Regular Paper
Generation of Hydroxyl Radical in Isolated Pea Root Cell Wall,
and the Role of Cell Wall-Bound Peroxidase, Mn-SOD and
Phenolics in Their Production
Biljana Kukavica1, Miloš Mojović 2, Željko Vučinić 1, Vuk Maksimović 1, Umeo Takahama3 and Sonja
Veljović Jovanović 1,*
1Institute
for Multidisciplinary Research, Kneza Višeslava 1, 11030 Belgrade, Serbia
of Physical Chemistry, University of Belgrade, Serbia
3Kyushu Dental College, Kitakyushu, 803-8580 Japan
2Faculty
The hydroxyl radical produced in the apoplast has been
demonstrated to facilitate cell wall loosening during cell
elongation. Cell wall-bound peroxidases (PODs) have been
implicated in hydroxyl radical formation. For this
mechanism, the apoplast or cell walls should contain the
electron donors for (i) H2O2 formation from dioxygen; and
(ii) the POD-catalyzed reduction of H2O2 to the hydroxyl
radical. The aim of the work was to identify the electron
donors in these reactions. In this report, hydroxyl radical
(·OH) generation in the cell wall isolated from pea roots
was detected in the absence of any exogenous reductants,
suggesting that the plant cell wall possesses the capacity
to generate ·OH in situ. Distinct POD and Mn-superoxide
dismutase (Mn-SOD) isoforms different from other
cellular isoforms were shown by native gel electrophoresis to be preferably bound to the cell walls. Electron
paramagnetic resonance (EPR) spectroscopy of cell
wall isolates containing the spin-trapping reagent,
5-diethoxyphosphoryl-5-methyl-1-pyrroline- N -oxide
(DEPMPO), was used for detection of and differentiation
between ·OH and the superoxide radical (O2–·). The data
obtained using POD inhibitors confirmed that tightly
bound cell wall PODs are involved in DEPMPO/OH adduct
formation. A decrease in DEPMPO/OH adduct formation
in the presence of H2O2 scavengers demonstrated that this
hydroxyl radical was derived from H2O2. During the
generation of ·OH, the concentration of quinhydrone
structures (as detected by EPR spectroscopy) increased,
suggesting that the H2O2 required for the formation of
·OH in isolated cell walls is produced during the reduction
of O2 by hydroxycinnamic acids. Cell wall isolates in which
the proteins have been denaturated (including the
endogenous POD and SOD) did not produce ·OH. Addition
of exogenous H2O2 again induced the production of ·OH,
and these were shown to originate from the Fenton
reaction with tightly bound metal ions. However, the
appearance of the DEPMPO/OOH adduct could also be
observed, due to the production of O2–· when endogenous
SOD has been inactivated. Also, O2–· was converted to ·OH
in an in vitro horseradish peroxidase (HRP)/H2O2 system
to which exogenous SOD has been added. Taken together
with the discovery of the cell wall-bound Mn-SOD isoform,
these results support the role of such a cell wall-bound
SOD in the formation of ·OH jointly with the cell wallbound POD. According to the above findings, it seems
that the hydroxycinnamic acids from the cell wall, acting
as reductants, contribute to the formation of H2O2 in the
presence of O2 in an autocatalytic manner, and that POD
and Mn-SOD coupled together generate ·OH from such
H2O2.
Keywords: Cell wall isolates • Hydroxycinnamic acids •
Hydroxyl radical • Pea root • Peroxidase • Quinhydrone
structures.
Abbreviations: CWPOD, cell wall peroxidase; CWSOD, cell
wall superoxide dismutase; DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; DETAPAC, diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid; EPR, electroparamagnetic resonance; G6PD, glucose-6-phosphate
dehydrogenase; HRP, horseradish peroxidase; PMSF,
phenylmethylsulfonyl fluoride; POD, peroxidase; SHAM,
salicylhydroxyamic acid; SOD, superoxide dismutase.
*Corresponding author: E-mail, [email protected]; Fax, +381-11-3055289.
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199, available online at www.pcp.oxfordjournals.org
© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Generation of ROS in pea root cell wall
Introduction
It has been shown that intact plant tissues and cells can produce reactive oxygen species, such as H2O2, O2–· and ·OH
(Aver’yanov 1985, Kuchitsu et al. 1995, Schopfer et al. 2001,
Rodriguez-Serrano et al. 2006), which can have important
physiological functions, especially in the oxidative metabolism of the cell wall. These are closely linked with the two
enzymes utilizing and producing these reactive oxygen species, the peroxidases (PODs) and superoxide dismutases
(SODs). It is well established that plant cell walls contain
class III POD (EC 1.11.1.7), and various POD isoforms are
present in the cell walls. Their physiological roles are related
to polymerization reactions such as lignification, suberization and cross-linking, consuming H2O2 (Kolattukudy 1980,
Lewis and Yamamoto 1990, Iiyama et al. 1994). It has been
shown, however, that PODs also possess the capacity to produce H2O2 while oxidizing different types of reductants
including phenolics in the presence of trace amounts of
metal ions (Vianello and Macri 1991, Pichorner et al. 1992,
Jiang and Miels 1993, Hadži-Tašković Šukalović et al. 2005).
CuZn-SOD has been found in apoplastic fluid (Streller
and Wingsle 1994, Ogawa et al. 1997, Schinkel et al. 1998,
Karpinska et al. 2001, Bogdanović et al. 2006) and the cell
wall (Karlsson et al. 2005). In addition, the presence of
Mn-SOD has been demonstrated in the cell wall of moss
(Yamahara et al. 1999). SOD has also been shown to be capable of catalyzing the formation of ·OH in the presence of
H2O2 (Yim et al. 1990, Yim et al. 1993).
A physiological role for ·OH has been proposed in loosening of the cell wall and cleavage of polysaccharide polymers
(Fry 1998, Schweikert et al. 2000). Schopfer and associates
(Chen and Schopfer 1999, Schopfer et al. 2002, Liszkay et al.
2003) have demonstrated on whole roots and coleoptiles a
POD-associated production of ·OH, in the presence of externally added reductants such as NADH or dihydroxyfumarate,
using spectrofluorimetry and electron parametric resonance
(EPR) spectroscopy.
The question of the naturally occurring reductant in the
apoplastic space, participating in the production of reactive
oxygen species, however, remains unclear. Involvement of
the superoxide anion radical in ·OH production by the cell
wall has been proposed in the literature (Chen and Schopfer
1999, Liszkay et al. 2004, Karkonen and Fry 2006). Various
cellular components including plasma membranes were
shown to be capable of generating O2–· (Vuletić et al. 2003,
Mojović et al. 2004). Plasma membrane-bound NADH oxidase has been implicated, based on an inhibitory effect of
iodonium compounds, as a crucial enzyme responsible for
the generation of O2–· in the apoplast (Doke 1985, Murphy
and Auh 1996, van Gestelen et al. 1997). The problem with
the membrane-associated sources of O2–· is the extreme
reactivity of this radical species, the half-lives of O2–· in water
being 0.2 and 20 ms at concentrations of 10 and 1 µM,
respectively (Bielski et al. 1985), which can be assumed to be
similar to that in the apoplastic fluid. SOD accelerates the
dismutation by 400-fold (rate constant, 2.4×109 M–1 s–1)
(Scandalios 1997). Thus, it is very hard to envisage that
plasma membrane-generated O2–· can participate in the
apoplastic space reactions in a controlled manner, and there
is no evidence in the available literature for the presence of
NAD(P)H in the apoplastic compartment.
In contrast to the adenylates, phenolics are ubiquitous
apoplastic components (Takahama 2004). Since some phenolics are auto-oxidizable and can function as reductants,
phenolics in the apoplast including the cell wall may contribute to the generation of H2O2 by reducing O2 (Takahama
2004). If H2O2 is generated in the cell walls by the auto-oxidation of phenolics, PODs and SODs in the walls may also be
able to catalyze the formation of ·OH in addition to its formation by the Fenton reaction (Fry 1998, Karkonen and Fry
2006).
The aim of this work was to elucidate the mechanism of
production of ·OH using cell wall isolated and purified from
pea roots and to determine whether endogenous phenolics
from the cell wall can act as reductants. To discuss the mechanism of ·OH production, mechanisms of production of O2–·
and H2O2 were also studied, taking O2-dependent oxidation
of hydroxycinnamic acids into consideration. ·OH and O2–·
generated in cell wall isolates were detected using a spintrapping reagent, 5-(diethoxyphosphoryl)-5-methyl-1-pyroline N-oxide (DEPMPO), due to its capacity to differentiate
between O2–· and ·OH (Frejaville et al. 1995, Mojović et al.
2004). The results obtained in this study suggest that cell
wall isolates could reduce O2 to O2–· by auto-oxidation of
hydroxycinnamic acids bound to the cell wall, which in turn
can be transformed by cell wall-bound SOD to H2O2. Subsequently, the cell wall-bound PODs can produce ·OH using
the H2O2 generated.
Results
Components of cell wall isolates
First, we examined the purity of cell wall isolates prepared
in this study. The total activity of glucose-6-phosphate dehydrogenase (G6PD), a cytosol marker enzyme, in the soluble
pea root fraction was 0.53 ± 0.072 µmol gFW–1 min–1 (means
± SD) and in the cell wall isolate was 0.0027 ± 0.0011 µmol
gFW–1 min–1 (means ± SD). The percentage of total activities
of G6PD in cell wall isolates relative to that in the soluble
root fraction was calculated to be 0.5 ± 0.06%, confirming
that cell wall isolates were not contaminated with intracellular components. In addition, we could not detect NAD(P)
H oxidase in the cell wall isolates; only the activity of ascorbate oxidase was observed (data not shown).
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
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B. Kukavica et al.
Table 1 The metal content (µg gDW–1) in cell wall isolates of pea
root (with intact and denatured proteins)
Cell wall isolate
Denatured cell
wall isolate
Fe
165 ± 45
188 ± 37
Mn
3.8 ± 1.4
3.4 ± 1.6
Zn
30 ± 3
14.1 ± 5
Cu
23 ± 6
17 ± 5.4
Cell wall isolates with denatured proteins were prepared by boiling the isolates in
2% SDS. Data are means ± SD (n = 3).
Table 2 The content of hydroxycinnamic acids (nmol gFW–1) in
roots, cell wall isolates and apoplastic fluid
Hydroxycinnamic acids
Ferulic acid
Isoferulic acid
Chlorogenic
acid
Caffeic acid
p-Coumaric
acid
Roots
9.8 ± 1
26 ± 3
n.d.a
Cell wall
isolates
14 ± 3
n.d.
20 ± 1.1
Apoplast
20 ± 10
2210 ± 220
26.1 ± 1.4
n.d.
n.d.
50 ± 2.3
60 ± 0.5
n.d
n.d.
Hydroxycinnamic acids were identified by comparing their retention times with
standard compounds. The retention times of chlorogenic, caffeic, p-coumaric,
ferulic and isoferulic acid were 6.32, 8.17, 9.58, 11.82 and 12.65 min, respectively.
Data are means ± SDs (n = 3)
aCould not be detected.
A
Control
H2O2
KCN
The content of metals and hydroxycinnamic acids and
the activities of SODs and PODs were also measured using
cell wall isolates. Table 1 gives the content of metals found
in cell wall isolates. The quantity of the redox active metals
(Fe and Cu) did not change significantly by treatment of cell
wall isolates with SDS/heat (see Materials and Methods),
suggesting that the metals were bound to the cell wall tightly.
Table 2 shows the content of hydroxycinnamic acid in three
root fractions. In cell wall isolates, ferulic, chlorogenic, caffeic
and p-coumaric acids were found. By comparing the amount
of each hydroxycinnamic acid in different fractions, it was
clear that isoferulic acid was present in root extract and apoplastic fluid but not in cell wall isolates. This result additionally suggests that cell wall isolates were not contaminated
with the soluble hydroxycinnamic acid contained in cytoplasm and apoplastic fluid. Fig. 1 shows SOD isoforms found
in root extracts and cell wall isolates. In root extracts, three
SOD isoforms were detected (Fig. 1A). H2O2 and KCN inhibited the activity of two isoforms, suggesting the presence of
two isoforms of Cu,Zn-SOD and one isoform of Mn-SOD.
After treatment with KCN, faint bands were detected, suggesting the presence of Fe-SOD. In cell wall isolates, several
isoforms of SOD were also separated and their activities were
not affected by either H2O2 or KCN (Fig. 1B). The latter indicates that the cell wall isolates contain several Mn-SOD isoforms
(CWSODs), as opposed to the soluble root fraction which
contains Cu,Zn-SOD.
B
Control
H2O2
KCN
MnSOD
MnSOD
MnSOD
CuZnSOD
FeSOD
FeSOD
MnSOD
FeSOD
MnSOD
CuZnSOD
FeSOD
Fig. 1 Separation of SOD isoforms in root extracts and cell wall isolates by native PAGE. (A) root extract; (B) cell wall isolate. Arrows indicate the presence of
SOD isoforms. The amount of protein applied to each well was equivalent to 50 µg of bovine serum albumin. H2O2, H2O2-treated gel; KCN, KCN-treated gel.
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Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
Generation of ROS in pea root cell wall
1
2
Formation of ·OH in cell wall isolates
3
Fig. 2 Separation of POD isoforms in root extracts and cell wall
isolates by native PAGE. 1, root extract; 2, cell wall isolate; 3, cell wall
isolate after treatment with cellulase and pectinase. Arrows indicate
the presence of POD isoforms.
POD isoforms were also found in root extracts and cell
wall isolates (Fig. 2). Three isoforms were detected in soluble
root extracts (lane 1). In cell wall isolates that were not
digested with cellulase and pectinase, POD activity was
bound to the cell wall isolates (lane 2). When cell wall isolates were pre-digested with cellulase and pectinase, only
one POD isoform could be detected, and this isoform was
different from those observed in the cytoplasm (lane 3).
These results suggest that the cell wall isolate posseses a
distinct POD isoform (CWPOD), and that such cell wall
isolates were not contaminated with cytoplasmic POD.
In addition to 4-chloro-α-naphtol used as a substrate for
the POD reaction, we also studied whether cell wall isolates
are capable of oxidizing hydroxycinnamic acids present normally in plants. Chlorogenic, caffeic and ferulic acids were
oxidized at rates of 571 ± 131 ∆A410 mg protein–1 min–1, 532
± 272 ∆A450 mg protein–1 min–1 and 227 ± 31 ∆A356 mg
protein–1 min–1 (means ± SD), respectively, when H2O2 was
added.
The results presented in Table 2 and Figs. 1 and 2 demonstrate that the cell wall isolates were not contaminated
with either cytoplasmic phenolics, cytoplasmic isoforms of
SOD or POD, and that the cell wall-bound POD could oxidize
endogenous hydroxycinnamic acids (Table 2).
When cell walls isolated from pea roots were suspended in
buffer solution in the presence of DEPMPO, an EPR spectrum
was observed, as shown in Fig. 3 (spectrum 3A). By comparing
the EPR spectra of DEPMPO/OH and DEPMPO/OOH
adducts, which were formed in chemical systems generating
·OH and O2–·, respectively, with spectrum 3A (Mojović et al.,
2004), we could conclude that the spectrum of the DEPMPO/
OH adduct was contained in spectrum 3A. Computer simulation of the spectra (dashed line in spectrum 3A) supported
the presence of EPR signals of the DEPMPO/OH adduct in
spectrum 3A. In addition, signals of DEPMPO/H and
DEPMPO/CH3 adducts were included in the spectrum. In
Fig. 3G, the simulated spectra of each of the possible
DEPMPO adducts are given. Protein denaturation of cell wall
isolates by SDS/heat treatment abolished the generation of
not only the DEPMPO/OH adduct but also other adducts
(Fig. 3D). This finding suggests the contribution of proteins
to the generation of spectrum 3A by cell wall isolates. The
addition of H2O2 to the denatured cell wall resulted in the
appearance of signal of DEPMPO/OOH in addition to
DEPMPO/OH, DEPMPO/H and DEPMPO/CH3 adducts
(spectra 3A and 3E). The signal of the DEPMPO/OOH adduct
was observed transiently during the initial period after the
addition of H2O2. The similarity of spectrum 3A to spectrum
3E except for the DEPMPO/OOH adduct suggests that H2O2
was generated during incubation of cell wall isolates.
The addition of H2O2 to native cell wall isolates resulted in
an increase in signal intensities from DEPMPO/OH and
DEPMPO/H adducts. No signal due to the DEPMPO/OOH
adduct could be observed in native cell wall isolates
(spectrum 3B).
In the case of SDS/heat-treated cell wall isolates, no EPR
signals could be observed in the absence of H2O2 (spectrum
3D). When H2O2 was added to such denaturated cell wall
isolates, the DEPMPO/OOH adduct could be observed, in
addition to the DEPMPO/OH and DEPMPO/H adducts
(spectrum 3E). When the chelator DETAPAC (diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid) was added to
SDS/heat-treated and intact cell wall isolates and H2O2
subsequently added, no EPR signal was observed in the case
of SDS/heat-treated isolates (spectrum 3F), as opposed to
intact isolates where DETAPAC partially suppressed the
generation of EPR signals due to the DEPMPO/OH adduct
(spectrum 3C). As DETAPAC can chelate transition metal
ions, the result suggests the formation of ·OH and O2–· by
metal ion-catalyzed reactions. The presence of transition
metals in cell wall isolates has been shown (Table 1). Thus,
the Fenton reaction might contribute to the formation of
·OH in cell wall isolates with denaturated proteins.
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
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B. Kukavica et al.
Cell wall fragments with
covalently bound protein
A
Cell wall fragments in which the
proteins have been denaturated
20 G
D
B
E
C
F
20 G
G
DEPMPO/OH
DEPMPO/H
DEPMPO/CH3
DEPMPO/OOH
Fig. 3 EPR spectra of DEPMPO/OH adducts and other adducts in cell wall isolates. (A–C) Cell wall isolates; (D–F) denatured cell wall isolates.
A and D, incubated for 5 min at room temperature; B and E, incubated for 5 min after the addition of 4 mM H2O2; C incubated for 5 min in
the presence of 3 mM DETAPAC and 4 mM H2O2; F, incubated for 5 min in the presence of 3 mM DETAPAC and 4 mM H2O2. Filled circles, a
characteristic peak of the DEPMPO/OH adduct; filled inverted triangles, a characteristic peak of the DEPMPO/H adduct; open inverted triangles,
a characteristic peak of the DEPMPO/CH3 adduct; open circles, a characteristic peak of the DEPMPO/OOH adduct. Spectral simulations of
spectra A, B and E were performed using the parameters given in Materials and Methods. (A) 50% DEPMPO/OH, 25% DEPMPO/CH3, 25%
DEPMPO/H. (B) 25% DEPMPO/OH, 50% DEPMPO/CH3, 25% DEPMPO/H. (E) 38.5% DEPMPO/OOH, 38.5% DEPMPO/OH, 11.5% DEPMPO/
CH3, 11.5% DEPMPO/H. (G) Spectral simulations of the four possible participating DEPMPO adducts (OH, OOH, CH3 and H).
Participation of POD/H2O2 in ·OH formation by cell
wall isolates
It was demonstrated in Fig. 3 that ·OH was formed in cell
wall isolates. To elucidate the mechanism of ·OH formation,
we studied the effects of POD inhibitors [KCN, NaN3 and
salicylhydroxyamic acid (SHAM)] and scavengers of H2O2
(pyruvate and catalase) on the formation of the DEPMPO/
OH adduct in cell wall isolates. Not only the inhibitors of
POD but also the scavengers of H2O2 suppressed the formation of the DEPMPO/OH adduct (Fig. 4). The result suggests
that H2O2 was generated in cell wall isolates and that the
H2O2 contributed to the formation of ·OH. Cell wall isolates
308
oxidized NADH in the presence of Mn2+ and SHAM, p-coumaric acid or ferulic acid, and rates of the oxidation were
0.90 ± 0.03, 0.77 ± 0.02 and 0.32 ± 0.09 mM mg protein–1
min–1 (means ± SD). As it has been reported that H2O2
is generated in a mixture of POD, Mn2+ and phenolics (Halliwell 1978), the reduction of cell wall isolates by exogenously
added reductant (in this experiment NADH) and the presence
of phenolics supports the concept of H2O2 generation by
cell wall isolates. As described above, POD and H2O2 might
participate in the formation of ·OH in cell walls as proposed
previously (Schopfer 2002) and, since we found a SOD isoform tightly bound to the cell wall, we studied the formation
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
Generation of ROS in pea root cell wall
20 G
A
20 G
A
B
B
C
C
D
E
F
Fig. 4 Effect of inhibitors of POD and scavengers of H2O2 on the generation
of the DEPMPO/OH adduct in cell wall isolates. Cell wall isolates were
incubated with DEPMPO for 5 min in the presence and absence of the
inhibitors or the scavengers: (A) no addition; (B) 3 mM KCN; (C) 3 mM
NaN3; (D) 3 mM SHAM; (E) 1,500 U of catalase ml–1; F, 3 mM pyruvate.
Filled cirles, DEPMPO/OH adduct; filled inverted triangles, DEPMPO/H
adduct; filled squares, quinhydrone structures (see Fig. 6).
of ·OH in an in vitro horseradish peroxidase (HRP)/H2O2
system in the presence and absence of bovine CuZn-SOD
(Fig. 5). The addition of H2O2 to HRP resulted in the appearance
of not only the DEPMPO/OH adduct but also the DEPMPO/
OOH adduct (spectrum 5B). Computer simulation also
suggested the formation of the two products, although the
concentration of the DEPMPO/OH adduct seemed to be
much lower than that of DEPMPO/OOH. The result indicates that O2–· as well as ·OH was produced in the HRP/H2O2
system. When both H2O2 and SOD were added to HRP, EPR
signals due to the DEPMPO/OH adduct increased (spectrum
5C), suggesting that scavenging of O2–· resulted in an increase
of the formation of ·OH. Control experiments with the
spin trap and HRP without H2O2 (spectrum 5A) or spin
trap and H2O2 without HRP (data not shown) did not generate
any adducts.
Fig. 5 H2O2-dependent generation of ·OH and O2–· by HRP. The
reaction mixture contained 2.63 U of HRP (type II) and 42.5 mM
DEPMPO in 100 mM potassium phosphate buffer (pH 7.0). (A) No
addition; (B) the reaction mixture contained 2.63 U of HRP (type
II) and 42.5 mM DEPMPO in 100 mM potassium phosphate buffer
(pH 7.0) and 10 mM H2O2; (C) the reaction mixture contained 2.63 U
of HRP (type II), 3.7 U of SOD and 42.5 mM DEPMPO in 100 mM
potassium phosphate buffer (pH 7.0); the spectrum was recorded
5 min after the addition of 10 mM H2O2. Filled circles, DEPMPO/OH
adduct; open circles, DEPMPO/OOH adduct. Spectral simulations
of spectra B and C were performed using the parameters given in
Materials and Methods: (B) 97% DEPMPO/OOH, 3% DEPMPO/OH.
(C) 35.5% DEPMPO/OOH, 64.5% DEPMPO/OH.
H2O2-induced formation of quinhydrone structures
in cell wall isolates
To clarify the cause of the differences between the HRP/
H2O2/SOD system (Fig. 5B) and cell wall-derived EPR spectra
(Fig. 3A), we analyzed the kinetics of the observed changes
after the addition of H2O2 to the cell wall (Fig. 6). On the
addition of DEPMPO to cell wall isolates, the signal of the
DEPMPO/OH adduct was detected (spectrum 6A). Addition of 20 mM H2O2 to cell wall isolates resulted in the
increase in signals of DEPMPO/OH and DEPMPO/H adducts
and a quinhydrone-derived radical (spectrum 6B). The EPR
spectrum in Fig. 6B changed as a function of time. The signal
intensity of DEPMPO/OH and DEPMPO/H adducts
decreased to nearly zero, and then the signal of the DEPMPO/
OH adduct reappeared gradually as a function of incubation
time. As the signals due to DEPMPO/OH and DEPMPO/H
adducts decreased, the EPR spectrum of the quinhydronederived radical became clear. The signal intensity of the
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
309
B. Kukavica et al.
20 G
A
20 G
A
Control
B
Oxidized CW
C
E
F
B
ECD response (nA)
D
G
14.00
12.00
10.00
8.00
6.00
Control
chlorogenic
caffeic
p-coumaric
ferulic
Oxidized CW
4.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
H
I
J
Fig. 6 Changes in EPR spectra after the addition of H2O2 to cell
wall isolates. (A) At 5 min after the addition of DEPMPO to cell wall
isolates; (B) 5 min after the addition of 20 mM H2O2 to A; (C–J) EPR
spectra measured every 2 min following the registration of spectrum
B. Filled circles, DEPMPO/OH adduct; filled inverted triangles,
DEPMPO/H adduct; filled squares, quinhydrone structures. Before
addition of DEPMPO to cell wall isolates, the EPR signal marked with
a square was also detected.
quinhydrone-derived radical increased 10- to 20-fold by
increasing the pH of the mixture to 12 after H2O2 treatment
(data not shown). The line shape was asymmetric and the
line width was about 0.48 mT. These findings suggest that
the signal was due to quinhydrone structures (Chio et al.
1982, Arnaud et al. 1983, Takahama et al. 2001). If the signal
was due to free phenoxyl radicals, the line width would
be smaller and hyperfine structures would be observed.
A similar EPR signal, the intensity of which increased under
alkaline conditions, was also observed in intact roots (data
not shown). It has been reported that the presence of
quinhydrone structures may be related to the reduction of
O2 by phenolics to O2–· and H2O2 (Furman 1986, Takahama
et al. 2001). After removing phenolic compounds by acid
and alkali hydrolysis of cell wall isolates, no EPR signal due
to quinhydrone structures could be detected (data not
shown). Oxidation of cell wall components by incubation of
cell wall isolates with a high concentration of H2O2 (0.1 M),
followed by thorough washing of the cell walls, resulted in a
significant decrease in the intensity of signals due to the
DEPMPO/OH adduct and quinhydrone structures (Fig. 7,
310
Time (min)
Fig. 7 Effects of pre-oxidation of cell wall-associated phenolics on
the formation of the DEPMPO/OH adduct in cell wall isolates. Box A:
upper EPR spectrum, control cell wall isolates plus DEPMPO; lower
EPR spectrum, oxidized cell wall isolates previously incubated with
0.1 M H2O2 for 30 min at 4°C and thoroughly washed before addition
of DEPMPO. Box B: HPLC analysis of the hydroxycinnamic acids
present in control and pre-oxidized cell wall isolates.
Table 3 The content of ascorbic and dehydroascorbic acid (µmol
gFW–1) in whole pea root extract and apoplastic fluid
Ascorbic acid +
dehydroascorbic
acid
Ascorbic acid (% of
total)
Root extract
0.98 ± 013
69 ± 5
Apoplastic fluid
0.240 ± 0.025
16 ± 4
Whole pea root extract and apoplastic fluid were prepared as described in
Materials and Methods. Data are means ± SDs (n = 4).
lower spectrum in box A). Such oxidized cell walls had greatly
reduced, or sometimes completely abolished, quantities of
hydroxycinnamic acids (Fig. 7, box B). However, reduction of
cell wall isolates by a high concentration of ascorbic acid
(20 mM) resulted in the disappearance of quinhydrone
structures as well as the DEPMPO/OH adduct, and the
appearance of the signal of the ascorbyl radical (data not
shown). Pea roots and the apoplastic fluid contained ascorbic
and dehydroascorbic acids, and about 69 and 16% of ascorbic
acid plus dehydroascorbic acid were present as the reduced
form in the roots and the apoplastic fluid, respectively (Table 3).
These results indicate that quinhydrone structures
observed in this study consisted of hydroquinone–quinone
couples in macromolecules. Caffeic and chlorogenic acids
and their quinone forms in the cell wall isolates may contribute
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
Generation of ROS in pea root cell wall
Discussion
Relative EPR signal intensity
80
To study biochemical reactions taking place in cell walls, it is
necessary to know how much the cell wall preparations were
contaminated with symplastic components. The cell wall
isolates used in this study seemed not to be contaminated
with hydroxycinnamic acids, SOD- and POD-derived symplast,
or NAD(P)H oxidase and G6PD (< 1% of symplast). Thus, the
cell wall isolates prepared in this study can be used as a
model system to simulate the reactions occurring between
POD, SOD, metals, hydroxycinnamic acids and O2 in cell walls.
A HRP/H2O2 in vitro system generated ·OH and O2–·, and
the generation of ·OH was enhanced by SOD (Fig. 5). The
generation of ·OH and O2–· might be explained if the following reactions were taken into consideration (Adediran and
Lambeir 1989, Wariishi and Gold 1990, Chen and Schopfer
1999, Hernandez-Ruiz et al. 2001, Liszkay et al. 2003, Furtmüller
et al. 2004, Liszkay et al. 2004):
70
60
50
20 G
40
pH 3
pH 5
30
pH 6.5
20
pH 7
10
pH 8
3
4
5
6
7
8
pH
Relative EPR signal intensity
2.7
2.4
(1)
2.1
1.8
(2)
pH 3
1.5
pH 5
1.2
(3)
pH 7
pH 8
0.9
3
4
5
6
7
8
(4)
pH
Fig. 8 Effects of pH on the formation of the DEPMPO/OH adduct
and quinhydrone structures in cell wall isolates. Cell wall isolates were
incubated for 5 min in buffer solutions (pH 3–8). Upper panel, cell
wall isolates with DEPMPO; lower panel, cell wall isolates without
DEPMPO. Signal intensities marked with closed circles (A) and closed
squares (B) were plotted as a function of pH.
to the formation of quinhydrone structures as quinhydrone
structures are easily formed when quinones and hydroquinones
are close to each other (Rex 1960, Steelink 1964, Furman
1986, Oniki 1998).
The effects of pH on the generation of the DEPMPO/OH
adduct and formation of quinhydrone structures were studied
using cell wall isolates (Fig. 8). The optimum pH for the generation of the DEPMPO/OH adduct was observed at about
pH 7 and that for the formation of quinhydrone structures
was also at pH 7. The result suggests that the production of
·OH was related to the oxidation of hydroxycinnamic acids
to their quinone forms, which were included in the formation
of quinhydrone structures.
(5)
SOD-dependent enhancement of the formation of the
DEPMPO/OH adduct might be explained by the inhibition
of reaction 3 and enhancement of reactions 2 and 4 producing
compound III and of reaction 5 producing hydroxyl radical.
Besides the above-mentioned chain of reactions, the metalcatalyzed Fenton reaction can be also a source of hydroxyl
radical production in the cell wall preparations (Fig. 3) via
the following reaction:
The DEPMPO/OH adduct was detected in cell wall isolates, (Fig. 3). As cell wall isolates could produce H2O2 by
auto-oxidation of hydroxycinnamic acids, the formation
of ·OH in cell wall isolates could also be explained by the
reaction between compound III and H2O2 (see also Fig. 9).
Cell wall-bound SOD in the isolates might facilitate the
production of ·OH similarly to SOD in the HRP/H2O2 in vitro
system. If SOD contributed to the formation of ·OH from
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
311
B. Kukavica et al.
MX+
−PhOH
M(X-1)+
−PhOH
−Ph=O
Quin hydrone
structure
−PhO• + H+
O2
−Ph=O
•O2−
H2O2
•OH2, O2, OH−
−PhOH
Mn-SOD
•O2−
−Ph=O
H2O2
Compound III
O2
POD
•O2−
−PhO•
+H+
−PhOH
H2O2
Compound II
Compound I
−PhOH
−PhO•
+H+
Fig. 9 Schematic diagram showing the possible reactions occurring
in the cell wall isolates. The scheme is a modification of the model
proposed by Chen and Schopfer (1999), based on the results obtained
in this study. M X+ and M (X–1)+ , oxidized and reduced forms of
a metal ion, respectively; -PhOH, -PhO· and -Ph = O, cell wall-bound
o-dihydroxyphenolic, phenoxyl radical and quinone, respectively.
H2O2, the presence of CWSOD would be advantageous for
the production of ·OH as this Mn-SOD isoform is resistant
to inactivation with H2O2 (Beauchamp and Fridovich 1971,
Weisiger and Fridovich 1973). DEPMPO/OOH was not
observed in cell wall isolates with inactivated proteins
unless H2O2 was added. Inactivation of enzymes (including
POD and SOD) in the cell wall isolates by SDS/heat
treatment demonstrated that besides the POD-associated
production of ·OH, tightly bound metal ions can also, in
the presence of H2O2, produce these radical species through
the activity of the Fenton reaction. This potential source
of hydroxyl radical species was abolished by the chelator
DETAPAC. What is interesting, however, is that in the
case of cell walls with inactivated proteins, one could also
observe the production of the superoxide radical (Fig. 3E).
This argues in favor of the participation of the CWSOD in
dismutation of O2–· and the contribution to ·OH production
in the cell wall. Indeed, our in vitro experiment with purified
HRP and SOD, which in the presence of O2–· produced ·OH,
supports such a mechanism.
The observed spectra generated by cell wall isolates are
more complex than simple in vitro reactions. Simulations
performed are best fitted if one assumes the participation
312
of DEPMPO/H and/CH3 adducts. This could be due to ·H
formation (marked by inverted triangles in Figs. 1, 2 and 7)
(Bačić et al. 2008). This would be in line with the proposal
of Ward et al. (2003) who studied the lignin peroxidasecatalyzed oxidation of a series of phenolic compounds in
detail and suggested the participation of phenoxyl radicals
(probably DEPMPO/CH3 adducts) in the formation of ·H
under certain pH and redox conditions.
As described above, H2O2 was generated in cell wall isolates.
We propose that the mechanism of its formation in cell wall
isolates is as follows. The initial step of the generation of
H2O2 may be the reduction of O2 to O2–· that can be transformed into H2O2. As the activity of NAD(P)H oxidase, which
reduces O2 to O2–·, was not detected in cell wall isolates used
in this study, other reductants for the formation of O2–·
should be considered. The phenolics contained in cell walls
are likely candidates because hydroxycinnamic acids can be
oxidized by Fe3+ and Cu2+ to the radicals, which can reduce
O2 to O2–· (Takahama 2004). The occurrence of metals
(Table 1) and o-dihydroxycinnamic acids such as caffeic and
chlorogenic acids (Table 2) in cell walls satisfies the requirements for this reaction. No formation of the DEPMPO/OH
adduct in the cell wall isolates after extensive oxidation of
their bound hydroxycinnamic acids (Fig. 7) supports the
necessity of hydroxycinnamic acids for the production of
H2O2, the source of the hydroxyl radical. Once H2O2 is
formed, the CWPOD/H2O2 system in the cell wall can oxidize
caffeic, chlorogenic and ferulic acids in the isolates, enhancing the production of radicals of the hydroxycinnamic acid,
which can react with O2, producing O2–· (Fig. 9). It has been
reported that o-dihydroxycinnamic acids found in cell wall
isolates are auto-oxidizable and that the oxidation products
(polymers with quinhydrone structures) are also autooxidizable (Takahama et al. 1999). The detection of an EPR
signal of quinhydrone structures (Fig. 6) not only in cell wall
isolates but also in intact roots (data not shown) suggests
the presence of auto-oxidizable polymers in intact cell walls
in addition to hydroxycinnamic acids. It has been reported
that ascorbate added to the cell wall isolates suppressed the
formation of ·OH (Veljović-Jovanović et al. 2005) and that
cell wall POD-catalyzed oxidation of phenolics is inhibited
by ascorbate (Takahama and Oniki 1992, Takahama 1993,
Takahama and Oniki 1994). In this study, EPR signals of the
DEPMPO/OH adduct and quinhydrone structures disappeared by adding ascorbate to the cell wall isolates (data not
shown). The result suggests that ascorbate could inhibit the
formation of ·OH, scavenge the DEPMPO/OH adduct and
reduce quinones in quinhydrone structures. Ascorbate in
the apoplast might be included in scavenging of ·OH generated
in cell walls, resulting in the inhibition of elongation growth
if ·OH participated in the loosening of cell walls on elongation
growth. This idea is supported by reports that the concentration of apoplastic ascorbate along hypocotyls shows a
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
Generation of ROS in pea root cell wall
significant negative correlation with the growth rate (CordobaPedregosa et al. 2003, Rodriguez-Serrano et al. 2006) and
that the concentration of ascorbate plus dehydroascorbate
in IAA-treated epicotyls of Vigna angularis was similar to
that in untreated hypocotyls, whereas the ratio of ascorbate
to ascorbate plus dehydro-ascorbate is somewhat decreased
by IAA (Takahama and Oniki 1994).
Conclusions
The data presented in this report demonstrate that hydroxycinnamic acids in cell walls can be electron donors of metalcatalyzed reduction of dioxygen to form superoxide, and that
cell wall-bound POD mediates the production of hydroxyl
radical from H2O2, which is formed from the superoxide.
Mn-superoxide dismutase isoforms (CWSOD) were found
in cell wall isolates, indicating their contribution to the
facilitated formation of H2O2 within the cell wall. The presence
of quinhydrone structures in cell wall isolates as well as in
excised root was also demonstrated. The quinhydrone structures disappeared by oxidation and reduction of cell wall
isolates, and the pH dependency of their formation was
similar to that of the formation of the DEPMPO/OH adduct.
The latter indicates that the reduction of O2 to H2O2 in
the cell walls might be related to the transformation of a
dihydroxycinnamic acid to its quinone form. Ascorbate
seems to have an important role in regulating the production of ·OH and quinhydrone structures, which in turn may
result in the regulation of root growth.
Materials and Methods
Reagents
DEPMPO was obtained from Alexis Biochemicals (Lausen,
Switzerland). DETAPAC, SHAM, CuZn-SOD from bovine
erythrocytes, HRP (type II), ascorbate oxidase and cellulase
were from Sigma (St Louis, MO, USA). Pectinase was obtained
from Serva (New York, USA). HPLC-grade acetonitrile and
methanol were from J. T. Baker (Deventer, The Netherlands)
and Carbo Reagenti (Milano, Italy), respectively. All solutions
were prepared daily by dissolving in 18 M redistilled and
deionized water (Millipore, Bedford, MA, USA).
Isolation of cell walls
Pea plants (Pisum sativum L.) were grown in hydroponic culture under an 8/16 h day/night regime. Fourteen-day-old
plants were used for analysis and cell wall isolation. Cell walls
were isolated from pea roots following the method described
by Carpita (1984) with some minor modifications. Roots
(60 g) were powdered in liquid N2 and homogenized in
120 ml of buffer [50 mM Tris–HCl (pH 7.2), 50 mM NaCl;
0.05% Tween-80; 1 mM phenylmethylsulfonyl fluoride
(PMSF)]. The homogenate was filtered through two layers of
cloth. The filtrate was sonicated for 1 min and centrifuged at
1,000×g for 20 min. The cell wall pellet was washed four
times in the above buffer without detergent and salt and
then suspended in 10 ml of 1 M NaCl, followed by incubation
for 30 min at 4°C and centrifugation at 1,000 × g for 15 min.
The pellet (2 g) (in the following, cell wall isolates) was
washed and centrifuged with 5 ml of 50 mM Tris–HCl (pH
7.2) several times and finally suspended in 5 ml of 50 mM
Tris–HCl (pH 7.2) for measurement of EPR spectra in the
presence and absence of DEPMPO. When the effects of pH
on the formation of radicals was determined, the pellet was
suspended in buffers with various pH values (see below). The
suspension was kept at 0°C until used for experiments.
Cell wall isolate (2 g) obtained as described above was
digested for 24 h at 4°C in 5 ml of a mixture containing 0.5%
cellulase and 2.5% pectinase in 50 mM Tris–HCl (pH 7.2),
and centrifuged at 10,000 × g for 10 min (Lin and Kao 2001).
POD and SOD isoforms bound to the cell wall isolates were
released by this procedure, and POD and SOD in the supernatant analyzed by native PAGE (see below).
Cell wall isolates with denatured proteins were prepared
by incubation of the isolates in 2% SDS for 30 min at 100°C,
which were then washed four times by suspension and
centrifugation at 1,000 × g for 15 min in 50 mM Tris–HCl
(pH 7.2).
Extraction of soluble proteins and metabolites
from roots
For extraction of soluble enzymes from roots, 0.5 g of frozen
roots was powdered in a mortar containing liquid N2 and
suspended in 100 mM potassium phosphate buffer (pH 6.5)
and 1 mM PMSF. The homogenate was centrifuged at
10,000 × g for 15 min at 4°C. The supernatant was used for
the separation of SOD isoforms by native PAGE (see below)
and measurement of the activity of G6DP. For determination of total ascorbate (ascorbate plus dehydroascorbate),
roots (0.5 g) were homogenized in 4 ml of 5% (v/v) HClO4
and the homogenate was centrifuged at 12,000 × g for 10 min
at 4°C to obtain the supernatant.
Extraction of apoplastic fluid
Freshly cut roots (20 root sections of length 3 mm) were
vacuum infiltrated with distilled water for 10 min, and, after
water on the surface was removed by filter paper, placed in 5 ml
Eppendorf tips, with the anterior ends down. The Eppendorf
tip was placed in a centrifugation tube containing 100 µl of
extraction medium for ascorbate [5% (v/v) HClO4] or for
proteins [100 mM phosphate buffer (pH 6.5)]. Upon centrifugation at 500 × g for 10 min, apoplastic fluid eluting from
the cut roots was instantly mixed with the extraction
medium. Protein extract was used for determination of the
G6PD activity.
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
313
B. Kukavica et al.
EPR spectroscopy
EPR measurements were made using a custom-constructed
Teflon flat cell (50 µl) with one side formed from oxygenpermeable thin Teflon foil. EPR spectra were recorded at
room temperature using a Varian E104-A EPR spectrometer
operating at the X-band (9.51 GHz) under the following settings: modulation amplitude, 0.2 mT; modulation frequency,
100 kHz; microwave power, 10 mW; centre of magnetic field,
341 mT; scan range, 20 mT; scan speed, 4 mT min–1. Spectra
were recorded and analyzed using EW software (Scientific
Software). DEPMPO (final concentration, 42.5 mM) was added
to cell wall isolates, apoplastic fluid or HRP. EPR signals of
quinhydrone structures and ascorbyl radicals were also measured using the settings for the measurement of DEPMPO/
OH and DEPMPO/OOH adducts. The pH dependence of the
formation of the DEPMPO/OH adduct and quinhydrone
structures was measured in the pH range from 3 to 8. The
buffer solutions used were 50 mM citrate/phosphate buffer
(pH range from 3 to 7) and 50 mM potassium phosphate
buffer (pH range from 7 to 8). Computer simulations of the
EPR spectra were performed using the WINEPR SinFonia
program. Parameters indicated in parentheses were used for
the simulation of the DEPMPO/OH adduct (aP = 46.70;
aN = 13.64; aHβ = 12.78) and the DEPMPO/OOH adduct
[isomer I (55%): aP = 50.15, aN = 13, aHβ = 11.3, aH_ = 0.85,
aH_ = 0.35, aH_(3) = 0.53; isomer II (37%): aP = 48.68,
aN = 13.8, aHβ = 0.88G, aH_ = 10.2, aH_ = 0.41, aH_ = 0.34;
isomer III (8.5%): aP = 40.8, aN = 13.3, aHβ = 1.5, aH_ = 10]
(Vasquez-Vivar et al. 2000; Mojović et al. 2004). The
DEPMPO/H adduct was simulated using the following
parameters (aP = 45.32; aN = 13.97; aHβ = 17.47; aHβ = 3.36)
from Bačić et al. (2008) and the DEPMPO/CH3 adduct was
simulated using the following parameters (aP = 46.26;
aN = 14.35; aHβ = 21.48) from Berliner et al (2002).
Analysis of phenolics by HPLC
Aliquots of root extract, apoplastic fluid and cell wall isolates
were boiled in methanol for 30 min to extract phenolics.
After cooling, the extracts were centrifuged at 10,000 × g for
15 min to remove methanol-insoluble components. For acid
hydrolysis, the pellet with cell wall isolates was suspended in
1 mol l–1 HCl and incubated at 37°C for 3 h in a water bath.
The rest of the cell wall isolates, after removing methanol
and acid-solubilized phenolics, were hydrolyzed in a similar
way using 100 g l–1 KOH in methanol (incubated at 60°C for
1 h in a water bath). Aliquots of samples (supernatants) prepared as above were injected in a Breeze HPLC system with a
Waters 2465 electrochemical detector equipped with 3 mm
gold working and hydrogen reference electrodes (Waters,
Milford, MA, USA). Signals were detected in the direct
scan mode at the constant potential of + 0.6 V. Phenolics
were separated on a Waters Symmetry C-18 RP column
314
(125×4 mm) with 5 µm particle size. The mobile phases were
0.1% phosphoric acid (adjusted to pH 2.4 in K2HPO4) (mobile
phase A) and acetonitrile (mobile phase B) with the following
gradient profile: in the first 10 min from 10 to 22% of mobile
phase B, followed by a 10 min linear rise up to 30% of mobile
phase B, ending with 5 min reversion to 10% of mobile phase
B. The flow rate was 1 ml min–1.
Native PAGE
Native PAGE was performed in a 10% polyacrylamide gel
with a reservoir buffer consisting of 0.025 M Tris and 0.192 M
glycine (pH 8.3) at 24 mA for 120 min. To detect POD activity
after electrophoresis, the gel was incubated with 10%
4-chloro-α-naphthol and 0.03% H2O2 in 100 mM potassium
phosphate buffer (pH 6.5). SOD separated by electrophoresis was detected according to Beauchamp and Fridovich
(1971) by incubating the gel in a reaction mixture containing 0.01 M EDTA, 0.098 mM nitroblue tetrazolium, 0.030 mM
riboflavin and 2 mM TEMED in 50 mM potassium phosphate
(pH 7.8) for 30 min in the dark, followed by washing
with distilled water and illumination by a fluorescent lamp
(30 µEm–2 s–1 for 15 min). CuZn-, Mn-and Fe-SODs were
distinguished from each other by incubating the gel with
5 mM KCN and/or 5 mM H2O2 before staining (Weisiger and
Fridovich 1973. Yamahara et al. 1999). Protein content was
measured according to Bradford (1976).
Enzyme assays
POD activities were measured in a reaction mixture (3 ml)
containing a 50 µl suspension of cell wall isolates, 3.3 mM
H2O2 and 4 mM chlorogenic, caffeic or ferulic acids in
100 mM potassium phosphate buffer (pH 6.5). Oxidation of
the above hydroxycinnamic acids was measured by the
increase in absorbance at 410, 450 and 356 nm, respectively,
according to Bestwick et al. (1998). The oxidation of NADH
by cell wall isolates was determined by measuring the absorbance decrease at 340 nm (ε = 6.22 mM–1 cm–1) (Halliwell
1978). The reaction mixture (3 ml) contained 50 µl of cell
wall isolates, 0.2 mM NADH, 0.25 mM MnCl2 and 0.2 mM
SHAM, p-coumaric acid or ferulic acid in 50 mM potassium
phosphate buffer (pH 5.5). Cytosolic contamination of apoplastic fluid and cell wall isolate was monitored by assaying
G6PD activity as marker enzyme. The activity of G6PD was
determined in a reaction mixture (3 ml) consisting of 5 mM
MgSO4 , 10 mM glucose-6-phosphate and 0.1 mM NADPH
in 100 mM potassium phosphate buffer (pH 8). The rate of
oxidation of NADPH was estimated by the absorbance
decrease at 340 nm.
Plant Cell Physiol. 50(2): 304–317 (2009) doi:10.1093/pcp/pcn199 © The Author 2008.
Generation of ROS in pea root cell wall
Determination of the concentration of ascorbic and
dehydroxyascorbic acids
The pH of the apoplastic fluid centrifuged down into 5%
HClO4 and roots extracted with 5% HClO4 was adjusted
to pH 4 by adding 5 M K2CO3, and insoluble components
were removed by centrifugation at 12,000 × g for 1 min at 4°C.
The reduced form of ascorbic acid was assayed in a reaction
mixture (3.5 ml) containing 100 µl of the root homogenate
or apoplastic fluid in 300 mM potassium phosphate buffer
(pH 5.6). The decrease in absorbance at 262 nm after addition
of 1 U of ascorbate oxidase was taken for calculation of the
amount of ascorbate (ε = 14.3 mM–1 cm–1). The concentration
of dehydroascorbate was estimated in a reaction mixture
(3.5 ml) containing 100 µl of root homogenate or apoplastic
fluid and 20 µl of 100 mM dithiothreitol in 300 mM potassium phosphate buffer (pH 7.6). The absorbance increase at
262 nm after the addition of dithiothreitol was used to calculate the concentration of dehydroascorbate.
Determination of metal contents
Cell wall isolates were dried at 60°C for 24 h and then milled.
Samples were digested in 1 M HNO3, and metal content was
determined by flame atomic absorption spectrometry
(Varian Spectra 220).
Funding
The Ministry of Science of Republic of Serbia (Project Nos.
143020B and 143016B).
Acknowledgments
The authors would like to thank the reviewers for their
helpful suggestions.
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(Received July 1, 2008; Accepted December 16, 2008)
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