Correlation Analysis of Root Physiological Indicators and Metabolites of Medicago sativa During Overwintering Period

Medicago sativa is a high-yielding and high-protein forage crop with the largest cultivation area worldwide. However, winter freezing injury severely limits its growth in high-latitude regions of Northeast China, particularly affecting the root system, which acts as a vital functional tissue for cold resistance. To date, the physiological characteristics and metabolic responses of M. sativa roots to low temperatures remain unclear. In this study, roots of the cold-tolerant cultivar ‘Longmu801′ and the cold-sensitive cultivar ‘Sardi’ were compared during the overwintering period. Root physiological indicators were measured, and metabolomic profiling was performed. The results showed that the contents of glutathione (GSH), sucrose (SUC), soluble sugar (SS), and proline (Pro) in Longmu801 roots were markedly higher than those in Sardi, while peroxidase and superoxide dismutase activities and the contents of H2O2 and malondialdehyde (MDA) were significantly lower (p < 0.05). Metabolomic analysis identified 304 differentially accumulated metabolites (DAMs). Correlation analysis between DAMs and physiological indicators showed that DAMs in the MEblue module were positively and significantly correlated with glutathione, proline, and soluble sugar (p < 0.05). These DAMs were mainly associated with pathways including arginine and proline metabolism, glutathione metabolism, the tricarboxylic acid cycle, and pyruvate metabolism. Four candidate DAMs related to cold tolerance were further identified, namely spermidine, N-acetyl-L-glutamate 5-semialdehyde, citric acid, and malic acid. These findings provide a theoretical basis and candidate metabolic markers for breeding cold-tolerance M. sativa cultivars and improving overwintering production in high-latitude cold regions.