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In this study, a genome-wide association study (GWAS) was conducted using 328 accessions of mei (Prunus mume) to identify single-nucleotide polymorphisms (SNPs) associated with red pigmentation in petals, filaments, and xylem. Based on these significant SNPs, we defined 2 haplotypes (bHLH162hap1 and bHLH162hap2) and identified PmbHLH162, a bHLH transcription factor gene responsible for anthocyanin biosynthesis regulation. Transient silencing of PmbHLH162 in mei petals via Agrobacterium-mediated transformation resulted in significant color fading, whereas its overexpression dramatically elevated anthocyanin levels. Haplotype analysis showed that 2 promoter variants in bHLH162hap2 (Chr03_2669885 A\/C and Chr03_2670272 A\/G) alter the binding affinity of transcription factors PmWRKY18 and PmWRKY70. Stronger binding to the G\/C alleles gave rise to higher PmbHLH162 expression in bHLH162hap2, thereby promoted red pigmentation in multiple tissues. By contrast, accessions carrying bHLH162hap1 displayed light\/colorless phenotype without accumulation of red pigment. Furthermore, PmbHLH162 interacted respectively with PmMYC2, PmTT8, and PmEGL1 to form heterodimers, and markedly enhanced PmMYC2-mediated transcriptional activation of the anthocyanin biosynthetic structural genes PmCHS and PmANS. Geographic haplotype analysis revealed that bHLH162hap2 was predominantly enriched in high-latitude northern populations but was declining markedly at lower latitudes. Collectively, our study reveals the genetic and molecular basis underlying anthocyanin accumulation in mei and identifies a PmbHLH162\u2013PmMYC2 regulatory module in which PmbHLH162 enhances PmMYC2-mediated activation of key anthocyanin biosynthetic genes. The additional interactions of PmbHLH162 with the MBW-associated bHLH factors PmTT8 and PmEGL1 further suggest potential crosstalk between this module and the canonical anthocyanin regulatory network.<\/jats:p>","DOI":"10.1093\/plphys\/kiag644","type":"journal-article","created":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T10:43:01Z","timestamp":1787827381000},"source":"Crossref","is-referenced-by-count":0,"title":["Natural variation in the\n                    <i>PmbHLH162<\/i>\n                    promoter regulates anthocyanin biosynthesis and accumulation in\n                    <i>Prunus mume<\/i>"],"prefix":"10.1093","volume":"202","author":[{"given":"Boda","family":"Liu","sequence":"first","affiliation":[{"name":"Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, Beijing Laboratory of Urban and Rural Ecological Environment, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, School of Landscape Architecture, Beijing Forestry University , Beijing 100083 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1178-9382","authenticated-orcid":false,"given":"Zhiyuan","family":"Ma","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, Beijing Laboratory of Urban and Rural Ecological Environment, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, School of Landscape Architecture, Beijing Forestry University , Beijing 100083 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-0961-9322","authenticated-orcid":false,"given":"Jing","family":"Pan","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory 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