1 Introduction
Rice (Oryza sativa ) is one of
the most important food crops and feeds over half of the world
population. Flowering time determines the cropping seasons and regions,
and appropriate flowering time will benefit for successful reproduction
(Izawa, 2007; Sun et al., 2014). Photoperiodic flowering is the major
flowering pathway in rice, which is controlled by both the internal
circadian rhythm and the environmental cue, such as day length and light
(Shim, Kubota, & Imaizumi, 2017). The regulations of circadian clock
and photoperiodic flowering have been widely studied in the model
long-day (LD) plant, Arabidopsis (Johansson & Staiger, 2015;
Shim et al., 2017; Song, Shim, Kinmonth-Schultz, & Imaizumi, 2015).
While it is limited study on the connection between circadian clock and
photoperiodic flowering in the model short-day (SD) plant, rice.
The Arabidopsis CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and its
functional paralog LATE ELONGATED HYPOCOTYL (LHY) are the important part
of circadian clock oscillator (Schaffer et al., 1998; Wang & Tobin,
1998). CCA1 and LHY are the hub components of multiple interlocked
feedback loops, which required to maintain circadian rhythms (Locke et
al., 2006; McClung, 2014). CCA1, LHY and PSEUDO-RESPONSE REGULATOR1
(PRR1) comprise the first central negative transcriptional feedback
loop. The CCA1 and LHY can be activated at dawn to repress
the transcription of evening-expressed PRR1 by directly binding
to its promoter. Whereas PRR1 peaks at dusk, which in inverse
inhibits the expression of CCA1 and LHY (Alabadi et al.,
2001; Gendron et al., 2012). Meanwhile, CCA1 , LHY and two
PRR family members PRR7 and PRR9 also form a negative
feedback loop. CCA1 and LHY repress the expression of PRR7 andPRR9 , and then PRR7 and PRR9 along with PRR5 directly suppress
the expression of CCA1 and LHY (Farre, Harmer, Harmon,
Yanovsky, & Kay, 2005; Kamioka et al., 2016; Nakamichi et al., 2010).
In Arabidopsis , GIGANTEA (GI ) acts as a positive
regulator of CONSTANS (CO ), which then activates the
florigen gene FLOWERING LOCUS T (FT ) under LD conditions
(Yano et al., 2000), which form the Arabidopsis major
photoperiodic flowering pathway. The Arabidopsis
GI -CO -FT photoperiodic flowering pathway is partially
conserved with the OsGI -Heading date 1(Hd1 )-Heading date 3a (Hd3a )/ RICE FLOWERING
LOCUS T 1 (RFT1 ) pathway in rice. OsGI activtates the expression
of CO ortholog Hd1 in the conserved pathway (Hayama,
Yokoi, Tamaki, Yano, & Shimamoto, 2003). However, the Hd1 has a
dual function in controlling flowering, which promotes photoperiodic
flowering under SD conditions, while inhibits photoperiodic flowering
under LD conditions (Yano et al., 2000). There are two florigen genesHd3a and RFT1 in rice, the Hd3a more likely induces
flowering under SD, and RFT1 induces flowering under LD
conditions (Kojima et al., 2002; Komiya, Ikegami, Tamaki, Yokoi, &
Shimamoto, 2008; Komiya, Yokoi, & Shimamoto, 2009). In addition, the
other photoperiodic flowering pathway: Grain number, plant height,
and heading date 7 (Ghd7 )-Early heading date 1(Ehd1 )-Hd3a /RFT1 pathway is unique present in rice.Ehd1 functions as flowering inducer through activating the
expression of Hd3a and RFT1 under both LD and SD
conditions (Doi et al., 2004). Under LD conditions, the expression ofGhd7 is enhanced and then Ghd7 suppresses the expression
of Ehd1 to delay floral transition, suggesting that Ghd7acts as a flowering repressor (Xue et al., 2008). Furthermore, Ghd7
could interact with Hd1 to inhibit flowering by repressing Ehd1 ,
which indicated that the two pathways are not strictly independent in
controlling photoperiodic flowering (Nemoto, Nonoue, Yano, & Izawa,
2016).
In Arabidopsis , CCA1 and LHY function in the
regulation network of photoperiodic flowering (Mizoguchi et al., 2002;
Z. Y. Wang & Tobin, 1998). Loss-of-function of CCA1 andLHY lead to early flowering (Mizoguchi et al., 2002) and
constitutive expression of CCA1 resulted in late flowering inArabidopsis (Wang & Tobin, 1998). CCA1 cooperates or acts in
parallel with ELF3 to regulate flowering time (Lu et al., 2012).
Moreover, CCA1/LHY also represses the transcription of GI andFT via directly binding to their promoters, and then suppresses
the photoperiodic flowering (Lu et al., 2012; Park, Kwon, Gil, & Park,
2016).
However, there is only single copy of LHY/CCA1 ortholog in rice
(Murakami, Tago, Yamashino, & Mizuno, 2007). And it is still unknown
how the function of OsLHY in photoperiodic flowering in rice.
Here, we generated OsLHY -defective mutants by CRISPR/Cas9 genome
editing system, which exhibited delayed flowering phenotypes under LD,
but not SD conditions. The circadian clock was significantly affected in
the oslhy under both LD and SD conditions, confirming that the
photoperiodic flowering was associated with circadian rhythm in high
plants (Johansson & Staiger, 2015; Song et al., 2015). As a positive
regulator of photoperiodic flowering in rice, OsLHY could suppress the
expression of OsGI , Hd1 and Ghd7 and activateEhd1 , but has transcriptional repression effect on Hd3aand RFT1 in tobacco leaves. Furthermore, OsLHY can
directly bind to RFT1 promoter in yeast, similar to that inArabidopsis . While actually, the transcription of Hd3a andRFT1 was significantly reduced in oslhy mutants under LD
conditions. These results indicated that OsLHY regulated the expression
of Hd3a and RFT1 to promote flowering mainly through
indirect manners in rice.