Molecular analysis of sulfate assimilation in higher plants: Effect of cysteine, sulfur and nitrogen nutrients, heavy metal stress; and genomic DNA cloning.

摘要

The effect of cysteine, sulfur and nitrogen nutrient, and heavy metals on plant sulfur metabolism was analyzed, and ATP sulfurylase and APS kinase genomic DNAs were cloned from A. thaliana. Cysteine represses APS reductase (AR) activity and mRNA level in Arabidopsis thaliana roots. The intracellular sulfate concentration is not affected by cysteine treatment. AR activity was more repressed than mRNA level. Cysteine was found to inhibit AR enzyme in vitro. The results indicate that cysteine represses AR by inhibiting mRNA expression and by directly inhibiting the enzyme. The regulation of ATP sulfurylase (AS) and AR by sulfur and nitrogen nutrient status was analyzed in Brassica juncea roots. AS and AR activity and mRNA level were decreased by nitrate starvation and increased by sulfate starvation. The activation of AS and AR activity by sulfate starvation was inhibited by sulfate/nitrate starvation. But the increase of steady state mRNA level for AS and AR by sulfate starvation was not affected by sulfate/nitrate starvation. Cysteine decreased AS and AR activity and mRNA level even when the plant were simultaneously starved for sulfate. This indicates that cysteine, the end-product of sulfate assimilation may be the key regulatory signal. The expression of mRNA and enzyme activity for AS and AR was measured in B. juncea exposed to Cd. AS mRNA increased only in roots, but there was no change in enzyme activity. AR mRNA level was unaffected in leaves but the enzyme activity declined. In roots, AR mRNA and enzyme increased asynchronously. Plants exposed for 3 hours to Cd and then transferred to Cd-free medium showed ∼ 5 fold more rapid increase of AR activity than continuously exposed plants. The cadmium-induced increase in AR activity was abolished by feeding of cysteine or glutathione. The asynchronous response of AR activity and mRNA expression after Cd treatment does not occur with other heavy metals. When treated with Zn, Pb, Cu, and Hg AR mRNA and enzyme activity increased synchronously. Transgenic A. thaliana under-expressing [S(--)] or over-expressing [S(+)] ATP sulfurylase was tested for Cd sensitivity. S(--) has ∼ 30% and S(+) has 500 ∼ 1700% AS activity compared with transgenic control plants. S(--) showed increased sensitivity to cadmium compared to control plant and S(+) were more resistant. Transgenic A. thaliana in which APS kinase (APK) expression was inhibited showed undetectable APK mRNA levels and were resistant to cadmium stress compared with control plants. APS kinase and ATP sulfurylase genomic DNAs from A. thaliana were cloned and sequenced. The APS kinase coding sequence was found to be interrupted by 6 introns. A transcription initiation site is located 120 bp 5' of the translation start codon. I found a new member of ATP sulfurylase gene (APS4) and four ATP sulfurylase genes were found to be interrupted by 4 introns and the position of introns in the coding region is the same.