Comparative analysis of the physiological and transcriptomic profiles reveals alfalfa drought resistance mechanisms

Background: Drought stress is a major limiting factor that affects forage yields, and understanding the drought resistance mechanism of plants is crucial for improving crop yields in arid areas. Alfalfa (Medicago sativa L.) is the most important legume plant, mainly planted in arid and semi-arid areas. However, the adaptability of alfalfa to drought stress and its physiological and molecular mechanisms of drought resistance remains unclear.

Results: In this study, we analyzed the physiological and transcriptome responses of alfalfa cultivars with different drought resistances (drought-sensitive Gannong No. 3 (G3), drought-resistant Gannong No. 8 (G8), and strong drought-resistant Longdong (LD)) under drought stress at 0, 6, 12, and 24 h. LD had higher catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities and a higher soluble protein content, lower malondialdehyde (MDA) content, a lower O₂^·- production rate, and a lower H₂O₂ content than G8 and G3 (P < 0.05). The functional enrichment analysis, temporal expression pattern analysis, and weighted gene co-expression network analysis (WGCNA) of the differentially expressed genes (DEGs) showed phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, glycolysis/gluconeogenesis, glutathione metabolism, and biosynthesis of amino acid responses to drought stress in alfalfa. The differential expression of genes during phenylpropanoid biosynthesis, starch and sucrose metabolism, and the glutathione metabolism pathway was further studied, and it was speculated that PAL, COMT, 4CL, CCR, CAD, HXK, INV, SUS, WAXY, AGP, GST, and APX1 played important roles in the alfalfa drought stress response.

Conclusions: The aim of this study was to enhance alfalfa drought resistance by overexpressing positively regulated genes and knocking out negatively regulated genes, providing genetic resources for the subsequent molecular-assisted breeding of drought-resistant alfalfa crops.