OsbHLH6 regulates OsMADS57 and amino acid metabolism to impair cold tolerance in rice.

key messageOsbHLH6 mediates a CBF-independent cold response through transcriptional repression and metabolic disruption. Cold stress severely impairs rice (Oryza sativa L.) productivity, yet the regulatory mechanisms underlying cold sensitivity remain incompletely understood. Here, we identify OsbHLH6 as a negative regulator of rice cold tolerance that acts through transcriptional repression and metabolic reprogramming. Transcriptomic profiling of OsbHLH6-overexpressing (OsbHLH6_OE) plants revealed suppression of key cold-resilience genes, including OsMADS57, a known positive regulator of cold adaptation. Yeast one-hybrid and electrophoretic mobility shift assays confirmed direct binding of OsbHLH6 to E-box motifs in the OsMADS57 promoter, repressing its transcription and downstream OsWRKY94 expression. Metabolomic analysis under cold stress demonstrated that OsbHLH6_OE plants accumulated excessive arginine (Arg) and histidine (His), exacerbating reactive oxygen species (ROS) accumulation and membrane damage. Exogenous application of Arg and His exacerbated cold sensitivity in OsbHLH6_OE seedlings, linking amino acid dysregulation to impaired cold tolerance. Haplotype analysis revealed distinct OsbHLH6 alleles in japonica and indica subspecies, which originated from wild progenitors and were conserved during domestication. Our findings reveal that cold-induced OsbHLH6 transcription suppresses OsMADS57 and disrupts amino acid metabolism, each contributing to impaired cold tolerance. This study provides novel insights into bHLH-mediated stress adaptation and positions OsbHLH6 as a target for breeding cold-tolerant rice varieties.