Genome-wide identification of the ADF gene family and expression analysis of responses to cold stress in bitter gourd.

BACKGROUND: Actin-depolymerizing factors (ADFs) maintain actin dynamics depending on the local concentration in cells, regulating the severing and disassembly of actin filaments at low concentrations, whereas they contribute to actin nucleation and Pi release in filaments at high concentrations. These activities enable ADFs to modulate diverse plant processes, including development, growth, defense responses, and stress adaptation. While genome-wide identification and characterization of the ADF gene family have been conducted in several plant species (e.g., Arabidopsis thaliana, wheat, Citrus sinensis, soybean, maize, and tomato), knowledge of ADFs in bitter gourd remains limited. This study aimed to identify and analyze ADF genes in bitter gourd (McADFs), investigate their structural and functional characteristics, and explore their roles in cold stress responses. Specifically, the research sought to clarify how McADFs are regulated under cold stress and whether they interact with salicylic acid signaling to enhance cold tolerance, providing a foundation for understanding their molecular mechanisms in bitter gourd. RESULTS: 11 McADF genes were obtained via the hidden Markov model and phylogenetic analysis, and classified into three major clades. The McADF gene structures, promoter regions, and protein properties were investigated. Expression profiling of the 11 McADFs under cold stress identified McADF09 as key candidate gene associated with cold acclimation. Overexpression of McADF09 in Arabidopsis significantly enhanced cold tolerance, confirming its role in stress adaptation. Exogenous salicylic acid (SA) treatment enhanced peroxidase activity, decreased MDA content, and the upregulated expression of McADF09 may be regulated by the SA signaling pathway, which accounts for cold tolerance. Furthermore, McADF09 was localized to the cytoplasm, consistent with its potential role in regulating cytoplasmic actin dynamics. CONCLUSIONS: These findings offer fundamental insights into the functions of McADFs in bitter gourd, particularly their roles in cold acclimation and SA-mediated signaling pathways, and establish a basis for in-depth investigations into the molecular mechanisms underlying the associations between ADFs, actin dynamics, and stress tolerance. Their potential application significance resides in the fact that modulation of McADF09 or related genes could enhance the cold hardiness of bitter gourd and other cash crops, thereby providing novel strategies for crop improvement. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-026-08271-w.