Functional Analysis Of OsRE1 And OsRIP1 In Regulating Rice Heading Date

Flowering(heading in crops)is an important turning point in the life process of plants,which determines the transition from vegetative growth stage to reproductive growth stage.In crops,heading date is particularly important.It not only determines the adaptability of different crop varieties to the environment of the cultivated area,but also affects the yield seriously.Rice(Oryza sativa)is one of the most important crops in the world.As the staple food for billions of people,the research on the heading date of rice is a hot spot.As a typical short-day plant,rice heading date is mainly regulated by photoperiod.At present,dozens of genes involved in the regulation of rice heading date have been cloned,and two major regulatory pathways,Hd1 pathway and Ehd1 pathway,have been formed.The rice OsGI-Hd1-Hd3a/RFT1 pathway is homologous to the GI-CO-FT pathway in Arabidopsis,as a prevalent flowering regulatory pathway in plants.Ehd1 is a rice-specific heading date gene that receives upstream light signals and directly regulates the expression of the rice florigen genes Hd3a/RFT1,forming a rice-specific heading date pathway.A series of genes have been found to regulate the expression of Ehd1,such as the flowering-promoting genes Ehd2,Ehd3,Ehd4 and flowering-suppressing genes Ghd7,DTH8.Less studies were reported about transcription factors which directly bind to the Ehd1 promoter,causing the loss of key factors in the Ehd1 pathway.In this study,we found a rice bZIP TF family gene OsRE1 and a B-box TF family gene OsRIP1 by reverse genetics,which are involved in the regulation of rice heading date.We analyzed the functions of OsRE1 and OsRIP1 by genetics,molecular biology and cell biology.The main results are as follows:1.OsRE1 can directly bind to the promoter of the heading date gene Ehd1.Through promoter fragmentation and protein-DNA binding experiments,it was confirmed that OsRE1 can bind to the A-box motif in the-700 site of the Ehd1 promoter,regulating the expression of Ehd1.2.OsRE1 delays the rice heading date.Under the natural long-day conditions in Beijing,the Osrel mutant plants flowered 3-5 days earlier than the wild-type Nipponbare,while the Osre1 ehd1 double mutants flowered later,like the ehd1 single mutants.This result proved that OsRE1 participates in the Ehd1 pathway.3.RT-qPCR assay showed that OsRE1 was constitutively expressed and mainly in leaves,sheaths and panicles,which was consistent with the tissue expression pattern of the genes participating in the rice heading date pathway.OsRE1 is a rhythmic expression gene under both long day and short day conditions.The expression of OsRE1 increased under dark and decreased under light,reaching the peak at dawn and the lowest point at dusk.The rhythmic expression of OsRE1 can be partically destroyed under continuous light and continuous dark conditions,which indicated that OsRE1 is regulated by light.4.Subcellular localization of OsRE1 revealed that OsRE1 is a nuclear localization protein.OsRIP1,the interacting protein of OsRE1,was found by yeast two-hybrid assay.The two proteins interacted with each other through their bZIP domain and the second B-box domain.The interaction was verified in vivo and in vitro by BiFC,CO-IP and Pull-down assays.5.OsRIP1 belongs to the B-box transcription factor family,which contains only two B-box domains but no CCT domain.The expression pattern of OsRIP1 is the same as OsRE1,mainly expressed in leaves.Like OsRE1,OsRIP1 is a rhythmic expression gene,regulated mainly by light.6.OsRIP1 functions as a transcription factor in the nucleus.The Osrip1 mutant flowered significantly earlier than the wild type for about 1 week.The expressions of Ehd1 and florin genes Hd3a/RFT1 in Osrip1 mutant were increased,indicating that OsRIP1 participates in the regulation of rice heading date by inhibiting the expression of Ehd1.7.In the qualitative transactivation assay,OsRIP1 significantly inhibited the expression of Ehd1 in the presence of OsRE1,demonstrated that OsRIP1 and OsRE1 regulate the expression of Ehd1 together.8.The sudy of OsREl and OsRIP1 that regulate the expression of Ehd1,provide new tools for molecular breeding through finly regulating rice heading date.