| Growing in a constantly changing natural environment,plants are often affected by abiotic and biological stresses.However,plants have evolved a series of complex mechanisms to cope with the adverse environments in the long-term process of interaction with the environments.It is of great significance for the genetic improvement and yield increase of crops to study the molecular mechanism of plant stress responses and explore the important functional genes.Glycosyltransferase is a type of enzymes to be considered as the key factor controlling cell metabolism balance,which catalyzes plant molecular glycosylation and plays a key role in adjusting the adaptive change of the metabolic pathway and the attainment of new balance.Therefore,glycosyltransferases are crucial for the regulation of plant growth and development and plant adaptability to environments.Their functions have been attracting the increasing attention.In this thesis,we studied the function of two glycosyltransferase genes in plant adaptation to heat and drought stress using rice as experimental materials.In addition,a kinase gene involved in plant disease resistance was also partially studied.The main research contents and results are as follows:1.Rice glycosyltransferase gene IAGT1 regulates development and response to heat stress through the auxin pathwayAuxin is an important hormone regulating plant growth and development,and its glycosylation is one of the important mechanisms regulating auxin homeostasis.The glycosylation of auxin in Arabidopsis thaliana has been extensively studied,but the auxin glucosyltransferase in rice is less researched.In this study,the rice glycosyltransferase gene IAGT1 involved in the glycosylation of auxin IAA was identified.HPLC and LC-MS analysis showed that IAGT1 could catalyze the glycosylation of auxin IAA in vitro and in vivo.Moreover,IAGT1 was induced by exogenous IAA and IBA treatment.In order to study the function of IAGT1 gene,the growth phenotypes and auxin changes of the overexpression lines and mutants of IAGT1 gene were analyzed.Although the mutants showed no changes in phenotype and auxin content compared with the wild type,possibly due to functional redundancy,the root length,bud length and plant height of the overexpression lines were found to be shorter than those of the wild type.It was found that the glycosylated product of IAA（IAA-Glc）was accumulated in overexpression lines,and the content of IAA was decreased,accompanied by down-regulation of expression levels of auxin response genes.But the auxin synthesis genes in overexpression lines was up-regulated,which may be the result of feedback regulation.These results suggest that IAGT1 can regulate the growth and development of rice by affecting auxin homeostasis.Furthermore,the key promoter element of IAGT1 gene and its upstream regulatory factors were analyzed.The heat shock transcription factor OsHSFA2d was identified and experiments showed that it can bind to the promoter of IAGT1 and negatively regulate the expression of IAGTI.This regulatory relationship was also demonstrated by ChIP assay and the expression level of IAGT1 in oshsfa2d mutants.Considering that OsHSFA2d is a heat shock transcription factor that plays a positive role in response to high temperature,heat stress experiments were performed on the overexpression lines and mutants of IAGT1.After 42℃ treatment,although the mutants showed little difference from WT,the overexpression lines showed more sensitive phenotype to high temperature than WT,accompanied by physiological changes such as increased ion permeability and ROS accumulation.qRT-PCR analysis showed that the transcription levels of some marker genes responding to high temperature were significantly down-regulated in overexpression lines.The changes of IAA-Glc in transgenic lines after high temperature treatment were detected.It was found that the accumulation of IAA-Glc in overexpression lines was increased compared with WT,and the expression level of auxin response gene ARFs in overexpression lines was decreased compared with WT.These results suggested that IAGT1 played an important role in the response to high temperature stress through the auxin pathway.In conclusion,IAGT1 acting as a rice auxin glycosyltransferase,not only regulates the growth and development of rice by mediating auxin homeostasis,but also participates in responding to high temperature stress through its interaction with OsHSFA2d.This discovery provides a new perspective for understanding the regulation of growth and development and the mechanism of response to high temperature stress in rice.2.Rice glycosyltransferase gene UGT85E1 participates in drought stress response through abscisic acid pathwayDrought is one of the most important environmental constraints affecting plant growth and development,and ultimately leads to crop yield reduction.Based on our finding that rice glycosyltransferase gene UGT85E1 can be induced by osmotic stress and ABA,the function and mechanism of UGT85E1 involved in the drought tolerance response was studied.Firstly,the gene was cloned,its overexpression vector was constructed,and two overexpression homozygous lines were obtained through genetic transformation.Meanwhile,homozygous mutants were obtained by CRISPR/Cas9 technique.Phenotype analysis of mutants and overexpression lines under osmotic stress and drought stress was then carried out.It was found that after mannitol treatment,the ugt85e1 mutants showed more sensitive phenotype to osmotic stress than WT,while the growth of the two overexpression lines were better than WT,and their survival rates were consistent with the phenotype observation.When testing the soilgrown rice plants,the ugt85e1 mutants were also found to be more sensitive than WT,while the overexpression lines were more resistant to drought stress.These results suggested that UGT85E1 gene was involved in responding to drought stress and positively regulates the drought resistance in rice.The stomatal opening and closing of the mutants and the overexpression lines were observed by scanning electron microscopy.It was found that the number and degree of stomatal closure of the overexpression lines were much more than those of the wild type after drought treatment,but the mutants were the opposite.In addition,the water loss rate of the overexpression lines was slower than WT.These results suggested that overexpression of UGT85E1 can promote stomatal closure and reduce the water loss under drought condition.By measuring endogenous ABA content,it was found that UGT85E1 overexpression lines accumulated much more ABA than WT after drought treatment,while ABA content in mutants decreased significantly.The expression levels of ABA synthesis and signaling genes in overexpression lines and mutants were consistent with their changes in ABA contents.The contents of proline and soluble sugar were also detected.The contents of proline and soluble sugar in overexpression lines were significantly higher than those of WT,while the mutants showed the lower contents.DAB staining showed that amount of H2O2 was less in overexpression lines,but the mutants accumulated more H2O2.The transcription levels of ROS scavenging enzymes were significantly increased in overexpression lines but decreased in mutants.In addition,after the detached leaves of transgenic lines were treated with H2O2 and MV,the mutants showed severe chlorosis and significantly decreased chlorophyll content.while the overexpression lines showed better green retention.These results indicated that overexpression of UGT85E1 increased the antioxidant capacity of rice.The transcription levels of drought stress responsive genes OsRAB16,OsRAB21,OsDREB2B and OsDREB1A in transgenic lines were detected.It was found that the expression of these genes in overexpression lines was higher than that in the wild type,but the mutants showed lower expression.These results suggested that UGT85E1 gene was involved in responding to drought stress and played a positive regulatory role through affecting ABA synthesis.The function of UGT85E1 was further verified by transforming UGT85E1 gene into Arabidopsis.It was found that transgenic Arabidopsis showed significant drought tolerance from the aspects of survival rate,water loss rate,ROS accumulation and transcription levels of drought tolerant genes.These results suggest that UGT85E1 plays a positive role in mediating the adaptive response to drought in rice and other plants.This study may provide a candidate gene for drought resistance breeding of dicotyledons and monocotyledons. |
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