Download Comparative Biochemistry of the Oxidative Burst Produced by Rose

Plant Physiol. (1998) 116: 1379–1385
Comparative Biochemistry of the Oxidative Burst Produced
by Rose and French Bean Cells Reveals Two
Distinct Mechanisms1
G. Paul Bolwell, Dewi R. Davies, Chris Gerrish, Chung-Kyoon Auh2, and Terence M. Murphy*
Division of Biochemistry, School of Biological Sciences, Royal Holloway, and Bedford New College, University
of London, Egham, Surrey TW20 0EX, United Kingdom (G.P.B., D.R.D., C.G.); and Section of Plant Biology,
Division of Biological Sciences, University of California, Davis, California 95616 (C.-K.A., T.M.M.)
Cultured cells of rose (Rosa damascena) treated with an elicitor
derived from Phytophthora spp. and suspension-cultured cells of
French bean (Phaseolus vulgaris) treated with an elicitor derived
from the cell walls of Colletotrichum lindemuthianum both produced H2O2. It has been hypothesized that in rose cells H2O2 is
produced by a plasma membrane NAD(P)H oxidase (superoxide
synthase), whereas in bean cells H2O2 is derived directly from cell
wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system
treated with N,N-diethyldithiocarbamate, superoxide was detected
by a N,N*-dimethyl-9,9*-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no
superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp.
elicitor, they accumulated a higher maximum concentration of
H2O2 than when treated without the washing procedure. In contrast, a washing treatment reduced the H2O2 accumulated by
French bean cells treated with C. lindemuthianum elicitor. Rose
cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H2O2 but did not have a
peroxidase capable of forming H2O2 in the presence of reductant.
Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.
An oxidative burst is a common response of plant cells to
physical or biological stress. The production of ROS such as
superoxide and H2O2 has been noted when plants are
challenged with particular viral, bacterial, or fungal pathogens (Mehdy, 1994; Low and Merida, 1996; Wojtaszek,
1997; Bolwell and Wojtaszek, 1998). It is generally considered a component of the hypersensitive response, an acute
defensive syndrome, and is often highly specific for particular genotypes of pathogen and host, but nonspecific
interactions can also be demonstrated. The oxidative burst
may be related to subsequent events in the challenged cells,
1
This work was supported in part by the U.S. Department of
Agriculture National Research Initiative-Cooperative State Research Service (grant no. 94-37100-0788) to T.M.M.
2
Present address: Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695.
* Corresponding author; e-mail [email protected]; fax
1–916 –752–5410.
such as programmed cell death, although the exact nature
of the relationship is unclear. Free radical oxidation of
plasma membrane lipids, induced by ROS, may kill cells
directly. Alternatively, superoxide (Jabs et al., 1996) or
H2O2 (Levine et al., 1994) may serve as signals leading
indirectly to mortality. However, in at least one case it has
been shown that an oxidative burst by itself is not sufficient
to trigger programmed cell death (Glazener et al., 1996).
The oxidative burst is often a very rapid response, occurring within seconds in some systems, such as cultured
cells of French bean (Phaseolus vulgaris) and soybean
(Bolwell et al., 1995). In other systems, such as rose (Rosa
damascena) cultured cells (Arnott and Murphy, 1991), it
may be delayed for minutes or hours, but in general it is
thought not to require de novo protein synthesis. Thus, it
involves the activation of pre-existing enzymes.
The source of the ROS is under study. There are several
hypotheses to explain the appearance of H2O2 in the medium of cultured cells and the apoplastic fluid of wholeplant tissues. The earliest, proposed to explain the origin of
H2O2 needed for formation of lignin in developing xylem
of horseradish (Armoracia lapathifolia), involves the reduction of O2 to superoxide by phenolic and NAD. radicals
produced by peroxidase (Yamazaki and Yokota, 1973;
Elstner and Heupel, 1976; Gross et al., 1977; Halliwell,
1978). In this model the source of electrons in the apoplast
is said to be malate, exported across the plasma membrane
by a malate/oxalacetate carrier and used to reduce NAD1
by apoplastic malate dehydrogenase. H2O2 is formed by the
dismutation of superoxide.
A second hypothesis, proposed for the oxidative burst
induced in French bean cultured cells by Colletotrichum
lindemuthianum cell wall elicitor, involves an apoplastic
peroxidase in a more direct way (Bolwell et al., 1995). The
O2-heme complex of peroxidase is reduced to compound
III by reductants exported from the cell. Under the proper
conditions, i.e. elevated pH, the complex is effectively hydrolyzed to release H2O2. In this model the source of
electrons has not been identified, but the release of a reductant from elicited cells has been observed.
Abbreviations: DDC, N,N-diethyldithiocarbamate; DPI, diphenyleneiodonium; lucigenin, N,N9-dimethyl-9,99-biacridium dinitrate; luminol, 5-amino-2,3-dihydro-1,4-phthalazinedione; ROS,
reactive oxygen species; SOD, superoxide dismutase.
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Bolwell et al.
A third hypothesis, proposed for the oxidative burst
induced in rose cultured cells by Phytophthora spp. elicitor
(Auh and Murphy, 1995) and for other systems, does not
involve peroxidase. Rather, a trans-plasma membrane superoxide synthase (NAD(P)H oxidase) transfers electrons
from NADH or NADPH in the cytoplasm to O2 to form
superoxide. H2O2 is formed by the dismutation of superoxide. Superoxide synthesis has been observed in purified
preparations of plasma membrane from several plant systems (Vianello and Macrı́, 1989; Qiu et al., 1994; Doke and
Miura, 1995; Murphy and Auh, 1996; Van Gestelen et al.,
1997).
The experimental systems used to derive the second and
third hypotheses involved relatively similar systems: cultured parenchymatous cells challenged with elicitors derived from fungal (or protistan) cell walls. However, not all
of the same experiments were used to develop the hypotheses. We believed it was important to determine whether
the differences in interpretation are due to fundamental
differences in the origin of H2O2 or to differences in approach. The objective of the present study was to compare
the two systems, French bean and rose, to resolve this
question.
MATERIALS AND METHODS
Chemicals
Luminol, lucigenin, and horseradish (Armoracia lapathifolia) peroxidase (EC 1.11.1.7) were obtained from Sigma.
DDC was from Sigma or Fisher Scientific and DPI
was from Cookson Chemicals, Ltd. (Southampton, UK) or
Calbiochem.
Cells and Elicitor
Cells of rose (Rosa damascena Mill. cv Gloire de Guilan)
were derived and grown in a suspension culture as previously described (Murphy et al., 1979). Elicitor was derived
from Phytophthora cinnamomea or Phytophthora megasperma,
as described by Auh and Murphy (1995), and used at a final
concentration of 25 or 15 mg mL21 Glc equivalents, respectively. Derivation and maintenance of cell cultures of
French bean (Phaseolus vulgaris L.) and preparation of elicitor from Colletotrichum lindemuthianum were as described
previously (Dixon and Lamb, 1979). Elicitor was used at a
final concentration of 30 mg mL21 Glc equivalents.
Determination of the Oxidative Burst
Lucigenin Assay for Superoxide
The accumulation of superoxide in the cell medium was
measured by lucigenin-dependent chemiluminescence. The
assay was conducted in a total volume of 2 mL by placing
0.2 mL of cell suspension and 0.2 mL of 1 mm lucigenin in
0.1 m Gly-NaOH buffer (pH 9.0) containing 1 mm EDTA
and 1 mm sodium salicylate. The SOD inhibitor Na-DDC
was added to the cell suspensions at 1 mm to block the
dismutation of superoxide to H2O2 by SOD. The chemi-
Plant Physiol. Vol. 116, 1998
luminescence was detected in the luminometer, which detects real-time luminosity, or by using the single-channel
mode in a scintillation spectrometer. This procedure has
been considered a specific assay of superoxide (Corbisier et
al., 1987), but recent reports (Liochev and Fridovich, 1997;
Vásques-Vivar et al., 1997) indicate that reduced lucigenin
can react with O2 to produce superoxide. Thus, enzymatic
or nonenzymatic reactions that reduce lucigenin can give a
false-positive reading.
Luminol Assay for H2O2
In rose cells, luminol-dependent chemiluminescence was
detected in a total volume of 1 mL by combining 0.2 mL of
cell-suspension medium and 0.01 mL of 1 mm luminol
solution in 50 mm Tris buffer (pH 8.0) in a scintillation vial.
Following the addition of 0.01 mL of 13 mm K3Fe(CN)6 the
scintillation vial was immediately placed in a scintillation
spectrometer (model LS8000, Beckman) and chemiluminescence was detected on single-channel mode. Counts were
reported every 12 s for 36 s and the last value was used.
Earlier experiments with this system (Auh and Murphy,
1995) used a similar technique but relied on the addition of
horseradish peroxidase together with the substrate H2O2
for the oxidation of luminol. In French bean cells, the
oxidative burst was routinely measured using a luminometer fitted with a rotating cuvette holder and an injection
port (model 1250, LKB Wallac, Broma, Sweden). A 1-mL
sample of suspension culture was loaded into the luminometer cuvette and continually stirred; 200 mL of 1 mm
luminol solution was then injected rapidly into the cuvette.
Real-time luminosity was recorded every second beginning
from 10 s before injection until the level returned to background. The time between sampling the suspension culture
and injecting the luminol was always less than 1 min. In
some experiments, specified in “Results,” a scintillation
counter was used to allow a more exact comparison between the rose and French bean systems. The luminometer
measures chemiluminescence immediately after the addition of luminol and with stirred cells, whereas there is a
delay of several seconds using the liquid-scintillation
counter. However, over the time scale of measurements of
the oxidative burst in cells, the latter delay is not significant. In each case, the assay was calibrated with a solution
containing a known concentration of H2O2.
Determination of Superoxide Production by NADH
Oxidase and H2O2 Production by Peroxidases
Superoxide production by a partially solubilized enzyme
preparation from rose cell plasma membrane was measured by Cyt c reduction. A reaction mixture contained
buffer (20 mm Tris-Cl, pH 7.5, and 3 mm MgCl2) to give a
total volume of 0.5 mL, 100 mm NADH, 0.02% (w/v) Triton
X-100, 100 mm Cyt c, DPI in concentrations as noted in
Figure 4, and 0.1 to 0.6 mg of enzyme preparation protein.
Change in A550 was measured over the 1st min in a DU-640
spectrophotometer (Beckman). The data were adjusted by
subtracting the background rate of reduction obtained in
the presence of 40 units of SOD.
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Two Mechanisms for the Oxidative Burst
Measurements of H2O2 production were determined for
purified peroxidases in the luminometer, the operation of
which was as described above. The reactions contained 1
mL of stirred buffer to which 3 mL of peroxidase containing
a known number of units (e.g. 3 units, as specified by
Sigma for horseradish peroxidase) was added. The buffers
used were at the pH optimum for the requisite peroxidase.
Borate buffer, pH 8.5, was used for horseradish peroxidase
(type XII, Sigma) and phosphate buffer, pH 7.2, was used
for French bean peroxidase 1 (FBP1) from French bean.
Two-hundred microliters of 1 mm luminol solution was
then added and the initial burst of chemiluminescence was
measured. Reductant such as Cys (0.5 mm) was then added
and the subsequent chemiluminescence monitored.
RESULTS
Comparison of the Effectiveness of Elicitors on Rose and
French Bean Cells in Generating H2O2
Both systems were capable of inducing rapid production
of H2O2. For rose cells and Phytophthora spp. elicitor, peak
concentrations of about 20 mm were reached at about 45 to
120 min after the initial addition of elicitor. In French bean
peak concentrations of about 130 mm H2O2 were reached 8
to 16 min after the addition of C. lindemuthiamum elicitor.
When C. lindemuthiamum elicitor was added to rose cells,
the cells produced variable, but generally weak, levels of
H2O2: the luminol-dependent chemiluminescence indicated a peak of about 7 mm H2O2 at 30 to 60 min (Fig. 1A).
Phytophthora spp. elicitor applied to French bean cells gave
a peak response at 16 min that was on average less than
15% of the peak response seen at 8 min with C. lindemuthiamum elicitor applied to the same batch of cells (Fig. 1B).
Thus,
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reciprocal experiments gave similar results in both systems: heterologous elicitors induced H2O2 production but
at reduced levels compared with the homologous elicitors.
In general, Phytophthora spp. elicitor stimulated a slower
response and C. lindemuthiamum elicitor stimulated a more
rapid response.
The Effect of Using Washed Cells with
Homologous Elicitor
In rose cells the appearance of H2O2 was increased by an
initial washing of the cells three times with a solution
containing 1 mm CaCl2 and 0.1 mm KCl (Qian et al., 1993).
The increase was ascribed to the loss of peroxidases (or
catalase) that consumed the H2O2. In contrast, prewashing
French bean cells reduced the subsequent luminoldependent chemiluminescence (Fig. 2). The contrasting effect of washing the cells suggests that the two cell species
produce H2O2 in different ways and that the French bean
system requires a component that is lost in the washing
step.
Comparative Effects of Elicitors on Superoxide Production
by Rose and French Bean Cells
The presumptive accumulation of superoxide, measured
by lucigenin-dependent chemiluminescence in the presence of DDC, was readily detected in rose cells when
elicited with Phytophthora spp. (Auh and Murphy, 1995). In
contrast, a significant accumulation of superoxide was
never observed in French bean cells treated with either C.
lindemuthianum or Phytophthora spp. elicitor, either with or
without DDC (data not shown).
Figure 1. Accumulation of H2O2 (luminol-dependent chemiluminescence) in the medium of rose (A) and French bean (B)
cells elicited by cell wall extract from Phytophthora spp. or C. lindemuthianum. Representative plots are shown. For rose
cells, the peak H2O2 concentrations were 20 6 8 mM (mean 6 SE; n 5 4) with Phytophthora spp. elicitor and 7 6 2 mM (n 5 4)
with C. lindemuthianum elicitor. For French bean cells, the peak H2O2 concentrations were 133 6 39 (n 5 5) with
C. linedmuthianum elicitor and 16 6 3 (n 5 3) with Phytophthora spp. elicitor.
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1382
Bolwell et al.
Plant Physiol. Vol. 116, 1998
in rose cells (in one experiment, more than 90% after an
overnight incubation) but not in French bean cells (in one
experiment, cells treated overnight with 100 mm DPI had
higher respiration rates than untreated controls).
The contrasting sensitivities of rose and French bean cells
were matched by the sensitivities of plasma membrane
NADH oxidase and peroxidase. DPI inhibited the production of superoxide by a rose plasma membrane NADH
oxidase more than 50% at 0.1 mm and of H2O2 by peroxidase, either from horseradish (pH optimum 8.5) or FBP1
(pH optimum 7.2), in the presence of the reducing agent
Cys, approximately 50% at concentrations up to 240 mm
(Fig. 4B).
The Induced Appearance of Reducing Agent
Figure 2. Effects of washing on the luminol-dependent chemiluminescence of rose cells elicited by cell wall extract from Phytophthora
spp. and of French bean cells elicited by cell wall extract from C.
lindemuthianum. The data for rose cells were calculated from Qian
et al. (1993). Results are means 6 SE (n 5 3). For rose cells, values
were normalized to the mean with washed cells; for French bean
cells, values were normalized to the mean with unwashed cells.
The Effect of KCN and DPI on Cell-Generated
Superoxide and H2O2
Peroxidases are exquisitely sensitive to micromolar concentrations of KCN (Saunders et al., 1964), whereas flavindependent enzymes, including the mammalian NADPH
oxidase, are strongly inhibited by DPI (Cross and Jones,
1986). These two inhibitors played a strong role in the
interpretation of the mechanism of H2O2 generation in the
French bean and rose systems (Auh and Murphy, 1995;
Bolwell et al., 1995). We expected that low concentrations
of KCN would inhibit synthesis of H2O2 in French bean
cells more strongly than in rose cells. However, it was not
possible to make such a distinction, apparently because
cells detoxified KCN over the short time in which they
synthesized H2O2. Figure 3 illustrates this point. The effect
of KCN on the accumulation of H2O2 by French bean cells
was substantially less 15 min after the addition of elicitor
than 10 min after the addition.
DPI inhibited H2O2 accumulation induced by Phytophthora spp. (or C. lindemuthianum) elicitor in rose cells with
50% inhibition at 2 mm, whereas in the French bean system
induced with C. lindemuthianum elicitor, 50% inhibition
required about 40 mm (Fig. 4A). Over the time course of the
oxidative burst (up to 150 min), 15 mm DPI inhibited respiration of rose cells by less than 20% and 100 mm inhibited
respiration by less than 30%; the respiration of French bean
cells was increased 12 and 43% by 15 and 100 mm DPI,
respectively. This indicates that metabolically generated
reducing power was available for the reduction of O2, and
energy was available for signal cascades initiated by elicitor. At longer times, DPI inhibited O2 uptake more strongly
The synthesis of H2O2 by French bean is stimulated by a
reducing agent released from cells challenged with C. lindemuthianum elicitor (Bolwell et al., 1995). Rose cells also
released an agent capable of stimulating H2O2 synthesis by
horseradish peroxidase, more when treated with C. lindemuthianum elicitor than with Phytophthora spp. elicitor (Fig.
5). However, the extracellular peroxidases collected in medium or high-salt eluate from rose cultures did not produce
detectable H2O2 when provided with Cys as a reducing
agent. In fact, peroxidases or catalases in the medium
and eluate from rose cultures reduced the accumulation
of H2O2 produced by horseradish peroxidase plus Cys
(Table I).
The Effect of DDC on the Detection of
Superoxide and H2O2
DDC is known as a chelator of Cu ions and an inhibitor
of the Cu/Zn isozyme of SOD (Heikkila et al., 1976; Kelner
and Alexander, 1986). It was added to rose cells in the
Figure 3. Effect of KCN on luminol-dependent chemiluminescence
of French bean cells, elicited by cell wall extract from C. lindemuthianum and assayed for 10 or 15 min after the addition of elicitor. The
points indicate means (bars are SEs) from two to three independent
experiments.
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Two Mechanisms for the Oxidative Burst
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Figure 4. A, H2O2 accumulation. Inhibition by DPI of the luminol-dependent chemiluminescence of rose cells elicited with
cell wall extract from Phytophthora spp. and of French bean cells elicited with cell wall extract from C. lindemuthianum.
B, Enzyme activity. Inhibition by DPI of the synthesis of superoxide by rose cell NADH oxidase and of H2O2 (luminoldependent chemiluminescence) by horseradish peroxidase in the presence of 0.5 mM Cys. The points indicate means (bars
are SEs) from at least three independent experiments.
expectation that the inhibition of SOD would allow the
accumulation of superoxide (observed through lucigenindependent chemiluminescence) and inhibit the accumulation of H2O2 (luminol-dependent chemiluminescence) during an oxidative burst, assuming that H2O2 was formed by
dismutation from superoxide. These expectations were fulfilled (Auh and Murphy, 1995). In the French bean cell
system, DDC abolished luminol-dependent chemiluminescence; however, it did not potentiate the detection of superoxide by lucigenin-dependent chemiluminescence. The
lack of production of superoxide in the French bean system, in contrast to results in the rose system, supports the
contention that the oxidative bursts in the two systems
have different origins. However, further experiments suggested that DDC reacts directly with H2O2: the addition of
H2O2 to DDC caused a rapid change in the UV absorption
spectrum of DDC (Fig. 6). This implies that DDC removes
Table I. Synthesis of H2O2 by peroxidase plus Cys
A mixture containing 50 mM sodium borate, pH 8.5, 2 mg mL21
luminol, and 5 mM Cys was placed in a scintillation counter and
counted on single-channel mode for 1 min, during which time
luminescence decayed to a low level. Volumes of samples containing peroxidase activity (commercial horseradish peroxidase, filtrate
from a 9-d-old rose cell culture, or 1 M KCl eluate from the filtered
rose cells) were then added and the mixtures were recounted. Values
show the increase in chemiluminescence relative to that seen with
horseradish peroxidase (alone). Guaiacol peroxidase activities of the
same volumes of each sample were measured spectrophometrically
at 470 nm in 50 mM phosphate buffer, pH 6.1, in 16 mM guaiacol,
and in 2 mM H2O2.
Sample
Figure 5. Induction by cell wall extracts of Phytophthora spp. (Ph.
elicitor) and C. lindemuthianum (C. elicitor) of secretion from rose
cells of an agent that stimulates H2O2 synthesis by horseradish
peroxidase. The data represent means from three independent experiments. By analysis of variance, variation among beakers was
shown to be significant at the 2% confidence level; variation among
sampling times was significant at the 0.2% level. Beaker 3 (C.
elicitor) at 30 min was significantly different from all other samples
(Student’s t test, 5% level).
Horseradish peroxidase
Rose filtrate
Rose eluate
Horseradish peroxidase followed
by rose filtrate
Horseradish peroxidase followed
by rose eluate
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H2O2 Synthesis
Guaiacol
Peroxidase
Activity
relative units
mM min21
100
0.4
20.05
8.3
20 6 4
6.3 6 0.4
3.0 6 0.1
12.5
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Bolwell et al.
Figure 6. Reaction of 0.1 mM DDC with 4 mM H2O2. The peaks at
258 and 283 nm represent DDC in the absence of H2O2; the spectrum for 4 mM H2O2 alone is also shown. The curves identified as
10 s, 3 min, and 10 min show the spectrum of the DDC sample at the
indicated times after the addition of H2O2.
H2O2 from solution. Thus, the role of DDC in the stimulation of lucigenin-dependent chemiluminescence and inhibition of luminol-dependent chemiluminescence must be
reinterpreted. Specifically, the inhibition of luminoldependent chemiluminescence by DDC cannot be interpreted to mean that the production of H2O2 proceeds by
the dismutation of superoxide.
DISCUSSION
Comparative biochemical studies of the oxidative burst
produced by rose and French bean cells have revealed two
distinct mechanisms. Both systems ultimately produced
H2O2 in response to elicitor treatment, but the intermediate
formation of superoxide was not as significant in French
bean as in rose cells.
Differences in the two systems include (a) the kinetics of
the oxidative burst and the peak concentrations of H2O2
that accumulated, (b) the effect of washing the cells, (c)
the detection of superoxide in the presence of DDC, (d) the
effect of DPI in inhibiting the oxidative burst, and (e)
the ability of extracellular peroxidases to generate H2O2 in
the presence of reducer.
Evidence related to the detection of superoxide in the
medium of elicited cells requires some qualification. As
noted in “Materials and Methods,” the reduction of lucigenin can lead to false-positive indications for the presence of
superoxide (Liochev and Fridovich, 1997; Vásques-Vivar et
al., 1997). Thus, the detection of superoxide (lucigenindependent luminescence) detected in the media of elicited
rose cells by Auh and Murphy (1995) could actually have
been produced by reducing substances released from the
cells. However, the presence of DDC-insensitive SOD in
the apoplast of French bean cells could have prevented a
positive indication of superoxide. The presence of superoxide in the apoplast is an important issue (Jabs et al.,
1996), but one that will require further investigation.
Plant Physiol. Vol. 116, 1998
The finding that different mechanisms are responsible
for the production of H2O2 in different elicited systems
may relate to systems other than the ones we studied. The
synthesis of H2O2 by tomato cells treated with elicitor from
Cladosporium fulvum is very sensitive to submicromolar
concentrations of DPI (K. Hammond-Kosack, personal
communication), whereas synthesis of H2O2 by lettuce cells
treated with Pseudomonas syringae pv phaseolicola was considered to be more sensitive to azide and KCN than to DPI
(Bestwick et al., 1997). The rapid production of H2O2 by
soybean cells treated with a Verticilium dahliae elicitor was
inhibited 60% by 6 mm KCN (Apostol et al., 1989). Allan
and Fluhr (1997) reported that ROS induced in tobacco
epidermal tissue by different agents arose by different
mechanisms: the elicitor cryptogein stimulated an oxidative burst, measured as oxidation of dichlorofluorescein,
that was sensitive to DPI but not to exogenous catalase, and
added Arg stimulated oxidation of dichlorofluorescein that
was sensitive to catalase but not to DPI.
The effects of DDC on the accumulation of H2O2 could
not be used to distinguish the two mechanisms. DDC
blocked the detection of H2O2 (luminol-dependent chemiluminescence) by reacting rapidly with H2O2. This effect
may also have been responsible, at least in part, for the
finding that DDC allowed an accumulation of superoxide.
H2O2 oxidizes ferrous ion, which in turn oxidizes superoxide. Since iron is a required component of plant cell culture
media, we would expect ferrous/ferric ions to be present at
the cation-exchange sites of the cell walls.
It is a common observation that evidence from inhibitor
studies is plagued with uncertainties, and our results reinforce that statement. DPI inhibits peroxidase-mediated
generation of H2O2, as well as NAD(P)H-oxidase-mediated
generation of superoxide, an observation also made by
Deme et al. (1994) and Dwyer et al. (1996). Thus, a careful
study of the concentration dependence of inhibition by DPI
is needed if such inhibition is to be used to distinguish
peroxidase- and flavoprotein-mediated synthesis of H2O2.
ACKNOWLEDGMENTS
The authors are grateful to Han Vu and Thuyhuong Nguyen,
University of California, Davis, for their excellent technical
assistance.
Received November 5, 1997; accepted December 4, 1997.
Copyright Clearance Center: 0032–0889/98/116/1379/07.
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