Functional characterization of VaMAPK9 in enhancing cold tolerance of Vitis amurensis

Mitogen-activated protein kinases (MAPKs) play pivotal roles in response to low-temperature stress, but their functional characterization under low-temperature stress in Vitis amurensis (V. amurensis) remains elusive. This study identified eleven MAPK genes through sequence analysis in V. amurensis. Phylogenetic tree analysis revealed that VaMAPK9 shares the closest evolutionary relationships with AtMAPK18 and OsMAPK9. Transcriptional profiling demonstrated significant upregulation of VaMAPK9 in 'Zuoshan 1' under low-temperature stress. Subcellular localization analysis confirmed that the VaMAPK9 protein was located in the cytoplasm. Functional validation through heterologous overexpression (OE) and RNA interference (RNAi) in grape calli and leaves demonstrated that transgenic VaMAPK9-OE lines exhibited significant reductions in electrolyte leakage (EL), malondialdehyde (MDA) accumulation, and hydrogen peroxide (H₂O₂) content under low-temperature treatment. Additionally, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and proline (Pro) content were significantly increased. Meanwhile, expression levels of cold-responsive (COR) genes, C-repeat binding factors (CBF1, CBF2, CBF3) and inducer of CBF expression (ICE1), were upregulated in VaMAPK9-OE lines, whereas VaMAPK9-RNAi lines manifested inverse phenotypic and biochemical trends. RNA sequencing (RNA-seq) analysis revealed that VaMAPK9 could enhance cold tolerance through the regulation of metabolic pathways, flavonoid biosynthesis, and phytohormone signaling transduction networks. Additionally, yeast two-hybrid (Y2H) and luciferase complementation assay (LCA), and Bimolecular Fluorescence Complementation (BiFC) experiments confirmed the interaction between VaMAPK9 and VaBCCP2 proteins. This study elucidates that VaMAPK9 enhances cold tolerance in V. amurensis by coordinating the CBF and ICE signaling pathways along with the antioxidant defense system. The findings provide a theoretical foundation for molecular breeding of low-temperature-resistant grapevines.