Plant Physiol. (1998) 117: 1103-1114

Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves

Hélène Vanacker, Tim L.W. Carver, and Christine H. Foyer^*

Department of Environmental Biology, Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, United Kingdom SY23 3EB

	ABSTRACT

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Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity.

	INTRODUCTION

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The mechanisms by which plant cells sense the presence of foreign organisms and transduce this information to the nucleus to elicit an appropriate response are largely unresolved. Appropriate contacts with useful organisms, such as mycorrhizal fungi or N₂-fixing bacteria, must evoke a rapid response to establish liaisons that facilitate mutual benefit. In contrast, identification of a harmful pathogen must cause adaptive responses that prevent the spread and limit the sustainability of the invasive agent. In recent years it has become clear that redox signals are integral to the transduction sequences by which changes in the environment modify metabolism and gene transcription.

The plasmalemma of plant cells produces bursts of H₂O₂ in response to both biotic and abiotic stimuli (Doke et al., 1994; Doke, 1997; Wojtaszek, 1997). The mechanisms involved in the regulation of the initiation, intensity, and duration of these bursts are largely unknown but it is clear that plants, like animals, use active oxygen species such as H₂O₂ to perturb the redox state of cells and allow controlled oxidation that may have an immediate antimicrobial effect (Segal and Abo, 1993; Groom et al., 1996; Lamb and Dixon, 1997). The damage caused to the metabolism and delicate fabric of the plant cells per se depends on the efficiency of the endogenous antioxidant defense system (Fig. 1). Although plant cells contain glutathione peroxidases similar to those present in animal cells, these enzymes in plants appear to catalyze the glutathione-dependent destruction of lipid peroxides rather than H₂O₂ (Eshdat et al., 1997; Roxas et al., 1997). In plants H₂O₂ is destroyed predominantly by APXs and catalases (Asada, 1997; Willekens et al., 1997). Although the catalases are restricted to the peroxisomes, and perhaps mitochondria, APXs have been found in every compartment of the plant cell in which they have been sought (Asada, 1997; Jiménez et al., 1997). Confirmation of the role of redox signals in the elicitation of defense reactions has come from studies on transformed plants; for example tobacco antisense transformants containing only 10% of the foliar catalase activity of the untransformed controls show enhanced expression of defense-related proteins and increased accumulation of the antioxidant glutathione (Chamnongpol et al., 1996).

View larger version (51K): [in this window] [in a new window]   Figure 1. Reduction of H2O2 by the ascorbate-glutathione cycle.

Incompatible plant-pathogen interactions lead to the phenomenon known as programmed cell death (Jones and Dangl, 1996; Mittler and Lam, 1996). Activation of programmed cell death during HR results in the formation of a zone of dead cells around the infection site. H₂O₂ produced around the developing papillae and surrounding halos of barley (Hordeum vulgare L.) leaves infected with the powdery mildew fungus (Thordal-Christensen et al., 1997) is considered to be involved in the orchestration of HR (Levine et al., 1994; Mehdy, 1994).

The production of H₂O₂ during the oxidative burst is involved in the integration of cellular processes and the adaptation to environmental stimuli. First, it leads to rapid cell wall reinforcement because it is involved in oxidative cross-linking (Bradley et al., 1992) and insolubilization of Hyp-rich proteins (Bradley et al., 1992; Wojtaszek et al., 1995; Otte and Barz, 1996). Second, it causes release of calcium into the cellular matrix, which may be central to the signal transduction process (Price et al., 1994). Third, it alters the concentrations and redox status of intracellular antioxidants, such as ascorbate and glutathione, which are also signal-transducing molecules (Foyer et al., 1997). Fourth, it is implicated in the induction of defense genes (Wu et al., 1997). Stimuli that induce defense-gene expression cause a reciprocal repression of cell-cycle-related genes (Logemann et al., 1995). The cellular redox state is a critical factor in the regulation of the G1/S transition in the eukaryotic cell cycle (Russo et al., 1995). The ability to reduce cell division under stress conditions allows conservation of energy and reduces the risk of heritable damage.

Sustained H₂O₂ production by the plasmalemma appears to be an integral feature of incompatible plant-pathogen interactions (Chai and Doke, 1987; Baker et al., 1991, 1993; Levine et al., 1994). The measurement of H₂O₂ production in whole-plant tissues or organs in situ is fraught with technical difficulties. However, using various cytochemical detection techniques, prolonged bursts of H₂O₂ production have been measured between 5 and 8 h after inoculation of lettuce by the phytopathogenic bacterium Pseudomonas syringae (Bestwick et al., 1997). Similarly, in barley inoculated with Blumeria graminis (originally called Erysiphe graminis), H₂O₂ production was detected in the epidermal cell wall adjacent to the mesophyll cells and subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium after 15 h (Thordal-Christensen et al., 1997). In contrast, accumulation of H₂O₂ was not detected in bean leaf discs infected with Botrytis cinerea until 48 h after inoculation, when the infection was spreading rapidly (Bestwick et al., 1997).

The oxidative burst is considered to produce such large quantities of H₂O₂ that the antioxidative defenses of the cell are overwhelmed at least temporarily (Lamb and Dixon, 1997; Wojtaszek, 1997). An early response to H₂O₂ addition, however, is the transcription of enzymes involved in antioxidant defense. Induction of glutathione S-transferase gene expression, for example, has been observed 30 to 60 min after addition of exogenous H₂O₂ to soybean cell cultures (Levine et al., 1994). The time required to modify gene expression in isolated cell cultures in response to H₂O₂ addition or pathogen infection may be quite different, however, from that in intact tissues and organs under pathogen attack. In tobacco leaves inoculated with tobacco mosaic virus, increases in antioxidant capacity were observed only after the onset of necrosis (Fodor et al., 1997). The temporal distribution of responses and the dynamic changes in the concentrations of active oxygen species within the cellular and extracellular compartments of intact tissues are key factors governing the outcome of plant-pathogen interactions (Tiedemann, 1997).

In leaves fungal infections have been shown to induce different components of the ascorbate-glutathione cycle (Fig. 1) and other antioxidant defenses (Gönner and Schlösser, 1993; El-Zahaby et al., 1995; Fodor et al., 1997). HR increases transcription of specific SOD and catalase genes, in addition to glutathione S-transferases and glutathione peroxidases, to ensure that maximal protection is maintained within the appropriate cellular compartments (Bowler et al., 1994). Glutathione, a major low-molecular-weight thiol in plant cells, is increased after pathogen infection (Edwards et al., 1991). Exogenous application of GSH increased Phe ammonia-lyase and chalcone synthase transcripts in bean cell suspensions (Wingate et al., 1988). In addition, GSH-responsive elements on the promoters of these genes have been identified (Dron et al., 1988). GSH was found to accumulate in bean and alfalfa cell-suspension cultures treated with fungal elicitor (Edwards et al., 1991) and in elicitor-treated liverwort cells (Nakagawara et al., 1993). In carrot inhibition of glutathione synthesis triggers phytoalexin accumulation, whereas the addition of H₂O₂ mimics this response (Guo et al., 1993).

B. graminis D.C. f. sp. hordei Marchal is a biotrophic fungal pathogen that causes barley powdery mildew. Recently, the processes of B. graminis development and host-cell response to attack have been reviewed (Aist and Bushnell, 1991; Carver et al., 1995). Epidermal cells of barley and other cereals attacked by appressoria of B. graminis may express two different forms of response correlating to resistance that prevent establishment of a biotrophic relationship: (a) infection is arrested before penetration of the attacked cells, and this is correlated to the deposition of papillae (wall appositions) by the living cell (Carver et al., 1996); (b) the host cell dies (HR), thus preventing establishment of biotrophy, and autofluorogens accumulate throughout the dead cell (Fig. 2) (Koga et al., 1990). In the present study two near-isogenic lines of barley, developed by J.G. Moseman (Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD) and differing at the Mla locus, were used to study the antioxidant response to pathogen attack 24 h after inoculation. Algerian/4* (F14) Man. (R) (hereafter referred to as Alg-R) has race-specific resistance to B. graminis conferred by the dominant Mla1 allele, and associated with hypersensitive cell death (HR) (Johnson et al., 1979; Zeyen and Bushnell, 1979; Koga et al., 1990; Hippe-Sanwald et al., 1992). Algerian/4* (F14) Man. (S) (hereafter referred to as Alg-S) carries the recessive mla1 allele for susceptibility to B. graminis. In Alg-R leaves about 50% of attacked cells express HR and show whole-cell autofluorescence as a result of attack by an avirulent fungal isolate (Zeyen et al., 1995). By contrast, Alg-S shows cell death at a very low frequency (about 1% of attacked cells).

View larger version (98K): [in this window] [in a new window]   Figure 2. Incident fluorescence micrograph of a B. graminis f. sp. hordei germling attacking Alg-R barley 24 h after inoculation. The germling produced a primary germ tube (PGT) and an appressorial germ tube (A). The epidermal cell attacked by the appressorium shows intense whole-cell autofluorescence (E+) indicative of cell death typical of the HR.

Previous studies with B. graminis infection of barley have demonstrated that maximum induction of genes coding for peroxidases, pathogenesis-related proteins, and enzymes associated with phytoalexin production occurred 24 h after inoculation (Boyd et al., 1994; Clark et al., 1994). To determine whether the induction of defense responses was associated with sustained oxidation, the oxidation states of the ascorbate and glutathione pools of whole barley leaves were examined 24 h after inoculation and compared with those of uninoculated leaves. Because the apoplast is the site of H₂O₂ production during the oxidative burst, our principle objective was to determine pathogen-induced changes in the antioxidant status of the apoplast. We examined the distribution of glutathione and ascorbate and associated antioxidant enzymes in whole-leaf and apoplastic extracts from inoculated leaves of resistant and susceptible barley. We documented pathogen-induced changes in the activities of antioxidant enzymes and the total pool sizes of ascorbate and glutathione and their degree of oxidation in the cellular and extracellular compartments of barley leaves 24 h after inoculation, when the infection peg had emerged from beneath the appressorium and attempted to penetrate the host epidermal cells. No extensive oxidation of either the foliar ascorbate or glutathione pool was observed, however, suggesting that H₂O₂ produced as a result of the plant-pathogen interaction was efficiently destroyed either during or before the 24-h time point.

	MATERIALS AND METHODS

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Plant and Pathogen Material

2

1

Blumeria graminis f. sp. hordei, isolate CC1 (avirulent to Mla1), was maintained on susceptible barley (Hordeum vulgare L.) seedlings in a spore-proof greenhouse. One day before the inoculum was required for experimentation, heavily sporulating plants were shaken to remove older conidia and to ensure a supply of vigorous young spores for experimentation.

Inoculation and Incubation of Experimental Material

2

Fixation and Clearing of Leaf Tissue for Microscopy

Extraction of Soluble Apoplastic Components (EWF)

Between 1.1 and 1.2 mL of EWF was obtained from 5 g of leaves (fresh weight). EWF was used immediately after isolation for the determination of enzymic activities or for metabolite measurements. In all cases the samples were kept at 4°C until assay.

Extraction of Enzymes from Whole Leaves

Extraction of Metabolites from Whole Leaves

Determination of Enzyme Activities

MDHAR was assayed at 340 nm by a modification of the method of Miyake and Asada (1992). Monodehydroascorbate was generated via the action of ascorbate oxidase (0.4 unit; 1 unit = 1 µmol of ascorbate oxidized per min) in a reaction mixture (1 mL) containing 100 mM Hepes/KOH (pH 7.6), 25 µM NADPH, 2.5 mM ascorbate, and 100 µL of extract.

DHAR was assayed in a reaction mixture (1 mL) consisting of 50 mM Hepes/KOH buffer (pH 7.0), 2.5 mM GSH, 0.2 mM DHA, 0.1 mM EDTA, and 10 to 50 µL of extract. Reaction rates were measured by monitoring the change in A₂₆₅ as ascorbate was generated (Miyake and Asada, 1992). DHAR activity was calculated using an extinction coefficient of 7.0 mM¹ cm¹.

APX was measured spectrophotometrically by a modification of the method of Nakano and Asada (1987). The reaction mixture (1 mL) contained 50 mM KH₂PO₄/K₂HPO₄ buffer (pH 7.0), 250 µM ascorbate, 1 mM H₂O₂, and 100 µL of extract. The decrease in A₂₉₀ was measured as ascorbate was oxidized. APX activity was calculated using an extinction coefficient of 2.8 mM¹ cm¹ for ascorbate at 290 nm.

SOD activity was measured as described by McCord and Fridovich (1969) by the inhibition of color formation at 560 nm in the presence of the extract in a reaction mixture (1 mL) containing 50 M Hepes/KOH buffer (pH 7.8), 0.5 mM EDTA buffer, 0.05 unit of xanthine oxidase, 0.5 mM nitroblue tetrazolium, and 4 mM xanthine. A concentration curve was produced for each sample to calculate activity.

Catalase was measured at 20°C in a liquid-phase O₂ electrode (Hansatech, Kings Lynn, UK). Total extractable catalase activity was measured via O₂ evolution in a reaction medium (1 mL) containing 100 mM Hepes/KOH (pH 7.4), 0.5 M H₂O₂, and 10 to 50 µL of extract (Clairborne, 1985).

Determination of Ascorbate and Glutathione

Statistical Analysis

	RESULTS

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Host Epidermal Cell Responses and B. graminis Development

The proportion of appressoria that formed haustoria was also quite different between Alg-R and Alg-S (Table I). In Alg-R, only approximately 3% had formed haustoria at 24 h, and this increased to only about 7% by 48 h. In Alg-S, 88% of appressoria had formed haustoria at 24 h, and this increased only slightly to 96% by 48 h. Thus, in both isolines success of attempted epidermal cell infection was largely determined by 24 h after inoculation.

Determination of Contamination in Apoplastic Extracts by Cytoplasmic Components and Calculation of Corrected Values for Apoplastic Components

Ascorbate and Glutathione Contents

View larger version (25K): [in this window] [in a new window]   Figure 3. The effect of powdery mildew attack on ascorbate (A and B) and glutathione (C and D) contents of barley leaves (A and C) and apoplastic extracts (B and D) 24 h after inoculation. Black bars, Inoculated leaves; white bars, noninoculated controls. Bars represent SE of means (n = 4, four repetitions from two independent experiments). *, **, and *** indicate values that differ significantly from the control at P < 0.05, P < 0.01, and P < 0.001, respectively. FW, Fresh weight.

Ascorbate

The apoplast contained 7% to 10% of the total leaf (oxidized plus reduced) ascorbate pool. In all cases the apoplastic ascorbate pool consisted almost entirely of DHA (Fig. 3B). To determinate whether this was the situation in vivo or whether ascorbate in the apoplast was oxidized upon extraction, recovery experiments were performed in which a known quantity of AA was infiltrated into the apoplast before extraction. DHA and AA were determined before and after extraction. After extraction, all of the added AA was recovered. Importantly, no DHA was found in the buffer after extraction. These results confirmed that AA was not oxidized during the extraction procedures. Furthermore, in the apoplastic extracts obtained from AA-infiltrated leaves, after correcting values for endogenous ascorbate, DHA was four times higher than AA, suggesting that AA was oxidized to DHA in the apoplast. This is in agreement with previous observations (Horemans, 1997).

The apoplast of inoculated barley leaves contained 7.0% ± 0.5% of the total leaf (oxidized plus reduced) ascorbate. This was slightly lower than in the apoplast of controls. After inoculation, a significant (P < 0.05) decrease (23%) in DHA was found in the apoplast of Alg-R but not in the apoplast of Alg-S (Fig. 3B).

Glutathione

Glutathione was a minor component of the apoplast of both lines (Fig. 3D). In noninoculated Alg-S and Alg-R, 2.05% and less than 1%, respectively, of the total glutathione pool was found in the apoplast. Both GSH and GSSG were found in the apoplast (Fig. 3D), but the percentage reduction state of the apoplastic pool was less than in whole leaves. Inoculation caused a significant (P < 0.001) increase (61%) of apoplastic GSH in Alg-R. By contrast, inoculation caused a significant (P < 0.01) decrease of both apoplastic GSH (42%) and GSSG (46%) in Alg-S.

Enzyme Activities

View larger version (18K): [in this window] [in a new window]   Figure 4. The effect of powdery mildew attack on SOD (A and B) and catalase (C and D) activities of barley leaves (A and C) and apoplastic extracts (B and D) 24 h after inoculation. Black bars, Inoculated leaves; white bars, noninoculated controls. Bars represent SE of means (n = 4, four repetitions from two independent experiments). *, **, and *** indicate values that differ significantly from the control at P < 0.05, P < 0.01, and P < 0.001, respectively. FW, Fresh weight.

Total SOD

Catalase

Less than 0.5% of the total catalase activity was found in the apoplast of noninoculated leaves (Fig. 4D). Inoculation caused a significant (P < 0.001) increase in apoplastic catalase activity in Alg-R (900%) and in Alg-S (2230%) (Fig. 4D). The apoplast of inoculated Alg-R and Alg-S leaves contained 2.52% and 1.31%, respectively, of the total leaf catalase activity.

APX

View larger version (18K): [in this window] [in a new window]   Figure 5. The effect of powdery mildew attack on APX (A and B) and GR (C and D) activities of barley leaves (A and C) and apoplastic extracts (B and D) 24 h after inoculation. Black bars, Inoculated leaves; white bars, noninoculated controls. Bars represent SE of means (n = 4, four repetitions from two independent experiments). *, **, and *** indicate values that differ significantly from the control at P < 0.05, P < 0.01, and P < 0.001, respectively. FW, Fresh weight.

GR

The apoplast of noninoculated leaves contained 0.5% (Alg-R) and 1.4% (Alg-S) of the total foliar APX activity. No significant change was found in apoplastic GR activity in either line after inoculation (Fig. 5D). As a consequence of the large inoculation-dependent decrease in total foliar GR in Alg-R, the percentage of GR in the apoplast of Alg-R increased by 3% but no change was observed in Alg-S.

MDHAR

View larger version (18K): [in this window] [in a new window]   Figure 6. The effect of powdery mildew attack on MDHAR (A and B) and DHAR (C and D) activities of barley leaves (A and C) and apoplastic extracts (B and D) 24 h after inoculation. Black bars, Inoculated leaves; white bars, noninoculated controls. Bars represent SE of means (n = 4, four repetitions from two independent experiments). *, **, and *** indicate values that differ significantly from the control at P < 0.05, P < 0.01, and P < 0.001, respectively. FW, Fresh weight.

DHAR

	DISCUSSION

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The development of B. graminis and host epidermal responses, shown in Figure 2 and Table I, were comparable with those observed in previous studies of Alg-R and Alg-S (Carver et al., 1994; Zeyen et al., 1995). Conidia of B. graminis germinate within 1 h of inoculation, producing first a short, aseptate primary germ tube, which attaches to the host epidermal cell surface and engenders localized host-cell responses (approximately 1-6 h). A second germ tube emerges (about 2-4 h), elongates, and differentiates a specialized infection structure, the appressorium (approximately 8-10 h). An infection peg emerges from beneath the appressorium and attempts to penetrate the host epidermal cell (about 12-15 h). If penetration succeeds, a feeding structure, the haustorium, is formed within the epidermal cell (approximately 15-18 h) and absorbs nutrients from the living host cell to supply the developing colony. The response of Alg-R to attack by the avirulent B. graminis isolate was extreme, leading to a very high frequency of epidermal cell HR (Table I). By contrast, in Alg-S cell death was extremely infrequent. The proportion of HR cells did not increase between 24 and 48 h, indicating that the majority of important plant cell responses were under way or accomplished by the time the biochemical assays were performed. The antioxidant status of the whole leaves and the leaf apoplast, therefore, were determined 24 h after inoculation, i.e. when near-maximal HR had been achieved (Table I). At this time the plasmalemma was no more "leaky" in any of the inoculated leaves than in the healthy leaves, as indicated by the low contamination (less than 2%) of the apoplast by the cytoplasmic marker (G6PDH) in EWF extracts from either Alg-S or Alg-R leaves. The changes in antioxidant status observed in this study, therefore, are a consequence of the reaction of the plant to penetration by the fungus.

The pathogen-induced responses observed in this study result not only from phenomena occurring in cells undergoing the HR, but also from those occurring in surrounding tissue alerted by signals derived from the cells undergoing the HR. No inoculation-dependent decreases in either the AA-to-DHA or GSH-to-GSSG ratios were observed in either the resistant or the susceptible line. The AA-to-DHA ratio was increased in Alg-S leaves 24 h after inoculation. This suggests that if general oxidation of the mesophyll tissues, attributable to H₂O₂ generation, had occurred, it was transient and reversed by 24 h after inoculation in both lines.

The total ascorbate pool size was decreased in inoculated Alg-R leaves relative to controls, suggesting that a change in turnover had occurred in the resistant isoline. This was not observed in the susceptible isoline. Catalase activity increased and GSH accumulation occurred after inoculation with B. graminis. Clear differences in the responses of these antioxidants to attack were observed in the resistant and susceptible lines. Total extractable catalase activity had dramatically increased (by about 400%) 24 h after inoculation in Alg-S, whereas catalase activity was unchanged in Alg-R. This suggests that there is an inverse relationship between catalase induction in barley leaves and resistance to B. graminis. Although the temporal sequence of events cannot be deduced from the present studies, it is interesting to note that H₂O₂-induced oxidation was found to cause expression of pathogenesis-related genes in transformed tobacco plants deficient in the major catalase isoform, Cat 1 (Chamnongpol et al., 1996). H₂O₂ per se did not accumulate in these transformants, but the total glutathione pool increased, and there was a strong decrease in the GSH-to-GSSG ratio (Willekens et al., 1997). In the present study the induction of catalase activity in the susceptible line may have occurred too late in the response sequence to afford protection. Alternatively, catalase induction may have limited signal transduction by effectively removing H₂O₂ as it was formed.

No inoculation-dependent increases in the activities of any of the other antioxidant enzymes were observed. Foliar APX activity decreased in Alg-R but not in Alg-S after inoculation, whereas DHAR activity was unchanged. Slight changes in APX and DHAR activities have been observed in susceptible barley lines 4 d after inoculation (El-Zahaby et al., 1995).

Foliar glutathione was increased in Alg-R but not in Alg-S after inoculation, indicating a positive relationship between glutathione accumulation and pathogen resistance, as has been observed previously (Dron et al., 1988; Wingate et al., 1988; Edwards et al., 1991). Glutathione synthesized in leaf cells is transported throughout the plant (Noctor et al., 1997a, 1997b) and therefore has been identified as a putative long-distance signaling molecule (Foyer et al., 1997). Glutathione accumulation must occur after engagement of Ca²⁺-dependent signal transduction pathways, such as have been described after H₂O₂ addition to aquorin-containing cells (Price et al., 1994). Differential antioxidant deployment, however, between Alg-R and Alg-S may be central to resistance strategies. Foliar APX and GR activities had decreased (62% and 80%, respectively) in Alg-R but not in Alg-S 24 h after inoculation. The GSH-to-GSSG ratio was high in both Alg-R and Alg-S at this time, but a decrease in GR activity could sensitize the system to later bursts of H₂O₂ production to allow perturbations in the GSH-to-GSSG ratio.

There has been an upsurge of interest in the antioxidant defenses of the apoplast in recent years as the importance of this compartment has become apparent (Penal and Castillo, 1991; Arrigoni, 1994; Luwe, 1996). Because ascorbate is a substrate for cell wall peroxidases, it may play a role in the regulation of cell wall lignification, particularly during the HR, through its capacity to inhibit the oxidation of phenolic compounds by peroxidases (Takahama and Oniki, 1992; de Cabo et al., 1996; Mehlhorn et al., 1996). The pathogen-induced increase in the peroxidase activity of the cell wall would be effective only in the absence of AA. In the barley leaves used in the present study, which were obtained from 9-d-old seedlings, only DHA was found in the apoplast. Similar results have been obtained with dicotyledonous leaves during the early stages of development (Luwe, 1996). The absence of AA from the apoplast may reflect the requirement for efficient functioning of cell wall peroxidases during leaf expansion. A strong plasmalemma-bound, ascorbate-oxidizing activity has been detected (Horemans et al., 1997).

Apoplastic DHA decreased after inoculation in Alg-R but not in Alg-S. This may suggest that more rapid import of DHA into the cytosol occurred after inoculation in Alg-R compared with Alg-S. In contrast, the net loss of total ascorbate observed in Alg-R leaves after inoculation is consistent with severe previous experience of oxidative stress during HR.

Although a substantial proportion (about 8%) of the total foliar (reduced plus oxidized) ascorbate pool was found in the apoplast, there was little or no glutathione (only 1%-2%). The differences in the apoplastic contents of ascorbate and glutathione are striking and may reflect the differential roles of these two antioxidants. It is interesting that the glutathione pool increased in the apoplast of the Alg-R leaves and decreased in Alg-S after inoculation, perhaps implying that increased synthesis and export of glutathione are resistance responses.

The activities of antioxidant enzymes in the apoplast are low compared with the activities in whole leaves, but because the volume of the aqueous apoplastic phase is only 4.5% of the total cell volume (Winter et al., 1993), these percentages reflect large activities per unit volume. Substantial amounts of SOD, APX, MDHAR, and GR were found in the apoplast of healthy leaves (1%-4%), suggesting that all of the enzymes required for destruction of superoxide and H₂O₂ (i.e. the ascorbate-glutathione cycle) were present in this compartment. The apoplastic antioxidant enzyme activities showed an almost universal increase in response to inoculation and were much greater (at least double) in the susceptible Alg-S line compared with Alg-R. This may suggest that increased apoplastic antioxidant defenses were a feature of the establishment of biotrophy in the susceptible host. The ascorbate-glutathione cycle, however, requires a source of reducing power in the form of NADPH to sustain detoxification. Although NADPH may be present in the apoplast, at least some DHA and GSSG produced by the action of the ascorbate-glutathione cycle may be returned to the cytosol for reduction, since transporters for these oxidized forms have been described (Foyer and Lelandais, 1996; Jamaï et al., 1996; Horemans et al., 1997).

	FOOTNOTES

   Received February 19, 1998; accepted April 20, 1998.

	ABBREVIATIONS

Abbreviations: AA, reduced ascorbate. Alg, Algerian. APX, ascorbate peroxidase. DHA, dehydroascorbate. DHAR, dehydroascorbate reductase. EWF, extracellular washing fluid. G6PDH, Glc-6-P dehydrogenase. GR, glutathione reductase. GSSG, glutathione disulfide. HR, hypersensitive response. MDHAR, monodehydroascorbate reductase. SOD, superoxide dismutase.

	ACKNOWLEDGMENTS

We thank Dr. Andrea Polle (Universität Göttingen, Germany) for her invaluable advice concerning the technique used to extract the apoplast. We also thank Dr. William Bushnell (U.S. Department of Agriculture, Cereal Rust Laboratory, St. Paul, MN) for providing the barley seed.

	LITERATURE  CITED

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