Study On The Transcription Factor NAC120 Regulating Rice Drought Stress Response Through ABA Signaling Pathway

Rice(Oryza sativa L.)is one of the four main crops in China,and it is also an ideal model plant for research.It is often affected by drought,low temperature and other adverse environments during growth.Therefore,it is of great significance to study the stress response of rice and reveal the molecular mechanism.In recent years,there are series of research on the molecular mechanism of stress responses in rice,including transcription factors and their molecular regulatory mechanisms.Transcription factors play a central regulatory role in the signal transduction pathway in rice stress responses.NAC(NAM,ATAF1/2 and CUC)is plant-specific transcription factors,which participate in a variety of biological processes in plant growth and development,and biological and abiotic stress responses.NAC120 is a member of rice NAC transcription factor family.Therefore,research on the biological function and regulation mechanism of NAC120 in rice stress response can provide scientific basis for enriching the function of NAC family.In this study,both T-DNA insertion mutants(nac120)and NAC120-overexpressing(NAC120-OE)plants were used to study the biological function of NAC120 and the molecular mechanism of NAC120 regulating rice drought stress response.The main research results are as follows:(1)Analysis the spatio-temporal expression profile of NAC120 in rice.The analysis showed that NAC120 was expressed in various tissues of rice,and the highest expression level was found in the root.(2)The subcellular localization analysis showed that NAC120 was mainly located in the nucleus.(3)The loss of function mutation of NAC120 affects the growth and development of rice.Compared with the wild type(DJ),at the seedling stage,the growth of shoot and root of the loss-of-function mutant nac120 was inhibited.Overexpression of NAC120 does not affect the growth and development of rice.(4)nac120 mutants and NAC120-OE plants were used to study the response of NAC120 to drought stress.Compared with the wild type(DJ),nac120 mutants showed the enhanced drought tolerance,while NAC120-OE plants exhibited the increased sensitivity to drought stress.When subjected to drought stress,the transcritp levels of drought-responsive genes were much higher in nac120 mutants but lower in NAC120-OE plants than that in wild type(DJ).(5)ABA-sensitivity assays demonstrated that,compared with wild type(DJ),nac120 mutants showed an increased sensitivity to exogenous ABA,while NAC120-OE plants displayed an insensitive phenotype to ABA,suggesting that NAC120 may negatively regulate rice response to drought stress through ABA signaling pathway.(6)Yeast two hybrid,GST pull-down,Bi FC(Bimolecular Fluorescent Complimentary,Bi FC)and Co IP(Coimmunoprecipitation)experiments indicated that NAC120 could physically interact with SAPK9,which is a core element in ABA signaling pathway.(7)Phosphorylation assays in vitro showed that SAPK9 can phosphorylate NAC120 in E.Coli.When Ser at position 40 and Thr at position 140 in the NAC120 were mutated to Ala,the phosphorylation of NAC120 by SAPK9 was significantly reduced,indicating that Ser40 and Thr140 may be phosphorylation sites recognized by SAPK9 on NAC120.(8)The results of cell-free degradation assay showed that,compared with the wild-type(DJ)protein extract,the degradation rate of the recombinant protein His-NAC120 in the sapk9 mutant protein extract was significantly reduced,indicating that SAPK9 affects the stability of the NAC120 protein.These results above exhibited that NAC120 affects the expression levels of drought response genes by interacting with SAPK9 and phsophorated by SAPK9,and thus affecting the response of rice plants to drought stress.This study can not only provide scientific basis and technical guidance for improving rice stress resistance in application,but also enrich the biological function and mechanism of NAC transcription factor family in theory.