Integrative Physiological, Metabolomic, and Transcriptomic Insights into MsCIPK2 Responses to Drought Stress in Alfalfa

The adaptability of plants to drought involves tolerance and recovery. Alfalfa (Medicago sativa L.), a key forage crop, is highly susceptible to water deficit. This study found that key pathways, including starch and sucrose metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis, were activated under drought and rewatering. Key module analysis identified 4 hub genes (CIPK2, MYB6, bZIP43, and NF-YB3), and revealed that the expression of CIPK2 continuously increased during drought stress. Functional validation in Arabidopsis confirmed that MsCIPK2 enhanced drought resistance by promoting root growth, lateral root development, and stomatal closure. Physiologically, transgenic lines exhibited increased superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, maximum quantum efficiency of PSII (Fv/Fm), photosynthetic efficiency (ΦPSII), photochemical quenching (qP), and nonphotochemical quenching (NPQ) and reduced malondialdehyde (MDA) and relative electrolyte conductivity (REC). Mechanistically, MsCIPK2 mediated these physiological alterations by modulating the expression of key genes such as APX1, PP2C, ARF7, LEA14 and others. These findings provide molecular insights into alfalfa drought adaptation and a biotechnological basis for improving its resistance.