Transcriptomic responses of rice to melatonin and nitrogen link altered carbon-nitrogen metabolism to methane emission dynamics.

Methane (CH4) emissions from rice paddies are a major source of greenhouse gases and are strongly influenced by nitrogen (N) fertilization. This study investigated how two N levels (75 kg/ha, N1; 150 kg/ha, N2) and water irrigation melatonin (100 μM) interactively affect CH4 dynamics, rice growth, and the leaf transcriptome. Methane concentrations peaked during the tillering stage (36-56 days after treatment), reaching 121 ± 11 ppm under N2 alone, and were reduced by 30-50% when melatonin was co-applied with N (p < 0.05). Nitrogen enhanced shoot biomass and chlorophyll content, whereas melatonin alone had modest effects on growth but significantly shifted biomass allocation toward roots. RNA-seq identified 1376 differentially expressed genes (DEGs) in the N2+Melatonin comparison (|log2 fold-change| > 2, adjusted p < 0.05). KEGG enrichment analysis highlighted amino sugar and nucleotide sugar metabolism, glycolysis/gluconeogenesis, nitrogen metabolism, and photosynthesis as the most affected pathways. Within these pathways, genes encoding UDP-N-acetylglucosamine diphosphorylase, fructokinase, pyruvate decarboxylase, nitrate transporters, and photosystem II components were significantly altered by melatonin. Quantitative RT-PCR validation of 18 DEGs showed a statistically significant positive correlation between RNA-seq and qRT-PCR log2 fold-changes (Pearson R = 0.307, R2 = 0.094, p = 0.003). These transcriptional signatures suggest that melatonin, particularly under high N, reprograms carbon and nitrogen metabolism in a direction that would reduce the availability of labile carbon substrates for methanogens. When interpreted together with a companion 16S rRNA microbiome study of the same experiment (which showed significant enrichment of methanotrophic bacteria), these findings provide a plant molecular framework for melatonin-mediated methane mitigation in rice paddies.