Molecular Mechanisms Of OsFKF1 On Regulating Flowering Time In Rice

Flowering time of plants is an important transition from vegetative growth to reproductive growth,which is regulated by the interaction between endogenous factors and environmental cues,such as photoperiod and temperature.Photoperiod is one of the most important factors to control flowering time of rice.Hd1 and Ehd1 are two pivotal factors of photoperiod-induced rice flowering.Up to now,many regulatory factors of Ehd1 have been discovered;however,there are few reports on the molecular mechanisms of Hd1 regulation.FKF1 is involved in the regulation of CO in Arabidopsis(a homologous protein of Hd1 in rice).Os FKF1 is the homologous protein of FKF1.Its function has been not known.In this study,the T-DNA insertion knockout mutant osfkf1-1 was used as material.The temporal and spatial expression patterns of Os FKF1 and the subcellular localization of its encoded protein are observed.And also the primary functions of Os FKF1 on rice flowering time control are revealed.The main results are described as the following.1.Os FKF1 is evolutionarily conserved.Phylogenetic analysis of FKF1 homologous proteins in rice,maize,sorghum,wheat,soybean,and Arabidopsis suggest that FKF1 has far relationship with ZTL and LKP2 which is not the same branch with them,and also Os FKF1 belongs to two different branches with Os ZTL1 and Os ZTL2.It's worth noting that Os FKF1 and FKF1 have higher homology.The amino acid sequence alignment shows that LOV,F-box and Kelch repeat are highly conserved.Through the homology modeling of Swissmodel,it is found that the tertiary structures of both FKF1 and Os FKF1 contain similar spherical conformations.2.Os FKF1 can complement the late flowering phenotype of Arabidopsis fkf1-2 mutant.Two Os FKF1-gene-containing vectors,35S:Os FKF1c-GFP and Os FKF1p:Os FKF1c-GFP,were constructed to transfer into fkf1-2 through Agrobacterium-mediated genetic transformation method.Flowering time of the transgenic lines is similar to WT,but earlier than that of fkf1-2 mutant,indicating that Os FKF1 is able to rescue the late flowering phenotype of fkf1-2.The expression of flowering promoters such as FT,SOC1 and CO is similar to WT,but higher than that in fkf1-2 under long-day conditions.These results suggest that the functions of Os FKF1 in flowering time control are conserved.3.Os FKF1 is expressed in the organs or tissues detected.Os FKF1 is expressed in all organs of rice detected by real time RT-PCR.The expression of Os FKF1 in leaves is the highest.Similar results are obtained by GUS staining methods.The results show that Os FKF1 is expressed in leaves,sheaths,stems,roots,seeds,and spikelets;especially in the vascular system.In addition,the expression of Os FKF1 increased first and then decreased slightly during its life cycle.Furthermore,the expression of Os FKF1 is regulated by circadian clock,which exhibits the rhythm expression in a 24-hour period,and peaks in the evening.4.Os FKF1 locates in the nucleus and cytoplasm.The subcellular localization of Os FKF1 was observed by using the transgenic plants of fkf1-2 transformed with 35S:Os FKF1c-GFP.The results show that Os FKF1 locates in both nucleus and cytoplasm.5.Os FKF1 is a positive regulator of flowering time in rice.T-DNA insertion mutant osfkf1-1 delays flowering time,but leaf emergence speed is not significantly different from wild type under different photoperiod conditions.The results indicate that Os FKF1 promotes flowering in rice.6.Os FKF1 regulates flowering time through regulating the expression of florigen genes and other genes in rice.q RT-PCR analysis shows that the transcription levels of florigen genes Hd3 a and RFT1 are decreased;the peak of Hd1 expression is delayed,and the expression of Hd1 upstream gene Os GI is unchanged;Ehd1 expression is decreased,and Ghd7,which is the transcription inhibitor of Ehd1,is up-regulated,and also,other Ehd1 upstream regulatory factors Ehd2 and Os LFL1 are changed under both photoperiod conditions.Therefore,Os FKF1 promotes flowering by regulating multiple transcriptional factors such as Hd1 and Ehd1,thereby promoting transcription of the genes and induce flowering.7.Os FKF1 can interact with Hd1 and Os GI to regulate rice flowering.Yeast two-hybrid assay shows that Os FKF1 can interact weakly with Hd1 mainly through the N-terminus of B-Box domain in Hd1;and Os FKF1 can also interact with the N-terminus region of Os GI(1-398 aa).However,we detect the weak self-activation effects of these proteins,thus we should have designed other experiment schemes to verify the true interaction.In summary,the research provides a certain theoretical basis for artificial regulation of rice flowering.