Physiological response and transcriptomic analysis of red-fleshed apple seedlings to low temperature stress

• LT stress significantly enhanced the accumulation of anthocyanins, soluble sugars, soluble proteins, and other metabolites in the red-fleshed apple seedlings. • Leveraging RNA-seq data, we identified a potential LT stress-responsive transcription factor, which we designated as MdbHLH51. • Functional characterization revealed that overexpression of MdbHLH51 in ‘Orin’ calli significantly promoted anthocyanin accumulation and upregulated the expression of all MdCBFs genes, thereby bolstering tolerance to cold stress. Low temperature (LT) stress negatively impacts the yield and quality of key horticultural crops such as apples. While previous research has predominantly focused on white-fleshed apple varieties with limited cold tolerance, the mechanisms underlying low temperature responses in red-fleshed apples remain largely unexplored. In this study, we used red-fleshed apple seedlings that we developed previously to investigate the biochemical responses to varying temperature conditions. Notably, LT stress significantly enhanced the accumulation of anthocyanins, soluble sugars, soluble proteins, and other metabolites in the red-fleshed apple seedlings. Leveraging RNA-seq data, we identified a potential LT stress-responsive transcription factor, which we designated as MdbHLH51. Functional characterization revealed that overexpression of MdbHLH51 in ‘Orin’ calli significantly promoted anthocyanin accumulation and up-regulated the expression of all MdCBFs genes, thereby bolstering tolerance to cold stress. These findings provide valuable insights into the cold stress response mechanisms in red-fleshed apples, offering a theoretical foundation for the genetic breeding of cold-resistant red apple varieties.