O-acetylserine Thiol Lyase Gene Research Articles

Biofortifying multiple micronutrients and decreasing arsenic accumulation in rice grain simultaneously by expressing a mutant allele of OAS-TL gene.

Rice grains typically contain relatively high levels of toxic arsenic (As) but low levels of essential micronutrients. Biofortification of essential micronutrients while decreasing As accumulation in rice would benefit human nutrition and health. We generated transgenic rice expressing a gain-of-function mutant allele astol1 driven by the OsGPX1 promoter. astol1 encodes a plastid-localized O-acetylserine (thiol) lyase (OAS-TL) with Ser189Asn substitution (OsASTOL1S189N), which enhances cysteine biosynthesis by forming an indissociable cysteine synthase complex with its partner serine acetyltransferase (SAT). The effects on growth, As tolerance, and nutrient and As accumulation in rice grain were evaluated in hydroponic, pot and field experiments. The expression of OsASTOL1S189N in pOsGPX1::astol1 transgenic lines enhanced SAT activity, sulphate uptake, biosynthesis of cysteine, glutathione, phytochelatins and nicotianamine, and enhanced tolerance to As. The expression of OsASTOL1S189N decreased As accumulation while increased the accumulation of multiple macronutrients (especially sulphur, nitrogen and potassium) and micronutrients (especially zinc and selenium) in rice grain in a pot experiment and two field experiments, and had little effect on plant growth and grain yield. Our study provides a new strategy to genetically engineer rice to biofortify multiple essential nutrients, reducing As accumulation in rice grain and enhancing As tolerance simultaneously.

Gene identification, expression analysis, and molecular docking of SAT and OASTL in the metabolic pathway of selenium in Cardamine hupingshanensis

Key messageIdentification of selenium stress-responsive expression and molecular docking of serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) in Cardamine hupingshanensis.A complex coupled with serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) is the key enzyme that catalyzes selenocysteine (Sec) synthesis in plants. The functions of SAT and OASTL genes were identified in some plants, but it is still unclear whether SAT and OASTL are involved in the selenium metabolic pathway in Cardamine hupingshanensis. In this study, genome-wide identification and comparative analysis of ChSATs and ChOASTLs were performed. The eight ChSAT genes were divided into three branches, and the thirteen ChOASTL genes were divided into four branches by phylogenetic analysis and sequence alignment, indicating the evolutionary conservation of the gene structure and its association with other plant species. qRT-PCR analysis showed that the ChSAT and ChOASTL genes were differentially expressed in different tissues under various selenium levels, suggesting their important roles in Sec synthesis. The ChSAT1;2 and ChOASTLA1;2 were silenced by the VIGS system to investigate their involvement in selenium metabolites in C. hupingshanensis. The findings contribute to understanding the gene functions of ChSATs and ChOASTLs in the selenium stress and provide a reference for further exploration of the selenium metabolic pathway in plants.

Cloning and functional analysis of four O-Acetylserine (thiol) lyase family genes from foxtail millet

Cysteine is the first organic molecule generated during the assimilation of sulfate. As such, cysteine and its derivatives are always essential signal molecules and thus have important roles in the regulation of many plant processes. O-acetylserine (thiol) lyase (OASTL) catalyzes the last step of the biosynthesis of cysteine. At present, detailed and comprehensive work about these enzymes has only been reported from the plant Arabidopsis thaliana, though sporadic studies on OASTL have been conducted on other dicots, such as spinach and soybean. However, few reports on the functions of OASTLs in monocots have been found in the literature. Here in this study, we obtained four SiOASTL genes (SiOASTL7, SiOASTL8, SiOASTL9 and SiOASTL10) from foxtail millet and analyzed their potential functions. Phylogenetically, the four SiOASTL genes did not belong to any published subfamily of the OASTL genes; instead they constituted a new subfamily specific to the OASTL genes from monocots. In sequencing, we found that with the exception of the pseudogene SiOASTL8, proteins encoded by the other three genes exhibited high similarity with OASTL proteins from Arabidopsis, though the critical PLP-binding sites of both SiOASTL7 and SiOASTL10 were missing. The enzymatic activity assays demonstrated that SiOASTL9 has the ability to catalyze the biosynthesis of both cysteine and S-sulfocysteine, while SiOASTL7 and SiOASTL10 did not possess any previously reported catalyzing abilities. In addition, the gene expression pattern analysis showed that all four genes were widely expressed in various tissues of foxtail millet, and all had a preference in the leaves. Under abiotic stresses, the expression of these genes could be induced by salt and drought stress. Our finding that cadmium could only up-regulate the transcription of SlOASTL8 and SlOASTL9, further indicates the diversified responses of SiOASTLs to abiotic stresses.

GRA78 encoding a putative S-sulfocysteine synthase is involved in chloroplast development at the early seedling stage of rice

Cysteine functions not only as an amino acid in proteins but also as a precursor for a large number of essential biomolecules. Cysteine is synthesized via the incorporation of sulfide to O-acetylserine under the catalysis of O-acetylserine(thiol)lyase (OASTL). In dicotyledonous Arabidopsis, nine OASTL genes have been reported. However, in their null mutants, only the mutant of CS26 encoding S-sulfocysteine synthase showed the visible phenotypic changes, displaying significantly small plants and pale-green leaves under long-day condition but not short-day condition. Up to now, no OASTL gene or mutant has been identified in monocotyledon. In this study, we isolated a green-revertible albino mutant gra78 in rice (Oryza sativa). Its albino phenotype at the early seedling stage was sensitive to temperature but independent of photoperiod. Map-based cloning revealed that candidate gene LOC_Os01g59920 of GRA78 encodes a putative S-sulfocysteine synthase showing significant similarity with Arabidopsis CS26. Complementation experiment confirmed that mutation in LOC_Os01g59920 accounted for the mutant phenotype of gra78. GRA78 is constitutively expressed in all tissues and its encoded protein is targeted to the chloroplast. In addition, qRT-PCR suggested that expression levels of four OASTL homolog genes and five photosynthetic genes were remarkably down-regulated.

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

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

Impact of Sulfur Starvation in Autotrophic and Heterotrophic Cultures of the Extremophilic Microalga Galdieria phlegrea (Cyanidiophyceae).

In plants and algae, sulfate assimilation and cysteine synthesis are regulated by sulfur (S) accessibility from the environment. This study reports the effects of S deprivation in autotrophic and heterotrophic cultures of Galdieria phlegrea (Cyanidiophyceae), a unicellular red alga isolated in the Solfatara crater located in Campi Flegrei (Naples, Italy), where H2S is the prevalent form of gaseous S in the fumarolic fluids and S is widespread in the soils near the fumaroles. This is the first report on the effects of S deprivation on a sulfurous microalga that is also able to grow heterotrophically in the dark. The removal of S from the culture medium of illuminated cells caused a decrease in the soluble protein content and a significant decrease in the intracellular levels of glutathione. Cells from heterotrophic cultures of G. phlegrea exhibited high levels of internal proteins and high glutathione content, which did not diminish during S starvation, but rather glutathione significantly increased. The activity of O-acetylserine(thiol)lyase (OASTL), the enzyme synthesizing cysteine, was enhanced under S deprivation in a time-dependent manner in autotrophic but not in heterotrophic cells. Analysis of the transcript abundance of the OASTL gene supports the OASTL activity increase in autotrophic cultures under S deprivation.

A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms

An enzyme, O-acetylserine(thiol)lyase (OASTL), also known as O-acetylserine sulfhydrylase or cysteine synthase (CSase), catalyses the incorporation of sulfide into O-acetylserine and produces cysteine. We previously identified a cDNA encoding an OASTL-like protein from Spinacia oleracea, (SoCSaseLP), but a recombinant SoCSaseLP produced in Escherichia coli did not show OASTL activity. The exon-intron structure of the SoCSaseLP gene shared conserved structures with other spinach OASTL genes. The SoCSaseLP and a Beta vulgaris homologue protein, KMT13462, comprise a unique clade in the phylogenetic tree of the OASTL family. Interestingly, when the SoCSaseLP gene was expressed in tobacco plants, total OASTL activity in tobacco leaves was reduced. This reduction in total OASTL activity was most likely caused by interference by SoCSaseLP with cytosolic OASTL. To investigate the possible interaction of SoCSaseLP with a spinach cytosolic OASTL isoform SoCSaseA, a pull-down assay was carried out. The recombinant glutathione S-transferase (GST)-SoCSaseLP fusion protein was expressed in E. coli together with the histidine-tagged SoCSaseA protein, and the protein extract was subjected to glutathione affinity chromatography. The histidine-tagged SoCSaseA was co-purified with the GST-SoCSaseLP fusion protein, indicating the binding of SoCSaseLP to SoCSaseA. Consistent with this interaction, the OASTL activity of the co-purified SoCSaseA was reduced compared with the activity of SoCSaseA that was purified on its own. These results strongly suggest that SoCSaseLP negatively regulates the activity of other cytosolic OASTL family members by direct interaction.

Implications of cysteine metabolism in the heavy metal response in Trichoderma harzianum and in three Fusarium species

We studied the ability of four different fungal species, Trichoderma harzianum, Fusarium antophyllum, Fusarium compactum and Fusarium phyllophilum, to grow in the presence of heavy metals, and monitored their cysteine and glutathione content and the activity of O-acetylserine(thiol)lyase (OASTL), which is involved in cysteine biosynthesis. Zn and Pb did not affect fungal growth or sporulation at the concentrations used, whereas Cd and Hg did. In most cases, cysteine and glutathione content was higher when fungi were grown in the presence of toxic metals. As T. harzianum and F. phyllophilum presented the best growth rate on Cd and Hg, they were selected to further analyse the accumulation of toxic metals. Both species accumulated the four metals in large amounts. Using degenerate oligonucleotides based on fungal OASTL genes present in databases, partial OASTL DNA sequences were cloned by PCR from T. harzianum and F. phyllophilum, and the regulation of the OASTL genes under metal stress was analyzed. Cd produced the strongest increase in OASTL activity and mRNA levels in both fungi. The results suggest a possible correlation between cysteine metabolism and the heavy metal response in fungi.