Comprehensive transcriptomics and widely targeted metabolomics analysis revealed the molecular mechanism of amino acid and flavonoid metabolism-induced freezing resistance during dormancy of oil crop almond

As an oil crop, almond plays an important role in the cultivation and trade worldwide. Wanfeng almond exhibits exceptional cold tolerance and economic value, yet its regulatory mechanism during freezing injury remains unclear. This study utilized one-year-old quiescent branches of Wanfeng almond as experimental material, subjecting them to six freezing stress temperatures: −5°C (CK), −10°C (T1), −15°C (T2), −20°C (T3), −25°C (T4), and −30°C (T5). A comprehensive analysis was conducted using integrated physiology, transcriptomics, and extensive targeted metabolomics. Results indicated that compared to the CK group, the electrolyte leakage index (ELI) increased rapidly at temperatures below −25°C, while malondialdehyde (MDA) and proline levels also rose. At −20°C treatment, soluble sugar and soluble protein content peaked, and peroxidase (POD) and superoxide dismutase (SOD) activities were highest. Transcriptome and metabolomics evaluation revealed significant enrichment of mitogen-activated protein kinase (MAPK) signaling pathway and ABC transporter biosynthesis pathway during plant freezing stress. Amino acid metabolism and flavonoid metabolism emerged as crucial metabolic pathways in Wanfeng during freezing stress, contributing to cell membrane integrity and peroxide removal, thereby reducing plant freezing damage. A gene co-expression network was constructed using weighted gene co-expression network analysis (WGCNA), identifying four biologically significant modules. MEE14 , XERICO , TOGT1 , YUC1 , GATL1 and TL were identified as hub genes in different modules. Combined analysis of metabolomics and transcriptomics revealed strong correlations between AOC , BAM1 , NIA1 , CH , PKS5 , TOGT1 , ANR and CK1 and differentially expressed metabolites under freezing stress. These findings contribute to elucidating the molecular mechanisms of frost resistance in Wanfeng almonds and provide new insights into the regulatory network of cold stress responses during dormancy. • Antioxidant and carbohydrate accumulation are potential sources of almond freezing response. • Amino acid and flavonoid metabolism are potential ways to reduce freezing injury. • The DEG and DEM in the MAPK signaling pathway are related to freezing stress. • AOC, CH , PKS5 and ANR are the key regulatory genes of freezing stress.