Insights into desirable plant cell wall traits for cold tolerance of alfalfa

Medicago varia Martyn. exhibited enhanced cold tolerance that was correlated with coordinated adjustments in root xylem structure, modulation of cell wall components, and reprogramming of secondary metabolism, suggesting an integrated adaptive mechanism linking structure, composition, and metabolism. Alfalfa, as one of the most valuable perennial forage crops, is cultivated worldwide. However, its productivity is being threatened by extreme cold events. This study tried to understand the mechanisms of Medicago varia Martyn. (MvM) and Medicago sativa L. (MS) to combat cold climate through chemical, anatomical, spectral, and metabolic analysis. The results showed that MvM had higher content of neutral detergent soluble (74.60%) and soluble proteins (0.15 mg/g), yet lower content of cellulose (10.45%) as compared to MS. Additionally, fourier transform infrared spectroscopy analysis also confirmed differences in functional groups associated with cellulose, hemicellulose, and lignin between MS and MvM. The smaller diameter and higher density of vessel in MvM were consistent with anatomical traits predicted to enhance hydraulic safety under freeze-thaw stress. Metabolomic profiling identified 831 and 604 differentially accumulated metabolites in MvM roots at flowering and senescence stages, with significant enrichment in pathways related to isoflavonoid biosynthesis, arginine/proline metabolism, and tryptophan metabolism. Key metabolites, such as Calceolarioside B, hydroxytyrosol, and Medicarpin, were markedly up-regulated in MvM. Hierarchical clustering highlighted species-specific accumulation of phenylpropanoids and alkaloids in MvM. These findings suggested that the enhanced cold tolerance of MvM might involve in coordinated structural adjustments in root xylem, modulation of cell wall composition, and reprogramming of secondary metabolism. This study provided new insights into the integrative mechanisms of cold adaptation in alfalfa and supported the development of cold-resistant varieties for cultivation in high-latitude regions.