Abstract
Nitric oxide (NO) is a short-lived radical gas that acts as a signaling molecule in all higher organisms, and that is involved in multiple plant processes, including germination, root growth, and fertility. Regulation of NO-levels is predominantly achieved by reaction of oxidation products of NO with glutathione to form S-nitrosoglutathione (GSNO), the principal bioactive form of NO. The enzyme S-nitrosoglutathione reductase (GSNOR) is a major route of NADH-dependent GSNO catabolism and is critical to NO homeostasis. Here, we performed a proteomic analysis examining changes in the total leaf proteome of an Arabidopsis thaliana GSNOR null mutant (hot5-2/gsnor1-3). Significant increases or decreases in proteins associated with chlorophyll metabolism and with redox and stress metabolism provide insight into phenotypes observed in hot5-2/gsnor1-3 plants. Importantly, we identified a significant increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8 and 9. Because specific AKRs have been linked to NO metabolism in mammals, we expressed and purified A. thaliana AKR4C8 and 9 and close homologs AKR4C10 and 11 and determined that they have NADPH-dependent activity in GSNO and S-nitroso-coenzyme A (SNO-CoA) reduction. Further, we found an increase of NADPH-dependent GSNO reduction activity in hot5-2/gsnor1-3 mutant plants. These data uncover a new, NADPH-dependent component of NO metabolism that may be integrated with NADH-dependent GSNOR activity to control NO homeostasis in plants.
Highlights
The ubiquitous signaling molecule nitric oxide (NO) is a free radical gas and constitutes the prominent reactive nitrogen species (RNS) in cells
We report that leaves of A. thaliana GSNOR-null hot5-2 mutant plants, which lack the central enzyme known to be involved in NO homeostasis, show an upregulation of specific aldo-keto reductase (AKR) proteins
We compared the total protein profile of a GSNOR null mutant, hot5-2, and WT A. thaliana leaf material in order to identify proteins that change in expression level as a consequence of accumulation of glutathione to form S-nitrosoglutathione (GSNO) and protein-SNOs and might, be involved in the regulation of these phenotypes
Summary
The ubiquitous signaling molecule nitric oxide (NO) is a free radical gas and constitutes the prominent reactive nitrogen species (RNS) in cells. S-nitrosation ( referred to as nitrosylation), the addition of a NO group to reactive thiols of Cys residues of proteins, is reported to be the most important NO-related PTM (Astier et al, 2012; Guerra et al, 2016; Zaffagnini et al, 2016; Stomberski et al, 2019b). Tyr nitration and nitrosylation of metal-containing proteins are other PTMs associated with nitro-oxidative stresses (see Gupta et al, 2019 for NO PTM terminology). These NO-mediated PTMs can modulate protein activity, alter structural stability, and result in changes of protein subcellular localization or interaction with other proteins (Kovacs and Lindermayr, 2013; Guerra et al, 2016; Zhan et al, 2018; Feng et al, 2019; Zaffagnini et al, 2019)
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