GhNMO-A12 plays a role in drought response by modulating nitrogen metabolism in cotton

• GhNMO-A12, a γ-PGA-induced nitronate monooxygenase-encoding gene in cotton, is significantly up-regulated under ABA treatment, PEG-simulated drought, and natural drought conditions, with specific binding capacity to three nitro compounds. • Functional validation via VIGS and overexpression confirms that GhNMO-A12 positively regulates plant drought tolerance: silencing it impairs cotton drought adaptation, while overexpressing it enhances Arabidopsis growth and physiological performance under drought stress. • GhNMO-A12 improves drought tolerance by promoting nitrogen metabolism, as evidenced by elevated NO content, NIR activity, and transcription of key nitrogen assimilation-related genes (NR, NIR) in overexpressing lines, especially under drought and nitrogen deficiency co-stress. Drought stress is a major abiotic constraint limiting crop production. Mining key stress-responsive genes and deciphering their mechanisms are crucial for breeding drought-resistant crop varieties. Building on previous findings that exogenous polyglutamic acid (γ-PGA) application improves drought tolerance in cotton, our transcriptome analysis revealed that GhNMO-A12 – a drought-inducible gene – is significantly upregulated in cotton under drought stress upon γ-PGA treatment. Molecular docking predictions indicated stable binding of the GhNMO-A12 protein to nitro compounds (10-nitrolinoleic acid, juglone, 2-nitrophenol), implying its potential link to nitrogen metabolism. Expression analysis showed that GhNMO-A12 is strongly induced by ABA, PEG‑simulated drought, and natural drought. Functional validation via virus‑induced gene silencing (VIGS) and transgenic overexpression in cotton and Arabidopsis demonstrated that silencing GhNMO-A12 significantly compromised drought tolerance, whereas its overexpression markedly improved survival and the accumulation of chlorophyll and soluble protein under drought stress. Phenotypic analysis further indicated that GhNMO-A12 overexpressing plants maintained better root growth under low‑nitrogen and combined mannitol-simulated drought and nitrogen deficiency stress. Mechanistic investigations revealed that under drought conditions, overexpression of GhNMO‑A12 up‑regulated the activities and corresponding gene expression of nitrate reductase (NR) and nitrite reductase (NiR), and significantly increased nitric oxide (NO) levels. In summary, GhNMO-A12 enhances drought tolerance by positively regulating the activity and expression of key nitrogen‑assimilation enzymes, thereby improving nitrogen‑use efficiency. This study provides a novel candidate gene and theoretical foundation for improving stress resilience in crops.