Fine-tuning Flowering Time via Genome Editing of Upstream Open Reading Frames of Heading Date 2 in Rice.

Flowering time of rice (Oryza sativa L.) is among the most important agronomic traits for region adaptation and grain yield. In the process of rice breeding, efficient and slightly modulating the flowering time of an elite cultivar would be more popular with breeder. Hence, we are interested in slightly increasing the expression of flowering repressors by CRISPR/Cas9 genome editing system. It was predicated there were three uORFs in 5' leader sequence of Hd2. In this study, through editing Hd2 uORFs, we got four homozygous mutant lines. Phenotypic analysis showed that the hd2 urf edited lines flowered later by 4.6-11.2 days relative to wild type SJ2. Supporting the later flowering phenotype, the expression of Ehd1, Hd3a, and RFT1 is significantly decreased in hd2 urf than that in wild type. Moreover, we found that the transcription level of Hd2 is not affected, whereas the Hd2 protein level was increased in hd2 urf compared with wild type, which indicated that Hd2 uORFs indeed affect the translation of a downstream Hd2 pORF. In summary, we developed a efficient approach for delaying rice heading date based on editing uORF region of flowering repressor, which is time and labor saving compared to traditional breeding. In future, uORF of other flowering time related genes, including flowering promoter and flowering repressor genes, can also be used as targets to fine-tune the flowering time of varieties.