The TaDof-2D-miR1832-TaP450-7A module regulates low-temperature-induced seed dormancy release in wheat

In wheat, exposure to low temperatures (LTs) during the middle and late stages of seed development induces dormancy release; however, the underlying regulatory mechanism remains unclear. Here, using whole-transcriptome sequencing, we identified a novel microRNA (miR1832) that is downregulated by LT and located at a key node of the associated regulatory network. Germination assays showed that overexpression of miR1832 enhanced seed dormancy, whereas its silencing reduced seed dormancy. Sequence variation and association analyses further indicated that an A/G mutation at -670 bp in the miR1832 promoter is significantly associated with phenotypic variation in seed dormancy among wheat varieties, with the A allele correlated with strong dormancy. Through yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase (LUC) reporter assays, we found that the LT-responsive Dof transcription factor TaDof-2D directly binds to the A site in the miR1832 promoter and inhibits its transcription. Subsequent expression analysis, dual-LUC assays, and 5' rapid amplification of cDNA ends confirmed that miR1832 targets the cytochrome P450 gene TaP450-7A, which is upregulated by LT and negatively regulates seed dormancy. Finally, physiological and biochemical analyses further demonstrated that the TaDof-2D-miR1832-TaP450-7A module participates in LT-induced dormancy release by modulating α-amylase activity and the abscisic acid and gibberellin pathways. These findings uncover a previously uncharacterized regulatory mechanism underlying LT-induced dormancy release and provide promising genetic resources and molecular markers for breeding wheat varieties with optimal dormancy levels.