Phenotypic Identification Of ABA Response Regulated By CRY2 And Analysis Of The Relationship Between CRY2 And ABI4 In Arabidopsis Thaliana

Light,as one of the main environmental factors,participates in regulating multiple aspect of plant growth and development.Plants experience light stimulation mainly through the photoreceptors.Three types of photoreceptors such as red/far red light photoreceptor,blue/UV-A light receptor,and UV-B light receptor have been identified in Arabidopsis thaliana.Cryptochromes(CRYs),the blue/UV-A light receptors,play important roles in promoting photomorphogenesis and regulating photoperiod flowering,and studies on the molecular mechanism of bule light signal transduction mediated by CRYs are more in-depth.More and more reports provide the evidence that CRYs are involved in the regulation of abiotic stress responses in plants,including drought,salt,high temperature and high-light stress.However,the intrinsic mechanism of cryptochrome-mediated abiotic stress response in plants is still unclear.In this study,the wild type(Col4),the loss-of-function mutant of CRY2(cry2)and CRY2-overexpressed transgenic Arabidopsis thaliana(GFP-CRY2)were used as materials to identify their phenetypes responding to ABA during their seed germination and seedling growth period.The results show that CRY2 plays a positive regulatory role in Abscisic acid(ABA)response.At the same time,the protein-protein interaction and protein-DNA interaction between CRY2 and ABI4(ABA INSENSITIVE 4,ABI4),an important transcriptional factor in ABA signaling pathway,were identified by in vitro and in vivo assays.The transgenic Arabidopsis overexpressing CRY2 in abi4(GFP-CRY2/abi4),a loss-of-function mutant of ABI4,was obtained and its phenotype responding to ABA was identified and the results indicats that the regulation of CRY2 on ABA response is at least dependent on ABI4.The detailed results are as follows:1.Compared with Col4,the seed germination and the seedling primary root elongation of cry2 were significantly less sensitive to exogenous ABA,while the sensitivity of GFP-CRY2 seed germination to ABA increased significantly,suggesting that CRY2 regulates ABA response positively.2.The results from the yeast two-hybrid and the BiFC assays confirmed the blue light-dependent interaction between CRY2 and ABI4.3.Through the yeast one-hybrid and dual luciferase reporter assays,the protein-DNA interaction between CRY2 and the promoter of ABI4 and CRY2-facilitated transcriptional activity of ABI4 promoter was confirmed.4.Statistical analysis of seed germination rate of T3 homozygotes of GFP-CRY2/abi4 induced by exogenous ABA showed that the seed germination rate was significantly lower than that of GFP-CRY2,but higher than that of abi4,indicating that CRY2 regulates ABA response at least depending on ABI4,and other factors supporting the function of CRY2 in this process exist.