Impact of sulfur starvation on cysteine biosynthesis in T-DNA mutants deficient for compartment-specific serine-acetyltransferase.

Sulfur plays a pivotal role in the cellular metabolism of many organisms. In plants, the uptake and assimilation of sulfate is strongly regulated at the transcriptional level. Regulatory factors are the demand of reduced sulfur in organic or non-organic form and the level of O-acetylserine (OAS), the carbon precursor for cysteine biosynthesis. In plants, cysteine is synthesized by action of the cysteine-synthase complex (CSC) containing serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL). Both enzymes are located in plastids, mitochondria and the cytosol. The function of the compartmentation of the CSC to regulate sulfate uptake and assimilation is still not clearly resolved. To address this question, we analyzed Arabidopsis thaliana mutants for the plastidic and cytosolic SAT isoenzymes under sulfur starvation conditions. In addition, subcellular metabolite analysis by non-aqueous fractionation revealed distinct changes in subcellular metabolite distribution upon short-term sulfur starvation. Metabolite and transcript analyses of SERAT1.1 and SERAT2.1 mutants [previously analyzed in Krueger et al. (Plant Cell Environ 32:349-367, 2009)] grown under sulfur starvation conditions indicate that both isoenzymes do not contribute directly to the transcriptional regulation of genes involved in sulfate uptake and assimilation. Here, we summarize the current knowledge about the regulation of cysteine biosynthesis and the contribution of the different compartments to this metabolic process. We relate hypotheses and views of the regulation of cysteine biosynthesis with our results of applying sulfur starvation to mutants impaired in compartment-specific cysteine biosynthetic enzymes.

Analysis of cytosolic and plastidic serine acetyltransferase mutants and subcellular metabolite distributions suggests interplay of the cellular compartments for cysteine biosynthesis in Arabidopsis.

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, gamma-glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine-synthase complex.

Knocking out cytosolic cysteine synthesis compromises the antioxidant capacity of the cytosol to maintain discrete concentrations of hydrogen peroxide in Arabidopsis.

Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in cysteine (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 Arabidopsis (Arabidopsis thaliana). Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favor 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 cadmium. 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 nonstress 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. Evidences that the mutation caused a perturbation in H2O2 homeostasis are that, in the knockout, H2O2 production was localized in shoots and roots; spontaneous cell death lesions occurred in the leaves; and lignification and guaiacol peroxidase activity were significantly increased. All these findings indicate that a deficiency of OAS-A1 in the cytosol promotes a perturbation in H2O2 homeostasis and that Cys is an important determinant of the antioxidative capacity of the cytosol in Arabidopsis.

Cytosolic cysteine in redox signaling.

CYSTEINE BIOSYNTHESIS IN PLANTS TAKES PLACE IN THE THREE CELLULAR COMPARTMENTS WITH AUTONOMOUS PROTEIN BIOSYNTHESIS MACHINERY: cytosol, plastids and mitochondria. This sulfur-containing molecule is synthesized sequentially in these compartments by two enzymatic families, the serine acetyltransferases and the O-acetylserine(thiol) lyases. Each family consists of several isoforms that differ in subcellular localization and abundance. Why so many isoforms are required in plant cell for cysteine biosynthesis has remained unknown to date. The characterization of gene-specific knockout mutants has started to address this question. In our recent work, we have performed a detailed analysis of the Arabidopsis oas-a1 null mutant and showed that the antioxidant capacity of the cytosol is compromised, highlighting the contribution of cytosolic Cys in redox signaling.

Increased cysteine availability is essential for cadmium tolerance and accumulation in Arabidopsis thaliana.

Employing genetic transformation using an Atcys-3A cDNA construct expressing the cytosolic O-acetylserine(thiol)lyase (OASTL), we obtained two Arabidopsis lines with different capabilities for supplying cysteine under metal stress conditions. Lines 1-2 and 10-10, grown under standard conditions, showed similar levels of cysteine and glutathione (GSH) to those of the wild-type. However, in the presence of cadmium, line 10-10 showed significantly higher levels. The increased thiol content allowed line 10-10 to survive under severe heavy metal stress conditions (up to 400 microm of cadmium in the growth medium), and resulted in an accumulation of cadmium in the leaves to a level similar to that of metal hyperaccumulator plants. Investigation of the epidermal leaf surface clearly showed that most of the cadmium had accumulated in the trichomes. Furthermore, line 10-10 was able to accumulate more cadmium in its trichomes than the wild-type, whereas line 1-2 showed a reduced capacity for cadmium accumulation. Our results suggest that an increased rate of cysteine biosynthesis is responsible for the enhanced cadmium tolerance and accumulation in trichome leaves. Thus, molecular engineering of the cysteine biosynthesis pathway, together with modification of the number of leaf trichomes, may have considerable potential in increasing heavy metal accumulation for phytoremediation purposes.