Functional Study Of COMPASS-like Complex In Rice Flowering Time Regulation

Rice is now the first major ceral crop of China as well as one of the three largest food crops in the world.Therefore,stabilizing and improving rice yield is of vital importance.Flowering time(heading date)is one of the most agronomic traits that determine rice production and histone mehtylation were shown to plays important roles in flowering time regulation.It has been reported that COMPASS-like complex in Arabidopsis thaliana was responsible for tri-methylation of histone 3 lysine 4(H3K4me3)mediated regulation of gene transcription,flowering time,and environmental stress responses.However,it’s still unclear that whether there exists the COMPASS-like complex in rice.Meanwhile,in the eukaryotes,the mechanism of how COMPASS and COMPASS-like complex recognize specific target genes is still poorly understood.In this study,we identified the core COMPASS-like complex in rice and further anaylsised its biological function in detail by the methods of molecular biology,biochemistry,genetics and physiology.The main results are listed as follows.1.By complementing rice OsTrx1/SDG723 to arabidopsis atx1-1 mutants,it was proved that rice OsTrx1 has conserved biological function with its arabidopsis homologous ATX1,the core component of Arabidopsis COMPASS-like complex.2.Through CRISPR/Cas9 and RNA interference methods,the knockout and knockdown mutants of rice OsTrx1 were obtained.Phenotypic analysis and statistics showed that the defection of OsTrx1 leds to the delayed flowering time under both long day and short day conditions.3.Yeast two-hybridation,Co-IP,pull-down,BIFC and other experiments were taken,the results showed that OsWDR5 a can interact with OsTrx1 through its Win motif region.OsWDR5 a can interact with histone H3 specifically and promote the methylation activity of OsTrx1 to histone H3 in vitro.4.Phenotypic analysis and statistics of T-DNA insertion and RNA interference mutants of OsWDR5 a showed that OsWDR5 a was involved in the regulation of embryo development of rice seeds and the loss function of OsWDR5 a was leathal,and knock down of OsWDR5 a leds to the delayed flowering time phenotype under both long day and short day conditions.5.Through methods of molecular biology,biochemistry and genetics,it was proved that Ehd1,the key player in rice specific flowering pathway,was the direct target gene of OsTrx1 and OsWDR5 a.6.Yeast two-hybridation,Co-IP,pull-down and BIFC were taken,and a zin-finger contained transcription factor SIP1(SDG723 interaction protein 1)was found to interact with OsTrx1 directly.7.Through methods of molecular biology and genetics,it was showed that loss function of SIP1 leds to the phenotype of delayed flowering time under both long day and short day conditions.The expression patterns of genes in flowering pathway of SIP1 mutants were similar with those of OsTrx1 and OsWDR5 a mutans.8.With GUS activity assay,yeast one-hybridation,EMSA and CHIP analysis,it was shown that SIP1 can specifically identify and binds to the 15 bp target sequence of Ehd1 promoter region,and recruit OsTrx1 to accomplish its H3K4 tri-methylation.Together,our results show that there exists COMPASS-like core complex in rice which consists of OsTrx1 and OsWDR5 a.The interaction of OsTrx1 and transcription factor SIP1 achieves the specific recognition and H3K4 tri-methylation of COMPASS-like complex to the target gene Ehd1,and thus promote rice flowering under both long day and short day conditions.