Unknown,Transcriptomics,Genomics,Proteomics

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An O-acetylserine(thiol)lyase homolog regulates cysteine homeostasis in Arabidopsis thaliana

ABSTRACT: Cysteine occupies a central position in plant metabolism due to its biochemical functions. Arabidopsis thaliana cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes that catalyze the biosynthesis of cysteine. Because they are localized in the cytosol, plastids and mitochondria, this results in multiple subcellular cysteine pools. Much progress has been made on the most abundant OASTL enzymes; however, information on the less abundant OASTL-like proteins has been scarce. To unequivocally establish the enzymatic reaction catalyzed by the minor cytosolic OASTL isoform CS-LIKE (AT5G28030), we expressed this enzyme in bacteria and characterized the purified recombinant protein. Our results demonstrate that CS-LIKE catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate. Thus, CS-LIKE is a novel L-cysteine desulfhydrase (EC 4.4.1.1), and we propose to designate it DES1. The impact and functionality of DES1 in cysteine metabolism was revealed by the phenotype of the T-DNA insertion mutants des1-1 and des1-2. Mutation of the DES1 gene leads to premature leaf senescence, as demonstrated by the increased expression of senescence-associated genes and transcription factors. Also, the absence of DES1 significantly reduces the total cysteine desulfuration activity in leaves, and there is a concomitant increase in the total cysteine content. As a consequence, the expression levels of sulfur-responsive genes are de-regulated, and the mutant plants show enhanced antioxidant defenses and tolerance to conditions that promote oxidative stress. Our results suggest that DES1 from Arabidopsis is an L-cysteine desulfhydrase involved in maintaining cysteine homeostasis, mainly at late developmental stages or under environmental perturbations. Using the Affymetrix ATH1 GeneChips, we performed a comparative transcriptomic analysis on leaves of the des1-1 and wild type plants grown on soil for three weeks. Three biological replicates were performed for each sample and hybridized to the chips. We made the comparison of des1-1 leaves versus wild-type leaves to classify the differently expressed genes in the mutant plant.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Cecilia Gotor

PROVIDER: E-GEOD-19244 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Cysteine homeostasis plays an essential role in plant immunity.

Álvarez Consolación C Ángeles Bermúdez M M Romero Luis C LC Gotor Cecilia C García Irene I

The New phytologist 20111011 1

• Cysteine is the metabolic precursor of essential biomolecules such as vitamins, cofactors, antioxidants and many defense compounds. The last step of cysteine metabolism is catalysed by O-acetylserine(thiol)lyase (OASTL), which incorporates reduced sulfur into O-acetylserine to produce cysteine. In Arabidopsis thaliana, the main OASTL isoform OAS-A1 and the cytosolic desulfhydrase DES1, which degrades cysteine, contribute to the cytosolic cysteine homeostasis. • Meta-analysis of the transcripto ...[more]

PMID: 21988475

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Project description:In bacteria, the biosynthesis of cysteine is accomplished by two enzymes that are encoged by the cysK and cysM genes. CysM is also able to incorporate thiosulfate to produce S-sulfocysteine. In plant cells, the biosynthesis of cysteine occurs in the cytosol, mitochondria and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of cs26 suppressed the S-sulfocysteine synthase activity that was detected in wild type; furthermore, the mutants exhibited a growth phenotype, but penetrance depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants), as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify ROS, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our finding reveals that S-sulfocysteine and the activity of S-sulfocysteine synthase play an important role in chloroplast function and are essential for light-dependent redox regulation within the chloroplast. Using the Affymetrix ATH1 GeneChips, we performed a comparative transcriptomic analysis on leaves of the cs26 and wild type plants under two different photoperiod conditions. Wild type and cs26 mutant plants were grown on soil under a long-day photoperiod (LD) or under a short-day photoperiod (SD). Total RNA was extracted from the leaves of 3-week-old plants grown under identical LD conditions, and from the leaves of 5-week-old plants grown under identical SD conditions. Three biological replicates were performed for each sample and hybridized to the chips. We made two different comparisons to classify the differently expressed genes in the mutant plant: cs26 leaves under LD versus wild-type leaves under LD and cs26 leaves under SD versus wild-type leaves under SD.

Project description:Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in Cys biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidposis thaliana. Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favour of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to Cd. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under non-stress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. To further elucidate the specific function(s) of the OAS-A1 isoform in the adaptation response to cadmium we extended the trasncriptome experiment to the wild type and oas-a1.1 mutant plants exposed to Cd. The comparison of transcriptomic profiles showed a higher proportion of genes with altered expression in the mutant than in the wild type, highlighting up-regulated genes identified as of the general oxidative stress response rather than metal-responsive genes. Wild type and oas-a1.1 mutant plants were grown hydroponically and, after a two-week acclimation period, the roots and shoots were harvested separately. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. We made two different comparisons to classify the differently expressed genes in the mutant plant: oas-a1.1 roots versus wild-type roots and oas-a1.1 shoots versus wild-type shoots. Hydroponically-grown wild type and oas-a1.1 mutant plants were further treated with 50µM CdCl2 and 18h-treated-roots and 24h-treated-shoots were harvested. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. Different comparisons were performed as follows: 18h Cd-treated wild type roots versus untreated wild type roots; 24h Cd-treated wild type shoots versus untreated wild type shoots; 18h Cd-treated oas-a1.1 roots versus untreated oas-a1.1 roots; 24h Cd-treated oas-a1.1 shoots versus untreated oas-a1.1 shoots; 18h Cd-treated oas-a1.1 roots versus 18h Cd-treated wild type roots; 24h Cd-treated oas-a1.1 shoots versus 24h Cd-treated wild type shoots

Project description:Arabidopsis thaliana cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes that catalyze the biosynthesis of cysteine. Recently, we have deeply investigated about one of the minor OASTL-like protein located in the cytosol, named DES1, highlighting some important clues about its metabolic function. We have demonstrated that DES1 catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate, instead of the biosynthesis of Cys, and thus, is a novel L-cysteine desulfhydrase (EC 4.4.1.1). The functionality of DES1 is being revealed by the phenotype of the T-DNA insertion mutants des1-1 and des1-2. We have performed a comparative transcriptomic analysis on leaves of the des1-1 and Col-0 wild type plants grown for 30 days under long-day conditions. The normalized data from the replicates showed differential expression of 1614 genes in the des1-1 mutant, with 701 genes down-regulated and 913 genes up-regulated by more than twofold, with a False Discovery Rate (FDR) of < 0.05 and an intensity signal restriction of lgSignal >7. This des1-1 transcriptional profile show a strong alteration when compared to a previous comparative transcriptomic analysis performed on leaves of the des1-1 and Col-0 wild type plants grown for 20 days under identical long-day conditions (GSE 19244). We have also performed a comparative transcriptomic analysis on leaves of the des1-1 and Col-0 wild type plants grown for 20 days and treated with sodium sulfide for 10 additional days. The comparison of the transcriptional profile of des1-1+Na2S versus Col-0+Na2S clearly shows that exogenous sulfide reversed the transcriptional level differences between the mutant and the wild type to reach similar transcriptional patterns as the array GSE19244. Our results suggest a role of sulfide as transcriptional regulator in the des1-1 mutant background. Using the Affymetrix ATH1 GeneChips, we performed two comparative transcriptomic analyses on leaves of the des1-1 and Col-0 wild type plants grown on soil under long-day conditions. One was performed on plants grown for 30 days and the other on plants grown for 20 days and treated for 10 additional days with 200 mM Na2S. Three biological replicates were performed for each sample and hybridized to the chips. We made the comparison of des1-1 versus wild-type and des1-1+Na2S versus wild-type+Na2S to classify the differently expressed genes in the mutant plant.

Dataset Information

An O-acetylserine(thiol)lyase homolog regulates cysteine homeostasis in Arabidopsis thaliana

Publications

Cysteine homeostasis plays an essential role in plant immunity.

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