Molecular Dissection Of OsWRKY53 In Negatively Regulating Rice Cold Tolerance At The Booting Stage

Cold tolerance at the booting stage will damage the reproductive organ function of rice(Oryza sativa),thus impair the development of anther,ultimately leading to a significant decrease in the seed setting rate and yield of rice.Heilongjiang Province is an important rice commercial grain area in China,and the rice yield accounts for about 1/9 of the whole country.Heilongjiang Province is the most northern rice growing area in China,and cold injury frequently occurs at booting stage,which seriously affects the rice production.Due to the difficulty in identifying cold tolerance phenotype at booting stage,only a few cold tolerance genes at booting stage have been identified at present.Therefore,excavating more cold-tolerance genes at booting stage and analyzing their regulatory mechanisms have Very important practical significance.Here,we characterize the function of the cold-induced transcription factor OsWRKY53.And OsWRKY53 could negatively regulate the cold tolerance of rice at booting stage.The theoretical basis and application value of OsWRKY53 in regulating cold tolerance at booting stage were confirmed by biochemical and genetic experiments.The specific research results of this paper are as follows:1.Through RNA-seq analysis and q RT-PCR assay,OsWRKY53 was clearly induced by cold treatment at booting stage.2.The phenotypes of Longjing 11,oswrky53 mutants and OsWRKY53-OE plants were observed and analyzed,and the cold tolerance phenotypes were identified at booting stage.The results showed that OsWRKY53 negatively regulates the cold tolerance of rice at booting stage.3.GA positively regulates cold tolerance of rice at booting stage confirmed by GA spraying experiment.GA content in the anther of oswrky53 mutant was significantly higher than that of the wild type Longjing 11 under cold treatment.Further q RT-PCR analysis showed that the expression level of GA biosynthesis genes(GA20ox1 、GA20ox3、GA3ox1)in oswrky53 mutant was significantly higher than Longjing 11 after low temperature treatment.These results suggest that OsWRKY53 may be involved in regulating cold tolerance at booting stage by negatively regulating the biosynthesis of GA in anthers and negatively regulating GA content in anthers.4.The results of EMSA,Ch IP and rice protoplast transient transformation showed that OsWRKY53 directly associates with the promoters of GA biosynthesis genes(GA20ox1、GA20ox3、GA3ox1)and transcriptionally represses their expression in anthers.Genetic experiments showed that OsWRKY53 was located upstream of GA biosynthesis genes during cold tolerance regulation at booting stage.5.OsWRKY53 could affect the difference of cold tolerance between indica and japonica rice by identifying the cold tolerance phenotypes of the Chromosome Segment Substitution Lines(CSSL)and Near Isogenic Lines(NIL)at booting stage of Nipponbare and 9311.And,discovered a functional SNP in the OsWRKY53 coding region.The variation of SNP-518 showed obvious differentiation between indica and japonica rice,and OsWRKY53-518 G type showed stronger transcriptional inhibitory activity against GA biosynthesis gene.The association analysis further proved that the cold tolerance of OsWRKY53-518 C type rice material was significantly stronger than that of OsWRKY53-518 G type rice material at booting stage.Evolution analysis showed that natural selection occurred at OsWRKY53-518C/G locus during the evolution of rice.6.Analysis of agronomic characters in the field showed that oswrky53 mutant plants in different varieties showed that the 1000-grain weight of oswrky53 mutant plants decreased due to the small grain size,but the tiller number of oswrky53 mutant plants increased,which had no effect on the yield.But,the yield per ten plants of OsWRKY53 mutant under low temperature stress was about 43.8% higher than that of the wild-type Longjing 11.OsWRKY53 rice materials were obtained from Daohuaxiang 2 and Longdao 16,the main cultivars in Heilongjiang Province,as background,and the cold tolerance phenotype was identified.The results showed that OsWRKY53 rice materials under different backgrounds could significantly improve the cold tolerance at booting stage.These results suggested that OsWRKY53 could negatively regulate GA content in anther by directly bind to GA biosynthesis gene and transcriptional inhibit its expression,thus negatively regulating cold tolerance of rice at booting stage.OsWRKY53 mutant could improve rice yield under low temperature stress.Together,these findings provide a target for improving cold tolerance at the booting stage in rice.