S-nitrosylation, however, was only confirmed for APX from GSNO-treated potato leaves Kato et al. In the same study DHAR was demonstrated to be S-nitrosylated and inhibited by NO.

A possible target Cys essential for enzymatic function was revealed by point mutation of candidate Cys residues. Human manganese SOD is a mitochondrial protein that undergoes site-specific nitration at Tyr34 during inflammation.

Inactivation of Mn-SOD by nitration provokes oxidative stress and ultimately dysfunction of mitochondria Radi, It would be interesting to elucidate if plant SODs are targets of nitrating species with possible roles e.

Collectively, the discussed data suggest that APX, CAT, and DHAR are good candidates for NO-regulated antioxidant enzymes in plants.

A systematic approach is needed for deciphering, which antioxidant enzymes are controlled by NO under stress conditions, and what are the underlying molecular mechanisms.

We mentioned before that NO bioactivity has been implicated both in increased as well as decreased antioxidant enzyme activities and ROS levels. One way of explaining the contradictory findings is based on the hypothesis that NO has a dose-dependent effect on the cellular redox status Figure 2 Thomas et al.

At low concentrations NO might stimulate the antioxidant system and promote cell survival while high concentrations of NO cause severe cell damage and even death.

Little damage and NO-induced signaling will be perceived by the cell triggering antioxidant defence and repair mechanisms.

This would shift conditions in the cell from weak oxidative stress toward heavy nitrosative stress, which—according to the hypothesis of Thomas et al. For some biological effects the duration of NO production is decisive because certain target molecules bind NO very slowly or need sequential NO and ROS modifications Thomas et al.

Figure 2. Hypothetical model on the dynamic interaction between NO, ROS and the antioxidant system under stress conditions. Weak stress triggers a moderate elevation of ROS reactive oxygen species and NO levels.

ROS act as signals inducing NO synthesis and activation of the antioxidant system for improved metabolic adaptation. If ROS is produced at a somewhat higher rate than NO there would be mainly formation of oxidizing and nitrating RNS reactive nitrogen species imposing a weak oxidative stress to the cell.

Heavy stress leads to a strong ROS and RNS burst. High NO levels promote formation of N 2 O 3 from NO 2 and NO and consequently nitrosative stress.

Under these conditions ROS and RNS inhibit the antoxidant system causing damage and ultimately death of plant cells.

The versatility of signaling by RNS and ROS is further extended by their interaction with antioxidants. Consequently, NO is released and AsA is converted to DHA Combet et al. DHA spontaneously decays to the ascorbyl radical, which can combine with NO to give O-nitrosoascorbate.

However, the high concentrations of GSH and AsA in plant cells could contribute to maintaining low levels of NO derivatives under non-stress conditions. Recently, cytokinins were demonstrated to be involved in controlling NO levels in A.

thaliana Liu et al. Continuous root-uptake of μM SNP severely inhibited growth of A. Further characterization of the mutant revealed a correlation between NO resistance and elevated cytokinin levels. Accordingly, WT plants infiltrated with the cytokinin zeatin displayed improved growth on SNP-loaded agar medium.

In vitro , zeatin was nitrated by peroxynitrite, which produced 8-nitro-zeatin. In vivo , SNP caused strong accumulation of 8-nitro-zeatin in cnu-1 as compared to WT. NO interacts with glutathione in various ways. At the transcriptional level SNP and GSNO stimulated genes involved in GSH synthesis causing elevated levels of total glutathione in Medicago truncatula roots Innocenti et al.

Accordingly, NO donor treatment triggered an increase in total glutathione in 8 of 10 studies summarized in Table 1. In contrast, SNP had no strong effect on GSH concentrations in tobacco BY-2 cells De Pinto et al. At the level of chemical interactions GSH binds NO by S-nitrosylation.

GSNO is formed either after 1 ROS-induced accumulation of glutathiyl radicals, which bind NO with rate constants near the diffusion-controlled limit Madej et al. GSNO then functions as storage and transport form of NO. Levels of the S-nitrosylated tripeptide GSNO are tightly controlled by the enzyme GSNOR.

This GSH-dependent formaldehyde dehydrogenase catalyzes the transformation of GSNO to GSSG and hydroxylamine NH 2 NO in the presence of GSH and NADH as the reducing species Figure 3 Liu et al. GSNOR1 deficient plants were severely affected in growth and development Kwon et al. They also showed increased resistance to the herbicide paraquat and altered responses toward heat stress and pathogen infection Diaz et al.

In addition to control of NO levels, GSNOR is also indirectly involved in protein denitrosylation because GSNO and S-nitrosylated proteins are in equilibrium Benhar et al. For more information on GSNOR functions refer to recent reviews Leitner et al.

Figure 3. Enzymatic regulation of NO homeostasis by 1 S-nitrosogutathione reductase GSNOR , 2 hemoglobin Hb , and 3 peroxiredoxin IIE PrxIIE. PrxIIE is reduced by thioredoxin Trx.

Another upcoming topic is the modulation of NO homeostasis by plant hemoglobins. Particularly, the role of alfalfa and A. thaliana Hb1 in hypoxia has been studied in more detail Dordas et al. It was shown that hypoxia triggered expression of the Hb1-coding gene in roots, probably for confining the stress-induced accumulation of NO.

Reduced expression of Hb1 in transgenic and mutant lines caused an increase in NO levels concomitant with decreased plant growth whereas Hb1 over-expression improved plant fitness during hypoxia. By scavenging NO the plant might suppress a costly defence response for saving energy and valuable nitrogen under limited oxygen availability Hebelstrup et al.

Recently, Hb1 was found to be involved in pathogen resistance. thaliana mutants defective in the Hb1-coding gene GLB1 were more resistant to the hemibiotrophic P. syringae and the necrotrophic fungus Botrytis cinerea Mur et al. The mutant phenotype was reversed by over-expression of GLB1 under control of the 35S promoter.

The enhanced resistance in the glb1 mutant correlated with accumulation of SA, JA, and ET. GLB1 was down-regulated in WT plants during infection, which probably facilitated the induction of defence responses by NO accumulation. Both enzymes are then reactivated by thioredoxin in a NADPH-consuming manner.

During severe hypoxia deoxygenated A. thaliana Hb1 might act as nitrite reductase although with rather slow kinetics Tiso et al. A more wide-spread phenomenon could be the nitration-promoting activity of peroxidases. For instance, three A.

Sakihama et al. All the above data on Hb1 acting as nitrite reductase and enzymatic nitration by peroxidases were obtained in vitro and it is difficult to draw any meaningful conclusions for the in vivo situation.

ROS and RNS are major players in plant stress signaling. In this section we will survey current knowledge on the roles of ROS, RNS and elements of the antioxidant system in cell death events induced by biotic and abiotic stressors.

Plants attacked by an avirulent pathogen develop HR, which is a defence mechanism for restricting the spread of pathogens by cell wall reinforcement, production of defensive secondary metabolites and ultimately cell death Mur et al.

Almost 20 years ago Chris Lamb and his co-workers discovered that soybean cells infected with avirulent Pseudomonas syringae pv. glycinea accumulated high levels of H 2 O 2 , which functioned as a cell death inducer during the HR Levine et al.

Suppression of the pathogen-induced H 2 O 2 burst by the NADPH oxidase inhibitor diphenylene iodonium DPI prevented cell death whereas low millimolar concentrations of exogenous H 2 O 2 triggered HR-PCD in a calcium-dependent manner Levine et al.

Later, researchers of the same group demonstrated that NO was another essential messenger in cell death execution Delledonne et al. Application of a NO scavenger and a NOS activity inhibitor both reduced HR-PCD of soybean suspension cells infected with avirulent bacterial pathogens.

Importantly, SNP triggered cell death most efficiently in conjunction with ROS but not in the presence of DPI or CAT. ROS donors in turn efficiently killed soybean cells only if applied together with SNP Delledonne et al. Comparable results were obtained with tobacco BY-2 cells.

Therefore, it was postulated that NO and ROS cooperate in cell death signaling Figure 2. Recent studies have begun to unravel the underlying modes of interactions between NO, ROS and the antioxidant system during PCD.

thaliana plants challenged by avirulent Pseudomonas syringae Gaupels et al. However, contrary to mammalian cells this RNS does not kill plant cells Delledonne et al. If this is a significant process in vivo remains to be proven. This particular ROS acts as an inducer of NO synthesis in tobacco cells De Pinto et al.

For instance, rice knock-out mutants defective in a CAT-coding gene showed increased H 2 O 2 levels, nitrate reductase-dependent accumulation of NO and spontaneous leaf cell death Lin et al.

Application of the NO scavenger PTIO mitigated the cell death phenotype. The importance of a down-regulation of ROS detoxifying enzymes during PCD was further corroborated by the finding that overexpression of thylakoidal APX led to a higher resistance against SNP induced cell death Murgia et al.

thaliana WT plants 5mM SNP triggered H 2 O 2 accumulation and cell death, which was both reduced in the transgenic line probably because H 2 O 2 was degraded by the elevated APX activity in these plants. The antioxidant enzymes CAT and APX control H 2 O 2 levels under mild stress conditions.

Severe cadmium stress triggered NO as well as H 2 O 2 accumulation and senescence-like PCD of A. thaliana suspension cultured cells De Michele et al. However, co-treatment with the NOS inhibitor L-NMMA prevented the NO-dependent inhibition of CAT and APX, which in turn reduced H 2 O 2 levels and increased cell viability under cadmium stress.

Mechanical wounding provokes cell damage, which could serve as a point of entry into the plant e. To avoid this, PCD is triggered in intact cells nearby the damaged cells for sealing the wound site.

In wounded leaves of Pelargonium peltatum NO accumulation was restricted to the site of injury Arasimowicz et al. Treatment with cPTIO confirmed that NO inhibited APX and CAT activity thereby temporarily enhancing the H 2 O 2 content at the edge of the wound.

Pre-treatment of leaves with NO donors before wounding prevented the H 2 O 2 burst and reduced necrotic cell death in sweet potato Lin et al. The exact mechanism of NO action was not determined but available data suggest that APX, GR, MDHAR and thioredoxin are S-nitrosylated during PCD, which could affect their activity Murgia et al.

Inhibition of GR and MDHAR would also impact on the redox status of the glutathione and ascorbate pools. It should be considered that enzymatic activity can also be influenced by ROS-dependent modifications, which was proposed for oxidation-triggered inhibition of APX Figure 2 De Pinto et al.

The latter enzyme was also suppressed in gene expression during PCD De Pinto et al. The role of NO in incompatible interactions between A. thaliana and avirulent Pseudomonas syringae was investigated using transgenic plant lines expressing a bacterial NO dioxygenase NOD, flavohemoglobin Zeier et al.

NOD expression attenuated the pathogen-induced NO accumulation. As a consequence the H 2 O 2 burst was diminished and transgenic plants developed less HR-PCD and were delayed in SA-dependent PR1 expression.

These results support again the hypothesis that high levels of NO amplify redox signaling during PCD by inhibiting the plant antioxidant machinery Zeier et al.

NO and H 2 O 2 might mutually enhance each other's accumulation by positive feed-back regulation. To this end, NO and ROS producing enzymes as well as elements of the antioxidant system must be regulated in a highly coordinate fashion for initiation of PCD.

The exact signaling pathways remain to be deciphered in future studies. However, the plant must also constrain stress signaling by NO, ROS and the antioxidant system for avoiding excessive damage by runaway cell death. Therefore, it is worth mentioning that both ROS as well as NO were found to induce genes involved in cell protection such as a gene coding for glutathione S-transferase Levine et al.

Yun and colleagues Yun et al. thaliana challenged by avirulent bacteria. The authors proposed a model, in which the early burst of ROS and NO initiates HR-PCD but at later stages of the defence response the SNO levels exceed a certain threshold and subsequently the AtRBOHD is inactivated by S-nitrosylation at Cys , which terminates the HR.

In contrast to R gene-mediated resistance against avirulent pathogens, bacterial lipopolysaccharides LPS elicit basal pathogen resistance without onset of HR-PCD.

LPS-induced NO synthesis by an arginine-dependent enzymatic source even protected plant cells against oxidative stress and cell death by enhancing the activities of CAT, SOD, and POD. The changed cellular redox status contributed to the regulation of NPR1-dependent expression of defence genes Sun et al.

In sum, NO can either act as an inducer or suppressor of plant PCD dependent on its local cellular levels and its tightly controlled interaction with ROS and elements of the antioxidant system Figure 2. ROS and NO are increasingly recognized signaling molecules in plant physiology.

While research on ROS has a long history NO came into focus only 15 years ago. In the present paper we reviewed recent literature dealing with the interaction between ROS, NO and the antioxidant system during stress defence.

As one interesting outcome we found that exposure of plants to unfavorable conditions inevitably induced ROS but not necessarily NO accumulation.

In contrast, NO is rather a highly specialized second messenger, which modifies ROS signaling or acts independently of ROS. Significantly, ROS and NO bursts are often triggered simultaneously—sometimes even in the same cellular compartment. Particularly chloroplasts and peroxisomes are hotspots of NO-ROS interactions.

More indirect interactions include induction of NO synthesis by H 2 O 2 and accumulation of ROS due to inhibition of antioxidant enzymes by NO-dependent protein modifications. Therefore, plants have developed efficient measures for controlling NO levels by GSNOR, hemoglobins and other RNS scavenging enzymes.

This review was also aimed at investigating the extreme versatility of possible reactions between NO, ROS and the antioxidant system. More basic research is urgently needed for defining chemical reactions and their products actually occurring in planta.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Werner Heller for helpful discussions and critical reading of the manuscript. Ahlfors, R. Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana.

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Nitric Oxide 6, — Maassen, A. Effect of Medicago sativa Mhb1gene expression on defense response of Arabidopsis thaliana plants. The Janus face of nitric oxide NO has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury.

There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO.

In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase.

In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion.

This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress. Mechanisms of the antioxidant effects of nitric oxide. Publication , Journal Article. Wink, DA; Miranda, KM; Espey, MG; Pluta, RM; Hewett, SJ; Colton, C; Vitek, M; Feelisch, M; Grisham, MB.

April Published version DOI Link to item. Author Carol Anne Colton Neurology, Behavioral Neurology. Author Michael P. Vitek Neurology, Behavioral Neurology.