CHH demethylation of the BcMAM1 promoter contributes to cold tolerance in autotetraploid wucai ( Brassica campestris L.)

Polyploidization has long been recognized as a driving force in plant adaptation to adverse environments. Chilling injury poses a major challenge to global crop production, and the regulatory hierarchies that govern cold adaptation in polyploid crops are unexplored. Here, we show that tetraploid wucai (Brassica campestris L.) displays greater cold tolerance than its diploid counterpart. Under prolonged cold stress, tetraploid plants displayed enhanced glucosinolate-associated transcriptional and metabolic responses, which may contribute to their improved stress adaptation. At the epigenetic level, diploid and tetraploid wucai showed distinct DNA methylation responses to short-term cold, with diploid plants tending toward greater CHH hypermethylation in promoter regions. This ploidy-specific increase in methylation correlates with transcriptional repression of multiple stress-responsive loci in diploids. Notably, expression of the glucosinolate biosynthesis gene METHYLTHIOALKYLMALATE SYNTHASE1 (BcMAM1) diverged between ploidies after cold treatment, inversely correlating with opposing CHH methylation (H = A, C, or T) trends in its promoter. Pharmacological demethylation using 5-azacytidine upregulated BcMAM1 and improved cold tolerance. Through a combination of functional assays and promoter-binding analyses, we identify the upstream transcription factor C-REPEAT BINDING FACTOR4 (BcCBF4), which binds to differentially methylated regions within the BcMAM1 promoter in a methylation-sensitive manner, supporting an association between the epigenetic state and transcriptional output. Together, our findings support a mechanistic model for the distinct cold response observed in the autotetraploid wucai line, involving coordinated changes in transcriptional activation and DNA methylation dynamics. The epigenetic and transcriptional targets identified in this study may inform breeding strategies for cold-resilient crops.