Specificity in nitric oxide signalling

Specificity in nitric oxide signalling Abstract Reactive nitrogen species (RNS) and their cognate redox signalling networks pervade almost all facets of plant growth, development, immunity, and environmental interactions. The emerging evidence implies that specificity in redox signalling is achieved by a multilayered molecular framework. This encompasses the production of redox cues in the locale of the given protein target and protein tertiary structures that convey the appropriate local chemical environment to support redox-based, post-translational modifications (PTMs). Nascent nitrosylases have also recently emerged that mediate the formation of redox-based PTMs. Reversal of these redox-based PTMs, rather than their formation, is also a major contributor of signalling specificity. In this context, the activities of S-nitrosoglutathione (GSNO) reductase and thioredoxin h5 (Trxh5) are a key feature. Redox signalling specificity is also conveyed by the unique chemistries of individual RNS which is overlaid on the structural constraints imposed by tertiary protein structure in gating access to given redox switches. Finally, the interactions between RNS and ROS (reactive oxygen species) can also indirectly establish signalling specificity through shaping the formation of appropriate redox cues. It is anticipated that some of these insights might function as primers to initiate their future translation into agricultural, horticultural, and industrial biological applications. Free radicals, nitric oxide, NO, plant signalling, plant signal transduction, reactive nitrogen species, reactive oxygen species, redox signalling, S-nitrosylation, S-nitrosothiols Introduction Reactive nitrogen species (RNS) are central to the regulation of a plethora of cellular processes in plants integral to development (Foreman et al., 2003; Kwon et al., 2012; Homem and Loake, 2013; Kaya et al., 2014; Rodríguez-Ruiz et al., 2017), immunity (Torres et al., 2002), and abiotic stress (Wrzaczek et al., 2013; Vermeirssen et al., 2014; Fancy et al., 2017; Mata-Pérez et al., 2017). Significantly, these small, redox-active molecules are key components of cellular signal transduction networks (del Río et al., 2006; Yun et al., 2012; Skelly and Loake, 2013). However, the molecular details underpinning exactly how signalling specificity is achieved by these small, redox-active molecules remains to be fully established. Most of the recent insights in this area have been achieved in mammals, where nitric oxide (NO) signalling is the subject of intensive investigation due to its links with both health and disease. Thus, in this review, we also highlight some of the recent mammalian literature in this area, as a primer for research in plant biology. In a protein-centric signalling pathway, the complementarity of macromolecular shapes largely guides non-covalent ligand binding to an associated receptor. Conversely, RNS predominantly transmit signals via their inherent chemistries, targeting specific atoms of proteins resulting in redox-based, post-translational modifications (PTMs) (Nathan, 2003). Therefore, RNS molecular recognition is conveyed at the atomic rather than the macromolecular level. Herein, we will discuss some of the features of RNS signalling, focusing on NO, which convey specificity onto this pivotal redox-based cue. NO synthesis in plants RNS is a collective term used to describe both free radicals with a short half-life and non-radicals, which either serve as oxidizing/reducing agents or become transformed into radicals. RNS include: NO, peroxynitrite (ONOO−), S-nitrosothiols (SNOs), higher oxides of nitrogen (NOx), and dinitrosyl–iron complexes. These molecules are largely derived from NO, which in mammals is synthesized via an oxidative mechanism utilizing NO synthase (NOS), which consists of three well-characterized isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) (Alderton et al., 2001). NO is produced by the NADPH-dependent oxidation of l-arginine, producing l-citrulline and NO (Palmer et al., 1988; Knowles and Moncada, 1994; Correa-Aragunde et al., 2013). A NOS has also been reported to function in the green alga, Ostreococcustauri (Foresi et al., 2010). However, a total of 1087 transcriptome sequences of land plants have been interrogated for the presence of canonical NOS sequences, but none was found (Jeandroz et al., 2016). Hence, unless plant NOS enzymes are assembled from multipolypeptides (Mata-Pérez et al., 2017), land plants generate NO by one or more mechanisms distinct from mammals. In this context, nitrate reductase (NR) is a key enzyme for NO production in plants, and NR also facilitates NO homeostasis (Chamizo-Ampudia et al., 2017). Moreover, various other sources for NO production have been proposed in higher plants, including a NOS-like activity, xanthine oxidoreductase, and apolyamine/hydroxylamine-mediated NO synthesis. Thus, the generation of NO still remains to be fully established in higher plants (Durner et al., 1998; Stöhr and Stremlau, 2006; Stoimenova et al., 2007; Gupta et al., 2011; Yu et al., 2014). In order to acknowledge the biological significance of NO, in vivo synthesis of SNOs must be known. Multiple in vitro studies have highlighted various mechanisms for SNO formation. NO as a free radical (NO) can lose or gain electrons to generate an oxidized nitrosonium cation (NO+) or a reduced nitroxyl anion (NO–), or NO can react with superoxide (O2–) and oxygen (O2) to generate ONOO– and NO oxides (N2O3/NOX), which all can act as S-nitrosylating agents (Fig. 1) (Arnelle and Stamler, 1995; Broniowska and Hogg, 2012). Furthermore, NO radicals can react directly with thiyl radicals (RS), metal–NO complexes (M-NO), or protein thiols to generate SNO. The thiol groups and thiyl radicals are part of either a protein or glutathione. Furthermore, hydrogen peroxide (H2O2), organic hydroperoxides, and peroxynitrite oxidizes reactive cysteine thiols (R-SHs) to form sulphenic acids, sulphenamides (R-SNRs), or sulfonamides (R-SONRs), and sulphonamides (R-SO2NRa) (Klomsiri et al., 2011). Fig. 1. View largeDownload slide Generation of S-nitrosothiols. Nitric oxide (NO) can lose or gain an electron to form an oxidized nitrosonium cation (NO+) or a reduced nitroxyl anion (NO–), respectively. NO can also react with superoxide (O2–) to form peroxynitrite (ONOO–). Further, NO can react with oxygen (O2) to form higher order nitrogen oxides (NOx), such as dinitrogen trioxide (N2O3). NO radicals can also react directly with transition metals in metal-containing proteins to form metal–nitrosyl complexes (M-NOs). Each of the described molecules can react with either thiol groups, typically as a thiolate (RS–) or thiyl (RS·) associated with either proteins or glutathione to generate an S-nitrosothiol (SNO). Fig. 1. View largeDownload slide Generation of S-nitrosothiols. Nitric oxide (NO) can lose or gain an electron to form an oxidized nitrosonium cation (NO+) or a reduced nitroxyl anion (NO–), respectively. NO can also react with superoxide (O2–) to form peroxynitrite (ONOO–). Further, NO can react with oxygen (O2) to form higher order nitrogen oxides (NOx), such as dinitrogen trioxide (N2O3). NO radicals can also react directly with transition metals in metal-containing proteins to form metal–nitrosyl complexes (M-NOs). Each of the described molecules can react with either thiol groups, typically as a thiolate (RS–) or thiyl (RS·) associated with either proteins or glutathione to generate an S-nitrosothiol (SNO). A redox code of cysteine residue modifications? Rare, highly reactive protein cysteine (Cys) thiols (SHs) embedded within protein structures are key atomic targets for RNS-dependent signalling (Boehning and Snyder, 2003; Poole et al., 2004). These Cys residues are solvent exposed with a low pKa sulphydryl group, which facilitates their efficient oxidation by RNS (Meng et al., 2002; Yu et al., 2014). Redox-based Cys modification is influenced by the ionization state of the reactive Cys: thiolate anions (-S–) have a greater tendency to donate electrons and become polarized relative to their corresponding protonated counterparts. Cys thiols undergo a redox continuum of modifications generating an array of possible outcomes, including S-nitrosylation, the addition of an NO moiety to form an SNO, S-glutathionylation (S-SG), S-sulphenation (S-OH), S-thiolation (S-S, i.e. disulphide formation), and S-sulphination (S-O2H) (Fig. 2) (Spadaro et al., 2010; Spoel and Loake, 2011, Klomsiri et al., 2011). A key feature of specificity in redox signalling is the translation of different redox-based modifications into discrete protein functions to generate different cellular outcomes. This has been proposed to provide the basis of a redox code that translates protein structure into function. For example, a single Cys residue in OxyR, a transcriptional activator of Escherichia coli, is modified to generate four stable, redox-related modifications (S-H, S-OH, S-SG, or SNO) (Kim et al., 2002). Each modification resulted in a differential conformational change in OxyR structure, conveying unique DNA binding affinities, promoter activities, and co-operative properties. These findings imply that fine-grained transcriptional regulation might therefore be controlled by exploiting distinct Cys modifications. Fig. 2. View largeDownload slide Schematic overview of redox-based cysteine (Cys) modifications. Cys thiol (SH), S-nitrosothiol (SNO), sulphenic acid (SOH), disulphide (S-S), S-glutathionylation (SSG), sulphinic acid (SO2H), and sulphonic acid formation (SO3H). Molecules in this sequence are formed following increasing oxidation. The formation of all these functional groups is thought to be reversible, except for sulphonic acid formation, which is irreversible. (Modified from Spadaro et al., 2010. The redox switch: dynamic regulation of protein function by cysteine modifications. Physiologia Plantarum 138, 360–371, with permission). Fig. 2. View largeDownload slide Schematic overview of redox-based cysteine (Cys) modifications. Cys thiol (SH), S-nitrosothiol (SNO), sulphenic acid (SOH), disulphide (S-S), S-glutathionylation (SSG), sulphinic acid (SO2H), and sulphonic acid formation (SO3H). Molecules in this sequence are formed following increasing oxidation. The formation of all these functional groups is thought to be reversible, except for sulphonic acid formation, which is irreversible. (Modified from Spadaro et al., 2010. The redox switch: dynamic regulation of protein function by cysteine modifications. Physiologia Plantarum 138, 360–371, with permission). Local chemical environments conducive to redox-based PTMs S-Nitrosylation has emerged as a prototypic redox-based, PTM across phylogenetic kingdoms (Foster et al., 2009; Yun et al., 2011). In plants, protein–SNO formation has been shown to help shape growth, development, immunity, and abiotic interactions (Lee et al., 2008; Kwon et al., 2012). Several studies have been undertaken to uncover a potentially conserved sequence motif for S-nitrosylation of protein targets, utilizing high-throughput proteomic approaches in conjunction with bioinformatic interrogation. Various factors have been reported to contribute to the selectivity of Cys residues for protein S-nitrosylation, including solvent accessibility, the presence of acidic–basic groups in their proximity (within 6–8 Å), the incidence of adjacent hydrophobic amino acids, increased nucleophilicity, and the presence of local α-helical domains (Chen et al., 2010; Doulias et al., 2010; Marino and Gladyshev, 2010; Seth and Stamler, 2011). The motif I/L-X-C-X2-D/E carries a Cys residue with a high nucleophilicity, surrounded by a hydrophobic residue, I/L, and an acidic residue, D/E. This motif therefore contains most, if not all, of the characteristics thought to be necessary for S-nitrosylation of a target Cys. In this context, this motif is associated with iNOS-dependent S-nitrosylation of S100A8/A9 (calprotectin/calcium-binding protein complex) and was present in 19 additional candidate SNO–proteins (Jia et al., 2012). However, analysis of 445 human proteins with 810 SNO–peptides revealed that only 15 peptides present in 15 proteins contain the I/L-X-C-X2-D/E motif (Jia et al., 2012; Lee et al., 2012). Hence, results based on large-scale data sets have cast doubt on the reliability of a specific motif based on a primary amino acid sequence underpinning S-nitrosylation. Interestingly, a computational tool, GPS-SNO 1.0, has been developed for the prediction of S-nitrosylation sites. This can provide a convenient and rapid strategy to generate useful information for subsequent experimental verification (Xue et al., 2010). The developers utilized 504 experimentally verified S-nitrosylation sites in 327 unique proteins. The developed GPS-SNO 1.0 tool predicted at least one potential S-nitrosylation site in 359 out of 485 S-nitrosylated proteins, with a previously unknown S-nitrosylation site. Further, a set of 46 proteins with 53 S-nitrosylation sites experimentally identified in Arabidopsis, were interrogated by GPS-SNO 1.0. A total of 60 S-nitrosylation sites were identified amongst these proteins, but only 11 of these sites had been confirmed experimentally (Chaki et al., 2014). Thus, simple motif recognition/computational tools may also be of only limited value as they only reveal information associated with common features responsible for specificity and patterns implemented in predictive algorithms. Consequently, these bioinformatics approaches have largely failed to identify a specific consensus sequence that directs site-selective S-nitrosylation. As the overall tertiary structure of proteins largely defines the activity of a given cognate amino acid residue, it may be naive to attempt the identification of target Cys residues based on a motif with only primary sequence information. In addition to protein three-dimensional structure, future bioinformatic tools should also consider the given redox cue, for example NO, GSNO, or even oxidized lipids (Mata-Pérez et al., 2017), the likely concentrations of the given RNS, and the physiological conditions under which the given PTM occurs, because all these variables can also influence Cys target specificity. Proximity-based RNS production and associated Cys modification Another potential mechanism thought to be integral for conveying specificity in redox signalling is the proximity of the target Cys to the source of either RNS. Thus, the subcellular organization of the source of redox-active molecules within cells alongside the spatial and temporal existence of given protein targets may be of prime importance to trigger a particular redox signalling pathway (Derakhshan et al., 2007). For example, in mammalian cells, eNOS and nNOS co-exist in cardiac myocytes but are located within distinct subcellular compartments: while eNOS is located in the plasma membrane, nNOS is found in the sarcoplasmic reticulum. Spatial confinement of these NO sources enables this redox-active molecule to trigger discriminate signalling pathways which determine distinct downstream effects. Thus, NO generated by eNOS limits myocardial contractility, whereas NO generated by nNOS increases contractility (Barouch et al., 2002). An alternative biological strategy for achieving specificity in redox signalling is through proximity of both the given RNS source and the cognate target Cys thiol. For example, eNOS binds to heat shock protein 90 (Hsp90) directly, which results in S-nitrosylation of Hsp90 at Cys597, affecting its ATPase activity (Martínez-Ruiz et al., 2005). Moreover, cycloxygenase-2 (COX-2) becomes activated following direct binding of iNOS, which results in the selective S-nitrosylation of COX-2 on Cys526 (Kim et al., 2005). A variation on this theme is the deployment of a scaffold protein to link the redox molecule generator with the target protein. In this context, NMDAR (N-methyl-d-aspartate receptor) binds indirectly to nNOS via the scaffold-protein PSD95 (post-synaptic density protein). The NMDAR–PSD95–nNOS ternary complex is essential for NMDAR activation, which ultimately also increases nNOS activity (Brenman et al., 1996; Christopherson et al., 1999). Interestingly, another adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), competes with PSD95 for binding with nNOS, positioning NO generation to drive S-nitrosylation of Dexras (Ras superfamily gene induced by dexamethasone) at Cys11, which facilitates the exchange of bound GDP for GTP (Jaffrey et al., 1998, 2002). While now well established in mammalian systems, this proximity-based production of redox cues has not to date been demonstrated in plants. At least in the case of NO and other RNS, this has been impeded by the continuing controversy regarding the source of NO production in plants(Corpas et al., 2006; Jeandroz et al., 2016; Mata-Pérez et al., 2017; Zemojtel et al., 2006). Specific protein trans-nitrosylation Interestingly, the accumulating evidence from mammalian systems suggests that some proteins might function to transfer their NO group linked to the protein Cys sulphur to specific protein substrates, effectively functioning as nitrosylase ‘enzymes’, akin to more established enzymes in PTMs such as protein kinases (Hess et al., 2005). To date, only a small number of S-nitrosylases have been reported in animals, but this list is steadily growing (Nakamura and Lipton, 2013). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a classic glycolytic enzyme, has been proposed to function as a nitrosylase. Upon apoptotic stimulation, mammalian GAPDH is modified at Cys150 by NO, generated by NOS. Subsequently, SNO–GAPDH is thought to bind to Siah1, an E3 ubiquitin ligase, which is then translocated to the nucleus (Hara et al., 2005). SNO–GAPDH binding to Siah1 stabilizes this protein against turnover, and in the nucleus Siah1 degrades its target substrates and also enhances p300/CBP-associated acetylation of nuclear proteins, including p53, which cause cell death (Sen et al., 2008). The SNO–GAPDH–Siah1 complex also mediates nuclear translocation of mutant Huntingtin (mHtt) protein which is associated with Huntington’s disease (Bae et al., 2006). Further, after nuclear translocation, SNO–GAPDH also transfers its NO group at Cys150 to specific Cys residues within targeted protein thiols including SIRT1, a histone deacetylase-2 (HDAC2), and DNA-activated protein kinase (DNA-PK) (Kornberg et al., 2011). Hence, SNO–GAPDH essentially also functions as an S-nitrosylase, transferring its NO to Cys thiols embedded in target proteins. However, under low levels of NO availability, GOSPEL (GAPDH’s competitor Of Siah Protein Enhances Life) is S-nitrosylated and competes with Siah1 for GAPDH binding, preventing the formation of a GAPDG–Siah1 complex (Sen et al., 2009). The S-nitrosylation of haemoglobin has been proposed to regulate human blood pressure (Jia et al., 1996). The central iron molecule of the haem group in haemoglobin can bind oxygen, facilitating the transfer of this molecule from the lungs to the rest of the body. Binding of NO at this haem group rapidly facilitates auto-S-nitrosylation of haemoglobin at its highly conserved Cysâ93 residue, by the transfer of the bound NO moiety, freeing the haem iron to bind to oxygen. In human hypoxia, haemoglobin–SNO has been proposed to function as a nitrosylase, where the NO group is transferred from SNO–Cysâ93 to the anion exchange protein (AE1/band3) in human red blood cells, following binding to the red blood cell membrane. Subsequently, SNO–AE1 is thought to release NO which can subsequently diffuse into smooth muscle cells, promoting the relaxation of blood vessels (Pawloski et al., 2001). In this scenario, allosteric regulation of haemoglobin is employed to propagate a vasodilatory signal via the trans-nitrosylative transfer of NO. Thus, protein trans-nitrosylation constitutes an additional strategy to convey specificity to NO-related redox signalling in biological systems. Removal of redox-based PTMs An important feature of the addition of PTMs to their protein targets associated with cellular signalling is their selective reversal to disengage the given signal networks. It is highly likely that specificity in redox signalling is achieved largely by rates of reversal of Cys modifications, rather than by their formation, as rapidly degraded redox modifications would probably have far less impact than more persistent ones (Derakhshan et al., 2007). In this context, it has been demonstrated that different protein–SNOs can have widely diverse biological lifetimes (Seth and Stamler, 2015).While a proportion of this can be attributed to the innate chemical stability of a given protein–SNO, this property is also influenced by potential non-enzymatic breakdown, for example by either of the key cellular antioxidant molecules, ascorbate or glutathione (Masella et al., 2005; Feechan et al., 2005; Benhar et al., 2008; Kneeshaw et al., 2014). In the green alga Chlamydomonas reinhardtii, haemoglobin affects the nitrogen assimilation pathway by simultaneously modulating NO levels and NR activity (Sanz-Luque et al., 2015). Interestingly, non-symbiotic plant haemoglobins (phytoglobins) have also been proposed to function as NO scavengers in plants, with their activity possibly impacting plant developmental programmes (Hebelstrup et al., 2013). However, these proteins are not thought to control the consequences of the pathogen-triggered, nitrosative burst and associated host cell death during plant immunity (Perazzolli et al., 2004). Ascorbic acid can reduce phytoglobins in support of NO scavenging, generating nitrate and monodehydroascorbate. The monodehydroascorbate is recycled back to ascorbic acid by monodehydroascorbate reductase using NADH. Phytoglobins scavenge NO by forming S-nitrosophytoglobin; however, how this is recycled back to the reduced phytoglobin remains to be determined, but recent evidence suggests that this does not involve ascorbate in planta (Wang and Hargrove, 2013). Significantly, in more established signal transduction mechanisms, such as phosphorylation, signalling and its associated specificity is the result of a delicate poise between the activities of kinase and phosphatase. The conceptual equivalent of protein phosphatases associated with redox signalling has recently started to come into sharper focus. Indirect and selective protein denitrosylation It is now well established that potential non-enzymatic mechanisms make a key contribution to protein–SNO homeostasis (Liu et al., 2001; Feechan et al., 2005). The antioxidant tripeptide glutathione (GSH) has been shown to access sites of S-nitrosylation in protein–SNOs, reducing the SNO group back to a Cys thiol and forming S-nitrosoglutathione (GSNO); GSH is S-nitrosylated at its Cys residue as a consequence (Corrales et al., 1999; Romero and Bizzozero, 2009). Arabidopsis GAPDH is S-nitrosylated at Cys149 and is denitrosylated selectively by GSH (Zaffagnini et al., 2013). However, in animals, SNO–GAPDH binding to Siah1, an E3 ubiquitin ligase, forms a complex which changes the conformation of SNO–GAPDH, precluding GAPDH–SNO-Cys149 access to GSH, abolishing GSH-mediated denitrosylation (Paige et al., 2008). Importantly, GSNO can function as a natural NO donor driving S-nitrosylation of reactive Cys thiols by trans-nitrosylation, resulting in specific S-nitrosylation of the target protein (Hess et al., 2005). GSNO is relatively stable and can therefore function as a cellular reservoir of NO bioactivity. Significantly, GSNO can be turned over by GSNO reductase (Feechan et al., 2005; Lee et al., 2008; Chen et al., 2009). Thus, S-nitrosylated proteins may be in dynamic equilibrium with their denitrosylated counterparts controlled by the activity of GSNOR. A growing body of evidence has demonstrated that GSNOR-mediated denitrosylation has essential roles in both plant immunity and development. Loss- or gain-of-function mutants in GSNOR1 in Arabidopsis result in either reduced or increased denitrosylation, respectively. While reduced denitrosylation promotes enhanced disease susceptibility, increased denitrosylation leads to enhanced disease resistance (Feechan et al., 2005; Tada et al., 2008). Further, the control of denitrosylation has also been shown to regulate pathogen-triggered cell death by controlling the extent of S-nitrosylation at Cys890 of the respiratory burst oxidase homologue D (RBOHD), and this mechanism may be evolutionarily conserved across kingdoms (Yun et al., 2011). In a similar fashion, dysregulation of denitrosylation also appears to impact a number of key plant developmental programmes (Lea et al., 2004; Lee et al., 2008; Kwon et al., 2012). How is the specificity in GSNOR function established? The emerging evidence suggests that the tertiary protein structure restricts the ability of GSH to function as a non-enzymatic denitrosylase to a subset of reactive protein Cys thiols (Foster et al., 2009; Yun et al., 2016). Thus, GSH might not be able to denitrosylate all possible cellular protein–SNOs, forming the free protein thiol and GSNO in the process. Consequently, only a subset of total cellular S-nitrosylated proteins will be under the indirect control of the protein denitrosylase, GSNOR. Direct and selective protein denitrosylation Thioredoxins (Trxs) are present in all living organisms, and their activity can be recycled by NADPH-dependent thioredoxin reductase (TrxR). Trx/TrxR-mediated denitrosylation has emerged as an important mechanism to control redox regulation. In animals, Trx-mediated denitrosylation of caspase-3 results in the activation of this enzyme (Benhar et al., 2008). Other targets for Trx-mediated selective denitrosylation in animals are caspase-9, protein tyrosine phosphatase 1B, and GAPDH (Benhar et al., 2008). In plant immunity, Trxh5 has been proposed to denitrosylate selectively the transcriptional co-activator, NPR1 (Kneeshaw et al., 2014). NPR1 is S-nitrosylated in response to increasing GSNO levels, leading to NPR1 oligomerization, thus sequestering this co-activator in the cytosol, preventing its nuclear translocation and the subsequent activation of pathogenesis related (PR) gene expression (Tada et al., 2008; Yu et al., 2014). Trxh5 and TrxR have been proposed to denitrosylate NPR1 directly, leading to the release of NPR1 monomers, their translocation to the nucleus, and the subsequent engagement of PR gene transcription (Kneeshaw et al., 2014). Thus, selective denitrosylation of NPR1 is an important feature of the plant immune response. However, it is noteworthy that NPR1 function is also impacted by GSNOR activity (Feechan et al., 2005; Tada et al., 2008), suggesting that the S-nitrosylation status of this transcriptional co-activator is regulated directly by Trxh5 but indirectly by GSNOR. The emerging biochemical and genetic evidence suggests that Trxh5 and TrxR can denitrosylate a subset of the plant SNO proteome directly and selectively in vivo (Kneeshaw et al., 2014). Thus, the regulation of denitrosylation at other key protein Cys thiols is also likely to be under the control of Trxh5 and TrxR. Further, Trxs are comprised of a large gene family in Arabidopsis; therefore, it is possible that other Trx family members might also function in combination with TrxR as direct and selective denitros ylases of a range of possible substrates. Interestingly, in mammals, two Trx-mimetic (TXM) peptides derived from the Trx conserved active site (CXXC) have been documented to act as denitrosylating catalysts (Kronenfeld et al., 2015). TXMs with TrxR can effectively reduce both low molecular weight SNOs and protein–SNO. GSNO is reduced to GSH through trans-nitrosylation, resulting in S-nitrosylated TXMs, which are cyclized by TrxR or other endogenous thiol reductases. TXMs have the ability to denitrosylate multiple classes of SNO proteins. TXM also protects TrxR from SNO-mediated loss of activity, which is important to conserve its function in redox signalling. Collectively, the current state-of-the-art suggests that Trx and TrxR can function as direct and selective denitrosylases to regulate a subset of S-nitrosylated proteins across kingdoms. Discrete activities of given RNS Recent studies have shown that NO and SNO might have discrete or overlapping protein targets in relation to redox signalling (Yun et al., 2016), providing an additional layer of RNS signalling specificity. NO has been proposed to have a more extensive range of biological activity relative to GSNO (Fig. 3). In addition to protein S-nitrosylation, NO is thought to be largely responsible for metal nitrosylation, the most rapid known reaction of NO with proteins. NO forms metal–nitrosyl complexes (M-NOs), with metals embedded within proteins, for instance zinc (Zn2+), iron (Fe2+ or Fe3+), or copper (Cu2+) (Leitner et al., 2009). Mammalian soluble guanylate cyclase is a prototypic example of a protein that is regulated by this type of PTM (Derbyshire and Marletta, 2012). NO can also nitrosylate amino acid side chains to form so-called N-NO (Stamler et al., 1992). However, NO cannot directly oxidize amino acid side chains at any significant biological rate. Hence, NO mediates N-NO via auto-oxidation to nitrogen dioxide, dinitrogen trioxide, or peroxynitrite. In contrast, GSNO is thought principally to mediate protein S-nitrosylation (Foster et al., 2009; Yun et al., 2016). Fig. 3. View largeDownload slide Schematic model showing discrete and overlapping activities of NO and GSNO associated with cellular signalling. NO can target metals embedded in proteins (1), amino acid side chains (2), GSH to form GSNO, which is reversible (3), and proteins to form S-nitrosothiols (4). NO can also S-nitrosylate GAPDH (5). NO can be metabolized by microbial FHb into NO3/N2O (6). GSNO can trans-nitrosylate selected protein targets (7). GSH can reverse S-nitrosylation at a subset of proteins, generating the denitrosylated protein and GSNO (8). GAPDH–SNO can function as a nitrosylase (in mammals) to S-nitrosylate target proteins (9). GSNO can S-nitrosylate GAPDH, forming GAPDH–SNO and GSH (10). GSH can denitrosylate GAPDH–SNO in Arabidopsis to generate GAPDH and GSNO (11). Trx (Trxh5 in Arabidopsis) can directly and selectively denitrosylate a subset of protein–SNO (12). Fig. 3. View largeDownload slide Schematic model showing discrete and overlapping activities of NO and GSNO associated with cellular signalling. NO can target metals embedded in proteins (1), amino acid side chains (2), GSH to form GSNO, which is reversible (3), and proteins to form S-nitrosothiols (4). NO can also S-nitrosylate GAPDH (5). NO can be metabolized by microbial FHb into NO3/N2O (6). GSNO can trans-nitrosylate selected protein targets (7). GSH can reverse S-nitrosylation at a subset of proteins, generating the denitrosylated protein and GSNO (8). GAPDH–SNO can function as a nitrosylase (in mammals) to S-nitrosylate target proteins (9). GSNO can S-nitrosylate GAPDH, forming GAPDH–SNO and GSH (10). GSH can denitrosylate GAPDH–SNO in Arabidopsis to generate GAPDH and GSNO (11). Trx (Trxh5 in Arabidopsis) can directly and selectively denitrosylate a subset of protein–SNO (12). A microbial flavohaemoglobin (FHb) has been shown to turn over NO in a number of bacteria and may function as a virulence factor to counter antimicrobial NO production by potential mammalian hosts produced via iNOS (Poole et al., 1996; Poole and Hughes, 2000). Loss-of-function mutations in a FHb gene present in the human pathogens Cryptococcus neoformans or Salmonella typhimurium reduced their virulence in mice. However, virulence was restored in mice lacking an iNOS (Bang et al., 2006; de Jesús-Berrı́os et al., 2003). Informatively, double mutants lacking both FHb and GSNOR function further reduced the virulence of C. neoformans, consistent with an additive antimicrobial function of NO and GSNO (Liu et al., 2004), suggesting that these RNS might have separable functions. Thus, NO and GSNO might target a different spectrum of microbial protein Cys thiols. Similarly, in Saccharomyces cerevisiae, an FHb knock-out mutant strain was deficient in NO turnover as compared with the wild type or GSNOR knock-out strains. Further, exogenous application of NO retarded the growth of the fhb mutant but had no impact on either the wild type or a gsnor mutant strain. However, exogenous application of GSNO had a greater impact on the fhb gsnor double mutant relative to either of the single mutant strains. Thus, GSNOR activity conveys greater protection against GSNO-induced nitrosative stress, rather than nitrosative stress established by NO (Foster et al., 2009). This is consistent with the proposal that NO and GSNO may have distinct and overlapping activities. Genetic evidence for this dichotomy between the biological activities of NO and GSNO has also recently been established in plants (Yun et al., 2016). The Arabidopsis mutants, no-over producer 1-1 (nox1-1) and gsnor1-3, exhibit increased levels of either NO or SNO in vivo, respectively (He et al., 2004; Feechan et al., 2005; Yun et al., 2016). Informatively, the double mutant, gsnor1-3 nox1-1, appears to be genetically additive, as it shows greater susceptibility to pathogens than either of the single mutants alone. Further, GSNOR1 overexpression in gsnor1 plants restored wild-type levels of immunity; however, this was not the case in nox1-1 plants. This may reflect the notion that NO can drive both M-NO and N-NO formation in addition to protein S-nitrosylation. In contrast, GSNO predominantly favours the generation of protein–SNOs. Interestingly, genetic analysis suggests that high in vivo SNO concentrations can facilitate pathogen-triggered hypersensitive response (HR) cell death independently of ROS synthesized by the NADPH oxidases, AtRBOHD and AtRBOHF, responsible for the extracellular oxidative burst (Yun et al., 2011). While NO was previously reported to require superoxide (O2−) in order to trigger the HR (Delledonne et al., 2001) (see next section). Collectively, these data suggest that NO and SNO might have discrete or overlapping functions in the context of redox signalling during the establishment of plant immunity (Yun et al., 2016) and perhaps other plant molecular systems, providing addition scope for specificity in redox regulation of cellular function. Specificity conveyed by interactions between RNS and ROS Chemical interactions between RNS and ROS are a prominent feature of redox signalling networks. These interactions have been proposed to modulate indirectly the activity of a series of proteins (Hausladen and Fridovich, 1994; Romero et al., 2003; Wang et al., 2013) and, by extension, this presents another way of achieving specificity in redox signalling. For example, NO is unable to influence the activity of the tricarboxylic acid cycle enzyme, aconitase, in Escherichia coli. However, the interaction of NO with superoxide (O2−) to form peroxynitrite (ONOO−) generates a powerful inhibitor of aconitase activity (Hausladen and Fridovich, 1994). In plant biology, interaction between NO and H2O2 has been reported to be a key factor associated with the development of the HR, leading to the programmed execution of pathogen-challenged plant cells (Mur et al., 2008). NO production following pathogen recognition has been posited to be insufficient to activate HR cell death. Rather, the HR has been proposed to be triggered only by a balanced production of NO and ROS (Delledonne et al., 2001; Lin et al., 2012). Thus, HR cell death is activated after interaction of NO not with superoxide (O2−), presumably produced by RBOHD and RBOHF (Torres et al., 2002), but with H2O2 generated from O2− by superoxide dismutase (Delledonne et al., 2001). Increasing the level of O2− reduced NO-mediated HR cell death, by converting more NO into ONOO−, which was proposed to have low levels of toxicity towards plant cells. However, this is counterintuitive, because it is well established that ONOO− exhibits an extremely high level of cellular toxicity (Pacher and Szabo, 2008). Perhaps plants can more effectively turn over ONOO−. In a similar fashion, both NO and GSNO have been proposed to be important mediators in the process of H2O2-induced leaf cell death in rice (Lin et al., 2012) and, in response to the fungal elicitor, cryptogein, the production of NO and H2O2 in tobacco appears to be reciprocally regulated (Kulik et al., 2015). Also, S-nitrosylation of RBOHD at Cys890 curbs ROS production limiting pathogen-triggered cell death development (Yun et al., 2011). An interaction between NO and H2O2 has also been reported to be required for stimulation of stomatal closure in Arabidopsis in response to UV-B exposure. An increase in H2O2 scavenging activity or an inhibition of H2O2 synthesis prevented NO generation and subsequent stomatal closure (He et al., 2013). In aggregate, this work demonstrates that either direct or indirect interactions between distinct redox molecules can shape the specificity of signalling outputs. Conclusions and future prospects The prevalence of redox signalling in the biology of plants and other organisms is being increasingly appreciated. However, there are many potential molecular switches, including but not limited to highly reactive Cys thiols and chemically co-ordinated metal cofactors. So how is specificity in redox regulation achieved? As discussed above, the emerging model suggests that redox specificity is mediated via a multilayered molecular framework. This might encompass the production of the signalling redox molecule(s) in the vicinity of the protein target. Tertiary protein structures subsequently provide a chemical environment compatible with oxidation/reduction of the target redox switch. Superimposed upon this molecular landscape, trans-nitrosylation, by a nascent but growing repertoire of nitrosylases, can function as an additional mechanism to deliver and then transfer an NO moiety to a target protein thiol. Reversal of redox modifications, rather than their formation, is also a major contributor to signalling specificity. In this context, selective, indirect protein denitrosylation conveyed by GSNOR-dependent GSNO turnover is a prominent feature. This mechanism is augmented by the selective and direct denitrosylation mediated by Trxh5 and TrxR. Signalling specificity is also mediated by the unique chemistries of individual RNS which is overlaid on the structural constraints imposed by tertiary protein structure gating access to given redox switches. Finally, the direct or indirect interactions between RNS and ROS can also indirectly establish signalling specificity through shaping the formation of appropriate redox cues. The emerging evidence suggests that redox signalling pervades almost all aspects of plant growth, development, immunity, and abiotic environmental interactions (Besson-Bard et al., 2008; Yu et al., 2012, 2014). 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Specificity in nitric oxide signalling

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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0022-0957
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Abstract

Abstract Reactive nitrogen species (RNS) and their cognate redox signalling networks pervade almost all facets of plant growth, development, immunity, and environmental interactions. The emerging evidence implies that specificity in redox signalling is achieved by a multilayered molecular framework. This encompasses the production of redox cues in the locale of the given protein target and protein tertiary structures that convey the appropriate local chemical environment to support redox-based, post-translational modifications (PTMs). Nascent nitrosylases have also recently emerged that mediate the formation of redox-based PTMs. Reversal of these redox-based PTMs, rather than their formation, is also a major contributor of signalling specificity. In this context, the activities of S-nitrosoglutathione (GSNO) reductase and thioredoxin h5 (Trxh5) are a key feature. Redox signalling specificity is also conveyed by the unique chemistries of individual RNS which is overlaid on the structural constraints imposed by tertiary protein structure in gating access to given redox switches. Finally, the interactions between RNS and ROS (reactive oxygen species) can also indirectly establish signalling specificity through shaping the formation of appropriate redox cues. It is anticipated that some of these insights might function as primers to initiate their future translation into agricultural, horticultural, and industrial biological applications. Free radicals, nitric oxide, NO, plant signalling, plant signal transduction, reactive nitrogen species, reactive oxygen species, redox signalling, S-nitrosylation, S-nitrosothiols Introduction Reactive nitrogen species (RNS) are central to the regulation of a plethora of cellular processes in plants integral to development (Foreman et al., 2003; Kwon et al., 2012; Homem and Loake, 2013; Kaya et al., 2014; Rodríguez-Ruiz et al., 2017), immunity (Torres et al., 2002), and abiotic stress (Wrzaczek et al., 2013; Vermeirssen et al., 2014; Fancy et al., 2017; Mata-Pérez et al., 2017). Significantly, these small, redox-active molecules are key components of cellular signal transduction networks (del Río et al., 2006; Yun et al., 2012; Skelly and Loake, 2013). However, the molecular details underpinning exactly how signalling specificity is achieved by these small, redox-active molecules remains to be fully established. Most of the recent insights in this area have been achieved in mammals, where nitric oxide (NO) signalling is the subject of intensive investigation due to its links with both health and disease. Thus, in this review, we also highlight some of the recent mammalian literature in this area, as a primer for research in plant biology. In a protein-centric signalling pathway, the complementarity of macromolecular shapes largely guides non-covalent ligand binding to an associated receptor. Conversely, RNS predominantly transmit signals via their inherent chemistries, targeting specific atoms of proteins resulting in redox-based, post-translational modifications (PTMs) (Nathan, 2003). Therefore, RNS molecular recognition is conveyed at the atomic rather than the macromolecular level. Herein, we will discuss some of the features of RNS signalling, focusing on NO, which convey specificity onto this pivotal redox-based cue. NO synthesis in plants RNS is a collective term used to describe both free radicals with a short half-life and non-radicals, which either serve as oxidizing/reducing agents or become transformed into radicals. RNS include: NO, peroxynitrite (ONOO−), S-nitrosothiols (SNOs), higher oxides of nitrogen (NOx), and dinitrosyl–iron complexes. These molecules are largely derived from NO, which in mammals is synthesized via an oxidative mechanism utilizing NO synthase (NOS), which consists of three well-characterized isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) (Alderton et al., 2001). NO is produced by the NADPH-dependent oxidation of l-arginine, producing l-citrulline and NO (Palmer et al., 1988; Knowles and Moncada, 1994; Correa-Aragunde et al., 2013). A NOS has also been reported to function in the green alga, Ostreococcustauri (Foresi et al., 2010). However, a total of 1087 transcriptome sequences of land plants have been interrogated for the presence of canonical NOS sequences, but none was found (Jeandroz et al., 2016). Hence, unless plant NOS enzymes are assembled from multipolypeptides (Mata-Pérez et al., 2017), land plants generate NO by one or more mechanisms distinct from mammals. In this context, nitrate reductase (NR) is a key enzyme for NO production in plants, and NR also facilitates NO homeostasis (Chamizo-Ampudia et al., 2017). Moreover, various other sources for NO production have been proposed in higher plants, including a NOS-like activity, xanthine oxidoreductase, and apolyamine/hydroxylamine-mediated NO synthesis. Thus, the generation of NO still remains to be fully established in higher plants (Durner et al., 1998; Stöhr and Stremlau, 2006; Stoimenova et al., 2007; Gupta et al., 2011; Yu et al., 2014). In order to acknowledge the biological significance of NO, in vivo synthesis of SNOs must be known. Multiple in vitro studies have highlighted various mechanisms for SNO formation. NO as a free radical (NO) can lose or gain electrons to generate an oxidized nitrosonium cation (NO+) or a reduced nitroxyl anion (NO–), or NO can react with superoxide (O2–) and oxygen (O2) to generate ONOO– and NO oxides (N2O3/NOX), which all can act as S-nitrosylating agents (Fig. 1) (Arnelle and Stamler, 1995; Broniowska and Hogg, 2012). Furthermore, NO radicals can react directly with thiyl radicals (RS), metal–NO complexes (M-NO), or protein thiols to generate SNO. The thiol groups and thiyl radicals are part of either a protein or glutathione. Furthermore, hydrogen peroxide (H2O2), organic hydroperoxides, and peroxynitrite oxidizes reactive cysteine thiols (R-SHs) to form sulphenic acids, sulphenamides (R-SNRs), or sulfonamides (R-SONRs), and sulphonamides (R-SO2NRa) (Klomsiri et al., 2011). Fig. 1. View largeDownload slide Generation of S-nitrosothiols. Nitric oxide (NO) can lose or gain an electron to form an oxidized nitrosonium cation (NO+) or a reduced nitroxyl anion (NO–), respectively. NO can also react with superoxide (O2–) to form peroxynitrite (ONOO–). Further, NO can react with oxygen (O2) to form higher order nitrogen oxides (NOx), such as dinitrogen trioxide (N2O3). NO radicals can also react directly with transition metals in metal-containing proteins to form metal–nitrosyl complexes (M-NOs). Each of the described molecules can react with either thiol groups, typically as a thiolate (RS–) or thiyl (RS·) associated with either proteins or glutathione to generate an S-nitrosothiol (SNO). Fig. 1. View largeDownload slide Generation of S-nitrosothiols. Nitric oxide (NO) can lose or gain an electron to form an oxidized nitrosonium cation (NO+) or a reduced nitroxyl anion (NO–), respectively. NO can also react with superoxide (O2–) to form peroxynitrite (ONOO–). Further, NO can react with oxygen (O2) to form higher order nitrogen oxides (NOx), such as dinitrogen trioxide (N2O3). NO radicals can also react directly with transition metals in metal-containing proteins to form metal–nitrosyl complexes (M-NOs). Each of the described molecules can react with either thiol groups, typically as a thiolate (RS–) or thiyl (RS·) associated with either proteins or glutathione to generate an S-nitrosothiol (SNO). A redox code of cysteine residue modifications? Rare, highly reactive protein cysteine (Cys) thiols (SHs) embedded within protein structures are key atomic targets for RNS-dependent signalling (Boehning and Snyder, 2003; Poole et al., 2004). These Cys residues are solvent exposed with a low pKa sulphydryl group, which facilitates their efficient oxidation by RNS (Meng et al., 2002; Yu et al., 2014). Redox-based Cys modification is influenced by the ionization state of the reactive Cys: thiolate anions (-S–) have a greater tendency to donate electrons and become polarized relative to their corresponding protonated counterparts. Cys thiols undergo a redox continuum of modifications generating an array of possible outcomes, including S-nitrosylation, the addition of an NO moiety to form an SNO, S-glutathionylation (S-SG), S-sulphenation (S-OH), S-thiolation (S-S, i.e. disulphide formation), and S-sulphination (S-O2H) (Fig. 2) (Spadaro et al., 2010; Spoel and Loake, 2011, Klomsiri et al., 2011). A key feature of specificity in redox signalling is the translation of different redox-based modifications into discrete protein functions to generate different cellular outcomes. This has been proposed to provide the basis of a redox code that translates protein structure into function. For example, a single Cys residue in OxyR, a transcriptional activator of Escherichia coli, is modified to generate four stable, redox-related modifications (S-H, S-OH, S-SG, or SNO) (Kim et al., 2002). Each modification resulted in a differential conformational change in OxyR structure, conveying unique DNA binding affinities, promoter activities, and co-operative properties. These findings imply that fine-grained transcriptional regulation might therefore be controlled by exploiting distinct Cys modifications. Fig. 2. View largeDownload slide Schematic overview of redox-based cysteine (Cys) modifications. Cys thiol (SH), S-nitrosothiol (SNO), sulphenic acid (SOH), disulphide (S-S), S-glutathionylation (SSG), sulphinic acid (SO2H), and sulphonic acid formation (SO3H). Molecules in this sequence are formed following increasing oxidation. The formation of all these functional groups is thought to be reversible, except for sulphonic acid formation, which is irreversible. (Modified from Spadaro et al., 2010. The redox switch: dynamic regulation of protein function by cysteine modifications. Physiologia Plantarum 138, 360–371, with permission). Fig. 2. View largeDownload slide Schematic overview of redox-based cysteine (Cys) modifications. Cys thiol (SH), S-nitrosothiol (SNO), sulphenic acid (SOH), disulphide (S-S), S-glutathionylation (SSG), sulphinic acid (SO2H), and sulphonic acid formation (SO3H). Molecules in this sequence are formed following increasing oxidation. The formation of all these functional groups is thought to be reversible, except for sulphonic acid formation, which is irreversible. (Modified from Spadaro et al., 2010. The redox switch: dynamic regulation of protein function by cysteine modifications. Physiologia Plantarum 138, 360–371, with permission). Local chemical environments conducive to redox-based PTMs S-Nitrosylation has emerged as a prototypic redox-based, PTM across phylogenetic kingdoms (Foster et al., 2009; Yun et al., 2011). In plants, protein–SNO formation has been shown to help shape growth, development, immunity, and abiotic interactions (Lee et al., 2008; Kwon et al., 2012). Several studies have been undertaken to uncover a potentially conserved sequence motif for S-nitrosylation of protein targets, utilizing high-throughput proteomic approaches in conjunction with bioinformatic interrogation. Various factors have been reported to contribute to the selectivity of Cys residues for protein S-nitrosylation, including solvent accessibility, the presence of acidic–basic groups in their proximity (within 6–8 Å), the incidence of adjacent hydrophobic amino acids, increased nucleophilicity, and the presence of local α-helical domains (Chen et al., 2010; Doulias et al., 2010; Marino and Gladyshev, 2010; Seth and Stamler, 2011). The motif I/L-X-C-X2-D/E carries a Cys residue with a high nucleophilicity, surrounded by a hydrophobic residue, I/L, and an acidic residue, D/E. This motif therefore contains most, if not all, of the characteristics thought to be necessary for S-nitrosylation of a target Cys. In this context, this motif is associated with iNOS-dependent S-nitrosylation of S100A8/A9 (calprotectin/calcium-binding protein complex) and was present in 19 additional candidate SNO–proteins (Jia et al., 2012). However, analysis of 445 human proteins with 810 SNO–peptides revealed that only 15 peptides present in 15 proteins contain the I/L-X-C-X2-D/E motif (Jia et al., 2012; Lee et al., 2012). Hence, results based on large-scale data sets have cast doubt on the reliability of a specific motif based on a primary amino acid sequence underpinning S-nitrosylation. Interestingly, a computational tool, GPS-SNO 1.0, has been developed for the prediction of S-nitrosylation sites. This can provide a convenient and rapid strategy to generate useful information for subsequent experimental verification (Xue et al., 2010). The developers utilized 504 experimentally verified S-nitrosylation sites in 327 unique proteins. The developed GPS-SNO 1.0 tool predicted at least one potential S-nitrosylation site in 359 out of 485 S-nitrosylated proteins, with a previously unknown S-nitrosylation site. Further, a set of 46 proteins with 53 S-nitrosylation sites experimentally identified in Arabidopsis, were interrogated by GPS-SNO 1.0. A total of 60 S-nitrosylation sites were identified amongst these proteins, but only 11 of these sites had been confirmed experimentally (Chaki et al., 2014). Thus, simple motif recognition/computational tools may also be of only limited value as they only reveal information associated with common features responsible for specificity and patterns implemented in predictive algorithms. Consequently, these bioinformatics approaches have largely failed to identify a specific consensus sequence that directs site-selective S-nitrosylation. As the overall tertiary structure of proteins largely defines the activity of a given cognate amino acid residue, it may be naive to attempt the identification of target Cys residues based on a motif with only primary sequence information. In addition to protein three-dimensional structure, future bioinformatic tools should also consider the given redox cue, for example NO, GSNO, or even oxidized lipids (Mata-Pérez et al., 2017), the likely concentrations of the given RNS, and the physiological conditions under which the given PTM occurs, because all these variables can also influence Cys target specificity. Proximity-based RNS production and associated Cys modification Another potential mechanism thought to be integral for conveying specificity in redox signalling is the proximity of the target Cys to the source of either RNS. Thus, the subcellular organization of the source of redox-active molecules within cells alongside the spatial and temporal existence of given protein targets may be of prime importance to trigger a particular redox signalling pathway (Derakhshan et al., 2007). For example, in mammalian cells, eNOS and nNOS co-exist in cardiac myocytes but are located within distinct subcellular compartments: while eNOS is located in the plasma membrane, nNOS is found in the sarcoplasmic reticulum. Spatial confinement of these NO sources enables this redox-active molecule to trigger discriminate signalling pathways which determine distinct downstream effects. Thus, NO generated by eNOS limits myocardial contractility, whereas NO generated by nNOS increases contractility (Barouch et al., 2002). An alternative biological strategy for achieving specificity in redox signalling is through proximity of both the given RNS source and the cognate target Cys thiol. For example, eNOS binds to heat shock protein 90 (Hsp90) directly, which results in S-nitrosylation of Hsp90 at Cys597, affecting its ATPase activity (Martínez-Ruiz et al., 2005). Moreover, cycloxygenase-2 (COX-2) becomes activated following direct binding of iNOS, which results in the selective S-nitrosylation of COX-2 on Cys526 (Kim et al., 2005). A variation on this theme is the deployment of a scaffold protein to link the redox molecule generator with the target protein. In this context, NMDAR (N-methyl-d-aspartate receptor) binds indirectly to nNOS via the scaffold-protein PSD95 (post-synaptic density protein). The NMDAR–PSD95–nNOS ternary complex is essential for NMDAR activation, which ultimately also increases nNOS activity (Brenman et al., 1996; Christopherson et al., 1999). Interestingly, another adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), competes with PSD95 for binding with nNOS, positioning NO generation to drive S-nitrosylation of Dexras (Ras superfamily gene induced by dexamethasone) at Cys11, which facilitates the exchange of bound GDP for GTP (Jaffrey et al., 1998, 2002). While now well established in mammalian systems, this proximity-based production of redox cues has not to date been demonstrated in plants. At least in the case of NO and other RNS, this has been impeded by the continuing controversy regarding the source of NO production in plants(Corpas et al., 2006; Jeandroz et al., 2016; Mata-Pérez et al., 2017; Zemojtel et al., 2006). Specific protein trans-nitrosylation Interestingly, the accumulating evidence from mammalian systems suggests that some proteins might function to transfer their NO group linked to the protein Cys sulphur to specific protein substrates, effectively functioning as nitrosylase ‘enzymes’, akin to more established enzymes in PTMs such as protein kinases (Hess et al., 2005). To date, only a small number of S-nitrosylases have been reported in animals, but this list is steadily growing (Nakamura and Lipton, 2013). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a classic glycolytic enzyme, has been proposed to function as a nitrosylase. Upon apoptotic stimulation, mammalian GAPDH is modified at Cys150 by NO, generated by NOS. Subsequently, SNO–GAPDH is thought to bind to Siah1, an E3 ubiquitin ligase, which is then translocated to the nucleus (Hara et al., 2005). SNO–GAPDH binding to Siah1 stabilizes this protein against turnover, and in the nucleus Siah1 degrades its target substrates and also enhances p300/CBP-associated acetylation of nuclear proteins, including p53, which cause cell death (Sen et al., 2008). The SNO–GAPDH–Siah1 complex also mediates nuclear translocation of mutant Huntingtin (mHtt) protein which is associated with Huntington’s disease (Bae et al., 2006). Further, after nuclear translocation, SNO–GAPDH also transfers its NO group at Cys150 to specific Cys residues within targeted protein thiols including SIRT1, a histone deacetylase-2 (HDAC2), and DNA-activated protein kinase (DNA-PK) (Kornberg et al., 2011). Hence, SNO–GAPDH essentially also functions as an S-nitrosylase, transferring its NO to Cys thiols embedded in target proteins. However, under low levels of NO availability, GOSPEL (GAPDH’s competitor Of Siah Protein Enhances Life) is S-nitrosylated and competes with Siah1 for GAPDH binding, preventing the formation of a GAPDG–Siah1 complex (Sen et al., 2009). The S-nitrosylation of haemoglobin has been proposed to regulate human blood pressure (Jia et al., 1996). The central iron molecule of the haem group in haemoglobin can bind oxygen, facilitating the transfer of this molecule from the lungs to the rest of the body. Binding of NO at this haem group rapidly facilitates auto-S-nitrosylation of haemoglobin at its highly conserved Cysâ93 residue, by the transfer of the bound NO moiety, freeing the haem iron to bind to oxygen. In human hypoxia, haemoglobin–SNO has been proposed to function as a nitrosylase, where the NO group is transferred from SNO–Cysâ93 to the anion exchange protein (AE1/band3) in human red blood cells, following binding to the red blood cell membrane. Subsequently, SNO–AE1 is thought to release NO which can subsequently diffuse into smooth muscle cells, promoting the relaxation of blood vessels (Pawloski et al., 2001). In this scenario, allosteric regulation of haemoglobin is employed to propagate a vasodilatory signal via the trans-nitrosylative transfer of NO. Thus, protein trans-nitrosylation constitutes an additional strategy to convey specificity to NO-related redox signalling in biological systems. Removal of redox-based PTMs An important feature of the addition of PTMs to their protein targets associated with cellular signalling is their selective reversal to disengage the given signal networks. It is highly likely that specificity in redox signalling is achieved largely by rates of reversal of Cys modifications, rather than by their formation, as rapidly degraded redox modifications would probably have far less impact than more persistent ones (Derakhshan et al., 2007). In this context, it has been demonstrated that different protein–SNOs can have widely diverse biological lifetimes (Seth and Stamler, 2015).While a proportion of this can be attributed to the innate chemical stability of a given protein–SNO, this property is also influenced by potential non-enzymatic breakdown, for example by either of the key cellular antioxidant molecules, ascorbate or glutathione (Masella et al., 2005; Feechan et al., 2005; Benhar et al., 2008; Kneeshaw et al., 2014). In the green alga Chlamydomonas reinhardtii, haemoglobin affects the nitrogen assimilation pathway by simultaneously modulating NO levels and NR activity (Sanz-Luque et al., 2015). Interestingly, non-symbiotic plant haemoglobins (phytoglobins) have also been proposed to function as NO scavengers in plants, with their activity possibly impacting plant developmental programmes (Hebelstrup et al., 2013). However, these proteins are not thought to control the consequences of the pathogen-triggered, nitrosative burst and associated host cell death during plant immunity (Perazzolli et al., 2004). Ascorbic acid can reduce phytoglobins in support of NO scavenging, generating nitrate and monodehydroascorbate. The monodehydroascorbate is recycled back to ascorbic acid by monodehydroascorbate reductase using NADH. Phytoglobins scavenge NO by forming S-nitrosophytoglobin; however, how this is recycled back to the reduced phytoglobin remains to be determined, but recent evidence suggests that this does not involve ascorbate in planta (Wang and Hargrove, 2013). Significantly, in more established signal transduction mechanisms, such as phosphorylation, signalling and its associated specificity is the result of a delicate poise between the activities of kinase and phosphatase. The conceptual equivalent of protein phosphatases associated with redox signalling has recently started to come into sharper focus. Indirect and selective protein denitrosylation It is now well established that potential non-enzymatic mechanisms make a key contribution to protein–SNO homeostasis (Liu et al., 2001; Feechan et al., 2005). The antioxidant tripeptide glutathione (GSH) has been shown to access sites of S-nitrosylation in protein–SNOs, reducing the SNO group back to a Cys thiol and forming S-nitrosoglutathione (GSNO); GSH is S-nitrosylated at its Cys residue as a consequence (Corrales et al., 1999; Romero and Bizzozero, 2009). Arabidopsis GAPDH is S-nitrosylated at Cys149 and is denitrosylated selectively by GSH (Zaffagnini et al., 2013). However, in animals, SNO–GAPDH binding to Siah1, an E3 ubiquitin ligase, forms a complex which changes the conformation of SNO–GAPDH, precluding GAPDH–SNO-Cys149 access to GSH, abolishing GSH-mediated denitrosylation (Paige et al., 2008). Importantly, GSNO can function as a natural NO donor driving S-nitrosylation of reactive Cys thiols by trans-nitrosylation, resulting in specific S-nitrosylation of the target protein (Hess et al., 2005). GSNO is relatively stable and can therefore function as a cellular reservoir of NO bioactivity. Significantly, GSNO can be turned over by GSNO reductase (Feechan et al., 2005; Lee et al., 2008; Chen et al., 2009). Thus, S-nitrosylated proteins may be in dynamic equilibrium with their denitrosylated counterparts controlled by the activity of GSNOR. A growing body of evidence has demonstrated that GSNOR-mediated denitrosylation has essential roles in both plant immunity and development. Loss- or gain-of-function mutants in GSNOR1 in Arabidopsis result in either reduced or increased denitrosylation, respectively. While reduced denitrosylation promotes enhanced disease susceptibility, increased denitrosylation leads to enhanced disease resistance (Feechan et al., 2005; Tada et al., 2008). Further, the control of denitrosylation has also been shown to regulate pathogen-triggered cell death by controlling the extent of S-nitrosylation at Cys890 of the respiratory burst oxidase homologue D (RBOHD), and this mechanism may be evolutionarily conserved across kingdoms (Yun et al., 2011). In a similar fashion, dysregulation of denitrosylation also appears to impact a number of key plant developmental programmes (Lea et al., 2004; Lee et al., 2008; Kwon et al., 2012). How is the specificity in GSNOR function established? The emerging evidence suggests that the tertiary protein structure restricts the ability of GSH to function as a non-enzymatic denitrosylase to a subset of reactive protein Cys thiols (Foster et al., 2009; Yun et al., 2016). Thus, GSH might not be able to denitrosylate all possible cellular protein–SNOs, forming the free protein thiol and GSNO in the process. Consequently, only a subset of total cellular S-nitrosylated proteins will be under the indirect control of the protein denitrosylase, GSNOR. Direct and selective protein denitrosylation Thioredoxins (Trxs) are present in all living organisms, and their activity can be recycled by NADPH-dependent thioredoxin reductase (TrxR). Trx/TrxR-mediated denitrosylation has emerged as an important mechanism to control redox regulation. In animals, Trx-mediated denitrosylation of caspase-3 results in the activation of this enzyme (Benhar et al., 2008). Other targets for Trx-mediated selective denitrosylation in animals are caspase-9, protein tyrosine phosphatase 1B, and GAPDH (Benhar et al., 2008). In plant immunity, Trxh5 has been proposed to denitrosylate selectively the transcriptional co-activator, NPR1 (Kneeshaw et al., 2014). NPR1 is S-nitrosylated in response to increasing GSNO levels, leading to NPR1 oligomerization, thus sequestering this co-activator in the cytosol, preventing its nuclear translocation and the subsequent activation of pathogenesis related (PR) gene expression (Tada et al., 2008; Yu et al., 2014). Trxh5 and TrxR have been proposed to denitrosylate NPR1 directly, leading to the release of NPR1 monomers, their translocation to the nucleus, and the subsequent engagement of PR gene transcription (Kneeshaw et al., 2014). Thus, selective denitrosylation of NPR1 is an important feature of the plant immune response. However, it is noteworthy that NPR1 function is also impacted by GSNOR activity (Feechan et al., 2005; Tada et al., 2008), suggesting that the S-nitrosylation status of this transcriptional co-activator is regulated directly by Trxh5 but indirectly by GSNOR. The emerging biochemical and genetic evidence suggests that Trxh5 and TrxR can denitrosylate a subset of the plant SNO proteome directly and selectively in vivo (Kneeshaw et al., 2014). Thus, the regulation of denitrosylation at other key protein Cys thiols is also likely to be under the control of Trxh5 and TrxR. Further, Trxs are comprised of a large gene family in Arabidopsis; therefore, it is possible that other Trx family members might also function in combination with TrxR as direct and selective denitros ylases of a range of possible substrates. Interestingly, in mammals, two Trx-mimetic (TXM) peptides derived from the Trx conserved active site (CXXC) have been documented to act as denitrosylating catalysts (Kronenfeld et al., 2015). TXMs with TrxR can effectively reduce both low molecular weight SNOs and protein–SNO. GSNO is reduced to GSH through trans-nitrosylation, resulting in S-nitrosylated TXMs, which are cyclized by TrxR or other endogenous thiol reductases. TXMs have the ability to denitrosylate multiple classes of SNO proteins. TXM also protects TrxR from SNO-mediated loss of activity, which is important to conserve its function in redox signalling. Collectively, the current state-of-the-art suggests that Trx and TrxR can function as direct and selective denitrosylases to regulate a subset of S-nitrosylated proteins across kingdoms. Discrete activities of given RNS Recent studies have shown that NO and SNO might have discrete or overlapping protein targets in relation to redox signalling (Yun et al., 2016), providing an additional layer of RNS signalling specificity. NO has been proposed to have a more extensive range of biological activity relative to GSNO (Fig. 3). In addition to protein S-nitrosylation, NO is thought to be largely responsible for metal nitrosylation, the most rapid known reaction of NO with proteins. NO forms metal–nitrosyl complexes (M-NOs), with metals embedded within proteins, for instance zinc (Zn2+), iron (Fe2+ or Fe3+), or copper (Cu2+) (Leitner et al., 2009). Mammalian soluble guanylate cyclase is a prototypic example of a protein that is regulated by this type of PTM (Derbyshire and Marletta, 2012). NO can also nitrosylate amino acid side chains to form so-called N-NO (Stamler et al., 1992). However, NO cannot directly oxidize amino acid side chains at any significant biological rate. Hence, NO mediates N-NO via auto-oxidation to nitrogen dioxide, dinitrogen trioxide, or peroxynitrite. In contrast, GSNO is thought principally to mediate protein S-nitrosylation (Foster et al., 2009; Yun et al., 2016). Fig. 3. View largeDownload slide Schematic model showing discrete and overlapping activities of NO and GSNO associated with cellular signalling. NO can target metals embedded in proteins (1), amino acid side chains (2), GSH to form GSNO, which is reversible (3), and proteins to form S-nitrosothiols (4). NO can also S-nitrosylate GAPDH (5). NO can be metabolized by microbial FHb into NO3/N2O (6). GSNO can trans-nitrosylate selected protein targets (7). GSH can reverse S-nitrosylation at a subset of proteins, generating the denitrosylated protein and GSNO (8). GAPDH–SNO can function as a nitrosylase (in mammals) to S-nitrosylate target proteins (9). GSNO can S-nitrosylate GAPDH, forming GAPDH–SNO and GSH (10). GSH can denitrosylate GAPDH–SNO in Arabidopsis to generate GAPDH and GSNO (11). Trx (Trxh5 in Arabidopsis) can directly and selectively denitrosylate a subset of protein–SNO (12). Fig. 3. View largeDownload slide Schematic model showing discrete and overlapping activities of NO and GSNO associated with cellular signalling. NO can target metals embedded in proteins (1), amino acid side chains (2), GSH to form GSNO, which is reversible (3), and proteins to form S-nitrosothiols (4). NO can also S-nitrosylate GAPDH (5). NO can be metabolized by microbial FHb into NO3/N2O (6). GSNO can trans-nitrosylate selected protein targets (7). GSH can reverse S-nitrosylation at a subset of proteins, generating the denitrosylated protein and GSNO (8). GAPDH–SNO can function as a nitrosylase (in mammals) to S-nitrosylate target proteins (9). GSNO can S-nitrosylate GAPDH, forming GAPDH–SNO and GSH (10). GSH can denitrosylate GAPDH–SNO in Arabidopsis to generate GAPDH and GSNO (11). Trx (Trxh5 in Arabidopsis) can directly and selectively denitrosylate a subset of protein–SNO (12). A microbial flavohaemoglobin (FHb) has been shown to turn over NO in a number of bacteria and may function as a virulence factor to counter antimicrobial NO production by potential mammalian hosts produced via iNOS (Poole et al., 1996; Poole and Hughes, 2000). Loss-of-function mutations in a FHb gene present in the human pathogens Cryptococcus neoformans or Salmonella typhimurium reduced their virulence in mice. However, virulence was restored in mice lacking an iNOS (Bang et al., 2006; de Jesús-Berrı́os et al., 2003). Informatively, double mutants lacking both FHb and GSNOR function further reduced the virulence of C. neoformans, consistent with an additive antimicrobial function of NO and GSNO (Liu et al., 2004), suggesting that these RNS might have separable functions. Thus, NO and GSNO might target a different spectrum of microbial protein Cys thiols. Similarly, in Saccharomyces cerevisiae, an FHb knock-out mutant strain was deficient in NO turnover as compared with the wild type or GSNOR knock-out strains. Further, exogenous application of NO retarded the growth of the fhb mutant but had no impact on either the wild type or a gsnor mutant strain. However, exogenous application of GSNO had a greater impact on the fhb gsnor double mutant relative to either of the single mutant strains. Thus, GSNOR activity conveys greater protection against GSNO-induced nitrosative stress, rather than nitrosative stress established by NO (Foster et al., 2009). This is consistent with the proposal that NO and GSNO may have distinct and overlapping activities. Genetic evidence for this dichotomy between the biological activities of NO and GSNO has also recently been established in plants (Yun et al., 2016). The Arabidopsis mutants, no-over producer 1-1 (nox1-1) and gsnor1-3, exhibit increased levels of either NO or SNO in vivo, respectively (He et al., 2004; Feechan et al., 2005; Yun et al., 2016). Informatively, the double mutant, gsnor1-3 nox1-1, appears to be genetically additive, as it shows greater susceptibility to pathogens than either of the single mutants alone. Further, GSNOR1 overexpression in gsnor1 plants restored wild-type levels of immunity; however, this was not the case in nox1-1 plants. This may reflect the notion that NO can drive both M-NO and N-NO formation in addition to protein S-nitrosylation. In contrast, GSNO predominantly favours the generation of protein–SNOs. Interestingly, genetic analysis suggests that high in vivo SNO concentrations can facilitate pathogen-triggered hypersensitive response (HR) cell death independently of ROS synthesized by the NADPH oxidases, AtRBOHD and AtRBOHF, responsible for the extracellular oxidative burst (Yun et al., 2011). While NO was previously reported to require superoxide (O2−) in order to trigger the HR (Delledonne et al., 2001) (see next section). Collectively, these data suggest that NO and SNO might have discrete or overlapping functions in the context of redox signalling during the establishment of plant immunity (Yun et al., 2016) and perhaps other plant molecular systems, providing addition scope for specificity in redox regulation of cellular function. Specificity conveyed by interactions between RNS and ROS Chemical interactions between RNS and ROS are a prominent feature of redox signalling networks. These interactions have been proposed to modulate indirectly the activity of a series of proteins (Hausladen and Fridovich, 1994; Romero et al., 2003; Wang et al., 2013) and, by extension, this presents another way of achieving specificity in redox signalling. For example, NO is unable to influence the activity of the tricarboxylic acid cycle enzyme, aconitase, in Escherichia coli. However, the interaction of NO with superoxide (O2−) to form peroxynitrite (ONOO−) generates a powerful inhibitor of aconitase activity (Hausladen and Fridovich, 1994). In plant biology, interaction between NO and H2O2 has been reported to be a key factor associated with the development of the HR, leading to the programmed execution of pathogen-challenged plant cells (Mur et al., 2008). NO production following pathogen recognition has been posited to be insufficient to activate HR cell death. Rather, the HR has been proposed to be triggered only by a balanced production of NO and ROS (Delledonne et al., 2001; Lin et al., 2012). Thus, HR cell death is activated after interaction of NO not with superoxide (O2−), presumably produced by RBOHD and RBOHF (Torres et al., 2002), but with H2O2 generated from O2− by superoxide dismutase (Delledonne et al., 2001). Increasing the level of O2− reduced NO-mediated HR cell death, by converting more NO into ONOO−, which was proposed to have low levels of toxicity towards plant cells. However, this is counterintuitive, because it is well established that ONOO− exhibits an extremely high level of cellular toxicity (Pacher and Szabo, 2008). Perhaps plants can more effectively turn over ONOO−. In a similar fashion, both NO and GSNO have been proposed to be important mediators in the process of H2O2-induced leaf cell death in rice (Lin et al., 2012) and, in response to the fungal elicitor, cryptogein, the production of NO and H2O2 in tobacco appears to be reciprocally regulated (Kulik et al., 2015). Also, S-nitrosylation of RBOHD at Cys890 curbs ROS production limiting pathogen-triggered cell death development (Yun et al., 2011). An interaction between NO and H2O2 has also been reported to be required for stimulation of stomatal closure in Arabidopsis in response to UV-B exposure. An increase in H2O2 scavenging activity or an inhibition of H2O2 synthesis prevented NO generation and subsequent stomatal closure (He et al., 2013). In aggregate, this work demonstrates that either direct or indirect interactions between distinct redox molecules can shape the specificity of signalling outputs. Conclusions and future prospects The prevalence of redox signalling in the biology of plants and other organisms is being increasingly appreciated. However, there are many potential molecular switches, including but not limited to highly reactive Cys thiols and chemically co-ordinated metal cofactors. So how is specificity in redox regulation achieved? As discussed above, the emerging model suggests that redox specificity is mediated via a multilayered molecular framework. This might encompass the production of the signalling redox molecule(s) in the vicinity of the protein target. Tertiary protein structures subsequently provide a chemical environment compatible with oxidation/reduction of the target redox switch. Superimposed upon this molecular landscape, trans-nitrosylation, by a nascent but growing repertoire of nitrosylases, can function as an additional mechanism to deliver and then transfer an NO moiety to a target protein thiol. Reversal of redox modifications, rather than their formation, is also a major contributor to signalling specificity. In this context, selective, indirect protein denitrosylation conveyed by GSNOR-dependent GSNO turnover is a prominent feature. This mechanism is augmented by the selective and direct denitrosylation mediated by Trxh5 and TrxR. Signalling specificity is also mediated by the unique chemistries of individual RNS which is overlaid on the structural constraints imposed by tertiary protein structure gating access to given redox switches. Finally, the direct or indirect interactions between RNS and ROS can also indirectly establish signalling specificity through shaping the formation of appropriate redox cues. The emerging evidence suggests that redox signalling pervades almost all aspects of plant growth, development, immunity, and abiotic environmental interactions (Besson-Bard et al., 2008; Yu et al., 2012, 2014). 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