Biochemical characterization of two differentially expressed polyphenol oxidases from hybrid poplar

Article abstract of DOI:10.1016/S0031-9422(03)00159-6

Two polyphenol oxidase isoforms with distinct expression patterns were identified in hybrid poplar (Populus trichocarpax P. deltoides). PPO-1, corresponding to the previously cloned PtdPPO (Constabel et al., Plant Physiol. 124: 285-295) was primarily leaf

Full text of DOI:10.1016/S0031-9422(03)00159-6

Phytochemistry 64 (2003) 115–121
www.elsevier.com/locate/phytochem
Biochemical characterization of two differentially expressed
polyphenol oxidases from hybrid poplar
Jiehua Wanga,b, C. Peter Constabela,*
aCenter for Forest Biology and Department of Biology, University of Victoria, Victoria, Canada V8W3N5
bDepartment of Biological Sciences, University of Alberta, Edmonton, Canada T6G 2E9
Received 8 November 2002; received in revised form 13 February 2003
Dedicated to the memory of Professor Jeffrey B. Harborne
Abstract
Two polyphenol oxidase isoforms with distinct expression patterns were identified in hybrid poplar (Populus trichocarpaÂ
P. deltoides). PPO-1, corresponding to the previously cloned PtdPPO (Constabel et al., Plant Physiol. 124: 285–295) was primarily
leaf tissue-specific and detected only after wounding. PPO-2 was expressed constitutively in all tissue types tested except mature
leaves, with highest expression in very young leaves and conducting tissues such as roots, stems and petioles. These two PPO isoforms
were partially purified from hybrid poplar by ammonium sulfate fractionation followed by hydrophobic interaction chromatography.
They were found to differ in stability, pH optimum, and activation by SDS. Tests with common phenolic substrates showed that
PPO-1 had a broader substrate specificity than PPO-2. The distinct enzymatic properties and expression patterns of these two PPO
isoforms suggest that they may have different physiological functions in hybrid poplar.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Populus; Salicaceae; Catechol oxidase; Phenolase; Catechin
1. Introduction
by Li and Steffens (2002) using PPO-overexpressing
transgenic tomato plants, which show enhanced resistance
to Pseudomonas syringae. An intriguing feature of PPO is
that while this enzyme has been found in many plant spe-
cies, patterns and levels of expression may differ widely
between species (Constabel et al., 1998). This suggests that
other physiological roles for PPO still remain to be dis-
covered; for example, a hydroxylation reaction during
aurone biosynthesis was recently found to be carried out
by a PPO-like enzyme (Nakayama et al., 2000).
Plant PPOs are usually encoded by mid-sized gene
families. For example, in tomato there are at least seven
PPO genes (Newman et al., 1993), and in potato, five
PPO genes were characterized (Thygesen et al., 1995).
The genes can have complex temporal and tissue-spe-
cific patterns of expression, with high levels of transcript
commonly found in flowers, young leaves, trichomes,
roots, and tubers (Thipyapong et al., 1997; Thygesen et
al., 1995). Only one member of the tomato PPO gene
family is wound- and stress-induced (Thipyapong and
Steffens, 1997). As in higher plants, multiple forms of
polyphenol oxidases have also been described in fungi
(Zhang and Flurkey, 1997).
Polyphenol oxidase (PPO, EC 1.10.3.1) is a common
enzyme which uses molecular oxygen to oxidize
o-diphenols to o-quinones, and in some plants also
hydroxylate monophenols to o-diphenols (mono-
phenolase activity; EC 1.14.18.1). The quinones are
reactive compounds which are responsible for the
damage-induced browning of many fruit and vegetables
(Steffens et al., 1994). Typically, plant PPOs have broad
substrate specificities and are able to oxidize a variety of
o-diphenols. While the physiological function of PPO in
many plants is unresolved, in some species there is
strong evidence for a role of PPO in defense against
insects as well as pathogens (reviewed in Steffens et al.,
1994; Constabel et al., 1996). In tomato, PPO induction
by the wound signal systemin and the octadecanoid
pathway provided strong evidence for a defensive func-
tion of PPO against insects (Constabel et al., 1995). A
clear role of PPO in pathogen defense was demonstrated
* Corresponding author. Tel.: +1-250-472-5140.
E-mail address: cpc@uvic.ca (C.P. Constabel).
0031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00159-6

116
J. Wang, C.P. Constabel / Phytochemistry 64 (2003) 115–121
Previously, a wound- and herbivore-inducible leaf spe-
The 59 kDa band was not present in petioles, stems,
and roots, and was only faintly visible in midvein sam-
ples of P. trichocarpaÂP. deltoides (Fig. 1A). In these
tissues, however, the antibody recognized a different
protein band, migrating at approximately 66 kDa. The
intensity of this band correlated well with PPO activity
in crude extracts made from these tissues (data not
shown), suggesting that this protein represents a second
PPO isoform. Additional western blots with the P. tri-
chocarpa parental clone, as well as an unrelated P.
tremulaÂP. nigra hybrid, also showed the induced 59
kDa band in wounded leaves and the 66 kDa PPO band
in petioles (Fig. 1B). In addition, the 66 kDa band was
present in control and wounded leaves of the P.
tremulaÂP. nigra hybrid, and small amounts of the 59
kDa band were present in control leaves of both this
and the P. trichocarpa genotype. We concluded that the
66 kDa PPO band does not represent a second parental
allele found only in hybrids, but rather represents a
second PPO isoform encoded by a second gene. The
wound-induced leaf isoform was named PPO-1, and the
constitutive isoform PPO-2. Wounding of the PPO-2
expressing tissues did not increase expression levels
discernably on western blots (data not shown).
cific PPO from hybrid poplar (Populus trichocarpaÂP.
deltoides) was cloned and proposed to be a component
of the inducible defense against leaf-eating herbivores
(Constabel et al., 2000). Additional experiments with
PPO-specific antibodies showed the presence of two
differentially expressed PPO proteins in hybrid poplar.
In order to learn more about these PPO isoforms and
their possible functions, both hybrid poplar PPOs were
partially purified and characterized.
2. Results
2.1. Hybrid poplar contains two major PPO isoforms
with distinct patterns of expression
In the course of characterizing a wound-induced PPO
cDNA, PtdPPO from Populus trichocarpaÂP. deltoides
(Constabel et al., 2000), we observed that in addition to
the inducible leaf PPO activity, hybrid poplar contained
high levels of PPO activity in petiole and stem tissue. To
further characterize this PPO activity, an antibody was
raised against recombinant PtdPPO (M. Christopher
and C.P. Constabel, unpublished data) and then used to
perform western blots with different poplar sapling tis-
sues (Fig. 1A). While there was no PPO band in control
leaves, the antibody recognized a protein migrating at
59 kDa in wounded leaves. Based on its induced
expression, size, and recognition by the PPO antibody,
this band likely represents the product of the wound-
induced PtdPPO gene. Furthermore, in PtdPPO-over-
expressing transgenic canola plants, the same 59 kDa
band was present while being absent in controls, pro-
viding direct proof that PtdPPO encodes the 59 kDa
PPO (C.P. Constabel, unpublished data).
2.2. Partial purification of PPO isoforms
The distinct patterns of expression of the two PPO
isoforms prompted us to undertake partial purification
of both proteins, in order to compare their enzymatic
properties. Wounded leaf tissue, and stems and petioles
were used as starting material for extraction of PPO-1
and PPO-2, respectively. Since Populus is known to
contain high levels of phenolic compounds, PVP-40,
Amberlite XAD-4, and PVPP were included in the
extraction buffer as phenolic adsorbants. Protease inhi-
bitor cocktails were also added, as these were found to
preserve the integrity of the PPO protein as visualized
on western blots.
Preliminary experiments suggested that while the
starting material for PPO extraction was highly enriched
for either of the PPOs, it contained small amounts of the
other isoform. Therefore, the protein extracts were
fractionated using ammonium sulfate precipitation, fol-
lowed by column chromatography for further purifica-
tion. Anion exchange chromatography on DEAE
cellulose was not successful in separating the isoforms
(data not shown). However, a hydrophobic interaction
column (Phenyl Sepharose CL-4B), was very useful for
separating the two PPO isoforms, and allowed us to
eliminate the small amount of PPO-2 from the leaf PPO-
1 preparation. Western blot analysis of the extracts
during this procedure confirmed that the final prepara-
tion contained only a single PPO protein band (Fig. 2).
The semi-purified PPO preparations were analyzed
using in situ PPO-staining in non-denaturing SDS-poly-
Fig. 1. Western blot analysis of PPO in different tissues of hybrid
poplar. Tissues were extracted and blotted as described in Experi-
mental, and analyzed using an antibody raised against recombinant
PPO-1. (A). Analysis of P. trichocarpaÂP. deltoides tissues. (B). Ana-
lysis of the P. trichocarpa parent and a P. tremulaÂP. nigra hybrid.
CL, Control leaf; WL, Wounded leaf; M, Midvein; P, Petiole; S, Stem;
R, Root.

J. Wang, C.P. Constabel / Phytochemistry 64 (2003) 115–121
117
ꢀ
were kept at À20 C, and PPO activity was measured
using 25 mM DOPA as substrate. PPO-1 activity
decreased to 43% after one week, and continued to
decrease until after 4 weeks at À20 ꢀC, only 8% of the ori-
ginal activity remained. By contrast, for PPO-2 no loss
of activity occurred in the same 4-week experimental
period. Thus, PPO-2 was more stable in storage than
PPO-1. Because of this differential stability, fresh PPO
preparations were used for all subsequent experiments.
In order to study the tolerance of the PPO isoforms to
heat treatment, both PPO preparations were incubated
at temperatures between 40–80 ꢀC. Aliquots were
removed periodically, cooled on ice, and then assayed
for PPO activity. Incubation at temperatures between
40–70 ꢀC stimulated PPO-1 activity. This transient
temperature-induced stimulation of PPO was not
observed for PPO-2, and PPO-2 activity decreased pro-
gressively during incubation at 70 ꢀC. Between 40–60 ꢀC
PPO-2 was not inactivated, but maintained 100% of its
activity for the full 1 h period. Both isoforms were
Fig. 2. Western blot analysis showing the major steps of the purifica-
tion process of both PPO isoforms. Aliquots of active fractions were
applied to SDS-PAGE gels, blotted, and analysed using the PPO
antibody. 1, crude extract; 2, 40–90% (NH4)2SO4 precipitate; 3, semi-
purified PPO from Phenyl Sepharose CL-4B column.
acrylamide gels. This technique is based on the obser-
vation that plant PPOs retain full activity in the pre-
sence of SDS; poplar PPO is extracted in a latent form
but is activated by SDS (Constabel et al., 2000). When
analyzed on non-denaturing SDS-polyacrylamide gels,
only single PPO activity bands were detected for both
PPO-1 and PPO-2 preparations, which confirmed suc-
cessful chromatographic separation of the PPO iso-
forms (Fig. 3). Interestingly, the difference in migration
between the two PPOs as seen on western blots was also
retained, with PPO-1 migrating faster than PPO-2. We
concluded that each of our semi-purified preparations
contained only one of the two PPO isoforms, and that
these were suitable for further comparisons.
ꢀ
inactivated rapidly at 80 C, again with PPO-2 being
more sensitive. Therefore, PPO-1 appeared to be more
tolerant of high temperature incubations, and was even
activated at high temperatures compared to PPO-2.
Plant PPOs generally appear to be robust enzymes;
recently, a latex PPO from Hevea brasilensis was also
shoꢀwn to be very resistant to heat treatment, and at
60 C this PPO retained 80% of the activity (Witisu-
wannakul et al., 2002).
When the pH activity profiles of the poplar PPO iso-
forms were compared, they both showed broad pH pre-
ferences (Fig. 4). Using catechol as the substrate, PPO-1
had the highest activity at pH 6, whereas the optimum
for PPO-2 was pH 7. PPO-1 had a somewhat narrower
pH range than PPO-2, which retained significant activity
even at pH 4. The only previous report of poplar PPO,
obtained from senescent P. nigra leaves, found the
highest activity at pH 7.5, but also reported a similarly
broad pH preference (Tremolieres and Bieth, 1984).
2.3. PPO stability, heat tolerance and pH optima
To investigate the stability of both PPO isoforms
during storage, aliquots of both enzyme preparations
Fig. 3. PPO activity-stained gel of the two partially purified PPO iso-
forms. Proteins were electrophoresed on 12% SDS-PAGE gels with-
out prior boiling and without ß-mercaptoethanol. Following
electrophoresis, PPO activity was visualized using 25 mM DOPA in
100 mM sodium phosphate buffer. MW indicates prestained molecular
weight marker.
Fig. 4. Effect of assay buffer pH on activity of PPO-1 and PPO-2. 100
mM citrate phosphate buffer was used from pH 4.0 to pH 6.5 and 100
mM sodium phosphate buffer was used from pH 6.5 to pH 8.0. The
pH optimum of PPO-1 and PPO-2 activity was measured using 10 mM
catechol and the TNB assay.

118
J. Wang, C.P. Constabel / Phytochemistry 64 (2003) 115–121
2.4. Substrate specificities and Km determinations
catechol, chlorogenic acid and methyl catechol. We
obtained similar values for PPO-1 (leaf PPO) as these
authors, with the exception of our calculated Km for
methyl catechol of 0.6 mM (Table 1). This difference
could be due to their use of senescent poplar leaves as a
source of PPO. In general, the Km values obtained are in
the range commonly observed in plant PPOs with these
substrates (Robert et al., 1996; Fujita et al., 1991; Bill-
aud et al., 1996). No appreciable enzyme activity was
detected with either PPO isoform using the monophenol
4-hydroxyanisol (Espın et al., 1998), suggesting that there
is no monophenolase activity associated with either PPO.
A major objective of this work was to identify poten-
tial differences in substrate preference between PPO-1
and PPO-2, since these may provide clues as to physi-
ological function. A number of substrates were tested
using the 2-nitro-5-thiobenzoic acid (TNB) assay, in
which the PPO-generated quinones react with TNB and
yield colorless Michael-type adducts (Esterbauer et al.,
1977). This spectrophotometric assay monitors the con-
sumption of TNB, and therefore allows for the direct
comparison of enzyme rates with different substrates.
PPO-1 showed the highest rates of oxidation with
methyl catechol, followed by catechin>chlorogenic
acid>DOPA>catechol>caffeic acid, in decreasing
order (data not shown). This substrate profile is generally
consistent with our previous analysis of PPO wound-
induced leaves (Constabel et al., 2000). Minor differ-
ences were observed, likely because earlier data were
obtained using crude leaf extracts. For PPO-2, the sub-
strate preferences were catecholꢁmethyl catechol> >
chlorogenic acid>caffeic acid> >catechin>DOPA
(data not shown). Methyl catechol was clearly a good
substrate for both enzymes, but surprisingly, catechol
was a much poorer substrate for PPO-1 than for PPO-2.
Conversely, catechin and DOPA were good substrates
for PPO-1 but not for PPO-2. Overall, we concluded
that PPO-2 has a greater substrate specificity than PPO-
1. As a control, for all experiments and substrates we
confirmed that the measured activity was inhibited by
the PPO inhibitor tropolone (Kahn and Andrawis,
1985).
2.5. Effects of inhibitors and activators
Tropolone is a very potent PPO inhibitor (Kahn and
Andrawis, 1985) and was the most effective inhibitor for
both PPOs, with less than 0.5% of either PPO activity
remaining at 200 mM (Table 2). Kojic acid (Chen et al.,
1991) and ferulic acid (Walker and McCallion, 1980)
were also effective inhibitors of both PPOs. Therefore,
both PPOs showed comparable sensitivity to common
PPO inhibitors. Cetyltrimethylammonium bromide
(CTAB), an inhibitor of laccase (p-diphenol oxidase),
did not have an inhibitory effect on either poplar PPO
isoform (data not shown).
To determine if PPO-1 and PPO-2 differ in their abil-
ity to be activated by SDS, both enzymes were assayed
at different SDS concentrations. PPO-1 is fully activated
with approximately 4 mM SDS, whereas PPO-2 had
already achieved maximal activity at 1 mM (Fig. 5).
Therefore, PPO-2 appears to be more sensitive to SDS.
The identical results were obtained when the experiment
was repeated using crude enzyme extracts, indicating
that this effect is not dependent on different total pro-
tein concentrations (data not shown). There was no
inhibitory effect of SDS concentrations greater than 10
mM (data not shown), although such a decrease in
activity has been reported for fungal tyrosinases (Perez-
Gilabert et al., 2001). Proteases such as trypsin are also
known to activate latent PPOs (Steffens et al., 1994),
and both poplar PPO isoforms were activated by trypsin
The pattern of substrate preference was confirmed by
Km values determined for the best PPO substrates
(Table 1). Both PPOs had the lowest Km with methyl
catechol, 0.6 mM and 0.5 mM, respectively. However,
PPO-2 had a much larger range of Km values compared
to PPO-1, consistent with earlier observations (see
above). Again, the most dramatic differences were
found for catechin, DOPA, and catechol, confirming
previous findings. Tremolieres and Bieth (1984) repor-
ted Km values for P. nigra leaf PPO of 5.0 mM for
Table 1
Michaelis–Menten constants (Km) for PPO-1 and PPO-2a
Table 2
Effects of inhibitors on PPO-1 and PPO-2 activitya
Km (mM)
PPO activity remaining (%)
Substrates
PPO-1
PPO-2
Inhibitors
PPO-1
PPO-2
4-Methyl catechol
Chlorogenic acid
Catechin
0.6
3.5
0.5
6.5
Tropolone (200 mM)
Kojic acid (1 mM)
Ferulic acid (1 mM)
None
0.43
6.1
0.25
13.5
68.8
3.7
6.6
24.3
0.9
Catechol
DOPA
38.4
100.0
21.1
79.5
100.0
a
a
Michaelis–Menten constants (Km) were determined using the TNB
assay. The Km for each substrate was calculated from a plot of 1/V vs.
1/[S] by the method of Lineweaver and Burk.
Enzyme inhibition was determined using the TNB assay with 25
mM DOPA as substrate. Inhibitors were added to final concentrations
as indicated.

J. Wang, C.P. Constabel / Phytochemistry 64 (2003) 115–121
119
(Table 1). Interestingly, PPOs from a number of plants
are not able to oxidize catechins (Ho, 1999; Witisu-
wannakul et al., 2002). We speculate that this substrate
difference between poplar PPO-1 and PPO-2 is indica-
tive of differences in function. PPO-1 is wound-induced
and active during tissue disruption due to insect feeding,
where cross-linking and amino acid alkylation by the
PPO-generated o-quinones can lead to a loss of amino
acid assimilation by the insect gut. Similarily, PPO-1
may also be involved in pathogen defense. In these
defense situations, one might expect selection for an
enzyme with a broad range of acceptable substrates,
such as PPO-1. Greater specificity, as observed with
PPO-2, might indicate a separate physiological function,
one that requires oxidation of particular phenolic sub-
strates. The in vivo substrates of hybrid poplar PPO are
as yet unknown. However, in P. tremuloides, there is
indirect evidence that the abundant phenolic glycosides
tremuloidin and salicortin are broken down in insect
guts, releasing catechol as a possible PPO substrate
(Clausen et al., 1989; Haruta et al., 2001). By contrast,
in tomato leaves the most likely in vivo PPO substrate is
chlorogenic acid (Li and Steffens, 2002).
Fig. 5. Effect of SDS on poplar PPO isoforms. SDS was added to the
assay buffer at different concentrations and PPO activity was deter-
mined immediately using a direct assay with 25 mM DOPA as
substrate.
to 70% of maximal activity (data not shown). Analysis
by SDS-PAGE and western blotting indicated that
trypsin-treatment of PPO resulted in migration on SDS-
PAGE at approximately 45 kDa. It thus appears that
poplar PPO, like the enzyme from grape, has a protease
sensitive site, and that removal of a C-terminal exten-
sion exposes the active site or activates it via a con-
formational shift (Dry and Robinson, 1992).
Although PPO has been purified from many sources,
in only a few studies have different PPO isoforms from
the same species been compared directly. Ho (1999)
characterized four PPO isoforms from orchid root, and
Witisuwannakul et al. (2002) compared two PPOs from
Hevea brasiliensis latex. In both of these studies, the
PPO isoforms compared were very similar. By contrast,
our data indicate that poplar PPO-1 and PPO-2 are
quite distinct and show clear differences in substrate
preference; together with their distinct patterns of
expression, this may be indicative of differences in func-
tion. While PPO-1 is hypothesized to be an inducible
anti-nutritive defense against folivore insects, the role of
PPO-2 is unknown. It is possible that PPO-2 has a role
in sealing of wounds, in pathogen defense, or other pro-
cesses, and experiments to study PPO expression in
poplar following stress treatments are under way. In
addition, other novel roles for PPO have recently been
discovered, for example in hydroxylation reactions dur-
ing the biosynthesis of aurones in snapdragon flower
pigments (Nakayama et al., 2000). Future work will
focus on the cell-type localization of both PPO isoforms
and on the identification of the PPO-2 encoding gene.
3. Discussion
Higher plant PPOs are often encoded by multigene
families with complex regulation, and different tissues
can express very distinct sets of PPO isoforms (Thipya-
pong et al., 1997; Thygesen et al., 1995). The sig-
nificance of this complexity is not clear, in particular as
the catalytic differences between these PPO isoforms
have rarely been investigated. In hybrid poplar there are
at least two PPO genes, and western blot analysis con-
firmed the presence of two major PPO protein isoforms
(Fig. 1). PPO-1 was detected primarily in wound-
induced leaves, whereas PPO-2 is expressed con-
stitutively in midveins, petioles, stems and roots. We
partially purified both PPO isoforms from poplar tissues
in order to characterize these enzymes biochemically
and determine differences which could provide clues as
to differences in function.
Both PPO isoforms differed in stability, heat sensitiv-
ity, pH optimum, and activation by SDS; overall, the
results are generally typical of plant PPOs, which are
stable enzymes and have broad pH optima. Although
both poplar PPOs are extracted in a latent form and
require activation, PPOs from other plant sources are
extracted in fully active form, for example from apricot
(Chevalier et al., 1999). The most striking differences
between PPO-1 and PPO-2 were observed in substrate
preference. Our results indicate that PPO-1 accepts a
broader range of substrates compared to PPO-2, as
both catechin and DOPA are good PPO-1 sustrates
4. Experimental
4.1. Plant material
Saplings of poplar hybrid H11-11 (Populus
trichocarpaÂP. deltoides) were propagated and grown in
environmental chambers with 16 h days at 21 ꢀC as
described previously (Constabel et al., 2000). P. tricho-

120
J. Wang, C.P. Constabel / Phytochemistry 64 (2003) 115–121
carpa (clone 93-968) was obtained from Dr. Carl Dou-
glas, University of British Columbia, Vancouver,
Canada, and P. tremulaÂP. nigra (clone 305-40) was
obtained from Dr. Michael Carlson, British Columbia
Ministry of Forests, Vernon, Canada. Plants were about
two months old when used. To obtain wounded leaf
samples, leaves were mechanically wounded by crushing
leaf blades at the margins with pliers. Each leaf was
wounded three times at 2 h intervals. Vascular tissue for
purification included the stem and petioles between
internode 1 and internode 10. All tissue samples were
tease inhibitor cocktails (Roche Molecular Biochem-
icals, Montreal), and was stirred on ice for 20ꢀmin. The
homogenate was centrifuged at 16,000 g at 4 C for 20
min and the supernatant used as the crude extract. Pro-
teins were precipitated sequentially from the crude
extract using 40 and 90% (NH4)2SO4. Following cen-
trifugation, the protein pellet was suspended in 10 ml of
0.5 M (NH4)2SO4 in 10 mM sodium phosphate buffer,
pH 7.0. The redissolved protein was loaded on a Phenyl
Sepharose CL-4B column (diameter 1.5 cm, length 13
cm) equilibrated with the same buffer and eluted at a
flow rate of 45 ml hÀ1. After washing away unbound
proteins with the equilibration buffer, the PPO activity
was eluted using a linear gradient (0.5–0 M) of
(NH4)2SO4 in 10 mM sodium phosphate buffer (pH
7.0), followed by distilled water. The absorbance at 280
nm was monitored with a single path UV monitor and
the PPO activity was determined on each 1.5 ml fraction
using the DOPA assay. Fractions containing PPO activity
were pooled, and the pooled fractions were dialyzed at
4 ꢀC overnight against 100 mM sodium phosphate buffers
pH 7.0. The dialyzed samples were aliquoted and kept at
À20 ꢀC for use in further experiments.
ꢀ
frozen in liquid N2 and stored at À80 C until use.
4.2. PPO assay and protein determination
Unless noted otherwise, all chemicals were purchased
from Sigma (Oakville, Ontario). For routine measure-
ments, PPO activity was measured spectrophotometrically
by following the conversion of DL-DOPA (dihydroxy-
phenylalanine) to dopaquinone at 490 nm using a Shi-
madzu Model UV-1601PC spectrophotometer. DOPA
(25 mM) was dissolved in 100 mM citrate phosphate
buffer (pH 5.0) with 0.15% SDS and 470 units/ml cata-
lase. Catechol was used as a substrate at a concentration
of 10 mM, and the change in absorbance monitored at
410 nm.The assay buffer was aerated for 5 min prior to
the assay. For some assays, a coupled TNB (2-nitro-5-
thiobenzoic acid) assay was used (Esterbauer et al.,
1977). The TNB standard reaction mixture contained
0.1 M citrate–phosphate buffer (pH 6.0), 0.05 mM
TNB, and 0.15% SDS. The reaction was initiated by the
addition of PPO enzyme, and the decrease in absor-
bance due to oxidation of TNB was followed at 412 nm.
Under the conditions used, reaction rates were linear for
at least 2 min. Approximately equal amounts of PPO-1
and PPO-2 activity were used for all comparative
experiments. Depending on the experiment, between 20
and 60 units of PPO were used per assay, where one unit
is defined as the amount of enzyme converting 1 mmol
DOPA to dopaquinone per min, calculated using a
molar extinction coefficient of 3600 MÀ1 cmÀ1 (Burton
and Kirchmann, 1997). Because the TNB assay is more
sensitive, lower amounts of enzyme activity were
required for this assay. Protein concentration was
determined with the Bradford reagent using bovine
serum albumin as a standard (Bradford, 1976). All PPO
assays were performed in triplicate and means reported.
Variation was less than 5% between assays. All experi-
ments were repeated at least three times.
4.4. Immunoblotting
SDS-PAGE and immunoblotting was carried out
using standard procedures (Harlow and Lane, 1988).
Polyclonal anti-PPO antiserum was raised against
recombinant PtdPPO protein (Christopher and Con-
stabel, unpublished data). The antiserum was diluted
1:15,000 for immunostaining of the blots. Antibody
binding was visualized using goat anti-rabbit IgG
(H+L) alkaline phosphatase conjugate in a dilution of
1:3000 (BioRad, USA) and alkaline phosphatase color
development reagents 5-bromo-4-chloro-3-indoyl phos-
phate (BCIP) and nitroblue tetrazolium chloride (NBT).
4.5. In-gel staining of PPO activity
Partially purified PPO-1 and PPO-2 (10 ml each),
unboiled and without reducing agent, were applied to
SDS-polyacrylamide gels and electrophoresed in the
presence of SDS. Following electrophoresis, PPO activ-
ity was visualized by staining the gels with 25 mM DL-
DOPA in 10 mM sodium phosphate buffer (pH 6.0)
containing 0.15% (w/v) SDS and 470 units/ml catalase.
Gels were stained until no further increase in color was
apparent, usually within 30 min. The gels were dried
between cellophane sheets and photographed.
4.3. Extraction and partial purification of PPO isoforms
Ground frozen plant material (3 g) was suspended in
30 ml of 100 mM sodium phosphate buffer (pH 7.0)
containing 1% Triton X-100, 80 mM ascorbic acid, 2%
PVP-40, 2% Amberlite XAD-4, 5% PVPP, and pro-
Acknowledgements
This work was funded by a Natural Sciences and
Engineering Research Council of Canada grant to

J. Wang, C.P. Constabel / Phytochemistry 64 (2003) 115–121
121
C.P.C. J.W. was recipient of a FS Chia Fellowship from
the University of Alberta.
Harlow, E., Lane, D., 1988. Antibodies. A Laboratory Manual. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor.
Haruta, M., Pederson, J.A., Constabel, C.P., 2001. Polyphenol
oxidase and herbivore defense in trembling aspen (Populus tremu-
loides): cDNA cloning, expression, and potential substrates. Phy-
siologia Plantarum 112, 552–558.
References
Ho, K.-K., 1999. Characterization of polyphenol oxidase from aerial
roots of an orchid, Aranda ‘Christine 130’. Plant Physiology and
Biochemistry 37, 841–848.
Billaud, C., Lecornu, D., Nicolas, J., 1996. Substrates and carboxylic
acid inhibitors of a partially purified polyphenol oxidase from gum
arabic. Journal of Agricultural and Food Chemistry 44, 1668–1675.
Burton, S.G., Kirchmann, S., 1997. Optimised detergent-based
method for extraction of a chloroplast membrane-bound enzyme:
polyphenol oxidase from tea (Camellia sinensis). Biotechnology
Techniques 11, 645–648.
Kahn, V., Andrawis, A., 1985. Inhibition of mushroom tyrosinase by
tropolone. Phytochemistry 24, 905–908.
Li, L., Steffens, J.C., 2002. Overexpression of polyphenol oxidase in
trangenic tomato plants results in enhanced bacterial disease resis-
tance. Planta 215, 239–247.
Bradford, M., 1976. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principles of pro-
tein-dye binding. Analytical Chemistry 72, 248–254.
Nakayama, T., Yonekura-Sakakibara, K., Sato, T., Kikuchi, S., Fukui,
Y., Fukuchi-Mizutani, M., Ueda, T., Nakao, M., Tanaka, Y., Kusumi,
T., Nishino, T., 2000. Aureusidin synthase: a polyphenol oxidase
homolog responsible for flower coloration. Science 290, 1163–1166.
Newman, S.M., Eannetta, N.T., Yu, H., Prince, J.P., De-Vicente,
M.C., Tanksley, S.D., Steffens, J.C., 1993. Organisation of the
tomato polyphenol oxidase gene family. Plant Molecular Biology
21, 1035–1051.
Chen, J.S., Wei, C.-I., Marshall, M., 1991. Inhibition mechanism of
kojic acid on polyphenol oxidase. Journal of Agricultural and Food
Chemistry 39, 1897–1901.
Chevalier, T., de Rigal, D., Mbeguie-A-Mbeguie, D., Gauillard, F.,
Richard-Forget, F., Fils-Lycaon, B.R., 1999. Molecular cloning and
characterization of apricot fruit polyphenol oxidase. Plant Physiol-
ogy 119, 1261–1269.
Perez-Gilabert, M., Morte, A., Honrubia, M., Garcıa-Carmona, F.,
2001. Partial purification, characterization, and histochemical loca-
lization of fully latent desert truffle (Terfezia claveryi chatin) poly-
phenol oxidase. Journal of Agricultural and Food Chemistry 49,
1922–1927.
Clausen, T.P., Reichardt, P.B., Bryant, J.P., Werner, R.A., Post, K.,
Frisby, K., 1989. Chemical model for short-term induction in
quaking aspen (Populus tremuloides) foliage against herbivores.
Journal of Chemical Ecology 15, 2335–2346.
Robert, C., Rouch, C., Richard-Forget, F., Pabion, M., Cadet, F.,
1996. Partial purification and physicochemical characterization of
Acanthophoenix rubra polyphenol oxidase. Plant Physiology and
Biochemistry 34, 369–375.
Constabel, C.P., Ryan, C.A., 1998. A survey of wound- and methyl
jasmonate-induced leaf polyphenol oxidase in crop plants. Phy-
tochemistry 47, 507–511.
Constabel, C.P., Bergey, D.R., Ryan, C.A., 1996. Polyphenol oxidase
as a component of the inducible defense response in tomato against
herbivores. In: Romeo, J.T., Saunder, J.A., Barbosa, P. (Eds.),
Phytochemical Diversity and Redundancy in Ecological Interac-
tions. Plenum Press, New York, pp. 231–252.
Steffens, J.C., Harel, E., Hunt, M.D., 1994. Polyphenol Oxidase. In:
Ellis, B.E., Kuroki, G.W., Stafford, H.A. (Eds.), Genetic Engineer-
ing of Plant Secondary Metabolism. Plenum Press, New York, pp.
275–312.
Thipyapong, P., Joel, D.M., Steffens, J.C., 1997. Differential expres-
sion and turnover of the tomato polyphenol oxidase gene family
during vegetative and reproductive development. Plant Physiology
113, 707–718.
Constabel, C.P., Bergey, D.R., Ryan, C.A., 1995. Systemin activates
synthesis of wound-inducible tomato leaf polyphenol oxidase via the
octadecanoid defense signaling pathway. Proceedings of the
National Academy of Sciences of U.S.A. 92, 407–411.
Thipyapong, P., Steffens, J.C., 1997. Tomato polyphenol oxidase.
Differential response of the polyphenol oxidase F promoter to inju-
ries and wound signals. Plant Physiology 115, 409–418.
Thygesen, P.W., Dry, I.B., Robinson, S.P., 1995. Polyphenol oxidase
in potato: a multigene family that exhibits differental expression
patterns. Plant Physiology 109, 525–531.
Constabel, C.P., Yip, L., Patton, J.J., Christopher, M.E., 2000. Poly-
phenol oxidase from hybrid poplar. Cloning and expression in
response to wounding and herbivory. Plant Physiology 124, 285–295.
Dry, I.B., Robinson, S.P., 1994. Molecular cloning and characteriza-
tion of grape berry polyphenol oxidase. Plant Molecular Biology 26,
495–502.
Tremolieres, M., Bieth, J.G., 1984. Isolation and characterization of
the polyphenoloxidase from senescent leaves of black poplar. Phy-
tochemistry 23, 501–505.
Espın, J.C., Tudela, J., Garcıa-Canovas, F., 1998. 4-Hydroxyanisole:
the most suitable monophenolic substrate for determining spectro-
photometrically the monophenolase activity of polyphenol oxidase
from fruits and vegetables. Analytical Biochemistry 259, 118–126.
Esterbauer, H., Schwarzl, E., Hayn, M., 1977. A rapid assay for cate-
chol oxidase and laccase using 2-nitro-5-thiobenzoic acid. Analytical
Biochemistry 77, 486–494.
Walker, J.R.L., McCallion, R.F., 1980. The selective inhibition of
ortho- and para-diphenol oxidases. Phytochemistry 19, 373–377.
Wititsuwannakul, D., Chareonthiphakorn, N., Pace, M., Wititsu-
wannakul, R., 2002. Polyphenol oxidase from latex of Hevea brasi-
liensis: purification and characterization. Phytochemistry 61, 115–
121.
Fujita, S., Tono, T., Kawahara, H., 1991. Purification and properties
of polyphenol oxidase in head lettuce (Lactuca sativa). Journal of
the Science of Food and Agriculture 55, 643–651.
Zhang, X., Flurkey, W.H., 1997. Phenoloxidases in Portabella mush-
rooms. Journal of Food Science 62, 97–100.