Melatonin bridges timekeeping and survival: coordinating diurnal rhythms, autophagy, and energy balance in upland cotton

Melatonin is widely recognized for its regulatory role in animal circadian rhythms, but its functional significance in plant chronobiology, especially its integration with energy metabolism and autophagy, remains poorly understood. To address this gap, we examined the interplay between melatonin oscillation, clock gene expression, carbohydrate metabolism, and autophagic activity in Gossypium hirsutum. Plants were subjected to four treatments: control, melatonin inhibition (melatonin inhibitor (Tip60 Histone Acetyltransferase inhibitor (MG-149), melatonin rescue (MG-149 + exogenous melatonin), and excess melatonin. Across a 24-hr cycle, we quantified melatonin levels, circadian gene expression-LATE ELONGATED HYPOCOTYL1 (LHY1), CIRCADIAN CLOCK ASSOCIATED1 (CCA1), PSEUDO-RESPONSE REGULATOR7 (PRR7), and TIMING OF CAB EXPRESSION 1 (TOC1), Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) pathway activity, autophagy markers, and carbohydrate dynamics. Control and melatonin-rescue treatments displayed robust diel oscillations in melatonin content, clock gene expression, energy signaling (SnRK1), and autophagy-related markers (ATG8-PE, ATG5/7/9), with starch and ATP levels following rhythmic trends. In contrast, melatonin-deficient and melatonin-excess treatments disrupted these rhythmic patterns, leading to deregulated sugar metabolism, suppressed SnRK1 expression, and loss of autophagic cycling. Notably, the rhythmicity, rather than mere presence of melatonin, emerged as critical for synchronizing internal timekeeping with cellular metabolic processes. This study identifies melatonin as a central coordinator of plant diurnal regulation, linking diel gene expression with energy and stress-adaptive homeostasis, and opens promising avenues for chronobiology-based crop improvement.