Integrated analysis of structure, physiology, transcriptome and metabolome reveals key metabolic pathway responses of cold-tolerant pear cultivar germplasms under low-temperature stress

Freezing injury during winter is a critical abiotic stress that severely impacts the growth, development and, fruit quality of deciduous fruit trees. Cold tolerance can be induced through seasonal cold acclimation, which involves coordinated adjustments in tissue structure, physiology, and biochemistry driven by natural low-temperature, with distinct strategies across plant species. However, the cold-tolerant mechanisms of pear trees during cold acclimation remain poorly understood. Here, one-year-old branches of 10 pear cultivar germplasms were evaluated for cold tolerance based on the semi-lethal low temperature (LT 50 ), with three biological replicates across two consecutive experimental years (2021–2022). LT 50 values varied significantly among these materials, ranging from -42.43 ℃ to -32.59 ℃ and exhibited a highly significant negative correlation with field freezing injury indices ( r = 0.86091, p < 0.0001). Subsequently, integrating anatomical structure observation, physiological index determination, metabolomics, and transcriptomics (with three biological replicates, each with three technical replicates for all omics and molecular experiments), we compared the low-temperature stress responses of cold-resistant ‘Shanli’ and cold-sensitive ‘Hanhong’, with statistical validation via one-way ANOVA, Duncan’s multiple range test, Pearson’s correlation analysis, and gray relational analysis. The results demonstrated that with decreasing temperature, the xylem ratio and lignin content in ‘Shanli’ branches increased markedly compared to ‘Hanhong’, and overwintering capability was correlated with branch lignin synthesis ( r = -0.7783, p < 0.01). Cold acclimation enhanced lignin accumulation in ‘Shanli’ branches by increasing guaiacyl (G) and syringyl (S) units, associated with increased activities of key enzymes (shikimate hydroxycinnamoyl transferase (HCT), caffeoyl shikimate esterase (CSE), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), peroxidase (POD)) and critical intermediate metabolites (phenylalanine, ferulic acid, sinapic acid) in the phenylpropanoid pathway. Transcriptomic analysis identified 75 differentially expressed genes (DEGs) (|log2FoldChange|≥1 and FDR < 0.05) mapped to the phenylpropanoid pathway. Five potential key genes from the HCT , CSE , F5H , CAD and POD gene families, together with their co-expressed genes (such as ERF105-like ) were identified as potentially associated with lignin content and composition modulationce in pear branches, providing novel insights into the regulatory network of lignin synthesis under natur under cold stress. Our findings reveal relationships between lignin synthesis pathways and cold toleranal low-temperature stress and candidate genes for cold-resistant pear breeding.