Plant Cell Physiol. 38(4): 456-462 (1997)
JSPP © 1997
Enhancement of Peroxidase-Dependent Oxidation of Sinapyl Alcohol by an
Apoplastic Component, 4-Coumaric Acid Ester Isolated from Epicotyls of
Vigna angularis L.
Umeo Takahama and Takayuki Oniki
Kyushu Dental College, Kitakyushu, 803 Japan
A water-soluble component that enhanced the peroxidase-dependent (POX-dependent) oxidation of sinapyl alcohol was isolated from epicotyls of Vigna angularis. This
compound was an ester of 4-coumaric acid and a hexose,
and it was found in both the apoplast and the symplast.
The ester was oxidized by a basic POX isozyme (Km, about
20 pM) and by an acidic POX isozyme (Km, about 40 fiM)
that had been partially purified from the apoplastic fraction of epicotyls of V. angularis. These POX isozymes oxidized sinapyl alcohol at only a very low rate, but a 15-fold
enhancement was observed upon addition of the ester. The
concentrations of the ester required for the half-maximal
enhancement were similar to the Km values of the ester for
its oxidation by the respective isozymes. The apoplastic concentration of the ester was higher than 130 fM, suggesting
that this ester might act as a donor of electrons to the apoplastic POX isozymes in situ. Coniferyl alcohol also enhanced the POX-catalyzed oxidation of sinapyl alcohol.
The concentrations of coniferyl alcohol required for halfmaximal enhancement of the oxidation of sinapyl alcohol
were about 23 and 250 /JM when reactions were catalyzed
by the basic and acidic POXs, respectively. These values
were similar to the Km values of coniferyl alcohol for its oxidation by the respective isozymes. These results suggest
that 4-coumaric acid ester and coniferyl alcohol, if it is
present in the apoplast, can enhance the POX-dependent
oxidation of sinapyl alcohol in the apoplast of epicotyls of
V. angularis.
to another. For example, apoplastic POX from some
plants [e.g., Acer (Sterjiades et al. 1993), Vigna (Chabanet
et al. 1993, 1994, Takahama and Oniki 1994) and Zinnia
(Church and Galston 1988, Sato et al. 1995)] cannot rapidly oxidize compounds with a syringyl group, such as
sinapyl alcohol, but the POX from other plants [e.g., Nicotiana (Takahama et al. 1996) and Populus (Tsutsumi et al.
1994)] can rapidly oxidize sinapyl alcohol. It has been
reported that plants in which the apoplastic POX cannot
rapidly oxidize sinapyl alcohol can use phenoxyl radicals,
such as coniferyl alcohol, 4-coumaric acid and ferulic acid
radicals, to carry out the oxidation (Takahama 1995, Takahama et al. 1996). However, 4-coumaric and ferulic acids
are usually present as esters in plants. Therefore, it is necessary to determine whether such esters are present in the
apoplast and whether such esters can enhance the POX-dependent oxidation of sinapyl alcohol.
In a previous study (Takahama et al. 1996), it was demonstrated that POX isozymes in the apoplast of Vigna angularis oxidize sinapyl alcohol more slowly than coniferyl alcohol, 4-coumaric acid, ferulic acid and an ester of ferulic
acid. However, the oxidation of sinapyl alcohol can be considerably enhanced by an apoplastic component of the epicotyls of V. angularis. This report describes the isolation of
an apoplastic component that enhances the POX-dependent oxidation of sinapyl alcohol, as well as kinetic analysis
of the oxidation of sinapyl alcohol by POX isozymes isolated from the apoplastic fraction of epicotyls of V. angularis.
Key words: Apoplastic peroxidase isozymes — 4-Coumaric
acid ester — Lignin biosynthesis — Oxidation of sinapyl alcohol — Vigna angularis.
Materials and Methods
Plant materials and preparation of IWF—-Seeds of Vigna
angularis Ohwi et Ohashi cv. erimoshouzu were grown as described previously (Takahama et al. 1996). Intercellular washing
fluid (IWF) that contained apoplastic components was prepared
from epicotyl segments between 0.5 and 4.5 cm from the first
foliage by vacuum infiltration and centrifugation (Takahama et
al. 1996). For infiltration, solutions of 10 mM sodium phosphate
(pH 6.0), with or without 0.3 M KC1, were used.
HPLC—HPLC was performed with a reverse-phase column
[Shim-Pack CLC-Cig (6 mm i.d. x 150 mm); Shimadzu, Kyoto]
combined with a spectrophotometric detector with a photodiode
array (SPD-M1A; Shimadzu). The mobile phase used to separate
components of the IWF and of methanol extracts of whole epicotyls was a mixture of methanol and 25 mM KH2PO4 (1 : 4, v/v). A
mixture of methanol and 25 mM KH2PO4 ( 2 : 3 , v/v) was used for
1
in every case.
Lignins are composed of 4-hydroxyphenyl, guaiacyl
and syringyl groups (Monties 1989), which are derived
mainly from 4-coumaryl, coniferyl and sinapyl alcohols.
Peroxidase (POX) and phenol oxidase in the apoplast can
oxidize these lignin monomers (Lewis and Yamamoto
1990, Davin and Lewis 1992). However, the substrate specificities of POX and phenol oxidase vary from one species
Abbreviations: 4-CA, 4-coumaric acid; HRP, horseradish
peroxidase; IWF, intercellular washing fluid; POX, peroxidase.
456
Mechanism of oxidation of sinapyl alcohol
Partial purification of 4-CA ester—Fresh epicotyls (32 g)
were homogenized in 300 ml of methanol and the homogenate was
filtered through filter paper. Then, 300 ml of ethyl acetate were
added to the filtrate and the mixture was filtered again through
filter paper. Three liters of ethyl acetate were added to this filtrate.
The mixture was allowed to stand for one night at room temperature (about 15°C) in darkness. A light brown precipitate formed
and was collected by centrifugation (3,500xg, 5 min, 10°C). It
was dissolved in 50 ml of methanol. The solution in methanol was
concentrated to about 3 ml under a stream of N2 gas at about
15°C. A portion of the solution (0.5 ml) was applied to a Lobar
column [RP-8 (10 mm i.d. x 240 mm); Merck, Darmstadt, Germany] that was combined with a spectrophotometric detector with
a photodiode array. The mobile phase was a mixture of methanol
and 25 mM KH2PO4 (1 : 9, v/v) and the flow rate was 1 ml min" 1 .
A peak with a retention time of about 25 min was detected when
the eluate monitored at 308 nm. The eluate around the peak was
collected. This fraction had an absorption maximum around 308
nm. This chromatographic procedure was repeated six times and
the total volume of pooled fractions was about 50 ml.
To remove potassium phosphate from this solution, 200 ml
of methanol and then 500 ml of ethyl acetate were added to the 50ml pooled fraction. After filtration of the mixture through filter
paper, the filtrate was evaporated to dryness in a rotary evaporator at about 40°C. The residue was dissolved in 10 ml of
methanol, and the solution was then concentrated to 1 ml under a
stream of N2 gas at about 15°C. The addition of 9 ml of ethyl
acetate to the solution in methanol resulted in the formation of a
white precipitate, which was then collected by centrifugation,
washed once with ethyl acetate and kept at — 20°C until required.
The compound was dissolved in 2 ml of methanol and referred to
as compound I.
Hydrolysis—The solution in methanol of compound I (20^1)
was added to 2 ml of 1 M NaOH and the mixture was incubated
for 1.5 h under a stream of N2 gas at 40°C. After the addition of 2
ml of 2 M HC1 to the alkaline solution, the mixture was extracted
twice with 1 ml of ethyl acetate. The ethyl acetate fractions were
combined and evaporated to dryness under a stream of N2 gas.
The residue was dissolved in methanol and used for identification
of phenolic components. To identify the sugar moiety of compound 1, we added 0.1 ml of the solution in methanol of compound I to 5 ml of 2 M trifluoroacetic acid, and then incubated the
mixture over boiling water for 1.25 h (Shibuya 1984). After three
extractions of the trifluoroacetic acid with ethyl acetate, the
remaining aqueous solution was evaporated to dryness in vacuo
over solid NaOH and used for identification of sugars by thinlayer chromatography on cellulose plates (Merck) with a mixture
of 1-butanol, pyridine and water ( 9 : 5 : 8 , v/v) as the mobile
phase and staining with aniline-hydrogen phthalate (Harborne
1988).
457
(pH6.0) and applied to a column (1.5 cm i.d. x 8 cm) of CM52
(Whatmann, Maidstone, England) that had been equilibrated
with the same buffer. Proteins bound to the column were eluted
successively by 15-ml aliquots of 0.1, 0.2, 0.3, 0.4 and 0.5 M NaCl
in 10 mM sodium phosphate (pH 6.0). High POX activity was detected in fractions 9 through 13. These fractions (3 ml each) were
combined and concentrated by Centricon-10 (Amicon, MA,
U.S.A.). The POX activity in the resultant fraction contained a
basic isozyme, and its specific activity was 10.4 nmol of tetraguaiacol formed fig protein" 1 min"1.
Proteins that had passed through the CM52 column were
loaded onto a column (1.5 cm i.d. x 5 cm) of DEAE-cellulose (Serva Feinbiochemical GmbH, Germany) that had been equilibrated
with 10 mM sodium phosphate (pH 6.0). Proteins were eluted as
described above. A single peak of POX activity was detected, and
the appropriate fractions were combined and concentrated as described above. The POX activity of the acidic isozyme was 3.4
nmol of tetraguaiacol formed ftg protein" 1 min" 1 .
Quantitation of the oxidation of sinapyl alcohol—The POXdependent oxidation of sinapyl alcohol was quantitated in a reaction mixture (1 ml) that contained 90 fiM. sinapyl alcohol, various
amounts of compound I or coniferyl alcohol, 1 mM hydrogen peroxide and fraction that contained a POX isozyme with a denned
activity in 10 mM sodium phosphate (pH 6.0). The oxidation of
sinapyl alcohol was monitored at 270 nm. The difference between
the absorption coefficients of sinapyl alcohol and the stable products of oxidation was taken as 7.5 mM" 1 cm" 1 (Takahama 1995).
Assay of glucose-6-phosphate dehydrogenase—The activity
of this enzyme was measured at 340 nm (£=6.2 mM" 1 cm"1) in
90 mM Tricine-NaOH (pH 8.0) with 1 mM glucose-6-phosphate,
0.2 mM NADP + , 5 mM MgCl2 and 10 ft\ of sample.
Reagents—Coniferyl alcohol and a basic HRP isozyme (Type
IX) were purchased from Sigma Chemical Co. (St. Louis, MO,
U.S.A.). 4-CA, ferulic acid and tryptophan were obtained from
Tokyo Kasei Kogyo Co. Ltd. (Tokyo) and sinapyl alcohol was
from Aldrich Chemical Co. (Milwaukee, WI, U.S.A.). Sinapyl alcohol was purified as described previously (Takahama et al. 1996)
by thin-layer chromatography on cellulose plates (Merck).
Results
Figure 1A (trace 1) shows profile of the elution of IWF
after HPLC in terms of the absorbance at 220 nm. Eight
peaks were detected. After incubation of IWF with 1 mM
hydrogen peroxide for 5 min at about 20°C, peak a disappeared completely, but the other peaks did not disappear
and only some decreased in height (Fig. 1A, trace 2). The
absorption spectrum of the material in peak a had a maximum around 308 nm (Fig. IB, curve 1), suggesting that
Partial purification of POX isozymes—IWF from epicotyls
this component might be an ester of hydroxycinnamic acid.
of V. angularis contains two acidic POX isozymes and a basic
The material in peak b was tentatively identified as tryptoPOX isozyme (Takahama et al. 1996). To prepare the IWF for
phan from a comparison of retention times and absorption
separation of the isozymes, we vacuum-infiltrated about 40 g of epspectra (data not shown).
icotyl segments in 0.3 M KC1 in 10 mM sodium phosphate (pH
6.0) with subsequent centrifugation at 800xg for 5 min at 4 C C.
Two peaks that corresponded to peaks a and b in trace
About 1.5 ml of IWF was obtained. POX activity of this IWF
1 were also found after HPLC of a methanol extract of
1
was equivalent to 1.6 nmol of tetraguaiacol formed fig protein"
whole epicotyls (Fig. 1A, trace 3). The absorption spectrum
min" 1 when measured in a reaction mixture (1 ml) that contained
of the material in peak a in trace 3 was identical to that of
2.5 mM guaiacol, 5 mM hydrogen peroxide and 10fi\ of enzyme
the material in peak a in trace 1 from 220 to 400 nm
solution in 10 mM sodium phosphate (pH 6.0). This value was calculated from an absorption coefficient of 26.6 mM" 1 cm" 1 at 470
(Fig. IB, curves 1 and 2), suggesting that peak a in trace 3
nm. The IWF was diluted to 15 ml with 10 mM sodium phosphate
represented a cinnamic acid ester and an unknown compo-
458
Mechanism of oxidation of sinapyl alcohol
nent that absorbed UV-light at wavelength below 220 nm.
This peak disappeared when the methanol extract was treated with HRP and hydrogen peroxide (data not shown). The
absorption spectrum of the material in peak b in trace 3
was identical with that of the material in peak b in trace 1
(data not shown), suggesting that this peak also included
tryptophan.
The levels of tryptophan in IWF and in the methanol
extract were estimated from the areas of peak b in traces 1
and 3, and the levels were, at most, 3.3 and 356 nmol per g
FW, respectively, indicating that about 1% of the total tryptophan was present in the apoplastic fraction. The areas
of peak a were 3.8 cm2 per g FW in trace 1 and 59.4 cm2 per
1
1
j
I
)
1
I, A
U
i
'
'
'®
b
g FW in trace 3, indicating that about 6.5% of the total
component of peak a was present in the apoplastic fraction
(for the levels of this component in the apoplast and in the
symplast, see below). The activity of glucose-6-phosphate
dehydrogenase in the apoplastic fraction was about 0.3%
of that in a cell-free extract prepared with 0.1 M sodium
phosphate (pH 6.8). If tryptophan and glucoses-phosphate dehydrogenase are present only in the symplast, we
can conclude that the material in peak a was present not
only in the symplast but also in the apoplast.
Compound I, isolated from methanol extracts of epicotyls, gave a single peak after HPLC, with a retention
time identical with that of peak a in trace 1 in Figure 1A
(data not shown). The absorption spectrum (Fig. IB, curve
3) was also identical to that of the material in peak a (curve
1). These results indicate that compound I corresponded to
the component in peak a in trace 1 in Figure 1A. Compound I was used in the following experiments as described
below.
b
„ A
b
IK
.1
1
6
9
Retention time (min)
1
0
1
12
15
200
<
300
18
1
Io
200
6
12
Retention time (min)
400
Wavelength (nm)
Fig. 1 Elution profiles after HPLC of IWF and a methanol extract of epicotyls and the absorption spectra of peak components.
IWF was prepared with 10 mM sodium phosphate (pH 6.0). Panel
A, elution profiles after HPLC. Trace 1, 5^1 IWF equivalent to
0.1 g FW of epicotyls; trace 2, 5 n\ IWF treated with 1 mM hydrogen peroxide for 5 min at about 20°C prior to the application
to the HPLC column; trace 3, 5 ft\ methanol extract equivalent to
0.0045 g FW of epicotyls. Panel B, absorption spectra. Curve 1,
the material in peak a in trace 1 in panel A; curve 2, the material in
peak a in trace 3 in panel A; curve 3 (dashed line), isolated compound I. AH spectra were normalized at 308 nm.
300
Wavelength (nm)
400
Fig. 2 Elution profiles after HPLC of products of alkaline hydrolysis of compound I and the absorption spectra of peak components. Panel A, elution profiles after HPLC. Trace 1, untreated
compound I; trace 2, hydrogen peroxide-treated compound I.
Twenty /A of a solution of compound I were incubated in a reaction mixture (lml) that contained 20fig of HRP and 10 mM
sodium phosphate (pH 6.0) with or without 15 ^M hydrogen
peroxide for 3 min at about 20°C. Alkaline hydrolysis was then
performed, and phenolic compounds were extracted with ethyl
acetate as described in Materials and Methods. Panel B, absorption spectra. Curve 1, the material in peak 1 of trace 1 in Panel A;
curve 2, the material in peak 2 of trace 1 in panel A; curve 3, 4coumaric acid; curve 4, ferulic acid. Spectra 1 and 3 were normalized at 280 nm.
Mechanism of oxidation of sinapyl alcohol
After the hydrolysis of compound I under alkaline conditions, components with retention times of 5.5 min (peak
1) and 6.4 min (peak 2) were detected (Fig. 2A, trace 1).
These components were almost undetectable after compound I had been treated with hydrogen peroxide plus
HRP prior to alkaline hydrolysis (trace 2). The retention
time of peak 2 was close to that of 4-CA and ferulic acid.
However, the absorption spectrum of the material in peak
2 (Fig. 2B, curve 1) was identical to that of 4-CA (curve 3)
but not to that of ferulic acid (curve 4). The absorbance
spectrum of the material in peak 1 (curve 2) had a maximum at 265 nm. This component was identified as a product of the degradation of 4-coumaric acid by alkaline hydrolysis because the same product was generated when
authentic 4-coumaric acid was hydrolyzed under alkaline
conditions. A brown spot with an Rf value close to those of
glucose and galactose was detected after thin-layer chromatography and staining for sugars. These results suggest that
compound I, namely, the material in peak a in trace 1 in
Figure 1A, was an ester of 4-CA and hexose.
When HRP (20//g) and hydrogen peroxide (1 mM)
were added to a solution of the 4-CA ester, the absorption
at 308 nm decreased from 0.3 to 0.2 in 5 min (Fig. 3). To
estimate the concentration of the 4-CA ester, the decrease
in absorbance was monitored as a function of the concentration of hydrogen peroxide in the presence of 20 fig of
HRP. A maximum decrease in absorption of 0.1 was observed when hydrogen peroxide was added at 17 ^M. Further addition of hydrogen peroxide did not result in any
f
i
l
l
1
1
1
1
where (4-CA ester)2 represents a stable dimer of the 4-CA
AA
0.05
244
-0.05
308
0.05
270
222
-0.05- - I
I
I
I
I
l_
ii-
1
308
1
\
-0.1
v \
- \\
\\
\\
/
/
- vv
\v
y"\
\
\
~NM
1 /
'/
\
-0.2
v
•
0
200
2(4-CA ester)+H2O2->- 2(4-CA ester radical)+ 2H2O
2(4-CA ester radical) -* (4-CA ester)2
_
0.4 - \
"l
further decrease in the absorbance. No peak that corresponded to the 4-CA ester was detected when the titrated
mixture was analyzed by HPLC (data not shown). From
these results, the concentration of the 4-CA ester in the experiment for which results are shown in Fig. 3 was estimated to be 34 /uM on the assumption that the product of the
oxidation of the ester was a stable dimer, as indicated
below:
-
1
I
V
0.2
1
459
I
.
1
1
1
200
300
400
Wavelength (nm)
1
300
Wavelength
V222
400
(nm)
Fig. 3 Changes in the absorbance spectrum after treatment with
HRP and H2O2. The reaction mixture (1 ml) contained 4-CA ester
with an absorption of 0.3 at 308 nm, 20 ftg of HRP and 1 mM hydrogen peroxide in 10 mM sodium phosphate (pH 6.0). Trace 1,
before the addition of HRP; trace 2, 5 min after the addition of
HRP. The light path of the measuring beam was 4 mm.
Fig. 4 Enhancement of the oxidation of sinapyl alcohol by 4CA ester. The reaction mixture (1 ml) contained 1 mM hydrogen
peroxide and a basic POX isozyme equivalent to 0.15/ig of protein in 10 mM sodium phosphate (pH 6.0). Panel A, 34 nM 4-CA
ester; panel B, 90//M sinapyl alcohol; panel C, 34/*M 4-CA ester
plus 90 nM sinapyl alcohol. Reactions were started by the addition
of POX after memorization of a reference spectrum. Scanning
was repeated at 1.8-min intervals at a speed of 120 nm min" 1 .
460
Mechanism of oxidation of sinapyl alcohol
_ 0
a.
m
c
55
10
[4-CA ester]
20
30
OMr
a
a
c
0.2
UJ
-0.1
0.1
0.2
1/[4-CA ester] (nM-1)
Fig. 5 Dose-response curves for the enhancement of the oxidation of sinapyl alcohol by 4-CA ester. The reaction mixture (1 ml)
contained 1 mM hydrogen peroxide, 90//M sinapyl alcohol and
various concentrations of 4-CA ester in 10 mM sodium phosphate
(pH 6.0). Panel A, oxidation of sinapyl alcohol as a function of
the concentration of 4-CA ester. Panel B, double-reciprocal plots
of the enhancement of oxidation versus the concentration of 4-CA
ester. Closed circles, a basic POX isozyme equivalent to 0.05 fig of
protein; open circles, an acidic POX isozyme equivalent to 0.32 fig
of protein.
ester. The absorption coefficient of the ester was then calculated to be 22.1 mM" 1 cm" 1 at 308 nm. Furthermore, the
difference between absorption coefficients of the ester and
the stable oxidation product was estimated to be 7.4 mM" 1
cm"' (308 nm) from the difference in absorbance before
and after the addition of hydrogen peroxide. Using the
above absorption coefficient, we estimated the average concentration of the ester in the IWF to be around 130 fiM,
and we estimated the levels of the ester in the apoplast and
the symplast to be about 7 and 92 nmol per g FW, respectively.
Figure 4 shows difference spectra for the oxidation of
the 4-CA ester (panel A) and sinapyl alcohol (panel B) by a
basic POX isozyme prepared from an apoplastic fraction
of epicotyls. During the oxidation of the 4-CA ester, absorption decreased at 308 nm and increased at 244 nm, with
an isosbestic point at 266 nm (panel A), suggesting the formation of a stable product(s) of oxidation. In the presence
of both the 4-CA ester and sinapyl alcohol, a change in absorption characteristic of the oxidation of sinapyl alcohol
was recorded (panel C), indicating the enhancement of the
oxidation of sinapyl alcohol by the 4-CA ester. The rate of
oxidation of sinapyl alcohol in the presence of the 4-CA
ester (Fig. 4C) was about 2.5 times the rate of oxidation of
the 4-CA ester itself (Fig. 4A) and much higher than the
rate of oxidation sinapyl alcohol. The oxidation of sinapyl
alcohol catalyzed by an acidic POX-isozyme was also enhanced by the 4-CA ester (Fig. 5).
Their rates of the oxidation of sinapyl alcohol catalyzed by basic and acidic POX isozymes depended on the
concentration of the 4-CA ester (Fig. 5). The maximum enhancement and the concentration of the 4-CA ester required for half-maximal enhancement were estimated for
each isozyme from double-reciprocal plots (Fig. 5B). The
results are listed in Table 1.
Since coniferyl alcohol might be present in the apoplast when lignins are synthesized (Davin and Lewis 1992),
we also examined the effects of coniferyl alcohol on the oxi-
Table 1 Concentrations of 4-CA ester and coniferyl alcohol required for half-maximal enhancement of the oxidation of
sinapyl alcohol and the Km values of these compounds
raicuucici
Maximal enhancement
Basic POX isozyme
Coniferyl alcohol
4-CA ester
Acidic POX isozyme
Coniferyl alcohol
4-CA ester
17-fold
15-fold
15-fold
20-fold
Concentration for Maxi /2 (fiM)
14
23
36
250
Km (JJM) for oxidation
22
36
38
330
Maximal enhancement of the oxidation of sinapyl alcohol by 4-CA and coniferyl alcohol and the concentrations of these compounds for
half-maximal enhancement were determined by the method indicated in Fig. 5. Km values were determined from double-reciprocal plots.
The reaction mixture (1 ml) contained 1 mM hydrogen peroxide, a basic POX isozyme equivalent to 0.05 fig of protein or an acidic POX
isozyme equivalent to 0.32 /ig of protein and various concentrations of 4-CA ester or coniferyl alcohol in 10 mM sodium phosphate (pH
6.0). Maxi/2, Half-maximal enhancement.
Mechanism of oxidation of sinapyl alcohol
dation of sinapyl alcohol by isolated POX isozymes (Table
1). The effects of coniferyl alcohol were similar to those of
the 4-CA ester when the oxidation of sinapyl alcohol was
catalyzed by the basic POX isozyme. However, when the
oxidation was catalyzed by the acidic POX isozyme, a
much higher concentration of coniferyl alcohol than that
of the 4-CA ester was required for half-maximal enhancement.
The Km values of each substrate for the oxidations catalyzed by the POX isozymes are also shown in Table 1. The
Km values are similar to the concentrations required for
half-maximal enhancement.
461
ester could serve as a donor of electrons to the POX isozymes in Vigna.
The enhancement of the basic and the acidic isozymedependent oxidations of sinapyl alcohol by the 4-CA ester
(Fig. 4, 5) suggests that, if the ester coexists with sinapyl alcohol in the apoplast of epicotyls, then the ester can enhance the oxidation of this alcohol in V. angularis. The rate
of oxidation of sinapyl alcohol in the presence of the 4-CA
ester (Fig. 4C) was about 2.5 times that of the 4-CA ester
(Fig. 4A). This result suggests that, if radicals of the 4-CA
ester are formed, such radicals react rapidly with sinapyl alcohol. The Km values of the ester were similar to the concentrations of the ester required for the half-maximal enhancement of the oxidation of sinapyl alcohol by such isozyme
Discussion
(Table 1). This result suggests that the affinity of the 4A compound that was identified as an ester of 4-CA CA ester for POX isozymes was not strongly affected by
was extracted from the apoplast of epicotyls by vacuum in- sinapyl alcohol.
filtration with a buffered aqueous solution and centrifugaThe data in Table 1 also indicate that coniferyl alcohol
tion. This result indicates that the ester was present in the radicals formed by the POX isozymes might also react
apoplast as a water-soluble component in the epicotyls of rapidly with sinapyl alcohol, as reported previously (TakaV. angularis. A water-soluble phenolic component of the hama 1995). The concentrations of coniferyl alcohol reapoplast has also been reported in spinach leaves (Taka- quired for half-maximal enhancement of the oxidation of
hama and Oniki 1992). In leaves of tobacco and broad sinapyl alcohol were about 23 and 250 ^M for the basic and
bean, chlorogenic acid (an ester of caffeic acid and quinic the acidic POX isozymes, respectively. The Km of coniferyl
acid) or compound related to chlorogenic acid and 3,4-dihy- alcohol for oxidation by the basic isozyme was also about
droxyphenylalanine, respectively, are present in the apo- ten times lower than the Km of coniferyl alcohol for oxidaplast as water-soluble phenolic components (unpublished tion by the acidic isozyme (Table 1). At present, it is
results). These compounds might be transported from the unclear why such a difference was observed. In Zinnia, the
symplast to the apoplast since the same compounds were Km values of coniferyl alcohol for the oxidation by basic
POX isozymes range from 100 to 500 fiM (Sato et al. 1995).
also found in cell-free extracts.
A possible function of the water-soluble phenolic com- Pedrefio et al. (1989) have reported that the Km of POX
pounds in the apoplast has been discussed in relation to the that was bound ionically to cell walls was about 500/iM.
scavenging of hydrogen peroxide in cooperation with ascorThe difference in substrate specificities between the
bate (Takahama and Oniki 1992). In addition, water-solu- basic and the acidic POX isozymes (Table 1) suggests that
ble phenolic esters derived from cell walls have also been they might play different roles in the biosynthesis of lignins
reported (Ishii and Saka 1992). On the other hand, 4-CA in the epicotyls of V. angularis. Additional studies are reand ferulic acid are found in water-insoluble fractions of quired to determine the concentrations of coniferyl and
cell walls prepared from various plants, such as A vena sinapyl alcohols in the apoplast for further discussions of
(Kamisaka et al. 1990), rice (Shibuya 1984) and forage the POX-dependent biosynthesis of lignins. Studies of the
plants (Lam et al. 1990). Such phenolics bind to polysaccha- localization of the substrates and the POX isozymes in
rides by ester linkages (Iiyama et al. 1994, Shibuya 1984) different tissues of the epicotyl are also necessary.
and to lignins by ether linkages (Iiyama et al. 1994, Ralph
The authors thank Dr. S. Houghton for correction of the
et al. 1992, Scalbert et al. 1985, 1986).
text,
and Drs. K.-J. Dietz and C. Cruz for stimulating discussions.
The concentration of the 4-CA ester in the IWF that
we prepared from epicotyls of V. angularis was estimated
to be 130/iM. This concentration might be equivalent to
References
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were no gas spaces in the apoplast. However, since we Chabanet, A., Catesson, A.M. and Goldberg, R. (1993) Peroxidase and
phenolase activities in mung bean hypocotyl cell walls. Phytochemistry
know that there are gas spaces in the apoplast, the concen33: 759-763.
tration in the apoplastic aqueous phase should be higher be- Chabanet, A., Goldberg, R., Catesson, A.M., Quinet-Szely, M., Delaunay, A.M. and Faye, L. (1994) Characterization and localization of a
cause the volume of the IWF includes not only the volume
phenol oxidase in mung bean hypocotyl cell walls. Plant Physiol. 106:
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(Received July 1, 1996; Accepted February 5, 1997)