Glutamine synthetase (GS) plays a central role in nitrogen metabolism in all plants. GS catalyzes the ATP dependent condensation of ammonia with glutamate (Glu), to yield glutamine (Gln). GS in plants is located either in the cytosol (GS 1) or in the chloroplast (GS 2). A positive correlation between GS/1 activity and yield has been observed in several crop plants, suggesting that GS/1 could represent a key component in nitrogen use efficiency and yield. Thus increasing GS/1 activity by genetic engineering is a viable approach to improve the nitrogen use efficiency in plants. However, there appears to be a wide range of responses to overexpression of GS/1 which appears to be species specific and may depend on the site of nitrogen assimilation in that plant. GS/1 is a very highly regulated enzyme and this regulation may account for the variability in the expression of the transgene. Thus, understanding the regulatory mechanism underlying the expression of GS/1 genes is critical in our strategies to increase GS/1 levels in transgenic plants. Alfalfa transformants with a GS/1 gene driven by a constitutive CaMV 35S promoter showed no increase in GS/1 polypeptide or GS activity. The transcript level for the transgene and the endogenous GS genes also showed a significant drop when the transformants were treated with nitrate. More recent work from our laboratory has shown that the removal of the 3'UTR from the soybean GS/1 transgene abolishes the regulatory constraints in the expression of the transgene in alfalfa and the transformants show increased transgene transcript, polypeptide and enzyme activity. Our hypothesis is that sequences in the 3'UTR of GS genes play a central role in the posttranscriptional regulation of GS/1 gene expression. The goal of this project is to understand the posttranscriptional regulatory mechanism underlying GS/1 expression in plants. The objectives of this proposal are: i. to identify cis-acting elements in GS/1 mRNA with a role in transcript turnover and/or translation initiation; ii. to identify the metabolic signal for this regulatory step; iii. to isolate and characterize the trans-factors that interact with these cis-elements.
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