Mechanism Analysis Of OsWRKY53 Regulating Rice Cold Tolerance At Booting Stage Through GA Pathway

Rice is an important food crop.Chilling damage at the booting stage will affect the function of flower organs,reduce fertility,and cause serious reduction in rice production.Therefore,it is of great significance to analyze the molecular mechanism of chilling injury at the booting stage of rice for rice production.Gibberellin(GA)is an important plant hormone that regulates multiple processes of plant growth and development and is involved in responding to various abiotic stresses.As a negative regulatory element of GA signaling,DELLA protein usually participates in the regulation of GA signaling-related physiological processes in the form of protein interaction.The laboratory obtained an oswrky53 knockout mutant in a previous study,which accumulated a higher level of active GA in anthers under low temperature stress,and significantly improved cold tolerance at booting stage,indicating that OsWRKY53 negatively regulates GA in anthers under low temperature stress.The content further improved the cold tolerance at booting stage.In order to further analyze the mechanism of GA regulating cold tolerance at booting stage,we comprehensively used various experimental methods to explore the rice DELLA protein SLR1(SLENDER RICE 1)and the key factors controlling tapetum development UDT1(UNDEVELOPED TAPETUM 1)and TDR(TAPETUM DEGENERATION).RETARDATION),and thus preliminarily analyzed the molecular mechanism of GA signaling involved in the regulation of cold tolerance at booting stage of rice by OsWRKY53.The main results are as follows:1.Through Western blot detection,it was found that the SLR1 protein content of oswrky53 in vivo was significantly lower than that of the wild type under cold treatment conditions,indicating that the change of SLR1 protein content was involved in the regulation of GA content by OsWRKY53.2.Through yeast two-hybrid,LCI,BiFC,and Pull-down experiments,it is proved that SLR1 and UDT1/TDR can interact in vivo and in vitro,indicating that GA signaling may be the interaction between SLR1 and UDT1/TDR,which regulates rice by regulating rice pollen fertility.3.Through the EMSA test,it was found that UDT1 and TDR can directly bind to the E-box element in the promoter region of OsCP1,and this binding can be interfered by SLR1;through the rice protoplast transient expression system,it is shown that both UDT1 and TDR can activate OsCP1 However,co-expression of SLR1 significantly reduced the transcriptional activation ability of UDT1 and TDR on OsCP1,which was relieved by exogenous GA3 treatment;in addition,the expression level of OsCP1 was significantly decreased in the ga20ox1 mutant.These results suggest that SLR1 can inhibit the transcriptional regulation of OsCP1 by UDT1/TDR,and the SLR1-UDT1/TDR module affects rice fertility by regulating the expression of OsCP1.4.Through qRT-PCR analysis,it was found that the dynamic expression level of OsCP1 decreased significantly under low temperature treatment,while the expression level of OsCP1 in the panicle of mutant oswrky53 was significantly higher than that of wild type.This indicated that OsWRKY53 may regulate the expression of OsCP1 through the SLR1-UDT1/TDR module,thereby regulating cold tolerance at booting stage of rice.5.Using plant overexpression transgenic technology and Agrobacterium-mediated genetic transformation technology,it was found that the overexpression of UDT1 or TDR damages the anther development of plants,affects the proportion of fertile pollen,led to a significant decrease in the seed setting rate,and even appeared sterile phenotype,indicating that UDT1/TDR overexpression affects rice anther development.