Decoding the role and regulatory network of the NcSnRK2 gene family in cold stress response of Nymphaea candida.

The white water lily (Nymphaea candida), native to the high-altitude regions of Xinjiang, China, serves as an excellent model for studying cold tolerance in aquatic plants. This study explored the sucrose non-fermenting related protein kinase 2 (SnRK2) family genes in Nymphaea candida (NcSnRK2), establishing a functional link between NcSnRK2 and cold tolerance. Phylogenetic analysis grouped NcSnRK2 family members into three evolutionarily conserved clades. NcSnRK2 proteins exhibited unique subcellular localization in the nucleus and plasma membrane, potentially compensating for regulatory deficits due to open stomata. Specific ABA signaling drove functional divergence in NcSnRK2, optimizing stress adaptation through temporal decoupling. Transient expression analysis in Nymphaea candida demonstrated that these genes play varying roles in enhancing cold tolerance, with NcSnRK2.2, NcSnRK2.6, and NcSnRK2.9 identified as the most stress-responsive genes, exhibiting a marked enhancement of cold tolerance. The analysis of transgenic Arabidopsis further confirmed that NcSnRK2.2, NcSnRK2.6, and NcSnRK2.9 expression significantly improved cold tolerance relative to wild-type plants. Protein interactions suggest that NcSnRK2.2 and NcSnRK2.9 mediate membrane-nuclear signaling through nuclear factors (NAC048, EIN3/RAP2-3) and membrane-anchored proteins (PP2C phosphatases, PIP2), resulting in a coordinated rapid response network. Overexpression of NcNAC048 reduced oxidative bursts and stabilized membranes, underscoring its role in the NcSnRK2-mediated cold response. Our findings highlight the pivotal role of the NcSnRK2 gene family in cold tolerance, providing strategic genetic targets to enhance resilience in aquatic crops and deepening our understanding of plant adaptation mechanisms to extreme environments.