Molecular Cloning, Characterization and Inhibition Studies on Sucrose-Phosphate Synthase (SPS) Gene Functions in the Indian Potato Cultivars

Abstract

Sucrose-phosphate synthase (SPS, EC 2.4.1.14) is a key enzyme in sucrose biosynthesis in both photosynthetic and nonphotosynthetic tissues of plants. It is encoded by different gene families. SPS exists in multiple forms which show differential distributions and functional specializations in the plant tissues. SPS activity is highly regulated by hierarchy of mechanisms including posttranslational modifications via phosphorylation, and allosteric control by the metabolic effectors, such as glucose-6-phosphate (Glc-6-P) (activator) and inorganic phosphate (Pi) (inhibitor). In this study, we report an isolation of a cDNA clone (designated SPS-C1; 3,591 bp) encoding a distinct full-length SPS A form consisting of 1,054 amino acids (designated KC-SPS1) from a commercially important Indian potato (Solanum tuberosum L.) cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. This was first report from an Indian potato cultivar. Sequence analyses and comparison of the SPS sequences from the Solanaceae family members namely, potato, tobacco and tomato revealed many distinct features within and between the SPS gene families not documented earlier. Phylogenetic tree was made based on a large number of full-length SPS sequences from taxonomically different plant species. SPS expression level was studied in the leaves and tubers from some of the Indian potato cultivars by semi-quantitative RT-PCR, protein blot analyses, and assaying extractable SPS activities. The role of SPS is well-recognized in cold-induced sweetening in the potato tubers. Sucrose accumulation in the cold-stored tubers is usually correlated with higher SPS activities. For inhibition of SPS gene function, a number of antisense/sense binary genetic constructs were made using the different regions of the cDNA clone, SPS-C1 under both constitutive CaMV 35S and tuber-specific GBSSI promoters. Based on the cv. Kufri Chipsona-1, transgenic potato plants were generated using these constructs. Some promising transformant potato lines were obtained corresponding to each binary construct. Since, in the cold-stored transformed tubers, the level of soluble sugars (both sucrose and reducing sugars) was reduced by 5080% as compared with control. In these tubers, sucrose, reducing sugar contents and extractable SPS activities were shown to be decreased significantly. Inhibition of SPS gene expression was demonstrated by semi-quantitative RT-PCR and protein blot analyses. This report would be useful for further in-depth studies on various SPS isoforms in potato and other Solanaceae family members. Moreover, various approaches as adopted in this study for improvement of the potato crop are quite relevant and promising.