ASHS 2025 Conference

Late spring frosts pose a recurrent threat to apple production, particularly under climate change scenarios that promote earlier bloom and heightened exposure to frost events. Recent evidence from our investigations on different apple-rootstock combinations suggested that rootstock genotype may influence scion resilience to frost, yet the underlying molecular mechanisms remain poorly understood. Field trials were conducted from 2021–2023 on 12-year-old ‘Gala’ and ‘Fuji’ trees grafted onto ten rootstocks, including ‘B.9’ and ‘M.26’, representing extremes of frost tolerance. Following three naturally occurring frost events, we have shown that ‘B.9’ rootstock consistently conferred lower bud mortality relative to ‘M.26’, independent of bloom timing. To dissect the molecular and biochemical mechanisms underlying the differential responses of these scion-rootstock combinations to frost, tissue-specific RNA-Seq analysis and untargeted metabolic profiling were conducted on floral buds, scion leaves, and rootstock sucker leaves from ‘Gala’-grafted trees. Samples were collected 12 h before and 6 h after the 2021 frost. Weighted gene co-expression network analysis (WGCNA) was employed to identify frost-responsive modules, and hub genes were validated via qRT-PCR in both ‘Gala’ and ‘Fuji’. Transcriptomic analyses revealed extensive gene regulation following frost exposure, including over 10,000 differentially expressed genes (DEGs). WGCNA identified three frost-correlated gene modules. Key hub genes were implicated in cold acclimation, ABA signaling, and cellular stress tolerance. GO and KEGG enrichment analyses highlighted significant representation of pathways associated with ABA response, cold response, and water deprivation stress. Metabolomic profiling of different tissues and genotypes further supported the transcriptomic findings and provided metabolic signatures associated with the identified gene networks. This work establishes a foundation for integrative omics approaches to dissect physiological cold tolerance mechanisms in apples and guides rootstock selection and germplasm improvement strategies.