MicroRNA159a targets BrMYB12 to antagonistically regulate thermotolerance in flowering Chinese cabbage through myo-inositol/raffinose biosynthesis

Heat stress poses a major threat to crop yield and quality. Although microRNAs are well-established regulators of stress-responsive networks, their mechanistic roles in modulating thermotolerance through the raffinose family oligosaccharide (RFO) biosynthesis pathway remain elusive. Here, integrated transcriptomic and metabolomic analyses reveal that microRNA159a (miR159a)-mediated thermotolerance is primarily achieved via the RFO biosynthetic pathway, with significant downregulation of BrMIPS1, BrGolS1.1, and BrRS2 in flowering Chinese cabbage seedlings at the five-leaf stage under high temperature stress (42 °C). miR159a cleaves the BrMYB12 transcript at a specific site between nucleotides 6 and 7 of the complementary sequence, consequently suppressing BrMYB12-mediated transcriptional activation of BrMIPS1, BrGolS1.1, and BrRS2. Overexpression of miR159a or knockout of BrMYB12 in flowering Chinese cabbage compromised thermotolerance, accompanied by exacerbated reactive oxygen species accumulation, membrane damage, and diminished myo-inositol and raffinose levels, whereas miR159a knockdown or BrMYB12-overexpression lines exhibited contrasting effects. Compared with BrMYB12-overexpression lines, silencing of any individual BrMIPS1, BrGolS1.1, or BrRS2 in the BrMYB12-overexpression background partially impaired thermotolerance, and triple co-silencing almost completely abolished the BrMYB12-conferred heat tolerance. Moreover, exogenous application of myo-inositol or raffinose alleviated the heat-sensitive defects of both miR159a-overexpression and brmyb12 plants. This work establishes a regulatory circuit in which miR159a functions as a thermosensing “switch” that represses BrMYB12, thereby attenuating BrMYB12-mediated activation of BrMIPS1, BrGolS1.1, and BrRS2, leading to reduced myo-inositol and raffinose accumulation and compromised thermotolerance. This pathway integrates miRNA-directed repression, transcriptional activation, and metabolic remodeling, offering promising molecular targets for breeding heat-resilient Brassica crops.