- Email: [email protected]
Plant antioxidant gene responses to fungal pathogens
O P I N I O N
Barbour, A.G. (1990) Mol. Microbiol. 4, 811-820 11 Kitten,T. and Barbour, A.G. (1990) Proc. Natl Acad. Sci. USA 87, 6077-6081 12 Hinnebusch,J. and Barbour, A.G. (1992) J. Bacteriol. 174, 5251-7525 13 Hayes,I,.J., Wright, D.J.M. and Archard, I,.C. (1988)]. Gen. Microbiol. 134, 1785-1793 14 Kitten, T. and Barbour, A.G. (1992) Genetics 132, 311-324 15 Hinnebusch,J. and Barbour, A.G. (1991) J. Bacteriol. 173, 7233-7239
16 Barbour, A.G. (1988).I. Clin. Microbiol. 26, 475-478 17 Schwan,T.G., Burgorfer,W. and Garon, C.I:. (1988) Infect. lmmun. 56, 1831-1836 18 Sadziene,A. et al. (1992)J. Exp. Med. 176, 799-809 19 Perng, G.C. and LeFebvre,R.B. (1990) Infect. Immun. 58, 1744-1748 20 Marconi, R.T. and Garon, C.F. (1993) ]. Bacteriol. 175, 926-932 21 Sadziene,A. et al. (1993) Infect. Immun. 61, 2192-2195 22 Jonsson, M. et al. (1992) Infect. hnmun.
60, 1845-1853 23 Rosa, P.A., Schwan,T. and Hogan, D. (1992) Mol. Microbiol. 6, 3031-3040 24 Burman, N. et al. (1990) Mol. Microbiol. 4, 1715-1726 25 Restrepo,B.I.et al. (1992) Mol. Microbiol. 6, 3299-3311 26 Barbour, A.G. et al. (1991) Mol. Microbiol. 5, 489-493 27 De Lange, T. and Borst, P. (1982) Nature 299, 451-453 28 Garon, C.F., Dorward, D.W. and Corwin, M.D. (1989) Scanning Microsc. $3, 109-115
Plant antioxidant gene responses to
fungal pathogens John D. Williamson and John G. Scandalios eing unable to avoid environmental stresses by running away or by physically altering their surroundings, plants respond to changing environmental conditions by being biochemically agile. This can result in responses that are more complex at the cellular level than animal responses to the same stimuli. For example, one of the most severe environmental challenges faced by a plant is undoubtedly that of pathogen attack. It is not surprising, therefore, that plant responses to fungal attack are complex, multilayered and often particular to individual species. There are several recent reviews on responses to pathogen attack ~ . For the most part, these focus on the expression of genes involved in the synthesis of isoflavonoid phytoalexins (e.g. phenylalanine ammonia-lyase, chaicone synthase and chalcone isomerase) or the response of the pathogenesisrelated (PR) proteins. This review focuses on aspects of the less studied response of plant antioxidant genes to fungal attack. Although we are, for the most Part, looking at specific, isolated responses, keep in mind that each response is part of an interconnected, layered response by the whole organism
B
Anhoxidant defense systems are a prominent element in plant responses to environmental stress. Activated oxygen species have themselves been implicated as both a part of the plant's defense against pathogen attack as well as the phytotoxic component of photosensitizing fungal toxins. Molecular analyses are just beginning to define how plant oxidant and antioxidant genes might integrate with other defense responses to provide effective protection against pathogen attack. i.D. Williamson and J.G. Scandalios are in the l)ept of Genetics, North Carolina State Universi~. , Raleigh, NC 27695-7614, USA.
to a complex stimulus, and that the interconnections are only now beginning to be elucidated. Several levels of response to pathogen attack have been demonstrated. Initial responses (0-8 min) involve production of activated oxygen species (e.g. H 2 0 2 ) 4"s, which act both as antimicrobial agents and as substrate for peroxidasemediated cell wall lignification. Other early reactions (stress responses) serve to stabilize the plant's own metabolic and physiological
functions. Chronologically (after 2-4 h), the next layer of defense involves the production of antimicrobials such as tannins, phytoalexins and proteinase inhibitors, followed somewhat later by the production of hydroxyprolinerich glycoproteins. These early responses retard the advance of pathogen invasion until more permanent defensive mechanisms can be mobilized. Final responses occur 12-48 h after elicitation and include reinforcement of the cell wall by lignification and suberization, which physically exclude pathogen ingress. An effective resistance response requires that critical levels of specific defense products be produced before pathogen colonization can be achieved. Resistant plants have been shown to respond faster at one or more levels than susceptible plants of the same species. The plant's antioxidant defense systems are involved at several distinct levels in plant-pathogen interactions. First, they respond directly to oxygen radicals produced by the plant itself in response to the attacking fungus. Next, antioxidant enzymes are involved in protective mechanisms such as H202mediated cell wall lignification.
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Box 1. Defenses against activated oxygen The evolution of organisms that use molecular oxygen (02) as a terminal electron acceptor for respiration (i.e. aerobic organisms) gave rise to a paradox. On the one hand, aerobic metabolism provides a much higher energy yield than anaerobic respiration, giving the aerobic organism a huge competitive advantage. On the other hand, the respiratory reduction of 02 to H20 leads to the stepwise formation of reactive oxygen intermediates35.36, the superoxide radical (-02-), hydrogen peroxide (H202) and the hydroxyl radical (.OH). In the presence of light, photosensitizer-mediated production of the reactive singlet form of oxygen (10 2) may also occur. 10 2
02
+e-
+e-
~ "02-
----"
2
Overall reaction :
H202
H+
+e-
---~
+e-
"OH
H"
H+
02 + 4 e- + 4 H*
-
H20
-~ 2 H20
-
Essentially the opposite process, the four-electron oxidation of water and the associated formation of oxygen radicals, occurs during photosynthesis. Normally these highly reactive species remain bound during the oxidation/ reduction reactions involved in respiration and photosynthesis. Occasionally, however, they are released and unless dealt with immediately are capable of causing extensive cell damage36.3L Of even more devastating effect,-02and H202 act as substrates for the transition-metal-catalysed Haber-Weiss reaction ~.39. One of the products of this reaction, the hydroxyl radical (.OH), is the most reactive of the oxygen radicals, and can directly and indiscriminately attack lipids, proteins and DNA. Activated oxygen species are also produced by normal metabolic processes and in response to various environmental stresses. The superoxide radical (-02-), for example, is generated by various enzymatic reactions in the cell (e.g. uricase and xanthine oxidase). Hydrogen peroxide may be produced by the dismutation reaction of two superoxide anions, by the ~-oxidation of fatty acids in the course of normal lipid metabolism and in response to stress. The production of activated oxygen species in plant tissues in response to pathogen attack is also well documented 4,5. Finally, at least one fungal species, Cercospora, produces activated oxygen species directly, as a means of disrupting host tissues. Disruptive effects of these activated oxygen species include direct inhibition by H202 of enzymes involved in C02 fixation4°.41, and peroxidation of membrane lipids. Damage to intracellular membranes can lead to leakage of cellular contents, loss of organellar function and cell death. To minimize the damaging effects of activated oxygen species, of both external and internal origin, aerobic organisms have evolved both enzymatic and nonenzymatic antioxidant defense systems. Nonenzymatic systems include compounds with intrinsic antioxidant properties such as vitamins C and E, glutathione and l~carotene42.43.These are often linked with enzymes such as ascorbate peroxidase (AP) and glutathione-reductase (GR), which regenerate the antioxidant capacity of the quenching molecule. Purely enzymatic systems, such as the catalases (CAT), peroxidases (Px) and superoxide dismutases (SOD), protect the organism by directly scavenging superoxide radicals and hydrogen peroxide and converting them to less reactive species: •02 + "02 + 2H + SOD 02 + H202
2H202 CAT 2H20 + 02
H202+R(OH)2 Px ) 2H20+R(O)2
Since the rate constants of reactions involving .OH radical attack are as fast as the fastest reactions catalysed by the antioxidant enzymes such as SOD, no specific enzyme systems are known that directly scavenge these reactive oxygen species. It is hypothesized that, instead, the superoxide dismutases, coupled with the catalases and peroxidases, act to limit the availability of substrate for the reactions leading to the formation of the hydroxyl radical.
Third, they respond to stressinduced changes in the plant's metabolism that lead to increased production of oxygen radicals. Finally, antioxidant defense systems may respond directly to activated oxygen species produced by photosensitizing fungal toxins. While we allude to a number of these responses as a means of
TRENDS
IN
emphasizing the layered nature of the plant's response to pathogen attack, this review will focus primarily on recent investigations on antioxidant enzyme induction by fungal pathogens, and the specific response of plant antioxidant defense systems to the best characterized of the fungal photosensitizers, cercosporin.
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Response of plant peroxidases to fungal attack
The plant peroxidases catalyse the donor-mediated reduction of H202 to HzO and oxidized donor (see Box 1). The biochemistry and cell biology of this complex family of enzymes has been reviewed elsewhere 6. Peroxidases are found in organelles and in the cytoplasm,
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hip
Fe3Fe2
LH
o I 02
L Fe2F 3
OH H2O2--o2 0 o.
H
OH"
H20
•L
0
LOOH
LO'O
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LOiH
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O2
I
I
Type I
Type II
Fig. 1. Lipid peroxidation by Type I and Type II photosensitizers (see Ref. 13). Absorption of a photon (hv) by the ground state photosensitizer (°S) yields the lowest excited singlet state (1S) followed by conversion to the less energetic triplet state (aS). Type I reaction initiation by hydrogen transfer from an unsaturated lipid substrate (LH) leads to the production of the lipid alkyl radical (.L), the sensitizer radical anion (-S) and H'. Subsequent propagation of lipid radicals (4_), lipid peroxides (LOOH) and lipid peroxyl radicals (LO-O) is illustrated. In the presence of free oxygen, autoxidation of .S- generates the superoxide radical (-O2-). This also leads to lipid radical formation by the pathway indicated. In Type II reactions singlet oxygen 002) is formed by direct energy transfer from sS to Or. Singlet oxygen reacts directly with unsaturated lipids (LH) to produce lipid hydroperoxides (LOOH). Initiation of radical proliferation via a metal-catalysed, Haber-Weiss type reaction to form lipid oxyl radical (L-O) is depicted.
as well as extracellularly, and have been widely implicated in the defense response of plants to fungal attack and stress. The increased expression of extracellular anionic peroxidases is perhaps the best studied fungaiinduced antioxidant gene response. These peroxidases are involved in cell wall lignification and suberization 6,7, and are thought to protect the plant by forming a physical barrier to pathogen ingress. It has been shown, for instance, that anionic peroxidases localized in the cell walls of suberizing potato and tomato cells are induced by fungal attack, wounding and abscisic acidS,L In resistant lines of tomato, expression of one of these peroxidases was induced earlier than in susceptible lines (24 versus 48 h after inoculation) '°. Induction of a wheat anionic peroxidase by fungal extracts has also been demonstrated ~t. Transcription of the wheat anionic peroxidase gene was induced about 3 h after inoculation, and intercellular peroxidase activity increased between 6 and 36 h after inoculation. In tomato at lcast three anionic peroxidases appear to be involved in cell wall suberization. In wound
healing studies using transgenic plants expressing antisense RNA to block the expression of two tomato anionic peroxidases, cell wall suberization was not measurably inhibited '2. Evaluating the ability of these transgenic plants to withstand pathogen attack will be illuminating. Cell wall lignification is a relatively slow response and other protective mechanisms must be deployed during the first hours of attack. Furthermore, there must be a source for the H,_O2 used by peroxidases as a substrate. The pathogen-induced production of activated oxygen species by plant tissues is well documented 4, with extracellular UzO2, presumably produced by an outer membrane oxido-reductase, appearing during the first minutes of pathogen attack s. It has been hypothesized that this response may serve an immediate antimicrobial role, as well as providing substrate for later peroxidase-mediated cell wall lignification. It may also account, at least in part, for the induction of cytosolic peroxidases, catalases and superoxide dismutases by non-cercosporin-containing fungal extracts (see below).
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Photosensltizlng fungal toxins Photosensitizers are a structurally diverse group of compounds with a common mechanism of action. When activated by light, the photosensitizer molecule is converted to an electronically excited triplet state, which can react with 02 to form activated oxygen species (Fig. 1). This reaction may be mediated through a reducing substrate to form the reactive oxygen species H202, "O2-, or .OH (Type I reaction), or may take the form of a direct energy transfer resulting in the formation of singlet oxygen (~O2, Type II reactionp s. Many plant pathogenic fungi are known to produce photosensitizers that presumably act as phytomxins ~4. The best studied of these is the light-induced polykedde toxin cercosporin produced by members of the genus Cercospora. Cercosporin itself does no damage. However, activated oxygen species produced by cercosporin in the presence of light are directly and indiscriminately toxic to living tissues, causing oxidation of lipids, proteins, carbohydrates and nucleic acids (Figs 1 and 2). Because of the nonspecific nature of the damage caused by activated
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"02- ~ Polyketide biosynthetic -7__ hv pathway
Plant cell
i02+
Excited triplet state
Membrane damage
02
/ Cercosporin Benefits to fungus (1) leakage of nutrients (2) physical access
Y,
Cell death
Fig. 2. In the presence of light (459-490 nm) cercosporin synthesis is induced and an excited form (triplet state) of the toxin molecule is generated. Depending on the reducing potential of the environment (see Fig. 1), this excited form reacts with 02 to produce singlet oxygen (102) and/or superoxide radicals (-Or-). These activated oxygen species cause rapid breakdown of plant cell membranes by lipid peroxidation, leading to leakage of cell components and cell death.
oxygen, a successful defense strategy must protect at several levels and, as with most plant responses, the specific responses of individual plant species to Cercospora and cercosporin are quite diverse. Cercosporin resistance in crop plants Many important crop species display little or no resistance to Cercospora infection. Although there have been numerous efforts to use biotechnology to produce plants that display general resistance to oxidative stress (ozone, etc.) and specific resistance to cercosporin, no single factor seems unambiguously to provide such universal protection against oxidative stress 1~-'7. The inescapable conclusion is that no single factor or gene protects the plant against all types of environmentally and/or metabolically generated oxidative stress. As with many multifactor traits, more has been achieved by traditional breeding, and cultivars of several crop species have been produced that show significant levels of Cercospora resistance (maize TM, rice 19 and sugarbeet2°). Cultivars of rice (Oryza sativa) with high levels of resistance to Cercospora oryzae have been grown commercially since the 1940s. In a recent study 19, rice varieties showing varying degrees of resistance to C. oryzae were examined. Resistance ranges from almost complete in a wild variety, Louisiana red rice, to only 2% in the com-
mercial variety Labelle. Because of the broad toxicity of cercosporin, it was felt that resistance to Cercospora was most likely based on resistance to the toxin itself. Sensitivity to the purified toxin was, therefore, assessed by inhibition of callus and seedling growth, chlorosis and necrosis in leaves, and ion leakage in cell suspension culture. By all measures, the susceptible cultivar Labelle was the most sensitive to cercosporin damage, while the resistant red rice was the least sensitive (others were intermediate). None of the tested varieties showed any reaction to the toxin in the dark, consistent with cercosporin's mode of action as a photosensitizer. Measurement of the cercosporin content of cells grown in suspension culture in the presence of cercosporin revealed that the resistant red rice cells contain less than one-tenth the level of cercosporin present in cells of the sensitive cultivar Labelle. The authors proposed that these differences might be due to (1) differential exclusion or uptake, (2) active export or (3) detoxification of cercosporin (or a combination of the three). Red rice also contained approximately twice the total carotenoid level of Labelle. Lutein and 13-carotene were present in both varieties, but red rice also contained several carotenoids absent in Labelle. Inhibition of carotenoid biosynthesis resulted in the loss of cercosporin resistance
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in red rice suspension cells. This is possibly because carotenoids are potent quenchers of oxygen radicals. So, although exclusion or detoxification of cercosporin might be important, the ability to synthesize carotenoids also appears to be necessary for cercosporin resistance. Sugarbeet varieties (Beta vulgaris) showing different levels of resistance to Cercospora beticola have also been analysed 2°. In this work response to infection was characterized by an immediate increase in the level of cytosolic peroxidases. This increase was consistently higher in resistant than in susceptible varieties. Later in the defense response, orthodiphenol oxidase, an organellar (mitochondrial and chloroplastic) enzyme that produces oxidized phenolic compounds toxic to C. beticola, was induced at higher levels in resistant varieties. It was concluded that a successful resistance response involves multiple factors acting at different times with different effects. Because Cercospora infection is likely first to influence the cytoplasm of the cell, the cytoplasmic localization of the peroxidase and its ability to oxidize a wide variety of donor compounds suggest that it is used in the initial defensive responses of the cell to oxidize toxin-derived H202 and to stabilize stressinduced metabolic changes. Response of SOD and CAT to fungi and cercosporin While the superoxide dismutase (SOD) and catalase (CAT) isozyme systems have been examined to some extent in other plants 1s,21, they have been most thoroughly described in maize. In corn (Zea mays), at least six structural genes
(Sod1, Sod2, Sod3, Sod4, Sod4A and Sod5) encode corresponding SOD isozymesn. The various superoxide dismutases are precisely compartmentalized, with the Cu/Zncontaining SODs being localized in chloroplasts (SOD-l) or in the cytosol (SOD-2, 4, 4A and 5), and the Mn-containing SOD-3 in mitochondria2L This specific compartmentalization presumably reflects specific defensive function(s). The
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three biochemically distinct catalase isozymes, CAT-I, CAT-2 and CAT-3, are encoded by three unlinked structural genes, Catl, Cat2 and Cat3 (Refs 22, 24, 25). In mature green leaves of maize, CAT-2 is localized in the peroxisomes of bundle-sheath cells, while CAT-1 and CAT-3 are found primarily in mesophyll cells25. Both CAT-1 and CAT-2 isozymes are localized in glyoxysomes/peroxisomes where present, or, in tissues and mutants lacking these organelles, in the cytosol. In contrast, maize CAT-3 coisolates exclusively with the mitochondrial cell fraction 26. Very little has been reported on the response of the superoxide dismutases or catalases to fungal attack (or pathogen attack in general). In two cases, where SOD response to non-toxin-producing fungi has been reported, changes in SOD activity did not appear to be a measurable early response. The first study27 examined SOD and peroxidase activities in sensitive and resistant (hypersensitive) varieties of Phaseolus vulgaris inoculated with Uromyces phaseoli. As seen in previous reports (above), peroxidase activity increased earlier in infected resistant plants than in infected susceptible plants (18 versus 48 h). However, 48 h after inoculation neither Cu/Zn nor Mn SOD activity had changed measurably in either infected line. Only after the onset of fungal spore formation (4-8 d after infection) did the Cu/Zn SOD activity in both susceptible and resistant lines begin to increase while Mn SOD activity increased only in susceptible lines. Given the time-frame, these changes are not likely to be a primary response to fungal elicitors, but are perhaps a reflection of the relative levels of general metabolic stress in each case. The second study 28 reported that total SOD activity in blastinfected leaves of susceptible and resistant rice was not measurably different to that in uninfected plants. Changes in RNA levels were not examined in either study. This is in contrast to the report 2~ of increased Mn SOl) protein and
RNA accumulation in Nicotiana plumbaginifolia inoculated with a bacterial pathogen (Pseudomonas syringae). In inoculated plants the level of Mn Sod RNA had increased significantly by 6 h, although increased Mn SOD protein was not detected until 48 h. However, a complete comparison with expression in uninoculated plants was not presented. The phytotoxic effect of photosensitizers probably results from both the immediate destructive effects of activated oxygen species and the radicals arising from lipid peroxidation ~.~(Fig. 1). Fungal attack also induces plant production of oxygen radicals ~4. So, while induction of Cat and Sod gene expression may not be the plant's sole defense against fungal attack in general, and Cercospora in particular, it seems reasonable that these critical antioxidant defensive systems must constitute an important q9mponent. In this laboratory we have, therefore, begun to examine more closely the response of SOD and CAT to Cercospora and cercosporin, and to explore the possibility of using cercosporin as a means of controlled application of external oxidative stress. In two recent papers ~°'3~ we reported on several aspects of maize CAT and SOD response to extracts of light- and dark-grown Cercospora and to purified cercosporin. Because numerous environmental factors influence the rate and type of oxygen radicals produced by cercosporin ~2'~3, a controlled plate assay was used employing excised embryos incubated on simple growth medium supplemented with purified cercosporin. Maize cmbryos incubated in the presence of increasing levels of purified cercosporin showed a dose-dependent increase in total catalase activity, individual isozyme protein levels and corresponding Cat transcript accumulation. The rcsponse of the Cat gene products to increasing doses of purified cercosporin seen in this study, in fact, closely mimics their response in maize embryos to increasing doses of hydrogen peroxide2L However, the most intriguing observation was
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that, while both total SOD activity and individual SOD isozyme protein levels remained constant in all toxin treatments during the 18-24 h test period, transcript accumulation for all Sods changed dramatically. One plausible explanation for this contrast in CAT and SOD protein response is the difference in the relative toxicities of the respective enzyme substrates and the method maize has evolved to deal with each. Because the superoxide radical, and the products of its peroxidation of the lipid bilayer, are highly and immediately toxic to the cell, maximal constitutive levels of the appropriate SODs might be required for adequate protection. Hydrogen peroxide, however, is less immediately damaging to the cell. The time required for de novo induction of catalase expression might, therefore, bc adequate for defense against this relatively less toxic species. The observed increase in Sod transcript levels in the absence of increased SOD protein accumulation also suggests that protein turnover plays a role in the response of SODs to activated oxygen species. Active transfer of highenergy free electrons probably eventually destroys or inactivates the mediating protein(s). Therefore, under high levels of oxidative stress, high SOD protein turnover rates would require new synthesis to maintain adequate SOD isozyme levels. Since the cercosporin plate assay developed for these studies is ideal for both controlled application of oxidative stress and for in vivo pulse-chase labeling studies, it should bc simple to determine whether or not this type of selective turnover does indeed occur.
In a subsequent study n we showed that Cercospora produces compounds, other than cercosporin, that affect Cat and Sod transcript accumulation. The synthesis as well as the activation of cercosporin is light depcndent '4. Thus we were able to compare the effects of cercosporin- and non-cercosporin-containing fungal extracts (from light- and darkgrown Cercospora kikuchii mycelia, respectively) with that of
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Table 1. Partial summary of antioxidant systems in higher plants" Non-enzymatic scavenging systems
Subcellular location
Type of active oxygen species
Source of active oxygenspecies
Enzymatic scavenging systems
Chloroplast
Superoxide
H202
Photosystem II Enzymatic
SOD Peroxidases
Mitochondria
Superoxide H202
Electron transport Enzymatic
SOD Peroxidase Catalase
H20~ H20 + oxidized donor H20 + 02
Cytosol
Superoxide H202
Enzymatic Enzymatic
SOD Catalase Peroxidase
H202 H20 + 02 H20 + oxidized donor
Glyoxysomes Peroxisomes
H202 H202
13-0xidation Photorespiration
Catalase Catalase
H20 + 02 H20 + 02
Extracellular
Superoxide
Enzymatic Enzymatic
None known Peroxidase
None known Lignin, suberin, hydroxyproline
H202
Products
H202 Glutathione, NADP÷, d ihydroascorbate
Ferredoxin, carotenoids, xanthophylls
"Reproduced from Ref. 35. SOD, superoxide dismutase.
purified cercosporin on the accumulation of Cat and Sod transcripts. Extracts from fungi grown in the dark (i.e. containing no cercosporin) elicited distinct changes in accumulation for several of the Cat and Sod transcripts. The response of Cat and Sod expression to extracts from light-grown Cercospora (i.e. containing cercosporin) appeared to be a combined response to purified cercosporin and one or more fungal compounds presumably present in both light- and dark-grown fungal culture extracts. Under the conditions used in these two studies, all of the Cat and Sod transcripts respond to purified cercosporin and cercosporin-containing fungal extracts. On the other hand, only two of the cytosolic (Cu/Zn) Sods (Sod2 and 4), the glyoxysomal/ peroxisomal Cat2 and mitochondrial coisolating Cat3 transcripts respond to non-cercosporin extracts. This might be a direct response to fungal elicitor(s) 2 or a response to oxygen radicals produced as part of the oxidative burst 4,~. It is, however, not yet possible unambiguously to attribute specific protective roles to the individual isozymes. Further manipulation of the experimental
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parameters (e.g. light intensity, toxin concentration and developmental stage) should be revealing. In these same studies we found that the catalases also show stagespecific changes in response to cercosporin-containing fungal extracts~L In the scutella of developing (immature) embryos, increasing levels of cercosporin result in decreased CAT-2 accumulation, while in the scutella of germinating (mature) embryos, CAT-2 isozyme levels increase. In addition, CAT-1 levels in developing embryos increase in response to fungal extracts regardless of cercosporin content, while in mature germinating embryos there is no marked response to either. In separate studies we observed that the response of the catalases to the plant growth regulator abscisic acid was different in developing and germinating maize embryos 34. Embryogenesis and germination represent two distinct, well-defined, alternative pathways in developmental programing. Different responses to the same treatment at these two stages may, therefore, reflect not only the profound changes in gene expression that occur when embryos undergo the developmental shift from embryo
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maturation to germination, but also the specific functions of these genes in cellular responses to oxidative stress. Concluding remarks
Individual plant stress responses do not occur in isolation, but are components of an integrated defense, with each part being necessary, but often insufficient by itself, to provide comprehensive protection. The antioxidant defense enzymes are a good example of the biochemical complexity of one element of the plant's response to environmental stressors (Table 1)36. While animals have a single catalase, most plants examined have three biochemically distinct and differently compartmentalized catalases. The plant SODs are even more complex, while the complexity of the peroxidases is stunning. This complexity is perhaps a reflection of the highly reactive nature of oxygen radicals. Superoxide, for instance, reacts on contact with cellular structures with immediate destruction of the affected molecule and the generation of additional radicals. The plant's defense response must, therefore, be appropriate and instantaneous. This means having
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maximal levels of SODs in exactly the right place before attack occurs. Hence, even though epigenetic expression of a mitochondrial SOD might increase resistance to paraquat, it wouldn't protect against cytosolic radicals. IJkewise, overexpression of a cytosolic CAT would not enhance resistance to photosensitizer-derived extracellular superoxide. Available information, therefore, suggests that efforts to engineer oxidative stress tolerant plants should perhaps focus less on overexpressing individual Cat or Sod genes and more on augmenting the balanced, coordinated expression of all the essential antioxidant enzymes in the various cell compartments. To maintain the critical equilibrium between the generation and removal of superoxide radicals, organisms have evolved elaborate regulatory mechanisms to control the synthesis, accumulation and compartmentalization of the appropriate SODs. At present, however, our knowledge of these underlying cellular and molecular mechanisms governing mobilization of antioxidant defenses is meager. Understanding the basis of the differential responses of the different SOD and CAT isozymes to given stressors or signals and their specific spatial and temporal compartmentalization will clarify the specific functions of multiple forms of these enzymes. The identification and characterization of cis-acting elements and associated transacting factors involved in the regulation of these genes will increase our understanding of the signal transduction pathway for the oxidative stress response and further our efforts to engineer organisms with higher resistance to oxidative stress. Molecular analysis of Sod and Cat gene/promoter structure and expression together with mutant and physiological analyses should help us unravel the mechanisms controlling this finely tuned system and thereby show us where changes will enhance plant stress resistance. Acknowledgements We gratefullyacknowledgeour colleaguesin the laboratoryfor theircontributionsoverthe
.N
years. We thank M. Dauband M. Ehrenshaft for an advancecopy of a reviewand helpful commentson the manuscript,and S. Ruzsafor assistancewith figurepreparation. References 1 Bowles,D.J. (1990) Annu. Rev. Biochem. 59, 873-907 2 Dixon,R.A.and Harrison,M.J. (1990) Adv. Genet. 28, 165-234 3 Dixon,R.A.and Lamb,C.J. (1990) Annu. Rev. Plant Physiol. Plant Mol. Biol. 41,339-367 4 Sutherland,M.W. (1991) Physiol. Mol. Plant Pathol. 39, 79-93 5 Apostol,I., Heinstein,P.F.and Low,P.S. (1989) Plant Physiol. 90, 109-116 6 Van-Huystee,R.B.(1987) Annu. Rev. Plant Physiol. 38,205-257 7 Kolattukudy,P.E. (1984) Can. ]. Bot. 62, 2918-2933 8 Roberts,E., Kutchan,T. and Kolattukudy,P.E. (1988) Plant Mol. Biol. 11, 15-26 9 Roberts,E. and Kolattukudy,P.E. (1989) Mol. Gen. Genet. 217, 223-232 10 Kolattukudy,P.E., Podila,G.K.and Mohan, R. (1989) Genome 31, 342-349 11 Sclrweizer,P., Hunziker,W. and Mosinger, E. (1989) Plant Mol. Biol. 12, 643-654 12 Sherf,B.A.,Bajar,A.M.and Kolattukudy,P.E. (1993) Plant Physiol. 101,201-208 13 Girotti,A.W.(1990) Photochem. Photobiol. 51,497-509 14 Daub, M. and Ehrenshaft,M. Physiol. Plant. (in press) 15 Bowler,C., Van Montagu,M. and lnze', D. (1992) Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 86-116 16 Furusawa,I. et al. (1984) Plant Cell Physiol. 25, 1247-1254 17 Tanaka, K. and Sugihara,K. (1980) Plant Cell Physiol. 21, 601-611 18 Malan,C., Greyling,M.M. and Gressel, J. (1990) Plant Sci. 69, 157-166 19 Batchvarova,R.B.,Reddy,V.S.and Bennett,J. (1992) Phytopathology 82, 642-646 20 Rautela,G.S.and Payne,M.G. (1970) Phytopathology 60, 239-245 21 Havir,E.A.and McHale,N.A. (1989)
Plant Physiol. 91, 812-815 22 Scandalios,J.G. (1992)in Molecular Biology of Free Radical Scavenging Systems (Scandalios,J.G., ed.), pp. 117-152, Cold SpringHarbor LaboratoryPress 23 Baum,J.A. and Scandalios,J.G. (1979) Differentiation 13, 133-140 24 Scandalios,J.G. (1965)Proc. Natl Acad. Sci. USA 53, 1035-1040 25 Scandalios,J.G. et al. (1984) Dev. Genet. 4, 281-293 26 Scandalios,J.G., Tong, W.F.and Roupakias, D.G. (1980) Mol. Gen. Genet. 179, 33-41 27 Buonaurio,R., DellaTorte, G. and Montalbini,P. (1987)Physiol.Mol. Plant Pathol. 31,173-184 28 Matsuyama,N. (1983) Ann. Phytopathol. Soc. Japan 49, 270-273 29 Bowler,C. etal. (1989) EMBO]. 8, 31-38 30 Williamson,J.D. and Scandalios,J.G. (1992) Plant]. 2, 351-358 31 Williamson,J.D. and Scandalios,J.G. (1993) Physiol. Plant. 88, 159-166 32 Macri,F. and Vianello,A. (1979) Plant Cell Environ. 2, 267-271 33 Daub, M.E. {1987)in Light Activated Pesticides (tieitz,J.R. and Downum, K.R., eds), pp. 271-280, The American ChemicalSociety 34 Williamson,J.D. and Scandalios,J.G. (1992) Proc. Natl Acad. Sci. USA 89, 8842-8846 35 Scandalios,J.G. (I 993) Plant Physiol. 101,7-12 36 Halliwell,B. and Gutteridge,J.M.C. (1985) Free Radicals in Biology and Medicine, ClarendonPress 37 Fridovich,I. (1986) Adv. Enzymol. 58, 62-97 38 Haber,F. and Weiss,J. (1934) Proc. R. Soc. London Ser. A 147, 332-351 39 Kappus,H. (1985)in Oxidative Stress (Sies, H., ed.), pp. 273-310, Academic Press 40 Kaiser,W. (1979) Planta 144, 19,3-200 41 Charles,S. and Halliwell,B. (1981) Planta 151,242-246 42 Krinsky,N.I. (1989) Free Radical Biol. Med. 7, 617-635 43 l,ars0n,R.A. (1988) Phytochemistry 27, 969-978
Writing for TIM Most of the articles published in Trends in Microbiology are specially commissioned by the Editor. However, other ideas for articles will also be considered. If you wish to write for TIM, please contact the Editor first with an outline of your intended article. Note that all articles are subject to rigorous peer and editorial review: commissioning does not guarantee publication.
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Barbour, A.G. (1990) Mol. Microbiol. 4, 811-820 11 Kitten,T. and Barbour, A.G. (1990) Proc. Natl Acad. Sci. USA 87, 6077-6081 12 Hinnebusch,J. and Barbour, A.G. (1992) J. Bacteriol. 174, 5251-7525 13 Hayes,I,.J., Wright, D.J.M. and Archard, I,.C. (1988)]. Gen. Microbiol. 134, 1785-1793 14 Kitten, T. and Barbour, A.G. (1992) Genetics 132, 311-324 15 Hinnebusch,J. and Barbour, A.G. (1991) J. Bacteriol. 173, 7233-7239
16 Barbour, A.G. (1988).I. Clin. Microbiol. 26, 475-478 17 Schwan,T.G., Burgorfer,W. and Garon, C.I:. (1988) Infect. lmmun. 56, 1831-1836 18 Sadziene,A. et al. (1992)J. Exp. Med. 176, 799-809 19 Perng, G.C. and LeFebvre,R.B. (1990) Infect. Immun. 58, 1744-1748 20 Marconi, R.T. and Garon, C.F. (1993) ]. Bacteriol. 175, 926-932 21 Sadziene,A. et al. (1993) Infect. Immun. 61, 2192-2195 22 Jonsson, M. et al. (1992) Infect. hnmun.
60, 1845-1853 23 Rosa, P.A., Schwan,T. and Hogan, D. (1992) Mol. Microbiol. 6, 3031-3040 24 Burman, N. et al. (1990) Mol. Microbiol. 4, 1715-1726 25 Restrepo,B.I.et al. (1992) Mol. Microbiol. 6, 3299-3311 26 Barbour, A.G. et al. (1991) Mol. Microbiol. 5, 489-493 27 De Lange, T. and Borst, P. (1982) Nature 299, 451-453 28 Garon, C.F., Dorward, D.W. and Corwin, M.D. (1989) Scanning Microsc. $3, 109-115
Plant antioxidant gene responses to
fungal pathogens John D. Williamson and John G. Scandalios eing unable to avoid environmental stresses by running away or by physically altering their surroundings, plants respond to changing environmental conditions by being biochemically agile. This can result in responses that are more complex at the cellular level than animal responses to the same stimuli. For example, one of the most severe environmental challenges faced by a plant is undoubtedly that of pathogen attack. It is not surprising, therefore, that plant responses to fungal attack are complex, multilayered and often particular to individual species. There are several recent reviews on responses to pathogen attack ~ . For the most part, these focus on the expression of genes involved in the synthesis of isoflavonoid phytoalexins (e.g. phenylalanine ammonia-lyase, chaicone synthase and chalcone isomerase) or the response of the pathogenesisrelated (PR) proteins. This review focuses on aspects of the less studied response of plant antioxidant genes to fungal attack. Although we are, for the most Part, looking at specific, isolated responses, keep in mind that each response is part of an interconnected, layered response by the whole organism
B
Anhoxidant defense systems are a prominent element in plant responses to environmental stress. Activated oxygen species have themselves been implicated as both a part of the plant's defense against pathogen attack as well as the phytotoxic component of photosensitizing fungal toxins. Molecular analyses are just beginning to define how plant oxidant and antioxidant genes might integrate with other defense responses to provide effective protection against pathogen attack. i.D. Williamson and J.G. Scandalios are in the l)ept of Genetics, North Carolina State Universi~. , Raleigh, NC 27695-7614, USA.
to a complex stimulus, and that the interconnections are only now beginning to be elucidated. Several levels of response to pathogen attack have been demonstrated. Initial responses (0-8 min) involve production of activated oxygen species (e.g. H 2 0 2 ) 4"s, which act both as antimicrobial agents and as substrate for peroxidasemediated cell wall lignification. Other early reactions (stress responses) serve to stabilize the plant's own metabolic and physiological
functions. Chronologically (after 2-4 h), the next layer of defense involves the production of antimicrobials such as tannins, phytoalexins and proteinase inhibitors, followed somewhat later by the production of hydroxyprolinerich glycoproteins. These early responses retard the advance of pathogen invasion until more permanent defensive mechanisms can be mobilized. Final responses occur 12-48 h after elicitation and include reinforcement of the cell wall by lignification and suberization, which physically exclude pathogen ingress. An effective resistance response requires that critical levels of specific defense products be produced before pathogen colonization can be achieved. Resistant plants have been shown to respond faster at one or more levels than susceptible plants of the same species. The plant's antioxidant defense systems are involved at several distinct levels in plant-pathogen interactions. First, they respond directly to oxygen radicals produced by the plant itself in response to the attacking fungus. Next, antioxidant enzymes are involved in protective mechanisms such as H202mediated cell wall lignification.
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Box 1. Defenses against activated oxygen The evolution of organisms that use molecular oxygen (02) as a terminal electron acceptor for respiration (i.e. aerobic organisms) gave rise to a paradox. On the one hand, aerobic metabolism provides a much higher energy yield than anaerobic respiration, giving the aerobic organism a huge competitive advantage. On the other hand, the respiratory reduction of 02 to H20 leads to the stepwise formation of reactive oxygen intermediates35.36, the superoxide radical (-02-), hydrogen peroxide (H202) and the hydroxyl radical (.OH). In the presence of light, photosensitizer-mediated production of the reactive singlet form of oxygen (10 2) may also occur. 10 2
02
+e-
+e-
~ "02-
----"
2
Overall reaction :
H202
H+
+e-
---~
+e-
"OH
H"
H+
02 + 4 e- + 4 H*
-
H20
-~ 2 H20
-
Essentially the opposite process, the four-electron oxidation of water and the associated formation of oxygen radicals, occurs during photosynthesis. Normally these highly reactive species remain bound during the oxidation/ reduction reactions involved in respiration and photosynthesis. Occasionally, however, they are released and unless dealt with immediately are capable of causing extensive cell damage36.3L Of even more devastating effect,-02and H202 act as substrates for the transition-metal-catalysed Haber-Weiss reaction ~.39. One of the products of this reaction, the hydroxyl radical (.OH), is the most reactive of the oxygen radicals, and can directly and indiscriminately attack lipids, proteins and DNA. Activated oxygen species are also produced by normal metabolic processes and in response to various environmental stresses. The superoxide radical (-02-), for example, is generated by various enzymatic reactions in the cell (e.g. uricase and xanthine oxidase). Hydrogen peroxide may be produced by the dismutation reaction of two superoxide anions, by the ~-oxidation of fatty acids in the course of normal lipid metabolism and in response to stress. The production of activated oxygen species in plant tissues in response to pathogen attack is also well documented 4,5. Finally, at least one fungal species, Cercospora, produces activated oxygen species directly, as a means of disrupting host tissues. Disruptive effects of these activated oxygen species include direct inhibition by H202 of enzymes involved in C02 fixation4°.41, and peroxidation of membrane lipids. Damage to intracellular membranes can lead to leakage of cellular contents, loss of organellar function and cell death. To minimize the damaging effects of activated oxygen species, of both external and internal origin, aerobic organisms have evolved both enzymatic and nonenzymatic antioxidant defense systems. Nonenzymatic systems include compounds with intrinsic antioxidant properties such as vitamins C and E, glutathione and l~carotene42.43.These are often linked with enzymes such as ascorbate peroxidase (AP) and glutathione-reductase (GR), which regenerate the antioxidant capacity of the quenching molecule. Purely enzymatic systems, such as the catalases (CAT), peroxidases (Px) and superoxide dismutases (SOD), protect the organism by directly scavenging superoxide radicals and hydrogen peroxide and converting them to less reactive species: •02 + "02 + 2H + SOD 02 + H202
2H202 CAT 2H20 + 02
H202+R(OH)2 Px ) 2H20+R(O)2
Since the rate constants of reactions involving .OH radical attack are as fast as the fastest reactions catalysed by the antioxidant enzymes such as SOD, no specific enzyme systems are known that directly scavenge these reactive oxygen species. It is hypothesized that, instead, the superoxide dismutases, coupled with the catalases and peroxidases, act to limit the availability of substrate for the reactions leading to the formation of the hydroxyl radical.
Third, they respond to stressinduced changes in the plant's metabolism that lead to increased production of oxygen radicals. Finally, antioxidant defense systems may respond directly to activated oxygen species produced by photosensitizing fungal toxins. While we allude to a number of these responses as a means of
TRENDS
IN
emphasizing the layered nature of the plant's response to pathogen attack, this review will focus primarily on recent investigations on antioxidant enzyme induction by fungal pathogens, and the specific response of plant antioxidant defense systems to the best characterized of the fungal photosensitizers, cercosporin.
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Response of plant peroxidases to fungal attack
The plant peroxidases catalyse the donor-mediated reduction of H202 to HzO and oxidized donor (see Box 1). The biochemistry and cell biology of this complex family of enzymes has been reviewed elsewhere 6. Peroxidases are found in organelles and in the cytoplasm,
SEPTEMBFA~.
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O P I N I O N
hip
Fe3Fe2
LH
o I 02
L Fe2F 3
OH H2O2--o2 0 o.
H
OH"
H20
•L
0
LOOH
LO'O
"S-
LOiH
15
"L
O2
I
I
Type I
Type II
Fig. 1. Lipid peroxidation by Type I and Type II photosensitizers (see Ref. 13). Absorption of a photon (hv) by the ground state photosensitizer (°S) yields the lowest excited singlet state (1S) followed by conversion to the less energetic triplet state (aS). Type I reaction initiation by hydrogen transfer from an unsaturated lipid substrate (LH) leads to the production of the lipid alkyl radical (.L), the sensitizer radical anion (-S) and H'. Subsequent propagation of lipid radicals (4_), lipid peroxides (LOOH) and lipid peroxyl radicals (LO-O) is illustrated. In the presence of free oxygen, autoxidation of .S- generates the superoxide radical (-O2-). This also leads to lipid radical formation by the pathway indicated. In Type II reactions singlet oxygen 002) is formed by direct energy transfer from sS to Or. Singlet oxygen reacts directly with unsaturated lipids (LH) to produce lipid hydroperoxides (LOOH). Initiation of radical proliferation via a metal-catalysed, Haber-Weiss type reaction to form lipid oxyl radical (L-O) is depicted.
as well as extracellularly, and have been widely implicated in the defense response of plants to fungal attack and stress. The increased expression of extracellular anionic peroxidases is perhaps the best studied fungaiinduced antioxidant gene response. These peroxidases are involved in cell wall lignification and suberization 6,7, and are thought to protect the plant by forming a physical barrier to pathogen ingress. It has been shown, for instance, that anionic peroxidases localized in the cell walls of suberizing potato and tomato cells are induced by fungal attack, wounding and abscisic acidS,L In resistant lines of tomato, expression of one of these peroxidases was induced earlier than in susceptible lines (24 versus 48 h after inoculation) '°. Induction of a wheat anionic peroxidase by fungal extracts has also been demonstrated ~t. Transcription of the wheat anionic peroxidase gene was induced about 3 h after inoculation, and intercellular peroxidase activity increased between 6 and 36 h after inoculation. In tomato at lcast three anionic peroxidases appear to be involved in cell wall suberization. In wound
healing studies using transgenic plants expressing antisense RNA to block the expression of two tomato anionic peroxidases, cell wall suberization was not measurably inhibited '2. Evaluating the ability of these transgenic plants to withstand pathogen attack will be illuminating. Cell wall lignification is a relatively slow response and other protective mechanisms must be deployed during the first hours of attack. Furthermore, there must be a source for the H,_O2 used by peroxidases as a substrate. The pathogen-induced production of activated oxygen species by plant tissues is well documented 4, with extracellular UzO2, presumably produced by an outer membrane oxido-reductase, appearing during the first minutes of pathogen attack s. It has been hypothesized that this response may serve an immediate antimicrobial role, as well as providing substrate for later peroxidase-mediated cell wall lignification. It may also account, at least in part, for the induction of cytosolic peroxidases, catalases and superoxide dismutases by non-cercosporin-containing fungal extracts (see below).
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Photosensltizlng fungal toxins Photosensitizers are a structurally diverse group of compounds with a common mechanism of action. When activated by light, the photosensitizer molecule is converted to an electronically excited triplet state, which can react with 02 to form activated oxygen species (Fig. 1). This reaction may be mediated through a reducing substrate to form the reactive oxygen species H202, "O2-, or .OH (Type I reaction), or may take the form of a direct energy transfer resulting in the formation of singlet oxygen (~O2, Type II reactionp s. Many plant pathogenic fungi are known to produce photosensitizers that presumably act as phytomxins ~4. The best studied of these is the light-induced polykedde toxin cercosporin produced by members of the genus Cercospora. Cercosporin itself does no damage. However, activated oxygen species produced by cercosporin in the presence of light are directly and indiscriminately toxic to living tissues, causing oxidation of lipids, proteins, carbohydrates and nucleic acids (Figs 1 and 2). Because of the nonspecific nature of the damage caused by activated
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O P I N I O N
"02- ~ Polyketide biosynthetic -7__ hv pathway
Plant cell
i02+
Excited triplet state
Membrane damage
02
/ Cercosporin Benefits to fungus (1) leakage of nutrients (2) physical access
Y,
Cell death
Fig. 2. In the presence of light (459-490 nm) cercosporin synthesis is induced and an excited form (triplet state) of the toxin molecule is generated. Depending on the reducing potential of the environment (see Fig. 1), this excited form reacts with 02 to produce singlet oxygen (102) and/or superoxide radicals (-Or-). These activated oxygen species cause rapid breakdown of plant cell membranes by lipid peroxidation, leading to leakage of cell components and cell death.
oxygen, a successful defense strategy must protect at several levels and, as with most plant responses, the specific responses of individual plant species to Cercospora and cercosporin are quite diverse. Cercosporin resistance in crop plants Many important crop species display little or no resistance to Cercospora infection. Although there have been numerous efforts to use biotechnology to produce plants that display general resistance to oxidative stress (ozone, etc.) and specific resistance to cercosporin, no single factor seems unambiguously to provide such universal protection against oxidative stress 1~-'7. The inescapable conclusion is that no single factor or gene protects the plant against all types of environmentally and/or metabolically generated oxidative stress. As with many multifactor traits, more has been achieved by traditional breeding, and cultivars of several crop species have been produced that show significant levels of Cercospora resistance (maize TM, rice 19 and sugarbeet2°). Cultivars of rice (Oryza sativa) with high levels of resistance to Cercospora oryzae have been grown commercially since the 1940s. In a recent study 19, rice varieties showing varying degrees of resistance to C. oryzae were examined. Resistance ranges from almost complete in a wild variety, Louisiana red rice, to only 2% in the com-
mercial variety Labelle. Because of the broad toxicity of cercosporin, it was felt that resistance to Cercospora was most likely based on resistance to the toxin itself. Sensitivity to the purified toxin was, therefore, assessed by inhibition of callus and seedling growth, chlorosis and necrosis in leaves, and ion leakage in cell suspension culture. By all measures, the susceptible cultivar Labelle was the most sensitive to cercosporin damage, while the resistant red rice was the least sensitive (others were intermediate). None of the tested varieties showed any reaction to the toxin in the dark, consistent with cercosporin's mode of action as a photosensitizer. Measurement of the cercosporin content of cells grown in suspension culture in the presence of cercosporin revealed that the resistant red rice cells contain less than one-tenth the level of cercosporin present in cells of the sensitive cultivar Labelle. The authors proposed that these differences might be due to (1) differential exclusion or uptake, (2) active export or (3) detoxification of cercosporin (or a combination of the three). Red rice also contained approximately twice the total carotenoid level of Labelle. Lutein and 13-carotene were present in both varieties, but red rice also contained several carotenoids absent in Labelle. Inhibition of carotenoid biosynthesis resulted in the loss of cercosporin resistance
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in red rice suspension cells. This is possibly because carotenoids are potent quenchers of oxygen radicals. So, although exclusion or detoxification of cercosporin might be important, the ability to synthesize carotenoids also appears to be necessary for cercosporin resistance. Sugarbeet varieties (Beta vulgaris) showing different levels of resistance to Cercospora beticola have also been analysed 2°. In this work response to infection was characterized by an immediate increase in the level of cytosolic peroxidases. This increase was consistently higher in resistant than in susceptible varieties. Later in the defense response, orthodiphenol oxidase, an organellar (mitochondrial and chloroplastic) enzyme that produces oxidized phenolic compounds toxic to C. beticola, was induced at higher levels in resistant varieties. It was concluded that a successful resistance response involves multiple factors acting at different times with different effects. Because Cercospora infection is likely first to influence the cytoplasm of the cell, the cytoplasmic localization of the peroxidase and its ability to oxidize a wide variety of donor compounds suggest that it is used in the initial defensive responses of the cell to oxidize toxin-derived H202 and to stabilize stressinduced metabolic changes. Response of SOD and CAT to fungi and cercosporin While the superoxide dismutase (SOD) and catalase (CAT) isozyme systems have been examined to some extent in other plants 1s,21, they have been most thoroughly described in maize. In corn (Zea mays), at least six structural genes
(Sod1, Sod2, Sod3, Sod4, Sod4A and Sod5) encode corresponding SOD isozymesn. The various superoxide dismutases are precisely compartmentalized, with the Cu/Zncontaining SODs being localized in chloroplasts (SOD-l) or in the cytosol (SOD-2, 4, 4A and 5), and the Mn-containing SOD-3 in mitochondria2L This specific compartmentalization presumably reflects specific defensive function(s). The
199.3
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three biochemically distinct catalase isozymes, CAT-I, CAT-2 and CAT-3, are encoded by three unlinked structural genes, Catl, Cat2 and Cat3 (Refs 22, 24, 25). In mature green leaves of maize, CAT-2 is localized in the peroxisomes of bundle-sheath cells, while CAT-1 and CAT-3 are found primarily in mesophyll cells25. Both CAT-1 and CAT-2 isozymes are localized in glyoxysomes/peroxisomes where present, or, in tissues and mutants lacking these organelles, in the cytosol. In contrast, maize CAT-3 coisolates exclusively with the mitochondrial cell fraction 26. Very little has been reported on the response of the superoxide dismutases or catalases to fungal attack (or pathogen attack in general). In two cases, where SOD response to non-toxin-producing fungi has been reported, changes in SOD activity did not appear to be a measurable early response. The first study27 examined SOD and peroxidase activities in sensitive and resistant (hypersensitive) varieties of Phaseolus vulgaris inoculated with Uromyces phaseoli. As seen in previous reports (above), peroxidase activity increased earlier in infected resistant plants than in infected susceptible plants (18 versus 48 h). However, 48 h after inoculation neither Cu/Zn nor Mn SOD activity had changed measurably in either infected line. Only after the onset of fungal spore formation (4-8 d after infection) did the Cu/Zn SOD activity in both susceptible and resistant lines begin to increase while Mn SOD activity increased only in susceptible lines. Given the time-frame, these changes are not likely to be a primary response to fungal elicitors, but are perhaps a reflection of the relative levels of general metabolic stress in each case. The second study 28 reported that total SOD activity in blastinfected leaves of susceptible and resistant rice was not measurably different to that in uninfected plants. Changes in RNA levels were not examined in either study. This is in contrast to the report 2~ of increased Mn SOl) protein and
RNA accumulation in Nicotiana plumbaginifolia inoculated with a bacterial pathogen (Pseudomonas syringae). In inoculated plants the level of Mn Sod RNA had increased significantly by 6 h, although increased Mn SOD protein was not detected until 48 h. However, a complete comparison with expression in uninoculated plants was not presented. The phytotoxic effect of photosensitizers probably results from both the immediate destructive effects of activated oxygen species and the radicals arising from lipid peroxidation ~.~(Fig. 1). Fungal attack also induces plant production of oxygen radicals ~4. So, while induction of Cat and Sod gene expression may not be the plant's sole defense against fungal attack in general, and Cercospora in particular, it seems reasonable that these critical antioxidant defensive systems must constitute an important q9mponent. In this laboratory we have, therefore, begun to examine more closely the response of SOD and CAT to Cercospora and cercosporin, and to explore the possibility of using cercosporin as a means of controlled application of external oxidative stress. In two recent papers ~°'3~ we reported on several aspects of maize CAT and SOD response to extracts of light- and dark-grown Cercospora and to purified cercosporin. Because numerous environmental factors influence the rate and type of oxygen radicals produced by cercosporin ~2'~3, a controlled plate assay was used employing excised embryos incubated on simple growth medium supplemented with purified cercosporin. Maize cmbryos incubated in the presence of increasing levels of purified cercosporin showed a dose-dependent increase in total catalase activity, individual isozyme protein levels and corresponding Cat transcript accumulation. The rcsponse of the Cat gene products to increasing doses of purified cercosporin seen in this study, in fact, closely mimics their response in maize embryos to increasing doses of hydrogen peroxide2L However, the most intriguing observation was
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that, while both total SOD activity and individual SOD isozyme protein levels remained constant in all toxin treatments during the 18-24 h test period, transcript accumulation for all Sods changed dramatically. One plausible explanation for this contrast in CAT and SOD protein response is the difference in the relative toxicities of the respective enzyme substrates and the method maize has evolved to deal with each. Because the superoxide radical, and the products of its peroxidation of the lipid bilayer, are highly and immediately toxic to the cell, maximal constitutive levels of the appropriate SODs might be required for adequate protection. Hydrogen peroxide, however, is less immediately damaging to the cell. The time required for de novo induction of catalase expression might, therefore, bc adequate for defense against this relatively less toxic species. The observed increase in Sod transcript levels in the absence of increased SOD protein accumulation also suggests that protein turnover plays a role in the response of SODs to activated oxygen species. Active transfer of highenergy free electrons probably eventually destroys or inactivates the mediating protein(s). Therefore, under high levels of oxidative stress, high SOD protein turnover rates would require new synthesis to maintain adequate SOD isozyme levels. Since the cercosporin plate assay developed for these studies is ideal for both controlled application of oxidative stress and for in vivo pulse-chase labeling studies, it should bc simple to determine whether or not this type of selective turnover does indeed occur.
In a subsequent study n we showed that Cercospora produces compounds, other than cercosporin, that affect Cat and Sod transcript accumulation. The synthesis as well as the activation of cercosporin is light depcndent '4. Thus we were able to compare the effects of cercosporin- and non-cercosporin-containing fungal extracts (from light- and darkgrown Cercospora kikuchii mycelia, respectively) with that of
SH"I'I:MBI~I'. 1993
O P I N I O N
Table 1. Partial summary of antioxidant systems in higher plants" Non-enzymatic scavenging systems
Subcellular location
Type of active oxygen species
Source of active oxygenspecies
Enzymatic scavenging systems
Chloroplast
Superoxide
H202
Photosystem II Enzymatic
SOD Peroxidases
Mitochondria
Superoxide H202
Electron transport Enzymatic
SOD Peroxidase Catalase
H20~ H20 + oxidized donor H20 + 02
Cytosol
Superoxide H202
Enzymatic Enzymatic
SOD Catalase Peroxidase
H202 H20 + 02 H20 + oxidized donor
Glyoxysomes Peroxisomes
H202 H202
13-0xidation Photorespiration
Catalase Catalase
H20 + 02 H20 + 02
Extracellular
Superoxide
Enzymatic Enzymatic
None known Peroxidase
None known Lignin, suberin, hydroxyproline
H202
Products
H202 Glutathione, NADP÷, d ihydroascorbate
Ferredoxin, carotenoids, xanthophylls
"Reproduced from Ref. 35. SOD, superoxide dismutase.
purified cercosporin on the accumulation of Cat and Sod transcripts. Extracts from fungi grown in the dark (i.e. containing no cercosporin) elicited distinct changes in accumulation for several of the Cat and Sod transcripts. The response of Cat and Sod expression to extracts from light-grown Cercospora (i.e. containing cercosporin) appeared to be a combined response to purified cercosporin and one or more fungal compounds presumably present in both light- and dark-grown fungal culture extracts. Under the conditions used in these two studies, all of the Cat and Sod transcripts respond to purified cercosporin and cercosporin-containing fungal extracts. On the other hand, only two of the cytosolic (Cu/Zn) Sods (Sod2 and 4), the glyoxysomal/ peroxisomal Cat2 and mitochondrial coisolating Cat3 transcripts respond to non-cercosporin extracts. This might be a direct response to fungal elicitor(s) 2 or a response to oxygen radicals produced as part of the oxidative burst 4,~. It is, however, not yet possible unambiguously to attribute specific protective roles to the individual isozymes. Further manipulation of the experimental
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parameters (e.g. light intensity, toxin concentration and developmental stage) should be revealing. In these same studies we found that the catalases also show stagespecific changes in response to cercosporin-containing fungal extracts~L In the scutella of developing (immature) embryos, increasing levels of cercosporin result in decreased CAT-2 accumulation, while in the scutella of germinating (mature) embryos, CAT-2 isozyme levels increase. In addition, CAT-1 levels in developing embryos increase in response to fungal extracts regardless of cercosporin content, while in mature germinating embryos there is no marked response to either. In separate studies we observed that the response of the catalases to the plant growth regulator abscisic acid was different in developing and germinating maize embryos 34. Embryogenesis and germination represent two distinct, well-defined, alternative pathways in developmental programing. Different responses to the same treatment at these two stages may, therefore, reflect not only the profound changes in gene expression that occur when embryos undergo the developmental shift from embryo
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maturation to germination, but also the specific functions of these genes in cellular responses to oxidative stress. Concluding remarks
Individual plant stress responses do not occur in isolation, but are components of an integrated defense, with each part being necessary, but often insufficient by itself, to provide comprehensive protection. The antioxidant defense enzymes are a good example of the biochemical complexity of one element of the plant's response to environmental stressors (Table 1)36. While animals have a single catalase, most plants examined have three biochemically distinct and differently compartmentalized catalases. The plant SODs are even more complex, while the complexity of the peroxidases is stunning. This complexity is perhaps a reflection of the highly reactive nature of oxygen radicals. Superoxide, for instance, reacts on contact with cellular structures with immediate destruction of the affected molecule and the generation of additional radicals. The plant's defense response must, therefore, be appropriate and instantaneous. This means having
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maximal levels of SODs in exactly the right place before attack occurs. Hence, even though epigenetic expression of a mitochondrial SOD might increase resistance to paraquat, it wouldn't protect against cytosolic radicals. IJkewise, overexpression of a cytosolic CAT would not enhance resistance to photosensitizer-derived extracellular superoxide. Available information, therefore, suggests that efforts to engineer oxidative stress tolerant plants should perhaps focus less on overexpressing individual Cat or Sod genes and more on augmenting the balanced, coordinated expression of all the essential antioxidant enzymes in the various cell compartments. To maintain the critical equilibrium between the generation and removal of superoxide radicals, organisms have evolved elaborate regulatory mechanisms to control the synthesis, accumulation and compartmentalization of the appropriate SODs. At present, however, our knowledge of these underlying cellular and molecular mechanisms governing mobilization of antioxidant defenses is meager. Understanding the basis of the differential responses of the different SOD and CAT isozymes to given stressors or signals and their specific spatial and temporal compartmentalization will clarify the specific functions of multiple forms of these enzymes. The identification and characterization of cis-acting elements and associated transacting factors involved in the regulation of these genes will increase our understanding of the signal transduction pathway for the oxidative stress response and further our efforts to engineer organisms with higher resistance to oxidative stress. Molecular analysis of Sod and Cat gene/promoter structure and expression together with mutant and physiological analyses should help us unravel the mechanisms controlling this finely tuned system and thereby show us where changes will enhance plant stress resistance. Acknowledgements We gratefullyacknowledgeour colleaguesin the laboratoryfor theircontributionsoverthe
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years. We thank M. Dauband M. Ehrenshaft for an advancecopy of a reviewand helpful commentson the manuscript,and S. Ruzsafor assistancewith figurepreparation. References 1 Bowles,D.J. (1990) Annu. Rev. Biochem. 59, 873-907 2 Dixon,R.A.and Harrison,M.J. (1990) Adv. Genet. 28, 165-234 3 Dixon,R.A.and Lamb,C.J. (1990) Annu. Rev. Plant Physiol. Plant Mol. Biol. 41,339-367 4 Sutherland,M.W. (1991) Physiol. Mol. Plant Pathol. 39, 79-93 5 Apostol,I., Heinstein,P.F.and Low,P.S. (1989) Plant Physiol. 90, 109-116 6 Van-Huystee,R.B.(1987) Annu. Rev. Plant Physiol. 38,205-257 7 Kolattukudy,P.E. (1984) Can. ]. Bot. 62, 2918-2933 8 Roberts,E., Kutchan,T. and Kolattukudy,P.E. (1988) Plant Mol. Biol. 11, 15-26 9 Roberts,E. and Kolattukudy,P.E. (1989) Mol. Gen. Genet. 217, 223-232 10 Kolattukudy,P.E., Podila,G.K.and Mohan, R. (1989) Genome 31, 342-349 11 Sclrweizer,P., Hunziker,W. and Mosinger, E. (1989) Plant Mol. Biol. 12, 643-654 12 Sherf,B.A.,Bajar,A.M.and Kolattukudy,P.E. (1993) Plant Physiol. 101,201-208 13 Girotti,A.W.(1990) Photochem. Photobiol. 51,497-509 14 Daub, M. and Ehrenshaft,M. Physiol. Plant. (in press) 15 Bowler,C., Van Montagu,M. and lnze', D. (1992) Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 86-116 16 Furusawa,I. et al. (1984) Plant Cell Physiol. 25, 1247-1254 17 Tanaka, K. and Sugihara,K. (1980) Plant Cell Physiol. 21, 601-611 18 Malan,C., Greyling,M.M. and Gressel, J. (1990) Plant Sci. 69, 157-166 19 Batchvarova,R.B.,Reddy,V.S.and Bennett,J. (1992) Phytopathology 82, 642-646 20 Rautela,G.S.and Payne,M.G. (1970) Phytopathology 60, 239-245 21 Havir,E.A.and McHale,N.A. (1989)
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