WRKY20 Coordinates With ABI5 To Regulate The ABA Signal Pathway In Arabidopsis

Abscisic acid(ABA)is an important phytohormone regulating plant growth,development,and stress responses.It has an essential role in multiple physiological processes of plants,such as seed germination,stomatal closure,flowering and leaf senescence.Under stress conditions like low temperature,drought and high salinity will induce ABA accumulation,ABA integrates a variety of stress signals and then controls stress-responsive gene expression.Thus ensuring plants adaptation to stress.ABI5(ABSCISIC ACID INSENSITIVE 5)is a key transcription factor in ABA signal pathway,and has an essential role in plant biological processes,such as seed germination and dormancy,plant growth and development,anthocyanin synthesis,and stress response.WRKY is a unique transcription factor family with zinc finger domain in plant,which takes part in the physiological processes of various stress responses and plant development.At present,the research mainly indicated that WRKY can participate in the response of various signal pathways,such as abscisic acid(ABA),jasmonate(JA),salicylic acid(SA),to fullfill its biological function.In this study,we found that WRKY20 plays an important role in ABA signal pathway through investigating the phenotype of wrky20 mutant and WRKY20 overexpression strains in Arabidopsis thaliana under exogenous ABA treatment.Further study by using reverse genetics and molecular biology found that,WRKY20 can interact with ABI5 to coordinate the regulation of downstream ABA response genes’ expression.The main results are as follows:Compared with the wild type,wrky20 mutant showed a significantly reduced anthocyanin synthesis phenotype,we speculated that WRKY20 is related to the sugar signal transduction,and then we found a sugar insensitive phenotype in wrky20 mutant after glucose treatment,but no significant change in further starch detection.It has been reported that sugar can promote the synthesis of ABA.Compared with wild type,wrky20 mutant has higher germination and regreening rate after exogenous ABA treatment,wrky20 mutant show an insensitive phenotype under exogenous ABA treatment.It proves that WRKY20 is involved in the response of plants to ABA;Through RT-qPCR,we found that WRKY20 could promote the expression of ABA synthesis,signal transduction and response genes;Through yeast one-hybrid screening,we found WRKY20 can directly bind to the promoter of Em6.Analysising the promoter of Em6 showed that there are three W-box sites within 700 bp upstream of ATG which WRKY can bind to.Yeast one-hybrid and chromatin immunocoprecipitation experiment shows that WRKY20 could bind to the third W-box of Em6 promoter.Em6 is a direct downstream gene regulated by ABI5,thus we speculated WRKY20 can interact with ABI5.Yeast two-hybrid and bimolecular fluorescence complementary experiments showed that WRKY20 could indeed interact with ABI5 to coordinate ABA signal regulation,and WRKY20 can promote the activation of ABI5 on Em6.In conclusion,we found that WRKY20,as a plant transcription factor,can directly bind to the promoter of ABA signal response gene Em6 to promote its expression;at the same time,WRKY20 can interact with ABI5 to promote the transcriptional activation of its downstream genes and coordinate the regulation of ABA signal pathway.