Abstract
Cold stress represents a significant environmental challenge that constrains the development of the grapevine industry. The basic helix-loop-helix (bHLH) transcription factor family plays pivotal regulatory roles in plant growth, development, and stress responses. This study identifies a novel member of the bHLH family, referred to as VabHLH015, isolated from Vitis amurensis 'Zuoshan-1'. The physicochemical properties, conserved domains, and evolutionary relationships of VabHLH015 were thoroughly analyzed. Additionally, Arabidopsis thaliana and Vitis vinifera 'Chardonnay' calli overexpressing VabHLH015 were generated to evaluate their cold stress tolerance. Amino acid sequence analysis indicated that the VabHLH015 protein comprises 491 amino acids, containing the bHLH-MYC_N and bHLH_AtAIB_like domains. Tissue-specific expression analysis demonstrated differential expression of VabHLH015 across various grapevine organs, with transcript levels markedly increasing in response to low temperature and multiple exogenous hormones. Subcellular localization confirmed that VabHLH015 is a nuclear protein. A transcriptional activation assay showed that it possesses autoactivation activity. Functional analyses revealed that transgenic A. thaliana and grape calli overexpressing VabHLH015 exhibited heightened sensitivity to cold stress. In comparison to wild-type controls, these transgenic lines displayed significantly elevated malondialdehyde (MDA) content and electrolyte leakage, alongside diminished activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as reduced proline (Pro) accumulation. Moreover, the expression levels of several cold-responsive genes were significantly downregulated in the transgenic lines. In summary, the overexpression of VabHLH015 attenuates plant tolerance to low temperatures by modulating the expression of cold tolerance-related genes and antioxidant enzyme activities. This study offers a novel candidate gene and provides a theoretical framework for elucidating the molecular mechanisms underlying cold tolerance, facilitating the breeding of cold-tolerant grapevine varieties.