Impairment of Root GS Activity in Alfalfa by Antesense RNA Technology and Its Effect on NO3-Assimilation, Nodulation and N2-Fixation

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This project is aimed at testing the hypotheses that impairing glutamine synthetase (GS) activity in roots of nitrate fed and N2- fixing alfalfa plants perform better than normal plants. The working hypothesis for the NO3-fed, root GS impaired plants is that the NO3-taken up by the root instead of being distributed between the root and leaves for assimilation is now exclusively assimilated in the leaves. Being energetically more efficient to assimilate NO3-in the photosynthetic tissue, the result is more efficient usage of nitrogen. In the case of the nodulated alfalfa plants, it is postulated that all the photosynthate normally distributed between the roots and the nodules is now directed primarily to the nodules. The increased flow of photosynthate to the nodules provides more energy substrate for the bacteroides to fix N2 and more C-skeleton for assimilating the fixed N2, resulting in increased N2-fixation rates and ammonia assimilation. The experimental approach to impair root GS activity is to use the antisense RNA technology. To specifically target the root GS, antisense gene constructs consisting of either the constitutive 35S promoter and a root GS specific sequence or a root specific/enhanced promoter and a GS coding sequence in antisense orientation have been introduced into alfalfa via Agrobacterium tumefaciens mediated transformation of leaf discs. To ensure that GS activity in nodules is not impaired, root GS impaired alfalfa transformants will be transformed with GS gene sense contructs behind the leghemoglobin gene promoter or the nodule-specific GS gene promoter. Since the effects of increased GS activity is not known, alfalfa plants have also been transformed with GS sense constructs under the control of constitutive and root specific/enhanced promoters. Both molecular and biochemical analysis of the different tissues will be performed to test for changes in expression of genes encoding key enzymes in N- and C- metabolism. Besides allowing us to understand why root GS impaired plants perform better, this analyses will allow us to determine if changes in N-metabolism has any effect on C-metabolism. Availability of root GS impaired plants will provide us with the necessary tools to test our hypothesis with and eventually allow us to manipulate plants for better usage of N and C.