Widely targeted metabolic profiling reveals drought resistance mechanisms in alfalfa leaves.

Drought is considered a primary factor constraining alfalfa (Medicago sativa L.) yield and acreage. To understand the internal drought resistance mechanisms of alfalfa is essential for breeding drought-resistant alfalfa varieties. Here, we compared the phenotypic characteristics, metabolic pathways and metabolites of drought-resistant (Longzhong, LZ) and drought-sensitive (Gannong No. 3, G3) varieties under drought stress. Phenotypic analysis revealed that drought stress reduced plant height, single plant fresh weight, single plant dry weight and leaf RWC in alfalfa, with a greater effect observed in G3. The root length of LZ increased under drought stress, whereas there was no significant change in the root length of G3. Widely targeted metabolomics revealed that LZ could maintain higher glycolysis/gluconeogenesis and tricarboxylic acid cycle under drought stress, which provided more ATP and substrates for amino acids biosynthesis, arginine and proline metabolism and phenylpropanoid metabolism. This allowed LZ to accumulate more amino acids, spermidine, spermine, 4-aminobutyric acid, naringenin, isoliquiritigenin, glycitein, glycitin, calycosin, ferulate, scopoline, scopoletin, sinapyl alcohol and coniferin, which favor the enhancement of drought resistance in alfalfa. Moreover, widely targeted metabolomics showed salicylic acid and trans-zeatin were key hormones involved in drought resistance in alfalfa. The results of this study provide useful insights into the enhancement of drought resistance in alfalfa through metabolic regulatory mechanisms, which provides a theoretical basis for the breeding drought-resistant alfalfa varieties.