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A dual role for glutathione transferase U7 in plant growth and protection from methyl viologen-induced oxidative stress

Abstract Plant glutathione S-transferases (GSTs) are glutathione-dependent enzymes with versatile functions, mainly related to detoxification of electrophilic xenobiotics and peroxides. The Arabidopsis (Arabidopsis thaliana) genome codes for 53 GSTs, divided into seven subclasses; however, understanding of their precise functions is limited. A recent study showed that class II TGA transcription factors TGA2, TGA5, and TGA6 are essential for tolerance of UV-B-induced oxidative stress and that this tolerance is associated with an antioxidative function of cytosolic tau-GSTs (GSTUs). Specifically, TGA2 controls the expression of several GSTUs under UV-B light, and constitutive expression of GSTU7 in the tga256 triple mutant is sufficient to revert the UV-B-susceptible phenotype of tga256. To further study the function of GSTU7, we characterized its role in mitigation of oxidative damage caused by the herbicide methyl viologen (MV). Under non-stress conditions, gstu7 null mutants were smaller than wild-type (WT) plants and delayed in the onset of the MV-induced antioxidative response, which led to accumulation of hydrogen peroxide and diminished seedling survival. Complementation of gstu7 by constitutive expression of GSTU7 rescued these phenotypes. Furthermore, live monitoring of the glutathione redox potential in intact cells with the fluorescent probe Grx1-roGFP2 revealed that GSTU7 overexpression completely abolished the MV-induced oxidation of the cytosolic glutathione buffer compared with WT plants. GSTU7 acted as a glutathione peroxidase able to complement the lack of peroxidase-type GSTs in yeast. Together, these findings show that GSTU7 is crucial in the antioxidative response by limiting oxidative damage and thus contributes to oxidative stress resistance in the cell.


Introduction
In plants, animals, and prokaryotes, glutathione S-transferases (GSTs) constitute a large family of enzymes with a wide range of cellular functions (Basantani and Srivastava, 2007). In plants, GSTs are mainly involved in the detoxification of exogenous xenobiotics and intracellular oxidized molecules, such as lipid peroxides, to alleviate chemical and oxidative damage (Sappl et al., 2009;Chronopoulou et al., 2017a) and anthocyanin transport to the vacuole (Alfenito et al., 1998;Mueller et al., 2000;Gomez et al., 2011). GST-mediated cellular detoxification is largely based on two enzymatic activities: the transferase and the peroxidase activities. As transferases, GSTs mediate the conjugation of the reduced tripeptide glutathione (GSH) with the electrophilic center of different organic molecules Labrou et al., 2015). The resulting conjugates are subsequently exported from the cytosol to the vacuole or the apoplast (Cummins et al., 2011). As peroxidases, some GSTs exhibit a GSHdependent glutathione peroxidase (GPOX) activity which allows the reduction of lipid peroxides to alcohols (Edwards et al., 2000;Dixon et al., 2009). Plant GSTs are also involved in other processes such as secondary metabolism and development (Roxas et al., 1997;Chen et al., 2007;. Expression of several GSTs is boosted under biotic and abiotic stresses, for example pathogen infection (Mauch and Dudler, 1993), high salt concentrations (Roxas et al., 2000;Du et al., 2019), hypoxia (Moons, 2003), UV-B radiation (Loyall et al., 2000), exposure to safeners (DeRidder et al., 2002;Cummins et al., 2009), herbicides , oxylipins Stotz et al., 2013), or salicylic acid (SA) (Chen et al., 1996;Sappl et al., 2009). The common denominator of these stress conditions is that they all trigger the production of reactive oxygen species (ROS) and hence cause oxidative stress. The induction of GSTs across this extensive span of conditions and treatments has strongly reinforced the idea of their protective role against oxidative stress (Chen et al., 2012;Chronopoulou et al., 2017b).
In Arabidopsis (Arabidopsis thaliana), the GST gene family counts 53 members of which only a few are functionally characterized (Sylvestre-Gonon et al., 2019). Based on their protein sequence and function, the GSTs are divided into seven distinct subclasses (Supplemental Figure S1, A; Wagner et al., 2002). The subclasses phi (F) and tau (U) are plant-specific and constitute the largest classes with 14 and 28 members, respectively (Chronopoulou et al., 2017a). Tolerance to herbicides mediated by GSTs relies on the capacity of GSTUs and GSTFs to conjugate electrophilic herbicides or their derivatives to GSH (Cummins et al., 1999;Edwards et al., 2000). Furthermore, studies on weeds indicate that herbicide resistance is mediated by GSTF isoforms and depends on preventing the accumulation of cytotoxic organic hydroperoxides caused by herbicide applications, rather than by the direct detoxification of the herbicide itself (Cummins et al., 1999).
The detoxification activities of GSTs strictly depend on GSH and thus are tightly interconnected with the cellular redox state . GSH is the most abundant low-molecular-weight thiol in the cell and is involved in a number of key cellular functions. Direct or indirect use of GSH as an electron donor for ROS scavenging leads to formation of glutathione disulfide (GSSG) (Noctor et al., 2012). Based on in vivo measurements with genetically encoded fluorescent probes, the GSH/GSSG ratio in subcellular compartments that contain a functional glutathione reductase has been calculated to be in the order of 50,000:1 (Schwarzländer et al., 2016). Stress situations and experimental systems like treatment with bacterial elicitors cause a pronounced oxidative shift (Nietzel et al., 2019). The shift in the GSH/GSSG ratio is a key feature of stress responses and is considered to be important for downstream signaling pathways and developmental programs (Meyer, 2008).
Methyl viologen (MV) is a broad-spectrum herbicide that acts in chloroplasts by redirecting electrons originating from photosystem I (PSI) to molecular oxygen (O 2 ), which generates superoxide (O 2 Á-) (Mano et al., 2001;Scarpeci et al., 2008). The enhanced production of O 2 Áhas made MV a model for photooxidative stress in plants and it has been shown recently that MV-induced photooxidative stress in plastids causes a pronounced release of H 2 O 2 into the cytosol . Similarly, in non-photosynthetic organisms, MV redirects electrons from complex I of the mitochondrial electron transport chain to O 2 , which also generates O 2 Á- (Cochemé and Murphy, 2008). Increased ROS production leads to lipid peroxidation, which is considered to be the main cause of MV toxicity in plant and mammalian cells (Bus et al., 1976;Misra and Gorsky, 1981;Liu et al., 2009).
Studies in plants have already demonstrated that GSTs contribute to MV tolerance. For instance, heterologous overexpression of cotton GST-cr1 in tobacco (Nicotiana tabacum) (Yu et al., 2003), a Suaeda salsa GST in rice (Zhao and Zhang, 2006), and rice GSTU4 in Arabidopsis (Sharma et al., 2014), as well as homologous overexpression of GSTU51 in poplar (Choi et al., 2013) and GSTU19 in Arabidopsis (Xu et al., 2016), consistently led to an improved MV tolerance when compared with wild-type (WT) controls. All these studies established a link between the tolerance phenotype and either an increased enzymatic antioxidant activity (GPOX, GST, CAT) and reduced oxidative damage upon MV treatment. The molecular function of GSTs in this context, however, remains unknown.
Expression of GSTU7 has been reported to be induced in plants exposed to a wide range of stress stimuli including the exposure to heavy metals (Becher et al., 2004), to oxylipins and phytoprostanes (Loeffler et al., 2005;Mueller et al., 2008;Stotz et al., 2013) and to SA (Blanco et al., 2009). SA also promotes the accumulation of GSTU7 protein in cell cultures (Sappl et al., 2004;Gruhler et al., 2005). For oxylipin treatment, expression of GSTU7 has been shown to be controlled by class II TGA transcription factors (TGA2, 5, and 6) (Stotz et al., 2013). Independent findings further support a role of class II TGA transcription factors in controlling the expression of a group of genes with putative antioxidant functions in response to SA (Blanco et al., 2009) andUV-B stress (Herrera-Vásquez et al., 2021). In the latter work, tga2 tga5 tga6 triple mutants (tga256) were more susceptible to oxidative stress caused by UV-B similar to the susceptibility of tga256 to SA established earlier (Zhang et al., 2003). Interestingly, at least 12 GSTUs including GSTU7 were induced by UV-B and shown to be controlled by the class II TGA transcription factors. The role of TGAs in expression of GSTU7 was further confirmed in ChIP assays showing binding of TGA2 to the palindromic TGA-box (TGACGTCA) in the GSTU7 promoter region under stress conditions in vivo (Herrera-Vásquez et al., 2021). It is unclear though why GSTU7 would be beneficial in stress situations that activate class II TGAs.
The large number of GSTs in plants and widespread functional redundancy among them make it challenging to study the function of specific GSTs and, therefore, little is known about their individual roles in Arabidopsis. In this study, we report a role of GSTU7 in plant growth and characterize GSTU7 as a key enzyme mediating the tolerance of Arabidopsis to oxidative stress induced by MV and explore its importance in cellular redox homeostasis.

Expression of GSTU7 is induced under different stress conditions
To determine regulation of GSTU7 expression induced by different stress conditions, Arabidopsis WT seedlings were treated with either 0.5 mM SA, UV-B radiation, or 50 mM MV and GSTU7 transcript levels determined at different time points (Figure 1, A-C). SA caused a six-fold induction of GSTU7 expression within 30 min, leading to a peak of seven-fold transcript level after 2 h compared with nontreated controls (Figure 1, A). Between 8 and 24 h after the start of the SA incubation, GSTU7 expression was again close to expression in control plants. UV-B radiation (Figure 1, B) induced a 2.3-fold increase in transcript levels after 2.5 h which slightly increased to three-fold at 24 h. Treatment of plants with MV resulted in the most pronounced induction of GSTU7 expression among the three treatments with a maximum of 25-fold after 24 h (Figure 1, C).
Recently, UV-B-induced expression of 12 GSTs including GSTU7 was found to be controlled by the class II TGA transcription factors TGA2, TGA5, and TGA6 (Herrera-Vásquez et al., 2021). To test whether the induction of GSTU7 in response to UV-B and MV (Figure 1, B and C) is dependent on class II TGAs or SA, UV-B-and MV-induced changes in GSTU7 transcript levels were compared between WT, the tga256 triple mutant, and the SA-deficient mutant sid2-2. In contrast to the response in WT plants, induction of GSTU7 expression was completely abolished in the tga256 triple mutant (Figure 1, D and E). In sid2-2 mutant, however, GSTU7 was still induced by treatments with UV-B and MV, Figure 1 GSTU7 gene expression is induced by SA, UV-B radiation (UV-B), and MV. A-C, mRNA accumulation of GSTU7 was determined by RT-qPCR in 2-week-old WT Arabidopsis seedlings treated with 0.5 mM SA in MS medium (A), UV-B radiation (B), or 50 lM MV in MS medium (C) (all shown by filled circles). For control treatments, seedlings were floated on MS medium (open circles) (A, C) or irradiated through a filter blocking UV-B (B). D and E, GSTU7 transcript levels measured by RT-qPCR in WT, tga256, and sid2-2 seedlings after 5 h of UV-B radiation (D) or after 24 h of incubation with 50 lM MV (E). Gene expression is expressed as fold change relative to a subunit of CLATHRIN ADAPTOR COMPLEX gene (for SA and MV) or the YLS8 (for UV-B) housekeeping genes and normalized to the expression at 0 h (A-C) or to the expression in untreated WT seedlings (D and E). Means ± SD (A-E) were calculated from three independent biological replicates (two to three seedlings pooled per replicate). The obtained data were tested for significant differences by ANOVA with Fisher's LSD test. * indicates differences compared with the expression at the start of the treatment (0 h; P 5 0.05). Different letters indicate statistically different groups (P 5 0.05). similarly to induction in WT plants (Figure 1,D and E). Altogether, the experiments showed that UV-B and MV stress-induced expression of GSTU7 is controlled by class II TGAs but not through a SA-dependent pathway.

Deletion of GSTU7 causes growth retardation
To further characterize the role of GSTU7, an insertional mutant, gstu7, was isolated and the T-DNA confirmed by sequencing to be inserted between A and T of the ATG start codon (Figure 2, A). Absence of detectable transcript confirmed gstu7 as a null mutant (Figure 2, B). For complementation, a UBQ10 pro :GSTU7-V5 construct was cloned and transformed into gstu7 plants. Two independent lines, gstu7/ GSTU7-V5 #2 and gstu7/GSTU7-V5 #5, were selected from a larger pool of 16 transformants based on high expression levels and protein abundance. As expected, treatment of WT plants with 50 mM MV caused increased GSTU7 expression, while in both complemented lines, GSTU7 transcripts were detected at higher levels than in the WT, without pronounced changes after MV treatment (Figure 2, B). Constitutive expression of GSTU7-V5 was confirmed by immunodetection of the V5 tag in both lines, albeit with much higher protein abundance in line #2 than in line #5 (Figure 2, C).
Homozygous gstu7 mutants developed a dwarf phenotype with shorter roots compared with WT seedlings (Figure 2, D and E), smaller rosettes ( Figure 2, F), and delayed bolting (Figure 2, G). Complementing the mutant with GSTU7-V5 restored gstu7 to a WT phenotype at all analyzed developmental stages. To confirm that the smaller phenotype of gstu7 was due to a single T-DNA insertion, gstu7 was backcrossed to a WT plant. The F2 progeny of this cross segregated with a 3:1 ratio for WT:short root mutant phenotypes (v 2 = 0.067; P = 0.79) (Figure 2, E).

GSTU7 mitigates oxidative stress induced by MV and 3-aminotriazole
The main function of GSTs is the detoxification of exogenously applied xenobiotics and endogenously generated organic peroxides, such as lipid peroxides (Sappl et al., 2009;Chronopoulou et al., 2017a). To further test whether GSTU7 is involved in developing plant tolerance to oxidative stress, the effect of MV on growth of WT, gstu7, and the two complemented lines #2 and #5 was investigated (Figure 3, A). Seedling tolerance to 0.1 mM MV was determined as survival rates after germination on plates containing MV and documented as the percentage of seedlings with green tissue relative to the total number of germinated seedlings following established protocols (Laporte et al., 2012). gstu7 showed a survival rate of 29% compared with WT seedlings of which 62% survived (Figure 3, A). Constitutive overexpression of GSTU7 cured the apparent increased susceptibility of gstu7 toward MV and led to a survival rate of about 90%, which is significantly higher than in WT plants (P = 0.0003). The same general pattern with increased sensitivity in gstu7 and diminished sensitivity in complemented gstu7 lines was found when seedlings were transferred to plates containing 50 nM MV after germination and then grown for 17 d (Figure 3, B).
The role of GSTU7 for alleviating herbicide-induced oxidative stress was also tested upon treatments with the catalase inhibitor 3-aminotriazole (3-AT), which is known to induce oxidative stress (May and Leaver, 1993; Figure 3, B and C). After germination on MS medium, WT, gstu7, and the complemented lines were transferred either to plates with MS or MS supplemented with 1 mM 3-AT (Figure 3, B). Herbicide tolerance was quantified for each line as the relative growth compared with the respective controls grown on MS without 3-AT. For WT seedlings, 3-AT restricted the growth to 74% compared with non-treated controls. This growth restriction was far more pronounced in gstu7, which reached only 30% of the biomass of the respective control seedlings grown without 3-AT (Figure 3, C). Both complemented lines on average reached 52% and 62% of the biomass of seedlings not treated with 3-AT and thus performed better than gstu7 on 3-AT.
Unlike the distinct sensitivity of gstu7 toward MV and 3-AT, the mutant did not show increased susceptibility toward pathogen infection with Pseudomonas syringae DC3000 AvrRpt2. While mutant lines, such as sid2-2 and tga256, which are known to be more susceptible (Zhang et al., 2003;Seguel et al., 2018), showed severe cell damage and ion leakage after infection and thus a high relative conductivity in the medium, the susceptibility of gstu7 mutants and the respective complementation lines were very similar to the WT plants (Supplemental Figure S2).
To further assess whether the increased susceptibility of gstu7 mutants to MV corresponds with a higher production of ROS, primary leaves were treated with a 2 mL drop of 30 lM MV, incubated under constant light conditions for 24 h and subsequently stained with 3,3 0 -diaminobenzidine (DAB) for hydrogen peroxide (H 2 O 2 ) detection ( Figure 3, D). For assessment of the staining, the DAB-stained areas on each leaf were masked out and set into relation to the whole area of the respective leaf ( Figure 3, D). After MV treatment, gstu7 seedlings had a three-fold larger area of intense staining than WT seedlings, which indicates the presence of increased amounts of H 2 O 2 . In contrast, complemented gstu7 seedlings after MV treatment showed even less DAB staining than WT seedlings. In this case, the staining was not significantly different from staining in control seedlings not treated with MV (#2, P = 0.2957; #5, P = 0.1257) (Figure 3, D).
As an orthogonal approach, MV-induced ROS production was detected with 2,7-dihydrodichlorofluorescein diacetate (H 2 DCF-DA) and quantified as fluorescence intensity per leaf area (Figure 3, E). While for most genotypes treatment with MV caused an increase in fluorescence the most pronounced increase in fluorescence was observed in gstu7 seedlings. Consistent with the earlier experiments, constitutive expression of GSTU7 in the gstu7 background was sufficient to revert the phenotype and limit the fluorescence to values observed in WT seedlings (Figure 3, E).
To better understand if the sensitivity of gstu7 to photooxidative stress is linked to an altered level of antioxidant capacity, GPOX activity was measured in protein extracts from WT, gstu7, and the complemented lines after a 4-h incubation in MV-containing solution. For all tested genotypes, MV consistently caused a decrease in GPOX activity. Surprisingly, protein extracts from gstu7 plants treated only with water contained less GPOX activity compared with WT or the complemented lines (Supplemental Figure S3, A). The diminished GPOX activity inversely corresponds with higher levels of lipid peroxides quantified as malondialdehyde (MDA) equivalents (Supplemental Figure S3, B). After treatment with MV both, WT and gstu7 seedlings contained increased amounts of MDA with values in gstu7 being significantly higher than in WT. Seedlings constitutively overexpressing GSTU7 had MDA levels similar to homozygous Figure 2 Phenotypic characterization of the Arabidopsis gstu7 mutant and gstu7/GSTU7-V5 complemented lines. A, Scheme of the GSTU7 gene indicating the site of T-DNA insertion in the gstu7 mutant line (SALK_086642). The TGA-box (gray) is around 50 bps upstream of the transcription start site ( + 1). Forward (Fw) and reverse (Rv) primer binding sites used for mRNA quantification are indicated. B, Quantification of GSTU7 transcripts in 2-week-old WT, gstu7, and two GSTU7 overexpression lines floated for 24 h in liquid MS medium alone (-) or supplemented with 50mM MV ( + ). As a negative control for the PCR, a reaction without cDNA was added (C-). A fragment of the YLS8 housekeeping gene was amplified as a reference. C, Immunoblot analysis of GSTU7 using an antibody against the V5 tag of the complement. Twenty micrograms of protein were loaded per lane. To verify even loading, the membrane was stained with Coomassie Blue (lower panel). D, Growth phenotype of 1-week-old seedlings. Photographs (upper panel) and quantification of the primary root length (lower panel), n = 24-35. E, Segregation of gstu7-linked short root phenotype. WT and gstu7 seedlings were grown vertically on plates and analyzed for their root length 5 d after germination. Segregation of a backcross resulted in a short root:normal WT root ratio of 1:3, n = 55-61. F, Rosette phenotypes of soil grown plants 4 weeks after germination, n = 12. G, Appearance of gstu7 and complemented lines 7 weeks after germination. For box plots in D, E, and F, Box = interquartile range between the lower and upper quartiles, center line = median, + = mean, whiskers = min and max values. Statistical analyses for D, E, and F were performed using ANOVA with Fisher's LSD test. Different letters indicate statistically different groups (P 5 0.05).
Figure 3 GSTU7 counteracts oxidative stress induced by MV and 3-AT. A, Phenotype of seedlings from WT, gstu7, and gstu7/GSTU7-V5 complemented lines (#2 and #5) grown on MS plates (Control) or MS plates supplemented with 0.1 mM MV. Tolerance of seedlings to MV was measured after 1 week on MV-containing plates as the survival rate, which is the percentage of plants with any green cotyledon over the total number of germinated seeds (n = 3, means + SD). B and C, Effect of 3-AT or MV on WT, gstu7, and gstu7/GSTU7 complemented seedlings. Seeds were germinated on MS plates and after 5 d transferred to new MS plates (Control) (B, left) or plates containing either 1 mM 3-AT or 50 nM MV (B, middle and right) on which plants were grown for 17 d. C, Fresh weight of the plants grown in MS or MS supplemented with 1 mM 3-AT or 50 nM MV (C, left plot) and the percentage of fresh weight of 3-AT or MV-treated plants relative to non-treated control plants (C, right plot), n = 7-8. D, Accumulation of H 2 O 2 in primary leaves exposed to MV. A 2 lL drop of 30 lM MV ( + ) or water (Control, -) was placed on the leaf surface of plants with different genotypes. Subsequently, the plants were kept under constant light for 24 h before H 2 O 2 accumulation was evaluated by staining with DAB. The intensity of DAB staining was quantified as the stained leaf area relative to the whole leave area (% DAB staining) (n = 9, means + SD). E, ROS accumulation in cotyledons of 7-d-old seedlings floated for 4 h in 100 mM MV ( + ) or water (control, -) under constant light. After the treatment, seedlings were stained for 30 min with 25 mM H 2 DCF-DA and imaged by confocal microscopy. Fluorescence was collected between 505 and 530 nm after excitation with 488 nm (upper panel). Signal intensity was quantified using FIJI and expressed as intensity per mm 2 of leaf area (lower panel) (n = 8-15). Statistical analyses were performed by ANOVA with Fisher's LSD test. Different letters indicate statistically different groups (P 5 0.05). For all box plots, Box = interquartile range between the lower and upper quartiles, center line = median, + = mean, whiskers = min and max values. gstu7 but did not show any further MDA accumulation after treatment with MV (Supplemental Figure S3, B).
A similar stress-induced increase in DAB staining was recently reported for tga256 after exposure to UV-B light (Herrera-Vásquez et al., 2021). Because the respective TGA transcriptions factors control the expression of GSTU7 (Figure 1, E), we tested whether tga256 is also more sensitive to MV and whether this sensitivity is mitigated by overexpression of GSTU7. Consistent with the hypothesis, tga256 mutants showed a survival rate of only about 4% compared with about 45% for the WT on plates containing 0.1 mM MV ( Figure 4). Constitutive overexpression of GSTU7 in tga256 increased the survival to WT level plants and thus rescued the hypersensitivity of tga256 to MV. Taken together, these results strongly suggest that GSTU7 plays a central role in mitigating the oxidative stress response triggered by MV.

GSTU7 is located in the cytosol and has GPOX activity
In Arabidopsis, most GSTs are predicted to be localized in the cytosol (Supplemental Figure S1). GSTU7 has been found in the Arabidopsis cytosolic proteome (Ito et al., 2011) and a GSTU7-GFP fusion has only been mentioned to be localized in the cytosol (Dixon et al., 2009). To further confirm these findings, we stably expressed a GSTU7-GFP fusion protein in Arabidopsis and visualized the localization on a confocal microscope ( Figure 5, A). All images consistently revealed cytosolic localization of the fusion protein. In all observations, the nuclear region was marked by a bright ring surrounding a dim region with low fluorescence indicating that the fusion protein is barred from the nucleoplasm.
Because of the pronounced mitigation of oxidative stress in GSTU7 overexpressing seedlings (Figure 3, B), the enzymatic activity of GSTU7 was further evaluated in vitro with the recombinant protein and 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate for GST activity and with either H 2 O 2 or cumene hydroperoxide (CHP) as substrates for a putative GPOX activity ( Figure 5, B and C). GSTU25, which has been reported to be the GST with the highest GPOX and GST activities in Arabidopsis (Dixon et al., 2009), was used as a reference. GSTU7 showed a minor GST activity and was outperformed 97-fold by GSTU25 ( Figure 5, B). Both GSTU7 and GSTU25 were capable of reducing H 2 O 2 with activities between 51.3 and 23.5 nmol NADPH min -1 mg -1 ( Figure 5, C). Activity of GSTU7 toward CHP was 87.3 nmol min -1 mg -1 and thus in the same order of magnitude as toward H 2 O 2 . In contrast, GSTU25 revealed a six-fold higher turnover of CHP compared with H 2 O 2 ( Figure 5, C).
To validate that the GPOX activity of GSTU7 observed in vitro has biological relevance in vivo, we tested whether GSTU7 can complement a yeast mutant deficient in the respective activity. The gtt1D gtt2D mutant in Saccharomyces cerevisiae lacks two key GSTs and has been reported to be sensitive to H 2 O 2 and organic peroxides (Choi et al., 1998;Collinson and Grant, 2003;Figure 5, D and E). This yeast strain was transformed with either GSTU7 or an empty vector control. In the absence of exogenous oxidants, transformed and non-transformed cultures showed the same logarithmic growth curves. Supplementing YPD with either 1.5 mM H 2 O 2 (Figure 5, D) or 0.09 mM CHP (Figure 5,E) abolished growth of the cultures almost completely. In medium supplemented with H 2 O 2 , growth resumed only after a lag phase of 10 h. Cells expressing GSTU7 showed an even longer lag phase of 14 h but reached the same density after 24 h ( Figure 5, D). Incubation with CHP completely abolished growth of non-transformed cultures while gtt1D gtt2D cells expressing GSTU7 were able to survive and regain growth after 20 h ( Figure 5, E). This supports the hypothesis that GSTU7 possesses GPOX activity in vivo, especially toward CHP.
Because selected glutathione peroxidase (GPX) proteins have been shown to act as protein thiol oxidases and thus can be exploited as H 2 O 2 sensors (Delaunay et al., 2002;Gutscher et al., 2009), we also tested whether GSTU7 also is capable of transferring the primary oxidation of its catalytic cysteine resulting from peroxide detoxification to roGFP2 as a target protein. In contrast to bovine GPX used as a positive control, GSTU7 was not able to mediate a H 2 O 2 -dependent oxidation of roGFP2 (Supplemental Figure S4).

GSTU7 protects cytosolic glutathione from MVinduced oxidation
Massive accumulation of ROS induced by MV treatment should also lead to a gradual oxidation of the glutathione redox buffer in the cell. With Grx1-roGFP2 as a fully dynamic probe for the local E GSH , the impact of MV and the role of GSTU7 on the oxidative load in the cytosol can be monitored in living cells over extended time periods. To test whether GSTU7 protects the cell from increased and ultimately deleterious oxidation, we monitored the cytosolic E GSH with Grx1-roGFP2 in vivo, using WT, gstu7, and gstu7/ GSTU7-V5 (#5 line) plants as genetic background and quantified by HPLC the levels of reduced and oxidized glutathione in the non-fluorescent genetic backgrounds. Stable homozygous roGFP2 reporter lines were selected and confirmed to show sufficiently high fluorescence intensities and typical spectral properties of the sensor with two excitations peaks and ratiometric properties using either a confocal microscope or a plate reader (Supplemental Figure S5, A-C).
Changes in the roGFP2 fluorescence ratio triggered by MV treatments were measured in leaf discs of WT, the gstu7 mutant, and one of the complemented lines all expressing the Grx1-roGFP2 sensor ( Figure 6, A-C). Considering that manipulation of the leaf discs itself can trigger a stress response resulting in an altered redox state (Rosenwasser et al., 2010), leaf samples were initially incubated with 10 mM dithiothreitol (DTT) to attain complete and uniform reduction of the sensor. With this precaution, all measurements started with the sensor at the same level of reduction, serving as the reference point for later ratio normalization. After pre-reduction of the sensor, DTT was removed and leaf discs were incubated either with 100 lM MV or with water as control. WT plants treated with MV showed a gradual increase in the fluorescence ratio over time indicating gradual oxidation of the glutathione pool ( Figure 6, A and Supplemental Figure S6). This increase in the fluorescence ratio was more pronounced in gstu7 plants (Figure 6, B and Supplemental Figure S6). In contrast, leaves expressing GSTU7 showed no MV-induced oxidation and Grx1-roGFP2 remained almost completely reduced throughout the entire time course of 15 h ( Figure 6, C and Supplemental Figure  S6).
Glutathione quantification in extracts of all lines confirmed that MV treatment caused a decrease in total glutathione compared with the control conditions. At the same time increased relative amounts of GSSG led to a decrease in the GSH/GSSH ratio, indicating an overall oxidation of the glutathione pool ( Figure 6, D and E). This oxidation was slightly more pronounced in gstu7 (1.71 ± 0.61) than in the WT (3.64 ± 1.29). In contrast, the line gstu7/GSTU7-V5 (#5) that constitutively express GSTU7 showed a consistently higher GSH/GSSG ratio compared with both WT and gstu7 (7.27 ± 3.80). The ratio in this case was not significantly different (ANOVA with Fisher's LSD test, P = 0.4639) between MV-treated samples and control seedlings (Figure 6, E).

Discussion
It is well established that GSTU7 is strongly induced under different abiotic stress conditions and that many GSTs are induced by SA and other phytohormones (Marrs, 1996; Sylvestre-Gonon et al., 2019). Our results confirm that SA Figure 5 GSTU7 is located in the cytosol and has GPOX activity. A, Confocal microscopy images of Arabidopsis pavement cells and stomata stably expressing 35S pro :GSTU7-GFP. Panels show GFP fluorescence (k ex = 488 nm; k em = 505-530 nm), chlorophyll autofluorescence (k ex = 488 nm; k em = 650-695 nm), and transmitted light. Merge images are a projection of the previous channels. The arrow head indicates the nucleus (n) lacking GFP signal. B, GST activity of recombinant GSTU7 and GSTU25 was measured as the conjugation of CDNB with GSH (means + SD of n = 21 for GSTU7 and n = 3 for GSTU25). C, GPOX activity of GSTU7 and GSTU25 upon incubation with H 2 O 2 or CHP was measured as consumption of NADPH in a coupled assay with glutathione reductase reducing the produced GSSG (means + SD of n = 4-9). Statistical analysis in panels B and C was performed using ANOVA with Fisher's LSD test. Different letters indicate statistically different groups (P 5 0.05). D and E, Heterologous expression of GSTU7 in a gtt1D gtt2D yeast strain deficient in the two dominant GSTs. Yeast growth was followed in cells incubated in liquid YPD media (closed symbols, solid line) or YPD supplemented with 1.5 mM H 2 O 2 (D) or 0.09 CHP (E) (open symbols; dashed lines). nt, non-transformed; ev, empty vector control. Data represent means + SEM, n = 4. induces the expression of GSTU7 within a few hours of application. In addition, there is also a medium to long-term response resulting in a very pronounced induction of GSTU7 expression 5 h after irradiation with UV-B or 24 h after application of MV (Figure 1, D). Both transcriptional responses are mediated by class II TGAs but are independent of the endogenous production of SA (Figure 1, D). This indicates that GSTU7 expression is controlled by at least two separate signaling pathways that are either SA-or TGA256dependent. A common response to stress situations is the formation of ROS, resulting in secondary responses like lipid peroxidation, which are generally summarized as oxidative stress (Waszczak et al., 2018;Smirnoff and Arnaud, 2019). UV-B triggers an accumulation of H 2 O 2 and the finding that GSTU7 can complement the UV-B-sensitive phenotype of tga256 led to the hypothesis that GSTU7 has an antioxidative function (Herrera-Vásquez et al., 2021). MV treatment is a classical means of inducing oxidative stress in green tissues because MV transfers electrons from PSI to molecular O 2 to form superoxide (O 2 Á-) (Mano et al., 2001). O 2 Áis subsequently converted to H 2 O 2 , which is not contained in plastids but also reaches the cytosol . Increased susceptibility of gstu7 null mutants to MV or 3-AT and the more intense MV-induced DAB and H 2 DCF-DA staining was completely reverted by overexpression of GSTU7 (Figure 3). Although it cannot be fully excluded that DAB staining is also responsive to increased level of phenolic compounds (Veljovic-Jovanovic et al., 2002), all evidences presented here are consistent with the hypothesis that more intense DAB staining is a reliable proxy for the increased production of H 2 O 2 . Even with the confirmatory results from H 2 DCF-DA staining, it cannot be distinguished though whether the increased production of H 2 O 2 is a primary effect caused by MV-induced photooxidative stress in plastids or a secondary effect due to the increase of intracellular lipid peroxides. Deletion of GSTU7 resulted in a dwarf phenotype, which was consistently smaller than WT plants throughout its life cycle (Figure 2, D-G). Delayed bolting of gstu7 is similar to phenotypes of gstf2 knockdown mutants and gstu17 Figure 6 GSTU7 prevents MV-induced oxidation of cytosolic glutathione. A-C, Grx1-roGFP2 was expressed in Arabidopsis WT, gstu7, and gstu7/ GSTU7-V5 #5 backgrounds. Leaf discs from stably transformed plants were placed in a 96-well plate and treated with 10 mM DTT to fully reduce the sensor (first black arrow). Subsequently, DTT was removed and the samples were treated with either 100 lM MV or water as control (second black arrow). The redox state of the sensor was followed over time as the ratio between the fluorescence excited at 410 and 480 nm. Ratio values were normalized to the fully reduced sensor after addition of DTT. The dotted lines indicate the ratios for fully reduced and fully oxidized roGFP2 measured at the end of the time course. All data indicate the mean fluorescence + SD of six samples from independent plants. D and E, HPLC analysis of reduced glutathione (GSH) and glutathione disulfide (GSSG) in WT, gstu7, and gstu7/GSTU7-V5 #5 seedlings. Samples were floated in water (-) or 100 mM MV ( + ) for 15 h under constant light. Samples were also collected before any treatment (0). Data represent means + SD, n = 3-4. null-mutants (Gong et al., 2005;Jiang et al., 2010;Chen et al., 2012). While loss of GSTU17 expression has no impact on the development of young seedlings under control conditions , loss of GSTU7 appears to affect the plant in a more general form resulting in defects in all developmental stages. Conversely, overexpression of a Brassica campestris GSTU closely related to Arabidopsis GSTU24 in Arabidopsis resulted in bigger rosettes, longer primary roots, and an increased number of lateral roots (Kao et al., 2016). At the molecular level, GSTU17 controls plant development by regulating far-red light signaling through interaction with phytochrome A (PHYA) . Recently, selective binding of natural products has been shown for GSTU19 and GSTF2 from Arabidopsis (Dixon and Edwards, 2018). Also immobilized recombinant GSTU7 was found to specifically interact with glutathionylated protoporphyrin after HPLC-MS analysis. However, no binding of unique ligands was found after heterologous expression in Nicotiana benthamiana leaves (Dixon and Edwards, 2018).
It is established that GSTUs and GSTFs are subject to complex transcriptional and post-translational regulation in response to infection, abiotic stress, and development (Gullner et al., 2018;Kumar and Trivedi, 2018;Sylvestre-Gonon et al., 2019). The list of abiotic stress factors affecting GST expression has recently expanded to UV-B, which induces the expression of 12 GSTUs including GSTU7 via class II TGA transcription factors (Herrera-Vásquez et al., 2021). Based on these and our own observations, it is tempting to speculate about a function of GSTU7 in ROS metabolism and signaling even though the exact mechanism remains unknown at this stage. Interestingly, the formation of H 2 O 2 has been associated with root development and a set of different peroxidases to control the transition from proliferation to differentiation in the root by balancing ROS between root zones (Tsukagoshi et al., 2010;Eljebbawi et al., 2021). It remains unknown though whether the observed growthinhibitory effects of MV in roots are a secondary effect caused by diminished carbohydrate supply from the shoot or whether it is a primary local effect. Like in mammalian cells, MV is known to also affect the mitochondrial electron transport chain in plants leaving the possibility for an alternative primary source of ROS (Shapiguzov et al., 2019).
Previous studies showed that GSTs mediate detoxification of several classes of herbicides by conjugating GSH to their electrophilic centers (Timmerman, 1989;Prade et al., 1998). Accordingly, plants overexpressing GSTs are frequently more resistant to herbicides (Cummins et al., 1999;Cummins et al., 2013). Furthermore, plant tolerance to the herbicide MV was increased by overexpressing thylakoid ascorbate peroxidase (Murgia et al., 2004), copper-zinc superoxide dismutases (Furusawa et al., 1984;Perl et al., 1993;Aono et al., 1995), as well as different Arabidopsis GSTs, like GSTU4 (Sharma et al., 2014) and GSTU19 (Xu et al., 2016). MV has no electrophilic site for conjugation with GSH and its elimination in plants is thought to take place primarily via photocatalytic degradation (Moctezuma et al., 1999) while in soil it is degraded through the yeast Lipomyces starkeyi (Carr et al., 1985). In mammals and plants, MV toxicity is associated with the accumulation of lipid peroxides (Misra and Gorsky, 1981;Liu et al., 2009). Consistent with these earlier findings, gstu7 seedlings exposed to MV accumulate more lipid peroxides than WT seedlings (Supplemental Figure S3, B). Efficient elimination of these MVinduced secondary cytotoxic compounds can be assumed to be a critical component of the cellular tolerance to the herbicide and could be a key function of GSTs in the defense response.
In order to generate less-toxic conjugates, GSTs need to interact with both, GSH and the respective toxic electrophilic substrate. Binding of GSH occurs through residues highly conserved among GSTUs, while the second substrate is recognized and bound at the H-sites. The latter are less conserved residues and might explain the diversity of these, toward a diverse array of ligands (e.g. xenobiotics, herbicides, or hormones) (Sylvestre-Gonon et al., 2019). To explore putative Hsites in GSTU7, a sequence alignment was made with Arabidopsis GSTUs from each cluster within the TAU subfamily plus other plant GSTUs with known substrates or resolved crystal structures (Supplemental Figure S1, B). The alignment revealed that the H-site of GSTU7 contains a Met-Trp-Ala motif that is also present in OsGSTU4 from rice. The overexpression of OsGSTU4 in Arabidopsis has proven to generate more tolerant plants toward salt stress and MVinduced photooxidative stress, while in silico characterization led to the hypothesis that OsGSTU4 might detoxify the herbicide metsulfuron (Sharma et al., 2014;Amador et al., 2020). These results together suggest a conserved function of distinct TAU GSTs and may provide leads for further analysis.
GSTs are mostly located in the cytosol, with only three family members predicted to be localized in plastids (Supplemental Figure S1). We confirmed the cytosolic localization of GSTU7 through stable expression and visualization of GSTU7-GFP fusion proteins in Arabidopsis ( Figure 5, A). In line with observations by (Dixon et al., 2009) we found GSTU7 being excluded from the nucleus. But in the absence of any obvious nuclear exclusion signal it is not clear how this exclusion is mediated or whether it is a consequence of the GFP fusion, which results in a larger fusion protein of 53 kDa. Indeed, GSTU7 might form homo-or heterodimeric protein complexes with molecular mass higher than 100 kDa, which is well above the size exclusion limit of about $40 kDa of nuclear pores for passive transport (Tamura and Hara-Nishimura, 2014).
Most GSTUs have been reported to have a GPOX activity with CPH as substrate (Dixon et al., 2009). This includes GSTU7, which, however, is among the family members with the lowest GPOX activity. Our data confirm this low activity also in comparison to the highly active GPOX GSTU25. Similarly, the about 100-fold lower GSTU7 activity toward CDNB as an electrophilic substrate compared with GSTU25 largely confirms the relative activities measured by Dixon et al. (2009) (Figure 5). Nonetheless, GPOX activity toward H 2 O 2 in total protein extracts proved to be diminished in gstu7 (Supplemental Figure S3). As a different approach, we tested for thiol oxidase activity of GSTU7 with roGFP2 as the reporting target protein for oxidation. All these attempts with recombinant GSTU7, however, failed and thus do not provide a clear lead for further biochemical characterization of the enzymatic activity (Supplemental Figure S4). This leaves us with the conundrum of an enzyme preventing cells from severe oxidation induced by MV or UV-B and a lack of biochemical information. A possible limitation of the biochemical in vitro assays may be that GSTs tend to form homo-or heterodimeric protein complexes (Cummins et al., 1997;Dixon et al., 1997;Gronwald and Plaisance, 1998), which may not occur with recombinant proteins. In this situation, we thought to get access to the function of GSTU7 by testing its activity in vivo. In a previous study that aimed at functionally characterizing the large family of plant GSTs for their role in fungicide detoxification, almost all Arabidopsis GSTs were expressed in a GST-deficient yeast strain (gtt1D gtt2D grx1D grx1D tef4D), which is hypersensitive to fungicides and CDNB (Krajewski et al., 2013). Complementation of this yeast strain with different GSTs, including GSTU7, resulted in increased tolerance toward fungicides. Here, overexpression of GSTU7 in the GSTdeficient yeast strain gtt1D gtt2D conferred at least partial tolerance to CHP, which further supports the notion that GSTU7 may be involved in lipid peroxide detoxification. roGFP2 is extremely sensitive to even minor increases in glutathione disulfide (GSSG) during detoxification of peroxides with GSH as electron donor (Marty et al., 2009). The completely abolished MV-induced oxidation in GSTU7 overexpressor plants strongly argues against the hypothesis for a GPOX activity and rather suggests that GSTU7 acts earlier in the pathway between MV and H 2 O 2 accumulation. The fact that GSTU7 also rescues tga256 mutants from UV-B stress (Herrera-Vásquez et al., 2021) strongly argues for a direct role of GSTU7 in peroxide detoxification.
Overall, our results reveal the importance of Arabidopsis GSTU7 as a protective enzyme for cellular detoxification, development, and the antioxidant response to herbicides, such as MV. Whether the identified phenotypes in development and under stress situations are dependent on different molecular functions of GSTU7 or whether these functions converge to the same mechanism remains unknown. Thus, further studies are needed to elucidate the mechanistic properties of GSTU7 in plant growth and during MVinduced oxidative stress. Beyond future work on the mechanism, the current findings may also open new research possibilities for breeding of more stress-tolerant plants, in which over-expression of GSTs may contribute to overcoming present and future challenging environmental conditions that would lead to formation of ROS in plants. (SALK_086642C, ABRC, characterized in this work), tga256/ GSTU7-V5 lines #1 and #2 (Herrera-Vásquez et al., 2021), and gstu7/GSTU7 lines #2 and #5 (obtained in this work) were in Columbia (Col-0) background. Seeds were surface sterilized with 70% ethanol and rinsed three times with sterile deionized water. Clean seeds were sown on plates with 0.5Â Murashige and Skoog (MS) growth medium (Murashige and Skoog, 1962) (Phyto Technology Laboratories, Shawnee Mission, KS) supplemented with 1 g L -1 sucrose and 3 g L -1 phytagel (Sigma, Steinheim, Germany) or 8 g L -1 agar (Winkler, Santiago, Chile). Plates were incubated in a growth chamber under a long day regime (16-h light, 100 lmoles m -2 s -1 , at 22 C ± 2 C; 8-h dark at 18 C ± 2 C). For experiments with leaf discs, 5-d-old seedlings were transferred to Jiffy-7 peat pellets (Jiffy, Oslo, Norway) and grown under the same controlled conditions for 4-5 weeks. Leaves were excised using a 7-mm-diameter cork borer.

Plant treatments for gene expression analysis
Treatments to evaluate gene expression were performed on 2-week-old seedlings grown under a long-day regime. Whole seedlings were collected at the indicated time points, frozen in liquid nitrogen, and stored at -80 C until processing. For SA treatments, seedlings were floated on a solution of 0.5 mM sodium salicylate (Sigma), 0.5Â MS, 1% (w/v) sucrose liquid medium or 0.5Â MS, and 1% (w/v) sucrose liquid medium as control. Treatments with MV (Sigma) were performed with seedlings floated on a solution of 50 lM MV, 0.5Â MS, 1% (w/v) sucrose liquid medium or using 0.5Â MS, and 1% (w/v) sucrose liquid medium as a control, for the indicated time points. MV stock aliquots (10 mM, dissolved in deionized water) were stored at -20 C in the dark and used within 1 month after the preparation. UV-B treatment was performed in a chamber equipped with two F8T5 UVP3400401 fluorescent tubes (k = 306 nm) (USHIO, Cypress, CA) at a distance of 53 cm from the samples, irradiating with an intensity of 0.220 mW cm -2 . As a control, a 2-mm-cellulose acetate polyester was used to filter UV-B radiation.

Gene expression analysis
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. cDNA was synthesized from 2 lg of total RNA with the MMLV kit (Promega, Madison, WI). Semi-quantitative PCR was performed within the linear amplification phase using a recombinant Taq Polymerase (Invitrogen) amplifying a fragment of 323 bp of the coding sequence of GSTU7 and a fragment of 294 bp of the YLS8 housekeeping gene. Quantitative PCR was done using the Brilliant III Ultra-Fast SYBR Green Master mix (Agilent Technologies, Santa Clara, CA) in an AriaMx (Agilent Technologies) or an MX3000P instrument (Stratagene San Diego, CA). Expression levels of GSTU7 were calculated as fold-change to the CLATHRIN ADAPTOR COMPLEX gene (CLA), for SA and MV treatments, or to YSL8 for UV-B treatments. Primer sequences are listed in Supplemental Table S1.
Oxidative stress tolerance assay and ROS detection MV tolerance assays were performed according to Laporte et al. (2012), with the following modifications. Surface sterilized seeds were stratified for 48 h in the dark at 4 C and then sown on MS-Phytagel plates supplemented with 0.1 mM MV. After 1 week, tolerance to MV was measured as survival rate that is the percentage of green seedlings/cotyledons compared with the total number of germinated seeds. For 3-AT (3-Amino-1,2,4-triazole, 3-AT) (Sigma-Aldrich) tolerance, seedlings were grown on MS-Agar plates for 5 d and then transferred to new MS-Agar plates supplemented with 1 mM 3-AT or MS-Agar plates as control. Subsequently, plants were allowed to grow for another 17 d before analysis for which plates were photographed and the fresh weight of individual plants determined. 3-AT tolerance was determined for each genotype as the percentage of fresh weight after 3-AT treatment relative to the weight under control conditions.
To evaluate the mitigation of MV-induced oxidative damage by GSTU7, either 2 lL of a 30 lM MV solution or 2 mL of water were placed as a single drop on the adaxial side of leaves of 2-week-old seedlings grown on plates. After a 24-h incubation period in constant light, the accumulation of H 2 O 2 was measured after staining with DAB (Sigma) according to Daudi et al. (2012). Leaves were detached and placed in six-well plates with 2 mL of staining solution. After a 5 min vacuum-infiltration, samples were incubated with constant orbital agitation for 6 h in the dark or until staining was visible. Chlorophyll was removed by boiling in 80% (v/v) ethanol and subsequent rinsing with distilled water. To assess the relative labeling intensity and enable a direct comparison between leaves from different genotypes and MVtreated and non-treated leaves, all stained leaves together were mounted in 20% glycerol between two SDS-page glass plates and documented with an Epson V850 Pro scanner. Quantification of the staining was performed using the FIJI software (Schindelin et al., 2012). Images were first converted to 8-bit grayscale dark pixels representing DAB staining. Subsequently, an arbitrary threshold was defined to mask out all obviously DAB-stained areas, that is pixels with most intense staining and hence the lowest gray values on the 8bit grayscale. In the current experiments, this accounted to 3% of all pixels in the whole scan. The area of the masked pixels for each leaf was then compared with the whole lamina size of the respective leaf to get a relative measure of the DAB staining that allowed direct comparison of all different leaves and treatments.
MV-dependent ROS production was also tested in 7-d-old seedlings immersed for 4 h in imaging buffer (10 mM MES, 10 mM MgCl 2 , 10 mM CaCl 2 , 5 mM KCl, pH 5.8) supplemented with 100 mM MV or just buffer as control and exposed to constant actinic light to induce photooxidative stress according to Ugalde et al. (2021). After the treatment, the seedlings were carefully transferred to a 25 mM H 2 DCF-DA (Sigma-Aldrich) staining solution prepared in 10 mM Tris-HCl (pH 7.4) buffer. Samples were incubated for 30 min and rinsed three times with Tris buffer before being imaged with a 5Â lens (EC-Plan-Neofluar, NA 0.16). Fluorescence resulting from H 2 DCF-DA labeling was collected at 505-530 nm prior excitation with 488 nm. Fluorescence intensity was quantified per mm 2 of cotyledon using the FIJI software and expressed as arbitrary units of fluorescence per mm 2 .

Bacterial infections and ion leakage
Pseudomonas syringae pv. tomato DC3000 avirulent strains expressing the AvrRpt2 effector were grown at 28 C under constant shaking on King B media supplemented with 50 lg/mL kanamycin and 50 mg/mL rifampicin. Cells were collected by centrifugation at 5,000 g for 7 min and washed twice with sterile water before resuspension in sterile 10 mM MgCl 2 solution to a final concentration of 1 Â 10 5 cfu/mL. Two-week-old plants grown in plates were inoculated by flooding (Ishiga et al., 2011) with 30 mL of the bacterial suspension for 3 min. After inoculation, the bacterial suspension was discarded and the plants were incubated in long day conditions for 72 h. Pathogen-induced cell death was measured as the ions leaked from the sample after infection. The aerial part of three infected plants was pooled and incubated for 20 min in 6 mL of deionized water under constant agitation. Water was discarded and samples were incubated in 6 mL of fresh deionized water for 16 h under constant agitation. The released ions were measured as conductivity using a Twin Cond B-173 conductometer (Horiba, Tokyo, Japan). The conductivity was expressed relative to the conductivity measured for autoclaved samples and presented as the ion leakage (%).

Cloning and plant transformation
For complementation of gstu7 and transient expression of the GSTU7-GFP fusion protein, the constructs UBQ10 pro :GSTU7-V5 and 35S pro :GSTU7-GFP were used, respectively. To obtain these constructs, a fragment of 681 bp corresponding to the full CDS of GSTU7 (stop codon excluded) was amplified by PCR using the forward and reverse pEN-GSTU7-CDS primers (see Supplemental Table S1 for sequences). The fragment was purified using a NucleoSpin kit (Macherey-Nagel, Düren, Germany) and cloned into the pENTR/SD/D-Topo vector (Invitrogen) following the manufacturer's instructions. The entry clone pEN-GSTU7 was recombined into the pB7m34gW destination vector (Karimi et al., 2002), together with pDONR-pUBQ and pDONR-V5-His (Herrera-Vásquez et al., 2021) which contained the sequences for the ubiquitin 10 promoter (UBQ10 pro ) and V5 tag (UBQ10 pro :GSTU7-V5), respectively. For subcellular localization, the CDS of GSTU7 cloned in the pEN-GSTU7 was fused to the GFP fluorescent protein sequence by recombination with the pK7FWG2.0 binary vector (Karimi et al., 2002), generating the 35S pro :GSTU7-GFP construct.
To express the Grx1-roGFP2 redox sensor in the different genetic backgrounds, gstu7 was crossed with a WT plant expressing Grx1-GFP2 in the cytosol, and the complemented line gstu7/GSTU7 #5 was transformed by floral dip with a plasmid containing the UBQ10 pro :Grx1-roGFP2 construct.
For protein purification, the plasmid pETG-GSTU7-His was generated. For this purpose, the CDS of GSTU7 was amplified by PCR with the forward and reverse pDon-GSTU7-CDS primers carrying the respective attB recombination sites (see Supplemental Table S1 for sequences); the fragment was then purified and cloned into the pDONR201 entry vector (Invitrogen) following the manufacturer's instructions and recombined with the pETG10A vector, adding a C-terminal His 6 tag to GSTU7. In parallel, the entry vector, pDONR-GSTU7, was recombined with the binary vector pAG415GPD-ccdB-HA (a gift from Susan Lindquist, Addgene plasmid # 14242) for GSTU7 expression in S. cerevisiae. All constructs were verified by sequencing.

Yeast complementation and growth
The S. cerevisiae mutant gtt1::TRP1 gtt2::URA3 (gtt1D gtt2D), in the genetic background W303 (Choi et al., 1998;Collinson and Grant, 2003), was transformed either with the empty vector (ev) pAG415GPD-ccdB-HA or a version harboring GSTU7 according to Gietz and Woods (2002 Aliquots of the saturated cultures were incubated for 2 h in 15 mL of fresh medium until they reached exponential growth and then diluted with YPD media (20 g L -1 tryptone/peptone, 10 g L -1 yeast extract) to a final OD 600 = 0.1. Oxidative conditions were generated with H 2 O 2 or CHP, added to the indicated final concentrations. Growth rates were measured as absorbance at 600 nm on a POLARstar plate reader (BMG Labtech, Ortenberg, Germany) in a final volume of 200 lL at 27.5 C.

Protein extraction and immunodetection
Protein extracts were obtained from 2-week-old frozen seedlings by grinding the material in a microfuge tube with a plastic pestle. The resulting powder was suspended in lysis buffer (50 mM sodium phosphate buffer, pH 7.5-8.0, 150 mM NaCl, 0.2% [w/v] IGEPAL, and 5 mM EDTA) and supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Samples were centrifuged at 2,350 g for 10 min at 4 C and the supernatant was collected. Protein concentration was determined by the Bradford method, using the BioRad Protein Assay (Bio-Rad Laboratories, Inc., CA, USA), and samples were stored at -80 C.

Protein purification and enzyme assays
A culture of Escherichia coli strain BL21 harboring pETG10A-GSTU7 was grown in LB medium supplemented with 100 lg mL -1 ampicillin at 37 C with constant shaking. Fresh cultures were prepared from saturated aliquots and grown until cells reached an OD 600 of 0.6-0.8. GSTU7-His expression was induced by incubating the cells for 24 h with isopropyl b-D-1-thiogalactopyranoside (IPTG) in a final concentration of 0.5 mM and 200 lL of anti-foaming solution (Sigma), with constant shaking at 22 C. Cells were collected by centrifugation at 4,000 g for 15 min at 4 C and the pellet was suspended in a buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5 mM PMSF) supplemented with 5 mg L -1 DNAse I and 0.5 g/L lysozyme (Roche Diagnostics). Cells were disrupted by sonication (3 Â 2 min, 40% power output, 50% duty cycle, in a SONOPULS Ultrasonic Homogenizer HD 2200, Bandelin, Berlin, Germany). The lysate was centrifuged at 19,000 g for 20 min at 4 C and the supernatant filtered through a sterile filter with 0.22 lm nominal size. The filtered fraction was loaded onto a Ni-NTA HisTrap column (GE Healthcare, Little Chalfont, UK) using a peristaltic pump at a rate of 1 mL min -1 . Proteins were eluted from the column with a 10-200 mM imidazole gradient (100 mM Tris-HCl, pH 8.0, 200 mM NaCl) using an Ä KTA Prime Plus chromatography system (GE Healthcare, Chicago, IL). Fractions were collected and stored at 4 C and the presence of GSTU7-His protein was confirmed by analyzing the collected fractions by SDS-PAGE.
GPOX enzyme activity was measured using a GR-coupled protocol adapted from Navrot et al. (2006) and expressed as the decrease in absorbance of NADPH at 340 nm in the presence or absence of a peroxide substrate. A final concentration of 10 lM recombinant GSTU7 protein, or no protein as the basal line, was incubated with 5 M yeast GR1, 400 lM NADPH, and 500 lM GSH as the electron donor. The assays were carried out in a final volume of 100 lL in assay buffer (100 mM Tris-HCl, pH 7.4, 5 mM EDTA) where the substrates, H 2 O 2 , or CHP, were automatically injected to a final concentration of 200 lM. The absorbance was recorded at 340 nm in a POLARstar plate reader. The test thiol oxidase activity of GSTs, 10 mM of purified GSTU7 were incubated with 1 mM of purified roGFP2 and 4 mM of either H 2 O 2 or CHP. Bovine GPX (Sigma) was used as a control. Oxidation of roGFP was followed on the plate reader exciting at 390 ± 5 and 480 ± 5 nm, and collecting the fluorescence from both channels at 520 ± 5 nm.
GPOX activity was determined as well in protein extracts from Arabidopsis plants. For this, 2-week-old WT, gstu7, gstu7/GSTU7-V5 #2, and #5 plants were incubated in deionized water or 100 mM MV for 4 h under constant actinic light. After treatment, 500 mg of tissue was collected and homogenized in liquid N 2 using a mortar and pestle. The grounded tissue was resuspended in 1 mL cold lysis buffer and centrifuged at 16,000 g for 20 min at 4 C, saving the supernatant in a new tube. After quantification, 60 mg of total protein extract was resuspended in final 500 mL of reaction mix (50 mM phosphate buffer pH 7.0, 1 mM GSH, 0.15 mM NADPH, and 5 U of glutathione reductase). In addition, 10 mM H 2 O 2 was added immediately before the measurements were performed. GPOX activity was measured as the decrease in absorbance at 340 nm during the first minute of the reaction after H 2 O 2 addition.
GST enzyme activity was measured as the conjugation of GSH to CDNB. The increase of GS-CDNB conjugate was followed as an increase in absorbance at 340 nm in a POLARstar plate reader. For preparing the stock solutions, 200 mM GSH was dissolved in assay buffer (100 mM Tris-HCl, pH 7.4, 5 mM EDTA) and 100 mM CDNB was dissolved in 96% (v/v) ethanol. The assay mix was prepared by adding 100 lL of each stock solution to 9.8 mL of assay buffer before use. For the plate reader measurements, 195 lL of assay buffer was added to 5 lL of diluted recombinant GSTU7 protein and measured for 10 min after 10 s of orbital agitation at 200 rpm. All measurements were done in triplicates with three different batches of purified protein.

Lipid peroxidation quantification
MV-induced lipid peroxidation was determined as the accumulation of MDA equivalents by the thiobarbituric acid reactive substances (TBARS) assay. For this 3-week-old WT, gstu7 and gstu7/GSTU7-V5 #2 plants were floated in distilled water or 100 mM MV for 16 h under constant actinic light. After treatment around 150 mg of tissue was ground in liquid N 2 and the homogenized, resuspended in 1 mL of precooled 0.1% (v/v) TCA. Samples were centrifuged at a maximum speed for 10 min at 4 C, and the supernatant was placed in a new tube on ice. About 300 mL of supernatant was added to a tube with 1 mL of reaction mix (20% [v/v] TCA, 0.5% [v/v] TBA) and to a no TBA mix (20% [v/v] TCA) for later background subtraction (Landi, 2017). The reaction was incubated for 30 min at 95 C, cooled down in ice, and centrifuged again at maximum speed for 10 min at 4 C. From the supernatant, MDA was measured as the absorbance at 532 nm and its concentration was calculated using MDA's extinction coefficient = 155 mM -1 cm -1 .

Confocal microscopy
Arabidopsis plants stably expressing the 35Spro:GSTU7-GFP construct were grown in MS-Agar plates for 4 d and imaged on a confocal laser scanning microscope (Zeiss LSM 780, connected to an Axio Observer.Z1; Carl Zeiss Microscopy, Jena, Germany) using a 40Â lens (C-Apochromat 40Â/1.2 W Korr). GFP and chlorophyll were excited at 488 nm and fluorescence emission was collected at 499-553 and 630-691 nm, respectively. Ratiometric imaging and analysis of WT and mutant lines harboring the Grx1-roGFP2 sensor were performed as described by Attacha et al. (2017) utilizing a custom-written MATLAB script (Fricker, 2016).

Monitoring of roGFP2 fluorescence in leaf discs
Fluorescence of the Grx1-roGFP2 sensor expressed in 4-5week-old WT, mutant, and complemented lines was initially confirmed in a stereomicroscope (M165 FC, Leica, Wetzlar, Germany) equipped with a GFP filter (470 ± 40 nm excitation and 525 ± 50 nm emission) and documented with the attached camera (DFC425 C, Leica). Leaf discs were cut from positive fluorescent plants using a 7-mm cork borer and placed with the abaxial side up in the bottom of transparent Nunc 96-wells plates (Sigma).
For spectral measurements, leaf samples were excited in the range of 360-496 nm with a step width of 1 nm and the emission was collected at 520 nm using a CLARIOstar plate reader (BMG Labtech). Non-fluorescent samples of each genotype and treatment were used to subtract the basal background fluorescence.
Ratiometric measurements of Grx1-roGFP2 in leaf samples were performed in a POLARstar plate reader equipped with two excitation filters, 410 ± 5 nm for the peak of the oxidized roGFP, and 480 ± 5 nm for the peak of the reduced roGFP; the emitted fluorescence was collected at 520 ± 5 nm. Samples were equally treated in 200 lL of final volume and the fully reduced and oxidized treatments were performed using a solution of 10 mM DTT or 5 mM 2,2 0 -Dipyridyldisulfide (DPS), respectively.
Leaf samples were treated with DTT to fully reduce the sensor, then rinsed twice with distilled water, and treated with 200 lL of 100 lM of MV or distilled water as control. Fluorescence was measured every 210 s for over 15 h at a constant temperature of 25 C. To assess the dynamic range of the sensor, at the end of each experiment the probe was fully reduced and then fully oxidized by discarding the solution containing MV, and replacing it with 200 lL of 10 mM DTT for 5 min and then replacing it with 200 lL of 5 mM DPS. For every measurement, non-fluorescent samples of each genotype were treated and recorded under the same conditions. These values were later used to subtract autofluorescence. The experiment was repeated six times and, for each experiment, six leaf discs from independent plants were used for the measurements.

Analysis of low-molecular-weight thiols under oxidative stress
Seeds from WT, gstu7, and gstu7/GSTU7-V5 #5 were surface sterilized and vertically grown on MS-Agar plate for 7 d. Seedlings were harvested and floated in 100 mM MV or distilled water as control, and incubated for 15 h under constant actinic light. After the treatment approximately 30 mg of fresh tissue was collected per replicate and snap frozen in liquid N 2 . Sample homogenization and thiol quantification were done as described in Bangash et al. (2019).

Sequence alignment and phylogenetic analysis
Protein sequences were aligned in Jalview (Waterhouse et al., 2009) using the Muscle algorithm with default settings. After manual curation of the sequence alignment, an unrooted phylogenetic tree of Arabidopsis GSTs (SSupplemental Figure S1) was constructed using Bayesian inference with MrBayes (Ronquist and Huelsenbeck, 2003). A graphical representation of the phylogenetic tree was created using the Figtree software (v1.4.2, A. Rambaut, http:// tree.bio.ed.ac.uk/software/figtree/).

Supplemental data
The following materials are available in the online version of this article.
Supplemental Figure S1. Phylogenetic analysis of the GST protein family from Arabidopsis.
Supplemental Figure S2. Cellular ion leakage under abiotic and biotic stress.
Supplemental Figure S3. GPOX activity and lipid peroxide accumulation in gstu7 under MV exposure.
Supplemental Figure S4. roGFP2 thiol oxidation assay. Supplemental Figure S5. Subcellular localization and spectral properties of the Grx1-roGFP2 sensor in Arabidopsis leaves.
Supplemental Figure S6. Biological replicates for measurement of the glutathione redox state in response to MV in Arabidopsis leaves.
Supplemental Table S1. List of primers used in this work.