Fig 1: Loss-of-function alleles of ossfl1 delayed flowering time in rice under SDs. (a) Schematic diagram of OsSFL1 mutant lines. The sgRNA mediating the CRISPR–Cas9 target sites of OsSFL1 was indicated by the dotted line. The T-DNA insertion mutant ossfl1-T was indicated with hollow triangle. (b) Phenotype of Nip and ossfl1 lines under SDs (9.5 h light/14.5 h dark). Bar = 10 cm. (c) Heading date of Nip and ossfl1 lines under SDs and LDs (14.5 h light/9.5 h dark). Bars indicated for standard deviation (s.d.); double asterisks indicated statistically significant differences revealed by two-tailed Student's t test (**, P < 0.01). (d) Heading date of DJ and ossfl1-T lines under SDs. (e) Diurnal expression patterns of OsSFL1 in wild-type Nip at SDs. OsActin1 was used as a reference gene. White and dark bars below the X-axis indicate light and dark periods, respectively. (f) Nuclear localization of the OsSFL1-GFP fusion protein in rice protoplast. OsSFL1-GFP and mCherry fluorescence were imaged using a laser scanning confocal microscope. The nuclear marker OsMADS3 fused with mCherry was used as a nuclear localization control. Scale bars = 50 µm. (g-h) Diurnal expression patterns of Hd1 (g) and Hd3a (h) under SDs. (i) Diurnal expression pattern of Hd1 under LDs. (j-k) Co-immunoprecipitation of OsSFL1 with OsSAP18 (j) or OsHDAC2 (k) in rice protoplasts. Total protein extracts from rice protoplasts transformed with OsSFL1:HA and OsSAP18:FLAG or OsHDAC2:FLAG, then fractionated through SDS-PAGE gel electrophoresis (input) and immunoprecipitated with anti-FLAG agarose (Sigma, Cat#: A2220). The asterisk indicated appearance of input band. (l) The Western blot results immunoblotted with anti-H3acetyl (K9 + K14 + K18 + K23 + K27) (Abcam, Cat#: ab47915) in OsSFL1 mutants and wild types. Band intensities were quantified by the ImageJ program. (m) Verification of synthesized OsSFL1 polyclonal antibody by Western blot (WB) assay (anti-Actin as a control). (n-o) Anti-OsSFL1 (n) and anti-H3 acetyl at K9 + K14 + K18 + K23 + K27 (o) enrichment at Hd1 loci under SDs. The amounts of immunoprecipitated genomic fragments were measured by real-time quantitative PCR and normalized to OsActin1 as an internal control.