Mechanism Study Of Rice Glycosyltransferase Genes UGT2 And UGT3 Involved In Responses To Abiotic Stresses

With the deterioration and instability of the current global environment,abiotic stresses（including drought,salinity and extreme temperature,etc.）greatly threaten the production of rice.Exploring rice stress-tolerant genes and understanding the molecular mechanisms in response to abiotic stresses are of great significance for breeding stress-tolerant rice varieties through molecular design breeding.Plant glycosyltransferase family 1 is a class of enzymes that catalyze the glycosylation modification of small molecules in secondary metabolism.Glycosylation is a process by which glycosyltransferase transfers sugar to a variety of small molecules,changing their activity and transportation and other characteristics,finally influencing their function.Molecular genetic studies in Arabidopsis and other model plants indicated that glycosyltransferases play an important role in regulating the growth and development of plants or the interaction with environments.However,the rice glycosyltransferase family 1 contains a huge number of members.The function for most of members are still unclear yet.In this study,rice glycosyltransferase genes UGT2 and UGT3 were analyzed toward their functions and their mechanism in response to abiotic stresses.1.The research on glycosyltransferase gene UGT2 of rice in response to salt stressBased on publicly available microarray data,it was found that the transcription of UGT2 was significantly induced by salt and ABA.The qRT-PCR analysis further confirmed this result,suggesting that this gene may be involved in the plant’s salt stress response.In order to investigate the function of this gene,the ugt2 mutant lines were prepared by CRISPR-Cas9 technology.At the same time,transgenic overexpression lines of UGT2 were obtained through genetic transformation.Firstly,the liquid medium-grown UGT2 transgenic lines were tested for abiotic stress tolerance.It was found that the growth of the UGT2 overexpression seedlings exhibited higher relative shoot growth and root growth than those of WT,while ugt2ko mutant lines were more inhibited.Subsequently,the soil-grown UGT2 transgenic plants were also subjected to salt stress.It was found that ugt2ko mutant lines were severely withered and most of them died,while the overexpression lines grew well and had low mortality.These results indicated that overexpression of UGT2 in rice can increase abiotic tolerance,but the loss of the gene’s function leads to the sensitivity to abiotic stress.The contents of free proline and soluble carbohydrates were measured following salt stress.Experimental results showed that the contents of proline and soluble sugar in UGT2 overexpression plants were higher than that of wild-type,the contents in ugt2ko mutant plants were less than that of wild-type.At the same time.the expression levels of abiotic stress-related genes and oxidative stress-related genes after salt stress treatment were also detected,and it was also found that the expression levels of these genes were higher in overexpression lines than in wild type.In order to better verify the function of UGT2,we overexpressed UGT2 in Arabidopsis,and found that ectopic expression of UGT2 in Arabidopsis enhances salt tolerance.These results fully proved that the rice glycosyltransferase gene UGT2 participates in the salt stress response of plants and positively regulates salt tolerance.In order to explore the upstream regulatory mechanism of UGT2 gene involved in salt stress response,the promoter region of UGT2 was analyzed,and it was found that the promoter region of UGT2 contains ABRE elements.By consulting the literature,some candidate transcription factors that can bind ABRE elements and positively regulate salt stress response have been identified.Yeast one-hybrid experiments,ChIP-qPCR experiments,and Dualluciferase reporter gene experiments further confirmed that OsbZIP23 directly binds to the UGT2 promoter and positively regulates UGT2 expression.This clarifies the upstream regulatory mechanism of the UGT2 gene and indicates that OsbZIP23-UGT2 module may play a major role in regulating the response of plants to abiotic stresses in this thesis.2.The research on glycosyltransferase gene UGT3 of rice in response to salt and drought stressesThe rice glycosyltransferase gene UGT3（Os02g0755900）was not only induced by high salt,but also induced by PEG in our qRT-PCR analysis,it was thus suggested that this gene was different from UGT2 and may be involved in both salt and drought stress responses.We generated UGT3 overexpression lines（OE lines）and knockout mutants（ko lines）,the growth phenotypes under salt and drought stress were also analyzed.These results indicated that overexpression of UGT3 in rice significantly increased salt tolerance compared with wild-type,and also showed an enhanced tolerance to osmotic and drought stresses.The ugt3ko mutant lines showed increased sensitivity to adversity treatments in terms of stress tolerance experiments.The rice glycosyltransferase gene UGT3 was introduced into Arabidopsis and the resulting UGT3 overexpression plants showed better resistance than WT under salt and drought stresses.These datas indicated that rice gene UGT3 participated in the salt and drought stress responses of plants and positively regulates the response of adversity in plants.This study determined some tolerance-related physiological indices of rice transgenic plants.Experimental results showed that the contents of proline and soluble sugar in UGT3 overexpression plants were higher than that of wild-type,the contents in ugt3ko mutant plants were less than that of wild-type in response to abiotic stresses.NBT staining experiments also indicated that the UGT3 overexpression enhanced the removal of the superoxide radicals produced under salt and drought stresses.The expression levels of antioxidant enzyme encoding genes and abiotic stress-related genes under stress conditions were tested,and the results showed that the expression levels of these genes in the overexpression lines were significantly higher than those in the wild type.The leaf stomatal apertures of UGT3 transgenic plants were investigated,and it was found that the overexpressing UGT3 promoted the stomatal closure under drought stress conditions,while mutants delayed the stomatal closure.Thus,the endogenous ABA levels in the leaves of UGT3 transgenic plants were measured.Compared with that of WT,the ABA levels were much higher in UGT3 overexpression seedlings and lower in ugt3ko mutant lines.At the same time,the transcript levels of several enzyme genes controlling ABA biosynthesis were examined.It was found that UGT3 could upregulate the expression of ABA biosynthesis genes.The result implied that overexpression of UGT3 could upregulate the expression of ABA biosynthesis genes when plants facing stress conditions,which might largely contribute to the enhanced plant stress tolerance.By exogenously applying with ABA,the sensitivity of rice seeds was observed during germination and post-germination growth.When applied with ABA,the seed germination rates of UGT3 transgenic overexpression lines were significantly lower than that of WT.In contrast,the germination rates of ugt3ko lines were much higher than WT.Thus,it is speculated that UGT3 may protect plants from stress damage through upregulating the expression level of ABA biosynthesis genes and enhancing ABA sensitivity.In conclusion,UGT2 and UGT3 can enhance the ability of plants to scavenge reactive oxygen species and the stress tolerance of plants by enhancing the expression levels of antioxidant enzymes-related genes and stress-related genes.