|
Plant Physiol. (1999) 121: 197-206
The Generation of Active Oxygen Species Differs in Tobacco and
Grapevine Mesophyll Protoplasts1
Anastasia K. Papadakis and
Kalliopi A. Roubelakis-Angelakis*
Department of Biology, University of Crete, P.O. Box 2280, 71 409 Heraklio, Greece
 |
ABSTRACT |
Our previous results have shown that
oxidative stress may reduce the regeneration potential of protoplasts,
but only protoplasts that are able to supply extracellularly
H2O2 can actually divide (C.I. Siminis, A.K.
Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87:
263-270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375-1383; A. de Marco, K.A.
Roubelakis-Angelakis [1996a] Plant Physiol 110: 137-145; A. de
Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149:
109-114). In the present study we have attempted to break down
the oxidative burst response into the individual active oxygen species
(AOS) superoxide (O2· ) and
H2O2, and into individual AOS-generating
systems during the isolation of regenerating tobacco (Nicotiana
tabacum L.) and non-regenerating grape (Vitis
vinifera L.) mesophyll protoplasts. Wounding leaf tissue or
applying purified cellulase did not elicit AOS production. However, the
application of non-purified cellulase during maceration induced a burst
of O2· and H2O2
accumulation in tobacco leaf, while in grape significantly lower levels
of both AOS accumulated. AOS were also generated when protoplasts
isolated with purified cellulase were treated with non-purified
cellulase. The response was rapid: after 5 min, AOS began to accumulate
in the culture medium, with significant quantitative differences
between the two species. In tobacco protoplasts and plasma membrane
vesicles, two different AOS synthase activities were revealed, one that
showed specificity to NADPH and sensitivity to diphenyleneiodonium
(DPI) and was responsible for O2·
production, and a second NAD(P)H activity that was sensitive to KCN and
NaN3, contributing to the production of both AOS. The first
activity probably corresponds to a mammalian-like NADPH oxidase and the
second to a NAD(P)H oxidase-peroxidase. In grape, only one
AOS-generating activity was detected, which corresponded to a NAD(P)H
oxidase-peroxidase responsible for the generation of both AOS.
| |
INTRODUCTION |
The active oxygen species (AOS), superoxide
(O2·),
H2O2, and the hydroxyl
radical are inevitably produced in higher plant cells during normal
metabolism (Scandalios, 1993 ). Their accumulation is enhanced by
exposure to environmental stresses, chemicals, and microbial factors
(Sutherland, 1991; Scandalios, 1993; Baker and Orlandi, 1995).
Overproduction of reduced and chemically reactive AOS results in
oxidative stress and cellular damage (Scandalios, 1993). On the
other hand, AOS also play a positive role in plant growth and
development. Some of the better studied downstream responses promoted
by H2O2 are its
participation in the polymerization of lignin, suberin, and possibly
other cell wall components, the induction of defense-related genes, the
stimulation of phytoalexin production, and the promotion of the
hypersensitive response (for review, see Low and Merida, 1996; Lamb and
Dixon, 1997, and refs. therein).
O2· and
H2O2 also act directly as
antimicrobial agents and may play a signaling role in inducing other
defense mechanisms (Levine et al., 1994; Jabs et al., 1997).
The mechanism(s) of synthesis of AOS is a topic of active discussion
(for review, see Sutherland, 1991; Low and Merida, 1996; Wojtaszek,
1997). The earliest hypothesis on the origin of apoplastic H2O2 during the
lignification of horseradish roots involved the reduction of
O2 to
O2· by phenolic and
NAD· radicals produced by peroxidase, and the
subsequent formation of
H2O2 by the dismutation of
O2· (Gross et al.,
1977; Elstner and Heupel, 1978; Halliwell, 1978). A second model was
proposed for the production of
H2O2 induced in cultured
French bean cells with a Colletotrichum lindemuthianum cell
wall elicitor and involved an apoplastic peroxidase. 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 hydrolyzed to release
H2O2 (Bolwell et al., 1995). The third hypothesis on AOS generation, which is gaining increased interest in the literature, involves a transmembrane O2· synthase, NAD(P)H
oxidase, which transfers electrons from cytoplasmic NAD(P)H to
O2 to form
O2·, which is then
dismutated to H2O2 (Doke
and Miura, 1995; Low and Merida, 1996; Murphy and Auh, 1996; Van
Gestelen et al., 1997).
Evidence supports the similarity of NAD(P)H oxidase in plant cells and
NADPH oxidase in phagocytic animal cells involved in H2O2 production. In
particular, antibodies raised against components of the mammalian
neutrophil NADPH oxidase cross-react with polypeptides of similar
molecular masses in plants (Desikan et al., 1996; Kieffer et al., 1997;
Xing et al., 1997). Moreover, the activity of plant NAD(P)H oxidase is
inhibited by diphenylene iodonium (DPI), a well-known inhibitor of the
mammalian plasma membrane oxidase (Levine et al., 1994; Auh and Murphy,
1995; Jabs et al., 1997). Furthermore, a cDNA from rice (Groom et al.,
1996) and a full-length clone of Arabidopsis (Keller et al., 1998)
homologous to one integral component of the mammalian NADPH oxidase
have recently been characterized.
Plant protoplasts undergo complex metabolic alterations during their
isolation and culture. The expression of totipotency in cultured
protoplasts is a prerequisite for plant regeneration; however, many
important agricultural plant genera, including Vitis, exhibit recalcitrance. Oxidative stress has been proposed to contribute to the recalcitrance of plant protoplasts (Cutler et al., 1991; Roubelakis-Angelakis, 1993; Siminis et al., 1993, 1994; de Marco and
Roubelakis-Angelakis, 1996a, 1996b, 1999). Reduced oxygen species were
generated during the enzymic maceration of cell walls during cereal
protoplast isolation (Ishii, 1987). Endoxylanase, an enzymic component
of some commercial cellulase and pectinase preparations, induced
alterations in membrane integrity in tobacco (Nicotiana
tabacum L.) (Bailey et al., 1992), and caused necrosis in cereal
cells (Ishii, 1988) and tobacco protoplasts (Sharon et al., 1993).
Cultured grape cells exhibited hypersensitive response-like necrosis
after the addition of non-purified cellulase (Calderon et al., 1994).
Ishii (1988) suggested that the treatment of cells with xylanase
generates O2· anions.
In an effort to understand the mechanism(s) governing the regeneration
of plant protoplasts, and to identify potential factors contributing to
their recalcitrance, we optimized the isolation and culture conditions
(Theodoropoulos and Roubelakis-Angelakis, 1990) and studied the
biochemical and ultrastructural characteristics of cell wall
reconstitution (Katsirdakis and Roubelakis-Angelakis, 1992a), the
protein profile, and the activities and profiles of several enzymes
contributing to cell defense against oxidative stress (Siminis et al.,
1993, 1994; de Marco and Roubelakis-Angelakis, 1996a, 1996b, 1999)
between regenerating tobacco and non-regenerating grape protoplasts. In
addition, by modifying the procedure of protoplast isolation, we were
able to identify two populations of tobacco protoplasts, one that
readily regenerated, and one that did not (Siminis et al., 1994). A
comparative study indicated a simultaneous higher scavenging activity
and H2O2 accumulation potential in regenerating tobacco protoplasts (de Marco and
Roubelakis-Angelakis, 1996a, 1996b).
In this work we have attempted to reveal the site and mechanism(s) of
AOS generation during isolation of protoplasts from two plant species,
grape and tobacco. We show that wounding of grape and tobacco leaf
tissue alone or the use of purified cellulase for protoplast isolation
does not induce AOS production. The addition of non-purified cellulase
to leaf strips induced a burst of
O2· and
H2O2 accumulation in
tobacco leaves, while in grape, significantly lower levels of both AOS
accumulated. Protoplasts isolated from tobacco and grape leaves with
purified cellulase responded differently to the addition of
non-purified cellulase with respect to the kinds and levels of
extracellular AOS accumulation. The use of inhibitors revealed the
operation of different enzymic systems of AOS production in the two
plant species.
| |
MATERIALS AND METHODS |
Plant Material
Protoplasts were isolated from fully expanded, but not senescent,
leaves of in vitro-grown grape (Vitis vinifera L. cv
Sultanina) (Roubelakis-Angelakis and Zivanovitc, 1991) and
greenhouse-grown tobacco (Nicotiana tabacum L. cv Xanthi)
plants. Small leaf segments (2 mm) were used in all experiments.
Isolation of Mesophyll Protoplasts
Grape and tobacco mesophyll protoplasts were isolated and cultured
as previously described (Koop and Schweiger, 1985; Katsirdakis and
Roubelakis-Angelakis, 1992b). The cell wall-hydrolyzing enzymic preparations used were 0.25% (w/v) purified cellulase (Worthington Biochemical, Lakewood, NJ) and 0.5% (w/v) Macerozyme (R-10 Onozuka, Yakult Honsha, Tokyo), and the duration of the maceration period was
4 h. The purified cellulase was free of detectable amounts of
xylanase (Fuchs et al., 1989). All treatments were performed in the
dark at 25°C.
Elicitation of AOS in Leaf Tissue and Protoplasts
Elicitation of AOS production was studied in leaf tissue and
protoplasts. Leaf strips were intensively wounded with a razor blade
and floated in the culture medium as previously described for
protoplast isolation (Koop and Schweiger, 1985; Katsirdakis and
Roubelakis-Angelakis, 1992b). At time zero, two different cellulase
preparations with similar enzymic activity were added to leaf segments:
1% (w/v) non-purified cellulase known to contain xylanase (Fuchs et
al., 1989) or 0.25% (w/v) purified cellulase. Wounded strips floated
in the same medium were used to study AOS production elicited by
wounding alone. AOS accumulation in the culture medium was monitored
over a period of 16 h.
Protoplasts were cultured as previously described (Katsirdakis and
Roubelakis-Angelakis, 1992b). At time zero, 1% (w/v) non-purified cellulase was added and the culture medium was used for determination of AOS accumulation over a period of 4 h.
Plasma Membrane Isolation
Plasma membranes were isolated from control protoplasts and from
protoplasts treated with 1% (w/v) non-purified cellulase for 30 min,
as previously described (de Marco and Roubelakis-Angelakis, 1996a).
Four volumes of the extraction buffer (50 mM Tris-HCl, pH
7.5, 20% [w/v] sorbitol, 1 mM ascorbate, 1 mM EDTA, 10 mM DDT, 10 µM
leupeptin, and 0.3% [v/v] Triton X-100) was added to protoplasts and, after intense vortexing, the homogenate was centrifuged for 20 min
at 13,000g. The resulting supernatant was further
centrifuged for 50 min at 85,000g to separate the microsomal
fraction, which was resuspended in 50 mM
Tris-HCl, pH 7.0, 250 mM Suc, 0.5 M KCl, and 10% (v/v) glycerol and re-centrifuged
twice to wash away any unspecifically bound enzyme. Plasma membranes
were isolated using a two-phase partitioning system, as previously
described (de Marco and Roubelakis-Angelakis, 1996a). Total protein of
the plasma membranes was measured by the method of Lowry et al. (1951),
with BSA as a standard.
Luminol-Dependent Chemiluminescence Assay for
H2O2
The production of H2O2
from leaf cells and protoplasts was determined by the chemiluminescence
assay of luminol described by Murphy and Huerta (1990). Leaf segments
(50 mg mL1) were floated in the culture medium
as described above, which was used for
H2O2 assays. The production
of H2O2 from protoplasts, corresponding to the same fresh weight of leaf tissue, was determined in the culture medium after centrifugation at 1,000 rpm for 15 s.
The assay was conducted in a total volume of 2 mL by placing 0.8 mL of
reaction buffer containing 10 mM Tris-Mes, pH 7.0, 1 mM CaCl2, 0.1 mM KCl, 0.2 mL of 1 mM luminol solution, 0.1 unit of peroxidase in 20 mM potassium phosphate buffer (pH 7.4), and 1 mL of culture
medium in a scintillation vial (Auh and Murphy, 1995). The vial was
immediately placed in a scintillation spectrometer (model LS 6000SE,
Beckman) and chemiluminescence was detected. Counts were reported every
15 s for 1 min, and the last two values were averaged. A standard
curve was produced correlating the chemiluminescence values with
standard concentrations of
H2O2. Proper controls were always used.
Lucigenin-Dependent Chemiluminescence Assay for
O2·
The accumulation of
O2· was measured by the
chemiluminescence of lucigenin, which is specific for
O2· (Corbisier et al.,
1987). Although the validity of lucigenin as a probe for detecting
O2· has recently been
questioned because of its ability to undergo redox cycling (Liochev and
Fridovich, 1997), Li et al. (1998) presented a detailed and careful
investigation in which the validity of lucigenin as a
chemilumigenic probe for detecting
O2· production by
enzymatic and cellular systems was established. The assay was conducted
in a total volume of 2 mL by placing 0.2 mL of 1 mM
lucigenin in 0.1 M Gly-NaOH buffer (pH 9.0) containing 1 mM EDTA (Auh and Murphy, 1995). All of the other conditions described for H2O2 assays
were also followed for the
O2· assays. Counts were
reported every 6 s for 30 s, and the last two values were
averaged. The xanthine/xanthine oxidase system described by Murphy
and Auh (1996) was used to convert chemiluminescence data to
production rates: 106 cpm was equivalent to
38.21 ± 2.16 pmol
O2· produced
min1 (mean ± SE of three
experiments).
Assay for O2· Synthase
The assay for O2·
synthase was also based on the chemiluminescence of lucigenin according
to the method of Murphy and Auh (1996) with minor modifications: 1 mL
of the reaction mixture containing 100 mM Gly-NaOH, pH 9.0, 1 mM EDTA, 200 µM NADH or 100 µM NADPH, 0.02% (v/v) Triton X-100, and 0.4 mM lucigenin, with or without inhibitors, was measured in a
liquid scintillation counter for 60 min. The last count ( 5 min) was
used for normalization of the samples. At time zero, 5 µg of plasma
membrane protein was added and the mixtures were counted for 1 min. The
results from experimental treatments were always compared with those
from untreated controls counted at the same time. In inhibitor
experiments, controls containing similar concentrations of inhibitors
were used.
| |
RESULTS |
Extracellular AOS Accumulation by Leaf Strips
The accumulation of
O2· and
H2O2 in the incubation
medium of tobacco and grape leaf strips was followed over a 16-h period with the lucigenin- and the luminol-dependent chemiluminescence assays,
respectively. O2· and
H2O2 accumulated at low
levels in the incubation medium in both systems (Fig.
1). The addition of purified cellulase
did not affect AOS accumulation, whereas the addition of non-purified cellulase to the incubation medium resulted in a significant increase in the accumulation of both AOS in tobacco, but only the accumulation of H2O2 in grape leaf
strips (Fig. 1). In tobacco, both AOS started to accumulate soon after
the addition of non-purified cellulase, reached a maximum after 4 h, and decreased thereafter. In grape, the accumulation of
O2· was very low but,
as in tobacco, H2O2 started
to increase rapidly after the addition of non-purified cellulase;
however, the level reached a maximum after 8 h and was 3-fold
lower than that of tobacco (Fig. 1). In tobacco the maximum
O2· and
H2O2 that accumulated in
the culture medium and corresponded to 1 mg fresh weight were
0.180 ± 0.013 nM and 0.030 ± 0.008 µM, whereas in grape the respective values were
0.012 ± 0.002 nM and 0.011 ± 0.003 µM.
View larger version (31K):
[in this window]
[in a new window]
| Figure 1.
Effect of cellulase preparations and wounding on
O2· and H2O2
accumulation in leaf tissue of tobacco (left) and grape (right). Leaf
strips (10 × 5 mm) were treated with 1% non-purified cellulase
or 0.25% purified cellulase, or were not treated. Cellulase
preparations were added at time zero to the incubation medium in which
the leaf strips were floated. Accumulation of
O2· and H2O2 was
also determined in leaf strips following intense wounding. Values are
means ± SE from seven independent experiments.
|
|
No increase in O2· or
H2O2 was found when tobacco
and grape leaf strips were either extensively wounded or incubated with
purified cellulase, suggesting that wounding and purified cellulase do not induce AOS production (Fig. 1). In both plant species, the accumulation of O2· and
H2O2 was dependent on the
amount of non-purified cellulase. This dose response of AOS production
by tobacco leaf segments treated with non-purified cellulase is shown
in Figure 2. The same effect was found in
grape leaves for H2O2
production (data not shown).
View larger version (20K):
[in this window]
[in a new window]
| Figure 2.
Dose-dependent accumulation of
O2· ( ) and H2O2
( ) in the incubation medium of tobacco leaf strips treated with
non-purified cellulase. Samples were taken 60 min after cellulase
addition. Values are means ± SE from three
independent experiments.
|
|
Extracellular Accumulation of AOS in the Culture Medium of
Protoplasts
Freshly isolated protoplasts from tobacco leaves using purified
cellulase contained 2.4 nM
O2· and 40 nM H2O2 per
105 protoplasts. The respective values for grape
protoplasts were 0.9 and 6 nM. These protoplasts were
cultured at a density of 105 protoplasts/mL. The
culture medium of freshly isolated tobacco protoplasts contained 0.36 and 6.6 nM
O2· and
H2O2, respectively, and
that of freshly isolated grape protoplasts 0.12 and 2.5 nM,
respectively (Fig. 3). The levels of both
AOS were elevated, possibly due to isolation procedures such as
physical pressure (Yahraus et al., 1995) and washing (Qian et al.,
1993). Non-purified cellulase was added to these protoplasts, and AOS accumulation in the culture medium was monitored over a 4-h period. The
detected levels of the two AOS in the culture medium were at the
nanomolar and micromolar range for
O2· and
H2O2, respectively.
View larger version (27K):
[in this window]
[in a new window]
| Figure 3.
Generation of AOS in tobacco (left) and grape
(right) mesophyll protoplasts treated with non-purified cellulase.
Protoplasts were isolated after 4 h of maceration with 0.25%
purified cellulase and 0.5% Macerozyme. Then, non-purified cellulase
was added to protoplasts (105 mL1) at time
zero, and the culture medium was assayed for
O2· and H2O2.
Values are means ± SE from five independent
experiments. White bars, Untreated protoplasts; black bars, treated
protoplasts.
|
|
As shown in Figure 3, a rapid accumulation of both AOS in the culture
medium of tobacco protoplasts was found soon after the treatment.
O2· accumulation in the
culture medium showed a 6-fold increase, reached a maximum after 15 min, and decreased slowly thereafter. H2O2, which was very low at
time zero, increased dramatically (330-fold increase), reaching a
maximum 2 h after treatment. In grape protoplasts, the addition of
non-purified cellulase resulted in a 2- and a 185-fold increase in the
extracellular level of O2· and
H2O2, respectively,
compared with the controls, reaching a maximum 2 h after treatment
(Fig. 3). The maximum extracellular concentrations in the culture
medium of tobacco and grape protoplasts were 2.3 and 0.23 nM for O2·
and 2.22 and 0.48 µM for
H2O2, respectively.
DDC (N,N-diethyldithiocarbamate), an inhibitor of Cu/Zn-SOD
(Heikkila et al., 1976), was added to tobacco and grape protoplasts treated with non-purified cellulase to determine whether the inhibition of SOD would result in the accumulation of
O2· and inhibit the
accumulation of H2O2 in the
culture medium, which would indicate that
H2O2 was formed by the
dismutation of O2· by
SOD. No such effect was observed in either system. The addition of
exogenous SOD to protoplasts strongly diminished the accumulation of
O2· caused by
non-purified cellulase and stimulated
H2O2 production in tobacco
and grape protoplasts (Fig. 4).
Furthermore, the addition of catalase to protoplasts resulted in the
reduction of H2O2
accumulation, supporting the specificity of the method used (Fig. 4).
View larger version (28K):
[in this window]
[in a new window]
| Figure 4.
Effects of inhibitors, exogenous SOD, and
catalase on the production of O2· (top) and
H2O2 (bottom) by tobacco (white bars) and grape
(black bars) protoplasts treated with non-purified or purified
cellulase. Inhibitors were added to protoplasts 10 min before the
addition of cellulase. Samples (culture medium) were taken 30 min after
treatment and assayed for O2· and
H2O2. Values are relative to control (100%).
Background activities (without protoplasts but with inhibitor) were
subtracted. The concentration of the inhibitors was 10 µM
DDC, 25 units/mL SOD, 100 units/mL CAT, 25 µM DPI,
0.5 mM quinacrine, 10 mM imidazole, 20 mM pyridine, 50 µM KCN, and 5 mM
NaN3.  , Negative values. Means ± SE
were calculated from six independent experiments.
|
|
Inhibitors of the mammalian neutrophil NADPH oxidase (DPI, quinacrine,
imidazole, and pyridine) and inhibitors of peroxidase (KCN and
NaN3) were used and their effect on extracellular
accumulation of AOS was tested. DPI and quinacrine directly inhibit
NADPH oxidase by binding to the flavoprotein component of the oxidase
complex (Cross and Jones, 1986, 1991). In tobacco protoplasts, 25 µM DPI and 0.5 mM quinacrine inhibited the
production of O2·
stimulated by non-purified cellulase by 50% and 69%, respectively (Fig. 4). Both imidazole (10 mM) and pyridine (20 mM), which bind to the Cyt b component of
neutrophil oxidase (Iizuka et al., 1985), inhibited
O2· production by about
55% and 65%, respectively (Fig. 4). None of the four inhibitors
affected H2O2 production
(Fig. 4).
The addition of peroxidase inhibitors to tobacco protoplasts resulted
in reduced accumulation of both AOS: 50 µM KCN and 5 mM NaN3 decreased
O2· accumulation by
about 40% and 35%, respectively, while both inhibitors completely
eradicated H2O2 production,
giving 100% inhibition (Fig. 4). In grape protoplasts, AOS production
was not affected by the first class of inhibitors, but was strongly
reduced by both KCN and NaN3;
O2· production was
inhibited by about 55% and 70% by the addition of KCN and
NaN3, respectively, and
H2O2 production was
completely inhibited by both agents (Fig. 4).
The above data indicate that different systems are responsible for AOS
generation elicited by non-purified cellulase in each plant species. In
tobacco, a mammalian-like oxidase seems to be responsible for
O2· production, since
its inhibitors were able to reduce the accumulation of
O2·, but this enzyme
had no effect on H2O2
generation. A peroxidase-like activity may contribute to
H2O2 generation and may
also have a role in O2·
production. These two enzymes probably either operate in tandem or have
different kinetics. In grape protoplasts, only peroxidase activity was
identified with the use of inhibitors, and was responsible for the
generation of both AOS (Fig. 4).
O2· Synthases
In an effort to further identify the nature of the
O2·-generating system
in tobacco and grape, plasma membranes were isolated from protoplasts
and assayed for NAD(P)H
O2· synthase activity
(Table I). The enzyme was assayed from
control protoplasts and from protoplasts treated with 1% non-purified cellulase for 30 min, when
O2· and
H2O2 had already started to
accumulate (Fig. 3). The
O2· synthase activity
was strongly dependent on the presence of Triton X-100 when either
NADPH or NADH was used as a substrate. The latency, given as 1 (activity without Triton)/(maximum activity with Triton), was 1.0 ± 0.02 (an average from six independent plasma membrane preparations).
The strongly stimulating effect of Triton X-100 on
O2· synthesis in plasma
membrane vesicles is consistent with the idea that the oxidizing sites
of O2· synthase(s) are
on the cytoplasmic side of the vesicles, unavailable to the NADPH or
NADH in the absence of the detergent (Murphy and Auh, 1996).
View this table:
[in this window]
[in a new window]
|
Table I.
O2 ·-synthase
activity in plasma membrane preparations from tobacco and grape leaf
protoplasts treated with non-purified cellulase or untreated
All the assays for synthase activity contained 100 mM
Gly-NaOH, pH 9.0, 1 mM EDTA, 0.02% (v/v) Triton X-100, 0.4 mM lucigenin, 100 µM NADPH or 200 µM NADH, and 5 µg of plasma membrane protein.
Km was calculated from curves fitted to the
Michaelis-Menten equation by the nonlinear regression procedure (data
not shown). Background activities (without enzyme) were subtracted.
Means ± SE were calculated using four independent
plasma membrane preparations.
|
|
In tobacco plasma membrane preparations, the
O2·-generating activity
was higher than in grape no matter which substrate used (Table I).
O2· production from
tobacco plasma membrane preparations showed differences in substrate
(NADPH or NADH) affinity, although both obeyed Michaelis-Menten kinetics (data not shown). The Km was
7.9 ± 2.7 µM for NADPH and 102.5 ± 6.1 µM for NADH. In grape, the respective
values for the Km were 32.6 ± 7.8 and 43.1 ± 8.3 µM (Table I). Plasma
membranes prepared from protoplasts treated with non-purified cellulase had the same specific activity as the control preparations in both
plant species (Table I).
Various inhibitors were tested for revealing the possible relationship
of tobacco and grape
O2· synthases to other
characterized oxidases. The plasma membrane vesicles used in the
inhibitor studies were isolated from protoplasts treated with
non-purified cellulase. In the presence of 100 µM NADPH,
25 µM DPI reduced the
O2· synthase activity
by 50% in tobacco but had no effect in grape (Fig.
5). Imidazole (10 mM),
another inhibitor of the mammalian NADPH oxidase, resulted in about
75% inhibition in tobacco plasma membrane preparations (Fig. 5). With
200 µM NADH, neither DPI nor imidazole affected
O2· production in
either plant species. In tobacco,
O2· production was
reduced by about 55% and 70% by 50 µM KCN and 5 mM NaN3, respectively, when NADPH was
used, and by 40% and 70%, respectively, when NADH was used as the
substrate. In grape, an approximately 40% inhibition was caused by
both KCN and NaN3 when NADPH was used, and 75%
and 80% inhibition, respectively, when NADH was used as the substrate
(Fig. 5).
View larger version (32K):
[in this window]
[in a new window]
| Figure 5.
Effect of inhibitors on
O2· synthase activity of plasma membrane
preparations from tobacco (white bars) and grape (black bars)
protoplasts. All assays contained 5 µg of plasma membrane protein,
plus 100 mM Gly-NaOH, pH 9.0, 1 mM EDTA, 0.02%
(v/v) Triton X-100, 0.4 mM lucigenin, and either 100 µM NADPH (top) or 200 µM NADH (bottom).
Values are relative to control (100%). Background activities (without
enzyme but with inhibitor) were subtracted. The concentration of the
inhibitors was 25 µM DPI, 10 mM imidazole, 50 µM KCN, and 5 mM NaN3. Means ± SE were calculated using five independent plasma
membrane preparations.
|
|
In tobacco plasma membrane preparations, two different
O2· synthase activities
were found: one that exhibited specificity for NADPH and sensitivity to
DPI and imidazole, and a second that could use either NADPH or NADH and
was sensitive to KCN and NaN3. The first activity
fits the profile of a mammalian-like oxidase (Doke and Miura, 1995;
Murphy and Auh, 1996; Van Gestelen et al., 1997) and the second that of
a a NAD(P)H oxidase-peroxidase (Askerlund et al., 1987;
Vera-Estrella et al., 1992; Bolwell et al., 1995; Bestwick et al.,
1997). In grape only the second activity was detected.
| |
DISCUSSION |
The expression of totipotency in cultured protoplasts seems to
involve at least two developmental pathways: the suppression of events
leading to cell death and the induction of cell elongation and cell
division. Results from previous comparative work using regenerating
tobacco and non-regenerating grape protoplasts indicated that
H2O2 participates in the
peroxidase-mediated intramolecular isotyrosine ether cross-linking of
reconstituted cell walls (Hahne and Hoffmann, 1984; Gaspar et
al., 1989; Iiyama et al., 1994; de Marco and Roubelakis-Angelakis,
1996a, 1996b, 1997). Total peroxidase was significantly higher in
regenerating than in non-regenerating protoplasts during culture,
comprising expression of specific isoenzymes (Siminis et al., 1993),
whereas NADH peroxidase activity, which has been proposed to generate
H2O2 (Gross et al., 1977; Elstner and Heupel, 1978), was not detectable at all in
non-regenerating tobacco protoplasts (Siminis et al., 1993; de Marco
and Roubelakis-Angelakis, 1996a). Protoplasts survived but lost their
dividing potential when peroxidase activity was inhibited by cyanide or
DTT and when exogenous catalase was added to the culture medium,
suggesting that division is dependent on modification of cell wall
plasticity (de Marco and Roubelakis-Angelakis, 1996a, 1996b).
On the other hand, oxidative stress can induce cell death. The
H2O2-scavenging activity of
catalase was higher in regenerating (tobacco) than in non-regenerating
(grape) protoplasts, with differences in the expression of specific
subunits (Siminis et al., 1994; de Marco and Roubelakis-Angelakis,
1996a). Ascorbate peroxidase activity and transcript were present only
in regenerating tobacco protoplasts, which did not survive when
ascorbate peroxidase activity was inhibited (de Marco and
Roubelakis-Angelakis, 1996b). The activities of antioxidant enzymes of
the Halliwell-Asada pathway were significantly lower in
non-regenerating protoplasts (de Marco and Roubelakis-Angelakis, 1996a,
1999).
In this work we have attempted to break down the oxidative burst
response into individual AOS species and individual AOS-generating systems in leaf strips, protoplasts, and plasma membrane vesicles from
tobacco and grape. O2·
can be generated by the plasma membrane mammalian-like NAD(P)H oxidase
(Askerlund et al., 1987; Vera-Estrella et al., 1992; Bolwell et al.,
1995). This enzyme in mammals is composed of membrane-bound and
cytosolic proteins. In the center of the NADPH oxidase lies the
heterodimeric NADPH-binding flavocytochrome
b558, which consists of the
glycosylated transmembrane protein gp91phox and
the nonglycosylated p22phox subunit. The
flavocytochrome contains the entire electron transport chain from NADPH
to O2. Upon activation, the cytosolic proteins p47phox and p67phox become
phosphorylated and translocate, together with
p40phox and p21rac, to the
membrane to form the active NADPH oxidase complex (Segal and Abo,
1993). The generated
O2· can then form
H2O2 by spontaneous or
SOD-mediated dismutation (Doke, 1983; Levine et al., 1994; Auh and
Murphy, 1995; Doke and Miura, 1995; Murphy and Auh, 1996) and/or by the
action of extracellular peroxidases (Askerlund et al., 1987;
Vera-Estrella et al., 1992; Bolwell et al., 1995, 1998; Bestwick et
al., 1997).
AOS generation by tobacco and grape leaf strips was not induced by
extensive wounding and/or addition of purified cellulase (Fig. 1); the
addition of non-purified cellulase known to contain, among other
things, xylanase and pectine lyase (Fuchs et al., 1989) resulted in a
dose-dependent (Fig. 2), significant increase in both AOS in tobacco
leaf strips (Fig. 1), whereas in grape the response was much less. The
addition of non-purified cellulase to freshly isolated (with purified
cellulase) protoplasts resulted in a 6- and 330-fold and 2- and
185-fold increase of
O2· and
H2O2 in tobacco and grape
protoplasts, respectively (Fig. 3).
H2O2 was the building force
of the oxidative burst, which agrees with other studies (Bolwell et
al., 1995; Desikan et al., 1996). On the other hand, Doke (1983) and
Auh and Murphy (1995) characterized
O2· as the major AOS in
potato and rose, respectively, whereas in tomato suspension cells both
AOS were detected after elicitation (Vera Estrella et al., 1992).
O2· and
H2O2 accumulation in
tobacco and grape protoplasts stimulated by non-purified cellulase was
not affected by DDC (Fig. 4), a chelator of Cu ions and an inhibitor of
Cu/Zn-SOD (Heikkila et al., 1976), indicating that there is no
extracellular SOD activity that could mask
O2· detection or be
involved in H2O2 production
through enzymic dismutation of
O2·. Also, diamine
or/and polyamine oxidases do not contribute to H2O2 production in tobacco
protoplasts (de Marco and Roubelakis-Angelakis, 1996a). The hypothesis
that phytotoxic factors from cell walls elicit AOS generation during
protoplast isolation (Hahne and Lorz, 1988) is not supported by our
results, because: (a) AOS accumulation occurred in protoplasts, which
lack cell walls; (b) the generated AOS by leaf strips treated with
non-purified cellulase were significantly lower than the AOS generated
by protoplasts; and (c) no AOS were generated by leaf strips treated
with purified cellulase (Figs. 1 and 3).
The induced O2·
generation by tobacco protoplasts was sensitive to four inhibitors of
the mammalian neutrophil NADPH oxidase, whereas none of them inhibited
O2· production in grape
protoplasts treated with non-purified cellulase (Fig. 4). In contrast,
KCN and NaN3, inhibitors of plant peroxidases, both affected O2·
production and completely inhibited
H2O2 generation in both
plant species (Fig. 4). These results, along with those from isolated plasma membranes (Table I; Fig. 5), support the idea that in tobacco
two different O2·
synthase activities are operating; one with specificity for NADPH and
sensitivity to DPI and imidazole, which fits the profile of the
mammalian-like oxidase, and a NAD(P)H oxidase-peroxidase, which can use
either NADPH or NADH and is inhibited by KCN and NaN3. In grape, only the second activity was
detected, and it was responsible for the synthesis of both AOS. Two
distinct sources of elicited AOS were also described in tobacco
epidermal cells: a flavin-containing oxidase system and a cell wall
peroxidase-like activity (Allan and Fluhr, 1997). In rose cells,
H2O2 is produced by a
plasma membrane NAD(P)H oxidase, whereas in bean cells it is derived
directly from cell wall peroxidases (Bolwell et al., 1998).
Plasma membranes isolated from protoplasts treated with non-purified
cellulase did not exhibit significantly higher
O2·-generating activity
compared with control preparations of both species (Table I), nor was
the addition of non-purified cellulase to isolated plasma membranes
from control protoplasts able to further stimulate
O2· synthesis. This
result is in agreement with the results of Murphy and Auh (1996) using
membranes from rose cells treated with Phytophthora infestans elicitor, and from results using potato (Doke and Miura, 1995), in which O2·
synthesis could be stimulated in vitro by P. infestans
elicitors only in the presence of cytosolic components. The
immunological and molecular evidence that the cytosolic proteins p47,
p67, and Rac1, which are components of the neutrophil NADPH oxidase,
also exist in plant cells and are prerequisites for activation and membrane assembly of the oxidase complex (Tenhaken et al., 1995; Dwyer
et al., 1996; Groom et al., 1996; Kieffer et al., 1997; Keller et al.,
1998) explain the above results.
The release of both AOS was extracellular (Figs. 1 and 3), which is
consistent with results from potato protoplasts (Doke, 1983) and rose
cells (Auh and Murphy, 1995), while
O2· synthesis by plasma
membrane vesicles was strongly stimulated by detergent, which indicates
a cytoplasmic site of
O2· generation (Murphy
and Auh, 1996). Allan and Fluhr (1997) suggested that AOS generation
takes place within the cell or in the apoplast, depending on the
elicitor used. Recently, a homolog of the neutrophil NADPH oxidase
gp91phox subunit gene that encodes for a
intrinsic plasma membrane protein was reported in Arabidopsis (Keller
et al., 1998), suggesting that the initial products of the NADPH
oxidase reaction will accumulate on the external face of the plasma
membrane, which is in keeping with the functions of AOS in cell wall
cross-linking and intercellular signaling (Lamb and Dixon,
1997).
The elicitor molecule for the induction of AOS generation remains
unknown, but several putative receptors have been identified and at
least partially characterized. Further interactions implicate the
involvement of GTP-binding proteins, ion channels, protein kinases and
phosphatases, phospholipases A and C, and possibly cAMP along the
signaling pathway leading to the activation of NADPH oxidase
(Wojtaszek, 1997, and refs. therein). Differences between tobacco and
grape in the component(s) of the enzyme(s) and/or the receptors and the
activators of AOS generation and removal, as well as their
physiological significance on the complex developmental phenomena of
viability and regenerating potentiality of protoplasts remain to be
elucidated with the use of currently available immunological and
molecular probes.
| |
FOOTNOTES |
1
This work was supported in part by the Interreg
II project (European Union and Greek Government).
*
Corresponding author; e-mail poproube{at}biology.uch.gr; fax
30-81-39459.
Received January 5, 1999;
accepted May 21, 1999.
| |
LITERATURE CITED |
Allan AC,
Fluhr R
(1997)
Two distinct sources of elicited reactive oxygen species in tobacco epidermal cells.
Plant Cell
9:
1559-1572
[Abstract]
Askerlund P,
Larson C,
Widell S,
Moller I
(1987)
NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences.
Physiol Plant
71:
9-19
[CrossRef]
Auh CK,
Murphy TM
(1995)
Plasma membrane redox enzyme is involved in the synthesis of O2 and H2O2 by Phytophthora elicitor-stimulated rose cells.
Plant Physiol
107:
1241-1247
[Abstract]
Bailey BA,
Korcak RF,
Anderson JD
(1992)
Alterations in Nicotiana tabacum L. cv Xanthi cell membrane function following treatment with an ethylene biosynthesis-inducing endoxylanase.
Plant Physiol
100:
749-755
[Abstract/Free Full Text]
Baker CJ,
Orlandi EW
(1995)
Active oxygen in plant pathogenesis.
Annu Rev Phytopathol
33:
299-321
[CrossRef][ISI]
Bestwick CS,
Brown IR,
Bennett MHR,
Mansfield JW
(1997)
Localization of hydrogen peroxide accumulation during the hypersensitive response of lettuce cells to Pseudomonas syringae pv phaseolicola.
Plant Cell
9:
209-221
[Abstract]
Bolwell GP,
Butt VS,
Davies DR,
Zimmerlin A
(1995)
The origin of the oxidative burst in plants.
Free Radic Res
23:
517-532
[ISI][Medline]
Bolwell GP,
Davies DR,
Gerrish C,
Auh C-K,
Murphy TM
(1998)
Comparative biochemistry of the oxidative burst produced by rose and French bean reveals two distinct mechanisms.
Plant Physiol
116:
1379-1385
[Abstract/Free Full Text]
Calderon AA,
Zapata JM,
Ros Barcelo A
(1994)
Peroxidase-mediated formation of resveratrol oxidation products during the hypersensitive-like reaction of grapevine cells to an elicitor from Trichoderma viride.
Physiol Mol Plant Pathol
44:
289-299
Corbisier P,
Houbion A,
Remacle J
(1987)
A new technique for highly sensitive detection of superoxide dismutase activity by chemiluminescence.
Anal Biochem
164:
240-247
[CrossRef][Medline]
Cross AR,
Jones OTG
(1986)
The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils: specific labeling of a component polypeptide of the oxidase.
Biochem J
237:
111-116
[ISI][Medline]
Cross AR,
Jones OTG
(1991)
Enzymic mechanisms of superoxide production.
Biochem Biophys Acta
1057:
281-298
[Medline]
Cutler A,
Saleem M,
Wang H
(1991)
Cereal protoplast recalcitrance.
In Vitro Cell Dev Biol
27:
104-111
de Marco A,
Roubelakis-Angelakis KA
(1996a)
The complexity of enzymic control of hydrogen peroxide concentration may affect the regeneration potential of plant protoplasts.
Plant Physiol
110:
137-145
[Abstract]
de Marco A,
Roubelakis-Angelakis KA
(1996b)
Hydrogen peroxide plays a bivalent role in the regeneration of protoplasts.
J Plant Physiol
149:
109-114
de Marco A,
Roubelakis-Angelakis KA
(1997)
Laccase activity could contribute to cell-wall reconstitution in regenerated protoplasts.
Phytochemistry
46:
421-425
[CrossRef]
de Marco A,
Roubelakis-Angelakis KA
(1999)
Specific features of the ascorbate/glutathione cycle in cultured protoplasts.
Plant Cell Rep
18:
406-411
[CrossRef]
Desikan R,
Hancock JT,
Coffey MJ,
Neill SJ
(1996)
Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH-oxidase-like enzyme.
FEBS Lett
382:
213-217
[CrossRef][ISI][Medline]
Doke N
(1983)
Generation of superoxide anion by potato tuber protoplasts during hypersensitive response to hyphal wall components of Phytophtora infestans and specific inhibition of the reaction with supressors of hypersensitivity.
Physiol Plant Pathol
23:
359-367
Doke N,
Miura Y
(1995)
In vitro activation of NADPH-dependent superoxide generating system in a plasma membrane-rich fraction of potato tuber tissues by treatment with an elicitor from Phytophtora infestans or with digitonin.
Physiol Mol Plant Pathol
46:
17-28
[CrossRef]
Dwyer SC,
Legendre L,
Loe PS,
Leto TL
(1996)
Plant and human neutrophil oxidative burst contain immunological related proteins.
Biochim Biophys Acta
1289:
231-237
[Medline]
Elstner EF,
Heupel A
(1978)
Formation of H2O2 by isolated cell-walls from horseradish (Armoracia lapathifolia).
Planta
130:
175-180
Fuchs Y,
Saxena YA,
Gamble HR,
Anderson JD
(1989)
Ethylene biosynthesis-inducing protein from cellulisin is an endoxylanase.
Plant Physiol
89:
138-143
[Abstract/Free Full Text]
Gaspar T, Penel C, Hagege D, Greppin H (1989) Peroxidase in plant
growth, differentiation and developmental processes. In J
Lobarzewski, H Greppin, C Penel, T Gaspar, eds, Biochemical, Molecular,
and Physiological Aspects of Plant Peroxidases. University M. Curie-Sklodowska, Lublin, Poland, and University of Geneva, pp 249-280
Groom QJ,
Torres MA,
Fordham-Skelton AP,
Hammond-Kosack KE,
Robinson NJ,
Jones JDG
(1996)
rbohA, a rice homologue to mammalian gp91phox respiratory burst oxidase gene.
Plant J
10:
515-522
[CrossRef][ISI][Medline]
Gross GG,
Janse C,
Elstner EF
(1977)
Involvement of malate, monophenols and the superoxide radical in hydrogen peroxide formation by isolated cell wall from horseradish (Armoracia lepathifolia Gilib).
Planta
136:
271-276
[CrossRef]
Hahne G,
Hoffmann F
(1984)
Dimethyl sulfoxide can initiate cell divisions of arrested callus protoplasts by promoting cortical microtubule assembly.
Proc Natl Acad Sci USA
81:
5449-5453
[Abstract/Free Full Text]
Hahne G,
Lorz H
(1988)
Release of phytotoxic factors from plant cell walls during protoplast isolation.
J Plant Physiol
132:
345-350
Halliwell B
(1978)
Lignin synthesis: the generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols.
Planta
140:
81-88
[CrossRef]
Heikkila RE,
Cabbat FS,
Cohen G
(1976)
In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate.
J Biol Chem
251:
2182-2185
[Abstract/Free Full Text]
Iiyama K,
Lam TB-T,
Stone BA
(1994)
Covalent cross-links in the cell wall.
Plant Physiol
104:
315-320
[ISI][Medline]
Iizuka T,
Kanegasaki S,
Makino R,
Tanaka T,
Ishimura Y
(1985)
Pyridine and imidazole reversibly inhibit the respiratory burst in porcine and human neutrophils: evidence for the involvement of cytochrome b558 in the reaction.
Biochem Biophys Res Commun
130:
621-626
[CrossRef][Medline]
Ishii S
(1987)
Generation of active oxygen species during enzymic isolation of protoplasts from oat leaves.
In Vitro Cell Dev Biol
23:
653-658
Ishii S
(1988)
Factors influencing protoplast viability of suspension-cultured rice cells during isolation process.
Plant Physiol
88:
26-29
[Abstract/Free Full Text]
Jabs T,
Tschope M,
Colling C,
Hahlbrock K,
Scheel D
(1997)
Elicitor-stimulated ion fluxes and superoxide from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley.
Proc Natl Acad Sci USA
94:
4800-4805
[Abstract/Free Full Text]
Katsirdakis KC,
Roubelakis-Angelakis KA
(1992a)
Ultrastructural and biochemical aspects of cell wall regeneration in recalcitrant and regenerating leaf protoplasts.
In Vitro Cell Dev Biol
28:
90-96
Katsirdakis KC,
Roubelakis-Angelakis KA
(1992b)
A modified culture medium and culture conditions increase viability and cell wall synthesis in grapevine (Vitis vinifera L. cv Sultanina) leaf protoplasts.
Plant Cell Tissue Org Cult
28:
255-260
Keller T,
Damude HG,
Werner D,
Doerner P,
Dixon R,
Lamb C
(1998)
A plant homologue of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs.
Plant Cell
10:
225-236
Kieffer F,
Simon-Plus F,
Maume B,
Blein J-P
(1997)
Tobacco cells contain a protein immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst.
FEBS Lett
403:
149-153
[CrossRef][ISI][Medline]
Koop HV,
Schweiger HG
(1985)
Regeneration of plants from individually cultivated protoplasts using an improved microculture system.
J Plant Physiol
121:
245-257
Lamb C,
Dixon R
(1997)
The oxidative burst in plant disease resistance.
Annu Rev Plant Physiol Plant Mol Biol
48:
251-275
[CrossRef][ISI]
Levine A,
Tenhaken R,
Dixon R,
Lamb C
(1994)
H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response.
Cell
79:
583-593
[CrossRef][ISI][Medline]
Li Y,
Zhu H,
Kuppusamy P,
Zweier JL,
Trush MA
(1998)
Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems.
J Biol Chem
273:
2015-2023
[Abstract/Free Full Text]
Liochev SI,
Fridovich I
(1997)
Lucigenin (bis-N-methylacridinium) as a mediator of superoxide anion production.
Arch Biochem Biophys
337:
115-120
[CrossRef][ISI][Medline]
Low PS,
Merida JR
(1996)
The oxidative burst in plant defense: function and signal transduction.
Physiol Plant
96:
533-542
[CrossRef]
Lowry OH,
Rosebrough NJ,
Farr AL,
Randall RJ
(1951)
Protein measurement with the Folin phenol reagent.
J Biol Chem
193:
265-275
[Free Full Text]
Murphy TM,
Auh CK
(1996)
The superoxide synthases of plasma membrane preparations from cultured rose cells.
Plant Physiol
110:
621-629
[Abstract]
Murphy TM,
Huerta AJ
(1990)
Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation.
Physiol Plant
78:
247-253
[CrossRef]
Qian YC,
Nguyen T,
Murphy TM
(1993)
Effects of washing on the plasma membrane and on stress reactions of cultured rose cells.
Plant Cell Tissue Org Cult
35:
245-252
Roubelakis-Angelakis KA (1993) An assessment of possible factors
contributing to recalcitrance of plant protoplasts. In KA
Roubelakis-Angelakis, K Tran Thanh Van, eds, Morphogenesis in Plants:
Molecular Approaches, Vol 253. Plenum Press, New York, pp 201-220
Roubelakis-Angelakis KA,
Zivanovitc S
(1991)
A new culture medium for in vitro rhizogenesis of grapevine (Vitis spp) genotypes.
HortScience
26:
1552-1555
Scandalios JG
(1993)
Oxygen stress and superoxide dismutases.
Plant Physiol
101:
7-12
[ISI][Medline]
Segal AW,
Abo AW
(1993)
The biochemical basis of the NADPH oxidase of phagocytes.
Trends Biochem Sci
18:
43-47
[CrossRef][ISI][Medline]
Sharon A,
Fuchs Y,
Anderson JD
(1993)
The elicitation of ethylene biosynthesis by a Trichoderma xylanase is not related to the cell wall degradation activity of the enzyme.
Plant Physiol
102:
1325-1329
[Abstract]
Siminis CI,
Kanellis AK,
Roubelakis-Angelakis KA
(1993)
Differences in protein synthesis and peroxidase isoenzymes between recalcitrant and regenerating protoplasts.
Physiol Plant
87:
263-270
[CrossRef]
Siminis CI,
Kanellis AK,
Roubelakis-Angelakis KA
(1994)
Catalase is differentially expressed in dividing and nondividing protoplasts.
Plant Physiol
105:
1375-1383
[Abstract]
Sutherland MW
(1991)
The generation of oxygen radicals during host plant responses to infection.
Physiol Mol Plant Pathol
39:
79-93
[CrossRef]
Tenhaken R,
Levine A,
Brisson LF,
Dixon RA,
Lamb C
(1995)
Function of the oxidative burst in hypersensitive disease resistance.
Proc Natl Acad Sci USA
92:
4158-4163
[Abstract/Free Full Text]
Theodoropoulos PA,
Roubelakis-Angelakis KA
(1990)
Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.
Plant Cell Tissue Org Cult
20:
15-23
Van Gestelen P,
Asard H,
Caubergs RJ
(1997)
Solubilization and separation of a plant plasma membrane NADPH-superoxide synthase from other NAD(P)H oxidoreductases.
Plant Physiol
115:
543-550
[Abstract]
Vera-Estrella R,
Blumwald E,
Higgins VJ
(1992)
Effect of specific elicitors of Cladosporium fulvum on tomato suspension cells.
Plant Physiol
99:
1208-1215
[Abstract/Free Full Text]
Wojtaszek P
(1997)
Oxidative burst: an early plant response to pathogen infection.
Biochem J
322:
681-692
Xing T,
Higgins VJ,
Blumwald E
(1997)
Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells.
Plant Cell
9:
249-259
[Abstract]
Yahraus T,
Chandra S,
Legendre L,
Low PS
(1995)
Evidence for a mechanically induced oxidative burst.
Plant Physiol
109:
1259-1266
|
Top
Abstract
Introduction