On the InsideMinorsky, Peter V.
doi: 10.1104/pp.19.01384pmid: N/A
Plasmodesmata Transport Is Regulated by the Circadian Clock Plasmodesmata (PD) are membrane-bound tunnels that connect the cytosols of neighboring plant cells. The rate of plasmodesmatal transport between cells changes during the course of plant development. Forward genetic screens to identify factors controlling transport through PD have repeatedly revealed that chloroplasts influence plasmodesmatal transport. This has led to a paradigm shift, focusing less on the role of structural changes directly at PD (e.g. callose deposition) and more on how cellular physiology influences the function of PD. Although many groups have now demonstrated that chloroplast function and plasmodesmatal transport are tightly connected, the biological significance of this relationship between chloroplasts and PD remains unresolved. Given the connection between plasmodesmatal transport and chloroplast physiology, Brunkard and Zambryski (pp. 1459–1467) have examined whether plasmodesmatal transport is sensitive to light. In the current study, they combine genetic and physiological approaches to show that PD transport is dynamically regulated by light and the circadian clock throughout the diurnal cycle. Light promotes plasmodesmatal transport during the day, but light is not sufficient to increase rates of plasmodesmatal transport at night, suggesting a circadian gating mechanism. Indeed, silencing the expression of the core circadian clock gene, LHY/CCA1, allows light to strongly promote plasmodesmatal transport during subjective night, confirming that the plant circadian clock controls the plasmodesmatal transport light response. The authors conclude that plasmodesmatal transport is dynamically regulated during the day/night cycle. Due to the many roles of PD in plant biology, this discovery has strong implications for plant development, physiology, and pathogenesis. Ray Parenchymal Cells Contribute to Lignification In conifers such as Norway spruce (Picea abies), lignin is a major cell wall constituent of secondary xylem (wood), forming ∼27% of the dry weight. Lignin enhances the structural stability of wood, as well as water transport through it. Lignification itself encompasses many steps, starting with the biosynthesis of monolignols in the cytosol and their transport across the plasma membrane. In the cell wall, monolignols are exposed to extracellular peroxidases and laccases, the enzymes that initiate polymerization of lignin. But from where do these monolignols originate? Is monolignol biosynthesis in conifers a cell-autonomous process occurring in xylem tracheids, the main cell type in conifer wood, or do neighboring cells (i.e. ray parenchymal cells) contribute to the production of monolignols? To answer this question, Blokhina et al. (pp. 1552–1572) used laser-capture microdissection to isolate parenchymal ray cells and upright tracheids from tangential cryosections of developing xylem of Norway spruce trees. The transcriptome analysis revealed that most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Since transcript abundances are not always reflective of protein abundances, enzymatic activities, and metabolite levels, single-cell metabolome analysis was conducted separately from developing ray cells and tracheids using picoliter pressure probe-electrospray ionization-mass spectrometry to investigate whether monolignol-related metabolites existed in developing ray cells. Both the transcriptomic and metabolomic data strongly suggest that developing ray cells contribute to lignification of cell walls of developing tracheids. Plant Calmodulin-Dependent NAD+ Kinase A common plant response to a variety of stresses is an influx of calcium (Ca2+) ions followed by an apoplastic burst of reactive oxygen species (ROS). This ROS burst is generated by Ca2+-dependent plasma membrane NADPH oxidases. A rapid increase in the NADP(H) pool size may be required to sustain the ROS burst. NADP+ production in plants has long been known to involve a calmodulin (CaM)/Ca2+-dependent NAD+ kinase. The NADP+ produced by this enzyme is converted to NADPH (the substrate of NADPH oxidases) by NADP-isocitrate dehydrogenase or by the reducing branch of the oxidative pentose phosphate pathway. Surprisingly, however, relatively little is known about CaM/Ca2+-dependent NAD+ kinases in plants. Here, Dell’Aglio et al. (pp. 1449–1458) report the characterization of an Arabidopsis (Arabidopsis thaliana) CaM/Ca2+-dependent NAD+ kinase that displays all the properties of the elusive enzyme. By means of proteomic, biochemical, molecular, and in vivo analyses, the authors have successfully identified an Arabidopsis protein that catalyzes NADP+ production exclusively in the presence of CaM/Ca2+. This enzyme, which they have named NAD kinase-CaM dependent (NADKc), has a CaM-binding peptide located in its N-terminal region and displays peculiar biochemical properties as well as different domain organization compared with other known plant NAD+ kinases. In response to a pathogen elicitor (flagellin22), the activity of NADKc, which is associated with the mitochondrial periphery, contributes to an increase in the cellular NADP+ concentration and to the amplification of the elicitor-induced oxidative burst. The authors propose that NADKc represents the missing link between Ca2+ signaling, metabolism, and the oxidative burst. Glyphosate Resistance in Barnyard Grass Glyphosate is the world's most commonly used herbicide owing to its high efficacy, broad spectrum, and systemic mode of action. Most plant species cannot significantly metabolize glyphosate, which is a major factor contributing to its lethality in plants. However, the widespread adoption of glyphosate-tolerant transgenic crops from 1996 onward imposed very high glyphosate selection pressure in agricultural systems, resulting in the extensive evolution of glyphosate resistance in weeds. Given the widespread occurrence and importance of glyphosate-resistant weed evolution, the biochemical and molecular basis of mechanisms endowing glyphosate resistance is under intensive study. It has been determined that specific mutations in the target enzyme of glyphosate, 5-enolpyruvylshikimate3-P synthase, often endow glyphosate resistance to plants. An Australian population of Echinochloa colona (awnless barnyard grass), however, has evolved resistance to glyphosate by a mechanism that does not involve 5-enolpyruvylshikimate3-P synthase or another common mechanism of resistance, that is, reduced glyphosate uptake or translocation at the tissue level. By isolating and comparing glyphosate-resistant (GR) and glyphosate-susceptible E. colona lines, Pan et al. (pp. 1519–1534), using RNA sequencing, have identified a novel AKR (Aldoketo Reductase) gene (designated as EcAKR4-1) in their GR E. colona population. Rice (Oryza sativa) calli and seedlings overexpressing EcAKR4-1 and displaying increased AKR activity were resistant to glyphosate. EcAKR4-1 expressed in Escherichia coli was found to metabolize glyphosate to produce aminomethylphosphonic acid and glyoxylate. Consistent with these results, GR E. colona plants exhibited enhanced capacity for detoxifying glyphosate into aminomethylphosphonic acid and glyoxylate. This study provides experimental evidence of the evolution of a plant AKR that metabolizes glyphosate and thereby confers glyphosate resistance. Jasmonic Acid and High Light and Heat Stress Plants often experience high light (HL) intensities in the field, many times in conjunction with elevated temperatures. Such conditions are a serious threat to agriculture production, because photosynthesis is highly sensitive to both HL intensities and high-temperature stress (HS). During HL stress, the reaction centers become saturated and the excess excitation energy can become harmful, because it can irreversibly damage PSII. In plants, PSII contains more than 20 subunits, including four major core subunits, one of which, termed D1 (PsbA), is the main site susceptible to damage by HS or HL. Damage to PSII leads to photoinhibition—a sustained decline in photosynthetic efficiency caused by the imbalance between the rate of photodamage to PSII and the rate of PSII repair. In addition to HL stress, HS can compromise PSII electron transport due to the increase in fluidity of the thylakoid membranes, which causes dislodging of PSII light harvesting complexes and decreased integrity of PSII. HS can also impair the repair process of PSII, exacerbating the effects of HL stress. Balfagón et al. (pp. 1668–1682) have studied the responses of Arabidopsis plants to a combination of HL and HS (HL+HS) conditions. Combined HL+HS was accompanied by irreversible damage to PSII, decreased D1 protein levels, and an enhanced transcriptional response indicative of PSII repair activation. They further identified several unique aspects of this stress combination that included enhanced accumulation of jasmonic acid (JA) and its conjugate form JA-Ile, as well as the elevated expression of >2,200 different transcripts that are unique to the stress combination (including many that are JA associated). HL-induced structural changes to chloroplasts included a decrease in the number of starch granules and enhanced stacking of thylakoids (number of thylakoid membranes). In contrast, chloroplasts of HS-treated plants have more starch granules and reduced granal stacking. A mutant deficient in JA biosynthesis (allene oxide synthase) displayed enhanced sensitivity to combined HL+HS. This study reveals that JA plays an important role in the acclimation of plants to a combination of HL+HS. The Functions of Retinoblastoma-Related Proteins in Rice RETINOBLASTOMA (RB) was originally identified as a tumor suppressor gene in animals. A basic and core function of its protein is to control cell proliferation via regulating cell cycle entry. RB protein is also involved in regulating cell differentiation and organ specification. In plants, RBR (RB-Related) genes are widely distributed. The mechanism of controlling the cell cycle is also highly conserved in RBR genes. However, plants possess a set of special cellular structures and developmental patterns that are distinguished from those of animals, such as the cell wall, cell totipotency, postembryonic organ formation, and the development of stem cells and meristem from a specific narrow area. These characteristics of plants may reflect differences between plant RBR and animal RB in regard to spatiotemporal expression pattern and functional mechanism, as well as specific molecular interactions and responses. Duan et al. (pp. 1600–1614) have characterized the functions of OsRBR1 and OsRBR2 in rice growth and development by means of forward- and reverse-genetics. The two genes were found to be coexpressed and to perform redundant roles in vegetative organs but exhibited separate functions in flowers. OsRBR1 was highly expressed in the floral meristem and regulated the expression of floral homeotic genes involved in floral organ formation. Mutation of OsRBR1 caused loss of floral meristem identity, resulting in diverse floral abnormalities. OsRBR2 was preferentially expressed in stamens and promoted pollen formation. Mutation of OsRBR2 led to deformed anthers without pollen. OsRBR1 and OsRBR2 physically associated with OsMSI1 (Multicopy Suppressors of the Iral), suggesting that their functions might be dependent on interaction with OsMSI1 at the protein level. This work further elucidates the functions of RBRs and improves our current understanding of specific regulatory pathways of floral specification and pollen formation in rice. Author notes www.plantphysiol.org/cgi/doi/10.1104/pp.19.01384 © 2019 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Keep it Steamy: Improved Quantification of the Humidity within the LeafJulkowska, Magdalena M.
doi: 10.1104/pp.19.01275pmid: 31767789
Stomata are the gatekeepers of plant water status, regulating the balance between plant CO2 uptake and water loss. Stomatal conductance (gs) can be estimated by microscopy of wax or plastic leaf surface imprints, but this technique is time consuming and labor intensive. Another method for the assessment of gs relies on measurements of (1) leaf water loss, (2) air humidity surrounding the leaf, and (3) leaf temperature. Typically, the responsiveness of the stomatal aperture is tested by gradually decreasing atmospheric water pressure surrounding the leaf (i.e. increasing the vapor-pressure deficit [VPD]) and recording the corresponding transpiration response. Assuming that the air inside the leaf is saturated, the humidity within the leaf can be calculated from the leaf temperature. This assumption can be tested using a recently developed method, where leaf gas exchange is coupled with measurements of the stable isotope compositions of CO2 and water vapor passing over the leaf, both of which become enriched in 18O during transpiration (Cernusak et al., 2018). Using this method, it was observed that the intercellular vapor pressure dropped below saturation when the leaves of two conifer species were exposed to increasing VPD (Cernusak et al., 2018), challenging the assumption that leaf vapor pressure is always saturated. In the work published in this issue of Plant Physiology, Cernusak et al. (2019) used the stable-isotope method to examine the changes in gs and intercellular vapor pressure in the leaves of wild-type poplar (Populus × canescens) and the abi1 mutant, which exhibits compromised stomatal closure. They observed that in the wild-type plants, gs and photosynthesis rate decreased with the increasing VPD, while the relative humidity inside the leaf was maintained. In the abi1 mutants, the humidity inside the leaf decreased with increasing VPD. This lower intercellular humidity in abi1 mutants had tremendous consequences, as no gas exchange could be observed above 2.5 kPa VPD, which corresponded to the drop of relative humidity inside the leaf below 60%. The results presented by Cernusak et al. (2019) demonstrate perfectly the essential role of stomata by illustrating the precision with which they function to maintain a moisture-saturated leaf interior and contrasts this with the narrow survival envelope of a mutant with impaired abscisic acid signaling. The new method where gas exchange is combined with stable isotope measurements provides an improved estimation of gs and internal leaf humidity. The method also allows quantification of the importance of functional stomata, preventing the drop in relative humidity with increasing VPD. Another article in this issue of Plant Physiology (Holloway-Phillips et al., 2019) takes a two-source δ18O technique one step further, using source gases of two different isotopic compositions, allowing simultaneous estimation of mesophyll conductance and intercellular humidity. While the methods described by Holloway-Phillips et al. (2019) and Cernusak et al. (2019) both allow addressing questions that were out of reach thus far, they require highly specialized equipment, careful calibration, and extensive expertise. However, as the technology continues to develop, the possibilities of scaling the simultaneous measurement of intercellular humidity and mesophyll conductance in a wider range of species will improve. Cernusak et al. already observed that the range of internal leaf humidity differed between poplar (Cernusak et al., 2019) and coniferous species tested in earlier studies (Cernusak et al., 2018). The stomata of vascular plants show wide diversity, from kidney-shaped guard cells in Arabidopsis (Arabidopsis thaliana) to dumbbell-shaped stomata in the grasses, with the stomatal size and density differing across the species, accessions, and growth conditions (Harrison et al., 2019). Future studies using the improved method for the quantification of intercellular humidity and mesophyll conductance will be able to show how these morphological and developmental differences in stomata affect humidity within the leaf, transpiration, and the photosynthetic rate. LITERATURE CITED Cernusak LA , Goldsmith G, Arend M, Siegwolf RTW ( 2019 ) Effect of vapor pressure deficit on gas exchange in wild-type and abscisic acid-insensitive plants . Plant Physiol 181 : 1573 – 1586 Google Scholar Crossref Search ADS PubMed WorldCat Cernusak LA , Ubierna N, Jenkins MW, Garrity SR, Rahn T, Powers HH, Hanson DT, Sevanto S, Wong SC, McDowell NG, et al. ( 2018 ) Unsaturation of vapour pressure inside leaves of two conifer species . Sci Rep 8 : 7667 Google Scholar Crossref Search ADS PubMed WorldCat Harrison EL , Arce Cubas L, Gray JE, Hepworth C ( 2019 ) The influence of stomatal morphology and distribution on photosynthetic gas exchange . Plant J doi:10.1111/tpj.14560 Google Scholar OpenURL Placeholder Text WorldCat Holloway-Phillips MM , Cernusak LA, Stuart-Williams H, Ubierna N, Farquhar GD ( 2019 ) Two-source δ18O method to validate the CO18O-photosynthetic discrimination model: Implications for mesophyll conductance . Plant Physiol 181 : 1175 – 1190 Google Scholar Crossref Search ADS PubMed WorldCat Author notes www.plantphysiol.org/cgi/doi/10.1104/pp.19.01275 © 2019 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Restriction Release: Improved Maize Transformation EfficiencyJulkowska, Magdalena M.
doi: 10.1104/pp.19.01311pmid: 31767790
Improvement of crops using traditional breeding is too slow to ensure food production able to sustain the growing human population, especially in the face of climate change (Hickey et al., 2019). Transformation methods for monocot crops depend on the availability of immature embryos and are effective only for a limited number of genotypes, oftentimes eliminating the most productive elite varieties, which are recalcitrant to transformation. Lowe et al. (2016) recently addressed the above issues by equipping a vector for DNA delivery with the embryogenesis-inducing genes Wuschel (WUS) and Baby Boom (Bbm). Expression of WUS and Bbm in the positive transformants promoted callus regeneration, thereby increasing the recovery rate and widening the range of cultivars available for transformation. In this issue of Plant Physiology, Zhang et al. (2019) introduce a new system that employs WUS and Bbm for efficient delivery of CRISPR/Cas9 in maize (Zea mays). They constructed a ternary vector system, carrying maize WUS2 and Bbm genes, two glyphosate-resistance genes (GAT and CP4EPSPS), and a Cre/lox module. The Cre/lox module is driven by a drought-inducible promoter (Rab17), which ensures that the cassette containing the morphogenesis regulators (WUS2 and Bbm) is removed upon desiccation treatment of the developing callus. The transformation efficiency for the plants transfected with the morphogenesis regulators increased from 20% to 60% compared with the vectors without the WUS2 and Bbm genes. The efficiency of gene editing with these vectors carrying a single guide RNA was similar to the traditional vector, but the increased transformation efficiency of the vector carrying morphogenic regulators resulted in an overall higher success rate. The authors further improved Agrobacterium tumefaciens-mediated DNA delivery by developing a new binary vector (pGreen3), which carries the T-DNA insertion and the replication origin pRK2 (oriV; Zhang et al., 2019). The helper plasmid (pVS1-VIR2) carrying the virulence genes ensures efficient transformation and serves as a replication helper for pGreen3. The two plasmids, pVS1 and pGreen3, showed improved stability in transformed A. tumefaciens, further enhancing transformation efficiency. Oftentimes, the availability of new methods is restricted by the limited availability of the resources; however, the tools developed by Zhang et al. (2019) are available in public repositories (Addgene and Molecular Cloud), and the guide RNA can be exchanged in a jiffy using a one-step Gateway reaction. The method developed by Zhang et al. (2019) illustrates that combining tissue dedifferentiation with compatible vectors can unlock a higher transformation efficiency, releasing the restrictions on the transformation of obstinate maize inbred lines like ND88, which can now be successfully transformed. This work further confirms that driving transformed tissue to a quasi-embryonic state provides an important contribution to the gains of the transformation efficiency. LITERATURE CITED Hickey LT , N Hafeez A, Robinson H, Jackson SA, Leal-Bertioli SCM, Tester M, Gao C, Godwin ID, Hayes BJ, Wulff BBH ( 2019 ) Breeding crops to feed 10 billion . Nat Biotechnol 37 : 744 – 754 Google Scholar Crossref Search ADS PubMed WorldCat Lowe K , Wu E, Wang N, Hoerster G, Hastings C, Cho MJ, Scelonge C, Lenderts B, Chamberlin M, Cushatt J, et al. ( 2016 ) Morphogenic regulators Baby boom and Wuschel improve monocot transformation . Plant Cell 28 : 1998 – 2015 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Q , Zhang Y, Lu MH, Chai YP, Jiang YY, Zhou Y, Wang XC, Chen QJ ( 2019 ) A novel ternary vector system united with morphogenic genes enhances CRISPR/Cas delivery in maize . Plant Physiol 181 : 1441 – 1448 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 2 Senior author. www.plantphysiol.org/cgi/doi/10.1104/pp.19.01311 © 2019 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
A Novel Upstream Regulator of Trichome Development InhibitorsCox, Naomi; Smith, Lisa M.
doi: 10.1104/pp.19.01269pmid: 31767791
Much like the spikes that deter birds from sitting on fences, trichomes (hair-like projections on the leaf surface) are the epidermis’ first line of defense, discouraging insects and other pests (Levin, 1973). In addition to their role protecting the plant, trichomes are an excellent marker to study developmental biology: they arise from the same progenitor cells as stomata and pavement cells, are easily visible, and their patterning is under strict genetic control. To better understand trichome development, we must understand two core processes: trichome patterning (or the fate determination of progenitor cells) and trichome differentiation, both of which have been most studied in Arabidopsis (Arabidopsis thaliana). The decision to adopt a trichome or other epidermal cell identity is regulated antagonistically by two protein complexes termed the initiator complex and the inhibitor complex (Fig. 1A). The initiator complex consists of GLABRA1 (GL1), GL3 or ENHANCER OF GL3 (EGL3), and TRANSPARENT TESTA GLABRA1 (TTG1; for review, see Pattanaik et al., 2014). This initiator complex activates the expression of GL2, whose transcription factor product initiates trichome morphogenesis (Szymanski et al., 1998). Replacement of GL1 by an inhibitor renders the complex unable to activate GL2 expression, preventing trichome morphogenesis. While the expression of most trichome inhibitors is regulated by the initiator complex, a small number are not, including TRICHOMELESS1 (TCL1) and TCL2. To date, the roles of TCL1 and TCL2 in trichome development in leaves have not been extensively studied, and their upstream regulators in this early stage of development are unknown. Figure 1. Open in new tabDownload slide Graphical summary of the article. A, Previous work has shown that GL2 positively regulates trichome initiation. B, This study uses several lines with altered TCP4 expression. Lines where TCP4 function is lost are tcp2;4;10 and jaw-D. Lines where TCP4 is overexpressed are TCP4:VP16, pBLS::rTCP4:GFP, inducible mTCP4 jawD;GR, inducible mTCP4 Col-0;GR, and p35S::mTCP4:GR. The wild-type (WT) Columbia-0 (Col-0) is used as a control. C, Several techniques were used to study the indirect effect of TCP4 on GL2 and thus trichome number, including the use of existing databases, quantitative PCR (qPCR), and GUS expression in stably transformed lines. D, Several techniques were used to analyze TCP4 binding and activity, including genetic screens, binding motif prediction, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), formaldehyde-assisted isolation of regulatory element (FAIRE), and transient luciferase assays. E, The authors propose that TCP4 affects trichome initiation through directly binding to the TCL1/2 promoter, which in turn inhibits GL2. Figure created with biorender.com. (Adapted from figure 6E ofVadde et al., 2019.) Figure 1. Open in new tabDownload slide Graphical summary of the article. A, Previous work has shown that GL2 positively regulates trichome initiation. B, This study uses several lines with altered TCP4 expression. Lines where TCP4 function is lost are tcp2;4;10 and jaw-D. Lines where TCP4 is overexpressed are TCP4:VP16, pBLS::rTCP4:GFP, inducible mTCP4 jawD;GR, inducible mTCP4 Col-0;GR, and p35S::mTCP4:GR. The wild-type (WT) Columbia-0 (Col-0) is used as a control. C, Several techniques were used to study the indirect effect of TCP4 on GL2 and thus trichome number, including the use of existing databases, quantitative PCR (qPCR), and GUS expression in stably transformed lines. D, Several techniques were used to analyze TCP4 binding and activity, including genetic screens, binding motif prediction, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), formaldehyde-assisted isolation of regulatory element (FAIRE), and transient luciferase assays. E, The authors propose that TCP4 affects trichome initiation through directly binding to the TCL1/2 promoter, which in turn inhibits GL2. Figure created with biorender.com. (Adapted from figure 6E ofVadde et al., 2019.) In this issue of Plant Physiology, Vadde et al. (2019), from the Indian Institute of Science, present their comprehensive characterization of the role of a class II TCP (a family named after the first four studied members: TEOSINTE BRANCHED1 [TB1; Zea mays], CYCLOIDIA [CYC; Antirrhinum majus], and PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1/2 [PCF1/2; Oryza sativa]) in the regulation of TCL1 and TCL2, and therefore its role in trichome development on Arabidopsis leaves. The authors hypothesized that upstream regulators of TCL1 and TCL2 would be transcription factors present for the duration of leaf morphogenesis. The TCPs are a family of plant-specific transcription factors that can be split broadly into two classes, which act antagonistically to promote (class I) or repress (class II) cell proliferation (for review, see Martín-Trillo and Cubas, 2010). TCPs are known to regulate several developmental processes, including leaf shape determination, petal and stamen development, and circadian clock function (Li, 2015). In addition, TCP expression overlaps both temporally and spatially with factors involved in trichome initiation, making them a potential candidate in this process. Plants with TCP4 (a class II TCP) gain of function have previously been observed to have fewer trichomes on their leaf surfaces compared with wild-type plants (Efroni et al., 2008). Vadde et al. (2019) confirmed this observation through examining two TCP4 gain-of-function mutants: TCP4:VP16, in which TCP4 is fused to a viral activation domain; and pBLS::rTCP4:GFP, which expresses a miR319-resistant form of TCP4 fused to GFP under an early leaf-specific promoter. Both plant lines showed reduced trichome density compared with the wild type. Conversely, two TCP4 loss-of-function lines, the triple mutant tcp2 tcp4 tcp10 and jawD (an overexpressor of miR319, whose targets include class II TCPs TCP2, TCP3, TCP4, TCP10, and TCP24), displayed an increased trichome density compared with the wild type (Fig. 1B). Publicly available gene expression data show that the positive regulators of trichome initiation GL1 and GL2 are significantly up-regulated in TCP loss-of-function mutants and vice versa in gain-of-function mutants. This was validated by Vadde et al. (2019) by quantitative PCR time-course experiments and GUS assays (Fig. 1C), demonstrating that TCP4 suppresses the expression of these two genes. As the class II TCPs are transcriptional activators, it was hypothesized that this down-regulation of GL1 and GL2 by TCP4 is an indirect effect. To identify which genes act downstream of TCP4 in GL2 transcriptional regulation, transcript levels of known trichome initiator and trichome inhibitor complex genes were compared between a TCP4 gain-of-function line and the wild type. Four genes, TCL1, TCL2, ENHANCER OF TRY AND CPC2 (ETC2), and ETC3, were up-regulated in the TCP4 gain-of-function line. Comparison with microarray data previously produced in the Nath lab revealed that only TCL1 and TCL2 are activated soon after TCP4 induction (Challa et al., 2016). TCL1 and TCL2 activation is associated with down-regulation of GL1, supporting the model that TCP4 prevents trichome morphogenesis via activation of TCL1 and TCL2, which constitute part of the trichome inhibition complex. Vadde et al. (2019) go on to show that TCP4 directly targets TCL1 and TCL2. In the absence of protein synthesis, induction of TCP4 increased TCL1 and TCL2 transcript levels by at least twofold. Putative TCP4 binding sites were identified in the regulatory regions of both TCL1 and TCL2. These in silico predictions were verified by in vitro electrophoretic mobility shift assays, which demonstrated that TCP4 binds specifically to these sequence motifs, and chromatin immunoprecipitation showed that TCP4 is recruited to these sites in the genome. Formaldehyde-assisted isolation of regulatory element assays confirmed that the presence of TCP4 increases chromatin accessibility in the promoters of TCP1 and TCP2. Furthermore, an in vivo luciferase assay in isolated protoplasts and an in planta GUS assay both confirmed that TCP4 binds to and activates the TCL1/2 genes, supporting the model that trichome morphogenesis is inhibited by TCP4 through direct activation of TCL1 and TCL2 (Fig. 1D). Further genetic experiments showed that TCL1 is required for the reduction in trichome number observed in the leaves of TCP4 overexpression lines. Here, examination of trichomes on the inflorescence stems and older leaves could have been included to determine whether TCP4 regulation of TCL1 is restricted to young leaves or is ubiquitous throughout plant development. The thorough work presented in this article is an important step forward in our understanding of trichome development, summarized in Figure 1E. For the first time, an upstream transcription factor controlling inhibitory regulators of trichome morphogenesis in leaves has been identified. Excitingly, TCP4 is also implicated in the repression of cell proliferation (Schommer et al., 2014) and in fundamental leaf patterning (Koyama et al., 2017), and thus may play an important role in the coordination of development. As there is some degree of functional redundancy within the TCP family (Danisman et al., 2013), whether additional TCPs are involved in trichome initiation and patterning must await further study. TCPs are ubiquitous across plant species; thus, advances in our understanding of their role in coordinating development will also be beneficial when considering economically important crop species. LITERATURE CITED Challa KR , Aggarwal P, Nath U ( 2016 ) Activation of YUCCA5 by the transcription factor TCP4 integrates developmental and environmental signals to promote hypocotyl elongation in Arabidopsis . Plant Cell 28 : 2117 – 2130 Google Scholar Crossref Search ADS PubMed WorldCat Danisman S , van Dijk ADJ, Bimbo A, van der Wal F, Hennig L, de Folter S, Angenent GC, Immink RGH ( 2013 ) Analysis of functional redundancies within the Arabidopsis TCP transcription factor family . J Exp Bot 64 : 5673 – 5685 Google Scholar Crossref Search ADS PubMed WorldCat Efroni I , Blum E, Goldshmidt A, Eshed Y ( 2008 ) A protracted and dynamic maturation schedule underlies Arabidopsis leaf development . Plant Cell 20 : 2293 – 2306 Google Scholar Crossref Search ADS PubMed WorldCat Koyama T , Sato F, Ohme-Takagi M ( 2017 ) Roles of miR319 and TCP transcription factors in leaf development . Plant Physiol 175 : 874 – 885 Google Scholar Crossref Search ADS PubMed WorldCat Levin DA ( 1973 ) The role of trichomes in plant defence . Q Rev Biol 48 : 3 – 15 Google Scholar Crossref Search ADS WorldCat Li S ( 2015 ) The Arabidopsis thaliana TCP transcription factors: A broadening horizon beyond development . Plant Signal Behav 10 : e1044192 Google Scholar Crossref Search ADS PubMed WorldCat Martín-Trillo M , Cubas P ( 2010 ) TCP genes: A family snapshot ten years later . Trends Plant Sci 15 : 31 – 39 Google Scholar Crossref Search ADS PubMed WorldCat Pattanaik S , Patra B, Singh SK, Yuan L ( 2014 ) An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis . Front Plant Sci 5 : 259 Google Scholar Crossref Search ADS PubMed WorldCat Schommer C , Debernardi JM, Bresso EG, Rodriguez RE, Palatnik JF ( 2014 ) Repression of cell proliferation by miR319-regulated TCP4 . Mol Plant 7 : 1533 – 1544 Google Scholar Crossref Search ADS PubMed WorldCat Szymanski DB , Jilk RA, Pollock SM, Marks MD ( 1998 ) Control of GL2 expression in Arabidopsis leaves and trichomes . Development 125 : 1161 – 1171 Google Scholar PubMed OpenURL Placeholder Text WorldCat Vadde BVL , Challa KR, Sunkara P, Hegde AS, Nath U ( 2019 ) The TCP4 transcription factor directly activates TRICHOMELESS1 and 2 and suppresses trichome initiation . Plant Physiol 181 : 1587 – 1599 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 2 Senior author. www.plantphysiol.org/cgi/doi/10.1104/pp.19.01269 © 2019 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Enhanced Metabolic Degradation: The Last Evolved Glyphosate Resistance Mechanism of Weeds?Duke, Stephen O.
doi: 10.1104/pp.19.01245pmid: 31767792
For the past two decades, glyphosate has been the most used herbicide worldwide, resulting in prolonged, extreme selection pressure for glyphosate-resistant (GR) weeds. Glyphosate's only target as a herbicide is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme of the shikimate pathway. It forms a stabile dead-end complex by binding EPSP to form a transition-state analog that is competitive with phosphoenolpyruvate (Sammons and Gaines, 2014). Its widespread use has been due both to its superior properties as a foliar-applied, nonselective herbicide and the widespread adoption of transgenic, GR crops (Duke and Powles, 2008). After glyphosate introduction as a commercial herbicide in 1974, the lag phase for evolution of GR weeds was more than 20 years. To date, both target site (TS) and non-target site (NTS) mechanisms of glyphosate resistance have evolved in 45 weed species (Table 1; Heap and Duke, 2018; Heap, 2019). This relatively long lag phase compared to that of some other herbicide classes has been partly due to the inability of a one-codon change (Pro-106 to Ser, Ala, Thr, or Leu and Thr-102Ser) in the gene for EPSPS to provide robust TS glyphosate resistance (Sammons and Gaines, 2014). Significantly higher levels of TS glyphosate resistance eventually evolved in some weed species via two- (Thr-102Ile and Pro-106Ser) or three-codon mutations (Thr-102Ile, Ala-103Val, and Pro-106Ser) in the EPSPS gene or by duplication of the EPSPS gene to produce higher concentrations of EPSPS in the plant than can be effectively inhibited by recommended doses of the herbicide (Gaines et al., 2019). The molecular mechanisms of gene duplication are understood in only two of the eight reported cases of gene duplication, and these two mechanisms differ (Table 1). No other cases of herbicide resistance involve multiple mutations in the same gene, and there is only one additional reported case of gene duplication-mediated TS herbicide resistance (Laforest et al., 2017), other than the eight species with this mechanism for glyphosate resistance (Gaines et al., 2019). The intense selection pressure by glyphosate over vast geographic areas annually for decades has resulted in TS mechanisms not seen or rarely seen in evolved resistance to other herbicides. Mechanisms of evolved resistance to glyphosate Table 1. Mechanisms of evolved resistance to glyphosate Mechanism . GR Species . Reference . TS Mechanisms Mutated EPSPS One-codon change—Pro-106 to Ser, Ala, Thr, or Leu Several—e.g. Eleusine indica and Lolium spp Sammons and Gaines, 2014 Thr-102Ile Tridax procumbens Li et al., 2018 Two-codon changes (Thr-102Ile and Pro-106Ser) E. indica Yu et al., 2015 Three-codon change (Thr-102Ile, Ala-103Val, and Pro- 106Ser) Amaranthus hybridus Perotti et al., 2019 EPSPS gene duplication Eight species—e.g. L. perrene, Bromus diandrus, Chlorus truncata Patterson et al., 2018 On an extrachromosomal circular DNA Amaranthus palmeri Koo et al., 2018 Tandem duplication at a single locus Kochia scoparia, A. tuberculatis Gaines et al., 2019 NTS mechanisms Reduced movement of glyphosate into the plant Several—e.g. Sorghum halepense, Leptochloa virgata Heap and Duke, 2018 Reduced translocation of glyphosate Several—Chloris elata, Conyza canadensis Heap and Duke, 2018 Vacuolar sequestration of glyphosate C. canadensis, Lolium spp Gaines et al., 2019 Phoenix phenomenon (rapid necrosis, followed by regeneration) A. trifida Van Horn et al., 2018 Enhanced degradation to AMPA and glyoxylate by elevated AKR activity E. colona Pan et al., 2019 TS plus NTS mechanisms For example, one-codon change in EPSPS and reduced translocation Several—e.g. A. tuberculatis, L. rigidum Sammons and Gaines, 2014 Mechanism . GR Species . Reference . TS Mechanisms Mutated EPSPS One-codon change—Pro-106 to Ser, Ala, Thr, or Leu Several—e.g. Eleusine indica and Lolium spp Sammons and Gaines, 2014 Thr-102Ile Tridax procumbens Li et al., 2018 Two-codon changes (Thr-102Ile and Pro-106Ser) E. indica Yu et al., 2015 Three-codon change (Thr-102Ile, Ala-103Val, and Pro- 106Ser) Amaranthus hybridus Perotti et al., 2019 EPSPS gene duplication Eight species—e.g. L. perrene, Bromus diandrus, Chlorus truncata Patterson et al., 2018 On an extrachromosomal circular DNA Amaranthus palmeri Koo et al., 2018 Tandem duplication at a single locus Kochia scoparia, A. tuberculatis Gaines et al., 2019 NTS mechanisms Reduced movement of glyphosate into the plant Several—e.g. Sorghum halepense, Leptochloa virgata Heap and Duke, 2018 Reduced translocation of glyphosate Several—Chloris elata, Conyza canadensis Heap and Duke, 2018 Vacuolar sequestration of glyphosate C. canadensis, Lolium spp Gaines et al., 2019 Phoenix phenomenon (rapid necrosis, followed by regeneration) A. trifida Van Horn et al., 2018 Enhanced degradation to AMPA and glyoxylate by elevated AKR activity E. colona Pan et al., 2019 TS plus NTS mechanisms For example, one-codon change in EPSPS and reduced translocation Several—e.g. A. tuberculatis, L. rigidum Sammons and Gaines, 2014 Open in new tab Table 1. Mechanisms of evolved resistance to glyphosate Mechanism . GR Species . Reference . TS Mechanisms Mutated EPSPS One-codon change—Pro-106 to Ser, Ala, Thr, or Leu Several—e.g. Eleusine indica and Lolium spp Sammons and Gaines, 2014 Thr-102Ile Tridax procumbens Li et al., 2018 Two-codon changes (Thr-102Ile and Pro-106Ser) E. indica Yu et al., 2015 Three-codon change (Thr-102Ile, Ala-103Val, and Pro- 106Ser) Amaranthus hybridus Perotti et al., 2019 EPSPS gene duplication Eight species—e.g. L. perrene, Bromus diandrus, Chlorus truncata Patterson et al., 2018 On an extrachromosomal circular DNA Amaranthus palmeri Koo et al., 2018 Tandem duplication at a single locus Kochia scoparia, A. tuberculatis Gaines et al., 2019 NTS mechanisms Reduced movement of glyphosate into the plant Several—e.g. Sorghum halepense, Leptochloa virgata Heap and Duke, 2018 Reduced translocation of glyphosate Several—Chloris elata, Conyza canadensis Heap and Duke, 2018 Vacuolar sequestration of glyphosate C. canadensis, Lolium spp Gaines et al., 2019 Phoenix phenomenon (rapid necrosis, followed by regeneration) A. trifida Van Horn et al., 2018 Enhanced degradation to AMPA and glyoxylate by elevated AKR activity E. colona Pan et al., 2019 TS plus NTS mechanisms For example, one-codon change in EPSPS and reduced translocation Several—e.g. A. tuberculatis, L. rigidum Sammons and Gaines, 2014 Mechanism . GR Species . Reference . TS Mechanisms Mutated EPSPS One-codon change—Pro-106 to Ser, Ala, Thr, or Leu Several—e.g. Eleusine indica and Lolium spp Sammons and Gaines, 2014 Thr-102Ile Tridax procumbens Li et al., 2018 Two-codon changes (Thr-102Ile and Pro-106Ser) E. indica Yu et al., 2015 Three-codon change (Thr-102Ile, Ala-103Val, and Pro- 106Ser) Amaranthus hybridus Perotti et al., 2019 EPSPS gene duplication Eight species—e.g. L. perrene, Bromus diandrus, Chlorus truncata Patterson et al., 2018 On an extrachromosomal circular DNA Amaranthus palmeri Koo et al., 2018 Tandem duplication at a single locus Kochia scoparia, A. tuberculatis Gaines et al., 2019 NTS mechanisms Reduced movement of glyphosate into the plant Several—e.g. Sorghum halepense, Leptochloa virgata Heap and Duke, 2018 Reduced translocation of glyphosate Several—Chloris elata, Conyza canadensis Heap and Duke, 2018 Vacuolar sequestration of glyphosate C. canadensis, Lolium spp Gaines et al., 2019 Phoenix phenomenon (rapid necrosis, followed by regeneration) A. trifida Van Horn et al., 2018 Enhanced degradation to AMPA and glyoxylate by elevated AKR activity E. colona Pan et al., 2019 TS plus NTS mechanisms For example, one-codon change in EPSPS and reduced translocation Several—e.g. A. tuberculatis, L. rigidum Sammons and Gaines, 2014 Open in new tab Considering the complications in evolving robust TS-based glyphosate resistance, NTS-based resistance might have been expected to predominate. Indeed, there are well-documented cases of glyphosate resistance in some species that are based upon reduced movement of the herbicide into the plant (Heap and Duke, 2018), reduced translocation, and/or enhanced sequestration into vacuoles (Gaines et al., 2019). Vacuolar sequestration is one mechanism for reduced translocation. TS and NTS mechanisms of resistance have been found in the same GR biotypes of several weed species (Sammons and Gaines, 2014). For example, some populations of Lolium rigidum have evolved both the single codon Pro-106Ser EPSPS mutation and reduced translocation of glyphosate in the same weed biotype (Bostamam et al., 2012), resulting in more robust resistance than either change alone would afford. An extreme mechanism of NTS-based resistance is that of Ambrosia trifida, in which the weed has evolved to respond rapidly to this normally slow-acting herbicide (Van Horn et al., 2018). In this GR weed, rapid necrosis occurs in foliage directly contacted by the herbicide spray, preventing its translocation to meristems. The plant then regrows from these unaffected meristems (representing the so-called “Phoenix” phenomenon) after the tissues containing glyphosate die. The NTS-based mechanism of resistance that is common to many weeds resistant to other classes of herbicides (e.g. diclofop resistance in L. rigidum) and for crops that are naturally tolerant to certain herbicides (e.g. propanil tolerance in rice [Oryza sativa]) is enhanced metabolic degradation of the herbicide to nonphytotoxic compounds. Metabolic degradation mechanisms of resistance or tolerance of crops can provide protection from even fast-acting herbicides, such as soybean (Glycine max) tolerance to the protoporphyrinogen oxidase inhibitor, acifluorfen. Until the work of Pan et al. (2019), none of the well-documented, evolved mechanisms of glyphosate resistance (Table 1) have represented enhanced metabolic degradation. Most higher plant species can metabolize glyphosate to aminomethylphosphonic acid (AMPA) and glyoxylate (Duke, 2011). Although a comprehensive taxonomic survey of higher plant metabolism of glyphosate has not been conducted, degradation of glyphosate to AMPA appears to be more pronounced in dicotyledonous than monocotyledonous species. The enzyme(s) that produce(s) AMPA in plants has/have been unknown, but it has been presumed to be similar to the glyphosate oxidoreductase (GOX) of some microbes that degrade glyphosate to AMPA and glyoxylate (Duke, 2011). The first commercial transgenic GR canola (Brassica napus) varieties contained transgenes from microbes for both GOX and GR EPSPS, so that glyphosate was metabolically degraded, and the plant could maintain the shikimate pathway with the GR EPSPS. Glyphosate is a slow-acting herbicide, which should provide the time needed to reduce the herbicide concentration to nonlethal levels by metabolic degradation, provided there is sufficient enzymatic activity. Pan et al. (2019) clearly document enhanced degradation of glyphosate as the mechanism of resistance in a GR weed (Echinocloa colona). Perhaps more important, this work identifies the enzyme (an aldo-keto reductase; AKR) that oxidizes glyphosate to AMPA and glyoxylate in an evolved GR weed. This AKR protein has little similarity to GOX and Gly oxidase of microbes that generate AMPA and glyoxylate from glyphosate. Vemanna et al. (2017) had already shown that overexpression of transgenes of Pseudomonas and rice encoding AKR provides glyphosate resistance in tobacco (Nicotiana tabacum), and that silencing or mutating AKR genes in both plants and microbes increased sensitivity to glyphosate. These findings provided the clue that evolution of enhanced expression of an AKR gene in a weed could provide glyphosate resistance. Now that enhanced expression of a plant degradation enzyme has been shown to provide evolved resistance to glyphosate, other questions might be more easily answered. Do plant species that naturally degrade significant amounts of glyphosate to AMPA and glyoxylate (e.g. soybean) do so with an AKR? Why does relatively rapid degradation of glyphosate by some plant species (e.g. soybean) not provide tolerance? Considering the fact that many plant species can degrade glyphosate, why has enhanced degradation been so rare as a mechanism of evolved glyphosate resistance? Is there a fitness penalty associated with high AKR activity? Is AKR the only plant enzyme involved in glyphosate degradation? The panoply of mechanisms of resistance to glyphosate now clearly includes enhanced metabolic degradation (Table 1). 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