Cold tolerance mechanisms in apple: integrative multi-omics reveals rootstock-mediated resilience via lipid remodeling and cryoprotectant-antioxidant flux.

Low temperatures threaten deciduous woody perennials, with floral tissues representing the most thermally vulnerable phenological stage. Although rootstocks are one of the primary determinants of scion thermal resilience, the systemic metabolic mechanisms conferring this protection remain poorly resolved. Here, we employed an integrated multi-omic framework combining metabolomics and transcriptomics, coupled with physiological validations of cellular redox status, to dissect the biochemical architecture of rootstock-mediated frost resilience in 'Gala' apple grafted onto cold-tolerant (B9) and cold-susceptible (M26) rootstocks. Spatiotemporal profiling of floral buds, scion leaves, and rootstocks revealed divergent metabolic reprogramming between genotypes. The B9 system orchestrated a coordinated adaptive response characterized by the selective accumulation of low-molecular-weight cryoprotectants (e.g., trehalose, melibiose, and Cinn-AraGlc), localized ROS accumulation, and hydroxyl-rich antioxidant flavonoids maintaining redox homeostasis. This was coupled with active membrane lipid remodeling evidenced by induction of glycerophospholipid DL-GPC and bioactive lysophospholipid LysoPA. Physiological quantification directly validated this compartmentalized oxidative environment, revealing that B9 rootstock tissues exhibited a sustained 4-fold elevation in basal O2•- and a 2.5-fold increase in H2O2 concentrations relative to M26, while scion floral buds maintained near-baseline levels (40% O2•- and 22% H2O2 elevations). Conversely, M26 exhibited a degradative lipid response marked by rapid declines in structural phosphatidylcholines, plasmalogen-type glycerophospholipids, and phenolic defenses, alongside pro-apoptotic ceramide induction. Intersectional network analysis highlighted a potential synchronized acropetal mobilization of cryoprotectants across the graft union exclusively in B9; however, this proposed vascular-mediated systemic priming mechanism necessitates functional validation. Multi-omic integration via DIABLO revealed that B9 resilience is mechanistically anchored in the co-activation of lipid metabolism and the TCA cycle, driven by linoleic acid metabolism and branched-chain amino acid catabolism. These findings suggest that genotype-specific membrane remodeling competency and cryoprotectant-antioxidant flux, rather than differential stress perception alone, play a critical role in modulating rootstock-mediated frost resilience in apple.