Decoding nitric oxide signals: The S-denitrosation machinery in plants

Decoding nitric oxide signals: The S-denitrosation machinery in plants Tereza Jedelsk´ a, Lenka Luhov´ a, Marek Petˇ rivalský * Department of Biochemistry, Faculty of Science, Palacký University in Olomouc, ˇ Slechtitelů 27, Olomouc 77900, Czech Republic ARTICLE INFO Keywords: Aldo-keto reductase Denitrosation S-nitrosation S-nitrosothiols S-nitrosoglutathione reductase Thioredoxins Thioredoxin reductase ABSTRACT S-nitrosation of protein cysteines has been recognised as a crucial mechanism mediating the biological activity of nitric oxide (NO). Here, we review the current knowledge on the enzymatic machinery mediating protein Sdenitrosation in plants, a key process that modulates S-nitrosothiol levels within NO redox signalling pathways. Three major enzymatic systems are characterised: the NADPH-dependent thioredoxin system, S-nitrosoglutathione reductase (GSNOR), and aldo-keto reductases (AKRs). Protein S-nitrosothiols are reduced via dithiol-disulfide exchange mechanisms catalysed by thioredoxins, which are re-reduced by NADPH-dependent thioredoxin reductases. GSNO, the principal low-molecular-weight S-nitrosothiol, is degraded by GSNOR, indirectly modulating the global S-nitrosation status. This process is tightly regulated via reversible oxidative and nitrosative modifications of GSNOR’s cysteine residues. In the absence or impairment of GSNOR activity, compensatory GSNO catabolism is mediated by upregulated AKR isoforms exhibiting NADPH-dependent GSNO reductase activity. The physiological and developmental relevance of protein denitrosation is examined in the context of root morphogenesis, gametophytic development, and immune responses, where S-denitrosation has been demonstrated to modulate the activity, stability, and subcellular localisation of key regulatory proteins. Moreover, pathogen-derived effectors targeting denitrosylases such as GSNOR have been implicated in virulence strategies to disrupt NO homeostasis. Denitrosation represents a critical regulatory node in NO redox signalling, with spatial and temporal specificity yet to be fully elucidated. Further elucidation of the enzymatic substrate specificity, subcellular localisation, and cross-regulatory mechanisms under both physiological and stress conditions is required to fully define the role of denitrosation in plant redox biology. 1. Introduction In the molecular mechanisms of redox signalling and regulation, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved as intracellular signalling molecules and effectors of responses to external signals. Posttranslational modifications of proteins, such as Cys S-nitrosation and tyrosine nitration, are essential components of nitric oxide (NO) signalling (Umbreen et al., 2018; Lundberg and Weitzberg, 2022). S-nitrosation regulates protein structure, functions, and localisation, and participates in diverse cellular processes, including metabolic pathways, maintaining redox balance, iron homeostasis, control of protein quality, gene transcription, and programmed cell death (Borrowman et al., 2023; Wei et al., 2024). Mechanisms of S-nitrosation and its effects on protein structures and activities have been described in detail in plants as well as in other organisms; however, the precise intracellular siteand time-dependent regulation of S-nitrosation within the complex network of redox signalling is still not fully understood (S´ anchez-Vicente et al., 2019; Feng et al., 2019; Massa et al., 2021; Chaudron et al., 2025). The available results in plant biochemistry suggest that controlling intracellular levels of S-nitrosoglutathione (GSNO), the major low-molecular S-nitrosothiol, represents a primary mechanism regulating the dynamic processes of S-nitrosation and denitrosation, and hence the protein nitrosation status (Liu et al., 2024; Wei et al., 2024). GSNO is involved in transnitrosation reactions through which the NO group is transferred to the thiol group of another Cys to form a new protein S-nitrosothiol. In plants, two key enzymes involved in the regulation of S-nitrosothiol levels have been described: NADH-dependent S-nitrosoglutathione reductase (GSNOR, EC 1.1.1.284) and NADPH-dependent thioredoxin (Trx) reductase (NTR, EC 1.8.1.9). The balance between low-molecular-weight S-nitrosothiols and S-nitrosated proteins is indirectly controlled by GSNOR-mediated degradation of GSNO. The NTR/Trx system performs highly specific and efficient direct protein denitrosation and occupies a prominent role in multiple processes related to regulating protein structure and activity * Corresponding author. E-mail address: [email protected] (M. Petˇ rivalský). Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci https://doi.org/10.1016/j.plantsci.2025.112801 Received 30 June 2025; Received in revised form 12 September 2025; Accepted 3 October 2025 Plant Science 362 (2026) 112801 Available online 3 October 2025 0168-9452/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). by post-translational modifications of protein Cys residues (Chakraborty et al., 2022; Chatterji and Sengupta, 2021). In addition, aldo-keto reductases (AKRs, EC 1.1.1.-) have been shown to act as GSNO reductases in animals and plants (Stomberski et al., 2019; Treffon et al., 2021) (Fig. 1). Elucidation of the molecular mechanisms of denitrosation in plants is essential to understand how signal transduction pathways are regulated within plant metabolism during growth and development. Moreover, protein denitrosation belongs to important components of plant response to nitrosative stress, induced by dysregulated production or removal of RNS and ROS. 2. Thioredoxin system in protein denitrosation 2.1. Crucial components of the plant thioredoxin system Thioredoxins (Trxs) are key proteins involved in the maintenance of cellular redox balance and redox regulation of diverse cellular processes. The Trx superfamily consists of three main subclasses of oxidoreductases: Trxs, glutaredoxins (Grxs), and protein disulfide isomerases (PDIs, EC 5.4.3.1). Trxs, ubiquitous low molecular weight proteins (12–14 kDa), are electron donors in various redox regulatory or metabolic dithiol-disulfide interchange reactions in cells (Sevilla et al., 2023; Jedelsk´ a et al., 2020). Trx fold comprises four β-sheets surrounded by four α -helices with a canonical WCGPC or noncanonical XCXXC catalytic motif located in a highly conserved fold at the protein periphery (Martí et al., 2020). Oxidised Trxs can be reduced by thioredoxin reductases (TR), which mainly include NADPH-dependent Trx reductase (NTR, EC 1.8.1.9) and ferredoxin (Fdx)-dependent Trx reductase (FTR), thus forming two active Trx systems in plants: NTR/Trx and FTR/Trx (Zaffagnini et al., 2019). Thirty-nine genes encoding putative Trxs have been described in Arabidopsis, where 21 of them are classified as typical Family I Trxs located in different subcellular compartments: Trxs h in the cytoplasm, nucleus, plasma membrane, and ER; Trxs m1–4, f1–2, x, y1–2, and z in the plastids; Trx s (symbiosis) in the ER; Trx o1–2 in the mitochondria; and Trx o1 also in the nucleus. Family II Trxs contains proteins with one or more Trx domains linked to other domains, such as plastid NTR and adenosine 5 ′ -phosphosulfate reductase (EC 1.8.4.13). It also includes atypical Cys-His-rich Trx. Plants’ Cys redox system also contains homologs of nucleocytoplasmic Trx subfamilies known as nucleoredoxins (Nrxs), which have different active site sequences (WCG/PPC or WCR/PPC/F) and are reduced by NTRs in both the cytoplasm and nucleus (Jim´ enez et al., 2024). 2.2. Overview of plant NTRs Oxidised Trxs are reduced to their dithiol form by NTRs, enzymes with a flavine cofactor, and a double Cys motif in the catalytic centre. Three main types of NTRs are present in plants: cytosolic (NTRA), mitochondrial (NTRB), and plastid (NTRC) (Foyer et al., 2020). Genome-wide analysis in Brassica juncea yielded 12 NTR genes, four each of NTRA, NTRB, and NTRC (Babuta et al., 2023). Plant NTRAs and NTRBs exhibit a TR motif with 82 % sequence similarity, whereas those between NTRA/NTRB and NTRC were 45 % and 43 %, respectively. NTRA and NTRB are functionally redundant (Reichheld et al., 2007). In contrast, NTRC contains both a C-terminal Trx and an N-terminal TR domain, forming a heterodimer in which the Trx domain of one subunit Fig. 1. Overview of protein denitrosation in plants Nitric oxide (NO), produced enzymatically and non-enzymatically, is involved in the processes of metal nitrosation, tyrosine nitration, and S-nitrosation. The principle of S-nitrosation is the binding of nitroso-group (-NO) to the free sulfhydryl group of reduced glutathione (GSH), producing stable S-nitrosoglutathione (GSNO), which can further interact with free cysteines of proteins. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO to form oxidised glutathione (GSSG), which is the substrate of glutathione reductase (GR), and GSH is regenerated. Plant GSNORs are inhibited by cysteine oxidative modifications or through cysteine S-nitrosation. GSNO is the most important low molecular weight S-nitrosothiol, and its specific reaction with free sulfhydryl groups of proteins results in S-transnitrosation. Trans-nitrosation reactions, which consist of the exchange of the NO group on the target cysteines, also occur among the proteins. Aldo-keto reductases (AKRs) are involved in the NADPH-dependent regulation of protein S-nitrosothiols and NO homeostasis in plant cells. Denitrosation mediated by the thioredoxin system (NTR/Trx) is shown on the left. The active site of thioredoxin (Trx) contains a dithiol motif, which is oxidised in a denitrosation reaction with S-nitrosated protein. Oxidised Trx is reduced by the NADPH-dependent reaction of the enzyme thioredoxin reductase (NTR), and active Trx is regenerated. The figure also shows the localisation of key denitrosation enzymes in the plant cell. Asterisks indicate predicted subcellular localisation sites. Created in https://BioRender.com. T. Jedelsk´ a et al. Plant Science 362 (2026) 112801 2 is reduced by the TR domain of the other subunit (Serrato et al., 2004; P´ erez-Ruiz et al., 2009). All NTRs are characterised by their signature active site, CxxC, which regulates their redox state (Babuta et al., 2023). Only one TR is present in mammalian mitochondria, and its knockout is lethal (Conrad et al., 2004). Due to genetic duplication, plants have two NTR proteins (NTRA and NTRB) (Reichheld et al., 2007). Arabidopsis plants lacking both NTRC or NTRA and NTRB show reduced growth compared to WT plants (Serrato et al., 2004; Reichheld et al., 2007; Daloso et al., 2015). Restricted growth of ntrab and ntrc mutants is linked to changes in cytosolic and mitochondrial carbon metabolism (Daloso et al., 2015) and reduced photosynthetic capacity under changing light conditions (Thorm¨ ahlen et al., 2017). Souza et al. (2023) obtained a triple NTR mutant (ntrabc) by crossing the ntrc single mutant with the double ntrab mutant in Arabidopsis. Ntrabc plants showed reduced growth but can complete their life cycle and produce fertile seeds. Porto et al. (2022) provide evidence that Trxh2 and the mitochondrial NTR/Trx system regulate in a light-independent manner the metabolic fluxes throughout the tricarboxylic acid cycle (TCA cycle) and associated pathways, including glycolysis, gluconeogenesis, and glutamine biosynthesis. NTRC acts as an electron donor for plastidial 2-Cys peroxiredoxins (Prx) and participates in an NADPH-dependent H 2 O 2 scavenging system in chloroplasts in the dark. NTRC is required for chlorophyll biosynthesis and biogenesis of the photosynthetic apparatus (Moon et al., 2006). Moreover, NTRC activates aerobic cyclase, which converts Mg-protoporphyrin monomethyl ester into protochlorophyllide (Stenbaek et al., 2008), and is also involved in a light-dependent regulation of starch biosynthesis by redox activation of ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27), the central enzyme of starch biosynthesis (Michalska et al., 2009). The role of the plant NTRC has also been uncovered in regulating sink leaf metabolism and plant acclimation to high CO 2 (Souza et al., 2023). Recently, Hou et al. (2024) used redox proteomics to investigate the dynamics of plants’ thiol–redox network in response to temporal changes in light availability and across genotypes lacking different parts of NTRC/Trx systems. They found light to lead to reduction and re-oxidation dynamics of photosynthetic proteins linked to the Fdx/Trx (f-, m-, and x-type Trxs) and the NTRC/2-Cys Prx systems (including Trx y2), respectively, which showed opposite changes in their light-responsive redox patterns. Many studies have described the link between NTRC and plant protection against oxidative stress (Serrato et al., 2004; Chae et al., 2013; Correa-Aragunde et al., 2015; Moon et al., 2015; Naranjo et al., 2016). In contrast, the importance of NTRA and NTRB under stress conditions has not been sufficiently studied, probably due to the functional redundancy between cytosolic and mitochondrial forms. On the other hand, plants overexpressing NTRA have been shown to have higher resistance to oxidative and drought stress through the regulation of ROS levels (Cha et al., 2014; Cha et al., 2015). NAD(H) and NADP(H) are coenzymes of oxidation-reduction reactions in the cell. The formation of NADPH and NADH occurs through different, regulated, independent pathways. Their balance is crucial for the control of metabolic pathways. The metabolism of pyridine dinucleotides is closely associated with the NTR system since NAD(P)H is used as an electron donor to reduce Trxs (Cejudo et al., 2021). The absence of changes in NADP + and NADPH concentrations in ntrc, ntrab, and ntrabc mutants suggests that NTRs are not critical for regulating enzymes related to NADPH production under the analysed conditions (da Fonseca-Pereira et al., 2021; Souza et al., 2023). The FTR-Trx system likely compensates for the absence of NTRC to maintain NADPH production through the chloroplast electron transport chain (cETC) and NADP-dependent malate dehydrogenase (EC 1.1.1.82) (Yoshida and Hisabori, 2016; Hashida et al., 2018). NADH/NAD + ratio changes affect the kinetics of glycine decarboxylase (EC 1.4.4.2) and enzymes of the TCA cycle. Souza et al. (2023) found that ntrabc plants had a lower % of NAD + and a higher % of NADH than WT. None of these results were found in ntrc or ntrab plants, suggesting that a cumulative effect derived from the deficiency of all NTRs disrupted the NADH/NAD + balance. Higher NADH levels could be related to higher production from glycolysis and the TCA cycle. This hypothesis is supported by findings where higher respiration rates and increased metabolic flux were observed in mitochondrial NTR/Trx mutants throughout the TCA cycle (Daloso et al., 2015; Lima et al., 2021). 2.3. Mechanisms of NTR/Trx-mediated denitrosation in plants For plants, two possible mechanisms by which NTR/Trx directly interacts with S-nitrosated proteins and catalyses their denitrosation have been proposed in plants (Treffon and Vierling, 2022). In the first mechanism–reductive pathway, the nucleophilic Cys located in the active site of reduced Trx attacks the nitrosated Cys of the target protein to form a disulfide intermediate and NO (Fig. 2A). The resulting intermediate is subsequently reduced by the action of the Cys of another Trx, and oxidised Trx is released. NTR then recycles oxidised Trx (Fig. 2B). In the second mechanism, the transnitrosation pathway, the initial attack leads to the formation of nitrosated Trx and reduced protein. Subsequently, NO is released, and disulfide bond formation occurs at Trx. Transnitrosation transfer of the NO group from the protein substrate to the enzyme active site was observed for Arabidopsis Trxh5. NTRA regulates Trxh5 activity. Protoplasts of the NO-donor-treated ntra mutants contained more protein S-nitrosothiols than WT and trxh3 trxh5 double mutant, suggesting that NTRA is essential for Trxh-mediated denitrosation in plants (Kneeshaw et al., 2014). In more detail, it was demonstrated that the denitrosation activity of Trxh5 depends on only one of the two Cys residues in the active site, and replacing Cys39 or Cys42 with a Ser did not affect denitrosylase activity. A combination of mutations of both Cys residues resulted in a loss of Trxh5 denitrosation activity. In contrast, elimination of the catalytic N-terminal Cys in the motif abolishes the denitrosylase activity of plant Trx (Kneeshaw et al., 2014). 3. GSNOR-mediated protein denitrosation 3.1. Plant GSNOR Enzyme GSNOR, also known as glutathione-dependent formaldehyde dehydrogenase (GSH-FALDH) or class III alcohol dehydrogenase (ADH3), is named in the original classification as S-(hydroxymethyl) glutathione dehydrogenase (EC 1.1.1.284), due to the discovery of its true substrate S-(hydroxymethyl)glutathione (HMGSH), the spontaneously formed adduct of glutathione and formaldehyde. HMGSH is converted by a NAD + -dependent oxidation to S-formylglutathione; this reaction is crucial in the formaldehyde detoxification in cells (Achkor et al., 2003; Staab et al., 2008a, 2008b). More physiologically relevant is the NADH-dependent reduction of GSNO to oxidised glutathione (GSSG) and ammonia (Jensen et al., 1998). The subcellular localisation of Arabidopsis GSNOR (AtGSNOR1) was described in the cytosol, peroxisomes, and nucleus, and surprisingly absent in the nucleolus (Reumann et al., 2007). A putative mitochondrial targeting signal was reported for Arabidopsis, rice, and Selaginella GSNORs (Xu et al., 2013). AtGSNOR1 was also located in the ER of Arabidopsis roots (Labudda et al., 2020; Qin et al., 2023). Moreover, the subcellular localisation prediction showed that GSNOR from B. juncea (BjGSNOR-4) possessed an ER signal peptide targeted to the Golgi apparatus and ER (Babuta et al., 2023). A single-copy gene was found in Arabidopsis (Martínez et al., 1996; Lee et al., 2008), tomato (Kubienov´ a et al., 2013), and tobacco (Dıaz et al., 2003). In silico studies suggest that gene duplication can lead to multiple gene copies (Xu et al., 2013): poplar (Populus trichocarpa), diploid cotton (Gossypium raimondii), and the moss Physcomitrella patens are predicted to have two gene copies. Legumes such as soybean (Glycine max), common bean (Phaseolus vulgaris), and barrel medic (Medicago truncatula) also contain two GSNOR copies. However, whether both T. Jedelsk´ a et al. Plant Science 362 (2026) 112801 3 codes for active proteins is uncertain (Xu et al., 2013). Two genes of GSNOR from wild legume Lotus japonicus have been identified and fully characterised (Matamoros et al., 2020). Recently, genome-wide identification revealed four genes of GSNOR in Indian mustard (B. juncea) (Babuta et al., 2023). In Arabidopsis, GSNOR is expressed in all plant organs except mature pollen (Martínez et al., 1996). The highest expression of GSNOR protein was determined in the root, rosette leaves, and flowers of Arabidopsis ecotype Columbia (Espunya et al., 2006). AtGSNOR1 gene mutations have pleiotropic effects on plant development, leading to stunted growth, stem and trichome branching defects, flowering defects, and reduced seed production (Feechan et al., 2005; Lee et al., 2008; Chen et al., 2009; Holzmeister et al., 2011; Kwon et al., 2012; Xu et al., 2013; Shi et al., 2015; Hussain et al., 2019). The Arabidopsis functional mutant gsnor1–3 is characterised by loss of GSNOR activity, increased S-nitrosothiol levels, and developmental defects manifested by reduced growth, branching, and fertility (Feechan et al., 2005). Similar defects have also been found in Arabidopsis hot5–2 and par2–1 mutant lines carrying two different gsnor1 alleles (Lee et al., 2008; Chen et al., 2009; Kwon et al., 2012; Xu et al., 2013). The involvement of GSNOR as a regulator of cell death was described through the characterisation of the par2–1 mutant (Chen et al., 2009). Using positional cloning, the PAR2 gene was identified as AtGSNOR1/HOT5. The par2–1 carries a nonsense mutation of a highly conserved glycine, which causes instability of the mutant protein. The function of the PAR2/AtGSNOR1 gene was studied after treatment with the nonselective herbicide paraquat (1,1 ′ -dimethyl-4,4 ′ -bipyridinium dichloride), which triggers cell death through increased ROS production. In par2–1, a lower superoxide level was demonstrated following paraquat treatment, suggesting a role for AtGSNOR1 in regulating cell death (Chen et al., 2009). (Kovacs et al., 2016b) demonstrated that paraquat inhibits GSNOR activity, subsequently leading to increased S-nitrosothiols and ROS levels. Higher resistance to paraquat and higher S-nitrosothiol levels were detected in gsnor mutants compared to WT. Dysfunction of AtGSNOR1 in the gsnor1/hot5 mutants led to the increased expression of ER stress-responsive genes (Labudda et al., 2020). A recent nitrosoproteome study revealed that AtGSNOR1 participates in ER stress and unfolded protein response by increasing the S-nitrosation of ER oxidoreductase 1 (ERO1) and other proteins (Qin et al., 2023). 3.2. Molecular properties of plant GSNOR The first crystal structure of plant GSNOR was obtained from the tomato (Solanum lycopersicum cv. Amateur, SlGSNOR). SlGSNOR is a homodimeric protein (MW=81.085 kDa) with two zinc atoms in each monomer; one has a catalytic function, whereas the second zinc is a structural component. The highly conserved active site is located in the cleft between the catalytic and coenzyme-binding domains. The catalytic domain is composed of residues 1–177 and 327–379. The central part of the subunit contact area is a small NAD + -binding Rossman fold composed of residues 178–326. The formation of the protein dimer is based on the interaction of twelve pseudo-continuous beta-sheets, which form the bulk of the coenzyme-binding domain. The zinc ion with catalytic function is bound to Cys177, Cys47, His69, and Glu70 or the hydroxide anion of water, depending on the specific complex. The zinc atom with structural function is bound to four Cys (Cys99, Cys102, Cys105, and Cys113). The binding of the coenzyme is associated with the movement of the zinc atom in the active site, and changes in its coordination occur (Kubienov´ a et al., 2013). Meloni et al. (2024) recently determined new crystal structures of Arabidopsis thaliana GSNOR in the apo-form (apo-GSNOR) to broaden knowledge of the structural landscape of ADHs and to conduct an in-depth structural comparison between the different apoand holo-forms (NAD + and NADH structures). Alcohol dehydrogenase class 3 (ADH3), which includes GSNOR, has a significantly different active site structure than class 1ADHs. Larger substrates such as GSNO, HMGSH, ω -hydroxybutyric acids, and longer chain alcohols (octanol, nonanol) can bind to the active site of ADH3. Alcohols with shorter chains than four-carbon alcohols are unsuitable substrates for plant GSNOR. The substrate specificity of plant GSNORs is strongly dependent on the substrate chain length concerning the size of the active site and interactions with relevant residues (Kubienov´ a et al., 2013; Tich´ a et al., 2017a; Meloni et al., 2024). In SlGSNOR, residues Thr49, Asp58, Glu60, Arg117, and Lys287 are conserved, with the exception of Gln112 and Tyr140. Like human GSNOR (hGSNOR), Arg117 binds the carboxyl group of the glycine HMGSH, and Asp58 and Glu60 bind the α -amino group of the γ-glutamate HMGSH. The S-hydroxymethyl group of HMGSH is hydrogen-bonded to Thr49. A significant difference from hGSNOR is the composition of the Fig. 2. a) Reaction mechanisms of Trx-mediated denitrosation. Studies have revealed two reaction mechanisms that involve either the formation of an intermolecular disulfide intermediate, in which Trx is covalently bound to the substrate protein via a disulfide bridge, or transnitrosation, where Trx is transiently Snitrosated. b) NADPH-dependent thioredoxin reductases (NTRs). NTRs are flavoenzymes that catalyse the reduction of oxidised thioredoxins (Trxs), thereby sustaining the cellular redox network. The reaction mechanism involves electron transfer from NADPH to the FAD cofactor within NTR, followed by transfer to a conserved disulfide active site. This reduced disulfide then donates electrons to the disulfide bond of Trx, generating the active dithiol form of Trx. The reduced Trx, in turn, regulates a wide spectrum of target proteins by reducing their disulfide bonds, thereby modulating enzyme activity, signalling, and stress responses. Through this cyclic mechanism, NTRs link the reducing power of NADPH to protein redox regulation in all plant compartments. T. Jedelsk´ a et al. Plant Science 362 (2026) 112801 4 anion-binding pocket that anchors the carboxyl group of the ω -hydroxybutyric acids. SlGSNOR lacks its boundary and comprises only two residues, Arg117 and Lys287. The glutamine residue, which forms hydrogen bonds with the oxygen atoms of the carboxyl in hGSNOR, is replaced by Gly114 in SlGSNOR. Despite this difference, SlGSNOR can convert ω -hydroxybutyric acids with a lower binding affinity (Kubienov´ a et al., 2013; Tich´ a et al., 2017a). Recently, both Arabidopsis GSNOR and ADH1 were shown to be capable of oxidising long-chain alcohols; however, GSNOR showed significantly lower activity with octanol and cinnamyl alcohol, likely due to its large catalytic cavity preventing efficient binding of long-chain alcohols (Meloni et al. 2024) For plant GSNOR, fatty acids (dodecanoic, decanoic and octanoic) and glutathione derivatives (S-methylglutathione, glutathionesulfonic acid, S-acetamidoglutathione, glutathione disulfide) were tested as possible inhibitors. Non-competitive inhibition of NADH-dependent reduction of GSNO was found to occur, with dodecanoic acid showing the highest inhibitory ability (Kubienov´ a et al., 2013; Tich´ a et al., 2017a; Babuta and Deswal, 2024). Compound N6022 (3-(5-(4-(1H-imidazol-1-yl)phenyl)-1-(4-- carbamoyl-2-methylphenyl)-1H-pyrrol-2-yl) propionic acid) has been developed and applied as a potent inhibitor of animal and plant GSNORs (Sun et al., 2011a–c; Kubienov´ a et al., 2013; Tich´ a et al., 2017a; Jedelsk´ a et al., 2019). N6022 inhibits GSNO-reductase activity at nanomolar concentrations due to the interaction of propionic acid, located in the side chain of its structure, with all three residues forming the anion-binding pocket (Green et al., 2012; Kubienov´ a et al., 2013). 3.3. Modifications of Cys residues regulate GSNOR activity GSNOR is a Cys-rich protein highly conserved from microorganisms to humans (Liu et al., 2001). A detailed bioinformatics study by Xu et al. (2013), focusing on the analysis of conserved and zinc-coordinating Cys residues of the AtGSNOR, revealed that residues Cys10, Cys271, and Cys370 may have a conserved function in the regulation of activity. Frungillo et al. (2014) suggest inhibiting AtGSNOR1 activity by binding an NO group to the corresponding Cys residue. Treatment of donors (DEA/NO and Cys-NO) on Arabidopsis crude leaf extract showed dose-dependent inhibition of AtGSNOR1 activity. In contrast, fumigation with NO (60 ppm) on Arabidopsis WT plants reduced the activity to 40 %. Using the biotin-switch technique (BST), they demonstrated S-nitrosation of AtGSNOR1 in vitro and in vivo. Similarly, mass spectrometry confirmed the S-nitrosation of Cys10, Cys271, and Cys370 residues in GSNOR1 (Guerra et al. 2016; Zhan et al., 2018). Treatment with NO donors has been shown to affect the activity of plant GSNORs. After the application of various NO donors, they significantly decreased GSNOR activity, which slightly increased after adding the reducing agent DTT, indicating the role of S-nitrosation in reducing the enzyme’s catalytic ability (Guerra et al., 2016; Tich´ a et al., 2017b; Zhan et al., 2018; Zhang et al., 2020; Babuta and Deswal, 2024). Zhan et al. (2018) revealed a unique mechanism by which S-nitrosation induces selective autophagy of AtGSNOR1 during hypoxia responses. S-nitrosation of AtGSNOR1 at Cys10 induces conformational changes, exposing its AUTOPHAGY-RELATED8 (ATG8)-interacting motif (AIM) accessible by autophagy machinery. Upon binding by ATG8, AtGSNOR1 is recruited into the autophagosome and degraded in an AIM-dependent manner. Meloni et al. (2024) examined the effect of the thiol-modifying agents on AtGSNOR activity. MMTS caused inhibition of AtGSNOR by altering the redox state of Cys residues, particularly those involved in zinc ion coordination, which resulted in the release of the metal. However, MMTS also caused a loss of the enzyme´s structural integrity. NEM and DTNB partially inactivated AtGSNOR, indicating that derivatisation of solvent-accessible Cys thiols affects the catalytic activity. Among possible Cys candidates, modified Cys271 could be responsible for the altered enzyme activity since it is located near the cofactor, and the modification of its thiol group (i.e., Cys-TNB disulfide or Cys-maleimide) could hinder proper binding of the cofactor. However, the reaction of DTNB/NEM with Cys370, which is also solvent-exposed, may also contribute to the inactivation of AtGSNOR. Hydrogen peroxide (H 2 O 2 ) is known to interact with reactive Cys thiols on proteins. In the study by (Kovacs et al., 2016b), inhibition of AtGSNOR1 activity by in vitro application of H 2 O 2 and in vivo application of paraquat was shown to be due to oxidative modifications of Cys47, Cys177, and Cys271. In other studies on plant GSNORs, it was demonstrated that H 2 O 2 -induced oxidation causes inhibition of plant GSNOR in a timeand concentration-dependent manner and for AtGSNOR destabilisation of zinc coordination without altering native protein folding (Tich´ a et al., 2017b; Meloni et al., 2024). Recently, Ol´ ah et al. (2023) reported that limited Zn supply inhibits the GSNOR enzyme at gene and protein levels but not at the activity level, slightly induces NO levels, and increases physiological nitrosoproteome in Arabidopsis. A hypothetical model for the role of GSNOR in NO homeostasis and signalling pathway has been proposed (Guerra et al., 2016): at low NO levels, GSNO is efficiently converted through the constitutive activity of GSNOR. Conversely, at high NO levels, there is a reversible inhibition of GSNOR activity, which results in the accumulation of GSNO in cells that may participate in the S-nitrosation of other target proteins. The reducing environment in the cytosol restores GSNOR activity, and GSNO levels are reduced to basal levels after NO levels are reduced. Together, S-nitrosothiol levels in cells under physiological or stress conditions can be controlled by modulating GSNOR activity through S-nitrosation or oxidative modifications of the corresponding Cys residues, indicating the presence of a feedback mechanism in regulating GSNOR activity. 3.4. Glutathione: key player on both sides of the S-nitrosation flow GSH, one of the most important low molecular weight antioxidants, belong to the crucial regulator of the overall cellular redox state. GSH is synthesised in the cytosol and plastids but functions in multiple plant cell compartments. Its high abundance and reduced thiol group enable GSH to act as a potent antioxidant, redox buffer, and regulator of posttranslational cysteine-based modifications. In plants, high GSH concentrations are observed, namely in meristematic tissues, where it supports developmental processes like cell division and differentiation (Cairns et al., 2006). GSH influences root architecture by modulating auxin transport and signalling, possibly through redox regulation of PIN proteins and auxin response factors (Pasternak et al., 2020). GSH also regulates programmed cell death during development and stress, acting in concert with NO and ROS signals (Koffler et al., 2014). NO/GSH balance impacts seed germination, flowering, and meristem activity, partly through denitrosation-controlled transcriptional networks (Albertos et al., 2015; S´ anchez-Vicente et al., 2019; Feng et al., 2019). Mutant plants deficient in GSH biosynthesis exhibit abnormal embryogenesis and impaired organ formation, highlighting its indispensable role. Under stress conditions, GSH roles include scavenging ROS directly or as a cofactor for glutathione peroxidases, regeneration of ascorbate via the ascorbate-glutathione cycle, and detoxification of xenobiotics mediated by glutathione S-transferases (Foyer and Kunert, 2024). The glutathione redox potential, dependent on the ratio of oxidised to reduced glutathione ([GSSG]/[GSH] 2 ), is an essential indicator of the cell’s physiological state and is usually reduced during oxidative stress (Foyer and Noctor, 2011). Moreover, the current experimental evidence points to localised modulations of glutathione redox potential within intracellular compartments, which are related to the rates of ROS production and catabolism, activities of GSH-dependent enzymes and redox potentials of NAD(P)H/NAD(P) + pairs. GSNO was proposed to be generated by S-nitrosation of GSH occurring indirectly via N 2 O 3 formation or by direct reaction of NO with glutathionyl radical as a reaction intermediate (Keszler et al., 2010; Broniowska et al., 2013). However, early studies on the biological roles of S-nitrosothiols noted that these rather labile NO-derived compounds were reported to occur in the intracellular environment with millimolar T. Jedelsk´ a et al. Plant Science 362 (2026) 112801 5 concentrations of GSH, which can decompose S-nitrosothiols by non-enzymatic reaction mechanisms. In vitro, the complex oxygen-dependent reactions of GSH and GSNO lead to GSSG and nitrite, whereas under an excess of GSH, the formation of ammonia is observed (Singh et al., 1996). Studies in mammalian tissues confirmed that GSH, as well as ascorbate, another cellular reductant, significantly affect GSNO bioactivity (Xu et al., 2000). In human neurons, the removal of GSNO results in a rapid disappearance of protein S-nitrosothiols. S-nitrosothiol decomposition is accelerated by increased GSH, while GSH depletion has a stabilising effect (Romero and Bizzozero, 2009). The authors concluded that most neuronal protein S-nitrosothiols are rapidly denitrosated via transnitrosylation with GSH, with denitrosation rates of individual proteins influenced by hitherto unknown structural factors. Advances in experimental techniques of S-nitrosoproteome dynamics have enabled to shed more light on the mechanisms and specificity of GSH-dependent protein denitrosation. The majority of S-nitrosated proteins are likely efficiently denitrosated by GSH; however, a rather small subset of proteins forms stable nitrosothiols, which also exhibit exceptional stability in vivo. Proteins with S-nitrosocysteines stable in the presence of millimolar GSH levels were suggested to represent components of NO signalling pathways (Paige et al., 2008). Approximately 80 % of protein S-nitrosothiols extracted from HepG2 cells could be denitrosated with 0.5–5 mM GSH. Interestingly, the S-nitrosothiol subset resistant to GSH reduction could be fully denitrosated by the Trx system, suggesting functional complementarity of these two denitrosating mechanisms (Stoyanovsky et al., 2013). In a subsequent study, S-nitrosated Trx1 and glutaredoxin 1 were found to be completely denitrosated by 5 mM GSH (Sircar et al., 2022). Similarly, S-nitrosation of the S1 subunit of ribonucleotide reductase, an important enzyme of the DNA biosynthesis pathway, was removed by GSH, whereas it could not be denitrosated by Trx because of the steric inaccessibility of the S-nitrosothiol in the narrow active site of the enzyme. Collectively, the reports from animal cell studies envisage a quantitatively important role of non-enzyme GSH-dependent denitrosation of most protein S-nitrosothiols; the specificity required for the involvement of reversible S-nitrosation within signalling pathways of NO is provided by selective and specific action of denitrosating enzyme systems, including Trxs, GSNOR and AKRs (Chakraborty et al., 2022). In contrast, it should be noted that our knowledge on the extent to which GSH or ascorbate levels modulate GSNO stability in plant cells is so far very limited. A pioneering study in this area identified cytoplasmic GAPDH from Arabidopsis thaliana to be effectively denitrosated by GSH in vitro (Zaffagnini et al., 2013). LC-ES/MS analysis of plant tissues determined that the GSNO level is in the same order of magnitude as the GSSG level in the roots, stems, and leaves of both pepper and Arabidopsis. The GSH/GSNO ratio was thus suggested as an important indicator of the plant cell redox state as a molecular link between ROS and RNS metabolism (Airaki et al., 2012; Zaffagnini et al., 2016). 3.5. GSNOR-mediated denitrosation: an indirect but key regulatory mechanism GSNOR plays a pivotal role in modulating cellular NO signalling through a regulatory mechanism centred on its ability to metabolise GSNO, a key intracellular reservoir of bioactive NO (Liu et al., 2001; Frungillo et al., 2014). GSNOR does not directly cleave the S–NO bond in proteins but exerts systemic control over protein S-nitrosation status by modulating the availability of low-molecular-weight S-nitrosothiols. GSNOR can effectively deplete the intracellular GSNO pool, which is in dynamic equilibrium with protein S-nitrosothiols through non-enzymatic transnitrosation reactions. GSNO depletion shifts the equilibrium of the transnitrosation reaction toward the formation of reduced thiol groups on proteins, thereby facilitating the indirect denitrosation of S-nitrosylated proteins (Feechan et al., 2005; Sengupta and Holmgren, A., 2012; Jahnov´ a et al., 2019). The significance of this regulatory mechanism is particularly evident under nitrosative stress conditions, where elevated GSNO levels can lead to widespread protein S-nitrosation, potentially disrupting redox-sensitive signalling pathways (Yun et al., 2016; Zhan et al., 2018). Moreover, GSNOR-deficient mutants consistently show elevated GSNO and protein S-nitrosothiol levels, highlighting the critical role of GSNOR enzyme activity in maintaining S-nitrosothiol homeostasis (Feechan et al., 2005; Xu et al., 2013). GSNOR activity ensures the reversibility and spatial precision of NO-based signalling, and this regulatory role has important implications for plant development, immunity, and stress responses (Borrowman et al., 2023; Liu et al., 2024). In summary, GSNOR functions as a gatekeeper of protein denitrosation, not by directly removing NO from proteins, but by controlling the intracellular S-nitrosation landscape. The GSNOR enzyme catalyses the NADH-dependent reduction of GSNO to form GSSG and ammonia, which regulates intracellular GSNO levels. The reduction of GSNO is influenced by several factors that affect the reaction, such as cellular redox potential and NADH coenzyme availability. In the cytoplasm, the ratio of NAD + /NADH is high, in contrast to the ratio for the coenzyme NADP + /NADPH, which is low. This low ratio allows the reduced form to act as a reductant in biosynthetic pathways. NADPH is not significantly utilised as a cofactor of GSNOR (Jensen et al., 1998; Hedberg et al., 2003). The reduction of GSNO itself results in the formation of N-hydroxysulfinamide (GSNHOH), which is unstable and tends to be converted to end products depending on the level of GSH (Fig. 3). In excess of GSH, a nucleophilic attack of GSH occurs, and hydroxylamine (NH 2 OH) is released to form GSSG. At low GSH concentrations, spontaneous rearrangement of GSNHOH results in the formation of glutathione sulfinamid (GSONH 2 ), which is further hydrolysed in an acidic environment to glutathione sulfinic acid (GSO 2 H) and ammonia (Kubienov´ a et al., 2013). GSNOR accelerates enzymatic denitrosation via GSH, thereby decreasing the concentration of protein S-nitrosothiols (Montagna et al., 2014). The extent of GSH-dependent denitrosation is proportional to the GSH/GSNO ratio when a decrease in GSNO results in an increased GSH/GSNO ratio, causing denitrosation of proteins (Zaffagnini et al., 2013). To assess the ability of purified plant GSNOR to denitrosate S-nitrosated proteins, a crude extract of B. juncea was S-nitrosated with Fig. 3. Reaction mechanism of S-nitrosoglutathione reductase (GSNOR) GSNOR catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO) to an unstable N-hydroxysulfinamide intermediate (GSNHOH). At high levels of glutathione (GSH), GSNHOH is decomposed into glutathione disulfide (GSSG) and hydroxylamine (NH 2 OH). Conversely, when GSH levels are low, GSNHOH spontaneously converts to glutathione sulfinamide (GSONH 2 ), which can be hydrolysed to glutathione sulfinic acid (GSOOH) and ammonia. GSNOR activity is tightly regulated by the cellular redox environment, and both reactive oxygen species (ROS) and nitric oxide (NO) can inhibit its function. Oxidative or nitrosative modifications, such as S-nitrosation or S-glutathionylation of critical cysteine residues, have been shown to suppress GSNOR activity, thereby contributing to the accumulation of GSNO and enhanced protein S-nitrosation. Created in https://BioRender.com. T. Jedelsk´ a et al. Plant Science 362 (2026) 112801 6 500 μ M GSNO and then denitrosated with purified BjGSNORs using BST. They showed that most S-nitrosated proteins were denitrosated in the presence of BjGSNOR-A and BjGSNOR-B with NADH as a cofactor. Among them, BjGSNOR-B showed higher denitrosation efficiency, which was revealed by the lesser intensities of S-nitrosated proteins. The results indicated that purified BjGSNORs possess variable denitrosation efficiencies (Babuta and Deswal, 2024). However, the precise manner in which GSNOR carries out selective denitrosation is still unknown. Remarkably, once inactivated, denitrosylase enzymes lead to a change in the NADPH/NADP and NADH/NAD + ratio, inhibiting cells’ ability to recover from oxidative damage and ultimately leading to stress-induced cell death (Liu et al., 2024). GSNOR, due to its ability to metabolise GSNO, is indirectly involved in regulating levels of S-nitrosated proteins. The specific reaction between GSNO and free sulfhydryl groups of proteins leads to S-transnitrosation (Liu et al., 2001; Malik et al., 2011). The rate of protein transnitrosation via GSNO is determined by the size of the GSNO pool and the current redox state of the cell (Borrowman et al., 2023). Protein transferring the NO moiety, termed a nitrosylase, is increasing both the efficiency and specificity of S-nitrosation in an enzyme-like fashion (Gupta et al., 2022). They have not yet been identified in plants, but several structurally distinct transnitrosylases have been identified in animals and Escherichia coli (Feng et al., 2019). Using genetic screening, Chen et al. (2020) identified a suppressor mutant rog1 of the Arabidopsis mutant gsnor1. ROG1 is the repressor of gsnor1, which regulates redox NO signalling. After identifying nine allelic mutants, they found that the rog1–1 mutation significantly reduced the elevated S-nitrosothiol levels in gsnor1, specifically suppressing the gsnor1 mutant phenotype. Interestingly, one of the substrates of the ROG1 transnitrosylase is AtGSNOR1 itself, and ROG1 specifically modifies the Cys10 site of AtGSNOR1. The transnitrosylase activity of ROG1 is controlled by the conserved Cys343 residue. The ROG1 C343T mutant exhibited increased catalase activity but decreased transnitrosylase activity. The rog1 mutant showed reduced sensitivity to NO under normal and stress conditions. Cys343 is essential for defining the primary activity of ROG1 as a transnitrosylase. Thus, these results represent a unique mechanism for regulating NO signalling in plants through ROG1-mediated transnitrosation. As the first plant transnitrosylase to be characterised, the structure of ROG1 and ROG1-like proteins differs from all other transnitrosylases found in bacteria and animals. Remarkably, all identified transnitrosylases exhibit unique structural features, and no common or conserved structural domains/primary sequences specific to this class of proteins have been identified. 4. Aldo-keto reductases Aldo-keto reductases (AKRs) represent a large family of enzymes that catalyse the reduction of aldehydes and ketones to alcohols using NADH or NADPH as a cofactor. AKRs are found in all phyla of life and play key roles in diverse biological processes, including steroid metabolism, xenobiotic detoxification, and response to cellular oxidative or electrophilic stresses (Jin and Penning, 2007; Penning, 2015). AKRs show a broad substrate specificity, acting on a wide range of endogenous and exogenous compounds, including sugars, steroids, and xenobiotics. Specifically in plants, AKRs are also involved in the biosynthesis of osmolytes and secondary metabolites and contribute to abiotic and biotic stress defence mechanisms (Sengupta et al., 2015; Yu et al., 2020; Guan et al., 2023; Guo et al., 2024). Fig. 4. Aldo-keto reductases (AKRs). role in denitrosation AKRs are versatile NADPH-dependent oxidoreductases that catalyse the reduction of diverse electrophilic substrates. 1. In the canonical AKR reaction, electrons from NADPH are transferred to the enzyme-bound carbonyl group of aldehydes or ketones, yielding less reactive alcohols and thereby detoxifying reactive carbonyl species generated under stress. 2. AKRs also function as SNO-CoA reductases, using NADPH to reduce S-nitrosylated coenzyme A (CoASNO), thus regulating thiol-based redox signalling and maintaining CoA homeostasis. 3. In addition, certain AKRs exhibit GSNO reductase activity, where NADPH serves as the electron donor for the reduction of S-nitrosoglutathione (GSNO), contributing to the control of NO bioavailability and protein S-nitrosylation. Through these complementary mechanisms, AKRs couple NADPH oxidation with the detoxification of reactive aldehydes and regulation of NO–derived signalling intermediates, positioning them as key enzymes at the interface of oxidative and nitrosative stress responses. Created in https://BioR ender.com. T. Jedelsk´ a et al. Plant Science 362 (2026) 112801 7 In pioneer studies by Stamler´s group, aldo-keto reductase family 1 member A1 (AKR1A1) was identified as an enzyme catalysing the NADPH-dependent GSNO reductase activity in mammalian tissues (Anand et al., 2014; Stomberski et al., 2019). Moreover, AKR1A1 was found to act as an archetypal mammalian S-nitroso-coenzyme A reductase (Fig. 4). In mice, AKR1A1 deletion resulted in strongly decreased NADPH-dependent GSNOR activity, whereas GSNOR deficiency was associated with elevated AKR1A1, suggesting a functional connection between GSNO catabolic enzymes. Up-regulation of Arabidopsis homologues of AKR1A, AKR4C8 and AKR4C9 was recently observed in GSNOR null mutant plants (Treffon et al., 2021). In vitro studies confirmed that all four members of the Arabidopsis 4 C clade of AKRs show NADPH-dependent GSNO reductase activity. Similar to mice, plants deficient in GSNOR compensate for this with upregulation of NADPH-dependent GSNO reductase activity. However, uncovering the significance of plant AKRs for regulating GSNO and protein S-nitrosothiol levels in vivo requires further research. 5. Functions of denitrosation in plant development and stress responses 5.1. Denitrosation in the root development Root development is a complex process regulated by various internal and external factors, including plant hormones, light, and nutrient availability. Auxin is the main phytohormone controlling root development in plants, regulating cell division, elongation, and differentiation within the root tissues (Roychoudhry and Kepinski, 2022). Several studies have uncovered involvement of S-nitrosation in the auxin signalling pathway (Fern´ andez-Marcos et al., 2012; Terrile et al., 2012; Shi et al., 2015; Correa-Aragunde et al., 2015; Correa-Aragunde et al., 2016). GSNOR contributes to intracellular maintenance of NO homeostasis, essential for regulating protein S-nitrosation in plant development and fertility under optimal growth conditions (Jahnov´ a et al., 2019). Atgsnor1–3 mutant showed delayed germination and increased auxin sensitivity, suggesting that AtGSNOR1-mediated denitrosation is involved in the auxin signalling pathway (Kwon et al., 2012). Similarly, participation of the NTR–Trx system and GSH in auxin signalling and transport was shown using the Arabidopsis triple mutant ntra ntrb cad2 (Bashandy et al., 2010). Later, NO was found essential for auxin-induced NTR activation and S-denitrosation of proteins during root development in Arabidopsis thaliana (Correa-Aragunde et al., 2015). The auxin-mediated induction of NTR activity was inhibited by the cPTIO (NO scavenger), suggesting that NO is downstream of auxin in this regulatory pathway. The NTR inhibitors prevent auxin-mediated activation of NTR and lateral root formation and cause increased total S-nitrosothiols. In agreement with these results, the levels of S-nitrosated proteins were increased in the Arabidopsis double mutant ntra ntrb compared to the wild-type. Correa-Aragunde et al. (2016) proposed a model where regulation of the auxinand NO-driven processes requires precise control of the redox balance involving the coordinated action of many redox-regulated proteins. For example, a general feature of auxin action is the NADPH-oxidase-dependent increase in H 2 O 2 concentration required for cell elongation, division, and differentiation. In contrast, auxin and NO simultaneously activate NTR, controlling excessive ROS-mediated oxidation of proteins and preventing damage to cellular components. Terrile et al. (2012) identified S-nitrosation of the Cys140 on "TRANSPORT INHIBITOR RESPONSE 1" protein (TIR1), an auxin receptor (Dharmasiri et al., 2005; Kepinski and Leyser, 2005). Upon auxin binding, TIR1 mediates the degradation of the auxin/IAA repressor in the 26S proteasome. By S-nitrosation of TIR1, auxin/IAA degradation is accelerated, leading to increased auxin signalling. Replacement of Cys140 with Ala resulted in reduced interactions between TIR1-IAA3 and TIR1-IAA7. Optimal cellular levels of IAA and NO act synergistically to induce the expression of the reporter genes BA3 and DR5 (Terrile et al., 2012). Conversely, higher concentrations of IAA and NO can induce the opposite effect, suggesting a fine regulation of the maintenance of the IAA/NO balance, an essential element in regulating gene transcription in response to auxin (Shi et al., 2015). PIN proteins are crucial for auxin export from cells; polar auxin transport is disrupted due to reduced levels (Luschnig and Friml, 2024). The absence of the AtGSNOR1 increases GSNO levels, leading to decreased accumulation of several PIN proteins independent of transcriptional regulation and degradation in the proteasome (Shi et al., 2015). It is also possible that direct S-nitrosation of PIN proteins decreases their activity, leading to reduced polar auxin transport. On the other hand, increased GSNO levels inhibit the auxin signalling pathway through S-nitrosation of TIR1 and the ubiquitin-conjugating enzyme E2. Collectively, disruption of polar auxin transport and its signalling pathways may contribute to the atgsnor1–3 phenotype, showing a similar phenotype to the typical auxin mutant (Shi et al., 2015). Recently, Jing et al. (2023) showed that S-nitrosation of IAA17 on Cys70 is key in regulating auxin response. NO inhibits auxin signalling by inhibiting IAA17 protein degradation. NO induces S-nitrosation of Cys70 located in the intrinsically disordered region of IAA17, which inhibits TIR1–IAA17 interactions, thereby inhibiting degradation of IAA17, leading to partial auxin resistance and defective lateral root development. 5.2. Denitrosation and plant reproduction NO plays a vital role in plant reproduction, acting as a signalling molecule regulating various processes from seed germination to floral development (ˇ Sírov´ a et al., 2011; Domingos et al., 2015; Kolbert et al., 2019). Accumulated evidence so far indicates that NO homeostasis, maintained by GSNOR activity, is crucial for the proper development of the female gametophyte and its function. In Arabidopsis, hot5–2/gsnor1 mutants show defects in female gametophyte development related to elevated levels of RNS and S-nitrosothiols (Lee et al., 2008; Xu et al., 2013). Recent data also suggest that functional NO homeostasis is required to maintain auxin transport and maternal control during the development of the female gametophyte, a vital determinant of the seed yield (Wang et al., 2024). Studies of female gametophyte development in Arabidopsis have shown that GSNOR, before fertilisation, is exclusively accumulated in sporophytic tissues and indirectly controls the gametophyte development. In contrast, in the GSNOR null mutant, RNS accumulation decreased auxin efflux, inhibited gametophyte development and reduced fertility. The proportion of normal female gametophytes and fertility could be restored by GSNOR expression in maternal tissues and by adding auxin efflux substrate, increasing fertility even during drought and salt stress conditions. Recently, a study of nitrosoproteome in Arabidopsis floral tissues combined with quantitative proteomic profiling identified 1049 Snitrosated proteins unique to hot5–2/gsnor1 mutant, with 728 novel Snitrosation targets not previously described in other plant tissues (Treffon and Vierling, 2025). Remarkably, specific UDP-glycosyltransferases and argonaute proteins were S-nitrosated in floral tissues and differentially regulated in pistils. The AGO protein family members, which play key roles in female reproductive development, were less abundant in hot5–2 pistils. It was suggested that specific AGO proteins are targets for S-nitrosation, leading to their selective degradation observed in hot5–2 pistils. Moreover, the observed S-nitrosation of multiple 26S proteasome subunits in reproductive tissues in this study suggests a potential link between protein quality control and fertility defects associated with disrupted NO homeostasis and S-nitrosation status. Collectively, advances in identifying S-nitrosation targets in plant reproductive tissues open new avenues of future research into the biological roles of protein S-nitrosation and denitrosation in plant reproduction. T. Jedelsk´ a et al. Plant Science 362 (2026) 112801 8 5.3. Denitrosation in the regulation of plant immune responses Multiple studies have described significant roles of GSNOR in controlling plant immunity through the regulation of S-nitrosation, which contributes to understanding how the cellular redox state controls and reprograms the immune functions of plants (Sedl´ aˇ rov´ a et al., 2025). The first evidence for the role of AtGSNOR1 in the cellular regulation of S-nitrosothiols and control of the biosynthesis of the immune activator salicylic acid (SA) was provided by the study of Feechan et al. (2005). S-nitrosation/denitrosation has a key regulatory role in SA-dependent defence responses, where increased levels of SA lead to the activation of defence genes at local and systemic levels, mediated by NPR1 (Nonexpressor of Pathogenesis-Related protein 1) and SABP3 (Salicylic Acid Binding Protein 3) proteins. The S-nitrosation process regulates the subcellular localisation and transcriptional activity of NPR1. The inactive protein is localised as an oligomer in the cytosol. In Arabidopsis, five out of ten Cys of the NPR1 protein are involved in the formation of intermolecular disulfide bridges (Mou et al., 2003). When a redox change is induced in the cell by SA produced after pathogen infection, disulfide bridges are broken down, and the resulting active NPR1 monomers are translocated to the nucleus and bind to specific TGA transcription factors, triggering the expression of pathogen-related (PR) defence genes (Dong, 2004). GSNO, as an NO donor, was observed to S-nitrosate Cys156 residues, causing oligomerisation of the NPR1 protein (Tada et al., 2008). It is suggested that S-nitrosation of Cys156 may induce conformational changes, facilitating disulfide bond formation between NPR1 monomers. GSNOR-mediated denitrosation is crucial in this context: in atgsnor1–3, S-nitrosation of NPR1 is enhanced, and the inactive oligomeric form is more abundant (Tada et al., 2008). In contrast, in Arabidopsis protoplasts treated with GSNO for 20 h, NPR1 localised predominantly as the active monomeric form in the nucleus (Lindermayr et al., 2010). These differences in the observed effects of S-nitrosation on NPR1 could be due precisely to the treatment with NO donors that induce the accumulation of SA (Durner et al., 1998), which has been shown to activate the translocation of NPR1 to the nucleus (Kinkema et al., 2000). Trxh5 role has also been described during SA induction in reducing disulfide bonds within NPR1 oligomers in the cytosol (Tada et al., 2008). Direct regulation of Trxh5 expression by the transcription factor WRKY33, interacting specifically with the W box (5 ′ -TTGAC[CT]-3 ′ ), has also been described, which could influence NPR1 activity (Birkenbihl et al., 2012). Furthermore, Trxh5 can also denitrosate NPR1 and facilitate monomerisation by preventing the formation of intermolecular disulfide bonds (Kneeshaw et al., 2014). Thus, the dynamic balance between NPR1 monomers and oligomers in the cytosol is an important point of control of plant immunity (Yu et al., 2014). Kovacs et al., 2016a showed that in Arabidopsis protoplasts treated with GSNO, NPR1 translocation to the nucleus is induced after 24 h. In contrast, NPR1 is translocated to the nucleus in SA-treated protoplasts after 2 h. These findings suggest that nuclear translocation of NPR1 may not directly result from its S-nitrosation. Increased GSH levels are linked to the activation of NPR1 after infection with the pathogen P. syringae pv. tomato and activate isochorismate synthase 1 (EC 5.4.4.2) and subsequent SA production. NPR1 monomerisation and translocation to the nucleus occur, and defence responses are activated with increased SA levels. TGA1, one of the transcription factors activated through NPR1 monomer binding, also undergoes S-nitrosation. TGA1, containing four Cys residues in its structure, is inactive when two disulfide bonds are formed. The addition of GSNO induces TGA1 binding capacity for sequence element 1 found in the promoters of several plant defence genes (Lindermayr et al., 2010). In the presence of NPR1, this binding capacity is enhanced by breaking the disulfide bond in the TGA1 protein between Cys172 and Cys287, and both residues undergo further S-nitrosation or glutathionylation. Thus, GSNO modification of the thiol prevents the formation of the TGA1 disulfide bond and stabilises a more favourable conformation for TGA1-NPR1 interaction. Kumar et al. (2022) provided a structural explanation for the direct role of SA in regulating NPR1-dependent gene expression. They reported cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form the enhanceosome, nucleoprotein complexes comprising multiple transcription factors binding at high density to short enhancer sequences (Kumar et al., 2022). However, it has not yet been shown to be redox-dependent or mediated by S-nitrosation (Borrowman et al., 2023). The chloroplast enzyme carbonic anhydrase (EC 4.2.1.1) is often referred to as SABP3 (salicylic acid binding protein 3) due to its strong binding capacity for SA. S-nitrosation of Cys280 in SABP3 causes loss of its enzyme activity and SA-binding capacity (Wang et al., 2009). S-nitrosation of SABP3 serves as a negative feedback loop modulating plant defence response by GSNOR-mediated denitrosation (Wang et al., 2009). NO and ROS are involved in hypersensitive response (HR) and programmed cell death at the site of pathogen-induced infection (Grant and Loake, 2000; Delledonne et al., 2001). Atgsnor1–3, with loss of GSNOR activity and increased S-nitrosothiol levels, exhibited faster cell death upon infection with P. syringae pv. tomato compared to the atgsnor1–1. Surprisingly, the levels of SA and ROS were reduced in atgsnor1–3 (Yun et al., 2011). Furthermore, AtRBOHD (Arabidopsis thaliana Respiratory Burst Oxidase Homolog D), key for ROS production, was found to undergo S-nitrosation of Cys890, resulting in decreased activity and thus ROS production during HR. In the later stages of HR development, redox modification by NO has a negative feedback function, limiting the extent of cell death due to lower ROS production (Yun et al., 2011). Recent studies of the tomato-Phytophthora capsica pathosystem provided new insights on the GSNOR role as a positive regulator of plant resistance (Li et al., 2025). As a novel mechanism, GSNOR activity was found to be blocked by the binding of proteinaceous virulence effector produced by the oomycete pathogen. The effector directly inhibits GSNOR activity and promotes its autophagy-mediated degradation, resulting in increased NO and S-nitrosothiol levels. Mutations in the effector binding site abolished its ability to induce NO levels and decreased its virulence, whereas mutations of GSNOR in the effector binding site enhanced plant resistance to the pathogen. These observations provide interesting insight into pathogen strategies which exploit disruption of NO homeostasis through manipulation of plant denitrosation capacity. NO signalling pathways, including protein nitrosylation, belong to important regulatory mechanisms in the growth and development of fungal and oomycete pathogens (Jedelsk´ a et al., 2021, Sedl´ aˇ rov´ a et al., 2025). A recent study on GSNOR in the phytopathogenic fungus Colletotrichum gloeosporioides confirmed its role as a critical regulator of NO homeostasis in the pathogen (Yang et al., 2025). The CgGSNOR deletion disrupted nitrosative homeostasis, resulting in increased NO and protein S-nitrosylation levels and mitochondrial dysfunction. Cytochrome c oxidase subunit 6B (COX6B) was identified as the key target of S-nitrosylation, required for fungal pathogenicity, host infection, and resistance to fungicides. Nevertheless, whether fungal GSNOR can be exploited as a molecular target for developing new antifungal strategies requires further investigation. 5.4. Where two or three gather: interplay of Trx, GSNOR, and AKRs denitrosation pathways in plant stress responses Plants are continually exposed to abiotic and biotic stimuli, which are perceived and responded to by complex mechanisms governed by stress signalling pathways, including redox signals of reactive oxygen and nitrogen species. Stress conditions that might eventually disrupt cellular redox balance can be effectively mitigated by activation and modulation of the redox regulatory cascade. Current evidence suggests that plant enzyme systems involved in the regulation of protein Snitrosative status play distinct yet intersecting roles in mediating stress T. Jedelsk´ a et al. Plant Science 362 (2026) 112801 9