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Plant Physiol. (1998) 118: 1379-1387
The Respiratory Burst and Electrolyte Leakage Induced by
Sulfhydryl Blockers in Egeria densa Leaves Are
Associated with H2O2 Production and Are
Dependent on Ca2+ Influx1
Maria Teresa Marrè*,
Enrica Amicucci,
Luisa Zingarelli,
Francesco Albergoni, and
Erasmo Marrè
Centro di Studio del Consiglio Nazionale delle Ricerche sulla
Biologia Cellulare e Molecolare delle Piante, via Celoria 26, 20133 Milan, Italy (M.T.M.); and Dipartimento di Biologia, Università
di Milano, via Celoria 26, 20133 Milan, Italy (E.A., L.Z., F.A., E.M.)
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ABSTRACT |
In leaves of Egeria
densa Planchon, N-ethylmaleimide (NEM) and other
sulfhydryl-binding reagents induce a temporary increase in
nonmitochondrial respiration ( QO2) that is inhibited by
diphenylene iodonium and quinacrine, two known inhibitors of the plasma
membrane NADPH oxidase, and are associated with a relevant increase in electrolyte leakage (M. Bellando, S. Sacco, F. Albergoni, P. Rocco, M.T. Marré [1997] Bot Acta 110: 388-394). In this paper we
report data indicating further analogies between the oxidative burst induced by sulfhydryl blockers in E. densa and that
induced by pathogen-derived elicitors in animal and plant cells: (a)
NEM- and Ag+-induced QO2 was associated with
H2O2 production and both effects depended on
the presence of external Ca2+; (b) Ca2+ influx
was markedly increased by treatment with NEM; (c) the Ca2+
channel blocker LaCl3 inhibited QO2,
electrolyte release, and membrane depolarization induced by the
sulfhydryl reagents; and (d) LaCl3 also inhibited
electrolyte leakage induced by the direct infiltration of the leaves
with H2O2. These results suggest a model in
which the interaction of sulfhydryl blockers with sulfhydryl groups of
cell components would primarily induce an increase in the
Ca2+ cytosolic concentration, followed by membrane
depolarization and activation of a plasma membrane NADPH oxidase. This
latter effect, producing active oxygen species, might further influence plasma membrane permeability, leading to the massive release of electrolytes from the tissue.
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INTRODUCTION |
The ability of NEM and a number of other sulfhydryl group
blockers to induce QO2 in Egeria
densa Planchon leaves has been reported previously (Albergoni et
al., 1996 ; Bellando et al., 1997; Marrè and Albergoni, 1998).
QO2 induced by these reagents is completely
insensitive to mitochondrial inhibitors such as cyanide,
salicylhydroxamic acid, and propyl gallate and to the uncoupler
carbonylcyanide-m-chlorphenylhydrazone, whereas it is suppressed by quinacrine and diphenylene iodonium, two inhibitors of
plasma membrane NADPH oxidase, an enzyme responsible for the oxidative
burst induced by pathogenic factors in granulocytes and in plants
(Babior, 1992; Doussière and Vignais, 1992; Auh and Murphy, 1995;
Desikan et al., 1996; Murphy and Auh, 1996; Van Gestelen et al., 1997).
The respiratory burst induced by sulfhydryl blockers is also associated
with an early depolarization of the transmembrane electrical potential
difference (Em), with a rapid increase in
electrolyte leakage (mainly K+, but also
HPO42 and
Cl), and with an increase in
H+ influx (Bellando et al., 1997; Marrè and
Albergoni, 1998). All of these features are similar to the effects of
pathogenic elicitors in higher plants in which the hypersensitive
reaction has been investigated. This suggests that a common general
mechanism is involved in the two orders of phenomena.
In addition to its intrinsic value, the elucidation of the mode of
action of sulfhydryl reagents on O2 uptake and
plasma membrane permeability might contribute to the understanding of
the mechanism of the pathogen-induced oxidative burst and the increase
in membrane permeability that are characteristic of the hypersensitive
response (Apostol et al., 1989; Conrath et al., 1991; Goodman and
Novacky, 1994). Two important observations concerning the
pathogen-derived, elicitor-induced oxidative burst are: (a) the
production of active oxygen species, mainly
O2 and
H2O2 (Doke, 1983;
Sutherland, 1991; Levine et al., 1994; Mehdy, 1994; Doke and Miura,
1995; Lamb and Dixon, 1997; Xing et al., 1997), which are potentially
responsible for changes at the plasma membrane; and (b) the
demonstration of a strict Ca2+ dependence of the
effects of treatments with a variety of pathogenic elicitors, leading
to the conclusion that an increase in the cytosolic Ca2+ level is a very early step in the sequence
of events involved in the phenomenon (Bach et al., 1993; Tavernier et
al., 1995; Levine et al., 1996; Gelli et al., 1997; Jabs et al., 1997;
Pugin et al., 1997).
The present investigation was undertaken to determine whether the
effects of sulfhydryl blockers in E. densa are associated with the production of active oxygen species, and whether in this case,
as well as in the induction of the respiratory burst, membrane depolarization and electrolyte leakage depend on the presence of
Ca2+ in the medium and on its influx into the
tissue. We also investigated the possibility that the production of
H2O2, as a product of the oxidative burst, could contribute to the changes in membrane
permeability associated with electrolyte leakage.
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MATERIALS AND METHODS |
Plant Material
Egeria densa Planchon plants were grown in flowing tap
water in a greenhouse. Young, fully expanded leaves were cut about 2 to
4 cm from the branch apex, randomized, and then grouped into samples.
Before the experiments, the samples were washed twice for 15 min with
20 mL of 0.5 mM CaSO4 and
preincubated for 30 min in 20 mL of 0.5 mM
CaSO4 and 5 µM DCMU, with
continuous shaking at 20°C in the dark. DCMU, a specific inhibitor of
photosynthetic O2 evolution and C fixation, was
added to prevent the eventual effects of photosynthesis caused by
occasional exposure to light (Marrè et al., 1989; Albergoni et
al., 1996).
QO2 Measurements
QO2 was measured on four leaves (about 30 mg
fresh weight) in 2 mL of medium using a Clark-type
O2 electrode (unit KW2, control box CB1,
Hansatech, Norfolk, UK). Measurements were carried out continuously, as
described by Marrè and Albergoni (1998), and medium was renewed
every 8 min to restore maximal concentration of dissolved
O2. The basal medium contained 5 µM
DCMU, 10 mM Mes buffer (pH 6.0), with BTP.
CaSO4 and other reagents were added at the
concentrations specified in the legends to the figures.
H2O2 Production
The low values of H2O2
accumulation in the samples revealed that there was a problem with the
measurement procedure. The widely used luminol method presented the
disadvantages of a relatively low sensitivity, together with a large
variability. After several trials using this method (as described by
Viard et al., 1994), we decided to follow the method described by Jiang
et al. (1990) using the peroxide-mediated oxidation of
Fe2+, followed by the
reaction of Fe3+ with xylenol orange. This method
produced reproducible results in the 0.1 to 1 µM
H2O2 concentration range
(Fig. 1).
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| Figure 1.
Standard curve of the H2O2
concentration determined by the xylenol method in the range of 0.1 to 1 µM. H2O2 was measured with 100 µM xylenol orange, 250 µM Fe2+,
and 100 µM sorbitol in 25 mM
H2SO4. A560 was read
after 45 min at room temperature. Data are the means of triplicate
determinations. SD did not exceed ±3%.
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The possibility of interference by other chemicals present in the
various assay media used in our experiments was excluded by a series of
tests, and the specificity for
H2O2 was tested by
determining the effects of
H2O2 elimination by
catalase. For the determination of
H2O2 with this method
500-µL aliquots of the incubation medium, drawn from the
O2 electrode chamber every 8 min, were added to
500 µL of the reaction mixture containing 500 µM
ammonium ferrous sulfate, 50 mM sulfuric acid, 200 µM xylenol orange, and 200 mM sorbitol. After
45 min at room temperature the changes in
A560 were evaluated with a Biochrom
Ultrospec 2 spectrophotometer (LKB, Bromma, Sweden). The addition of
catalase (1600 units) to the sample aliquots for 5 min decreased the
absorbance changes to less than 10%. The
H2O2 values reported in
Figures 1 and 6 are those left after subtraction of the small
values found after treatment with catalase.
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| Figure 6.
Effects of the presence of 0.25 mM
La3+ (added at time 0) on the rate of
H2O2 production induced by 0.2 mM
NEM and 5 µM Ag+. Experimental conditions
were as in Figure 2. Data are from one representative experiment out of
three. FW, Fresh weight.
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Electrophysiological Measurements
A single leaf held in a 5-mL plexiglass chamber was irrigated with
a continuously flowing (approximately 15 mL
min1) aerated medium thermoregulated at 20°C.
The basal medium was the same as that described above, except that 0.5 mM CaSO4 was added to the solution.
Other reagents were added at the concentrations reported in the legends
to the figures. Micropipettes (tip resistance, 10 M ) filled with 1 M KCl were used as microsalt bridges of Ag/AgCl electrodes
and were inserted vertically into the tissue by means of a
micromanipulator (Leitz, Wetzlar, Germany).
Em was measured with a high-impedance
electrometer amplifier (model WPI K5-700, World Precision
Instruments, New Haven, CT) connected to a chart recorder. The
data presented represent one of at least three experiments for each set
of experimental conditions.
Measurement of Electrolyte Leakage
After pretreatment, samples of 10 leaves (approximately 100 mg
fresh weight) were incubated in 10 mL of a stirred solution thermoregulated at 20°C. Basal medium contained 5 µM
DCMU and 10 mM Mes buffer (pH 6.0), with BTP. Other
reagents were added at the concentrations specified in the legends to
the figures. The conductivity of the medium was measured continuously
by a conductivity meter (model MO101, Analytical Control, Milan, Italy) connected to a chart recorder for the required time. The data represent
the changes in medium conductivity during the treatments, calculated
per gram fresh weight, taking 0 as the value of conductivity measured
at the end of the equilibration period before starting the treatment.
Ca2+ Uptake and Net Flux Measurements
Ca2+ influx was measured as
45Ca2+ uptake, and the
radioactivity was provided as
45CaCl2 (1.48 GBq
mol1). After pretreatment, samples of leaves
(100 mg fresh weight) were incubated in 20 mL of basal medium
containing 0.5 mM CaSO4, 10 mM Mes-BTP (pH 6.0), and 5 µM DCMU, with or
without 0.2 mM NEM. To remove by exchange the
45Ca2+ contained in the
free space and in the cell wall, after 30 and 90 min of treatment the
samples were briefly rinsed twice in 20 mL of an unlabeled ice-cold
solution containing 10 mM CaSO4, 10 mM Mes-BTP (pH 6.0), and 5 µM DCMU. The
leaves were then transferred twice (for 2.5 and 18 min, respectively)
in 20 mL of the same unlabeled solution and finally collected, blotted
on filter paper, and placed into scintillation vials. Digestion and
bleaching of the tissue were performed as described by Romani and
Beffagna (1991). The radioactivity was measured by liquid-scintillation counting in a TriCarb counter (Packard Instruments, Meriden, CT) after
addition of 10 mL of scintillation liquid (Hionic Fluor, Packard
Instruments).
Net Ca2+ fluxes were measured in samples of
leaves incubated in the same experimental conditions described above,
without 45Ca2+. The changes
in Ca2+ concentrations in the assay media were
measured after 90 min of incubation using a
Ca2+-selective electrode (MI-600,
Microelectrodes, Bedford, NH).
SOD Assay
E. densa leaves (130 mg/mL) were homogenized in 50 mM potassium phosphate (pH 7.8) containing 0.1 mM EDTA and 0.3% (w/v) Triton X-100 at 4°C. The
homogenate was filtered through four layers of cheesecloth and promptly
used for the SOD assay. SOD activity was determined
spectrophotometrically as the inhibition of xanthine-xanthine oxidase-mediated reduction of Cyt c (McCord and Fridovich,
1969). The assay was performed at 25°C in a 3-mL cuvette containing
50 mM potassium phosphate (pH 7.8), 0.1 mM
EDTA, 50 µM Cyt c, and 1 mM
xanthine. The reduction of Cyt c was followed by a
double-beam spectrophotometer (ZWS II, Sigma). The data are means of
three determinations on two different leaf extracts.
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RESULTS |
H2O2 Production in NEM and the
Ag+-Induced Oxidative Burst
Previously, it was shown that the QO2
induced by NEM or by Ag+ was insensitive to
cyanide and salicylhydroxamic acid but completely inhibited by
diphenylene iodonium and quinacrine. This suggested that it was
mediated by a type of NADPH oxidase operating in the oxidative burst
induced by pathogen-derived elicitors, both in granulocytes and in
plants, and responsible for the production of
O2 and (through SOD)
H2O2 (Mehdy, 1994; Doke and
Miura, 1995; Desikan et al., 1996). To confirm this conclusion we
investigated whether H2O2
was the product of the oxidative reaction, as in the case of treatment
with the sulfhydryl blockers. Figure 2
shows that NEM-induced QO2 was indeed closely
associated with and parallel to the production of
H2O2. Similar results were
obtained on leaves in which the oxidative burst had been induced by
treatment with 5 µM Ag+. Attempts
to demonstrate the appearance of
O2 in the incubation medium
failed to give significant and reproducible results. This might be a
consequence of SOD rapidly metabolizing the products of NADPH oxidase
activity. Measurements of SOD activity in E. densa leaves
gave a very high value of this enzyme (1.3 ± 0.15 µmol
min1 g1 fresh weight;
see ``Materials and Methods'').
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| Figure 2.
Effects of 0.2 mM NEM on the rate of
O2 uptake and of H2O2 production by
E. densa leaves. The incubation medium contained 10 mM Mes buffer (pH 6.0) with BTP, 5 µM DCMU,
and 0.5 mM CaSO4. Aliquots for
QO2 measurements and H2O2
determination came from the same incubation medium. Data are from one
representative experiment out of five. FW, Fresh weight.
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Ca2+ Dependence of Sulfhydryl Reagent-Induced
QO2, Membrane Depolarization, and Electrolyte Leakage
Effects of Ca2+ Concentration in the Medium
In our previous studies on the oxidative burst induced by the
sulfhydryl blockers in aquatic plants, 0.5 mM
Ca2+ was always present in the medium (Albergoni
et al., 1996; Bellando et al., 1997; Marrè and Albergoni, 1998).
In this study the relevance of extracellular Ca2+
concentration on the NEM- and Ag+-induced
increase in O2 consumption was investigated at
Ca2+ concentrations ranging from 0 to 0.5 mM. The results shown in Figure
3A indicate that the ability of NEM to
induce the respiratory burst was completely abolished by the absence of
extracellular Ca2+ (control) or by its presence
at 15 µM, whereas it was barely appreciable at 50 µM, and steadily increased from 0.1 to 0.5 mM Ca2+. Figure 3B shows that the absence of
Ca2+ in the medium completely suppressed the
NEM-induced electrolyte leakage, an aspect of sulfhydryl-blocker action
similar to that reported for the pathogenic elicitor-induced
respiratory burst (Atkinson at al., 1990; Bach et al., 1993; Goodman
and Novacky, 1994; Jabs et al., 1997).
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| Figure 3.
Effects of increasing concentrations of
Ca2+ on 0.2 mM NEM-induced respiratory burst
(A) and Ca2+ dependence of NEM-induced electrolyte leakage
(B). Experimental conditions were as in Figure 2. QO2
data (A) are from one representative experiment out of three for each
Ca2+ concentration tested. Conductivity change data (B) are
the means of three experiments. SE did not exceed ±8%. gr
FW-1, Per gram fresh weight.
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The effect of Ca2+ in conditioning the
NEM-induced oxidative burst and electrolyte leakage might have depended
on an increase of its influx in the cells. To test this hypothesis, we
measured the effects of NEM on Ca2+ influx in a
medium containing 45Ca2+
(Fig. 4) after 30 and 90 min of
incubation. The results shown in Figure 4A demonstrate a very large
increase of Ca2+ influx in the NEM-treated
leaves. This finding was confirmed by measurements of
Ca2+ concentration changes in the incubation
medium, which showed a decrease of 20 µmol g1
fresh weight (Fig. 4B). Although changes in the
Ca2+ content in the cell walls contributed
somewhat to these results, these data are interpreted as indicating a
marked stimulation of Ca2+ influx in the
NEM-treated leaves.
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| Figure 4.
45Ca2+ influx in E. densa leaves (A) and changes in external Ca2+
concentration (B) induced by 0.2 mM NEM. The
45Ca2+ uptake was determined after 30 and 90 min of incubation, as described in ``Materials and Methods''. Changes
in external Ca2+ concentration were measured in the medium
with a Ca2+-selective electrode after 90 min of incubation.
Samples were incubated under the same conditions described for the
influx, except for the absence of 45Ca2+. The
data represent the means of two experiments run in triplicate.
SE did not exceed ±10%. FW, Fresh weight.
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Effects of La3+ on QO2 and
H2O2 Production
In several plant systems the ability to respond to a stress
stimulus (i.e. temperature shock, drought, salinity, or
pathogen-derived elicitors) requires the presence of extracellular
Ca2+. The inhibition by
Ca2+-channel blockers such as
La3+ suggests that Ca2+
influx increases the cytosolic Ca2+ concentration
(Atkinson et al., 1990; Bach et al., 1993; Price et al., 1994;
Tavernier et al., 1995; Gelli et al., 1997; Pugin et al., 1997;
Takahashi et al., 1997). The results shown in Figures 5 and 6
indicate that in our system the induction of
QO2 and the production of
H2O2 by NEM or
Ag+ were completely inhibited by the presence of
0.25 mM La3+. After treatment with
NEM, the onset of the oxidative burst was seen after a lag of about 20 min, and the addition of La3+ was still able to
counteract the appearance of QO2. As shown in
Figure 7, the development of the
oxidative burst was completely prevented when
La3+ was added 15 min after NEM, and was
interrupted almost immediately when La3+ was
added at the QO2 already initiated.
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| Figure 5.
Effects of 0.25 mM La3+ on
0.2 mM NEM-induced (A) or 5 µM
Ag+-induced (B) respiratory burst. Experimental conditions
were as in Figure 2. Data are from one representative experiment out of
three. gr FW-1, Per gram fresh weight.
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| Figure 7.
Effects of the addition of La3+ 15 and
25 min after the addition of NEM on the NEM-induced respiratory burst.
Experimental conditions were as in Figure 2. Data are from one
representative experiment out of three. FW, Fresh weight.
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Effect of La3+ on the Em and
on Electrolyte Leakage
Previous experiments have shown that sulfhydryl blockers induced a
respiratory burst and an increase in electrolyte leakage that were
preceded by an early, strong depolarization of the plasma membrane
(Bellando et al., 1997; Marrè and Albergoni, 1998). As shown in
Figure 8, the addition of 0.25 mM LaCl3 to the flowing solution
before the addition of either NEM or Ag+
completely suppressed the depolarizing effect of the two drugs (Fig.
8B). In other experiments La3+ was added 20 min
after NEM, i.e. toward the end of the lag period between the addition
of NEM and the onset of the respiratory burst, but when the
depolarization of NEM had fully developed and electrolyte leakage had
already started. In this case La3+ completely
reversed the NEM-induced depolarization (Fig. 8C) (for a similar effect
of La3+ on elicitor-induced depolarization in
tobacco cells, compare Pugin et al., 1997). La3+
alone did not significantly change the basal
Em value of E. densa cells,
indicating a weak ability of this cation to enter the cells. The
La3+-induced inhibition of the membrane
depolarization caused by NEM or Ag+ was
associated with an equally complete suppression of the increase in
electrolyte leakage (Fig. 9).
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| Figure 8.
Effects of the presence of 0.25 mM
La3+ on 5 µM Ag+-induced (A and
B) and 0.2 mM NEM-induced (C and D) membrane
depolarization. Note that in C, La3+ was added after NEM
addition, so that NEM-induced depolarization had already reached its
maximum (about 15 min) but QO2 had not yet started
(compare Fig. 7). Data are from one representative experiment out of
three. A, Depolarization by 5 µM Ag+; B, its
inhibition by La3+; C, depolarization by 0.2 mM
NEM and its reversal by La3+; and D, inhibition by the
presence of La3+ of the NEM-induced depolarization.
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| Figure 9.
Effects of 0.25 mM La3+ on
0.2 mM NEM-induced (A) and 5 µM
Ag+-induced (B) electrolyte leakage. Basal medium was as in
Figure 2 with 0.5 mM CaSO4 always present; 0.25 mM La3+ was added at time 0. Data are the means
of three experiments. SE did not exceed ±10%. µS,
Change in medium conductivity.
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Effect of La3+ on H2O2-Induced
Electrolyte Leakage
A role has been proposed for
H2O2 in the changes in the
plasma membrane leading to electrolyte leakage in the oxidative burst induced by pathogen-derived elicitors (Levine et al., 1994; Mehdy, 1994; Lamb and Dixon, 1997). This also appears to be true in the case
of sulfhydryl-blocker-induced
H2O2 production in E. densa. Figure 10 shows that
infiltration of E. densa leaves with 1 and 10 mM
H2O2 induced a rapid
increase in electrolyte leakage.
H2O2 concentrations less
than 0.5 mM did not induce any detectable response.
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| Figure 10.
Effects of the presence of 1 and 10 mM H2O2 in the incubation medium on
electrolyte leakage in the presence and absence of 0.25 mM
La3+. To facilitate H2O2 entry,
before the experiment was started leaves were rapidly infiltrated with
the same medium used in the treatment. Experimental conditions were as
in Figure 2. Data are from one representative experiment out of four.
µS, Change in medium conductivity.
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The ability of H2O2 to
induce electrolyte leakage might depend on the activation of
Ca2+ influx (Price et al., 1994; Levine et al.,
1996). Thus, one might expect an effect of La3+
on the effect of H2O2 on
membrane permeability and electrolyte leakage. In our experiments the
presence of La3+ completely inhibited the
H2O2 effect on electrolyte
leakage, suggesting the involvement of some step influenced by
Ca2+ extracellular concentration (for a similar
conclusion in soybean cells, see Levine et al., 1996).
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DISCUSSION |
The results presented in this paper integrate the previously
reported evidence concerning the effects of sulfhydryl reagents on
respiration and ion transport in E. densa leaves. These new data demonstrate that the respiratory burst induced by sulfhydryl blockers (NEM or Ag+) in E. densa
leaves was associated with a parallel production of
H2O2. The presence of
Ca2+ in the incubation medium was strictly
required for the effects of NEM and Ag+ on
transmembrane potential, electrolyte leakage, O2
uptake, and H2O2
production, and Ca2+ influx was markedly
stimulated by NEM. With 0.5 mM Ca2+
present, the addition of the Ca2+-channel blocker
La3+ completely inhibited all of these effects of
NEM or Ag+, suggesting that
Ca2+ action depends on its influx. The addition
of H2O2 to the medium induced an electrolyte leakage of the same order as that induced by NEM
or Ag+, and this effect was completely inhibited
by La3+.
The integration of these results with those of previous studies allows
a coherent interpretation of the mechanism of action of the sulfhydryl
blockers on respiration and membrane activities in E. densa
leaves. According to a model covering the available evidence to date,
the primary interaction of the sulfhydryl blockers with the sulfhydryl
groups of some cell component would lead to a rapid increase in
Ca2+ influx. The consequent increase in cytosolic
Ca2+ concentration would influence ion-transport
systems at the plasma membrane (for a similar interpretation of the
effects of Ca2+, see Schroeder and
Hagiwara, 1989; Price et al., 1994; Tavernier et al., 1995;
Pugin et al., 1997), and would therefore explain the observed rapid
membrane depolarization and increased release of electrolytes.
According to our previous data, the main effects on ionic fluxes
induced by sulfhydryl reagents are the effluxes of
K+, Cl, and
H2PO4
and the influx of H+ and
Ca2+ (according to the present results). In guard
cells an increase in cytosolic Ca2+ concentration
has been reported to favor the opening of anion (Cl and
H2PO4)
channels and the closure of inwardly rectified K+
channels (Schroeder and Hagiwara, 1989), which would explain the
changes in anion fluxes in our system. Membrane depolarization would
depend at least in part on anion efflux and might be responsible for
the opening of outward K+ channels and for
K+ efflux. Thus, one might propose that all
changes in the fluxes of these ions and in the
Em are consequences of
Ca2+ entry into the cell, whereas the increase of
H+ net uptake (this is potentially important
because of its influence on cytosolic pH and the membrane potential)
would depend on a Ca2+-induced increase in
passive membrane permeability to H+ and perhaps
also on the inhibition of the H+-ATPase by the
sulfhydryl blockers (Brooker and Slayman, 1982).
The identification of H2O2
production during the QO2 burst confirms the
previous conclusion that an
O2-producing NADPH oxidase of
the type speculated to be involved in the hypersensitive reaction
mediates the oxidative burst induced by the sulfhydryl blockers
(Doussière and Vignais, 1992; Felix et al., 1994; Auh and Murphy,
1995; Van Gestelen et al., 1997; Xing et al., 1997), a conclusion
previously drawn from the inhibition of the
QO2 effect by diphenylene iodonium and
quinacrine (Bellando et al., 1997). The activation mechanism of the
NADPH oxidase by the sulfhydryl blockers remains open to investigation
and probably involves various steps, as proposed for NADPH oxidase
activation by pathogen-derived elicitors (Legendre et al., 1992;
Morrè et al., 1993; Mehdy, 1994; Vera-Estrella et al.,
1994). The conclusion that the responses of membrane potential and
electrolyte leakage are largely independent of the activation of the
NADPH oxidase is suggested by the findings that: (a) after treatment
with NEM, the two responses are clearly separated in time; (b) NADPH
oxidase inhibitors completely suppress the oxidative burst, whereas
they inhibit electrolyte leakage only partially (Bellando et al., 1997; Marrè and Albergoni, 1998); and (c) the addition of
La3+ 15 to 20 min after that of NEM completely
prevents the oxidative burst, although membrane depolarization has
reached its maximum and electrolyte leakage has already started. The
findings that the addition of La3+ completely
suppresses NEM-induced QO2 and
H2O2 production and that it
reverses the previous effects of NEM on membrane potential suggests
that all of the leaf responses to the sulfhydryl blockers require the
continuous influx of Ca2+ into the cells.
These experiments demonstrate that Ca2+ influx is
strictly required for sulfhydryl reagent-induced changes in both ion
transport and oxidative metabolism. However, an unresolved question is
whether the influx of Ca2+ by itself is
sufficient to induce the oxidative burst. The necessary involvement of
ion fluxes other than that of Ca2+ in the
induction of the oxidative burst has been suggested by Jabs et al.
(1997). Further data are requested to define the situation in our
system.
The model shown in Figure 11 summarizes
the main lines of our interpretation of the action of the sulfhydryl
blockers in E. densa leaves. A strict similarity is evident
between this model and that proposed for the action of pathogen-derived
elicitors in the induction of the oxidative burst (for review, see Lamb and Dixon, 1997). Our model is similar to that presented by Pugin et
al. (1997) in a recent and very complete investigation regarding the
action of cryptogein in tobacco cells, in which the effects of this
elicitor on membrane depolarization, ion transport, and oxidative
metabolism appeared to be very similar to those induced by the
sulfhydryl blockers in E. densa leaves. According to our model, the H2O2 produced by
NADPH oxidase might converge with the primary effect of the sulfhydryl
reagent increasing the passive permeability of the plasma membrane to
ions. Such an effect of active oxygen species produced by
pathogen-derived elicitors in various plant materials has been proposed
by various authors (Keppler and Novacky, 1987; Levine et al., 1994;
Mehdy, 1994; Lamb and Dixon, 1997). In our case an interesting feature
of the activation of electrolyte leakage by
H2O2 was its inhibition by
La3+, suggesting that Ca2+
influx was involved. A stimulation of Ca2+ influx
by exogenous H2O2 has been
reported by Price et al. (1994) in tobacco cells, and a stimulating
effect of Ca2+ on anion efflux (and thus plasma
membrane depolarization) has been reported by Schroeder and Hagiwara
(1989). Thus, it seems possible that the effect of
H2O2 depends on its nature
as a sulfhydryl-oxidizing agent, endowed with a mode of action on
Ca2+ influx similar to that of
Ag+ or NEM, rather than on its direct interaction
with the plasma membrane.
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| Figure 11.
Proposed model for the action of the sulfhydryl
(SH) blockers and La3+ on membrane potential, electrolyte
leakage, H+ influx, O2 uptake, and
H2O2 production burst in E. densa leaves. According to this scheme, Ca2+ influx
would influence the effects on NADPH oxidase independently of its
action on ion transport and transmembrane potential. However, the
present evidence is compatible with the alternative interpretation,
that the activation of NADPH oxidase is a consequence of the changes at
the ion-transport level (see Jabs et al., 1997).
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The concentrations of H2O2
in the medium required to induce a consistent electrolyte leakage are
much higher than those that accumulated in the medium after treatment
with NEM. This might be attributable to the fact that in the first
case, H2O2 reaches the cell
from the outside, whereas in the second case, it is produced inside of
the cell. A high H2O2
concentration has been used in studies of the possible relevance of
H2O2 in pathogen-derived effects on ion transport (Apostol et al., 1989; Levine et al., 1994,
1996; Price et al., 1994). However, the very high
H2O2 concentration required
to induce leakage raises some doubt about its possible role in NEM
action.
Taken together, these new results emphasize the similarity between the
effect of the sulfhydryl blockers in E. densa and the effects of pathogen-derived elicitors on plasma membrane activities and
oxidative metabolism in a number of plant materials. This underscores
the need for a comparative analysis of the possibly common steps in the
mechanism of action of the sulfhydryl reagents and the pathogen-derived
elicitors. Useful information in this regard should come from the
investigation of the effects of nonspecific pathogen-derived elicitors
in the E. densa system, an approach that we plan to develop
in the near future. Progress in the study of the responses to the
sulfhydryl blockers is of interest not only for the understanding of
the mode of action of the sulfhydryl reagents, the nature of
plasmalemma redox systems, and the role of Ca2+
in the regulation of other ion fluxes, but also for the elucidation of
the mechanism of defense reactions in plants.
| |
FOOTNOTES |
1
This work was supported in part by the Ministero
Italiano dell' Università e della Ricerca Scientifica e
Tecnologica.
*
Corresponding author; e-mail teamarre{at}imiucca.unimi.it; fax
39-2-26604399.
Received April 22, 1998;
accepted August 31, 1998.
| |
ABBREVIATIONS |
Abbreviations:
BTP, 1,3-bis[tris(hydroxymethyl)methylamino]
propane.
Em, transmembrane electric
potential difference.
NEM, N-ethylmaleimide.
QO2, oxygen uptake.
QO2, increase in
nonmitochondrial respiration.
SOD, superoxide dismutase.
| |
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Top
Abstract
Introduction