Identification And Functional Analysis Of An Auxin Glycosyltransferase Gene UGT74D1in Arabidopsis Thaliana

Auxin is one type of phytohormones that plays important roles in nearly all aspects of plant growth and developmental processes, such as the development of root, shoot and leaf, vascular initiation and differentiation, geotropism and phototropism, organic senescence etc. The homeostasis of endogenous auxins is the maintenance of a steady state concentration of the auxins in the receptive tissue appropriate to any fixed environmental condition. Since their discovery, although numerous work have been done to study the auxins’ physiological function during plant growth and development by genetics, biochemistry and physiology, the significance of their modification by glycosylation is still largely unknown. Glycoslation of auxins takes place on the carboxyl group of the side chain of indole ring, and usually uses UDP-Glc as activated sugar donor. Glycosylation is the most universal modification in plant and it is thought to be one of most important mechanisms to precisely control auxin homeostasis for plant in order to keep normal growth and development under different developmental stage and various environments.Glycosyltransferases can typically transfer single or multiple activated sugars from a donor (usually nucleotide sugar UDP-glucose) to a wide range of small molecular acceptors including hormones, secondary metabolites and abiotic chemicals and toxins from the environment. Glycosylation modification will often change the plant molecules in their biological activity, water-solubility, stability, transport characteristics, subcellular localization and binding properties with receptors, and also can reduce or eliminate the toxicity of endogenous and exogenous substances. Therefore, glycosylation plays a key role in maintaining cell homeostasis, thus likely participating in the regulation of plant growth and development. This research project was supported by grants from the National Natural Science Foundation of China. In this study, the in vitro biochemical screening of the group L of Arabidopsis thaliana glycosyltransferase superfamily was firstly carried out for the enzymatic activity toward auxins. UGT74D1was identified to be a novel auxin glycosyltransferase. Next, the physiological role of auxin glucosyltransferase UGT74D1in planta was investigated through a molecular genetic approach. Our data obtained from this study provide an important reference for further understanding of the auxin regulation by glycosylation in plants.The main contents and results of this study are as follows:1. Several candidate glycosyltransferases were expressed using prokaryotic system and UGT74D1was identified to be a novel auxin glycosyltransferase.Candidate genes were constructed to pGEX prokaryotic expression vectors, expressed in E.coli XL1-Blue, and then recombinant proteins were purified. The enzyme activities of the recombinant proteins were analyzed by incubating them separately with7auxins and analogs used as substrates. Following HPLC and LC-MS analyses of the reaction products, it was found that the recombinant UGT74D1had a strong activity toward natural auxins IBA and IAA.2. The enzymatic properties of glycosyltranferase UGT74D1were studied.Under the same condition, relative conversion rates of these7auxin substrates were calculated and it was found that UGT74D1had a highest enzymatic activity toward IBA in vitro. Taking IBA as an example, the influence factors of enzymatic activity including temperature, pH were tested. The results showed that37centigrade degree was the best temperature for maximum enzyme activity. The pH optimum was HEPES buffer with pH6.0. Enzyme kinetic parameters of UGT74D1toward IBA were also investigated.3. The expression pattern and subcellular localization of UGT74D1were analyzed.UGT74D1promoter::GUS reporter construct was introduced into Arabidosis and GUS histochemical assay was used for UGT74D1expression pattern analysis. UGT74D1was intensively expressed in cotyledons, hypocotyl-root junction and primary root tip for the first7days of seedlings. UGT74D1was also expressed in young leaves, leaf vein, primary root tip and lateral roots for the one to two week old seedlings. For the5-week-old seedlings, UGT74D1was highly expressed in inflorescences, young silique capsule, cauline leaf vein and root. Insterestly, the UGT74D1expression was observed to concentrate at the marginal region of developed leaves.Green fluorescent protein fusion vector of UGT74D1was introduced into onion epidermal cells by particle bombardment technology. The observation of green fluorescence showed that the auxins glycosyltransferase UGT74D1was localized in cytoplasm and nucleus, indicating its subcellular action sites.4. UGT74D1can glucosylate auxins in planta, thus regulate plant response to auxins and impact on the plant growth and development.In order to know the physiological role of UGT74D1, all the T-DNA mutant (ugt74d1), double mutant (ugt74dlugt74e2) and overexpression lines (UGT74D1OEs) were obtained. Crude protein extracts from seedlings of different lines were analyzed for enzyme activity toward auxins. The results showed that lines with higher UGT74D1transcripts also displayed stronger enzyme activity toward tested auxins to form their glucose conjugates. When exogenous IBA was applied to different plant lines, much higher level of IBA-glucose in overexpression lines than in mutants and wild type plants can be observed. These data indicated that over-production of the UGT74D1in the plants indeed led to increased level of the glucose conjugate of IBA, suggesting an in vivo glucosylating role of UGT74D1to plant auxins.Plant response to auxins was investigated by measuring root elongation and lateral root density under the application of exogenous auxins IBA and IAA to different lines described above. Mutants showed a more sensitive phenotype both in root elongation and lateral root density than wild type. In contrast, overexpression lines were relatively insensitive than wild type, suggesting that UGT74D1regulated plant responses to auxins possibly by deactivating auxins. Furthermore, we employed the pDR5:GUS reporter construct to explore the possible role of UGT74D1in auxin signaling. It was found that auxin signaling was much reduced in UGT74D1overexpression lines than in wild type plants, indicating that UGT74D1might regulate the auxin signaling through glucosylating and thus deactivating auxin approach.More interesting finding in this study is the changes in plant phenotype. Under normal growth conditions, it was found that UGT74D1overexpression lines displayed less erect petioles and curling leaves. These phenotypic changes were well maintained under different light intensity. These findings implicated that UGT74D1had an important physiological role in modulating plant growth and development.In summary, a novel auxin glucosyltransferase, UGT74D1, was biochemically identified from Arabidopsis in this study. Further, the auxin glucosylating activiyt of glucosyltransferase UGT74D1in planta was demonstrated through a molecular genetic approach. Moreover, the physiological role of UGT74D1was found in regulating auxin response and modulating plant growth and development. All these results provide theoretical basis for a better understanding of molecular mechanisms of auxin glucosylation and auxin regulation in planta.