Cross Talk between H2O2 and Interacting Signal Molecules under Plant Stress Response

1In plants, reactive oxygen species (ROS) are continuously produced in different cellular compartments as byproducts of various metabolic pathways such as respiration and photosynthesis. All aerobic organisms use molecular oxygen as terminal oxidant during respiration. Oxygen is neither very reactive nor harmful, but it has the potential to be only partially reduced, leading to the formation of very reactive and therefore toxic intermediates like singlet oxygen (1O2), superoxide radical, hydrogen peroxide (H2O2) and hydroxyl radical (∙OH). All ROS are extremely reactive, causing damage to membranes and other cellular components. ROS also have strong affinities toward membrane, DNA, proteins, carbohydrates and lipids in plant cells. Hence, these molecules are constantly scavenged by different non-enzymatic and enzymatic detoxification mechanisms that are often confined to particular compartments. It is important to remove ROS from the plant system in order to abate stress response, taking also into account that the final consequence of an eventual disequilibrium due to adverse environmental factors is the rapid increase of intracellular ROS levels, the so-called “oxidative burst". However, the balance between frequent production and scavenging of ROS may be disturbed by a number of adverse environmental factors such as light, temperature, drought, salinity, cold, heavy metal ions, UV exposure and water. The usual external stress factors that affect ROS production in plants can be biotic (executed by other organisms) or abiotic (arising from changes in chemical of physical environment). However, in plants the constant regulation of the ROS concentration, including H2O2, is controlled by the enzymes such as catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferases (GSTs), glutathione reductase (GR), and peroxyredoxin; and non-enzymatic compounds, like ascorbate, glutathione (GSH), α-tocopherol and flavonoids.

2Recent studies have elucidated that under different stress conditions, plants react in a very complex manner which includes various physiological and cellular changes. In order to combat stress response, plants use various signaling mechanisms derived from hormonal regulations. Nevertheless, recent studies indicate that plants also make use of ROS as signaling molecules for regulating development and various physiological responses. There is also increasing focus on ROS production and its integration with various hormonal signaling pathway in regulation of plant growth and stress tolerance.

3Amongst all, superoxide and H2O2 are two ROS that have been given more importance in recent studies. The main focus of this review is on H2O2. H2O2 is freely diffusible across membranes, which enables it to diffuse the damage. It is relatively long lived and it acts as a central player in stress signal transduction pathways. Thenard was the first in 1818 to isolate H2O2 which later came across as a cell damaging molecule when accumulated at higher concentrations in the cell. In early 90 s Ievinsh and Tiberg also predicted the role of H2O2 as a signaling molecule. Based on earlier studies, it is certain that H2O2 is part of oxidative metabolism and is involved in various metabolism and signaling cascades which are essential for plant growth and development, such as development of root hair, reinforcement of plant cell wall, xylem differentiation, resistance enhancement, cell wall structural cross linking and cell wall loosening in stomatal control.

4H2O2 being a versatile molecule acts as an important signal at normal levels, whereas under abiotic or biotic stress conditions it induces oxidative stress. Its unique property of stability and less reactivity differentiates H2O2 from the rest of the ROS molecules, thus making it a good signaling molecule. In plants, H2O2 works as a key factor in non-toxic level concentration. As a signaling molecule, it shows tolerance to biotic and abiotic stress by getting involved in various pathways. At toxic concentration levels H2O2 showed involvement in programmed cell death (PCD; ). In a recent article, the dual nature of H2O2 has been studied where 600 μM H2O2 treatment caused an increase in the vase life of hybrid lily “Manissa,” while an increase in concentrations resulted in negative effects.

5Several studies have indicated that H2O2 interplays with other signaling molecules such as abscisic acid (ABA) and ethylene which are important for plant development and senescence. Table 1 shows recent studies unveiling the mechanism by which H2O2 is involved in various biological processes. A recent study indicated the involvement of nitric oxide (NO) and H2O2 in salicylic acid (SA)-induced salvianolic acid B production in Salvia miltiorrhiza cell culture. Increase in H2O2 production has been observed by SA despite being inhibited by IMD (an inhibitor of NADPH oxidase) or DMTU (a quencher of H2O2) which further increases NO production and Sal B accumulation. In mung bean seedlings SA also plays roles in adventitious root formation and its effect on H2O2 accumulation has been observed. It has been concluded from the study that H2O2 accumulation acts as a downstream process in regulation with SA induced adventitious root formation.

6H2O2 pretreatment in maize leaves significantly increased ABA content. Pre-treatment of sodium nitroprusside (SNP) and SA in wheat seedlings decreased the effect of osmotic stress. It was also observed that pre-treatment of seedlings with methylene blue (a guanylatecyclase inhibitor) diminished the protective effects of both SA and SNP. Therefore, it is concluded that protective effect may only be limited to NO. Another study on jasmonate revealed that 12-oxo phytodienoic acid is involved in reduced H2O2 accumulation., proposed a model describing three pathways in modulating the transcription factor AtERF73/HRE1 which includes ethylene dependent, ethylene-independent/H2O2-dependent pathway, and an ethylene and H2O2-independent pathway. This study also proposes involvement of H2O2 and ethylene in AtERF73/HRE1 and ADH1 gene expression under stress. There is another study stating the involvement of ethylene in H2O2 accumulation during PCD. In drought stress, calcium-dependent protein kinase (CPK8) has been involved in ABA-mediated stomatal regulation via CAT3 (CPK8-interacting protein) activity. It also has been observed that cpk8 and cat3 plants showed reduced CAT activity and higher H2O2 accumulation.

7studied the possibility of SA involvement in H2O2 and NO induced salvianolic acid B accumulation; where the main function of NO is to downstream SA signaling which results in reduced oxidative stress. Exogenous application of H2O2 and its induction in high light showed different effects on gene expression. It was shown that H2O2 could be involved in the signaling of plant growth regulators such as ethephon. The application of ethephon results in an elevation in H2O2 levels, which is accompanied by the increased expression of sweet potato CAT.

8H2O2 is produced in photosynthesizing cells, majorly in peroxisomes (photosynthetic carbon oxidation cycle) and minorly in choroloplast, mitochondria (respiratory electron transport chain) (Figure 1), the endoplasmic reticulum (ER), nucleus and plasma membranes. Peroxisomes and chloroplasts may accumulate 30–100 times higher H2O2 as compared to mitochondria. H2O2 production occurs during the oxidative burst when reduction of molecular oxygen (O2) into superoxide anion occurs. At the cell wall this undergoes spontaneous dismutation at a higher rate and at an acidic pH. Nicotinamide adenine dinucleotide (NADH) undergoes oxidation to form NAD+ by cell wall peroxidase with further reduction of O2 to resulting in the production of O2 and H2O2. In apoplast, amine oxidase and germin-like oxidase have been proposed to generate H2O2. Cell membrane NADPH-dependent oxidase serves as a H2O2 source. H2O2 production occurs in the cell via reaction between the oxygen molecules (O2), forming superoxide anion. During the stress response O2 is reduced to which undergoes spontaneous dismutation. is also catalyzed and reduced by superoxide dismutase (SOD) and protein kinase C (PKC) to form H2O2. SOD enzyme catalyzes which mainly occurs in cytosol, chloroplast and mitochondria. Rather than superoxide disproportionation, H2O2 is also produced by reduction by reductants such as ferredoxins, thiols, ascorbate. PKC also shows involvement in H2O2 production. OH is produced in the reaction of H2O2 with Fe2+. To maintain the balance between H2O2-scavenging enzymes and SODs, equilibrium for H2O2 concentration in cells should be attained.

9H2O2 production in chloroplasts originates from several locations and occurs mainly in Chl associated with the photosynthetic electron transport chain (PET) which is the primary source of O2. In chloroplast, molecular oxygen (O2) uptake is associated with photoreduction of O2 to superoxide radical. Singlet oxygen (1O2) is produced by energy transfer to triplet oxygen (3O2) in photosystem II (PSII). Photosystem II excitation results in the oxidation of water (H2O) to O2 (Figure 1A). The reductant formed by this process donates electrons (e–) to plastoquinone (PQ). These e– eventually passes through the cytochrome f (Cyt f) complex, plastocyanin (PC), and then to photosystem I (PSI). The PET chain includes a number of enzymes on the reducing (acceptor) side of photo system I (PSI): Fe–S centers, reduced thioredoxin (TRX), and ferredoxin. When ferredoxin (Fd) is over reduced during photosynthesis electron transfer, electron may be transferred from photosystem I (PSI) to O2 to form by the process called Mehler reaction. The formed then undergoes dismutation to hydrogen peroxide (H2O2) either spontaneously or facilitated by SOD (Figure 1A).

10In plants, the main function of peroxisome is photorespiration which involves O2 uptake (light-mediated) and the emission of CO2 in simulation with H2O2 formation. H2O2 production in peroxisome results from the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). It is being stated that plants exposed to favorable conditions for oxygenation are more subjected to produce H2O2. H2O2 is generated during the oxidation of glycolate in the C2 cycle of peroxisomes (Figure 1B). Production of H2O2 is attributed to the matrix-localized enzyme, xanthine oxidase (XOD), which catalyses the oxidation of xanthine or hypoxanthine to uric acid, and is a well-known producer of in the process. This is then converted into H2O2 by Cu, Zn-SOD (Figure 1B). An NAD(P)H-dependent production site which uses as an electron acceptor is present in the peroxisomal membrane and it releases the into the cytosol. This site appears to be formed by a small ETC which is composed of a flavoprotein NADH and Cyt b.

11Two major mitochondrial sites for superoxide radical production in electron transport chain are cytochrome bc1 complex and NAD(P)H dehydrogenases. The mitochondrial respiratory electron transport (RET) harbors electrons with sufficient free energy to directly reduce O2, which is considered as a primary source of H2O2 generation (Figure 1C). This direct reduction of O2 to takes place in the flavoprotein region of the NADH dehydrogenase segment, which is responsible for the O2 consumption. During RET, the oxygen radical is markedly enhanced in the presence of antimycin A, which blocks the electron flow after ubiquinone (UQ). Autooxidation of reduced UQ results in the production of which is a major precursor of H2O2 production in UQ location of the RET. Completely reduced UQ donates e– to cytochrome c (Cyt c) and leaves an unstable, highly reducing semiquinone species, which would normally reduce cytochrome b (Cyt b), which instead reduces the O2 to (Figure 1C). This is further reduced by manganese SOD (Mn-SOD) dismutation to H2O2.

12Some other sources of H2O2 production are plasma membrane, cytoplasm and the extracellular matrix (ECM). There are various enzymes responsible for H2O2 production in plasma membrane. H2O2 production in cytoplasm occurs by the electron transport chain which is associated with the ER. This oxidation and hydroxylation process involve cytochrome P450 and cytochrome P450 reductase whereas, fatty acid desaturation involves cytochrome b5 and cytochrome b5 reductase, which donate electrons for H2O2 formation.

13Studies have shown that NADPH oxidase at the plasma membrane in the plant cell is involved in plant defense reactions to pathogen attack and in response to various abiotic stresses. The NADPH-dependent oxidase system sometimes referred to as rboh (for respiratory burst oxidase homolog), similar to that present in mammalian neutrophils, has received the most attention. It catalyzes the production of by one-electron reduction of oxygen using NADPH as the electron donor. The superoxide anion radical is most likely located in the apoplastic space and is converted to H2O2 either spontaneously or by extracellular SOD (Figure 1D). There are other plant species which NADPH oxidase or rboh genes have been cloned.

14Abscisic acid is one of the crucial phytohormones which play important roles under various environmental cues. It accumulates as a response to stressful environmental conditions such as dehydration, cold temperature or shortened day length. The application of ABA plays fundamental role in different physiological processes and biological pathways such as seed dormancy and delay in germination, development of seeds, promotion of stomatal closure, embryo morphogenesis, synthesis of storage proteins and lipids, leaf senescence and also defense against pathogen.

15It has been reported that the ABA signaling pathway is identified as a central regulator of abiotic stress response in plants, triggering major changes in gene expression and adaptive physiological responses (Figure 2). Recently, MAPK (mitogen activated protein kinase) cascades have also been shown to be implicated in ABA signaling. External ABA treated plants induced the transcriptional regulations, protein accumulation and stability, and kinase activity of several components of distinct MAPK signaling cascades in many plant species. These existing evidences suggest that MAPK cascades are actively involved in several ABA responses, including abiotic stress defense mechanisms and guard cell signaling. reported that the MAPK cascade is activated by the exogenous H2O2 which in turn is mediated by the hormones like ABA, Jasmonic acid (JA) and SA.

16In plants, MAPK cascades are generally organized pathways in which the upstream activated MAPK kinase kinase (MAPKKK) undergoes the sequential phosphorylation and subsequent activation of downstream MAPK kinases (MAPKKs) and MAPKs. This MAPK cascades translate incoming environmental signals into post-translational modifications of target proteins that ultimately reorganize gene expression and stress adaptation. In Arabidopsis, H2O2 activates the MAPKs (MPK3 and MPK6) via the MAPK kinase kinase (MAPKKK) ANP1. MKP2 is a key regulator of the MPK3 and MPK6 networks that are involved in controlling both abiotic and biotic stress responses (. Treatment with ABA in Arabidopsis plants induced the transcriptional regulation of MPK3, MPK5, MPK7, MPK18, MPK20, MKK9, MAPKKK1 (ANP1), MAPKKK10 (MEKK3), MAPKKK14, MAPKKK15, MAPKKK16, MAPKKK17, MAPKKK18, MAPKKK19, Raf6, Raf12, and Raf35 signifying a promising role in ABA signaling. Recently, used a cell type-specific functional genomics approach and identified that the MAPKs, MPK9 and MPK12 were preferentially expressed in guard cells. It was also reported that MPK12 were activated by ABA and H2O2. MPK9 and MPK12 mediate ABA signals in guard cells. In Arabidopsis, a T-DNA oxi1 null mutant is impaired in the activation of the two MAPKs, MPK3 and MPK6, upon oxidative stress, suggesting that serine/threonine kinase OXI1 functions downstream of ROS but upstream of the MAPK module. H2O2 also increases the expression of the Arabidopsis nucleoside diphosphate (NDP) kinase 2. H2O2 accumulation was reduced by the over expression of At-NDPK2 and enhanced tolerance to multiple stresses, including cold, salt, and oxidative stress. Moreover, the MAPKs, MPK3 and MPK6 are activated by H2O2 induced NDPK2. identified a transcription factor ANAC42 and reported that it was highly regulated by H2O2 which in turn is achieved through OXI1/MPK3 and MPK6 activation (Figure 2). Interestingly, ANAC42 plays a role in senescence and H2O2 induced PCD. Also, the MAPKKK can interact directly with WRKY53, a transcription factor that involved in senescence induced PCD (Figure 2), thus bypassing the downstream kinases. Nevertheless, it is clear from the past reports that MPK3 and MPK6 are two important integrating points of signals from cellular developmental programs and the external environment changes. Even though most of the connections between ABA and MAPKs are poorly understood, it is evident that these pathways are part of the complex cellular signaling network of plants to integrate various environmental signals.

17One of the most important functions of ABA is to induce stomatal closure by reducing the turgor of guard cells under water deficit. There is evidence that H2O2 may function as an intermediate in ABA signaling in Vicia faba guard cells. There are reports which suggest that H2O2 is one of the major ROS and plays an important role as a second messenger in ABA induced stomatal closure. While ABA induced the synthesis of H2O2 and superoxide under stressful condition and caused oxidative stress. However, ABA is a natural growth regulator which accumulates in plants under plant stress conditions. Results of provide evidence for H2O2 involvement in ABA induced stomatal movement in V. faba. Major findings of this study showed stomatal closure by exogenous application of H2O2 with ABA whereas, reverse action of H2O2 generation by scavenging its activity results in reduced stomatal closure. Overproduction of ABA induces H2O2 accumulation which enhances the stomatal closure by activating plasma membrane calcium channels. Plasma membrane-located NADPH oxidases Atrboh D and Atrboh F were found to be required for ABA-induced stomatal ROS production. It has been reported that during oxidative burst, NADPH oxidase can trigger Ca2+, and mitogen-activated protein kinase (MAPK) signaling pathways as well as suppress the hormone signal transfer routes such as SA, JA, and ethylene (Figure 2). Abiotic stress condition enhances ABA/gibberellic acid (GA) ratio supporting DELLA protein accumulation which in turn lowers H2O2 levels. In rice seeds, ABA decreased ROS production, which leads to a repression of ascorbate and GA accumulation. It was also reported that H2O2 is involved in GA/ABA signaling in barley aleurone cells. Calcium and calmodulin (CaM)-dependent protein kinase (CCaMK) belongs to calcium/CaM-dependent protein kinase superfamily; activation by free Ca2+ and Ca2+ bound to CaM has been indicated to be involved in ABA signaling during abiotic stress responses and ABA-induced antioxidant defense by functioning upstream of ABA-activated MAPK.

18These available pieces of evidence clearly indicate that the ABA is a key hormone in inducing abiotic stress responses. Downstream events mediated by MAPK cascade, alterations in Ca2+ fluxes and the activation of ion channels changes the redox state of the cell. All these actions lead to transcriptional reprogramming, which results in target gene expression such as cell death specific nucleases and proteases, and eventually PCD (Figure 2). Most of the genes (NADPH oxidases, SOD and extracellular peroxidases) expressed in the early signals are involved in the ROS generation, essential for triggering PCD. While other genes are responsible for maintaining ROS levels (CAT and APXs) (Figure 2).

19Calcium (Ca2+) is important for robust cell wall formation. It is also crucial for the root system and in young, growing cells. Alteration in Ca2+ level is observed under shifting environmental conditions, including changes in light, temperature, and drought. Ca2+ is significant for cross-linking acidic pectin residues and permeability of the plasma membrane. As a secondary messenger, Ca2+ concentration is balanced by storing it in vacuoles and is released whenever necessary. It is indispensable for all important signaling pathways. Plant signaling network has the capability to alter its functioning under various environmental challenges. The plant cell primary response under stress condition is a modification in plasma membrane permeability leading to calcium and proton influx that appears to be necessary and sufficient for the induction of H2O2. Plant metabolism involves various Ca2+/CaM proteins having different functions out of which some are involved in H2O2 signaling such as NAD kinase. CAT is also important for H2O2 regulation and its deficiency can lead to H2O2 accumulation. H2O2 and Ca2+ interrelation study has been shown by in Arabidopsis. Another study on Arabidopsis seedlings suggests the role of H2O2 in biphasic Ca2+ elevation, with the first peak located in cotyledons and the second in the root. The antioxidant system may also be considered as a target of Ca2+ influence, for example, the efficiency of H2O2 scavenging in Arabidopsis plants depends on the peroxisomal Ca2+ concentration.

20Continuous Ca2+ invasion is most importantly required for H2O2 accumulation which also activates NADPH oxidase located in the plasma membrane. There is another study suggesting the role of biphasic (Ca2+) and H2O2 in aequorin tobacco cell culture’s expression. Pollen tube growth has been enhanced by H2O2 regulated spermidine oxidase, which also induces Ca2+ channel. H2O2 involvements in Ca2+ influx via Ca2+ permeable channel and partial stomatal closure were observed in the study. Significant induction in nuclear and cytosolic Ca2+ level by free sphingoid Long Chain Base (LCB) sphinganine has been observed in simulation with decreased accumulation of H2O2 in tobacco cells. Later studies have revealed that CAT can scavenge H2O2 production which is likely mediated by Ca2+ homeostasis in Arabidopsis. In this case, cytoplasmic Ca2+ was shown to bind to rboh at the N-terminal region and thus to promote the activation of rboh and produce H2O2. H2O2 mediated rapid gene expression (LeCDPK1) in tomato leaves has been observed whereas, H2O2 treatment in wheat plant also leads to enhanced responsiveness in eight out of 20 studied calcium-dependent protein kinase (CDPKs). Induction in gene expression (GST1) by Ca2+response in association with H2O2 may be due to changes in redox balance.

21Increasing evidence based on experiments has shown a vital role of NO in protecting plants against stress conditions. It is generated in higher plants through oxidative (arginine or hydroxylamine-dependent) and reductive (nitrate-dependent) pathways. NO being part of various physiological processes in plant system makes it one of the major signaling molecules. Initially NO was considered to be a toxic gas. However, this idea changed after the discovery of the signaling role of NO in regulating the cardiovascular system. One of the major areas in the study of NO is its involvement in coordinating several defense responses during both biotic and abiotic stress conditions in the plants. In the past 2 decades, much focus was given to NO related studies toward its role in plant defense system under oxidative stress. Studies on adaptive mechanisms of plants have shown an increased basal level of NO in water and heat stressed plants, suggesting its importance in abating stress. The defensive mechanism of NO in plants under oxidative stress is associated with its ability to function as an antioxidant by directly scavenging the ROS, thus reducing cellular damage and acting as a signaling molecule which eventually results in changes in gene expression.

22A study focused on H2O2 generation in simultaneous correlation with NO production was shown during the hypersensitive response (HR) in which both cooperates to trigger hypersensitive cell death. The function of NO is tightly linked to ROS and it has a half-life of only a few seconds, once produced, interacts rapidly with ROS, giving rise to a number of reactive nitrogen species, such as nitrogen dioxide (NO2), which degrades to nitrite and nitrate in aqueous solutions. There were studies showing involvement of both NO and H2O2 in bacterially induced PCD in soybean where both signals work synergistically and in Arabidopsis they work as additive. H2O2 formation may occur via superoxide radical. There is a probability that NO reacts with to form highly reactive peroxynitrite anion ONOO- and subsequent cellular effects may then be induced by peroxynitrite.

23In mammals, NO has been shown to react with glutathione to form S-nitrosoglutathione (GSNO) which serves as a systemic source of NO and a similar situation has been suggested for plants. It is clear that both H2O2 and NO can mediate transcription, but the involved processes remain unclear. There is a possibility of both H2O2 and NO having a direct effect on transcription factors by S-nitrosylation and oxidation of cysteine. A recent study suggests characterization of redox-sensitive factor in yeast where H2O2 oxidation alters the activity of this protein. Phosphorylation of cascade such as the mitogen-activated protein kinases (MAPK) is suggested to play another important role on H2O2 and NO. There is another study on tobacco Bright Yellow-2 (TBY-2) cells, suggesting an involvement of both H2O2 and NO in the activation of PCD, and treatment of scavenger for both the signaling molecules restores the cell viability.

24In a new study, the cloning of rice NOE1, a gene whose knockout enhances NO production, revealed that this is indeed the rice CAT OsCATC. Increase in leaf H2O2 content leads to a characterization of mutant NOE1 which in turn leads to nitrate reductase (NR) dependent NO production. Increased H2O2 concentrations provoked by ABA may in turn trigger NO generation by NR and nitrogen oxide synthase (NOS)-like enzymes. NO accumulation under abiotic stress is similar to the events seen in H2O2 production. In Arabidopsis, both H2O2 and NO showed similar function which influences the induction and reduction in root growth stimulated by various concentrations of nucleotides. reported that NO and H2O2 are also involved in the stimulation of stomatal closure in Arabidopsis in response to ultraviolet-B exposure. Exclusion of H2O2 with antioxidants or inhibition of its synthesis by inhibiting NADPH oxidase activity prevents NO generation and stomatal closure. Wang et al. indicated the idea that H2O2-induced synthesis of NO might be mediated by MPK6 in Arabidopsis.

25Salicylic acid is one of the key phytohormones involved in both abiotic and biotic stress adaptation. The discovery of the salicylate role in thermotolerance during potato tissue culture research was mere coincidence. Inclusion of the artificial SA analog acetyl salicylic acid (ASA) in the culture medium of microplants of potato (Solanum tuberosum L.) causes potentially useful effects such as tuberization. It has been shown to play a central role as a signaling molecule involved in both local defense reactions and in the induction of systemic resistance. Another important aspect is gene regulation of antifungal hydrolases by SA, such as pathogenesis-related (PR) encoding PR1 and PR proteins which target to the plant cell wall. Reduced synthesis of SA in transgenic plants due to disruption of SA pathways results in vulnerability toward fungal (Phytophtora parasitica, Cercospora nicotianae), bacterial (Pseudomonas syringae), and viral (tobacco mosaic virus) pathogens.

26There are various reports suggesting involvement of SA in various biotic and abiotic stresses. Various environmental factors such as temperature, salinity, drought and high light exposure are responsible for ROS generation in cell organelles (peroxisomes, chloroplast). SA can be directly or indirectly involved in signaling pathways and interplays with ROS and GSH in stressed plants. Under drought stress, increased level of SA has been observed in oat plants, whereas another study also stated the same condition in peroxisome and chloroplast for the gene cat2 knockout and thylakoidal ascorbate PRX gene silencing, respectively. H2O2 stimulated PCD, SA accumulation and sesquiterpene production in cultured cell suspensions of Aquilaria sinensis. H2O2 production induced SA up-regulated the mRNA transcription of heat shock protein (Hsp) genes through AtHsfA2, a key component of acquiring thermotolerance in Arabidopsis.

27Salicylic acid and H2O2 interrelation is suggested by Leon, since the pathway involved benzoic acid (the immediate precursor of SA) leads to activation of benzoic acid 2-hydroxylase which is H2O2 dependent. Relative pathway of this case suggest that H2O2 production in cell organelles (peroxisome and chloroplast) induces SA synthesis, and leads to protective mechanism such as stomatal closure and cell death. Salicylate can increase H2O2 levels in plant tissues, on the contrary SA accumulation can be induced by elevated H2O2 levels. The germination of sid2 seeds under high salinity is hypersensitive to H2O2, but the physiological concentrations of SA modulate antioxidant activity to prevent oxidative damage. There is another study suggesting exogenous application of SA relieves Cd toxicity by reducing the H2O2 accumulation in root apoplasts of the legumes Phaseolus aureus and Vicia sativa.

28The HR to pathogens exhibits an early ‘oxidative burst’ of superoxide which rapidly dismutates to H2O2. This mechanism involves key interactive roles for SA and H2O2, as the HR was impaired in tobacco plants with an H2O2-inducible SA-hydroxylase transgene. It is considered that mammalian plasma membrane NADPH oxidase is a homolog of oxidative stress enzyme and it may be that this enzyme is potentiated by SA. Increased accumulation of SA and enhancement in H2O2 concentration in simultaneous pathogenesis gene induction were observed in GR1 dependent glutathione.

29Ascorbic acid (AsA) is a critical water soluble phytohormone found in plant and animals. It acts as a signal for plant growth and development, and regulates cell division, growth and signal transduction. In the mitochondria plants synthesize AsA which is then transported to other parts of the plants. There can be a direct or indirect reaction of H2O2 with AsA, which is catalyzed by APX. APX is responsible for scavenging H2O2 hyperaccumulation found in higher plants (cytosol, chloroplast and mitochondria).

30H2O2 detoxification can be done by various antioxidants in peroxisomes such as CAT in the matrix, APX and monodehydroascorbate reductase (MDAR) in association with AsA, resulting in a decrease in the accumulation of H2O2. In the chloroplast stroma, where the pH is higher during the day time, there is a consequence of AsA consumption during H2O2 reduction. A rate limiting amount of dehydroascorbate reductase (DHAR) efficiently catalyzes the recycling of AsA. The signaling function of H2O2 in guard cells is controlled by the rate of its production and the rate of its removal, in which AsA and DHAR play a critical role. The slower responsiveness of guard cells of DHAR over expressing tobacco allows more ozone to diffuse into the leaf interior. However, the increase of AsA content in all cells and consequent increase in their ability to detoxify entered ozone, reduce the oxidative load of the leaf (i.e., lower levels of foliar and apoplectic H2O2). From past reports, it is clear that the oxidative stress induced ROS level increases monodehydroascorbate (MDA) accumulation which is being converted into L-ascorbate (AsA) and dehydroascorbate (DHA) (Figure 3A). The accumulated H2O2 is reduced to H2O by oxidation of AsA to MDA radical, which is catalyzed by APX. The MDHA is subsequently reduced back to AsA by either ferredoxin reduction or NAD(P)H catalyzed monodehydroascorbate reductase (MDHAR). In GPX cycle, similar to APX, GPX uses GSH as a reducing agent to detoxify H2O2 to H2O. In addition to GPX, the organellar redox state is regulated by different enzymatic antioxidants like GR, MDHAR in addition to GPX. Disproportionation in L-ascorbate and MDHA is maintained by GSH (Figure 3A).

31Phytohormones act as a major factor responsible for plant growth and development. Oxylipin is considered to be one of the most important signaling molecules, i.e., plant hormone JA. Due to the unique physiological properties and abundance, jasmonate and its derivate (methyl jasmonate) came into the limelight as bioactive in nature. Chloroplast membrane is considered to be the initial site for JA synthesis where membrane phospholipids acts as a source for alfa-linolenic acid (C18:3) and hexadecatrienoic acid (C16:3) production. Major pathway for JA synthesis in plants is supposed to be an octadecanoid pathway with the involvement of alfa-linolenic acid as a substrate.

32The defensive mechanism of JA has been observed in tomato against tobacco hornworm larvae whereas in Arabidopsis against the fly Bradysia. There are studies confirming the role of JA as protective agent against pathogen (Pythium mastophorum) attack in Arabidopsis., came across with the result that both H2O2 and JA are primary signaling molecules during the cellular response involved in saponin biosynthesis mediated by oligogalacturonic acid (OGA) which also leads to the H2O2 mediated upregulation of JA. JA derivate (methyl jasmonate) has also been studied for its involvement in the induction of H2O2 accumulation in parsley suspension-cultured cells, whereas another study suggested its role in inducing defensive genes of tomato.

33Jasmonic acid induces glutathione, an important antioxidant for redox balance. Increased expression of nuclear factor erythroid 2-related factor 2 (NrF2) has also been observed, which reduces the ROS level induced by H2O2. In association with this study, increased expression of glutamyl cysteine ligase with an increase in NrF2 helps in regulating enzymes reducing oxidative stress.

34Ethylene has long been regarded as a stress hormone. It is not only involved in plant growth and development, but also involved in plant responses to biotic stress, such as pathogen attack; and abiotic stress, such as wounding, ozone, and salinity. Ethylene regulates many different processes in plants and has shown response in defense mechanism as well. In order to evaluate the defensive role of ethylene against various environmental conditions signal transduction pathways for ethylene has been studied with mutants.

35The roles of ethylene have been established in damage control caused by virulent bacteria or fungal pathogens when it is being inoculated but its importance against avirulent bacteria infected plants has yet to be proven. The most important signaling molecules in the ethylene pathway are ETR1 and EIN2. Change in gene expression of the ethylene receptor (ETR1) results in reduced ethylene response.

36Environmental stress affects many signaling pathways in plants which also includes an alternative pathway (AP). Despite slight evidence about H2O2 and ethylene playing roles in inducing AP, there is no clear picture of how these signaling molecules are inducing the AP under various environmental conditions. Results of showed the possibility of involvement of H2O2 and ethylene mediated induction of AP under salt stress as it shows activity in wild-type callus whereas no activity was observed with ETR1-3 callus. In recent years, an increasing number of positive results on ethylene toward mutants in Arabidopsis have confirmed its role in signaling pathways. In another study H2O2 accumulation in simultaneous production of ethylene has been observed in tobacco plant stressed with ozone. In plants, oxygen-deficient conditions shifts energy metabolism from aerobic to anaerobic, which in turn adversely affects nutrient and water uptake. Eventually, hypoxiasignalling triggers the production of both hydrogen peroxide (H2O2) and ethylene. H2O2 and ethylene interplay has an effect on AtERF73/HRE1 and ADH1 expression during the early stages of hypoxia signaling in Arabidopsis. Hypoxia signaling induces the ethylene biosynthesis enzymes such as ACC synthase (ACS) and ACC oxidase (ACO) (Figure 3B). Arabidopsis AtERF73/HRE1 is very similar to the rice Sub1A and SNORKEL genes, which belongs to the group VII ERF (ethylene responsive factor) subfamily. They play major roles in the submergence tolerance of lowland and deepwater rice. According to, AtERF73/HRE1 positively regulates ADH1 genes as well as negatively regulates peroxidase and cytochrome P450 genes in hypoxia signaling (Figure 3B).

37Increasing urge to identify the role of hydrogen peroxide as a signaling molecule has gathered the interest of researchers to focus their work on the mechanisms regulating the generation of hydrogen peroxide, and this is certainly an important growing area of research. Significant scientific effort in the last 10 years has determined the position of H2O2 in signal transduction networks in plants, demonstrating that it is essential for both the communication between external biotic and abiotic stimuli, and the control of developmentally regulated processes. There are many signaling pathways for H2O2 mediated stress and defense responses that have been studied, but it remains a large scope of additional research unexplored, which can further clarify the mechanism involved in these pathways. The focus should be imposed on a clear description of roles of endogenous compounds which modify the plant responses. It has been reported that the phytohormones like ABA, SA, JA, GA, and ethylene regulates the protective responses in plants under abiotic stress by involving in different H2O2 induced signaling. Despite of its regular activities in plant growth and development, ABA plays crucial role in H2O2 mediated stress cues. indicated that ABA-induced H2O2 production mediates NO generation, which in turn, activates MAPK cascade and results in the over expression and up regulation in of antioxidant enzyme activities in ABA signaling. However, there are some contradictory roles of NO. According to, NO has been shown to negatively modulate wound signaling in tomato plant blocking H2O2 production and proteinase inhibitor synthesis by JA, contradicting with previous study in which NO has been considered to show positive response in abiotic stress. Nevertheless, there are many studies suggesting H2O2 response in association with NO generation under biotic/abiotic stress.

38Due to different results suggesting various roles of H2O2, it is important to focus future studies in getting a clear picture of signaling pathways during stress response in various conditions. Interactions between different signaling molecules and their biological functioning with the involvement in various pathways still needs to be cataloged. Another important aspect that should be focused on is the role and localization of enzymes, which are involved in signaling pathways. Some important factors for future research should be the identification of the site for H2O2 production in the cell and the major factors influencing its interaction with other signaling molecules.

39ZC initiated the project. SS produced the figures. IS, SS, and ZC wrote the manuscript. SS and ZC revised the manuscript.

40The 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.