Molecular Mechanism Of OsWRKY63 Regulating Cold Tolerance In Rice (Oryza Sativa L.)

Low temperature is one of the important factors limiting the yield and quality of rice,which seriously affects global food security and the living quality of human beings.It is an urgent problem for rice cultivars to reveal the molecular mechanism of cold tolerance and improve cold tolerance in rice.Cold tolerance is a complex trait controlled by multiple genes.Transcription factors(TFs)as regulatory factors are important components in the signal transduction process of cold tolerance.Previously,we constructed a transgenic rice library of the japonica cv Kitaake background overexpressing different TFs.We screened OsWRKY63-overexpressing lines decreased cold tolerance compared with wild-type Kitaake.OsWRKY63 is a WRKY transcription factor with an unknown function.Therefore,we deeply explored the molecular mechanism of OsWRKY63 in rice cold tolerance and constructed a transcriptional regulatory cascade model of OsWRKY63 in response to cold stress.The main research results are as follows:1.Analysis of cold tolerance of OsWRKY63Bioinformatics analysis showed that OsWRKY63 had two WRKY domains and belonged to group I of the WRKY TFs,and its promoter region contained many stress response elements.In OsWRKY63pro:GUS transgenic plants,GUS staining analysis showed that OsWRKY63 was expressed in multiple tissues tested.Dual-luciferase assay system analysis showed that OsWRKY63 functioned a transcriptional repressor by binding to the W-box(TTGACC/T)in rice protoplasts.Jada718 is a high-quality national authorized japonica rice We generated OsWRKY63-overexpressing plants and OsWRKY63-knockout mutants obtained by a CRISPR/Cas9 genome editing approach in the Jada718 background.OsWRKY63 overexpression caused serious damage to the membrane of rice,increased electrolyte extravasation,increased ion leakage,and reduced the content of osmotic regulatory substance proline,thus reducing the survival rate of the seedlings at low temperature and weakening the cold tolerance of rice at seedling stage.OsWRKY63-knockout rice mutants attenuated the membrane damage caused by low temperature,kept ion leakage,and significantly increased the proline content,so as to improve the survival rate of the seedlings under low temperature and enhance the cold tolerance of rice at seedling stage.These results indicate that OsWRKY63 is a negative regulator of cold tolerance.2.Transcriptome analysis of OsWRKY63-overexpressing plantsTranscriptome analysis showed that OsWRKY63 overexpression resulted in the differential expression of a large number of genes under normal conditions and cold treatment.GO analysis showed that differentially expressed genes after cold treatment were enriched in biological processes,including metabolic process,response to stress,response to oxidative stress,response to abiotic stimulus,and response to cold.Heat map of reactive oxygen species(ROS)scavenging-related genes analysis showed that OsWRKY63 overexpression significantly downregulated the expressions of 20 ROS scavenging-related genes after cold treatment.NBT staining and SOD activity analysis showed that OsWRKY63 could significantly decrease the SOD activity and increase the ROS content under cold treatment.In addition,heat map of resistance genes analysis showed that OsWRKY63 overexpression significantly inhibited the expression of OsWRKY71,MYBS3,OsWRKY76 and other resistance genes under normal conditions and cold treatment.These results indicate that OsWRKY63 could inhibit the expression of ROS scavenging-related genes,decreased the SOD activity,and increased the ROS content,thus weakening cold tolerance in rice.3.Analysis of transcriptional regulatory cascade of OsWRKY63 in response to cold stressChIP-qPCR and EMSA assays showed that OsWRKY63 could bind to the OsWRKY76 promoter containing the W-box element.q RT-PCR analysis showed that OsWRKY63 overexpression inhibited the expression of OsWRKY76 and OsWRKY63 knockout promoted the expression of OsWRKY76 under normal conditions and cold treatment.OsWRKY76-knockout rice mutants caused serious damage to the membrane of rice,increased electrolyte extravasation,increased ion leakage,and reduced the proline content and SOD activity,thus reducing the survival rate of the seedlings at low temperature and weakening the cold tolerance of rice at seedling stage.These results indicate that OsWRKY76 is a direct target of OsWRKY63.qRT-PCR analysis showed that OsWRKY76-knockout rice mutants repressed the cold-induced expression of Os DREB1 A,Os DREB1 B,Os DREB1 C,Os DREB1 D,and Os DREB1 J.Yeast one hybrid,EMSA and dual-luciferase assays showed that OsWRKY76 could bind to the Os DREB1 B promoter and activate its expression.Based on these findings,we revealed that OsWRKY63 is a negative regulator of cold tolerance by the OsWRKY63–OsWRKY76–Os DREB1 B transcriptional regulatory cascade.4.OsbHLH148 participates in the transcriptional regulatory cascade of OsWRKY63OsbHLH148 interacting with OsWRKY76 was predicted by STRING database.Yeast two hybrid,bimolecular fluorescence complementation,and pull-down assays showed that OsWRKY76 interacted with OsbHLH148.The Gal4-dependent chimeric transactivation assay showed that OsWRKY76 and OsbHLH148 formed a heterodimer in rice protoplasts,and the transcriptional activation activity of the heterodimer was significantly increased compared with that of OsWRKY76 or OsbHLH148.Dual-luciferase assays showed that OsWRKY76 cooperated with OsbHLH148 to activate the expression of Os DREB1 B and that the function of OsbHLH148 was largely dependent on OsWRKY76 in rice protoplasts.OsbHLH148-knockout rice mutants caused serious damage to the membrane of rice,increased electrolyte extravasation,and increased ion leakage,thus weakening the cold tolerance of rice at seedling stage.These results indicate that OsWRKY76 interacted with OsbHLH148,transactivating the expression of Os DREB1 B to enhance chilling tolerance in rice.In addition,yeast two hybrid and bimolecular fluorescence complementation assays showed that OsWRKY76 also interacts with Os JAZ12,the interacting protein of OsbHLH148.The Gal4-dependent chimeric transactivation assay showed that Os JAZ12 protein seriously repressed the transactivation activity of OsbHLH148,and this repression could be partly recovered by OsWRKY76.By measuring seed germination rates,seedling height,and root length under exogenous application of Me JA,we analyzed that OsWRKY76-knockout mutant decreased Me JA sensitivity.To sum up,we found that OsWRKY63 is a negative regulator of cold tolerance by the OsWRKY63–OsWRKY76/OsbHLH148–Os DREB1 B transcriptional regulatory cascade,which provides a new clue to reveal the molecular mechanism of cold tolerance in rice.OsWRKY63-knockout mutants obtained by a CRISPR/Cas9 genome editing approach in the Jada718 background observably improved the cold tolerance of rice,which provides a backup cold tolerance high-quality rice variety resource for practical production.