4.1 OsLHY has essential effect on the circadian clock
As one of the central clock oscillator components, loss-of-function ofOsLHY obviously affected the rhythmic expression pattern of circadian clock-related genes. In Arabidopsis , LHY andCCA1 defective mutants showed shortened circadian periods (Green & Tobin, 1999; Mizoguchi et al., 2002; Niwa et al., 2007). In theoslhy mutant, the expression peaks of OsPRR73 ,OsPRR37 , OsPRR95 , OsPRR59 , OsPRR1 andOsGI were shifted early (Figure 3), suggested that OsLHYdefective mutants could also result in shortened circadian periods.HY and CCA1 can inhibit the transcription of almost all circadian clock-related genes in the morning (Nagel & Kay, 2012; Sanchez & Kay, 2016). The expression of OsPRR73 andOsPRR37 was significantly reduced under LD conditions, but increased under SD conditions in oslhy mutants. WhileOsPRR95 , OsPRR59 , OsPRR1 , OsGI andOsELF3 was increased in oslhy mutants under LD conditions (Figure 3), which indicated that OsLHY may suppress these genes in early time of the daytime. The expression of OsPRR1 was increased inoslhy mutants (Figure 3K and 3L), because OsLHY (orOsCCA1 ) could repress its expression by directly binding to its promoter (Wang et al., 2020). In addition, OsLHY was highly expressed in the osprr1 mutant (Wang et al., 2020), which may confirm the speculation that OsLHY and OsPRR1 are components of the central negative feedback loop in rice (Yang et al., 2013). Overexpression of OsPRR37 suppressed the expression ofOsLHY under LD conditions (Liu et al., 2018), indicated that reciprocally regulation between OsLHY and OsPRR37 is complicated. In Arabidopsis, CCA1 suppresses GI and ELF3 by interacting directly with their promoters, which add additional connections between the circadian clock and the flowering pathways (Lu et al., 2012). The expression of OsGI was increased at some early daytime points (Figure 3M and 3N), and the tobacco transient expression and Y1H showed that OsLHY can directly repress the transcription ofOsGI and OsELF3 (Figure 5A, Supplemental Figure 4 and Figure 6). These data revealed that the additional connections among circadian clock and the flowering pathways are conserved between rice and Arabidopsis .