WRKY1 Transcription Factor Regulating Leaf Senescence Through Salicylic Acid Pathway In Arabidopsis Thaliana

Leaf senescence is the last stage of leaf development,which is affected not only by internal factors such as age,hormones and reactive oxygen species(ROS),and also by environmental factors such as drought,high temperature and pathogens.SA is a phenolic hormone in plants,which affects seed germination,cell growth,stomatal opening,leaf senescence,fruit yield,and has the ability to defend against biological and abiotic stresses.Previous studies have shown that the SA in the plant through Isochorisic acid(ICS)and Phenylalanine(PAL)way to synthesis and in precise regulation network,different hormones or regulatory factors through regulating the SA metabolism related genes expression of space and time to regulate the content and distribution of SA,but SA metabolism molecular mechanism remains to be further study of transcriptional regulation.We studied a transcription factor WRKY1 that regulates SA metabolism.The main research conclusions are as follows:(1)35S: WRKY1-GFP expression vector was constructed and transformed into Nicotiana benthamiana.The transient expression of WRKY1-GFP fusion protein was observed by laser confocal microscopy,and it was found that WRKY1-GFP co-localized with the nuclear localization marker protein,indicating that WRKY1 is a nuclear protein.The subcellular localization of WRKY1 is consistent with the function of its transcription factors.(2)qRT-PCR was used to detect the expression of WRKY1 in wide type leaves at different aging stages,and to detect the expression of WRKY1 in wide type leaves treated with SA and Pseudomonas syringae pv tomato DC3000(Pst DC3000)pathogens,respectively.It was found that WRKY1 expression was higher in aging leaves and was induced by SA and pathogens at the same time.We further constructed a WRKY1 promoter-driven GUS reporter vector Pro WRKY1: GUS.GUS staining of transgenic plants showed that WRKY1 expression increased with seedling age,SA and Pst DC3000,which was consistent with the q RT-PCR results.These results indicated that WRKY1 was induced by SA,pathogens and leaf senescence signals.(3)Phenotypic observation showed that wrky1 T-DNA mutants exhibited late leaf senescence phenotype,while WRKY1-OE plants exhibited premature leaf senescence phenotype.Treatment with Pst DC3000 showed that wrky1 was sensitive to pathogens,while WRKY1-OE showed strong disease resistance.These results indicated that WRKY1 transcription factor positively regulates leaf senescence and plant bacterial resistance.(4)Compared with the wild type,the contents of SA and 2,5-DHBA increased in WRKY1-OE plants,but decreased in wrky1 mutants.Therefore,we speculated that WRKY1 positively regulates SA and 2,5-DHBA production.(5)The Dual-luciferase reporter assay,chromatin immunoprecipitation PCR(Ch IP-PCR)and electrophoretic mobility shift assays(EMSAs)were used to demonstrate that WRKY1 can directly bind to the promoter of SID2 and S5 H,respectively,and positively regulate their transcription to promote the synthesis and dynamic homeostatic of SA,thus regulating the normal aging of leaves.(6)sid2 mutant was crossbred with WRKY1-OE,and it was found that sid2 mutant could inhibit WRKY1-induced leaf senescence phenotype,suggesting that WRKY1 regulates leaf senescence through the SA synthesis pathway.(7)Construction of WRKY1-OE1×S5H-OE hybrid plants,phenotypic analysis and SA content detection found that WRKY1-OE1×S5H-OE hybrid plants showed a delayed senescence phenotype,and its 2,5-DHBA content was significantly higher than that of S5H-OE,which means that WRKY1 promotes SA hydroxylation to generate 2,5-DHBA by promoting S5 H transcription.In conclusion,WRKY1 transcription factors are localized in the nucleus,induced by SA and pathogens,and directly bind to the promoter regions of SID2 and S5 H to induce gene expression,regulate the dynamic balance of SA through the synthesis of SA and 2,5-DHBA,positively regulate the process of leaf aging,and improve plant bacterial resistance at the same time.Clarification of WRKY1 gene function is of great significance for further understanding the regulation of leaf senescence and bacterial resistance by SA.