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