Exogenous Melatonin Regulates the Flavonoid Biosynthesis Pathway to Alleviate Saline–Alkali Stress in Ulmus pumila ‘Zhonghua Jinye’

Melatonin, a potent endogenous antioxidant, holds promise for enhancing stress tolerance in woody plants, yet its molecular mechanism under saline–alkali stress remains poorly understood. This study systematically investigated the effects of exogenous melatonin on Ulmus pumila ‘Zhonghua Jinye’ by integrating physiological assays, transcriptomics, and metabolomics. Two-year-old cuttings were subjected to 150 mmol·L−1 saline–alkali stress and treated with varying melatonin concentrations (0, 50, 100, 200, 400 μmol·L−1; three replicates). Physiological evaluations identified 100 μmol·L−1 melatonin (SMT100) as optimal, significantly enhancing antioxidant enzyme activities (SOD, CAT, APX, GR) by 28.7–41.5% and reducing reactive oxygen species (H2O2 by 31.5%; O2− by 38.2%) compared to untreated stressed controls. Integrated omics analysis (CK, S, SMT100 groups) revealed that saline–alkali stress suppressed the flavonoid biosynthesis pathway, down-regulating key genes such as UpANS1 (10.74-fold), UpANS2, UpHCT1, and UpDFR2, thereby reducing the accumulation of protective flavonoids like quercetin and kaempferol. Conversely, melatonin treatment reactivated this pathway, significantly up-regulating UpANS1 (17.36-fold induction), UpDFR2 (5.55-fold), UpCHS1, UpF3H6, and UpLAR2. This genetic reconfiguration promoted the synthesis of antioxidant flavonoids, enhancing the plant’s overall stress resilience, thus identifying UpANS1 as candidates associated with treatment response. The study provides a scientific basis for cultivating U. pumila ‘Zhonghua Jinye’ in saline–alkali soils and clarifies the molecular mechanism by which melatonin alleviates combined saline–alkali stress via flavonoid pathway regulation.