| Summary: | Drought stress severely impacts the survival of <i>Platycrater arguta</i>, an endangered tertiary relict plant. This study investigated the mechanism by which exogenous melatonin enhances drought tolerance in <i>P. arguta</i> seedlings through integrated physiological, transcriptomic, and metabolomic analyses. Under 30% PEG-6000-induced drought, seedlings exhibited leaf wilting, reduced relative water content (RWC: 78.6% vs. 84.8% in controls), and elevated oxidative damage (malondialdehyde increased by 62.94%, H<sub>2</sub>O<sub>2</sub> by 83.78%). Exogenous melatonin application, particularly at 100 μM (T3), significantly alleviated drought symptoms, restoring RWC to 83.7%, reducing membrane permeability (relative electrical conductivity 1.38-fold vs. CK vs. 2.55-fold in PEG), and lowering oxidative markers (MDA and H<sub>2</sub>O<sub>2</sub> accumulation by 28.33% and 27.84%, respectively). T3 treatment also enhanced osmolyte synthesis, increasing proline content by 90.14% and soluble sugars by 47.25% compared to the controls. Transcriptome sequencing revealed 31,870 differentially expressed genes in T3, predominantly enriched in carbohydrate metabolism, oxidative phosphorylation, and tryptophan metabolism pathways. Metabolomic profiling demonstrated that 100 μM melatonin elevated endogenous melatonin levels 19.62-fold and modulated tryptophan-derived metabolites, including indole derivatives and benzoic acid compounds. These findings indicate that melatonin mitigates drought stress by enhancing antioxidant capacity, osmotic adjustment, and metabolic reprogramming, with 100 μM as the optimal concentration. This study provides critical insights into melatonin-mediated drought resistance mechanisms, offering a theoretical foundation for conserving and restoring <i>P. arguta</i> populations in arid habitats.
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