Abstract: Abiotic stress is the major limiting factor of plant growth and crop yield. Better understanding of plant stress responses and tolerance is very important in the light of increasing intensities of stressors like salinity, drought, flooding, heavy metal, temperature extremes, high-light intensities, UB-radiation, herbicides, ozone and others, due to global climatic and other environmental changes. The role of Nitric oxide (NO) in stress responses in plants came in the focus of plant science in the last decade. NO is an important signaling molecule with diverse physiological and biochemical functions involving the induction of different intracellular plants processes, including the expression of defense-related and redox regulated genes against abiotic and biotic stress induced reactive oxygen species (ROS) detoxification. In spite of the significant progress that has been made in understanding NO biosynthesis and signaling in plant, several crucial questions remain unanswered. In this study, we reviewed the recent progress in NO research to reveal its diverse role in the physiological and biochemical processes in plants and the protective mechanisms towards abiotic stress tolerance.
INTRODUCTION
Abiotic stresses viz. salinity, drought, flooding, heavy metal, temperature extremes, high-light intensities, UB-radiation, herbicides, ozone are the major causes of yield loss in cultivated crops worldwide. The survival of plants under such a stressful condition depends on the plant's ability to perceive the stimulus, generate and transmit the signals and to initiate various physiological and biochemical changes (Bohnert and Jensen, 1996; Hossain and Fujita, 2009b; Hasanuzzaman et al., 2009). Nitric oxide (NO) is a highly reactive, membrane-permeable free radical which was earlier considered as a highly toxic compound. However, the discovery of NO signaling role in regulation of cardiovascular system has changed the paradigm concerning the cytotoxicity. Later, the discovery of its biological functions has been elucidated. Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signaling molecule in different intracellular processes in plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defense-related genes against abiotic and biotic stress and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and so on. NO can be produced in plants by non-enzymatic and enzymatic systems (Del Rio et al., 2004; Crawford and Guo, 2005; Delledonne, 2005; Arasimowicz and Floryszak-Wieczorek, 2007). However, the effects of NO on different types of cells have been proved to be either protective or toxic, depending on the concentration and situation.
NO triggers many kinds of redox-regulated defense-related gene expression directly or indirectly to establish plant stress tolerance (Polverari et al., 2003; Sung and Hong, 2010). Different reports have been published in recent years on the physiological function of NO (Bolwell, 1999; Wojtaszek, 2000; Beligni and Lamattina, 2001; Wendehenne et al., 2001; Neill et al., 2003; Lamattina et al., 2003) and particularly on NO signaling in the induction of cell death, defence genes and interaction with Reactive Oxygen Species (ROS) during plant defense (Van Camp et al., 1998; Durner and Klessig, 1999; Klessig et al., 2000; Delledonne et al., 2001; Wendehenne et al., 2001; Neill et al., 2003; Romero-Puertas and Delledonne, 2003). Exogenous application of NO confers tolerance to various abiotic stresses in plants by enhancing both enzymatic and non-enzymatic antioxidant defense system (Neill et al., 2002; Tian and Lei, 2006; Sheokand et al., 2008; Zheng et al., 2009; Singh et al., 2009; Xu et al., 2010). Several lines of study have shown that the protective effect of NO against abiotic stress is closely related to the NO-mediated reduction of ROS in plants (Beligni and Lamattina, 1999a; Wang and Yang, 2005).
In this review, we discuss recent progress in understanding the function of NO in plant responses and tolerance to abiotic stresses and in plant development. We explore the physiological and biochemical mechanisms of NO induced abiotic stress tolerance and the mechanisms by which it transduce signals into cellular responses towards stress tolerance.
HISTORICAL PERSPECTIVE OF NO
Nitric oxide was first described by Joseph Priestley in 1772, when it was considered as a highly toxic compound; indeed, it is a component of exhaust gas and industrial wastes. However, the discovery in the late 1980s of NO signaling role in regulation of cardiovascular system by R.F. Furchgott, L.J. Ignarro and F. Murad (Nobel Prize winners in Physiology and Medicine, 1998) has changed the paradigm concerning the cytotoxicity of free radical substances. The discovery and elucidation of its biological functions in the 1980s came as a surprise. NO was named Molecule of the Year in 1992 by the journal Science, a NO Society was founded and a scientific journal devoted entirely to NO was created (Delledonne, 2005). NO is a diffusible gaseous free radical. Its emission from plants has been reported several years ago in soybean plants (Klepper, 1979). Later, in vivo and in vitro Nitrate Reductase (NR) dependent NO production has been found in other plants such as sunflower and maize (Rockel et al., 2002). Although, NO synthase, the main enzyme that catalyses the in vivo synthesis of NO in animals has not been isolated in plants yet, NO has proved to be a functional metabolite in plants (Neill et al., 2002).
PRODUCTION OR GENERATION OF NO IN PLANTS
There are several sources of NO in nature and environment. As a pollutant, NO is produced by both automobile engines and power stations. NO is also emitted from plants under stress situations, such as herbicide treatment or pathogen attack, as well as under normal growth conditions (Wendehenne et al., 2004). In pea plants, wilting intensified the NO emission (Leshem and Haramaty, 1996) and in tobacco cells under heat, osmotic and salinity stresses, a rapid increase in NO production was observed (Gould et al., 2003). In leaves of Arabidopsis, wounding induced a fast accumulation of NO, as checked by Confocal Laser Scanning Microscopy (CLSM) and spin trapping Electron Paramagnetic Resonance (EPR) (Huang et al., 2004). These data led to postulate that NO could be a useful marker of plant stress (Magalhaes et al., 1999) and that NO generation, like that of the ROS, can occur naturally as a generalized response to different types of stress (Magalhaes et al., 1999; Gould et al., 2003). In cells, NO can exist in the form of three interconverting compounds: a free-radical nitric oxide (NO●), a nitrosonium cation (NO+) and a nitroxyl anion (NO¯) (Hong et al., 2008).
In biological systems, NO can be generated enzymatically or non-enzymatically. The most extensively described NO-producing enzymes have been Nitric Oxide Synthase (NOS) and Nitrate Reductase (NR). Much early effort by plant scientists focused on searching for a plant NOS. The enzymic oxidation of L-arginine to yield NO and L-citrulline has been reported in extracts from pea (Leshem and Harmaty, 1996), lupin (Cueto et al., 1996), soybean (Delledonne et al., 1998), tobacco (Durner et al., 1998) and maize (Ribeiro et al., 1999). Competitive inhibitors based on L-arginine have been used to suppress NO production in soybean, Arabidopsis and tobacco (Delledonne et al., 1998; Durner et al., 1998) implicating NOS activity. NOS (Moncada et al., 1991) catalyses the two-step oxidation of L-arginine to NO and citrulline (L-arginine+NADPH+H+O2 → Nω hydroxyarginine +NADP++H2O and thereafter Nω hydroxyarginine + ½ NADPH + ½ H+ → Citrulline+NO+ ½ NADP+ H2O), a reaction that might also be catalysed by a cytochrome P450 (Boucher et al., 1992; Wojtaszek, 2000). NR generates NO from nitrite with NADPH as electron donor (Kaiser et al., 2002; Yamasaki et al., 1999). Zemojtel et al. (2004) postulated the discovery of a novel conserved family of NOS. The authors showed significant homology in NOS sequence in as divergent organisms as plants, snails and mammals. The discovery of a new class of NOS in Arabidopsis thaliana is a real breakthrough in the studies on NO occurrence and function in plants. Furthermore, it is now obvious that plants have evolved multiple routes of NO synthesis, different from those found in animals (Kopyra and Gwozdz, 2004).
Another enzyme involved in NO production is Nitrate Reductase (NR). Despite the tentative identification of a plant NOS gene, clear evidence shows that plants can produce NO from nitrite via NADPH-dependent NR (NO3 → NO2 → •NO+O2). The application of high nitrite levels under conditions of anoxia increased NO production (Rockel et al., 2002). The formation of NO due to NR activity was reported in many plant species, such as sunflower, spinach, maize (Rockel et al., 2002), cucumber (De la Haba et al., 2001), Arabidopsis thaliana (Desikan et al., 2002), green alga Chlamydomonas reinhardtii (Sakihama et al., 2002) wheat, orchid and aloe (Xu and Zhao, 2003). Desikan et al. (2002) provided good evidence that NR-mediated NO generation in guard cells is required for abscisic acid-induced stomatal closure in A. thaliana. Xu and Zhao (2003) postulated that NR is a main source of endogenous NO in higher non-leguminous plants. NO content in leaves of wheat, orchid and aloe was reduced by 90% following the heat or microwave treatment, which indicates that NO is mostly enzymatically produced. Moreover, the reduction of NR activity and concomitant decrease in NO content were observed in wheat seedlings growing in a medium lacking molybdenum, which is the NR cofactor and after treatment with sodium tungstate, the NR inhibitor (Xu and Zhao, 2003).
| Fig. 1: | Possible sources of NO in environment. NO is generated by the action of nitric oxide synthase (NOS). Major origins of NO are the reactions utilizing NO2¯: non-enzymatic reductions either at acidic pH or light-driven in the presence of carotenoids and enzymatic catalysed by NAD(P)H-dependent Nitrate Reductase (NR) or nitrite reductases (NiR). It could also be a by-product of denitrification, nitrate assimilation and/or respiration. Nitrification of NH4+ is the major source of N2O emitted to the atmosphere where it might be further oxidized to NO and NO2 (Wojtaszek, 2000) |
Other enzymes that can generate NO are nitrite: NO-reductase (Ni-NOR), probably situated in the plasma membrane (Stöhr and Ullrich, 2002) and xanthine oxidoreductase (XOR) operating at low oxygen tensions and requiring molybdenum as a co factor (Neill et al., 2003).
In plants, NO can also be generated by non-enzymatic reduction of nitrite (2NO2¯ +2H+ → 2HNO2 → NO+NO2 +H2O), but this is favored only under acid conditions such as found in the barley aleurone cell (Beligni et al., 2002; Bethke et al., 2004). Nitrification /denitrification cycles provide NO as a by-product of NO2 oxidation into the atmosphere (Wojtaszek, 2000). Nitrite can also be chemically reduced by ascorbic acid at pH 3–6 to yield NO and dehydroascorbic acid (Henry et al., 1997). This reaction could occur at microlocalized pH conditions in the chloroplast and apoplastic space where ascorbic acid is known to be present (Horemans et al., 2000). Another non-enzymatic mechanism proposed of NO formation is the light-mediated reduction of NO2 by carotenoids (Cooney et al., 1994). The possible sources of NO in the environment are illustrated in Fig. 1.
PROTECTION MECHANISM OF NO IN PLANT
Two mechanisms by which NO might abate stress have been postulated by Radi et al. (1991). First, NO might function as an antioxidant, by directly scavenging the ROS, such as superoxide radicals (O2¯), to form peroxynitrite (Radi et al., 1991), which is considerably less toxic than peroxides and thus limit cellular damage. Second, NO could function as a signaling molecule in the cascade of events leading to changes of gene expression (Lamattina et al., 2003). Whereas some authors considered NO as a stress inducing agent (Leshem, 1996), others have reported its protective role (Beligni and Lamattina, 1999a, b; Hsu and Kao, 2004), depending on its concentration, the plant tissue or age and the type of stress. When present at low amounts, NO acts as signals for the activation of defense responses, however, higher concentrations produced by uncontrolled ROS generation cause severe injury. The presence of an unpaired electron within the NO molecule makes it a reactive species and is also the origin of its duality. NO is generally toxic and in these conditions, when combined with low amounts of O2¯, the formation of peroxynitrite (ONOO¯) was reported to be deleterious to lipids, proteins and DNA (Wink et al., 1993). However, whenever toxicity is incurred as a result of ROS damage, NO might act as a chain breaker and thus limit damage. In these situations, peroxides have proven to be much more toxic than NO and ONOO¯ and NO is considered to have a protective function (Wink et al., 1993). In addition, the reaction of NO with lipid alcoxyl (LO•) and peroxyl (LOO•) radicals is rapid, giving rise to the expectation that NO could also stop the propagation of radical-mediated lipid oxidation.
The NO-producing enzymes identified in plants are NR and several NOS-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different available evidence indicated that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450 and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought (Del Rio et al., 2004). Most of the work on NO action in plant cells has focused on its ability to act in the same direction as ROS. This concept explains NO participation in the hypersensitive response (Van Camp et al., 1998), in the regulation of the expression of defense genes and in the increase in chlorophyll fluorescence (Leshem, 1996). Consequently, the participation of NO in the antioxidant cellular system of plants, as in animals, is a strong possibility. The main sources for NO-mediated cytoprotection within plant cells are shown in Fig. 2.
| Fig. 2: | Probable chemical reactions of NO in related to cytoprotection. NO reactivity with reactive oxygen species accounts for direct sources of both toxicity and protection. Indirect protection would come from the interaction between NO and the cellular antioxidant system. Signaling pathways, together with direct modification of target molecules could be mechanisms for other physiological functions of NO in plants. [Abbreviations: R•, non-oxygen free radicals; RO•, alcoxyl radicals; ROO•, peroxyl radicals] (Beligni and Lamattina, 1999b) |
NO AS SIGNALING MOLECULE
NO acts as a signaling molecule within species from every biological kingdom, a feature reflecting its physical properties which give it an exceptionally rich chemistry. NO is highly reactive due to the presence of an unpaired electron and, as with oxygen, it can exist in a variety of reduced states, NO¯ (nitroxyl ion), NO and NO+ (nitrosonium ion), with each Reactive Nitrogen Intermediate (RNI) able to undergo specific interactions (Gow and Ischiropoulos, 2001). Every stressor triggers in the cell a signaling cascade leading to the triggering of specific defense responses. Recognition of the stress stimulus by the cell membrane receptor results in the formation of signaling molecules, which in turn leads to a change in the concentration or modulation of the so-called second messengers and as a consequence to the triggering of defense response.
In biological systems, NO affects signaling through a range of actions. Many NO effects are mediated by oxidative damage associated with the formation of the potent oxidant peroxynitrite via interaction with superoxide (NO+O2¯ → ONOO¯). A more subtle action is the electrophilic attack by NO¯ on thiol groups, particularly cysteine residues, resulting in S-nitrosylation of molecules such as glutathione or proteins. Protein S-nitrosylation can modulate protein activity (Lander et al., 1996) while, the kinase is inactivated (Park et al., 2000). Alternatively, NO can modify proteins by nitration, particularly of tyrosine residues (Radi, 2004). The effects induced by NO may be independent of cellular second messengers, although the biochemical mechanism of this effect has not been comprehensively clarified. The chemical nature of NO results in transition metals (e.g., Fe, Cu, Zn) and proteins containing thiol groups being important targets for this molecule (Wendehenne et al., 2001). Analogously as in the NO-guanylate cyclase interaction, NO may interact with iron present in other proteins. In this way, NO modifies activity of aconitase and Fe-S enzyme catalysing isomerization of citrate to isocitrate, in tobacco (Navarre et al., 2000). Inactivation of this enzyme decreases the cellular energy metabolism, which may results in reduced electron flow through the mitochondrial chain and a subsequent decrease in the ROS generation. Moreover, tobacco cytosolic aconitases have reasonably high homology to human iron regulatory protein (IRP-1), which suggests that it may possesses IRP activity and affect iron homeostasis in plants (Navarre et al., 2000). NO periodically inhibits also catalase and peroxidase, containing the haem system, which may potentially regulate ROS level in the cell, e.g., during programmed cell death (PCD) in xylem formation (Ferrer and Barcelo, 1999; Clark et al., 2000).
PHYSIOLOGICAL ASPECTS OF NO INDUCED ABIOTIC STRESS TOLERANCE
Nitric oxide (NO) is a relatively stable free radical gas which may act as a key signaling molecule in plants and mediates various physiological, biochemical and developmental processes including seed germination, stomatal closure, root development and hypersensitive responses (Delledonne et al., 2001; Neill et al., 2003). Garcia-Mata and Lamattina (2001) showed that exogenous NO (applied as sodium nitroprusside, SNP) reduced transpiration and induced stomatal closure in several species such as Vicia faba, Salpichroa and Tradescantia sp. and NO was indicated to be a component of ABA signaling pathways in ABA-induced stomatal closure. On the other hand, NO can also mediate plant growth regulators and ROS metabolism and increasing evidence has shown it is involved in signal transduction and responses to abiotic stress such as drought, low and high temperatures, UV-radiation. Some physiologican role of NO under abiotic stress condition are presented in Table 1.
| Table 1: | Outline of some important NO-mediating effect during abiotic stresses |
As a mediator of physiological processes, NO has an incredible number of beneficial effects; for example, it functions as a messenger in immune responses. But it can become very toxic under certain complex conditions determined, for example, by its rate of production and diffusion and the redox state of the cell (Murphy, 1999). In plant cells, NO and NO-derived molecules are involved in response to many abiotic stresses. Consequently, when a specific abiotic stress alters physiological NO metabolism causing damage to biological molecules, a nitrosative stress is generated (Corpas et al., 2007; Valderrama et al., 2007). In fact, NO interacts with ROS in various ways and might serve an antioxidant function during various stresses (Beligni and Lamattina, 1999b). Modulation by NO of superoxide formation (Caro and Puntarulo, 1998) and inhibition of lipid peroxidation (Boveris et al., 2000) also illustrate its potential antioxidant role, mainly due to its ability to maintain the cellular redox homeostasis and regulate ROS toxicity. Another key role of NO in abiotic stress response relies on its properties as a signaling molecule as described in previous heading.
SALINITY
Salinity is one of the most important stress factors which limit the growth and development of plant by altering their morphological, physiological and biochemical attributes. Under saline conditions, tolerant plant cells achieve ion homeostasis by extruding Na to the external medium and/or compartmentalizing into vacuoles, maintaining K uptake and high K and low Na in the cytosol. It has been proven that the activity of the plasma membrane H+-ATPase is a key index of plant adaptation to salt stress (Hasanuzzaman et al., 2009; Nahar and Hasanuzzaman, 2009). The protective role of NO in salt tolerance of plants is well documented. Zhang et al. (2004) reported that NO enhanced salt tolerance in maize seedlings, through increasing K+ accumulation in roots, leaves and sheathes, while decreasing Na+ accumulation (Zhang et al., 2004). Similarly, NO induced salt resistance of calluses from Populus euphratica also found by increasing the K+/Na+ ratio and this process was mediated by H2O2 and was dependent on the increased plasma membrane H+-ATPase activity (Zhang et al., 2007). In maize, addition of exogenous NO increases tolerance to salt stress by elevating the activities of the proton-pump and Na+/H+ antiport of the tonoplast (Zhang et al., 2004). Additionally, pretreatment with NO donor (SNP) protected young rice seedlings, resulting in better plant growth and viability (Uchida et al., 2002), promoted seed germination and root growth of yellow lupine seedlings (Kopyra and Gwozdz, 2003) and increased growth and dry weight of maize seedlings (Zhang et al., 2006) under salt stress conditions. NO treated wheat (Triticum aestivum L.) leaves also showed less destruction of chlorophyll and plasma membrane permeability induced by NaCl treatment (Ruan et al., 2002). There is a wealth of evidence that NO induced salt tolerance is due to profound increase in both non enzymatic and enzymatic components.
DROUGHT
Drought is one of the most important abiotic stresses that causes significant reductions in crop yield and thus hinders the food security. Upon exposure to drought stress, plants exhibit a wide range of responses at the whole plant, cellular and molecular levels (Chaves et al., 2003; Shinozaki and Yamaguchi-Shinozaki, 2007; Hossain and Fujita, 2009b). The NO-synthesizing activity in wheat plants was found to increase under drought conditions. The newly synthesized NO together with H2O2 participated in the regulation of ABA-induced closing of stomata in various plant species (Neill et al., 2008). In addition, the protective role of NO in drought-stressed plants has been reported by several researchers. In a recent work, the activity of NOS in the cytosolic and microsomal fractions of maize leaves was determined (Sang et al., 2008). The results showed that water stress induced increases in NOS activity in the cytosolic and microsomal fractions and the NOS activity in the microsomal fraction was higher and more susceptible to water stress treatment than that in the cytosolic fraction of maize leaves. It was observed that exogenously applied NO, reduced water loss from detached wheat leaves and seedlings subjected to drought conditions, decreased ion leakage and transpiration rate and induced stomatal closure, thereby enhancing plant tolerance to drought stress (Garcia-Mata and Lamattina, 2001). Interestingly, a specific NO scavenger, cPTIO, reverted the above actions of NO (Garcia-Mata and Lamattina, 2001). Results of this experiment suggest that exogenous application of NO donors might confer on plants an increased tolerance to severe drought stress conditions. It was shown that treatment of plants with exogenous NO enhanced drought tolerance of cut leaves and seedlings of wheat (Tian and Lei, 2006). In addition, NO treatment enhanced wheat seedling growth and maintained relatively high water content and alleviated oxidative damage (Hao and Zhang, 2010). However, higher dose (2 mM SNP) aggravated the stress as a result of uncontrolled generation of ROS and ineffectiveness of antioxidant systems. Exogenous NO increased the activities of water stress induced subcellular antioxidant enzymes, which decreased accumulation of H2O2. These results suggest that NOS and NR are involved in water stress-induced NO production and NOS is the major source of NO. The potential ability of NO to scavenge H2O2 is at least in part due to the induction of a subcellular antioxidant defense mechanism. NO alleviates the ROS-mediated cytotoxic process in potato leaves (Beligni and Lamattina, 1999a). The ROS-mediated damages caused by drought, including cell death, ion leakage and DNA fragmentation, were inhibited by exogenous NO and all of the protective effects were abolished by the treatment with PTIO (Beligni and Lamattina, 1999a). The protective effect of NO in osmotic stress was recently confirmed in two ecotypes of reed suspension cultures. Zhao et al. (2008) suggested that polyethylene glycol (PEG-6000) induced NO release in stress-tolerant but not sensitive ecotype reed, effectively protecting against oxidative damage and conferring an increased tolerance to osmotic stress (Zhao et al., 2008). In wheat seedlings, the osmotic stress produced by treatment with 0.4 M manitol reduced leaf water loss while increasing the leaf ABA content. These effects were partially reversed by NO scavengers and NOS activity inhibitors (Xing et al., 2004). In tomato detached leaves, the application of NO donors inhibited the synthesis of proteinase inhibitor I and the generation of H2O2 in response to mechanical wounding (Orozco-Cárdenas and Ryan, 2002).
EXTREME TEMPERATURE
Every plant has a critical temperature for its growth and development. Temperature, either very high or low, is harmful for plants. Research results indicated that NO also participates in plant response to high and low temperature stress. For example, high temperature treatment of lucerne cells resulted in an increase of NO synthesis, whereas, the application of exogenous NO increased cold tolerance in tomato, wheat and maize (Neill et al., 2003). It was shown that both in tobacco leaf peels and suspension cells, high temperature generated a rapid and significant surge in NO levels (Gould et al., 2003). Leshem (2001) reported that short term heat stress increased the NO production in alfalfa, which negatively correlated with ethylene production. NO pretreatment reduced heat-induced damage in rice seedlings and prevented the impairment of photosystem II (PSII). Additionally, NO pretreatment induces not only active oxygen scavenging enzyme activities but also expression of transcripts for stress related genes encoding sucrose-phosphate synthase and small heat shock protein (Uchida et al., 2002). Lamattina et al. (2001) observed that treatment with NO increased the survival rate of leaves of wheat and maize seedlings (Lamattina et al., 2001). The role of NO during extreme temperature stress might be to decrease the ROS level caused by heat or lower temperature (Neill et al., 2002).
HEAVY METALS AND ALUMINUM
Heavy metal contamination of soils is an increasing problem worldwide and great environmental threats to biota as these metals are being accumulating in soils and plants in undesirable amounts. Heavy metal cause oxidative damages to plants when its concentration exceeds the limit (Hossain et al., 2010). Interestingly, under heavy metal stress plant produces NO which further may protect the plants against damages due to stress (Hsu and Kao, 2004). In order to demonstrate the possible role of NO in response to heavy metals in the metal accumulator Brassica juncea and the crop plant Pisum sativum, researchers grew these plants in presence of 100 μM cadmium (Cd), copper (Cu), or zinc (Zn) (Bartha et al., 2005). They obtained different NO levels with different heavy metal loads; the most effective metals were copper and cadmium, where the NO production doubled after 1 week of treatment. In case of copper treatment, two-phase kinetics was found, that is, a rapid NO burst in the first 6 h was followed by a slower, gradual increase. The fast appearance of NO in the presence of cupric ions suggests that this can be a novel reaction hitherto not studied in plants under heavy metal stress. In relation to other abiotic stresses it was documented that exogenous NO reduces the destructive action of heavy metals, ethylene and herbicides on plants (Hung et al., 2002; Kopyra and Gwozdz, 2003).
In soybean plants exposed to an acute level of CdCl2 (200 μM), the exogenous application of NO protected against oxidative damage caused by this metal stress, elevated levels of heme oxygenase-1 expression, as it occurs with other genes involved in the antioxidant defense system (Rao and Davis, 2001). In contrast, pretreatment of seedlings with 100 mM SNP protected sunflower leaves against Cd-induced oxidative stress (Orozco-Cardenas and Ryan, 2002). A similar effect has been described in Lupinus roots grown with 50 μM Cd2+ and it was proposed that the protective effect of NO could consist of stimulation of superoxide dismutase activity to counteract overproduction of superoxide radicals, thus avoiding formation of peroxynitrite from NO and O2¯ (Uchida et al., 2002). Hibiscus moscheutos exposed to 100 μM AlCl3 experienced inhibition of root growth. This effect was accompanied by inhibition of NOS activity and reduced NO concentrations (Neill et al., 2008). Using fluorescent and laser scanning confocal microscopy, Kopyra and Gwozdz (2003) found that NO pretreatment significantly reduced O2¯-induced specific fluorescence in Lupinus luteus roots under heavy metals treatment. These results suggest that NO antioxidant function may be carried out by a scavenging of O2¯. The detoxifying effect and antioxidative role of NO were also found in soybean cell cultures exposed to cadmium and copper treated Chlorella (Singh et al., 2004). Furthermore, a recent finding showed that NO alleviated the Al3+ toxicity in root elongation of Hibiscus moschetuos (Tian et al., 2007). In addition, mechanical damage was reported to elicit NO production from NOS activity in Arabidopsis leaves (Garces et al., 2001). In another study, Cassia tora plants pretreated for 12 h with 0.4 mM SNP and subsequently exposed for 24 h to 10 μM Al exhibited a significantly greater root elongation and a decrease in Al accumulation in root apexes as compared with plants without SNP treatment (Wang and Yang, 2005). All these data indicate the importance of exogenous NO in the uptake of micronutrients and in the protection against deleterious effects of toxic heavy metals such as Cd or Al (Corpas et al., 2006; Zhang et al., 2008a). Recently, Cu-induced NO generation and its relationship with proline synthesis in Chlamydomonas reinhardtii were investigated (Zhang et al., 2008b). However, the physiological implication of the plant endogenous NO in the response to heavy metal stress is still not well-known (Corpas et al., 2006).
HERBICIDES
More than 30 years ago, it was shown that the application of herbicides to soybean plants increased the NO production (Klepper, 1979). More recently, several studies have confirmed that the treatment with NO donors (SNP) protect plants from the deleterious herbicidal effects (Beligni and Lamattina, 1999c, 2002; Huang et al., 2002). NO treatment also protects chloroplast membrane, the diquat induced chlorophyll loss (Beligni and Lamattina, 1999c). Additionally, the paraquat induced protein content reduction was prevented by NO Cell death, ion leakage and DNA fragmentation, which are ROS-mediated damages resulting from Phytophthora infestans infection, were inhibited by NO donor and all those protective effects were abolished after treatment with cPTIO (Beligni and Lamattina, 1999c).
UV-RADIATION AND OZONE
The stratospheric ozone (O3) layer is vital to life on earth because it is the principal agent absorbing the ultraviolet radiation in the earth's atmosphere. Since, 1990, the depletion of the stratosphere O3 layer due to anthropogenic and natural destruction is leading to increasing levels of solar ultraviolet-B (UV-B: 280-320 nm) radiation reaching the earth's surface (Kerr and McElroy, 1993; Russell et al., 1996). Ambient UV-B irradiance at low latitudes is also high due to the high solar angle and a relatively low stratospheric O3 amount (Baker et al., 1980). Like other stress, exposure to UV-B leads to the generation of ROS. In addition,O3 effects on plants are primarily induced by an increased production of ROS, both outside and inside the plant cell, which is a common feature of in plants.
Mackerness et al. (2001) showed the participation of NO in plant response to UV-B radiation, demonstrating an increase in NOS-type enzymatic activity and an elevation of NO level. Shi et al. (2005) suggested that NO may effectively protect plants against UV-B radiation, most probably through the increased activity of antioxidative enzymes. Qu et al. (2006) showed that UV-B radiation significantly induced NOS activity and promoted NO release. Zhang et al. (2003b) found that NO was a second messenger associated with developmental growth under UV-B radiation. It was found that UV-B radiation significantly induced NOS activities and accelerated the release of apparent NO of mesocotyl and that rhizospheric treatments to exogenous NO donors may mimic the response of the mesocotyl to UV-B radiation. Bean seedlings subjected to UV-B radiation, exogenous NO partially alleviated the UV-B effect characterized by a decrease in chlorophyll content and oxidative damage to the thylakoid membrane (Shi et al., 2005). Moreover, UV-B induced stomatal closure, which was mediated by NO and H2O2 and the generation of NO was caused by a NOS-like activity (Ruan et al., 2004). However, other authors reported that NO generated in guard cells were produced by NR activity (Shi et al., 2007). NO treatment has been shown to increase levels of O3-induced ethylene production and increase leaf injury (Rao and Davis, 2001). In tobacco plants, fumigated with O3, the accumulation of hydrogen peroxide in mitochondria and early accumulation of NO and ethylene in leaf tissues have been described. He et al. (2005) reported that UV-B radiation induced stomatal closure by promoting NO and H2O2 production.
The treatment of Vicia faba leaves with SNP alleviated the injurious effect of UV-B, leading to the increased chlorophyll content and to the increase in potential and effective quantum yields of electron flow in photosystem II; the oxidative damage to thylakoid membranes was reduced (Shi et al., 2005). The alleviating effects of NO were also observed in experiments with an algal culture of Spirulina platensis, which was evident from protective action on total biomass and physiological parameters, such as the content of chlorophyll a and proline (Xue et al., 2006). Although SNP mitigated the inhibitory effect of UV-B irradiation, the endogenous NO was found to be the main factor responsible for inhibition of mesocotyle growth upon UV-B irradiation (Ederli et al., 2009).
BIOCHEMICAL MECHANISM OF NO INDUCED ABIOTIC STRESS TOLERANCE
A great variety of abiotic stresses including drought, salinity, ultraviolet light, heat, chilling, air pollutants and heavy metals cause molecular damage to plants, either directly or indirectly through ROS formation (Laspina et al., 2005; Ferreira and Cataneo, 2010), such as superoxide (O2¯) and hydroxyl (·OH) radicals, hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Therond et al., 2000). Literature data supply evidence showing that plant response to such stressors as drought (Garcia-Mata and Lamattina, 2001; Zhao et al., 2001; Neill et al., 2002), salinity (Zhao et al., 2004, 2007), heavy metal (Kopyra and Gwozdz, 2003; Hsu and Kao, 2004; Wang et al., 2004), high light (Xu et al., 2010), UV-radiation (Mackerness et al., 2001), high temperature (Leshem et al., 1998; Yang et al., 2006), herbicide (Mallick et al., 2000; Huang et al., 2002) is regulated by NO. In order to avoid ROS toxicity, aerobic cells are provided with a flexible set of enzymes and metabolites involved in ROS catabolism, which often acts at the site of ROS production (Shigeoka et al., 2002; Foyer, 2004; Mittler et al., 2004; De Pinto et al., 2006). Survival under these conditions depends on the capability of plants to increase specific pathways involved in ROS removal (Noctor and Foyer, 1998; Asada, 1999).
One of the most intriguing issues in NO biology is its dual function as a potent oxidant and an effective antioxidant (Beligni and Lamattina, 1999b). This dual role of NO might depend on differences in dose, bioprocesses, development stages, or species (Ferrer and Bacelo, 1999; Clark et al., 2000; Zeier et al., 2004). The cytoprotective role of NO is mainly based on its ability to maintain the cellular redox homeostasis and to regulate the level and toxicity of ROS. NO exerts a protective function against oxidative stress mediated by (1) reaction with lipid radicals, which stops the propagation of lipid oxidation; (2) scavenging the superoxide anion (O2¯) and formation of peroxynitrite (ONOO-) that is toxic for plants but can be neutralized by ascorbate and glutathione; (3) activation of antioxidant enzymes (SOD, CAT and POX etc.). One of the fastest reactions of NO within a biological system is its combination with superoxide anion (O2¯) that leads to the formation of strong oxidant peroxynitrite (ONOO-) (Neill et al., 2003; Wendehenne et al., 2001) that is one of the major toxic reactive nitrogen species that exerts deleterious effects on DNA, lipids and proteins (Stamler et al., 1992; Pryor and Squadrito, 1995; Yamasaki et al., 1999). The antioxidative protection offered by NO can be described under following headings:
REGULATION OF NON-ENZYMATIC ANTIOXIDANT CONTENT IN PLANTS BY NO AND STRESS TOLERANCE
Plants possess a variety of non-enzymatic molecules which play a substantial role in counteracting oxidative stress caused by stress. The non-enzymatic antioxidants include ascorbate, glutathione, tocopherols, carotenoids and flavanoids etc. (Noctor and Foyer, 1998; Tausz and Grill, 2000). They act coordinately with antioxidant enzymes to maintain the cellular redox state of the cell under stressful conditions.
Ascorbate(AsA)
In plant cell, AsA is the most abundant antioxidant and serves as a major
contributor to the cellular redox state and protects plant against oxidative
damage resulting from a range of biotic and abiotic stresses (Smirnoff,
2000; Hossain and Fujita, 2010). Due to the ability
of AsA to donate electrons in a number of enzymatic and non-enzymatic reactions,
it is considering to be the most popular and powerful ROS detoxifying compound.
It is the substrate of cAPX and the corresponding organellar isoforms, which
are critical components of the AsA-GSH cycle for H2O2
detoxification (Nakano and Asada, 1981; Dalton
et al., 1986). AsA can directly quench 1O2,
O2¯ and ·OH and regenerate α-tocopherol from α-chromanoxyl
radical thereby providing protection to membranes. Elevated levels of endogenous
AsA in plants are necessary to offset oxidative stress in addition to regulating
other plant metabolic processes (Smirnoff, 2000). Hsu
and Kao (2004) reported that NO increase the levels AsA as a result of an
increase in the capacity of NO to scavenge ROS in rice leaves treated with NO
and CdCl2 and might account in part for the lower contents of H2O2
observed in rice leaves treated with NO and CdCl2. Laspina
et al. (2005) reported that NO pretreatment before Cd exposure returned
AsA contents to values close to the controls and NO-treated plants showed AsA
content similar to controls.
Glutathione (GSH)
In higher plants, the redox active tripeptide glutathione (GSH) fulfils
a plethora of functions. The chemical reactivity and high water solubility of
the thiol group of GSH makes it particularly suitable to serve a broad range
of biochemical functions to protect plants against oxidative stress (Hossain
and Fujita, 2010; Hossain et al., 2010). These
include its pivotal role for maintaining the cellular redox poise and its involvement
in detoxification of heavy metals and xenobiotics. Intimately linked to these
functions, GSH also acts as a cellular signal, mediating control of enzyme and/or
regulatory protein activities, either directly or via glutaredoxins. GSH can
participate not only in scavenging H2O2 through the AsA-GSH
cycle but also in a direct reaction with other active oxygen species (May
et al., 1998).
NO protects plant cells against oxidative processes by stimulating GSH synthesis. Increasing evidence indicates that the GSH biosynthetic pathway is stimulated in response to NO in plant and animal cells and increases oxidative stress tolerance (Moellering et al., 1998; Kim et al., 2004; Innocenti et al., 2007). The regulation of GSH synthesis by NO raises the question of the physiological roles that may be sustained by such a modulation. Several studies have evidenced the capacity of NO to counteract oxidative damages (Beligni and Lamattina, 1999c; Beligni et al., 2002; Wang and Wu, 2005). GSH may also play an important role in regulating NO bioactivity. Indeed, it can readily react with NO to form GSNO, which serves as a NO reservoir and a long-distance NO vector in mammals (Zhang and Hogg, 2004). In regard of recent reports indicating the importance of nitrosothiols in controlling plant responses to pathogens (Feechan et al., 2005), the stimulation of GSH synthesis by NO may provide an important regulatory loop for NO bioactivity. Laspina et al. (2005) observed a decrease in GSH level by Cd in sunflower leaves, but NO was able to counteract efficiently GSH depletion. In fact, Cd forms stable complexes with thiol groups such as GSH and phytochelatins (Cobbett, 2000) and this might be explaining, at least in part, GSH decrease. NO could be acting simply as an antioxidant (Beligni and Lamattina, 1999a; Beligni et al., 2002; Neill et al., 2002) or could be playing a role in the elevation of GSH levels, either by increasing the biosynthesis rate of this metabolite or through an increased supply of cysteine, the limiting substrate (Li et al., 1999). It was observed that NO pre-treated salt-stressed seedlings showed significant increase in GSH level as compared to the seedlings subjected to salt stress alone because GSH synthesis is enhanced by NO treatment (Moellering et al., 1998; Innocenti et al., 2007). The increased GSH pool was also found in wheat roots in response to NO (Groppa et al., 2008) which could be attributed to the increased NO levels. Several studies have evidenced that GSH biosynthetic pathway is stimulated in response to NO in animal cells and yeast (Moellering et al., 1998; Kim et al., 2004) and very recently, Innocenti et al. (2007) reported the effect of NO on the GSH/hGSH synthesis pathway was examined in roots of Medicago truncatula. Generation of NO was achieved by treatment of roots with NO and GSNO, two different NO donors with unrelated structures, which have been widely used to analyse gene expression in plants (Durner et al., 1998; Polverari et al., 2003; Murgia et al., 2004). This result provided the evidence that GSH synthesis is stimulated by NO in plants. This result is in resemblance with those of De Pinto et al. (2002), who reported a decrease of GSH content in NO treated BY-2 tobacco cells, suggesting a different response between cell culture and roots. Nevertheless, a similar response was previously reported for fission yeast and animal cells (Kuo and Abe, 1996; Moellering et al., 1998; Kim et al., 2004) and the present report extends the effect of NO on GSH synthesis pathway to plants. As for yeast and animals, NO triggered an increase of the endogenous GSH amount above control in Medicago truncatula roots through the stimulation of GSH synthesis gene transcript accumulation. Whereas only γ-ecs gene stimulation was tested for GSH accumulation upon NO treatment in yeast and animals (Kuo and Abe, 1996; Kim et al., 2004).
REGULATION OF ANTIOXIDANT ENZYMATIC ACTIVITIES BY NO AND OXIDATIVE STRESS TOLERANCE
Apart from non-enzymatic antioxidant plant possess an array of antioxidant enzymes that maintains ROS homeostasis in all cellular compartments and regulates the adjustment of ROS levels according to the cellular needs at a particular time (Apel and Hirt, 2004; Gechev et al., 2006). These antioxidants include the enzymes, superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione S-transferases (GST; EC 2.5.1.18), ascorbate peroxidase (APX; EC 1.11.1.11), dehydroascorbate reductase (DHAR; EC 1.8.5.1), glutathione reductase (GR; EC 1.6.4.2) and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4). Extensive research findings supported the idea that coordinated induction and regulation of both enzymatic and non-enzymatic antioxidant defense pathway is necessary to obtain substantial tolerance against oxidative stress in plants.
Superoxide Dismutase (SOD)
Superoxide dismutase (SOD) is an important antioxidant enzyme and is the
first line defense against oxidative stress in plants. SOD catalyses the dismutation
of O2¯ to molecular oxygen (O2) and H2O2
(Yu et al., 2005). It plays an important part
in determining the concentration of O2¯ and H2O2
in plants hence performs a key role in the defense mechanism against free radical
toxicity (Bowler et al., 1992). The induction
of SOD in plant cells in response to different stressful conditions reflects
its important role in the defense mechanism of plants. Stress tolerant plants
have higher SOD activity as compared to sensitive plants (Shalata
et al., 2001; Sekmen et al., 2007).
As a signal molecule NO induces/stabilizes the expression of many antioxidative
enzymes including SOD (Frank et al., 2000). Huaifu
et al. (2007) found that exogenous NO increased the SOD activity
of leaves under NaCl stress. Similarly, Shi et al.
(2007) showed that application of NO significantly decreased the inhibition
of SOD activity by salt stress, which suggested that application of NO could
promote the conversion from O2- into H2O2
and O2, which is an important step in protecting the cell. Additionally,
Cheng et al. (2002) concluded that the inhibition
of osmotic stress- and dehydration-enhanced senescences of rice leaves by NO
is most likely mediated through an increase in SOD activity and a decrease in
lipid peroxidation. In contrast, Laspina et al. (2005)
observed 110% increase in SOD activity in Cd-treated plants, while plants treated
with NO and subjected to Cd stress the SOD activity was also increased, but
only to 59% over the control. NO prevented the paraquat-induced reduction in
protein content, increase in level of MDA and decline in the activities of antioxidant
enzymes including SOD. Therefore, increased SOD activity enhances stress tolerance
of plants when other important antioxidant enzymes (APX, DHAR, MDHAR, GR, GSH
and AsA) are also present in high levels. Because the over produced H2O2
must be scavenged efficiently, otherwise it can interact with O2-
to form highly reactive hydroxyl radicals (·OH) that are thought to be
primarily responsible for oxygen toxicity in the cell. There is a plenty of
evidence that NO not only increases the SOD activity but also significantly
increase the H2O2 degrading enzymes to maintain its level
to perform intracellular signaling roles.
Ascorbate Peroxidase (APX)
Scavenging of H2O2 by APX is the first step of the
AsA-GSH cycle (Asada, 1994). In the AsA-GSH cycle, APX
catalyzes the reduction of H2O2 into H2O with
AsA serving as an electronic donor (Zhang et al.,
2008a, b). On the other hand, it is known that APX
is more efficient than CAT to detoxify H2O2, since it
is widely distributed inside the cell and has high substrate affinities in the
presence of AsA as reductant. In addition to H2O2 detoxification,
cAPX isozymes have a dynamic function in redox signal modulation and gene expression
under oxidative stress condition by modulating the concentration of H2O2
to adjust its activity for expression to a level sufficient for second
messenger activity. NO could participate in a series of resistant physiological
reaction by adjusting activities of APX and other relative enzymes containing
hemachrome iron or by restraining activity of aconitase without hemachrome iron
(Wang et al., 2004). The increase of APX activities
reduced much production of ROS which makes it possible to increase osmotic adjustment
ability and salt tolerance (Zhu, 2002). Exogenous NO
reduced the membrane permeability and membrane lipid peroxidation and prevented
the electrolyte leakage, which suggested exogenous NO possessed the functions
of repairing and protecting the cell membrane to alleviate the injury in the
cell membrane system. Farooq et al. (2009) reported
that NO application improved the APX under drought stress conditions.
Chen et al. (2010) reported that NO treatment significantly elevated the depressed APX activity in barley seedlings after 10 and 15 days of CdCl2 treatment. Thus, it might be deduced that NO indirectly scavenges ROS accumulation by elevating Cd-decreased APX activity which may account in part for its alleviating effect on Cd-induced oxidative damage in barley seedlings. In sunflower leaves treated with 0.5 mM Cd, APX activity increased 76% over the controls, but NO+Cd and NO treatments increased APX activity even more, 163 and 106% over the controls, respectively (Laspina et al., 2005). Additionally, pretreatment with SNP or SNAP resulted in remarkable increase in the activities of APX in the callus of Reed (Song et al., 2006). With cPTIO (NO scavenger) or in combination with SNP or SNAP treatments, the activities of APX kept at the level of heat treatment alone in callus whereas they declined markedly in callus compared with those under heat stress alone. Moreover, some antioxidant genes including APX were also found to be induced by NO in Arabidopsis suspension cells (Huang et al., 2002). Yang et al. (2006) reported that after heat shock, activity of APX decreased in water presoaked leaf discs and partially or fully recuperated due to SNP presoaking. Because the physiological role of APX is to break down H2O2 in the cell, decreases in activities of these two enzymes would result in H2O2 accumulation. A remarkable increase in the activity of APX was also observed with the treatment with NO in UV-B stressed bean plant (Shi et al., 2005). Mackerness et al. (2001) reported that indeed NO, upon UV-B treatment, can up- or downregulate different genes involved in the defense response or photosynthetic genes. Thus, it is highly possible that the protective effect of NO may be mediated by increased level of expression of genes encoding active oxygen scavenging enzymes under UV-B radiation. However, the effect of NO on peroxidase is somewhat controvertible; the lower concentration of NO increases peroxidase activity in Brassica whereas higher concentration proved inhibitory (Zanardo et al., 2005). Similarly, APX activity was inhibited by higher NO concentration in tobacco and canola (Ferrer and Barcelo, 1999). Generation of NO in Arabidopsis plants induces a decrease in the thylakoidal APX transcript accumulation; consistently, Arabidopsis plants over- or underexpressing on thylakoidal APX gene show increased or decreased sensitivity, respectively, to both NO-induced cell death and paraquat-induced oxidative stress (Murgia et al., 2004; Tarantino et al., 2005).
Monodehydroascorbate Reductase (MDHAR)
AsA is present in most cellular compartment and several pathways exist to
ensure AsA recycling. With its ability to directly regenerate AsA from MDHA,
MDHAR plays an important role in maintaining reduced pool of AsA and ascorbate
redox state (Hossain et al., 2010). It has been
suggested that regeneration of AsA is regulated in this cycle mainly by NADPH-dependent
MDHAR activity (Mittova et al., 2000; Shalata
et al., 2001). Recent studies showed that both MDHAR and DHAR are
equally important in regulating AsA level and its redox state under oxidative
stress condition (Eltayeb et al., 2006, 2007;
Wang et al., 2010). There are very few reports
about NO action on MDHAR activity in plants subjected to abiotic stresses. In
H2O2-treated floral petals of Phalaenopsis, Tewari
et al. (2009) reported that exogenous application of NO donors significantly
enhanced the activity MDHAR.
Dehydroascorbate Reductase (DHAR)
Elevated AsA levels through increased DHAR activity as well as overexpression
of DHAR in different sub-cellular compartments contribute significantly in enhancing
plants tolerance to oxidative stress. In the absence of sufficient DHAR activity,
DHA undergoes irreversible hydrolysis to 2, 3-dikitoglunic acid. DHAR allows
the plant to recycle DHA, thereby capturing AsA before it is lost. Thus DHAR
is a physiologically important reducing enzyme in the AsA-GSH cycle in higher
plants (Hossain and Fujita, 2010). Ding
et al. (2008) found a strong synergistic effect of NO under Fe deficiency
in Chinese cabbage and concluded that addition of NO dramatically induced DHAR
activity. Similarly, significant increase in DHAR activity was also observed
in cucumber roots subjected to salt stress (Shi et al.,
2007). However, Sheokand et al. (2008) reported
that DHAR activity remained unchanged when treated with NO under salt stress
conditions.
Glutathione Reductase (GR)
Biochemical and molecular studies have shown that GR plays an essential
role in cell defense against reactive oxygen metabolites by sustaining the reduced
state of GSH and AsA pools which in tern maintain cellular redox state under
stress. It has been observed that stress-tolerant plants tend to have high activities
of GR (Mittova et al., 2003; Sekmen
et al., 2007). Additionally, overexpression of GR increases antioxidant
activity and improves tolerance to oxidative stress (Potters
et al., 2004). In contrast, decreased GR activity results in increased
stress sensitivity (Noctor and Foyer, 1998). Increases
in GR activity in NO treated seedlings were also reported in plants under various
abiotic stress conditions (Sang et al., 2008;
Xu et al., 2010). Application of NO significantly
increased GR activity in salt-treated cucumber roots (Shi
et al., 2007). Xu et al. (2010) reported
that addition of exogenous NO significantly enhanced the GR activity under high-light
stress and whereas a reversed pattern was found when the NO scavenger, PTIO
was applied in tall fescue leaves. They also postulated the role of NO as a
signaling molecule involved in inducing increases in the activities of antioxidant
enzymes. However, Singh et al. (2008) showed
lesser induction of GR activity by NO under short-term Cd-stress. In contrast,
Sheokand et al. (2008) and Laspina
et al. (2005) reported that GR activity was unchanged by exogenous
NO pretreatment under salt and Cd-stress conditions.
Glutathione Peroxidase (GPX)
In addition to CAT and APX, GPX is also reported to be the major H2O2-utilizing
enzymes in plants (Asada, 1994). Laspina
et al. (2005) observed that GPX activity, either in Cd or NO+Cd-treated
plants, increased 31% over the controls. Similar increase of GPX activity by
NO was observed in our laboratory in wheat seedlings subjected to salt stress
(Hossain and Fujita, 2010). Shi
et al. (2007) reported that application of NO did not increase GPX
activity under salt stress on the 4th d of treatment but significantly
enhanced GPX activity on the 8th d of treatment. Under normal conditions,
application of NO also significantly increased GPX activity on both the 4th
and 8th d of treatment (Shi et al., 2007).
In contrast, Singh et al. (2008) found a decrease
in GPX activity when Cd-stressed wheat roots were treated with NO.
Catalase (CAT)
Catalase is a key antioxidant enzyme present exclusively in perxoisomes
which decomposes H2O2. The regulatory role of NO on CAT
activity under abiotic stress condition has studied by several researchers.
NO could significantly enhance antioxidative capacity by increasing the activities
of CAT during wheat seed germination under osmotic stress was reported by
Zhang et al. (2003a) and Farooq et al.
(2009). Tu et al. (2003) reported that wheat
leaves treated with NO reduced H2O2 by activating CAT
in ageing wheat leaves. Ding et al. (2008) observed
that addition of NO dramatically induced CAT activity under Fe deficiency whose
activities were even beyond control. Huaifu et al.
(2007) observed that NO treatment significantly increased the CAT activity
when it was subjected to salt stress as compared to the seedlings exposed to
salt alone. Exogenous NO treatment also significantly increased CAT activity
in the mitochondria during germination under salt stress which might have contributed
to the alleviated oxidative stress in the mitochondria of germinating wheat
seeds and thereby improved germinating rate under salt stress (Zheng
et al., 2009). However, Laspina et al.
(2005) reported that CAT activity was strongly inhibited in sunflower plants
treated with 0.5 mM Cd, showing a decay of 44% as compared to the controls.
However, pretreatment with NO restored completely CAT activity, increasing its
value 21% over the controls. NO-treated plants also increased the enzyme activity
22% over the controls. According to Yang et al.
(2006), after heat shock, activity of CAT decreased in water presoaked leaf
discs and partially or fully recuperated due to presoaking with NO. Because
the physiological role of CAT is to break down H2O2 in
the cell, decreases in activities of these two enzymes would result in H2O2
accumulation. In rice seedlings, the supplementation of NO to Cd-treatment solution
resulted in a significant decrease in induction levels of these scavenging enzymes
compared to Cd treatment alone (Singh et al., 2008).
A large number of researches indicating that NO significantly increased the enzymatic and non-enzymatic components of antioxidant defense. However, Singh et al. (2009) reported that apart from upregulation of antioxidant enzymes, upon SNP addition the induction level of these scavenging enzymes were significantly lesser than in the treatments without SNP indicating its direct involvement as an antioxidant and quenching the ROS. However, the observed trend of changes in antioxidant enzymes upon NO supplement paralleled the changes in ROS species (O2¯, H2O2 and MDA content).
INTERACTION OF NO WITH OTHER SIGNALING MOLECULES
Although, it is well established that H2O2 induces NO synthesis, there has been some inconsistencies in the reports regarding NO regulation of H2O2 synthesis (Lum et al., 2002; She et al., 2004; He et al., 2005; Bright et al., 2006). NO affected H2O2 concentration due to the inhibition of CAT and APX (Clark et al., 2000), whereas exogenous H2O2 activated NO synthesis in tobacco (De Pinto et al., 2006), suggesting a bidirectional interaction between the two compounds. An understanding of the signaling events that lie upstream and downstream of H2O2 or NO may be a clue in determining the relationships between these two molecules in the regulation of stomatal movements. Removal of H2O2 using antioxidants or inhibition of its synthesis by inhibiting NAD(P)H oxidase activity prevented both NO production and stomatal closure. Similarly, removal of NO by PTIO compromised the induction of stomatal closure by H2O2 or ABA. It is generally accepted that ABA-induced stomatal closure typically requires elevations in cytosolic calcium (Allen et al., 2000). Synthesis and action of calcium-mobilizing molecules such as cADPR regulate these elevations in calcium (Leckie et al., 1998). There are some evidences that both H2O2 and NO actions in the regulation of stomatal closure require calcium. Both NO and H2O2 were reported to activate calcium channels and inactivate K+ channels in Arabidopsis or Vicia faba guard cells (Garcia-Mata et al., 2003; Pei et al., 2000; Zhang et al., 2001; Kohler et al., 2003). NO also influenced GSH synthesis, as demonstrated in Medicago truncatula roots in which the levels of GSH and γ-EC and Glutathione Synthetase (GS) gene expressions were increased by NO (Innocenti et al., 2007; Xu et al., 2010). During the interaction of GSH with NO, S-nitrosoglutathione (GSNO) is formed in a reaction that may interconnect the ROS- and NOS-based signaling pathways (Neill et al., 2002).
CONCLUSIONS
The roles of NO in plant responses to abiotic stresses are studied through investigating the effects on plant physiological and biochemical changes under stress. NO has been found to play a crucial role in plant growth and development, starting from cell cycle regulation, differentiation and morphogenesis, including flowering and root formation. However, the most important and best documented function of NO is the up-regulation of antioxidant defense or directly functions as an antioxidant. Although several NO synthesis pathways in plants have been suggested, biochemical and molecular details of each pathway remain obscure and it is unclear how these identified pathways cooperate with each other in plants and which pathway operates in each particular tissue or organ or at a specific time. Regarding NO biosynthesis, future studies should focus on how NO is produced in a particular tissue or organ (and in which pathway), at what time scale NO production is elicited by a developmental or environmental stimulus and how the above described pathways work in concert when/if they all work in the same tissue or organ at the same time scale.
In the last few years NO and H2O2 have emerged to be central players in the world of plant cell signaling, particularly under various stressful situations. The full range of biological functions for these two signaling molecules remain to be catalogued and determining the ways in which they interact, both together and with the ever-increasing array of signals known to be recognized by plants, will need to be elucidated (Neill et al., 2002). Other research priorities must include full characterization of the enzymes through which the intracellular concentrations of H2O2 and NO are regulated and where these enzymes are located in different cells and tissues. The intracellular signaling cascades that transduce H2O2 and NO perceptions into cellular responses have so far been characterized only superficially. Finally, there arises the question how H2O2 and NO are detected by cells. Such perception could conceivably involve direct interaction of H2O2 and NO with various cellular proteins, such as transcription factors, ion channels or enzymes. H2O2- and NO-sensitive enzymes could include signaling enzymes such as protein kinases and phosphatases (Neill et al., 2002). NOS deficient mutant and/or gene knock-out mutant are now available. Genomics tools are accelerating the discovery of NO producing genes on a global scale and are expanding our understanding of the oxidative stress response and the pleiotropic roles of NO in signaling, gene expression and plant stress tolerance.
ACKNOWLEDGMENT
We specially thank Prof. Dr. Kamal Uddin Ahamed and Mrs. Kamrun Nahar, Lecturer, Department of Agricultural Botany, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh for critical reading and invaluable language improvement of this manuscript.