CRISPR/Cas9-mediated Mutagenesis of OsERF94 Enhances Pre-harvest Sprouting via Regulation of GA Biosynthesis and Deactivation in Rice
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Pre-harvest sprouting (PHS), where seeds germinate on panicles before harvest under humid conditions, is a serious global issue in cereal crop production, including rice. OsERF94 was previously identified as a candidate gene associated with PHS through a genome-wide association study. In this study, we investigated the role of OsERF94 in PHS using CRISPR/Cas9 gene editing. The CRISPR/Cas9-mediated mutagenesis of OsERF94 induced frameshift mutations, resulting in a loss-of-function of OsERF94 in the 1-I-ET and 2-D-ET lines. The 1-I-ET and 2-D-ET lines exhibited significantly higher germination rates under PHS conditions compared to the wild type, indicating increased susceptibility to PHS. Whole-genome re-sequencing confirmed that few or no mutations could be detected at off-target candidate sites in both edited lines, ensuring the precision of the CRISPR/Cas9 gene editing. A transcriptome analysis revealed that OsERF94 modulates the expression of key GA biosynthetic and catabolic genes, including OsLOL1 , OsKO3 , OsGA3ox2 , and OsGA2ox5 , during both seed development and the early germination stages of PHS. The up-regulation of GA biosynthetic genes and the down-regulation of GA deactivation genes in both gene-edited lines likely led to elevated endogenous GA levels at 0 and 1 days after PHS, promoting germination under PHS conditions. These findings suggest that OsERF94 acts as a negative regulator of germination by modulating both GA biosynthesis and deactivation. Our findings contribute to expanding our knowledge of the molecular mechanisms of OsERF94 in PHS and highlight OsERF94 as a promising target for the genetic improvement of PHS resistance in rice-breeding programs.