Nitric oxide buffering and conditional nitric oxide release in stress response

Nitric oxide buffering and conditional nitric oxide release in stress response Abstract Nitric oxide (NO) has emerged as an essential biological messenger in plant biology that usually transmits its bioactivity by post-translational modifications such as S-nitrosylation, the reversible addition of an NO group to a protein cysteine residue leading to S-nitrosothiols (SNOs). In recent years, SNOs have risen as key signalling molecules mainly involved in plant response to stress. Chief among SNOs is S-nitrosoglutathione (GSNO), generated by S-nitrosylation of the key antioxidant glutathione (GSH). GSNO is considered the major NO reservoir and a phloem mobile signal that confers to NO the capacity to be a long-distance signalling molecule. GSNO is able to regulate protein function and gene expression, resulting in a key role for GSNO in fundamental processes in plants, such as development and response to a wide range of environmental stresses. In addition, GSNO is also able to regulate the total SNO pool and, consequently, it could be considered the storage of NO in cells that may control NO signalling under basal and stress-related responses. Thus, GSNO function could be crucial during plant response to environmental stresses. Besides the importance of GSNO in plant biology, its mode of action has not been widely discussed in the literature. In this review, we will first discuss the GSNO turnover in cells and secondly the role of GSNO as a mediator of physiological and stress-related processes in plants, highlighting those aspects for which there is still some controversy. nitric oxide, nitric oxide signalling, plant stress, S-nitrosoglutathione Introduction As sessile organisms, plants cannot move to find the best conditions for their growth and development, and consequently are continuously exposed to adverse environmental changes. To face these environmental attacks, they have to perceive these alterations and initiate complex signalling mechanisms leading to an effective response to the threats detected. Redox changes are usually a cue to trigger plant responses to these environmental fluctuations (Begara-Morales et al., 2016a). In this context, different signalling molecules have emerged as key regulators of plant biology, chief among these being reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydrogen peroxide (H2O2) and nitric oxide (NO), respectively. Although the connection between ROS and RNS signalling pathways is becoming well established (Yun et al., 2011; Groß et al., 2013; Lindermayr and Durner, 2015; Arora et al., 2016; Begara-Morales et al., 2016b; Farnese et al., 2016), this review will focus only on the role of NO and especially S-nitrosoglutathione (GSNO; a central NO-derived molecule) in plant response to stress. However, excellent reviews on ROS signalling pathways are available (Tripathy and Oelmüller, 2012; Choudhury et al., 2013; Baxter et al., 2014). NO is a small, short-lived biological messenger acting as a key regulator of a wide range of processes in animals and plants (Mur et al., 2013). For instance, in plants, NO has been shown to be involved in seed germination (Albertos et al., 2015; Krasuska et al., 2015; Wang et al., 2015b), flowering (He et al., 2004), cotyledon senescence (Du et al., 2014), stomatal closure (Wang et al., 2015a), and plant response to stress (reviewed by Yu et al., 2014; Fancy et al., 2016). The radical nature of NO and its ability to cross lipid membranes promote its interaction with different macromolecules. Therefore, NO bioactivity is transmitted mainly by post-translational modifications (NO-PTMs) such as tyrosine nitration and S-nitrosylation, which can regulate the function of the target proteins (Astier and Lindermayr, 2012; Corpas et al., 2015). Tyrosine nitration consists of the binding of an NO2 moiety to the aromatic ring of a tyrosine residue, leading to 3-nitrotyrosine (Gow et al., 2004; Radi, 2004). This NO-PTM is usually mediated by peroxynitrite (ONOO–) (Szabó et al., 2007), suggesting that this modification takes place during severe oxidative stress conditions (Mur et al., 2013; Zaffagnini et al., 2016). To function effectively as a molecular cue, this modification should be specific and transient. In this regard, a potential denitrase activity has been proposed in mammalian cells (Görg et al., 2007; Deeb et al., 2013). In this sense, protein extracts from rat spleen and cerebral cortex were able to decrease the in vitro nitration of glutamine synthetase (Görg et al., 2007). Similarly, a potential denitration of cyclooxygenase 1 was reported in different murine cells (Deeb et al., 2013). However, although in these studies a decrease in nitrotyrosine content is described after incubation with protein extracts, there is no clear evidence of the existence of a denitrase protein, and no information is available in plants (Kolbert et al., 2017). Therefore, this NO-PTM has previously been considered to be an irreversible modification involved in nitrosative stress (Corpas et al., 2007). Consequently, major efforts have been focused on the study of the other main NO-PTM, S-nitrosylation, which consists of the reversible attachment of NO to a cysteine residue, leading to S-nitrosothiols (SNOs) (Hess et al., 2005; Astier et al., 2011). In recent years, the metabolism of SNOs has gained special relevance since numerous pieces of evidence have shown that SNOs are integral to plant biology. In this regard, SNOs are key mediators in the plant response to stress (Yu et al., 2014; Fancy et al., 2016) and, interestingly, this action can be independent of NO production (Feechan et al., 2005; Chaki et al., 2011a, b; Yun et al., 2016). Additionally, the half-life of NO in vivo is very short (~5–15 s; Lancaster, 1997), whereas SNOs are generally more stable in solution (Williams, 1999; Hogg, 2002). Therefore, SNOs may prolong temporally while spatially extending the in vivo actions of locally produced NO (Hogg, 2002). One of the main signalling molecules among SNOs is GSNO, the major low molecular weight (LMW) SNO that is considered as a stable store of NO with a lifetime of hours in aqueous solution (Floryszak-Wieczorek et al., 2006) and an essential molecule involved in NO-dependent signalling (Leitner et al., 2009; Broniowska et al., 2013; Mur et al., 2013). The presence of endogenous GSNO in vascular tissues under basal conditions and its regulation in response to stress (Barroso et al., 2006; Valderrama et al., 2007; Chaki et al., 2011a, b) suggest a function as a signal molecule because it would be ready to travel throughout the plant in response to external signals. In line with this assumption, phloem can propagate redox messengers, including different RNS, during plant defence (Gaupels et al., 2017), and GSNO is involved as a key player in the systemic response to wounding stress (Espunya et al., 2012). Thus, GSNO could act as an NO carrier throughout the plant, thereby conferring to NO with the capacity to be a long-distance signalling molecule. Consequently, GSNO could act as an NO buffer from which NO is released to orchestrate the plant’s response to stress; the NO liberation rate from GSNO has been reported to be 1.22 µM min–1 (Ederli et al., 2009). Thus, GSNO can carry out transnitrosylation reactions, transferring its NO group to a protein cysteine thiol (Hess et al., 2005; Broniowska et al., 2013), thereby regulating the function of the target proteins (Astier et al., 2011). In addition, the enzyme S-nitrosoglutathione reductase (GSNOR) can break down GSNO (for further details, see later) and indirectly controls the overall levels of SNOs in cells (Liu et al., 2001; Feechan et al., 2005), suggesting that GSNO could be crucial in regulating the pool of total SNOs. In this context, GSNO may be considered to have a central role within NO-dependent signal transduction, acting as a pivotal modulator of metabolism of NO/SNOs in plant response to stress. In this regard, different stress conditions can alter GSNO levels, thus affecting NO-dependent signalling responses. In this review, given this importance of GSNO, we will describe current knowledge of the GSNO turnover mechanisms and especially the role of GSNO as a modulator of NO-dependent signalling in physiological and stress conditions in plants. GSNO turnover mechanisms Total SNO levels have been reported in different plant species (Table 1). As shown in Table 1, there is a great difference in the values of published SNOs according to the plant species/tissue analysed and especially to the detection method used. In this context, the chemiluminescence assay using a nitric oxide autoanalyser (NOA) is considered a much more sensitive and reliable method than Saville–Griess to obtain results of NO and SNO levels. Anyway, it is advisable to use at least two different detection methods to verify NO/SNO levels in plant biology (Yamasaki et al., 2016). In contrast, there are only few data on the GSNO content in plants (Table 2). GSNO is considered the most abundant LMW SNO. Thus, the first attempt at GSNO quantification in plants was carried out in a sample fraction that was previously passed through a 5 kDa cut-off membrane to obtain LMW SNOs (Feechan et al., 2005, Rustérucci et al., 2007). Using this approach, the LMW SNO content in Arabidopsis leaves was reported to be ~5.5 pmol mg–1 protein (Table 2; Feechan et al., 2005). It is assumed thus that almost all LMW SNOs are GSNO, but other LMW SNOs cannot really be ruled out. For this reason, a method to analyse GSNO, glutathione (GSH), and GSSG under acidic extraction by LC-ES/MS was proposed in plants (Airaki et al., 2011). However, the artefactual formation of GSNO from GSH is possible under acidic conditions in the presence of nitrite. Thus, acidification of the sample should be avoided when both nitrite and GSH are present (Broniowska et al., 2013). This limitation can be circumvented by pre-treatment with ammonium sulphamate or sulphanilamide to remove nitrite and N-ethylmaleimide (NEM) to block thiol groups on GSH. It would be necessary to confirm the observed GSNO changes under acidic extractions through a clear quantitative assay that uses these molecules. Table 1. Total S-nitrosothiol (SNO) content in plant tissues Plant species Organs [SNO] basal conditions (pmol mg–1protein) Detection methods Stress/effect on SNO levels References Arabidopsis thaliana Leaves 50 NOA Pseudomonas syringae pv tomato (avrB)/+ Feechan et al. (2005) Arabidopsis thaliana Leaves 350 NOA Heat stress/– Lee et al. (2008) Arabidopsis thaliana Leaves 15 NOA Paraquat/+ Kovacs et al. (2016) Arabidopsis thaliana Leaves 150 Saville–Griess Pseudomonas syringae pv maculicola (avrRpm1)/+ Rustérucci et al. (2007) Arabidopsis thaliana Leaves 250 Saville–Griess Wounding/+ Espunya et al. (2012) Arabidopsis thaliana Roots 3000 Saville–Griess ND Correa-Aragunde et al. (2015) Arabidopsis thaliana Seedlings 25000 Saville-Griess Cold/+ Puyaubert et al. (2014) Arabidopsis thaliana Cell cultures 22.6 Saville–Griess Nutritional stress/– Frungillo et al. (2013) Solanum lycopersicum Leaves 1750 Saville–Griess Cadmium/+ Hasan et al. (2016) Roots 750 Saville–Griess Cadmium/= Hasan et al. (2016) Solanum lycopersicum Leaves 22000 Saville–Griess Chilling/+ Chen et al. (2017) Hellianthus annus Hypocotyl 1300 NOA High temperature/+ Mechanical wounding/+ Low temperature/= High light intensity/= Continuous light/= Continuous darkness/= Chaki et al. (2011b) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Pisum sativum Leaves 300 NOA High light intensity/+Low temperature/+Continuous light/=Continuous darkness/+Wounding/+ Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008) Oryza sativa Leaves 4000 Saville–Griess ND Lin et al. (2012) Brassica juncea Seedlings 140 pM mg–1 proteina Saville–Griess Low temperature/+High temperature/+Salinity/+Drought/+ Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009) Boehmeria nivea Leaves 46 NOA Cadmium/= D. Wang et al. (2015) Plant species Organs [SNO] basal conditions (pmol mg–1protein) Detection methods Stress/effect on SNO levels References Arabidopsis thaliana Leaves 50 NOA Pseudomonas syringae pv tomato (avrB)/+ Feechan et al. (2005) Arabidopsis thaliana Leaves 350 NOA Heat stress/– Lee et al. (2008) Arabidopsis thaliana Leaves 15 NOA Paraquat/+ Kovacs et al. (2016) Arabidopsis thaliana Leaves 150 Saville–Griess Pseudomonas syringae pv maculicola (avrRpm1)/+ Rustérucci et al. (2007) Arabidopsis thaliana Leaves 250 Saville–Griess Wounding/+ Espunya et al. (2012) Arabidopsis thaliana Roots 3000 Saville–Griess ND Correa-Aragunde et al. (2015) Arabidopsis thaliana Seedlings 25000 Saville-Griess Cold/+ Puyaubert et al. (2014) Arabidopsis thaliana Cell cultures 22.6 Saville–Griess Nutritional stress/– Frungillo et al. (2013) Solanum lycopersicum Leaves 1750 Saville–Griess Cadmium/+ Hasan et al. (2016) Roots 750 Saville–Griess Cadmium/= Hasan et al. (2016) Solanum lycopersicum Leaves 22000 Saville–Griess Chilling/+ Chen et al. (2017) Hellianthus annus Hypocotyl 1300 NOA High temperature/+ Mechanical wounding/+ Low temperature/= High light intensity/= Continuous light/= Continuous darkness/= Chaki et al. (2011b) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Pisum sativum Leaves 300 NOA High light intensity/+Low temperature/+Continuous light/=Continuous darkness/+Wounding/+ Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008) Oryza sativa Leaves 4000 Saville–Griess ND Lin et al. (2012) Brassica juncea Seedlings 140 pM mg–1 proteina Saville–Griess Low temperature/+High temperature/+Salinity/+Drought/+ Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009) Boehmeria nivea Leaves 46 NOA Cadmium/= D. Wang et al. (2015) NOA, nitric oxide autoanalyser; ND, not determined; +, increased; –, decreased; =, not changed. a The conversion to pmol mg–1 protein was not possible. View Large Table 1. Total S-nitrosothiol (SNO) content in plant tissues Plant species Organs [SNO] basal conditions (pmol mg–1protein) Detection methods Stress/effect on SNO levels References Arabidopsis thaliana Leaves 50 NOA Pseudomonas syringae pv tomato (avrB)/+ Feechan et al. (2005) Arabidopsis thaliana Leaves 350 NOA Heat stress/– Lee et al. (2008) Arabidopsis thaliana Leaves 15 NOA Paraquat/+ Kovacs et al. (2016) Arabidopsis thaliana Leaves 150 Saville–Griess Pseudomonas syringae pv maculicola (avrRpm1)/+ Rustérucci et al. (2007) Arabidopsis thaliana Leaves 250 Saville–Griess Wounding/+ Espunya et al. (2012) Arabidopsis thaliana Roots 3000 Saville–Griess ND Correa-Aragunde et al. (2015) Arabidopsis thaliana Seedlings 25000 Saville-Griess Cold/+ Puyaubert et al. (2014) Arabidopsis thaliana Cell cultures 22.6 Saville–Griess Nutritional stress/– Frungillo et al. (2013) Solanum lycopersicum Leaves 1750 Saville–Griess Cadmium/+ Hasan et al. (2016) Roots 750 Saville–Griess Cadmium/= Hasan et al. (2016) Solanum lycopersicum Leaves 22000 Saville–Griess Chilling/+ Chen et al. (2017) Hellianthus annus Hypocotyl 1300 NOA High temperature/+ Mechanical wounding/+ Low temperature/= High light intensity/= Continuous light/= Continuous darkness/= Chaki et al. (2011b) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Pisum sativum Leaves 300 NOA High light intensity/+Low temperature/+Continuous light/=Continuous darkness/+Wounding/+ Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008) Oryza sativa Leaves 4000 Saville–Griess ND Lin et al. (2012) Brassica juncea Seedlings 140 pM mg–1 proteina Saville–Griess Low temperature/+High temperature/+Salinity/+Drought/+ Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009) Boehmeria nivea Leaves 46 NOA Cadmium/= D. Wang et al. (2015) Plant species Organs [SNO] basal conditions (pmol mg–1protein) Detection methods Stress/effect on SNO levels References Arabidopsis thaliana Leaves 50 NOA Pseudomonas syringae pv tomato (avrB)/+ Feechan et al. (2005) Arabidopsis thaliana Leaves 350 NOA Heat stress/– Lee et al. (2008) Arabidopsis thaliana Leaves 15 NOA Paraquat/+ Kovacs et al. (2016) Arabidopsis thaliana Leaves 150 Saville–Griess Pseudomonas syringae pv maculicola (avrRpm1)/+ Rustérucci et al. (2007) Arabidopsis thaliana Leaves 250 Saville–Griess Wounding/+ Espunya et al. (2012) Arabidopsis thaliana Roots 3000 Saville–Griess ND Correa-Aragunde et al. (2015) Arabidopsis thaliana Seedlings 25000 Saville-Griess Cold/+ Puyaubert et al. (2014) Arabidopsis thaliana Cell cultures 22.6 Saville–Griess Nutritional stress/– Frungillo et al. (2013) Solanum lycopersicum Leaves 1750 Saville–Griess Cadmium/+ Hasan et al. (2016) Roots 750 Saville–Griess Cadmium/= Hasan et al. (2016) Solanum lycopersicum Leaves 22000 Saville–Griess Chilling/+ Chen et al. (2017) Hellianthus annus Hypocotyl 1300 NOA High temperature/+ Mechanical wounding/+ Low temperature/= High light intensity/= Continuous light/= Continuous darkness/= Chaki et al. (2011b) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Pisum sativum Leaves 300 NOA High light intensity/+Low temperature/+Continuous light/=Continuous darkness/+Wounding/+ Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008) Oryza sativa Leaves 4000 Saville–Griess ND Lin et al. (2012) Brassica juncea Seedlings 140 pM mg–1 proteina Saville–Griess Low temperature/+High temperature/+Salinity/+Drought/+ Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009) Boehmeria nivea Leaves 46 NOA Cadmium/= D. Wang et al. (2015) NOA, nitric oxide autoanalyser; ND, not determined; +, increased; –, decreased; =, not changed. a The conversion to pmol mg–1 protein was not possible. View Large Table 2. Content of GSNO in plants Plant species Tissue types GSNO levels References Arabidopsis thaliana Leaves 3.7 nmol g–1 FW Airaki et al. (2011) Leaves 5.5 pmol mg–1 protein Feechan et al. (2005) Capsicum annuum Leaves 5.5 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Roots 7.9 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Stem 4.2 nmol g–1 FW Airaki et al. (2011) Solanum lycopersicum Leaves 5 nmol g–1 FW Chen et al. (2017) Plant species Tissue types GSNO levels References Arabidopsis thaliana Leaves 3.7 nmol g–1 FW Airaki et al. (2011) Leaves 5.5 pmol mg–1 protein Feechan et al. (2005) Capsicum annuum Leaves 5.5 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Roots 7.9 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Stem 4.2 nmol g–1 FW Airaki et al. (2011) Solanum lycopersicum Leaves 5 nmol g–1 FW Chen et al. (2017) View Large Table 2. Content of GSNO in plants Plant species Tissue types GSNO levels References Arabidopsis thaliana Leaves 3.7 nmol g–1 FW Airaki et al. (2011) Leaves 5.5 pmol mg–1 protein Feechan et al. (2005) Capsicum annuum Leaves 5.5 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Roots 7.9 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Stem 4.2 nmol g–1 FW Airaki et al. (2011) Solanum lycopersicum Leaves 5 nmol g–1 FW Chen et al. (2017) Plant species Tissue types GSNO levels References Arabidopsis thaliana Leaves 3.7 nmol g–1 FW Airaki et al. (2011) Leaves 5.5 pmol mg–1 protein Feechan et al. (2005) Capsicum annuum Leaves 5.5 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Roots 7.9 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Stem 4.2 nmol g–1 FW Airaki et al. (2011) Solanum lycopersicum Leaves 5 nmol g–1 FW Chen et al. (2017) View Large It is important to quantify GSNO in plants because as a crucial molecule that may control NO-dependent signalling, the modulation of GSNO content could be an essential regulatory step in regulation of NO levels in cells (Mur et al., 2013). In the following sections, we will briefly describe the mechanisms involved in GSNO turnover that may consequently affect NO function under basal or stress conditions in plants. GSNO biosynthesis processes Like other SNOs (Williams, 1999) (Equation 1), GSNO can be chemically synthesized in acid media using nitrous acid, as outlined in Equation 2 (Broniowska et al., 2013). We can take advantage of this reaction to synthesize GSNO to be used as an NO donor in the lab (Hart, 1985). RSH+HNO2→RSNO+H2O (1) GSH+HNO2→GSNO+H2O (2) However, the exact mechanism of in vivo GSNO formation remains unclear (Zaffagnini et al., 2016). GSNO synthesis is usually described as the reaction of NO with GSH. However, it is important to note that NO itself is relatively unreactive with non-radical molecules and therefore it does not directly react with GSH to produce GSNO (Williams, 1999; Hogg, 2002; Broniowska et al., 2013; Zaffagnini et al., 2016). The direct interaction between NO and thiols (SH) yields a thiol disulphide but no SNOs; however, the presence of oxygen can generate some oxygen intermediates that give rise to SNOs (Hogg, 2002). In this context, as in SNO formation (Lindermayr and Durner, 2009), GSNO appears to be generated through two main pathways (Fig. 1): reaction of NO with a glutathionyl radical (GS·), generated probably under a stress condition, or S-nitrosylation of GSH by N2O3 (Hogg, 2002; Broniowska and Hogg, 2012; Broniowska et al., 2013). In addition, GSNO can also be generated by transnitrosylation reactions between SNOs and GSH (Fig. 1). It is worth noting that GSH is present at millimolar concentrations and it is widely distributed in cells (Noctor et al., 2012); thus the pool of GSH is ready to be S-nitrosylated, leading to GSNO generation (Mur et al., 2013). However, the source of NO in plants is still under debate (Gupta et al., 2011; Yu et al., 2014). Currently, there are several pathways proposed to generate NO that can be classified either as reductive or oxidative pathways (Gupta et al., 2011; Yu et al., 2014; Sahay and Gupta, 2017). Nitrate reductase (NR) and mitochondria or plasma membrane-associated NO production are components of the reductive route, whereas nitric oxide synthase (NOS)-like activity and polyamines are oxidative components. NR catalyses the reduction of nitrate to nitrite using NADH as an electron donor. In addition, this enzyme has also been proposed to generate NO by a reductive pathway in which nitrite can be converted into NO. However, the yield of this reaction appears to be only 1–2% of its activity with nitrate (Dean and Harper, 1986; Yamasaki and Sakihama, 2000; Gupta et al., 2011). Keeping in mind that NR may not be the main NO source but that it is involved in NO scavenging and recycling, NR has recently been proposed to play a role in the NO homeostasis in plants (Chamizo-Ampudia et al., 2017). In this context, it has recently been established in Clamydomonas that a dual system between NR and the molybdoenzyme amidoxime-reducing component (ARC) is able to synthesize NO from nitrite in vivo and in vitro. In this system, NR would transfer electrons to ARC for reducing nitrite to NO (Chamizo-Ampudia et al., 2016). Interestingly, NR can also promote the removal of NO by the truncated haemoglobin THB1. Consequently, NR may be involved in both the synthesis and the elimination of NO, therefore controlling cellular NO levels (Chamizo-Ampudia et al., 2017) Fig. 1. View largeDownload slide S-Nitrosoglutathione (GSNO) turnover in plants. The GSNO level is regulated by synthesis processes (blue arrows) and mechanisms that lead to its degradation (red arrows). The main sources of NO in plants are NOS-like activity and nitrate reductase (NR) (green arrows). However, NO itself does not directly interact with GSH to form GSNO, but two different routes allow its production. NO can interact with glutathionyl radicals (GS·) (1), probably generated by oxidative radicals produced under stress conditions. In addition, the generation of N2O3 can S-nitrosylate GSH (2). Another source of GSNO generation is mediated by the SNO pool that can donate its NO moiety to GSH (3). In contrast, GSNO can be decomposed by redox changes, light, or temperature (4), by the action of the GSNOR enzyme (5), or by transnitrosylation processes (6). See text for more details. Fig. 1. View largeDownload slide S-Nitrosoglutathione (GSNO) turnover in plants. The GSNO level is regulated by synthesis processes (blue arrows) and mechanisms that lead to its degradation (red arrows). The main sources of NO in plants are NOS-like activity and nitrate reductase (NR) (green arrows). However, NO itself does not directly interact with GSH to form GSNO, but two different routes allow its production. NO can interact with glutathionyl radicals (GS·) (1), probably generated by oxidative radicals produced under stress conditions. In addition, the generation of N2O3 can S-nitrosylate GSH (2). Another source of GSNO generation is mediated by the SNO pool that can donate its NO moiety to GSH (3). In contrast, GSNO can be decomposed by redox changes, light, or temperature (4), by the action of the GSNOR enzyme (5), or by transnitrosylation processes (6). See text for more details. In addition to NR, a membrane-bound nitrite reductase (Ni:NOR) has been proposed to generate NO in tobacco roots (Stöhr et al., 2001). Furthermore, mitochondria under low oxygen conditions appear to be a major source of NO production in plants (Planchet et al., 2005; Gupta et al., 2005; Gupta and Igamberdiev, 2011; Stoimenova et al., 2007). In this case, NO is generated by nitrite reduction in the mitochondrial inner membrane, probably via complex III (Gupta and Igamberdiev, 2011). Conversely, NO production via oxidation of l-arginine by a NOS-like protein generates a strong controversy because, although a NOS-like activity has been well characterized in higher plants (Barroso et al., 1999; Corpas et al., 2009; Corpas and Barroso, 2014, 2017; Santolini et al., 2017), a specific protein similar to mammalian NOS has not yet been identified. Recently, an elegant bioinformatic approach analysing the sequence of a high number of land plants has proposed that a homologue of mammalian NOS does not exist in higher plants, but researchers do not rule out that different peptides may work together to produce this NOS-like activity (Jeandroz et al., 2016). Along this line, research on plant NO should be focused on the task of definitively elucidating the NOS-like pathway (Corpas and Barroso, 2017). It is worth noting that these NO sources could not act independently and they may be inter-related, establishing a complex mechanism of controlling NO production in plants. For instance, NOS-like-dependent NO production under an enriched CO2 environment could differentially regulate the activity of NR under different N concentrations (Du et al., 2016). It is also important to note that non-symbiotic haemoglobins (phytoglobins) are essential players in NO turnover given that they are endogenous NO scavengers. Consequently, overexpressing and silenced mutant lines exhibit lower and higher NO levels, respectively (Hill, 2012). In this context, non- symbiotic haemoglobins may have a crucial role in the NO signalling pathway. Mechanisms for GSNO degradation GSNO, like other SNOs, is light and redox sensitive and therefore can be non-enzymatically decomposed. Reducing agents such as GSH and ascorbic acid or temperature can lead to GSNO degradation (Singh et al., 1996; Williams, 1999; Hogg, 2002) (Fig. 1). However, the presence of metal ions is probably the most critical for non-enzymatic decomposition of SNOs/GSNOs, and consequently buffers containing metal chelators are usually used for stabilizing and analysing these compounds (Williams, 1999; Hogg, 2002). However, SNO/GSNO levels, as potential molecular cues involved in signalling pathways, have to be tightly regulated and not left to the random decomposition mentioned above (Broniowska and Hogg, 2012). The thermal spontaneous S–N bond homolysis of GSNO is unlikely to be a biologically meaningful reaction, and consequently GSNO does not spontaneously homolyse to form NO (Broniowska et al., 2013). In addition, GSNO is more stable in the presence of metal ions than other SNOs (Broniowska et al., 2013). Consequently, some specific enzymatic mechanisms that drive denitrosylation have been described in animals and plants (Benhar et al., 2009; Anand and Stamler, 2012; Begara-Morales and Loake, 2016). Although there is a growing body of evidence suggesting that thioredoxins can mediate protein denitrosylation (Benhar et al., 2009; Kneeshaw et al., 2014; Begara-Morales and Loake, 2016), we will focus on the mechanism that specifically degrades GSNO. In this regard, GSNOR has emerged as the key enzyme regulating the GSNO pool (Mur et al., 2013) since it specifically breaks down GSNO (Jensen et al., 1998; Liu et al., 2001) (Fig. 1). Consequently, GSNOR does not directly act as a protein denitrosylase, but it controls intracellular levels of GSNO and, indirectly, total SNO (Feechan et al., 2005). Consequently, GSNOR regulates the GSNO pool and therefore it would also be able to modulate cellular NO levels given that GSNO is a main source of NO. In line with this assumption is the fact that GSNOR loss-of-function mutants accumulate GSNO but also exhibit increased NO levels (Lee et al., 2008; Kawabe et al., 2017). GSNOR is an evolutionarily conserved enzyme, from bacteria to mammals (Liu et al., 2001), suggesting its relevance in cell function. At the same time, GSNOR activity can be regulated by S-nitrosylation via GSNO/NO (Frungillo et al., 2014; Guerra et al., 2016), suggesting that GSNO could regulate its own scavenging and therefore establish a loop finely regulating NO homeostasis in the cell. The structure of GSNOR from tomato plants was recently characterized (Kubienová et al., 2013), allowing researchers to understand how GSNOR activity and structure can be modified under different environmental stresses. GSNOR has been related to different physiological and pathological conditions in animals (Liu et al., 2004; Benhar et al., 2009; Anand and Stamler, 2012; Beigi et al., 2012; Cao et al., 2015). In plants, the use of transgenic lines where GSNOR is either blocked (atgsnor1-3) or enhanced (atgsnor1-1 or 35S::FLAG-GSNOR) (Feechan et al., 2005) has allowed the direct analysis of the importance of GSNO/SNOs in physiological and stress conditions in plants (see later). This is possible because these mutants are affected in the main GSNO enzymatic degradation pathway that indirectly controls the total SNO pool, assuming that the observed effects in these plants are a direct reflection of GSNO bioactivity. Another important mechanism leading to the decomposition of GSNO is transnitrosylation, which in turn is probably the main mode of action of GSNO (Fig. 1). This pathway is feasible due to the capacity of GSNO to release NO and transfer it to a protein thiol (Singh et al., 1996; Broniowska and Hogg, 2012). In this regard, GSNO has traditionally been used as a physiological NO donor to analyse the impact of S-nitrosylation on target proteins. In gsnor1-3 mutants, the augmentation of GSNO can trigger transnitrosylation reactions and therefore increase the total S-nitrosylated proteins (Feechan et al., 2005), indicating that GSNO is a potent cell NO donor (Frungillo et al., 2014) and therefore has an essential role in NO homeostasis. In this context, GSNO is much more stable than NO (Stamler et al., 1992), and this molecule acting as an NO reservoir could enable plants to use NO more efficiently (Frungillo et al., 2014), modulating the function of target proteins via transnitrosylation reactions in response to stress conditions and ultimately regulating the NO signalling. GSNO-dependent signalling As stated above, the relevance of GSNO as a mediator of NO-dependent responses has been established using GSNOR-transgenic plants that modulate GSNO levels and consequently total SNOs. In this situation, it is difficult to differentiate between the direct effect of GSNO or protein-SNO, but one can assume that the down-regulation of GSNOR in atgsnor1-3 plants allows the accumulation of GSNO, so that this mutant would be the reflection of GSNO bioactivity. GSNO could thus act as a central molecule involved in crucial physiological and stress response processes (Fig. 2) that will be addressed in the following sections. Fig. 2. View largeDownload slide S-Nitrosoglutathione (GSNO) signalling in plants. GSNO plays a crucial role in different physiological and stress-related processes in plants. In the former, GSNO regulates specific enzymes involved in the photosynthesis process and it is also related to the hormone network, particularly auxin metabolism. At the level of nitrogen metabolism, GSNO modulates nitrate uptake and assimilation, regulating the activity of the nitrate reductase (NR) enzyme. In the latter, GSNO modulates the function of key players in plant response to abiotic stress and plant immunity. See the text for more details. FBPase, fructose-1,6-bisphosphatase; PRK, phosphoribulokinase; SABP, salicylic acid-binding protein 3; NPR1, non-expressor of pathogenesis-related genes 1. Fig. 2. View largeDownload slide S-Nitrosoglutathione (GSNO) signalling in plants. GSNO plays a crucial role in different physiological and stress-related processes in plants. In the former, GSNO regulates specific enzymes involved in the photosynthesis process and it is also related to the hormone network, particularly auxin metabolism. At the level of nitrogen metabolism, GSNO modulates nitrate uptake and assimilation, regulating the activity of the nitrate reductase (NR) enzyme. In the latter, GSNO modulates the function of key players in plant response to abiotic stress and plant immunity. See the text for more details. FBPase, fructose-1,6-bisphosphatase; PRK, phosphoribulokinase; SABP, salicylic acid-binding protein 3; NPR1, non-expressor of pathogenesis-related genes 1. GSNO regulates key processes under basal conditions in plants GSNO can regulate a wide range of processes under physiological conditions through the control of specific targets. In this context, some works indicate that the photosynthesis process could be regulated by GSNO. For instance, a novel GSNO chromatography using an affinity support GSNO-vinyl sulphone has enabled the identification of several protein targets of GSNO under non-stress conditions, some of which are related to the photosynthesis process (Begara-Morales et al., 2013). Similarly, the use of atgsnor1-3 transgenic lines has led to the identification of a high number of endogenously S-nitrosylated proteins and, notably, the alteration of the photosynthetic properties (Hu et al., 2015). However, the specific impact of S-nitrosylation on photosynthesis remains to be elucidated, and therefore future research is required in order to move forward in this field. The down-regulation of enzymatic GSNO breakdown has a significant impact on plant development, affecting processes such as hypocotyl elongation, seed production, flowering time, and root development (Lee et al., 2008; Kwon et al., 2012; Xu et al., 2013; Shi et al., 2015). It bears noting that some of these phenotypic effects are present in auxin-deficient plants (Shi et al., 2015), suggesting that the accumulation of GSNO/SNOs could have a potential role in auxin signalling. It has been shown (Kwon et al., 2012) that atgsnor1-3 plants have an specific auxin-sensitive phenotype compared with the wild type, whereas other hormones have no effects. In addition, the auxin-related phenotypes of atgsnor1-3 are a consequence of impaired auxin signalling and compromised auxin transport (Shi et al., 2015). The use of mutants with altered root hair formation (RHF) treated with auxin or GSNO has shown that GSNO is a crucial signalling molecule involved in cell wall remodelling in auxin-mediated RHF, suggesting that GSNO/NO and auxin may alter cell wall composition during root hair development (Moro et al., 2017). A growing body of evidence indicates that not only does the auxin signalling pathway interact with NO, but there is also crosstalk between NO and hormones to control plant development under different abiotic stress conditions, extensively reviewed by Nawaz et al., (2017). Supporting this assumption, the plant hormone network has been proposed to be modulated by S-nitrosylation (París et al., 2013). GSNO/NO appears to be involved in another vital metabolic pathway for plant growth and development, namely nitrogen (N) homeostasis. In an elegant work (Frungillo et al., 2014), it was shown that the accumulation of GSNO/SNO in atgsnor1-3 plants leads to modulation of NR activity, and N assimilation and reduction depending on N availability. Consequently, the aberrant growth of atgsnor1-3 plants could be related to the shortage of N in these plants, indicating that (S)NOs are key modulators of nitrate assimilation and therefore of plant growth and development (Frungillo et al., 2014). The authors even showed that GSNO inhibits but free NO does not affect NR activity, suggesting that these two types of RNS can modulate different targets. Recently, it has been reported that NO and GSNO function additively and may have different or overlapping molecular targets during development and immune response (Yun et al., 2016). Furthermore, nitrate-derived NO inhibits GSNOR by S-nitrosylation, modulating GSNO levels that in turn regulate nitrate uptake and assimilation (Frungillo et al., 2014). Similarly, using a GSNOR-overexpressing system in Medicago truncatula roots leads to an intensified NR activity (Thalineau et al., 2016). However, this effect on NR activity in both studies occurs with a different background of total SNO levels since the GSNOR-overexpressing system strangely increased total SNO in Medicago roots (Thalineau et al., 2016), whereas this strategy usually lowers SNO levels (Feechan et al., 2005; Frungillo et al., 2014; Yun et al., 2016). This discrepancy may be derived from the different strategies to eliminate/reduce GSNOR function (discussed below) or from the different plant species analysed. However, the results of these studies point to a key relationship between NO and N homeostasis. In this scenario, NO has emerged as a pivotal signalling molecule involved in regulating nutrient uptake and assimilation by plants (Simontacchi et al., 2015; Begara-Morales, 2016). The results of these works may be of great relevance to agricultural practices since they may allow an accurate management of nitrate-containing fertilizers to improve plant growth in N-deficient soils. In addition to the regulation of protein function by S-nitrosylation, GSNO is also able to induce changes in gene expression that lead to a transcriptional reprogramming in different plants (Ferrarini et al., 2008; Begara-Morales et al., 2014). The exogenous GSNO under non-stress conditions was applied in Arabidopsis roots, triggering an organ-specific modulation of gene expression determined by RNA-sequencing (Begara-Morales et al., 2014). In this study, although the analysis was carried out under non-stress conditions, an important set of GSNO-responsive genes were related to stress signalling pathways. Additionally, very recently, GSNO/NO has been proposed to be a potential modulator of gene expression in controlling the chromatin acetylation process (Mengel et al., 2017). GSNO treatment induces histone hyperacetylation by inhibiting total histone deacetylase activity and therefore controlling the expression of target genes (Mengel et al., 2017), most of which are related to stress response. Moreover, the authors demonstrated that salicylic acid (SA), a key hormone in the regulation of plant response to stress, induced endogenous NO production that had a negative effect on histone deacetylase activity. The potential role of GSNO/NO in epigenetic modifications as a consequence of environmental stresses is a promising niche for future research. In addition, very recently a connection has been established between S-nitrosylation and protein methylation under stress responses, highlighting that these two post-translational modifications may act together in response to adverse conditions (Hu et al., 2017). All these studies point towards the contention that GSNO can be perceived as a molecular cue to trigger defence mechanisms, leading to orchestratation of an NO-dependent response to several stress conditions. Consequently, these results support the idea that NO and/or GSNO as its main reservoir act as signal molecules involved in transcriptional modulation leading to the adaptive response to several plant environmental stresses (Ferrarini et al., 2008; Besson-Bard et al., 2009; Begara-Morales et al., 2014; Mengel et al., 2017; Singh et al., 2017). GSNO metabolism during plant response to abiotic stress NO function in plant abiotic stress is becoming well established (reviewed by Corpas et al., 2011; Siddiqui et al., 2011; Procházková et al., 2014; Fancy et al., 2016), but less attention has been focused on the regulation of endogenous GSNO as the main source of NO under basal and stress conditions (Corpas et al., 2013). GSNOR activity and consequently GSNO levels are modulated during development and plant response to different environmental stresses (Ziogas et al. 2013; Kubienová et al., 2014; Tichá et al., 2017). Furthermore, NO and GSNO can act independently in response to abiotic stress, reinforcing the idea that GSNO is a key modulator of plant response to stress. Thus, in this section we will discuss the role of GSNO as the potential pivotal regulator of NO signalling that orchestrates plant response to environmental insults. As mentioned above, GSNO is considered a mobile signal molecule of the phloem involved in plant responses to stress. Thus, using GSNOR transgenic lines, GSNO has been proposed to have a pivotal role in wound- and SA-induced systemic responses in Arabidopsis, where basal GSNO is localized mainly in vascular tissue and parenchyma cells (Espunya et al., 2012). Mechanical wounding at different time points increased GSNO in the injured and systemic leaves, first in the vascular tissue and later in parenchyma cells, suggesting that GSNO is involved in the transmission of the wound signal from injured to systemic tissues through the vascular tissue (Espunya et al., 2012). In sunflower hypocotyls, none of the several enzymatic and non-enzymatic sources of NO analysed (NOS-like and NR activities and total levels of nitrate and nitrite) was altered under mechanical injury, and consequently NO production did not change under this stress condition (Chaki et al., 2011a). However, wounding depletes GSNOR (Díaz et al., 2003; Chaki et al., 2011a) and thus increases GSNO and total SNOs that mediate a nitrosative stress by increasing peroxynitrite and the total nitrotyrosine content (Chaki et al., 2011a). A similar effect was found in the same plant organ under heat stress. In this case, the increased tyrosine nitration mediated by GSNO/SNO leads to the inhibition of two enzymes involved in photosynthesis, namely ferredoxin-NADP-reductase and carbonic anhydrase (CA), suggesting that GSNO/SNO could have a key regulatory role in this process during high temperature stress (Chaki et al., 2011b, 2013). Notably, in both studies, endogenous GSNO was localized mainly in vascular tissues and, after the stress application, there was a redistribution to the cortex and epidermal cells. The authors suggest that this situation may be a consequence of a protective mechanism since these cells are in direct contact with the external environment, highlighting the importance of GSNO in modulating the adaptive response of sunflower plants to these stresses. As a general rule, when there is a down-regulation of GSNOR, GSNO/SNOs usually increase, and vice versa. However, there are some examples in which this correlation is disrupted. For instance, in pea plants subjected to different types of abiotic stress (Barroso et al., 2006; Corpas et al., 2008) and in the roots of Medicago plants overexpressing GSNOR (Thalineau et al., 2016), total SNOs increase concomitantly with greater GSNOR activity. In the former, NO increases in some stresses but not in others, and NO levels do not change in the latter. Similarly, the accumulation of SNOs, independent of NO production, together with an increase of GSNOR activity and transient tyrosine nitration may also be related to the acclimation response of pepper plants to low temperatures (Airaki et al., 2012). Unfortunately, the GSNO levels were not analysed in these works, but it can be hypothesized that in these cases the accumulation of total SNOs may be caused by a GSNO transnitrosylation-independent mechanism. This would be a worthwhile field to explore fully. Informatively, nox1 mutants (accumulate NO) could alter SNOs levels but to a lesser extent than a GSNOR loss- of-function mutant. It is worth noting that the latter could also slightly increase NO levels (Yun et al., 2016). These results suggest that it would be crucial to measure GSNO levels to establish whether the NO-dependent response is mediated mainly by GSNO or NO, since both could have an additive function during development and disease resistance (Yun et al., 2016). However, the quantification of SNOs is not an easy task because, although different methods have been developed (reviewed by Broniowska et al., 2013; Zaffagnini et al., 2016), it is still difficult to differentiate total SNOs from GSNO. To resolve this, some methods specifically to detect and quantify GSNO have been proposed in animals (reviewed in Broniowska et al., 2013), but in plants this field remains to be explored in more depth. Next, we address other potential roles of GSNO/SNOs that have been established using GSNOR transgenic mutant lines. This strategy has been employed mainly to characterize the role of GSNO/SNOs during plant immunity (for more details, see below). Along the same lines, a wealth of evidence has established a direct role for GSNO/SNOs in plant responses to abiotic stress, such as thermotolerance, cell death, alkaline stress (Lee et al., 2008; Chen et al., 2009; Gong et al., 2015; Kovacs et al., 2015), or the aforementioned wounding stress (Espunya et al., 2012). Lee et al. (2008) identified different Arabidopsis thermotolerance-defective mutants, collectively called hot5 (sensitive to hot temperature 5). It is worth noting that HOT5 was subsequently found to encode a GSNOR1 gene. The null hot 5-2 mutants (identical to atgsnor1-3 mutants) exhibited higher total SNO levels and heat sensitivity, highlighting the importance of GSNO metabolism in Arabidopsis thermotolerance (Lee et al., 2008). Paraquat is a herbicide that triggers cell death via increased ROS production (Chen et al., 2009). Using an extensive genetic approach, the paraquat resistance 2 (PAR2) gene was found to be identical to GSNOR1. Therefore, the loss of function of PAR2, generally named par2-1, is a mutated allele of GSNOR1 that expresses an unstable GSNOR protein, leading to the resistance to paraquat-induced cell death (Chen et al., 2009). This resistance was related to the higher SNO levels present in these mutants compared with the wild-type plants, but the exact mechanisms underlying this protection remained unclear. However, some information on these protective mechanisms has recently been reported (Kovacs et al., 2016). This study showed that GSNOR can be oxidized by H2O2in vitro, and paraquat induced oxidative stress in vivo, inhibiting its activity and therefore accumulating SNOs. Notably, the authors showed that in a atgsnor1 background, genes involved in antioxidant responses are up-regulated. At the same time, the high level of GSNO/SNOs induced a greater GSH-dependent antioxidant capacity compared with wild-type plants, and therefore led to a protective effect against the paraquat-generated oxidative stress (Kovacs et al., 2016). Supporting these results is the fact that GSNO/NO has been proposed to modulate the key antioxidant capacity of plant tissues (Begara-Morales et al., 2016b) under aluminium (Sun et al., 2014), cadmium (D. Wang et al., 2015), drought (Shan et al., 2015), and water-deficient (Silveira et al., 2017) stress conditions that ultimately are stress conditions usually leading to an oxidative stress. Moreover, GSNO metabolism is also crucial for regulating the plant response to alkaline stress (Gong et al., 2015). Loss-of-function mutation in GSNOR enhances sensitivity to alkaline stress as a consequence of elevated SNO/NO levels. In this context, there was a rise in ROS levels, causing damage to the photosynthetic apparatus. However, GSNOR overexpression lines showed more resistance to this stress, revealing the crucial importance of regulating GSNO levels in tomato plant tolerance to this abiotic stress (Gong et al., 2015). All the above demonstrate the relevance of regulating GSNO/SNO levels as key mediators of NO-dependent signalling events during the adaptive response of plants to several abiotic stress conditions. GSNO is a key player in plant immunity Although the involvement of GSNO/SNOs in the plant immune response after pathogen challenge has been widely addressed (Malik et al., 2011; Yu et al., 2014; Fig. 3), a certain controversy persists in this field, as will be highlighted and discussed in this section. Fig. 3. View largeDownload slide Role of GSNO in salicylic acid (SA)-dependent immune signalling. NPR1 homeostasis is essential in plant immunity, so the equilibrium between its monomeric and oligomeric form is crucial for plant survival after pathogen attack. Under basal conditions, NPR1 is sequestered as an oligomeric complex in the cytoplasm, a conformation that is also promoted by S-nitrosylation. Upon pathogen challenge, the increase in GSH and SA, together with TRX-h5, favours the nuclear accumulation of NPR1 that in turn mediates the expression of defence genes. NPR1 equilibrium can be modified in atgsnor1-3 plants that exhibit an excessive accumulation of GSNO/SNOs, hindering NPR1 nuclear translocation and inhibiting SABP3 function, and therefore compromising plant survival. We differentiate the effect of exogenous GSNO (green route) from endogenous GSNO in atgsnor1-3 plants (red route) that apparently generate contradictory effects. See text for a detailed discussion. Fig. 3. View largeDownload slide Role of GSNO in salicylic acid (SA)-dependent immune signalling. NPR1 homeostasis is essential in plant immunity, so the equilibrium between its monomeric and oligomeric form is crucial for plant survival after pathogen attack. Under basal conditions, NPR1 is sequestered as an oligomeric complex in the cytoplasm, a conformation that is also promoted by S-nitrosylation. Upon pathogen challenge, the increase in GSH and SA, together with TRX-h5, favours the nuclear accumulation of NPR1 that in turn mediates the expression of defence genes. NPR1 equilibrium can be modified in atgsnor1-3 plants that exhibit an excessive accumulation of GSNO/SNOs, hindering NPR1 nuclear translocation and inhibiting SABP3 function, and therefore compromising plant survival. We differentiate the effect of exogenous GSNO (green route) from endogenous GSNO in atgsnor1-3 plants (red route) that apparently generate contradictory effects. See text for a detailed discussion. First, it would be essential to differentiate between the potential roles of NO or GSNO during the establishment of the immune response. As mentioned above, GSNO is a stable reservoir of NO and therefore could be a major factor responsible for NO signalling after pathogen attack. It has in fact been shown that GSNO and NO act additively and may have distinct or overlapping molecular targets during development and immune response (Yun et al., 2016), confirming that atgsnor1-3 plants can be a good background to analyse the effect of GSNO content during plant immunity (Feechan et al., 2005). It has recently been shown that thioredoxin-h5 (TRXh5) can specifically act as a denitrosylase enzyme mainly through a transnitrosylation mechanism using a single active cysteine (Kneeshaw et al. 2014). Further, TRXh5 rescued immunity after pathogen challenge in nox1 plants that accumulate NO but fails in restoring immunity in atgsnor1 mutants that exhibit high GSNO levels. Based on these results, TRXh5 could discriminate between different protein-SNO substrates, highlighting that TRXh5 and GSNOR may denitrosylate a different set of protein-SNOs (Kneeshaw et al., 2014). Accumulation of GSNO/SNOs has been reported to have different effects on plant resistance to pathogens that ultimately could be a consequence of different SA accumulation in the distinct Arabidopsis transgenic lines. In a pioneer study, Feechan et al. (2005) demonstrated that GSNO/SNO accumulation in atgsnor1-3 Arabidopsis transgenic lines compromises the expression of multiple modes of plant disease resistance. In this scenario, the high level of S-nitrosylation negatively regulates SA biosynthesis and signalling, implying a pivotal role for GSNO/SNO during the establishment of SA-dependent immune response (Feechan et al., 2005) (Fig. 3). Conversely, a positive regulation of plant defence against pathogens was reported in Arabidopsis as a consequence of GSNO/SNO accumulation in plants with a defective GSNOR activity (Rustérucci et al., 2007). These apparently contradictory results could be explained by at least two features of the transgenic lines. (i) In atgsnor1-3 lines, there is a significant decrease in free SA, whereas in the other transgenic line SA levels remain unaffected (Rustérucci et al., 2007; Espunya et al., 2012). Thus, the decline in SA production could compromise plant defence in atgsnor1 as this hormone is a pivotal regulator of plant immunity (Rustérucci et al., 2007). (ii) These different responses may be derived from the different strategies to eliminate/reduce GSNOR function. Informatively, atgsnor1-3 plants are null mutants with a T-DNA insertion (Feechan et al., 2005) whereas, in the other case, an antisense approach was taken to develop a mutant that still conserves 50% of GSNOR transcript accumulation and activity (Rustérucci et al., 2007; Espunya et al., 2012). Thus, GSNO levels could differ between these transgenic lines and consequently exhibit a different response upon pathogen challenge. The main relevance of GSNO as a regulator of plant immunity is related to the control of the function of two crucial players in plant response to pathogens, SA-binding protein 3 (SABP3) and especially non-expressor of pathogenesis-related genes 1 (NPR1) (Fig. 3). S-Nitrosylation of AtSABP3 at Cys280 impairs both SA binding and CA activity, impacting plant defence against pathogens (Fig. 3). The inhibition of its CA activity has been proposed to contribute to a negative feedback loop that may modulate plant defence response (Wang et al., 2009). Furthermore, in atgsnor1-3 plants there is an increase in SNO-SABP3, and the CA activity of this protein is compromised after pathogen challenge, establishing a key role for GSNO-mediated transnitrosylation in the SA-dependent immune response in plants (Wang et al., 2009). NPR1 is a redox-sensitive transcriptional co-regulator involved in controlling SA-dependent immune signalling in plants (Pajerowska-Mukhtar et al., 2013). GSNO has emerged as a key regulator of NPR1 during plant immune response, but its exact impact on NPR1 function has raised some controversy. Once again, the differences observed could be a consequence of the different SA accumulation in the different strategies used to analyse NPR1 function. Under basal conditions, NPR1 is usually sequestered in the cytoplasm as an inactive oligomeric complex formed via disulphide bonds between conserved cysteines (Mou et al., 2003). Upon pathogen challenge, cellular redox changes lead to higher levels of SA and TRXh5 that mediate the release of the NPR1 monomer. At this time, NPR1 can be translocated to the nucleus and regulate the expression of defence genes involved in plant immunity (Kinkema et al., 2000) (Fig. 3). However, after pathogen challenge, the GSNO-mediated S-nitrosylation of NPR1 at Cys156 could favour the formation of the inactive oligomeric form, therefore compromising plant immune response (Tada et al., 2008). In this regard, both the oligomeric and monomeric form of NPR1 could be induced after pathogen challenge, suggesting that the equilibrium between these two forms of NPR1 is a crucial regulatory point of plant immunity (Tada et al., 2008). Therefore, the oligomer formation by S-nitrosylation could be a prior step in order to avoid NPR1 depletion, thereby maintaining the NPR1 homeostasis (Tada et al., 2008). When there is an excess of GSNO/SNOs, as in atgsnor1-3 plants, this balance would be shifted towards the formation of NPR1 oligomers, consequently compromising plant immune response. Conversely, the treatment of Arabidopsis protoplasts with exogenous GSNO favours the translocation of NPR1 to the nucleus, promoting its interaction with TGA1 and thereby positively regulating the plant immune response (Lindermayr et al., 2010) (Fig. 3). These different effects of GSNO on NPR1 conformation might be a consequence only of different SA accumulation, which is necessary for NPR1 monomerization, given that atgsnor1-3 plants show reduced levels of SA (Feechan et al., 2005). This result was recently confirmed in a study showing that nuclear translocation of NPR1 in Arabidopsis protoplasts after GSNO treatment could be a consequence of increased GSH levels that subsequently trigger SA accumulation. Thus, this increase in SA levels would be responsible for NPR1 monomerization and translocation to the nucleus in GSNO-treated protoplasts (Kovacs et al., 2015) (Fig. 3). Other causes that could explain these differences emerge from a technical standpoint. In this regard, this controversy could reflect the use of plant material with different physiology, such as the protoplast (Lindermayr et al., 2010; Kovacs et al., 2015) and whole leaves (Tada et al., 2008). In addition, the differences could be a consequence of the different GSNO source: endogenous (in atgsnor1-3 plants) versus exogenous (GSNO treatment). These differences could be a reflection only of the GSNO concentration reached at the cellular level in both cases. The use of exogenous GSNO at 100 µM in protoplasts could be excessive, given that the concentration of GSNO in Arabidopsis leaves was reported to be ~3.7 nmol g–1 FW (Airaki et al., 2011). Similarly, the concentrations of total SNO and LMW SNO in atgsnor1-3 plants have been previously reported to be ~50 pmol mg–1 protein and 5 pmol mg–1 protein, respectively (Feechan et al., 2005). However, as stated above, the exact concentration of GSNO in these plants remains to be elucidated. Thus, the development of a robust method to determine the endogenous and pathogen-induced GSNO levels is required, since if there were significant differences, a GSNO threshold might be established from which plant response to pathogens could be compromised. Collectively, these results on NPR1 function appear to be contradictory, but they highlight that GSNO/SNOs play an essential role in the fine-tuned regulation of NPR1 that is vital for plant survival upon pathogen challenge. Finally, GSNO/SNOs also have an impact in the control of the hypersensitive response (HR), a programmed execution of plant cells at sites of attempted infection. In this context, NO and reactive oxygen intermediates have been shown to be involved in the development of HR (Delledonne et al., 2001). When GSNO/SNO levels are elevated as a consequence of GSNOR mutation, pathogen-triggered cell death is accelerated (Yun et al., 2011). In this scenario, excessive S-nitrosylation upon pathogen challenge leads to the inactivation of the NADPH oxidase AtRBOHD, diminishing its ability to synthesize reactive oxygen intermediates. This enzymatic inhibition is thought to be a mechanism regulating a negative feedback loop that limits the extent of cell death in Arabidopsis (Yun et al., 2011). Final remarks NO is an important biological messenger acting as a key regulator of a wide range of physiological and stress response processes in plants. NO usually transmits its bioactivity via PTMs such as the reversible S-nitrosylation. The S-nitrosylation of GSH leads to generation of GSNO, the major LMW SNO and, more importantly, a stable and mobile reservoir of NO acting as relevant NO buffering in the plant system. Thus, GSNO can be considered a key player during plant response to stress, regulating the NO-dependent signalling response. Besides the presence of GSNO in vascular tissues, there is growing evidence suggesting that it can act as a long-distance signalling molecule. GSNO has been involved in plant response to abiotic stress and plant immunity, but certain contradictory results remain to be clarified. These conflicting data, which are related mainly to the effect of GSNO on NPR1 conformation during plant immunity, could be related to the GSNO concentration reached during treatments. Therefore, the development of a reliable and accurate method to quantify GSNO specifically under basal and stress response conditions is needed. 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Nitric oxide buffering and conditional nitric oxide release in stress response

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Abstract

Abstract Nitric oxide (NO) has emerged as an essential biological messenger in plant biology that usually transmits its bioactivity by post-translational modifications such as S-nitrosylation, the reversible addition of an NO group to a protein cysteine residue leading to S-nitrosothiols (SNOs). In recent years, SNOs have risen as key signalling molecules mainly involved in plant response to stress. Chief among SNOs is S-nitrosoglutathione (GSNO), generated by S-nitrosylation of the key antioxidant glutathione (GSH). GSNO is considered the major NO reservoir and a phloem mobile signal that confers to NO the capacity to be a long-distance signalling molecule. GSNO is able to regulate protein function and gene expression, resulting in a key role for GSNO in fundamental processes in plants, such as development and response to a wide range of environmental stresses. In addition, GSNO is also able to regulate the total SNO pool and, consequently, it could be considered the storage of NO in cells that may control NO signalling under basal and stress-related responses. Thus, GSNO function could be crucial during plant response to environmental stresses. Besides the importance of GSNO in plant biology, its mode of action has not been widely discussed in the literature. In this review, we will first discuss the GSNO turnover in cells and secondly the role of GSNO as a mediator of physiological and stress-related processes in plants, highlighting those aspects for which there is still some controversy. nitric oxide, nitric oxide signalling, plant stress, S-nitrosoglutathione Introduction As sessile organisms, plants cannot move to find the best conditions for their growth and development, and consequently are continuously exposed to adverse environmental changes. To face these environmental attacks, they have to perceive these alterations and initiate complex signalling mechanisms leading to an effective response to the threats detected. Redox changes are usually a cue to trigger plant responses to these environmental fluctuations (Begara-Morales et al., 2016a). In this context, different signalling molecules have emerged as key regulators of plant biology, chief among these being reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydrogen peroxide (H2O2) and nitric oxide (NO), respectively. Although the connection between ROS and RNS signalling pathways is becoming well established (Yun et al., 2011; Groß et al., 2013; Lindermayr and Durner, 2015; Arora et al., 2016; Begara-Morales et al., 2016b; Farnese et al., 2016), this review will focus only on the role of NO and especially S-nitrosoglutathione (GSNO; a central NO-derived molecule) in plant response to stress. However, excellent reviews on ROS signalling pathways are available (Tripathy and Oelmüller, 2012; Choudhury et al., 2013; Baxter et al., 2014). NO is a small, short-lived biological messenger acting as a key regulator of a wide range of processes in animals and plants (Mur et al., 2013). For instance, in plants, NO has been shown to be involved in seed germination (Albertos et al., 2015; Krasuska et al., 2015; Wang et al., 2015b), flowering (He et al., 2004), cotyledon senescence (Du et al., 2014), stomatal closure (Wang et al., 2015a), and plant response to stress (reviewed by Yu et al., 2014; Fancy et al., 2016). The radical nature of NO and its ability to cross lipid membranes promote its interaction with different macromolecules. Therefore, NO bioactivity is transmitted mainly by post-translational modifications (NO-PTMs) such as tyrosine nitration and S-nitrosylation, which can regulate the function of the target proteins (Astier and Lindermayr, 2012; Corpas et al., 2015). Tyrosine nitration consists of the binding of an NO2 moiety to the aromatic ring of a tyrosine residue, leading to 3-nitrotyrosine (Gow et al., 2004; Radi, 2004). This NO-PTM is usually mediated by peroxynitrite (ONOO–) (Szabó et al., 2007), suggesting that this modification takes place during severe oxidative stress conditions (Mur et al., 2013; Zaffagnini et al., 2016). To function effectively as a molecular cue, this modification should be specific and transient. In this regard, a potential denitrase activity has been proposed in mammalian cells (Görg et al., 2007; Deeb et al., 2013). In this sense, protein extracts from rat spleen and cerebral cortex were able to decrease the in vitro nitration of glutamine synthetase (Görg et al., 2007). Similarly, a potential denitration of cyclooxygenase 1 was reported in different murine cells (Deeb et al., 2013). However, although in these studies a decrease in nitrotyrosine content is described after incubation with protein extracts, there is no clear evidence of the existence of a denitrase protein, and no information is available in plants (Kolbert et al., 2017). Therefore, this NO-PTM has previously been considered to be an irreversible modification involved in nitrosative stress (Corpas et al., 2007). Consequently, major efforts have been focused on the study of the other main NO-PTM, S-nitrosylation, which consists of the reversible attachment of NO to a cysteine residue, leading to S-nitrosothiols (SNOs) (Hess et al., 2005; Astier et al., 2011). In recent years, the metabolism of SNOs has gained special relevance since numerous pieces of evidence have shown that SNOs are integral to plant biology. In this regard, SNOs are key mediators in the plant response to stress (Yu et al., 2014; Fancy et al., 2016) and, interestingly, this action can be independent of NO production (Feechan et al., 2005; Chaki et al., 2011a, b; Yun et al., 2016). Additionally, the half-life of NO in vivo is very short (~5–15 s; Lancaster, 1997), whereas SNOs are generally more stable in solution (Williams, 1999; Hogg, 2002). Therefore, SNOs may prolong temporally while spatially extending the in vivo actions of locally produced NO (Hogg, 2002). One of the main signalling molecules among SNOs is GSNO, the major low molecular weight (LMW) SNO that is considered as a stable store of NO with a lifetime of hours in aqueous solution (Floryszak-Wieczorek et al., 2006) and an essential molecule involved in NO-dependent signalling (Leitner et al., 2009; Broniowska et al., 2013; Mur et al., 2013). The presence of endogenous GSNO in vascular tissues under basal conditions and its regulation in response to stress (Barroso et al., 2006; Valderrama et al., 2007; Chaki et al., 2011a, b) suggest a function as a signal molecule because it would be ready to travel throughout the plant in response to external signals. In line with this assumption, phloem can propagate redox messengers, including different RNS, during plant defence (Gaupels et al., 2017), and GSNO is involved as a key player in the systemic response to wounding stress (Espunya et al., 2012). Thus, GSNO could act as an NO carrier throughout the plant, thereby conferring to NO with the capacity to be a long-distance signalling molecule. Consequently, GSNO could act as an NO buffer from which NO is released to orchestrate the plant’s response to stress; the NO liberation rate from GSNO has been reported to be 1.22 µM min–1 (Ederli et al., 2009). Thus, GSNO can carry out transnitrosylation reactions, transferring its NO group to a protein cysteine thiol (Hess et al., 2005; Broniowska et al., 2013), thereby regulating the function of the target proteins (Astier et al., 2011). In addition, the enzyme S-nitrosoglutathione reductase (GSNOR) can break down GSNO (for further details, see later) and indirectly controls the overall levels of SNOs in cells (Liu et al., 2001; Feechan et al., 2005), suggesting that GSNO could be crucial in regulating the pool of total SNOs. In this context, GSNO may be considered to have a central role within NO-dependent signal transduction, acting as a pivotal modulator of metabolism of NO/SNOs in plant response to stress. In this regard, different stress conditions can alter GSNO levels, thus affecting NO-dependent signalling responses. In this review, given this importance of GSNO, we will describe current knowledge of the GSNO turnover mechanisms and especially the role of GSNO as a modulator of NO-dependent signalling in physiological and stress conditions in plants. GSNO turnover mechanisms Total SNO levels have been reported in different plant species (Table 1). As shown in Table 1, there is a great difference in the values of published SNOs according to the plant species/tissue analysed and especially to the detection method used. In this context, the chemiluminescence assay using a nitric oxide autoanalyser (NOA) is considered a much more sensitive and reliable method than Saville–Griess to obtain results of NO and SNO levels. Anyway, it is advisable to use at least two different detection methods to verify NO/SNO levels in plant biology (Yamasaki et al., 2016). In contrast, there are only few data on the GSNO content in plants (Table 2). GSNO is considered the most abundant LMW SNO. Thus, the first attempt at GSNO quantification in plants was carried out in a sample fraction that was previously passed through a 5 kDa cut-off membrane to obtain LMW SNOs (Feechan et al., 2005, Rustérucci et al., 2007). Using this approach, the LMW SNO content in Arabidopsis leaves was reported to be ~5.5 pmol mg–1 protein (Table 2; Feechan et al., 2005). It is assumed thus that almost all LMW SNOs are GSNO, but other LMW SNOs cannot really be ruled out. For this reason, a method to analyse GSNO, glutathione (GSH), and GSSG under acidic extraction by LC-ES/MS was proposed in plants (Airaki et al., 2011). However, the artefactual formation of GSNO from GSH is possible under acidic conditions in the presence of nitrite. Thus, acidification of the sample should be avoided when both nitrite and GSH are present (Broniowska et al., 2013). This limitation can be circumvented by pre-treatment with ammonium sulphamate or sulphanilamide to remove nitrite and N-ethylmaleimide (NEM) to block thiol groups on GSH. It would be necessary to confirm the observed GSNO changes under acidic extractions through a clear quantitative assay that uses these molecules. Table 1. Total S-nitrosothiol (SNO) content in plant tissues Plant species Organs [SNO] basal conditions (pmol mg–1protein) Detection methods Stress/effect on SNO levels References Arabidopsis thaliana Leaves 50 NOA Pseudomonas syringae pv tomato (avrB)/+ Feechan et al. (2005) Arabidopsis thaliana Leaves 350 NOA Heat stress/– Lee et al. (2008) Arabidopsis thaliana Leaves 15 NOA Paraquat/+ Kovacs et al. (2016) Arabidopsis thaliana Leaves 150 Saville–Griess Pseudomonas syringae pv maculicola (avrRpm1)/+ Rustérucci et al. (2007) Arabidopsis thaliana Leaves 250 Saville–Griess Wounding/+ Espunya et al. (2012) Arabidopsis thaliana Roots 3000 Saville–Griess ND Correa-Aragunde et al. (2015) Arabidopsis thaliana Seedlings 25000 Saville-Griess Cold/+ Puyaubert et al. (2014) Arabidopsis thaliana Cell cultures 22.6 Saville–Griess Nutritional stress/– Frungillo et al. (2013) Solanum lycopersicum Leaves 1750 Saville–Griess Cadmium/+ Hasan et al. (2016) Roots 750 Saville–Griess Cadmium/= Hasan et al. (2016) Solanum lycopersicum Leaves 22000 Saville–Griess Chilling/+ Chen et al. (2017) Hellianthus annus Hypocotyl 1300 NOA High temperature/+ Mechanical wounding/+ Low temperature/= High light intensity/= Continuous light/= Continuous darkness/= Chaki et al. (2011b) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Pisum sativum Leaves 300 NOA High light intensity/+Low temperature/+Continuous light/=Continuous darkness/+Wounding/+ Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008) Oryza sativa Leaves 4000 Saville–Griess ND Lin et al. (2012) Brassica juncea Seedlings 140 pM mg–1 proteina Saville–Griess Low temperature/+High temperature/+Salinity/+Drought/+ Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009) Boehmeria nivea Leaves 46 NOA Cadmium/= D. Wang et al. (2015) Plant species Organs [SNO] basal conditions (pmol mg–1protein) Detection methods Stress/effect on SNO levels References Arabidopsis thaliana Leaves 50 NOA Pseudomonas syringae pv tomato (avrB)/+ Feechan et al. (2005) Arabidopsis thaliana Leaves 350 NOA Heat stress/– Lee et al. (2008) Arabidopsis thaliana Leaves 15 NOA Paraquat/+ Kovacs et al. (2016) Arabidopsis thaliana Leaves 150 Saville–Griess Pseudomonas syringae pv maculicola (avrRpm1)/+ Rustérucci et al. (2007) Arabidopsis thaliana Leaves 250 Saville–Griess Wounding/+ Espunya et al. (2012) Arabidopsis thaliana Roots 3000 Saville–Griess ND Correa-Aragunde et al. (2015) Arabidopsis thaliana Seedlings 25000 Saville-Griess Cold/+ Puyaubert et al. (2014) Arabidopsis thaliana Cell cultures 22.6 Saville–Griess Nutritional stress/– Frungillo et al. (2013) Solanum lycopersicum Leaves 1750 Saville–Griess Cadmium/+ Hasan et al. (2016) Roots 750 Saville–Griess Cadmium/= Hasan et al. (2016) Solanum lycopersicum Leaves 22000 Saville–Griess Chilling/+ Chen et al. (2017) Hellianthus annus Hypocotyl 1300 NOA High temperature/+ Mechanical wounding/+ Low temperature/= High light intensity/= Continuous light/= Continuous darkness/= Chaki et al. (2011b) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Pisum sativum Leaves 300 NOA High light intensity/+Low temperature/+Continuous light/=Continuous darkness/+Wounding/+ Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008) Oryza sativa Leaves 4000 Saville–Griess ND Lin et al. (2012) Brassica juncea Seedlings 140 pM mg–1 proteina Saville–Griess Low temperature/+High temperature/+Salinity/+Drought/+ Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009) Boehmeria nivea Leaves 46 NOA Cadmium/= D. Wang et al. (2015) NOA, nitric oxide autoanalyser; ND, not determined; +, increased; –, decreased; =, not changed. a The conversion to pmol mg–1 protein was not possible. View Large Table 1. Total S-nitrosothiol (SNO) content in plant tissues Plant species Organs [SNO] basal conditions (pmol mg–1protein) Detection methods Stress/effect on SNO levels References Arabidopsis thaliana Leaves 50 NOA Pseudomonas syringae pv tomato (avrB)/+ Feechan et al. (2005) Arabidopsis thaliana Leaves 350 NOA Heat stress/– Lee et al. (2008) Arabidopsis thaliana Leaves 15 NOA Paraquat/+ Kovacs et al. (2016) Arabidopsis thaliana Leaves 150 Saville–Griess Pseudomonas syringae pv maculicola (avrRpm1)/+ Rustérucci et al. (2007) Arabidopsis thaliana Leaves 250 Saville–Griess Wounding/+ Espunya et al. (2012) Arabidopsis thaliana Roots 3000 Saville–Griess ND Correa-Aragunde et al. (2015) Arabidopsis thaliana Seedlings 25000 Saville-Griess Cold/+ Puyaubert et al. (2014) Arabidopsis thaliana Cell cultures 22.6 Saville–Griess Nutritional stress/– Frungillo et al. (2013) Solanum lycopersicum Leaves 1750 Saville–Griess Cadmium/+ Hasan et al. (2016) Roots 750 Saville–Griess Cadmium/= Hasan et al. (2016) Solanum lycopersicum Leaves 22000 Saville–Griess Chilling/+ Chen et al. (2017) Hellianthus annus Hypocotyl 1300 NOA High temperature/+ Mechanical wounding/+ Low temperature/= High light intensity/= Continuous light/= Continuous darkness/= Chaki et al. (2011b) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Pisum sativum Leaves 300 NOA High light intensity/+Low temperature/+Continuous light/=Continuous darkness/+Wounding/+ Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008) Oryza sativa Leaves 4000 Saville–Griess ND Lin et al. (2012) Brassica juncea Seedlings 140 pM mg–1 proteina Saville–Griess Low temperature/+High temperature/+Salinity/+Drought/+ Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009) Boehmeria nivea Leaves 46 NOA Cadmium/= D. Wang et al. (2015) Plant species Organs [SNO] basal conditions (pmol mg–1protein) Detection methods Stress/effect on SNO levels References Arabidopsis thaliana Leaves 50 NOA Pseudomonas syringae pv tomato (avrB)/+ Feechan et al. (2005) Arabidopsis thaliana Leaves 350 NOA Heat stress/– Lee et al. (2008) Arabidopsis thaliana Leaves 15 NOA Paraquat/+ Kovacs et al. (2016) Arabidopsis thaliana Leaves 150 Saville–Griess Pseudomonas syringae pv maculicola (avrRpm1)/+ Rustérucci et al. (2007) Arabidopsis thaliana Leaves 250 Saville–Griess Wounding/+ Espunya et al. (2012) Arabidopsis thaliana Roots 3000 Saville–Griess ND Correa-Aragunde et al. (2015) Arabidopsis thaliana Seedlings 25000 Saville-Griess Cold/+ Puyaubert et al. (2014) Arabidopsis thaliana Cell cultures 22.6 Saville–Griess Nutritional stress/– Frungillo et al. (2013) Solanum lycopersicum Leaves 1750 Saville–Griess Cadmium/+ Hasan et al. (2016) Roots 750 Saville–Griess Cadmium/= Hasan et al. (2016) Solanum lycopersicum Leaves 22000 Saville–Griess Chilling/+ Chen et al. (2017) Hellianthus annus Hypocotyl 1300 NOA High temperature/+ Mechanical wounding/+ Low temperature/= High light intensity/= Continuous light/= Continuous darkness/= Chaki et al. (2011b) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Chaki et al. (2011a) Pisum sativum Leaves 300 NOA High light intensity/+Low temperature/+Continuous light/=Continuous darkness/+Wounding/+ Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008),Corpas et al. (2008) Oryza sativa Leaves 4000 Saville–Griess ND Lin et al. (2012) Brassica juncea Seedlings 140 pM mg–1 proteina Saville–Griess Low temperature/+High temperature/+Salinity/+Drought/+ Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009),Abat and Deswal (2009) Boehmeria nivea Leaves 46 NOA Cadmium/= D. Wang et al. (2015) NOA, nitric oxide autoanalyser; ND, not determined; +, increased; –, decreased; =, not changed. a The conversion to pmol mg–1 protein was not possible. View Large Table 2. Content of GSNO in plants Plant species Tissue types GSNO levels References Arabidopsis thaliana Leaves 3.7 nmol g–1 FW Airaki et al. (2011) Leaves 5.5 pmol mg–1 protein Feechan et al. (2005) Capsicum annuum Leaves 5.5 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Roots 7.9 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Stem 4.2 nmol g–1 FW Airaki et al. (2011) Solanum lycopersicum Leaves 5 nmol g–1 FW Chen et al. (2017) Plant species Tissue types GSNO levels References Arabidopsis thaliana Leaves 3.7 nmol g–1 FW Airaki et al. (2011) Leaves 5.5 pmol mg–1 protein Feechan et al. (2005) Capsicum annuum Leaves 5.5 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Roots 7.9 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Stem 4.2 nmol g–1 FW Airaki et al. (2011) Solanum lycopersicum Leaves 5 nmol g–1 FW Chen et al. (2017) View Large Table 2. Content of GSNO in plants Plant species Tissue types GSNO levels References Arabidopsis thaliana Leaves 3.7 nmol g–1 FW Airaki et al. (2011) Leaves 5.5 pmol mg–1 protein Feechan et al. (2005) Capsicum annuum Leaves 5.5 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Roots 7.9 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Stem 4.2 nmol g–1 FW Airaki et al. (2011) Solanum lycopersicum Leaves 5 nmol g–1 FW Chen et al. (2017) Plant species Tissue types GSNO levels References Arabidopsis thaliana Leaves 3.7 nmol g–1 FW Airaki et al. (2011) Leaves 5.5 pmol mg–1 protein Feechan et al. (2005) Capsicum annuum Leaves 5.5 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Roots 7.9 nmol g–1 FW Airaki et al. (2011) Capsicum annuum Stem 4.2 nmol g–1 FW Airaki et al. (2011) Solanum lycopersicum Leaves 5 nmol g–1 FW Chen et al. (2017) View Large It is important to quantify GSNO in plants because as a crucial molecule that may control NO-dependent signalling, the modulation of GSNO content could be an essential regulatory step in regulation of NO levels in cells (Mur et al., 2013). In the following sections, we will briefly describe the mechanisms involved in GSNO turnover that may consequently affect NO function under basal or stress conditions in plants. GSNO biosynthesis processes Like other SNOs (Williams, 1999) (Equation 1), GSNO can be chemically synthesized in acid media using nitrous acid, as outlined in Equation 2 (Broniowska et al., 2013). We can take advantage of this reaction to synthesize GSNO to be used as an NO donor in the lab (Hart, 1985). RSH+HNO2→RSNO+H2O (1) GSH+HNO2→GSNO+H2O (2) However, the exact mechanism of in vivo GSNO formation remains unclear (Zaffagnini et al., 2016). GSNO synthesis is usually described as the reaction of NO with GSH. However, it is important to note that NO itself is relatively unreactive with non-radical molecules and therefore it does not directly react with GSH to produce GSNO (Williams, 1999; Hogg, 2002; Broniowska et al., 2013; Zaffagnini et al., 2016). The direct interaction between NO and thiols (SH) yields a thiol disulphide but no SNOs; however, the presence of oxygen can generate some oxygen intermediates that give rise to SNOs (Hogg, 2002). In this context, as in SNO formation (Lindermayr and Durner, 2009), GSNO appears to be generated through two main pathways (Fig. 1): reaction of NO with a glutathionyl radical (GS·), generated probably under a stress condition, or S-nitrosylation of GSH by N2O3 (Hogg, 2002; Broniowska and Hogg, 2012; Broniowska et al., 2013). In addition, GSNO can also be generated by transnitrosylation reactions between SNOs and GSH (Fig. 1). It is worth noting that GSH is present at millimolar concentrations and it is widely distributed in cells (Noctor et al., 2012); thus the pool of GSH is ready to be S-nitrosylated, leading to GSNO generation (Mur et al., 2013). However, the source of NO in plants is still under debate (Gupta et al., 2011; Yu et al., 2014). Currently, there are several pathways proposed to generate NO that can be classified either as reductive or oxidative pathways (Gupta et al., 2011; Yu et al., 2014; Sahay and Gupta, 2017). Nitrate reductase (NR) and mitochondria or plasma membrane-associated NO production are components of the reductive route, whereas nitric oxide synthase (NOS)-like activity and polyamines are oxidative components. NR catalyses the reduction of nitrate to nitrite using NADH as an electron donor. In addition, this enzyme has also been proposed to generate NO by a reductive pathway in which nitrite can be converted into NO. However, the yield of this reaction appears to be only 1–2% of its activity with nitrate (Dean and Harper, 1986; Yamasaki and Sakihama, 2000; Gupta et al., 2011). Keeping in mind that NR may not be the main NO source but that it is involved in NO scavenging and recycling, NR has recently been proposed to play a role in the NO homeostasis in plants (Chamizo-Ampudia et al., 2017). In this context, it has recently been established in Clamydomonas that a dual system between NR and the molybdoenzyme amidoxime-reducing component (ARC) is able to synthesize NO from nitrite in vivo and in vitro. In this system, NR would transfer electrons to ARC for reducing nitrite to NO (Chamizo-Ampudia et al., 2016). Interestingly, NR can also promote the removal of NO by the truncated haemoglobin THB1. Consequently, NR may be involved in both the synthesis and the elimination of NO, therefore controlling cellular NO levels (Chamizo-Ampudia et al., 2017) Fig. 1. View largeDownload slide S-Nitrosoglutathione (GSNO) turnover in plants. The GSNO level is regulated by synthesis processes (blue arrows) and mechanisms that lead to its degradation (red arrows). The main sources of NO in plants are NOS-like activity and nitrate reductase (NR) (green arrows). However, NO itself does not directly interact with GSH to form GSNO, but two different routes allow its production. NO can interact with glutathionyl radicals (GS·) (1), probably generated by oxidative radicals produced under stress conditions. In addition, the generation of N2O3 can S-nitrosylate GSH (2). Another source of GSNO generation is mediated by the SNO pool that can donate its NO moiety to GSH (3). In contrast, GSNO can be decomposed by redox changes, light, or temperature (4), by the action of the GSNOR enzyme (5), or by transnitrosylation processes (6). See text for more details. Fig. 1. View largeDownload slide S-Nitrosoglutathione (GSNO) turnover in plants. The GSNO level is regulated by synthesis processes (blue arrows) and mechanisms that lead to its degradation (red arrows). The main sources of NO in plants are NOS-like activity and nitrate reductase (NR) (green arrows). However, NO itself does not directly interact with GSH to form GSNO, but two different routes allow its production. NO can interact with glutathionyl radicals (GS·) (1), probably generated by oxidative radicals produced under stress conditions. In addition, the generation of N2O3 can S-nitrosylate GSH (2). Another source of GSNO generation is mediated by the SNO pool that can donate its NO moiety to GSH (3). In contrast, GSNO can be decomposed by redox changes, light, or temperature (4), by the action of the GSNOR enzyme (5), or by transnitrosylation processes (6). See text for more details. In addition to NR, a membrane-bound nitrite reductase (Ni:NOR) has been proposed to generate NO in tobacco roots (Stöhr et al., 2001). Furthermore, mitochondria under low oxygen conditions appear to be a major source of NO production in plants (Planchet et al., 2005; Gupta et al., 2005; Gupta and Igamberdiev, 2011; Stoimenova et al., 2007). In this case, NO is generated by nitrite reduction in the mitochondrial inner membrane, probably via complex III (Gupta and Igamberdiev, 2011). Conversely, NO production via oxidation of l-arginine by a NOS-like protein generates a strong controversy because, although a NOS-like activity has been well characterized in higher plants (Barroso et al., 1999; Corpas et al., 2009; Corpas and Barroso, 2014, 2017; Santolini et al., 2017), a specific protein similar to mammalian NOS has not yet been identified. Recently, an elegant bioinformatic approach analysing the sequence of a high number of land plants has proposed that a homologue of mammalian NOS does not exist in higher plants, but researchers do not rule out that different peptides may work together to produce this NOS-like activity (Jeandroz et al., 2016). Along this line, research on plant NO should be focused on the task of definitively elucidating the NOS-like pathway (Corpas and Barroso, 2017). It is worth noting that these NO sources could not act independently and they may be inter-related, establishing a complex mechanism of controlling NO production in plants. For instance, NOS-like-dependent NO production under an enriched CO2 environment could differentially regulate the activity of NR under different N concentrations (Du et al., 2016). It is also important to note that non-symbiotic haemoglobins (phytoglobins) are essential players in NO turnover given that they are endogenous NO scavengers. Consequently, overexpressing and silenced mutant lines exhibit lower and higher NO levels, respectively (Hill, 2012). In this context, non- symbiotic haemoglobins may have a crucial role in the NO signalling pathway. Mechanisms for GSNO degradation GSNO, like other SNOs, is light and redox sensitive and therefore can be non-enzymatically decomposed. Reducing agents such as GSH and ascorbic acid or temperature can lead to GSNO degradation (Singh et al., 1996; Williams, 1999; Hogg, 2002) (Fig. 1). However, the presence of metal ions is probably the most critical for non-enzymatic decomposition of SNOs/GSNOs, and consequently buffers containing metal chelators are usually used for stabilizing and analysing these compounds (Williams, 1999; Hogg, 2002). However, SNO/GSNO levels, as potential molecular cues involved in signalling pathways, have to be tightly regulated and not left to the random decomposition mentioned above (Broniowska and Hogg, 2012). The thermal spontaneous S–N bond homolysis of GSNO is unlikely to be a biologically meaningful reaction, and consequently GSNO does not spontaneously homolyse to form NO (Broniowska et al., 2013). In addition, GSNO is more stable in the presence of metal ions than other SNOs (Broniowska et al., 2013). Consequently, some specific enzymatic mechanisms that drive denitrosylation have been described in animals and plants (Benhar et al., 2009; Anand and Stamler, 2012; Begara-Morales and Loake, 2016). Although there is a growing body of evidence suggesting that thioredoxins can mediate protein denitrosylation (Benhar et al., 2009; Kneeshaw et al., 2014; Begara-Morales and Loake, 2016), we will focus on the mechanism that specifically degrades GSNO. In this regard, GSNOR has emerged as the key enzyme regulating the GSNO pool (Mur et al., 2013) since it specifically breaks down GSNO (Jensen et al., 1998; Liu et al., 2001) (Fig. 1). Consequently, GSNOR does not directly act as a protein denitrosylase, but it controls intracellular levels of GSNO and, indirectly, total SNO (Feechan et al., 2005). Consequently, GSNOR regulates the GSNO pool and therefore it would also be able to modulate cellular NO levels given that GSNO is a main source of NO. In line with this assumption is the fact that GSNOR loss-of-function mutants accumulate GSNO but also exhibit increased NO levels (Lee et al., 2008; Kawabe et al., 2017). GSNOR is an evolutionarily conserved enzyme, from bacteria to mammals (Liu et al., 2001), suggesting its relevance in cell function. At the same time, GSNOR activity can be regulated by S-nitrosylation via GSNO/NO (Frungillo et al., 2014; Guerra et al., 2016), suggesting that GSNO could regulate its own scavenging and therefore establish a loop finely regulating NO homeostasis in the cell. The structure of GSNOR from tomato plants was recently characterized (Kubienová et al., 2013), allowing researchers to understand how GSNOR activity and structure can be modified under different environmental stresses. GSNOR has been related to different physiological and pathological conditions in animals (Liu et al., 2004; Benhar et al., 2009; Anand and Stamler, 2012; Beigi et al., 2012; Cao et al., 2015). In plants, the use of transgenic lines where GSNOR is either blocked (atgsnor1-3) or enhanced (atgsnor1-1 or 35S::FLAG-GSNOR) (Feechan et al., 2005) has allowed the direct analysis of the importance of GSNO/SNOs in physiological and stress conditions in plants (see later). This is possible because these mutants are affected in the main GSNO enzymatic degradation pathway that indirectly controls the total SNO pool, assuming that the observed effects in these plants are a direct reflection of GSNO bioactivity. Another important mechanism leading to the decomposition of GSNO is transnitrosylation, which in turn is probably the main mode of action of GSNO (Fig. 1). This pathway is feasible due to the capacity of GSNO to release NO and transfer it to a protein thiol (Singh et al., 1996; Broniowska and Hogg, 2012). In this regard, GSNO has traditionally been used as a physiological NO donor to analyse the impact of S-nitrosylation on target proteins. In gsnor1-3 mutants, the augmentation of GSNO can trigger transnitrosylation reactions and therefore increase the total S-nitrosylated proteins (Feechan et al., 2005), indicating that GSNO is a potent cell NO donor (Frungillo et al., 2014) and therefore has an essential role in NO homeostasis. In this context, GSNO is much more stable than NO (Stamler et al., 1992), and this molecule acting as an NO reservoir could enable plants to use NO more efficiently (Frungillo et al., 2014), modulating the function of target proteins via transnitrosylation reactions in response to stress conditions and ultimately regulating the NO signalling. GSNO-dependent signalling As stated above, the relevance of GSNO as a mediator of NO-dependent responses has been established using GSNOR-transgenic plants that modulate GSNO levels and consequently total SNOs. In this situation, it is difficult to differentiate between the direct effect of GSNO or protein-SNO, but one can assume that the down-regulation of GSNOR in atgsnor1-3 plants allows the accumulation of GSNO, so that this mutant would be the reflection of GSNO bioactivity. GSNO could thus act as a central molecule involved in crucial physiological and stress response processes (Fig. 2) that will be addressed in the following sections. Fig. 2. View largeDownload slide S-Nitrosoglutathione (GSNO) signalling in plants. GSNO plays a crucial role in different physiological and stress-related processes in plants. In the former, GSNO regulates specific enzymes involved in the photosynthesis process and it is also related to the hormone network, particularly auxin metabolism. At the level of nitrogen metabolism, GSNO modulates nitrate uptake and assimilation, regulating the activity of the nitrate reductase (NR) enzyme. In the latter, GSNO modulates the function of key players in plant response to abiotic stress and plant immunity. See the text for more details. FBPase, fructose-1,6-bisphosphatase; PRK, phosphoribulokinase; SABP, salicylic acid-binding protein 3; NPR1, non-expressor of pathogenesis-related genes 1. Fig. 2. View largeDownload slide S-Nitrosoglutathione (GSNO) signalling in plants. GSNO plays a crucial role in different physiological and stress-related processes in plants. In the former, GSNO regulates specific enzymes involved in the photosynthesis process and it is also related to the hormone network, particularly auxin metabolism. At the level of nitrogen metabolism, GSNO modulates nitrate uptake and assimilation, regulating the activity of the nitrate reductase (NR) enzyme. In the latter, GSNO modulates the function of key players in plant response to abiotic stress and plant immunity. See the text for more details. FBPase, fructose-1,6-bisphosphatase; PRK, phosphoribulokinase; SABP, salicylic acid-binding protein 3; NPR1, non-expressor of pathogenesis-related genes 1. GSNO regulates key processes under basal conditions in plants GSNO can regulate a wide range of processes under physiological conditions through the control of specific targets. In this context, some works indicate that the photosynthesis process could be regulated by GSNO. For instance, a novel GSNO chromatography using an affinity support GSNO-vinyl sulphone has enabled the identification of several protein targets of GSNO under non-stress conditions, some of which are related to the photosynthesis process (Begara-Morales et al., 2013). Similarly, the use of atgsnor1-3 transgenic lines has led to the identification of a high number of endogenously S-nitrosylated proteins and, notably, the alteration of the photosynthetic properties (Hu et al., 2015). However, the specific impact of S-nitrosylation on photosynthesis remains to be elucidated, and therefore future research is required in order to move forward in this field. The down-regulation of enzymatic GSNO breakdown has a significant impact on plant development, affecting processes such as hypocotyl elongation, seed production, flowering time, and root development (Lee et al., 2008; Kwon et al., 2012; Xu et al., 2013; Shi et al., 2015). It bears noting that some of these phenotypic effects are present in auxin-deficient plants (Shi et al., 2015), suggesting that the accumulation of GSNO/SNOs could have a potential role in auxin signalling. It has been shown (Kwon et al., 2012) that atgsnor1-3 plants have an specific auxin-sensitive phenotype compared with the wild type, whereas other hormones have no effects. In addition, the auxin-related phenotypes of atgsnor1-3 are a consequence of impaired auxin signalling and compromised auxin transport (Shi et al., 2015). The use of mutants with altered root hair formation (RHF) treated with auxin or GSNO has shown that GSNO is a crucial signalling molecule involved in cell wall remodelling in auxin-mediated RHF, suggesting that GSNO/NO and auxin may alter cell wall composition during root hair development (Moro et al., 2017). A growing body of evidence indicates that not only does the auxin signalling pathway interact with NO, but there is also crosstalk between NO and hormones to control plant development under different abiotic stress conditions, extensively reviewed by Nawaz et al., (2017). Supporting this assumption, the plant hormone network has been proposed to be modulated by S-nitrosylation (París et al., 2013). GSNO/NO appears to be involved in another vital metabolic pathway for plant growth and development, namely nitrogen (N) homeostasis. In an elegant work (Frungillo et al., 2014), it was shown that the accumulation of GSNO/SNO in atgsnor1-3 plants leads to modulation of NR activity, and N assimilation and reduction depending on N availability. Consequently, the aberrant growth of atgsnor1-3 plants could be related to the shortage of N in these plants, indicating that (S)NOs are key modulators of nitrate assimilation and therefore of plant growth and development (Frungillo et al., 2014). The authors even showed that GSNO inhibits but free NO does not affect NR activity, suggesting that these two types of RNS can modulate different targets. Recently, it has been reported that NO and GSNO function additively and may have different or overlapping molecular targets during development and immune response (Yun et al., 2016). Furthermore, nitrate-derived NO inhibits GSNOR by S-nitrosylation, modulating GSNO levels that in turn regulate nitrate uptake and assimilation (Frungillo et al., 2014). Similarly, using a GSNOR-overexpressing system in Medicago truncatula roots leads to an intensified NR activity (Thalineau et al., 2016). However, this effect on NR activity in both studies occurs with a different background of total SNO levels since the GSNOR-overexpressing system strangely increased total SNO in Medicago roots (Thalineau et al., 2016), whereas this strategy usually lowers SNO levels (Feechan et al., 2005; Frungillo et al., 2014; Yun et al., 2016). This discrepancy may be derived from the different strategies to eliminate/reduce GSNOR function (discussed below) or from the different plant species analysed. However, the results of these studies point to a key relationship between NO and N homeostasis. In this scenario, NO has emerged as a pivotal signalling molecule involved in regulating nutrient uptake and assimilation by plants (Simontacchi et al., 2015; Begara-Morales, 2016). The results of these works may be of great relevance to agricultural practices since they may allow an accurate management of nitrate-containing fertilizers to improve plant growth in N-deficient soils. In addition to the regulation of protein function by S-nitrosylation, GSNO is also able to induce changes in gene expression that lead to a transcriptional reprogramming in different plants (Ferrarini et al., 2008; Begara-Morales et al., 2014). The exogenous GSNO under non-stress conditions was applied in Arabidopsis roots, triggering an organ-specific modulation of gene expression determined by RNA-sequencing (Begara-Morales et al., 2014). In this study, although the analysis was carried out under non-stress conditions, an important set of GSNO-responsive genes were related to stress signalling pathways. Additionally, very recently, GSNO/NO has been proposed to be a potential modulator of gene expression in controlling the chromatin acetylation process (Mengel et al., 2017). GSNO treatment induces histone hyperacetylation by inhibiting total histone deacetylase activity and therefore controlling the expression of target genes (Mengel et al., 2017), most of which are related to stress response. Moreover, the authors demonstrated that salicylic acid (SA), a key hormone in the regulation of plant response to stress, induced endogenous NO production that had a negative effect on histone deacetylase activity. The potential role of GSNO/NO in epigenetic modifications as a consequence of environmental stresses is a promising niche for future research. In addition, very recently a connection has been established between S-nitrosylation and protein methylation under stress responses, highlighting that these two post-translational modifications may act together in response to adverse conditions (Hu et al., 2017). All these studies point towards the contention that GSNO can be perceived as a molecular cue to trigger defence mechanisms, leading to orchestratation of an NO-dependent response to several stress conditions. Consequently, these results support the idea that NO and/or GSNO as its main reservoir act as signal molecules involved in transcriptional modulation leading to the adaptive response to several plant environmental stresses (Ferrarini et al., 2008; Besson-Bard et al., 2009; Begara-Morales et al., 2014; Mengel et al., 2017; Singh et al., 2017). GSNO metabolism during plant response to abiotic stress NO function in plant abiotic stress is becoming well established (reviewed by Corpas et al., 2011; Siddiqui et al., 2011; Procházková et al., 2014; Fancy et al., 2016), but less attention has been focused on the regulation of endogenous GSNO as the main source of NO under basal and stress conditions (Corpas et al., 2013). GSNOR activity and consequently GSNO levels are modulated during development and plant response to different environmental stresses (Ziogas et al. 2013; Kubienová et al., 2014; Tichá et al., 2017). Furthermore, NO and GSNO can act independently in response to abiotic stress, reinforcing the idea that GSNO is a key modulator of plant response to stress. Thus, in this section we will discuss the role of GSNO as the potential pivotal regulator of NO signalling that orchestrates plant response to environmental insults. As mentioned above, GSNO is considered a mobile signal molecule of the phloem involved in plant responses to stress. Thus, using GSNOR transgenic lines, GSNO has been proposed to have a pivotal role in wound- and SA-induced systemic responses in Arabidopsis, where basal GSNO is localized mainly in vascular tissue and parenchyma cells (Espunya et al., 2012). Mechanical wounding at different time points increased GSNO in the injured and systemic leaves, first in the vascular tissue and later in parenchyma cells, suggesting that GSNO is involved in the transmission of the wound signal from injured to systemic tissues through the vascular tissue (Espunya et al., 2012). In sunflower hypocotyls, none of the several enzymatic and non-enzymatic sources of NO analysed (NOS-like and NR activities and total levels of nitrate and nitrite) was altered under mechanical injury, and consequently NO production did not change under this stress condition (Chaki et al., 2011a). However, wounding depletes GSNOR (Díaz et al., 2003; Chaki et al., 2011a) and thus increases GSNO and total SNOs that mediate a nitrosative stress by increasing peroxynitrite and the total nitrotyrosine content (Chaki et al., 2011a). A similar effect was found in the same plant organ under heat stress. In this case, the increased tyrosine nitration mediated by GSNO/SNO leads to the inhibition of two enzymes involved in photosynthesis, namely ferredoxin-NADP-reductase and carbonic anhydrase (CA), suggesting that GSNO/SNO could have a key regulatory role in this process during high temperature stress (Chaki et al., 2011b, 2013). Notably, in both studies, endogenous GSNO was localized mainly in vascular tissues and, after the stress application, there was a redistribution to the cortex and epidermal cells. The authors suggest that this situation may be a consequence of a protective mechanism since these cells are in direct contact with the external environment, highlighting the importance of GSNO in modulating the adaptive response of sunflower plants to these stresses. As a general rule, when there is a down-regulation of GSNOR, GSNO/SNOs usually increase, and vice versa. However, there are some examples in which this correlation is disrupted. For instance, in pea plants subjected to different types of abiotic stress (Barroso et al., 2006; Corpas et al., 2008) and in the roots of Medicago plants overexpressing GSNOR (Thalineau et al., 2016), total SNOs increase concomitantly with greater GSNOR activity. In the former, NO increases in some stresses but not in others, and NO levels do not change in the latter. Similarly, the accumulation of SNOs, independent of NO production, together with an increase of GSNOR activity and transient tyrosine nitration may also be related to the acclimation response of pepper plants to low temperatures (Airaki et al., 2012). Unfortunately, the GSNO levels were not analysed in these works, but it can be hypothesized that in these cases the accumulation of total SNOs may be caused by a GSNO transnitrosylation-independent mechanism. This would be a worthwhile field to explore fully. Informatively, nox1 mutants (accumulate NO) could alter SNOs levels but to a lesser extent than a GSNOR loss- of-function mutant. It is worth noting that the latter could also slightly increase NO levels (Yun et al., 2016). These results suggest that it would be crucial to measure GSNO levels to establish whether the NO-dependent response is mediated mainly by GSNO or NO, since both could have an additive function during development and disease resistance (Yun et al., 2016). However, the quantification of SNOs is not an easy task because, although different methods have been developed (reviewed by Broniowska et al., 2013; Zaffagnini et al., 2016), it is still difficult to differentiate total SNOs from GSNO. To resolve this, some methods specifically to detect and quantify GSNO have been proposed in animals (reviewed in Broniowska et al., 2013), but in plants this field remains to be explored in more depth. Next, we address other potential roles of GSNO/SNOs that have been established using GSNOR transgenic mutant lines. This strategy has been employed mainly to characterize the role of GSNO/SNOs during plant immunity (for more details, see below). Along the same lines, a wealth of evidence has established a direct role for GSNO/SNOs in plant responses to abiotic stress, such as thermotolerance, cell death, alkaline stress (Lee et al., 2008; Chen et al., 2009; Gong et al., 2015; Kovacs et al., 2015), or the aforementioned wounding stress (Espunya et al., 2012). Lee et al. (2008) identified different Arabidopsis thermotolerance-defective mutants, collectively called hot5 (sensitive to hot temperature 5). It is worth noting that HOT5 was subsequently found to encode a GSNOR1 gene. The null hot 5-2 mutants (identical to atgsnor1-3 mutants) exhibited higher total SNO levels and heat sensitivity, highlighting the importance of GSNO metabolism in Arabidopsis thermotolerance (Lee et al., 2008). Paraquat is a herbicide that triggers cell death via increased ROS production (Chen et al., 2009). Using an extensive genetic approach, the paraquat resistance 2 (PAR2) gene was found to be identical to GSNOR1. Therefore, the loss of function of PAR2, generally named par2-1, is a mutated allele of GSNOR1 that expresses an unstable GSNOR protein, leading to the resistance to paraquat-induced cell death (Chen et al., 2009). This resistance was related to the higher SNO levels present in these mutants compared with the wild-type plants, but the exact mechanisms underlying this protection remained unclear. However, some information on these protective mechanisms has recently been reported (Kovacs et al., 2016). This study showed that GSNOR can be oxidized by H2O2in vitro, and paraquat induced oxidative stress in vivo, inhibiting its activity and therefore accumulating SNOs. Notably, the authors showed that in a atgsnor1 background, genes involved in antioxidant responses are up-regulated. At the same time, the high level of GSNO/SNOs induced a greater GSH-dependent antioxidant capacity compared with wild-type plants, and therefore led to a protective effect against the paraquat-generated oxidative stress (Kovacs et al., 2016). Supporting these results is the fact that GSNO/NO has been proposed to modulate the key antioxidant capacity of plant tissues (Begara-Morales et al., 2016b) under aluminium (Sun et al., 2014), cadmium (D. Wang et al., 2015), drought (Shan et al., 2015), and water-deficient (Silveira et al., 2017) stress conditions that ultimately are stress conditions usually leading to an oxidative stress. Moreover, GSNO metabolism is also crucial for regulating the plant response to alkaline stress (Gong et al., 2015). Loss-of-function mutation in GSNOR enhances sensitivity to alkaline stress as a consequence of elevated SNO/NO levels. In this context, there was a rise in ROS levels, causing damage to the photosynthetic apparatus. However, GSNOR overexpression lines showed more resistance to this stress, revealing the crucial importance of regulating GSNO levels in tomato plant tolerance to this abiotic stress (Gong et al., 2015). All the above demonstrate the relevance of regulating GSNO/SNO levels as key mediators of NO-dependent signalling events during the adaptive response of plants to several abiotic stress conditions. GSNO is a key player in plant immunity Although the involvement of GSNO/SNOs in the plant immune response after pathogen challenge has been widely addressed (Malik et al., 2011; Yu et al., 2014; Fig. 3), a certain controversy persists in this field, as will be highlighted and discussed in this section. Fig. 3. View largeDownload slide Role of GSNO in salicylic acid (SA)-dependent immune signalling. NPR1 homeostasis is essential in plant immunity, so the equilibrium between its monomeric and oligomeric form is crucial for plant survival after pathogen attack. Under basal conditions, NPR1 is sequestered as an oligomeric complex in the cytoplasm, a conformation that is also promoted by S-nitrosylation. Upon pathogen challenge, the increase in GSH and SA, together with TRX-h5, favours the nuclear accumulation of NPR1 that in turn mediates the expression of defence genes. NPR1 equilibrium can be modified in atgsnor1-3 plants that exhibit an excessive accumulation of GSNO/SNOs, hindering NPR1 nuclear translocation and inhibiting SABP3 function, and therefore compromising plant survival. We differentiate the effect of exogenous GSNO (green route) from endogenous GSNO in atgsnor1-3 plants (red route) that apparently generate contradictory effects. See text for a detailed discussion. Fig. 3. View largeDownload slide Role of GSNO in salicylic acid (SA)-dependent immune signalling. NPR1 homeostasis is essential in plant immunity, so the equilibrium between its monomeric and oligomeric form is crucial for plant survival after pathogen attack. Under basal conditions, NPR1 is sequestered as an oligomeric complex in the cytoplasm, a conformation that is also promoted by S-nitrosylation. Upon pathogen challenge, the increase in GSH and SA, together with TRX-h5, favours the nuclear accumulation of NPR1 that in turn mediates the expression of defence genes. NPR1 equilibrium can be modified in atgsnor1-3 plants that exhibit an excessive accumulation of GSNO/SNOs, hindering NPR1 nuclear translocation and inhibiting SABP3 function, and therefore compromising plant survival. We differentiate the effect of exogenous GSNO (green route) from endogenous GSNO in atgsnor1-3 plants (red route) that apparently generate contradictory effects. See text for a detailed discussion. First, it would be essential to differentiate between the potential roles of NO or GSNO during the establishment of the immune response. As mentioned above, GSNO is a stable reservoir of NO and therefore could be a major factor responsible for NO signalling after pathogen attack. It has in fact been shown that GSNO and NO act additively and may have distinct or overlapping molecular targets during development and immune response (Yun et al., 2016), confirming that atgsnor1-3 plants can be a good background to analyse the effect of GSNO content during plant immunity (Feechan et al., 2005). It has recently been shown that thioredoxin-h5 (TRXh5) can specifically act as a denitrosylase enzyme mainly through a transnitrosylation mechanism using a single active cysteine (Kneeshaw et al. 2014). Further, TRXh5 rescued immunity after pathogen challenge in nox1 plants that accumulate NO but fails in restoring immunity in atgsnor1 mutants that exhibit high GSNO levels. Based on these results, TRXh5 could discriminate between different protein-SNO substrates, highlighting that TRXh5 and GSNOR may denitrosylate a different set of protein-SNOs (Kneeshaw et al., 2014). Accumulation of GSNO/SNOs has been reported to have different effects on plant resistance to pathogens that ultimately could be a consequence of different SA accumulation in the distinct Arabidopsis transgenic lines. In a pioneer study, Feechan et al. (2005) demonstrated that GSNO/SNO accumulation in atgsnor1-3 Arabidopsis transgenic lines compromises the expression of multiple modes of plant disease resistance. In this scenario, the high level of S-nitrosylation negatively regulates SA biosynthesis and signalling, implying a pivotal role for GSNO/SNO during the establishment of SA-dependent immune response (Feechan et al., 2005) (Fig. 3). Conversely, a positive regulation of plant defence against pathogens was reported in Arabidopsis as a consequence of GSNO/SNO accumulation in plants with a defective GSNOR activity (Rustérucci et al., 2007). These apparently contradictory results could be explained by at least two features of the transgenic lines. (i) In atgsnor1-3 lines, there is a significant decrease in free SA, whereas in the other transgenic line SA levels remain unaffected (Rustérucci et al., 2007; Espunya et al., 2012). Thus, the decline in SA production could compromise plant defence in atgsnor1 as this hormone is a pivotal regulator of plant immunity (Rustérucci et al., 2007). (ii) These different responses may be derived from the different strategies to eliminate/reduce GSNOR function. Informatively, atgsnor1-3 plants are null mutants with a T-DNA insertion (Feechan et al., 2005) whereas, in the other case, an antisense approach was taken to develop a mutant that still conserves 50% of GSNOR transcript accumulation and activity (Rustérucci et al., 2007; Espunya et al., 2012). Thus, GSNO levels could differ between these transgenic lines and consequently exhibit a different response upon pathogen challenge. The main relevance of GSNO as a regulator of plant immunity is related to the control of the function of two crucial players in plant response to pathogens, SA-binding protein 3 (SABP3) and especially non-expressor of pathogenesis-related genes 1 (NPR1) (Fig. 3). S-Nitrosylation of AtSABP3 at Cys280 impairs both SA binding and CA activity, impacting plant defence against pathogens (Fig. 3). The inhibition of its CA activity has been proposed to contribute to a negative feedback loop that may modulate plant defence response (Wang et al., 2009). Furthermore, in atgsnor1-3 plants there is an increase in SNO-SABP3, and the CA activity of this protein is compromised after pathogen challenge, establishing a key role for GSNO-mediated transnitrosylation in the SA-dependent immune response in plants (Wang et al., 2009). NPR1 is a redox-sensitive transcriptional co-regulator involved in controlling SA-dependent immune signalling in plants (Pajerowska-Mukhtar et al., 2013). GSNO has emerged as a key regulator of NPR1 during plant immune response, but its exact impact on NPR1 function has raised some controversy. Once again, the differences observed could be a consequence of the different SA accumulation in the different strategies used to analyse NPR1 function. Under basal conditions, NPR1 is usually sequestered in the cytoplasm as an inactive oligomeric complex formed via disulphide bonds between conserved cysteines (Mou et al., 2003). Upon pathogen challenge, cellular redox changes lead to higher levels of SA and TRXh5 that mediate the release of the NPR1 monomer. At this time, NPR1 can be translocated to the nucleus and regulate the expression of defence genes involved in plant immunity (Kinkema et al., 2000) (Fig. 3). However, after pathogen challenge, the GSNO-mediated S-nitrosylation of NPR1 at Cys156 could favour the formation of the inactive oligomeric form, therefore compromising plant immune response (Tada et al., 2008). In this regard, both the oligomeric and monomeric form of NPR1 could be induced after pathogen challenge, suggesting that the equilibrium between these two forms of NPR1 is a crucial regulatory point of plant immunity (Tada et al., 2008). Therefore, the oligomer formation by S-nitrosylation could be a prior step in order to avoid NPR1 depletion, thereby maintaining the NPR1 homeostasis (Tada et al., 2008). When there is an excess of GSNO/SNOs, as in atgsnor1-3 plants, this balance would be shifted towards the formation of NPR1 oligomers, consequently compromising plant immune response. Conversely, the treatment of Arabidopsis protoplasts with exogenous GSNO favours the translocation of NPR1 to the nucleus, promoting its interaction with TGA1 and thereby positively regulating the plant immune response (Lindermayr et al., 2010) (Fig. 3). These different effects of GSNO on NPR1 conformation might be a consequence only of different SA accumulation, which is necessary for NPR1 monomerization, given that atgsnor1-3 plants show reduced levels of SA (Feechan et al., 2005). This result was recently confirmed in a study showing that nuclear translocation of NPR1 in Arabidopsis protoplasts after GSNO treatment could be a consequence of increased GSH levels that subsequently trigger SA accumulation. Thus, this increase in SA levels would be responsible for NPR1 monomerization and translocation to the nucleus in GSNO-treated protoplasts (Kovacs et al., 2015) (Fig. 3). Other causes that could explain these differences emerge from a technical standpoint. In this regard, this controversy could reflect the use of plant material with different physiology, such as the protoplast (Lindermayr et al., 2010; Kovacs et al., 2015) and whole leaves (Tada et al., 2008). In addition, the differences could be a consequence of the different GSNO source: endogenous (in atgsnor1-3 plants) versus exogenous (GSNO treatment). These differences could be a reflection only of the GSNO concentration reached at the cellular level in both cases. The use of exogenous GSNO at 100 µM in protoplasts could be excessive, given that the concentration of GSNO in Arabidopsis leaves was reported to be ~3.7 nmol g–1 FW (Airaki et al., 2011). Similarly, the concentrations of total SNO and LMW SNO in atgsnor1-3 plants have been previously reported to be ~50 pmol mg–1 protein and 5 pmol mg–1 protein, respectively (Feechan et al., 2005). However, as stated above, the exact concentration of GSNO in these plants remains to be elucidated. Thus, the development of a robust method to determine the endogenous and pathogen-induced GSNO levels is required, since if there were significant differences, a GSNO threshold might be established from which plant response to pathogens could be compromised. Collectively, these results on NPR1 function appear to be contradictory, but they highlight that GSNO/SNOs play an essential role in the fine-tuned regulation of NPR1 that is vital for plant survival upon pathogen challenge. Finally, GSNO/SNOs also have an impact in the control of the hypersensitive response (HR), a programmed execution of plant cells at sites of attempted infection. In this context, NO and reactive oxygen intermediates have been shown to be involved in the development of HR (Delledonne et al., 2001). When GSNO/SNO levels are elevated as a consequence of GSNOR mutation, pathogen-triggered cell death is accelerated (Yun et al., 2011). In this scenario, excessive S-nitrosylation upon pathogen challenge leads to the inactivation of the NADPH oxidase AtRBOHD, diminishing its ability to synthesize reactive oxygen intermediates. This enzymatic inhibition is thought to be a mechanism regulating a negative feedback loop that limits the extent of cell death in Arabidopsis (Yun et al., 2011). Final remarks NO is an important biological messenger acting as a key regulator of a wide range of physiological and stress response processes in plants. NO usually transmits its bioactivity via PTMs such as the reversible S-nitrosylation. The S-nitrosylation of GSH leads to generation of GSNO, the major LMW SNO and, more importantly, a stable and mobile reservoir of NO acting as relevant NO buffering in the plant system. Thus, GSNO can be considered a key player during plant response to stress, regulating the NO-dependent signalling response. Besides the presence of GSNO in vascular tissues, there is growing evidence suggesting that it can act as a long-distance signalling molecule. GSNO has been involved in plant response to abiotic stress and plant immunity, but certain contradictory results remain to be clarified. These conflicting data, which are related mainly to the effect of GSNO on NPR1 conformation during plant immunity, could be related to the GSNO concentration reached during treatments. Therefore, the development of a reliable and accurate method to quantify GSNO specifically under basal and stress response conditions is needed. 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Journal of Experimental BotanyOxford University Press

Published: Mar 1, 2018

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