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Nitric oxide imbalance provokes a nitrosative response in plants under abiotic stress

By Betty Jones,2014-09-24 19:01
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Nitric oxide imbalance provokes a nitrosative response in plants under abiotic stressa,in,oxide,under,plant,Oxide,Plant

    Nitric oxide imbalance provokes a nitrosative response in plants under abiotic stress Francisco J. Corpasa, , , Marina Leterriera, Raquel Valderramab, Morad Airakia, Mounira Chakia, José M. Palmaa and Juan B. Barrosob

    a Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín (EEZ), CSIC, Granada, Spain

    b Grupo de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al CSIC (EEZ), Área de Bioquímica y Biología Molecular, Universidad de Jaén, Spain Received 20 January 2011;

    revised 11 March 2011;

    accepted 12 April 2011.

    Available online 22 April 2011.

    Abstract

    Nitric oxide (NO), a free radical generated in plant cells, belongs to a family of related molecules designated as reactive nitrogen species (RNS). When an imbalance of RNS takes place for any adverse environmental circumstances, some of these molecules can cause direct or indirect damage at the cellular or molecular level, promoting a phenomenon of nitrosative stress. Thus, this review will emphasize the recent progress in understanding the function of NO and its production under adverse environmental conditions.

    Graphical abstract

Full-size image (13K)

Highlights

    ? Nitric oxide has a family of NO-related molecules designated as reactive nitrogen species (RNS) such as S-nitrosothiols or peroxynitrite. ? RNS participate in the mechanism of response against environmental stresses and can provoke post-translational modifications. ? A rise of

    protein tyrosine nitration is a reliable footprint of nitrosative stress. Keywords: Abiotic stress; Nitric oxide; Nitrosative stress; Nitrotyrosine; Reactive nitrogen species; Salinity

    Abbreviations: ABA, abscisic acid; CLSM, confocal laser scanning microscopy; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DAF-2 DA, 4,5-diaminofluorescein diacetate; DAF-FM DA, 4-aminomethyl-2′,7′-difluorofluorescein diacetate;

    DPI, diphenyleneiodonium; GSNO, nitrosoglutathione; JA, jasmonic acid; L-NAME, L-NG-nitroarginine methyl ester; L-NNA, NG-nitro-l-arginine; L-NMMA, L-NG-monomethyl

arginine acetate; NR, nitrate reductase; NO, nitric oxide; NOS, nitric oxide synthase; O2?,

    superoxide radical; ONOO?, peroxynitrite ion; PTIO,

    2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; RNS, reactive nitrogen species; ROS, reactive oxygen species; SA, salicylic acid; SNAP, S-nitroso-N-acetyl-penicillamine; SNOs, S-nitrosothiols; SOD, superoxide dismutase; SNP, sodium nitroprusside

    Article Outline

    1.

    Introduction

    2.

    Metabolism of NO in plant cells

    2.1. Potential sources of NO

    2.2. Reactive nitrogen species and nitrosative stress

    3.

    Nitric oxide and environmental abiotic stress

    3.1. Salinity

    3.2. Water stress

    3.3. Extreme heat and cold

    3.4. Mechanical wounding

    3.5. UV radiation

    3.6. Ozone

    3.7. Heavy metal

    4.

    Concluding remarks

    Acknowledgements

    References

    1. Introduction

    The gases nitric oxide (NO) and nitrogen dioxide (NO2) are commonly known under the term nitrogen oxides (NOx). The main interest of these gases concerns their involvement in air pollution because they contribute to acid rain, and the depletion of the ozone layer and they have harmful effects on human heath. However, the attention on NO suffered a significant change when it was identified, separately by Moncada and Ignarro, as the endothelium-derived relaxing factor (EDRF) [1] and [2]. Later, it was demonstrated that this gas was generated in mammalian cells from the amino acid l-arginine by a family of enzymes designated as nitric oxide synthases (NOS) [3] and [4]. Thus, in animals NO participates in a broad spectrum of functions in the cardiovascular, immune, and nervous systems [4]. However, NO is also involved in a wide array of human pathologies such as tumours, heart disease, asthma, infection diseases, diabetes, hypertension, atopic dermatitis, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis, among others. In higher plants, NO also has an important function in plant growth and development, including seed germination, primary and lateral root growth, flowering, pollen-tube growth regulation, fruit ripening, senescence, defence response, and abiotic stress, in addition to being a key signalling molecule in different intracellular processes [5], [6], [7], [8], [9] and [10]. 2. Metabolism of NO in plant cells

    Nitric oxide or nitrogen monoxide is a colourless gas which belongs to the free radical-type molecules because it has an unpaired electron in its π orbital, making NO a special molecule.

    Among these properties, NO has a solubility of 1.9 mM in aqueous solutions at 1 atm pressure; it diffuses at a rate of 50 μm per s, its in vivo lifetime is relatively short (less than 10 s), and it is a

    lipophilic radical that can diffuse across cell membranes as well as through the cytoplasm, reacting with a certain number of macromolecules (proteins, lipids, nucleic acids, etc.). Another relevant aspect of NO or NO-derived molecules is that directly or indirectly they may be involved in post-translational changes including binding to metal centres, S-nitrosylation of thiol groups, and the nitration of tyrosine, which may be involved in cell signalling under physiological and stress conditions.

    2.1. Potential sources of NO

    Even when the relevance of NO in plants is completely recognized, at present, a major question is still to determine which endogenous NO source(s) is (are) involved in a given physiological or stress process. Hence, plants can generate NO by non-enzymatic and enzymatic mechanisms [11], [12], [13] and [14], the l-arginine and nitrite pathways being the most plausible routes. The production of NO for l-arginine-dependent nitric oxide synthase (NOS) activity has been reported in different plant species having similar cofactor requirements, identified in mammalian systems (for a review see Corpas et al. [12]). Fig. 1A shows the biochemical characterization of a NOS activity in sunflower, where NO production depends strictly on the l-Arg concentration, BH4, with calmodulin and calcium activity strongly being reduced (8297%) in the absence of its cofactors

    (NADPH, FAD and FMN). In addition, NOS activity is also reduced a 97% in the presence of the animal NOS inhibitors AG or L-NMMA [15]. However, at this moment, search for the protein and gene of plant NOS continues [16] and [17]. Very recently the sequences of two l-arginine dependent NOS enzymes have been described from two green algal species of the genus Ostreococcus [18]. Furthermore, nitrate reductase (NR) is another NO producer, using NO2? and NADH as substrates [19], and it has been proposed that it is involved in NO production in some processes such as stomatal closure [11]. However, there is little information on the direct production of NO from NR in plant stress and the available information came from NR mutants that are impaired in NO production [20].

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Fig. 1.

    Biochemical characterization of l-arginine-dependent nitric oxide synthase (NOS) activity in hypocotyls of sunflower (Helianthus annuus L). Reaction mixtures containing hypocotyl samples were incubated in the absence and presence of l-arginine (1 mM or 0.1 mM), NADPH (1 mM), EGTA (0.5 mM), cofactors (10 μM FAD, 10 μM FMN and 10 μM BH4), calmodulin (CaM),

    L-NG-monomethyl arginine acetate (L-NMMA, competitive and irreversible inhibitor of all three animal NOS isoforms) and 1 mM aminoguanidine (AG, an animal NOS activity inhibitor). The

    NOS activity was quantified from the NO produced, which was determined by the ozone chemiluminescence method. l-Arg, l-arginine [15].

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    Additionally, it is known that the non-enzymatic reduction of nitrite can lead to the formation of NO, and this reaction is favoured at acidic pH values, when nitrite can convert to NO and nitrate. Nitrite can also be chemically reduced by ascorbic acid at pH 36 to yield NO and

    dehydroascorbic acid. This reaction could occur under microlocalized pH conditions in barley aleurone layers and in the chloroplast and apoplastic space where ascorbic acid is known to be present. Another non-enzymatic mechanism proposed for NO formation is the light-mediated reduction of NO2 by carotenoids [21] and [22].

    Polyamines include molecules such as putrescine, spermidine or spermine, which are synthesised from l-arginine. They are aliphatic nitrogen compounds positively charged at physiological pH, allowing its interaction with molecules having a negative charge such as proteins, nucleic acids or phospholipids. Recently, it has been shown that polyamines can induce the production of NO [23], but the mechanism is unknown. Future analysis in this sense could clarify the new signalling pathway considering that polyamines are also involved in the response mechanism against stress conditions [24].

    2.2. Reactive nitrogen species and nitrosative stress

    The term reactive nitrogen species (RNS) was introduced in the biological literature to designate NO and other NO-related molecules, such as S-nitrosothiols (SNOs), S-nitrosoglutathione (GSNO), peroxynitrite (ONOO?), dinitrogen trioxide (N2O3) and nitrogen dioxide (NO2), which

    have relevant roles in multiple physiological processes of animal and plant cells [7], [25] and [26]. S-Nitrosothiols, and particularly GSNO, seem to have a relevant role in the biochemistry of NO because they may function both as an intracellular NO reservoir and as a vehicle of NO throughout the cell [27]. In this context, a reaction of S-transnitrosation is considered to be the main mechanism for the biological effects of SNOs [28]. Consequently, they may be involved in post-translational changes in cell signalling under physiological and phytopathological conditions. However, in higher plants the information available on the metabolism of SNOs and RNS is still limited compared to that in animal systems.

    The term “oxidative stress” describes the cellular damage caused by overproduction of ROS, and the term nitrosative stress was introduced to describe a similar process caused by RNS in plants under stress conditions. Consequently, it is considered that under a specific situation the plant undergoes nitrosative stress when there is a de-regulated synthesis or overproduction of NO and NO-derived products that can have toxic physiological consequences. In this context, it is also important to define a reliable marker or footprints of this type of stress [29]. So far, a body of data has revealed that a rise of protein tyrosine nitration could be a good marker to evaluate a process of nitrosative stress [15], [30] and [31].

    S-Nitrosoglutathione is formed by the reaction of NO with reduced glutathione (GSH) in the presence of oxygen and, as mentioned previously, it can function as a mobile reservoir of NO bioactivity in animal and plant cells [32], [33], [34] and [35]. Liu et al. [36] reported that the enzyme designated as class III alcohol dehydrogenase (ADH3) is available to catalyse the NADH-dependent reduction of GSNO to oxidized glutathione (GSSG) and NH3, being also

    designed as glutathione-dependent enzyme formaldehyde dehydrogenase (FALDH; EC 1.2.1.1) or GSNO reductase (GSNOR) activity. So far, the presence of GSNOR activity in plants has been reported in different plant species [37] including arabidopsis [38] and [39], tobacco [34], pea [40] and [41] and sunflower [42], among others.

    Fig. 2A shows a schematic model of NO metabolism in plant cells under environmental stress conditions. NO can be generated enzymatically either by l-arginine-dependent nitric oxide synthase (NOS) activity using NADPH as an electron donor or from nitrite/nitrate by nitrate reductase (NR) using NADH. Alternatively, NO can be induced by polyamines which are generated also from l-arginine. This NO can react with reduced glutathione (GSH) in the presence of O2 to form GSNO. This metabolite can be converted by the enzyme GSNOR into oxidized glutathione (GSSG) and NH3. However, alternatively, GSNO in the presence of reductants, such as GSH or ascorbate, and Cu+, can be broken down to NO [43], [44] and [45]. On the other hand, NO can react with the thiol group of cysteine residues of proteins (S-nitrosylation) altering its function. This covalent modification is not a direct reaction and it could probably be performed through the formation N2O3 in the presence of O2, by nitrosonium (NO+) or by a process of transnitrosylation from GSNO among others [46], [47] and [48]. Moreover, NO can react with superoxide radicals (O2?) to generate ONOO?, a powerful oxidant than can mediate the tyrosine nitration of proteins. This post-translational modification involves the addition of a nitro (NO2)

    group to one of the two equivalent ortho-carbons of the aromatic ring of tyrosine residues [49]. Under stress conditions, a boost could be expected in the number of proteins or an intensification of specific protein targets of tyrosine nitration that it can be considered an indicator of nitrosative stress in plants [29].

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Fig. 2.

    (A) Model of NO metabolism in plant cells under abiotic stress. GSNOR, nitrosoglutathione reductase. NO+, nitrosonium. NOS, l-arginine-dependent nitric oxide synthase activity although the corresponding protein and gene have not be identified yet. NR, nitrate reductase. (B) Representative images illustrating the confocal laser-scanning microscopy (CLSM) detection of endogenous NO with 10 μM DAF-FM DA in cross-sections of leaves of control pea plants

    subjected to mechanical wounding. Bright-green fluorescence corresponds to NO detection. The red-orange colour corresponds to the chlorophyll autofluorescence. Adaxial epidermis (E1), abaxial epidermis (E2), main vein (V), palisade mesophyll (Pm), and spongy mesophyll (Sm). Scale bar = 150 μm.

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3. Nitric oxide and environmental abiotic stress

    Plants are exposed to a plethora of stress conditions such as salinity, extreme temperatures, drought, and heavy metals, this seriously threatening crop production and deteriorating environments. Thus, abiotic stress is estimated to be the primary cause of worldwide crop loss, which exceeds 50% [50]. The direct or indirect implication of NO in the mechanism of response against some environmental stress has been reported in different plant species. Here, we review how NO and other RNS interacts and how they are modulated under certain environmental/abiotic stress conditions.

    3.1. Salinity

    Salinity affects plant productivity due of its negative effects on plant growth, ion balance, and water relations. In many plant species it has been shown that NaCl provokes oxidative stress [51], [52], [53] and [54].

    The involvement of NO in the mechanism of response to salinity has begun to be studied, although the data available can sometimes be contradictory, depending of the plant species and the severity of the salinity treatment. In olive plants grown under in vitro conditions, salt stress (200 mM NaCl) augmented the l-arginine-dependent production of NO, total SNOs, and the number of proteins that underwent tyrosine nitration in the molecular-mass range between 44 and 60 kDa. Moreover, confocal laser scanning microscopy (CLSM) analysis using either specific fluorescent probes for NO and SNOs or antibodies to GSNO and 3-nitrotyrosine also showed a general increase in these RNS mainly in vascular tissue [31]. Thus, these findings appear to indicate that in olive leaves, salinity induces nitrosative stress, while vascular tissues could play an important role in the redistribution of NO-derived molecules throughout the different organs of the plants.

    There are also studies using indirect approaches by the exogenous application of NO donors and the effects in plants exposed to salinity stress. Thus, in the calluses of reed (Phragmites communis) under 200 mM NaCl treatment, the addition of SNP (a NO donor) stimulated the expression of the plasma membrane H+-ATPase, indicating that NO serves as a signal-inducing salt resistance by increasing the K+ to Na+ ratio [55]. In this model, the effect of NO was reversed by the application of L-NNA and PTIO (NOS activity inhibitors and NO scavenger, respectively), suggesting that NO was produced from NOS-like activity and not as a by-product of NR [55]. Similar results were found in maize, where the addition of exogenous NO also booted the salt-stress tolerance by elevating the activities of the proton-pump and the Na+/H+ antiport of the tonoplast [56].

    In sunflower seedlings, it has been demonstrated that NO provoked biochemical adaptation during the seedling growth under salinity conditions. Thus, the Na/K ratio increased 4-fold in roots, and Na+ was rapidly transported to the cotyledons, which registered a concomitant increase in this ratio. The origin of this endogenous generation of NO appears to be mediated by NOS activity. On the other hand, in response to 120 mM NaCl treatment, oil bodies from the cotyledons of 2-d-old seedlings exhibited a stronger NO signal [57].

    In Lupinus luteus, 200 mM NaCl inhibited germination, but seed preincubation with the NO donor SNP restored germination [58]. Similarly, in the succulent shrub Suaeda salsa, a halophyte, NO

    stimulates seed germination more efficiently than nitrate under salt stress [59]. In wheat plants exposed to 150 mM NaCl the interaction between carbon monoxide (CO) and NO has been reported. Accordingly, NaCl treatment increased CO accumulation in wheat-seedling roots, boosting the activity of haem oxygenase involved in CO synthesis with a concomitant rise in the NO production. Under these circumstances the exogenous application of CO plus NO to wheat seedlings attenuated the salinity toxicity which was mediated by up-regulation of antioxidant defence and maintenance of ion homeostasis. Thus, in this work, it is suggested that NO must be a downstream signal of the CO action [60].

    In 8-d-old rice plants pre-treated with 1 μM SNP or 10 μM H2O2 in the hydroponic solution for

    2 d, salt (100 mM NaCl) tolerance increased. This pre-treatment induced the activity of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase) as well some stress-related genes (sucrose-phosphate synthase, Δ′-pyrroline-5-carboxylate synthase, and small

    heat-shock protein 26). Moreover, similar behaviour was found under heat stress [61]. Similar observations have been reported in 5-month-old bitter orange (Citrus aurantium L.) trees, root pre-treatment with H2O2 or SNP induced major antioxidant defence (SOD, catalase, APX, and GR) responses in the leaves of citrus plants grown both in the absence or presence of 150 mM NaCl for 16 d. This induction of antioxidant system provided major resistance to salinity stress [62]. Moreover, a proteomic analysis in leaves from pre-treatment citrus plants showed that these treatments reduced protein carbonylation and modified the accumulation levels of leaf S-nitrosylated proteins. These results suggest an overlap between NO and H2O2 signalling pathways in salinity acclimation [63].

    Another component that appears to be involved in the signalling process mediated by NO during the salinity response is the family of protein kinases. Thus, in tobacco-cell suspensions exposed to salt stress, the osmotic stress-activated protein kinase (designated as NtOSAK) is activated by NO. A fuller analysis of this kinase revealed that the activation was not due to a process of S-nitrosylation, as might be expected, but rather a process of phosphorylation of two residues located in the kinase activation loop was found. Additionally, in this situation, it was observed that NtOSAK interacted with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the enzyme involved in glycolysis to provide energy and carbon molecules, which underwent a process of S-nitrosylation which affected neither GAPDH activity or nor interaction with NtOSAK [64]. Recently, in arabidopsis wild type and mutants expressing green fluorescent protein (GFP) through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, it has been shown that under salinity stress (100 mM NaCl), peroxisomes are required for NO accumulation in the cytosol of root cells, thereby participating in the generation of peroxynitrite (ONOO?) and in increasing protein tyrosine nitration. In this case the generation of NO in peroxisomes seems to be mediated by a putative calcium-dependent NOS activity [65]. 3.2. Water stress

    Water stress, frequently caused by drought, has major impact also on plant growth and development. Thus, the low water accessibility cause physical limitations, and the typical mechanism is the stomatal closure to conserve water. Then, the pathway for the exchange of water, CO2, and O2 are also closed, resulting in a decrease in photosynthesis. In this sense, it is now well known that NO-induced stomatal closure enhanced the adaptive plant responses against drought stress [66].

    In maize, the treatment with 10% polyethylene glycol induces water stress, leading to a rapid

    increase of NO in mesophyll cells of leaves. The NOS activity was remarkably induced in cytosolic and microsomal fractions by water stress, the activity of NOS in the microsomal fraction being higher. This behaviour was accompanied by an increase of antioxidant enzymes including superoxide dismutase, ascorbate peroxidase, and glutathione reductase [67]. Using a NO treatment with SNP, additional studies showed that this could alleviate the water loss and oxidative damage of maize leaves observed under water-deficit stress and a pharmacological analysis using a NOS-activity inhibitor seems to indicate that a NOS-like activity is also responsible of NO generation in this plant specie [68].

    In the roots of cucumber seedlings, a slightly enhanced of NO synthesis in the cells of root tips and in the surrounding elongation zone has been described. This NO production was reduced by pre-treatment with NOS and NR inhibitors. Exogenous applications of NO by either SNP or GSNO indicate the adaptive response of roots to water stress. Additionally, the induction of lipoxygenase observed in cucumber roots under water stress that mediates lipid peroxidation was also reduced with this exogenous NO application [69].

    3.3. Extreme heat and cold

    Extreme temperatures are the main factors limiting plant growth. High temperature (HT) is considered one of the major abiotic stresses that negatively affect both vegetative and reproductive growth. In general, HT can generate heat stress, which in turn produces specific families of proteins known as heat-shock proteins (HSPs) [70]. However, HT can also cause an overproduction of ROS, which could be involved in triggering defence responses against potentially damaging temperatures [71], [72], [73] and [74].

    In this sense, there is a set of data showing a rise in NO production under HT. For example, in alfalfa sprouts, heat stress (37 ?C for 2 h) resulted in a doubling of the rate of NO emission [75]. In tobacco, HT generates a rapid and significant rise in NO in adaxial epidermal cells after 7 min of heat treatment at 40 ?C and in suspension cells after 5 min of heat treatment at 45 ?C, evaluated by the fluorescence probe DAF-2 DA [76]. Conversely, pea plants exposed at 38 ?C for 4 h reduced the NO content of leaves but it did not significantly affect the NOS-like activity. However, it was found that the SNO content increased 3-fold and that some nitrated proteins intensified [30]. In sunflower seedlings under the same experimental conditions (38 ?C for 4 h) have been reported to undergo oxidative stress, which was accompanied by a reduction of NO production, inhibition of GSNOR activity, accumulation of S-nitrothiols, formation of peroxynitrite, and rise of protein nitration. A proteomic analysis of some specific targets identified nitration as causing an inhibition of the activities both of carbonic anhydrase and of ferredoxin-NADP reductase, proteins involved in photosynthesis [15]. In tobacco Bright Yellow-2 (TBY-2) suspension cells exposed for 10 min to 35 ?C or 55 ?C and then 27 ?C, the analysis of the released NO showed that 35 ?C heat-shocked cells had a low production of NO compared to cells exposed to 55 ?C, the latter showing fast-increasing NO production. This production was well correlated with the cell viability and DNA integrity as well with the presence of oxidative stress markers [72]. In arabidopsis, several mutants have been identified to have impairment in the GSNOR1 gene, showing the involvement of this gene in the mechanism of response against HT. Thus, the mutant HOT5 (sensitive to hot temperatures) showed that GSNOR modulates the intracellular level of SNOs, enabling thermo tolerance as well the regulation of plant growth and development [77]. In calluses of reed (P. communis), the exogenous application of SNP or ABA elevated thermo-tolerance by alleviating ion leakage, lipid peroxidation, and growth suppression induced by heat stress (45 ?C for 2 h).

    However, the pretreatment with L-NNA or cPTIO aggravated the damages caused by heat stress and blocked the protective effect of exogenous ABA. On the other hand, exogenous ABA notably activated NOS activity and increased NO release, maintaining the heat tolerance [78]. Taken together, the data indicate that the heat stress affects NO metabolism but the increase or decrease of this molecule depends on the degree of the treatment and plant tissues or species. Low temperature (LT), an environmental stress that affects plant growth and consequently crop production and quality, has been shown to regulate the expression of many genes, as well as the level of certain proteins and metabolites [79]. An analysis of NO and other RNS in pea plants exposed to LT (8 ?C for 48 h) showed activation of l-arginine NOS and GSNOR activities as well an increase in the content of SNOs. Moreover, in control plants, a pattern of six immunoreactive bands that underwent tyrosine nitration with molecular masses of 59, 42, 39, 37, 33, and 29 kDa was found, and after LT treatment an intensification of the immunoreactive bands in the range 2959 kDa was observed. Therefore, these results indicate that metabolism of RNS is triggered in response to LT and this implies a connection between NO and this stress [41]. In leaves of pepper plants, LT (8 ?C for 24 h) caused cold stress characterized by a general imbalance of the ROS and RNS metabolism, triggering a rise in the lipid oxidation and the protein tyrosine nitration. This point towards an induction of oxidative and nitrosative stress promoted by LT [80]. Similar behaviour has been observed in Arabidopsis thaliana exposed to 4 ?C for 14 h [81] or during cold

    acclimation [82], where the NO content increased. In Brassica juncea seedlings, LT stress (4 ?C for 148 h) provoked a rise of SNOs. Moreover, a proteomic analyse showed that LT induced differentially nitrosylated proteins being involved in photosynthesis, plant defence, glycolysis and signalling process. Notably, both subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) showed an increase as well as a decrease in nitrosylation by LT. Thus, LT inactivated Rubisco carboxylase by a process of S-nitrosylation which is well correlated with the photosynthetic inhibition detected under this type of stress [83].

    Consequently, all these results indicate a connection between NO metabolism and stress caused by extreme temperatures.

    3.4. Mechanical wounding

    Mechanical wounding in plants can be a consequence of diverse environmental stresses such as wind, sand, rain, or herbivores. In general, plants respond with the induction of numerous genes because the open wound could be a potential infection site for pathogens. Therefore, the expression of defence genes at the wound site is a barrier against opportunistic microorganisms [84] and [85].

    In tomato plants, the application of NO donors such as SNP or SNAP reportedly inhibited the expression of wound-inducible proteinase inhibitors [86]. In A. thaliana, mechanical wounding was found to induce a rapid accumulation of NO that could be involved in

    jasmonic-acid-associated defence responses and adjustments [87]. In pea leaves, mechanical wounding also prompted an accumulation of NO after 4 h evaluated by the fluorescence probe DAF-FM DA (Fig. 2B), and this was accompanied by a general induction of NOS and GSNOR activities as well an increase in the content of SNOs [41]. However, the pattern of proteins that undergo tyrosine nitration did not appear to be affected.

    In sunflower hypocotyls, mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while protein tyrosine nitration increases. Consequently, a process of nitrosative stress is induced in sunflower seedlings and S-nitrosothiols

could constitute a new wound signal in plants [15].

    3.5. UV radiation

    The UV-B radiation (280320 nm) has increased as a consequence of the destruction of the ozone layer, and this radiation clearly affects plant growth and usually induces oxidative stress (reduced photosynthesis, increased damage to DNA).

    In A. thaliana a NOS inhibitor and an NO scavenger partially blocked the induction by UV-B of the chalcone synthase gene involved in producing chalcones, molecules that participate in defence mechanisms and in the production of protective pigments such as flavonoids [88]. In maize seedlings, UV-B radiation strongly stimulated NOS activity while lowering both leaf biomass and depressing exo- and endo-β-glucanase activity [89]. Moreover, UV-B triggered a rise in ROS

    widely distributed in chloroplasts and mesophyll cells, causing cell damage. A noteworthy finding has been reported regarding the pre-treatment with apocynin, a natural organic compound structurally related to vanillin and used as an inhibitor of NADPH oxidase. That is, it was observed that apocynin reduces UV-B-induced oxidative damage because it reduces the chlorophyll breakdown caused by H2O2, and this was correlated with NO production mediated by a NOS activity [90]. In the case of maize leaves, the UV-B irradiation provoked a rise in the concentration of ABA, H2O2, and NO in leaves, and this ABA appears to be required for the NO-mediated attenuation of deleterious effect of this stress. Pre-treatment with DPI (inhibitors of NADPH oxidase) and L-NAME partially blocked the NO accumulation. On the other hand, the accumulation of endogenous NO in maize leaves in response to UV-B radiation is ABA-dependent and is paralleled by greater tolerance to high doses of UV-B radiation [91].

    In bean seedlings subjected to UV-B radiation, exogenous NO partially alleviated the UV-B effect characterized by a decrease in chlorophyll contents and oxidative damage to the thylakoid membrane [92]. Moreover, UV-B induced stomatal closure, which was mediated by NO and H2O2, and NO was generated by a NOS-like activity [93]. However, other authors have reported that the NO generated in the guard cells is produced by NR activity [94]. In excised leaves of kidney bean (Phaseolus vulgaris) under UV-B stress, NO and H2O2 production has also been detected. In this situation, the treatment with L-NNA blocked the NO release. In the same way, pre-treatment with catalase not only eliminated the production of H2O2 but also inhibited the activity of NOS and the emission of NO. By contrast, treatment with exogenous H2O2 increased both events. Therefore, the authors suggest that, under UV-B stress, NO production is mediated by H2O2 through greater NOS activity [95]. In soybean plants exposed to low UV-B doses, it has been shown that ROS mediates heme-oxygenase (HO) up-regulation, which exerts a protective action against oxidative damage. Moreover, it has been observed that the induction mechanism is mediated by NO, this being produced by a NOS-like activity [96]. In stems of pea (Pisum sativum L.) seedlings exposed to UV-B radiation, the release of NO through the induction of a NOS activity has been shown. The consequence was the inhibition of stem elongation because the xyloglucan-degrading activity involved in this process was inhibited. This inhibition was reversed by the application of L-NNA and PTIO. A notable finding was that the exogenous application of NO to the rhizosphere of pea seedlings mimicked the responses of stems to UV-B radiation [97]. A similar inhibitory effect of UV-B radiation in growth has been observed in reproductive organs, specifically in pollen germination and tube growth of Paulownia tomentosa, in which, after the treatment, NO production was found to be a consequence of NOS activity. This behaviour was also observed after the application of exogenous NO by either GSNO or SNP. Moreover, the

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