Fig. 1 Analysis of phenotypic (A) and physiological indices (B, C) of P. davidiana under freezing temperature stressData are means ± standard deviation of three independent experiments. Different letters indicate the significance of differences between groups as determined by one-way analysis of variance (ANOVA) and Duncan’s multiple comparison test (P<0.05). The same below

Fig. 2 Analysis of differentially accumulated metabolites in P. davidiana under different freezing stressesA: Principal component analysis (PCA) of the identified metabolites at different temperatures. B: Heat maps of all differential metabolites (DEMs) at four different temperature treatments. Colours indicate the relative content level of each differential metabolite, with red indicating high content and green indicating low content. C: Volcano plots of CK vs T5, CK vs T15 and CK vs T25 differential metabolites. D: Cluster analysis of all differential metabolites according to the trend of changes, and the detailed clustering results are shown in Figure S2. E: Venn diagrams of CK vs T5, CK vs T15 and CK vs T25 differential metabolites. F: Heat maps of a total of 109 differential metabolites in the three groups of CK vs T5, CK vs T15 and CK vs T25, and the detailed results of differential metabolites are shown in Table S5

Fig. 3 KEGG pathway enrichment analysis of differentially expressed metabolites (DEMs)A-C: KEGG pathway enrichment analysis of the comparisons CK vs T5, CK vs T15, and CK vs T25. D-F: Top 10 differentially expressed metabolites in the comparing CK vs T5, CK vs T15, and CK vs T25

Fig. 4 Expression patterns of differentially expressed genes (DEGs) and GO and KEGG enrichment analyses of co-expressed genesA: Up- and down-regulation of DEGs. B: Venn map of DEGs expression among different treatment groups. C: Co-regulatory relationships of DEGs in all comparison groups. D: Results of KEGG and GO enrichment analyses of top 20 KEGGs and GOs for the 510 co-expressed genes. E: The 510 co-expressed DEGs were classified into 9 clusters. The number of genes in each cluster is shown at the top, the green line indicates the average of the relative expression in each subcluster, and the pink line indicates the relative expressions of individual genes in each subcluster. F: KEGG pathways significantly enriched in each cluster (P<0.05)

Fig. 5 RT-qPCR validation of RNA-Seq dataData showed the means ± SE of three independent biological replicates

Fig. 6 Association of gene WGCNA analysis with physiological traitsA: Hierarchical clustering tree analysis based on WGCNA, with the main branch divided into 14 modules of different colours. B: Heatmap of module-trait correlation, each row corresponds to one module (colours are consistent with Figure A), and the colours at the intersections of the rows and columns reflect the correlation between the modules and the physiological traits, the black, green, salmon red, red, brownish-brown and yellow modules labelled with an asterisk are significantly (P<0.05) correlated with the electrolyte leakage rate (EL), malondialdehyde (MDA) and peroxidase activity (POD) are significantly correlated (P<0.05). C: Temperature response trend and KEGG pathway enrichment analysis of DEGs in the above six modules (achieved by R language pHYPER function, P<0.05)

Fig. 7 Metabolome-transcriptome association analysis of P. davidiana in response to different intensities of freezing stressA: Nine-quadrant plot demonstrates metabolite-gene associations, red indicates that both genes and metabolites are upregulated, green indicates that both genes and metabolites are downregulated, black indicates a statistically insignificant association, and blue indicates that the expression trends of genes and metabolites are opposite. B: Clustered heatmap of correlation coefficients of DAMs (PCCs>0.8) between treatment (CK vs T5/T15/T25) and control groups. C: Top 20 DEMs in the comparison of the three groups CK vs T5/T15/T25 KEGG-enriched pathway summary analysis

Fig. 8 Metabolic pathways involved in differentially expressed metabolites (DEMs) and differentially expressed genes (DEGs) in P. davidiana under freezing stressA: Starch and sugar metabolism. B: Organic acid metabolism. C: Flavonoid synthesis. Circles indicate regulated metabolites (green down-regulation, red up-regulation), rectangles indicate regulated genes (green down-regulation, red up-regulation)