Rapid Accumulation of Glutathione During Light Stress in Arabidopsis

Rapid Accumulation of Glutathione During Light Stress in Arabidopsis Abstract Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. We previously reported that in response to rapid changes in light intensity (0–60 s), Arabidopsis thaliana plants mount a large-scale transcriptomic response that includes several different transcripts essential for light stress acclimation. Here, we expand our analysis of the rapid response of Arabidopsis to light stress using a metabolomics approach and identify 111 metabolites that show a significant alteration in their level during the first 90 s of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is associated with an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in these responses. Introduction Light is the primary energy source for plants, as well as a key player in determining the plant’s diurnal cycles, growth, development and responses to different environmental stimuli. To optimize growth and development, and to avoid some of the damaging effects of excess light (Li et al. 2009, Takahashi and Badger 2011), plants need to constantly to adjust their metabolism and light-harvesting antennae in a process that is generally referred to as acclimation (Mullineaux et al. 2000, Kleine et al. 2007, Rossel et al. 2007, Galvez-Valdivieso et al. 2009, Gorecka et al. 2014, Dietz 2015, Oelze et al. 2012). Recent studies revealed that plant acclimation to light stress plays a major role in determining yield, as well as affecting crop productivity in the field (Mittler and Blumwald 2010, Kromdijk et al. 2016). In addition to diurnal and seasonal changes in light intensity and quality, under field conditions, plants are also subjected to random and rapid changes in light intensity and quality, sometimes referred to as sun flecks (Pastenes et al. 2005, Szechyńska-Hebda and Karpiński 2013, Raven 2014). These sudden changes in light intensity are primarily due to rapid changes in cloud cover or changes in canopy shading, and could result in enhanced photosynthesis or photoinhibition (Mittler and Blumwald 2010, Karpiński et al. 2013, Mittler, 2006). We recently reported on the molecular responses of Arabidopsis thaliana plants to rapid changes in light intensity, occurring within 0–90 s of plant exposure to high light stress (Suzuki et al. 2015). In that study, we identified a rapid transcriptomic response to light stress that included an ordered and clustered change in the steady-state level of 731 transcripts, occurring as early as 20–60 s following light stress application. We further determined that several of the transcripts identified by our analysis are required for plant acclimation to light stress, as well as demonstrating that at least some of the transcripts identified were transcriptionally regulated (Suzuki et al. 2015). Because many of the ultra-fast light stress response transcripts we identified in Arabidopsis were H2O2 or ABA response transcripts, we also studied the response of mutants impaired in ABA metabolism and sensing to light stress and found that the abi1-1 mutant that is impaired in ABA perception due to a constitutively active ABI1 protein (type-2C protein phosphatase) is more tolerant to light stress than the wild type. Our findings revealed that transcriptome reprogramming in plants occurs at a much faster rate than previously thought, and/or typically studied, and that this response could involve known as well as unknown transcripts and pathways (Suzuki et al. 2015). In addition to rapid alterations in the steady-state level of many transcripts during early stages of light stress, we identified rapid alterations in several different metabolites during light stress (Suzuki et al. 2013). These include rapid changes in the levels of ascorbic acid (decrease), and glycine, glycerate and sucrose (increase). These findings support the hypothesis that rapid alterations in transcript and metabolite levels play a key role in the acclimation of plants to ultra-fast changes in light stress (Suzuki et al. 2013, Dietz 2015, Suzuki et al. 2015). Excess light stress is known to affect the cellular redox status of cells, as well as potentially to result in the enhanced accumulation of reactive oxygen species (ROS; Asada 1999, Mullineaux et al. 2000, Galvez-Valdivieso et al. 2009, Takahashi and Badger 2011, Gorecka et al. 2014, Dietz 2015, Van Aken and Van Breusegem 2015). One of the key molecules at the crossroads between redox and ROS levels is the tripeptide antioxidant glutathione (GSH; Fig. 1; Queval et al. 2011, Foyer and Noctor 2013). Glutathione was found to play a central role in the response of plants to different biotic and abiotic stresses (including light stress), as well as in the regulation of different redox reactions and transcriptional responses (Noctor et al. 2012). In its free form it appears in cells as an oxidized (GSSG) or a reduced (GSH) molecule, and its conversion from oxidized to reduced, mediated by glutathione reductases (GRs) at the expense of NAD(P)H, keeps a pool of reductants that can be used for many purposes in cells (Maughan and Foyer 2006, Foyer and Noctor 2013). Reduced GSH can be used for the direct scavenging of H2O2, or for the indirect scavenging of ROS via maintaining the pools of ascorbate reduced via dehydroascorbate reductase (Asada–Foyer–Halliwell pathway; Foyer and Noctor 2011). Glutathione can also facilitate ROS removal by reducing glutaredoxins (GRXs) and thioredoxins (TRXs), used in H2O2 scavenging via peroxiredoxins (Mittler et al. 2004, Hanschmann et al. 2010). In addition, GSH is used in the regulation of redox-dependent transcription factors and the repair of oxidized proteins via GRXs (Rouhier 2010). Another important role for GSH during light stress could be its involvement in anthocyanin metabolism via glutathione S-transferase (GST; Rouhier et al. 2008). Fig. 1 View largeDownload slide Involvement of glutathione in key redox, signal transduction, metabolic and regulatory processes in plants. GSH and GSSG, reduced and oxidized glutathione, respectively; GR, glutathione reductase; TF, transcription factor; GSNO, S-nitrosoglutathione; GRX, glutaredoxin; TRX, thioredoxin; GST, glutathione S-transferase; ASC, ascorbate; APX, ascorbate peroxidase; DHA, dehydroascorbate; GPX, glutathione peroxidase; PRX, peroxiredoxin. Fig. 1 View largeDownload slide Involvement of glutathione in key redox, signal transduction, metabolic and regulatory processes in plants. GSH and GSSG, reduced and oxidized glutathione, respectively; GR, glutathione reductase; TF, transcription factor; GSNO, S-nitrosoglutathione; GRX, glutaredoxin; TRX, thioredoxin; GST, glutathione S-transferase; ASC, ascorbate; APX, ascorbate peroxidase; DHA, dehydroascorbate; GPX, glutathione peroxidase; PRX, peroxiredoxin. To address the role of GSH in the rapid response of plants to light stress (Suzuki et al. 2013, Suzuki et al. 2015), and to complement our transcriptomics studies with a metabolomics approach, we conducted a metabolomics study of the rapid response of plants to light stress, as well as studying changes in free GSH and GSSG levels, intermediates of GSH biosynthesis and degradation, and total GSH levels (free and bound GSH) during the first 90 s (0, 20, 60, 90 s time course) of excess light stress exposure in A. thaliana. Here we report that the rapid response of plants to light stress is accompanied by rapid alteration in the levels of 111 metabolites. We further report that free and total glutathione accumulate rapidly during light stress and that the accumulation of total GSH is dependent on nitric oxide (NO) accumulation. We additionally suggest that the increase in precursors for GSH biosynthesis could result from by-products of photorespiration, and that phosphoenolpyruvate (PEP) could function as a major energy/carbon source for rapid metabolic responses to light stress in Arabidopsis. Lastly, we show that a mutant altered in its responses to light stress (apx1) is also altered in its NO and glutathione accumulation during light stress. Results Rapid alterations in glutathione metabolism during light stress in Arabidopsis To study rapid metabolic changes in response to light stress, we subjected Arabidopsis plants grown under low light (50 μmol m−2 s−1) to light stress (1,000 μmol m−2 s−1) for 0, 20, 60 and 90 s, and immediately flash-froze their shoots and leaves in liquid nitrogen (Suzuki et al. 2015). Samples were subsequently subjected to metabolic analysis as described (Suzuki et al. 2013, Suzuki et al. 2015, Holt et al. 2016; Supplementary Fig. S1; Table S1). The light stress treatment described above resulted in a metabolic response that included 111 different metabolites significantly altered during rapid responses to light stress in Arabidopsis (Supplementary Fig. S1; Table S1). Of these, the steady-state levels of 45 metabolites increased, whereas the steady-state levels of 66 metabolites decreased (Supplementary Fig. S1). Because changes in glutathione levels are one of the major indicators of the redox state of the cell (Fig. 1; Foyer and Noctor 2011, Foyer and Noctor 2013), we focused our analysis on glutathione metabolism. As shown in Fig. 2, the level of free reduced glutathione (GSH) significantly increased in response to light stress, whereas the level of free oxidized glutathione (GSSG) transiently increased at 20 and 60 s, but then decreased back to control levels at 90 s. These results suggest that the rapid response of plants to excess light involves alterations in the level of free GSH in cells, reflecting oxidizing conditions at 20 and 60 s, but that at 90 s the plant could recover from these oxidizing conditions and even accumulate GSH to higher levels than those before the exposure to light stress (Fig. 2). The enhanced accumulation of free GSH was accompanied by enhanced accumulation of glycine and γ-glutamylcysteine, two of the immediate precursors of GSH biosynthesis (Fig. 2). In addition, while the level of γ-glutamylcysteine increased, the level of two of its precursors, glutamate and cysteine, decreased (Fig. 2). Taken together, the findings shown in Fig. 2 suggest that GSH biosynthesis or release from conjugated forms is enhanced during the rapid response to light stress in Arabidopsis. Fig. 2 View largeDownload slide Rapid changes in the level of glutathione and metabolites involved in glutathione biosynthesis and degradation in Arabidopsis plants subjected to light stress. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). GSH and GSSG, reduced and oxidized glutathione, respectively; γ-glutamyl-AA, γ -glutamyl amino acid. Fig. 2 View largeDownload slide Rapid changes in the level of glutathione and metabolites involved in glutathione biosynthesis and degradation in Arabidopsis plants subjected to light stress. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). GSH and GSSG, reduced and oxidized glutathione, respectively; γ-glutamyl-AA, γ -glutamyl amino acid. Responses of phosphoenolpyruvate, glycolysis, photorespiration and the TCA cycle to the rapid increase in light intensity To produce more GSH in the cell, at least two major determinants are needed: energy and building blocks. We therefore studied the response of different energy-producing pathways to light stress in Arabidopsis. As shown in Fig. 3, the rapid metabolic response to light stress in Arabidopsis included a decrease in glucose and glucose-6-phosphate, two entry metabolites for the glycolysis pathway, and an increase in 3-phosphoglycerate, a possible intermediate in glycolysis (but also involved in photorespiration; Supplementary Fig. S2). These changes were coupled to a very rapid decrease in PEP and a decrease in pyruvate that is one of the entry compounds into the tricarboxylic acid (TCA) cycle (Fig. 3). Because the conversion of PEP to pyruvate results in the phosphorylation of ADP to ATP, the rapid decrease in PEP levels could suggest that the PEP to pyruvate conversion is being used as an immediate source of ATP during the rapid response to light stress (Fig. 3). The changes observed in the glycolytic pathway during the rapid response to light stress were also followed by an overall decrease in the level of TCA cycle intermediates (Supplementary Fig. S3). Photorespiration plays a major role in alleviating excess light stress in C3 plants and could also be involved, together with serine and threonine, in the production of glycine, one of the major precursors of GSH (Supplementary Fig. S2; Mullineaux and Rausch et al., 2005). We therefore studied changes in serine, threonine and different metabolites involved in photorespiration (Supplementary Figs. S2, S4). As shown in Supplementary Fig. S2, the levels of glycolate, glycine and 3-phosphoglycerate increased during light stress in Arabidopsis, whereas the level of serine decreased. Because enhanced photorespiration is typically accompanied by enhanced levels of glycine and serine (Bauwe et al. 2010), the results shown in Supplementary Fig. S2 could suggest that only part of the photorespiration pathway is activated, or that serine is being utilized by other pathways in the cell (e.g. for the biosynthesis of glycine; Supplementary Fig. S4). In addition, the level of threonine, also involved in glycine production, in cells decreased during light stress in Arabidopsis (Supplementary Fig. S4). The rapid changes in the level of metabolites such as glucose, glucose-6-phosphate and PEP, which are typically kept at a relatively stable level during normal metabolism in cells, demonstrate that these compounds could respond promptly to changes in abiotic conditions. It is likely that if measured over longer periods of times, these metabolites will return to higher levels once the plant acclimates to the stress. Fig. 3 View largeDownload slide Rapid depletion of phosphoenolpyruvate (PEP) in Arabidopsis plants subjected to light stress. Changes in PEP and different metabolites involved in glycolysis during rapid responses to light stress in Arabidopsis. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). PEP, phosphoenolpyruvate. Fig. 3 View largeDownload slide Rapid depletion of phosphoenolpyruvate (PEP) in Arabidopsis plants subjected to light stress. Changes in PEP and different metabolites involved in glycolysis during rapid responses to light stress in Arabidopsis. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). PEP, phosphoenolpyruvate. Potential regulation of GSH accumulation by NO Because NO was shown to regulate different enzymes involved in the photorespiration pathway (Abat et al. 2008, Palmieri et al. 2010, Ortega-Galisteo et al. 2012), it might be involved in some of the observed alterations in this pathway during rapid responses to light stress (Supplementary Fig. S2). We therefore measured NO levels in plants during rapid responses to light stress (Fig. 4). As shown in Fig. 4A, NO levels accumulated in Arabidopsis plants subjected to light stress as early as 60 s following light stress application. The accumulation of NO during the rapid response to light stress was accompanied by a decrease in aspartate, and an increase in AMP and argininosuccinate (Supplementary Fig. S5), suggesting that the enhanced levels of NO during light stress (Fig. 4A) could result from the activation of the arginine–citrulline pathway (Supplementary Fig. S5). The findings shown in Fig. 4 and Supplementary Fig. S5 suggest that NO, produced during light stress in Arabidopsis, could regulate different pathways such as the photorespiration pathway and enhance the availability of precursors such as glycine and energy for glutathione biosynthesis. Alternatively, NO could directly regulate glutathione biosynthesis or degradation enzymes or alter their expression (Fig. 2). To examine whether NO is required for GSH accumulation during light stress, we measured total GSH accumulation in water- or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide- (cPTIO; NO scavenger; Fig. 4B;Groß et al. 2017) treated plants subjected to light stress (in contrast to the measurements of free GSH and GSSG shown in Fig. 2, measurements of total GSH, shown in Fig. 4B, includes the free and bound forms of GSH). As shown in Fig. 4B, pre-treatment of plants with cPTIO inhibited the accumulation of total GSH in response to light stress. It should be noted that although cPTIO is widely used by the plant community as an NO scavenger, and it suppressed NO accumulation in response to light stress (Fig. 4A), it could have additional effects on plants (D’Alessandro et al. 2013). Taken together, the results shown in Fig. 4 suggest that an increase in NO levels is associated with the accumulation of total GSH during light stress in Arabidopsis. Fig. 4 View largeDownload slide Potential regulation of glutathione accumulation by nitric oxide (NO) in Arabidopsis plants subjected to light stress. (A) Measurements of NO levels in control and cPTIO- (NO scavenger; 200 µM) treated Arabidopsis seedlings subjected to light stress. Representative images (top), and a graph of the mean ± SE of fluorescence measurement of three individual experiments each having 10–12 seedlings (bottom) are shown. (B) Measurements of total glutathione levels in control and cPTIO- (200 µM) treated Arabidopsis plants subjected to light stress. A representative graph is shown with the mean ± SE of fluorescence measurement of five individual experiments each performed with three technical repeats having 8–12 plants each. Different letters denote significant differences (P < 0.05) among means at different time points within a group (control and cPTIO) according to ANOVA (Duncan’s test). cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Scale bar = 1 mm. Fig. 4 View largeDownload slide Potential regulation of glutathione accumulation by nitric oxide (NO) in Arabidopsis plants subjected to light stress. (A) Measurements of NO levels in control and cPTIO- (NO scavenger; 200 µM) treated Arabidopsis seedlings subjected to light stress. Representative images (top), and a graph of the mean ± SE of fluorescence measurement of three individual experiments each having 10–12 seedlings (bottom) are shown. (B) Measurements of total glutathione levels in control and cPTIO- (200 µM) treated Arabidopsis plants subjected to light stress. A representative graph is shown with the mean ± SE of fluorescence measurement of five individual experiments each performed with three technical repeats having 8–12 plants each. Different letters denote significant differences (P < 0.05) among means at different time points within a group (control and cPTIO) according to ANOVA (Duncan’s test). cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Scale bar = 1 mm. The apx1 mutant does not accumulate glutathione and NO during its rapid response to light stress To study further the rapid response of Arabidopsis to light stress, we compared wild-type plants and a mutant deficient in cytosolic ascorbate peroxidase 1 (apx1). The apx1 mutant is deficient in H2O2 scavenging and accumulates high levels of H2O2 during excess light stress, compared with the wild type (Pnueli et al. 2003, Davletova et al. 2005). In addition, a large number of proteins in apx1 undergo carbonylation (oxidative damage) during light stress, and the expression of a large number of transcripts, including many different transcription factors, is augmented in apx1 during light stress (Davletova et al. 2005). Interestingly, the apx1 mutant does not appear to oxidize ascorbic acid during light stress, suggesting that the function of the ascorbate–glutathione cycle in this mutant during light stress is altered (Suzuki et al. 2013). A comparison of the rapid metabolic response to light stress between wild-type plants and apx1 is shown in Fig. 5. In the absence of light stress, 84 metabolites were altered in the apx1 mutant compared with the wild type (Supplementary Tables S1, S2). These belonged to several different pathways, including pathways involved in glutathione and ascorbic acid metabolism (Fig. 5A;Supplementary Tables S1, S2). As shown in Fig. 5B, the response of the apx1 mutant to light stress was different from that of the wild type, with only 26 compounds shared between the wild type and apx1 during the light stress response. As shown in Fig. 6, in contrast to the wild type, free GSH levels decreased during light stress, and free GSSG levels declined and then increased (opposite to the wild type) during light stress in apx1. These results suggest that apx1 plants do not accumulate significant levels of free glutathione during light stress. Because we observed a link between NO and GSH accumulation in wild-type plants (Fig. 4), we also measured the accumulation of NO in apx1 plants during light stress. As shown in Supplementary Fig. S6, NO levels did not accumulate in apx1 plants during early stages of light stress. This observation was also supported by mainly non-significant changes in the levels of the arginine–citrulline pathway metabolites (Supplementary Fig. S7). Fig. 5 View largeDownload slide Differences between the rapid metabolic response of the wild type and the apx1 mutant to light stress. (A) Number of metabolites associated with different pathways significantly altered between the wild type and the apx1 mutant at time 0. Significant differences (P ≤ 0.05) were calculated based on Student’s two-tailed t-test and pathway involvement was analyzed using the KEGG database. (B) Venn diagrams showing the total number of metabolites commonly or differentially altered between the wild type and the apx1 mutant during the response to light stress (top), the number of metabolites commonly or differentially up-regulated between the wild type and the apx1 mutant during the response to light stress (middle) and the number of metabolites commonly or differentially down-regulated between the wild type and the apx1 mutant during the response to light stress (bottom). Significant difference (P ≤ 0.05) was calculated based on ANOVA (Duncan’s test). A list of all metabolites altered in the wild type and the apx1 mutant is shown in Supplementary Table S1. Fig. 5 View largeDownload slide Differences between the rapid metabolic response of the wild type and the apx1 mutant to light stress. (A) Number of metabolites associated with different pathways significantly altered between the wild type and the apx1 mutant at time 0. Significant differences (P ≤ 0.05) were calculated based on Student’s two-tailed t-test and pathway involvement was analyzed using the KEGG database. (B) Venn diagrams showing the total number of metabolites commonly or differentially altered between the wild type and the apx1 mutant during the response to light stress (top), the number of metabolites commonly or differentially up-regulated between the wild type and the apx1 mutant during the response to light stress (middle) and the number of metabolites commonly or differentially down-regulated between the wild type and the apx1 mutant during the response to light stress (bottom). Significant difference (P ≤ 0.05) was calculated based on ANOVA (Duncan’s test). A list of all metabolites altered in the wild type and the apx1 mutant is shown in Supplementary Table S1. Fig. 6 View largeDownload slide Rapid changes in the level of glutathione and metabolites involved in glutathione biosynthesis and degradation in apx1 plants subjected to light stress. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). GSH and GSSG, reduced and oxidized glutathione, respectively; γ-glutamyl-AA, γ-glutamyl amino acid. Fig. 6 View largeDownload slide Rapid changes in the level of glutathione and metabolites involved in glutathione biosynthesis and degradation in apx1 plants subjected to light stress. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). GSH and GSSG, reduced and oxidized glutathione, respectively; γ-glutamyl-AA, γ-glutamyl amino acid. As shown in Fig. 6, the decrease in free GSH levels in apx1 plants was accompanied by a decrease in the level of the glutathione precursor cysteine, as well as slower accumulation of glycine. In addition, the levels of cysteinyl-glycine that gives rise to cysteine and glycine were suppressed (Fig. 6). These results suggest that glutathione biosynthesis is suppressed in the apx1 mutant during light stress. Discussion We previously reported that in response to light stress, plants mount a rapid transcriptional response, and that at least five of the transcripts that are specifically up-regulated at 20–60 s post-light stress application are essential for plant acclimation to light stress (Suzuki et al. 2015). Here we expanded on our analysis of the ultra-fast response of plants to light stress and identified rapid accumulation in the free and total levels of glutathione, a key regulator of redox reactions, acclimation and defense responses in plants (Foyer and Noctor 2011, Noctor et al. 2012, Foyer and Noctor 2013), as part of this rapid response (Figs. 2, 4B; Supplementary Fig. S8). The changes in free and total GSH during light stress (Figs. 2, 4B) could reflect a response of the glutathione pool to the oxidative conditions that are thought to accompany the initial stages of light stress in plants (Dietz 2015, Suzuki et al. 2015). Thus, glutathione could play an important role in redox regulation during early events of light stress acclimation. This possibility is supported by studies that showed altered tolerance to light stress in plants with altered content of glutathione (Creissen et al. 1999). A meta-analysis of the rapid transcriptional response of Arabidopsis to light stress (20–60 s; Suzuki et al. 2015) also supports this possibility, revealing that the steady-state transcript level of at least eight different transcripts associated with glutathione function in plants, including glutathione peroxidase 1 (GPX1), is significantly altered during early responses to light stress (Supplementary Fig. S8). Interestingly, in contrast to the level of free GSH that peaked at 90 s (Fig. 2), the level of total GSH, that includes both bound and free glutathione, peaked at 60 s (Fig. 4B), suggesting that the rapid response to light stress in Arabidopsis involved alterations in the ratio between free and bound GSH and/or GSSG in cells. These alterations could include release of glutathione from conjugated forms to increase the level of free GSH/GSSG (Fig. 2), and/or binding of glutathione to different molecules and proteins in cells that will decrease the level of free GSH/GSSG (Fig. 2), but enhance the level of total glutathione in cells (Fig. 4B). The reactions outlined above could simultaneously alter the levels of free and/or conjugated glutathione in similar or opposing manners in different subcellular compartments differentially affecting the total levels of free and bound glutathione in cells (Figs. 2, 4B; Noctor et al. 2011). As expected in C3 plants, the rapid metabolic response to light stress appeared to activate the photorespiration pathway (Supplementary Fig. S2). This activation was accompanied by the accumulation of glycine and the depletion of serine (Supplementary Fig. S2). Coupled with the depletion of threonine (Supplementary Fig. S4), these changes that occur during early stages of light stress in Arabidopsis might promote the biosynthesis of glutathione (Fig. 2; Mullineaux and Rausch 2005). It is interesting to note that in contrast to our earlier study conducted with more mature plants (Suzuki et al. 2013), the level of serine declined rather than increased during light stress. This finding could suggest that in contrast to glycine, the level of serine during early responses to light stress may depend on the age and developmental stage of the plant. Although the availability of glycine is generally believed to be one of the main rate-limiting factors in glutathione biosynthesis, our comparison between wild-type plants that accumulated glutathione and apx1 plants that did not (Figs. 2, 6) revealed that the level of γ-glutamylcysteine could also play an important role in this context. Thus, although glycine levels accumulate in apx1 plants during light stress, glutathione did not accumulate in these plants (Fig. 6). In contrast, γ-glutamylcysteine that accumulated in wild-type plants (Fig. 2) did not accumulate in apx1 mutants, raising the possibility that at least in apx1 plants the availability of γ-glutamylcysteine may play an important role in regulating glutathione biosynthesis. The plant hormone NO regulates the activity of many different enzymes and proteins in plants during stress (Wilson et al. 2008). These include different enzymes of the photorespiration pathway (Supplementary Fig. S2; Abat et al. 2008, Palmieri et al. 2010, Ortega-Galisteo et al. 2012). Because NO could inhibit the carboxylation reaction of Rubisco (Abat and Deswal 2009), it could drive photorespiration in the direction of glycolate and glycine accumulation (Supplementary Fig. S2). Further enhancement in glycine could also occur if NO inhibits glycine decarboxylase (Palmieri et al. 2010) and prevents glycine from being converted into serine (partially explaining the decrease in serine levels; Supplementary Fig. S2). The potential outcome of these effects of NO on photorespiration could therefore be a rapid enhancement in glycine accumulation that could be used in the biosynthesis of glutathione (Fig. 2; Mullineaux and Rausch 2005). Our findings that NO accumulates during the rapid response of Arabidopsis to light stress (Fig. 4A), and that when the accumulation of NO is prevented, total glutathione does not accumulate during this response (Fig. 4B), provide further support for the possibility that NO regulates glutathione accumulation during the rapid response of plants to light stress, potentially via its effects on photorespiration. Of course further studies are needed to address this possibility. A possible role for NO in regulating GSH biosynthesis was previously reported in Medicago truncatula roots (Innocenti et al. 2007), and in Arabidopsis plants subjected to pathogen attack (Kovacs et al. 2015), further supporting this possibility. In addition, our analysis of the response of apx1 plants to rapid changes in light intensity demonstrated that both glutathione and NO accumulation are impaired in this mutant that is sensitive to light stress (Figs. 6;Supplementary Figs. S6, S7; Pnueli et al. 2003, Davletova et al. 2005), further linking glutathione and NO accumulation to light stress acclimation. An interesting finding originating from our metabolic analysis of rapid responses to light stress, presented in this study, is the rapid depletion in the pool of PEP in cells (Fig. 3). PEP is an essential metabolite that is at the crossroads between several different metabolic pathways leading to energy production in the mitochondria, jasmonic acid and salicylic acid biosynthesis, organic and amino acid biosynthesis and production of phenolic compounds (Prabhakar et al. 2010, Dizengremel et al. 2012). From the standpoint of immediate energy production, the conversion of PEP to pyruvate generates ATP that could be used as an energy source to maintain the cell at a high redox state. Thus, PEP could play an important role in plant acclimation to rapid changes in light conditions. Taken together, our findings should be viewed as an initial metabolic analysis of the response of plants to rapid (0–90 s) changes in their environment. At least three main findings stem from our analysis and could lead to further studies. They include a role for glutathione as a redox and signaling molecule, NO as a signaling hormone and PEP as an immediate energy and carbon source. Our study further poses an interesting question: is it possible to augment the initial metabolic response of plants to rapid changes in environmental conditions by altering the expression of particular genes and/or the level of particular metabolites? For example, would it be possible to improve on the pool, or fluxes controlling the pool, of PEP or other metabolites that are rapidly depleted during rapid response to stress? Strengthening, or augmenting, the rapid response of plants to rapid changes in abiotic stress conditions, such as light intensity, could have a far-reaching effect on the overall acclimation process and tolerance of plants to stress (Suzuki et al. 2015), thereby increasing yield under field conditions (Kromdijk et al. 2016). Further studies are needed to address these questions. In addition, because many of the compounds measured by our analysis are involved in several different pathways in the cell and could be simultaneously altered in different manners (up or down) in different cellular compartments, further studies, in particular compartment-specific and pulse–chase flux analyses, are needed to resolve the role and association of different compounds with each other and with the metabolic pathways they are connected with. Our analysis should therefore be viewed as an initial identification of different pathways and metabolites that could be used to augment the rapid response of plants to abiotic stress. In particular, it highlights the important role of glutathione, PEP and NO for these responses. Materials and Methods Plant material and growth condition Arabidopsis thaliana ecotype Columbia and apx1 (Davletova et al. 2005) were grown in peat pellets (Jiffy-7, Jiffy, http://www.jiffygroup.com/en/) at 23°C under constant low light (50 μmol m−2 s−1) as previously described (Davletova et al. 2005, Suzuki et al. 2011, Luhua et al. 2013). Light stress treatment For metabolite profiling (free GSH and GSSG), and total glutathione assays, 4–5 plants grown in a peat pellet, as described in Suzuki et al. (2015), were exposed to a light intensity of 1,000 µmol m–2 s–1 at 22°C for a period of 0, 20, 60 and 90 s (Suzuki et al. 2015). From 20 to 25 different plants were used for each time point, and the experiment was conducted with a total of five biological repeats. Samples were collected by dipping the plants in liquid nitrogen immediately after stress treatment. Frozen tissue was collected into a falcon tube, ground into a fine powder and transferred into a 1.5 ml tube (about 250–300 mg per tube). Samples were kept frozen during the whole collection process and stored at –80°C. For treatment with the NO scavenger cPTIO (Sigma-Aldrich), wild-type plants grown in cookie were sprayed with water (control) or 200 µM cPTIO, incubated for 2 h and exposed to light stress for 0, 20, 60 and 90 s. Plants were then flash-frozen in liquid nitrogen (Suzuki et al. 2015) and assayed for their total glutathione content as described below. Eight to twelve different plants were used for each time point in three technical repeats, and the experiment was conducted with a total of five biological repeats. Metabolic analysis Metabolic analysis using ultra performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) analysis was conducted as a service by Metabolon, Inc. (Evans et al. 2009, Holt et al. 2016). The liquid chromatography (LC)-MS) analysis was performed using a Waters ACQUITY UPLC and a Thermo-Finnigan LTQ mass spectrometer. Raw data extraction, peak identification and processing for quality control were performed using Metabolon’s hardware and software. Compounds were identified by comparison with library entries of purified standards. Metabolon maintains a compound library containing the retention time/index (RI), mass to charge ratio (m/z) and MS/ MS spectral data based on an authenticated standard. Compounds are identified based on three criteria: retention index within a narrow RI window of the proposed identification, nominal mass match to the library ±0.4 amu and the MS/MS spectral similarity between experimental data and authentic standards. The combination of three categories of data gives enough deciding power to differentiate and distinguish between the compounds even in cases where there is similarity in one of these factors. This analysis identified 413 compounds of known identity and their peak areas were taken as a measure of intensity. The intensity of each compound was rescaled to set the median value equal to 1. The natural log of the scaled intensity was then calculated for each compound for graphical representation and statistical analysis. Quantitation of NO NO content was measured by a modified protocol from Gross et al. (2017). Five-day-old wild-type and apx1 seedlings grown in 1/2 Murashige and Skoog (MS) media were stained with 15 µM DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; Sigma-Aldrich) for 1 h. Staining was performed in 1/2 MS media with continuous shaking. Seedlings were then washed with the 1/2 MS media by continuous shaking for 15 min. As a control, the NO scavenger cPTIO (200 µM) was added along with DAF-FM DA during staining in duplicate samples. Seedlings were then exposed to a light intensity of 1,000 µmol m–2 s–1 for a period of 0, 1, 2 and 5 min, and immediately used for imaging. Imaging was performed with a fluorescence microscope (AMG Evos, X4 objective) using green fluorescence protein (GFP; excitation filter 495 nm and emission filter 515 nm) filter settings. Images were further analyzed with ImageJ software to quantify fluorescence intensity. Four biological experiments were performed with 10–12 technical repeats per time point each. Total glutathione measurement Total glutathione was measured using the glutathione reductase recycling method described in Queval and Noctor (2007) with modifications. Ground samples were freeze dried for 72 h at –40 °C in a Labconco freeze drier. A 2 mg aliquot of the freeze-dried tissue was collected in a 1.5 ml tube and total glutathione was extracted with 600 μl of 0.2 N HCl. The extract was neutralized with 0.2 M NaH2PO4 (pH 5.6) and 0.2 M NaOH. The assay was performed in a 96-well plate in three technical replicates, and absorbance (412 nm) was measured in a plate reader (Synergy BioTek) equipped with Gene5 software in a kinetic setting. To calculate total GSH content, the rate of the reaction was measured for the first 2 min. Statistical analysis and pathway analysis Analysis of variance (ANOVA) followed by a post-hoc Duncan’s test was used to identify compounds significantly altered over time using SPSS software (Paulose et al. 2013, Suzuki et al. 2015, Holt et al. 2016). Significant differences between mean values of the wild type and the apx1 mutant at the 0 s time point were assessed by a Student’s two-tailed t-test (Suzuki et al. 2015). Differences at P < 0.05 were considered significant. Pathway analysis was performed using TAIR (https://www.arabidopsis.org/) and KEGG (http://www.kegg.jp/) pathway analysis tools to generate different metabolic maps and figures as described in Holt et al. (2016). Funding This work was supported the National Science Foundation [IOS-1353886, IOS-1063287, IOS-1557787, MCB-1613462] and the University of North Texas, College of Science. Disclosures The authors have no conflicts of interest to declare. References Abat J.K. , Deswal R. ( 2009 ) Differential modulation of S-nitrosoproteome of Brassica juncea by low temperature: change in S-nitrosylation of Rubisco is responsible for the inactivation of its carboxylase activity . Proteomics 9 : 4368 – 4380 . Google Scholar Crossref Search ADS PubMed Abat J.K. , Mattoo A.K. , Deswal R. ( 2008 ) S-nitrosylated proteins of a medicinal CAM plant Kalanchoe pinnata—ribulose-1,5-bisphosphate carboxylase/oxygenase activity targeted for inhibition . FEBS J. 275 : 2862 – 2872 . Google Scholar Crossref Search ADS PubMed Asada K. ( 1999 ) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons . Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 : 601 – 639 . 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Rapid Accumulation of Glutathione During Light Stress in Arabidopsis

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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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Abstract

Abstract Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. We previously reported that in response to rapid changes in light intensity (0–60 s), Arabidopsis thaliana plants mount a large-scale transcriptomic response that includes several different transcripts essential for light stress acclimation. Here, we expand our analysis of the rapid response of Arabidopsis to light stress using a metabolomics approach and identify 111 metabolites that show a significant alteration in their level during the first 90 s of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is associated with an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in these responses. Introduction Light is the primary energy source for plants, as well as a key player in determining the plant’s diurnal cycles, growth, development and responses to different environmental stimuli. To optimize growth and development, and to avoid some of the damaging effects of excess light (Li et al. 2009, Takahashi and Badger 2011), plants need to constantly to adjust their metabolism and light-harvesting antennae in a process that is generally referred to as acclimation (Mullineaux et al. 2000, Kleine et al. 2007, Rossel et al. 2007, Galvez-Valdivieso et al. 2009, Gorecka et al. 2014, Dietz 2015, Oelze et al. 2012). Recent studies revealed that plant acclimation to light stress plays a major role in determining yield, as well as affecting crop productivity in the field (Mittler and Blumwald 2010, Kromdijk et al. 2016). In addition to diurnal and seasonal changes in light intensity and quality, under field conditions, plants are also subjected to random and rapid changes in light intensity and quality, sometimes referred to as sun flecks (Pastenes et al. 2005, Szechyńska-Hebda and Karpiński 2013, Raven 2014). These sudden changes in light intensity are primarily due to rapid changes in cloud cover or changes in canopy shading, and could result in enhanced photosynthesis or photoinhibition (Mittler and Blumwald 2010, Karpiński et al. 2013, Mittler, 2006). We recently reported on the molecular responses of Arabidopsis thaliana plants to rapid changes in light intensity, occurring within 0–90 s of plant exposure to high light stress (Suzuki et al. 2015). In that study, we identified a rapid transcriptomic response to light stress that included an ordered and clustered change in the steady-state level of 731 transcripts, occurring as early as 20–60 s following light stress application. We further determined that several of the transcripts identified by our analysis are required for plant acclimation to light stress, as well as demonstrating that at least some of the transcripts identified were transcriptionally regulated (Suzuki et al. 2015). Because many of the ultra-fast light stress response transcripts we identified in Arabidopsis were H2O2 or ABA response transcripts, we also studied the response of mutants impaired in ABA metabolism and sensing to light stress and found that the abi1-1 mutant that is impaired in ABA perception due to a constitutively active ABI1 protein (type-2C protein phosphatase) is more tolerant to light stress than the wild type. Our findings revealed that transcriptome reprogramming in plants occurs at a much faster rate than previously thought, and/or typically studied, and that this response could involve known as well as unknown transcripts and pathways (Suzuki et al. 2015). In addition to rapid alterations in the steady-state level of many transcripts during early stages of light stress, we identified rapid alterations in several different metabolites during light stress (Suzuki et al. 2013). These include rapid changes in the levels of ascorbic acid (decrease), and glycine, glycerate and sucrose (increase). These findings support the hypothesis that rapid alterations in transcript and metabolite levels play a key role in the acclimation of plants to ultra-fast changes in light stress (Suzuki et al. 2013, Dietz 2015, Suzuki et al. 2015). Excess light stress is known to affect the cellular redox status of cells, as well as potentially to result in the enhanced accumulation of reactive oxygen species (ROS; Asada 1999, Mullineaux et al. 2000, Galvez-Valdivieso et al. 2009, Takahashi and Badger 2011, Gorecka et al. 2014, Dietz 2015, Van Aken and Van Breusegem 2015). One of the key molecules at the crossroads between redox and ROS levels is the tripeptide antioxidant glutathione (GSH; Fig. 1; Queval et al. 2011, Foyer and Noctor 2013). Glutathione was found to play a central role in the response of plants to different biotic and abiotic stresses (including light stress), as well as in the regulation of different redox reactions and transcriptional responses (Noctor et al. 2012). In its free form it appears in cells as an oxidized (GSSG) or a reduced (GSH) molecule, and its conversion from oxidized to reduced, mediated by glutathione reductases (GRs) at the expense of NAD(P)H, keeps a pool of reductants that can be used for many purposes in cells (Maughan and Foyer 2006, Foyer and Noctor 2013). Reduced GSH can be used for the direct scavenging of H2O2, or for the indirect scavenging of ROS via maintaining the pools of ascorbate reduced via dehydroascorbate reductase (Asada–Foyer–Halliwell pathway; Foyer and Noctor 2011). Glutathione can also facilitate ROS removal by reducing glutaredoxins (GRXs) and thioredoxins (TRXs), used in H2O2 scavenging via peroxiredoxins (Mittler et al. 2004, Hanschmann et al. 2010). In addition, GSH is used in the regulation of redox-dependent transcription factors and the repair of oxidized proteins via GRXs (Rouhier 2010). Another important role for GSH during light stress could be its involvement in anthocyanin metabolism via glutathione S-transferase (GST; Rouhier et al. 2008). Fig. 1 View largeDownload slide Involvement of glutathione in key redox, signal transduction, metabolic and regulatory processes in plants. GSH and GSSG, reduced and oxidized glutathione, respectively; GR, glutathione reductase; TF, transcription factor; GSNO, S-nitrosoglutathione; GRX, glutaredoxin; TRX, thioredoxin; GST, glutathione S-transferase; ASC, ascorbate; APX, ascorbate peroxidase; DHA, dehydroascorbate; GPX, glutathione peroxidase; PRX, peroxiredoxin. Fig. 1 View largeDownload slide Involvement of glutathione in key redox, signal transduction, metabolic and regulatory processes in plants. GSH and GSSG, reduced and oxidized glutathione, respectively; GR, glutathione reductase; TF, transcription factor; GSNO, S-nitrosoglutathione; GRX, glutaredoxin; TRX, thioredoxin; GST, glutathione S-transferase; ASC, ascorbate; APX, ascorbate peroxidase; DHA, dehydroascorbate; GPX, glutathione peroxidase; PRX, peroxiredoxin. To address the role of GSH in the rapid response of plants to light stress (Suzuki et al. 2013, Suzuki et al. 2015), and to complement our transcriptomics studies with a metabolomics approach, we conducted a metabolomics study of the rapid response of plants to light stress, as well as studying changes in free GSH and GSSG levels, intermediates of GSH biosynthesis and degradation, and total GSH levels (free and bound GSH) during the first 90 s (0, 20, 60, 90 s time course) of excess light stress exposure in A. thaliana. Here we report that the rapid response of plants to light stress is accompanied by rapid alteration in the levels of 111 metabolites. We further report that free and total glutathione accumulate rapidly during light stress and that the accumulation of total GSH is dependent on nitric oxide (NO) accumulation. We additionally suggest that the increase in precursors for GSH biosynthesis could result from by-products of photorespiration, and that phosphoenolpyruvate (PEP) could function as a major energy/carbon source for rapid metabolic responses to light stress in Arabidopsis. Lastly, we show that a mutant altered in its responses to light stress (apx1) is also altered in its NO and glutathione accumulation during light stress. Results Rapid alterations in glutathione metabolism during light stress in Arabidopsis To study rapid metabolic changes in response to light stress, we subjected Arabidopsis plants grown under low light (50 μmol m−2 s−1) to light stress (1,000 μmol m−2 s−1) for 0, 20, 60 and 90 s, and immediately flash-froze their shoots and leaves in liquid nitrogen (Suzuki et al. 2015). Samples were subsequently subjected to metabolic analysis as described (Suzuki et al. 2013, Suzuki et al. 2015, Holt et al. 2016; Supplementary Fig. S1; Table S1). The light stress treatment described above resulted in a metabolic response that included 111 different metabolites significantly altered during rapid responses to light stress in Arabidopsis (Supplementary Fig. S1; Table S1). Of these, the steady-state levels of 45 metabolites increased, whereas the steady-state levels of 66 metabolites decreased (Supplementary Fig. S1). Because changes in glutathione levels are one of the major indicators of the redox state of the cell (Fig. 1; Foyer and Noctor 2011, Foyer and Noctor 2013), we focused our analysis on glutathione metabolism. As shown in Fig. 2, the level of free reduced glutathione (GSH) significantly increased in response to light stress, whereas the level of free oxidized glutathione (GSSG) transiently increased at 20 and 60 s, but then decreased back to control levels at 90 s. These results suggest that the rapid response of plants to excess light involves alterations in the level of free GSH in cells, reflecting oxidizing conditions at 20 and 60 s, but that at 90 s the plant could recover from these oxidizing conditions and even accumulate GSH to higher levels than those before the exposure to light stress (Fig. 2). The enhanced accumulation of free GSH was accompanied by enhanced accumulation of glycine and γ-glutamylcysteine, two of the immediate precursors of GSH biosynthesis (Fig. 2). In addition, while the level of γ-glutamylcysteine increased, the level of two of its precursors, glutamate and cysteine, decreased (Fig. 2). Taken together, the findings shown in Fig. 2 suggest that GSH biosynthesis or release from conjugated forms is enhanced during the rapid response to light stress in Arabidopsis. Fig. 2 View largeDownload slide Rapid changes in the level of glutathione and metabolites involved in glutathione biosynthesis and degradation in Arabidopsis plants subjected to light stress. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). GSH and GSSG, reduced and oxidized glutathione, respectively; γ-glutamyl-AA, γ -glutamyl amino acid. Fig. 2 View largeDownload slide Rapid changes in the level of glutathione and metabolites involved in glutathione biosynthesis and degradation in Arabidopsis plants subjected to light stress. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). GSH and GSSG, reduced and oxidized glutathione, respectively; γ-glutamyl-AA, γ -glutamyl amino acid. Responses of phosphoenolpyruvate, glycolysis, photorespiration and the TCA cycle to the rapid increase in light intensity To produce more GSH in the cell, at least two major determinants are needed: energy and building blocks. We therefore studied the response of different energy-producing pathways to light stress in Arabidopsis. As shown in Fig. 3, the rapid metabolic response to light stress in Arabidopsis included a decrease in glucose and glucose-6-phosphate, two entry metabolites for the glycolysis pathway, and an increase in 3-phosphoglycerate, a possible intermediate in glycolysis (but also involved in photorespiration; Supplementary Fig. S2). These changes were coupled to a very rapid decrease in PEP and a decrease in pyruvate that is one of the entry compounds into the tricarboxylic acid (TCA) cycle (Fig. 3). Because the conversion of PEP to pyruvate results in the phosphorylation of ADP to ATP, the rapid decrease in PEP levels could suggest that the PEP to pyruvate conversion is being used as an immediate source of ATP during the rapid response to light stress (Fig. 3). The changes observed in the glycolytic pathway during the rapid response to light stress were also followed by an overall decrease in the level of TCA cycle intermediates (Supplementary Fig. S3). Photorespiration plays a major role in alleviating excess light stress in C3 plants and could also be involved, together with serine and threonine, in the production of glycine, one of the major precursors of GSH (Supplementary Fig. S2; Mullineaux and Rausch et al., 2005). We therefore studied changes in serine, threonine and different metabolites involved in photorespiration (Supplementary Figs. S2, S4). As shown in Supplementary Fig. S2, the levels of glycolate, glycine and 3-phosphoglycerate increased during light stress in Arabidopsis, whereas the level of serine decreased. Because enhanced photorespiration is typically accompanied by enhanced levels of glycine and serine (Bauwe et al. 2010), the results shown in Supplementary Fig. S2 could suggest that only part of the photorespiration pathway is activated, or that serine is being utilized by other pathways in the cell (e.g. for the biosynthesis of glycine; Supplementary Fig. S4). In addition, the level of threonine, also involved in glycine production, in cells decreased during light stress in Arabidopsis (Supplementary Fig. S4). The rapid changes in the level of metabolites such as glucose, glucose-6-phosphate and PEP, which are typically kept at a relatively stable level during normal metabolism in cells, demonstrate that these compounds could respond promptly to changes in abiotic conditions. It is likely that if measured over longer periods of times, these metabolites will return to higher levels once the plant acclimates to the stress. Fig. 3 View largeDownload slide Rapid depletion of phosphoenolpyruvate (PEP) in Arabidopsis plants subjected to light stress. Changes in PEP and different metabolites involved in glycolysis during rapid responses to light stress in Arabidopsis. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). PEP, phosphoenolpyruvate. Fig. 3 View largeDownload slide Rapid depletion of phosphoenolpyruvate (PEP) in Arabidopsis plants subjected to light stress. Changes in PEP and different metabolites involved in glycolysis during rapid responses to light stress in Arabidopsis. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). PEP, phosphoenolpyruvate. Potential regulation of GSH accumulation by NO Because NO was shown to regulate different enzymes involved in the photorespiration pathway (Abat et al. 2008, Palmieri et al. 2010, Ortega-Galisteo et al. 2012), it might be involved in some of the observed alterations in this pathway during rapid responses to light stress (Supplementary Fig. S2). We therefore measured NO levels in plants during rapid responses to light stress (Fig. 4). As shown in Fig. 4A, NO levels accumulated in Arabidopsis plants subjected to light stress as early as 60 s following light stress application. The accumulation of NO during the rapid response to light stress was accompanied by a decrease in aspartate, and an increase in AMP and argininosuccinate (Supplementary Fig. S5), suggesting that the enhanced levels of NO during light stress (Fig. 4A) could result from the activation of the arginine–citrulline pathway (Supplementary Fig. S5). The findings shown in Fig. 4 and Supplementary Fig. S5 suggest that NO, produced during light stress in Arabidopsis, could regulate different pathways such as the photorespiration pathway and enhance the availability of precursors such as glycine and energy for glutathione biosynthesis. Alternatively, NO could directly regulate glutathione biosynthesis or degradation enzymes or alter their expression (Fig. 2). To examine whether NO is required for GSH accumulation during light stress, we measured total GSH accumulation in water- or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide- (cPTIO; NO scavenger; Fig. 4B;Groß et al. 2017) treated plants subjected to light stress (in contrast to the measurements of free GSH and GSSG shown in Fig. 2, measurements of total GSH, shown in Fig. 4B, includes the free and bound forms of GSH). As shown in Fig. 4B, pre-treatment of plants with cPTIO inhibited the accumulation of total GSH in response to light stress. It should be noted that although cPTIO is widely used by the plant community as an NO scavenger, and it suppressed NO accumulation in response to light stress (Fig. 4A), it could have additional effects on plants (D’Alessandro et al. 2013). Taken together, the results shown in Fig. 4 suggest that an increase in NO levels is associated with the accumulation of total GSH during light stress in Arabidopsis. Fig. 4 View largeDownload slide Potential regulation of glutathione accumulation by nitric oxide (NO) in Arabidopsis plants subjected to light stress. (A) Measurements of NO levels in control and cPTIO- (NO scavenger; 200 µM) treated Arabidopsis seedlings subjected to light stress. Representative images (top), and a graph of the mean ± SE of fluorescence measurement of three individual experiments each having 10–12 seedlings (bottom) are shown. (B) Measurements of total glutathione levels in control and cPTIO- (200 µM) treated Arabidopsis plants subjected to light stress. A representative graph is shown with the mean ± SE of fluorescence measurement of five individual experiments each performed with three technical repeats having 8–12 plants each. Different letters denote significant differences (P < 0.05) among means at different time points within a group (control and cPTIO) according to ANOVA (Duncan’s test). cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Scale bar = 1 mm. Fig. 4 View largeDownload slide Potential regulation of glutathione accumulation by nitric oxide (NO) in Arabidopsis plants subjected to light stress. (A) Measurements of NO levels in control and cPTIO- (NO scavenger; 200 µM) treated Arabidopsis seedlings subjected to light stress. Representative images (top), and a graph of the mean ± SE of fluorescence measurement of three individual experiments each having 10–12 seedlings (bottom) are shown. (B) Measurements of total glutathione levels in control and cPTIO- (200 µM) treated Arabidopsis plants subjected to light stress. A representative graph is shown with the mean ± SE of fluorescence measurement of five individual experiments each performed with three technical repeats having 8–12 plants each. Different letters denote significant differences (P < 0.05) among means at different time points within a group (control and cPTIO) according to ANOVA (Duncan’s test). cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Scale bar = 1 mm. The apx1 mutant does not accumulate glutathione and NO during its rapid response to light stress To study further the rapid response of Arabidopsis to light stress, we compared wild-type plants and a mutant deficient in cytosolic ascorbate peroxidase 1 (apx1). The apx1 mutant is deficient in H2O2 scavenging and accumulates high levels of H2O2 during excess light stress, compared with the wild type (Pnueli et al. 2003, Davletova et al. 2005). In addition, a large number of proteins in apx1 undergo carbonylation (oxidative damage) during light stress, and the expression of a large number of transcripts, including many different transcription factors, is augmented in apx1 during light stress (Davletova et al. 2005). Interestingly, the apx1 mutant does not appear to oxidize ascorbic acid during light stress, suggesting that the function of the ascorbate–glutathione cycle in this mutant during light stress is altered (Suzuki et al. 2013). A comparison of the rapid metabolic response to light stress between wild-type plants and apx1 is shown in Fig. 5. In the absence of light stress, 84 metabolites were altered in the apx1 mutant compared with the wild type (Supplementary Tables S1, S2). These belonged to several different pathways, including pathways involved in glutathione and ascorbic acid metabolism (Fig. 5A;Supplementary Tables S1, S2). As shown in Fig. 5B, the response of the apx1 mutant to light stress was different from that of the wild type, with only 26 compounds shared between the wild type and apx1 during the light stress response. As shown in Fig. 6, in contrast to the wild type, free GSH levels decreased during light stress, and free GSSG levels declined and then increased (opposite to the wild type) during light stress in apx1. These results suggest that apx1 plants do not accumulate significant levels of free glutathione during light stress. Because we observed a link between NO and GSH accumulation in wild-type plants (Fig. 4), we also measured the accumulation of NO in apx1 plants during light stress. As shown in Supplementary Fig. S6, NO levels did not accumulate in apx1 plants during early stages of light stress. This observation was also supported by mainly non-significant changes in the levels of the arginine–citrulline pathway metabolites (Supplementary Fig. S7). Fig. 5 View largeDownload slide Differences between the rapid metabolic response of the wild type and the apx1 mutant to light stress. (A) Number of metabolites associated with different pathways significantly altered between the wild type and the apx1 mutant at time 0. Significant differences (P ≤ 0.05) were calculated based on Student’s two-tailed t-test and pathway involvement was analyzed using the KEGG database. (B) Venn diagrams showing the total number of metabolites commonly or differentially altered between the wild type and the apx1 mutant during the response to light stress (top), the number of metabolites commonly or differentially up-regulated between the wild type and the apx1 mutant during the response to light stress (middle) and the number of metabolites commonly or differentially down-regulated between the wild type and the apx1 mutant during the response to light stress (bottom). Significant difference (P ≤ 0.05) was calculated based on ANOVA (Duncan’s test). A list of all metabolites altered in the wild type and the apx1 mutant is shown in Supplementary Table S1. Fig. 5 View largeDownload slide Differences between the rapid metabolic response of the wild type and the apx1 mutant to light stress. (A) Number of metabolites associated with different pathways significantly altered between the wild type and the apx1 mutant at time 0. Significant differences (P ≤ 0.05) were calculated based on Student’s two-tailed t-test and pathway involvement was analyzed using the KEGG database. (B) Venn diagrams showing the total number of metabolites commonly or differentially altered between the wild type and the apx1 mutant during the response to light stress (top), the number of metabolites commonly or differentially up-regulated between the wild type and the apx1 mutant during the response to light stress (middle) and the number of metabolites commonly or differentially down-regulated between the wild type and the apx1 mutant during the response to light stress (bottom). Significant difference (P ≤ 0.05) was calculated based on ANOVA (Duncan’s test). A list of all metabolites altered in the wild type and the apx1 mutant is shown in Supplementary Table S1. Fig. 6 View largeDownload slide Rapid changes in the level of glutathione and metabolites involved in glutathione biosynthesis and degradation in apx1 plants subjected to light stress. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). GSH and GSSG, reduced and oxidized glutathione, respectively; γ-glutamyl-AA, γ-glutamyl amino acid. Fig. 6 View largeDownload slide Rapid changes in the level of glutathione and metabolites involved in glutathione biosynthesis and degradation in apx1 plants subjected to light stress. Line graphs show the mean ± SE of relative abundance (natural log of the scaled intensity) of metabolites over different periods of light stress treatment from five individual experiments (n = 5). Different letters denote significant differences among means at P < 0.05 according to ANOVA (Duncan’s test). GSH and GSSG, reduced and oxidized glutathione, respectively; γ-glutamyl-AA, γ-glutamyl amino acid. As shown in Fig. 6, the decrease in free GSH levels in apx1 plants was accompanied by a decrease in the level of the glutathione precursor cysteine, as well as slower accumulation of glycine. In addition, the levels of cysteinyl-glycine that gives rise to cysteine and glycine were suppressed (Fig. 6). These results suggest that glutathione biosynthesis is suppressed in the apx1 mutant during light stress. Discussion We previously reported that in response to light stress, plants mount a rapid transcriptional response, and that at least five of the transcripts that are specifically up-regulated at 20–60 s post-light stress application are essential for plant acclimation to light stress (Suzuki et al. 2015). Here we expanded on our analysis of the ultra-fast response of plants to light stress and identified rapid accumulation in the free and total levels of glutathione, a key regulator of redox reactions, acclimation and defense responses in plants (Foyer and Noctor 2011, Noctor et al. 2012, Foyer and Noctor 2013), as part of this rapid response (Figs. 2, 4B; Supplementary Fig. S8). The changes in free and total GSH during light stress (Figs. 2, 4B) could reflect a response of the glutathione pool to the oxidative conditions that are thought to accompany the initial stages of light stress in plants (Dietz 2015, Suzuki et al. 2015). Thus, glutathione could play an important role in redox regulation during early events of light stress acclimation. This possibility is supported by studies that showed altered tolerance to light stress in plants with altered content of glutathione (Creissen et al. 1999). A meta-analysis of the rapid transcriptional response of Arabidopsis to light stress (20–60 s; Suzuki et al. 2015) also supports this possibility, revealing that the steady-state transcript level of at least eight different transcripts associated with glutathione function in plants, including glutathione peroxidase 1 (GPX1), is significantly altered during early responses to light stress (Supplementary Fig. S8). Interestingly, in contrast to the level of free GSH that peaked at 90 s (Fig. 2), the level of total GSH, that includes both bound and free glutathione, peaked at 60 s (Fig. 4B), suggesting that the rapid response to light stress in Arabidopsis involved alterations in the ratio between free and bound GSH and/or GSSG in cells. These alterations could include release of glutathione from conjugated forms to increase the level of free GSH/GSSG (Fig. 2), and/or binding of glutathione to different molecules and proteins in cells that will decrease the level of free GSH/GSSG (Fig. 2), but enhance the level of total glutathione in cells (Fig. 4B). The reactions outlined above could simultaneously alter the levels of free and/or conjugated glutathione in similar or opposing manners in different subcellular compartments differentially affecting the total levels of free and bound glutathione in cells (Figs. 2, 4B; Noctor et al. 2011). As expected in C3 plants, the rapid metabolic response to light stress appeared to activate the photorespiration pathway (Supplementary Fig. S2). This activation was accompanied by the accumulation of glycine and the depletion of serine (Supplementary Fig. S2). Coupled with the depletion of threonine (Supplementary Fig. S4), these changes that occur during early stages of light stress in Arabidopsis might promote the biosynthesis of glutathione (Fig. 2; Mullineaux and Rausch 2005). It is interesting to note that in contrast to our earlier study conducted with more mature plants (Suzuki et al. 2013), the level of serine declined rather than increased during light stress. This finding could suggest that in contrast to glycine, the level of serine during early responses to light stress may depend on the age and developmental stage of the plant. Although the availability of glycine is generally believed to be one of the main rate-limiting factors in glutathione biosynthesis, our comparison between wild-type plants that accumulated glutathione and apx1 plants that did not (Figs. 2, 6) revealed that the level of γ-glutamylcysteine could also play an important role in this context. Thus, although glycine levels accumulate in apx1 plants during light stress, glutathione did not accumulate in these plants (Fig. 6). In contrast, γ-glutamylcysteine that accumulated in wild-type plants (Fig. 2) did not accumulate in apx1 mutants, raising the possibility that at least in apx1 plants the availability of γ-glutamylcysteine may play an important role in regulating glutathione biosynthesis. The plant hormone NO regulates the activity of many different enzymes and proteins in plants during stress (Wilson et al. 2008). These include different enzymes of the photorespiration pathway (Supplementary Fig. S2; Abat et al. 2008, Palmieri et al. 2010, Ortega-Galisteo et al. 2012). Because NO could inhibit the carboxylation reaction of Rubisco (Abat and Deswal 2009), it could drive photorespiration in the direction of glycolate and glycine accumulation (Supplementary Fig. S2). Further enhancement in glycine could also occur if NO inhibits glycine decarboxylase (Palmieri et al. 2010) and prevents glycine from being converted into serine (partially explaining the decrease in serine levels; Supplementary Fig. S2). The potential outcome of these effects of NO on photorespiration could therefore be a rapid enhancement in glycine accumulation that could be used in the biosynthesis of glutathione (Fig. 2; Mullineaux and Rausch 2005). Our findings that NO accumulates during the rapid response of Arabidopsis to light stress (Fig. 4A), and that when the accumulation of NO is prevented, total glutathione does not accumulate during this response (Fig. 4B), provide further support for the possibility that NO regulates glutathione accumulation during the rapid response of plants to light stress, potentially via its effects on photorespiration. Of course further studies are needed to address this possibility. A possible role for NO in regulating GSH biosynthesis was previously reported in Medicago truncatula roots (Innocenti et al. 2007), and in Arabidopsis plants subjected to pathogen attack (Kovacs et al. 2015), further supporting this possibility. In addition, our analysis of the response of apx1 plants to rapid changes in light intensity demonstrated that both glutathione and NO accumulation are impaired in this mutant that is sensitive to light stress (Figs. 6;Supplementary Figs. S6, S7; Pnueli et al. 2003, Davletova et al. 2005), further linking glutathione and NO accumulation to light stress acclimation. An interesting finding originating from our metabolic analysis of rapid responses to light stress, presented in this study, is the rapid depletion in the pool of PEP in cells (Fig. 3). PEP is an essential metabolite that is at the crossroads between several different metabolic pathways leading to energy production in the mitochondria, jasmonic acid and salicylic acid biosynthesis, organic and amino acid biosynthesis and production of phenolic compounds (Prabhakar et al. 2010, Dizengremel et al. 2012). From the standpoint of immediate energy production, the conversion of PEP to pyruvate generates ATP that could be used as an energy source to maintain the cell at a high redox state. Thus, PEP could play an important role in plant acclimation to rapid changes in light conditions. Taken together, our findings should be viewed as an initial metabolic analysis of the response of plants to rapid (0–90 s) changes in their environment. At least three main findings stem from our analysis and could lead to further studies. They include a role for glutathione as a redox and signaling molecule, NO as a signaling hormone and PEP as an immediate energy and carbon source. Our study further poses an interesting question: is it possible to augment the initial metabolic response of plants to rapid changes in environmental conditions by altering the expression of particular genes and/or the level of particular metabolites? For example, would it be possible to improve on the pool, or fluxes controlling the pool, of PEP or other metabolites that are rapidly depleted during rapid response to stress? Strengthening, or augmenting, the rapid response of plants to rapid changes in abiotic stress conditions, such as light intensity, could have a far-reaching effect on the overall acclimation process and tolerance of plants to stress (Suzuki et al. 2015), thereby increasing yield under field conditions (Kromdijk et al. 2016). Further studies are needed to address these questions. In addition, because many of the compounds measured by our analysis are involved in several different pathways in the cell and could be simultaneously altered in different manners (up or down) in different cellular compartments, further studies, in particular compartment-specific and pulse–chase flux analyses, are needed to resolve the role and association of different compounds with each other and with the metabolic pathways they are connected with. Our analysis should therefore be viewed as an initial identification of different pathways and metabolites that could be used to augment the rapid response of plants to abiotic stress. In particular, it highlights the important role of glutathione, PEP and NO for these responses. Materials and Methods Plant material and growth condition Arabidopsis thaliana ecotype Columbia and apx1 (Davletova et al. 2005) were grown in peat pellets (Jiffy-7, Jiffy, http://www.jiffygroup.com/en/) at 23°C under constant low light (50 μmol m−2 s−1) as previously described (Davletova et al. 2005, Suzuki et al. 2011, Luhua et al. 2013). Light stress treatment For metabolite profiling (free GSH and GSSG), and total glutathione assays, 4–5 plants grown in a peat pellet, as described in Suzuki et al. (2015), were exposed to a light intensity of 1,000 µmol m–2 s–1 at 22°C for a period of 0, 20, 60 and 90 s (Suzuki et al. 2015). From 20 to 25 different plants were used for each time point, and the experiment was conducted with a total of five biological repeats. Samples were collected by dipping the plants in liquid nitrogen immediately after stress treatment. Frozen tissue was collected into a falcon tube, ground into a fine powder and transferred into a 1.5 ml tube (about 250–300 mg per tube). Samples were kept frozen during the whole collection process and stored at –80°C. For treatment with the NO scavenger cPTIO (Sigma-Aldrich), wild-type plants grown in cookie were sprayed with water (control) or 200 µM cPTIO, incubated for 2 h and exposed to light stress for 0, 20, 60 and 90 s. Plants were then flash-frozen in liquid nitrogen (Suzuki et al. 2015) and assayed for their total glutathione content as described below. Eight to twelve different plants were used for each time point in three technical repeats, and the experiment was conducted with a total of five biological repeats. Metabolic analysis Metabolic analysis using ultra performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) analysis was conducted as a service by Metabolon, Inc. (Evans et al. 2009, Holt et al. 2016). The liquid chromatography (LC)-MS) analysis was performed using a Waters ACQUITY UPLC and a Thermo-Finnigan LTQ mass spectrometer. Raw data extraction, peak identification and processing for quality control were performed using Metabolon’s hardware and software. Compounds were identified by comparison with library entries of purified standards. Metabolon maintains a compound library containing the retention time/index (RI), mass to charge ratio (m/z) and MS/ MS spectral data based on an authenticated standard. Compounds are identified based on three criteria: retention index within a narrow RI window of the proposed identification, nominal mass match to the library ±0.4 amu and the MS/MS spectral similarity between experimental data and authentic standards. The combination of three categories of data gives enough deciding power to differentiate and distinguish between the compounds even in cases where there is similarity in one of these factors. This analysis identified 413 compounds of known identity and their peak areas were taken as a measure of intensity. The intensity of each compound was rescaled to set the median value equal to 1. The natural log of the scaled intensity was then calculated for each compound for graphical representation and statistical analysis. Quantitation of NO NO content was measured by a modified protocol from Gross et al. (2017). Five-day-old wild-type and apx1 seedlings grown in 1/2 Murashige and Skoog (MS) media were stained with 15 µM DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; Sigma-Aldrich) for 1 h. Staining was performed in 1/2 MS media with continuous shaking. Seedlings were then washed with the 1/2 MS media by continuous shaking for 15 min. As a control, the NO scavenger cPTIO (200 µM) was added along with DAF-FM DA during staining in duplicate samples. Seedlings were then exposed to a light intensity of 1,000 µmol m–2 s–1 for a period of 0, 1, 2 and 5 min, and immediately used for imaging. Imaging was performed with a fluorescence microscope (AMG Evos, X4 objective) using green fluorescence protein (GFP; excitation filter 495 nm and emission filter 515 nm) filter settings. Images were further analyzed with ImageJ software to quantify fluorescence intensity. Four biological experiments were performed with 10–12 technical repeats per time point each. Total glutathione measurement Total glutathione was measured using the glutathione reductase recycling method described in Queval and Noctor (2007) with modifications. Ground samples were freeze dried for 72 h at –40 °C in a Labconco freeze drier. A 2 mg aliquot of the freeze-dried tissue was collected in a 1.5 ml tube and total glutathione was extracted with 600 μl of 0.2 N HCl. The extract was neutralized with 0.2 M NaH2PO4 (pH 5.6) and 0.2 M NaOH. The assay was performed in a 96-well plate in three technical replicates, and absorbance (412 nm) was measured in a plate reader (Synergy BioTek) equipped with Gene5 software in a kinetic setting. To calculate total GSH content, the rate of the reaction was measured for the first 2 min. Statistical analysis and pathway analysis Analysis of variance (ANOVA) followed by a post-hoc Duncan’s test was used to identify compounds significantly altered over time using SPSS software (Paulose et al. 2013, Suzuki et al. 2015, Holt et al. 2016). Significant differences between mean values of the wild type and the apx1 mutant at the 0 s time point were assessed by a Student’s two-tailed t-test (Suzuki et al. 2015). Differences at P < 0.05 were considered significant. Pathway analysis was performed using TAIR (https://www.arabidopsis.org/) and KEGG (http://www.kegg.jp/) pathway analysis tools to generate different metabolic maps and figures as described in Holt et al. (2016). Funding This work was supported the National Science Foundation [IOS-1353886, IOS-1063287, IOS-1557787, MCB-1613462] and the University of North Texas, College of Science. Disclosures The authors have no conflicts of interest to declare. References Abat J.K. , Deswal R. ( 2009 ) Differential modulation of S-nitrosoproteome of Brassica juncea by low temperature: change in S-nitrosylation of Rubisco is responsible for the inactivation of its carboxylase activity . Proteomics 9 : 4368 – 4380 . Google Scholar Crossref Search ADS PubMed Abat J.K. , Mattoo A.K. , Deswal R. ( 2008 ) S-nitrosylated proteins of a medicinal CAM plant Kalanchoe pinnata—ribulose-1,5-bisphosphate carboxylase/oxygenase activity targeted for inhibition . FEBS J. 275 : 2862 – 2872 . Google Scholar Crossref Search ADS PubMed Asada K. ( 1999 ) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons . Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 : 601 – 639 . 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Journal

Plant and Cell PhysiologyOxford University Press

Published: Sep 1, 2018

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