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

半胱氨酸是植物中形成含硫代谢物如蛋氨酸和谷胱甘肽的第一个有机前体。半胱氨酸形成的两种调节机制已被我们最近的研究所阐明;一种是在酶活性水平，另一种是在稳态mRNA水平。丝氨酸乙酰转移酶催化丝氨酸和乙酰辅酶A形成O -乙酰丝氨酸的酶活性在小于10 μ M的浓度下被L-半胱氨酸抑制。在来自西瓜、菠菜和拟南芥的同工型中观察到这种抑制。然而，两种可能定位于细胞器的拟南芥同工酶对L-半胱氨酸不敏感，表明不同亚细胞定位的同工酶之间存在不同的调节。北方杂交分析表明，稳态mRNA水平的硫酸盐转运蛋白（AST 68）的异构体增加，特别是在根中的硫酸盐饥饿高达-9倍。在所有的结构基因编码的蛋白质的硫酸盐同化，硫酸盐转运蛋白（AST 68）和APS还原酶是诱导硫酸盐饥饿在拟南芥。原位杂交和转基因植株的实验表明，AST 68基因在根的中心柱和茎的维管组织中表达。AST 68反义构建体的转基因植株表现出子叶缺硫症状，同时也表现出对硒的耐受性。这些结果表明，AST 68起着核心作用，在拟南芥的硫酸盐同化。从马铃薯中分离到编码蒜氨酸酶的cDNA，该酶负责蒜氨酸转化为次磺酸。重组蛋白在酵母细胞中的表达具有蒜氨酸酶活性。

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.