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bioRxiv · 10.64898/2026.07.28.741153

Integrative genetic and structural analyses reveal nitric oxide-driven regulation of ERFVII stability in Arabidopsis under low O2

Das, A. K.·Ismail, H.·Lee, D.-S.·Hameed, M.·Kang, S.-M.·Mostofa, M. G.·Yun, B.-W.
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Abstract

Nitric oxide (NO) is a key signaling molecule that regulates diverse physiological responses, including adaptation to hypoxia in plants. Although stabilization of group-VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors under low O2 is known to be facilitated by the N-dragon pathway, whether NO directly mediates N-terminal cysteine (Cys) oxidation to regulate ERFVII stability remains unresolved. Here, we combined genetic, computational, and structural approaches to investigate NOs role in proteasomal degradation of ERFVII during flooding stress. Arabidopsis mutants with elevated S-Nitrosoglutathione (GSNO) levels compromised ERFVII activation during dark submergence but increased expression of genes encoding N-degron pathway enzymes. Although direct detection of in vivo NO-mediated S-nitrosylated proteins remains technically challenging, the GPS-SNO 1.0 tool predicted the conserved N-terminal second Cys residue as a high-confidence S-nitrosylation site. Structural modeling using the Schrodinger Suite 2024-4 further revealed that conversion of the Cys thiol (Cys-SH) to S-nitrosothiol (Cys-SNO) induced notable conformational changes in ERFVII TFs. Molecular docking further demonstrated that Cys-SNO-modified ERFVII peptides exhibited stronger binding affinities and altered interaction networks with N-degron pathway enzymes, supporting a role for NO-mediated structural remodeling in ERFVII degradation. Elevated GSNO also disrupted energy balance efficiency for overcoming O2 deficiency, altered the expression of sugar starvation-responsive genes, and impaired ATP binding capacity of Cys-SNO ERFVII proteins, as evidenced by docking and molecular dynamics simulations. Collectively, these results support a model in which NO-mediated modification of the conserved N-terminal Cys promotes ERFVII degradation, thereby linking NO signaling to hypoxia-responsive transcriptional regulation and metabolic adaptation during flooding stress.

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Das, A. K., Ismail, H., Lee, D.-S., Hameed, M., Kang, S.-M., Mostofa, M. G., Yun, B.-W.. 2026-07-28. Integrative genetic and structural analyses reveal nitric oxide-driven regulation of ERFVII stability in Arabidopsis under low O2. https://doi.org/10.64898/2026.07.28.741153

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