Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana

! 1! Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana Consolación Álvarez, Irene García, Luis C. Romero and Cecilia Gotor Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas y Universidad de Sevilla, Avda. Américo Vespucio, 49, 41092 Sevilla, Spain. Running title: Functionality of OAS-C Author for correspondence: Cecilia Gotor: [email protected]. Tel: 34.954489516. Fax: 34.954460065. ! 2! ABSTRACT In non-cyanogenic species, the main source of cyanide derives from ethylene and camalexin biosyntheses. In mitochondria, cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolised by the β-Cyanoalanine synthase CYS-C1, catalysing the conversion of cysteine and cyanide to hydrogen sulfide and βcyanoalanine. The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine(thiol)lyase mitochondrial isoform, OAS-C, which catalyses the incorporation of sulfide to O-acetylserine to produce cysteine, thus generating a cyclic pathway in the mitochondria. The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme, showing defects in root hair formation. Genetic complementation with the OAS-C gene rescues the impairment of root hair elongation restoring the wild type phenotype. The mitochondria compromise their capacity to proper detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant. Consequently, we observe an accumulation of sulfide and cyanide and of the alternative oxidase, which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules. Our results allow us to suggest that the significance of OAS-C is related with its role in the proper sulfide and cyanide detoxification in mitochondria. Keywords: Alternative oxidase, Arabidopsis thaliana, Cysteine, Cyanide, βCyanoalanine synthase, O-Acetylserine(thiol)lyase, Reactive oxygen species, Root hair, Sulfide ! 3! INTRODUCTION Ethylene is involved in many aspects of the plant life cycle, including seed germination, root hair development, seedling growth, leaf and petal abscission, climacteric fruit ripening, organ senescence, and the modulation of plant responses to stresses. Ethylene is synthesized in the cytosol from methionine via S-adenosyl-Lmethionine (AdoMet), which is first converted to 1-aminocyclopropane-1-carboxylic acid (ACC) catalyzed by the enzyme S-adenosyl-L-methionine methylthioadenosinelyase (ACC synthase) and then ACC is converted to ethylene by the enzyme ACC oxidase (Bleecker and Kende, 2000). This second reaction generates equimolecular amounts of ethylene and cyanoformic acid, the latter of which is spontaneously degraded to carbon dioxide and cyanide (Peiser et al., 1984). In non-cyanogenic species, such as Arabidopsis thaliana, ethylene biosynthesis is the main source of cyanide. However, it has recently been demonstrated that the biosynthesis of camalexin, the characteristic phytoalexin of A. thaliana, is linked to cyanide formation. Therefore, during the biosynthesis of camalexin, the tryptophan-derived intermediate indole-3-acetonitrile is conjugated with cysteine to serve as substrate for the cytochrome P450 enzyme CYP71B15. This enzyme catalyzes the formation of the thiazoline ring as well as the release of cyanide and subsequent oxidative decarboxylation of dihydrocamalexic acid to camalexin (Bottcher et al., 2009). Therefore, under certain developmental or environmental conditions, plant cells produce significant amount of cyanide that may be harmful to those cells. Cyanide is a phytotoxic molecule that potently inhibits many important metalloenzymes, such as Cu/Zn superoxide dismutase, catalase, nitrate and nitrite reductase, nitrogenase and peroxidases (Siegien and Bogatek, 2006). In mitochondria, cyanide is a potent inhibitor of cytochrome c oxidase, which is complex IV of the mitochondrial respiratory chain (Cooper and Brown, 2008). Therefore, cyanide accumulation must be prevented during an ethylene biosynthesis burst. The main cyanide detoxification process described in plants is the conversion of cyanide to βcyanoalanine, which is converted to Asn, Asp and ammonia by NIT4 class nitrilases, ! 4! allowing the recycling of nitrogen (Piotrowski, 2008). Cyanide can also be detoxified to a lesser extent by thiosulfate sulfurtransferase (rhodanese) and mercaptopyruvate sulfurtransferase, which catalyses the transfer of sulfur ions from thiosulfate or mercaptopyruvate, respectively, to cyanide ions (Nakamura et al., 2000; Papenbrock and Schmidt, 2000; Papenbrock et al., 2011). β-Cyanoalanine synthase (CAS) catalyses the conversion of cysteine and cyanide to hydrogen sulfide and β-cyanoalanine. In A. thaliana, the most abundant CAS enzyme, located in the mitochondria, is encoded by CYS-C1 (At3g61440) (Hatzfeld et al., 2000; Yamaguchi et al., 2000), and contributes to most of the CAS activity in root and leaf tissues (Watanabe et al., 2008). The Arabidopsis CYS-C1 enzyme belongs to the β-substituted alanine synthase family that also comprises the three major O-acetylserine(thiol)lyase enzymes OAS-A1 (At4g14880), OAS-B (At2g43750) and OAS-C (At3g59760) (Watanabe et al., 2008), the L-cysteine desulfhydrase DES1 (At5g28030) (Alvarez et al., 2010) and the S-sulfocysteine synthase CS26 (At3g03630) (Bermudez et al., 2010). Phylogenetic analysis shows that the isoforms CYS-D1 and CYS-D2 are putative CAS enzymes (Jost et al., 2000); however, β-cyanoalanine synthase activity has not been observed in vitro for these enzymes (Hatzfeld et al., 2000; Yamaguchi et al., 2000). Recently, the analysis of T-DNA insertional mutants defective in the CYS-C1 enzyme has demonstrated that mitochondrial CAS activity is essential to maintain a low level of cyanide for proper root hair development (Garcia et al., 2010). Although cyanide is adequately detoxified in mitochondria through the formation of β-cyanoalanine, a co-product of the CAS enzymatic activity is hydrogen sulfide, which also inhibits oxygen consumption by inhibition of the mitochondrial cytochrome c oxidase. In mammalian systems, both HCN and H2S are noncompetitive inhibitors with respect to oxygen and show similar inhibition constants (Cooper and Brown, 2008). Therefore, the hydrogen sulfide produced by the detoxification of cyanide needs to be detoxified in the mitochondria by the mitochondrial isoform of the OASTL family. The mitochondrial enzyme OAS-C catalyses the incorporation of sulfide to O-acetylserine to produce cysteine, which in ! 5! turn could be used by CYS-C1 to detoxify cyanide, thus generating a cyclic pathway for cyanide detoxification in the mitochondria (Figure 1). The most highly expressed OASTL isoforms in Arabidopsis cells including OAS-C have been studied from the point of view of their involvement in the primary sulfate assimilation pathway and cysteine biosynthesis. Mitochondrial OAS-C contributes only 5% of total OASTL activity, but it has been suggested that OAS-C plays a much more important role than assumed (Heeg et al., 2008). Expression analysis has shown that OAS-C expression is higher in the roots than in the leaves and suggests that OAS-C plays a significant role in the roots (Watanabe et al., 2008). In this work, our main goal has been to characterize in detail T-DNA-tagged OASC deficient mutants in relation to its possible role in the detoxification of sulfide and cyanide in Arabidopsis mitochondria, acting jointly with CYS-C1. We have confirmed the involvement of OAS-C in the maintenance of low levels of sulfide and cyanide in mitochondria. RESULTS Identification and Characterization of T-DNA Insertion Mutants Interrupting Different Arabidopsis OAS-C Gene Models To deepen the understanding of the role of the OAS-C enzyme, we attempted the identification of new T-DNA insertion mutant alleles that are different from those previously described. Only one allele from the SALK collection, SALK_000860, has been previously characterized (Figure 2A) (Heeg et al., 2008; Watanabe et al., 2008). A screening of different T-DNA mutant collections revealed a different allele from the WiscDsLox T-DNA lines generated by Dr. Patrick Krysan and Dr. Sandra AustinPhillips at the University of Wisconsin-Madison. The T-DNA insertion in this allele, WiscDsLox381A8, is located in the tenth intron of the OAS-C sequence that curiously is only present in the predicted OAS-C gene model one, whereas the T- ! 6! DNA insertion in the SALK allele is located in the first intron of the OAS-C sequence and consequently interrupts the three predicted splice variants (Figure 2A). We performed RT-PCR analysis of the kanamycin-resistant seedlings from the SALK_000860 line and isolated three different plants knockouts for the three OAS-C gene models. We also analyzed nine Basta and hygromycin-resistant seedlings from the WiscDsLox381A8 line, and four of them were knockouts for splice variant one and wild type for splice variants two and three (data not shown). One line of each TDNA-tagged mutant was selected for real-time RT-PCR analysis using specific primers designed to discriminate between splice variant 1 and splice variants 2 and 3 (Supplemental Table 1). The results obtained corroborated the previous RT-PCR analysis (Figure 2B). Biochemical characterization of both mutants showed that the intracellular Cys and glutathione contents in the leaves of the SALK_000860 mutant were similar to the values obtained in wild type leaves, but significant reductions of 30% of both cysteine and glutathione contents were observed in the roots as previously described (Heeg et al., 2008; Watanabe et al., 2008). However, the WiscDsLox381A8 mutant showed wild type levels of Cys and glutathione contents even in the roots (Table 1). Moreover, we performed real-time RT-PCR on the OASTL gene family in the both mutants in leaf and root tissues. When compared with wild type, the transcript levels of the different gene members were identical in the WiscDsLox381A8 mutant, whereas in the both tissues we observed induction of OAS-A1 and OAS-B gene expression in the SALK_000860 mutant, the latter being more significant, and repression of the three β-cyanoalanine synthase-like encoding genes CYS-C1, CYSD1 and CYS-D2 (Figure 3). Phenotypic Traits of the SALK_000860 Mutant Are Dependent on Growth Conditions Phenotypic differences between both mutants and wild type were not observed in the aerial parts of the plants under normal long-day growth conditions either in soil or ! 7! solid MS medium. However, we could observe important differences in root tissues when grown on vertical MS medium without sucrose. There was appreciable that the roots of the SALK_000860 mutant showed shorter root hairs than either wild type or WiscDsLox381A8 mutant roots. This defect in hair root elongation in the SALK_000860 mutant was consistently observable in different batches of seedlings that were analyzed. Furthermore, genetic complementation of the mutant with the OAS-C gene resulted in wild type hair roots, thus confirming that the observed phenotype was indeed due to the mutation of the OAS-C gene (Figure 4). Due to the discrepancy of the oas-c mutant phenotype with previous reports (Heeg et al., 2008; Watanabe et al., 2008a), we explored the phenotype traits growing the seedlings in different media. When seedlings were grown on vertical MS medium with one-fourth strength of macronutrients phenotypic differences were observed. The whole seedlings of the SALK_000860 mutant showed a strong reduction in size when compared with either wild type or WiscDsLox381A8 mutant, and this defect was also rescued by OAS-C gene complementation (Figure 5). This growth phenotype was in agreement with previously reported smaller size of oas-c mutant lines (Heeg et al., 2008). When grown with sucrose in any of the growth media, nor shorter root hairs nor smaller seedlings were observable (Supplementary Figure 1 online). The SALK_000860 Mutant Accumulates Cyanide and Sulfide Because the root phenotype observed in the SALK_000860 mutant resembled that observed in the null cys-c1 mutant (Garcia et al., 2010) and also showed the repression of the CYS-C1 gene, we measured the total CAS activity in the leaf and root tissues. Compared with the activity levels in wild type plants, we determined a reduction in CAS activity of 15% in the leaves and a more significant reduction of 30% in the roots of the SALK_000860 mutant, while the WiscDsLox381A8 mutant showed wild type levels of CAS activity in both leaf and root tissues. Consistent with the reduction of CAS activity, there was a significant increase of cyanide in the SALK_000860 mutant, mainly in roots, which accumulated 25 % CNover the basal ! 8! levels of wild type (Table 2). We also measured the total level of sulfide and found a very significant increase of sulfide in leaves of the SALK_000860 mutant, which accumulated 76% more S2-, and even a stronger increase in roots that showed 2-fold accumulation over the basal levels of wild type and WisDsLox381A8 mutant (Table 2). The SALK_000860 Mutant Has Induced the Alternative Oxidase Pathway and the ROS Production To determine if the measured cyanide accumulation in the SALK_000860 mutant lead to induction of the alternative oxidase pathway, we analyzed the expression level of the AOX1a gene in both oas-c mutants. The qRT-PCR showed a significant increase in AOX1a expression in the SALK_000860 mutant that was not observed in the WiscDsLox381A8 mutant compared to wild type (Figure 6). We further measured the total respiration rates of leaf and root tissues and the proportions of the cytochrome and the alternative oxidase respiration pathways in the two mutants. We observed similar behavior in both the leaves and roots. Whereas the SALK_000860 mutant showed an induction of 50% in the respiration rate in the roots and slight and not significant increase in the leaves, the WiscDsLox381A8 mutant did not show significant differences in the respiration rates compared to wild type either in the leaves (92% of the wild type rate) or in the roots (94% of the wild type rate) (Figure 7). The increase in respiration of the SALK_000860 mutant was completely abolished even to rates below the rates of the wild type and the WiscDsLox381A8 mutant when SHAM, an inhibitor of the alternative oxidase pathway, was added (Figure 7). Our results clearly show an induction of the alternative oxidase respiration pathway in the SALK_000860 mutant, probably as a protection to prevent overreduction that can lead to excessive ROS production. Accordingly, we used the histochemical DAB method to examine the production of ROS in mature leaves of wild type and the two oas-c mutant plants. We clearly observed accumulation of ! 9! H2O2 in the SALK_000860 mutant leaves as a brown staining distributed along the leaf that was not present in the wild type and the WiscDsLox381A8 mutant leaves (Figure 8A). Furthermore, we quantified the amount of H2O2 in root tissues by spectrofluorimetry, and observed a significant increase of 25% in the concentration of H2O2 in the SALK_000860 mutant roots compared to wild type, whereas the amount of peroxide was not different in the WiscDsLox381A8 mutant (Figure 8B). Thus, the induction of alternative oxidase seems not to be sufficient to avoid the production of oxygen peroxide in the SALK_000860 mutant. Besides, the H2O2 detected in this mutant is signaled programmed cell death as we consistently observed lesions that are characteristic of spontaneous cell death in the leaves of the SALK_000860 mutant after staining with trypan blue that were not observed in the wild type or the WiscDsLox381A8 mutant (Figure 8C). DISCUSSION Several research groups have independently demonstrated in recent years that the cytosol is the most important compartment for cysteine synthesis in Arabidopsis. Free sulfide released from the plastids and OAS from the mitochondria are incorporated into Cys in the cytosol (Haas et al., 2008; Heeg et al., 2008; Watanabe et al., 2008; Watanabe et al., 2008b; Krueger et al., 2009). These results highlight the cytosolic OASTL isoform, OAS-A1, as the major contributor to Cys synthesis (Lopez-Martin et al., 2008). However, it is intriguing that the null oas-c mutant of the mitochondrial OAS-C, which contributes to 5% of the total OASTL activity, shows a growth phenotype when grown on soil under short-day conditions, which has been suggested is not linked to a limited capacity for Cys synthesis (Heeg et al., 2008). The presented results allow us to suggest that the significance of OAS-C is related to its role in proper sulfide detoxification in mitochondria, which is released by the enzyme activity of β-cyanoalanine synthase C1 (Garcia et al., 2010). The loss of a functional ! 16! For the histochemical detection of H2O2, mature leaves were immersed in 1 mg ml-1 DAB (Sigma-Aldrich), fixed with a solution of 3:1:1 (v/v/v) ethanol:lactic acid:glycerol and photographed. The quantification of H2O2 was performed following the protocol previously described (Joo et al., 2005). Basically, frozen plant tissue was hand ground in liquid nitrogen, and immediately resuspended in 10 mM Tris-HCl buffer, pH 7.5. The extract was centrifuged twice at 15,000 g for 10 min. We performed each measurement on two equal aliquots; one of which we added 100 mM ascorbate, and they were allowed to react for 15 min. Then ROS levels were assayed by adding H2DCFDA in DMSO to both aliquots to a final concentration of 25 µM and incubating at 30ºC for 30 min. Fluorescence was measured using a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.) with excitation/emission wavelengths set to 485 and 525 nm, respectively. We then subtracted the ascorbate insensitive background from each experimental value. Total protein was quantified using the Bradford method (Bradford, 1976). The average fluorescence value obtained from three successive measurements was divided by the protein content and expressed as relative fluorescence units per milligram of protein. Detection of Cell Death Trypan blue (Sigma-Aldrich) staining for dead cells in the leaves was performed by incubating the leaves in a lactic acid-phenol-trypan blue solution (2.5 mg ml-1 trypan blue, 25% [w/v] lactic acid, 23% phenol, 25% glycerol), then heating over boiling water for 1 min and finally destaining using a 2.5 g ml-1 chloral hydrate solution before photographing the leaves. Respiration Measurements Wild type and mutant plants were grown for 15 days on vertical MS-plates. Around 50 mg of separated leaves and root tissues were cut, dried on paper towels and transferred into airtight cuvettes containing 20 mM Hepes, pH 7.2 and 0.2 mM CaCl2, and oxygen uptake was measured as a decrease in O2 concentration in the dark using a Clark-type electrode. Cyanide-resistant O2 uptake was measured in the presence of ! 17! 0.5 mM KCN. The respiration component due to the alternative oxidase pathway was measured in the presence of 4 mM of the inhibitor SHAM. The results were expressed as the mean ± SD from at least three replica samples, and the experiment was repeated three times using independent samples. Supplementary Data Supplementary Figure 1 online. Phenotypes of the oas-c mutants in the presence of sucrose. Supplementary Figure 2 online. Expression levels of the three OAS-C gene models in different tissues at different growth stages of A. thaliana plants. Supplementary Table 1 online. Oligonucleotides used in this work ACKNOWLEDGMENTS This work was funded in part by the European Regional Development Fund (ERDF) through the Ministerio de Ciencia e Innovación (grant no. BIO2010-15201) and the Junta de Andalucía (grant nos. BIO–273). This work was also funded by the CONSOLIDER CSD2007–00057, Spain, and by JAE program (CSIC) to C.A. for fellowship support. We thank Inmaculada Moreno for technical help with the research work. ! 18! FIGURE LEGENDS Figure 1. Enzymatic reactions involved in cyanide detoxification in A. thaliana mitochondria. The CAS enzyme CYS-C1 catalyses the conversion of cyanide plus cysteine to form β-cyanoalanine (CN-ALA) plus sulfide, which is used by the OASTL enzyme OASC to incorporate it to O-acetylserine (OAS) to form cysteine. Figure 2. Identification of OAS-C tagged T-DNA mutants. (A) Intron-exon organization of the three OAS-C gene models and location of the TDNA insertion in the SALK_000860 and WiscDsLox381A8 insertion mutants. The 3’-end has been magnified to show the location and direction of the primers used for the mutant analysis: qOAS-CF1 (F1), qOAS-CR1 (R1), qOAS-CF2-3 (F2-3) and qOAS-CR2-3 (R2-3). (B) Real-time RT-PCR analysis of the SALK_000860 and WiscDsLox381A8 mutant plants. RNA samples were prepared from the leaves of wild type, SALK_000860 and WiscDsLox381A8 mutant plants, and the primers specific for the OAS-C gene (pairs F1/R1 for variant 1 and F2-3/R2-3 for variants 2 and 3) and for the constitutive UBQ10 (qUBQ10F/ qUBQ10R) transcripts were used. The transcript levels were normalized to the constitutive UBQ10 gene. Data shown are means + SD of three independent experiments. ND: non-detected. Figure 3. Relative expression level of the OASTL gene family in the SALK_000860 mutant plant. Real-time RT-PCR analysis of the expression of the OAS-A1 (At4g14880), OAS-B (At2g43750), DES1 (At5g28030), CS26 (At3g03630), CYS-C1 (At3g61440), CYS-D1 (At3g04940), and CYS-D2 (At5g28020) genes was performed in the leaves and roots from the wild type and the SALK_000860 mutant plants grown for three weeks under control conditions. The transcript levels were normalized to the constitutive UBQ10 gene. Data shown are means + SD of three independent experiments, and ! 19! they represent the transcript level of each gene in the mutant plants relative to the transcript level in the wild type plants. ANOVA was performed using the software OriginPro 8. *, P < 0.05 Figure 4. Root phenotypes of the oas-c mutants. Representative bright field images of roots from wild type, SALK_000860 and WiscDsLox381A8 insertion mutants and the SALK_00860 complementation line seedlings, growing for eight days on MS medium without sucrose. Two different roots from different batches of seedlings are shown for each line. Figure 5. Whole seedling phenotypes of the oas-c mutants. Representative bright field images of eight-day-old wild type, SALK_000860 and WiscDsLox381A8 insertion mutants and the SALK_00860 complementation line seedlings, growing on MS medium with one-fourth of macronutrients without sucrose. Two different batches of seedlings are shown. Figure 6. Relative expression level of the alternative oxidase gene AOX1a in the oas-c mutants. Real-time RT-PCR analysis of the expression of the AOX1a (At3g22370) gene was performed in the leaves from 15-day-old wild type and the oas-c mutants. The transcript levels were normalized to the constitutive UBQ10 gene. Data shown are means + SD of three independent experiments and they represent the transcript level of each gene in the mutant plants relative to the transcript level in the wild type plants. ANOVA was performed using the software OriginPro 8. *, P < 0.05. Figure 7. Respiration rates in the leaves and roots of the oas-c mutants. The respiration rate was measured in the leaf and root tissues of 15-days-old wild type and the oas-c mutant plant lines with an oxygen electrode. Alternative oxidase respiration was determined in the presence of 4 mM SHAM. The results were expressed as the mean + SD of three independent analyses. ANOVA was performed ! 20! using the software OriginPro 8. Significant differences between wild type and mutant plants in different conditions are indicated with one asterisk (P < 0.05). Figure 8. Accumulation of H2O2 and lesion formation in the oas-c mutants. (A) Histochemical detection of H2O2 in detached leaves from three-week-old plants by DAB staining. The experiment was repeated at least three times with similar results. (B) Quantification of H2O2 in root tissues from three-week-old wild type and mutant plants by H2DCFDA fluorescence as described in methods. The results were expressed as the mean + SD of three independent analyses. ANOVA was performed using the software OriginPro 8. *, P < 0.05. (C) Spontaneous cell death detection in detached leaves from three-week-old plants by trypan blue staining. The insert shows a magnified photo of a lesion in the leaf from SALK_000860 mutant plant. The experiment was repeated at least three times with similar results. ! 21! Table 1. Thiol content of the oas-c mutants. Plant line Total Cys (nmol g-1 FW) Total Glutathione (nmol g-1 FW) Leaves Roots Leaves Roots Wt 26.97 + 1.49 65.41 + 4.14 307.3 + 8.1 201.2 + 4.1 SALK_000860 28.41 + 1.12 46.37 + 2.24 * 328.9 + 3.8 144.2 + 7.5* WiscDsLox381A8 25.65 + 0.98 60.19 + 4.05 312.5 + 3.8 185.3 + 20.6 Total levels of Cys and glutathione were measured in the leaves and roots of wild type and oas-c mutant plants grown for three weeks in soil. Values are means + SD of three independent experiments. ANOVA was performed using the software OriginPro 8. *, P < 0.05. Table 2. CAS activity and cyanide and sulfide contents in the oas-c mutants. CAS enzyme activity and total levels of sulfide and cyanide were measured in the leaves and roots of wild type and oas-c mutant plants grown for three weeks in soil. Values are means + SD of three independent experiments. ANOVA was performed using the software OriginPro 8. *, P < 0.05. ! Plant line CAS activity (nmol min-1 mg-1) CN content (nmol g-1FW) Sulfide content (nmol g-1FW) Leaves Roots Leaves Roots Leaves Roots Wt 3.50 + 0.26 11.05 + 0.57 0.80 + 0.02 4.14 + 0.11 12.18 + 1.48 2.10 + 0.33 SALK_000860 2.97 + 0.25 7.75 + 0.23* 0.86 + 0.03 5.18 + 0.18* 21.50 + 0.94* 4.35 + 0.13* WiscDsLox381A8 3.65 + 0.17 11.25 + 0.55 0.76 + 0.03 4.28 + 0.22 11.80 + 1.28 2.01 + 0.69 ! 23! REFERENCES Alonso,'J.M.,'et'al.!(2003).!Genome=wide!insertional!mutagenesis!of!Arabidopsis! thaliana.!Science.!301,'653=657.! Alvarez,' C.,' Calo,' L.,' Romero,' L.C.,' Garcia,' I.,' and' Gotor,' C.! (2010).! An! O= acetylserine(thiol)lyase! homolog! with! L=cysteine! desulfhydrase! activity! regulates! cysteine! homeostasis! in! Arabidopsis.! Plant! Physiol.! 152,' 656= 669.! Alvarez,' C.,' Lozano?Juste,' J.,' Romero,' L.C.,' Garcia,' I.,' Gotor,' C.,' and' Leon,' J.! (2011).! Inhibition! of! Arabidopsis! O=acetylserine(thiol)lyase! A1! by! tyrosine!nitration.!J.!Biol.!Chem.!286,'578=586.! 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(2008).!Physiological!roles!of!the!beta=substituted!alanine!synthase!gene! family!in!Arabidopsis.!Plant!Physiol.!146,'310=320.! 0,00# 0,40# 0,80# 1,20# 1,60# 2,00# SALK WiscDsLox 2.0 1.6 1.2 0.8 0.4 0.0 AOX1A expression (relative to wt) Figure#6# * 0,000 0,100 0,200 0,300 wt SALK Wisc wt+ SHAM SALK + SHAM Wisc + SHAM 0,000 0,200 0,400 0,600 wt SALK Wisc wt+ SHAM SALK + SHAM Wisc + SHAM Leaves& 0.3& 0.2& 0.1& 0.0& Respira0on&rate& (nmol&O2&min81&mg81FW)& *" Roots& Respira0on&rate& (nmol&O2&min81&mg81FW)& 0.6& 0.4& 0.2& 0.0& *" *" Figure"7" Wt SALK Wisc (A) (C) Figure'8' (B) 200 150 100 50 0 Wt SALK WiscDsLox Relative fluorescence (units/mg) *