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.