Functional Analysis Of Cytokinin N-glycosyltransferase Genes In Arabidopsis Thaliana

Cytokinins are a class of important plant hormones that play a role in almost all aspects of plant growth and developments, including cell division, shoot initiation and growth, leaf senescence, seed germination, bud formation and photomorphogenic development. Since their discovery, although numerous work have been done to study their physiological function during plant growth and development by genetics, biochemistry and physiology, the significance of their modification by glycosylation is still largely unknown. Glycosylation is the most universal modification in plant and it is thought to be one of the most important mechanisms to precisely control cytokinins 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 nucleotide sugar donors to a wide range of small molecular acceptors including hormones, secondary metabolites and abiotic chemicals and toxins from the environment. The transfer of a sugar onto a lipophilic acceptor changes acceptor in its stability, chemical properties, bioactivity and enables the acceptor to access to membrane transporter systems.Glycoslation of cytokinins takes place on their adenine ring and N6-isoprenoid side chain, and usually uses UDP-Glc as activated sugar donor. Glucosylation of the adenine ring can occur at the 3-, the 7-, or 9-position to form N-glycosides. Glycosylation of the N6-isoprenoid side chain usually occur at-OH of trans-zeatin, cis-zeatin and dihydrozeatin to form O-glycosides.Genes encoding GTs that recognize cytokinins to form cytokinin O-glucosides have been identified from several plant species, such as tobacco, Arabidopsis and Zea mays. The gene functions were studied in vitro and in vivo of plant by expressed recombinant enzymes or transgenic technology. Compared with O-glycosylation, little was known about N-glycosylation of cytokinin. Although it was found, by biochemical analysis in vitro, that UGT76C1 and UGT76C2 of Arabidopsis could glycosylate cytokinins at the 7-,9-position of the adenine ring, the in vivo physiological function of UGT76C1 and UGT76C2 needs to be further confirmed in Planta.In this study, based on the results from in vitro study previously, the physiological role of cytokinin N-glucosyltransferases UGT76C1 and UGT76C2 in planta were investigated for the first time by exploiting their T-DNA insertion mutants and over-expressing homozygous transgenic lines.The main contents and results of this study are as follows:1. UGT76C1 and UGT76C2 can deactivate cytokinin and regulate cytokinin response of plant by N-glycosylationUGT76C1 and UGT76C2 T-DNA insertion mutants （ugt76cl and ugt76c2, SALK₁44355 and SALK₁35793） were ordered from NASC （http://nasc.life.nott.ac.uk/） and T-DNA insertion homozygote were selected by PCR and RT-PCR. UGT76C1 and UGT76C2 over-expressing homozygous transgenic lines （UGT76C1OE and UGT76C2OE） were obtained by transgenic technology from wild type Arabidopsis thaliana Col-0. Several experiments including root elongation, lateral root density （number of lateral root/root length）, chlorophyll retention, anthocyanin accumulation were conducted with application of exogenous cytokinin 6-BA among mutants, over-expressing homozygous transgenic lines and wild type. Results demonstrated that mutants （especially ugt76c2） were hypersensitive to exogenous cytokinin 6-BA, while over-expressing homozygous transgenic lines （especially UGT76C2OE） were relatively insensitive than wild type, suggesting that UGT76C1 and UGT76C2 can deactivate cytokinin and regulate cytokinin response of plant by glycosylation. Although no clear phenotype was observed in mutants and overexpressors grown under normal conditions when compared with wild type, it was found that the grain weight of ugt76c2 mutant was much lower than wild type, suggesting the possible role of UGT76C2 in seed development. The quantification of endogenous cytokinins and their conjugates among mutants, over-expressing homozygous transgenic lines and wild type grown under normal condition provided a solid evidence for the role of UGT76C1 and UGT76C2. Our data showed that although active cytokinin concentration was almost unchanged, mutants had much lower concentration of cytokinin N-glucosides than wild type as well as over-expressing homozygous transgenic lines had much higher concentration of cytokinin N-glucosides than wild type. These results confirmed the capacity of UGT76C1 and UGT76C2 to glucosylating cytokinins and forming their N-glucosides in planta.Expression levels of cytokinin related genes in the mutants and wild type were analyzed by RT-PCR and Real-time PCR. The results showed that those genes including cytokinin receptors （AHK2, AHK3） and cytokinin synthesis gene （IPT5） were down-regulated in mutants, while the other genes including cytokinin oxidase genes （CKX3, CKX4, CKX6） were up-regulated in mutants. These results suggest that the cytokinin homeostasis in plant may be controlled by the coordinated action of cytokinin N-glycosyltransferase genes and other cytokinin related genes. 2. The action sites of UGT76C1 and UGT76C2 are overlapping but divergingTo investigate the expression patterns of UGT76C1 and UGT76C2, their promoters were cloned into pBI121 vector to replace the cauliflower mosaic virus 35S promoter and generate the UGT promoter::β-glucuronidase （GUS） fusion construct. After the transgenic plants were obtained, GUS activity staining was conducted to investigate the expression pattern of two N-glycosyltransferases. Our results showed that both of the UGT76C1 and UGT76C2 were expressed in early development stages including germinating seed, primary root, cotyledon and hypocotyl within about 4 days. However, in later development stages, UGT76C1 was only slightly detected while UGT76C2 was still clearly detected. The Real-time PCR analysis of UGT76C1 and UGT76C2 expression patterns gave the similar results with GUS staining experiments. These results strongly suggest that the action sites of UGT76C1 and UGT76C2 are overlapping but diverging in some tissues at certain developmental stage.3. UGT76C1 and UGT76C2 are cytoplasmic enzymeGreen fluorescent protein fusion vectors of UGT76C1 and UGT76C2 were constructed and introduced into onion epidermal cells by particle bombardment technology. The observation of green fluorescence showed that UGT76C1 and UGT76C2 proteins were located in cytoplasm, indicating the subcellular action sites of UGT76C1 and UGT76C2 are all in cytoplasm.In summary, based on the results from in vitro study previously, the physiological role of cytokinin N-glucosyltransferases UGT76C1 and UGT76C2 in planta were investigated for the first time. Our results confirmed that UGT76C1 and UGT76C2 are cytokinin N-glucosyltransferases that can deactivate cytokinin and regulate cytokinin response of plant by N-glycosylation. Meanwhile, this study showed that UGT76C1 and UGT76C2 together with other cytokinin related genes constitute a complicated cytokinin metabolism network to maintain cytokinin homeostasis in plant. Furthermore, UGT76C1 and UGT76C2 are cytoplasmic enzyme and their temporal-spatial expression patterns are overlapping but differential, suggesting the functional divergence of these two cytokinin N-glycosyltransferases. All these results provided theoretical basis for the further understanding into physiological significance of cytokinin N-glucosylation in planta.