Unraveling the complexities of sucrose metabolism in alfalfa (Medicago sativa): A pursuit to increase sucrose-phosphate synthase activity

Sucrose, the major end product of photosynthesis plays a crucial role in plants. Sucrose synthesis is highly regulated with much of the control operating at the first step in sucrose formation, catalyzed by sucrose-phosphate synthase (SPS). The discovery of four SPS gene families has added an additional layer of complexity to an already complicated picture involving the regulation of SPS activity. The major goal of this dissertation is to understand the regulatory mechanism underlying the expression of SPS in alfalfa and to determine its function in root nodules that act as "metabolic sinks." An additional goat is to determine if it is possible to alter nitrogen assimilation in the nodules of alfalfa by overexpressing SPS.; We have identified cDNA clones for two SPS families (spsA and spsB) that are differentially expressed. SpsA is predominantly expressed in nodules, while spsB is almost exclusively expressed in the leaves. Bioinformatic analysis of several SPS promoters shows differences with regards to cis-elements identified, that emphasize differential regulation of the two SPS families. Based on the analysis of expression and activity of SPS during the diurnal cycle and exposure to cold, we conclude that SPS in alfalfa is mostly regulated at a posttranslational level under these conditions. Furthermore, transcript analysis showed that SPS is upregulated under N-deficient conditions in both the leaves and nodules, thus suggesting some kind of crosstalk between carbon and nitrogen metabolism.; We introduced the spinach SPS gene driven by a constitutive promoter (CaMV 35S) into alfalfa and shown accumulation of transcript for the transgene in the leaves but not in the nodules. No increase in SPS protein or activity in the transformants suggests that the transgene is posttranslationally regulated. To determine if SPS genes from different families show some variation with regards to posttranslational regulation, the SPS genes from spinach (A-family) and maize (B-family) driven by the 35S promoter was introduced into tobacco. Analysis of the transformants showed much higher level of posttranslational regulation of the spinach sps compared to the maize sps. The maize sps transformants showed increased glutamine synthetase activity suggesting that increased SPS activity may affect nitrogen assimilation.