not only the production of ethylene but other phytohormones are also affected by excessive HM. Upon exposure to the stress, the levels of jasmonic acid (JA), salicylic acid (SA), abscisic acid, and ethylene increase, while the contents of GA3 and auxin decrease in plants (Metwally et al., 2003; Cánovas et al., 2004; Atici et al., 2005; Maksymiec et al., 2005). Taking a case study of aluminum (Al) application in Arabidopsis as an example, it was observed that Al treatment led to the increased expression of ethylene biosynthesis-related genes, including both AtACS (AtACS2, AtACS6, and AtACS8) and AtACO (AtACO1 and AtACO2) genes (Sun et al., 2010). Moreover, in wild-type plants, this Al treatment also increased the transcript of AUXIN RESISTANT1 (AtAUX1) and PINFORMED2 (AtPIN2), yet the ethylene synthesis inhibitors Co and AVG, and the ethylene perception inhibitor silver, abolished this Al-induced expression of AtAUX1 and AtPIN2. In the auxin-insensitive single mutants aux1-7 and pin2, the Al-induced inhibition of root elongation was lower than that in the wild type. These data suggested that Al-induced ethylene production may lead to auxin redistribution by affecting auxin polar transport systems through AUX1 and PIN2 (Sun et al., 2010), which is an indicator of possible cross talk between ethylene and auxin in plant responses to HM stress. Interestingly, it was not PIN2 or AUX1 but PIN1 that was reported to be required for Cu-induced auxin redistribution in Arabidopsis (Yuan et al., 2013). Furthermore, the study of Yuan et al. (2013) also showed that both ein2-1 and wild-type plants exhibited similar effects on the inhibition of primary root elongation under Cu stress, indicating that ethylene-mediated signaling is not required for the Cu-inhibited primary root elongation. Together, these findings suggested that genes involved in the control of auxin redistribution might be specific, and they act dependently or independently of ethylene/ethylene signaling, depending on the type of HMs to which the plants are exposed. Recently, the ethylene and JA signaling pathways have been shown to converge at two ethylene-stabilized transcription factors, EIN3 and EIL1, and to function synergistically in the regulation of gene expression in Arabidopsis (Zhu et al., 2011). Moreover, other studies further indicated that the posttranslational regulation of ERFs by ethylene and JA was independent of EIN3/EIL1 (Bethke et al., 2009; Van der Does et al., 2013). When Arabidopsis plants were exposed to excessive Cd, these two hormone signaling pathways were activated, leading to the up-regulation of NITRATE TRANSPORTER1.8 (NRT1.8) and the down-regulation of NRT1.5, which mediated the stress-initiated nitrate allocation to roots to enhance the tolerance to Cd stress (Zhang et al., 2014). By studying the gibberellin insensitive ethylene overproducing2-1 double mutant, a functional GA3 signaling pathway was shown to be required for the increased ethylene biosynthesis in Arabidopsis, suggesting a possible link between ethylene and GA3 (De Grauwe et al., 2008). More recently, Masood and Khan (2013) suggested that treatment with GA3 and/or sulfur (S) at sufficient levels reduced undesirable stress ethylene induction, resulting in the alleviation of photosynthetic inhibition caused by Cd stress. It is well established that S assimilation leads to Cys biosynthesis, which is required for both ethylene and GSH biosyntheses under normal conditions (De Grauwe et al., 2008; Iqbal et al., 2013). On the other hand, under HM stress, application of S to Cd-treated plants was reported to adjust stress-induced ethylene content to an optimized level, which subsequently led to a maximal GSH content, thereby providing effective protection again oxidative stress and, thus, alleviating unbeneficial Cd-induced symptoms in plants (Asgher et al., 2014). Furthermore, both ethylene and S assimilation pathways were also affected by Cd stress and were shown to regulate GSH biosynthesis under Cd stress (Masood et al., 2012). This further suggested the role of the GSH pathway in the mitigation of HM stress through ethylene and ethylene signaling that might also involve the S pathway (i.e. the GSH pathway might be the check point of the cross talk between S and ethylene in plant responses to HM stress). The role of GSH in HM detoxification might be explained by numerous physiological, biochemical, and genetic studies that have confirmed that GSH is the substrate for phytochelatin (PC) biosynthesis (Cobbett, 2000). In Arabidopsis and fission yeast (Schizosaccharomyces pombe), PCs were shown to play an important role in Cd and arsenic detoxification by using PC synthase-deficient mutants (Ha et al., 1999). Down-regulation of GSH1 and a decrease in GSH content were observed in the Arabidopsis ein2-1 mutant, which led to the impaired GSH-dependent Pb tolerance (Cao et al., 2009), indicating that ethylene signaling positively regulates HM responses through the GSH pathway. On the other hand, there was also evidence that the EIN2 gene mediates Pb resistance through a GSH-independent PLEIOTROPIC DRUG RESISTANCE TRANSPORTER12 (AtPDR12)-mediated mechanism (Cao et al., 2009). PDR12, which is a member of the ATP-binding cassette transporter G family and is induced by auxin, abscisic acid, ethylene, JA, and SA, was reported to be up-regulated in Arabidopsis plants treated with AuCl−4 (Shukla et al., 2014). ROS itself was also reported to have an interaction with ethylene in plant responses to HMs. Ethylene and hydrogen peroxide were believed to act in a synergistic manner in tomato, and hydrogen peroxide plays an important role in ethylene-related Cd-induced cell death (Liu et al., 2008). Several studies have shown that HMs, such as Cd, Cu, Fe, Zn, Hg, manganese, and Al, can induce ROS production and alter the activities of antioxidant enzymes, including catalase, superoxide dismutase (SOD), peroxidase, ascorbate peroxidase (APX), and glutathione reductase (GR), in plants (Sun et al., 2010; Yuan et al., 2013; Montero-Palmero et al., 2014a; Khan et al., 2015b; Mostofa et al., 2015b). It was found that the application of ethephon or NBD could somehow adjust the stress-induced ethylene, thereby alleviating photosynthetic inhibition and decreasing oxidative stress, perhaps by the enhancement of SOD, APX, and GR metabolism, in mustard plants treated with Ni and Zn (Khan and Khan, 2014). More recently, Liu et al. (2010) reported that pretreatment of Cd-stressed Arabidopsis plants with GSH, a ROS scavenger, inhibited the activation of MAPK3 and MAPK6, which had been activated by Cd-induced ROS accumulation. MAPK3 and MAPK6 have been demonstrated to be involved in the regulation of ethylene biosynthesis and potentially in the ethylene signaling pathway, although this last possibility remains controversial (Ecker, 2004; Hahn and Harter, 2009), providing a hint about the potential interaction between ROS and ethylene through these MAPKs in the regulation of plant responses to HM stress. In response to HMs, not only ethylene but other hormones, including brassinosteroids, auxin, SA, GA3, and cytokinin, were shown to stimulate the antioxidant responses in order to scavenge different ROS when plants were grown under Cd, Cu, or Pb stress (Hayat et al., 2007; Noriega et al., 2012; Piotrowska-Niczyporuk et al., 2012). SA treatment increased the GSH content and resulted in an induction of antioxidant and metal detoxification systems, which led to Cd stress tolerance in wheat (Triticum aestivum) and pea (Pisum sativum) as well as amelioration of the negative effects of Cu stress in Brassica napus (Srivastava and Dwivedi, 1998; Khademi et al., 2014; Kovács et al., 2014). In contrast, JA was found to increase metal biosorption and ROS generation in the green microalga Chlorella vulgaris (Chlorophyceae) exposed to excessive Cd, Cu, or Pb (Piotrowska-Niczyporuk et al., 2012). Moreover, ROS production was triggered by JA in Arabidopsis treated with Cu or Cd (Maksymiec and Krupa, 2006). However, it has also been reported that JA-induced ROS is mediated by the oxidative status of GSH and that JA induced the expression of GSH metabolic genes (Xiang and Oliver, 1998; Mhamdi et al., 2010). Thus, the mechanism of how JA is involved in HM-induced oxidative stress and plant tolerance still requires further experiments. It would be interesting to see the changes in the levels of all other hormones, ROS, and antioxidant systems in ethylene-deficient or -overproducing plants under normal and HM stress conditions to learn more about the cross talk between ethylene and other hormones in plant responses to HM stress. Nitric oxide (NO), another signaling molecule, is well known to have a regulatory role in various plant responses, including ethylene emission (Leshem and Haramaty, 1996), biotic and abiotic responses (Leshem and Haramaty, 1996; Clark et al., 1998; Durner et al., 1998; Delledonne et al., 2001; Mostofa et al., 2015a), cell proliferation and plant development (Ribeiro et al., 1999), senescence (Corpas et al., 2004), programmed cell death (Magalhaes et al., 1999; Clarke et al., 2000; Pedroso et al., 2000), and stomatal closure (García-Mata and Lamattina, 2002; Neill et al., 2002). However, similar to ethylene, NO plays a controversial role in HM tolerance. Exogenous NO was shown to contribute to the enhancement of plant tolerance to excessive Cd, Ni, and Al (Laspina et al., 2005; Wang and Yang, 2005; Singh et al., 2008; Kazemi et al., 2010), whereas endogenous NO was reported to be involved in Cd toxicity in plants (Groppa et al., 2008; Besson-Bard et al., 2009; Ma et al., 2010). Recently, it was reported that the Cd-induced activation of MAPK6 is mediated by NO (Hahn and Harter, 2009; Ye et al., 2013), which might suggest a link between NO and ethylene through MAPK6 in plant responses to HM stress. NO could act as an antioxidant to scavenge ROS and, directly or indirectly, increase the activity of antioxidant enzymes in leaves of plants treated with Ni or Cd (Kazemi et al., 2010; Ye et al., 2013). The accumulation of ethylene and ROS, and the diminution of NO, led to Cd-induced senescence processes in pea (Rodríguez-Serrano et al., 2006). Moreover, ethylene, polyamines, NO, MAPKs, and several transcription factors, including MYBZ2, bZIP62, and DOF1, were proposed to integrate the responses to short-term Cd stress in young soybean seedlings (Chmielowska-Bąk et al., 2014). Together, these findings further suggest a possible role of NO in the HM-induced ethylene pathway. On the other hand, under Ni stress, application of both NO and SA significantly reduced Pro accumulation, lipid peroxidation, and ROS level in Brassica napus leaves as well as improved the chlorophyll content, thus reducing the toxic effects of Ni on this crop plant (Kazemi et al., 2010). These findings collectively indicate a complex mechanism of how phytohormones, including ethylene, and signaling molecules interact in response to HMs (Fig. 2). Figure 2. Open in new tabDownload slide Generalized model of ethylene biosynthesis and signaling pathways under HM stress in cross talk with other phytohormones and signaling molecules. Different colors show different networks of ethylene, auxin, SA, JA, GA3, abscisic acid (ABA), ROS, NO, and S assimilation in plants under HM stress. Arrows and T-bars indicate positive and negative regulatory interaction, respectively. Dashed lines indicate possible regulation under HM stress. The cross represents release from inhibition. Au, Gold; CAT, catalase; Mn, manganese. Figure 2. Open in new tabDownload slide Generalized model of ethylene biosynthesis and signaling pathways under HM stress in cross talk with other phytohormones and signaling molecules. Different colors show different networks of ethylene, auxin, SA, JA, GA3, abscisic acid (ABA), ROS, NO, and S assimilation in plants under HM stress. Arrows and T-bars indicate positive and negative regulatory interaction, respectively. Dashed lines indicate possible regulation under HM stress. The cross represents release from inhibition. Au, Gold; CAT, catalase; Mn, manganese. IMPROVEMENT OF PLANT TOLERANCE TO HM: AN APPROACH OF MODIFYING ETHYLENE ACTION HM stress has become a significant concern because of its severe impact on human health and plant productivity (Thapa et al., 2012). Understanding the changes in ethylene biosynthesis and signaling triggered by HMs at the molecular level may help identify gene(s) responsible for the expression of an HM-tolerant genotype, thus providing biotechnological approaches to improve plant fitness in HM-polluted areas. Manipulation of ethylene response/signaling and/or ethylene endogenous production plays an important role in the improvement of plant HM tolerance (Asgher et al., 2014; Khan and Khan, 2014; Khan et al., 2015b; Table I). Several studies have proved that the application of ethylene biosynthesis modulators adjusted endogenous stress-induced ethylene content to an optimized level and, consequently, resulted in beneficial effects in plants treated with Ni and Zn (Iqbal et al., 2012; Khan and Khan, 2014), Cd (Iakimova et al., 2008; Sun et al., 2010; Chmielowska-Bąk et al., 2014), or Al (Sun et al., 2010). Additionally, S application has proved to be effective in the alleviation of Cd stress, which was related to the reduction of undesirable stress-induced ethylene production in mustard, suggesting that S might be used to optimize the ethylene level for developing HM stress-tolerant cultivars as well (Asgher et al., 2014; Khan et al., 2015a). Furthermore, a combined treatment of mustard plants with GA3 and/or S decreased Cd-induced stress ethylene production and promoted a photosynthetic response to Cd stress (Masood and Khan, 2013). As supportive evidence for the approach of reducing stress ethylene levels to improve HM tolerance, Schellingen et al. (2014) reported that the ethylene-deficient acs2-1 acs6-1 double mutant showed alleviated growth inhibition of leaves in Cd-exposed Arabidopsis plants, as discussed earlier. These findings together suggest that the alteration of endogenous levels of ethylene can be used to mitigate the HM toxicity of plants, and the manipulation of endogenous ethylene levels, therefore, can be considered as a potential biotechnological approach for the development of crop cultivars with improved HM tolerance. Summary of the experimental manipulation of ethylene levels and the ethylene signaling pathway in plant responses to HM stress Table I. Summary of the experimental manipulation of ethylene levels and the ethylene signaling pathway in plant responses to HM stress The ↓ and ↑ arrows indicate decrease and increase, respectively. Nr, Never ripe. Stress . Species . Genetic Approaches . Physiological Traits . References . Al Arabidopsis etr1-3 mutant ↓ Inhibition of root elongation Sun et al. (2010) Al Arabidopsis ein2-1 mutant ↓ Inhibition of root elongation Sun et al. (2010) Cd Arabidopsis acs2-1 acs6-1 double mutants ↓ Inhibition of leaf biomass Schellingen et al. (2014) Cd Tomato Nr (LeETR3) mutant ↓ Root diameter Gratao et al. (2009) Cd Tomato Nr (LeETR3) mutant Maintenance of pigment content; ↓ leaf senescence Monteiro et al. (2011) Cd + S B. juncea None Optimization of ethylene level; ↓ undesirable Cd-induced symptoms Asgher et al. (2014) Cd + GA3 + S B. juncea None Optimization of ethylene level; ↓ undesirable Cd-induced symptoms Masood and Khan (2013) Cd + ethephon + S B. juncea None ↑ Ethylene sensitivity; ↑ photosynthesis Masood et al. (2012) Cd + STS P. coccineus None ↓ Inhibition of leaf growth Maksymiec (2011) Cu Arabidopsis ein2-1 mutant Similar inhibition of root elongation relative to the wild type Yuan et al. (2013) Hg Arabidopsis ein2-5 mutant ↓ Inhibition of root growth Montero-Palmero et al. (2014a) Ni + Zn + ethephon B. juncea None Optimization of ethylene level; ↓ photosynthetic inhibition Khan and Khan (2014) Pb Arabidopsis ein2-1 mutant Inhibition of root length; ↑ Pb content; ↓ GSH content Cao et al. (2009) Stress . Species . Genetic Approaches . Physiological Traits . References . Al Arabidopsis etr1-3 mutant ↓ Inhibition of root elongation Sun et al. (2010) Al Arabidopsis ein2-1 mutant ↓ Inhibition of root elongation Sun et al. (2010) Cd Arabidopsis acs2-1 acs6-1 double mutants ↓ Inhibition of leaf biomass Schellingen et al. (2014) Cd Tomato Nr (LeETR3) mutant ↓ Root diameter Gratao et al. (2009) Cd Tomato Nr (LeETR3) mutant Maintenance of pigment content; ↓ leaf senescence Monteiro et al. (2011) Cd + S B. juncea None Optimization of ethylene level; ↓ undesirable Cd-induced symptoms Asgher et al. (2014) Cd + GA3 + S B. juncea None Optimization of ethylene level; ↓ undesirable Cd-induced symptoms Masood and Khan (2013) Cd + ethephon + S B. juncea None ↑ Ethylene sensitivity; ↑ photosynthesis Masood et al. (2012) Cd + STS P. coccineus None ↓ Inhibition of leaf growth Maksymiec (2011) Cu Arabidopsis ein2-1 mutant Similar inhibition of root elongation relative to the wild type Yuan et al. (2013) Hg Arabidopsis ein2-5 mutant ↓ Inhibition of root growth Montero-Palmero et al. (2014a) Ni + Zn + ethephon B. juncea None Optimization of ethylene level; ↓ photosynthetic inhibition Khan and Khan (2014) Pb Arabidopsis ein2-1 mutant Inhibition of root length; ↑ Pb content; ↓ GSH content Cao et al. (2009) Open in new tab Table I. Summary of the experimental manipulation of ethylene levels and the ethylene signaling pathway in plant responses to HM stress The ↓ and ↑ arrows indicate decrease and increase, respectively. Nr, Never ripe. Stress . Species . Genetic Approaches . Physiological Traits . References . Al Arabidopsis etr1-3 mutant ↓ Inhibition of root elongation Sun et al. (2010) Al Arabidopsis ein2-1 mutant ↓ Inhibition of root elongation Sun et al. (2010) Cd Arabidopsis acs2-1 acs6-1 double mutants ↓ Inhibition of leaf biomass Schellingen et al. (2014) Cd Tomato Nr (LeETR3) mutant ↓ Root diameter Gratao et al. (2009) Cd Tomato Nr (LeETR3) mutant Maintenance of pigment content; ↓ leaf senescence Monteiro et al. (2011) Cd + S B. juncea None Optimization of ethylene level; ↓ undesirable Cd-induced symptoms Asgher et al. (2014) Cd + GA3 + S B. juncea None Optimization of ethylene level; ↓ undesirable Cd-induced symptoms Masood and Khan (2013) Cd + ethephon + S B. juncea None ↑ Ethylene sensitivity; ↑ photosynthesis Masood et al. (2012) Cd + STS P. coccineus None ↓ Inhibition of leaf growth Maksymiec (2011) Cu Arabidopsis ein2-1 mutant Similar inhibition of root elongation relative to the wild type Yuan et al. (2013) Hg Arabidopsis ein2-5 mutant ↓ Inhibition of root growth Montero-Palmero et al. (2014a) Ni + Zn + ethephon B. juncea None Optimization of ethylene level; ↓ photosynthetic inhibition Khan and Khan (2014) Pb Arabidopsis ein2-1 mutant Inhibition of root length; ↑ Pb content; ↓ GSH content Cao et al. (2009) Stress . Species . Genetic Approaches . Physiological Traits . References . Al Arabidopsis etr1-3 mutant ↓ Inhibition of root elongation Sun et al. (2010) Al Arabidopsis ein2-1 mutant ↓ Inhibition of root elongation Sun et al. (2010) Cd Arabidopsis acs2-1 acs6-1 double mutants ↓ Inhibition of leaf biomass Schellingen et al. (2014) Cd Tomato Nr (LeETR3) mutant ↓ Root diameter Gratao et al. (2009) Cd Tomato Nr (LeETR3) mutant Maintenance of pigment content; ↓ leaf senescence Monteiro et al. (2011) Cd + S B. juncea None Optimization of ethylene level; ↓ undesirable Cd-induced symptoms Asgher et al. (2014) Cd + GA3 + S B. juncea None Optimization of ethylene level; ↓ undesirable Cd-induced symptoms Masood and Khan (2013) Cd + ethephon + S B. juncea None ↑ Ethylene sensitivity; ↑ photosynthesis Masood et al. (2012) Cd + STS P. coccineus None ↓ Inhibition of leaf growth Maksymiec (2011) Cu Arabidopsis ein2-1 mutant Similar inhibition of root elongation relative to the wild type Yuan et al. (2013) Hg Arabidopsis ein2-5 mutant ↓ Inhibition of root growth Montero-Palmero et al. (2014a) Ni + Zn + ethephon B. juncea None Optimization of ethylene level; ↓ photosynthetic inhibition Khan and Khan (2014) Pb Arabidopsis ein2-1 mutant Inhibition of root length; ↑ Pb content; ↓ GSH content Cao et al. (2009) Open in new tab However, in many floral plants, targeting the ethylene signal transduction pathway is a preferred strategy (Ma et al., 2014). The ethylene-insensitive Nr mutant of tomato avoided or withstood Cd-induced stress by increasing antioxidant enzymes and affecting the intercellular spaces and the size of the mesophyll (Gratao et al., 2009; Monteiro et al., 2011). A single amino acid change in the sensor domain of Nr (LeETR3), which shows high homology to the Arabidopsis ethylene receptor ETR1, resulted in the loss of its capacity to respond to either endogenously generated or exogenously applied ethylene (Lanahan et al., 1994; Wilkinson et al., 1995). This observation in the Nr mutant has suggested that not only the manipulation of ethylene production but also of ethylene perception can be used to control plant responses to HM stress. Other studies also suggested that an appropriate control of ethylene signaling could be used as a biotechnological approach to improve HM stress tolerance. In Arabidopsis, EIN2 gene function was found to be required for plant Al and Hg sensitivities, as root growth inhibition under HM stress was alleviated in all the Arabidopsis ein2-1, ein2-5, and etr1-3 single mutants (Sun et al., 2010; Montero-Palmero et al., 2014a). By contrast, the EIN2 gene was reported to be important for Pb resistance in Arabidopsis plants (Cao et al., 2009), suggesting that the role of ethylene in plant responses to HM stress is complex and, perhaps, depends on the types of HMs to which the plants are exposed. It is noteworthy that the manipulation of ethylene signaling-related genes encoding upper components in the ethylene pathway, between the receptor and EIN2, such as knocking out OsETR2 or OsCTR2, normally causes a pleiotropic phenotype (Wuriyanghan et al., 2009; Wang et al., 2013). The tissue-specific or stress-inducible promoter should be considered for use to alleviate these side effects (Ma et al., 2014). Additionally, ERF transcription factors were reported to play an important role in regulating the expression of specific stress-related genes under Cd stress (DalCorso et al., 2010). Because each form of ERFs is likely to be involved in a specific response mechanism pathway to cope with stress, ERF genes are highly considered as ideal targets for a genetic engineering approach on ethylene action in order to improve plant tolerance while conferring minimal pleiotropic effects (Ma et al., 2014). In addition, the use of ethylene action inhibitors to alleviate stress symptoms in plants exposed to various HM stresses, including Al (Sun et al., 2010), Hg (Montero-Palmero et al., 2014b), Cd (Maksymiec, 2011), and Ni or Zn (Khan and Khan, 2014), has been discussed previously in this review. An integrated approach for the improvement of plant tolerance to HM stress is presented in Figure 3. Figure 3. Open in new tabDownload slide Potential targets for biotechnological applications to improve crop tolerance to HM stress. Figure 3. Open in new tabDownload slide Potential targets for biotechnological applications to improve crop tolerance to HM stress. CONCLUSION AND FUTURE PERSPECTIVES HM contamination and its toxicity have been recognized as a substantial threat to sustainable agriculture worldwide. Current research has shown a significant contribution of ethylene in the regulation of physiological processes and the mediation of HM tolerance in plants. However, a clear model of ethylene under HM stress is not easy to be drawn, since its regulatory role in plant responses to HM stress may lead to positive or negative effects on plant growth and reproduction. Since most up-to-date studies about the roles of ethylene and its signaling under HM stress have involved mostly physiological aspects, a molecular approach using mutants should take the lead in future studies in order to gain an in-depth understanding of the regulatory functions of ethylene in plant responses to HM stress at the molecular level. This will enable us to appropriately control the homeostasis of ethylene for the improvement of plant adaptation to HM stress as well as to open potential opportunities to select appropriate ethylene-related genes and promoters as promising candidates for genetic engineering aimed at developing HM stress-tolerant crop varieties. In addition, as the conventional plant breeding methods for improving plant tolerance to HM stress are time consuming and costly, the use of ethylene modulators for optimizing ethylene can be a wise strategy to enhance HM tolerance with minimal side effects. To effectively apply this strategy, knowledge of the relationship (antagonism/synergism) between ethylene and ethylene-responsive genes, or between ethylene and other factors (other phytohormones/other signaling molecules) for HM stress tolerance, is equally valuable. Therefore, more efforts should be made to gain a better understanding of ethylene biology, ethylene cross talk with other signaling molecules, and HM stress tolerance in the whole context, which will surely bring more benefits for both basic and applied research in the future. Glossary HM heavy metal ROS reactive oxygen species Cd cadmium Cu copper Fe iron Ni nickel Zn zinc ACC 1-aminocyclopropane-1-carboxylic acid Pb lead GSH reduced glutathione SAM S-adenosyl-methionine STS silver thiosulfate Co cobalt AVG aminoethoxyvinylglycine 1-MCP 1-methylcyclopropene Hg mercury NBD norbornadiene JA jasmonic acid SA salicylic acid Al aluminum S sulfur PC phytochelatin NO nitric oxide LITERATURE CITED Ahmad P , Sarwat M, Bhat NA, Wani MR, Kazi AG, Tran LS ( 2015 ) Alleviation of cadmium toxicity in Brassica juncea L. (Czern. & Coss.) by calcium application involves various physiological and biochemical strategies . PLoS ONE 10 : e0114571 Google Scholar Crossref Search ADS PubMed WorldCat Anjum NA , Ahmad I, Mohmood I, Pacheco M, Duarte AC, Pereira E, Umar S, Ahmad A, Khan NA, Iqbal M ( 2012 ) Modulation of glutathione and its related enzymes in plants’ responses to toxic metals and metalloids: a review . Environ Exp Bot 75 : 307 – 324 Google Scholar OpenURL Placeholder Text WorldCat Arteca RN , Arteca JM ( 2007 ) Heavy-metal-induced ethylene production in Arabidopsis thaliana . J Plant Physiol 164 : 1480 – 1488 Google Scholar Crossref Search ADS PubMed WorldCat Asgher M , Khan MIR, Anjum NA, Khan NA ( 2015 ) Minimising toxicity of cadmium in plants: role of plant growth regulators . Protoplasma 252 : 399 – 413 Google Scholar Crossref Search ADS PubMed WorldCat Asgher M , Khan NA, Khan MIR, Fatma M, Masood A ( 2014 ) Ethylene production is associated with alleviation of cadmium-induced oxidative stress by sulfur in mustard types differing in ethylene sensitivity . Ecotoxicol Environ Saf 106 : 54 – 61 Google Scholar Crossref Search ADS PubMed WorldCat Atici Ö , Ağar G, Battal P ( 2005 ) Changes in phytohormone contents in chickpea seeds germinating under lead or zinc stress . Biol Plant 49 : 215 – 222 Google Scholar Crossref Search ADS WorldCat Besson-Bard A , Gravot A, Richaud P, Auroy P, Duc C, Gaymard F, Taconnat L, Renou JP, Pugin A, Wendehenne D ( 2009 ) Nitric oxide contributes to cadmium toxicity in Arabidopsis by promoting cadmium accumulation in roots and by up-regulating genes related to iron uptake . Plant Physiol 149 : 1302 – 1315 Google Scholar Crossref Search ADS PubMed WorldCat Bethke G , Unthan T, Uhrig JF, Pöschl Y, Gust AA, Scheel D, Lee J ( 2009 ) Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling . Proc Natl Acad Sci USA 106 : 8067 – 8072 Google Scholar Crossref Search ADS PubMed WorldCat Binder BM , Walker JM, Gagne JM, Emborg TJ, Hemmann G, Bleecker AB, Vierstra RD ( 2007 ) The Arabidopsis EIN3 binding F-box proteins EBF1 and EBF2 have distinct but overlapping roles in ethylene signaling . Plant Cell 19 : 509 – 523 Google Scholar Crossref Search ADS PubMed WorldCat Blankenship SM , Dole JM ( 2003 ) 1-Methylcyclopropene: a review . Postharvest Biol Technol 28 : 1 – 25 Google Scholar Crossref Search ADS WorldCat Cánovas D , Vooijs R, Schat H, de Lorenzo V ( 2004 ) The role of thiol species in the hypertolerance of Aspergillus sp. P37 to arsenic . J Biol Chem 279 : 51234 – 51240 Google Scholar Crossref Search ADS PubMed WorldCat Cao S , Chen Z, Liu G, Jiang L, Yuan H, Ren G, Bian X, Jian H, Ma X ( 2009 ) The Arabidopsis Ethylene-Insensitive 2 gene is required for lead resistance . Plant Physiol Biochem 47 : 308 – 312 Google Scholar Crossref Search ADS PubMed WorldCat Carrió-Seguí A , Garcia-Molina A, Sanz A, Peñarrubia L ( 2015 ) Defective copper transport in the copt5 mutant affects cadmium tolerance . Plant Cell Physiol 56 : 442 – 454 Google Scholar Crossref Search ADS PubMed WorldCat Chmielowska-Bąk J , Lefèvre I, Lutts S, Kulik A, Deckert J ( 2014 ) Effect of cobalt chloride on soybean seedlings subjected to cadmium stress . Acta Soc Bot Pol 83 : 201 – 207 Google Scholar Crossref Search ADS WorldCat Clark KL , Larsen PB, Wang X, Chang C ( 1998 ) Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors . Proc Natl Acad Sci USA 95 : 5401 – 5406 Google Scholar Crossref Search ADS PubMed WorldCat Clarke A , Desikan R, Hurst RD, Hancock JT, Neill SJ ( 2000 ) NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures . Plant J 24 : 667 – 677 Google Scholar Crossref Search ADS PubMed WorldCat Cobbett CS ( 2000 ) Phytochelatins and their roles in heavy metal detoxification . Plant Physiol 123 : 825 – 832 Google Scholar Crossref Search ADS PubMed WorldCat Cooke AR , Randall DI ( 1968 ) 2-Haloethanephosphonic acids as ethylene releasing agents for the induction of flowering in pineapples . Nature 218 : 974 – 975 Google Scholar Crossref Search ADS PubMed WorldCat Corpas FJ , Barroso JB, Carreras A, Quirós M, León AM, Romero-Puertas MC, Esteban FJ, Valderrama R, Palma JM, Sandalio LM, et al. ( 2004 ) Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants . Plant Physiol 136 : 2722 – 2733 Google Scholar Crossref Search ADS PubMed WorldCat DalCorso G , Farinati S, Furini A ( 2010 ) Regulatory networks of cadmium stress in plants . Plant Signal Behav 5 : 663 – 667 Google Scholar Crossref Search ADS PubMed WorldCat De Grauwe L , Dugardeyn J, Van Der Straeten D ( 2008 ) Novel mechanisms of ethylene-gibberellin crosstalk revealed by the gai eto2-1 double mutant . Plant Signal Behav 3 : 1113 – 1115 Google Scholar Crossref Search ADS PubMed WorldCat Delledonne M , Zeier J, Marocco A, Lamb C ( 2001 ) Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response . Proc Natl Acad Sci USA 98 : 13454 – 13459 Google Scholar Crossref Search ADS PubMed WorldCat Dugardeyn J , Van Der Straeten D ( 2008 ) Ethylene: fine-tuning plant growth and development by stimulation and inhibition of elongation . Plant Sci 175 : 59 – 70 Google Scholar Crossref Search ADS WorldCat Durner J , Wendehenne D, Klessig DF ( 1998 ) Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose . Proc Natl Acad Sci USA 95 : 10328 – 10333 Google Scholar Crossref Search ADS PubMed WorldCat Ecker JR ( 2004 ) Reentry of the ethylene MPK6 module . Plant Cell 16 : 3169 – 3173 Google Scholar Crossref Search ADS WorldCat Feng X , Apelbaum A, Sisler EC, Goren R ( 2000 ) Control of ethylene responses in avocado fruit with 1-methylcyclopropene . Postharvest Biol Technol 20 : 143 – 150 Google Scholar Crossref Search ADS WorldCat Franchin C , Fossati T, Pasquini E, Lingua G, Castiglione S, Torrigiani P, Biondi S ( 2007 ) High concentrations of zinc and copper induce differential polyamine responses in micropropagated white poplar (Populus alba) . Physiol Plant 130 : 77 – 90 Google Scholar Crossref Search ADS WorldCat Fuhrer J ( 1982 ) Ethylene biosynthesis and cadmium toxicity in leaf tissue of beans (Phaseolus vulgaris L.) . Plant Physiol 70 : 162 – 167 Google Scholar Crossref Search ADS PubMed WorldCat García-Mata C , Lamattina L ( 2002 ) Nitric oxide and abscisic acid cross talk in guard cells . Plant Physiol 128 : 790 – 792 Google Scholar Crossref Search ADS PubMed WorldCat Gazzarrini S , McCourt P ( 2003 ) Cross-talk in plant hormone signalling: what Arabidopsis mutants are telling us . Ann Bot (Lond) 91 : 605 – 612 Google Scholar Crossref Search ADS WorldCat Gill SS , Tuteja N ( 2010 ) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants . Plant Physiol Biochem 48 : 909 – 930 Google Scholar Crossref Search ADS PubMed WorldCat Gratao PL , Monteiro CC, Rossi ML, Martinelli AP, Peres LE, Medici LO, Lea PJ, Azevedo RA ( 2009 ) Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium . Environ Exp Bot 67 : 387 – 394 Google Scholar Crossref Search ADS WorldCat Grimmig B , Gonzalez-Perez MN, Leubner-Metzger G, Vögeli-Lange R, Meins F Jr , Hain R, Penuelas J, Heidenreich B, Langebartels C, Ernst D, et al. ( 2003 ) Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling . Plant Mol Biol 51 : 599 – 607 Google Scholar Crossref Search ADS PubMed WorldCat Groppa MD , Rosales EP, Iannone MF, Benavides MP ( 2008 ) Nitric oxide, polyamines and Cd-induced phytotoxicity in wheat roots . Phytochemistry 69 : 2609 – 2615 Google Scholar Crossref Search ADS PubMed WorldCat Guo H , Ecker JR ( 2004 ) The ethylene signaling pathway: new insights . Curr Opin Plant Biol 7 : 40 – 49 Google Scholar Crossref Search ADS PubMed WorldCat Ha SB , Smith AP, Howden R, Dietrich WM, Bugg S, O’Connell MJ, Goldsbrough PB, Cobbett CS ( 1999 ) Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe . Plant Cell 11 : 1153 – 1164 Google Scholar Crossref Search ADS PubMed WorldCat Hahn A , Harter K ( 2009 ) Mitogen-activated protein kinase cascades and ethylene: signaling, biosynthesis, or both? Plant Physiol 149 : 1207 – 1210 Google Scholar Crossref Search ADS PubMed WorldCat Hasan SA , Hayat S, Wani AS, Ahmad A ( 2011 ) Establishment of sensitive and resistant variety of tomato on the basis of photosynthesis and antioxidative enzymes in the presence of cobalt applied as shotgun approach . Braz J Plant Physiol 23 : 175 – 185 Google Scholar Crossref Search ADS WorldCat Hayat S , Ali B, Hasan SA, Ahmad A ( 2007 ) Brassinosteroid enhanced the level of antioxidants under cadmium stress in Brassica juncea . Environ Exp Bot 60 : 33 – 41 Google Scholar Crossref Search ADS WorldCat Hossain MA , Piyatida P, da Silva JAT, Fujita M ( 2012 ) Molecular mechanism of heavy metal toxicity and tolerance in plants: central role of glutathione in detoxification of reactive oxygen species and methylglyoxal and in heavy metal chelation . J Bot 2012 : 872875 Google Scholar OpenURL Placeholder Text WorldCat Hua J , Meyerowitz EM ( 1998 ) Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana . Cell 94 : 261 – 271 Google Scholar Crossref Search ADS PubMed WorldCat Huang JY , Lin CH ( 2003 ) Cold water treatment promotes ethylene production and dwarfing in tomato seedlings . Plant Physiol Biochem 41 : 283 – 288 Google Scholar Crossref Search ADS WorldCat Iakimova ET , Woltering EJ, Kapchina-Toteva VM, Harren FJ, Cristescu SM ( 2008 ) Cadmium toxicity in cultured tomato cells: role of ethylene, proteases and oxidative stress in cell death signaling . Cell Biol Int 32 : 1521 – 1529 Google Scholar Crossref Search ADS PubMed WorldCat Ichimura K , Niki T ( 2014 ) Ethylene production associated with petal senescence in carnation flowers is induced irrespective of the gynoecium . J Plant Physiol 171 : 1679 – 1684 Google Scholar Crossref Search ADS PubMed WorldCat Iqbal N , Khan NA, Nazar R, da Silva JAT ( 2012 ) Ethylene-stimulated photosynthesis results from increased nitrogen and sulfur assimilation in mustard types that differ in photosynthetic capacity . Environ Exp Bot 78 : 84 – 90 Google Scholar Crossref Search ADS WorldCat Iqbal N , Masood A, Khan MIR, Asgher M, Fatma M, Khan NA ( 2013 ) Cross-talk between sulfur assimilation and ethylene signaling in plants . Plant Signal Behav 8 : e22478 Google Scholar Crossref Search ADS PubMed WorldCat Jakubowicz M , Gałgańska H, Nowak W, Sadowski J ( 2010 ) Exogenously induced expression of ethylene biosynthesis, ethylene perception, phospholipase D, and Rboh-oxidase genes in broccoli seedlings . J Exp Bot 61 : 3475 – 3491 Google Scholar Crossref Search ADS PubMed WorldCat Ju C , Yoon GM, Shemansky JM, Lin DY, Ying ZI, Chang J, Garrett WM, Kessenbrock M, Groth G, Tucker ML, et al. ( 2012 ) CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis . Proc Natl Acad Sci USA 109 : 19486 – 19491 Google Scholar Crossref Search ADS PubMed WorldCat Kazemi N , Khavari-Nejad RA, Fahimi H, Saadatmand S, Nejad-Sattari T ( 2010 ) Effects of exogenous salicylic acid and nitric oxide on lipid peroxidation and antioxidant enzyme activities in leaves of Brassica napus L. under nickel stress . Sci Hortic (Amsterdam) 126 : 402 – 407 Google Scholar Crossref Search ADS WorldCat Khademi S , Khavari-Nejad RA, Saadatmand S, Najafi F ( 2014 ) The effects of exogenous salicylic acid in the antioxidant defense system in canola plants (Brassica napus L.) exposed to copper . Int J Biosci 5 : 64 – 73 Google Scholar OpenURL Placeholder Text WorldCat Khan MIR , Iqbal N, Masood A, Mobin M, Anjum N, Khan NA (May 8, 2015 a ) Modulation and significance of nitrogen and sulfur metabolism in cadmium challenged plants . Plant Growth Regul http://dx.doi.org/10.1007/s10725-015-0071-9 Google Scholar OpenURL Placeholder Text WorldCat Khan MIR , Khan NA ( 2014 ) Ethylene reverses photosynthetic inhibition by nickel and zinc in mustard through changes in PS II activity, photosynthetic nitrogen use efficiency, and antioxidant metabolism . Protoplasma 251 : 1007 – 1019 Google Scholar Crossref Search ADS PubMed WorldCat Khan MIR , Nazir F, Asgher M, Per TS, Khan NA ( 2015 b ) Selenium and sulfur influence ethylene formation and alleviate cadmium-induced oxidative stress by improving proline and glutathione production in wheat . J Plant Physiol 173 : 9 – 18 Google Scholar Crossref Search ADS PubMed WorldCat Khan NA ( 2004 ) An evaluation of the effects of exogenous ethephon, an ethylene releasing compound, on photosynthesis of mustard (Brassica juncea) cultivars that differ in photosynthetic capacity . BMC Plant Biol 4 : 21 Google Scholar Crossref Search ADS PubMed WorldCat Kim HJ , Lynch JP, Brown KM ( 2008 ) Ethylene insensitivity impedes a subset of responses to phosphorus deficiency in tomato and petunia . Plant Cell Environ 31 : 1744 – 1755 Google Scholar Crossref Search ADS PubMed WorldCat Klee HJ ( 2004 ) Ethylene signal transduction: moving beyond Arabidopsis . Plant Physiol 135 : 660 – 667 Google Scholar Crossref Search ADS PubMed WorldCat Kovács V , Gondor OK, Szalai G, Darkó E, Majláth I, Janda T, Pál M ( 2014 ) Synthesis and role of salicylic acid in wheat varieties with different levels of cadmium tolerance . J Hazard Mater 280 : 12 – 19 Google Scholar Crossref Search ADS PubMed WorldCat Lanahan MB , Yen HC, Giovannoni JJ, Klee HJ ( 1994 ) The never ripe mutation blocks ethylene perception in tomato . Plant Cell 6 : 521 – 530 Google Scholar PubMed OpenURL Placeholder Text WorldCat Laspina N , Groppa M, Tomaro M, Benavides M ( 2005 ) Nitric oxide protects sunflower leaves against Cd-induced oxidative stress . Plant Sci 169 : 323 – 330 Google Scholar Crossref Search ADS WorldCat Lequeux H , Hermans C, Lutts S, Verbruggen N ( 2010 ) Response to copper excess in Arabidopsis thaliana: impact on the root system architecture, hormone distribution, lignin accumulation and mineral profile . Plant Physiol Biochem 48 : 673 – 682 Google Scholar Crossref Search ADS PubMed WorldCat Leshem Y , Haramaty E ( 1996 ) Plant aging: the emission of NO and ethylene and effect of NO-releasing compounds on growth of pea (Pisum sativum) foliage . J Plant Physiol 148 : 258 – 263 Google Scholar Crossref Search ADS WorldCat Li G , Meng X, Wang R, Mao G, Han L, Liu Y, Zhang S ( 2012 ) Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis . PLoS Genet 8 : e1002767 Google Scholar Crossref Search ADS PubMed WorldCat Liu KL , Shen L, Wang JQ, Sheng JP ( 2008 ) Rapid inactivation of chloroplastic ascorbate peroxidase is responsible for oxidative modification to Rubisco in tomato (Lycopersicon esculentum) under cadmium stress . J Integr Plant Biol 50 : 415 – 426 Google Scholar Crossref Search ADS PubMed WorldCat Liu X-M , Kim KE, Kim KC, Nguyen XC, Han HJ, Jung MS, Kim HS, Kim SH, Park HC, Yun DJ, et al. ( 2010 ) Cadmium activates Arabidopsis MPK3 and MPK6 via accumulation of reactive oxygen species . Phytochemistry 71 : 614 – 618 Google Scholar Crossref Search ADS PubMed WorldCat Liu Y , Zhang S ( 2004 ) Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis . Plant Cell 16 : 3386 – 3399 Google Scholar Crossref Search ADS PubMed WorldCat Ma B , Chen H, Chen SY, Zhang JS ( 2014 ) Roles of ethylene in plant growth and responses to stresses . In LSP Tran , S Pal , eds, Phytohormones: A Window to Metabolism, Signaling and Biotechnological Applications. Springer-Verlag , New York , pp 81 – 118 Google Scholar Crossref Search ADS Ma W , Xu W, Xu H, Chen Y, He Z, Ma M ( 2010 ) Nitric oxide modulates cadmium influx during cadmium-induced programmed cell death in tobacco BY-2 cells . Planta 232 : 325 – 335 Google Scholar Crossref Search ADS PubMed WorldCat Magalhaes JR , Pedroso MC, Durzan D ( 1999 ) Nitric oxide apoptosis and plant stresses . Physiol Mol Biol Plants 5 : 115 – 125 Google Scholar OpenURL Placeholder Text WorldCat Maksymiec W ( 2007 ) Signaling responses in plants to heavy metal stress . Acta Physiol Plant 29 : 177 – 187 Google Scholar Crossref Search ADS WorldCat Maksymiec W ( 2011 ) Effects of jasmonate and some other signalling factors on bean and onion growth during the initial phase of cadmium action . Biol Plant 55 : 112 – 118 Google Scholar Crossref Search ADS WorldCat Maksymiec W , Baszyński T ( 1996 ) Chlorophyll fluorescence in primary leaves of excess Cu-treated runner bean plants depends on their growth stages and the duration of Cu-action . J Plant Physiol 149 : 196 – 200 Google Scholar Crossref Search ADS WorldCat Maksymiec W , Krupa Z ( 2006 ) The effects of short-term exposition to Cd, excess Cu ions and jasmonate on oxidative stress appearing in Arabidopsis thaliana . Environ Exp Bot 57 : 187 – 194 Google Scholar Crossref Search ADS WorldCat Maksymiec W , Krupa Z ( 2007 ) Effects of methyl jasmonate and excess copper on root and leaf growth . Biol Plant 51 : 322 – 326 Google Scholar Crossref Search ADS WorldCat Maksymiec W , Wianowska D, Dawidowicz AL, Radkiewicz S, Mardarowicz M, Krupa Z ( 2005 ) The level of jasmonic acid in Arabidopsis thaliana and Phaseolus coccineus plants under heavy metal stress . J Plant Physiol 162 : 1338 – 1346 Google Scholar Crossref Search ADS PubMed WorldCat Malik A ( 2004 ) Metal bioremediation through growing cells . Environ Int 30 : 261 – 278 Google Scholar Crossref Search ADS PubMed WorldCat Masood A , Iqbal N, Khan NA ( 2012 ) Role of ethylene in alleviation of cadmium-induced photosynthetic capacity inhibition by sulphur in mustard . Plant Cell Environ 35 : 524 – 533 Google Scholar Crossref Search ADS PubMed WorldCat Masood A , Khan N ( 2013 ) Ethylene and gibberellic acid interplay in regulation of photosynthetic capacity inhibition by cadmium . J Plant Biochem Physiol 1 : 111 Google Scholar OpenURL Placeholder Text WorldCat Metwally A , Finkemeier I, Georgi M, Dietz K-J ( 2003 ) Salicylic acid alleviates the cadmium toxicity in barley seedlings . Plant Physiol 132 : 272 – 281 Google Scholar Crossref Search ADS PubMed WorldCat Mhamdi A , Hager J, Chaouch S, Queval G, Han Y, Taconnat L, Saindrenan P, Gouia H, Issakidis-Bourguet E, Renou JP, et al. ( 2010 ) Arabidopsis GLUTATHIONE REDUCTASE1 plays a crucial role in leaf responses to intracellular hydrogen peroxide and in ensuring appropriate gene expression through both salicylic acid and jasmonic acid signaling pathways . Plant Physiol 153 : 1144 – 1160 Google Scholar Crossref Search ADS PubMed WorldCat Monteiro CC , Carvalho RF, Gratão PL, Carvalho G, Tezotto T, Medici LO, Peres LE, Azevedo RA ( 2011 ) Biochemical responses of the ethylene-insensitive never ripe tomato mutant subjected to cadmium and sodium stresses . Environ Exp Bot 71 : 306 – 320 Google Scholar Crossref Search ADS WorldCat Montero-Palmero MB , Martín-Barranco A, Escobar C, Hernández LE ( 2014 a ) Early transcriptional responses to mercury: a role for ethylene in mercury-induced stress . New Phytol 201 : 116 – 130 Google Scholar Crossref Search ADS PubMed WorldCat Montero-Palmero MB , Ortega-Villasante C, Escobar C, Hernández LE ( 2014 b ) Are plant endogenous factors like ethylene modulators of the early oxidative stress induced by mercury? Environ Toxicol 2 : 34 Google Scholar OpenURL Placeholder Text WorldCat Mostofa M , Fujita M, Tran LS (April 17, 2015 a ) Nitric oxide mediates hydrogen peroxide- and salicylic acid-induced salt tolerance in rice (Oryza sativa L.) seedlings . Plant Growth Regul http://dx.doi.org/10.1007/s10725-015-0061-y Google Scholar OpenURL Placeholder Text WorldCat Mostofa MG , Hossain MA, Fujita M, Tran LSP ( 2015 b ) Physiological and biochemical mechanisms associated with trehalose-induced copper-stress tolerance in rice . Sci Rep 5 : 11433 Google Scholar Crossref Search ADS PubMed WorldCat Nagajyoti P , Lee K, Sreekanth T ( 2010 ) Heavy metals, occurrence and toxicity for plants: a review . Environ Chem Lett 8 : 199 – 216 Google Scholar Crossref Search ADS WorldCat Neill SJ , Desikan R, Clarke A, Hancock JT ( 2002 ) Nitric oxide is a novel component of abscisic acid signaling in stomatal guard cells . Plant Physiol 128 : 13 – 16 Google Scholar Crossref Search ADS PubMed WorldCat Nies DH ( 1999 ) Microbial heavy-metal resistance . Appl Microbiol Biotechnol 51 : 730 – 750 Google Scholar Crossref Search ADS PubMed WorldCat Noriega G , Caggiano E, Lecube ML, Cruz DS, Batlle A, Tomaro M, Balestrasse KB ( 2012 ) The role of salicylic acid in the prevention of oxidative stress elicited by cadmium in soybean plants . Biometals 25 : 1155 – 1165 Google Scholar Crossref Search ADS PubMed WorldCat Opdenakker K , Remans T, Vangronsveld J, Cuypers A ( 2012 ) Mitogen-Activated Protein (MAP) kinases in plant metal stress: regulation and responses in comparison to other biotic and abiotic stresses . Int J Mol Sci 13 : 7828 – 7853 Google Scholar Crossref Search ADS PubMed WorldCat Pacifici S , Prisa D, Burchi G, van Doorn WG ( 2014 ) Pollination increases ethylene production in Lilium hybrida cv. Brindisi flowers but does not affect the time to tepal senescence or tepal abscission . J Plant Physiol 173C : 116 – 119 Google Scholar OpenURL Placeholder Text WorldCat Palit S , Sharma A, Talukder G ( 1994 ) Effects of cobalt on plants . Bot Rev 60 : 149 – 181 Google Scholar Crossref Search ADS WorldCat Pedroso MC , Magalhaes JR, Durzan D ( 2000 ) Nitric oxide induces cell death in Taxus cells . Plant Sci 157 : 173 – 180 Google Scholar Crossref Search ADS PubMed WorldCat Peñarrubia L , Romero P, Carrió-Seguí A, Andrés-Bordería A, Moreno J, Sanz A ( 2015 ) Temporal aspects of copper homeostasis and its crosstalk with hormones . Front Plant Sci 6 : 255 Google Scholar Crossref Search ADS PubMed WorldCat Pierik R , Tholen D, Poorter H, Visser EJ, Voesenek LA ( 2006 ) The Janus face of ethylene: growth inhibition and stimulation . Trends Plant Sci 11 : 176 – 183 Google Scholar Crossref Search ADS PubMed WorldCat Piotrowska-Niczyporuk A , Bajguz A, Zambrzycka E, Godlewska-Żyłkiewicz B ( 2012 ) Phytohormones as regulators of heavy metal biosorption and toxicity in green alga Chlorella vulgaris (Chlorophyceae) . Plant Physiol Biochem 52 : 52 – 65 Google Scholar Crossref Search ADS PubMed WorldCat Qiao H , Shen Z, Huang SS, Schmitz RJ, Urich MA, Briggs SP, Ecker JR ( 2012 ) Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas . Science 338 : 390 – 393 Google Scholar Crossref Search ADS PubMed WorldCat Ribeiro EA Jr , Cunha FQ, Tamashiro WM, Martins IS ( 1999 ) Growth phase-dependent subcellular localization of nitric oxide synthase in maize cells . FEBS Lett 445 : 283 – 286 Google Scholar Crossref Search ADS PubMed WorldCat Rodecap KD , Tingey DT, Tibbs JH ( 1981 ) Cadmium-induced ethylene production in bean plants . Z Pflanzenphysiol 105 : 65 – 74 Google Scholar Crossref Search ADS WorldCat Rodríguez FI , Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB ( 1999 ) A copper cofactor for the ethylene receptor ETR1 from Arabidopsis . Science 283 : 996 – 998 Google Scholar Crossref Search ADS PubMed WorldCat Rodríguez-Serrano M , Romero-Puertas MC, Zabalza A, Corpas FJ, Gómez M, Del Río LA, Sandalio LM ( 2006 ) Cadmium effect on oxidative metabolism of pea (Pisum sativum L.) roots: imaging of reactive oxygen species and nitric oxide accumulation in vivo . Plant Cell Environ 29 : 1532 – 1544 Google Scholar Crossref Search ADS PubMed WorldCat Sandmann G , Böger P ( 1980 ) Copper-mediated lipid peroxidation processes in photosynthetic membranes . Plant Physiol 66 : 797 – 800 Google Scholar Crossref Search ADS PubMed WorldCat Schellingen K , Van Der Straeten D, Vandenbussche F, Prinsen E, Remans T, Vangronsveld J, Cuypers A ( 2014 ) Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression . BMC Plant Biol 14 : 214 Google Scholar Crossref Search ADS PubMed WorldCat Schlagnhaufer CD , Arteca RN ( 1997 ) Ozone‐induced oxidative stress: mechanisms of action and reaction . Physiol Plant 100 : 264 – 273 Google Scholar OpenURL Placeholder Text WorldCat Schlagnhaufer CD , Arteca RN, Pell EJ ( 1997 ) Sequential expression of two 1-aminocyclopropane-1-carboxylate synthase genes in response to biotic and abiotic stresses in potato (Solanum tuberosum L.) leaves . Plant Mol Biol 35 : 683 – 688 Google Scholar Crossref Search ADS PubMed WorldCat Serek M , Woltering EJ, Sisler EC, Frello S, Sriskandarajah S ( 2006 ) Controlling ethylene responses in flowers at the receptor level . Biotechnol Adv 24 : 368 – 381 Google Scholar Crossref Search ADS PubMed WorldCat Shan X , Yan J, Xie D ( 2012 ) Comparison of phytohormone signaling mechanisms . Curr Opin Plant Biol 15 : 84 – 91 Google Scholar Crossref Search ADS PubMed WorldCat Sharma J , Chakraverty N ( 2013 ) Mechanism of plant tolerance in response to heavy metals . In GR Rout , AB Das , eds, Molecular Stress Physiology of Plants. Springer India , New Delhi , pp 289 – 308 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Shukla D , Krishnamurthy S, Sahi SV ( 2014 ) Genome wide transcriptome analysis reveals ABA mediated response in Arabidopsis during gold (AuCl−4) treatment . Front Plant Sci 5 : 652 Google Scholar Crossref Search ADS PubMed WorldCat Singh HP , Batish DR, Kaur G, Arora K, Kohli RK ( 2008 ) Nitric oxide (as sodium nitroprusside) supplementation ameliorates Cd toxicity in hydroponically grown wheat roots . Environ Exp Bot 63 : 158 – 167 Google Scholar Crossref Search ADS WorldCat Sisler E , Serek M ( 1997 ) Inhibitors of ethylene responses in plants at the receptor level: recent developments . Physiol Plant 100 : 577 – 582 Google Scholar Crossref Search ADS WorldCat Srivastava AK , Penna S, Nguyen DV, Tran LSP ( 2014 ) Multifaceted roles of aquaporins as molecular conduits in plant responses to abiotic stresses . Crit Rev Biotechnol 1 – 10 Google Scholar OpenURL Placeholder Text WorldCat Srivastava MK , Dwivedi UN ( 1998 ) Salicylic acid modulates glutathione metabolism in pea seedlings . J Plant Physiol 153 : 409 – 414 Google Scholar Crossref Search ADS WorldCat Steffens B ( 2014 ) The role of ethylene and ROS in salinity, heavy metal, and flooding responses in rice . Front Plant Sci 5 : 685 Google Scholar Crossref Search ADS PubMed WorldCat Sun P , Tian QY, Chen J, Zhang WH ( 2010 ) Aluminium-induced inhibition of root elongation in Arabidopsis is mediated by ethylene and auxin . J Exp Bot 61 : 347 – 356 Google Scholar Crossref Search ADS PubMed WorldCat Takahashi F , Yoshida R, Ichimura K, Mizoguchi T, Seo S, Yonezawa M, Maruyama K, Yamaguchi-Shinozaki K, Shinozaki K ( 2007 ) The mitogen-activated protein kinase cascade MKK3-MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis . Plant Cell 19 : 805 – 818 Google Scholar Crossref Search ADS PubMed WorldCat Thao NP , Tran LSP ( 2012 ) Potentials toward genetic engineering of drought-tolerant soybean . Crit Rev Biotechnol 32 : 349 – 362 Google Scholar Crossref Search ADS PubMed WorldCat Thapa G , Sadhukhan A, Panda SK, Sahoo L ( 2012 ) Molecular mechanistic model of plant heavy metal tolerance . Biometals 25 : 489 – 505 Google Scholar Crossref Search ADS PubMed WorldCat Trinh NN , Huang TL, Chi WC, Fu SF, Chen CC, Huang HJ ( 2014 ) Chromium stress response effect on signal transduction and expression of signaling genes in rice . Physiol Plant 150 : 205 – 224 Google Scholar Crossref Search ADS PubMed WorldCat Van der Does D , Leon-Reyes A, Koornneef A, Van Verk MC, Rodenburg N, Pauwels L, Goossens A, Körbes AP, Memelink J, Ritsema T, et al. ( 2013 ) Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59 . Plant Cell 25 : 744 – 761 Google Scholar Crossref Search ADS PubMed WorldCat Verstraeten SV , Aimo L, Oteiza PI ( 2008 ) Aluminium and lead: molecular mechanisms of brain toxicity . Arch Toxicol 82 : 789 – 802 Google Scholar Crossref Search ADS PubMed WorldCat Wang Q , Zhang W, Yin Z, Wen CK ( 2013 ) Rice CONSTITUTIVE TRIPLE-RESPONSE2 is involved in the ethylene-receptor signalling and regulation of various aspects of rice growth and development . J Exp Bot 64 : 4863 – 4875 Google Scholar Crossref Search ADS PubMed WorldCat Wang YS , Yang ZM ( 2005 ) Nitric oxide reduces aluminum toxicity by preventing oxidative stress in the roots of Cassia tora L . Plant Cell Physiol 46 : 1915 – 1923 Google Scholar Crossref Search ADS PubMed WorldCat Wen X , Zhang C, Ji Y, Zhao Q, He W, An F, Jiang L, Guo H ( 2012 ) Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus . Cell Res 22 : 1613 – 1616 Google Scholar Crossref Search ADS PubMed WorldCat Wilkinson JQ , Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ ( 1995 ) An ethylene-inducible component of signal transduction encoded by never-ripe . Science 270 : 1807 – 1809 Google Scholar Crossref Search ADS PubMed WorldCat Wise R , Naylor A ( 1988 ) Stress ethylene does not originate directly from lipid peroxidation during chilling-enhanced photooxidation . J Plant Physiol 133 : 62 – 66 Google Scholar Crossref Search ADS WorldCat Wuriyanghan H , Zhang B, Cao WH, Ma B, Lei G, Liu YF, Wei W, Wu HJ, Chen LJ, Chen HW, et al. ( 2009 ) The ethylene receptor ETR2 delays floral transition and affects starch accumulation in rice . Plant Cell 21 : 1473 – 1494 Google Scholar Crossref Search ADS PubMed WorldCat Xia XJ , Zhou YH, Shi K, Zhou J, Foyer CH, Yu JQ ( 2015 ) Interplay between reactive oxygen species and hormones in the control of plant development and stress tolerance . J Exp Bot 66 : 2839 – 2856 Google Scholar Crossref Search ADS PubMed WorldCat Xiang C , Oliver DJ ( 1998 ) Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis . Plant Cell 10 : 1539 – 1550 Google Scholar Crossref Search ADS PubMed WorldCat Xu J , Zhang S ( 2015 ) Ethylene biosynthesis and regulation in plants . In CK Wen , ed, Ethylene in Plants. Springer, Dordrecht , The Netherlands , pp 1 – 25 Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Yadav S ( 2010 ) Heavy metals toxicity in plants: an overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants . S Afr J Bot 76 : 167 – 179 Google Scholar Crossref Search ADS WorldCat Ye Y , Li Z, Xing D ( 2013 ) Nitric oxide promotes MPK6-mediated caspase-3-like activation in cadmium-induced Arabidopsis thaliana programmed cell death . Plant Cell Environ 36 : 1 – 15 Google Scholar Crossref Search ADS PubMed WorldCat Yıldız M , Ciğerci İH, Konuk M, Fidan AF, Terzi H ( 2009 ) Determination of genotoxic effects of copper sulphate and cobalt chloride in Allium cepa root cells by chromosome aberration and comet assays . Chemosphere 75 : 934 – 938 Google Scholar Crossref Search ADS PubMed WorldCat Yokotani N , Tamura S, Nakano R, Inaba A, McGlasson WB, Kubo Y ( 2004 ) Comparison of ethylene- and wound-induced responses in fruit of wild-type, rin and nor tomatoes . Postharvest Biol Technol 32 : 247 – 252 Google Scholar Crossref Search ADS WorldCat Yoo SD , Cho Y, Sheen J ( 2009 ) Emerging connections in the ethylene signaling network . Trends Plant Sci 14 : 270 – 279 Google Scholar Crossref Search ADS PubMed WorldCat Yuan HM , Xu HH, Liu WC, Lu YT ( 2013 ) Copper regulates primary root elongation through PIN1-mediated auxin redistribution . Plant Cell Physiol 54 : 766 – 778 Google Scholar Crossref Search ADS PubMed WorldCat Zapata PJ , Pretel MT, Amorós A, Botella MÁ ( 2003 ) Changes in ethylene evolution and polyamine profiles of seedlings of nine cultivars of Lactuca sativa L. in response to salt stress during germination . Plant Sci 164 : 557 – 563 Google Scholar Crossref Search ADS WorldCat Zhang GB , Yi HY, Gong JM ( 2014 ) The Arabidopsis ethylene/jasmonic acid-NRT signaling module coordinates nitrate reallocation and the trade-off between growth and environmental adaptation . Plant Cell 26 : 3984 – 3998 Google Scholar Crossref Search ADS PubMed WorldCat Zhang W , Wen CK ( 2010 ) Preparation of ethylene gas and comparison of ethylene responses induced by ethylene, ACC, and ethephon . Plant Physiol Biochem 48 : 45 – 53 Google Scholar Crossref Search ADS PubMed WorldCat Zhao XC , Qu X, Mathews DE, Schaller GE ( 2002 ) Effect of ethylene pathway mutations upon expression of the ethylene receptor ETR1 from Arabidopsis . Plant Physiol 130 : 1983 – 1991 Google Scholar Crossref Search ADS PubMed WorldCat Zhu Z , An F, Feng Y, Li P, Xue L, A M, Jiang Z, Kim JM, To TK, Li W, et al. ( 2011 ) Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis . Proc Natl Acad Sci USA 108 : 12539 – 12544 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by Vietnam National University (grant no. C2014–28–07 to N.P.T.) and by the University Grants Commission, New Delhi [grant no. F.40–3(M/S)/2009 (SA–III/MANF) to M.I.R.K. and N.A.K.]. 2 These authors contributed equally to the article. * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00663 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Ju, Chuanli; Chang, Caren

doi: 10.1104/pp.15.00845pmid: 26246449

Abstract The gaseous hormone ethylene profoundly affects plant growth, development, and stress responses. Ethylene perception occurs at the endoplasmic reticulum membrane, and signal transduction leads to a transcriptional cascade that initiates diverse responses, often in conjunction with other signals. Recent findings provide a more complete picture of the components and mechanisms in ethylene signaling, now rendering a more dynamic view of this conserved pathway. This includes newly identified protein-protein interactions at the endoplasmic reticulum membrane, as well as the major discoveries that the central regulator ETHYLENE INSENSITIVE2 (EIN2) is the long-sought phosphorylation substrate for the CONSTITUTIVE RESPONSE1 protein kinase, and that cleavage of EIN2 transmits the signal to the nucleus. In the nucleus, hundreds of potential gene targets of the EIN3 master transcription factor have been identified and found to be induced in transcriptional waves, and transcriptional coregulation has been shown to be a mechanism of ethylene cross talk. Ethylene, the first gaseous plant hormone that was identified, regulates numerous developmental processes and stress responses in plants. Ethylene is best known for its effects on agronomically important processes such as ripening in climacteric fruits and organ senescence and abscission, but ethylene also mediates numerous other aspects of plant growth and development, including seed germination, root initiation, leaf expansion, flower development, and sex determination (Abeles et al., 1992; Lin et al., 2009). Ethylene also functions as a stress hormone, as its production is elicited in response to biotic and abiotic challenges, such as wounding, flooding, cold, nutrient stress, and pathogen attack (Lin et al., 2009; Iqbal et al., 2013). Ethylene signal transduction has received intense research attention, in large part due to its wide-ranging effects and critical roles in agronomically important plants. Over the past several decades, the mechanisms of ethylene perception and response have been extensively investigated in the model plant Arabidopsis (Arabidopsis thaliana), as well as in fruit species such as tomato (Solanum lycopersicum; Klee, 2004) and more recently in the rice (Oryza sativa) crop (Yang et al., 2015). The molecular genetic dissection of the ethylene signaling pathway in Arabidopsis has provided major breakthroughs in our understanding of the pathway. The isolation of ethylene response mutants in Arabidopsis was facilitated by a simple genetic screen based on the etiolated seedling triple response phenotype, consisting of a shortened and thickened hypocotyl and root and an exaggerated apical hook (Bleecker et al., 1988; Guzmán and Ecker, 1990; Fig. 1). Mutants that lack the triple response when treated with exogenous ethylene are ethylene insensitive, and mutants that exhibit the triple response even in the absence of ethylene are constitutive response mutants (Fig. 1). The order of action for many of these components in the pathway was determined using double mutant (epistasis) analysis (e.g. Roman et al., 1995). Figure 1. Open in new tabDownload slide Genetic diagram of the core ethylene signaling pathway. The ethylene signal represses the function of the five ethylene receptor genes (ETHYLENE RESPONSE1 [ETR1], ETHYLENE RESPONSE SENSOR1 [ERS1], ETR2, ETHYLENE INSENSITIVE4 [EIN4], and ERS2), which otherwise repress ethylene responses through the negative regulator CONSTITUTIVE RESPONSE1 (CTR1). Ethylene responses are positively regulated by EIN2, EIN3, and downstream primary and secondary ethylene-responsive genes, such as ETHYLENE RESPONSE FACTOR1 (ERF1). Representative seeding phenotypes in the triple response assay (ethylene insensitivity or constitutive ethylene response) are shown for the dominant gain-of-function mutations in the ethylene receptor genes and the loss-of-function mutations in CTR1, EIN2, and EIN3/ETHYLENE INSENSITIVE3-LIKE1 (EIL1). Arrows indicate activation, and T-bars indicate repression of the pathway. Figure 1. Open in new tabDownload slide Genetic diagram of the core ethylene signaling pathway. The ethylene signal represses the function of the five ethylene receptor genes (ETHYLENE RESPONSE1 [ETR1], ETHYLENE RESPONSE SENSOR1 [ERS1], ETR2, ETHYLENE INSENSITIVE4 [EIN4], and ERS2), which otherwise repress ethylene responses through the negative regulator CONSTITUTIVE RESPONSE1 (CTR1). Ethylene responses are positively regulated by EIN2, EIN3, and downstream primary and secondary ethylene-responsive genes, such as ETHYLENE RESPONSE FACTOR1 (ERF1). Representative seeding phenotypes in the triple response assay (ethylene insensitivity or constitutive ethylene response) are shown for the dominant gain-of-function mutations in the ethylene receptor genes and the loss-of-function mutations in CTR1, EIN2, and EIN3/ETHYLENE INSENSITIVE3-LIKE1 (EIL1). Arrows indicate activation, and T-bars indicate repression of the pathway. The key components of the pathway, their mutant phenotypes, and their genetic actions are shown in Figure 1. Notably, Arabidopsis has five ethylene receptor genes (ETR1, ERS1, ETR2, EIN4, and ERS2). As established by genetic studies, the ethylene receptors are negative regulators of ethylene responses (Fig. 1). Dominant gain-of-function mutations in each receptor gene confer ethylene insensitivity (Chang et al., 1993; Hua et al., 1995; Wilkinson et al., 1995; Sakai et al., 1998; Wang et al., 2006; Fig. 1). In contrast, loss-of-function mutations in individual receptor genes confer little or no phenotypes, indicating a significant degree of functional redundancy among the receptors (Hua and Meyerowitz, 1998; Qu et al., 2007). The combination of at least two or three loss-of-function ethylene receptor mutations, however, confers constitutive ethylene responses (Hua and Meyerowitz, 1998). In the ethylene-signaling pathway, the ethylene molecule represses ethylene receptor signaling (Hua and Meyerowitz, 1998), thereby preventing the next downstream component, negative regulator CTR1 (Kieber et al., 1993), from repressing the pathway (Fig. 1); EIN2, a central positive regulator of ethylene responses (Alonso et al., 1999), is then free to signal to the nucleus where the transcription factors EIN3 and its homolog EIL1 (Chao et al., 1997; An et al., 2010) initiate a transcriptional cascade involving ERF1 (Solano et al., 1998), while also directly controlling the expression of many other genes (Chang et al., 2013; Fig. 1). Mechanistic details of this pathway, described in this Update, were brought into view through molecular cloning followed by extensive characterization of the corresponding genes and proteins (Fig. 2). This revealed that ethylene perception takes place at the ER membrane and regulates a transcriptional cascade in the nucleus, leading to diverse cellular, physiological, and metabolic responses. Figure 2. Open in new tabDownload slide Schematic model of the ethylene signaling pathway. In the absence of ethylene perception (left), the formation of functional ethylene receptors depends on a copper cofactor provided by the copper transporter RESPONSIVE TO ANTAGONIST1 (RAN1), as well as activation by REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), which depends on cytochrome b5 (Cb5). The ethylene receptors (represented here by ETR1 and ERS1 homodimers) at the endoplasmic reticulum (ER) membrane are in a protein complex with downstream components EIN2 and CTR1. The receptors associate with and activate (by an undefined signaling mechanism) the CTR1 protein kinase domain (KD), which phosphorylates the EIN2 C-terminal domain. Phosphorylation prevents EIN2 from signaling, and EIN2 is targeted for 26S proteasomal degradation by F-box proteins ETHYLENE INSENSITIVE2 TARGETING PROTEIN1 (ETP1) and ETP2. Meanwhile, in the nucleus, the F-box proteins ETHYLENE INSENSITIVE3 BINDING F-BOX1 (EBF1) and EBF2 target the EIN3/EIL1 transcription factors for 26S proteasomal degradation (only EIN3 is shown), preventing induction of gene expression such that there is no ethylene response. Additionally, there is a postulated secondary pathway from the receptors involving autophosphorylation of the His by the receptor His kinase (HK) domain, and transfer of the phosphate to the receiver (R) domain, followed by transfer of the phosphate to ARABIDOPSIS HIS PHOSPHOTRANSFER (AHP), which is released by a conformational change in the receptors (indicated by the altered shapes of the HK and R domains between the left and right sides) when they bind ethylene (right). The binding of ethylene (right) inactivates ethylene receptor signaling (indicated by the altered shapes of the HK and R domains between the left and right sides). In addition, the levels of ERS1 and other ethylene receptor isoforms (not shown) increase (represented by the darker color on the right side relative to the left side) due to transcriptional induction, but reach an equilibrium state due to being degraded by the 26S proteasome. CTR1 levels increase in the complex as well (represented by the darker color on the right side relative to the left side) due to the increased level of ethylene receptors and protect the ETR1 receptor from proteolysis. However, the ethylene receptors no longer activate CTR1, and therefore, EIN2 is no longer phosphorylated. Instead, a cytoplasmic portion of EIN2 is proteolytically cleaved by an unidentified protease, and the liberated C-terminal portion of EIN2 (C-END) moves into the nucleus where signal transmission results in EIN2-dependent 26S proteasomal degradation of the F-box proteins EBF1/2 and, consequently, the stabilization and accumulation of master transcription factors EIN3/EIL1. EIN3/EIL1 activate a transcriptional cascade that includes the downstream ERF1 transcription factor gene. An exoribonuclease (EXORIBONUCLEASE4 [XRN4]) also plays an indirect role in the repression of EBF1/2 mRNA. Figure 2. Open in new tabDownload slide Schematic model of the ethylene signaling pathway. In the absence of ethylene perception (left), the formation of functional ethylene receptors depends on a copper cofactor provided by the copper transporter RESPONSIVE TO ANTAGONIST1 (RAN1), as well as activation by REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), which depends on cytochrome b5 (Cb5). The ethylene receptors (represented here by ETR1 and ERS1 homodimers) at the endoplasmic reticulum (ER) membrane are in a protein complex with downstream components EIN2 and CTR1. The receptors associate with and activate (by an undefined signaling mechanism) the CTR1 protein kinase domain (KD), which phosphorylates the EIN2 C-terminal domain. Phosphorylation prevents EIN2 from signaling, and EIN2 is targeted for 26S proteasomal degradation by F-box proteins ETHYLENE INSENSITIVE2 TARGETING PROTEIN1 (ETP1) and ETP2. Meanwhile, in the nucleus, the F-box proteins ETHYLENE INSENSITIVE3 BINDING F-BOX1 (EBF1) and EBF2 target the EIN3/EIL1 transcription factors for 26S proteasomal degradation (only EIN3 is shown), preventing induction of gene expression such that there is no ethylene response. Additionally, there is a postulated secondary pathway from the receptors involving autophosphorylation of the His by the receptor His kinase (HK) domain, and transfer of the phosphate to the receiver (R) domain, followed by transfer of the phosphate to ARABIDOPSIS HIS PHOSPHOTRANSFER (AHP), which is released by a conformational change in the receptors (indicated by the altered shapes of the HK and R domains between the left and right sides) when they bind ethylene (right). The binding of ethylene (right) inactivates ethylene receptor signaling (indicated by the altered shapes of the HK and R domains between the left and right sides). In addition, the levels of ERS1 and other ethylene receptor isoforms (not shown) increase (represented by the darker color on the right side relative to the left side) due to transcriptional induction, but reach an equilibrium state due to being degraded by the 26S proteasome. CTR1 levels increase in the complex as well (represented by the darker color on the right side relative to the left side) due to the increased level of ethylene receptors and protect the ETR1 receptor from proteolysis. However, the ethylene receptors no longer activate CTR1, and therefore, EIN2 is no longer phosphorylated. Instead, a cytoplasmic portion of EIN2 is proteolytically cleaved by an unidentified protease, and the liberated C-terminal portion of EIN2 (C-END) moves into the nucleus where signal transmission results in EIN2-dependent 26S proteasomal degradation of the F-box proteins EBF1/2 and, consequently, the stabilization and accumulation of master transcription factors EIN3/EIL1. EIN3/EIL1 activate a transcriptional cascade that includes the downstream ERF1 transcription factor gene. An exoribonuclease (EXORIBONUCLEASE4 [XRN4]) also plays an indirect role in the repression of EBF1/2 mRNA. The ethylene signaling pathway outlined above is highly conserved in plants. Moreover, it was recently found that this pathway is functionally conserved in the charophyte alga Spirogyra pratensis (Ju et al., 2015). Charophytes, which are freshwater algae, gave rise to land plants at least 450 million years ago (Sanderson et al., 2004). The conservation of the ethylene-signaling pathway in S. pratensis suggests that this pathway had been present in the aquatic ancestor of land plants prior to the colonization of land. Evolutionarily, this is the first known appearance of the ethylene-signaling pathway. Despite the conservation of the ethylene-signaling pathway, the responses mediated by the pathway are often quite different among various species. Recent examples include differences in seedling growth kinetics and other ethylene responses between eudicots and monocots (Kim et al., 2012; Yang et al., 2015). Although the seedling triple response is highly specific to ethylene, the response involves coordinated regulation with other hormones, including auxin, GA, brassinosteroid, and jasmonate (JA) in apical hook development (Li et al., 2004; Vriezen et al., 2004; Vandenbussche et al., 2010; Zádníková et al., 2010; An et al., 2012; Muday et al., 2012; Song et al., 2014; Zhang et al., 2014) and auxin in hypocotyl and root elongation (Swarup et al., 2007; Strader et al., 2010; Muday et al., 2012; Xu et al., 2012). An understanding of ethylene signal transduction therefore provides a foundation for identifying mechanisms of hormone cross talk. This Update summarizes recent advances in the ethylene-signaling pathway with an emphasis on mechanisms in ethylene signaling and downstream cross talk. ETHYLENE RECEPTOR SIGNALING AT THE ER MEMBRANE Ethylene Receptor Domain Structure Plants possess a small family of ethylene receptors (Gallie, 2015) related to prokaryotic two-component His protein kinase (HK) receptors (Schaller et al., 2011). As in typical HK receptors, the ethylene receptors have an N-terminal ligand-binding domain that is connected to a HK-like domain via a GAF domain (named for the proteins in which it was first characterized: cyclic guanosine monophosphate phosphodiesterase, adenylyl cyclase, and formate hydrogen lyase transcriptional activator). At the C terminus of some ethylene receptors, there is also a receiver domain (the second component of the two-component system; Gallie, 2015). The ethylene receptors fall into two subfamilies based on their phylogeny and shared sequence features. In contrast to subfamily I members, subfamily II receptors generally have degenerate HK domains and typically have an additional transmembrane domain at the N terminus that possibly serves as a signal sequence (for review, see Binder et al., 2012; Shakeel et al., 2013). In Arabidopsis, ETR1 and ERS1 are in subfamily I, and ETR2, EIN4, and ERS2 are in subfamily II (Chang et al., 1993; Hua et al., 1995; Hua and Meyerowitz, 1998; Sakai et al., 1998). One receptor in each Arabidopsis subfamily (ERS1 and ERS2) lacks the receiver domain, but this arrangement varies among plant species (Gallie, 2015). The ethylene receptors are disulfide-linked homodimers (Schaller et al., 1995; Müller-Dieckmann et al., 1999; Hall et al., 2000; Mayerhofer et al., 2015) with the ethylene-binding domain lying within the ER membrane and the signaling domains residing in the cytoplasm (Chen et al., 2002, 2007; Ma et al., 2006; Fig. 2). Three conserved N-terminal transmembrane domains in each monomer form the ethylene-binding domain (Schaller and Bleecker, 1995; Hall et al., 2000; Ma et al., 2006). Ethylene binding requires a copper ion cofactor (Rodríguez et al., 1999), which is supplied by the P-type adenosine triphosphatase copper transporter RAN1 (Hirayama et al., 1999; Fig. 2). The copper cofactor is also required for ethylene receptor biogenesis (Hirayama et al., 1999; Woeste and Kieber, 2000; Binder et al., 2010). The receptor dimers form clusters that are mediated in part by GAF-GAF domain interactions (Gao et al., 2008; Grefen et al., 2008). Besides interacting among themselves, the receptors interact with other components in the ethylene-signaling pathway, such as CTR1 and EIN2 (Gao et al., 2003; Bisson et al., 2009; Bisson and Groth, 2010; Dong et al., 2010; Ju et al., 2012; Shakeel et al., 2015; Fig. 2). The receptors have been observed in high-molecular-mass protein complexes (Chen et al., 2010), indicating the likely presence of additional unidentified proteins. Although the ethylene receptor isoforms have been shown to have overlapping functions (e.g. Hua and Meyerowitz, 1998), it has become increasingly clear that the isoforms also have distinct roles (Shakeel et al., 2013). For example, Arabidopsis subfamily II receptors cannot functionally substitute for subfamily I receptors (Wang et al., 2003). Arabidopsis subfamily I has a larger role than subfamily II (Qu et al., 2007), whereas in tobacco (Nicotiana tabacum), subfamily II appears to play a greater role than subfamily I (Chen et al., 2009). Within Arabidopsis subfamily I, ETR1 and ERS1 differentially repress ethylene responses (Liu and Wen, 2012), and ERS1 even appears to positively regulate ethylene responses in an ETR1-dependent manner (Liu et al., 2010). Among the Arabidopsis ethylene receptors, only ETR1 is required for ethylene-induced nutational bending in the hypocotyl (Kim et al., 2011), and ETR1 plays a major role in mediating the effects of silver, which blocks ethylene responses (McDaniel and Binder, 2012). In addition, Arabidopsis ETR1 and ETR2 act oppositely in abscisic acid-mediated seed germination under salt stress (Wilson et al., 2014). How Do the Ethylene Receptors Signal? An important question that remains largely unanswered concerns the biochemical mechanism(s) of ethylene receptor signaling. In the canonical two-component signaling system, His autophosphorylation by the receptor is followed by phosphotransfer to an Asp residue in the associated receiver domain, which then mediates downstream responses (Schaller et al., 2011). Although Arabidopsis ETR1 displays HK activity in vitro (Gamble et al., 1998; Moussatche and Klee, 2004), there is no evidence that this is the primary receptor signaling mechanism (Binder et al., 2012; Shakeel et al., 2013). In fact, the HK activity of ETR1 is dispensable in ethylene signaling (Wang et al., 2003), playing only a minor role in modulating the level of ethylene receptor signaling (Hall et al., 2012). There is also no evidence that phosphotransfer to the receiver domain of the receptors is required for ethylene responses. The presence of the receiver domain is, however, important in the growth recovery response upon the removal/dispersal of ethylene (Binder et al., 2004; Kim et al., 2011). Adding to the confusion, ETR1 exhibits in vitro association with ARABIDOPSIS HIS PHOSPHOTRANSFER (AHP) proteins (Scharein et al., 2008), which are downstream elements in the two-component multistep (His-Asp-His-Asp) phosphorelay (Schaller et al., 2011), raising the possibility of a two-component multistep phosphorelay downstream of the ethylene receptors (Fig. 2). Interestingly, the ETR1-AHP1 interaction appears to be controlled by the phosphorylation state of ETR1 (Scharein and Groth, 2011). Given that the cytokinin pathway utilizes His-Asp-His-Asp pathway components (Schaller et al., 2011), an intriguing possibility is that the AHP pathway is involved in ethylene cross talk with cytokinin signaling. Instead of HK activity, subfamily II receptors in Arabidopsis (Moussatche and Klee, 2004), tobacco (Chen et al., 2009), and rice (Wuriyanghan et al., 2009) all exhibit Ser/Thr kinase activity in vitro, whereas Arabidopsis ERS1 (in subfamily I) is bifunctional, displaying both His and Ser/Thr kinase activity in vitro (Moussatche and Klee, 2004). Overexpression of the tobacco subfamily II receptor NTHK1 in Arabidopsis conferred ethylene insensitivity that was abolished by a mutation blocking kinase activity (Chen et al., 2009), suggesting that Ser/Thr kinase activity plays a role in tobacco ethylene signaling. Moreover, upon ethylene treatment or salt stress, NTHK1 phosphorylates an ankyrin repeat protein named NTHK1 ETHYLENE RECEPTOR-INTERACTING PROTEIN2, which inhibits ethylene responses and improves plant growth through its interaction with NTHK1 (Cao et al., 2015). Another question is the relation between receptor kinase activity and ethylene binding. In vitro biochemical data suggest that ethylene binding inhibits HK activity in Arabidopsis ETR1 (Voet-van-Vormizeele and Groth, 2008), whereas genetic data indicate that ethylene perception activates ETR1 kinase activity (Hall et al., 2012). In tomato, the ethylene receptors SlETR4 (in subfamily II) and Never Ripe (in subfamily I) were found to be phosphorylated on multiple sites in vivo, and the level of phosphorylation was reduced during ethylene treatment and fruit ripening (Kamiyoshihara et al., 2012), supporting an inverse relationship between ethylene binding and kinase activity. What remains unclear is whether the observed phosphorylation is due to autokinase activity of SlETR4 and Never Ripe. Overall, further experimentation is needed to reconcile the biochemical versus genetic relationship between ethylene binding and receptor signaling activity, and to resolve the significance of receptor kinase activity in ethylene signaling. Toward this goal, a crystal structure of the ethylene-binding domain would be highly valuable. REVERSION-TO-ETHYLENE SENSITIVITY1/GREEN-RIPE and Cytochrome b5 Promote ETR1 Receptor Signaling ETR1 signaling activity in Arabidopsis is dependent on an upstream unique protein called RTE1. RTE1 localizes to the Golgi/ER membrane (Dong et al., 2008) and represses ethylene responses by promoting ETR1 signaling (Resnick et al., 2006, 2008). Although the biochemical function of RTE1 is unknown, its action in ethylene signaling seems to involve a physical interaction with ETR1 (Dong et al., 2010) and requires the N-terminal domain of ETR1 (Zhou et al., 2007; Qiu et al., 2012). Interestingly, RTE1 appears to have little or no effect on the other Arabidopsis ethylene receptors (Resnick et al., 2006; Rivarola et al., 2009), but the basis for this specificity is unknown. Expression of the RTE1 gene is somehow regulated by HYPER RECOMBINATION1 (HPR1), a component of the THO/Transcription Export complex that is related to mRNA processing (Xu et al., 2015). HPR1 has been previously implicated in ethylene responses (Pan et al., 2012). Recently, Arabidopsis RTE1 was found to interact with ER-localized isoforms of Cb5. Genetic analyses suggest that Cb5 proteins act via RTE1 to promote ETR1 signaling (Chang et al., 2014; Fig. 2.). Since Cb5 proteins carry out oxidation/reduction reactions in other organisms (Schenkman and Jansson, 2003), RTE1 might be activated by Cb5-mediated redox modification. Another possibility is that Cb5s and RTE1 are involved in redox modification of ETR1, providing a mechanistic link between oxidative stress and ethylene signaling. Plants carry two or three members of an RTE1 gene family. Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1-homolog (AtRTH) has no apparent role in ethylene signaling and, instead, might share an unidentified function with that of metazoan RTE1 homologs. The tomato RTE1 homologs GREEN-RIPE (GR) and GREEN-RIPE LIKE1 (SlGRL1; Barry and Giovannoni, 2006) repress distinct but overlapping sets of ethylene responses, whereas SlGRL2, which is more similar in sequence to AtRTH, does not appear to have a role in ethylene responses (Ma et al., 2012). Among the three rice RTE1 homologs (OsRTHs), only OsRTH1, which is most similar to AtRTE1, conferred ethylene-insensitive phenotypes when overexpressed in rice (Zhang et al., 2012). In petunia (Petunia hybrida), a knock-down mutant of PhGRL2, which lies in the AtRTH/SlGRL2 group, conferred enhanced flower senescence; although this phenotype would be consistent with RTE1 function, the authors have raised the possibility that PhGRL2 prevents ethylene biosynthesis (Tan et al., 2014). Dynamic Associations of the Ethylene Receptors with the Downstream Proteins CTR1 and EIN2 The ethylene receptors signal to CTR1, a Ser/Thr protein kinase that negatively regulates ethylene responses (Kieber et al., 1993; Wang et al., 2013; Fig. 2). Genetic evidence indicates that the CTR1 kinase is activated by the receptors in the absence of ethylene and is inactive in the presence of ethylene (Kieber et al., 1993; Huang et al., 2003). CTR1 has an N-terminal regulatory domain and a C-terminal protein kinase domain (Kieber et al., 1993). A physical association between the receptor signaling domains (the HK-like and receiver domains) and the CTR1 regulatory domain is essential for activating the CTR1 kinase domain (Gao et al., 2003; Huang et al., 2003), but the mechanism of activation is unknown. Since ETR1 HK activity is nonessential for ethylene signaling and there is no evidence that the receptors phosphorylate CTR1, the activation of CTR1 potentially involves a noncatalytic steric interaction between the receptors and CTR1. Crystal structure analysis suggests that the Arabidopsis CTR1 kinase domain is active when dimerized, and oligomerization of the CTR1 dimer might help to bring the ethylene receptors together, reinforcing the receptor complex (Mayerhofer et al., 2012). Increased levels of ER-associated CTR1 are correlated with ethylene-induced expression of ethylene receptors due to their physical interaction (Shakeel et al., 2015). The increase in ethylene receptors is countered by ethylene-induced receptor turnover, except for the ETR1 receptor, which appears to be protected from turnover by its tight association with CTR1 (Shakeel et al., 2015). Several Arabidopsis and tomato ethylene receptors have been shown to undergo ligand-induced degradation by the 26S proteasome-dependent pathway (Chen et al., 2007; Kevany et al., 2007; Shakeel et al., 2015; Fig. 2). The ethylene receptors also interact with EIN2 (Bisson et al., 2009; Bisson and Groth, 2010, 2015), a central positive regulator in the ethylene-signaling pathway (Alonso et al., 1999) that acts downstream of CTR1 (Roman et al., 1995; Ju et al., 2012). Arabidopsis ein2 mutants are completely insensitive to ethylene (Alonso et al., 1999), and mutants of EIN2 orthologs in rice (Ma et al., 2013), Medicago truncatula (Penmetsa et al., 2008), and Lotus japonicus (Miyata et al., 2013) also display ethylene-insensitive phenotypes. EIN2 has an ER membrane-localized N-terminal domain that has sequence similarity to the widely conserved Nramp (Natural resistance-associated macrophage protein) metal ion transporters, although metal transport has not been demonstrated for EIN2. The C-terminal portion of EIN2 consists of a plant-specific hydrophilic domain of unknown biochemical function that is required for the activation of ethylene responses (Alonso et al., 1999). In Arabidopsis, it was shown that the HK-like domain of the ethylene receptors interacts with the C-terminal domain of EIN2 (Bisson et al., 2009; Bisson and Groth, 2010). Blocking or mimicking His phosphorylation on ETR1 resulted in reduced or increased affinity with EIN2, respectively (Bisson and Groth, 2010), and a nuclear localization signal in the EIN2 C-terminal domain is important for this interaction (Bisson and Groth, 2015). The direct interaction of EIN2 with the receptors could be consistent with an alternative pathway of ethylene receptor signaling that bypasses CTR1; such a pathway has been implicated by the finding that overexpression of the ETR1 N-terminal domain partially suppresses the constitutive ethylene response phenotype of the Arabidopsis ctr1-1 mutant (Qiu et al., 2012). The above findings indicate that there is dynamic regulation of proteins within the ethylene receptor complex in response to ethylene. Recently obtained crystal structures for the Arabidopsis ETR1 cytosolic domain and catalytic ATP-binding domain, as well as for the dimerization domain of the Arabidopsis ERS1 HK domain (Mayerhofer et al., 2015), should facilitate the modeling of conformational states of ethylene receptor signaling and receptor interactions with CTR1, EIN2, AHPs, and other proteins. THE CENTRAL REGULATOR, EIN2, IS CONTROLLED BY THE CTR1 PROTEIN KINASE AND DELIVERS THE SIGNAL FROM THE ER TO THE NUCLEUS Until recently, there have been major gaps in our understanding of ethylene signaling between CTR1 and events in the nucleus. Because CTR1 is most similar in sequence to the Rapidly accelerated fibrosarcoma family of mitogen-activated protein kinase (MAPK) kinase kinases (Kieber et al., 1993), the expectation for many years had been that EIN2, which is the next known downstream element in the pathway, would be regulated by a MAPK pathway. To date, however, there are no known MAPK kinases or MAPKs acting together with CTR1. Meanwhile, it had long been proposed that EIN2 resides at the nuclear membrane to signal to the nucleus where changes in gene expression were known to take place. When EIN2 was instead found to localize to the ER membrane (Bisson et al., 2009), this raised the key question of how the ethylene signal could be transmitted from EIN2 into the nucleus. Recently, there has been substantial progress in understanding how EIN2 both receives and then relays the ethylene signal, even as the biochemical functions of EIN2 remain elusive. In terms of how EIN2 is regulated, proteomic studies of ethylene responses in Arabidopsis revealed that the EIN2 C-terminal portion is phosphorylated on multiple Ser and Thr residues in the absence, but not presence, of ethylene (Chen et al., 2011; Qiao et al., 2012). Considering that CTR1 is known to be active in the absence of ethylene, CTR1 was a likely candidate for being the kinase responsible for phosphorylating EIN2. Indeed, CTR1 was found to phosphorylate the C-terminal portion of EIN2 on multiple residues in vitro (Ju et al., 2012), and these residues matched those that had been identified in vivo by Chen et al. (2011). Ala substitutions blocking phosphorylation at two highly conserved serines at positions 645 and 924 resulted in constitutive ethylene responses similar to those exhibited by ctr1 mutants (Ju et al., 2012; Qiao et al., 2012), leading to the conclusion that EIN2 signaling is repressed by phosphorylation at these residues in the absence of ethylene (Fig. 2). These findings also suggest that signaling from CTR1 to EIN2 does not require a MAPK pathway. It will be interesting to see whether this phosphorylation is connected to the proteasomal degradation of EIN2 via two F-box proteins, ETP1/2, observed in the absence of ethylene (Qiao et al., 2009). Closing the physical gap in ethylene signaling from the ER membrane to the nucleus, it was discovered that a portion of the EIN2 C terminus (C-END) is proteolytically cleaved from the ER-anchored N-terminal domain of EIN2 and then translocated into the nucleus (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012; Fig. 2). Nuclear localization of the C-END is required for the activation of ethylene responses (Qiao et al., 2012; Wen et al., 2012), and the phosphorylation of EIN2 by CTR1 is a key regulatory mechanism of this translocation, as the translocation occurs constitutively in ctr1 mutants or when Ser phosphorylation of EIN2 is blocked by Ala substitutions (Ju et al., 2012; Qiao et al., 2012). Cleavage of EIN2 reportedly occurs at Ser-645 (Qiao et al., 2012), although there is some conflicting evidence on the importance of this specific site (Cooper, 2013; Qiao et al., 2013). Final confirmation of the cleavage site(s) will likely require the identification of the protease responsible for the cleavage. It will also be interesting to see how both the EIN2 N-terminal domain and dynamic interactions between the ethylene receptors and EIN2 (described in the previous section) are involved in regulating EIN2 signaling. Once in the nucleus, the biochemical mechanisms of EIN2 C-END signaling are still unknown. EIN2 might be involved in either directly activating the transcription factors EIN3/EIL1 or stabilizing EIN3/EIL1 via repression of EBF1 and EBF2, which target EIN3/EIL1 for degradation (described in the next section). Interestingly, the EIN2 C-terminal domain can interact with subunits of the CONSTITUTIVE PHOTOMORPHOGENIC9 signalosome (Christians et al., 2008), although the biological significance of these interactions is unknown. In the current model of EIN2 regulation, CTR1 phosphorylates the EIN2 C-terminal domain at the ER to prevent ethylene signaling in the absence of ethylene. Protein turnover of EIN2 also appears to play a role in preventing EIN2 from signaling (Qiao et al., 2009). In the presence of ethylene, the absence of EIN2 phosphorylation results in the activation of downstream signaling via the cleavage of EIN2 and translocation of the C-END into the nucleus (Fig. 2). THE EIN3/EIL1 TRANSCRIPTION FACTORS INITIATE A TRANSCRIPTIONAL CASCADE The EIN3 transcription factor and its homolog EIL1 are master positive regulators of ethylene responses in Arabidopsis (Chao et al., 1997; Solano et al., 1998; An et al., 2010; Chang et al., 2013). Together, EIN3 and EIL1 cooperatively and differentially regulate the full array of ethylene responses, with EIN3 mainly controlling seedling responses and EIL1 having a greater role in adult leaves and stems (An et al., 2010). EIN3/EIL1 activate a transcriptional cascade by binding as homodimers (Solano et al., 1998; Yamasaki et al., 2005) to the promoters of ERF genes, such as ERF1 (Solano et al., 1998), ETHYLENE AND SALT INDUCIBLE1 (Zhang et al., 2011), and other ERF genes (Chang et al., 2013; Figs. 2 and 3A) in the APETALA2-ETHYLENE RESPONSE ELEMENT BINDING PROTEIN transcription factor family. The ERFs in turn bind to the GCC box element in the promoters of additional ethylene-responsive genes (Fujimoto et al., 2000; Fig. 2), such as stress-response genes (e.g. Wu et al., 2008; Zhang et al., 2011). Additionally, EIN3 has been found to bind to the promoter of a microRNA, miR164, repressing its transcription and thereby hastening the progression of leaf senescence in response to ethylene (Li et al., 2013; Fig. 3A). EIN3/EIL1 also directly control the expression of FLAGELLIN SENSITIVE2 (FLS2; Fig. 3A), which encodes a Leu-rich repeat receptor kinase in plant immunity (Boutrot et al., 2010). Additional EIN3/EIL1 targets are described in the next section. Recently, a chromatin immunoprecipitation sequencing (ChIP-seq) study of Arabidopsis EIN3-binding targets during a time course of ethylene treatment identified nearly 1,500 candidate binding regions, while also revealing a dynamic response in which EIN3 temporally controls four transcriptional waves including a negative feedback loop for ethylene signaling (Chang et al., 2013). Figure 3. Open in new tabDownload slide Examples of the EIN3-mediated regulatory network involving ethylene and other signals. A, Ethylene-stabilized EIN3 directly binds to the promoters of various target genes (described in the text) that control a diverse array of responses. B, The EIN3 protein can physically associate with other transcriptional activators or repressors, including DELLA, JASMONATE ZIM-DOMAINs (JAZs), MYC2, and FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), which are regulated by GA, JA, and iron, respectively, to coactivate (arrows) or corepress (T-bars) transcription in various processes. Figure 3. Open in new tabDownload slide Examples of the EIN3-mediated regulatory network involving ethylene and other signals. A, Ethylene-stabilized EIN3 directly binds to the promoters of various target genes (described in the text) that control a diverse array of responses. B, The EIN3 protein can physically associate with other transcriptional activators or repressors, including DELLA, JASMONATE ZIM-DOMAINs (JAZs), MYC2, and FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), which are regulated by GA, JA, and iron, respectively, to coactivate (arrows) or corepress (T-bars) transcription in various processes. Although EIN3/EIL1 are constitutively expressed, their protein products are degraded in the absence of ethylene via the 26S proteasomal pathway (Guo and Ecker, 2003; An et al., 2010; Fig. 2). Two F-box proteins, EBF1/2 (mentioned earlier), are responsible for targeting EIN3/EIL1 for degradation (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004; Binder et al., 2007; An et al., 2010). When ethylene is perceived, the EBF1/2 F-box proteins themselves are turned over, resulting in the stabilization and accumulation of EIN3/EIL1 (An et al., 2010). The ethylene-induced degradation of EBF1/2 and concomitant EIN3/EIL1 stabilization are EIN2 dependent (An et al., 2010; Fig. 2). At the mRNA level, EIN3 activates the expression of EBF2, providing a negative feedback (Konishi and Yanagisawa, 2008; Chang et al., 2013). XRN4 represses the level of EBF1/2 transcripts (Fig. 2), although the mechanism appears to be indirect (Olmedo et al., 2006; Potuschak et al., 2006). EIN3 turnover and stability might involve phosphorylation (Yoo et al., 2008); a conserved phosphorylation site in tomato SlEIL1 has been implicated in having a role in SlEIL1 dimerization and signaling (Li et al., 2012). EIN3/EIL1 ARE AN INTEGRATION NODE FOR SIGNAL CROSS TALK The EIN3/EIL1 transcription factors serve as a major integration point for ethylene cross talk with other signals, and this could largely explain the involvement of ethylene in diverse responses (Fig. 3). The Chang et al. (2013) ChIP-seq study of EIN3 transcriptional targets revealed numerous genes either within or downstream of essentially all other plant hormone pathways (e.g. Fig. 3A). For example, EIN3/EIL1 bind to the promoters of HOOKLESS1 (HLS1) and HLS1-LIKE HOMOLOG2 (HLH2; An et al., 2012; Chang et al., 2013), which are positive regulators of apical hook formation and thought to be part of the mechanism underlying the coregulation of ethylene and auxin in plant growth and development (Chang et al., 2013; Fig. 3A). Additional studies have identified EIN3/EIL1 target genes involved in cross talk with abiotic signals. For example, ethylene plays an essential role in seedling deetiolation in coordination with light; EIN3/EIL1 activates expression of the PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A and B (PORA/B) genes, which encode rate-limiting enzymes in the chlorophyll biosynthesis pathway (Zhong et al., 2009; Fig. 3A). In freezing tolerance, which is negatively regulated by ethylene (e.g. Zhao et al., 2014), EIN3 binds to the promoters of cold-induced C-repeat Binding Factor/Dehydration-Responsive Element (DRE) Binding Factor (CBF) genes and type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes to prevent their transcription (Shi et al., 2012; Fig. 3A). High salinity enhances proteasomal degradation of F-box proteins EBF1/2, resulting in the accumulation of EIN3/EIL1 proteins, which then activate expression of salt tolerance genes (Zhang et al., 2011; Peng et al., 2014), as well as peroxidase genes whose products scavenge reactive oxygen species to reduce the damage imposed by high salt (Peng et al., 2014). Recent studies have also uncovered a mechanism of transcriptional coregulation that involves physical interactions between EIN3/EIL1 and other transcription factors (Fig. 3B). For example, in the coordinated regulation of apical hook formation in Arabidopsis seedlings by ethylene and GA3, the GA3-repressible DELLA transcriptional regulators interact with the DNA-binding domains of EIN3/EIL1 to attenuate transcription of HLS1 in the absence of GA3, and EIN3/EIL1 are derepressed in the presence of GA3 (An et al., 2012). Similarly, there is evidence that JA signaling leads to the removal of JAZ transcriptional repressors that physically interact with and repress EIN3/EIL1 activity in root development and necrotrophic pathogen defense (Zhu et al., 2011). On the other hand, a reciprocal inhibitory interaction between EIN3 and the JA-activated transcription factor MYC2 was found to underlie the antagonistic roles of ethylene and JA in the regulation of apical hook curvature and herbivory defense (Song et al., 2014). In contrast, EIN3/EIL1 play a stabilizing role, rather than an inhibitory role, in the ethylene-dependent response to iron deficiency in roots by interacting with FIT, a positive regulator of iron uptake (Lingam et al., 2011). CONCLUSION Critical advances in recent years have elucidated several key aspects of ethylene signal transduction, providing a more comprehensive view of the pathway, particularly with respect to mechanistic and dynamic properties. We now have greater insight into (1) the ethylene receptors, their roles, and their dynamic interactions with other signaling proteins; (2) the direct role of CTR1 in regulating EIN2; (3) the dynamics of EIN2 regulation and shuttling to the nucleus; (4) how diverse ethylene responses are achieved via an extensive EIN3-regulated transcriptional network; and (5) how cross talk signaling is achieved via EIN3/EIL1. There are still a number of underlying mechanisms in the pathway that are poorly understood. Major questions surround EIN2, its biochemical activities, the relationship between the EIN2 N-terminal Nramp-like domain and ethylene signaling by the C-END, how EIN2 is cleaved, and the functions of EIN2 C-END in the nucleus. In addition, the signaling mechanisms leading to the turnover of F-box proteins EBF1/2, which are critical to controlling EIN3/EIL1 levels, are unknown. Mechanistic details need to be resolved in the early part of the pathway as well, such as the biochemical mechanisms of ethylene receptor signaling and the regulation of CTR1. Structural protein analyses will be essential in providing insight into the signaling dynamics of the ethylene receptor complex, which could also advance our understanding of how plants respond to ethylene with various sensitivities depending on the tissue and/or developmental stage. Together with mechanistic insights, additional signaling elements are likely to be discovered, such as the protease responsible for cleaving EIN2, the signaling pathway(s) that bypass CTR1, and as yet uncloned genes (e.g. ENHANCING CTR1-10 ETHYLENE RESPONSE2, reported by Xu et al., 2014). A broader challenge that is gaining more attention is to understand how ethylene signaling results in so many diverse responses in different species in a variety of tissues and stages. This can be addressed by further studies to elucidate the transcriptional networks of ethylene signaling while also carrying out in vivo analyses of the numerous targets already uncovered (e.g. by EIN3 ChIP-seq). Continued technological improvements for transcriptomic and proteomic analyses will also help to elucidate ethylene signaling networks, and greater insights into these networks should facilitate the eventual modeling of specific ethylene responses, integrated with a diversity of signals, including environmental stresses and other plant hormones. The application of such knowledge has the potential to tremendously impact agriculture and to provide ways of addressing pressing global concerns, such as the changing climate and the increasing demand for food. Glossary Cb5 cytochrome b5 ER endoplasmic reticulum HK His kinase R receiver C-END C-terminal portion of ETHYLENE INSENSITIVE2 JA jasmonate LITERATURE CITED Abeles FB , Morgan PW, Saltveit ME ( 1992 ) Ethylene in Plant Biology. (2nd edn), Academic Press , San Diego, CA Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Alonso JM , Hirayama T, Roman G, Nourizadeh S, Ecker JR ( 1999 ) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis . Science 284 : 2148 – 2152 Google Scholar Crossref Search ADS PubMed WorldCat An F , Zhang X, Zhu Z, Ji Y, He W, Jiang Z, Li M, Guo H ( 2012 ) Coordinated regulation of apical hook development by gibberellins and ethylene in etiolated Arabidopsis seedlings . Cell Res 22 : 915 – 927 Google Scholar Crossref Search ADS PubMed WorldCat An F , Zhao Q, Ji Y, Li W, Jiang Z, Yu X, Zhang C, Han Y, He W, Liu Y, et al. . ( 2010 ) Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis. Plant Cell 22 : 2384 – 2401 Google Scholar Crossref Search ADS PubMed WorldCat Barry CS , Giovannoni JJ ( 2006 ) Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling . Proc Natl Acad Sci USA 103 : 7923 – 7928 Google Scholar Crossref Search ADS PubMed WorldCat Binder BM , Chang C, Schaller GE ( 2012 ) Perception of ethylene by plants: ethylene receptors . In MT McManus, ed, Annual Plant Reviews: The Plant Hormone Ethylene, Vol 44. Wiley-Blackwell, Oxford, UK, pp 117–145 OpenURL Placeholder Text WorldCat Binder BM , O’malley RC, Wang W, Moore JM, Parks BM, Spalding EP, Bleecker AB ( 2004 ) Arabidopsis seedling growth response and recovery to ethylene: a kinetic analysis . Plant Physiol 136 : 2913 – 2920 Google Scholar Crossref Search ADS PubMed WorldCat Binder BM , Rodríguez FI, Bleecker AB ( 2010 ) The copper transporter RAN1 is essential for biogenesis of ethylene receptors in Arabidopsis. J Biol Chem 285 : 37263 – 37270 Google Scholar Crossref Search ADS PubMed WorldCat Binder BM , Walker JM, Gagne JM, Emborg TJ, Hemmann G, Bleecker AB, Vierstra RD ( 2007 ) The Arabidopsis EIN3 binding F-Box proteins EBF1 and EBF2 have distinct but overlapping roles in ethylene signaling . Plant Cell 19 : 509 – 523 Google Scholar Crossref Search ADS PubMed WorldCat Bisson MMA , Bleckmann A, Allekotte S, Groth G ( 2009 ) EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1 . Biochem J 424 : 1 – 6 Google Scholar Crossref Search ADS PubMed WorldCat Bisson MMA , Groth G ( 2010 ) New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2 . Mol Plant 3 : 882 – 889 Google Scholar Crossref Search ADS PubMed WorldCat Bisson MMA , Groth G ( 2015 ) Targeting plant ethylene responses by controlling essential protein-protein interactions in the ethylene pathway . Mol Plant 8 : 1165 – 1174 Google Scholar Crossref Search ADS PubMed WorldCat Bleecker AB , Estelle MA, Somerville C, Kende H ( 1988 ) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana . Science 241 : 1086 – 1089 Google Scholar Crossref Search ADS PubMed WorldCat Boutrot F , Segonzac C, Chang KN, Qiao H, Ecker JR, Zipfel C, Rathjen JP ( 2010 ) Direct transcriptional control of the Arabidopsis immune receptor FLS2 by the ethylene-dependent transcription factors EIN3 and EIL1 . Proc Natl Acad Sci USA 107 : 14502 – 14507 Google Scholar Crossref Search ADS PubMed WorldCat Cao YR , Chen HW, Li ZG, Tao JJ, Ma B, Zhang WK, Chen SY, Zhang JS ( 2015 ) Tobacco ankyrin protein NEIP2 interacts with ethylene receptor NTHK1 and regulates plant growth and stress responses . Plant Cell Physiol 56 : 803 – 818 Google Scholar Crossref Search ADS PubMed WorldCat Chang C , Kwok SF, Bleecker AB, Meyerowitz EM ( 1993 ) Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators . Science 262 : 539 – 544 Google Scholar Crossref Search ADS PubMed WorldCat Chang J , Clay JM, Chang C ( 2014 ) Association of cytochrome b5 with ETR1 ethylene receptor signaling through RTE1 in Arabidopsis . Plant J 77 : 558 – 567 Google Scholar Crossref Search ADS PubMed WorldCat Chang KN , Zhong S, Weirauch MT, Hon G, Pelizzola M, Li H, Huang SS, Schmitz RJ, Urich MA, Kuo D, et al. . ( 2013 ) Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis. eLife 2 : e00675 Google Scholar Crossref Search ADS PubMed WorldCat Chao Q , Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR ( 1997 ) Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins . Cell 89 : 1133 – 1144 Google Scholar Crossref Search ADS PubMed WorldCat Chen R , Binder BM, Garrett WM, Tucker ML, Chang C, Cooper B ( 2011 ) Proteomic responses in Arabidopsis thaliana seedlings treated with ethylene . Mol Biosyst 7 : 2637 – 2650 Google Scholar Crossref Search ADS PubMed WorldCat Chen T , Liu J, Lei G, Liu YF, Li ZG, Tao JJ, Hao YJ, Cao YR, Lin Q, Zhang WK, et al. . ( 2009 ) Effects of tobacco ethylene receptor mutations on receptor kinase activity, plant growth and stress responses . Plant Cell Physiol 50 : 1636 – 1650 Google Scholar Crossref Search ADS PubMed WorldCat Chen YF , Gao Z, Kerris III RJ, Wang W, Binder BM, Schaller GE ( 2010 ) Ethylene receptors function as components of high-molecular-mass protein complexes in Arabidopsis. PLoS One 5 : e8640 Google Scholar Crossref Search ADS PubMed WorldCat Chen YF , Randlett MD, Findell JL, Schaller GE ( 2002 ) Localization of the ethylene receptor ETR1 to the endoplasmic reticulum of Arabidopsis. J Biol Chem 277 : 19861 – 19866 Google Scholar Crossref Search ADS PubMed WorldCat Chen YF , Shakeel SN, Bowers J, Zhao XC, Etheridge N, Schaller GE ( 2007 ) Ligand-induced degradation of the ethylene receptor ETR2 through a proteasome-dependent pathway in Arabidopsis. J Biol Chem 282 : 24752 – 24758 Google Scholar Crossref Search ADS PubMed WorldCat Christians MJ , Robles LM, Zeller SM, Larsen PB ( 2008 ) The eer5 mutation, which affects a novel proteasome-related subunit, indicates a prominent role for the COP9 signalosome in resetting the ethylene-signaling pathway in Arabidopsis . Plant J 55 : 467 – 477 Google Scholar Crossref Search ADS PubMed WorldCat Cooper B ( 2013 ) Separation anxiety: an analysis of ethylene-induced cleavage of EIN2 . Plant Signal Behav 8 : e24721 Google Scholar Crossref Search ADS PubMed WorldCat Dong CH , Jang M, Scharein B, Malach A, Rivarola M, Liesch J, Groth G, Hwang I, Chang C ( 2010 ) Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1 . J Biol Chem 285 : 40706 – 40713 Google Scholar Crossref Search ADS PubMed WorldCat Dong CH , Rivarola M, Resnick JS, Maggin BD, Chang C ( 2008 ) Subcellular co-localization of Arabidopsis RTE1 and ETR1 supports a regulatory role for RTE1 in ETR1 ethylene signaling . Plant J 53 : 275 – 286 Google Scholar Crossref Search ADS PubMed WorldCat Fujimoto SY , Ohta M, Usui A, Shinshi H, Ohme-Takagi M ( 2000 ) Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box–mediated gene expression . Plant Cell 12 : 393 – 404 Google Scholar PubMed OpenURL Placeholder Text WorldCat Gagne JM , Smalle J, Gingerich DJ, Walker JM, Yoo SD, Yanagisawa S, Vierstra RD ( 2004 ) Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action and promote growth by directing EIN3 degradation . Proc Natl Acad Sci USA 101 : 6803 – 6808 Google Scholar Crossref Search ADS PubMed WorldCat Gallie DR ( 2015 ) Appearance and elaboration of the ethylene receptor family during land plant evolution . Plant Mol Biol 87 : 521 – 539 Google Scholar Crossref Search ADS PubMed WorldCat Gamble RL , Coonfield ML, Schaller GE ( 1998 ) Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis . Proc Natl Acad Sci USA 95 : 7825 – 7829 Google Scholar Crossref Search ADS PubMed WorldCat Gao Z , Chen YF, Randlett MD, Zhao XC, Findell JL, Kieber JJ, Schaller GE ( 2003 ) Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes . J Biol Chem 278 : 34725 – 34732 Google Scholar Crossref Search ADS PubMed WorldCat Gao Z , Wen CK, Binder BM, Chen YF, Chang J, Chiang YH, Kerris III RJ, Chang C, Schaller GE ( 2008 ) Heteromeric interactions among ethylene receptors mediate signaling in Arabidopsis. J Biol Chem 283 : 23801 – 23810 Google Scholar Crossref Search ADS PubMed WorldCat Grefen C , Städele K, Růzicka K, Obrdlik P, Harter K, Horák J ( 2008 ) Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene receptor family members . Mol Plant 1 : 308 – 320 Google Scholar Crossref Search ADS PubMed WorldCat Guo H , Ecker JR ( 2003 ) Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor . Cell 115 : 667 – 677 Google Scholar Crossref Search ADS PubMed WorldCat Guzmán P , Ecker JR ( 1990 ) Exploiting the triple response of Arabidopsis to identify ethylene-related mutants . Plant Cell 2 : 513 – 523 Google Scholar PubMed OpenURL Placeholder Text WorldCat Hall AE , Findell JL, Schaller GE, Sisler EC, Bleecker AB ( 2000 ) Ethylene perception by the ERS1 protein in Arabidopsis . Plant Physiol 123 : 1449 – 1458 Google Scholar Crossref Search ADS PubMed WorldCat Hall BP , Shakeel SN, Amir M, Ul Haq N, Qu X, Schaller GE ( 2012 ) Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis . Plant Physiol 159 : 682 – 695 Google Scholar Crossref Search ADS PubMed WorldCat Hirayama T , Kieber JJ, Hirayama N, Kogan M, Guzman P, Nourizadeh S, Alonso JM, Dailey WP, Dancis A, Ecker JR ( 1999 ) RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis. Cell 97 : 383 – 393 Google Scholar Crossref Search ADS PubMed WorldCat Hua J , Chang C, Sun Q, Meyerowitz EM ( 1995 ) Ethylene insensitivity conferred by Arabidopsis ERS gene . Science 269 : 1712 – 1714 Google Scholar Crossref Search ADS PubMed WorldCat Hua J , Meyerowitz EM ( 1998 ) Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94 : 261 – 271 Google Scholar Crossref Search ADS PubMed WorldCat Huang Y , Li H, Hutchison CE, Laskey J, Kieber JJ ( 2003 ) Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis. Plant J 33 : 221 – 233 Google Scholar Crossref Search ADS PubMed WorldCat Iqbal N , Trivellini A, Masood A, Ferrante A, Khan NA ( 2013 ) Current understanding on ethylene signaling in plants: the influence of nutrient availability . Plant Physiol Biochem 73 : 128 – 138 Google Scholar Crossref Search ADS PubMed WorldCat Ju C , Van de Poel B, Cooper ED, Thierer JH, Gibbons TR, Delwiche CF, Chang C ( 2015 ) Conservation of ethylene as a plant hormone over 450 million years of evolution . Nature Plants 1 : 14004 Ju C , Yoon GM, Shemansky JM, Lin DY, Ying ZI, Chang J, Garrett WM, Kessenbrock M, Groth G, Tucker ML, et al. . ( 2012 ) CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc Natl Acad Sci USA 109 : 19486 – 19491 Google Scholar Crossref Search ADS PubMed WorldCat Kamiyoshihara Y , Tieman DM, Huber DJ, Klee HJ ( 2012 ) Ligand-induced alterations in the phosphorylation state of ethylene receptors in tomato fruit . Plant Physiol 160 : 488 – 497 Google Scholar Crossref Search ADS PubMed WorldCat Kevany BM , Tieman DM, Taylor MG, Cin VD, Klee HJ ( 2007 ) Ethylene receptor degradation controls the timing of ripening in tomato fruit . Plant J 51 : 458 – 467 Google Scholar Crossref Search ADS PubMed WorldCat Kieber JJ , Rothenberg M, Roman G, Feldmann KA, Ecker JR ( 1993 ) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases . Cell 72 : 427 – 441 Google Scholar Crossref Search ADS PubMed WorldCat Kim H , Helmbrecht EE, Stalans MB, Schmitt C, Patel N, Wen CK, Wang W, Binder BM ( 2011 ) Ethylene receptor ETHYLENE RECEPTOR1 domain requirements for ethylene responses in Arabidopsis seedlings . Plant Physiol 156 : 417 – 429 Google Scholar Crossref Search ADS PubMed WorldCat Kim J , Wilson RL, Case JB, Binder BM ( 2012 ) A comparative study of ethylene growth response kinetics in eudicots and monocots reveals a role for gibberellin in growth inhibition and recovery . Plant Physiol 160 : 1567 – 1580 Google Scholar Crossref Search ADS PubMed WorldCat Klee HJ ( 2004 ) Ethylene signal transduction: moving beyond Arabidopsis . Plant Physiol 135 : 660 – 667 Google Scholar Crossref Search ADS PubMed WorldCat Konishi M , Yanagisawa S ( 2008 ) Ethylene signaling in Arabidopsis involves feedback regulation via the elaborate control of EBF2 expression by EIN3 . Plant J 55 : 821 – 831 Google Scholar Crossref Search ADS PubMed WorldCat Li H , Johnson P, Stepanova A, Alonso JM, Ecker JR ( 2004 ) Convergence of signaling pathways in the control of differential cell growth in Arabidopsis. Dev Cell 7 : 193 – 204 Google Scholar Crossref Search ADS PubMed WorldCat Li J , Li Z, Tang L, Yang Y, Zouine M, Bouzayen M ( 2012 ) A conserved phosphorylation site regulates the transcriptional function of ETHYLENE-INSENSITIVE3-like1 in tomato . J Exp Bot 63 : 427 – 439 Google Scholar Crossref Search ADS PubMed WorldCat Li Z , Peng J, Wen X, Guo H ( 2013 ) ETHYLENE-INSENSITIVE3 is a senescence-associated gene that accelerates age-dependent leaf senescence by directly repressing miR164 transcription in Arabidopsis . Plant Cell 25 : 3311 – 3328 Google Scholar Crossref Search ADS PubMed WorldCat Lin Z , Zhong S, Grierson D ( 2009 ) Recent advances in ethylene research . J Exp Bot 60 : 3311 – 3336 Google Scholar Crossref Search ADS PubMed WorldCat Lingam S , Mohrbacher J, Brumbarova T, Potuschak T, Fink-Straube C, Blondet E, Genschik P, Bauer P ( 2011 ) Interaction between the bHLH transcription factor FIT and ETHYLENE INSENSITIVE3/ETHYLENE INSENSITIVE3-LIKE1 reveals molecular linkage between the regulation of iron acquisition and ethylene signaling in Arabidopsis . Plant Cell 23 : 1815 – 1829 Google Scholar Crossref Search ADS PubMed WorldCat Liu Q , Wen CK ( 2012 ) Arabidopsis ETR1 and ERS1 differentially repress the ethylene response in combination with other ethylene receptor genes . Plant Physiol 158 : 1193 – 1207 Google Scholar Crossref Search ADS PubMed WorldCat Liu Q , Xu C, Wen CK ( 2010 ) Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis . BMC Plant Biol 10 : 60 Google Scholar Crossref Search ADS PubMed WorldCat Ma B , Cui ML, Sun HJ, Takada K, Mori H, Kamada H, Ezura H ( 2006 ) Subcellular localization and membrane topology of the melon ethylene receptor CmERS1 . Plant Physiol 141 : 587 – 597 Google Scholar Crossref Search ADS PubMed WorldCat Ma B , He SJ, Duan KX, Yin CC, Chen H, Yang C, Xiong Q, Song QX, Lu X, Chen HW, et al. . ( 2013 ) Identification of rice ethylene-response mutants and characterization of MHZ7/OsEIN2 in distinct ethylene response and yield trait regulation . Mol Plant 6 : 1830 – 1848 Google Scholar Crossref Search ADS PubMed WorldCat Ma Q , Du W, Brandizzi F, Giovannoni JJ, Barry CS ( 2012 ) Differential control of ethylene responses by GREEN-RIPE and GREEN-RIPE LIKE1 provides evidence for distinct ethylene signaling modules in tomato . Plant Physiol 160 : 1968 – 1984 Google Scholar Crossref Search ADS PubMed WorldCat Mayerhofer H , Panneerselvam S, Kaljunen H, Tuukkanen A, Mertens HDT, Mueller-Dieckmann J ( 2015 ) Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1) . J Biol Chem 290 : 2644 – 2658 Google Scholar Crossref Search ADS PubMed WorldCat Mayerhofer H , Panneerselvam S, Mueller-Dieckmann J ( 2012 ) Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk . J Mol Biol 415 : 768 – 779 Google Scholar Crossref Search ADS PubMed WorldCat McDaniel BK , Binder BM ( 2012 ) Ethylene receptor 1 (etr1) is sufficient and has the predominant role in mediating inhibition of ethylene response by silver in Arabidopsis thaliana. J Biol Chem 287 : 26094 – 26103 Google Scholar Crossref Search ADS PubMed WorldCat Miyata K , Kawaguchi M, Nakagawa T ( 2013 ) Two distinct EIN2 genes cooperatively regulate ethylene signaling in Lotus japonicus. Plant Cell Physiol 54 : 1469 – 1477 Google Scholar Crossref Search ADS PubMed WorldCat Moussatche P , Klee HJ ( 2004 ) Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family . J Biol Chem 279 : 48734 – 48741 Google Scholar Crossref Search ADS PubMed WorldCat Muday GK , Rahman A, Binder BM ( 2012 ) Auxin and ethylene: collaborators or competitors? Trends Plant Sci 17 : 181 – 195 Google Scholar Crossref Search ADS PubMed WorldCat Müller-Dieckmann HJ , Grantz AA, Kim SH ( 1999 ) The structure of the signal receiver domain of the Arabidopsis thaliana ethylene receptor ETR1 . Structure 7 : 1547 – 1556 Google Scholar Crossref Search ADS PubMed WorldCat Olmedo G , Guo H, Gregory BD, Nourizadeh SD, Aguilar-Henonin L, Li H, An F, Guzman P, Ecker JR ( 2006 ) ETHYLENE-INSENSITIVE5 encodes a 5′-->3′ exoribonuclease required for regulation of the EIN3-targeting F-box proteins EBF1/2 . Proc Natl Acad Sci USA 103 : 13286 – 13293 Google Scholar Crossref Search ADS PubMed WorldCat Pan H , Liu S, Tang D ( 2012 ) The THO/TREX complex functions in disease resistance in Arabidopsis . Plant Signal Behav 7 : 422 – 424 Google Scholar Crossref Search ADS PubMed WorldCat Peng J , Li Z, Wen X, Li W, Shi H, Yang L, Zhu H, Guo H ( 2014 ) Salt-induced stabilization of EIN3/EIL1 confers salinity tolerance by deterring ROS accumulation in Arabidopsis . PLoS Genet 10 : e1004664 Google Scholar Crossref Search ADS PubMed WorldCat Penmetsa RV , Uribe P, Anderson J, Lichtenzveig J, Gish JC, Nam YW, Engstrom E, Xu K, Sckisel G, Pereira M, et al. . ( 2008 ) The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations . Plant J 55 : 580 – 595 Google Scholar Crossref Search ADS PubMed WorldCat Potuschak T , Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C, Genschik P ( 2003 ) EIN3-dependent regulation of plant ethylene hormone signaling by two arabidopsis F box proteins: EBF1 and EBF2 . Cell 115 : 679 – 689 Google Scholar Crossref Search ADS PubMed WorldCat Potuschak T , Vansiri A, Binder BM, Lechner E, Vierstra RD, Genschik P ( 2006 ) The exoribonuclease XRN4 is a component of the ethylene response pathway in Arabidopsis . Plant Cell 18 : 3047 – 3057 Google Scholar Crossref Search ADS PubMed WorldCat Qiao H , Chang KN, Yazaki J, Ecker JR ( 2009 ) Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis . Genes Dev 23 : 512 – 521 Google Scholar Crossref Search ADS PubMed WorldCat Qiao H , Shen Z, Huang SS, Schmitz RJ, Urich MA, Briggs SP, Ecker JR ( 2012 ) Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas . Science 338 : 390 – 393 Google Scholar Crossref Search ADS PubMed WorldCat Qiao H , Shen Z, Huang SS, Schmitz RJ, Urich MA, Briggs SP, Ecker JR ( 2013 ) Response to perspective: “separation anxiety: an analysis of ethylene-induced cleavage of EIN2” . Plant Signal Behav 8 : e25037 Google Scholar Crossref Search ADS PubMed WorldCat Qiu L , Xie F, Yu J, Wen CK ( 2012 ) Arabidopsis RTE1 is essential to ethylene receptor ETR1 amino-terminal signaling independent of CTR1 . Plant Physiol 159 : 1263 – 1276 Google Scholar Crossref Search ADS PubMed WorldCat Qu X , Hall BP, Gao Z, Schaller GE ( 2007 ) A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors ETR1 and ERS1. BMC Plant Biol 7 : 3 Google Scholar Crossref Search ADS PubMed WorldCat Resnick JS , Rivarola M, Chang C ( 2008 ) Involvement of RTE1 in conformational changes promoting ETR1 ethylene receptor signaling in Arabidopsis . Plant J 56 : 423 – 431 Google Scholar Crossref Search ADS PubMed WorldCat Resnick JS , Wen CK, Shockey JA, Chang C ( 2006 ) REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis . Proc Natl Acad Sci USA 103 : 7917 – 7922 Google Scholar Crossref Search ADS PubMed WorldCat Rivarola M , McClellan CA, Resnick JS, Chang C ( 2009 ) ETR1-specific mutations distinguish ETR1 from other Arabidopsis ethylene receptors as revealed by genetic interaction with RTE1 . Plant Physiol 150 : 547 – 551 Google Scholar Crossref Search ADS PubMed WorldCat Rodríguez FI , Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB ( 1999 ) A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science 283 : 996 – 998 Google Scholar Crossref Search ADS PubMed WorldCat Roman G , Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR ( 1995 ) Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway . Genetics 139 : 1393 – 1409 Google Scholar Crossref Search ADS PubMed WorldCat Sakai H , Hua J, Chen QG, Chang C, Medrano LJ, Bleecker AB, Meyerowitz EM ( 1998 ) ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis. Proc Natl Acad Sci USA 95 : 5812 – 5817 Google Scholar Crossref Search ADS PubMed WorldCat Sanderson MJ , Thorne JL, Wikström N, Bremer K ( 2004 ) Molecular evidence on plant divergence times . Am J Bot 91 : 1656 – 1665 Google Scholar Crossref Search ADS PubMed WorldCat Schaller GE , Bleecker AB ( 1995 ) Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene . Science 270 : 1809 – 1811 Google Scholar Crossref Search ADS PubMed WorldCat Schaller GE , Ladd AN, Lanahan MB, Spanbauer JM, Bleecker AB ( 1995 ) The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer . J Biol Chem 270 : 12526 – 12530 Google Scholar Crossref Search ADS PubMed WorldCat Schaller GE , Shiu SH, Armitage JP ( 2011 ) Two-component systems and their co-option for eukaryotic signal transduction . Curr Biol 21 : R320 – R330 Google Scholar Crossref Search ADS PubMed WorldCat Scharein B , Groth G ( 2011 ) Phosphorylation alters the interaction of the Arabidopsis phosphotransfer protein AHP1 with its sensor kinase ETR1 . PLoS One 6 : e24173 Google Scholar Crossref Search ADS PubMed WorldCat Scharein B , Voet-van-Vormizeele J, Harter K, Groth G ( 2008 ) Ethylene signaling: identification of a putative ETR1-AHP1 phosphorelay complex by fluorescence spectroscopy . Anal Biochem 377 : 72 – 76 Google Scholar Crossref Search ADS PubMed WorldCat Schenkman JB , Jansson I ( 2003 ) The many roles of cytochrome b5 . Pharmacol Ther 97 : 139 – 152 Google Scholar Crossref Search ADS PubMed WorldCat Shakeel SN , Gao Z, Amir M, Chen Y-F, Rai MI, Haq NU, Schaller GE ( 2015 ) Ethylene regulates levels of ethylene receptor/CTR1 signaling complexes in Arabidopsis thaliana. J Biol Chem 290 : 12415 – 12424 Google Scholar Crossref Search ADS PubMed WorldCat Shakeel SN , Wang X, Binder BM, Schaller GE ( 2013 ) Mechanisms of signal transduction by ethylene: overlapping and non-overlapping signalling roles in a receptor family . AoB Plants 5 : plt010 Google Scholar Crossref Search ADS PubMed WorldCat Shi Y , Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S ( 2012 ) Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. Plant Cell 24 : 2578 – 2595 Google Scholar Crossref Search ADS PubMed WorldCat Solano R , Stepanova A, Chao Q, Ecker JR ( 1998 ) Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1 . Genes Dev 12 : 3703 – 3714 Google Scholar Crossref Search ADS PubMed WorldCat Song S , Huang H, Gao H, Wang J, Wu D, Liu X, Yang S, Zhai Q, Li C, Qi T , et al. ( 2014 ) Interaction between MYC2 and ETHYLENE INSENSITIVE3 modulates antagonism between jasmonate and ethylene signaling in Arabidopsis. Plant Cell 26 : 263 – 279 Google Scholar Crossref Search ADS PubMed WorldCat Strader LC , Chen GL, Bartel B ( 2010 ) Ethylene directs auxin to control root cell expansion . Plant J 64 : 874 – 884 Google Scholar Crossref Search ADS PubMed WorldCat Swarup R , Perry P, Hagenbeek D, Van Der Straeten D, Beemster GTS, Sandberg G, Bhalerao R, Ljung K, Bennett MJ ( 2007 ) Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation . Plant Cell 19 : 2186 – 2196 Google Scholar Crossref Search ADS PubMed WorldCat Tan Y , Liu J, Huang F, Guan J, Zhong S, Tang N, Zhao J, Yang W, Yu Y ( 2014 ) PhGRL2 protein, interacting with PhACO1, is involved in flower senescence in the petunia . Mol Plant 7 : 1384 – 1387 Google Scholar Crossref Search ADS PubMed WorldCat Vandenbussche F , Petrásek J, Zádníková P, Hoyerová K, Pesek B, Raz V, Swarup R, Bennett M, Zazímalová E, Benková E , et al. ( 2010 ) The auxin influx carriers AUX1 and LAX3 are involved in auxin-ethylene interactions during apical hook development in Arabidopsis thaliana seedlings . Development 137 : 597 – 606 Google Scholar Crossref Search ADS PubMed WorldCat Voet-van-Vormizeele J , Groth G ( 2008 ) Ethylene controls autophosphorylation of the histidine kinase domain in ethylene receptor ETR1 . Mol Plant 1 : 380 – 387 Google Scholar Crossref Search ADS PubMed WorldCat Vriezen WH , Achard P, Harberd NP, Van Der Straeten D ( 2004 ) Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent . Plant J 37 : 505 – 516 Google Scholar Crossref Search ADS PubMed WorldCat Wang Q , Zhang W, Yin Z, Wen CK ( 2013 ) Rice CONSTITUTIVE TRIPLE-RESPONSE2 is involved in the ethylene-receptor signalling and regulation of various aspects of rice growth and development . J Exp Bot 64 : 4863 – 4875 Google Scholar Crossref Search ADS PubMed WorldCat Wang W , Esch JJ, Shiu SH, Agula H, Binder BM, Chang C, Patterson SE, Bleecker AB ( 2006 ) Identification of important regions for ethylene binding and signaling in the transmembrane domain of the ETR1 ethylene receptor of Arabidopsis . Plant Cell 18 : 3429 – 3442 Google Scholar Crossref Search ADS PubMed WorldCat Wang W , Hall AE, O’Malley R, Bleecker AB ( 2003 ) Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission . Proc Natl Acad Sci USA 100 : 352 – 357 Google Scholar Crossref Search ADS PubMed WorldCat Wen X , Zhang C, Ji Y, Zhao Q, He W, An F, Jiang L, Guo H ( 2012 ) Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus . Cell Res 22 : 1613 – 1616 Google Scholar Crossref Search ADS PubMed WorldCat Wilkinson JQ , Lanahan MB, Yen H-C, Giovannoni JJ, Klee HJ ( 1995 ) An ethylene-inducible component of signal transduction encoded by never-ripe. Science 270 : 1807 – 1809 Google Scholar Crossref Search ADS PubMed WorldCat Wilson RL , Kim H, Bakshi A, Binder BM ( 2014 ) The ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE2 have contrasting roles in seed germination of Arabidopsis during salt stress . Plant Physiol 165 : 1353 – 1366 Google Scholar Crossref Search ADS PubMed WorldCat Woeste KE , Kieber JJ ( 2000 ) A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype . Plant Cell 12 : 443 – 455 Google Scholar Crossref Search ADS PubMed WorldCat Wu L , Zhang Z, Zhang H, Wang XC, Huang R ( 2008 ) Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt, drought, and freezing . Plant Physiol 148 : 1953 – 1963 Google Scholar Crossref Search ADS PubMed WorldCat Wuriyanghan H , Zhang B, Cao WH, Ma B, Lei G, Liu YF, Wei W, Wu HJ, Chen LJ, Chen HW, et al. . ( 2009 ) The ethylene receptor ETR2 delays floral transition and affects starch accumulation in rice . Plant Cell 21 : 1473 – 1494 Google Scholar Crossref Search ADS PubMed WorldCat Xu A , Zhang W, Wen CK ( 2014 ) ENHANCING ctr1-10 ETHYLENE RESPONSE2 is a novel allele involved in CONSTITUTIVE TRIPLE-RESPONSE1-mediated ethylene receptor signaling in Arabidopsis . BMC Plant Biol 14 : 48 Google Scholar Crossref Search ADS PubMed WorldCat Xu C , Gao X, Sun X, Wen CK ( 2012 ) The basal level ethylene response is important to the wall and endomembrane structure in the hypocotyl cells of etiolated Arabidopsis seedlings . J Integr Plant Biol 54 : 434 – 455 Google Scholar Crossref Search ADS PubMed WorldCat Xu C , Zhou X, Wen CK ( 2015 ) HYPER RECOMBINATION1 of the THO/TREX complex plays a role in controlling transcription of the REVERSION-TO-ETHYLENE SENSITIVITY1 gene in Arabidopsis . PLoS Genet 11 : e1004956 Google Scholar Crossref Search ADS PubMed WorldCat Yamasaki K , Kigawa T, Inoue M, Yamasaki T, Yabuki T, Aoki M, Seki E, Matsuda T, Tomo Y, Terada T, et al. . ( 2005 ) Solution structure of the major DNA-binding domain of Arabidopsis thaliana ethylene-insensitive3-like3 . J Mol Biol 348 : 253 – 264 Google Scholar Crossref Search ADS PubMed WorldCat Yang C , Lu X, Ma B, Chen SY, Zhang JS ( 2015 ) Ethylene signaling in rice and Arabidopsis: conserved and diverged aspects . Mol Plant 8 : 495 – 505 Google Scholar Crossref Search ADS PubMed WorldCat Yoo SD , Cho YH, Tena G, Xiong Y, Sheen J ( 2008 ) Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling . Nature 451 : 789 – 795 Google Scholar Crossref Search ADS PubMed WorldCat Zádníková P , Petrásek J, Marhavy P, Raz V, Vandenbussche F, Ding Z, Schwarzerová K, Morita MT, Tasaka M, Hejátko J, et al. . ( 2010 ) Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana. Development 137 : 607 – 617 Google Scholar Crossref Search ADS PubMed WorldCat Zhang L , Li Z, Quan R, Li G, Wang R, Huang R ( 2011 ) An AP2 domain-containing gene, ESE1, targeted by the ethylene signaling component EIN3 is important for the salt response in Arabidopsis . Plant Physiol 157 : 854 – 865 Google Scholar Crossref Search ADS PubMed WorldCat Zhang W , Zhou X, Wen CK ( 2012 ) Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development . J Exp Bot 63 : 4151 – 4164 Google Scholar Crossref Search ADS PubMed WorldCat Zhang X , Zhu Z, An F, Hao D, Li P, Song J, Yi C, Guo H ( 2014 ) Jasmonate-activated MYC2 represses ETHYLENE INSENSITIVE3 activity to antagonize ethylene-promoted apical hook formation in Arabidopsis . Plant Cell 26 : 1105 – 1117 Google Scholar Crossref Search ADS PubMed WorldCat Zhao M , Liu W, Xia X, Wang T, Zhang WH ( 2014 ) Cold acclimation-induced freezing tolerance of Medicago truncatula seedlings is negatively regulated by ethylene . Physiol Plant 152 : 115 – 129 Google Scholar Crossref Search ADS PubMed WorldCat Zhong S , Zhao M, Shi T, Shi H, An F, Zhao Q, Guo H ( 2009 ) EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings . Proc Natl Acad Sci USA 106 : 21431 – 21436 Google Scholar Crossref Search ADS PubMed WorldCat Zhou X , Liu Q, Xie F, Wen C-K ( 2007 ) RTE1 is a Golgi-associated and ETR1-dependent negative regulator of ethylene responses . Plant Physiol 145 : 75 – 86 Google Scholar Crossref Search ADS PubMed WorldCat Zhu Z , An F, Feng Y, Li P, Xue L, A M, Jiang Z, Kim JM, To TK, Li W, et al. . ( 2011 ) Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis . Proc Natl Acad Sci USA 108 : 12539 – 12544 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the National Science Foundation (grant nos. 0923796 and 1244303 to C.C.) and in part by the Maryland Agricultural Experiment Station (to C.C.). * Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00845 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)