Regulation of chloroplastic glutamine synthetase in alfalfa.

摘要

Nitrate is the major nitrogen source in most plants. Nitrate is reduced to ammonia via the joint action of nitrate reductase (NR) and nitrite reductase. Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to yield glutamine. There are two GS isoenzymes: GS₁ is located in the cytoplasm and GS₂ in the plastids of plant cells where they play different roles in the assimilation of ammonia from different sources. The goal of this study is to understand the regulation of GS₂ genes in alfalfa and the effects of modulating the levels of this enzyme on the overall status of the plant. A full-length GS₂ gene was cloned from an alfalfa cDNA library and characterized. Our data indicates that GS₂ is encoded by a single gene that is highly expressed in green tissues and at lower levels, in roots and nodules. Two-dimensional polyacrylamide gel electrophoresis analysis of GS₂ protein followed by western analysis, shows multiple GS₂ forms, indicating that GS₂ protein is subjected to post-translational modifications. GS ₂ gene expression is regulated by metabolic and environmental factors. Light is the main factor that regulates GS₂ expression in the leaves. Light induces GS₂ directly via phytochrome and indirectly through photosynthesis (sucrose production). Nitrate feeding experiments have shown that GS₂ gene is induced in the roots as a primary response to nitrate, while a product of nitrate assimilation regulates GS₂ gene in the leaves. To determine what regulatory events are involved in the expression of GS₂ genes, we introduced the GS₂ gene driven by the constitutive CaMV35S promoter into alfalfa. The GS₂ transgenic plants showed high GS₂ mRNA levels in leaves, stems, roots and nodules, while at the protein level they showed accumulation of GS₂ polypeptide and GS activity only in leaves and stems. Feeding nitrate resulted in higher transcript accumulation of both the transgene and the endogenous GS₂ gene in the different organs. The nitrate fed transformants also showed higher accumulation of GS₂ proteins. The results suggest that there is post-transcriptional and post-translational regulation of GS₂ mediated by nitrate or a by-product of ammonia assimilation.