The Oxidative and Nitrosative Nature of Abiotic Stress

Abiotic stresses represent the most limiting factors for agricultural productivity.1 Plants have developed unique strategies for responding to abiotic stress by adjusting their metabolic systems to maintain cellular homeostasis.2–4 As a consequence, diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of reactive oxygen species (ROS), including superoxide radical (O₂^·−), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH^·), together with singlet oxygen (¹O₂).5–8 Oxidative stress occurs due to excessive accumulation of ROS under various abiotic stress conditions and may provoke oxidative damage,9 ultimately leading to cell death.10 However, almost two decades of oxidative stress studies have yielded a concept of ROS as being ubiquitous stress markers and signaling species.7 Today, several parameters regarding ROS generation, scavenging and signaling have been uncovered, but how their role is being controlled is largely unknown.2–12 Also, it is becoming increasingly apparent that ROS signal molecules can cross-talk with other signaling pathways to affect multiple physiological processes in plants.6,13

Over the past years the free radical nitric oxide (NO) has emerged as a signal molecule in many important physiological processes in higher plants.14 The term reactive nitrogen species (RNS) has been formulated to designate NO and the NO-derived molecules such as dinitrogen tridoxide (N₂O₃), nitrogen dioxide (^·NO₂), peroxynitrite (ONOO⁻), S-nitrosothiols (RSNOs), S-nitrosoglutathione (GSNO), etc.15 Nitrosative stress is induced by pathophysiological levels of NO and S-nitrosothiols and it mainly arises from the nitrosylation of critical protein cysteine (Cys) thiols (S-nitrosylation) and metal co-factors.16 It has been increasingly evident that NO plays important roles in many aspects of developmental processes in a similar fashion to ROS, including seed germination and dormancy, root growth, root gravitropism, xylogenesis, leaf expansion, photomorphogenesis, floral transition, stomatal closure, senescence and programmed cell death.17–19 It is also apparent that NO and NO-derived molecules are involved in plants' response to a multitude of abiotic stresses such as salinity, high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding.20–22 Overwhelming evidence also suggests that plants use both ROS and RNS as signal transduction molecules during basic biological and cellular processes.23 The present mini-review organizes the information in order to illustrate the connection between oxidative and nitrosative ‘branches’ in the ‘tree’ of signaling network in plants.

Evidence for Oxidative and Nitrosative Signaling Cross-Talk

The best-characterized relationship between NO and ROS is their role in plant defense against pathogen attack, in particular in establishing the hypersensitive response (HR). It was shown that the oxidative burst that occurs during plant infection by pathogens is accompanied by a parallel nitrosative burst.24,25 Using two sunflower (Helianthus annuus L.) cultivars with different sensitivity to infection by the pathogen Plasmopara halstedii, Chaki et al.26 showed that the susceptible cultivar underwent nitrosative stress, as evidenced by the increase in nitrated proteins and by the induction of RSNO content. In contrast, no increase of RSNOs or tyrosine nitration of proteins was observed in the resistant cultivar, implying an absence of nitrosative stress. These observations provide solid evidence suggesting that the nitrosative responses are tightly linked with tolerance mechanisms against biotic stress conditions in plants. Since tolerance to stressful conditions is intimately connected to the capacity of plants to tolerate oxidative stress, it is obvious that oxidative and nitrosative syndromes are closely linked and coordinated during the overall response of plants to environmental stimuli. This is also supported by the fact that applications of various NO donors at whole plants or cell cultures revealed that NO can counteract oxidative damage and stimulate the activities of antioxidant enzymes.27,28

Due to the paramount significance of ROS and RNS in plant physiology, issues on ROS and RNS formation, chemistry of their interconversion and utilization have been studied. The key element in ROS and RNS interaction is the diffusion-limited reaction of NO with O₂^·− (k = 6.7 × 10⁹ litermol⁻¹s⁻¹) to form ONOO⁻. This reaction between NO and O₂^·− along with the ROS-scavenging system therefore determines the steady-state level of ROS and RNS, and the different developmental, metabolic, and/or defense pathways. The ROS/RNS mode of action during the induction of hypersensitive cell death proposed by Delledonne et al.29 describes a system in which NO is being scavenged before it can react with H₂O₂ if the balance between NO and O₂^·− production is in favor of O₂^·−, whereas if the balance is in favor of NO, the O₂^·− is scavenged before it can de dismutated to H₂O₂. Although ONOO⁻ is not a free radical in nature, it is much more reactive than its parent molecules NO and O₂^·−. Even though the half-life of ONOO⁻ is short (10–20 ms), it is sufficiently long-lived so as to cross biological membranes and to interact with critical biomolecules. Recent studies in plants identified an important signaling mechanism by which ONOO⁻ amplifies biological responses, including defense responses against abiotic stress and nitrosylation of peroxiredoxins.30,31 Therefore, ONOO⁻ through its formation from O₂^·− and NO, may function as a cellular rheostat of ROS/RNS homeostasis and functions.

Apart from ONOO⁻ generation there are many experimental data indicating that there are other active interactions between ROS and RNS, especially as evidenced in the case of H₂O₂ and NO. Both signaling molecules are simultaneously present during various physiological processes and sometimes NO and H₂O₂ production is interdependent; therefore, the close interplay between these two signals is very plausible. The most well-characterized H₂O₂ and NO interplay is the spatial and temporal production of both molecules in guard cells following ABA challenge.32,33 In addition, epidermal cells of stigmas from a range of different angiosperms accumulate relatively large amounts of ROS, principally H₂O₂, whereas pollen produces NO,34,35 suggesting a potential signaling role for ROS/H₂O₂ and RNS/NO in pollen-stigma recognition processes. More recently, Zafra et al. showed that both ROS and NO are actively produced in the reproductive tissues of olive plants throughout flower development, while Filippou et al. demonstrated the spatiotemporal induction of H₂O₂ and NO in roots and leaves of Medicago truncatula plants subjected to drought stress. These observations raise the question of why the production of H₂O₂ and NO is topologically linked. However, a full understanding of this phenomenon requires information about mechanisms that are involved in the production of H₂O₂ and NO. Although major H₂O₂-producing enzymes are well-characterized (e.g., plasma membrane NADPH oxidase, apoplastic amine, diamine and polyamine oxidase-type enzymes etc.,) the source of NO in plants is under continuous debate. The activity and biological function of AtNOS1 in Arabidopsis was questioned and there is still no strong evidence to indicate the occurrence of an animal-like NOS in plants.38 Interestingly, the discovery that hydroxylamines (R-NHOH) can be oxidized to NO by superoxide- or hydrogen peroxide-generating systems, has led to the recent proposal of an oxidative-associated pathway for NO synthesis.39 To gain a wide understanding of oxidative and nitrosative signaling in plants, it is therefore essential to obtain data concerning the relationship between H₂O₂ and NO production at both a cellular and a whole plant level. In addition, it is now clear that both H₂O₂ and NO act in parallel with multiple phytohormones signaling pathways in response to abiotic stress.40 Also Tun et al. provided evidence that polyamines (PAs) induce NO production in Arabidopsis and reported that NO could be a link between polyamine-mediated stress responses and other stress mediators. Since polyamines have also been proposed as important substrates for H₂O₂ production, being degraded by copper-containing amine oxidases,42 it is possible that PAs may participate in loops involving interaction with signal transduction pathways that may control oxidative and nitrosative events. Also, it is likely that many new signaling components and mechanisms involved in ROS and RNS cross-talk have yet to be unraveled. For example, Wang et al.43 recently showed that Arabidopsis prohibitin gene PHB3 affects the NO homeostasis system in response to H₂O₂. Asai and colleagues,44 using virus-induced gene silencing, also identified NbRibA encoding a bifunctional enzyme, guanosine triphosphate cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase, which participates in the biosynthesis of flavin and further showed that flavin biosynthesis participates in regulating NO and ROS production, as well as HR cell death. These data clearly suggest that there are new players in the word of ROS-RNS signaling and their biological roles need further investigation.

Paradigms for RNS and ROS Interplay at Gene and Protein Level

During the last decade advances in microarray technology, along with proteomic and metabolomic analyses have opened up novel possibilities in understanding ROS and RNS cross-talk in plants. Analysis of the integrated cellular response to oxidative stress is an area that is particularly suited for the use of genome- and proteome-wide approaches. In Arabidopsis thaliana, a network of more than 150 genes is involved in managing the level of ROS in cells. This network is highly dynamic and encodes for ROS sensing, scavenging and producing proteins.6 In addition, several reports provided a global analysis of oxidative stress-responsive genes and proteins in plants (reviewed in ref. 45 and 46). On the other hand, the NO-dependent changes in gene expression have been addressed with the use of different NO-releasing chemicals. Experiments using microarray and cDNA-AFLP approaches have shown that NO-responsive genes serve a variety of functions ranging from plant defense, oxidative stress responses and hormone interplay to metabolism and development.47 Notably, a number of these genes including glutathione- and redox-related enzymes such as glutathione peroxidase and l-ascorbate peroxidase, alternative oxidase, proteins of the cytochrome P450 family, PGIP1, CHIV, GLP5, DIN10 and AT3G55430 encoding a beta-1,3-glucanase-like protein were also commonly identified in oxidative stress studies indicating that NO functions as a signal involved in the plant's adaptive response to environmental-derived oxidative events.48–50 The first direct evidence indicating that there is a considerable overlap between H₂O₂- and NO-inducible genes was reported by Zago et al. in catalase-deficient (CAT1AS) plants during the induction of programmed cell death. These authors used a transcriptional approach to demonstrate that H₂O₂ and NO elicit common signaling pathways which might be attributed to the interplay of both molecules with other phytohormones, as evidenced by the upregulation of transcripts (e.g., lipoxygenase, cytochrome P450-dependent fatty acid hydroxylase, diacylglycerol kinase, lipase, 12-oxophytodienoate reductase) involved in the biosynthesis of oxylipins and jasmonic acid. The likelihood of a correlation between oxidative and nitrosative signaling and gene expression was further supported by the fact that NO- and O₃-induced gene expression largely overlaps in Arabidopsis.52 This study demonstrated that SNP application activated the expression of a large set of defense-related genes whereas SNP-regulated genes were also strongly activated by O₃, indicating a significant overlap between NO and O₃ signaling pathways.

A recent proteomic study on the regulatory role of oxidative and nitrosative episodes during the acclimation of citrus plants to salinity documented strong interplay between H₂O₂- and NO-responsive proteins in NaCl-stressed plants.53 Furthermore, identification of downstream targets of H₂O₂ and NO in citrus plants grown under physiological conditions uncover an interlinked H₂O₂ and NO protein network which mainly belongs to photosynthesis and especially to the Calvin-Benson cycle and, hence, provides a deeper understanding of oxidative and nitrosative signaling in plants.54 From these observations, it is clear that identification and characterization of the ROS and RNS targets under physiological and stress conditions will help to understand how these molecules participate in the cellular response to environmental stimuli. Besides systems biology approaches, individual proteins have also been characterized as common targets for RNS and ROS. For example, Wang et al.55 reported that mitogen-activated protein kinase 6 (MPK6) was involved in regulating H₂O₂-induced synthesis of NO in Arabidopsis. The authors also identified a direct interaction between MPK6 and one nitrate reductase isoform (NIA2) in vitro and in vivo and demonstrated that MPK6 could phosphorylate NIA2 resulting in the production of NO during root development. This observation together with data reported previously by Zhang et al.27 suggest that the MAPK cascade could represent an amplification loop between H₂O₂ and NO signaling during development process. In support, oxidative and nitrosative stress signals can be rapidly dispersed throughout Arabidopsis plants via activation of MAPKs.56

ROS and RNS-Based Post-Translational Modification

An intriguing and important question in our understanding of ROS and RNS interplay is what kind of mechanisms plant cells use for the sensing, transmission and integration of oxidative and nitrosative signals. Redox-based signaling by ROS and RNS is largely governed by their targeted modifications of key reactive Cys amino acids in proteins, including S-nitrosylation and S-glutathionylation as well as sulphenic acid, sulphinic acid and disulphide formation. Among them, the covalent attachment of a NO group to a reactive Cys thiol to form an S-nitrosothiol (SNO), a process known as nitrosylation, is now emerging as a key NO-driven signaling mechanism.57 Recent proteomic-based publications utilizing the biotin-switch procedure, which identifies S-nitrosylated Cys in vivo,58 have characterized nitrosylated proteins in plants and allowed to obtain valuable information regarding how this post-translational protein modification affects some physiological processes.31,59 Since Cys residues are also sensitive to ROS-derived protein carbonylation,60 it is obvious that there is a negative relationship between different Cys modifications produced by ROS and RNS. Experimental evidence indicates that exogenous application of NO and/or H₂O₂ directly affects protein nitrosylation and carbonylation, suggesting both a physiological role for ROS and RNS and a link between oxidative/nitrosative signaling and specific protein modifications.28,53 In recent years, it has also become common to use nitrotyrosine as a marker of nitrosative stress in animals and plants.61 Tyrosine nitration is a covalent protein modification, resulting in the incorporation of a nitrotriatomic group at position 3 of the phenolic ring of a tyrosine residue, forming 3-nitro-tyrosine.62 Recently, a number of proteins which have been shown to undergo tyrosine nitration in plants were indentified which are mainly involved in photosynthesis, as well as in antioxidant, ATP, carbohydrate and nitrogen metabolism.63 Since this now well-established post-translational modification seems to be mediated by ONOO⁻,30 it represents a candidate mechanism that could contribute to oxidative and nitrosative cross-talk signaling. Supportively, Radi showed that tyrosine nitration is also mediated by oxidants including O₂^·− and H₂O₂, and transition metal centers.64 These observations indicate that ROS and RNS-based post-translational modifications are key regulators of the oxidative and nitrosative signal transduction mechanism.