Sugar and nitrogen metabolisms help plants maintain cellular homeostasis, stress tolerance, and sustainable growth in drought conditions. Melatonin, a potent antioxidant and signaling molecule, appears to mitigate the negative impacts of drought on plants. This study aimed to investigate the potential role of foliar-applied melatonin in ameliorating drought-induced alterations in leaf sugar and nitrogen metabolisms' enzyme activities during cotton flowering and boll formation. To date, no study has examined drought-induced sugar and nitrogen metabolisms' enzyme activity changes in cotton treated with foliar melatonin. Drought levels (FC1 = 75 ± 5%, FC2 = 60 ± 5%, and FC3 = 45 ± 5%) were maintained between 3 and 35 days after flowering (DAF), and melatonin (M) concentrations (0, 25, 50, and 100 μmol L-1 ) were applied at 3 and 21 DAF in a completely randomized design. M100 concentrations at low FC levels significantly enhanced leaf sugar and N-metabolic enzyme activities, such as sucrose synthase (65.56%) and glutamine synthetase (55.24%), compared to plants not treated with melatonin; peaking between 7 and 21 DAF and declining gradually with crop growth. Moreover, the M100 concentrations at all FC levels, particularly FC3, significantly increased the relative expression of GhSusB, GhSusC, SPS1, and SPS3 genes, indicating that melatonin improves leaf sugar and N-metabolism enzymatic activities under drought stress. Therefore, applying M100 concentrations to cotton foliage under FC3 conditions during reproductive stages improves leaf water status, sugar, and N-metabolism enzyme activities, demonstrating melatonin's potent anti-stress, osmoregulatory, and growth-promoting properties in overcoming drought stress in cotton crops. Future research into the molecular mechanisms of melatonin-mediated sugar and nitrogen metabolism enzyme activities in cotton leaves may lead to biotechnological methods to improve drought resilience in cotton and other crops.
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