|
Plant Physiol. (1998) 116: 659-669
Oxidative Burst and Hypoosmotic Stress in Tobacco Cell
Suspensions
Anne-Claire Cazalé,
Marie-Aude Rouet-Mayer,
Hélène Barbier-Brygoo,
Yves Mathieu, and
Christiane Laurière*
Institut des Sciences Végétales, Centre National de la
Recherche Scientifique, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France
 |
ABSTRACT |
Oxidative burst constitutes an early
response in plant defense reactions toward pathogens, but active oxygen
production may also be induced by other stimuli. The oxidative response
of suspension-cultured tobacco (Nicotiana tabacum cv
Xanthi) cells to hypoosmotic and mechanical stresses was characterized.
The oxidase involved in the hypoosmotic stress response showed
similarities by its NADPH dependence and its inhibition by iodonium
diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative
response by hypoosmotic stress needed protein phosphorylation and anion
effluxes, as well as opening of Ca2+ channels. Inhibition
of the oxidative response impaired Cl efflux,
K+ efflux, and extracellular alkalinization, suggesting
that the oxidative burst may play a role in ionic flux regulation.
Active oxygen species also induced the cross-linking of a cell wall
protein, homologous to a soybean (Glycine max L.)
extensin, that may act as part of cell volume and turgor regulation
through modification of the physical properties of the cell wall.
| |
INTRODUCTION |
Plants have to cope with a large variety of environmental and
developmental stimuli. Oxidative burst, which corresponds to a
transient production of AOS, such as superoxide anion and
H2O2, is a part of the
response to pathogen attack in plants. The plant oxidative response
plays direct and indirect roles in plant defense (for reviews, see Low
and Merida, 1996 ; Mehdy et al., 1996). The produced AOS can act as an
antibiotic toward the pathogen (Mehdy et al., 1996) and reinforce the
cell wall by catalyzing cross-linking of cell wall proteins through a
peroxidase-dependent reaction (Bradley et al., 1992; Brisson et al.,
1994; Tenhaken et al., 1995; Otte and Barz, 1996). AOS are also second
messengers that activate downstream defense reactions, such as
synthesis of pathogenesis-related proteins (Chen et al., 1993),
glutathione S-transferase, glutathione peroxidase, and
ubiquitin (Levine et al., 1994), as well as phytoalexin accumulation
(Devlin and Gustine, 1992).
Structurally and chemically distinct compounds have been isolated from
fungi, bacteria, and plant cell walls that were effective elicitors of
oxidative burst in different plant models. In tobacco (Nicotiana
tabacum L.) cells, several molecules are able to induce oxidative
burst, such as the bacterial protein harpin (Baker et al., 1993),
oomycete elicitins (Yu, 1995) including cryptogein (Bottin et al.,
1994), and plant cell wall-derived oligogalacturonides (Mathieu et al.,
1996). Eliciting compounds seem to be recognized by receptors at the
plasma membrane, because specific binding sites have been visualized
(Nurnberger et al., 1995; Wendehenne et al., 1995). Transduction
pathways appear to differ according to the plant-elicitor model, and
only a few steps have been identified. The oxidative burst involves
protein phosphorylations (Schwacke and Hager, 1992; Viard et al., 1994;
Chandra and Low, 1995; Mathieu et al., 1996). The Ser/Thr kinase
encoded by the Pto-resistance gene in tomato induces a
specific oxidative burst in response to infection by an avirulent
pathogen expressing avrPto (Chandra et al., 1996b). The
oxidative burst activation by cryptogein in tobacco cell suspension and
by fungal extracts in spruce cells was shown to be
Ca2+ dependent (Schwacke and Hager, 1992;
Tavernier et al., 1995). GTP-binding protein and inositol
trisphosphate-mediated transduction was observed in soybean
(Glycine max L.) cells in response to oligogalacturonides
(Legendre et al., 1992, 1993b). Phospholipase A involvement was
reported in soybean cells elicited by extracts from Verticillium
dahliae (Chandra et al., 1996a).
The AOS-producing machinery activated in response to elicitor molecules
displays similarities with the neutrophil plasma membrane oxidase
involved in phagocytosis (Henderson and Chappell, 1996). The oxidative
burst in plants can be inhibited by IDP, an inhibitor of the neutrophil
NADPH oxidase (Levine et al., 1994; Dwyer et al., 1996; Rouet-Mayer et
al., 1997), and is dependent on NADPH (Pugin et al., 1997). Moreover,
there are immunochemical (Dwyer et al., 1996) and functional data
(Coffey et al., 1995) that suggest the existence of an analogous
enzymatic complex in plants. A cDNA has also been isolated from rice,
which is homologous to one integral membrane component of the mammalian
NADPH oxidase (Groom et al., 1996).
AOS production was also shown to be induced by physical stresses in
plants and animals. Swelling of neutrophils induces anion superoxide
production (Miyahara et al., 1993), and in soybean suspension cells,
AOS production was activated by osmotic shock, physical pressure
(Yahraus et al., 1995), and vigorous stirring of the suspension
(Legendre et al., 1993a). The transduction pathway mediating this
oxidative response activation has yet to be elucidated. Yahraus et al.
(1995) showed that mechanically induced oxidative burst in soybean
cells was prevented by Gd, an inhibitor of stretch-activated channels,
therefore suggesting the involvement of these channels in oxidase
activation. Ion, organic solute, and water fluxes caused by hypoosmotic
stress may represent additional elements of the mechanical stress
response. They are key elements of the osmoregulation process (Hallows
and Knauf, 1994) in which oxidative burst may take part but the role of
which has yet to be defined.
In this study we aimed to identify steps of the signaling pathway that
are involved in the activation of the oxidative burst by osmotic stress
and to bring information about the possible role of this response in
osmoregulation, using suspension cells of tobacco. The oxidative burst
induced by a hypoosmotic stress was characterized. Transduction events
involved in oxidative burst activation were studied:
Ca2+ requirement, opening of stretch-activated
channels, and phosphorylation processes. Anion fluxes have been shown
in parsley cells to play a key role in elicitor signaling, because the
whole set of Phytophthora-induced defense responses,
including oxidative burst, was inhibited by anion channel blockers
(Jabs et al., 1997). Also, anion fluxes contribute to osmoregulation in
guard cells, where they mediate the response to ABA by regulation of
K+ fluxes (Schroeder, 1995; Ward et al., 1995).
Involvement of anion channels in the activation of oxidative burst by
osmotic stress was thus tested. A possible role of oxidative burst in
osmotic stress response through modulation of ion fluxes, which
constitute driving forces of osmoregulation in guard cells (Schroeder
and Hedrich, 1989) or through cell wall modifications, was also
evaluated.
| |
MATERIALS AND METHODS |
Tobacco (Nicotiana tabacum cv Xanthi) cells were
cultured in B5 Gamborg's medium with 1 µm 2,4-D and 60 nm kinetin in constant light. Suspension cells were used
after 4 d of subculturing with 60 to 100 mg fresh weight
mL1 cell density.
Inhibitors
Stock solutions of A9C and NPPB (100 mm) were prepared
in DMSO and in water for DIDS. Stock solutions of GdCl3 and
La(NO3)3 (500 and 250 mm) were
prepared in water. Stock solutions of staurosporine, DMAP, and apigenin
(2, 100, and 500 mm) were prepared in DMSO. Stock solutions
of IDP chloride and glucosamine sulfate (20 mm and 1 m) were prepared in DMSO and water, respectively.
Osmotic Stress
Osmolarity was monitored using a freezing-point osmometer
(Roebling, Berlin, Germany) on 100-µL aliquots.
Cells were washed and equilibrated for 3 h after repartition in
aliquots in an ion-poor medium (160 mOsm), which is isoosmotic to the
culture medium, containing 10 mm Mes-Tris, pH 5.2, 1 mm CaSO4, and 150 mm Suc.
This medium allowed ion efflux measurements. Afterward,
extracellular medium was replaced by the same volume of hypoosmotic
medium (10 mm Mes-Tris, pH 5.2, 1 mm
CaSO4, Suc-free, 15 mOsm), hyperosmotic medium
(10 mm Mes-Tris, pH 5.2, 1 mm
CaSO4, and 500 mm Suc, 640 mOsm), or
fresh isoosmotic medium for control cells. The final osmotic strengths
of extracellular mediums after transfer of cells were about 40 and 600 mOsm, respectively, for hypo- or hyperosmotic conditions. These
variations from 15 to 40 or 640 to 600 mOsm were due to the presence of
cells previously equilibrated in the isoosmotic medium. Hyperosmotic
medium corresponds to a plasmolysis-inducing medium for tobacco cells.
For extracellular pH measurements, 10 mm Mes-Tris was
replaced by 1 mm Mes-Tris.
For the study of glucosamine effects, cells were equilibrated in their
culture medium in the presence of 10 mm Mes-Tris, pH 5.2. One hour before the shock, 100 mm glucosamine solution,
corresponding to a dilution in isoosmotic medium of the 1 m
stock adjusted to pH 5.2 by NaOH, was added to the medium to get a 10 mm final concentration in glucosamine (an equal volume of
extracellular medium was removed before addition), whereas Man was
added in corresponding controls to reach the same osmolarity (180 mOsm)
in both cases. Cells were then transferred either in isoosmotic medium
composed of 150 mm Man, 10 mm Mes-Tris, and 1 mm CaSO4 for controls or in
hypoosmotic medium containing 10 mm Mes-Tris and 1 mm CaSO4. The inhibitor (10 mm glucosamine) or the osmotic equivalent (30 mm Man) was also present after transfer. Glucosamine
addition induced a very limited pH effect upon addition in the 10 mm buffered medium (lower than 0.1 pH unit) during
pretreatment, but this effect was not detectable during the time of the
experiments following transfer in a fresh medium containing 1 mm Mes-Tris (Fig. 6D, Iso-Gl).
View larger version (29K):
[in this window]
[in a new window]
| Figure 6.
Effect of inhibitors of
H2O2 production on the ion fluxes induced by
hypoosmotic stress. Aliquots of cell suspension were treated by IDP
(A-C, Hypo-IDP and Iso-IDP) or glucosamine (D, Hypo-Gl and Iso-Gl) as
described in the legend of Figure 2 for each of the inhibitors, except
the replacement of 10 mm by 1 mm Mes-Tris, pH
5.2, for the pH measurement experiments. Means ± se
of at least two independent experiments are reported in each case. FW,
Fresh weight.
|
|
Mechanical Stress
To generate a mechanical stress to cells, a cross-section rod was
maintained in open vials containing aliquots of cells previously equilibrated for 2 h in culture medium containing 10 mm Mes-Tris, pH 5.2. The method that we used does not imply
a physical pressure on the cells, which may damage the cells and induce
oxidative burst. Introduction of the piston only allows increased
contact of cells in the agitated culture. The contact surface with the cells was about 2-fold higher after introduction of the piston. It was
verified that the cell viability was not modified after the application
of mechanical stress.
Extracellular Alkalinization
Extracellular pH was monitored with a glass electrode in 6 mL of
cell suspension aliquots previously equilibrated in isoosmotic medium
containing 1 mm Mes-Tris, pH 5.2, in open vials on a
shaker.
Oxidative Burst Assay
H2O2 concentration was
measured using scopoletin fluorescence oxidative quenching
(excitation wavelength 350 nm; emission 460 nm). To measure the
H2O2 production rate, 500 mm stock solution of scopoletin in DMSO (240 µm final) and 2 mg mL1 stock
solution of peroxidase (10 µg mL1 final) were
added to 5-mL aliquots of a cell suspension previously equilibrated in
buffered isoosmotic medium. Scopoletin was progressively oxidized, and
the production of AOS was calculated from the fluorescence decrease
using a calibration curve established in the presence of
H2O2. Aliquots of medium
were taken at various intervals and monitored by a fluorimeter
(FluoroskanII, Labsystems, Helsinki, Finland) or a spectrofluorimeter
SFM25 (Kontron Instruments, Montigny Le Bretonneux, France). To measure
AOS accumulation, aliquots of extracellular medium were taken at
various intervals and mixed with scopoletin and peroxidase to reach 1 µm and 8 µg mL1 final
concentrations, respectively.
Inhibition of soybean (Glycine max L.) peroxidase by IDP,
excluding use of the peroxidase-dependent assay for
H2O2 determination, has
been reported (Dwyer et al., 1996). The effect of IDP on
H2O2 determination using
scopoletin was tested in tobacco cells. In usual conditions of assay
(see above) the addition of IDP to obtain final concentrations of 5, 20, 50, or 100 µm did not modify the decrease of
scopoletin fluorescence (154 ± 6 units) because of the addition
of H2O2 (150 µm final). No inhibition of peroxidase by IDP could thus
be detected in these assay conditions, allowing the use of scopoletin
for H2O2 determination. It
was verified that the other molecules, protein kinase inhibitors and
anion channel blockers, did not modify the
H2O2 determination assay in
the same way.
Ion Concentration Measurements
Aliquots of cells (30 mL) were equilibrated in buffered isoosmotic
medium for 3 h. After transfer to iso- or hypoosmotic medium, samples of the cell suspension were taken at various times and rapidly
filtered. Aliquots were used for monitoring by specific K+ and Cl electrodes
(D821 and XS21, Radiometer, Copenhagen, Denmark) at a constant ionic
strength in 0.1 m NaCl or 0.1 m
K2SO4, respectively.
Cell Wall Protein Preparation
Cell wall proteins were extracted from untreated cells or from
cells 5 min after transfer to iso- or hypoosmotic medium, 20 min after
mechanical stress onset, or 5 min after treatment with 1 mm
H2O2. To test the effect of
IDP, 20 µm IDP was added for the last 15 min of
equilibration and then during the mechanical or hypoosmotic stresses.
H2O2 (2 mm) was
also added for 5 min to the cell wall pellet from untreated cells.
Cells were filtered and plasmolyzed (15 min) at 4°C in 50 mm Mes-Tris, pH 6.0, 20% glycerol, 0.5 m Suc,
1 mm MgCl2, 10 mm ascorbic acid, 5 mm DTT, 0.6% PVP (w/v), and 0.5 µg
mL1 leupeptin. After the homogenate was ground
in a pressure crusher, it was centrifuged at 10,000g for 10 min, resuspended in 10 mm Mes-Tris, pH 5.2, and centrifuged
again (all centrifugations were held in the same conditions). The cell
wall 10,000g pellet was frozen in liquid nitrogen, ground in
a mortar, and homogenized resuspended with a Potter-Elvehjem
homogenizer in 2% SDS, 80 mm Tris, and 1 m
 -mercaptoethanol, pH 6.8. After centrifugation, proteins of the
supernatant were precipitated in acetone overnight at  20°C. The
resulting pellet was washed in 80% acetone and dried, and the proteins
were resuspended in denaturating buffer without a reducing agent. They
were stored at 20°C until use.
SDS-PAGE and Western-Blot Analysis
Cell wall proteins were analyzed by SDS-PAGE (10% polyacrylamide)
and silver staining (Pharmacia-modified method from Blum et al., 1987).
For the western blot, the SDS-PAGE gel was transferred using
electrotransfer with a Mini Trans-blot Cell (Bio-Rad) in the presence
of Tris-Gly buffer, 20% (v/v) ethanol, for 1 h at 100 V and 50 mA. The PVDF membrane was then saturated with 5% defatted milk in TBS
containing 0.1% (v/v) Tween 20 (TBS-T) for 1 h at 37°C. The
membrane was rinsed for 5 min three times in TBS-T. Hybridoma cell
culture supernatant of monoclonal antibody MAC265 directed against p100
soybean extensin (Bradley et al., 1992) was a gift from Nick Brewin
(John Innes Center, Norwich, UK) and was used at a final dilution of
1:10 in TBS-T for 3 h at room temperature. The membrane was then
washed three times for 10 min in TBS-T before incubation with anti-rat
IgG (1 mg mL1) conjugated to alkaline
phosphatase diluted 1:1000 in TBS-T for 1 h at room temperature.
The membrane was washed for 5 min in TBS-T twice before alkaline
phosphatase activity revelation by 5-bromo-4-chloro-3-indolyl phosphate
staining in the dark at room temperature.
| |
RESULTS |
Induction of Oxidative Burst by Hypoosmotic and Mechanical Stimuli
Production of H2O2 in
suspension-cultured tobacco cells was assayed by following the
scopoletin fluorescence decrease after osmotic or mechanical stresses.
To study the effect of osmolarity, cells were transferred in media that
were lypo-, iso-, or hyperosmotic relative to the cell culture medium.
After the hypoosmotic shock, significant production of
H2O2 was observed (Fig.
1A). AOS produced in hypoosmotic
conditions accumulated in the extracellular medium, reaching a maximum
after about 10 min (Fig. 1B). Afterward,
H2O2 degradation was higher
than the corresponding production, leading to the disappearance of
H2O2 in the medium (Fig.
1B) In contrast, cells transferred to isoosmotic or hyperosmotic medium
showed only weak H2O2
production (Fig. 1A) and no detectable accumulation (Fig. 1B). The
oxidative burst could be stopped by return to isoosmotic conditions
either by transfer back to isoosmotic medium or by addition of Suc
(data not shown). The magnitude of the hypoosmotically induced
oxidative burst was highly dependent on the osmotic strength of the
transfer medium (Fig. 1C). The use of Man, sorbitol, PEG, or NaCl
instead of Suc in the isoosmotic medium induced a similar oxidative
burst for the similar osmotic strength decrease (data not shown).
Increased AOS production was also observed when cells in their culture
medium were subjected to a mechanical stress (Fig. 1D), in agreement
with the possibility that the response to hypoosmotic stress may
involve mechanical signaling. Increasing the intensity of the
mechanical stress by use of syringe pistons differing in size, and thus
differing in contact surface with cells, increased AOS production (data
not shown).
View larger version (28K):
[in this window]
[in a new window]
| Figure 1.
Oxidative burst induced by hypoosmotic
(A-C) or mechanical (D) stress in suspension-cultured
cells. H2O2 production (A) and accumulation in
extracellular medium (B) by cells transferred at 0 time to hypoosmotic
(Hypo, 40 mOsm), isoosmotic (Iso, 160 mOsm), or hyperosmotic (Hyper,
600 mOsm) medium buffered with 10 mm Mes-Tris, pH 5.2. One
representative experiment of three independent experiments is
illustrated in each case. C, Osmotic strength dependence of the
oxidative burst triggered by transfer in hypoosmotic conditions.
Aliquots of cells were transferred at 0 time in hypoosmotic media
differing in osmotic strength (corresponding to differing Suc contents)
and AOS production was followed. Maximal rate (100%) corresponds to
H2O2 production rate for maximal osmotic shock.
Means ± se of two independent experiments are
reported. D, H2O2 production of cells subjected
to a mechanical stress (see ``Materials and Methods''). Means ± se of four independent experiments are reported. Aliquots of cell suspension were equilibrated for 3 h in isoosmotic (A-C) or in culture medium (D) buffered with 10 mm Mes-Tris, pH
5.2, before stress treatments. FW, Fresh weight.
|
|
Involvement of a Plasma Membrane NADPH Oxidase
A possible homology between the AOS-generating system involved in
the hypoosmotic response and the plasma membrane NADPH oxidase of
phagocytic cells was tested for two properties: sensitivity to IDP and
NADPH dependence. AOS production induced by hypoosmotic shock was
completely prevented by 20 µm IDP (Fig.
2A). Complete inhibition was also found
for the mechanically induced oxidative burst (data not shown). It was
verified that IDP did not modify the
H2O2 determination in the
assay conditions used (see "Oxidative Burst Assay"). To test NADPH
dependence, glucosamine was used to inhibit the pentose phosphate
pathway, as previously shown on tobacco cells (Pugin et al., 1997).
These authors demonstrated that in glucosamine-treated cells the
NADPH-to-NADP+ ratio determined by
31P-NMR was largely decreased and that the
elicitor-induced oxidative burst was completely prevented. The
oxidative burst induced by hypoosmotic shock was clearly inhibited by
10 mm glucosamine (Fig. 2B). These data suggest that the
AOS production system that was activated following hypoosmotic stress
shares two properties with the mammalian NADPH oxidase: IDP sensitivity
and NADPH dependence.
View larger version (23K):
[in this window]
[in a new window]
| Figure 2.
Inhibition by IDP (A) and glucosamine (B) of the
oxidative burst induced by hypoosmotic stress in tobacco cell
suspensions. A, Aliquots of cell suspension were equilibrated for
3 h in isoosmotic medium before transfer at 0 time to hypoosmotic
(Hypo), isoosmotic (Iso), hypoosmotic containing 20 µm
IDP (Hypo-IDP), or isoosmotic containing 20 µm IDP
(Iso-IDP) media. The inhibitor was also added during the last 15 min of
the equilibration time at the concentration used after transfer. B,
Aliquots of cell suspension were equilibrated for 2 h in buffered
culture medium and for an additional 1 h in the same medium
containing 10 mm glucosamine, before transfer in
hypoosmotic medium containing glucosamine (Hypo-Gl) or isoosmotic medium containing glucosamine (Iso-Gl). Corresponding controls were
treated similarly, except for the replacement of glucosamine by 30 mm Man during the last 1 h of equilibration and after
transfer in hypoosmotic (Hypo) or isoosmotic (Iso) conditions.
Means ± se of two independent experiments are
reported in each case. FW, Fresh weight.
|
|
Transduction Events Involved in Oxidative Burst Induction
Activation of the oxidative burst by hypoosmotic and mechanical
stresses suggested involvement of stretch forces at the plasma membrane
level. Gd, reported as an inhibitor of stretch-activated channels (Yang
and Sachs, 1989; Ding and Pickard, 1993), was thus tested. In tobacco
cell suspensions, Gd was able to prevent the oxidative burst induced by
hypoosmotic shock in a dose-dependent manner, with a nearly total
inhibition efficiency for 500 µm Gd (Fig.
3A). Oxidative burst activation by
mechanical stress was also inhibited by 500 µm Gd (data
not shown). These results suggest that stretch-activated channels may
be involved in oxidative burst activation.
View larger version (39K):
[in this window]
[in a new window]
| Figure 3.
Inhibition of the hypoosmotically induced
oxidative burst by the presence of Gd (A) or La (B) and by the lack of
extracellular Ca2+ (C and D) in cell suspensions. A and B,
Aliquots of cell suspension were equilibrated for 3 h in
isoosmotic medium before transfer at time 0 to hypoosmotic (Hypo),
isoosmotic (Iso), or hypoosmotic medium containing 250 or 500 µm La(NO3)3 or GdCl3
(Hypo-La and Hypo-Gd) or isoosmotic medium containing the same
inhibitor concentrations (Iso-La and Iso-Gd). The inhibitor
La(NO3)3 or GdCl3 was also added
during the last 15 min of the equilibration time, at the concentration
used after transfer. C and D, Aliquots of cell suspension were
equilibrated for 3 h in isoosmotic medium deprived of
Ca2+ before transfer at 0 time in hypoosmotic medium
containing 5 or 10 mm EGTA (Hypo-EGTA) or without EGTA
(Hypo) and in isoosmotic medium containing 5 or 10 mm EGTA
(Iso-EGTA) or without EGTA (Iso). In D, 3 mm
CaCl2 was added after about 30 min to the cells previously transferred in hypoosmotic (Hypo-Ca) or isoosmotic (Iso-Ca) media containing 10 mm EGTA. One representative experiment out of
three independent experiments is illustrated in each part of the
figure. FW, Fresh weight.
|
|
Two methods were used to investigate the role of
Ca2+ in the induction of oxidative burst by
hypoosmotic shock. The first one used La, which has been described as
an inhibitor of Ca2+ channels (Ding and Pickard,
1993). The presence of La inhibited the oxidative burst with a maximal
inhibition for 250 µm La (Fig. 3B). The second method was
based on extracellular Ca2+ depletion by EGTA.
Cells were equilibrated for 3 h in Ca2+-free
medium and then transferred into a Ca2+-free
hypoosmotic medium containing EGTA. The oxidative response was clearly
reduced in a dose-dependent manner (Fig. 3C). The oxidative burst could
be reactivated after 10 mm EGTA treatment for 30 min by
addition of 3 mm CaCl2 (Fig. 3D).
These data indicate the involvement of Ca2+ in
the oxidative burst activation.
Hypoosmotic stress induced rapid Cl efflux (Fig.
4A). The possibility that anion efflux
involved in osmoregulation may play a role in the induction of the
oxidative burst was investigated. Three structurally unrelated
inhibitors of anion channels, NPPB, A9C, and DIDS, shown to be active
on guard cells (Marten et al., 1992; Ward et al., 1995) and tobacco
cell suspensions (S. Zimmerman, J.M. Frachisse, S. Thomine, H. Barbier-Brygoo, and J. Guern, unpublished data), were tested on
hypoosmotically stressed cells. These molecules were able to inhibit
the Cl effluxes induced by hypoosmotic shock with varying
efficiencies for a same inhibitor concentration (Fig. 4B). The
oxidative response was clearly susceptible to the three anion channel
blockers, with about 100, 80, and 60% inhibition for NPPB, A9C, and
DIDS, respectively. These products did not interfere with the
H2O2 determination in the
assay conditions used (data not shown). These results suggest that
Cl efflux could be a part of the signaling pathway for
hypoosmotically induced oxidative burst.
View larger version (41K):
[in this window]
[in a new window]
| Figure 4.
Cl efflux induced by hypoosmotic
stress (A) and inhibition of hypoosmotically induced responses by anion
channel blockers NPPB, A9C, and DIDS (B). A, Aliquots of cell
suspension were equilibrated for 3 h in isoosmotic medium before
transfer at 0 time to hypoosmotic (Hypo) or isoosmotic (Iso) medium.
Means ± se of two independent experiments are
reported. B, Aliquots of cell suspension were equilibrated for 3 h
in isoosmotic medium before transfer at time 0 to hypoosmotic medium
containing 100 µm NPPB, 100 µm A9C, or 100 µm DIDS. Inhibitions by each molecule of the
Cl efflux (left part) and H2O2
production (right part) initial rates, in comparison with control cells
deprived of inhibitor, are reported. Each inhibitor was also added
during the last 15 min of the equilibration time at the concentration
used after transfer. Means ± se of at least two
independent experiments are reported in each case. FW, Fresh weight.
|
|
Involvement of protein phosphorylation in the hypoosmotically induced
oxidative response was tested with various protein kinase inhibitors,
among which three structurally unrelated protein kinase inhibitors,
staurosporine, DMAP, and apigenin, were effective. All three inhibitors
were able to inhibit completely the oxidative burst, at 0.5 µm for staurosporine and 500 µm for the
other two molecules (Fig. 5). Although
these inhibitors were reported to block specifically protein kinase C
for staurosporine, cyclin-dependent kinases for DMAP, and MAP kinases
for apigenin, it is most probable that these compounds may have broader
specificities at these concentrations.
View larger version (17K):
[in this window]
[in a new window]
| Figure 5.
Involvement of protein phosphorylation in the
activation of oxidative burst by hypoosmotic stress. Aliquots of cell
suspension were equilibrated for 3 h in isoosmotic medium before
transfer at 0 time to hypoosmotic (Hypo), isoosmotic (Iso), or
hypoosmotic medium containing 0.5 µm staurosporine (A,
Hypo-Stau), 500 µm DMAP (B, Hypo-DMAP), 500 µm apigenin (C, Hypo-Api), or isoosmotic medium
containing the same inhibitor concentrations (A, Iso-Stau; B, Iso-DMAP;
and C, Iso-Api). One representative experiment of three independent
experiments is illustrated in each case. FW, Fresh weight.
|
|
Effect of the Oxidative Burst on Ion Fluxes
Hypoosmotic stress induced K+ and
Cl effluxes, as well as extracellular
alkalinization (Fig. 6, Hypo and Iso
curves). IDP and glucosamine were shown to inhibit the oxidative
response to hypoosmotic stress (Fig. 2). The possibility that AOS
production may play a role in the activation of ionic responses to the
hypoosmotic signal was evaluated. First, oxidative burst inhibition by
IDP resulted in a slight decrease of K+ efflux
(Fig. 6A), Cl efflux (Fig. 6B), and
extracellular alkalinization (Fig. 6C) induced by hypoosmotic shock.
Second, extracellular alkalinization was partially inhibited in the
presence of glucosamine (Fig. 6D). Although the effect seems weak
(20-30% inhibition), it was highly reproducible.
Effect of the Oxidative Burst on Cell Wall Protein
Cross-Linking
Insolubilization of cell wall proteins in response to hypoosmotic
and mechanical stresses was evaluated. Analysis by SDS-PAGE and silver
staining of SDS-extracted cell wall proteins (Fig. 7A) revealed the disappearance of a
protein of approximately 115 kD in hypoosmotically (lane c) or
mechanically (lane e) stressed cells compared with untreated cells
(lane a) or isoosmotically transferred cells (lane b). The protein was
not insolubilized after hypoosmotic shock or mechanical stress when the
oxidative burst was inhibited by 20 µm IDP (lanes d and
f, respectively). AOS involvement in the 115-kD protein
insolubilization was also confirmed by addition of
H2O2 to cells (lane g) or
to cell walls (lane h). These results indicate that the oxidative burst
activated by hypoosmotic shock or by mechanical stress induces the
cross-linking of a 115-kD protein. The MAC265 antibody directed against
a 100-kD extensin in soybean (Bradley et al., 1992) was used to
identify the 115-kD protein. Analysis by western blot (Fig. 7B)
revealed that a large band at approximately 115 kD, like that in silver staining, was recognized by the MAC265 antibody in control cells (lane
a) and that the protein disappeared in extracts from hypoosmotically stressed cells (lane b).
View larger version (62K):
[in this window]
[in a new window]
| Figure 7.
Cross-linking of cell wall proteins by oxidative
burst induced by hypoosmotic or mechanical stress in tobacco cells. A,
SDS-PAGE and silver staining of cell wall proteins extracted from
untreated cells (lane a), 5 min after cell transfer in isoosmotic
medium (lane b), hypoosmotic medium (lane c), hypoosmotic medium
containing 20 µm IDP (lane d), after 20 min of mechanical
stress in culture medium (lane e) or culture medium containing 20 µm IDP (lane f), after 5 min of in vivo treatment of
cells by 1 mm H2O2 (lane g) or 5 min of in vitro treatment of proteins by 2 mm
H2O2 (lane h). B, Western blot of cell wall
proteins corresponding to cells transferred in hypoosmotic medium
containing 20 µm IDP (a) or hypoosmotic medium without
IDP (b). St, Molecular mass standards (in kilodaltons). Arrowheads in A
and B indicate the 115-kD bands.
|
|
| |
DISCUSSION |
The present study documents the induction of an oxidative burst in
suspension-cultured tobacco cells transferred to hypoosmotic medium.
Specificity of the rapid production and extracellular accumulation of
AOS was shown by comparison with transfer experiments in iso- or
hyperosmotic conditions (Fig. 1, A and B). These results extend a
previous demonstration of oxidative burst induction by dilution with
water of soybean cell suspension (Yahraus et al., 1995). A similar
production of AOS was measured when cells were subjected to a
mechanical stress, independently of any change in the medium
composition (Fig. 1D). This result suggests that physical perturbations
at the plasma membrane level may take part in the transduction pathway
originating from the hypoosmotic signal. In agreement with this idea,
Gd, known as a stretch-activated channel inhibitor, was efficient to
prevent the activation of the hypoosmotically induced oxidative burst
(Fig. 3A). However, it cannot be excluded that Gd may prevent oxidative
burst by inhibition of Ca2+ channels as a
lanthanide-type element. It would be interesting to further assess the
parallel between transduction steps that correspond to hypoosmotic
shock and mechanical stimuli, to approach the eventual complexity of
the osmotic signal.
Several transduction events located upstream of the oxidase activated
by hypoosmotic stress could be identified, even if the sequence of the
steps was not yet determined within the cascade. The induction of the
oxidative burst was dependent on Ca2+ channels
(Fig. 3B) and external Ca2+ (Fig. 3, C and D).
The role of Ca2+ in the activation of oxidative
burst in plants was previously demonstrated in response to several
elicitors (for review, see Low and Merida, 1996) and, therefore, seems
common in these two different kinds of stimuli.
Ca2+ was evidenced as a response to hypoosmotic
shock in Lamprothamium succinctum cells (Okasaki and Tazawa,
1986), and intracellular Ca2+ increase in BY2
tobacco cells following hypoosmotic shock has recently been reported
(Takahashi et al., 1997a). Ca2+ influx was also
evidenced as a response to mechanical stress in plants (Braam and
Davis, 1990; Ding and Pickard, 1993). Ca2+ may
transduce the hypoosmotic signal through
Ca2+-dependent protein kinases. This hypothesis
has been tested in algae (Yuasa and Muto, 1992, 1996) and in BY2
tobacco cells (Takahashi et al., 1997b). Activations of protein kinases
were evidenced in the hypoosmotic response, but these kinases do not
require Ca2+ for their activities and the
postulated Ca2+-dependent protein kinases have
not yet been identified. Phosphorylations are also key elements in the
responses to mechanical stress (Piotrowski et al., 1996). In the
present study staurosporine, DMAP, and apigenin, three structurally
unrelated inhibitors of protein kinases, prevented oxidative burst
activation following hypoosmotic shock (Fig. 5), and the nature of the
involved protein kinases is currently being investigated. Inhibition of
Cl fluxes by different anion channel blockers, NPPB, A9C,
and DIDS, is concomitant with corresponding oxidative burst prevention
(Fig. 4). It suggests a role of Cl effluxes as a step in
hypoosmotic signal transduction in tobacco cells, leading to oxidase
activation. Similarly, in parsley cell suspensions, various anion
channel blockers were shown to inhibit with the same efficiency
elicitor-induced Cl and K+
effluxes, H2O2 production,
and phytoalexin accumulation, thus placing anion channels as the most
upstream elements so far identified within the elicitor-signaling
cascade (Jabs et al., 1997). The mechanisms by which anion fluxes are
required to initiate and/or maintain the oxidative burst are not yet
understood.
The AOS-producing system activated by hypoosmotic shock was shown to be
IDP sensitive (Fig. 2A) and NADPH dependent (Fig. 2B), thus sharing
these properties with NADPH oxidase from neutrophils. The
AOS-generating oxidase activated by elicitors in plants is also IDP
sensitive and was recently shown to be NADPH dependent by the original
approach, allowing NADPH depletion, as established by Pugin et al.
(1997). Therefore, it seems that this system can be activated by
different kinds of signals, such as elicitor and hypoosmotic shock.
However, involved signal transduction pathways are likely to differ
from one signal to another (Chandra et al., 1996b).
The AOS-generating system, as a component of a highly regulated system
of membrane transporters, and the produced AOS, through modification of
cell wall structure, could play a role in cell volume and turgor
regulation. Ion, organic solute, and water fluxes drive osmoregulation,
and it was shown here that oxidative burst contributes to a small but
significant part of Cl and K+
effluxes and extracellular alkalinization induced by hypoosmotic stress
(Fig. 6). Because oxidative burst induction depends on anion effluxes,
it suggests a feedback regulation of oxidative burst on anion
effluxes. Strong reduction of oligogalacturonide-induced extracellular
alkalinization by oxidative burst inhibition was also previously
observed (Rouet-Mayer et al., 1997). The way oxidative burst
participates in the regulation of these fluxes is still in question.
Oxidative burst could cause membrane lipid peroxidation, which would
modify plasma membrane permeability. Activated oxygen species could
interact with ion channel activity and oxidase-dependent electron
transfer could drive a depolarization, both effects leading to channel
activation. The regulation of K+ channels by
H2O2 (Vega-Saenz de Miera
and Rudy, 1992; Duprat et al., 1995) or by a membrane oxidoreductase
activity (Fehlau et al., 1989) has been reported in animals.
Electrophysiological studies should give new insights into the possible
role of the oxidative burst in osmoregulation.
Oxidative burst could also induce reinforcement of the cell wall. It
was shown that a 115-kD protein was cross-linked within 5 min upon
hypoosmotic stress, and this cross-linking was also observed upon
addition of exogenous H2O2
(Fig. 7). This protein was recognized by an antibody directed against
soybean extensin. Extensin, a Hyp-rich protein, is one of the major
cell wall proteins. Its cross-linking could contribute to cell wall
reinforcement, along with other cross-linked cell wall components,
leading to cell volume increase limitation after hypoosmotic stress.
Cross-linking may also contribute to turgor pressure increase and,
thus, contribute to the induction of osmosensing.
High-osmolarity-adapted cell suspension displayed diminished cell
volume, as well as cell wall-insoluble protein increase, and it was
also shown that Hyp-rich proteins are responsible for the cell wall
tensile strength (Iraki et al., 1989). Cell volume and turgor
regulations are also involved in cellular elongation and growth (Hohl
and Schopfer, 1995). At the plant level, oxidative burst could limit
growth by inducing protein peroxidase-dependent cross-linking and
lignification. Inhibition of peroxidase secretion was observed in the
case of gibberellin-stimulated growth (Fry, 1979), and reticulation of
Hyp-rich proteins increased with diminished growth (Taiz, 1984).
H2O2 inhibited elongation of coleoptiles (Schopfer, 1996) and was associated with lignification (Olson and Varner, 1993). Bradley et al. (1992) showed a correlation between the end of growth and protein cross-linking in bean hypocotyls (Bradley et al., 1992), suggesting that it could be one of the molecular bases for the measured modification of cell wall elasticity and plasticity. Cross-linking not only limits growth but also contributes to the plant morphology (Carpita and Gibeaut, 1993). Oxidative burst induction by mechanical stress suggested that it could
be involved in thigmomorphogenesis (Legendre et al., 1993a). From these
results, a general role of oxidative burst in plant development is
suggested.
| |
FOOTNOTES |
*
Corresponding author; e-mail
christiane.lauriere{at}isv.cnrs-gif.fr; fax 33-1-69-82-37-68.
Received August 15, 1997;
accepted November 7, 1997.
| |
ABBREVIATIONS |
Abbreviations:
AOS, active oxygen species.
A9C, anthracene-9-carboxylic acid.
DIDS, 4,4 -diisothiocyanostilbene-2,2-disulfonic acid.
DMAP, 6-dimethylaminopurine.
IDP, iodonium diphenyl.
NPPB, 5-nitro-2-(3-phenyl propylamino)-benzoic acid.
| |
ACKNOWLEDGMENTS |
We wish to warmly thank Jean Guern, who initiated and showed
constant interest for this work. We also thank Nick Brewin for the gift
of monoclonal antibody.
| |
LITERATURE CITED |
Baker CJ,
Orlandi EW,
Mock NM
(1993)
Harpin, an elicitor of the hypersensitive response in tobacco caused by Erwinia amylovora, elicits active oxygen production in suspension cells.
Plant Physiol
102:
1341-1344
[Abstract]
Blum H,
Beier H,
Gross HJ
(1987)
Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels.
Electrophoresis
8:
93-99
[CrossRef][ISI]
Bottin A,
Véronési C,
Pontier D,
Esquerré-Tugayé MT,
Blein JP,
Rusterucci C,
Ricci P
(1994)
Differential responses of tobacco cells to elicitors from two Phytophtora species.
Plant Physiol Biochem
32:
373-378
Braam J,
Davis RW
(1990)
Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes in Arabidopsis.
Cell
60:
357-364
[CrossRef][ISI][Medline]
Bradley DJ,
Kjellbom P,
Lamb CJ
(1992)
Elicitor and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response.
Cell
70:
21-30
[CrossRef][ISI][Medline]
Brisson LF,
Tenhaken R,
Lamb C
(1994)
Function of oxidative cross-linking of cell wall structural proteins in plant disease resistance.
Plant Cell
6:
1703-1712
[Abstract/Free Full Text]
Carpita NC,
Gibeaut DM
(1993)
Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth.
Plant J
3:
1-30
[CrossRef][ISI][Medline]
Chandra S,
Heinstein PF,
Low PS
(1996a)
Activation of phospholipase A by plant defense elicitors.
Plant Physiol
110:
979-986
[Abstract]
Chandra S,
Low PS
(1995)
Role of phosphorylation in elicitation of the oxidative burst in cultured soybean cells.
Proc Natl Acad Sci USA
92:
4120-4123
[Abstract/Free Full Text]
Chandra S,
Martin GB,
Low PS
(1996b)
The Pto kinase mediates a signaling pathway leading to the oxidative burst in tomato.
Proc Natl Acad Sci USA
93:
13393-13397
[Abstract/Free Full Text]
Chen Z,
Silva H,
Klessig DF
(1993)
Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid.
Science
262:
1883-1886
[Abstract/Free Full Text]
Desikam 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]
Devlin WS,
Gustine DL
(1992)
Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction.
Plant Physiol
100:
1189-1195
[Abstract/Free Full Text]
Ding JP,
Pickard BG
(1993)
Mechanosensory calcium-selective cation channels in epidermal cells.
Plant J
3:
83-110
[CrossRef][ISI][Medline]
Duprat F,
Guillemare E,
Romey G,
Fink M,
Lesage F,
Lazdunski M,
Honore E
(1995)
Susceptibility of cloned K+ channels to reactive oxygen species.
Proc Natl Acad Sci USA
92:
11796-11800
[Abstract/Free Full Text]
Dwyer SC,
Legendre L,
Low PS,
Leto TL
(1996)
Plant and human neutrophil oxidative burst complexes contain immunologically related proteins.
Biochim Biophys Acta
1289:
231-237
[Medline]
Fehlau R,
Grygorczyk R,
Fuhrmann GF,
Schwarz W
(1989)
Modulation of the Ca2+- or Pb2+-activated K+-selective channels in human red cells. II. Parallelisms to modulation of the activity of a membrane-bound oxidoreductase.
Biochim Biophys Acta
978:
37-42
[Medline]
Fry SC
(1979)
Phenolic components of the primary cell wall and their possible role in the hormonal regulation of growth.
Planta
146:
343-351
[CrossRef]
Groom QJ,
Torres MA,
Fordham-Skelton AP,
Hammond-Kosack KE,
Robinson NJ,
Jones JDG
(1996)
rbohA, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene.
Plant J
10:
515-522
[CrossRef][ISI][Medline]
Hallows KR,
Knauf PA
(1994)
Principles of cell volume regulation.
In
K Strange,
eds, Cellular and Molecular Physiology of Cell Volume Regulation.
CRC Press, Boca Raton, FL, pp 3-29
Henderson LM,
Chappell JB
(1996)
NADPH oxidase of neutrophils.
Biochim Biophys Acta
1273:
87-107
[Medline]
Hohl M,
Schopfer P
(1995)
Rheological analysis of viscoelastic cell wall changes in maize coleoptiles as affected by auxin and osmotic stress.
Physiol Plant
94:
499-505
[CrossRef]
Iraki NM,
Bressan RA,
Hasegawa PM,
Carpita NC
(1989)
Alteration of the physical and chemical structure of the primary cell wall of growth-limited plant cells adapted to osmotic stress.
Plant Physiol
91:
39-47
[Abstract/Free Full Text]
Jabs T,
Tschöpe M,
Colling C,
Hahlbrock K,
Scheel D
(1997)
Elicitor-stimulated ion fluxes and O2 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]
Legendre L,
Heinstein PF,
Low PS
(1992)
Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells.
J Biol Chem
267:
20140-20147
[Abstract/Free Full Text]
Legendre L,
Rueter S,
Heinstein PF,
Low PS
(1993a)
Characterization of the oligogalacturonide-induced oxidative burst in cultured soybean (Glycine max) cells.
Plant Physiol
102:
233-240
[Abstract]
Legendre L,
Yueh YG,
Crain R,
Haddock N,
Heinstein PF,
Low PS
(1993b)
Phospholipase C activation during elicitation of the oxidative burst in cultured plant cells.
J Biol Chem
268:
24559-24563
[Abstract/Free Full Text]
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]
Low PS,
Merida JR
(1996)
The oxidative burst in plant defense: function and signal transduction.
Physiol Plant
96:
533-542
[CrossRef]
Marten I,
Zeilinger C,
Redhead C,
Landry DW,
AI-Awqati Q,
Hedrich R
(1992)
Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.
EMBO J
11:
3569-3575
[ISI][Medline]
Mathieu Y,
Sanchez FJ,
Droillard M-J,
Lapous D,
Laurière C,
Guern J
(1996)
Involvement of protein phosphorylation in the early steps of transduction of the oligogalacturonide signal in tobacco cells.
Plant Physiol Biochem
34:
399-408
Mehdy MC,
Sharma YK,
Sathasivan K,
Bays NW
(1996)
The role of activated oxygen species in plant disease resistance.
Physiol Plant
98:
365-374
[CrossRef]
Miyahara M,
Watanabe Y,
Edashige K,
Yagyu KI
(1993)
Swelling-induced O2-generation in guinea-pig neutrophils.
Biochim Biophys Acta
1177:
61-70
[Medline]
Nurnberger T,
Nennstiel D,
Hahlbrock K,
Scheel D
(1995)
Covalent cross-linking of the Phytophtora megasperma oligopeptide elicitor to its receptor in parsley membranes.
Proc Natl Acad Sci USA
92:
2338-2342
[Abstract/Free Full Text]
Okasaki Y,
Tazawa M
(1986)
Involvement of calcium ion in turgor regulation upon hypotonic treatment in Lamprothamnium succinctum.
Plant Cell Environ
9:
185-190
Olson PD,
Varner JE
(1993)
Hydrogen peroxide and lignification.
Plant J
4:
887-892
[CrossRef]
Otte O,
Barz W
(1996)
The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubilization of two cell wall structural proteins.
Planta
200:
238-246
Piotrowski M,
Liss H,
Weiler EW
(1996)
Touch-induced protein phosphorylation in mechanosensitive tendrils of Bryonia dioica Jacq.
J Plant Physiol
147:
539-546
Pugin A,
Frachisse JM,
Tavernier E,
Bligny R,
Gout E,
Douce R,
Guern J
(1997)
Early events induced by the elicitor cryptogein in tobacco cells. Involvement of a plasma membrane NADPH oxidase and activation of glycolysis and the pentose phosphate pathway.
Plant Cell
11:
2077-2091
Rouet-Mayer MA,
Mathieu Y,
Cazalé AC,
Guern J,
Lauriè C
(1997)
Extracellular alkalinization and oxidative burst induced by fungal pectin lyase in tobacco cells are not due to the perception of oligogalacturonide fragments.
Plant Physiol Biochem
35:
321-330
Schopfer P
(1996)
Hydrogen peroxide-mediated cell-wall stiffening in vitro in maize coleoptiles.
Planta
199:
43-49
Schroeder J
(1995)
Anion channels as central mechanisms for signal transduction in guard cells and putative functions in roots for plant-soil interactions.
Plant Mol Biol
28:
353-361
[CrossRef][ISI][Medline]
Schroeder JI,
Hedrich R
(1989)
Involvement of ion channels and active transport in osmoregulation and signaling of higher plant cells.
Trends Biochem Sci
14:
187-192
[CrossRef][Medline]
Schwacke R,
Hager A
(1992)
Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca2+ and protein-kinase activity.
Planta
187:
136-141
Taiz L
(1984)
Plant cell expansion: regulation of cell wall mechanical properties.
Annu Rev Plant Physiol
35:
585-657
[CrossRef][ISI]
Takahashi K,
Isobe M,
Knight MR,
Trewavas AJ,
Muto S
(1997a)
Hypoosmotic shock induces increases in cytosolic Ca2+ in tobacco suspension-culture cells.
Plant Physiol
113:
587-594
[Abstract]
Takahashi K,
Isobe M,
Muto S
(1997b)
An increase in cytosolic calcium ion concentration precedes hypoosmotic shock-induced activation of protein kinases in tobacco suspension culture cells.
FEBS Lett
401:
202-206
[CrossRef][ISI][Medline]
Tavernier E,
Wendehenne D,
Blein JP,
Pugin A
(1995)
Involvement of free calcium in action of cryptogein, a proteinaceous elicitor of hypersensitive reaction, in tobacco cells.
Plant Physiol
109:
1025-1031
[Abstract]
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]
Vega-Saenz de Miera E,
Rudy B
(1992)
Modulation of K+ channels by hydrogen peroxide.
Biochem Biophys Res Commun
186:
1681-1687
[CrossRef][ISI][Medline]
Viard MP,
Martin F,
Pugin A,
Ricci P,
Blein JP
(1994)
Protein phosphorylation is induced in tobacco cells by the elicitor cryptogein.
Plant Physiol
104:
1245-1249
[Abstract]
Ward JM,
Pei Z-M,
Schroeder JI
(1995)
Roles of ion channels in initiation of signal transduction in higher plants.
Plant Cell
7:
833-844
[CrossRef][ISI][Medline]
Wendehenne D,
Binet MN,
Blein JP,
Ricci P,
Pugin A
(1995)
Evidence for specific high affinity binding sites for a proteinaceous elicitor in tobacco plasma membrane.
FEBS Lett
374:
203-207
[CrossRef][ISI][Medline]
Yahraus T,
Chandra S,
Legendre L,
Low PS
(1995)
Evidence for a mechanically induced oxidative burst.
Plant Physiol
109:
1259-1266
[Abstract]
Yang XC,
Sachs F
(1989)
Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions.
Science
243:
1068-1071
[Abstract/Free Full Text]
Yu LM
(1995)
Elicitins from Phytophotora and basic resistance in tobacco.
Proc Natl Acad Sci USA
92:
4088-4094
[Abstract/Free Full Text]
Yuasa T,
Muto S
(1992)
Change of protein phosphorylation of Dunaliella tertiolecta cells in osmotic shock.
In
M Murata,
eds, Research in Photosynthesis, Vol 4.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 263-266
Yuasa T,
Muto S
(1996)
Plant Cell Physiol
37:
35-42
[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
D. Bustos, R. Lascano, A. L. Villasuso, E. Machado, M. E. Senn, A. Cordoba, and E. Taleisnik
Reductions in Maize Root-tip Elongation by Salt and Osmotic Stress do not Correlate with Apoplastic O2*- Levels
Ann. Bot.,
October 1, 2008;
102(4):
551 - 559.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Trouverie, G. Vidal, Z. Zhang, C. Sirichandra, K. Madiona, Z. Amiar, J.-L. Prioul, E. Jeannette, J.-P. Rona, and M. Brault
Anion Channel Activation and Proton Pumping Inhibition Involved in the Plasma Membrane Depolarization Induced by ABA in Arabidopsis thaliana Suspension Cells are Both ROS Dependent
Plant Cell Physiol.,
October 1, 2008;
49(10):
1495 - 1507.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Beffagna and I. Lutzu
Inhibition of catalase activity as an early response of Arabidopsis thaliana cultured cells to the phytotoxin fusicoccin
J. Exp. Bot.,
December 1, 2007;
58(15-16):
4183 - 4194.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction