Integrated analysis of transcriptomic and metabolomic reveal insights into low-temperature stress in sainfoin (Onobrychis viciifolia) seedlings

Sainfoin, a valuable native legume forage grass in northwest China, is commonly used as livestock feed and exhibits resistance to environmental stressors. Although sainfoin displays some resistance, low temperature remains a significant limiting factor in its geographical distribution. Notably, previous studies have predominantly focused on more morphological adaptations, whereas the dynamic gene expression profiles and metabolic regulatory networks underlying sainfoin’s response to low-temperature stress remain poorly understood. In this study, we collected the leaves of the cold-tolerant new strain P4 (RC) and the cold-sensitive material 13,709 (SC) after low-temperature (4 °C) treatment. Through transcriptomic and metabolomic analyses, we aimed to elucidate the molecular mechanisms underlying sainfoin’s response to low-temperature stress. The UPLC-MS/MS analysis and Illumina HiSeq system were employed to detect 6619 metabolites and annotate 6939 genes. The metabolomic analysis revealed 26 common differential accumulated metabolites (DAMs) in the cold-tolerant and cold-sensitive sainfoin at different comparisons. These DAMs were primarily categorized into lipids, phenylpropanoids, and polyketides. Transcriptome analysis identified 1045, 1412, 5010, and 3119 differentially expressed genes (DEGs) in different comparison groups. By integrating the transcriptomic and metabolomic datasets, it was observed that several DAMs were closely associated with DEGs. KEGG enrichment analysis revealed the involvement of numerous genes and metabolites in anthocyanin biosynthesis and amino acid metabolism. Six candidate genes were validated via qRT-PCR, providing novel genetic markers for low- temperature stress. In this study, we analyzed the molecular response of sainfoin to low-temperature stress using integrated transcriptomics and metabolomics and identified the relevant regulatory networks of the anthocyanin biosynthesis pathway, amino acid pathway, and tricarboxylic acid cycle (TCA) pathway, which were closely related to sainfoin stress tolerance. The findings offer profound insights into the growth and development of sainfoin under low-temperature stress, as well as guide the bolstering of its resistance and the selection of cold-resistant varieties.