Proteome profiling of a S-Nitrosoglutathione reductase (GSNOR) null mutant reveals that aldo-keto reductases form a new class of enzymes involved in nitric oxide homeostasis.

Nitric oxide (NO) is a short-lived gas that acts as a signaling molecule in all higher organisms, including plants. Despite the involvement of NO in multiple plant processes, including germination, root growth and fertility, a basic understanding of the mechanisms by which NO exerts its effects is lacking. NO and its derivatives impact these physiological processes through reversible S-nitrosation of critical protein cysteines. In cells, regulation of NO-levels is predominantly achieved by reaction of reactive nitrogen species (RNS) with glutathione (GSH), thereby forming S-nitrosoglutathione (GSNO), a principal NO reservoir. Mutation of Arabidopsis thaliana S-nitrosoglutathione reductase (GSNOR) leads to higher intracellular concentrations of S-nitrosothiols, confirming that the GSNOR reduction of GSNO is a major route of GSNO catabolism in plants and other eukaryotes . We demonstrate in Arabidopsisthat absence of GSNOR results in differential regulation of proteins involved in chlorophyll metabolism, the general stress response and photosynthesis . In addition, our proteomic analysis identified a significant increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8 and 9. Since specific AKRs have been linked to NO metabolism in mammals, we expressed and purified ArabidopsisAKR4C8 and 9 and close homologues AKR4C10 and 11 and determined that they have NADPH-dependent activity in GSNO and S-nitroso-coenzyme A (SNO-CoA) reduction. Plants lacking GSNOR also show increased activity of NADPH-dependent GSNO reduction, consistent with increased AKR activity . To address the in vivo role of these AKRs, CRISPR/Cas9-mediated mutant lines will be generated, and their capacity to metabolize RNS assessed. In addition, by subjecting AKR mutant plants to different abiotic and nitrosative stresses, results will uncover the importance of AKR proteins in NO homeostasis during both optimal growth and stress conditions. Taken together, these data define a new, NADPH-dependent component of NO metabolism that may be integrated with NADH-dependent GSNOR activity to control NO homeostasis in plants and other organisms. 1. Lee, U., Wie, C., Fernandez, B. O., Feelisch, M. & Vierling, E. Modulation of nitrosative stress by S-nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis. Plant Cell 20, 786-802 (2008). 2. Treffon, P., Rossi, J., Gabellini, G., Trost, P., Zaffagnini, M., Vierling, E. Quantitative proteome profiling of a S-nitrosoglutathione reductase (GSNOR) null mutant reveals a new class of enzymes involved in nitric oxide homeostasis in plants. Accepted. Front. Plant Sci. (2021).