Aqueous phase-mediated lipid and ROS regulation confers low-temperature tolerance and regrowth in cryopreserved apple dormant buds

Apple ( Malus domestica Borkh.) is a high-value horticultural crop. Cryopreservation of dormant buds is a vital method for the long-term conservation of woody plant germplasm resources. However, the mechanisms underlying cell integrity maintenance, survival and subsequent regrowth after cryopreservation remain poorly understood. In this study, dormant buds adjusted to moisture content of 40%, 30%, and 20%, were cryopreserved by dehydration, controlled cooling, exposure to liquid nitrogen (LN), followed by rehydration, then grafted for regrowth assessment. Comprehensive analyses were performed to evaluate water status, membrane integrity, reactive oxygen species metabolism, lipidomic profiles, phospholipase activities and the corresponding expression of key lipid metabolism-related genes. Results showed that buds dehydrated to 30% MC exhibited the highest regrowth levels (93.33%). Lipid remodeling occurred during cryopreservation, with distinct patterns across MC treatments. In 30% MC buds, phosphatidic acid (PA), phosphatidyl choline (PC), and phosphatidyl ethanolamine (PE) showed a coordinated response before and after cryopreservation. Membrane fluidity in these buds decreased prior to LN exposure but increased upon rehydration, a shift that facilitated recovery and regrowth. Expressions of the key genes MdDGK4 , which regulated PA biosynthesis, were significantly up-regulated in 30% MC buds, potentially enhancing PA synthesis. Genes involved in PC and PE turnover, including the synthesis gene MdLPCAT2 and hydrolysis genes ( MdPLD3/5/17 , MdPDAT , and MdPLA2 ) also showed treatment-specific expression patterns. These findings uncover moisture content-dependent regulatory mechanisms involving membrane lipid remodeling and gene expression reprogramming that underpins cryopreservation success in apple dormant buds, providing theoretical and practical guidance for the conservation of woody germplasm resources via optimized cryopreservation procedures.