The term reactive oxygen species (ROS) usually encompasses singlet oxygen (¹O₂), superoxide ion (O2•–), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH⋅); however, other molecules, such as organic hydroperoxides (ROOH), are also included in this group (Simon et al., 2000). ROS can be generated in all cellular compartments through non-enzymatic mechanisms, such as electron flow in electron transport chains (ETC), or as byproducts of enzymatic reactions (Desikan et al., 2005). ROS are reactive and cause damage to macromolecules (proteins, lipids, and nucleic acids) affecting cellular functioning, but more recent evidence indicates that ROS play important roles in signaling and cell adaptation to stress. Mechanisms of intracellular ROS production and ROS detoxification have been extensively reviewed (Halliwell, 2006; Das and Roychoudhury, 2014); however, apoplastic ROS (apROS) metabolism has received less attention.

Only a small quantity of ROS is localized within the apoplast, apROS have an important role in plant development and plant responses to various stress conditions. We elucidate how apROS are engaged in signal transduction from extracellular spaces to the cell interior and may directly eliminate invading pathogens. Moreover, apROS metabolism regulates the plant cell division rate and cell elongation, and consequently whole plant growth (Barceló and Laura, 2009). It was shown that one of the components regulating cell proliferation is apoplast-localized low-mass antioxidants (Kato and Esaka, 1999; Potters et al., 2000). Growth by elongation is limited by the extension of cell walls in what is the result of two processes in which ROS are engaged: loosening of load-bearing bonds in the wall matrix and stiffening through the insertion of stabilizing cross-links between load-bearing polymers (Chen and Schopfer, 1999; Pedreira et al., 2004; Kärkönen and Kuchitsu, 2015). The presence of ROS in apoplastic spaces has mainly been demonstrated using microscopic methods (e.g., deposits of cerium peroxides observed in TEM or probes that became fluorescent when oxidized by ROS), but precise concentrations of apROS were also estimated in some plant species. The concentration of H₂O₂ in apoplastic fluid was about 10–25 pmol⋅g^-1 FW under control conditions and increased several fold under stress conditions (Hernández et al., 2001; Diaz-Vivancos et al., 2006). The question remains how the apoplastic respiratory burst does not become toxic for plants. Our aim in this update is to provide a concise overview of current knowledge about long known ROS producing mechanisms and also propose less prominent apoplastic ROS sources. Furthermore we discuss data regarding antioxidant capacity in the apoplast.

The combined action of ascorbate peroxidase (APX, EC1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), and glutathione reductase (GR, EC 1.6.4.2) can decompose H₂O₂ using ascorbate (AsA) and glutathione (GSH) as electron donors, via a pathway called the ascorbate-glutathione cycle (AGC, Figure 2). In the AGC the reduction of H₂O₂ is ultimately linked to NAD(P)H oxidation. All AGC enzymes were found in the apoplasts of barley, oat, and pea leaves, but their activity was estimated at only 0.2–2.8% of symplastic activity (Vanacker et al., 1998a,b; Hernández et al., 2001). However, in the apoplasts of other plants not all AGC enzymes were found to be active; therefore, it is also possible that the whole cycle is not operating in the apoplast, but only certain enzymatic reactions (De Pinto and De Gara, 2004).

The first enzyme of the AGC, APX, is a class I peroxidase that uses AsA as an electron donor to scavenge H₂O₂ to water. In the Arabidopsis genome eight types of APX were found, but only APX1 (At1g07890), which is considered a cytosolic enzyme, might also be secreted to the apoplast. APX1 was identified in several reports of extracted plasmalemma or cell wall proteins (Marmagne et al., 2007; O’Brien et al., 2012a). APX showed the highest activity of all AGC enzymes (Diaz-Vivancos et al., 2006), and in roots of sunflower seedlings apoplastic APX activity was even higher than that detected in the soluble symplastic fraction (Parra-Lobato et al., 2009). APX has an even higher affinity for H₂O₂ (μM range) than CATs and class III POXs (mM range).

The regeneration of AsA from its oxidized state is necessary for APX activity. MDHAR is a key enzyme for the recovery of AsA within plant cells, using NAD(P)H directly to reduce monodehydroascorbate (MDHA). The MDHAR multigene family is encoded by the MDHAR1-6 Arabidopsis genes that are targeted to most cellular compartments. Among these proteins MDHAR1 (At3g52880) seems to be localized in organelles and also in the apoplastic space (Bindschedler et al., 2008). Additionally, a plasma membrane-associated MDHAR was found in spinach and identified by NMR (Bérczi and Møller, 1998). However, the in vivo localization of MDHAR was shown to be on the cytosolic side of the plasma membrane.

Dehydroascorbate reductase catalyzes the reduction of dehydroascorbate (DHA) to AsA, but the enzyme activity requires GSH as an electron donor. DHAR is thought to be responsible for the regulation of the symplastic and apoplastic AsA redox state. In Arabidopsis DHAR is encoded by DHAR1-3 genes. DHAR1 (At1g19570) and DHAR2 (At1g75270) were found to also be associated with the plasma membrane (Marmagne et al., 2007). The reduction of DHA by GR simultaneously results in GSH oxidation.

Glutathione reductase catalyzes the NADPH-dependent reduction of glutathione disulfide (GSSG) to the sulfhydryl form of GSH. GR regenerates GSH and thereby fuels the AGC; therefore, GR is thought to be the rate-limiting step of the cycle. In the apoplast of barley and oat this enzyme has the lowest activity among AGC enzymes in EWF (Vanacker et al., 1998a,b). There seem to be only two genes representing GR in Arabidopsis, GR1 and GR2; however, to date no extracellular isoform has been identified in Arabidopsis.

The in vivo activity of GR and MDHAR in the apoplast remains a matter of debate since their substrate, NAD(P)H, seems to be present at very low concentrations in this space. In fact, there is no evidence in the available literature of reduced pyridine nucleotides being extracted from apoplastic fluids. However, the oxidized form of pyridine nucleotides (NAD⁺) which usually constitutes the major amount of nucleotides, was detected in EWF of needles of Norway spruce at a level up to 1.4 nmol⋅g^-1 DW (Otter and Polle, 1997). O’Brien et al. (2012b) proposed a cycle in which NADH can be produced, involving the action of apoplastic malate dehydrogenase (MDH) or lactate dehydrogenase (LDH) using NAD⁺ as a substrate.

Glutathione peroxidases (GPXs, EC 1.11.1.9) are important for the detoxification of peroxides, such as H₂O₂, alkyl hydroperoxides, and peroxynitrite, using GSH as a substrate. In Arabidopsis eight isoforms of GPXs can be found (GPX1-8); of these GPX2 (At2g31570), GPX5 (At3g63080), and GPX6 (At4g11600) were found in the plasma membrane (Marmagne et al., 2007). In sunflower seedlings GPX activity in the apoplast did not account for even 10% of symplastic enzyme activity (Parra-Lobato et al., 2009). Some peroxidases favor thioredoxin (TRX, EC 1.8.1.9) over GSH as a substrate in their regeneration system. Moreover, in the antioxidative system, 2 further thiol-dependent peroxidases are important: peroxiredoxins (PRXs) and glutaredoxins (GRXs). PRXs are reduced by TRX while GRX reduction depends on reduced GSH. These thiol-based POXs cannot only eliminate hydroxyperoxides, but GPXs, PRXs, and GRXs can revert protein disulfides to dithiols and can therefore protect protein thiols from oxidation. Some of these PRXs and GRXs might be secreted to the extracellular space, but detailed characterization of these enzymes is lacking (Meyer et al., 2008).

In the apoplast other low-mass antioxidants are also present, such as phenolics and polyamines, which might be important for ROS detoxification. Phenolics are a heterogeneous category of molecules containing a phenol group. Almost 10000 compounds can be assigned to this group, including diverse secondary metabolites. Many of these phenolics (flavonoids, tannins, hydroxycinnamic acid, and lignin) possess antioxidant properties. As electron donors, phenolic compounds can quench ROS because they are easily oxidized, and the resulting phenoxyl radicals are less reactive than oxygen radicals (Kagan and Tyurina, 1998). Polyphenols were tested in vitro and found to be the most efficient low-mass antioxidants in plant cells (more effective than AsA or tocopherols) but were not as effective as SOD (Taubert et al., 2003). Moreover, phenolics are also able to scavenge lipid radicals and prevent the propagation of lipid peroxidation. The major form in which phenolics can be detected is bound to the cell wall (Wallace and Fry, 1994; Hutzler et al., 1998). However, free phenolic compounds were also detected in EWF (Maksimović et al., 2008). Beyond that it was found that the composition of phenolics in the apoplast can change in response to different elicitors (Baker et al., 2015). Phenolics can be released into the apoplastic space either through vesicle-mediated transport or specific membrane transporters, including GST-flavonoid complexes. However, the ROS scavenging capacity of phenolic compounds in the apoplastic space was estimated not to be efficient enough, probably because of the low concentration (Baker and Mock, 2004). It is known that phenols can activate POX activity, since they serve as their substrate. The reaction products – phenoxyl radicals – have to be further detoxified via reduction by AsA. Therefore, phenols that are oxidized by POXs can form a ROS-scavenging phenolic/ASA/POX system, in which the action of MDHAR is also necessary to reduce the resulting MDHA (Takahama and Oniki, 1992). Among phenolic compounds, POXs can utilize flavonoids as their substrate. Flavonoids are one of the major classes of phenolics, with the ability to serve as direct electron donors; therefore, they can scavenge all kinds of ROS (Bors et al., 1990). In plant cells flavonoids can be transported via transporters or vesicles to the apoplast and accumulate mainly within cell walls (Zhao, 2016). Another category of phenolics includes acidic derivatives such as hydroxycinnamic acid and hydroxybenzoic acid. Hydroxycinnamic acid is of great interest because of its antioxidant features (hydrogen- or electron-donating ability). Hydroxycinnamic acids (such as ferulic, caffeic, sinapic, and p-coumaric acids) are structural and functional constituents of plant cell walls (Wallace and Fry, 1994). The oxidation of derivatives of hydroxycinnamic acid by peroxidases is one of the features that make them important in the apoplast.

Polyamines were assumed to be protective compounds (Bouchereau et al., 1999). The most abundant plant polyamines include Putrescine, Spermidine, and Spermine. However, the catabolism of polyamines occurs in the apoplast; therefore, their content can be very low or even at undetectable levels. The concentration of different polyamines in EWFs of tobacco and barley was determined to be around 1–10 μM (Marina et al., 2008; Sobieszczuk-Nowicka et al., 2016). Due to their cationic nature at physiological pH, polyamines are able to interact with negatively charged macromolecules in a reversible manner, which suggests that they may be free radical scavengers (Ha et al., 1998; Das and Misra, 2004). However, the oxidative degradation of polyamines in the apoplast by AOs can produce significant quantities of H₂O₂; therefore, their role as antioxidants is questionable (Rea et al., 2004). The direct beneficial effect of polyamines for plant development or defense responses is still a matter of research.

The amino acid proline (Pro) is not only an important osmolyte in plant cells, but also an important scavenger of ROS. Pro utilizes OH⋅ and ¹O₂, and can inhibit the damage caused by the chain reaction of lipid peroxidation (Matysik et al., 2002). The reactivity of Pro with H₂O₂ and O2•– seems very low; therefore, the role of Pro as an efficient cellular antioxidant has been questioned (Kaul et al., 2008). The content of Pro in apoplasts was estimated at around 1–20 μM in the EWF of bean and sugarcane (Tejera et al., 2006; Sobahan et al., 2009).

Some hormones belonging to the indole family show high reactivity with ROS, allowing the indoleamine to function as an electron donor and serve as a trap for ROS chain reactions. Even though these molecules are mostly known as mammalian hormones, some indole derivatives, such as tryptophan, melatonin, and auxins, are present in plants. The most prominent mammalian indole is melatonin, and its role as an antioxidant in plants has also been recognized (Arnao and Hernández-Ruiz, 2006). To date, indole derivatives have shown various different biological effects, other than acting as antioxidants. In plants auxins are the most studied among indole derivatives that regulate cell growth (Leyser, 2010). In recent years, their ROS decomposing activity became more apparent, and some indoles are even more active than GSH (Cano et al., 2003). The most valuable feature of indoles is their small size. Moreover, they are both water and lipid soluble; hence these molecules can easily penetrate the plasma membrane, but their antioxidant potential in the apoplast has to be further elucidated.

The plasma membrane has a lipophobic center that most antioxidants cannot enter; therefore, lipid-soluble vitamins are important for membrane protection. Two of the different forms of vitamin E are tocopherols and tocotrienols. In contrast to other low-mass antioxidants, tocopherols are hydrophobic and lipid-soluble and because of their proximity to membranes tocopherols can therefore protect lipids and other membrane components from lipid peroxidation (Munné-Bosch and Alegre, 2002). Among the α, β, γ, and δ forms of tocopherols, α-tocopherol is most abundant in leaves and is also the most active form (Kaiser et al., 1990). Tocopherols can directly quench ROS, especially ¹O₂ which is irreversibly oxidized. Regeneration of the resulting tocopheroxyl radical to its reduced state is performed by AsA or GSH (Fryer, 1992). Moreover, tocopherols can scavenge lipid peroxyl radicals considerably faster, before they are able to attack the target lipid substrate. The concentration of α-tocopherol in membranes is relatively low (less than 2 mol/mol phospholipid), and since no transport mechanism of these molecules from plastids (where they are synthesized) has been detected to date, its content in the plasma membrane is questionable. Nevertheless, it was shown that the lack of tocopherols in mutants resulted in cell wall defects such as callose deposition (Maeda et al., 2006).

The electrons from vitamin K1 might also be used to reduce oxidized lipids in plasma membranes, terminating lipid peroxidation chain reactions. A possible electron acceptor might be cytochrome b561, simultaneously causing AsA regeneration.

The main method to isolate the apoplastic fluids is the infiltration of leaves and extraction of EWF. In previous studies where the concentration of ascorbate and glutathione or antioxidant enzyme activity was determined, it is important to be aware of the possibility of contamination of this EWF fraction with cytosolic components. It is not possible for these low mass antioxidants to freely diffuse through the membranes to the apoplast, but small disruption of the plasma membrane cannot be excluded during the experimental procedure extracting EWF. The presence of glucose 6-phosphate dehydrogenase (G6PDH) activity can be used as a marker for cytoplasmic contamination of EWF. In all the described studies less than 2% of total extractable foliar G6PDH activity was found in the EWF.

Large scale proteomics of EWF revealed that most proteins are stress-related or cell wall-modifying enzymes (Rodríguez-Celma et al., 2016); therefore, there is no doubt over the apoplast’s function in defense. Nevertheless, a complex antioxidant network has evolved in the apoplastic spaces of plants, although the overall antioxidant defense in this space is generally low compared to that in the intracellular space. The low ROS detoxification capacity in the apoplastic space provides a fast overflow mechanism that allows ROS to accumulate. The aim of this respiratory burst may be the precondition for ROS signaling.

It is typically assumed that the plasma membrane serves as the first site of perception of environmental changes. Compartment-specific ROS accumulation can activate diverse signal transduction pathways (Figure 3). ROS signaling is not only controlled by ROS production and scavenging; the sequence of events is far more complicated. However, the signaling intermediates between stress perception in the apoplast and the physiological responses are still not fully understood (reviewed by Mittler et al., 2011; Shapiguzov et al., 2012; Baxter et al., 2014; Kangasjärvi and Kangasjärvi, 2014).

When H₂O₂ generation in the apoplast exceeds the relatively low scavenging capacity of this space ROS can accumulate. The first possibility is that H₂O₂, which is a neutral molecule, can diffuse through the plasma membrane to the cell, boosting the intracellular ROS pool and activating symplastic ROS signaling. Bienert et al. (2007) provided evidence for H₂O₂ diffusion through specific aquaporins. The second possibility is that the H₂O₂ wave can move within the apoplast from cell to cell of the same tissue (Mittler et al., 2011). For long distance transport ROS can move through the vascular system of plants. It is not surprising that the highest content of H₂O₂ within a leaf was detected in veins (Orozco-Cardenas and Ryan, 1999). Therefore, ROS participate in long distance communication, where the information has to be carried systematically and might be transformed into another signal to reach more distant plant organs (Baxter et al., 2014). The speed of the ROS wave was estimated to be approximately 8 cm⋅min^-1 (Miller et al., 2009).

A major function for RBOHD of responsibility for generating fast-moving ROS signals during stressful conditions was proposed. RBOH activity is crucial for both long distance ROS signaling and the ROS burst that is transduced to the nucleus. ROS produced by RBOHs are thought to activate calcium plasma membrane hyperpolarization-activated Ca²⁺ channels (Foreman et al., 2003). Opening of these channels leads to increases in cytosolic Ca²⁺ content and causes membrane depolarization (Mittler et al., 2011). In turn, increased symplastic Ca²⁺ content can activate RBOH activity by binding the cytosolic EF-hand motif creating a feedback loop (Sagi and Fluhr, 2001). Elevated cytosolic Ca²⁺ concentrations can initiate further responses via the action of Ca²⁺-binding proteins. In plant cells these proteins can be considered calcium sensors, including calmodulins (CaMs) and CaM-like proteins (McCormack et al., 2005), calcineurin B-like (CBL) proteins (Luan, 2009), and calcium-dependent protein kinases (CDPKs or CPKs) (Harmon et al., 2000). Similar to ROS, the ubiquitous second messenger Ca²⁺ seems to be another critical step toward initiating defense signaling (Steinhorst and Kudla, 2013). The Ca²⁺ influx to the cytosol can be accompanied by other ion fluxes through dedicated channels [H⁺ pump, anion channel, stelar K+ outward rectifier (SKOR)] or permeable membranes. Hence H⁺ influx and K⁺ or Cl^- efflux is one of the earliest responses in ROS-mediated signaling. It was shown that K⁺ flow through SKORs is a rapid response to apoplastic H₂O₂ (Garcia-Mata et al., 2010). Alterations in ion conductance might be a more general signaling pathway under oxidative stress. Not only the ion balance but also the pH of the apoplast may vary since the buffering capacity in this compartment is not as efficient as that of the symplast (Grignon and Sentenac, 1991). The pH of the apoplast, which is usually between 4.5 and 5.5, may fluctuate in response to external and internal stimuli and signals (Felle, 2001). Changes in extracellular pH impacts several enzyme activities, for instance cell wall POX. Moreover, a shift in the external pH was found to be a rapid modulator of plant gene expression (Lager et al., 2010).

The major antioxidants regulate the steady-state level of ROS and therefore determine signal transduction. Since the redox state of ascorbate and glutathione pools is directly linked to ROS levels, these low-mass antioxidants might be a redox link between the apoplast and the cytosol (Sierla et al., 2013). This checkpoint might control cell cycle progression, and thereby cell growth, during stressful conditions as indicated by Reichheld et al. (1999). The ascorbate pool is not only the most important low-mass antioxidant but is a well-established element of ROS signaling (Pastori et al., 2003; Pignocchi and Foyer, 2003). Since AsA can be directly oxidized by ROS, the increased levels of DHA in the apoplast can function as signaling molecules. It was shown that the redox state of ascorbate varies throughout the cell cycle phases (Horemans et al., 2000; Pignocchi and Foyer, 2003). Apoplastic ascorbate is engaged in the regulation of the cell cycle (Kato and Esaka, 1999; Potters et al., 2000). It was proposed that increased DHA oxidation in the apoplast and elevated import to the cell can affect cell cycle progression in tobacco cell cultures (De Pinto et al., 1999; Potters et al., 2000). Furthermore, apoplastic DHA seems to inhibit cell proliferation in several plant species (Paciolla et al., 2001; Potters et al., 2004). Moreover, soluble thiol-containing low-molecular-mass compounds, mainly GSH, are important regulatory compounds in plant cells. Li et al. (2013) proposed that extracellular GSH in plants has a similar function to that of a neurotransmitter in animal cells. Moreover, GSH itself was proposed to be an activator of TFs (Tron et al., 2002). There are also several redox-sensitive proteins that operate through switching “on” and “off” depending on the cellular redox state or ROS action. All enzymes that have sulfur residues (particularly PRXs, GRXs, and TRXs) are susceptible to reversible oxidation/reduction (Spadaro et al., 2010). These redox active compounds directly modify cellular metabolism or can further execute their function via downstream signaling components.

When ROS content in the apoplast is not effectively scavenged and exceeds a certain level, it might have a toxic effect and cause tissue damage, such as protein oxidation or non-enzymatic breakdown of lipids. Oxidized PUFAs, which may be embedded in the surface of the cell as free PUFA, represent the first step in the chain reaction of lipid peroxidation. Further lipid peroxidation products from the cellular membrane, called oxylipins, can act as signaling elements (Farmer and Mueller, 2013). ROS can also oxidize extracellular peptides or domains of receptor proteins in the apoplastic space. Putative ROS receptors were speculated to exist, but to date specific apoplastic receptor proteins that can be directly modified by ROS have not been identified (Kangasjärvi and Kangasjärvi, 2014). However, there are many receptor proteins at the outer surface of the plasma membrane, including hormone receptors, innate immune receptors and others that are sensitive to ROS. For instance, animal G protein signaling is also involved in many stress-associated physiological processes in plants. The heterotrimer present in the plasma membrane after perceiving extracellular stimuli, such as apoplastic ROS accumulation, can transfer the signal to the cytosol. It was proposed that RBOHs received initial signals from G proteins working in the same signaling network. In general, the increased level of apoplastic ROS can be directly monitored by oxidizing putative receptors present in the plasma membrane that can trigger downstream signaling events. Receptor-like kinases (RLKs) in the plasma membrane can be activated by apoplastic ROS. RLKs are cysteine-rich transmembrane proteins that contain an extracellular ligand binding domain and a conserved intracellular protein kinase domain. This could be one of the mechanisms by which extracellular ROS can directly activate intracellular signaling cascades via protein phosphatases and kinases (histidine kinases) (Neill et al., 2002). Reversible protein phosphorylation is a fast signaling mechanism that can amplify the response to ROS (Baxter et al., 2014). The mitogen-activated protein kinase (MAPK) pathway is the most prominent and best studied signaling cascade. These proteins have kinase activity and are thereby interlinked. Phosphorylated (activated) MAPK interacts with and alters the phosphorylation status of target proteins, including TFs, enzymes, and other proteins, ultimately influencing gene expression; specific intermediates are still being identified. Arabidopsis encodes 10 MAPKKKKs, 80 MAPKKKs, 10 MAPKKs, and 23 MAPKs which form complex signaling networks (Jonak et al., 2002). Some kinases were identified as playing specific roles in ROS-mediated signaling; these include the ROS-responsive MAPKKK, MEKK1, MPK3, MPK4, and MPK6 (Colcombet and Hirt, 2008). The application of H₂O₂ to Arabidopsis protoplasts was found to activate the phosphorylation cascade involving MPK3 and MPK6 (Kovtun et al., 2000). MEKK1–MPK4 kinase activity is also activated by exogenous H₂O₂ treatment of Arabidopsis seedlings (Nakagami et al., 2006). In many conditions, proteins from the WRKY family work downstream of MAPK and can directly activate gene transcription (Eulgem and Somssich, 2007; Chen et al., 2012). A specific characteristic of plant transduction pathways is negative regulation, where a response is induced by inactivating repressor proteins. This mechanism includes protein dephosphorylation or ubiquitination, and only then can transcription proceed. Rosenfeld et al. (2002) suggested that this negative regulation provides a faster transduction system. ROS can also directly oxidize diverse proteins triggering a metabolic response. Some oxidized proteins are targeted to be degraded via the ubiquitination pathway. Other oxidized peptides were found to act directly as signaling molecules. Therefore, the abundance of diverse oxidized proteins may regulate plant responses.

Apoplastic ROS can elicit plant hormone signaling. H₂O₂ interaction with specific hormonal compounds, mainly salicylic acid (SA), ethylene, jasmonic acid (JA), and abscisic acid (ABA), is well established (Xia et al., 2015). All these hormones may be perceived by plasmalemma-bound hormone receptors or can further activate gene expression or MAPK signaling (Santner and Estelle, 2009). Most genes that were sensitive to apoplastic ROS were also responsive to ABA signaling (Blomster et al., 2011). ABA is localized in all cellular compartments, but it was shown with immunogold labeling that the apoplast is a major site of free ABA accumulation (Bertrand et al., 1992) or transport (Sauter and Hartung, 2000). ABA has an important role in signal transduction pathways (Wilkinson and Davies, 2002). Most reports have demonstrated that exogenous application of ABA may promote H₂O₂ accumulation in the apoplast, which is dependent on RBOH activity and Ca²⁺ channels (Kwak et al., 2003; Hu et al., 2005). Interaction of ABA with other hormones was also shown in the regulation of diverse physiological processes, e.g., stomatal closure. JA, SA, and ethylene are considered second messengers in oxidative signal transduction; ROS act upstream and downstream of these hormones. On one hand ethylene and SA are generally induced by oxidative bursts, on the other hand ethylene and SA signaling promote enhanced ROS production forming a self-amplifying loop (Watanabe et al., 2001; Rao et al., 2002). Experimental work showed that the application of SA can induce extracellular O2•– production, which is probably catalyzed by cell wall POX (Kawano et al., 1998; Mori et al., 2001). Auxins can also induce O2•– production in the apoplast, and subsequent OH⋅ release can promote elongation growth in a POX-dependent reaction (Schopfer et al., 2002). However, JA has the opposite effect than ethylene and SA, acting as an antagonist to in the regulation of different stress responses (Lorenzo and Solano, 2005). Moreover, JA was found to have the ability to reduce RBOH-induced ROS generation (Denness et al., 2011).

All of the elements described above can activate specific TFs in the nucleus. Important TF for intracellular signaling pathways that can be activated in response to ROS can belong to different families including: WRKY, NAC (NAM, ATAF1/2, and CUC2), zinc finger transcription factors (ZAT), basic-domain leucine-zipper proteins (bZIP), heat shock proteins (HSP), and others (Singh et al., 2002; Van Breusegem et al., 2008). ROS are thought to activate TFs via direct oxidation. H₂O₂, O2•–, and ¹O₂ ROS can all induce transcription of specific sets of genes in plant cells (Neill et al., 2002; Gill and Tuteja, 2010). A common TF binding site for all oxidative stress-responsive genes has never been identified, but in several ROS-induced genes specific redox-sensitive promoters have been found by Desikan et al. (2001). More specific cis-regulatory elements (CRE) in gene promoter regions, functioning as binding sites for TFs, have been identified in the Arabidopsis genome using a universal algorithm (Geisler et al., 2006). In this microarray study well-characterized motifs such as the ABA-responsive CRE (ABRE), drought responsive element (DRE), and ethylene responsive element (ERE) were analyzed upon H₂O₂ treatment and let to the identification of 128 putative motifs in these functional CRE, that are engaged in abiotic stress signaling.

Extracellular ROS, in particular, can control several genetic defense or developmental programs. Several approaches have been used to simulate ROS production in the apoplastic space in order to reveal the expression of specific genes. A microarray study showed that mainly cellular antioxidant enzymes, but also apoplastic proteins including RBOHs and ascorbate oxidases, were activated by H₂O₂ treatment, while others such as POXs and expansins were downregulated (Stanley Kim et al., 2005). Interestingly, genes that are potentially involved in signaling, including TFs of the WRKY family, were downregulated. Using mass spectrometry Wan and Liu (2008) identified different protein spots in 2DE-gel that were responsive to H₂O₂ treatment in rice. Upregulated proteins were mainly engaged in cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis, and carbohydrate/energy metabolism. Rice seedling proteome changes were also analyzed in the apoplasts of roots, revealing that 45% of proteins that were responsive to H₂O₂ treatment were mainly involved in carbohydrate metabolism, redox homeostasis, cell defense, and signal transduction (Zhou et al., 2011). However, it should be kept in mind that exogenous treatment with H₂O₂ cannot be compared to in vivo apoplastic ROS generation.

For decades, there has been speculation as to how responses to apoplastic ROS can be so highly specific, since so many levels are engaged in signaling, and coordinated changes in gene expression and optimization of metabolic pathways are involved. This enables optimization of growth and development and survival upon environmental challenges. The ROS burst in the apoplast may be a fast on/off mechanism to induce signaling, but other signaling elements represent the specificity of signaling to different stimuli since these signaling pathways are not linear.

Plants have developed diverse mechanisms to perceive and react to abiotic and biotic stresses (Baxter et al., 2014). In particular, an apoplastic ROS burst induced by extracellular stimuli is one of the earliest events in many plant defense responses. Environmental stresses that particularly affect the apoplastic space of plants are explored below.

Further research is needed to understand apROS metabolism in detail. In this review, we have tried to emphasize how important this specific aspect is for plant metabolism. ApROS are produced and detoxified in enzymatic and non-enzymatic reaction depending on numerous factors, including apoplast pH, metal ion concentration, or availability of metabolites in the extracellular space. ApROS allow rearrangements of the cell wall, and in consequence the growth of plants. Products of apROS metabolism regulate the rate of cells division. The apoplast is the first place to receive signals coming from the outside, and apROS are involved in both local defense and the signal transmission into the cell and other plant organs. The results of experiments performed using plants with altered expression of apROS producing enzymes (Table 2) clearly indicate the importance of apROS in plant development and plant–pathogen interaction. It can be assumed that genetic manipulation of the expression of enzymes involved in apROS metabolism may allow the breeding of crop plants more resistant to pathogens, with greater biomass, or a desired lignin profile. However, it should be noted that apROS metabolism is a very complex multi-piece puzzle. Changing one of its elements may not lead to the desired effect because of (i) the multitude of enzyme isoforms (e.g., 10 RBOHs and over 70 isoforms of POX exist in Arabidopsis) that can take over its functions, (ii) individual compound cross-regulation (action of RBOHs can be regulated by POX activity), and (iii) the opposing reactions in which they are involved (e.g., POXs are ROS-producing and ROS-detoxifying enzymes; polyamines are antioxidants but also substrates for ROS- producing enzymes).

AP, MB, and BS wrote the manuscript. All authors read and approved the manuscript.

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.