Multiomics Analyses Reveal MsC3H29 Positively Regulates Flavonoid Biosynthesis to Improve Drought Resistance of Autotetraploid Cultivated Alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa subsp. sativa), the “queen of forage,” is the most important perennial legume, with high productivity and an excellent nutritional profile. Medicago sativa subsp. falcata is a subspecies of the alfalfa complex and exhibits better drought tolerance. However, drought stress significantly hampers their development and yield. The molecular mechanisms underlying the aboveground and underground tissues of sativa and falcata responding to drought stress remain obscure. Here, we performed a comprehensive comparative analysis of the physiological and transcriptomic responses of sativa and falcata under drought stress. The results showed that photosynthesis was inhibited, and antioxidant enzymes were activated under drought stress. MsC3H29, a CCCH-type zinc finger protein, was identified as a hub gene through weighted gene coexpression network analysis (WGCNA) and was significantly induced by drought in underground tissue. The MsC3H29 protein was localized in the nucleus. Overexpression (OE) of MsC3H29 can increase the primary root length and fresh weight of transgenic alfalfa hairy roots, while RNA interference (RNAi) decreases them under drought stress. The 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) staining revealed that MsC3H29 promoted drought tolerance of alfalfa hairy roots through decreasing ROS accumulation. The targeted metabolome analysis showed that the overexpression of MsC3H29 resulted in higher levels of accumulation for flavonoid monomers, including vicenin, daidzein, apigenin, isorhamnetin, quercetin, and tricin, in transgenic alfalfa hairy roots before and after drought stress, while RNAi led to a reduction. Our study provided a key candidate gene for molecular breeding to improve drought resistance in alfalfa.