3 Results
3.1 Loss-of-function of OsLHY leads to delayed heading date
To investigate the function of OsLHY in photoperiodic flowering in rice, we generated gene editing mutants using CRISPR/Cas9 technology. Totally, nine editing mutants were obtained (Supplemental Figure 1) and they all displayed late heading date phenotype in the growth chamber at 30°C with LD conditions (data not shown). Then in T1 generation, three independent homozygotes oslhy-1 , oslhy-4 andoslhy-5 were selected for further analyses. These three mutants caused open reading frame shift and resulted in premature stop codons. As expected, the oslhy mutants exhibited delayed flowering phenotype when grown under natural LD conditions. The heading date was delayed by approximately 14 d, 12 d and 15 d related to WT, respectively (Figures 1B and 1C). However, no significantly difference was observed between oslhy mutants and WT under SD conditions (Supplemental Figure 2A and 2B). Theoslhy mutants also showed more tiller numbers, shorter panicle length, less thousand grain weight and higher seed setting rate than that of WT (Supplemental Table 2), which was consistent to previous studies (Chaudhury, Dalal, & Sheoran, 2019; Wang et al., 2020). The expression of OsLHY was detected by RT-PCR and showed declined expression in oslhy mutants when compared to WT (Figure 1D).
3.2 OsLHY expression patterns and subcellular localization
The qRT-PCR was performed to understand the expression patterns of OsLHY in various tissues from root, stem, leaf blade, glum, pistil, anther and eight different panicle developmental stages. The results showed that OsLHY was expressed in all investigated tissues, with the relatively highest mRNA transcription level in leaf (Figure 2A), in accordance with the fact that OsLHY could involve in the regulation of photoperiodic flowering. Since OsLHY encodes a putative MYB transcription factor, the rice protoplast transient expression was performed to investigate the subcellular localization. The fluorescence signal of OsLHY-GFP overlapped with the fluorescence signal of nuclear mark (RPL1) (Figure 2B), indicating that OsLHY was localized in the nucleus, which was consistent with the LHY/CCA1 in Arabidopsis (Liu, Covington, Fankhauser, Chory, & Wagner, 2001).