Founction And Mechanism Research Of OsABF1 Regulating Rice Flowering

Rice is one of the main food crops in China.Heading date is an important physiological process of rice,which is affected by light,temperature and basic vegetative growth.Different regions choose rice varieties with different heading date to make full use of local climate conditions to achieve high quality and high yield.Therefore,discovery and utilization of heading date genes have important guiding significance for the genetic improvement of rice varieties.In this study,we used molecular biology methods such as CRISPR/Cas9,overexpression,subcellular location,yeast two hybridization,pull down,bimolecular fluorescence complementation assay and other molecular biological methods were used to identify a signaling pathway of"SAPK8-ABF1-Ehd1/Ehd2",which independent of photoperiod that inhibited rice heading through ABA.In response to exogenous ABA signal,SAPK8 upregulated the transcription of OsABF1,and phosphorylated OsABF1,finally enhanced its DNA binding ability.In addition,OsABF1 interacts with FIE2 to recruit PRC2 complex on the promoter region of Ehd1 and Ehd2 implement H3K27me3 inhibition modification,thereby inhibiting downstream Ehd1 and Ehd2transcription,and ultimately delayed rice heading.The results of this study provided a favorable theoretical basis for discovering of new heading date genes and elucidating of their molecular mechanisms.The main results of this study were as follows:1.OsABF1 regulated the heading date of rice and has functional redundancy with b ZIP40.In this study,overexpression technology with template of Nipponbare CDS,and created the OsABF1-OE^NIP and OsABF1-OE^ZS97 overexpression lines respectively.Compared with the wild type,the heading date of OsABF1-OE^NIP and OsABF1-OE^ZS97were delayed under natural long-day condition in Hangzhou.Compared with the wild type,the heading date of abf1 had no change.The heading date of abf1/bzip40 was earlier than that of the wild type under long and short day conditions.These results indicated that OsABF1 affected rice heading date and had functional redundancy with b ZIP40.2.OsABF1 participant in regulation of molecular network of rice heading date.In this study,yeast two hybridization analysis,pull down and bimolecular fluorescence complementation assay were applied,and verified that OsABF1 interacted with protein kinase SAPK8and could phosphorylated by SAPK8.Compared with the wild type,the heading date of SAPK8-OE were delayed under natural long-day and short-day conditions.However,the heading date of sapk8 was the same as wild type.These results indicated that OsABF1 could interact with SAPK8,and both were participate in the regulation of rice heading date.Further,EMSA,Luciferase assay and Ch IP-PCR experiments were used to verify that Ehd1 and Ehd2 could be directly negative regulated by OsABF1.The heading date of ehd1 and ehd2 were delayed compared with the wild type under natural long-day in Hangzhou.Overexpression of Ehd1 in the background of OsABF1-OE could restore the late heading phenotype.These results indicated that OsABF1 affected the heading date by directly regulated Ehd1.Yeast two hybridization analysis,pull down and bimolecular fluorescence complementation assay were applied to verify that OsABF1 could interact with FIE2(a member of PRC2 in rice).The heading date of fie2 was earlier compared with the wild type under long-day condition.FIE2 may recruit PRC2complex to implement H3K27me3 inhibition modification on the promoters of Ehd1 and Ehd2,inhibiting transcription level of Ehd1 and Ehd2 and finally delayed rice heading date.3.Exogenous ABA effected rice heading date.Compareing with the control,after treatment with ABA,the expression levels of OsABF1increased significantly in wild-type,OsABF1-OE and abf1/bzip40.The enrichment sites of OsABF1,FIE2 and H3K27me3 were co-located in the G-box region on the promoters of Ehd1 and Ehd2,and exogenous ABA could enhance the enrichment density.These results showed that ABA could enhance the inhibition of Ehd1 and Ehd2 through OsABF1.