Research On The Role Of Arabidopsis Glycosyltransferase Genes In Abiotic Stress Tolerance

The responses of plants to abiotic stress are involved in a complex series of physiological and biochemical processes, and they are often the results of interactions among multiple genes. Until now what we have found may simply reflect some aspects of the whole abiotic stress-tolerant mechanisms of plants. Therefore, studying the plant tolerance to abiotic stress from a new angle could help us to better understand plant abiotic stress tolerance mechanisms in a comprehensive level. Current researches on plant tolerance to abiotic stress mainly focus on salt and drought stress tolerance. To endow the plants with greater flexibility and plasticity under stress conditions, it is essential to make dramatic modifications to a variety of small molecules. In particular, glycosylation catalyzed by a superfamily of glycosyltransferases plays significant roles in modulating the solubility, stability, bioavailability and bioactivity of various small molecules. Thus, plant glycosyltransferases are closely related to plant abiotic stress tolerance.Glycosyltransferases are enzymes responsible for glycosylation of plant compounds, which catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, thus change their biological activity, water-solubility, transport characteristics, subcellular localization and binding properties with receptors. Therefore, glycosyltransferases play significant roles in maintaining cell homeostasis, regulating plant growth and development. Researches on plant glycosyltransferases, especially the family1glycosyltransferases of Arabidopsis thaliana in recent years, have demonstrated the wide range of biological functions of plant glycosyltransferases. Recent studies have shown that plant glycosyltransferases were involved in the regulation of plant responses to abiotic stresses, but their specific mechanisms of action remain to be determined. In our previous study, it was found that Arabidopsis glycosyltransferase gene UGT85A5was significantly induced by salt stress, indicating that it was involved in plant salt tolerance. In this thesis, we use molecular biology and reverse genetics methods to study the function of UGT85A5gene, trying to investigate the relationship between plant glycosyltransferase and stress tolerance through the model plants Arabidopsis thaliana and tobacco. In addition, we selected additional three glycosyltransferase genes (UGT85A2, UGT76E1, UGT73B1) as candidate stress-tolerant genes through microarray data, and studied their functions in stress tolerance. The main contents and results of this study are summarized as follows:1. The glycosyltransferase gene UGT85A5was for the first time demonstrated to play a significant role in enhancing plant salt tolerance.The results of a series of salt stress experiments showed that there were higher seed germination rates, better plant growth and less chlorophyll loss in UGT85A5transgenic tobacco compared to wild type plants under salt stress, suggesting the role of UGT85A5in enhancing plant salt tolerance.A series of salt-related physiological index were determined in transgenic tobacco. The results showed that the accumulations of proline and soluble sugars were increased, but the malondialdehyde accumulation and Na+/K+ratio were decreases in UGT85A5-expressing transgenic tobacco compared to wild type plants, indicating that UGT85A5transgenic tobacco could better protect the cell structure from damage under salt stress condition.The expression level of several salt-related genes was analyzed in tobacco. During salt stress, expression of some carbohydrate metabolism-related genes including those for sucrose synthase, sucrose-phosphate synthase, hexose transporter and a group2LEA protein were obviously upregulated in UG785A5-expressing transgenic plants compared with wild type controls, which were correlated with the physiological index described above. Furthermore, we analyzed the UGT85A5promoter and found that it has many binding sites of transcription factors responding to environment signals, such as ABRE binding site motif (ABF binding site), G-box promoter motif and the ACGT elements, which had been reported to be involved in responses to dehydration and ABA treatment. All these results suggest that UGT85A5may be a target of regulatory networks that control abiotic stress responses and paly a role through its interactions with upstream and downstream genes.2. The Arabidopsis overexpression lines and mutant lines of additional three candidate stress-tolerant glycosyltransferases genes were obtained, and stress tolerance analyses toward these materials were carried out.The full-length cDNA of UGT85A2, UGT76E1and UGT73B1were cloned from Arabidopsis thaliana using RT-PCR method, and their plant expression vectors were constructed. The Arabidopsis overexpression plants of these three genes were obtained through the Agrobacteium-mediaXed genetic transformation, kanamycin resistance screening and RT-PCR detection. The T-DNA insertion mutation homozygote of UGT73B1was identified using three-prime PCR method. Salt and drought stress tolerance analyses using these materials were carried out.In summary, this research demonstrated for the first time that the Arabidopsis glycosyltransferase gene UGT85A5plays a significant role in enhancing plant salt tolerance, thus providing a new gene for the crop breeding. In addition, brief stress tolerance analyses of the other three glycosyltransferase genes were also carried out. However, the detailed molecular mechanisms involved in plant abiotic stress tolerance of glycosyltransferases genes need further investigation.