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Plant Physiol. (1999) 120: 217-226
Salicylic Acid Induces Rapid Inhibition of Mitochondrial Electron
Transport and Oxidative Phosphorylation
in Tobacco
Cells1
Zhixin Xie and
Zhixiang Chen*
Department of Microbiology, Molecular Biology and Biochemistry,
University of Idaho, Moscow, Idaho 83844-3052
 |
ABSTRACT |
Salicylic acid (SA) is known to
induce alternative pathway respiration by activating expression of the
alternative oxidase gene. In the present study we report a rapid mode
of action by SA on plant mitochondrial functions. SA at concentrations
as low as 20 µM induced inhibition of both ATP synthesis
and respiratory O2 uptake within minutes of incubation in
tobacco (Nicotiana tabacum) cell cultures. Biologically
active SA analogs capable of inducing pathogenesis-related genes and
enhanced resistance also caused rapid inhibition of ATP synthesis and
respiratory O2 uptake, whereas biologically inactive
analogs did not. Inhibition of ATP synthesis and respiratory
O2 uptake by SA was insensitive to the protein synthesis
inhibitor cycloheximide, but was substantially reduced by the
antioxidant N-acetylcysteine, suggesting a possible role for reactive oxygen species in the inhibition of mitochondrial functions. With exogenous NADH as the respiratory substrate,
mitochondria isolated from SA-treated tobacco cell cultures were found
to have normal capacities for both ATP synthesis and respiratory
O2 uptake; direct incubation of isolated mitochondria with
SA had no significant effect on these mitochondrial functions. These
results indicate that (a) the respiration capacities of isolated
mitochondria do not correspond to the in vivo respiration activities in
SA-treated cell cultures and (b) the SA-induced inhibition of
respiration in tobacco cell cultures may involve other components that
are not present in isolated mitochondria. Given the recently
demonstrated roles of mitochondria in plant disease resistance and
animal apoptosis, this rapid inhibition by SA of mitochondrial
functions may play a role in SA-mediated biological processes,
including plant defense responses.
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INTRODUCTION |
Studies during the last several years have established that SA
plays an essential role in plant disease resistance (Klessig and
Malamy, 1994 ; Hunt et al., 1996; Yang et al., 1997). Application of
exogenous SA has long been known to activate expression of PR genes and
to induce resistance to plant diseases (White, 1979; Ward et al.,
1991). In an increasing number of plant species, elevated levels of SA
have been associated with resistance of the infected plant to the
invading pathogen (Malamy et al., 1990; Metraux et al., 1990; Uknes et
al., 1992). A large body of evidence documents the importance of this
systemic increase in SA levels for the induction of systemic acquired
resistance (Gaffney et al., 1993; Delaney et al., 1994). More recently,
genetic and molecular studies have indicated that SA also participates
in the development of the localized, hypersensitive disease resistance
induced by pathogen infection (Levine et al., 1994; Weyman et al.,
1995; Shirasu et al., 1997).
The signal transduction mechanism involved in the action of SA in plant
defense responses is poorly understood. Biochemical studies have shown
that SA and its analogs capable of inducing PR genes and disease
resistance bind to and inhibit certain heme-containing catalases and
peroxidases (Chen et al., 1993a, 1993b; Durner and Klessig, 1995).
Further studies have suggested that SA may serve as
one-electron-donating substrates for catalases/peroxidases and, in
doing so, are converted into SA free radicals in the presence of
H2O2 as electron acceptors
(Durner and Klessig, 1996; Kvaratskhelia et al., 1997). More recently,
Kawano et al. (1998) have demonstrated that in tobacco
(Nicotiana tabacum) cell cultures SA induces
extracellular superoxide generation by interacting with a
SHAM-sensitive extracellular peroxidase. Based on their established
roles in signal transduction, these SA-mediated ROS could be involved
in certain aspects of action by SA in plant defense responses. Other
studies on SA signaling pathways have focused on the regulation of the
genes induced by SA. These studies have provided evidence for the
possible involvement of protein phosphorylation in the transcriptional
regulation of at least some of these SA-responsive genes (Jupin and
Chua, 1996; Stange et al., 1997). A role for protein phosphorylation in
SA signal transduction is supported by the observed suppression of SA
action by protein kinases/phosphatase inhibitors and by the recent
identification of an SA-activated MAP kinase (Conrath et al., 1997;
Shirasu et al., 1997; Zhang and Klessig, 1997).
In addition to biochemical and molecular studies, genetic strategies
have been applied to the dissection of SA signal transduction pathways
by isolating and characterizing SA-insensitive mutants in Arabidopsis.
These screenings have identified a number of mutants that fail to
express PR genes and exhibit enhanced resistance to bacterial and
fungal pathogens in response to treatment with SA or its functional
analogs, 2,5-dichloroisonicotinic acid and benzothiodiazole (Cao et
al., 1997; Ryals et al., 1997; Shah et al., 1997). It is interesting
that all of these reported SA-insensitive mutants are allelic, caused
by mutations in a gene encoding a 60-kD protein with ankyrin repeats
and some homology to the animal I B protein (Cao et al., 1997; Ryals
et al., 1997). It is possible that other genes involved in this signal
transduction pathway have not been identified by screening for
SA-insensitive mutants because of their functional redundancy. In
addition, some of these regulatory components may be essential for
other important biological processes, so that mutations causing severe
reduction in their biological activities may be deleterious or even
lethal to the plants.
It has recently been demonstrated that SA-induced tobacco resistance to
TMV is sensitive to SHAM, an inhibitor of the terminal oxidase of the
mitochondrial alternative pathway (Chivasa et al., 1997). Moreover, the
respiratory inhibitors antimycin A and cyanide induced alternative
oxidase transcript accumulation and resistance to TMV (Chivasa and
Carr, 1998). Furthermore, cyanide restores N gene-mediated resistance
to TMV in transgenic tobacco that expresses the salicylate hydroxylase
(nahG) gene (Chivasa and Carr, 1998). These results suggest
that certain functions of plant mitochondria may play an important role
not only in SA-induced thermogenesis, as previously demonstrated
(Raskin et al., 1987), but also in SA-induced disease resistance.
To pursue these studies of the involvement of plant mitochondria in the
response to SA, we have recently examined the effect of SA on the most
important function of this organelle: oxidative phosphorylation (ATP
synthesis). We have discovered that SA induces rapid inhibition of ATP
synthesis when it is incubated with tobacco cell cultures. Based on the
concomitant inhibition of respiratory O2 uptake,
SA-induced inhibition of ATP synthesis appears to be caused by
inhibited mitochondrial electron transport, rather than by enhanced
alternative pathway respiration, which is known to be induced by SA
(Kapulnik et al., 1992; Rhoads and McIntosh, 1992, 1993; Wagner, 1995;
Lennon et al., 1997). Thus, in addition to the induction of alternative
pathway respiration, which is dependent on the expression of the
alternative oxidase gene, SA has a rapid mode of action on electron
transport and oxidative phosphorylation of plant mitochondria. In the
present paper, we discuss this rapid inhibition of plant mitochondrial
functions by SA with respect to its possible mechanisms and biological
roles in SA-induced biological processes, including plant defense
responses.
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MATERIALS AND METHODS |
Chemicals and Plant Culture
We obtained [ -32P]ATP and
32Pi from New England Nuclear and SA, SA analogs,
and other common chemicals from Sigma. SA and its analogs were
dissolved in water as 100 mM stock solutions and adjusted
to pH 6.5 with KOH.
All experiments were carried out with tobacco (Nicotiana
tabacum cv Xanthi-nc) cell cultures. The cell-suspension cultures were grown at room temperature on a rotary shaker at 110 rpm in the
dark in Murashige and Skoog medium supplemented with 1 µg mL 1  -NAA, 0.1 µg
mL1 2,4-D, and 0.1 µg
mL1 BA (Xie et al., 1998). Cells were
maintained by a 10-fold dilution with fresh medium every 5 to 6 d.
Two days after dilution the cells were used for all of the
experiments.
Isolation of Mitochondria
We adopted the method of Rhoads and McIntosh (1991), which is a
modification of the procedure of Schwitzguebel and Siegenthaler (1984),
to prepare the mitochondria. We washed the tobacco suspension-culture cells (approximately 280 mL) twice with fresh medium and ground them
with glass beads in a mortar and pestle in 20 mL of mitochondrial grinding buffer (0.35 M mannitol, 30 mM Mops,
pH 7.5, 4 mM Cys, 1 mM EDTA, 0.2% BSA, and
0.6% PVP). The homogenate was centrifuged for 2 min at
5,000g. The supernatant was then centrifuged again for 10 min at 20,000g, and the pellet was resuspended directly in a
reaction medium (250 mM Suc and 30 mM Mops, pH 6.8). The quality of the isolated
mitochondria was determined by demonstrating the dependence of ATP
synthesis and respiratory O2 uptake on the addition of an electron-donating substrate (e.g. NADH), which could be
further enhanced by the addition of ADP and Pi.
Analysis of ATP Synthesis
ATP synthesis of tobacco cell cultures after chemical treatment
was determined by direct labeling of [32P]Pi,
followed by homogenization, extraction, and TLC separation. Cell
cultures (1 mL) were incubated with SA at various concentrations for 10 to 30 min before the addition of 32Pi (4 µCi in
5 µL). After labeling for 10 min, the cells were washed with 1 mL of
cold medium three times and resuspended in 200 µL of medium. After
adding an equal volume of 6% perchloric acid, the cells were briefly
sonicated. We then centrifuged the resulting homogenates for 10 min in
a microcentrifuge to collect the supernatant. To every 300 µL of
supernatant, we added 66 µL of 2 N KOH/0.5
M triethanolamine to neutralize the pH. After incubation on
ice for 30 min followed by centrifugation in a microcentrifuge for 5 min, the supernatants (10 µL) with the same amount of radioactivity were loaded onto a TLC plate precoated with silica gel (Analtech, Newark, DE). Adenine nucleotides and 32Pi were
separated using an elution medium containing dioxane:isopropanol:25% NH4OH:H2O (4:2:3:4, v/v)
and 10 mM EDTA (Bronnikov and Zakharov, 1983). We
identified the unmetabolized 32Pi and the
synthesized [32P]ATP on the plates using
[32P]ATP and 32Pi as the
standards after autoradiography. ATP synthesis of isolated mitochondria
was determined in the mitochondrial reaction buffer in the presence of
1 mM NADH.
Analysis of Total Cellular ATP Levels
We determined total cellular ATP levels using the
luciferin-luciferase assay. After treatment we added 1 mL of cell
cultures and 1 mL of 6% ice-cold perchloric acid. The cells were
sonicated for 1 min and centrifuged for 10 min in a microcentrifuge. To adjust the pH from 7.5 to 8.0, we added 220 µL of 2 N
KOH/0.5 M triethanolamine to 1 mL of supernatant. The tubes
were placed on ice for 30 min to let the potassium perchloric acid
precipitate. After centrifugation for 5 min, the supernatant was
collected. To assay ATP levels, we added 100 µL of
luciferine-luciferase buffer (50 mM Gly, pH 8.0, 7.5 mM DTT, 1 mM EDTA, 2 mM
MgSO4, 15 µM luciferine, and 5 µg
mL1 luciferase) to 200 µL of 1:100 diluted
supernatant. The signals were integrated for 10 s in a LUMAT
luminometer (model LB9507, EG&G, Berthold, Germany). The actual ATP
levels were calculated from an ATP standard curve constructed with
commercially purchased ATP.
Respiration
Cells (5 mL, approximately 100 mg fresh weight) in the culture
medium were placed in an O2 electrode unit (YSI,
Yellow Springs, OH) at room temperature to measure respiratory
O2 uptake. The respiratory
O2 uptake of the isolated mitochondria was
determined in the mitochondrial reaction buffer in the presence of 1 mM NADH. We assumed the O2 in the
air-saturated medium to be 240 µM (Schwitzguebel and
Siegenthaler, 1984). We repeated all of the assays for respiratory O2 uptake three times with either independently
subcultured cells or prepared mitochondria.
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RESULTS |
Rapid Inhibition of ATP by SA
We made our initial observation on SA-induced rapid inhibition of
ATP synthesis from experiments designed to test for a possible change
in protein phosphorylation in tobacco cell cultures after treatment
with SA. In these experiments, tobacco cell cultures were treated with
SA for 30 min and labeled with 32Pi. The
treated/labeled cells were homogenized and subjected to SDS-PAGE to
detect proteins with altered phosphorylation as a result of SA
treatment. These experiments revealed drastically decreased intensities
of a low-Mr species in SA-treated samples that migrated behind 32Pi molecules, but
ahead of all detectable protein molecules. Based on the migration rate
and the high abundance in untreated cell cultures, we suspect that this
band corresponded to the labeled ATP molecules, which appeared to be
drastically decreased in the SA-treated samples. To verify this
possibility, we used more reliable TLC procedures with
[-32P]ATP and 32Pi as
the standards to establish the identity of the labeled band. As shown
in Figure 1A (lane 1), brief labeling of
untreated cells with 32Pi produced a band on the
TLC plates that comigrated with the [-32P]ATP standard, in addition to the much
more abundant band that corresponded to the unmetabolized
32Pi molecules. The identity of the minor band as
32P-labeled ATP was further established by its
susceptibility to treatment with ATP-hydrolyzing apyrase (Fig. 1A, lane
2). Apyrase catalyzes the hydrolysis of phosphoanhydride bonds of
nucleoside tri- and diphosphates (e.g. ATP and ADP), but not the
hydrolysis of phosphomonoester bonds of nucleoside monophosphate (e.g.
AMP) or sugar phosphates (Cori et al., 1965).
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| Figure 1.
Inhibition of ATP synthesis of tobacco cell
cultures by SA. A, Separation and identification of ATP and
32Pi on TLC plates. Separations were for supernatants from
untreated cell cultures (lane 1) or the same supernatants that had been
incubated at 30°C for 30 min in the presence (lane 2) or absence
(lane 3) of 10 units of the ATP-hydrolyzing apyrase. B, Time course of
inhibition of ATP synthesis by 1 mM SA. C, Concentration
curve for inhibition of ATP synthesis after incubation for 10 min with
20 to 500 µM SA. The first two lanes in A and B are
commercially purchased [-32P]ATP and 32Pi
used as the standards.
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Treatment with 1 mM SA for as short as 10 min effectively
inhibited the synthesis of cellular ATP in the tobacco cell cultures (Fig. 1B). In fact, treatment of the cell cultures for 10 min with SA
at concentrations as low as 20 µM inhibited ATP
synthesis, although a high level of inhibition was observed only at SA
concentrations higher than 50 µM (Fig. 1C). To determine
the specificity, we tested SA analogs for their ability to inhibit ATP
synthesis in tobacco cell cultures. Among these analogs,
5-chlorosalicylic acid and acetylsalicylic acid (aspirin) are
biologically active analogs that induce both PR gene expression and
disease resistance in tobacco, whereas 3-hydroxybenzoic acid and
4-hydroxybenzoic acid are not biologically active in inducing these
defense responses (Conrath et al., 1995). As shown in Figure
2, although biologically active SA,
acetylsalicylic acid, and 5-chlorosalicylic acid effectively inhibited
ATP synthesis at a similar potency, biologically inactive 3hydroxybenzoic acid and 4-hydroxybenzoic acid failed to have any
significant effect on ATP synthesis. Two additional biologically inactive benzoic acid derivatives (2,3-dihydroxybenzoic acid, and
2,4-dihydroxybenzoic acid) have also been tested and found to be
ineffective in inhibiting ATP synthesis of tobacco cell cultures (data
not shown).
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| Figure 2.
Inhibition of ATP synthesis by SA analogs. Tobacco
cell cultures were incubated with 0.5 mM SA,
acetylsalicylic acid (ASA), 5-chlorosalicylic acid (5-CSA),
3-hydroxybenzoic acid (3-HBA), or 4-hydroxybenzoic acid (4-HBA) for 30 min before assay for ATP synthesis.
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The decreased levels of 32P-labeled ATP in
SA-treated cells could result from a decreased uptake of
32Pi, rather than from inhibited ATP synthesis.
To test this possibility we directly measured the uptake of
32Pi by tobacco cell cultures. After treatment
with SA or its analogs for 30 min, these tobacco cells were labeled
with 32Pi for 10 min, washed three times in cold
medium, and counted for retained radioactivities. These experiments
established that treatment with SA or its analogs had no significant
effect on the uptake of 32Pi by tobacco cell
cultures (Fig. 3).
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| Figure 3.
Uptake of 32Pi by tobacco cells after
pretreatment with SA and analogs. Tobacco cell cultures were
pretreated with 0.5 mM SA, acetylsalicylic acid (ASA),
5-chlorosalicylic acid (5-CSA), 3-hydroxybenzoic acid (3-HBA), or
4-hydroxybenzoic acid (4-HBA) for 30 min followed by addition of
32Pi. After labeling for 10 min, the cells were washed
three times with cold medium, and the radioactivity retained by cells
was determined by scintillation counting and reported (+SE
calculated from three independent experiments).
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To further confirm the inhibition of ATP synthesis by SA, we
directly measured the total cellular ATP levels in the tobacco cell
cultures after SA treatment. As shown in Figure
4A, treatment with SA at concentrations
from 50 to 200 µM significantly decreased total ATP
levels. Treatment with 500 µM SA decreased ATP levels by
50% within the first 30 min of incubation, after which the ATP level
continued to decrease to as low as 15% of the control levels at the
end of assays (Fig. 4A). Furthermore, those SA analogs (acetylsalicylic acid and 5-chlorosalicylic acid) capable of inhibiting ATP synthesis were also able to deplete total cellular ATP levels, whereas the SA analogs (3-hydroxybenzoic acid and 4-hydroxybenzoic acid) incapable of inhibiting ATP synthesis did not significantly affect the cellular ATP levels of tobacco cell cultures (Fig. 4B).
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| Figure 4.
Depletion of total cellular ATP in tobacco cell
cultures after treatment with SA or its analogs. A, Tobacco cell
cultures were incubated with various concentrations of SA. At indicated
time points, cell samples were taken for determining total cellular ATP
levels. B, Tobacco cell cultures were incubated with 0.5 mM
SA, acetylsalicylic acid (ASA), 5-chlorosalicylic acid (5-CSA),
3-hydroxybenzoic acid (3-HBA), or 4-hydroxybenzoic acid (4-HBA). ATP
levels were determined after 6 h of incubation and reported
(+SE calculated from three independent assays). FW, Fresh
weight.
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Rapid Inhibition of Respiratory O2 Uptake by SA
Inhibition of ATP synthesis in SA-treated tobacco cell cultures
could be caused by (a) blockage of mitochondrial electron transport,
(b) conversion of electron transport from the cytochrome oxidase
pathway to the alternative oxidase pathway, which differ in their
capacity for ATP synthesis, and/or (c) uncoupling of electron transport
from oxidative phosphorylation as a result, for example, of
depolarization of the mitochondria inner membrane potential. For the
first mechanism, inhibited ATP synthesis is associated with decreased
respiratory O2 uptake. For the second or third
mechanism, however, inhibition of ATP synthesis may not necessarily
lead to a decrease in respiratory O2 uptake. To
distinguish between these possibilities, we directly measured the
respiratory O2 uptake of tobacco cell cultures
after SA treatment. As shown in Figure
5A, treatment of the cell cultures for 10 min with SA decreased respiratory O2 uptake with
a dose response similar to that observed for inhibition of ATP
synthesis (Fig. 1C). Furthermore, those SA analogs that inhibited ATP
synthesis also inhibited respiratory O2 uptake,
whereas those analogs incapable of inhibiting ATP synthesis failed to
inhibit respiratory O2 uptake (Fig. 5B). Thus,
inhibition of ATP synthesis by SA or its analogs was associated with
inhibited respiratory O2 uptake. In an earlier
report, Kapulnik et al. (1992) showed that 200 µM SA
inhibited respiratory O2 uptake of tobacco cell
cultures by approximately 50%. This relatively low sensitivity of
respiratory O2 uptake to SA may have been due to
the old age (7 d) of the cells used in the earlier study.
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| Figure 5.
Inhibition of respiratory O2 uptake by
SA and its analogs. A, Tobacco cell cultures were incubated with
various concentrations of SA for 10 min before the measurement for
respiratory O2 uptake. B, Tobacco cell cultures were
incubated with 0.5 mM SA, acetylsalicylic acid (ASA),
5-chlorosalicylic acid (5-CSA), 3-hydroxybenzoic acid (3-HBA), or
4-hydroxybenzoic acid (4-HBA) for 30 min before the measurement for
respiratory O2 uptake. FW, Fresh weight.
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Effect of the Protein Synthesis Inhibitor Cycloheximide
It has been shown previously that SA induced alternative pathway
respiration in tobacco cell cultures by activating expression of the
alternative oxidase gene (Kapulnik et al., 1992; Rhoads and McIntosh,
1993). Thus SA-induced alternative pathway respiration was sensitive to
inhibitors of transcription and protein synthesis (Rhoads and McIntosh,
1993). To test whether the rapid inhibition of ATP synthesis and
respiratory O2 uptake by SA is also dependent upon protein synthesis, we tested the sensitivity of SA inhibition effects to the protein synthesis inhibitor cycloheximide. Tobacco cell
cultures were pretreated with 100 µg mL1
cycloheximide for 30 min before treatment with SA for 30 min; both the
ATP synthesis and the respiratory O2 uptake were
then analyzed and compared with cell cultures without the pretreatment. As shown in Figure 6, pretreatment with
cycloheximide did not block SA-induced inhibition of ATP synthesis or
respiratory O2 uptake. Rhoads and McIntosh (1993)
showed that cycloheximide at this concentration effectively blocked
SA-induced expression of the alternative oxidase gene and alternative
pathway respiration in tobacco cell cultures.
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| Figure 6.
Effects of cycloheximide (CHX) on SA-induced
inhibition of ATP synthesis (A) and respiratory O2 uptake
(B). Tobacco cell cultures were preincubated with 100 µg
mL1 cycloheximide for 30 min before treatment with 100 to
500 µM SA. After SA treatment for 30 min, ATP synthesis
and respiration of treated cells were determined. FW, Fresh weight;
lanes  , no CHX; lanes +, with CHX.
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Effect of the Antioxidant NAC
Several recent studies have shown that SA and its biologically
active analogs inhibit catalases/peroxidases and induce lipid peroxidation and generation of ROS in tobacco cell cultures (Chen et
al., 1993b; Conrath et al., 1995; Durner and Klessig, 1995, 1996; Kvaratskhelia et al., 1997; Kawano et al., 1998). To test for the
possible involvement of ROS in SA-induced inhibition of ATP synthesis
and respiratory O2 uptake, we tested the effects of a commonly used antioxidant, NAC, which was previously shown to
block SA- and BTH-induced PR-1 protein synthesis in tobacco (Wendehenne
et al., 1998). Tobacco cell cultures were pretreated with 20 mM NAC for 1 h before treatment with SA for 30 min;
both the ATP synthesis and the respiratory O2
uptake were then analyzed and compared with cell cultures without
antioxidant pretreatment. As shown in Figure
7, pretreatment with NAC without
subsequent treatment with SA had no significant effect on either ATP
synthesis or respiratory O2 uptake in control
cell cultures. However, for cell cultures that were subsequently
treated with SA, NAC pretreatment decreased SA-induced inhibition of
ATP synthesis and respiratory O2 uptake by
approximately 50%. This result demonstrates that ROS production
probably plays a significant role in the SA-induced rapid inhibition of
ATP synthesis and respiratory O2 uptake.
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| Figure 7.
Effects of NAC on SA-induced inhibition of ATP
synthesis (A) and respiratory O2 uptake (B). Tobacco cell
cultures were preincubated with 20 mM NAC for 1 h
before treatment with 100 to 500 µM SA. After SA
treatment for 30 min, ATP synthesis and respiration of treated cells
was determined. FW, Fresh weight; lanes , no NAC; lanes +, with
NAC.
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Isolated Mitochondria
The rapid inhibition of ATP synthesis and respiration by SA in the
tobacco cell cultures observed in this study was not consistent with an
earlier report that treatment of tobacco cell cultures with 1 mM SA resulted in increased capacities of alternative
pathway respiration without significant change in cytochrome pathway
respiration (Rhoads and McIntosh, 1993). However, unlike the current
study, which measured ATP synthesis and respiratory
O2 uptake directly in cell cultures, the earlier
report measured respiration in mitochondria isolated from SA-treated
cell cultures. Thus, the mitochondria isolated from SA-treated tobacco
cell cultures may be capable of respiratory O2
uptake. To confirm this, we isolated mitochondria from SA-treated cell
cultures and determined their capacities for both ATP synthesis and
respiratory O2 uptake with exogenously added NADH
as the respiratory substrate. As shown in Figure
8, SA-treated tobacco cell cultures
showed inhibition of both ATP synthesis and respiratory
O2 uptake, whereas the mitochondria isolated from
these respiration-suppressed cell cultures exhibited normal capacities
in both ATP synthesis and respiratory O2 uptake. Thus, the high capacities in respiration found in mitochondria isolated
from SA-treated cells did not reflect the in vivo rates of respiration.
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| Figure 8.
Capacities of ATP synthesis and respiratory
O2 uptake of isolated mitochondria. A, Capacity of ATP
synthesis for mitochondria isolated from cell cultures treated with 0 mM (lanes 1 and 2) or 0.5 mM (lanes 3 and 4) SA
for 30 min. Before ATP synthesis assays, the isolated mitochondria were
again treated with 0 mM (lanes 1 and 3) or 0.5 mM (lanes 2 and 4) SA for 30 min. B, Respiratory
O2 uptake for isolated mitochondria. , No SA; +, with
SA.
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Inhibition of respiration in SA-treated cell cultures could be caused
by the direct effects of SA on the components of mitochondria that are
important for electron transport and oxidative phosphorylation. Alternatively, SA may induce generation of certain molecules such as
ROS, which subsequently inhibit respiration in SA-treated cells. To
distinguish between these two possibilities, we tested the effects of
the direct incubation of isolated mitochondria with SA on their
capacities of ATP synthesis and respiratory O2
uptake. As shown in Figure 8, no significant inhibition of respiration was observed in SA-treated mitochondria when assayed with exogenous NADH as the reducing substrates.
| |
DISCUSSION |
In the present study we show that 20 to 500 µM SA
inhibited ATP synthesis in tobacco cell cultures. Two lines of evidence indicated that this effect did not appear to depend on the induction of
alternative pathway respiration by SA that had previously been documented (Kapulnik et al., 1992; Rhoads and McIntosh, 1992, 1993;
Wagner, 1995). First, inhibition of ATP synthesis by SA required only
minutes of SA incubation and was insensitive to the protein synthesis
inhibitor cycloheximide. In contrast, SA-induced alternative
respiration is associated with de novo synthesis of the alternative
oxidase protein and requires hours to reach maximum levels (Kapulnik et
al., 1992; Rhoads and McIntosh, 1993). Second, the rapid inhibition of
ATP synthesis by SA is associated with decreased respiratory
O2 uptake, suggesting the blockage of electron transport for both the cytochrome oxidase and alternative oxidase pathways.
How SA incubation leads to such a rapid inhibition of ATP synthesis and
respiratory O2 uptake in tobacco cell cultures is unclear. Because inhibition of respiratory O2
uptake was nearly complete at relatively high SA concentrations, SA
appears to inhibit both the cytochrome oxidase pathway and the
alternative oxidase pathway. Therefore, the potential SA target(s) must
be important for both respiratory pathways. One possible candidate is
complex I of the mitochondrial electron transport chain, which provides electrons for both electron transport pathways. Alternatively, SA may
target components in the TCA cycle that provide the reducing substrates
for electron transport. Aconitase, an enzyme in the TCA cycle, was
previously shown to bind SA at relatively high affinity (Rüffer
et al., 1995). Either of these two mechanisms should predict normal
respiratory O2 uptake in mitochondria isolated from SA-treated cells, as long as exogenously added NADH is available to provide electrons directly to ubiquinone pools through the external
NADH dehydrogenase. This route of electron transport bypasses the TCA
cycle and/or complex I of the electron transport chain. This mechanism
may also explain why respiratory O2 uptake in
tobacco leaves did not change after SA treatment (Lennon et al., 1997):
cytosolic NADPH or NADH generated from photosynthesis would feed into
the mitochondrial electron chain without passing through inhibited SA
target(s).
The observed antagonism of the SA-induced inhibition of ATP synthesis
and respiratory O2 uptake by the antioxidant NAC
suggests that SA-induced inhibition of mitochondrial functions may
involve ROS. Very recently, Kawano et al. (1998) have shown that SA
induces rapid generation of extracellular superoxides in tobacco cell cultures. This rapid generation of ROS was found to be caused by the
interaction of SA with a SHAM-sensitive extracellular
guaiacol-utilizing peroxidase in tobacco cells. Because SA can serve as
one-electron-donating substrate for heme-containing
peroxidases/catalases (Durner and Klessig, 1996; Kvaratskhelia et al.,
1997), this interaction of SA with extracellular peroxidase should lead
to the oxidation of SA into its free radical form and, consequently,
other ROS (Kawano et al., 1998). Because intracellular heme-containing
catalases and peroxidases are known to interact with SA (Chen et al.,
1993b; Conrath et al., 1995; Durner and Klessig, 1995),
intracellular generation of superoxides is also possible through a
similar mechanism. These SA-mediated ROS could lead to the inhibition
of key enzymes involved in the TCA cycles and/or the mitochondrial
electron transport. For example, Hausladen and Fridovich (1994) found
that several dehydratases bearing Fe-S prosthetic groups were
inactivated by superoxide. Aconitase in the TCA cycle and several
components in the electron transport chain of mitochondria contained
Fe-S prosthetic groups and, therefore, could serve as potential targets for ROS.
SA is cytotoxic to plant cells at high concentrations (Allan and Fluhr,
1997; Anderson et al., 1998; Kawano et al., 1998). The rapid depletion
of ATP in cell cultures incubated with high levels of SA (>200
µM) could be one of the mechanisms responsible for the
cytotoxicity of SA. At relatively low levels of SA (20-100 µM, close to the physiological levels found in
pathogen-infected plants), perturbation of mitochondrial functions may
not be sufficient to cause cell death directly but could still play a
role in SA-mediated defense responses. The observation that only SA and
its analogs capable of inducing PR genes and disease resistance can
also inhibit these mitochondrial functions is consistent with a
possible role for the rapid inhibition of ATP synthesis and respiratory
O2 uptake in SA-mediated plant defense responses.
In TMV-infected resistant tobacco plants, SA levels rose to
approximately 50 to 100 µM in the cells immediately
surrounding the lesions (Enyedi et al., 1992). These levels of SA
should lead to significant inhibition of mitochondrial functions. In
upper uninfected leaves, however, SA levels increased by only 1.2- to 4-fold to approximately 0.06 to 0.2 µg g1
fresh weight or 0.5 to 1.5 µM (Enyedi et al., 1992;
Vernooij et al., 1994). At these low levels, inhibition of
mitochondrial functions would be insignificant based on the
dose-response curve established in this study (Figs. 1 and 4). However,
a comparison of exogenously applied SA concentrations with endogenously
produced SA levels as a means for determining physiological relevance
is misleading, because it does not take into consideration a number of
important factors that affect SA uptake, metabolism, and
compartmentalization by plant cells. In fact, although TMV-induced,
systemic-acquired resistance in resistant tobacco plants is associated
with only a small increase of endogenous SA levels in upper uninfected
leaves, much higher levels of exogenous SA (typically in the range of 1-5 mM) are often required to induce a similar level of
resistance and defense gene expression. For determining the
physiological relevance of a possible biochemical mechanism induced by
exogenous SA, it may be more appropriate to compare it with the
concentrations of exogenous SA required for inducing disease resistance
and defense gene expression.
The most direct evidence for a role of mitochondrial functions in SA
signal transduction comes from the studies on the effects of oxidative
respiration inhibitors on SA-induced resistance to viral pathogens
(Chivasa et al., 1997; Chivasa and Carr, 1998). These studies suggest
that SA-induced resistance to virus may be mediated by an induced
SHAM-sensitive signaling pathway, which probably involves alternative
oxidases. Because inhibitors of electron transport and certain enzymes
(e.g. aconitase) in the TCA cycle induced alternative oxidase gene
expression (Vanlerberghe and McIntosh, 1994, 1996), SA may induce
alternative oxidase gene expression through its rapid inhibitory effect
on mitochondrial electron transport. Furthermore, inhibition of
aconitase by SA or SA-induced ROS may lead to the increased levels of
citrate that serve as a signal for inducing alternative oxidase gene
expression and, consequently, the capacity of alternative pathway
respiration (Vanlerberghe and McIntosh, 1996).
SA-induced rapid inhibition of ATP synthesis and respiratory
O2 uptake may also play a role in the involvement
of SA in pathogen-induced hypersensitive response. Recent studies have
shown that pathogen-induced hypersensitive cell death in plants is a
form of programmed cell death and shares many mechanistic features with
animal apoptosis (Greenberg, 1996; Mittler et al., 1997). A variety of
key events in animal apoptosis are associated with mitochondria,
including the release of caspase activators (such as cytochrome
c), changes in electron transport, disruption of oxidative
phosphorylation, loss of mitochondrial transmembrane potential, and
altered cellular reduction-oxidation (redox) potential (Green and Reed,
1998). Based on the present studies of the rapid alteration of
mitochondrial functions and the previously demonstrated induction by SA
of ROS (Chen et al., 1993b; Rao et al., 1997; Kawano et al.,
1998), the involvement of SA in pathogen-induced hypersensitive cell
death could be mediated in part by these interrelated events associated with the functions of plant mitochondria.
| |
FOOTNOTES |
1
This work was supported in part by Idaho
Agricultural Experimental Station and U.S. Department of Agriculture
grant no. 96-36301-3316 to Z.C. Z.X. was supported by a Plant
Biotechnology Graduate Assistantship from the University of Idaho
Institute for Molecular and Agricultural Genetic Engineering.
*
Corresponding author; e-mail zchen{at}uidaho.edu; fax
1-208-885-6518.
Received October 15, 1998;
accepted February 2, 1999.
| |
ABBREVIATIONS |
Abbreviations:
apyrase, ATP-diphosphohydrolase.
NAC, N-acetylcysteine.
PR, pathogenesis-related.
ROS, reactive oxygen species.
SA, salicylic acid.
SHAM, salicylhydroxamic
acid.
TMV, tobacco mosaic virus.
| |
ACKNOWLEDGMENTS |
We would like to thank Dr. Allan Caplan for critically reading
the manuscript and Dr. Rolf Ingermann for providing access to the
luminometer for ATP assays.
| |
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Abstract
Introduction