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

Abstract

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.

Genetic basis of the early heading of high-latitude weedy rice

“…In this study, we performed a GWAS and QTL mapping to reveal the genetic basis of the early-heading feature of weedy rice. Our research has found that Hd1 and OsCCT22 may be responsible for the heading date variations in the natural population by GWAS (Takahashi et al, 2009;Zhang et al, 2021). Furthermore, Hd1, Dth7, and Hd16 were colocated using the ICIM-ADD model in the RIL population, and the three genes do have variations between parents.…”

“…Dth7 ( OsCCT28 ) is a major gene regulating the flowering time in the northeast of China, which also regulates the plant height and grains per panicle. Dth7 acts downstream of phyB, inhibiting the expression of Ehd1 (upstream regulator of rice florigen genes Hd3a and RFT1 ), thereby delaying flowering under day length ( Liu et al., 2013 ; Gao et al., 2014 ; Liu et al., 2021 ). We speculate that the Dth7 haplotype found in WR04-6 is non-functional because the additive effect of Dth7 is -1.8338 in the RIL population ( Table 2 ); the non-functional Dth7 leads to the early heading of weedy rice by reduced suppression of Ehd1 and promotes the expression of florigen genes Hd3a / RFT1 in the shoot apical meristem.…”

Research Trends and Challenges of Using CRISPR/Cas9 for Improving Rice Productivity

“…Four homozygous lines were modified to boost the expression of flowering repressors using the CRISPR/Cas9 genome editing technique by modifying Hd2 uORFs. A high-efficiency breeding technique for manipulating rice heading date was created using CRISPR/Cas9-mediated genome editing, which saves time and labor compared to conventional breeding [82,83]. The CRISPR/Cas9 technique was used to identify OsCIPK3.…”

Targeted insertion of regulatory elements enables translational enhancement in rice

“…For example, upstream open reading frames (uORFs) are typical translational elements that are thought to potentially inhibit the translation of main ORFs. And by editing of uORFs, several studies have successfully increased the protein abundance of target genes without transcriptional perturbation (Xu et al, 2017;Zhang et al, 2018;Liu et al, 2021b). In addition to uORF, many cis-acting RNA elements within the 5'UTR, known as translational enhancers, also contribute to the translational regulation of mRNAs (Leppek et al, 2018).…”