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).