4.2 OsLHY promotes flowering by regulating Hd1- andEhd1-mediated pathways under LD conditions
Overexpression of OsLHY in Arabidopsis displayed the late flowering independent of the day length, which is consistent with the phenotype of overexpression of LHY or CCA1 (Murakami et al., 2007; Wang & Tobin, 1998). Dysfunction of LHY andCCA1 in Arabidopsis exhibited an early flowering phenotype under both LD and SD conditions (Mizoguchi et al., 2002). Whereas, late flowering was observed in oslhy mutants specially under LD conditions (Figure 1B). The mutation in OsCCA1 leads to the delayed flowering phenotype (Wang et al., 2020), suggested that the role of LHY/CCA1 is not fully conserved between Arabidopsis and rice in controlling of photoperiodic flowering. This distinct role may result from partially conserved photoperiodic pathway betweenArabidopsis and rice. In Arabidopsis , CO induces flowering through activating the expression of FT under LD conditions (Kobayashi, Kaya, Goto, Iwabuchi, & Araki, 1999). CCA1 and LHY could directly repress the transcription of GI to inhibit flowering (Lu et al., 2012; Park et al., 2016). And CCA1 also directly repressesFT via binding to its promoter (Park et al., 2016). Additionally,ELF3 is directly suppressed by CCA1, in contrast, CCA1 is indirectly activated by ELF3 through PRR9 (Lu et al., 2012). Under LD conditions, ELF3 functions mainly upstream of CCA1 to delay flowering (Lu et al., 2012). There are two major photoperiodic flowering pathways in rice: the Hd1 - and Ehd1 -mediated pathways (Komiya et al., 2009; Tsuji, Taoka, & Shimamoto, 2011). TheHd1 -mediated pathway is conserved with the CO-mediated pathway inArabidopsis (Yano et al., 2000). OsGI promotes the expression ofHd1 , then Hd1 induce the transcription of Hd3a andRFT1 to promote flowering under SD conditions, but represses it by down-regulated of Hd3a and RFT1 under LD conditions (Figure 7) (Kojima et al., 2002; Yano et al., 2000). Besides, Hd1also participates in the Ehd1 -mediated pathway (Doi et al., 2004). It can interact with Ghd7 to form a complex, which then represses the transcription of Ehd1 through binding to its promoter under LD conditions (Nemoto et al., 2016). In the Ehd1 -mediated pathway, Ghd7 and Ehd1 are two unique flowering regulators which act as the flowering suppressor and flowering activator, respectively (Doi et al., 2004; Xue et al., 2008). OsELF3 functions as a flowering activator upstream of OsGI and Ghd7 under LD conditions and it may promote the expression of OsLHY by directly repress OsPRRs(Figure 7) (Yang et al., 2013). The dual luciferase assays showed that OsLHY has transcriptional suppression effect on OsGI , Hd1 ,Ghd7 , Hd3a , RFT1 and OsELF3 , while it has transcriptional activation effect on Ehd1 (Figure 5 and Supplemental Figure 3). The Y1H showed that OsLHY also could bind to promoters of OsGI , RFT1 and OsELF3 (Figure 6), suggested that though OsLHY directly represses the expression ofRFT1, it could increase the expression of Hd3a andRFT1 via both Hd1 - and Ehd1 -mediated pathways under LD conditions. As OsLHY could mitigate the inhibition effect of Hd1 onHd3a and RFT1 by OsGI-dependent or OsGI-independent suppression. Furthermore, OsLHY could repress the transcription of Ghd7 and promote the transcription of Ehd1 , which will result in increased expression of Hd3a and RFT1 (Figure 7).
Although OsLHY repressed the transcription of OsGI ,OsELF3 , Hd3a and RFT1 in tobacco leaves (Figure 5A, E, F and Supplemental Figure 3), the expression of OsGI ,Hd3a , RFT1 and OsELF3 was all significantly reduced under LD conditions in the oslhy mutant related to WT (Figure 3M, 3O and Figure 4A). However, the expression of Hd1 and Ghd7(Figure 4A) was increased and Ehd1 was decreased in oslhymutants under LD conditions, which was consistent with the results that OsLHY repressed the transcription of Hd1 and Ghd7 and activated the transcription Ehd1 in tobacco leaves (Figure 5B-D). It indicated that OsLHY may mainly regulate the expression ofHd3a and RFT1 through Hd1 - andEhd1 -medicated pathways in rice.
The expression pattern of OsGI under SD is similar with that under LD conditions, and the expression of Ghd7 was increased inoslhy mutants under SD conditions. However, the expression ofHd1 , Ehd1 , Hd3a and RFT1 was not severely impaired in oslhy mutant under SD conditions. It may explain whyoslhy mutants showed no obviously phenotype on flowering time under SD conditions. Taken together, it allowed us to deduce that OsLHY acts as the positive flowering activator by modulating the transcription of Hd1 and Ehd1 under LD conditions.