Characterization of the O-acetylserine(thiol)lyase gene family in Solanum lycopersicum L.

Key message

This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L.

Abstract

Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a β-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.

     

Identification of O-acetylserine(thiol)lyase (OASTL) genes in sorghum (Sorghum bicolor) and gene expression analysis under cadmium stress

Cysteine (Cys) is the first identified molecule in plant metabolism which includes both sulfur and nitrogen. It can be synthesized in three cellular compartments, containing chloroplast, cytoplasm and mitochondrion. The final step of cysteine biosynthesis is catalyzed by the O-acetylserine(thiol)lyase enzyme (OASTL, E.C. 4.2.99). In the present study, seven members of the OASTL gene family in the sorghum (Sorghum bicolor) genome were identified at a genome-wide scale and comparative bioinformatics analyses were performed between sorghum and Arabidopsis OASTLs. In all OASTL proteins, a pyridoxal-phosphate dependent domain structure (PALP, PF00291) was identified. The gene ontology annotations also revealed that all sorghum OASTL genes have KOG1252 (Cystathionine beta-synthase and related enzyme) and K01738 (cysteine synthase A) activities. In promotor sequences of OASTL genes, diverse cis-acting elements were found, including hormone and light responsiveness, abiotic stress responsiveness, and tissue-specific ones (meristem and endosperm). Sorghum OASTL genes demonstrated medium or high level expressions in anatomical parts and developmental stages based on the digital expression data. Expression of OASTL genes were also analyzed under cadmium (Cd) stress in sorghum by Real Time-quantitative PCR (RT-qPCR). The results exclusively showed that OASTL A1-2 gene was 1.12 fold up-regulated in roots, whereas cysteine synthase 26 was 2.25 fold down-regulated in leaves. The predicted 3D structure of OASTLs indicated some structural diversities as well as variations in the secondary structures.

     

The Second Serine Acetyltransferase, Bacterial-type O-Acetylserine (thiol) Lyase and Eukaryotic-type O-Acetylserine (thiol) Lyase from the Primitive Red Alga Cyanidioschyzon merolae

O -acetylserine (thiol) lyase (OASTL) genes were isolated by screening a genomic library of the primitive red alga Cyanidioschyzon merolae and named cmSAT2, and cmOASTL1 and cmOASTL2, respectively. cmSAT2 encoded a polypeptide of 406 amino acids. cmSAT2 was encoded on chromosome IX. cmOASTL1 and cmOASTL2 encoded for polypeptides of 389 and 390 amino acids, respectively. A molecular phylogenic tree of the amino acid sequences suggested that cmOASTL1 belongs to the group of eukaryotic plant OASTL while cmOASTL2 belongs to the bacterial type OASTL. cmOASTL1 and cmOASTL2 were encoded on chromosomes XVII and VIII, respectively. In Northern blot analyses, the probes for cmOASTL1 and cmOASTL2 hybridized with 1.4 kb and 1.3 kb bands, respectively. The identity of cmOASTL1 and cmOASTL2 was confirmed by genetic complementation in an OASTL- deficient mutant of Escherichia coli NK3 using sulfate or sulfide as a sole source of sulfur. Received 19 March 2001/ Accepted in revised form 7 May 2001     

Impact of reduced O-acetylserine(thiol)lyase isoform contents on potato plant metabolism

Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H(2)S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed.     

Mutational analysis of O-acetylserine (thiol) lyase conducted in yeast two-hybrid system

Cysteine biosynthesis, achieved by the sequential reaction of two enzymes, serine acetyltransferase and O-acetylserine (thiol) lyase (OASTL), represents the final step of sulfur assimilation pathway in plants and bacteria. The two enzymes form a bi-enzymatic cysteine synthase complex through specific protein-protein interactions. To identify the amino acids important for cysteine synthase complex formation, several mutations in bacterial OASTL were designed. Effects of mutagenesis were verified in a yeast two-hybrid model that allowed monitoring both, protein-protein interactions and the enzymatic activity of OASTL.     

Aerobic transformation of zinc into metal sulfide by photosynthetic microorganisms

Industrial activity over the last two centuries has increased heavy metal contamination worldwide, leading to greater human exposure. Zinc is particularly common in industrial effluents and although an essential nutrient, it is highly toxic at elevated concentrations. Photoautotrophic microbes hold promise for heavy metal bioremediation applications because of their ease of culture and their ability to produce sulfide through metabolic processes that in turn are known to complex with the metal ion, Hg(II). The green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the cyanobacterium Synechococcus leopoliensis were all able to synthesize sulfide and form zinc sulfide when exposed to Zn(II). Supplementation of their respective media with sulfite and cysteine had deleterious effects on growth, although ZnS still formed in Cyanidioschyzon cells to the same extent as in unsupplemented cells. The simultaneous addition of sulfate and Zn(II) had similar effects to that of Zn(II) alone in all three species, whereas supplying sulfate prior to exposure to Zn(II) enhanced metal sulfide production. The coupled activities of serine acetyltransferase and O-acetylserine(thiol)lyase (SAT/OASTL) did not increase significantly in response to conditions in which enhanced ZnS formation occurred; sulfate added prior to and simultaneously with Zn(II). However, even low activity could provide sufficient sulfate assimilation over this relatively long-term study. Because the extractable activity of cysteine desulfhydrase was elevated in cells that produced higher amounts of zinc sulfide, cysteine is the probable source of the sulfide in this aerobic process.     

Regulation of sulfate assimilation by nitrogen and sulfur nutrition in poplar trees

Plants cover their need for sulfur by taking up inorganic sulfate, reducing it to sulfide, and incorporating it into the amino acid cysteine. In herbaceous plants the pathway of assimilatory sulfate reduction is highly regulated by the availability of the nutrients sulfate and nitrate. To investigate the regulation of sulfate assimilation in deciduous trees we used the poplar hybrid Populus tremula × P. alba as a model. The enzymes of the pathway are present in several isoforms, except for sulfite reductase and -glutamylcysteine synthetase; the genomic organization of the pathway is thus similar to herbaceous plants. The mRNA level of APS reductase, the key enzyme of the pathway, was induced by 3 days of sulfur deficiency and reduced by nitrogen deficiency in the roots, whereas in the leaves it was affected only by the withdrawal of nitrogen. When both nutrients were absent, the mRNA levels did not differ from those in control plants. Four weeks of sulfur deficiency did not affect growth of the poplar plants, but the content of glutathione, the most abundant low molecular thiol, was reduced compared to control plants. Sulfur limitation resulted in an increase in mRNA levels of ATP sulfurylase, APS reductase, and sulfite reductase, probably as an adaptation mechanism to increase the efficiency of the sulfate assimilation pathway. Altogether, although distinct differences were found, e.g. no effect of sulfate deficiency on APR in poplar leaves, the regulation of sulfate assimilation by nutrient availability observed in poplar was similar to the regulation described for herbaceous plants.     

Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase [O-acetylserine(thiol)-lyase].

Cysteine synthase [O-acetyl-L-serine(thiol)-lyase, EC 4.2.99.8] (CSase), which is responsible for the terminal step of cysteine biosynthesis, catalyzes the formation of L-cysteine from O-acetyl-L-serine (OAS) and hydrogen sulfide. Three T-DNA vectors carrying a spinach (Spinacia oleracea) cytoplasmic CSase A cDNA (K. Saito, N. Miura, M. Yamazaki, H. Horano, I. Murakoshi [1992] Proc Natl Acad Sci USA 89: 8078-8082) were constructed as follows: pCSK3F, cDNA driven by the cauliflower mosaic virus (CaMV) 35S RNA promoter with a sense orientation; pCSK3R, cDNA driven by the CaMV 355 promoter with an antisense orientation; pCSK4F, cDNA fused with the sequence for chloroplast-targeting transit peptide of pea ribulose-1,5-biphosphate carboxylase small subunit driven by the CaMV 35S promoter with a sense orientation. These chimeric genes were transferred into tobacco (Nicotiana tabacum) with Agrobacterium-mediated transformation, and self-fertilized progeny were obtained. CSase activities in cell-free extracts of pCSK3F and pCSK4F transformants were 2- to 3-fold higher than those of control and pCSK3R plants. CSase activities in chloroplasts of pCSK4F transformants were severalfold higher than those of control and pCSK3F plants, indicating that the foreign CSase protein is transported and accumulated in a functionally active form in chloroplasts of pCSK4F plants. Isolated chloroplasts of a pCSK4F transformant had a more pronounced ability to form cysteine in response to addition of OAS and sulfur compounds than those of a control plant. In particular, feeding of OAS and sulfite resulted in enhanced cysteine formation, which required photoreduction of sulfite in chloroplasts. The enhanced cysteine formation in a pCSK4F plant responding to sulfite was also observed in leaf discs. In addition, these leaf discs were partially resistant to sulfite toxicity, possibly due to metabolic detoxification of sulfite by fixing into cysteine. These results suggested that overaccumulated foreign CSase in chloroplasts could modulate biosynthetic flow of cysteine in response to sulfur stress.     

Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces cerevisiae exhibit enhanced levels of cysteine and glutathione and increased tolerance to oxidative stress

Summary. The cysteine biosynthesis pathway differs between plants and the yeast Saccharomyces cerevisiae. The yeast MET25 gene encoded to O-acetylhomoserine sulfhydrylase (AHS) catalyzed the reaction that form homocysteine, which later can be converted into cystiene. In vitro studies show that this enzyme possesses also the activity of O-acetyl(thiol)lyase (OASTL) that catalyzes synthesis of cysteine in plants. In this study, we generated transgenic tobacco plants expressing the yeast MET25 gene under the control of a constitutive promoter and targeted the yeast protein to the cytosol or to the chloroplasts. Both sets of transgenic plants were taller and greener than wild-type plants. Addition of SO₂, the substrate of the yeast enzyme caused a significant elevation of the glutathione content in representative plants from each of the two sets of transgenic plants expressing the yeast gene. Determination of non-protein thiol content indicated up to four-folds higher cysteine and 2.5-fold glutathione levels in these plants. In addition, the leaf discs of the transgenic plants were more tolerant to toxic levels of sulphite, and to paraquat, an herbicide generating active oxygen species.     

Regulation of the O-acetyl-L-serine(thiol)lyase activity in Monoraphidium braunii

Total level of O-acetyl-L-serine(thiol)lyase (OASTL) activity observed in Monoraphidium braunii fed-repleted cells decreases up to 40% after 24 h the carbon source was removed from the culture; however, no significant change in the activity is observed in N-starved cells. On the other hand, sulfur starvation induces OASTL activity in M. braunii, which may increase 2.5-fold after 36 h. Normal intracellular level of the activity is restored when a sulfur source, such as sulfate, sulfite, L-cysteine, L-methionine or glutathione is added to the culture. The induction of the OASTL activity requires de novo synthesis of protein, and thus the presence in the culture of adequate carbon and nitrogen sources. The OASTL isoenzymes from M. braunii cells are differently affected by S-starvation.     

Signaling in the plant cytosol: cysteine or sulfide?

Cysteine (Cys) is the first organic compound containing reduced sulfur that is synthesized in the last stage of plant photosynthetic assimilation of sulfate. It is a very important metabolite not only because it is crucial for the structure, function and regulation of proteins but also because it is the precursor molecule of an enormous number of sulfur-containing metabolites essential for plant health and development. The biosynthesis of Cys is accomplished by the sequential reaction of serine acetyltransferase (SAT) and O-acetylserine(thiol)synthase (OASTL). In Arabidopsis thaliana, the analysis of specific mutants of members of the SAT and OASTL families has demonstrated that the cytosol is the compartment where the bulk of Cys synthesis takes place and that the cytosolic OASTL enzyme OAS-A1 is the responsible enzyme. Another member of the OASTL family is DES1, a novel l-cysteine desulfhydrase that catalyzes the desulfuration of Cys to produce sulfide, thus acting in a manner opposite to that of OAS-A1. Detailed studies of the oas-a1 and des1 null mutants have revealed the involvement of the DES1 and OAS-A1 proteins in coordinate regulation of Cys homeostasis and the generation of sulfide in the cytosol for signaling purposes. Thus, the levels of Cys in the cytosol strongly affect plant responses to both abiotic and biotic stress conditions, while sulfide specifically generated from the degradation of Cys negatively regulates autophagy induced in different situations. In conclusion, modulation of the levels of Cys and sulfide is likely critical for plant performance.     

Plant serine acetyltransferase: new insights for regulation of sulphur metabolism in plant cells

Plant serine acetyltransferase (SAT, E.C. 2.3.1.30) catalyses the first connecting reaction between nitrogen/carbon and sulphate metabolism. SAT is associated with the second committed enzyme, O-acetylserine(thiol)lyase (OASTL, E.C. 4.2.99.8), in a bi-enzyme complex called cysteine synthase (CS). Metabolic regulation of SAT-bound OASTL in the presence of cysteine (Cys) is analysed with the extracts from the leaf cell compartments of Pisum sativum. To this end, a high performance liquid chromatography (HPLC) technique is developed to measure the rate of O-acetylserine (OAS) formation by SAT. Under physiological experimental conditions, L-Cys specifically inhibits chloroplast-SAT activity, which is linked to the sulphate assimilation network. This metabolic feedback control does not apply to the SAT activity located in the cytosol. The non-physiological range of L-Cys inhibits the mitochondrial isoform. L-Cys in a non-competitive manner in presence of L-serine or acetyl-CoA (K_i of 12–20 μM) inhibits partially purified chloroplast SAT, free of bound OASTL. The K_i values are in the range of Cys concentrations estimated in this compartment. Furthermore, we report for the first time that the multi-enzyme complex, CS dissociates in the presence of Cys as previously described with OAS. From this study, and with the integration of data previously reported in the literature, we hypothesize a new model for the regulation of Cys synthesis in plant cells containing a chloroplastic Cys-sensitive SAT.     

Binding of Cysteine Synthase to the STAS Domain of Sulfate Transporter and Its Regulatory Consequences

The sulfate ion (SO₄²⁻) is transported into plant root cells by SO₄²⁻ transporters and then mostly reduced to sulfide (S²⁻). The S²⁻ is then bonded to O-acetylserine through the activity of cysteine synthase (O-acetylserine (thiol)lyase or OASTL) to form cysteine, the first organic molecule of the SO₄²⁻ assimilation pathway. Here, we show that a root plasma membrane SO₄²⁻ transporter of Arabidopsis, SULTR1;2, physically interacts with OASTL. The interaction was initially demonstrated using a yeast two-hybrid system and corroborated by both in vivo and in vitro binding assays. The domain of SULTR1;2 shown to be important for association with OASTL is called the STAS domain. This domain is at the C terminus of the transporter and extends from the plasma membrane into the cytoplasm. The functional relevance of the OASTL-STAS interaction was investigated using yeast mutant cells devoid of endogenous SO₄²⁻ uptake activity but co-expressing SULTR1;2 and OASTL. The analysis of SO₄²⁻ transport in these cells suggests that the binding of OASTL to the STAS domain in this heterologous system negatively impacts transporter activity. In contrast, the activity of purified OASTL measured in vitro was enhanced by co-incubation with the STAS domain of SULTR1;2 but not with the analogous domain of the SO₄²⁻ transporter isoform SULTR1;1, even though the SULTR1;1 STAS peptide also interacts with OASTL based on the yeast two-hybrid system and in vitro binding assays. These observations suggest a regulatory model in which interactions between SULTR1;2 and OASTL coordinate internalization of SO₄²⁻ with the energetic/metabolic state of plant root cells.     

Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants

Summary. Serine acetyltransferase (SATase) and cysteine synthase (O-acetylserine (thiol)-lyase) (CSase) are committed in the final step of cysteine biosynthesis. Six cDNA clones encoding SATase have been isolated from several plants, e.g. watermelon, spinach, Chinese chive and Arabidopsis thaliana. Feedback-inhibition pattern and subcellular localization of plant SATases were evaluated. Two types of SATase that differ in their sensitivity to the feedback inhibition by l-cysteine were found in plants. In Arabidopsis, cytosolic SATase was inhibited by l-cysteine at a physiological concentration in an allosteric manner, but the plastidic and mitochondrial forms were not subjected to this feedback regulation. These results suggest that the regulation of cysteine biosynthesis through feedback inhibition may differ depending on the subcellular compartment. The allosteric domain responsible for l-cysteine inhibition was characterized, using several SATase mutants. The single change of amino acid residue, glycine-277 to cysteine, in the C-terminal region of watermelon SATase caused a significant decrease of the feedback-inhibition sensitivity of watermelon SATase. We made the transgenic Arabidopsis overexpressing point-mutated watermelon SATase gene whose product was not inhibited by l-cysteine. The contents of OAS, cysteine, and glutathione in transgenic Arabidopsis were significantly increased as compared to the wild-type Arabidopsis. Transgenic tobacco (Nicotiana tabacum) (F₁) plants with enhanced CSase activities both in the cytosol and in the chloroplasts were generated by cross-fertilization of two transgenic tobacco expressing either cytosolic CSase or chloroplastic CSase. Upon fumigation with 0.1 μL L⁻¹ sulfur dioxide, both the cysteine and glutathione contents in leaves of F₁ plants were increased significantly, but not in leaves of non-transformed control plants. These results indicated that both SATase and CSase play important roles in cysteine biosynthesis and its regulation in plants. Received November 27, 2001 Accepted December 21, 2001