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Plant Physiol. (1998) 117: 1301-1305
The Superoxide Synthases of Rose Cells1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In an effort to identify the
enzymatic mechanism responsible for the synthesis of reactive oxygen
species produced during the hypersensitive response, preparations of
rose (Rosa damascena) cell plasma membranes, partially
solubilized plasma membrane protein, and cytosol were assayed for the
NADH- and NADPH-dependent synthesis of superoxide using assays for the
reduction of cytochrome c (Cyt c), assays
for the reduction of nitroblue tetrazolium, and assays for the
chemiluminescence of
N,N
-dimethyl-9,9-biacridium dinitrate (lucigenin). Each assay ascribed the highest activity to a different preparation: the Cyt c assay to cytosol, the nitroblue
tetrazolium assay to plasma membrane, and the lucigenin assay to the
partially solubilized plasma membrane protein (with NADH). This
suggests that no two assays measure the same set of enzymes and that
none of the assays is suitable for comparisons of superoxide synthesis among different cell fractions. With the plasma membrane preparation, the presence of large amounts of superoxide-dismutase-insensitive Cyt
c reductase confounded attempts to use Cyt
c to measure superoxide synthesis. With the partially
solubilized membrane protein, direct reduction of lucigenin probably
contributed to the chemiluminescence. Superoxide synthesis detected
with lucigenin should be confirmed by superoxide-dismutase-sensitive
Cyt c reduction.
In plant systems the synthesis of ROS, including superoxide,
hydrogen peroxide, and the hydroxyl radical, occurs as a by-product of
normal metabolism. However, excesses of ROS are produced during particular periods of development and in response to various stresses. There has been a recent focus on ROS produced during the hypersensitive response to pathogen infection and to the presence of noninfective elicitors from pathogenic and nonpathogenic microbes.
One mechanism for the production of ROS is the single-electron
reduction of O2 to form superoxide. Dismutation
of superoxide forms hydrogen peroxide and in the presence of transition
metals, principally ferrous iron, the Fenton reaction produces hydroxyl radicals from the substrate hydrogen peroxide. Thus, the formation of
superoxide leads to the other species of ROS. The detection of
superoxide and the quantification of superoxide synthesis is a
challenge, because traditional spectrophotometric methods are not
especially sensitive. A sensitive method that depends on the chemiluminescence of lucigenin has been used extensively; it is considered to be a specific indicator of superoxide because it shows
little signal from hydrogen peroxide (Corbisier et al., 1987 Using lucigenin, our laboratory (Auh and Murphy, 1995 Recent reports (Faulkner and Fridovich, 1993 Because of the importance of ROS production to many aspects of plant
cell physiology, we considered it necessary to use other assays to
confirm our earlier report of superoxide synthesis from rose plasma
membranes. The following data demonstrate the difference in the
characteristics and rates of NADH- and NADPH-dependent superoxide-synthetic enzymes in three fractions from cultured rose
cells, as assayed by lucigenin luminescence, Cyt c
reduction, and NBT reduction.
Chemicals
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
) and has
been used in a large number of studies of ROS production (for refs.,
see Faulkner and Fridovich, 1993).
) reported the
accumulation of superoxide in suspension cultures of rose (Rosa
damascena) cells treated with a cell wall elicitor from Phytophthora cinnamomea. Subsequently, Murphy and Auh (1996)
used lucigenin to measure the rate of synthesis of superoxide by
enzymes in rose cell plasma membranes.
; Liochev and Fridovich,
1997; Vásquez-Vivar et al., 1997) have cast doubt on the utility
of lucigenin as a specific indicator of the presence or synthesis of
superoxide. Liochev and Fridovich (1997) pointed out that the
chemiluminescence of lucigenin requires an initial reduction followed
by the reaction with superoxide. The ability of potassium
superoxide alone to induce a transient chemiluminescence from
lucigenin (Liochev and Fridovich, 1997) suggests that the superoxide
radical can itself accomplish the initial reduction, the reduced form
then reacting with an additional superoxide molecule. However, the
ability of endothelial nitric oxide synthase (Vásquez-Vivar et
al., 1997) and xanthine oxidase (Liochev and Fridovich, 1997) to elicit
chemiluminescence from lucigenin under conditions in which these
enzymes are not known to produce superoxide shows that reduction of
lucigenin is sufficient to give a positive signal: reduced lucigenin
can reduce oxygen and the resulting superoxide reacts with additional
reduced lucigenin to give chemiluminescence. Because the reaction
sequence still involves superoxide, chemiluminescence is inhibited by
SOD, even though the enzyme activity that is measured is not superoxide
synthesis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Cell Preparation
Cells of rose (Rosa damascena Mill. cv Gloire de Guilan) were grown in suspension in liquid medium as described by Murphy et al. (1979).
Extraction and Preparation of the S100, Plasma Membrane, and Supernatant II
Four-day suspension-cultured rose cells were washed once with water and filtered on Whatman 3MM paper. Polyvinylpolypyrrolidone (approximately 1 g g
1 fresh weight of
cells) and grinding buffer (25 mM Tris-Mes, pH 6.5, 3 mM Na2EDTA, 4.94 mM DTT,
250 mM Suc, and 2 mg/mL BSA) were added to the cells. The
cells were then sonicated four times for 15 s each, and the
resulting solution was filtered through four layers of cheesecloth.
PMSF was added to the filtered solution at 0.25 mM. The
filtrate was centrifuged at 10,000g for 15 min. The
supernatant was collected and centrifuged for 40 min at
100,000g. The supernatant from this centrifugation, labeled
S100, was dialyzed against grinding buffer minus DTT and PMSF before it
was used in assays. Pellets were resuspended in phase buffer (5 mM potassium phosphate, pH 7.6, 4 mM potassium
chloride, and 250 mM Suc) and subjected to three cycles of
phase partitioning using the PEG-dextran method of Larsson and
co-workers as described previously (Murphy and Auh, 1996).
Lucigenin Assay for Superoxide Synthesis
A standard assay mixture contained approximately 3 µg of plasma membrane protein, 6 µg of S100 protein, or 0.3 µg of supernatant II protein, 100 µM NADH or 30 µM NADPH, 0.02% (w/v) Triton X-100, 0.4 mM lucigenin, and buffer (0.1 M Gly-sodium hydroxide, 1 mM EDTA, pH 9.0) to make a total volume of 1 mL. Additionally, 40 units of SOD (as defined by the manufacturer) or 15 µM DPI was added to appropriate reaction mixtures. Chemiluminescence was measured in a scintillation counter (model 9800, Beckman) in the single-photon counting mode for 1 min at 15-min intervals (six cycles total). Calibration of this assay using xanthine and xanthine oxidase was described previously by Murphy and Auh (1996).
Cyt c Assay for Superoxide Synthesis
A standard assay mixture contained plasma membrane, S100, or supernatant II protein (amounts as given above), 100 µM NADH or 40 µM NADPH, 0.02% (w/v) Triton X-100, 100 µM Cyt c, and buffer (20 mM Tris-chloride, pH 7.5, 3 mM magnesium chloride) to make a total volume of 0.5 mL in a quartz cuvette. Additionally, 40 units of SOD or 30 µM DPI was added to appropriate reaction mixtures. Change in A550 was measured over the 1st min in a spectrophotometer (model DU-640, Beckman). Calculation of specific activity assumed an absorption coefficient of 21 mM1 cm1.
NBT Assay for Superoxide Synthesis
A standard assay mixture contained plasma membrane, S100, or supernatant II protein (amounts as given above), 100 µM NADH or 40 µM NADPH, 0.02% (w/v) Triton X-100, 100 µM NBT, and buffer (20 mM Tris-chloride, pH 7.5, 3 mM magnesium chloride) to make a total volume of 0.5 mL in a quartz cuvette. Additionally, 40 units of SOD or 30 µM DPI was added to the reaction mixture. Change in A530 was measured over the 1st min in the spectrophotometer. Calculation of specific activity assumed an absorption coefficient of 12.8 mM1
cm1.
Protein Assay
Protein concentration was measured for each sample with the Coomassie brilliant blue protein-staining reagent (Bradford, 1976). BSA
was used as a protein standard.
Calculations
To calculate specific activity, background values were obtained with all reaction components except enzyme. These values were then subtracted from the results with corresponding reaction mixtures including enzyme, and the difference was divided by the amount of protein added to the reaction mixture.SOD Assay
The SOD activities in representative preparations of plasma membrane, S100, and supernatant II were measured on microtiter plates using illuminated riboflavin as a source of superoxide and NBT as an indicator. Each well contained 100 µL of a defined dilution of sample (3-fold dilution series in 20 mM bis-Tris-propane-chloride buffer, pH 7.5, 0.075% dodecylmaltoside, and 250 mM Suc) plus 200 µL of reaction mixture (150 mM Tris-chloride, 300 mM EDTA, pH 7.5, 0.1 mM NBT, and 3 µM riboflavin). The plate was illuminated with intense white light until wells without SOD achieved a moderate blue color. The optical absorption of the wells was measured with an automatic plate reader (green filter). One unit was defined as the amount of activity that reduced the amount of colored reaction product by one-half. The sensitivity of the assay was compared with the nominal activity of commercial preparations of SOD and found to give values 3-fold higher than expected.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Plasma membrane-enriched preparations stimulated lucigenin
chemiluminescence, Cyt c reduction, and reduction of NBT
with both NADH and NADPH as electron donors (Fig.
1). The values for lucigenin chemiluminescence agreed with those reported previously (Murphy and
Auh, 1996). As reported, chemiluminescence tended to increase for 15 to
30 min, then slowly decreased (Fig. 2).
Also as found previously, luminescence was strongly reduced by the
addition of SOD or DPI. The results for NBT reduction were similar in
that the reactions were substantially reduced by SOD; however, NBT reduction was only partly sensitive to 30 µM DPI when
NADH was used as the electron source, and was not sensitive to DPI when NADPH was used. The activity of plasma membrane in the Cyt c
assay was higher than in either of the other assays. However, neither NADH- nor NADPH-dependent reduction of Cyt c was very
sensitive to SOD or DPI. It is likely that the plasma membrane
preparation contained substantial amounts of a Cyt c
reductase that transferred electrons to Cyt c without
forming superoxide. It is not known whether this enzyme was a component
of the plasma membrane or a contaminant, perhaps the
antimycin-A-insensitive Cyt c reductase of the ER.
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It is clear from the results presented in Figures 1, 3, and 4 that
success in detecting and quantifying a superoxide-producing enzyme
depends on the assay used. Each assay ascribed the highest activity to
a different preparation: the Cyt c assay to S100, the NBT
assay to plasma membrane, and the lucigenin assay to supernatant II
(with NADH). This suggests that no two assays measure the same set of
enzymes. The differences may not be only in superoxide-generating enzymes. The three assay reagents will respond differently to the
presence of SOD in the preparations. Liochev and Fridovich (1997) Received December 15, 1997;
accepted April 30, 1998.
Abbreviations:
DPI, diphenyleneiodonium.
lucigenin, N,N
Auh C-K,
Murphy TM
(1995)
Plasma membrane redox enzyme is involved in the synthesis of O2
Bolwell GP,
Davies DR,
Gerrish C,
Auh C-K,
Murphy TM
(1998)
Comparative biochemistry of the oxidative burst produced by rose and French bean cells reveals two distinct mechanisms.
Plant Physiol
116:
1379-1385
Bradford MM
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
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 labelling of a component polypeptide of the oxidase.
Biochem J
237:
111-116
[ISI][Medline]
Deme D,
Doussiere J,
De Sandro V,
Dupuy C,
Pommier J,
Virion A
(1994)
The Ca2+/NADPH-dependent H2O2 generation in thyroid plasma membrane: inhibition by diphenyleneiodonium.
Biochem J
301:
75-81
Faulkner K,
Fridovich I
(1993)
Luminol and lucigenin as detectors for O2·
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]
Murphy TM,
Auh C-K
(1996)
The superoxide synthases of plasma membrane preparations from cultured rose cells.
Plant Physiol
110:
621-629
[Abstract]
Murphy TM,
Hamilton CM,
Street HE
(1979)
A strain of Rosa damascena cultured cells resistant to ultraviolet light.
Plant Physiol
64:
936-941
O'Donnell VB,
Smith GCM,
Jones OTG
(1994)
Involvement of phenyl radicals in iodonium compound inhibition of flavoenzymes.
Mol Pharmacol
46:
778-785
[Abstract]
O'Donnell VB,
Tew DG,
Jones OTG,
England PJ
(1993)
Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.
Biochem J
290:
41-49
Picker SD,
Fridovich I
(1984)
On the mechanism of production of superoxide radical by reaction mixtures containing NAD, phenazine methosulfate, and nitroblue tetrazolium.
Arch Biochem Biophys
228:
155-158
[CrossRef][ISI][Medline]
Quail PH
(1979)
Plant cell fractionation.
Annu Rev Plant Physiol
30:
425-484
Vásquez-Vivar J,
Hogg N,
Pritchard KA Jr,
Martasek P,
Kalyanaraman B
(1997)
Superoxide anion formation from lucigenin: an electron spin resonance spin-trapping study.
FEBS Lett
403:
127-130
[CrossRef][ISI][Medline]
View larger version (25K):
[in a new window]
Figure 3.
Specific activity of superoxide synthases from
supernatant II. Reaction mixtures were supplemented with either 100 µM NADH or 30 µM NADPH. Where indicated,
reactions were conducted in the presence of 40 units of SOD or 30 µM DPI. Values are means ± SE of at
least five trials (Cyt c) or seven trials (NBT and
lucigenin).
View larger version (28K):
[in a new window]
Figure 4.
Specific activity of superoxide synthases from
S100. Reaction mixtures were supplemented with either 100 µM NADH or 30 µM NADPH. Where indicated,
reactions were conducted in the presence of 40 units of SOD or 30 µM DPI. Values are means ± SE of two
preparations, each assayed in triplicate.
0.02 unit µL1,
supernatant II had no detectable activity, and the two samples of S100
had 0.02 and 0.06 units µL1. All of the
assays of superoxide synthesis described above used 10 µL of
preparation.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
noted
that superoxide reacts faster with Cyt c than with reduced
lucigenin, and thus the Cyt c assay is less sensitive to
SOD. Therefore, none of the assays is suitable for comparing superoxide
synthesis in different cell fractions.
as is customarythe
difference in rates with and without SOD are measured. An
antimycin-A-resistant Cyt c reductase activity is generally
considered a marker for the ER (Quail, 1979). It is not known whether
the activity in our plasma membrane preparation represents
contamination from residual ER or from a plasma membrane-specific
enzyme.
and Vásquez-Vivar et al. (1997), the reduced lucigenin would react with oxygen to produce superoxide. As noted by these authors, the
sensitivity of the luminescence to SOD is no indication that the
enzymes are producing superoxide, because superoxide is essential to
the luminescence whether it is formed by the enzyme or by the nonenzymatic oxidation of reduced lucigenin. The gradual increase in
activity often observed over the first 15 min with plasma membrane (Fig. 2; figure 1 of Murphy and Auh, 1996) and occasionally with supernatant II (data not shown) is consistent with a two-step reaction
mechanism, the increase reflecting the accumulation of the reduced form
of either lucigenin, superoxide, or both. This problem can also apply
to NBT (Picker and Fridovich, 1984).
; Deme et al., 1994). The
inhibition of activity by DPI depended on the assay used. The NADH- and
NADPH-stimulated activities of all three preparations were sensitive to
30 µM DPI when assayed with lucigenin but less so or not
at all when assayed with NBT or Cyt c (with the exception of
supernatant II and NADPH). In some cases, e.g. with Cyt c
reductase of plasma membrane, an enzyme activity clearly different from activities that are sensitive to SOD, this is easy to understand. It is
more difficult to understand why plasma membrane activities assayed
with NBT and lucigenin, which are both sensitive to SOD, should be more
sensitive to DPI with lucigenin than with NBT. Because DPI must be
converted to a free radical before it is effective (O'Donnell et al.,
1993, 1994), the possibility of interaction between DPI and the assay
reagents should be investigated. In the mean time, the results of the
present study confirm the utility of DPI in investigations of
superoxide synthesis to this extent: because the generation of ROS by
rose cells is strongly inhibited by less than 1 µM DPI
(Bolwell et al., 1998), this process is unlikely to depend on an enzyme
that is insensitive to this compound at 30 µM.
1
This work was supported in part by U.S.
Department of Agriculture National Research Initiative-Cooperative
State Research Service grant no. 94-37100-0788 to T.M.M.
FOOTNOTES
*
Corresponding author; e-mail tmmurphy{at}ucdavis.edu; fax
1-916-752-5410.
ABBREVIATIONS
-dimethyl-9,9-biacridium dinitrate.
NBT, nitroblue tetrazolium.
ROS, reactive oxygen species.
SOD, superoxide dismutase.
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
and H2O2 by Phytophthora elicitor-stimulated rose cells.
Plant Physiol
107:
1241-1247
[Abstract].
Free Radical Biol Med
15:
447-451
[CrossRef][ISI][Medline]
Copyright Clearance Center: 0032-0889/98/117//05
© 1998 American Society of Plant Physiologists
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