Arabidopsis WRKY2 And WRKY10 Enhance Targeting Of CCA1 And LHY To PIF4 And Positively Regulate Its Expression And Regulate Plant Photomorphogenesis Under Light

Plant growth and development is influnced by multitudinous factors,including intrinsic factors such as hormone,and environmental factors such as light and temperature.Plants are sessile and evolve a complex molecular regulatory network to better adapt to the surrounding environment.In response to the changes of the external environment,plants adjust their own growth and development to mainatain an equilibrium status with their outside environment.Light,as one of the most important environment factors,not only provides the energy for plants photosynthesis but also functions as a signal to regulate various signaling pathways throughout their whole life cycle.Too strong or weak light intensity likely cause damages to plants,and plants evolved photoinhibition and shade avoidance responses to adapted to the rapidly changing environmental conditions.Therefore,studies on light signaling system is very important for both theoretical consideration and agricultural application.This study used model plant Arabidopsis thaliana and focused on the function of WRKY2/WRKY10(WRKY DNA-binding protein 2/10)-CCA1(CIRCADIAN CLOCK ASSOCIATED 1)/LHY(LATE LONGATED HYPOCOTYL)-SHB1(SHORT HYPOCOTYL UNDER BLUE 1)complex in the up-regulation of PIF4(PHYTOCHROME INTERACTING FACTOR 4)expression.PIF4 is a basic bHLH transcription factor,a negative regulator of plant photomorphogenesis,and involved in the regulation of many other aspects of plant growth and development.SHB1 is involved in plant blue light signal transduction and also influences seed size by associating with MINISEED 3(MINI3)and HAIKU 2(IKU2)promoters and up-regulating their expression.CCA1 and its homologous gene LHY are key components of the plant circadian clock and regulate multiple signal transduction passways.WRKY2 is one of the WRKY family transcription factors and regulates seed germination and pollen development.WRKY10 is another member of the WRKY family transcription factors,expressed in seed endosperm and aids recruiting SHB1 to the promoters of WRKY10 and IKU2 to up-regulate their expression and influence endosperm development and seed size.In this study,we found that WRKY2 is a homolog of WRKY10,and facilitates an up-regulated expression of PIF4 by CCA1/LHY and SHB1 under red light.This regulatory step provides a desensitization strategy under strong light intensity and maintains a balance state of plant photomorphogenesis.The major discoveries are as follows:(1)WRKY2 and WRKY10 are involved in the regulation of plant photomorphogenesisGUS assays with pWRKY10::GUS plants indicate that WRKY10 was also expressed in cotyledon,hypocotyl and root of young seedlings in the dark and under red light.Loss-offunction wrky10 mutant showed a shorter hypocotyl phenotype under red light but not in the dark or under blue and far-red light compared to that of wild type.WRKY2 is homologous to WRKY10,and double wrky2 wrky10+/-mutant showed a much shorter hypocotyl phenotype than that of wrky10 single mutant.Furthermore,PIF4 expression was down-regulated in the mutants.(2)WRKY2 and WRKY10 bind to the promoter of PIF4.As the WRKY family members,WRKY2 and WRKY10 contain the conserved WRKY DNA binding domain.ChIP results showed that both WRKY2 and WRKY10 bind to the promoter of PIF4 under red light but not in the dark.Y1 H and EMSA assays also confirmed that WRKY2 and WRKY10 bind to the promoter of PIF4.(3)WRKY2 and WRKY10 interact with CCA1/LHY and enhance the binding affinity of CCA1/LHY to the PIF4 promoter.The binding site of WRKY2 and WRKY10 is adjacent to the bingding site of CCA1/LHY in the promoter of PIF4.Co-IP assays indicate that WRKY2 and WRKY10 can interact with CCA1/LHY both in the dark and under red light.Y2 H and BiFC assays also showed the same result.Through ChIP assays,the binding ability of CCA1 and LHY to PIF4 promoter is enhanced in 35S::WRKY10 overexpression transgenic lines compared to that of wild type.Trans-activition assays with Arabidopsis mesophyll cell protoplasts showed the same result.In EMSA assays,the binding affinity of CCA1 or LHY was enhanced by WRKY2 or WRKY10 and a super-shifted was obvious.These results indicate that the promotion of PIF4 expression by WRKY2 and WRKY10 is tied to their enhancement over the binding ability of CCA1 and LHY to the PIF4 promoter.(4)WRKY10-CCA1/LHY/SHB1 form a complex and SHB1 up-regulates WRKY10 expression.WRKY10 interacted with SHB1 in the presence of CCA1/LHY but not in the absence of CCA1/LHY in Co-IP assays.WRKY10 and CCA1/LHY and SHB1 likely form a complex and WRKY10 interacts with SHB1 indirectly.SHB1 also binds the genomic region of WRKY10 and up-regulate its expression.(5)WRKY10 function relies on CCA1/LHY to regulate PIF4 expression.Under red light,the hypocotyl length of pWRKY10::WRKY10-MYC/Ws was longer than that of wild type.By contrast,the hypocotyl length of pWRKY10::WRKY10-MYC/cca1 lhy had no difference with that of cca1 lhy mutant.PIF4 expression was up-regulated in pWRKY10::WRKY10-MYC/Ws background compared to that in Ws background.PIF4 expression had no difference in cca1 lhy mutant and pWRKY10::WRKY10-MYC/cca1 lhy transgenic lines.In conclusion,WRKY2 and WRKY10 are involved in the regulation of plant photomorphogenesis.Under red light,WRKY2 and WRKY10 bind to PIF4 promoter,interact with CCA1/LHY and enhance their binding ability to the PIF4 promoter,thus recruiting more SHB1 protein to the PIF4 regulatory region.SHB1 in turn also associates with the WRKY10 genomic locus and up-regulates its expression.This regulatory process desensitizes light response under strong light condition and maintains a balance state of photomorphogenesis.