Promoter variation in BraHDAC1 modulates stomatal regulation and thermotolerance in Brassica rapa

Heat stress severely limits the productivity of Brassica rapa, a key vegetable crop worldwide. Deciphering the genetic basis of thermotolerance is essential for developing climate-resilient cultivars. By integrating genome-wide association studies (GWAS) with bulked segregant analysis (BSA), we identified two single nucleotide polymorphism (SNP1 and SNP2) in the promoter of the histone deacetylase gene BraHDAC1, which underlies natural variation in heat tolerance. These SNPs create a functional ABRE-CE cis-regulatory module exclusively in thermotolerant haplotypes (Hap1 and Hap3), enabling heat-induced transcription via binding of the transcription factor BraAREB1. Enhanced BraHDAC1 activity in turn represses the pectin methylesterase (PME) gene BraPME1 through histone H3K9/K14 deacetylation at its promoter. This repression, together with the coordinated regulation of additional PME and pectin methylesterase inhibitors (PMEI) genes, is associated with altered total PME activity and broader changes in pectin methylesterification, which may contribute to temporal stomatal regulation. This regulation involves early stomatal opening and increased transpiration associated with lower leaf temperature, consistent with enhanced transpirational cooling, followed by later closure to conserve water. Our findings define a BraAREB1-BraHDAC1 regulatory module in which BraPME1 is one functionally validated downstream target, illustrating how a cis-regulatory variation coordinates transcriptional, epigenetic, and cell wall modification responses to heat stress. The SNP and its associated regulatory module provide direct targets for marker-assisted breeding and genetic improvement of thermotolerant B. rapa.