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 |
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 |
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
).
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).

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| 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 |
Plant and Pathogen Material
Seedlings of Alg-R (resistant; Mla1 allele) and Alg-S
(susceptible; mla1 allele) were grown for 9 d in seed
trays of John Innes No. 3 compost (J and P Peat, Ltd., Tollund House,
Carlisle, UK) in controlled-environment chambers with a 16-h
photoperiod at 340 µmol m
2
s
1, at 20°C day/15°C night, and at a
constant (70%) RH.
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
To inoculate experimental plants, trays of 9-d-old seedlings were
taken at 9 AM to the spore-proof greenhouse, where they were inoculated with B. graminis conidia by shaking heavily
infected plants over them. It was impossible to ensure total uniformity of inoculum distribution, but glass slides placed among the seedlings showed an average density of approximately 10 conidia
mm
2. Inoculations were completed within 15 min.
Seed trays were immediately returned to the controlled-environment
chambers and incubated for 24 h before harvesting for biochemical
analyses. Equivalent trays of uninoculated seedlings provided control
material. In Alg-R, the HR can be first detected about 18 h after
inoculation (e.g. Bushnell and Liu, 1994
), and increases in frequency
up to 30 h. Therefore, it was assumed that in the present study,
in which material was harvested for biochemical analyses 24 h
after inoculation, most cells destined to express the HR would have done so. To check this assumption, three inoculated leaves of each
isoline were fixed for light microscopy 24 and 48 h after inoculation for assessment of pathogen development and epidermal host-cell responses to attack.
Fixation and Clearing of Leaf Tissue for Microscopy
Leaves were fixed and prepared for microscopy by a procedure that
avoids displacement of the fungus (Carver et al., 1991
). They were
mounted without a coverslip and observed with a "no-coverslip" 40×
objective lens (Carver et al., 1991
). To assess the success of
attempted primary infection by B. graminis, on each leaf 25 germlings with appressoria were examined by transmitted light microscopy to determine whether they had penetrated the host epidermal cell successfully to form a primary haustorium. Autofluorescent host-cell responses to B. graminis attack have been
described many times previously (e.g. see Carver et al., 1995
). For
each germling, the presence at appressorium contact of whole-cell
fluorescence, indicative of HR, was determined using incident
fluorescence microscopy (blue exciter filter, maximum transmittance 400 nm; dichroic mirror and barrier filter transmittance, 500-800
nm).
Extraction of Soluble Apoplastic Components (EWF)
Soluble apoplastic enzymes and metabolites were extracted by
vacuum infiltration by a method similar to that described by Polle et
al. (1990)
. Freshly cut leaves (5 g) were washed three times with
distilled water, placed in aluminum foil dishes containing 30 mL of
infiltration solution consisting of either 50 mM Mes/KOH buffer (pH 6.0), 40 mM KCl, and 2 mM
CaCl2 (for the extraction of enzymes), or 50 mM acetate buffer (pH 4.5), 100 mM KCl, and 2 mM CaCl2 (for the extraction of
metabolites). To ensure immersion in the infiltration solution, a
second perforated aluminum dish was placed on top of the leaves. The
infiltration dishes were placed in a vacuum desiccator and the leaves
were infiltrated for 20 periods of 30 s at
70 kPa. They were
then blotted gently, loaded into a perforated centrifuge tube (9 mL,
1.5 cm in diameter), and placed in an Eppendorf tube (1.5 mL). EWF was
recovered by centrifugation (10 min, 2900g, 4°C). For the
extraction of the metabolites, EWF was centrifuged directly in
Eppendorf tubes containing 500 µL of 0.1 M
HClO4 to immediately stop metabolism. Before
analysis, sufficient K2CO3
(5 M) was added to each sample of EWF to adjust the pH to
either 4.0 to 5.0 (for ascorbate determination) or 6.0 to 7.0 (for
glutathione determination). The exact conditions required for
extraction of EWF from barley leaves were optimized. Before the studies
reported here appropriate recovery experiments were performed with
known quantities of metabolites to ensure that any oxidation arising
from extraction procedures was taken into account.
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
Freshly cut leaves (0.3 g) were weighed, immersed in liquid
N2, and ground to a fine powder in the same
buffer as that used for EWF extractions of enzymes. When the mixtures
had thawed, they were ground again. Because SOD and APX have
membrane-bound isoenzyme forms, the extracts were not centrifuged and
assays were performed on the crude leaf homogenates. The extracts were analyzed for the cytoplasmic marker enzyme G6PDH, and for the antioxidant enzymes SOD, GR, APX, MDHAR, DHAR, and catalase.
Extraction of Metabolites from Whole Leaves
Leaves (3.0 g) were ground in liquid N2 to a
fine powder and 1 mL of cold HClO4 (2.5 M) was added. After the homogenates had thawed they were
ground again. The crude extracts were centrifuged at 16,000g
for 5 min at 4°C, and the supernatant was divided into two aliquots
of 400 µL and stored on ice before neutralization. For
ascorbate determinations, 100 µL of 0.1 M
NaH2PO4/NaOH buffer (pH
5.6) and sufficient 5 M
K2CO3 were added to bring
the pH to 4.0 to 5.0 at 4°C. For glutathione determination, 100 µL
of 0.1 M Hepes/KOH buffer (pH 7.0) was added and the pH was
adjusted with 5 M
K2CO3 to 6.0 to 7.0. The
mixtures were centrifuged (5 min, 16,000g, 4°C) to remove
insoluble potassium perchlorate and the clear supernatants were used
for assay.
Determination of Enzyme Activities
All measurements were made at 25°C (except for measurement of
catalase, which was done at 20°C) and were performed four times for
each sample. G6PDH, which was used as a cytoplasmic marker, was
determined as described by Weimar and Rothe (1986)
. GR was measured
spectrophotometrically at 340 nm by a modification of the method of
Foyer and Halliwell (1976)
. The assay contained 50 mM Hepes
(pH 8.0), 0.5 mM EDTA, 500 µM GSSG, 100 µL of extract, and 250 µM NADPH. Control rates were
obtained in the absence of GSSG.
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 mM
1
cm
1.
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 mM
1
cm
1 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
Ascorbate and DHA were measured as described by Foyer et al.
(1983)
via the decrease in A265 after the
addition of ascorbate oxidase. GSH was measured by the method of
Griffiths (1980)
. GSSG was determined as described by Griffiths (1980)
except that endogenous GSH was measured before GSSG in the same
cuvette; total glutathione (GSH plus GSSG) was estimated via the
increase in A412 after the addition of GR
and NADPH. GSSG was determined by the difference of the two values.
Statistical Analysis
The significance of differences between mean values obtained
from four samples produced in two independent experiments was determined by one-way analysis of variance.
 |
RESULTS |
Host Epidermal Cell Responses and B. graminis
Development
Table I shows the proportions of
B. graminis appressoria that stimulated whole-cell
autofluorescence indicative of the HR in leaves of the resistant Alg-R
and the susceptible Alg-S barley isolines. In both isolines there was
little change between 24 and 48 h after inoculation in the
proportions of appressoria that caused cell death. However, there were
very great differences between the isolines in the proportions of
appressoria that stimulated this response. In Alg-R, at 24 h more
than 60% of the cells attacked by B. graminis appressoria
showed whole-cell autofluorescence indicative of the HR, and this
proportion did not increase in the later sample. In Alg-S, less than
3% of cells died in response to attack. Thus, near-maximal expression
of the HR had been achieved by 24 h after inoculation, which is
when tissues were harvested for biochemical analyses.
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|
Table I.
Meana percentages of B. graminis f. sp.
hordei appressoria that were associated with a localized
autofluorescent host-cell response, cell death (HR) indicated by
whole-cell autofluorescence, and that penetrated epidermal cells
successfully to form haustoria in barley fixed 24 and 48 h after
inoculation
|
|
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
G6PDH, a cytoplasmic enzyme, was used to calculate cytoplasmic
contamination of the apoplastic extracts. On average for all of the
samples collected, less than 2% of the total extractable foliar G6PDH
activity was found in the apoplast fluid of both inoculated and control
leaves (Table II). Exact values for the contamination of each sample were obtained, and this allowed an accurate determination of cytoplasmic contamination in each apoplastic sample. From this, all of the following data relating to analysis of
apoplastic constituents were corrected to allow for cytoplasmic contamination of each sample.
Ascorbate and Glutathione Contents
Ascorbate and glutathione were measured in whole-leaf and
apoplastic extracts from control (noninoculated) and inoculated leaves
(Fig. 3).

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| 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
Total foliar ascorbate content (reduced plus oxidized) was similar
in both barley lines (Fig. 3A). In both lines the total ascorbate
pool was largely reduced (57%-62%) in inoculated and in control
leaves. A significant (P < 0.05) increase (23%) in AA was found
in inoculated Alg-S leaves compared with the controls; in Alg-R the
increase was slight and insignificant. In both lines a significant
(P < 0.05) decrease (20%-30%) in DHA was found in inoculated
leaves compared with the controls.
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
Noninoculated leaves of Alg-R and Alg-S had similar total
glutathione (Fig. 3C). The glutathione pool was largely (>99%)
reduced in noninoculated and inoculated leaves of both lines.
Inoculation caused a significant (P < 0.05) increase (36%) in
GSH in Alg-R but not in Alg-S. In contrast, inoculation caused a
significant (P < 0.05) decrease (37%) in GSSG only in Alg-S
(Fig. 3C).
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
The maximum extractable activities of the antioxidant enzymes SOD,
APX, MDHAR, DHAR, GR, and catalase measured in whole-leaf and
apoplastic extracts of noninoculated and inoculated leaves are shown in
Figures 4-6.

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| 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
Total SOD activities were similar in whole-leaf and apoplastic
extracts from noninoculated leaves of both lines (Fig.
4, A and B). After inoculation,
whole-leaf SOD activity did not change (Fig. 4A), but a significant
increase in apoplastic SOD was found in both resistant (150%) and
susceptible (300%) lines compared with noninoculated controls (Fig.
4B). The apoplast of inoculated leaves contained 2.5% to 3% of the
total foliar SOD activity, but only 0.8% to 1.3% was found in the
controls.
Catalase
Noninoculated whole-leaf extracts of both resistant and
susceptible lines had similar catalase activities (Fig. 4C). In Alg-S inoculation caused a massive (400%) and significant (P < 0.001) increase in catalase activity in these extracts. In contrast, there was
no significant change in Alg-R after inoculation (Fig. 4C).
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
Noninoculated whole-leaf extracts of both resistant and
susceptible lines had similar APX activities (Fig.
5A). Inoculation caused a significant
(P < 0.01) decrease (62%) of APX activity in whole-leaf Alg-R
extracts, but caused no significant change in inoculated Alg-S (Fig.
5A). The apoplast of noninoculated leaves contained 0.7% to 1% of the
total foliar APX activity. Inoculation caused a significant increase of
apoplastic APX activity in Alg-R (56%; P < 0.05) and Alg-S
(130%; P < 0.001) (Fig. 4B). After inoculation, the apoplast of
both lines contained 3% to 3.5% of the total foliar APX activity.

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
In noninoculated whole-leaf extracts, GR activity in Alg-R was
double that in Alg-S (Fig. 5C). Inoculation caused a significant (P < 0.01) decrease (80%) only in the resistant Alg-R line,
whereas no significant change was found in Alg-S (Fig. 5C).
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
MDHAR activity was similar in whole-leaf extracts of both lines
and no significant changes resulted from inoculation (Fig. 6A). The apoplast of barley contained 2%
to 3% of the total foliar MDHAR activity. As for whole-leaf extracts,
in apoplastic extracts MDHAR activity was similar between both lines
and was not affected significantly by inoculation (Fig. 6B).

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[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
DHAR activity was similar in whole-leaf extracts of both lines and
no significant changes resulted from inoculation (Fig. 6C). About 0.5%
of total foliar DHAR activity was found in the apoplast of
noninoculated leaves. In both lines inoculation appeared to cause an
increase in apoplastic DHAR activity, but the variation between samples
was high; thus, although the increase in apoplastic DHAR was
significant (P < 0.05) in Alg-R, it was not significant in Alg-S
(Fig. 6D).
 |
DISCUSSION |
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 |
*
Corresponding author; e-mail christine.foyer{at}bbsrc.ac.uk;
fax 44-1-970-828357.
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.
 |
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