From the Faculty of Pharmaceutical Sciences, We identified a rapid and novel system to
effectively metabolize a large amount of
H2O2 in the suspension cells of
Scutellaria baicalensis Georgi. In response to an elicitor,
the cells immediately initiate the hydrolysis of baicalein
7-O- Plants display a broad range of defense responses to protect
themselves against mechanical damage or pathogen attack. One of the
earliest responses is the production of large amounts of reactive
oxygen species (ROS),1 which
is called the oxidative burst (1-4). The physiological roles of the
oxidative burst have been well examined to date. For example, several
studies demonstrated that ROS directly reduce pathogen viability (5)
and that micromolar concentrations of H2O2
inhibit spore germination of a number of fungal pathogens (6). In
soybean and tomato, cell wall proteins such as hydroxyproline-rich proteins (extensin) are rapidly immobilized by the action of
peroxidases as soon as H2O2 is produced,
resulting in strengthening of the cell walls to pathogen attack (7, 8).
Moreover, it has been reported that the oxidative burst can induce
lipid peroxidation, leading to loss of membrane integrity and finally
to the death of host plant cells, which is known as the hypersensitive
response (5, 9). Thus, although the oxidative burst plays important roles in plant defense, the plant cells themselves are consequently exposed to serious oxidative stress. Most plant cells produce ROS as
by-products of redox reactions under normal conditions, but they are
maintained at a low level by ROS-metabolizing enzymes such as catalase
and ascorbate peroxidase (10, 11). However, the detoxification
mechanism of ROS produced by the oxidative burst has not been clearly
understood. In this work, we establish that the suspension cells of
Scutellaria baicalensis Georgi (skullcap plants) have a
rapid and novel system to detoxify a large amount of
H2O2.
S. baicalensis contains numerous flavones, and their
pharmacological properties have been extensively investigated. Among them, baicalein (BA) has attracted considerable attention, as it has a
variety of interesting activities such as antibacterial (12), antiviral
(13), anticancer (14), and lipoxygenase-inhibitory effects (15). In
addition, this plant has long been known to possess a
Plant Materials--
The calluses were induced from the shoot
stem segments of S. baicalensis, as described previously
(17). The suspension cells were obtained by incubating the 4-week-old
calluses in liquid Murashige-Skoog medium (18) containing
2,4-dichlorophenoxyacetic acid (0.5 mg/liter) and
N6-benzyladenine (0.5 mg/liter) at 25 ± 1 °C under
16-h light conditions. The suspension cells were subcultured every 4 weeks in liquid Murashige-Skoog medium under the same culture
conditions.
Flavones--
Apigenin and luteolin were purchased from Wako
Pure Chemical Industries (Osaka). Oroxylin A was donated by Dr.
Yamamoto (Hokuriku University, Japan). BAG, BA, and wogonin were
isolated from the dried roots of S. baicalensis (Uchida
Wakanyaku, Tokyo) as described previously (17).
Structural Determination of 6,7-Dehydrobaicalein--
Because
the amount of the unknown compound produced from the elicited cells by
oxidative burst was not enough to analyze its structure, we
enzymatically synthesized this compound from BA using CM-52 eluate as a
crude peroxidase preparation. The CM-52 eluate (50 ml), which was
prepared from 4-week-old cells (300 g) as described below, was added to
50 mM citrate buffer (pH 4.0, 200 ml) containing 2 mM H2O2. BA (30 mg) dissolved in
dimethyl sulfoxide (1 ml) was slowly added to the enzyme solution and
stirred at room temperature for 5 min. We confirmed by HPLC analysis
that the reaction product had the same retention time as the unknown compound produced by the oxidative burst. The reaction mixture was
applied to the reversed-phase polystyrene gel, MCI-gel CHP 20P
(Mitsubishi Chemical Co., Tokyo, 2.0 × 20.0 cm) previously equilibrated with distilled water. After the column was washed with 100 ml of distilled water, fractions containing the unknown compound alone
were eluted with 100 ml of methanol. However, we did not concentrate
this methanol solution because the unknown compound was unstable to
concentration procedures, including evaporation and lyophilization.
Therefore, NMR analysis could not be carried out, but the methanol
solution showed an [M+H]+ ion peak at m/z 269 in the fast atom bombardment mass spectrum. Finally, the unknown
compound could be isolated as a phenazine derivative. After
o-phenylenediamine (150 mg) was dissolved in the above
methanol solution containing the unknown compound, 1 ml of acetic acid
was added. The reaction mixture was stirred at room temperature for 10 min, and the methanol was then removed by evaporation. The residue was
loaded on an MCI-gel CHP 20P column (2.0 × 15.0 cm)
pre-equilibrated with 50% (v/v) aqueous methanol. After washing the
column with 100 ml of the same solvent, elution with 80% (v/v) aqueous
methanol afforded a phenazine derivative (19 mg). This compound was
characterized as the 6,7-phenazine derivative of BA by its NMR
analysis. Because o-phenylenediamine reacts with compounds
that have an ortho- or a 1,2-diketone structure to form
phenazine derivatives (19), the unknown compound was determined as
6,7-dehydrobaicalein (DBA). Structures of these compounds are shown as
insets in Fig. 3.
Elicitor Treatment of Suspension Cells--
The 4-week-old
suspension cells were harvested by filtration and washed with fresh
growth medium. Aliquots (each 5 g) of the cells were subdivided into
100-ml flasks, and 20 ml of the growth medium was added to each flask.
After 200 µl of 10% (w/v) yeast extract (Nacalei Tesque, Kyoto)
dissolved in the growth medium was added (final concentration of yeast
extract, 0.1%), each cell suspension was incubated at 25 °C for
0-180 min. The saccharic acid 1,4-lactone (SAL) treatment of the
elicited cells was carried out as described above, except a yeast
extract solution containing 100 mM SAL (final SAL
concentration, 1 mM, Sigma) was used instead of the yeast
extract solution alone. All experiments were repeated with three
replicates in each samples.
Determination of BAG, BA, and DBA in the Elicited
Cells--
Elicited samples incubated for different times were
homogenized with methanol (20 ml). After centrifugation of the
homogenate at 20,000 × g for 5 min, the supernatant
was used to determine the amounts of BAG, BA, and DBA. The
quantification of these flavones was carried out with an HPLC system
(Tosoh, Tokyo) composed of a CCPM pump and a UV-8000 absorbance
detector equipped with a Cosmosil 5C18 AR column (0.46 × 15.0 cm,
Nacalai Tesque). BAG, BA, and DBA were eluted at a flow rate of 1 ml/min with 25, 35, and 15% (v/v) aqueous acetonitrile containing 50 mM phosphoric acid, respectively. The effluent was
monitored by absorption at 254 nm, and the peak intensity was
determined with a Chromatocorder 21 (Tosoh). The amount of BAG and BA
was calculated from the standard curves obtained with authentic
samples. As we could not isolate DBA in a free form, the following
standard curve was used for the estimation of DBA levels. Various known
amounts of BA were enzymatically oxidized to DBA, and each peak
intensity of DBA was measured. The molar concentrations of DBA were
considered to be equal to those of BA because we had confirmed that the
oxidation of BA to DBA proceeds quantitatively under the conditions
used. Therefore, the standard curve for DBA was constructed with the concentrations of BA and the peak intensities of DBA.
Determination of Extracellular
H2O2--
After the elicited samples were
centrifuged at 20,000 × g for 5 min, the supernatant
(1.0 ml) was passed over a Pasteur pipette column containing 0.1 g of
Cosmosil 75C18 OPN (Nacalei Tesque) to remove phenolic compounds, which
interfere with the precise quantification of
H2O2. The column eluent was used to determine the amount of H2O2. We confirmed that
H2O2 is quantitatively recovered by this
chromatography method. The column eluent (100 µl) was incubated in a
buffer (100 µl) containing purified peroxidase 1 (0.2 µg), 400 µM BA, 0.6% (w/v) Triton X-100, and 200 mM
sodium citrate buffer (pH 4.0) at 30 °C for 10 min. Under these
assay conditions, the amount of H2O2 was shown
to be linear with respect to the DBA production. Therefore, the peak
intensity of DBA in the reaction mixture was measured by HPLC as
described above; the amount of H2O2 was
calculated from the calibration curve, which was constructed with known
amounts of H2O2 and the peak intensities of DBA
produced by the enzymatic reaction.
Assay of Assay of Preparation of Protoplasts--
The suspension cells (4.0 g)
were soaked in 0.6 M mannitol (50 ml) containing 1.0%
(w/v) Cellulase Onozuka R-10 (Yakult, Tokyo) and 0.1% (w/v) Macerozyme
R-10 (Yakult). After incubation at 30 °C for 120 min, the reaction
mixture was centrifuged at 100 × g for 10 min. The
resulting protoplasts were washed twice with 0.6 M mannitol
and centrifuged as described before.
Purification of Cell Wall Peroxidases--
All procedures were
carried out at 4 °C, unless otherwise indicated. Four-week-old
suspension cells (300 g) were shaken in 1 M NaCl (1,000 ml)
at 100 rpm for 60 min and then filtered with Nylon screen. After the
filtrate was centrifuged at 20,000 × g for 15 min, the
supernatant was fractionated by addition of ammonium sulfate. Proteins
precipitating between 10 and 75% saturation were collected by
centrifugation at 20,000 × g for 30 min and then
dialyzed overnight against three changes of 10 mM sodium phosphate buffer (pH 6.0). The dialyzed sample was applied to a Whatman
CM-cellulose column (2.5 × 25.0 cm) equilibrated with the same
buffer. After the column was washed with the above buffer (200 ml),
bound proteins were eluted with a 1,000-ml linear gradient of NaCl
(0-0.4 M) at a flow rate of 1.0 ml/min. The eluent was collected in 20-ml fractions. The most active fractions (fractions 23-25) were concentrated by ultrafiltration (Advantec, Tokyo) and
dialyzed against 10 mM sodium phosphate buffer (pH 7.0).
The dialysate was applied to a 1.0 × 20.0-cm column containing
hydroxylapatite (Nacalai Tesque, Kyoto) pre-equilibrated with 10 mM sodium phosphate buffer (pH 7.0). Fractions containing
peroxidase 1 were eluted with 150 ml of the same buffer at a flow rate
of 1.0 ml/min. The most active fractions (fractions 5-7, each 20 ml)
were pooled and concentrated by ultrafiltration. Peroxidase 2 was
eluted from the same column with a 400-ml linear gradient of ionic
strength sodium phosphate buffer (pH 7.0, 10-100 mM). The
most active fractions (fractions 12-14) were pooled, concentrated by
ultrafiltration, and dialyzed against 10 mM sodium
phosphate buffer.
Protein Assay--
Protein concentrations were measured
according to Bradford (20) using bovine serum albumin as the
standard.
Determination of Molecular Mass and Isoelectric
Point--
SDS-PAGE analysis was carried out with the system of
Laemmli (21) in a 12.5% acrylamide gel of 0.75-mm thickness (Bio-Rad). The subunit molecular mass of the enzyme was determined by comparison with low molecular mass protein standards (Sigma). Isoelectric focusing
was conducted according to O'Farrell (22) using 7.5-mm glass tubes
(Atto, Tokyo). The pI of the purified enzyme was determined by
comparison with marker proteins (Sigma). The native molecular masses of
peroxidases 1 and 2 were estimated by gel filtration chromatography on
a 1.5 × 75.0-cm column of Sephacryl S-200 HR (Amersham Pharmacia
Biotech) equilibrated with 10 mM phosphate buffer (pH 7.0)
at a flow rate of 0.3 ml/min. Fractions of 15 ml were collected.
Molecular mass markers from 29 to 700 kDa were resolved under the same
conditions prior to running the fractions containing peroxidase
activity.
Elicitor Treatment of Cell Suspension of S. baicalensis in
the Presence of
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-D-glucuronide by
-glucuronidase, and
the released baicalein is then quickly oxidized to 6,7-dehydrobaicalein by peroxidases. Hydrogen peroxide is effectively consumed during the
peroxidase reaction. The
-glucuronidase inhibitor, saccharic acid
1,4-lactone, significantly reduced the
H2O2-metabolizing ability of the
Scutellaria cells, indicating that
-glucuronidase, which
does not catalyze the H2O2 degradation, plays
an important role in the H2O2 metabolism. As
H2O2-metabolizing enzymes, we purified two
peroxidases using ammonium sulfate precipitation followed by sequential
chromatography on CM-cellulose and hydroxylapatite. Both peroxidases
show high H2O2-metabolizing activity using
baicalein, whereas other endogenous flavones are not substrates of the
peroxidase reaction. Therefore, baicalein predominantly contributed to
H2O2 metabolism. Because
-glucuronidase,
cell wall peroxidases, and baicalein pre-exist in
Scutellaria cells, their constitutive presence enables the
cells to rapidly induce the H2O2-metabolizing
system.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glucuronidase, called baicalinase (16). Previously, we purified
-glucuronidase from the callus culture and demonstrated that this
enzyme displays high activity for baicalein
7-O-
-D-glucuronide (BAG), the main flavonoid
of this plant (17). To establish the physiological roles of
-glucuronidase and BAG, we attempted further studies. Consequently,
we found that the suspension cells of S. baicalensis
effectively metabolize H2O2 by a sequential
reaction, including the hydrolysis of BAG to BA by
-glucuronidase
and then the oxidation of BA by cell wall peroxidases. In this paper,
we describe the detoxification mechanism of
H2O2 in Scutellaria cells. We also
report on the purification of the peroxidases involved in
H2O2 degradation and their kinetic
properties.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-Mannosidase--
The assay consisted of 100 mM sodium citrate buffer (pH 5.5), 2 mM
4-nitrophenol-
-D-mannoside, 1 mM
mercaptoethanol, and the enzyme solution (500 µl) in a final volume
of 1 ml. The samples were incubated at 30 °C for 60 min, and the
reaction terminated with 100 µl of 0.5 N NaOH. The
absorption of the sample was measured at 415 nm. The amount of
4-nitrophenol liberated was calculated from the standard curve.
-Glucuronidase and the Cell Wall Peroxidase--
The
assay for
-glucuronidase was conducted, as described previously
(17). The peroxidase activity was determined by incubating an assay
mixture containing the enzyme solution, together with 200 µM BA, 200 µM H2O2,
0.3% (v/w) Triton X-100, and 100 mM sodium citrate buffer
(pH 4.0) at 30 °C for 10 min in a final volume of 200 µl. The
amount of H2O2 in the reaction mixture was
measured as described above. The enzyme activity (katal) was defined as the amount (mole) of H2O2 consumed per
second.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-Glucuronidase Inhibitor--
S.
baicalensis is known to contain flavonoid-specific
-glucuronidase, although its physiological importance is not
understood (16). To reveal the roles of this enzyme in S. baicalensis, we first investigated effects on the cells induced by
inhibition of
-glucuronidase. In this study, SAL was used as the
-glucuronidase inhibitor.
-glucuronidase inhibition was observed in
the elicited cells but not in the nonelicited cells. When Scutellaria cells were treated with the elicitor yeast
extract in the presence of SAL, numerous components that should exist within the cells were released to the medium (Fig.
1A). In addition,
-mannosidase (0.60 nanokatal/g fresh cells), which is reported to be
present in the vacuoles and cytosol (23), was also detected in the
medium. Accordingly, the elicitation in the presence of SAL was thought
to result in serious damage to the cells. In contrast to SAL treatment,
elicitation in the absence of SAL resulted in much less damage; the
elicited cells released only several components (Fig. 1B)
and a lower level of
-mannosidase (0.09 nanokatal/g fresh cells) to
the medium. Similar experiments using H2O2
instead of the elicitor also demonstrated that the elicited cells
undergo more serious damage in the presence of SAL. These results
indicated that Scutellaria cells have a rapid
H2O2-detoxifying system that is inhibited by
SAL.
View larger version (19K):
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Fig. 1.
HPLC analysis of the extracellular fluid
after elicitation. The cells were incubated with 0.1% yeast
extract at 25 °C for 1 h in the presence (A) or
absence (B) of 1 mM SAL. After removal of the
cells by filtration, the media were analyzed using an HPLC system as
described under "Experimental Procedures." The column was initially
equilibrated with 5% aqueous acetonitrile. After injection of the
samples, the concentration of acetonitrile was linearly increased to
70% in 30 min at a flow rate of 1 ml/min.
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Production of 6,7-Dehydrobaicalein in Elicited Cells-- HPLC analysis showed that the elicited cells in the absence of SAL released an unknown compound (retention time, ~8 min) to the medium (Fig. 1B). In contrast, this unknown peak was much smaller on the chromatogram of the SAL-treated cells (Fig. 1A). As this compound was also assumed to be involved in the H2O2 metabolism, we attempted a structural elucidation. After several unsuccessful attempts, its structure was determined as DBA by the method as described under "Experimental Procedures." DBA is the first flavone with an ortho-diketone moiety. Judging from its structure, this flavone was considered to be derived from the oxidation of BA.
Changes in the Amounts of BAG, BA, and DBA during Elicitation-- We assessed changes in the amounts of BAG, BA, and DBA during elicitation. In the absence of SAL, the cells immediately initiated the hydrolysis of BAG after addition of the elicitor, and BAG continued to decrease until 30 min after incubation (Fig. 3A). The decrease of BAG correlated well with the H2O2 degradation such that H2O2 induced by the oxidative burst also decreased until 30 min after incubation, as shown in Fig. 2. On the other hand, BA increased rapidly with BAG hydrolysis, and the BA amount at 30 min was about two times higher than that before elicitation (Fig. 3B). Although at least ~2.5 µmol of BAG should be hydrolyzed after elicitation, only a slight increase (~0.06 µmol) in the BA amount was observed even at the maximum level, suggesting that most of the BA is immediately metabolized. SAL-untreated cells produced more DBA than BA, and its amount reached a maximum level after 60 min of incubation (Fig. 3C). Thus, the production of DBA also occurred rapidly in response to the elicitor. In contrast, addition of SAL extensively inhibited the hydrolysis of BAG (Fig. 3A) and the production of DBA (Fig. 3C), indicating that the hydrolysis of BAG is essential for the production of DBA. Therefore, we concluded that DBA is produced by the hydrolysis of BAG to BA, followed by the oxidation of BA. Hydrogen peroxide may be consumed during the conversion of BA to DBA because this step is an oxidative reaction. DBA was also assumed to undergo further metabolism, based on the facts that the decrease in the DBA amount was observed on incubation over 120 min and that only ~15% of BAG hydrolyzed was recovered as DBA (0.38 µmol at the maximum level). However, the mechanism of DBA metabolism was not established in this study.
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Identification of the H2O2-metabolizing Enzyme-- We investigated whether the oxidation of BA to DBA required H2O2. Consequently, crude enzyme extract from Scutellaria cells was shown to catalyze the formation of DBA from BA by consuming H2O2, indicating that H2O2 is metabolized by peroxidase. Furthermore, we confirmed that BA metabolism by enzymes except for peroxidase is ineffective because the crude enzyme extract did not metabolize BA in the absence of H2O2. To extract this peroxidase effectively, extraction conditions were optimized. Crude enzyme extracts were prepared using various solvents, and then the peroxidase activity in each extract was measured by quantifying the amount of H2O2 consumed by the oxidation of BA. The higher activity was observed in the extract prepared with 1 M NaCl, which is often used for solubilization of proteins ionically bound to the cell walls. Moreover, the protoplasts prepared by digestion of the cell walls displayed much lower metabolizing activity for H2O2 (0.5 nanokatal/g cells) as compared with the intact Scutellaria cells (11.5 nanokatal/g cells), and most of the activity was recovered in the digestion medium. These results apparently indicate that the H2O2-metabolizing enzymes exist in the cell walls.
The Activity of -Glucuronidase and the Cell Wall Peroxidase
after Elicitation--
Because it was confirmed that
-glucuronidase
and the cell wall peroxidase contribute to the detoxification of
H2O2, we measured the activity of both enzymes
after elicitation. As shown in Fig. 4,
neither activity increased during the incubation period (180 min)
tested, and the peroxidase activity decreased more than the
-glucuronidase activity. Hence, we concluded that the metabolism of
H2O2 produced by the oxidative burst is
catalyzed by these enzymes that are present consititutively in
Scutellaria cells.
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Purification of Cell Wall Peroxidases--
Previously, we purified
-glucuronidase from the Scutellaria calluses and
characterized its properties (17). At present, there is no information
on the cell wall peroxidases in this plant. Therefore, to characterize
precisely the cell wall peroxidases, we attempted to purify them. The
4-week-old cells of S. baicalensis were shaken in 1 M NaCl and then filtered. The filtrate was fractionated by
ammonium sulfate saturation. More than 70% of the peroxidase activity
was precipitated between 10 and 75% saturation of ammonium sulfate,
resulting in a 3-fold purification. As a first chromatographic step,
the solubilized ammonium sulfate fraction was applied to a CM-cellulose
(CM-52) column, where a peroxidase was eluted with a linearly
increasing gradient of NaCl (0-0.4 M). A lower level of
peroxidase activity was recovered in the void volume, whereas most of
the peroxidase activity eluted at ~0.2 M NaCl. This step increased the specific activity in the latter fractions by a factor of
23-fold with a recovery of 43%. Because the total enzyme activity in
the latter fractions was about 50 times higher than that in the former
fraction, further purification of the former fractions was not carried
out. As a final purification step, the latter CM-52 eluate was applied
to a hydroxylapatite column. A peroxidase activity was eluted with 10 mM phosphate buffer, and SDS-PAGE showed that the
peroxidase (termed peroxidase 1 in this paper) in these fractions was
purified to homogeneity (Fig. 5). Further elution with an increasing gradient of the ionic strength of phosphate buffer (10-100 mM) gave another peroxidase, named
peroxidase 2. The purification of peroxidase 2 to homogeneity was also
confirmed by SDS-PAGE. Peroxidases 1 and 2 were purified 121- and
89-fold by the three-step procedure with a final recovery of 17 and 8% of the enzyme activity, respectively. Both peroxidases are the first
flavone-metabolizing enzymes to be purified.
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Molecular Mass and Isoelectric Point of Peroxidases 1 and 2-- SDS-PAGE of peroxidases 1 and 2 showed subunit molecular masses of 38 and 34 kDa, respectively (Fig. 5). The native molecular masses were estimated from the elution volume of both peroxidases on Sephacryl S-200 HR chromatography, where peroxidases 1 and 2 eluted as a single molecular species with a molecular mass of about 35 kDa in each case. These results suggested that each peroxidase exists as a monomeric enzyme. The pI values for peroxidases 1 and 2 were determined to be 8.7 and 8.9, respectively by comparison with marker proteins of known pI on isoelectric focusing gels.
Standard Assay Conditions of Peroxidases 1 and 2-- We determined the optimum pH of each peroxidase using BA. The activity of peroxidase 1 was maximum between pH 4.0 and 4.5, with half-maximal activities at pH values around 3.0 and 6.0. Peroxidase 2 also showed the maximum activity between pH 4.0 and 4.5, although its activity was slightly lower than that of peroxidase 1. Based on these results, standard assays were carried out with citrate buffer (pH 4.0).
Effects of Various Flavones on H2O2-metabolizing Activity of Peroxidases 1 and 2-- In S. baicalensis, wogonin and oroxylin A are also biosynthesized as minor flavones (24). To reveal whether these endogenous flavones are involved in the detoxification of H2O2, the substrate specificity was examined with them as well as BA. As shown in Table I, peroxidases 1 and 2 showed the high H2O2-metabolizing activity using BA with peroxidase 1 showing somewhat higher activity than peroxidase 2. In contrast, BAG does not undergo oxidation by either peroxidase. Scutellaria cells contain a much lower level of BA (~0.06 µmol/g fresh cells) than BAG (~9.5 µmol/g fresh cells), indicating that the cells have to hydrolyze BAG to effectively degrade H2O2. In addition, peroxidases 1 and 2 could not oxidize any of the endogenous flavones except for BA. In contrast to BA, these flavones possess a methoxyl or a glucuronyl group in their molecule; therefore, it is assumed that both enzymes can only oxidize flavones with hydroxyl groups. Moreover, we evaluated the peroxidase activity using apigenin (5,7,4'-trihydroxyflavone) and luteolin (5,7,3',4'-tetrahydroxyflavone), which have not been identified in S. baicalensis. Both peroxidases showed a high activity with luteolin, whereas apigenin was not oxidized by either peroxidase. Like BA, luteolin has an ortho-dihydroxyl moiety in its molecule, thus suggesting that an ortho-dihydroxyl group is required for the peroxidase reaction.
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DISCUSSION |
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ROS play important roles in plant defense such as the pathogen growth inhibition, cell strengthening, and the hypersensitive reaction. However, they are also thought to inflict serious damage on the host plant cells. In particular, huge amounts of ROS are quickly produced by the oxidative burst, although their detoxification mechanism is not fully understood. As results of our studies on flavonoid metabolism, we identified in Scutellaria cells a novel H2O2-metabolizing system that is closely linked with the metabolism of the flavone BAG. Such a H2O2-metabolizing system has not been hitherto reported. Like Scutellaria cells, rye leaves (23), Pueraria lobata cells (25, 26), apple leaves (27), and garbanzo plants (28) also possess endogenous-flavonoid-specific glycosidase in addition to flavonoid glycosides. Interestingly, their aglycones are assumed to be oxidized by a peroxidase reaction or to have a potent antioxidant activity, suggesting that these plants may have ROS-detoxifying systems similar to that in Scutellaria cells.
The first step in the metabolic pathway of
H2O2 is the hydrolysis of BAG by
-glucuronidase. Previously, we reported that
-glucuronidase in
S. baicalensis shows a high activity for the endogenous
flavone BAG, but its roles have remained unclear (17). In the present
study, we found that
-glucuronidase catalyzes the production of an
antioxidant flavone BA. Keppler and Novacky reported that exogenous
addition of antioxidants reduces death of hypersensitively responding
cells (29), but surprisingly, Scutellaria cells can produce
the antioxidant flavone BA in response to an elicitor or
H2O2. To our knowledge, hydrolases involved in
H2O2 metabolism have not been reported so far.
Under normal conditions, BAG and
-glucuronidase are thought to exist
in the different cellular compartments, because despite the presence of
high
-glucuronidase activity, the amount of BA is much lower than
that of BAG. We assumed that the oxidative burst inflicted serious
damage on the compartmentation, resulting in the hydrolysis of BAG,
based on the fact that after addition of an elicitor or H2O2, the cells released components that should
exist within the cells into the extracellular medium. Flavonoid
hydrolysis initiated by stress has also been reported for other plants.
In P. lobata the hydrolysis of isoflavone glucosides is
initiated by the elicitor treatment (25, 26), whereas the infection of
pathogens to apple leaves causes hydrolysis of chalchone glucoside
(27).
As a second step, released BA was rapidly converted to DBA by cell wall peroxidases, and H2O2 was confirmed to be detoxified at this step. To characterize the properties of the enzymes in pure forms, we attempted to purify them from Scutellaria cells. After a combination of ammonium-sulfate precipitation and two chromatographic steps, two ionic forms of peroxidases (peroxidases 1 and 2) were resolved. The structural properties of peroxidases 1 and 2 are similar to each other. The molecular mass (38 and 34 kDa) of each peroxidase resembles those of ascorbate peroxidase (34 kDa), guaiacol peroxidase (33 kDa), and extensin peroxidases (34-37 kDa) (10). On the other hand, pI values (8.7 and 8.9) of peroxidases 1 and 2 indicated that they are cationic peroxidases.
Among endogenous flavones tested, peroxidases 1 and 2 showed the
highest H2O2-metabolizing activity with BA, and
peroxidase 1 displayed somewhat higher activity than peroxidase 2. In
contrast, both peroxidases could not oxidize other endogenous flavones
including BAG. Scutellaria cells contain much more BAG than
BA, indicating that H2O2 metabolism depends
significantly on the hydrolysis of BAG. These findings provided a
reasonable elucidation for the result that SAL treatment extensively
reduced the ability of Scutellaria cells to metabolize
H2O2. It is notable that
H2O2 is effectively metabolized using luteolin
as a proton donor. Neither luteolin nor its glycosides have been
identified in S. baicalensis, but they do occur in numerous
plants (30), thus suggesting that a similar detoxification of
H2O2 may occur commonly in the plant kingdom.
Anhalt and Weissenböck (23) reported a metabolic pathway of
luteolin glucuronide in rye leaves, where luteolin
7-O-di-glucuronyl-4'-O-glucuronide is hydrolyzed
by endogenous -glucuronidase, and the hydrolysate luteolin
7-O-diglucuronide is finally polymerized by a peroxidase reaction.
Flavonoid polymerization by peroxidase is also reported for daidzein, the isoflavone of P. lobata. Park et al. (26) demonstrated that in vitro peroxidase reaction with daidzein gives dimeric daidzein and unidentified polymers. We attempted similar reaction with BA and peroxidases, but such polymerization was not recognized, and only DBA was quantitatively produced. Therefore, we concluded that neither BA nor DBA is polymerized by peroxidases, contrary to the metabolism of luteolin 7-O-diglucuronide and daidzein, although the precise mechanism of DBA metabolism has remained still unclear.
Concerning the roles of cell wall peroxidases in other plants, the
insolubilization of extensin (7, 8) and the biosynthesis of the cell
wall including lignification (31) have been hitherto reported. Our
study unequivocally demonstrated a novel function in which cell wall
peroxidases rapidly metabolize a huge amount of
H2O2 produced by the oxidative burst. These
peroxidases and -glucuronidase pre-exist in Scutellaria
cells, and neither activity was increased by the oxidative burst. The
constitutive presence of these enzymes, in addition to a large amount
of BAG, possibly enables cells to immediately induce the
H2O2 degradation system.
Ascorbate peroxidase and catalase are well known as H2O2-metabolizing enzymes. SAL is not an inhibitor for both enzymes, although the SAL treatment induces a serious damage in the elicited Scutellaria cells. Therefore, H2O2 detoxification by ascorbate peroxidase and catalase seems less effective as compared with the cell wall peroxidases. BA belongs to a quite different class of natural products from ascorbic acid, which is required for H2O2 metabolism by ascorbate peroxidases, but it is interesting that both compounds act as a proton donor. The cell wall peroxidases of S. baicalensis and ascorbate peroxidase may metabolize H2O2 by a similar mechanism because both peroxidase reactions afforded a product (DBA and dehydroascorbic acid) with a diketone moiety.
In conclusion, BAG and BA were shown to be involved in the protection of Scutellaria cells against oxidative stress, whereas other interesting roles were also suggested for BAG metabolism. It seems particularly important that BA is rapidly formed in response to the elicitor because BA has antibacterial (12) and antiviral (13) effects. We also confirmed that BA shows antibacterial activity for Clavibactor michiganensis subsup. nebraskense and C. michiganensis subsup. michiganensis,2 suggesting that BA may contribute to a chemical defense against pathogens. Because more DBA was produced than BA during the oxidative burst, we are now examining the antimicrobial activity of DBA as well as BA using various pathogens.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-92-642-6581;
Fax: 81-92-642-6545; E-mail: morimoto{at}shoyaku.phar.kyushuu.ac.jp.
1
The abbreviations used are: ROS, reactive oxygen
species; BA, baicalein; BAG, baicalein
7-O--D-glucuronide; DBA,
6,7-dehydrobaicalein; SAL, saccharic acid 1,4-lactone; HPLC, high
performance liquid chromatography; PAGE, polyacrylamide gel
electrophoresis.
2 N. Furuya, N. Matsuyama, S. Morimoto, and Y. Shoyama, unpublished data.
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REFERENCES |
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