Fig. 1

Editing of Hd2 uORFs delays rice heading date. A There are three uORFs in 5’ leader sequence(blue) of Hd2, and uORFs putative initiation codon is underlined.pORF is shown in bold with underlined. B Schematic diagram of the CRISPR/Cas9 vector. Three target sequences (uORF-1,uORF-2,uORF-3) fused with sgRNA scaffolds were driven by respective U3 or U6 promoters. The three sgRNA expression cassettes were sequentially inserted into the binary vector pYLCRISPR/Cas9P_ubi-H. Red boxes indicate three target sequences and green boxes indicate sgRNA scaffolds. HPT, hygromycin phosphotransferase gene. C Four homozygous mutants (T₂ generation) of hd2 uorf1 to hd2 uorf4 obtained by CRISPR/Cas9 editing. The uORF sequence (blue) is shown with the sgRNA target site underlined and the protospacer-adjacent motif shown in red. The nucleotide changes are labeled in red. ‘-’ means deletion and sub. means substitution. D Representative flowering image of mutants uorf hd2-1 to uorf hd2-4 indicated genotypes under NLD in summer at Harbin. Wide type SJ2 was used as control. E Flowering time of each genotype under NLD conditions. Data are means ± SE (n = 20). F and G qRT-PCR analysis of Hd3a (F) and RFT1 (G) transcription level in indicated lines and SJ2. Rice UBIQUITIN gene was used as the internal control. Means and standard deviations were obtained from three biological replicates. Data are means ± SE (n = 3). H Schematic diagrams of the reporter plasmids used in rice protoplasts transient assay. REN, Renilla luciferase; LUC, firefly luciferase. I The LUC activity in rice protoplasts with indicated reporter plasmids. Data are means±SE (n=3). Statistically significant differences are indicated by different lowercase letters (P < 0.05, one-way ANOVA with Tukey’s significant difference test)