Function Analysis Of A Cytokinin O-glycosyltransferase Gene In Arabidopsis Thaliana

Cytokinins are a class of active plant hormones with a wide range of biological activity. It plays a crucial role in the regulation of cell proliferation and development, and control various stages of plant growth and development such as aging, shoot and root balance, nutrition, signal transduction, and crop production. Cytokinin glycosylation is an important style of cytokinin modification.Cytokinin glycosylation mainly includes O-glycosylated and N-glycosylated forms. In general, the cytokinins N-glycosylation is a permanent deactivation of the hormone, the activity of cytokinins can not be reversed; while O-glycosylation is a temporary inactivation of hormones, the activity of glycosylated cytokinin can be restored by removing the sugar-base under certain conditions. Both kinds of cytokinin glycosylations play an important role in the homeostasis of cytokinin in plants.Cytokinin glycosyltransferases can catalyze the glycosylation of cytokinin. In previous studies, two cytokinin N-glycosyltransferase genes and three cytokinin O-glycosyltransferase genes of Arabidopsis have been identified in vitro. But the physiological fuction and roles of these glycosyltransferases in planta remains largely unclear.The Arabidopsis O-glycosyltransferase gene UGT85A1, which has the highest activity towards cytokinin among the three identified O-glycosyltransferase genes, was selected in this research to study the effects of cytokinin O-glycosylation. The model plant Arabidopsis thaliana and tobacco were taken as the experimental materials.This research compared the morphology, physiology and other aspects of Arabidopsis mutants ugt85al,UGT85A1 overexpressing transgenic Arabidopsis and tobacco with their wild types. By taking advantage of the report system of GUS, expression pattern of the gene UGT85A1 in tissue-specific and development stage-specific manner were also analyzed. Through the report system of GFP fluorescence, subcellular localization of the glycosyltransferase UGT85A1 was studied. The results present in this study laid the foundation for the further understanding of the physiological significance of cytokinin O-glycosylation.The main results in this research were summarized as follows:1. Homozygote of the Arabidopsis mutant ugt85al was identified and was characterized as compared with wild-type. No significant differences were found between both genotypes in morphological and physiological characters like root length, lateral root number, chlorophyll content, free cytokinins and their glucosides contents.2. Plant overexpression vector 35Spro:UGT85A1 was constructed, and transformed into tobacco and Arabidopsis by Agrobacterium-mediated method. Under normal growth condition, the Arabidopsis overexpressors did not show clear phenotypic changes. When cultured on plates with application of exogenous trans-Zeatin (tZ), the UGT85A1 overexpressing Arabidopsis showed longer root length extension, much more lateral root numbers and lower chlorophyll content, suggesting relatively insensitive responses to cytokinin. The free cytokinins and their glucosides contents were measured for the UGT85A1 overexpressors and wild type grown in normal conditions. The results showed that although no changes in free cytokinins contents, the UGT85A1 overexpressors had much higher (4～6 folds) tZOG than wild type plants, suggesting a role of UGT85A1 in the cytokinin O-glucosylation. The UGT85A1 overexpressing tobacco showed phenotypes of earlier flowering, thinner stem and more lateral buds.The chlorophyll content of detached leaves from the transgenic tobacco was lower than the wild type after one week of keeping in dark. In tissue culture experiments, it was found that the leaf discs of the UGT85A1 overexpressing tobacco regenerated less shoots than wild-type on each tested media containing different concentrations of exogenous tZ. These results suggested that the function of UGT85A1 gene in plants is the O-glucosylation of cytokinins, inactivation of cytokinins and the regulation of plant responses to cytokinins.3. The vector harboring UGT85A1 pro::GUS construct was introduced into Arabidopsis and the spatial and temporal expression patterns of UGT85A1 gene in plants were studied. Our data showed that UGT85A1 gene mainly expressed in the germinated seeds and young seedlings such as the cotyledons, hypocotyls, primary roots and root tips. In addition, UGT85A1 also expressed in the late developing embryos. Experiment results indicated that the expression of UGT85A1 gene is in the temporal-spacial specific manner.4. To know the subcellular localization of UGT85A1 protein, the vector 35Spro:UGT85Al-GFP was constructed and transformed into onion epidermis by particle bombardment. The distribution of green fluorescence in cell indicated that UGT85A1 protein is located in both cytoplasm and nucleus.The present experimental results suggested that cytokinin O-glycosylation takes a part in keeping cytokinin homeostasis and regulating the plant response to cytokinin in growth and development, thus deepening our understanding of the role of cytokinin O-glycosylation in plants. At the same time, the results present in this study provided references for the comparison of the different roles in plants of cytokinin O-glycosylation and N-glycosylation. Due to the complexity of cytokinin metabolism and time limit, the gene UGT85A1 was not yet fully studied from every angles in this paper. Further research needs to be made to get more comprehensive insight into the molecular mechanisms and the roles of this gene.