The Molecular Mechanism Of BRINSENSITIVE1-EMS-SUPPRESSOR1 Interacting With ABSCISIC ACID INSENSITIVE5 In The Regulation Of Arabidopsis Seed Germination

Seeds dormancy and germination,which are extremely significant for the survival of plants,are tightly regulated by multiple external environmental factors and internal hormones.Under stresses like high salt,drought,low temperature and others,endogenous phytohormone abscisic acid(ABA)accumulates in seeds,and inhibits the germination process to help plants against the adverse environment.However,when the stress is released or the environment is suitable for plant growth,it is crucial to reduce the stress responses in seeds,and promote the seed germination process for plant survival and reproduction.Proper regulation of seed germination is necessary for successful propagation of plants.Therefore,the study on the regulation mechanisms of seed germination is especially important.ABA signaling pathway plays an important role in suppressing seed germination.Previous studies showed that phytohormone brassinosteroids(BRs)plays a role in antagonizing ABA signaling to promote seed germination.det2-1,a mutant which is deficient in BR biosynthesis showed ABA-sensitive phenotype,while bes1-D with constitutively activated BR signaling showed ABA-insensitive phenotype during seed germination.However,the molecular mechanism of ABA-BR antagonism remains to be elucidated.ABSCISIC ACID INSENSITIVE5(ABI5)is an important transcription factor in the ABA signaling pathway which plays a central role in suppressing seed germination.In this study,the result of yeast two-hybrid(Y2H)assay showed that the key transcription factor in the BR signaling pathway BRINSENSITIVE1(BRI1)-EMS-SUPPRESSOR1(BES1)specifically interacts with ABI5.In vitro pull-down assays,in vivo co-immunoprecipitation(Co-IP)assays,bimolecular fluorescence complementation(BiFC)and firefly luciferase complementation-imaging(LCI)assays further demonstrated that ABI5 can directly bind to BES1.As a member of the basic leucine zipper(bZIP)transcription factor family,ABI5 contains four conserved domains,including C1,C2,C3,C4,and a bZIP domain which is involved in DNA binding.Accordingly,truncated mutants of ABI5 were constructed and expressed for further experiments.The results of yeast two-hybrid,in vitro pull-down and LCI assays showed that BES1 binds to ABI5 through the bZIP domain of ABI5.The bZIP domain of ABI5 is the region through where ABI5 binds to the ABA-responsive elements of the downstream genes' promoters.Further experiments of electrophoretic gel mobility shift assays(EMSA)and transient expression in tobacco leaves were performed and the results showed that the interaction between BES1 and ABI5 negatively regulates the binding of ABI5 to the promoters of downstream genes.Previous studies showed that ABI3,another transcription factor in the ABA signaling pathway,can interact with ABI5 and form a complex with ABI5 to regulate the expression of downstream ABA-responsive genes.In vitro pull-down assay showed that the amount of ABI3 binding to ABI5 decreases gradually with the increase of BES1,indicating that BES1 can specifically compete with ABIB to bind ABI5.Yeast two-hybrid competition experiments further demonstrated that BES1 interferes with the interaction between ABI3 and ABI5.What is more,the expression of ABI5 downstream genes was examined and the result showed that the interaction between BES1 and ABI5 eventually leads to the down-regulation of ABI5 downstream genes,thereby promoting the seed germination.The environmental stress results in the accumulation of ABA in seeds,and induces the expression of ABA-regulated genes that inhibit seed germination,thus facilitating the seed dormancy.However,when the stress is released or maintained at a low level,seeds need to break the dormancy and germinate to complete their life cycles.This process requires the existence of an antagonistic mechanism to the ABA signaling.This study demonstrated that BES1 directly interacts with ABI5 and interferes with the transcriptional regulation function of ABI5,therefore affecting the expression of ABA responsive genes,and further promoting the transition from seed dormancy to seed germination.This study unravels a novel mechanism of BES1 regulating seed germination through its interaction with ABI5 at the protein level,broadens our understanding of the BR-ABA antagonism to regulate seed germination and provides new insights into the coordination mechanisms of various hormone signals during seed germination and plant development.