3.5 OsLHY affects the transcriptional activity of flowering related genes
To further explore the effects of OsLHY on the transcription of flowering related genes, the dual-luciferase detections were performed in tobacco leaves. The signals of LUC triggered by the OsGI , Hd1 , Ghd7 , Hd3a,RFT1 and OsELF3 promoters in tobacco leaves were weakened by introducing the OsLHY protein (Figure 5A-C, 5E-F and Supplemental Figure 3). While the signal of LUC triggered by Ehd1 promoter in tobacco leaves was strongly increased when the OsLHY was co-expressed (Figure 5D). These results indicated that OsLHY has transcriptional suppression activity on OsGI , Hd1 , Ghd7 ,Hd3a and RFT1 promoters, while has transcriptional activation activity on the Ehd1 promoter.
3.6 OsLHY physically binds to the promoters of flowering related genes
The clock genes LHY and CCA1 regulate multiple genes mainly though directly binding to their promoters in Arabidopsis(Alabadi et al., 2001; Lu et al., 2012; Park et al., 2016). The EE expanded sequences were predicted to be bound by OsLHY using web tools JASPAR (http://jaspar.genereg.net/) (Supplemental Figure 4A) (Fornes et al., 2020). The 2 kb promoter regions of seven flowering-related genes (OsELF3 , OsGI ,Hd1, Ghd7 , Ehd1, Hd3a and RFT1 ) were scanned using web tools MEME (http://meme-suite.org/tools/fimo) (Bailey et al., 2009; Grant, Bailey, & Noble, 2011), showed that their promoters contain the EE expanded binding sites (Supplemental Figure 4B and Supplementary Table 3). Therefore, it was speculated thatOsLHY could regulate flowering-related genes through directly interacting with their promoters.
Y1H was performed to confirm their interactions, and found that the co-transformants of pGADT7-OsLHY along with pHIS2-POsGI , pHIS2-POsELF3 and pHIS2-PRFT1 could grow on the selection medium SD/-Leu/-Trp/-His in the presence of 50 mM 3-AT, similar to the positive control (pHis2-P53 and pGADT7-53), whereas the co-transformants of pGADT7-OsLHY along with pHIS2-PHd1 , pHIS2-PGhd7 , pHIS2-PEhd1 and pHIS2-PHd3a were similar to the negative control (pHis2-P53 and pGADT7- OsLHY), failed to grow on the selection medium (Figure 6). These results indicated that OsLHY can physically bind to OsGI , OsELF3and RFT1 promoters in vivo . While it cannot directly bind to the promoters of Hd1 , Ghd7 , Ehd1 and