Analysis Of Interactions Between SAPKs And BZIPs And Functional Characterization Of SAPK10 Gene In Rice Flowering

Heading date is an important agronomic trait which affects yield,regional and seasonal adaptability in rice.Flowering time also means the normal transformation from vegetative growth to reproductive growth.The occurrence of this transformation is determined by the external environment and its genetic factors.Environmental factors,such as photoperiod,temperature and plant hormones,are important conditions in rice heading date.As a critical plant hormone in rice,ABA is involved in regulating seed development,flowering time and responding to biotic and abiotic stresses.Endogenous ABA is a key factor in rice heading date,and the concentration of exogenous ABA could promote or suppress flowering time in rice.The ABA signaling pathway is transduced by SnRK2s-ABF/AREB which is more conserved in rice thus SnRK2 is the core component in ABA signaling pathway.SnRK2 family contains 10 members denoted as SAPK1 to SAPK10.The bZIP transcription factor family is widely distributed and conserved in plants,with 89 members being identified in rice.However,it is still largely unknown which SAPKs pair with bZIPs respond to heading date in rice.According to the tissue expression profile,we discovered that nine bZIP transcription factors were induced by ABA treatment.We therefore performed qRT-PCR to confirm the transcriptional pattern of the nine ABA-induced bZIPs and SAPK1 to SAPK10.We systematically screened the protein-protein interactions between SAPKs and ABA-inducible bZIPs using yeast two hybrid system(Y2H)and also verified the result by in vitro pull-down assay.By using kinase assay,we demonstrated that SAPK10 could phosphorylate bZIP77 and also found out that overexpression of SAPK10 conferred early flowering.The main results are described as follows:(1)We established that 9 bZIPs and SnRK2 s were sensitivity to ABA by qRT-PCR at different time course.We also obtained 14 positive interactions by performing Y2 H analysis between 10 SAPKs and 9 ABA-induced bZIPs.The key flowering regulator(bZIP77)was found to form a functional floret-activated complex(FAC)with 14-3-3 and Hd3 a in the nucleus.The FAC could then activate MADS15 to promote rice flowering time,we therefore used in vitro pull-down assay to investigate the interaction between bZIP77 with SAPK9 and SAPK10 respectively in other to verify the reliability of the result obtained from Y2 H.(2)SAPK10 was found to phosphorylate bZIP77 in in-vitro kinase assay and overexpressed SAPK10 plants showed early flowering time in rice during long-day and short-day conditions.We also observed that the expression levels of bZIP77 and MADS15 was improved in over-expressed SAPK10 plants.Therefore,we resolved that SAPK10 phosphorylates bZIP77 to form FAC which could then activate MADS15 to promote early flowering in rice.Conclusively,our results provided an overall view of the role of ABA-inducible bZIP-SAPK interaction in regulating rice flowering time.