Cell signalling following plant/pathogen interactions involves the generation of reactive oxygen and reactive nitrogen species

It is now clear that reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), and reactive nitrogen species, such as nitric oxide (NO), are produced by plant cells in response to a variety of stresses, including pathogen challenge. Such molecules may be involved in direct defence mechanisms, such as cross-linking of plant cell walls, or as antimicrobial agents. However, it is also apparent that cells generate such reactive species as signalling molecules, produced at controlled levels, and leading to defined responses. Signalling responses to ROS and NO include the activation of mitogen-activated protein kinases, and the up- and down-regulation of gene expression, often leading to localised programmed cell death, characteristic of the hypersensitive response. Therefore, ROS and NO are key molecules which may help to orchestrate events following pathogen challenge. Here we review the generation and role of both reactive oxygen and reactive nitrogen species in plant cells.     

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv. Marketer. Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected. Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth. Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density. In contrast, cell density was significantly decreased following the addition of catalase to the medium. Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division. Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation.     

Signaling on the Stigma: Potential New Roles for ROS and NO in Plant Cell Signaling

Reactive oxygen species (ROS) and reactive nitrogen species, particularly NO, are key components of diverse signaling networks in animals and plants. We have recently shown that epidermal cells of stigmas from a range of different angiosperms accumulate relatively large amounts of ROS, principally H₂O₂, whereas pollen produces NO. Importantly, ROS/H₂O₂ levels appeared reduced in stigma cells supporting developing pollen grains compared to cells without pollen grains attached. To explore a possible link between pollen NO production and reduced levels of stigmatic ROS/H₂O₂, we supplied stigmas with NO and observed an overall reduction in levels of stigmatic ROS/H₂O₂. These new and unexpected data suggest a potential new signaling role for ROS/H₂O₂ and NO in pollen-stigma recognition processes.Key Words: stigma, pollen, reactive oxygen species, hydrogen peroxide, nitric oxide, signaling, defense     

Mechanisms of nitric oxide crosstalk with reactive oxygen species scavenging enzymes during abiotic stress tolerance in plants

Nitric oxide (NO) acts in a concentration and redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging reactive oxygen species (ROS), such as superoxide anions (O₂^?*), to form peroxynitrite (ONOO^?) or by acting as a signaling molecule, thereby altering gene expression. NO can interact with different metal centres in proteins, such as heme-iron, zinc–sulfur clusters, iron–sulfur clusters, and copper, resulting in the formation of a stable metal–nitrosyl complex or production of varied biochemical signals, which ultimately leads to modification of protein structure/function. The thiols (ferrous iron–thiol complex and nitrosothiols) are also involved in the metabolism and mobilization of NO. Thiols bind to NO and transport it to the site of action whereas nitrosothiols release NO after intercellular diffusion and uptake into the target cells. S-nitrosoglutathione (GSNO) also has the ability to transnitrosylate proteins. It is an NO˙ reservoir and a long-distance signaling molecule. Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins. The exact molecular mechanism(s) by which exogenous and endogenously generated NO (or reactive nitrogen species) modulate the induction of various genes affecting redox homeostasis, are being extensively investigated currently by various research groups. Present review provides an in-depth analysis of the mechanisms by which NO interacts with and modulates the activity of various ROS scavenging enzymes, particularly accompanying ROS generation in plants in response to varied abiotic stress.     

Candida albicans Suppresses Nitric Oxide Generation from Macrophages via a Secreted Molecule

Macrophages and neutrophils generate a potent burst of reactive oxygen and nitrogen species as a key aspect of the antimicrobial response. While most successful pathogens, including the fungus Candida albicans, encode enzymes for the detoxification of these compounds and repair of the resulting cellular damage, some species actively modulate immune function to suppress the generation of these toxic compounds. We report here that C. albicans actively inhibits macrophage production of nitric oxide (NO). NO production was blocked in a dose-dependent manner when live C. albicans were incubated with either cultured or bone marrow-derived mouse macrophages. While filamentous growth is a key virulence trait, yeast-locked fungal cells were still capable of dose-dependent NO suppression. C. albicans suppresses NO production from macrophages stimulated by exposure to IFN-γ and LPS or cells of the non-pathogenic Saccharomyces cerevisiae. The NO inhibitory activity was produced only when the fungal cells were in direct contact with macrophages, but the compound itself was secreted into the culture media. LPS/IFNγ stimulated macrophages cultured in cell-free conditioned media from co-cultures showed reduced levels of iNOS enzymatic activity and lower amounts of iNOS protein. Initial biochemical characterization of this activity indicates that the inhibitor is a small, aqueous, heat-stable compound. In summary, C. albicans actively blocks NO production by macrophages via a secreted mediator; these findings expand our understanding of phagocyte modulation by this important fungal pathogen and represent a potential target for intervention to enhance antifungal immune responses.     

Dormancy removal of apple seeds by cold stratification is associated with fluctuation in H2O2, NO production and protein carbonylation level

Reactive oxygen (ROS) and nitrogen (RNS) species play a signaling role in seed dormancy alleviation and germination. Their action may be described by the oxidative/nitrosative “window/door”. ROS accumulation in embryos could lead to oxidative modification of protein through carbonylation. Mature apple (Malus domestica Borkh.) seeds are dormant and do not germinate. Their dormancy may be overcome by 70–90 days long cold stratification. The aim of this work was to analyze the relationship between germinability of embryos isolated from cold (5 °C) or warm (25 °C) stratified apple seeds and ROS or nitric oxide (NO) production and accumulation of protein carbonyl groups. A biphasic pattern of variation in H₂O₂ concentration in the embryos during cold stratification was detected. H₂O₂ content increased markedly after 7 days of seeds imbibition at 5 °C. After an additional two months of cold stratification, the H₂O₂ concentration in embryos reached the maximum. NO production by the embryos was low during entire period of stratification, but increased significantly in germination sensu stricto (i.e. phase II of the germination process). The highest content of protein carbonyl groups was detected after 6 weeks of cold stratification treatment. Fluctuation of H₂O₂ and protein carbonylation seems to play a pivotal role in seed dormancy alleviation by cold stratification, while NO appears to be necessary for seed germination.     

Optimization of macrophage isolation from the Persian sturgeon and the Caspian kutum fish: a comparative study

The aim of this research was a comparative study on the isolation and culture of head kidney macrophages derived from Acipenser persicous and Rutilus frisii kutum as teleost and chondrostei species of fish. The macrophages were isolated by density gradient sedimentation, followed by adherence to a plastic surface. They exhibited strong phagocytic activity against bacteria. The effect of cell density, incubation time, FBS percentage, pH and temperatures on the cell number and viability were determined and compared. Also, the effect of light/dark regimen on viability, adherence, release of reactive oxygen species (ROS) and nitric oxide (NO) in the macrophages was determined. The results showed that the Caspian kutum macrophages were more sensitive to FBS percentage and cell density whereas the Persian sturgeon macrophages were more sensitive to pH of the cell culture media. The adherence and viability of the macrophages from both fish species firstly increased (P?<?0.05) after exposure to a light/dark regimen, but then significantly decreased as did ROS and NO productions. For the first time, this study has determined the optimal conditions for primary culture of macrophages derived from sturgeons, and shows the unique effect of light on the biology of fish immune cells.     

<Emphasis Type="Italic">Mycobacterium avium</Emphasis> MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨ_m), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨ_m loss, and apoptosis were all significantly reduced in TLR4^?/? macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.     

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.     

Cytosolic alkalinization is a common and early messenger preceding the production of ROS and NO during stomatal closure by variable signals,including abscisic acid,methyl jasmonate and chitosan

Stomata are unique that they sense and respond to several internal and external stimuli, by modulating signaling components in guard cells. The levels of reactive oxygen species (ROS), nitric oxide (NO) and cytosolic calcium (Ca²⁺) increase significantly during stomatal closure by not only plant hormones [such as abscisic acid (ABA) or methyl jasmonate (MJ)] but also elicitors (such as chitosan). We observed that cytosolic alkalinization preceded the production of ROS as well as NO during ABA induced stomatal closure. We therefore propose that besides ROS and NO, the cytosolic pH is an important secondary messenger during stomatal closure by ABA or MJ. We also noticed that there is either a cross talk or feedback regulation by cytosolic Ca²⁺ and ROS (mostly H₂O₂). Further experiments on the interactions between cytosolic pH, ROS, NO and Ca²⁺ would yield interesting results.Key words: abscisic acid, methyl jasmonate, chitosan, cytosolic pH, reactive oxygen species, H₂O₂, nitric oxide, cytosolic calcium     

Reactive oxygen and nitrogen species are involved in sorbitol-induced apoptosis of human erithroleukaemia cells K562

In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (ΔΨ), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of ΔΨ and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.     

Reactive oxygen and nitrogen species are involved in sorbitol-induced apoptosis of human erithroleukaemia cells K562

In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (ΔΨ), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of ΔΨ and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.     

A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria

In this paper we propose and provide evidence for a mechanism, oxidative nitrogen scavenging (ONS), whereby seedlings of some grass species may extract nitrogen from symbiotic diazotrophic bacteria through oxidation by plant-secreted reactive oxygen species (ROS). Experiments on this proposed mechanism employ tall fescue (Festuca arundinaceae) seedlings to elucidate features of the oxidative mechanism. We employed 15N₂ gas assimilation experiments to demonstrate nitrogen fixation, direct microscopic visualization of bacteria on seedling surfaces to visualize the bacterial oxidation process, reactive oxygen probes to test for the presence of H₂O₂ and cultural experiments to assess conditions under which H₂O₂ is secreted by seedlings. We also made surveys of the seedlings of several grass species to assess the distribution of the phenomenon of microbial oxidation in the Poaceae. Key elements of the proposed mechanism for nitrogen acquisition in seedlings include: 1) diazotrophic bacteria are vectored on or within seeds; 2) at seed germination bacteria colonize seedling roots and shoots; 3) seedling tissues secrete ROS onto bacteria; 4) bacterial cell walls, membranes, nucleic acids, proteins and other biological molecules are oxidized; 5) nitrates and/or smaller fragments of organic nitrogen-containing molecules resulting from oxidation may be absorbed by seedling tissues and larger peptide fragments may be further processed by secreted or cell wall plant proteases until they are small enough for transport into cells. Hydrogen peroxide secretion from seedling roots and bacterial oxidation was observed in several species in subfamily Pooideae where seeds possessed adherent paleas and lemmas, but was not seen in grasses that lacked this feature or long-cultivated crop species.     

Reactive oxygen and nitrogen species during meiotic resumption from diplotene arrest in mammalian oocytes

Mammalian ovary is metabolically active organ and generates by‐products such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) on an extraordinary scale. Both follicular somatic cells as well as oocyte generate ROS and RNS synchronously and their effects are neutralized by intricate array of antioxidants. ROS such as hydrogen peroxide (H₂O₂) and RNS such as nitric oxide (NO) act as signaling molecules and modulate various aspects of oocyte physiology including meiotic cell cycle arrest and resumption. Generation of intraoocyte H₂O₂ can induce meiotic resumption from diplotene arrest probably by the activation of adenosine monophosphate (AMP)‐activated protein kinase A (PRKA)—or Ca²⁺‐mediated pathway. However, reduced intraoocyte NO level may inactivate guanylyl cyclase‐mediated pathway that results in the reduced production of cyclic 3′,5′‐guanosine monophosphate (cGMP). The reduced level of cGMP results in the activation of cyclic 3′,5′‐adenosine monophosphate (cAMP)‐phosphodiesterase 3A (PDE3A), which hydrolyses cAMP. The reduced intraoocyte cAMP results in the activation of maturation promoting factor (MPF) that finally induces meiotic resumption. Thus, a transient increase of intraoocyte H₂O₂ level and decrease of NO level may signal meiotic resumption from diplotene arrest in mammalian oocytes. J. Cell. Biochem. 111: 521–528, 2010. © 2010 Wiley‐Liss, Inc.     

Reactive oxygen species in vascular biology: implications in hypertension

Reactive oxygen species (ROS), including superoxide (·O₂^–), hydrogen peroxide (H₂O₂), and hydroxyl anion (OH-), and reactive nitrogen species, such as nitric oxide (NO) and peroxynitrite (ONOO^–), are biologically important O₂ derivatives that are increasingly recognized to be important in vascular biology through their oxidation/reduction (redox) potential. All vascular cell types (endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts) produce ROS, primarily via cell membrane-associated NAD(P)H oxidase. Reactive oxygen species regulate vascular function by modulating cell growth, apoptosis/anoikis, migration, inflammation, secretion, and extracellular matrix protein production. An imbalance in redox state where pro-oxidants overwhelm anti-oxidant capacity results in oxidative stress. Oxidative stress and associated oxidative damage are mediators of vascular injury and inflammation in many cardiovascular diseases, including hypertension, hyperlipidemia, and diabetes. Increased generation of ROS has been demonstrated in experimental and human hypertension. Anti-oxidants and agents that interrupt NAD(P)H oxidase-driven ·O₂^– production regress vascular remodeling, improve endothelial function, reduce inflammation, and decrease blood pressure in hypertensive models. This experimental evidence has evoked considerable interest because of the possibilities that therapies targeted against reactive oxygen intermediates, by decreasing generation of ROS and/or by increasing availability of antioxidants, may be useful in minimizing vascular injury and hypertensive end organ damage. The present chapter focuses on the importance of ROS in vascular biology and discusses the role of oxidative stress in vascular damage in hypertension.     

Immune cell-induced synthesis of NO and reactive oxygen species in lymphoma cells causes their death by apoptosis

Induction of apoptosis in a lymphoma cell line using immune cell-conditioned medium, etoposide or an nitric oxide (NO) donor, resulted in the production of reactive oxygen species (ROS). Agents that inhibited NO production or scavenged ROS or species formed by reaction of NO with ROS, protected the cells from apoptosis. These data support the suggestion that immune rejection of an immunogenic derivative of this lymphoma in vivo involves the induced synthesis of both NO and ROS by the tumour cells.     

Phospholipase Dα1 and Phosphatidic Acid Regulate NADPH Oxidase Activity and Production of Reactive Oxygen Species in ABA-Mediated Stomatal Closure in Arabidopsis

We determined the role of Phospholipase Dα1 (PLDα1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldα1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pldα1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pldα1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA–ABI1 interaction was required for stomatal closure induced by ABA, H₂O₂, or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.     

Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involves reactive oxygen species

Matrix metalloproteinases (MMPs) participate in cell migration and remodeling processes by affecting the extracellular matrix. MMP-2 is thought to be involved in cancer cell invasiveness. It has been proposed that the activity of MMP-2 can be modulated by intracellular reactive oxygen species (ROS)/reactive nitrogen species. We hypothesized that manganese superoxide dismutase (MnSOD) could mediate MMP-2 activity by changing the intracellular ROS level and that nitric oxide ((.)NO) may be involved in this process. Human breast cancer MCF-7 cells were stably transfected with plasmids containing MnSOD cDNA. A 2-30-fold increase of MnSOD protein and activity was observed in four clones. Our data demonstrated that overexpression of MnSOD stimulated the activation of MMP-2 with a corresponding elevation of ROS. A decrease in ROS by ebselen, a glutathione peroxidase mimetic, or by transduction of adenovirus containing human catalase or glutathione peroxidase cDNA abolished the effect of MnSOD on MMP-2 activation. Treatment of MCF-7 cells with antimycin A or rotenone increased intracellular ROS production and MMP-2 activation simultaneously. Our data also showed a suppression of endothelial nitric-oxide synthase expression that was accompanied by decreased (.)NO production in MnSOD-overexpressing cells. However, the changes in endothelial nitric-oxide synthase and (.)NO did not correlate with the MnSOD activity. Corresponding changes of MMP-2 activity after the addition of a NOS inhibitor (N(G)-amino-l-arginine) or a (.)NO donor ((Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate) to the cells suggested the possibility that (.)NO may be involved in the MnSOD-mediated MMP-2 activation pathway. These results indicate that MnSOD induces MMP-2 activity by regulation of intracellular ROS and imply that signaling pathways involving (.)NO may also be involved in the MnSOD mediation of MMP-2 activity.