Abstract: Seed dormancy (SD) is the primary genetic determinant of pre-harvest sprouting (PHS) resistance. However, the molecular mechanisms underlying SD remain incompletely understood. Here, we identified a wheat cytochrome P450 gene, TaCYP94-A1, that is expressed at significantly higher levels in weak-dormancy varieties than in strong-dormancy varieties. TaCYP94-A1 expression increased during SD release and decreased during dormancy establishment. Knockout of TaCYP94-A1 markedly enhanced SD and PHS resistance without adversely affecting yield-related traits. Two key single-nucleotide polymorphisms (T/C at –1,895 bp and T/C at –1,225 bp) in the TaCYP94-A1 promoter were significantly associated with SD variation, with the TaCYP94-A1^1,895C and TaCYP94-A1^1,225C allele combination (haplotype Hap4) strongly associated with enhanced dormancy. Two transcription factors, TaABI4 and TaNAC-A1, bind directly to the 5′-ACCGC-3′ (C, –1,895 bp) and 5′-GACTTC-3′ (C, –1,225 bp) motifs in the TaCYP94-A1 promoter, respectively, and regulate its transcription through antagonistic protein–protein interactions in the nucleus. Physiological, biochemical, and gene expression analyses revealed that the TaABI4/TaNAC-A1–TaCYP94-A1 module regulates SD through crosstalk with the gibberellic acid, abscisic acid, and jasmonic acid pathways. Together, these findings uncover a previously uncharacterized regulatory module controlling SD and provide valuable genetic resources and molecular markers for developing PHS-resistant wheat cultivars through molecular design breeding.

Key words: cytochrome P450, plant hormones, pre-harvest sprouting, seed dormancy, wheat