Ethylene modulates root cortical senescence in barley

Ethylene modulates root cortical senescence in barley Abstract Background and Aims Root cortical senescence (RCS) is a poorly understood phenomenon with implications for adaptation to edaphic stress. It was hypothesized that RCS in barley (Hordeum vulgare L.) is (1) accelerated by exogenous ethylene exposure; (2) accompanied by differential expression of ethylene synthesis and signalling genes; and (3) associated with differential expression of programmed cell death (PCD) genes. Methods Gene expression of root segments from four barley genotypes with and without RCS was evaluated using quantitative real-time PCR (qRT-PCR). The progression of RCS was manipulated with root zone ethylene and ethylene inhibitor applications. Key Results The results demonstrate that ethylene modulates RCS. Four genes related to ethylene synthesis and signalling were upregulated during RCS in optimal, low nitrogen and low phosphorus nutrient regimes. RCS was accelerated by root zone ethylene treatment, and this effect was reversed by an ethylene action inhibitor. Roots treated with exogenous ethylene had 35 and 46 % more cortical senescence compared with the control aeration treatment in seminal and nodal roots, respectively. RCS was correlated with expression of two genes related to programmed cell death (PCD). Conclusions The development of RCS is similar to root cortical aerenchyma formation with respect to ethylene modulation of the PCD process. Barley, ethylene, Hordeum vulgare, programmed cell death, root, root cortical senescence INTRODUCTION Root cortical senescence (RCS) is an anatomical phene with potential for improving plant performance and soil resource acquisition efficiency in edaphic stress conditions (Schneider and Lynch, 2017; Schneider et al., 2017a, b). RCS begins in the outer cortical cell files and progresses inwards, increasing in frequency basipetally (Schneider et al., 2017b), and is distinct from the loss of the cortex due to secondary root growth in many dicotyledonous plants (Bingham, 2007). RCS has only been identified in monocots, including several important agronomic species such as barley (Hordeum vulgare), wheat (Triticum aestivum), triticale (Triticosecale) (Yeates and Parker, 1985; Liljeroth, 1995), rye (Secale cereale) (Deacon and Mitchell, 1985; Jupp and Newman, 1987) and oat (Avena sativa) (Yeates and Parker, 1985). Variation in the extent of RCS can be affected by factors such as species (Deacon and Mitchell, 1985), genotype (Liljeroth, 1995), root class and position along the root axis (Schneider et al., 2017b). While to our knowledge RCS occurs in all barley cultivars regardless of growth conditions, the rate of RCS is enhanced by abiotic edaphic stresses including low availabilities of nitrogen and phosphorus (Schneider et al., 2017b). The senescence of cortical cells during RCS causes significant physiological and morphological changes in plants, since root anatomical traits directly affect the cost and physiological functions of roots (Marschner, 1995; Tombesi et al., 2010; Chimungu et al., 2014a, b; Lynch et al., 2014; Saengwilai et al., 2014). RCS and root cortical aerenchyma (RCA), another phene involving cortical cell lysis, influence physiological processes central to plant function including reducing radial nutrient transport (Hu et al., 2014; Schneider et al., 2017b), reducing radial hydraulic conductivity (Fan et al., 2007; Schneider et al., 2017b) and, exclusively in RCS, increasing aliphatic suberin in the endodermis (Schneider et al., 2017b). Many of these physiological changes are due to modifications in cellular organization and barriers between symplastic and apoplastic spaces, affecting the kinetics and radial pathways of nutrient and water transport (Peterson and Perumalla, 1984; Marschner, 1995; Steudle and Peterson, 1998). We have proposed that RCS has physiological implications similar to RCA formation. RCA and RCS both reduce root metabolic costs (Zhu et al., 2010; Saengwilai et al., 2014; Schneider et al., 2017b), reduce radial water and nutrient transport (Hu et al., 2014; Schneider et al., 2017b), are accelerated by mineral nutrient deficiencies (Gillespie and Deacon, 1988; Drew et al., 1989; Elliott et al., 1993; Saengwilai et al., 2014; Schneider et al., 2017b; Galindo-Castañeda et al., 2018) and improve plant growth in edaphic stress conditions (Postma and Lynch, 2011; Saengwilai et al., 2014; Chimungu et al., 2015; Schneider et al., 2017a). However, the mechansisms of RCA formation have been extensively studied, while very little information is available on RCS. We will gain insights into and a better understanding of RCS by examining whether known mechanisms of RCA formation are involved in RCS. Despite the similarities, RCS is a phenomenon that is distinct from RCA formation (Deacon et al., 1986). The formation of RCA begins in the mid-cortex and progresses radially, leaving spokes of connected cortical tissue (Kawai et al., 1998; Lenochová et al., 2009), in contrast to RCS which consistently begins in the outer cortical cell files and progresses inward towards the endodermis (Holden, 1975; Henry and Deacon, 1981). RCS seems to be constitutive and its development is fairly limited to small temperate grains including barley, wheat, rye, oat and triticale (Deacon and Mitchell, 1985; Yeates and Parker, 1985; Jupp and Newman, 1987; Liljeroth, 1995). RCA is formed by many species, including those which also develop RCS such as barley and wheat. Root cortical aerenchyma formation is a type of programmed cell death (PCD) (Drew et al., 2000; Gunawardena et al., 2001) and it has been suggested that RCS may also be a type of PCD (Liljeroth and Bryngelsson, 2001). RCS develops at the same rate in sterile and non-sterile conditions and is not enhanced by the presence of soil micro-organisms (Henry and Deacon, 1981; Lewis and Deacon, 1982). RCS has apparent mechanistic similarities to apoptosis and forms in a predictable pattern in nodal and seminal roots (Schneider et al., 2017b). DNA fragmentation, an indicator of PCD, occurred in wheat and barley seminal roots coinciding with the progression of RCS in wheat as assessed by histological methods (Henry and Deacon, 1981; Liljeroth and Bryngelsson, 2001; Deng et al., 2010). Further evidence is needed in order to confirm that RCS is formed through PCD processes. Ethylene regulates many plant processes including PCD and the development of anatomical and architectural root traits (Tanimoto et al., 1995; He et al., 1996b; Borch et al., 1999; Fan et al., 2003; Zhang et al., 2003; Dahleen et al., 2012). PCD processes including petal senescence (Wagstaff et al., 2005), emergence of adventitious roots in rice (Steffens and Rasmussen, 2016) and the hypersensitive response to bacterial leaf spot in tomato (Ciardi et al., 2001) are all regulated by ethylene. A recent study demonstrated that >200 ethylene-related genes were differentially expressed during RCA formation, with ethylene perception occurring in root cortical and stellar cells (Takahashi et al., 2015). Similar to RCA formation, ethylene has been suggested to regulate the rate of RCS (Lascaris and Deacon, 1991). Cobalt, a known inhibitor of ethylene synthesis (Lau and Yang, 1976; Yu and Yang, 1979), reduced the rate of RCS in root pieces on an agar medium (Lascaris and Deacon, 1991). AgNO3, an early inhibitor of ethylene action (Beyer, 1976), inhibited the formation of RCA induced by ethylene or oxygen deficiency (Drew et al., 1981); however, at concentrations that inhibit RCA formation, the rate of RCS was unaffected in root segments on an agar medium (Lascaris and Deacon, 1991). However, cobalt and silver are old inhibitors and probably have non-specific effects. Since the role of ethylene in RCS remains unclear, we examined the role of ethylene in RCS in intact plants. We hypothesize that (1) exogenous ethylene exposure accelerates RCS; (2) ethylene-related genes are differentially expressed during RCS; and (3) PCD genes are differentially expressed during RCS. RCS was manipulated by using genotypic contrasts and nutrient deficiency in addition to treatments expected to alter ethylene responses. Understanding the role of ethylene in RCS is important in order to gain insights into the genetic regulation and adaptive significance of RCS. MATERIALS AND METHODS Plant materials and growing system Four barley (Hordeum vulgare L.) genotypes (landraces Nuernberg and Tkn24b, and modern varieties Arena and Golf) were selected for this study based on contrasting RCS (Schneider et al., 2017b). Landraces have accelerated RCS compared with modern varieties (Schneider et al., 2017b). Seeds were obtained from IPK, Gatersleben, Germany. Seeds were surface-sterilized in 1.5 % NaOCl in water, rolled into tubes of germination paper (76 lb, Anchor Paper, St Paul, MN, USA) and placed for 4 d in covered beakers containing 0.5 mm CaSO4 in a dark climate chamber at 28 °C. The beakers containing germinated seedlings were then placed under a constant fluorescent light (350 µmol photons m–2 s–1) at 28 °C for 1 d in a climate chamber before transplanting into aerated solution culture. Six randomly assigned seedlings were transplanted in foam plugs suspended above each 33 L solution culture tank. Plants were grown for 30 d in a climate chamber [22 °C, 12 h daylight (Agro high pressure sodium lamp), 50 % relative humidity]. The nutrient solution (pH 5.5, adjusted daily with KOH) was completely replaced every 7 d. The control nutrient solution contained 2.5 mm KNO3, 2.5 mm Ca(NO3)2, 1 mm MgSO4, 0.5 mm KH2PO4, 50 µm NaFeEDTA, 0.2 µm Na2MoO4, 10 µm H3BO3, 0.2 µm NiSO4, 1 µm ZnSO4, 2 µm MnCl2, 0.5 µm CuSO4 and 0.2 µm CoCl2. The solution was modified for two additional treatments (1) low nitrogen [200 µmol N L–1, KNO3 and Ca(NO3)2 were reduced from the nutrient solution and supplemented with K2SO4 and CaCl] and (2) low alumina-buffered phosphorus (buffered at 2 µmol P L–1, omitting KH2PO4) (Lynch et al., 1990). Ethylene treatment Barley plants were grown in optimal, low nitrogen and low phosphorus conditions in solution culture (as described above) with the addition of four aeration treatments for each nutrient condition to manipulate RCS. Treatments comprised (1) root zone air (control); (2) root zone ethylene; (3) root zone 1-MCP (1-methylcyclopropene, an ethylene inhibitor, approx. 3.8 % active ingredient, AgroFresh, Germany); and (4) combined root zone ethylene and 1-MCP application, all applied continuously beginning at seedling transfer to solution culture. Solution culture tanks in the control nutrient treatment were bubbled at 30 mL min–1 with ambient air in 99 L (three tanks of 33 L each) of solution culture. In the ethylene treatment, compressed ethylene (1 µL L–1 in air, as used by Gunawardena et al., 2001) was bubbled through 99 L (three tanks of 33 L each) of solution culture at 30 mL min–1. For the 1-MCP treatment, 1-MCP was volatilized by dissolving 0.17 g in 5 mL of water in a glass scintillation vial, and then transferred into a 2 L sidearm flask. An open-cell foam plug enclosed the mouth of the flask, and the headspace containing 1-MCP gas was bubbled through 99 L (three tanks of 33 L each) of solution culture using an air pump fitted with a one-way check valve. The inlet tubing was connected to the sidearm flask and the outlet tubing was connected to air diffusers submerged in solution culture tanks. The 1-MCP was bubbled at a rate of 30 mL min–1 to achieve a 1-MCP concentration of 7.7 µL L–1 solution, assuming all 1-MCP dissolved in solution. The air pump ran continuously, and the 1-MCP was replenished daily into the sidearm flask. For the combined ethylene and 1-MCP treatment, compressed ethylene was bubbled through 99 L (three tanks of 33 L each) of solution culture at 30 mL min–1 and 1-MCP gas was bubbled at a rate of 30 mL min–1 in the same tank. The ambient air in the climate chamber was circulated and completely exchanged every 10 min; therefore, no contamination between treatments was present. Harvest and sampling After 35 d of growth, root samples were harvested in four replications per genotype and treatment for ethylene experiments. For each root, one sample position was collected. Segments of 1 cm from five positions on nodal roots (2, 3, 8, 13 and 18 cm from the apex) and from ten positions on seminal roots (2, 6, 12, 18, 24, 30, 36, 42, 48 and 54 cm from the apex) were preserved in 70 % ethanol for acridine orange staining and anatomical analysis. At 35 d after germination (vegetative growth, tillering stage), barley plants grown in optimal, low nitrogen and low phosphorus conditions in the control aeration treatment were harvested from solution culture and root segments were sampled for qRT-PCR (quantitative real-time PCR) studies and anatomical analysis. Five representative seminal and nodal roots from each plant were excised and their lengths were measured. Three locations on each root were selected for gene expression studies based on the degree of RCS [3–4 cm (distal, no RCS), 8–9 cm (mid-root, partial RCS) and 34–36 cm (basal, complete cortical senescence) from the root apex]. Although root elongation rates were reduced in sub-optimal nutrient availability, previous studies have demonstrated that minor differences in elongation rates and root length had no significant effect on the quantification of RCS compared with absolute distances from the root apex (Schneider et al., 2017b). Root segments for each position were bulked per plant and were immediately contained in envelopes of aluminium foil and suspended in liquid nitrogen. A 1 cm segment directly basally adjacent to each sample position and a 1 cm segment at 2 cm from the root apex were collected for each root sampled and preserved in 70 % ethanol for acridine orange staining and anatomical analysis. Samples for anatomical analysis and qRT-PCR were collected from four replicate plants per genotype and treatment. Shoot dry biomass, root system dry biomass, tiller number and total root system length of plants were collected from four replicate plants per genotype and treatment for all experiments. Root length was measured by scanning and analysing whole root systems using WinRHIZO Pro (Régent 389 Instruments, Québec City, Québec, Canada). The ethylene and 1-MCP root zone treatments had no significant effect on root elongation rates (Supplementary DataTable S1). In addition to absolute distance from the apex, RCS was also evaluated based on the relative distance to the root apex, with similar results (data not shown). Gene expression Samples suspended in liquid nitrogen were transferred to the laboratory where they were ground with a pestle and mortar while kept frozen with a continuous application of liquid nitrogen. For every sample, 100 mg of ground sample was transferred to a pre-cooled 2 mL epitube and kept frozen at –80 °C until RNA extraction. RNA extraction was performed with the RNeasy Plant Mini Kit (Qiagen, Hildesheim, Germany) and cDNA was transcribed with the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). The qRT-PCR was performed with iQ SYBR Green Supermix (BioRad) according to the manufacturer’s instructions. Gene expression of four ethylene-related genes, Root Abundant Factor (HvRAF), Ethylene Response Factor 1 (BERF1), ACC (aminocyclopropane-1-carboxylic acid) synthase (HvACS6b) and ACC oxidase (HvACO7), and two genes related to PCD, Defender Against Death 2 (DAD2) and Defender Against Death 1 (DAD1) were evaluated. Ethylene-related genes were selected based on involvement in ethylene synthesis or signalling, annotation in barley, expression in barley roots and known involvement in PCD and hypoxia tolerance. PCD-related genes were selected based on their involvement in PCD and annotation in barley. Primers used for amplification are described in the supplementary material, and are unique to each gene target within a gene family and conserved among plant genotypes (Supplementary DataTable S2). For normalization, endogenous reference genes, 26S rRNA and ubiquitin, were averaged and used as internal standards. The PCR cycle conditions were: 95 °C for 4 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and at 72 °C for 30 s. Subsequently, melting curves were recorded to detect non-specific amplification by increasing the temperature from 60 to 95 °C in 0.5 °C increments. Semi-quantitative PCR was performed by 40 cycles of amplification (95 °C for 5 min, 58 °C for 1 min, 72 °C for 1 min). Anatomical characterization Root segments in 70 % ethanol were stained with acridine orange according to Henry and Deacon (1981) except that the staining time was extended to 30 min. Acridine orange-stained root segments were embedded with Tissue-Tek CRYO-OCT compound (Thermo Fisher Scientific, Waltham, MA, USA) in a gelatin capsule and frozen at –20 °C for 15 min. Transverse sections 60 µm thick were cut using a Kryostat 2800 Frigocut –E (Reichert-Jung, Leica Instruments GmbH, Nussloch, Germany) and sections were immediately transferred to a microscope slide and imaged. Images of root cross-sections were acquired on a compound microscope (Zeiss Axioplan 2, mounted with an AxioCam ICc 5, Filter 09: Blue 450–490 nm Carl Zeiss Jena GmbH, Jena, Germany; ×20 magnification). Cross-sectional images were phenotyped for root cross-sectional area and area of the stele using ImageJ (Rasband, 2015). Root segments collected at 2 cm from the root apex served as a reference for root cortical area in regions with no RCS on the same root. The percentage of the cortex senesced was calculated based on the difference in cortical cross-sectional areas (total cross-sectional area minus stele area) at 2 cm from the root apex and at the sample position. The shrinkage of the root cortex, compared with the cortical area at the root apex, is a direct indicator of RCS (Schneider et al., 2017b). Statistical methods Data were analysed by Tukey’s HSD (honest significant difference) using R 3.1.2 (R Core Team, 2014). For gene expression experiments, data analysis and relative expression was calculated using the Relative Expression Software Tool (REST) (Pfaffl et al., 2002). RESULTS Ethylene application significantly accelerated RCS development (Fig. 1). In seminal roots of modern cultivars and landraces grown in optimal nutrient conditions, root segments with ethylene treatment increased RCS (i.e. a greater percentage of cortical area senesced) by 52 % at 6 cm from the root apex, 33 % at 12 cm from the root apex and 16 % at 18 cm from the root apex compared with the control aeration treatment (Fig. 1A, B). As previously reported, low nitrogen and low phosphorus conditions accelerated RCS (Schneider et al., 2017b) (Fig. 1E–H). With low nitrogen and phosphorus availability, ethylene treatment increased RCS by 30 % at 6 cm from the root apex, 24 % at 12 cm from the root apex and 16 % at 18 cm from the root apex compared with the control aeration treatment (Fig. 1E, F). In all nutrient regimes, at 24 cm from the root apex, RCS had progressed to 70 % of the cortex senesced and there were no significant differences in RCS development in the ethylene-treated roots compared with the control or 1-MCP aeration treatment. The ethylene inhibitor 1-MCP completely reversed the ethylene effect but did not reduce RCS in the absence of exogenous ethylene. In all nutrient regimes, no significant differences were observed in RCS between the 1-MCP, control (air) and the 1-MCP plus ethylene treatments (Fig. 1A, B, E, F). Fig. 1. View largeDownload slide The progression of RCS in plants grown with optimal nutrient availability in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. Progression of RCS in plants grown in low nitrogen and low phosphorus treatments in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. No differences in RCS were observed in low nitrogen and phosphorus treatments, so data were combined for analysis. Points show the means of four replicates for each of two genotypes ± s.e. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. Fig. 1. View largeDownload slide The progression of RCS in plants grown with optimal nutrient availability in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. Progression of RCS in plants grown in low nitrogen and low phosphorus treatments in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. No differences in RCS were observed in low nitrogen and phosphorus treatments, so data were combined for analysis. Points show the means of four replicates for each of two genotypes ± s.e. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. In optimal nutrient conditions, nodal root segments of modern cultivars and landraces exposed to ethylene had 61 % more cortex senesced at 3 cm from the root apex, 41 % more cortex senesced at 8 cm from the root apex, 34 % more cortex senesced at 13 cm from the root apex and 19 % more cortex senesced from the root apex compared with the control aeration treatment (Fig. 1C, D). With low nitrogen and phosphorus availability, nodal root segments exposed to ethylene had 34 % more RCS at 3 cm from the root apex, 29 % more RCS at 8 cm from the root apex, 19 % more RCS at 13 cm from the root apex and 12% more RCS at 18 cm from the root apex compared with the aeration treatment (Fig. 1G, H). As with seminal roots, in all nutrient regimes the degree of RCS development was not significantly different between the 1-MCP, control (air) and 1-MCP plus ethylene treatments, but nodal roots showed a greater separation between ethylene-treated roots and those of the other treatments, and therefore may be more sensitive to ethylene when compared with seminal roots (Fig. 1). Ethylene treatment increased the rate of RCS on the entire root, but the ethylene and/or 1-MCP treatments did not affect shoot dry weight, tiller number, root dry weight and total root length in all nutrient regimes (Supplementary DataTable S1). Four genes related to ethylene synthesis and signalling were upregulated in the mid-root segments where RCS was ongoing, an effect that was enhanced by low nitrogen and low phosphorus treatments (Fig. 2). Expression of all four genes was very low in basal root segments, which had maximal RCS (90–100 % cortical senescence, complete senescence of the cortex), and in distal root segments, which had no RCS (Figs 3 and 4). In optimal nutrient conditions, HvACO7, a gene that encodes an enzyme involved in the synthesis of ethylene, was upregulated 1.8 log-fold (P < 0.001) in mid-root segments, where RCS is ongoing, compared with distal root segments with no RCS. HvACS6b, a gene that encodes an enzyme involved in the synthesis of ethylene, was upregulated 1.7 log-fold (P < 0.001) in mid-root segments compared with distal root segments. An ethylene response transcription factor gene, BERF1, was upregulated 2.9 log-fold (P < 0.001) in mid-root segments compared with distal root segments. HvRAF, encoding an ethylene response factor, was upregulated 2 log-fold (P < 0.001) in mid-root sections compared with distal sections (Fig. 2). No differences in gene expression were observed between nodal and seminal roots, among genotypes (data not shown), or between distal and basal root segments (Fig. 2). Fig. 2. View largeDownload slide Expression of ethylene-related genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments had no RCS, mid-root segments had partial RCS, and basal root segments had complete cortical senescence. (A) HvACS6, (B) HvRAF, (C) HvACO7 and (D) BERF1 were significantly upregulated in mid-root sections compared with distal and basal root segments. In mid-root segments, HvACO7 and HvRAF were significantly upregulated in low nitrogen compared with optimal nutrient conditions. In low phosphorus, expression of HvACO7 was significantly upregulated in mid-root segments compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Relative expression is the ratio of expression of the target gene compared with the reference genes. Fig. 2. View largeDownload slide Expression of ethylene-related genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments had no RCS, mid-root segments had partial RCS, and basal root segments had complete cortical senescence. (A) HvACS6, (B) HvRAF, (C) HvACO7 and (D) BERF1 were significantly upregulated in mid-root sections compared with distal and basal root segments. In mid-root segments, HvACO7 and HvRAF were significantly upregulated in low nitrogen compared with optimal nutrient conditions. In low phosphorus, expression of HvACO7 was significantly upregulated in mid-root segments compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Relative expression is the ratio of expression of the target gene compared with the reference genes. Fig. 3. View largeDownload slide Cross-sections of barley seminal root stained with acridine orange. (A) Root cross-section from a plant grown in optimal nutrient conditions with no RCS from the distal portion of the root (3 cm from the apex). (B) Root cross-section from a plant grown in optimal nutrient conditions with partial RCS from the mid-root section (8 cm from the apex). (C) Root cross-section from a plant grown in optimal nutrient conditions with maximal RCS from the basal portion of the root (34 cm from the apex). (D) Root cross-section from an ethylene-treated plant with partial RCS from the mid-root section. (E) Root cross-section from a 1-MCP-treated plant with partial RCS from the mid-root section. Scale bar =100 µm. Fig. 3. View largeDownload slide Cross-sections of barley seminal root stained with acridine orange. (A) Root cross-section from a plant grown in optimal nutrient conditions with no RCS from the distal portion of the root (3 cm from the apex). (B) Root cross-section from a plant grown in optimal nutrient conditions with partial RCS from the mid-root section (8 cm from the apex). (C) Root cross-section from a plant grown in optimal nutrient conditions with maximal RCS from the basal portion of the root (34 cm from the apex). (D) Root cross-section from an ethylene-treated plant with partial RCS from the mid-root section. (E) Root cross-section from a 1-MCP-treated plant with partial RCS from the mid-root section. Scale bar =100 µm. Fig. 4. View largeDownload slide RCS in optimal, low nitrogen and low phosphorus treatments in (A) seminal roots of modern cultivars, (B) nodal roots of modern cultivars, (C) seminal roots of landraces and (D) nodal roots of landraces. In seminal and nodal roots of modern cultivars and landraces, mid-root segments in low nitrogen and phosphorus conditions had significantly greater RCS compared with optimal nutrient conditions. Distal segments (no RCS) are 3–4 cm from the root apex, mid-root segments (RCS forming) are 8–9 cm from the apex and basal segments (complete RCS) are 34–46 cm from the apex. Points show the means of four replicates for each of two genotypes ± s.e. since no significant differences within the landraces or modern cultivar classes were observed. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Fig. 4. View largeDownload slide RCS in optimal, low nitrogen and low phosphorus treatments in (A) seminal roots of modern cultivars, (B) nodal roots of modern cultivars, (C) seminal roots of landraces and (D) nodal roots of landraces. In seminal and nodal roots of modern cultivars and landraces, mid-root segments in low nitrogen and phosphorus conditions had significantly greater RCS compared with optimal nutrient conditions. Distal segments (no RCS) are 3–4 cm from the root apex, mid-root segments (RCS forming) are 8–9 cm from the apex and basal segments (complete RCS) are 34–46 cm from the apex. Points show the means of four replicates for each of two genotypes ± s.e. since no significant differences within the landraces or modern cultivar classes were observed. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). In low nitrogen treatments, RCS was accelerated in mid-root segments by 28 % (i.e. a greater percentage of cortical area senesced) (P < 0.001) in modern cultivars (Fig. 4A, B) and 43 % (P < 0.001) in landraces compared with optimal nutrient conditions (Fig. 4C, D). Increased expression of ethylene-related genes coincided with the increase in RCS. Distal root segments had no RCS (Fig. 4). Mid-root segments of landraces had 47 % of the cortex senesced, which was significantly greater than the 38 % of the cortex senesced in mid-root segments of modern cultivars. Basal root segments had maximal cortical senescence (Figs 3 and 4). HvACO7, HvACS6b, BERF1 and HvRAF were upregulated 2.2, 2.5, 3.9 and 2.5 log-fold (P < 0.001), respectively, in mid-root segments undergoing RCS compared with distal root segments with no RCS (Fig. 2). No significant differences in gene expression were observed between distal and basal root segments. Plants grown in low nitrogen conditions had significantly greater HvACO7 and HvRAF expression in mid-root segments compared with mid-root segments of plants grown in optimal nutrient conditions, but expression of HvACS6b and BERF1 in mid-root sections was not significantly different from that of mid-root segments of plants grown in optimal nutrient conditions (Fig. 2). Nodal and seminal roots and different genotypes within these root classes had no significant differences in gene expression (data not shown). In roots grown under low phosphorus availability, RCS was accelerated in mid-root segments by 37 % (P < 0.001) in modern cultivars and 46 % (P < 0.001) in landraces compared with optimal nutrient conditions (Fig. 4). Distal root segments had no RCS, mid-root segments had 43 % of the cortex senesced in modern cultivars and 46 % cortical senescence in landraces, and basal root segments had maximal RCS. Landraces had significantly greater RCS in mid-root segments compared with modern cultivars. HvACO7, HvACS6b, BERF1 and HvRAF were upregulated 2, 2, 2 and 3 log-fold (P < 0.001), respectively, in mid-root segments undergoing RCS compared with distal root segments with no RCS (Fig. 2). In low phosphorus conditions, expression of HvACO7 was significantly upregulated in mid-root segments compared with optimal nutrient conditions. Although mean gene expression values of HvACS6b, BERF1 and HvRAF appeared higher in mid-root sections of plants grown in low phosphorus, variability was also high, so they did not have significantly different expression compared with mid-root segments of plants grown in optimal nutrient conditions. No significant differences in gene expression were observed between nodal and seminal roots or among genotypes within or between the modern cultivar and landrace classes (data not shown). Gene expression was not significantly different between distal and basal root segments (Fig. 2). Low nitrogen and low phosphorus conditions reduced shoot dry biomass by 32 % (Supplementary DataTable S1). Landraces (Nuernberg and Tkn24b) had significantly greater tiller number compared with modern cultivars (Arena and Golf) in control (air), ethylene, 1-MCP and ethylene plus 1-MCP aeration treatments. Significantly greater tiller numbers corresponded to significantly greater shoot dry biomass. No significant differences in the number of tillers or shoot dry biomass were observed among genotypes in low nitrogen or low phosphorus conditions. No significant differences were observed among genotypes or treatments in root dry biomass or total root length (Supplementary Data Table S1). Programmed cell death-related genes were differentially expressed during RCS. One gene indicating PCD, DAD2, was significantly upregulated in root segments beginning RCS compared with root segments with complete senescence or no senescence. In mid-root segments grown in optimal nutrient conditions, DAD2 was upregulated 2 log-fold (P < 0.001) compared with distal and basal root segments. In mid-root segments grown in low nitrogen conditions, DAD2 was upregulated 3.2 log-fold (P < 0.001) compared with distal and basal root segments, and expression was not significantly different compared with optimal nutrient conditions. In mid-root segments grown in low phosphorus conditions, DAD2 was upregulated 3.1 log-fold (P < 0.001) compared with distal and basal root segments and upregulated 2.7 log-fold (P < 0.001) compared with optimal nutrient conditions (Fig. 5B). Another gene indicating PCD, DAD1, was significantly downregulated in root segments with any degree of RCS. In mid-root and basal root segments, DAD1 had a –3.5, –3 and –3.3 log-fold decline (P < 0.001) compared with distal root segments in optimal, low nitrogen and low phosphorus nutrient treatments, respectively (Fig. 5A). Variability of gene expression data was high and therefore no differences between genotypes and – in many cases – treatments were observed (Figs 2 and 5), although differences in RCS were observed between genotypes and treatments (Fig. 4). Fig. 5. View largeDownload slide Expression of PCD genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments (3–4 cm from root apex) had no RCS, mid-root segments (8–9 cm from the root apex) had partial RCS and basal root segments (34–36 cm from the root apex) had complete cortical senescence. (A) Relative expression of DAD1. Relative expression is the ratio of expression of the target gene compared with the reference genes. DAD1 was significantly downregulated in mid- and basal root segments compared with distal root segments. DAD1 was significantly upregulated in low nitrogen and low phosphorus conditions compared with optimal nutrient conditions. (B) Relative expression of DAD2. DAD2 was significantly upregulated in mid-root segments compared with distal root segments. DAD2 was significantly upregulated in low phosphorus conditions compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Fig. 5. View largeDownload slide Expression of PCD genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments (3–4 cm from root apex) had no RCS, mid-root segments (8–9 cm from the root apex) had partial RCS and basal root segments (34–36 cm from the root apex) had complete cortical senescence. (A) Relative expression of DAD1. Relative expression is the ratio of expression of the target gene compared with the reference genes. DAD1 was significantly downregulated in mid- and basal root segments compared with distal root segments. DAD1 was significantly upregulated in low nitrogen and low phosphorus conditions compared with optimal nutrient conditions. (B) Relative expression of DAD2. DAD2 was significantly upregulated in mid-root segments compared with distal root segments. DAD2 was significantly upregulated in low phosphorus conditions compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). DISCUSSION Our results support the hypothesis that ethylene modulates, but is not required for, the development of RCS. In low nitrogen, low phosphorus and optimal nutrient regimes, RCS was accelerated by root zone ethylene treatment, an effect that was blocked by the ethylene inhibitor 1-MCP (Fig. 1). Genes involved in ethylene synthesis and signalling were significantly upregulated during RCS (Fig. 2). When RCS was manipulated by low nitrogen and low phosphorus treatments, differential expression of ethylene-related and PCD genes coincided with the distribution of RCS along the root axes in optimal nutrient, low nitrogen and low phosphorus treatments (Figs 2, 4 and 5). Although RCS development was accelerated by exogenous ethylene application, 1-MCP did not reduce RCS in the absence of ethylene. This suggests that ethylene action is not strictly required for RCS, but instead acts as a modulator of RCS initiation and progression. The 1-MCP concentration used here acted as an effective ethylene action inhibitor, since the treatment containing both ethylene and 1-MCP completely prevented the increase in RCS caused by ethylene treatment. The lack of reduction in RCS may not be due to the performance of the inhibitor, but rather to the limited action of ethylene in control conditions. Ethylene synthesis plays a role in RCS development, as shown by enhanced expression of ACC synthase and ACC oxidase genes in the mid-root segments during RCS (Fig. 2A, C). As the two enzymes limit ethylene synthesis, ACC oxidase and ACC synthase have known roles in root responses to stresses including hypoxia and nutrient stress (Olson et al., 1995; He et al., 1996a; Roldan et al., 2013). Upregulation of ACC synthase and oxidase genes has been associated with PCD responses, including petal senescence in tomato (Llop-Tous et al., 2000), hypoxia in maize (Geisler-Lee et al., 2010) and cell death of tomato leaves following ozone exposure (Moeder, 2002). The closest orthologous gene of HvACO7 in rice (OsACO7) (Dahleen et al., 2012) has been linked to increased ethylene biosynthesis associated with the hypersensitive response, another type of PCD (Iwai et al., 2006). Expression of HvACO7 was 213 times greater in root segments undergoing RCS compared with root segments with no RCS (Fig. 2C). In maize grown in waterlogged conditions, expression of the HvACO7 orthologue (GRMZM2G013448, 1-aminocyclopropane-1-carboxylate oxidase 1, ZM08G22670) was 19 times greater in hypoxic conditions (that induce aerenchyma formation) compared with hypoxic conditions plus 1-MCP treatment that reduced aerenchymya formation (Rajhi et al., 2011). The upregulation of ACC oxidase expression is another mechanism common to RCA in rice (Yamauchi et al., 2016) and RCS. The role of ethylene in RCS is further supported by the upregulation of ethylene response-related transcripts. BERF1 is a member of, and HvRAF is a close relative of, the group VII ERF subfamily (Hinz et al., 2010; Mendiondo et al., 2016). Group VII ethylene response factors are plant specific and play an important role in biotic and abiotic stress response and response to ethylene, gibberellin and abscisic acid (Gibbs et al., 2015). BERF1 is expressed in barley roots, upregulated in barley leaves exposed to ethylene (Osnato et al., 2010) and was upregulated during RCS (Fig. 2D). Another gene involved in ethylene signalling, HvRAF, encodes an ERF-type transcription factor with roles in regulating abiotic and biotic stress. In barley, HvRAF transcripts were more abundant in roots compared with leaves, and various treatments, including the ethylene-generating compound ethephon, could induce expression in seedlings (Jung et al., 2007). In maize, the closest orthologous gene of HvRAF (GRMZM2G053503, ERF-like 1, ZM08G07220) was expressed 4.3 times more highly in waterlogged conditions (which induced aerenchyma formation) compared with waterlogged conditions plus 1-MCP treatment, which reduced, but did not totally suppress, aerenchyma formation (Rajhi et al., 2011; Yamauchi et al., 2016). This is similar to the 5.8 times greater expression of the HvRAF gene in barley segments undergoing RCS compared with areas of the root with no RCS. Of the four ethylene-related genes evaluated in the current study, two gene orthologues (RAF and ACO7) were shown to be significantly upregulated during RCA formation in maize (Rajhi et al., 2011). This is evidence that RCS is regulated by ethylene and that RCS and RCA may be regulated by common endogenous, genetic or environmental cues. It has been suggested that different environmental factors (e.g. hypoxia, UV light or disease) induce PCD via common endogenous mechanisms (Deacon et al., 1986). The development of RCS and RCA has been shown to be accelerated by mineral nutrient deficiencies (Gillespie and Deacon, 1988; Drew et al., 1989; Elliott et al., 1993; Schneider et al., 2017b). With low phosphorus or nitrogen availability, ethylene production and the ACC synthase and ACC oxidase content and activity in excised apical segments of maize roots were decreased, indicating that nutrient deficiencies may slow the biosynthetic pathway of ethylene (Drew et al., 1989). Authors of previous studies in maize have suggested that the increased formation of RCA in low nutrient conditions is related to increased ethylene sensitivity of root tissue (Drew et al., 1989; He et al., 1992). In our study, RCS showed a greater response to ethylene in control conditions compared with nutrient stress conditions (Fig. 1) which suggests that accelerated RCS in low nutrient conditions is not due to increased ethylene sensitivity. Instead, increased RCS with low nitrogen and low phosphorus coincided with upregulation of ethylene synthesis and signalling genes. This study supports involvement of ethylene synthesis and signalling in RCS at the transcript level, and the lack of difference in ethylene sensitivity in low nutrient conditions suggests that these pathways are important for the greater extent of RCS with sub-optimal nutrient availability. Programmed cell death describes genetically controlled and ordered death and lysis of cells (Gunawardena et al., 2001). Ethylene alone does not induce PCD; however, it is considered to be a modulator of the cell death process (Greenberg, 1997; Drew et al., 2000). Evidence for a regulatory role for ethylene has been shown for several types of PCD (Orzaez and Granell, 1997b; Young et al., 1997; De Jong et al., 2002). RCS is considered to be an example of PCD with mechanistic similarities to apoptosis. Seminal root segments of older barley plants had more extensive DNA fragmentation compared with root segments of younger plants, coinciding with the progression of RCS (Liljeroth and Bryngelsson, 2001). DNA fragmentation is characteristic of PCD (Domínguez et al., 2018). Ethylene may regulate RCS through modulation of PCD or additional, unknown mechanisms. During RCS, DAD1 is significantly downregulated (Fig. 5A). It has been suggested that DAD1 is a universal negative regulator of PCD (Moharikar et al., 2007), and downregulation of DAD1 has been linked to PCD in animals and plants (Nakashima et al., 1993; Gallois et al., 1997; Tanaka et al., 1997). DAD1 interacts with MCL1, a member of the BCL2 protein family (notable for their regulation of apoptosis at the mitochondrion), suggesting a role for DAD1 in apoptotic death (Makishima et al., 2000). Previous studies have demonstrated that expression of DAD1 is suppressed upon the onset of DNA fragmentation during several types of PCD in plant tissues (Orzaez and Granell, 1997a, b; Lindholm et al., 2000; Yamada et al., 2004; Adamakis et al., 2011). The DAD1 homologue in rice can rescue temperature-sensitive dad1 mutants from apoptotic death, suggesting that it functions as a suppressor of PCD (Tanaka et al., 1997). In addition, in heat-stressed arabidopsis roots, a DAD1-overexpressing line had reduced apoptotic-like PCD in root hairs (Hogg et al., 2011) and overexpression of DAD1 in arabidopsis can suppress DNA fragmentation and slow the progression of UV-B-induced cell death (Danon et al., 2004). Other studies have suggested that DAD1 is not an early regulator of PCD, but rather may be required for the programmed dismantling of cells, since decreases in expression in carnation and iris leaves were observed just prior to or concomitant with cell death (van der Kop et al., 2003). The downregulation of DAD1 in low phosphorus and nitrogen treatments, coinciding with the onset of RCS (in mid-root segments), indicates that RCS is a type of apoptotic-like PCD. Another regulator of PCD, DAD2, was significantly upregulated in root segments beginning RCS, especially in low phosphorus treatments (Fig. 5B). DAD2 increases in germinating barley scutella before the onset of DNA fragmentation (Lindholm et al., 2000), and was highly expressed during the hypersensitive response in wheat upon exposure to wheat stripe rust, indicating that it may play a role in modulating the process of PCD (Ma et al., 2009). We demonstrate that ethylene is associated with the development of RCS. RCS and RCA are distinct processes in terms of development and spatial pattern of cell death, but share some common mechanisms including the involvement of typical PCD processes. In the present study, RCA formation was rarely observed and RCS was observed in every root examined at 35 d of growth (Lascaris and Deacon, 1991; Lenochová et al., 2009; this study). 1-MCP exposure in the absence of ethylene reduced aerenchyma formation up to 80 % in maize (Fan et al., 2003; Hu et al., 2014); however, it did not reduce RCS in barley. Further studies are needed in order to examine further physiological, genetic and functional similarities and differences between RCS and RCA including the molecular mechanisms controlling these phenes. These results support the hypothesis that ethylene enhances, but is not required for, RCS, i.e. RCS is not regulated by basal endogenous production of ethylene; however, increases in ethylene increase RCS. Understanding the mechanisms and the role of ethylene and PCD in RCS has important implications for breeding and understanding the development of root anatomical traits. Ethylene signalling may serve to modulate the extent and timing of RCS so that root function and metabolic cost can be adjusted in response to environmental conditions such as nutrient deficiencies. The predictable patterns in RCS distribution would permit breeding efforts utilizing RCS for increased edaphic stress tolerance (Schneider et al., 2017b). Genetic variation exists in RCS (Liljeroth, 1995; Schneider et al., 2017b) which could be exploited in breeding programmes. Further insights into the regulation of RCS will enable further evaluation of the benefits and costs of RCS. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: plant growth parameters from four barley genotypes grown in solution culture. 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Ethylene modulates root cortical senescence in barley

Annals of Botany , Volume Advance Article (1) – Apr 20, 2018

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Abstract

Abstract Background and Aims Root cortical senescence (RCS) is a poorly understood phenomenon with implications for adaptation to edaphic stress. It was hypothesized that RCS in barley (Hordeum vulgare L.) is (1) accelerated by exogenous ethylene exposure; (2) accompanied by differential expression of ethylene synthesis and signalling genes; and (3) associated with differential expression of programmed cell death (PCD) genes. Methods Gene expression of root segments from four barley genotypes with and without RCS was evaluated using quantitative real-time PCR (qRT-PCR). The progression of RCS was manipulated with root zone ethylene and ethylene inhibitor applications. Key Results The results demonstrate that ethylene modulates RCS. Four genes related to ethylene synthesis and signalling were upregulated during RCS in optimal, low nitrogen and low phosphorus nutrient regimes. RCS was accelerated by root zone ethylene treatment, and this effect was reversed by an ethylene action inhibitor. Roots treated with exogenous ethylene had 35 and 46 % more cortical senescence compared with the control aeration treatment in seminal and nodal roots, respectively. RCS was correlated with expression of two genes related to programmed cell death (PCD). Conclusions The development of RCS is similar to root cortical aerenchyma formation with respect to ethylene modulation of the PCD process. Barley, ethylene, Hordeum vulgare, programmed cell death, root, root cortical senescence INTRODUCTION Root cortical senescence (RCS) is an anatomical phene with potential for improving plant performance and soil resource acquisition efficiency in edaphic stress conditions (Schneider and Lynch, 2017; Schneider et al., 2017a, b). RCS begins in the outer cortical cell files and progresses inwards, increasing in frequency basipetally (Schneider et al., 2017b), and is distinct from the loss of the cortex due to secondary root growth in many dicotyledonous plants (Bingham, 2007). RCS has only been identified in monocots, including several important agronomic species such as barley (Hordeum vulgare), wheat (Triticum aestivum), triticale (Triticosecale) (Yeates and Parker, 1985; Liljeroth, 1995), rye (Secale cereale) (Deacon and Mitchell, 1985; Jupp and Newman, 1987) and oat (Avena sativa) (Yeates and Parker, 1985). Variation in the extent of RCS can be affected by factors such as species (Deacon and Mitchell, 1985), genotype (Liljeroth, 1995), root class and position along the root axis (Schneider et al., 2017b). While to our knowledge RCS occurs in all barley cultivars regardless of growth conditions, the rate of RCS is enhanced by abiotic edaphic stresses including low availabilities of nitrogen and phosphorus (Schneider et al., 2017b). The senescence of cortical cells during RCS causes significant physiological and morphological changes in plants, since root anatomical traits directly affect the cost and physiological functions of roots (Marschner, 1995; Tombesi et al., 2010; Chimungu et al., 2014a, b; Lynch et al., 2014; Saengwilai et al., 2014). RCS and root cortical aerenchyma (RCA), another phene involving cortical cell lysis, influence physiological processes central to plant function including reducing radial nutrient transport (Hu et al., 2014; Schneider et al., 2017b), reducing radial hydraulic conductivity (Fan et al., 2007; Schneider et al., 2017b) and, exclusively in RCS, increasing aliphatic suberin in the endodermis (Schneider et al., 2017b). Many of these physiological changes are due to modifications in cellular organization and barriers between symplastic and apoplastic spaces, affecting the kinetics and radial pathways of nutrient and water transport (Peterson and Perumalla, 1984; Marschner, 1995; Steudle and Peterson, 1998). We have proposed that RCS has physiological implications similar to RCA formation. RCA and RCS both reduce root metabolic costs (Zhu et al., 2010; Saengwilai et al., 2014; Schneider et al., 2017b), reduce radial water and nutrient transport (Hu et al., 2014; Schneider et al., 2017b), are accelerated by mineral nutrient deficiencies (Gillespie and Deacon, 1988; Drew et al., 1989; Elliott et al., 1993; Saengwilai et al., 2014; Schneider et al., 2017b; Galindo-Castañeda et al., 2018) and improve plant growth in edaphic stress conditions (Postma and Lynch, 2011; Saengwilai et al., 2014; Chimungu et al., 2015; Schneider et al., 2017a). However, the mechansisms of RCA formation have been extensively studied, while very little information is available on RCS. We will gain insights into and a better understanding of RCS by examining whether known mechanisms of RCA formation are involved in RCS. Despite the similarities, RCS is a phenomenon that is distinct from RCA formation (Deacon et al., 1986). The formation of RCA begins in the mid-cortex and progresses radially, leaving spokes of connected cortical tissue (Kawai et al., 1998; Lenochová et al., 2009), in contrast to RCS which consistently begins in the outer cortical cell files and progresses inward towards the endodermis (Holden, 1975; Henry and Deacon, 1981). RCS seems to be constitutive and its development is fairly limited to small temperate grains including barley, wheat, rye, oat and triticale (Deacon and Mitchell, 1985; Yeates and Parker, 1985; Jupp and Newman, 1987; Liljeroth, 1995). RCA is formed by many species, including those which also develop RCS such as barley and wheat. Root cortical aerenchyma formation is a type of programmed cell death (PCD) (Drew et al., 2000; Gunawardena et al., 2001) and it has been suggested that RCS may also be a type of PCD (Liljeroth and Bryngelsson, 2001). RCS develops at the same rate in sterile and non-sterile conditions and is not enhanced by the presence of soil micro-organisms (Henry and Deacon, 1981; Lewis and Deacon, 1982). RCS has apparent mechanistic similarities to apoptosis and forms in a predictable pattern in nodal and seminal roots (Schneider et al., 2017b). DNA fragmentation, an indicator of PCD, occurred in wheat and barley seminal roots coinciding with the progression of RCS in wheat as assessed by histological methods (Henry and Deacon, 1981; Liljeroth and Bryngelsson, 2001; Deng et al., 2010). Further evidence is needed in order to confirm that RCS is formed through PCD processes. Ethylene regulates many plant processes including PCD and the development of anatomical and architectural root traits (Tanimoto et al., 1995; He et al., 1996b; Borch et al., 1999; Fan et al., 2003; Zhang et al., 2003; Dahleen et al., 2012). PCD processes including petal senescence (Wagstaff et al., 2005), emergence of adventitious roots in rice (Steffens and Rasmussen, 2016) and the hypersensitive response to bacterial leaf spot in tomato (Ciardi et al., 2001) are all regulated by ethylene. A recent study demonstrated that >200 ethylene-related genes were differentially expressed during RCA formation, with ethylene perception occurring in root cortical and stellar cells (Takahashi et al., 2015). Similar to RCA formation, ethylene has been suggested to regulate the rate of RCS (Lascaris and Deacon, 1991). Cobalt, a known inhibitor of ethylene synthesis (Lau and Yang, 1976; Yu and Yang, 1979), reduced the rate of RCS in root pieces on an agar medium (Lascaris and Deacon, 1991). AgNO3, an early inhibitor of ethylene action (Beyer, 1976), inhibited the formation of RCA induced by ethylene or oxygen deficiency (Drew et al., 1981); however, at concentrations that inhibit RCA formation, the rate of RCS was unaffected in root segments on an agar medium (Lascaris and Deacon, 1991). However, cobalt and silver are old inhibitors and probably have non-specific effects. Since the role of ethylene in RCS remains unclear, we examined the role of ethylene in RCS in intact plants. We hypothesize that (1) exogenous ethylene exposure accelerates RCS; (2) ethylene-related genes are differentially expressed during RCS; and (3) PCD genes are differentially expressed during RCS. RCS was manipulated by using genotypic contrasts and nutrient deficiency in addition to treatments expected to alter ethylene responses. Understanding the role of ethylene in RCS is important in order to gain insights into the genetic regulation and adaptive significance of RCS. MATERIALS AND METHODS Plant materials and growing system Four barley (Hordeum vulgare L.) genotypes (landraces Nuernberg and Tkn24b, and modern varieties Arena and Golf) were selected for this study based on contrasting RCS (Schneider et al., 2017b). Landraces have accelerated RCS compared with modern varieties (Schneider et al., 2017b). Seeds were obtained from IPK, Gatersleben, Germany. Seeds were surface-sterilized in 1.5 % NaOCl in water, rolled into tubes of germination paper (76 lb, Anchor Paper, St Paul, MN, USA) and placed for 4 d in covered beakers containing 0.5 mm CaSO4 in a dark climate chamber at 28 °C. The beakers containing germinated seedlings were then placed under a constant fluorescent light (350 µmol photons m–2 s–1) at 28 °C for 1 d in a climate chamber before transplanting into aerated solution culture. Six randomly assigned seedlings were transplanted in foam plugs suspended above each 33 L solution culture tank. Plants were grown for 30 d in a climate chamber [22 °C, 12 h daylight (Agro high pressure sodium lamp), 50 % relative humidity]. The nutrient solution (pH 5.5, adjusted daily with KOH) was completely replaced every 7 d. The control nutrient solution contained 2.5 mm KNO3, 2.5 mm Ca(NO3)2, 1 mm MgSO4, 0.5 mm KH2PO4, 50 µm NaFeEDTA, 0.2 µm Na2MoO4, 10 µm H3BO3, 0.2 µm NiSO4, 1 µm ZnSO4, 2 µm MnCl2, 0.5 µm CuSO4 and 0.2 µm CoCl2. The solution was modified for two additional treatments (1) low nitrogen [200 µmol N L–1, KNO3 and Ca(NO3)2 were reduced from the nutrient solution and supplemented with K2SO4 and CaCl] and (2) low alumina-buffered phosphorus (buffered at 2 µmol P L–1, omitting KH2PO4) (Lynch et al., 1990). Ethylene treatment Barley plants were grown in optimal, low nitrogen and low phosphorus conditions in solution culture (as described above) with the addition of four aeration treatments for each nutrient condition to manipulate RCS. Treatments comprised (1) root zone air (control); (2) root zone ethylene; (3) root zone 1-MCP (1-methylcyclopropene, an ethylene inhibitor, approx. 3.8 % active ingredient, AgroFresh, Germany); and (4) combined root zone ethylene and 1-MCP application, all applied continuously beginning at seedling transfer to solution culture. Solution culture tanks in the control nutrient treatment were bubbled at 30 mL min–1 with ambient air in 99 L (three tanks of 33 L each) of solution culture. In the ethylene treatment, compressed ethylene (1 µL L–1 in air, as used by Gunawardena et al., 2001) was bubbled through 99 L (three tanks of 33 L each) of solution culture at 30 mL min–1. For the 1-MCP treatment, 1-MCP was volatilized by dissolving 0.17 g in 5 mL of water in a glass scintillation vial, and then transferred into a 2 L sidearm flask. An open-cell foam plug enclosed the mouth of the flask, and the headspace containing 1-MCP gas was bubbled through 99 L (three tanks of 33 L each) of solution culture using an air pump fitted with a one-way check valve. The inlet tubing was connected to the sidearm flask and the outlet tubing was connected to air diffusers submerged in solution culture tanks. The 1-MCP was bubbled at a rate of 30 mL min–1 to achieve a 1-MCP concentration of 7.7 µL L–1 solution, assuming all 1-MCP dissolved in solution. The air pump ran continuously, and the 1-MCP was replenished daily into the sidearm flask. For the combined ethylene and 1-MCP treatment, compressed ethylene was bubbled through 99 L (three tanks of 33 L each) of solution culture at 30 mL min–1 and 1-MCP gas was bubbled at a rate of 30 mL min–1 in the same tank. The ambient air in the climate chamber was circulated and completely exchanged every 10 min; therefore, no contamination between treatments was present. Harvest and sampling After 35 d of growth, root samples were harvested in four replications per genotype and treatment for ethylene experiments. For each root, one sample position was collected. Segments of 1 cm from five positions on nodal roots (2, 3, 8, 13 and 18 cm from the apex) and from ten positions on seminal roots (2, 6, 12, 18, 24, 30, 36, 42, 48 and 54 cm from the apex) were preserved in 70 % ethanol for acridine orange staining and anatomical analysis. At 35 d after germination (vegetative growth, tillering stage), barley plants grown in optimal, low nitrogen and low phosphorus conditions in the control aeration treatment were harvested from solution culture and root segments were sampled for qRT-PCR (quantitative real-time PCR) studies and anatomical analysis. Five representative seminal and nodal roots from each plant were excised and their lengths were measured. Three locations on each root were selected for gene expression studies based on the degree of RCS [3–4 cm (distal, no RCS), 8–9 cm (mid-root, partial RCS) and 34–36 cm (basal, complete cortical senescence) from the root apex]. Although root elongation rates were reduced in sub-optimal nutrient availability, previous studies have demonstrated that minor differences in elongation rates and root length had no significant effect on the quantification of RCS compared with absolute distances from the root apex (Schneider et al., 2017b). Root segments for each position were bulked per plant and were immediately contained in envelopes of aluminium foil and suspended in liquid nitrogen. A 1 cm segment directly basally adjacent to each sample position and a 1 cm segment at 2 cm from the root apex were collected for each root sampled and preserved in 70 % ethanol for acridine orange staining and anatomical analysis. Samples for anatomical analysis and qRT-PCR were collected from four replicate plants per genotype and treatment. Shoot dry biomass, root system dry biomass, tiller number and total root system length of plants were collected from four replicate plants per genotype and treatment for all experiments. Root length was measured by scanning and analysing whole root systems using WinRHIZO Pro (Régent 389 Instruments, Québec City, Québec, Canada). The ethylene and 1-MCP root zone treatments had no significant effect on root elongation rates (Supplementary DataTable S1). In addition to absolute distance from the apex, RCS was also evaluated based on the relative distance to the root apex, with similar results (data not shown). Gene expression Samples suspended in liquid nitrogen were transferred to the laboratory where they were ground with a pestle and mortar while kept frozen with a continuous application of liquid nitrogen. For every sample, 100 mg of ground sample was transferred to a pre-cooled 2 mL epitube and kept frozen at –80 °C until RNA extraction. RNA extraction was performed with the RNeasy Plant Mini Kit (Qiagen, Hildesheim, Germany) and cDNA was transcribed with the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). The qRT-PCR was performed with iQ SYBR Green Supermix (BioRad) according to the manufacturer’s instructions. Gene expression of four ethylene-related genes, Root Abundant Factor (HvRAF), Ethylene Response Factor 1 (BERF1), ACC (aminocyclopropane-1-carboxylic acid) synthase (HvACS6b) and ACC oxidase (HvACO7), and two genes related to PCD, Defender Against Death 2 (DAD2) and Defender Against Death 1 (DAD1) were evaluated. Ethylene-related genes were selected based on involvement in ethylene synthesis or signalling, annotation in barley, expression in barley roots and known involvement in PCD and hypoxia tolerance. PCD-related genes were selected based on their involvement in PCD and annotation in barley. Primers used for amplification are described in the supplementary material, and are unique to each gene target within a gene family and conserved among plant genotypes (Supplementary DataTable S2). For normalization, endogenous reference genes, 26S rRNA and ubiquitin, were averaged and used as internal standards. The PCR cycle conditions were: 95 °C for 4 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and at 72 °C for 30 s. Subsequently, melting curves were recorded to detect non-specific amplification by increasing the temperature from 60 to 95 °C in 0.5 °C increments. Semi-quantitative PCR was performed by 40 cycles of amplification (95 °C for 5 min, 58 °C for 1 min, 72 °C for 1 min). Anatomical characterization Root segments in 70 % ethanol were stained with acridine orange according to Henry and Deacon (1981) except that the staining time was extended to 30 min. Acridine orange-stained root segments were embedded with Tissue-Tek CRYO-OCT compound (Thermo Fisher Scientific, Waltham, MA, USA) in a gelatin capsule and frozen at –20 °C for 15 min. Transverse sections 60 µm thick were cut using a Kryostat 2800 Frigocut –E (Reichert-Jung, Leica Instruments GmbH, Nussloch, Germany) and sections were immediately transferred to a microscope slide and imaged. Images of root cross-sections were acquired on a compound microscope (Zeiss Axioplan 2, mounted with an AxioCam ICc 5, Filter 09: Blue 450–490 nm Carl Zeiss Jena GmbH, Jena, Germany; ×20 magnification). Cross-sectional images were phenotyped for root cross-sectional area and area of the stele using ImageJ (Rasband, 2015). Root segments collected at 2 cm from the root apex served as a reference for root cortical area in regions with no RCS on the same root. The percentage of the cortex senesced was calculated based on the difference in cortical cross-sectional areas (total cross-sectional area minus stele area) at 2 cm from the root apex and at the sample position. The shrinkage of the root cortex, compared with the cortical area at the root apex, is a direct indicator of RCS (Schneider et al., 2017b). Statistical methods Data were analysed by Tukey’s HSD (honest significant difference) using R 3.1.2 (R Core Team, 2014). For gene expression experiments, data analysis and relative expression was calculated using the Relative Expression Software Tool (REST) (Pfaffl et al., 2002). RESULTS Ethylene application significantly accelerated RCS development (Fig. 1). In seminal roots of modern cultivars and landraces grown in optimal nutrient conditions, root segments with ethylene treatment increased RCS (i.e. a greater percentage of cortical area senesced) by 52 % at 6 cm from the root apex, 33 % at 12 cm from the root apex and 16 % at 18 cm from the root apex compared with the control aeration treatment (Fig. 1A, B). As previously reported, low nitrogen and low phosphorus conditions accelerated RCS (Schneider et al., 2017b) (Fig. 1E–H). With low nitrogen and phosphorus availability, ethylene treatment increased RCS by 30 % at 6 cm from the root apex, 24 % at 12 cm from the root apex and 16 % at 18 cm from the root apex compared with the control aeration treatment (Fig. 1E, F). In all nutrient regimes, at 24 cm from the root apex, RCS had progressed to 70 % of the cortex senesced and there were no significant differences in RCS development in the ethylene-treated roots compared with the control or 1-MCP aeration treatment. The ethylene inhibitor 1-MCP completely reversed the ethylene effect but did not reduce RCS in the absence of exogenous ethylene. In all nutrient regimes, no significant differences were observed in RCS between the 1-MCP, control (air) and the 1-MCP plus ethylene treatments (Fig. 1A, B, E, F). Fig. 1. View largeDownload slide The progression of RCS in plants grown with optimal nutrient availability in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. Progression of RCS in plants grown in low nitrogen and low phosphorus treatments in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. No differences in RCS were observed in low nitrogen and phosphorus treatments, so data were combined for analysis. Points show the means of four replicates for each of two genotypes ± s.e. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. Fig. 1. View largeDownload slide The progression of RCS in plants grown with optimal nutrient availability in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. Progression of RCS in plants grown in low nitrogen and low phosphorus treatments in control (air), ethylene and 1-MCP aeration treatments in barley seminal and nodal roots of modern cultivars and landraces. No differences in RCS were observed in low nitrogen and phosphorus treatments, so data were combined for analysis. Points show the means of four replicates for each of two genotypes ± s.e. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. In optimal nutrient conditions, nodal root segments of modern cultivars and landraces exposed to ethylene had 61 % more cortex senesced at 3 cm from the root apex, 41 % more cortex senesced at 8 cm from the root apex, 34 % more cortex senesced at 13 cm from the root apex and 19 % more cortex senesced from the root apex compared with the control aeration treatment (Fig. 1C, D). With low nitrogen and phosphorus availability, nodal root segments exposed to ethylene had 34 % more RCS at 3 cm from the root apex, 29 % more RCS at 8 cm from the root apex, 19 % more RCS at 13 cm from the root apex and 12% more RCS at 18 cm from the root apex compared with the aeration treatment (Fig. 1G, H). As with seminal roots, in all nutrient regimes the degree of RCS development was not significantly different between the 1-MCP, control (air) and 1-MCP plus ethylene treatments, but nodal roots showed a greater separation between ethylene-treated roots and those of the other treatments, and therefore may be more sensitive to ethylene when compared with seminal roots (Fig. 1). Ethylene treatment increased the rate of RCS on the entire root, but the ethylene and/or 1-MCP treatments did not affect shoot dry weight, tiller number, root dry weight and total root length in all nutrient regimes (Supplementary DataTable S1). Four genes related to ethylene synthesis and signalling were upregulated in the mid-root segments where RCS was ongoing, an effect that was enhanced by low nitrogen and low phosphorus treatments (Fig. 2). Expression of all four genes was very low in basal root segments, which had maximal RCS (90–100 % cortical senescence, complete senescence of the cortex), and in distal root segments, which had no RCS (Figs 3 and 4). In optimal nutrient conditions, HvACO7, a gene that encodes an enzyme involved in the synthesis of ethylene, was upregulated 1.8 log-fold (P < 0.001) in mid-root segments, where RCS is ongoing, compared with distal root segments with no RCS. HvACS6b, a gene that encodes an enzyme involved in the synthesis of ethylene, was upregulated 1.7 log-fold (P < 0.001) in mid-root segments compared with distal root segments. An ethylene response transcription factor gene, BERF1, was upregulated 2.9 log-fold (P < 0.001) in mid-root segments compared with distal root segments. HvRAF, encoding an ethylene response factor, was upregulated 2 log-fold (P < 0.001) in mid-root sections compared with distal sections (Fig. 2). No differences in gene expression were observed between nodal and seminal roots, among genotypes (data not shown), or between distal and basal root segments (Fig. 2). Fig. 2. View largeDownload slide Expression of ethylene-related genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments had no RCS, mid-root segments had partial RCS, and basal root segments had complete cortical senescence. (A) HvACS6, (B) HvRAF, (C) HvACO7 and (D) BERF1 were significantly upregulated in mid-root sections compared with distal and basal root segments. In mid-root segments, HvACO7 and HvRAF were significantly upregulated in low nitrogen compared with optimal nutrient conditions. In low phosphorus, expression of HvACO7 was significantly upregulated in mid-root segments compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Relative expression is the ratio of expression of the target gene compared with the reference genes. Fig. 2. View largeDownload slide Expression of ethylene-related genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments had no RCS, mid-root segments had partial RCS, and basal root segments had complete cortical senescence. (A) HvACS6, (B) HvRAF, (C) HvACO7 and (D) BERF1 were significantly upregulated in mid-root sections compared with distal and basal root segments. In mid-root segments, HvACO7 and HvRAF were significantly upregulated in low nitrogen compared with optimal nutrient conditions. In low phosphorus, expression of HvACO7 was significantly upregulated in mid-root segments compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Relative expression is the ratio of expression of the target gene compared with the reference genes. Fig. 3. View largeDownload slide Cross-sections of barley seminal root stained with acridine orange. (A) Root cross-section from a plant grown in optimal nutrient conditions with no RCS from the distal portion of the root (3 cm from the apex). (B) Root cross-section from a plant grown in optimal nutrient conditions with partial RCS from the mid-root section (8 cm from the apex). (C) Root cross-section from a plant grown in optimal nutrient conditions with maximal RCS from the basal portion of the root (34 cm from the apex). (D) Root cross-section from an ethylene-treated plant with partial RCS from the mid-root section. (E) Root cross-section from a 1-MCP-treated plant with partial RCS from the mid-root section. Scale bar =100 µm. Fig. 3. View largeDownload slide Cross-sections of barley seminal root stained with acridine orange. (A) Root cross-section from a plant grown in optimal nutrient conditions with no RCS from the distal portion of the root (3 cm from the apex). (B) Root cross-section from a plant grown in optimal nutrient conditions with partial RCS from the mid-root section (8 cm from the apex). (C) Root cross-section from a plant grown in optimal nutrient conditions with maximal RCS from the basal portion of the root (34 cm from the apex). (D) Root cross-section from an ethylene-treated plant with partial RCS from the mid-root section. (E) Root cross-section from a 1-MCP-treated plant with partial RCS from the mid-root section. Scale bar =100 µm. Fig. 4. View largeDownload slide RCS in optimal, low nitrogen and low phosphorus treatments in (A) seminal roots of modern cultivars, (B) nodal roots of modern cultivars, (C) seminal roots of landraces and (D) nodal roots of landraces. In seminal and nodal roots of modern cultivars and landraces, mid-root segments in low nitrogen and phosphorus conditions had significantly greater RCS compared with optimal nutrient conditions. Distal segments (no RCS) are 3–4 cm from the root apex, mid-root segments (RCS forming) are 8–9 cm from the apex and basal segments (complete RCS) are 34–46 cm from the apex. Points show the means of four replicates for each of two genotypes ± s.e. since no significant differences within the landraces or modern cultivar classes were observed. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Fig. 4. View largeDownload slide RCS in optimal, low nitrogen and low phosphorus treatments in (A) seminal roots of modern cultivars, (B) nodal roots of modern cultivars, (C) seminal roots of landraces and (D) nodal roots of landraces. In seminal and nodal roots of modern cultivars and landraces, mid-root segments in low nitrogen and phosphorus conditions had significantly greater RCS compared with optimal nutrient conditions. Distal segments (no RCS) are 3–4 cm from the root apex, mid-root segments (RCS forming) are 8–9 cm from the apex and basal segments (complete RCS) are 34–46 cm from the apex. Points show the means of four replicates for each of two genotypes ± s.e. since no significant differences within the landraces or modern cultivar classes were observed. RCS was quantified by disappearance of the cortex compared with a non-senesced root segment on the same root. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). In low nitrogen treatments, RCS was accelerated in mid-root segments by 28 % (i.e. a greater percentage of cortical area senesced) (P < 0.001) in modern cultivars (Fig. 4A, B) and 43 % (P < 0.001) in landraces compared with optimal nutrient conditions (Fig. 4C, D). Increased expression of ethylene-related genes coincided with the increase in RCS. Distal root segments had no RCS (Fig. 4). Mid-root segments of landraces had 47 % of the cortex senesced, which was significantly greater than the 38 % of the cortex senesced in mid-root segments of modern cultivars. Basal root segments had maximal cortical senescence (Figs 3 and 4). HvACO7, HvACS6b, BERF1 and HvRAF were upregulated 2.2, 2.5, 3.9 and 2.5 log-fold (P < 0.001), respectively, in mid-root segments undergoing RCS compared with distal root segments with no RCS (Fig. 2). No significant differences in gene expression were observed between distal and basal root segments. Plants grown in low nitrogen conditions had significantly greater HvACO7 and HvRAF expression in mid-root segments compared with mid-root segments of plants grown in optimal nutrient conditions, but expression of HvACS6b and BERF1 in mid-root sections was not significantly different from that of mid-root segments of plants grown in optimal nutrient conditions (Fig. 2). Nodal and seminal roots and different genotypes within these root classes had no significant differences in gene expression (data not shown). In roots grown under low phosphorus availability, RCS was accelerated in mid-root segments by 37 % (P < 0.001) in modern cultivars and 46 % (P < 0.001) in landraces compared with optimal nutrient conditions (Fig. 4). Distal root segments had no RCS, mid-root segments had 43 % of the cortex senesced in modern cultivars and 46 % cortical senescence in landraces, and basal root segments had maximal RCS. Landraces had significantly greater RCS in mid-root segments compared with modern cultivars. HvACO7, HvACS6b, BERF1 and HvRAF were upregulated 2, 2, 2 and 3 log-fold (P < 0.001), respectively, in mid-root segments undergoing RCS compared with distal root segments with no RCS (Fig. 2). In low phosphorus conditions, expression of HvACO7 was significantly upregulated in mid-root segments compared with optimal nutrient conditions. Although mean gene expression values of HvACS6b, BERF1 and HvRAF appeared higher in mid-root sections of plants grown in low phosphorus, variability was also high, so they did not have significantly different expression compared with mid-root segments of plants grown in optimal nutrient conditions. No significant differences in gene expression were observed between nodal and seminal roots or among genotypes within or between the modern cultivar and landrace classes (data not shown). Gene expression was not significantly different between distal and basal root segments (Fig. 2). Low nitrogen and low phosphorus conditions reduced shoot dry biomass by 32 % (Supplementary DataTable S1). Landraces (Nuernberg and Tkn24b) had significantly greater tiller number compared with modern cultivars (Arena and Golf) in control (air), ethylene, 1-MCP and ethylene plus 1-MCP aeration treatments. Significantly greater tiller numbers corresponded to significantly greater shoot dry biomass. No significant differences in the number of tillers or shoot dry biomass were observed among genotypes in low nitrogen or low phosphorus conditions. No significant differences were observed among genotypes or treatments in root dry biomass or total root length (Supplementary Data Table S1). Programmed cell death-related genes were differentially expressed during RCS. One gene indicating PCD, DAD2, was significantly upregulated in root segments beginning RCS compared with root segments with complete senescence or no senescence. In mid-root segments grown in optimal nutrient conditions, DAD2 was upregulated 2 log-fold (P < 0.001) compared with distal and basal root segments. In mid-root segments grown in low nitrogen conditions, DAD2 was upregulated 3.2 log-fold (P < 0.001) compared with distal and basal root segments, and expression was not significantly different compared with optimal nutrient conditions. In mid-root segments grown in low phosphorus conditions, DAD2 was upregulated 3.1 log-fold (P < 0.001) compared with distal and basal root segments and upregulated 2.7 log-fold (P < 0.001) compared with optimal nutrient conditions (Fig. 5B). Another gene indicating PCD, DAD1, was significantly downregulated in root segments with any degree of RCS. In mid-root and basal root segments, DAD1 had a –3.5, –3 and –3.3 log-fold decline (P < 0.001) compared with distal root segments in optimal, low nitrogen and low phosphorus nutrient treatments, respectively (Fig. 5A). Variability of gene expression data was high and therefore no differences between genotypes and – in many cases – treatments were observed (Figs 2 and 5), although differences in RCS were observed between genotypes and treatments (Fig. 4). Fig. 5. View largeDownload slide Expression of PCD genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments (3–4 cm from root apex) had no RCS, mid-root segments (8–9 cm from the root apex) had partial RCS and basal root segments (34–36 cm from the root apex) had complete cortical senescence. (A) Relative expression of DAD1. Relative expression is the ratio of expression of the target gene compared with the reference genes. DAD1 was significantly downregulated in mid- and basal root segments compared with distal root segments. DAD1 was significantly upregulated in low nitrogen and low phosphorus conditions compared with optimal nutrient conditions. (B) Relative expression of DAD2. DAD2 was significantly upregulated in mid-root segments compared with distal root segments. DAD2 was significantly upregulated in low phosphorus conditions compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). Fig. 5. View largeDownload slide Expression of PCD genes in optimal, low nitrogen and low phosphorus conditions in root segments with different levels of RCS. Distal root segments (3–4 cm from root apex) had no RCS, mid-root segments (8–9 cm from the root apex) had partial RCS and basal root segments (34–36 cm from the root apex) had complete cortical senescence. (A) Relative expression of DAD1. Relative expression is the ratio of expression of the target gene compared with the reference genes. DAD1 was significantly downregulated in mid- and basal root segments compared with distal root segments. DAD1 was significantly upregulated in low nitrogen and low phosphorus conditions compared with optimal nutrient conditions. (B) Relative expression of DAD2. DAD2 was significantly upregulated in mid-root segments compared with distal root segments. DAD2 was significantly upregulated in low phosphorus conditions compared with optimal nutrient conditions. Points show the means of four replicates for each of four genotypes ± s.e. since no significant differences among genotypes were observed. Different letters represent RCS differences among distal, mid- and basal root segments in different nutrient treatments as determined by a Tukey’s test (P < 0.05). DISCUSSION Our results support the hypothesis that ethylene modulates, but is not required for, the development of RCS. In low nitrogen, low phosphorus and optimal nutrient regimes, RCS was accelerated by root zone ethylene treatment, an effect that was blocked by the ethylene inhibitor 1-MCP (Fig. 1). Genes involved in ethylene synthesis and signalling were significantly upregulated during RCS (Fig. 2). When RCS was manipulated by low nitrogen and low phosphorus treatments, differential expression of ethylene-related and PCD genes coincided with the distribution of RCS along the root axes in optimal nutrient, low nitrogen and low phosphorus treatments (Figs 2, 4 and 5). Although RCS development was accelerated by exogenous ethylene application, 1-MCP did not reduce RCS in the absence of ethylene. This suggests that ethylene action is not strictly required for RCS, but instead acts as a modulator of RCS initiation and progression. The 1-MCP concentration used here acted as an effective ethylene action inhibitor, since the treatment containing both ethylene and 1-MCP completely prevented the increase in RCS caused by ethylene treatment. The lack of reduction in RCS may not be due to the performance of the inhibitor, but rather to the limited action of ethylene in control conditions. Ethylene synthesis plays a role in RCS development, as shown by enhanced expression of ACC synthase and ACC oxidase genes in the mid-root segments during RCS (Fig. 2A, C). As the two enzymes limit ethylene synthesis, ACC oxidase and ACC synthase have known roles in root responses to stresses including hypoxia and nutrient stress (Olson et al., 1995; He et al., 1996a; Roldan et al., 2013). Upregulation of ACC synthase and oxidase genes has been associated with PCD responses, including petal senescence in tomato (Llop-Tous et al., 2000), hypoxia in maize (Geisler-Lee et al., 2010) and cell death of tomato leaves following ozone exposure (Moeder, 2002). The closest orthologous gene of HvACO7 in rice (OsACO7) (Dahleen et al., 2012) has been linked to increased ethylene biosynthesis associated with the hypersensitive response, another type of PCD (Iwai et al., 2006). Expression of HvACO7 was 213 times greater in root segments undergoing RCS compared with root segments with no RCS (Fig. 2C). In maize grown in waterlogged conditions, expression of the HvACO7 orthologue (GRMZM2G013448, 1-aminocyclopropane-1-carboxylate oxidase 1, ZM08G22670) was 19 times greater in hypoxic conditions (that induce aerenchyma formation) compared with hypoxic conditions plus 1-MCP treatment that reduced aerenchymya formation (Rajhi et al., 2011). The upregulation of ACC oxidase expression is another mechanism common to RCA in rice (Yamauchi et al., 2016) and RCS. The role of ethylene in RCS is further supported by the upregulation of ethylene response-related transcripts. BERF1 is a member of, and HvRAF is a close relative of, the group VII ERF subfamily (Hinz et al., 2010; Mendiondo et al., 2016). Group VII ethylene response factors are plant specific and play an important role in biotic and abiotic stress response and response to ethylene, gibberellin and abscisic acid (Gibbs et al., 2015). BERF1 is expressed in barley roots, upregulated in barley leaves exposed to ethylene (Osnato et al., 2010) and was upregulated during RCS (Fig. 2D). Another gene involved in ethylene signalling, HvRAF, encodes an ERF-type transcription factor with roles in regulating abiotic and biotic stress. In barley, HvRAF transcripts were more abundant in roots compared with leaves, and various treatments, including the ethylene-generating compound ethephon, could induce expression in seedlings (Jung et al., 2007). In maize, the closest orthologous gene of HvRAF (GRMZM2G053503, ERF-like 1, ZM08G07220) was expressed 4.3 times more highly in waterlogged conditions (which induced aerenchyma formation) compared with waterlogged conditions plus 1-MCP treatment, which reduced, but did not totally suppress, aerenchyma formation (Rajhi et al., 2011; Yamauchi et al., 2016). This is similar to the 5.8 times greater expression of the HvRAF gene in barley segments undergoing RCS compared with areas of the root with no RCS. Of the four ethylene-related genes evaluated in the current study, two gene orthologues (RAF and ACO7) were shown to be significantly upregulated during RCA formation in maize (Rajhi et al., 2011). This is evidence that RCS is regulated by ethylene and that RCS and RCA may be regulated by common endogenous, genetic or environmental cues. It has been suggested that different environmental factors (e.g. hypoxia, UV light or disease) induce PCD via common endogenous mechanisms (Deacon et al., 1986). The development of RCS and RCA has been shown to be accelerated by mineral nutrient deficiencies (Gillespie and Deacon, 1988; Drew et al., 1989; Elliott et al., 1993; Schneider et al., 2017b). With low phosphorus or nitrogen availability, ethylene production and the ACC synthase and ACC oxidase content and activity in excised apical segments of maize roots were decreased, indicating that nutrient deficiencies may slow the biosynthetic pathway of ethylene (Drew et al., 1989). Authors of previous studies in maize have suggested that the increased formation of RCA in low nutrient conditions is related to increased ethylene sensitivity of root tissue (Drew et al., 1989; He et al., 1992). In our study, RCS showed a greater response to ethylene in control conditions compared with nutrient stress conditions (Fig. 1) which suggests that accelerated RCS in low nutrient conditions is not due to increased ethylene sensitivity. Instead, increased RCS with low nitrogen and low phosphorus coincided with upregulation of ethylene synthesis and signalling genes. This study supports involvement of ethylene synthesis and signalling in RCS at the transcript level, and the lack of difference in ethylene sensitivity in low nutrient conditions suggests that these pathways are important for the greater extent of RCS with sub-optimal nutrient availability. Programmed cell death describes genetically controlled and ordered death and lysis of cells (Gunawardena et al., 2001). Ethylene alone does not induce PCD; however, it is considered to be a modulator of the cell death process (Greenberg, 1997; Drew et al., 2000). Evidence for a regulatory role for ethylene has been shown for several types of PCD (Orzaez and Granell, 1997b; Young et al., 1997; De Jong et al., 2002). RCS is considered to be an example of PCD with mechanistic similarities to apoptosis. Seminal root segments of older barley plants had more extensive DNA fragmentation compared with root segments of younger plants, coinciding with the progression of RCS (Liljeroth and Bryngelsson, 2001). DNA fragmentation is characteristic of PCD (Domínguez et al., 2018). Ethylene may regulate RCS through modulation of PCD or additional, unknown mechanisms. During RCS, DAD1 is significantly downregulated (Fig. 5A). It has been suggested that DAD1 is a universal negative regulator of PCD (Moharikar et al., 2007), and downregulation of DAD1 has been linked to PCD in animals and plants (Nakashima et al., 1993; Gallois et al., 1997; Tanaka et al., 1997). DAD1 interacts with MCL1, a member of the BCL2 protein family (notable for their regulation of apoptosis at the mitochondrion), suggesting a role for DAD1 in apoptotic death (Makishima et al., 2000). Previous studies have demonstrated that expression of DAD1 is suppressed upon the onset of DNA fragmentation during several types of PCD in plant tissues (Orzaez and Granell, 1997a, b; Lindholm et al., 2000; Yamada et al., 2004; Adamakis et al., 2011). The DAD1 homologue in rice can rescue temperature-sensitive dad1 mutants from apoptotic death, suggesting that it functions as a suppressor of PCD (Tanaka et al., 1997). In addition, in heat-stressed arabidopsis roots, a DAD1-overexpressing line had reduced apoptotic-like PCD in root hairs (Hogg et al., 2011) and overexpression of DAD1 in arabidopsis can suppress DNA fragmentation and slow the progression of UV-B-induced cell death (Danon et al., 2004). Other studies have suggested that DAD1 is not an early regulator of PCD, but rather may be required for the programmed dismantling of cells, since decreases in expression in carnation and iris leaves were observed just prior to or concomitant with cell death (van der Kop et al., 2003). The downregulation of DAD1 in low phosphorus and nitrogen treatments, coinciding with the onset of RCS (in mid-root segments), indicates that RCS is a type of apoptotic-like PCD. Another regulator of PCD, DAD2, was significantly upregulated in root segments beginning RCS, especially in low phosphorus treatments (Fig. 5B). DAD2 increases in germinating barley scutella before the onset of DNA fragmentation (Lindholm et al., 2000), and was highly expressed during the hypersensitive response in wheat upon exposure to wheat stripe rust, indicating that it may play a role in modulating the process of PCD (Ma et al., 2009). We demonstrate that ethylene is associated with the development of RCS. RCS and RCA are distinct processes in terms of development and spatial pattern of cell death, but share some common mechanisms including the involvement of typical PCD processes. In the present study, RCA formation was rarely observed and RCS was observed in every root examined at 35 d of growth (Lascaris and Deacon, 1991; Lenochová et al., 2009; this study). 1-MCP exposure in the absence of ethylene reduced aerenchyma formation up to 80 % in maize (Fan et al., 2003; Hu et al., 2014); however, it did not reduce RCS in barley. Further studies are needed in order to examine further physiological, genetic and functional similarities and differences between RCS and RCA including the molecular mechanisms controlling these phenes. These results support the hypothesis that ethylene enhances, but is not required for, RCS, i.e. RCS is not regulated by basal endogenous production of ethylene; however, increases in ethylene increase RCS. Understanding the mechanisms and the role of ethylene and PCD in RCS has important implications for breeding and understanding the development of root anatomical traits. Ethylene signalling may serve to modulate the extent and timing of RCS so that root function and metabolic cost can be adjusted in response to environmental conditions such as nutrient deficiencies. The predictable patterns in RCS distribution would permit breeding efforts utilizing RCS for increased edaphic stress tolerance (Schneider et al., 2017b). Genetic variation exists in RCS (Liljeroth, 1995; Schneider et al., 2017b) which could be exploited in breeding programmes. Further insights into the regulation of RCS will enable further evaluation of the benefits and costs of RCS. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: plant growth parameters from four barley genotypes grown in solution culture. 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Journal

Annals of BotanyOxford University Press

Published: Apr 20, 2018

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