Project Details

Description

9728215 Leustek The topic of this project is the developmental regulation of Met biosynthesis in higher plants, focusing primarily on the key enzyme cystathionine (-synthase (CS), and with some emphasis on threonine synthase (TS) and homoserine kinase (HK). The goal is to study how Met synthesis is regulated in cells and tissues during development in the model vascular plant Arabidopsis thaliana. Using a reporter gene method it was determined that expression from the CS promoter is not equal in all cells. In seedlings the CS promoter is highly active in meristems and the vascular tissues of roots and cotyledons. The activity is significantly lower in mesophyll cells. But in this cell type it is induced by treatment of seedlings with Thr & Lys (inhibitors of Met synthesis), and is repressed by Met-feeding. Promoter activity in the vascular tissues is unaffected by these treatments. In mature plants the CS promoter is active primarily in vascular tissue of leaves, the flower stalk, floral receptacle and anthers; and is inactive in siliques and developing seeds. Inhibition of CS expression with antisense-RNA causes a severe growth defect that can be partially restored by Met-feeding. The phenotype of these plants resembles that of A. thaliana with reduced levels of DNA methylation. The next phase of the project will center on further exploration of the reporter gene results. The possible role of substrate competition in regulating Met synthesis will be explored by studying CS and TS expression in parallel. Both will be analyzed by measurement of in situ mRNA and protein. The affect of Met and its metabolites on cell specific expression will be studied in two ways: through analysis of CS and TS in plants that have been treated with agents that perturb Met synthesis, and through analysis of A. thaliana mutant lines with elevated levels of Met or with lower levels of S-adenosylmethionine. The subcellular localization of the CS enzyme in plant cells will be explored and the kinetic propert ies of the recombinant CS enzyme will be analyzed in order to determine whether enzyme regulation plays any role regulating Met synthesis. Finally, the cDNA encoding HK will be cloned, the goal being to study the HK enzyme and to study how expression of HK is regulated. The amino acid methionine is of vital importance to all living organisms being intimately involved in every aspect of metabolism. Animals are unable to produce methionine and they must obtain it from their diet. Yet, most crop plants do not contain sufficient amounts of this key nutrient. One reason may be that plants control precisely the amount of methionine that they make. Understanding how this regulation takes place could lead to methods for improving the nutritional value of crops by increasing their methionine content.

StatusFinished
Effective start/end date3/1/982/28/02

Funding

  • National Science Foundation: $306,255.00

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