The Molecules that Cause Oxidative Stress

Oxidative stress is caused by a group of extremely reactive molecules comprising free radicals of oxygen, singlet oxygen, and hydrogen peroxide; these are collectively known as active oxygen species (AOS). Singlet oxygen (*O2) is formed when the two unpaired

Plant Cell Death Processes

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Figure 13-1. Formation of active oxygen species.

electrons of molecular oxygen become paired, thus increasing the reactivity by making O2 amenable to donation of electrons in different spin configurations. Addition of electrons to molecular oxygen forms free radicals [see Fig. 13-1; also reviewed in Halliwell and Gutteridge (1985), Cadenas (1989) and Wojtaszek (1997)]. Transfer of a single electron, often catalyzed by transition metals such as Fe and Cu, to O2 forms the superoxide radical, O2—. In aqueous solutions, O2— disproportionates to hydrogen peroxide, H2O2. The decomposition of H2O2 can form the extremely reactive hydroxyl radical, " OH; indeed, this is one of the main sources of H2O2 toxicity (Cadenas, 1989).

The reactivity of free radicals causes them to either abstract H+ from or transfer electrons to other molecules, thus forming more free radicals. AOS have different reactivities and different abilities to move throughout the cell. For example, the hydroxyl radical is extremely reactive, but singlet oxygen is less reactive (Halliwell and Gutteridge, 1985). Of all the AOS, H2O2 is the least reactive, but is uncharged and thus can move through membranes. The reactivity and mobility of superoxide varies depending on the pH; the pro-tonated species reacts faster, can move through membranes, and dismutes to H2O2 faster than the unprotonated form. O2 — is more likely to be protonated in the apoplast, which has a pH around 5 to 6, than in the neutral cytoplasm (Felle, 1998; Green and Hill, 1984; Muhling et al., 1995). Thus, H2O2 generated at any location in the cell can move through membranes to other locations, but the subcellular location of O2— generation influences its reactivity and mobility.

III. Generating Active Oxygen Species

Free radicals and reactive oxygen compounds form during normal aerobic metabolism, environmental stress, senescence, and the plant defense response to pathogen attack. For example, an oxidative burst in response to pathogen attack is produced in the apoplast (Wojtaszek, 1997). Metabolic sources of AOS include the chloroplast, the mitochondrion, and the peroxisome (Del Río et al., 1992); these organelles contain antioxidant enzymes to remove the AOS produced there (Foyer and Halliwell, 1976). The defenses against AOS target each of the different reactive species and localize to the subcellular regions of AOS formation.

A. Metabolic Byproducts

1. Photosynthesis and respiration

The light reactions of photosynthesis involve a long chain of electron transfers, beginning with the removal of an electron from a water molecule and eventually depositing the electron with NADP+. Molecular oxygen can compete with NADP+ to receive the electron from ferredoxin. Formation of O2 -" in the chloroplast stroma by this reaction is termed the Mehler reaction (Mehler, 1951) and is a major source of superoxide in the plant cell (Polle, 1996).

Similarly, mitochondrial electron transfers can produce superoxide when molecular oxygen competes with other electron acceptors. Also, chlorophyll and other porphyrins can interact with oxygen by triplet-triplet interchange, in which an electron donated from oxygen returns with its spin reversed, forming singlet oxygen. Thus, in an aerobic environment, reactions essential for the life of the cell also produce AOS as a byproduct.

2. Peroxisomes

Peroxisomes are small, membrane-bound organelles that conduct multiple oxidative functions, including photorespiration and the glyoxylate cycle, depending on tissue location (Del Rio et al., 1992). Leaf peroxisomes contain the enzymes to conduct the oxidative photosynthetic carbon cycle of photorespiration, as well as many antioxidant enzymes to detoxify the active oxygen produced. Multiple AOS are produced in peroxisomes, including H2O2 from flavin oxidases and photorespiration, and O2-' from both xanthine oxidases in the matrix and NADH-dependent electron transport components in the membrane (Del Rio et al., 1992). One of the major routes of reactive oxygen formation in the peroxisome is transfer of electrons from NADH to a flavoprotein, cytochrome b5. Oxygen can compete with the normal electron acceptors, thus forming superoxide radicals.

B. Abiotic Stresses

Oxidative stress plays a major role in environmental stresses such as chilling, drought, wounding, and exposure to sulfur dioxide, heavy metals, ozone, or excess light (reviewed in (Noctor and Foyer, 1998). AOS may be generated by different mechanisms during the stress, but cause the induction of common adaptive responses, such as increased production of antioxidant enzymes. The survival of abiotic stress can depend, not on the severity of the oxidative stress, but on the alacrity of the antioxidant response. For example, in chilling stress, acclimation at 14°C induces increases in antioxidant enzyme levels and allows maize seedlings to survive subsequent exposure to the usually lethal temperature of 4°C (Prasad,

1996). Both the low and the moderate temperature cause a pulse of H2O2, but at the lower temperature, antioxidant defenses such as catalase and ascorbate peroxidase do not increase (Prasad et al., 1994). Thus, the difference between a survivable and a lethal temperature may be the induction of protective enzymes.

Excess light causes oxidative stress in leaves when the ability of photosystem II (PSII) to use energy from the light-harvesting complex (LHC) is overloaded and electrons transfer to oxygen. AOS generated in this process can damage PSII and the LHC, causing photoinhibition of photosynthesis. The plant's response to this stress is very quick: plants grown in low light and exposed to excess light show expression of AOS scavengers such as ascorbate peroxidases (APX1 and APX2) after less than 15 minutes of stress (Karpinski et al.,

1997). Experiments with an APX2-luciferase fusion show that H2O2 is important for the response to excess excitation energy: vacuum infiltration of catalase into leaves to remove H2O2 blocks the induction of APX2 by excess light (Karpinski et al., 1999). Treatment with exogenous H2O2 also induced APX2, but not to the same levels as excess light, which may reflect differences in flux between H2O2 applied to the plant and H2O2 produced by the plant, or an additional signal that acts synergistically within the plant. Thus, excess light activates the expression of antioxidant genes through H2O2.

The pollutant ozone (O3) causes ozonolysis of lipids and the formation of radicals such as superoxide and the hydroxyl radical (Mudd, 1996). Ozone reacts primarily with components of the extracellular space (Van Camp et al, 1994). Comparison of sensitive and insensitive cultivars of tobacco may have uncovered a different mechanism for response to abiotic stress: amplification of AOS production. Exposure of the sensitive cultivar to 150 nL L-1 O3 caused the induction of cell death (Schrauder et al., 1998). In both the sensitive and insensitive cultivars, ozone exposure triggered an increase in AOS in the apoplast and similar induction of antioxidant defences; only in the sensitive cultivar did the initial exposure lead to prolonged production of AOS in the apoplast and cell death (Schrauder et al., 1998).

C. Oxidative Burst

As part of the plant disease resistance response, plant cells release O2-' and H2O2 in response to recognition of a specific pathogen elicitor. The reactive oxygen species produced may act directly to kill pathogens; they also induce several responses in the plant, including cell wall protein crosslinking, programmed cell death (hypersensitive response, or HR), and expression of defense genes in the surrounding area. These are discussed in the next section.

Evidence exists for two mechanisms to generate the oxidative burst. First, Bolwell et al. (1995) propose H2O2 generation by a pH-dependent, cell wall linked peroxidase. In french bean cells treated with purified elicitor, they find no detectable generation of superoxide, but do find KCN-inhibitable generation of H2O2. One caution in the interpretation of these results is that KCN can itself act as a H2O2 scavenger (Barcelo, 1998). Even disregarding inhibitor studies, the generation of H2O2 by a cell wall linked peroxidase whose activity is dependent on an increase in pH is supported by a body of evidence (Bestwick et al., 1999). For example, in lettuce cells undergoing HR, H2O2 accumulates within the cell wall proximal to attached Pseudomonas syringae (Bestwick et al., 1997).

The second mechanism for generation of AOS is generation of superoxide from aplasma-membrane NADPH oxidase, similar to the phagocyte oxidative burst. NADPH-dependent superoxide synthesis can be observed in purified plasma membrane from cultured rose cells (Murphy and Auh, 1996). Moreover, diphenylene iodonium (DPI), a suicide substrate of mammalian NADPH oxidase (Doussiere et al., 1999), inhibits the production of H2O2 in soybean suspension cells treated with purified elicitor (Levine et al., 1994). As with many inhibitor studies, others (Bestwick et al., 1999) caution in the interpretation of these results, as DPI is not specific for NADPH oxidase and may be acting on a different target in plants. More evidence comes from the isolation, in both rice and Arabidopsis, of a protein similar to gp91phox, a subunit of the mammalian NADPH oxidase (Keller et al, 1998).

H2O2 is likely produced by both mechanisms, but to different degrees in different systems (Bestwick et al., 1999). Moreover, both systems may operate synergistically in the oxidative burst, with the H+ consumption required for dismutation of superoxide causing an increase in extracellular pH (Wojtaszek, 1997). This pH increase, along with that produced by open ion channels, activates the pH-dependent peroxidase on the cell wall. A further interesting suggestion is that an ATP-dependent proton pump, activated more slowly than the oxidative burst, may acidify the extracellular space and terminate production of reactive oxygen.

D. Photosensitizing Compounds

Many compounds can absorb light and emit active electrons, producing AOS; however, intriguing evidence indicates that some of these molecules may act as specific signals, rather than simply as toxic molecules. For a review of phototoxicity, refer to Tyystjarvi

(this volume, Chapter 18). One striking example of a photoactive molecule is chlorophyll. Chlorophyll precursors and breakdown products also absorb light, but have no productive outlet for high-energy electrons. Mutants in chlorophyll synthesis enzymes cause the accumulation of phototoxic porphyrin compounds and a light-induced cell death pheno-type (Hu et al., 1998). A mutation red chlorophyll catabolite (RCC) reductase, an enzyme in the chlorophyll breakdown pathway, causes the light-dependent accelerated cell death phenotype of acd2 mutants (Mach etal., 2001). Interestingly, overexpression of the ACD2 protein, which is predicted to reduce the amount of chlorophyll breakdown products, makes Arabidopsis tolerant to bacterial infection: the bacteria grow, but the plant shows reduced cell death and other disease symptoms. Thus, chlorophyll breakdown products may enhance or activate cell death in disease. However, not all cases of porphyrin accumulation cause cell death; plants that accumulate the porphyrin pheophorbide a, the precursor to RCC, show delayed senescence, remaining green longer than usual (Thomas, 1987; Thomas and Matile, 1988; Thomas etal., 1996).

IV. Defending Against Oxidative Stress

A. Cellular Damage by Active Oxygen Species

AOS cause protein oxidation, DNA damage, and lipid peroxidation. Oxidization of proteins occurs through multiple reactions, including side chain alterations and backbone cleavage, and disrupts protein structure, causing denaturation, aggregation, and susceptibility to degradation (reviewed in Dean et al., 1997). Oxidative stress causes increased frequencies of mutation and illegitimate recombination in purple photosynthetic bacteria (Ouchane et al., 1997). Lipid peroxidation can result in membrane damage. Thus, the extreme reactivity of AOS causes damage to all the structures of the cell; to protect against this damage, plants use both enzymes and small molecule antioxidants in all subcellular compartments.

B. Enzymatic Detoxification

Because of the variety of AOS types and subcellular locations, many enzymes are required to remove active oxygen species (Noctor and Foyer, 1998) (diagrammed in Fig. 13-2). Superoxide dismutase (SOD) converts superoxide into H2O2, which subsequently must be detoxified. Two of the major enzymatic scavengers of H2O2 are catalase (CAT) and ascor-bate peroxidase (APX). Both enzymes remove H2O2, but catalyze the reaction differently and are localized to different subcellular compartments. Catalase is primarily found in per-oxisomes; APX is primarily found in chloroplasts and the cytosol, indicating that they may have non-overlapping functions.

Antioxidant enzymes are essential for survival of oxidative stress. For example, under high light conditions, catalase-deficient plants develop bleached areas of cell death on their leaves and accumulate high levels of oxidized glutathione (Willekens etal., 1997). Catalase-deficient leaves are also very sensitive to exogenous H2O2, showing photobleaching and membrane damage.

C. Small Molecule Antioxidants

Much of the control of oxidative stress in the plant cell comes from large quantities of small molecule oxidants, namely ascorbic acid (vitamin C) and glutathione (reviewed in

Figure 13-2. Detoxification of active oxygen species.

Figure 13-2. Detoxification of active oxygen species.

(Noctor and Foyer, 1998). Glutathione and ascorbate can react directly with free radicals, or can be used by antioxidant enzymes as a source of reducing power (see Fig. 13-2). Glutathione (y Glu-Cys-Gly) exists as either a reduced form (GSH) or as an oxidized form in which two molecules are joined through a disulfide bond between the cysteines (GSSG). GSH reacts readily with free radicals, forming a glutathione radical (GS') that can react in pairs to form GSSG. The oxidized form can then be reduced to GSH by the enzyme glutathione reductase, expending NADPH and H+ (Foyer and Halliwell, 1976).

Ascorbate also acts as a reducing agent and is regenerated by GSH oxidation. H+ donation by ascorbate forms the monodehydroxyascorbate radical, which then disproportionates to form dehydroxyascorbate (DHA). This can then be regenerated to ascorbic acid by the enzyme dehydroascorbate reductase, which uses GSH. Thus, ascorbate and glutathione can reduce each other, further buffering the antioxidant capacity of the cell in what is called the ascorbate-glutathione cycle (Foyer and Halliwell, 1976). The small molecule antioxidants can react with active oxygen species, detoxify free radicals, and be regenerated into their reducing forms.

Although glutathione and ascorbic acid are major antioxidants, other molecules also act to protect the plant from oxidative stress, especially in specialized cellular environments. Alpha tocopherol, also known as vitamin E, can act in non-aqueous environments to detoxify lipid radicals. The vitamin E radical formed subsequently reacts with ascorbic acid, thus removing radicals from the membrane and reducing lipid peroxidation. Carotenoids act to prevent the activated triplet state of chlorophyll from reacting with oxygen to form singlet oxygen. Carotenoid-deficient mutants of a purple photosynthetic mutant showed drastic reduction of survival and increased frequency of mutations and illegitimate recombination when grown in aerobic photosynthetic conditions, as compared to anaerobic conditions (Ouchane et al, 1997).

The antioxidant capacity of the cell is critical for the survival of environmental stress. For example, the ascorbate-deficient vtc (Vitamin C) mutant of Arabidopsis (Conklin et al., 1996, 1997) accumulates only 30% of the normal ascorbate concentration and shows increased sensitivity to ozone stress, sulfur dioxide and UVB irradiation. Thus, small molecule antioxidants are just as critical as enzymatic antioxidants.

The localization of antioxidant defenses follows the localization of sources of AOS. For example, the chloroplast contains many antioxidants, including glutathione and glutathione reductase (Foyer and Halliwell, 1976). Also the apoplast contains many antioxidants that are modulated during the oxidative burst; these antioxidants may prevent killing of the cell by the extreme amounts of AOS produced. For example, in barley inoculated with powdery mildew, whole-leaf SOD did not change, but apoplastic SOD activity increased by 150-300% (Vanacker etal., 1998).

V. Active Oxygen in Resistance and Senescence

Plants have harnessed AOS in their defense against pathogens. The oxidative burst in plants may have several functions, including the action of reactive oxygen species in killing pathogens, in crosslinking cell wall components, and in signaling plant cells to induce endogenous defenses.

A. Crosslinking of Cell Wall Proteins

One of the defenses mediated by H2O2 is crosslinking of proteins of the cell wall. In cultured soybean cells treated with fungal elicitors, a glycine-rich cell wall protein with simple repeats (Val-Tyr-Lys-Pro-Pro), disappears from the SDS-extractable fraction on polyacryl-amide gels, but is still detectable by in situ immunofluorescence (Bradley et al., 1992). This activity can also be stimulated by H2O2, and is blocked by the addition of catalase or ascorbate. Crosslinking toughens cell walls and may limit pathogen ingress; indeed, elicitor-treated soybean culture cells are highly resistant to protoplasting by microbial cellulysin and pectolyase (Brisson et al., 1994).

B. Triggering of Programmed Cell Death

AOS from the oxidative burst trigger programmed cell death in the immediate vicinity of the infection and trigger gene expression in a wider area and at lower concentrations. In soybean suspension cultured cells, cell death was induced at 6-8 mM exogenous H2O2 and glutathione S-transferase (GST) expression was induced at 1-2 mM (Levine et al., 1994), indicating that reactive oxygen can act as a long-range signal. Moreover, H2O2 acts synergistically with NO (Delledone et al., 1998) in the induction of cell death. It is unclear whether this PCD is triggered by overwhelming of the cell's antioxidative defenses or by activation of a specific genetic program, as H2O2 and NO are both oxidative stressors. Suggestively, a brief pulse of H2O2 is all that is required to trigger cell death, and additional pulses do not potentiate the cell death response (Levine et al., 1994).

C. Lesion Mimic Mutants

Mutations with a phenotype that resembles a hypersensitive response may help elucidate the genetic control of cell death and the relationship between AOS and cell death. These mutants, called lesion mimics, have been isolated in many plants [Les and other mutants of maize, and accelerated cell death, or acd mutants, and lesions simulating disease resistance, or lsd mutants of Arabidopsis (Dietrich etal., 1994; Greenberg andAusubel, 1993; Greenberg et al., 1994; Walbot et al., 1983)]. Lesion mimic mutants can be roughly classified by whether the patches of dead cells spread or not; this may reflect an effect on two different processes, lesion initiation and lesion propagation (Walbot et al., 1983).

The connection between AOS and the lesion mimic phenotype has been explored in the case of lsd1. The lsd1 phenotype includes spreading patches of cell death that appear spontaneously in mutant plants under restrictive conditions; these lesions can be induced under permissive conditions by infiltration of a superoxide-generating system (Jabs et al., 1996). Also, superoxide accumulates at the border of spreading lesions. Interestingly, infiltration of a H2O2-generating system does not induce the lesions. lsdl mutants do not induce copper-zinc superoxide dismutase properly in response to the signal molecule salicylic acid (Kliebenstein et al., 1999). The inability to detoxify superoxide likely triggers cell death.

Another intriguing gene with a lesion mimic phenotype is the lethal leaf spot (llsl) gene of maize, which encodes a protein similar to aromatic ring-hydroxylating dioxygenases (Gray et al., 1997). LLS1 may enzymatically remove an aromatic compound; another possibility is that the iron centers act to sense the redox state. Indeed, the bacterial regulator SoxR contains iron-sulfur clusters whose redox state regulates the activity of the protein (Ding and Demple, 1997). A more complete understanding of the relationship between AOS and the lesion mimic phenotype awaits the development of better tools for visualizing and manipulating AOS, so that both the localization and the timing of AOS production can be examined and perturbed in these mutants.

D. Systemic Acquired Resistance and Salicylic Acid

The formation of H2O2 in the epicenter of the infection induces gene expression in the surrounding area and in distal tissues, such as uninfected leaves on the same plant. The distal leaves will subsequently be resistant to pathogen challenge, in a process known as systemic acquired resistance, or SAR. The induction of SAR may act in a reiteration of the induction of gene expression in the tissue surrounding an infection. Indeed, the presence of an HR induces "microbursts" of reactive oxygen production and cell death in distal tissues, which then induce systemic gene expression (Alvarez et al., 1998). This phenomenon can also be triggered by the infiltration of an H2O2-generating system.

SAR also requires the signaling molecule salicylic acid (SA) (Gaffney et al., 1993). SA acts to potentiate the hypersensitive burst in response to pathogen: application of physiological concentrations of SA to cultured soybean cells does not induce H2O2 production, but when pathogen is also added, the oxidative burst reaches its maximum much faster (Shirasu etal., 1997). SA binds one form of tobacco catalase and reduces its activity (Chen etal., 1993); this may account for the potentiation ofH2O2 production by SA. Thus, SA acts synergistically with other signals in pathogen recognition to increase the oxidative burst.

E. Active Oxygen and Senescence

Leaf senescence can be induced by multiple environmental, developmental, and hormonal stimuli, the most important of which may be a decrease in photosynthetic capacity. Whether this is caused by lack of light, or lack of photosystem capacity, it induces a series of events, starting with breakdown of chloroplasts, then loss of proteins, and finally, nuclear breakdown and programmed cell death (reviewed in Gan and Amasino, 1997; Noodén, 1988). The orderly disassembly of cellular components allows the cellular nutrients to be recycled into growing leaves, seeds, or storage tissue.

During senescence, the production of AOS increases and some antioxidant defenses are increased while others are decreased. The peroxisome may play a key role in this process: peroxisomes increase in senescence and their enzyme composition changes. For example, during senescence in pea leaves, purified peroxisomes contained increased activity of superoxide-producing enzymes such as xanthine oxidase, as well as increased superoxide dismutase activity, but decreased catalase activity (Pastori and del Rio, 1997). This study also found increased production of superoxide from the peroxisomal membrane, as well as increased production of H2O2. This increase of AOS, specifically H2O2, suggests that AOS may act as a signal to induce gene expression and trigger programmed cell death in senescence similarly to disease resistance. One striking difference is the speed of execution of cell death in these two processes, with the rapid cell death in disease resistance allowing for leaf dessication and halting of pathogen spread, and the delayed cell death in senescence allowing for the dismantling and recycling of cellular components.

VI. Mechanisms of Signal Transduction

Although reactive oxygen species are often seen as toxic byproducts of stress or metabolism, they also induce many cellular responses. Indeed, the role of antioxidants in the plant may be both to detoxify these compounds and to modulate their levels for signaling. In a complex signaling network such as this, it is often difficult to separate cause from effect; is the plant responding to reactive oxygen, or responding through reactive oxygen? Many possible mechanisms for sensing the levels of active oxygen species can be imagined, such as receptors for oxidized cell components, or the degradation of the oxidized form of a particularly sensitive protein. One mechanism that has been seen in bacteria, mammals, and plants, is transcriptional control through sensing the redox state of a specific protein. Although the specific mechanisms of sensing reactive oxygen are unknown, the following examples may prove instructive.

A. Control of Transcription by Plastoquinone Redox State

Plastoquinone is one of a chain of electron carriers that transfer electrons from photosystem II (PSII), which removes them from water, to photosystem I (PSI), which eventually transfers them to NADPH+. If an excess of electrons is being pumped in from PSII, then the reduced form of plastoquinone, PQH2, accumulates; if an excess of electrons is being removed by PSI, then the oxidized form, PQ, accumulates. This balance can be changed by changing the light wavelength to favor either PS I or II. Transfer of electrons to plastoquinone can also be blocked by the inhibitor DCMU [3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea], leading to an accumulation of PQH2. Transfer of electrons from plastoquinone can be blocked by the inhibitor DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), leading to an accumulation of PQ. Using inhibitors and light changes, Pfannschmidt et al. (1999) found that the redox state of plastoquinones affects the transcription of the chloroplast-encoded genes for proteins in the reaction center of PSI, psaAB. The transcription of psaAB decreased with DCMU treatment, which inhibits electron flow into the plastoquinone pool, and increased with DBMIB treatment, which inhibits electron flow out of the plastoquinone pool. This transcriptional regulation serves to maintain the balance between PS I and II; in conditions favoring the passage of electrons through PSII, reduced plastoquinones will accumulate, causing an increase in transcription of PSI genes to compensate.

B. MAP Kinase Pathway Signaling

Although the exact mechanisms of sensing oxidative stress remain unknown, that signal is transduced into gene transcription and other cellular responses by mitogen-activated protein kinase (MAPK) signaling. This mechanism involves a cascade of protein phosphorylation, starting with the activation of upstream protein kinases and is involved in the plant response to abiotic stresses, pathogens, and plant hormones (Jonak et al., 1999). The upstream protein kinases NPK (Nicotiana protein kinase) from tobacco and ANP (Arabidopsis NPK-like protein kinase) activate stress-induced MAPK expression and gene expression (Kovtun et al., 2000). ANP activity is specifically induced by H2O2 treatment. Interestingly, transformation of constitutively active NPK into tobacco produced enhanced tolerance to environmental stress without the activation of some known drought and cold response pathways (Kovtun et al., 2000). This approach can tease apart cause from effect in the complex response to oxidative stress.

VII. Conclusions

Active oxygen species form during normal metabolism, during the response to abiotic stresses, during senescence and during the response to pathogen attack. Maintaining oxida-tive balance is essential for the survival of the cell, so in addition to having antioxidant defenses localized to the area of AOS production, the cells also have mechanisms for sensing and responding to oxidative imbalances. These mechanisms control many aspects of metabolism, such as the balance of photosystem I and II components. Beyond metabolism, AOS act in the plant defense response, to induce defence responses such as crosslinking of cell wall proteins, programmed cell death, and gene transcription. Response to AOS may involve such diverse mechanisms as direct chemical modifications of cell wall proteins, indirect triggering of general responses, and possibly PCD, by disruption of the oxidative balance of the cell, and signal transduction in response to changes in the redox state of unknown signaling molecules. Determination of the mechanisms of signal transduction in response to AOS provides many opportunities for exciting future research.


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