Functional Analysis Of Glycosyltransferase Genes Of Arabidopsis Thaliana And Zea Mays In Tolerance To Abiotic Stresses

In the process of plant growth and development,the secondary metabolism plays an important role.As one kind of secondary metabolism,glycosylation has an irreplaceable role in modulating molecules in plants.Glycosylation is a process by which glycosyltransferase transfers sugar to a variety of small molecules,changing their characteristics,influencing their functions,and finally affecting the growth and development of plants or the interaction with environments.However,the superfamily of glycosyltransferases contains a huge number of members.The functions for most members of this family are unknown yet.In this research,glycosyltransferase genes UGT79B2/79B3 of Arabidopsis thaliana and UGT787 of Zea Mays were investigated toward their functions and molecular mechanisms in plant tolerance to abiotic stresses.The main research contents and results are as follows:1.The function of glycosyl transferase genes UGT79B2/79B3 of Arabidopsis thaliana.Based on publicly available microarray data,it is suggested that the glycosyltransferase genes UGT79B2/79B3 may be involved in the plant's abiotic stress response.In order to investigate the function of these two genes,the expression patterns of these two genes were studied by qRT-PCR firstly.It was found that the transcriptions of UGT79B2/79B3 were significantly induced by salt,mannitol,ABA,low temperature and other stresses.After the glycosyltransferase genes UGT79B2/79B3 were cloned and their overexpression vectors were constructed,transgenic overexpression lines of UGT79B2/79B3 were obtained by using 35S promoter to drive the target genes.At the same time,the ugt79b2/79b3 double mutant lines prepared by CRISPR/Cas9 technology and ugt79b2/79b3 double knock-down lines prepared by RNAi interference technology were also obtained.The overexpression lines of UGT79B2/79B3 and double mutant lines of ugt79b2/79b3 cultured in media were subjected to stress treatments such as salt,mannitol,low temperature and ABA.Their growth phenotypes were investigated in terms of germination rate,cotyledons greening and root length.The overexpression lines showed an enhanced tolerance to those abitic stresses,while the double mutant lines were more sensitive to the applied stresses.The transgenic plants grown in soil were also tested for salt tolerance and drought tolerance.It was found that the overexpression lines had significantly increased salt tolerance and drought tolerance compared to wild type,whereas the stress tolerance of mutant lines were significantly reduced.Freezing stress experiments indicated that the anti-freezing ability of overexpression lines was obviously enhanced,while the mutant lines were less resistant to freezing when compared to wild type.Proline and soluble sugars are usually used as physiological indicators to monitor the osmotic effects of plant cells in abiotic stress conditions.It was found that the content of proline and soluble sugar in UGT79B2/79B3 overexpression lines was significantly higher than wild type,while the contents in double mutants were significantly reduced.The expression changes of stress related genes were also detected by qRT-PCR.DREB1B,DREB2B,AREB2,RD29A and COR47 were found to be significantly up-regulated in overexpression lines,and significantly down-regulated in the mutant lines.In order to investigate how the UGT79B2/79B3 genes respond to abiotic stresses,the promoter region of UGT79B2/79B3 were analyzed.It was found that the promoter region of UGT79B2/79B3 contains multiple stress-related response elements such as DRE.By using the site-directed mutagenesis,EMSA,yeast one hybrid and ChIP experiments,it was unequivocally demonstrated that CBF1/DREB1B is able to directly bind to the DRE element of UGT79B2/79B3 and regulate the expression level of UGT79B2/79B3 under abiotic stress conditions.Moreover,UGT79B2/79B3 expression regulation by CBF1 was also demonstrated in cbfl mutants and CBF1 overexpression lines.From the downstream of UGT79B2/79B3,how they play the role in tolerance to abiotic stresses?Looking for their substrates will be the key to unlock the mystery.UGT79B2/79B3 belongs to the member of the family I of the Arabidopsis thaliana glycosyltransferases.Theoretically,they would catalyze the glycosylation of small molecules.Thus a collection of small molecules naturally occurring in plants were screened by in vitro enzymatic reaction.It was found that UGT79B2/79B3 could specifically transfer the rhamnose to cyanidin and 3-O glucose cyanidin,but only a very weak reaction was observed toward quercetin and kaempferol.In vivo analyses showed that the accumulation of total anthocyanin in UGT79B2/79B3 overexpression lines was significantly increased,whereas decreased significantly in ugt79b2/79b3 double mutant lines.Under the continuous light conditions,the UGT79B2/B3 overexpression lines exhibited a deeper purple color at the hypocotyls and leaves,whereas the ugt79b2/b3 RNAi and CRISPR/Cas9 mutant lines showed lighter color.In addition,the transcription levels of key genes of anthocyanin synthesis pathway were significantly increased in overexpression lines under stress treatments,while significantly reduced in double mutants when compared to wild type.These results indicated that UGT79B2/79B3 may confer plant tolerance to abiotic stresses through the anthocyanin pathway.The hypothesis was further validated by using the mutant tt18 of the anthocyanin synthesis pathway.When the UGT79B2/79B3 was overexpressed in the mutant tt18 background,it was found that the plant tolerance to abiotic stresses disappeared.In contrast,when overexpressing UGT79B2/79B3 in the background of the mutant tt7(a mutant of flavonoid synthesis pathway),the plants remained tolerant to abiotic stresses.These results strongly suggested that UGT79B2/79B3 play a tolerance role under stress environments through anthocyanin pathway rather than flavonoid pathway.It was reported that plant anthocyanins are able to remove reactive oxygen and enhance stress resistance.In this research,it was found that the total antioxidant capacity was enhanced in UGT79B2/79B3 overexpression lines while reduced in double mutants.DAB and NBT staining experiments also indicated that the accumulation of reactive oxygen in UGT79B2/79B3 overexpression lines was much less than in wild type,however,much more accumulation of reactive oxygen was observed in double mutant lines.In summary,we proposed a working model for UGT79B2/79B3 as follows.Under abiotic stress conditions,the transcription factor CBF1 and others would directly bind to promoter of UGT79B2/79B3 and up-regulate their expressions.The enhanced anthocyanin glycosylation reaction by UGT79B2/79B3 would promote the anthocyanin pathway and cause anthocyanin accumulation.And then through the enhanced scavenging of reactive oxygen,plants were conferred an increased tolerance to abiotic stresses.2.The research on glycosyltransferase gene ugt787 of Zea Mays in response to adversity stressesThe maize GT1 glycosyltransferase family is also a large gene family that contains 147 members.However,the function has been identified very few.In this paper,a number of candidate genes of maize glycosyltransferase were selected by chip data analysis.We found that UGT787 has catalytic activity to flavonoids like quercetin,kaempferol,naringenin and others,which the catalytic activity of querctin and kaempferol are very high.Flavonoids and plants to adversity are colsely related,we analyzed the expression of UGT787 under adversity and found that it was significantly induced by salt and drought.We also found that UGT787 also was induced by small molecules such as quercetin and kaempferol.We also analyzed the expression of UGT787 in different growth stages and different parts of maize,and found that the expression of UGT787 is very extensive.Therefore,we predict that UGT787 has an important function in plants.We obtained the two transposons of UGT787 from the uniform Mu mutant library to insert mutants 689 and 943,which were subjected to salt and drought stress treatments and observed their growth situation.Two mutants all were found to show a sensitive phenotype in seed germination,root length,seedling resistance.The total flavonoids were extracted from the seedlings of wild type,689 and 943,it was found that both 689 and 943 had less flavonoids compared with wild type,no mater under normal conditions or stress conditions,maybe this is the reason why mutants are more sensitive under abiotic stress.In order to better verify the function of UGT787,we overexpressed UGT787 in Arabidopsis thaliana,and found that 35S::UGT787 transgenic Arabidopsis thaliana always showed enhanced stress in the seed germination,cotyledon green rate,root length and soil salt and drought and so on.In combination with UGT787 in vitro enzymatic reaction,the total flavonoid content was analysised in transgenic Arabidopsis thaliana,we found that transgenic Arabidopsis thaliana have accumulated more flavonoids than wild type no mater under normal growth conditions or under stress treatment conditions.The accumulation of flavonoids in plants was consistent with the ability of plants to resist stress.In order to further explore the mechanism of maize glycosyltransferase UGT787,under the high salt and drought stress,we determined some tolerant physiplogical indexes of maize mutant 689,943 and wild type.Found that the content of proline,soluble sugar,POD,SOD,CAT,H2O2 were significantly reduced when compared to wild type.At the same time,we also determined some of the stress tolerance physiological indexes in overexpressing lines of Arabidopsis thaliana,it was also found that their content in overexpression lines were significantly higher than wild type.DAB staining,under abiotic stresses,the color of maize mutants were shallower than wild type,the color of transgenic Arabidopsis thaliana were deeper than wild type.These results showed that the mutant lines have weaker ability to scavenge active oxygen than wild type,the overexpression lines of Arabidopsis thaliana have stronger ability to scavenge active oxygen than wild type.We also detected the genes that related to flavonoid pathway in maize mutant lines and transgenic Arabidopsis thaliana,the results showed that these genes were down-regulated in maize mutant lines,however,were significantly up-regulated in transgenic Arabidopsis thaliana.At the same time,we detected the genes that related to abiotic stress,the expression of KIN1,RD29A,RD29B,COR47,POD,CAT,etc in the overexpression of Arabidopsis thaliana were found up-regulated,while their expression in the maize mutant lines were showed down-regulated.According to previous reports,we know that the secondary metabolites flavonoids in plants are related to the antioxidant activity.The content of flavonoids in the maize mutant ugt787 are reduced,and its ability to scavenge reactive oxygen is reduced,and also showed sensitive to abiotic stress.However,and the content of flavonoids in Arabidopsis thaliana overexpression lines are increased,and its ability to scavenge active oxygen was also enhanced,and the tolerance to stress was enhanced,too.Therefore,we conclude that overexpression of UGT787 in maize can promote the process of flavonoid metabolic pathways throught the glycosylated flavonoid metabolic pathway of quercetin and kaempferol,and thus accumulate more plant secondary metabolit flavonoids,enhanced the ability of removal of toxic substances of active oxygen in plants,and thus enhanced the resistance ability to abiotic stress.