Abstract
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. Fine-mapping of the previously reported chromosome 4 locus identified
OsERF94
as a strong candidate gene for functional validation. 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 altered expression patterns of several GA-related genes, including
OsLOL1
,
OsKO3
,
OsGA3ox2
, and
OsGA2ox5
in the
OsERF94
mutant lines. The up-regulation of GA biosynthetic genes and the down-regulation of GA deactivation genes observed in both the
OsERF94
mutant lines suggest possible alterations in GA metabolism during the early stages of PHS. Transient luciferase reporter assays using a single-luciferase system suggested that
OsERF94
may be associated with changes in the promoter activities of several GA- and ethylene-related genes. These findings suggest that
OsERF94
may contribute to the regulation of PHS, potentially through moderation of GA- and ethylene-related pathways. Overall, this study improves our understanding of the molecular role of
OsERF94
in PHS and highlights its potential as a target for the genetic improvement of PHS resistance in rice-breeding programs.