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

In wheat, exposure to low temperatures (LT) during the middle and late stages of seed development induces the release of dormancy. However, the underlying regulatory mechanism remains unclear. Here, we identified a novel microRNA (miR1832), which is downregulated by LT and located at a key node of the related regulatory network, using the whole-transcriptome sequencing technology. Germination experiments showed that overexpression of miR1832 enhanced seed dormancy (SD), while its silencing repressed seed dormancy. Further sequence variation and association analysis indicated that an A/G mutation at -670 bp in the miR1832 promoter was significantly associated with phenotypic difference in seed dormancy across wheat varieties, with A associated with strong dormancy. Combining yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase (LUC) reporter assays, we found that the LT-responsive Dof transcription factor TaDof-2D binds directly to the A site in the miR1832 promoter and inhibits its transcription. Subsequently, through expression analysis, dual-LUC assay, and 5'-rapid amplification of cDNA ends (5'-RACE), we confirmed that miR1832 targets the cytochrome P450 gene TaP450-7A, which is upregulated by LT and negatively regulates SD. Finally, physiological and biochemical analysis further demonstrated that the TaDof-2D-miR1832-TaP450-7A module appears to participate in LT-induced dormancy release by influencing α-amylase activity as well as the abscisic acid (ABA) and gibberellin (GA) pathways. These findings not only demonstrate a new regulatory mechanism underlying LT-induced dormancy release, but also provide promising genetic resources and molecular markers for breeding wheat varieties with optimal dormancy levels.