Identification And Functional Analysis Of O-Aceylserine(Thiol) Lyase Family Genes In Tomato

sulfur(S)is an important nutrient element for plants,which is involved in many biological processes.Plants assimilate sulfur elements from the environment into sulfur compounds,such as cysteine,to provide basic nutrients for plant growth and development.Cysteine is the first organic sulfurcontaining compounds generated during the process of sulfur assimilation,and plays important roles during the development of plants.Moreover,the cysteine per se and most of its derivatives are essential signal molecules involved in many aspects of plant development.In plants,cysteine synthesis is catalyzed by two kinds of enzymes sequentially.They are O-acetylserine(thiol)lyase(O-acetylserine(thiol)lyase,OASTL)and Serine-acetyltransferase(Serine acetyltransferase,SAT),which constitute the cysteine synthase complex,and maintain the balance of sulfur.Among the two kinds of proteins,OASTL catalyzes the last step of biosynthesis Cysteine,and occupies an essential position during the assimilation of sulfur.Tomato is a kind of very popular fruit and vegetable in our daily life,and it is also an important model plant in the field of botany research.The study of the OASTL family genes in tomato will facilitate us to investigate the mechanism of sulfur assimilation in this species,and also provide more gene information to help us improve the quality,as well as the ability of stress-tolerance in tomato.In this work,we conducted a detailed study on the OASTL family genes in tomato from the aspects of bioinformatics,biochemistry,and the molecular cytobiology.The main results are as follows:1.Eight Sl OAS genes were cloned.Among the eight genes,Sl OAS3 was different from its prediction version in the database,in which all introns were retained.The results of the bioinformatics analysis indicated that the Sl OAS3 might encode several small peptides,but not an intact functional OASTL protein.The results of the protein sequence analysis showed that all the Sl OAS proteins are conserved at the PLP binding site and contained the amino acids necessary for PLP anchoring.Among the tomato OASTL proteins,Sl OAS9 was distinct from other family members,as its protein sequence was much longer than other proteins.In the substrate binding site(TSGNT),there were two amino acid mutants,which might affect its catalytic ability.The phylogenetic analysis further indicated that Sl OAS9 did not belong to any published OASTL subfamily.And meanwhile,the tomato OASTL proteins might have lost the function to be a S-sulfocysteine synthase,as no members from the tomato OASTL family was classified into the same subfamily with SCS from the Arabidopsis.2.The subcellular localization results showed that the Sl OAS family proteins were expressed in various parts of the cell.Sl OAS2,Sl OAS4,and Sl OAS6 were localized in the membrane,nucleus and cytoplasm;Sl OAS5 was localized in the chloroplasts;Sl OAS7 was co-localized with the autophagosomes;Sl OAS8 was expressed in the mitochondria;and Sl OAS9 was expressed in the peroxisomes.3.The yeast two-hybrid results showed that Sl OAS5 and Sl OAS6 could interact with SAT proteins,indicating that these two proteins might be authentic OASTL.Meanwhile,the results of the transactivation assay showed that Sl OAS7 had the activity of transactivation,indicating that Sl OAS7 might have taken a novel role in tomato.The enzymatic activity assay showed that Sl OAS5 and Sl OAS6 have the ability to synthesize cysteine,which is consistent with the result of yeast two-hybrid.It was also showed that Sl OAS2/4/5/6 has the ability to catalyze the biosynthesis of H2 S,but the activity of S-Sulfocysteine synthase was lost in tomato,which was in accordance with the results of the phylogenetic analysis.4.The results of the expression pattern analysis showed that the expression patterns of Sl OAS family genes were different in different parts of plant tissues,indicating that Sl OAS family genes played different roles in different stages of tomato development.To sum up,in this work,eight OASTL family genes were characterized and their potential functions were studied preliminarily.These results will facilitate our further study of the functions of this family genes,and lay a foundation for the deeper investigation of the process of sulfur assimilation in tomato.