Alfalfa is a high-quality forage legume species that is widely cultivated at high latitudes worldwide. However, a decrease in cold tolerance in early spring seriously affects regrowth and persistence of alfalfa. There has been limited research on the metabolomic changes that occur during de-acclimation. In this study, a liquid chromatography-mass spectrometry system was used to compare the metabolites in two alfalfa cultivars during a simulated overwintering treatment. In four pairwise comparisons, 367 differential metabolites were identified, of which 31 were annotated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Many of these metabolites were peptides, carbohydrates, and lipids. At the subclass level, 17 major pathways were revealed to be significantly enriched (P < 0.05). The main differential metabolites included amino acids, peptides and analogs, carbohydrates, and glycerol phosphocholines. A metabolomic analysis showed that the up-regulation of unsaturated fatty acids and amino acids as well as the enhancement of the related metabolic pathways might be an effective strategy for increasing alfalfa cold tolerance. Furthermore, glycerophospholipid metabolism affects alfalfa cold tolerance in early spring. Study results provide new insights about the changes in alfalfa metabolites that occur during de-acclimation, with potential implications for the selection and breeding of cold-tolerant cultivars.
© 2022. The Author(s).