Transcriptome‐Metabolome Integration Uncovers Salt Stress Effects on Flavonoid Biosynthesis in Two Self‐Selected Breeding Alfalfa Varieties

Salt stress constrains plant distribution and productivity, posing challenges to agriculture and ecosystems. Alfalfa (Medicago sativa L.) is one of the most important forages in the world. Seed germination, epigenetic physiology, transcriptome, metabolome, and the common regulatory mechanism of transcriptome and metabolome were investigated in Xizang's first independently bred alfalfa, ZangMu 1 (ZM1, highly salt-resistant type) and ZangMu 2 (ZM2, salt-sensitive type), under the treatments of 0, 100, and 200 mmol L^-1 NaCl. The results showed that the salt tolerance of ZM1 was significantly better than that of ZM2. Additionally, the seed germination and physiological indices of both varieties exhibited a trend of low promotion and high inhibition. The joint transcriptome and metabolome analyses revealed that the flavonoid biosynthesis pathway was the core pathway in response to salt stress, and ZM1 enhanced stress tolerance by significantly upregulating more differential genes and metabolites. The levels of five key antioxidant metabolites (naringenin (NAR), apigenin (API), dihydroquercetin (DHQ), galangin (GAL), and epigallocatechin (EGC)) were significantly changed under salt stress, indicating that the free radical scavenging system of the plant was regulated. The expression levels of the core genes (CHI1, FL3H, CYP9B16, CYP75A1, FLS, and LAR) showed a synergistic regulation pattern with the salt tolerance metabolites, and the results of qRT-PCR validation were highly consistent with the transcriptome data. This study systematically analysed the flavonoid metabolic network of salt tolerance in Xizang alfalfa, providing molecular targets and a theoretical basis for the selection and breeding of salt-tolerant varieties.