Molecular Biology and Engineering of Biosynthesis of Sulfur-containing Metabolites in Plants

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Cysteine is the first organic precursor for the formation of sulfur-containing metabolites in plants such as methionine and glutathione. Two regulatory mechanisms for cysteine formation have been clarified by our recent studies ; one at the level of enzyme activity and the other at the steady-state mRNA level. The enzyme activity of serine acetyltransferase catalyzing the formation of O -acetylserine from serine and acetyl - CoA is inhibited by L-cysteine at the concentration of less 10 _ M.This inhibition was observed in the isoforms from watermelon, spinach and Arabidopsis thaliana. However, two isozymes from A.thaliana presumably localizing in organelles were insensitive to L-cysteine, suggesting different regulation among isozymes of different subcellular localization. Northern hybridization analysis of sulfate starved plants indicated that the steady-state mRNA level of an isoform of sulfate transporter (AST68) increased specifically in roots up to -9 folds by sulfate starvation Among all the structural genes encoding the proteins for sulfate assimilation, sulfate transporter (AST68) and APS reductase are inducible by sulfate starvation in A.thaliana. The experiments of in situ hybridization and transgenic plants with the promoter-gus fusion indicated that AST68 is expressed in the central cylinder of roots and the vascular tissues of shoots. The transgenic plants of AST68 anti-sense construct exhibited sulfur deficient symptom in cotyledons and also tolerance to selenate. These results indicating that AST68 plays a central role in sulfate assimilation in A.thaliana. The cDNA encoding alliinase responsible for conversion of alliin to sulfenic acid was isolated from A Ilium tuberosum. The recombinant protein expressed in yeast cells exhibited the alliinase activity.