A 37-kDa peroxidase secreted from liverworts in response to chemical stress

Article abstract of DOI:10.1016/S0031-9422(00)00262-4

A peroxidase was purified from the culture medium of a suspension culture of Marchantia polymorpha (liverwort) after treatment with bornyl acetate, which acts as a chemical stress agent to the cells. The peroxidase was characterised as a glycoprotein of molecular mass 37-kDa having a pI of about 10 and an optimal pH of 6.5. The peroxidase was thermally stable at 50°C for up to 60 min. The partial amino acid sequence of the peroxidase was determined and found to be dissimilar to the amino acid sequences of other higher plant peroxidases. The oxidative polymerization of lunularin by this peroxidase was examined and the formation of a dimer, a trimer and a tetramer was demonstrated by negative ion Fast Atom Bombardment (FAB)-mass spectroscopy of the reaction products. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00262-4

Phytochemistry 55 (2000) 197±202
www.elsevier.com/locate/phytochem
A 37-kDa peroxidase secreted from liverworts in response
to chemical stress
a,
a
a
a
Toshifumi Hirata *, Yoshiyuki Ashida , Hideyuki Mori , Daisuke Yoshinaga ,
Lionel J. Goad b
aDepartment of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama,
Higashi-Hiroshima 739-8526, Japan
bSchool of Biological Sciences, Life Sciences Building, University of Liverpool, Liverpool L69 7ZB, UK
Received 15 November 1999; received in revised form 19 May 2000
Abstract
A peroxidase was puri®ed from the culture medium of a suspension culture of Marchantia polymorpha (liverwort) after treatment
with bornyl acetate, which acts as a chemical stress agent to the cells. The peroxidase was characterised as a glycoprotein of mole-
ꢀ
cular mass 37-kDa having a pI of about 10 and an optimal pH of 6.5. The peroxidase was thermally stable at 50 C for up to 60 min.
The partial amino acid sequence of the peroxidase was determined and found to be dissimilar to the amino acid sequences of other
higher plant peroxidases. The oxidative polymerization of lunularin by this peroxidase was examined and the formation of a dimer,
a trimer and a tetramer was demonstrated by negative ion Fast Atom Bombardment (FAB)-mass spectroscopy of the reaction
products. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Marchantia polymorpha; Hepaticae; Liverworts; Chemical stress; Secretion; Cationic peroxidase; Bornyl acetate
1
. Introduction
donors at the expense of hydrogen peroxide. In this
paper we describe the puri®cation of a peroxidase
secreted from cells of M. polymorpha cultured with the
chemical stress agent bornyl acetate and the oxidative
coupling of lunularin (1) with the peroxidase, in the
presence of H O .
It is well known that the attack on higher plants by
many phytopathogens can lead to induction of forma-
tion of biologically active substances (phytoalexins) and
the secretion of enzymes such as peroxidase (Chibbar et
al., 1984), phosphatase (Nishizaki et al., 1996) and
esterase (Izumi et al., 1995). Ishida et al. (1985, 1987)
reported that most of the peroxidase activity was found
in the cell walls of cultured cells of Marchantia poly-
morpha and peroxidase was released into the cultured
medium from the cell. On the other hand, we recently
found that cultured cells of M. polymorpha secreted
lunularin (1) and hydrogen peroxide when the cells were
treated with bornyl acetate as a chemical stress agent
2
2
2. Results and discussion
2.1. Puri®cation and characterization of Marchantia
polymorpha peroxidase
In order to demonstrate the secretion of peroxidase by
cells of M. polymorpha cultured under chemical stress,
the suspension cultures of M. polymorpha were treated
with bornyl acetate and peroxidase activity in the culture
medium was measured at regular time intervals. When
the cells were cultured with 1.3 mM bornyl acetate the
peroxidase activity (measured using guaiacol as the
substrate) was observed after 24 h cultivation which
corresponded to the start of exponential phase of the
cell growth. The peroxidase activity in the culture medium
(
Chen et al., 1996). The peroxidase activity in the cell
walls of plants is presumed to aid resistance against
disease and help wound healing. The primary function
of this peroxidase may be to oxidize a variety of hydrogen
* Corresponding author. Tel.: +81-824-247435; fax: +81-824-247435.
E-mail address: thirata@sci.hiroshima-u.ac.jp (T. Hirata).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00262-4

1
98
T. Hirata et al. / Phytochemistry 55 (2000) 197±202
subsequently increased during the culture period. The
presence of several peroxidase isoenzymes in the medium
of the 4-day old cultures, with or without bornyl acetate
treatment, was revealed by the analysis using sodium
dodecyl sulfate±polyacrylamide gel electrophoresis
(SDS±PAGE) and ion exchange chromatography.
The bornyl acetate-induced peroxidase was further
puri®ed using the procedures indicated in Table 1. The
Fig. 1. Puri®cation of peroxidase secreted from Marchantia polymorpha. Chromatography pro®les of butyl-Toyopearl (A), DEAE-Toyopearl (B), and Con-
A Sepharose (C) columns used for the puri®cation. Closed and open circles indicate the peroxidase activity and protein concentration pro®le, respectively.

T. Hirata et al. / Phytochemistry 55 (2000) 197±202
199
combination of the puri®cation steps resulted in a fur-
ther 69-fold puri®cation of the initial ammonium sulfate
fraction, as shown in Fig. 1 and Table 1. SDS±PAGE
analysis of the puri®ed peroxidase showed a single band
corresponding to a molecular mass of 37-kDa when
stained with guaiacol as a substrate or Coomassie Blue
R-250 (Fig. 2). Gel ®ltration analysis showed that the
peroxidase was eluted in a volume corresponding to a
molecular mass of about 35±40 kDa, indicating that the
native enzyme consisted of a monomer. Isoelectric
focusing revealed ꢁpI 10 for the peroxidase. The pre-
sence of other isoenzymes was excluded after isoelectric
focusing, because only one narrow colored band was
seen after guaiacol staining. The peroxidase showed a
Since it has been reported that most of the plant per-
oxidases have oligosaccharide chains linked to aspar-
agine (Wan and van Huystee, 1993; Yang et al., 1996),
the peroxidase was examined by both SDS±PAGE and
native PAGE with periodate/Schi€ staining to deter-
mine the presence of a sugar component. The analyses
showed that the peroxidase was indeed a glycoprotein.
The characterization of the glycoprotein was con®rmed
by the puri®cation step employing a concanavalin-A
column; the peroxidase was released with a methyl d-
glucopyranoside solution (Fig. 1C). Furthermore, when
the peroxidase bound to the concanavalin-A column
was treated with a-mannosidase (O'Donnell et al., 1992)
there was a loss of molecular mass unit of about 2-kDa,
whilst the enzyme activity was retained completely. These
observations suggested that the 37-kDa peroxidase is a
glycoprotein, and the linked oligosaccharide in the per-
oxidase is not essential for peroxidase activity.
The peroxidase was subjected to micro-sequencing
analysis without any treatment for removing the linked
saccharides, but it was found that the N-terminal amino
acid of the peroxidase was blocked. However, the analysis
of a peptide obtained by digestion of the enzyme with V8
endopeptidase gave some structural information of the
following partial amino acid sequence: Ala-Gln-Leu-
Leu-Ala-Thr-Leu-Lys-Lys-Thr-X-Pro-Asn-Gly-Gly-Gly.
This sequence shows a 50% identity to bovine gluta-
thione peroxidase (Martin-Alonso et al., 1993). However,
this sequence has no similarity with the sequences of
other glutathione peroxidases from higher plants, such
as Arabidopsis thaliana (Bartling et al., 1993; Mullineaux
et al., 1998; Sugimoto and Sakamoto, 1997), Helianthus
annuus (Roeckel-Drevet et al., 1998), Pisum sativum
ꢀ
minimal loss of activity by treatment at 50 C for 2 h,
indicating the enzyme to be very thermally stable. The
peroxidase showed an optimum pH of 6.5 in phosphate
and Tris±HCl bu€ers with guaiacol as substrate. The
peroxidase was stable at acidic pH; the activity at pH 4
was about 80% of the maximal activity, whereas the
activity at pH 8 was about 50%.
(
Mullineaux et al., 1998), Spinacia oleracea (Sugimoto
et al., 1997), Lycopersicon esculentum (Depege et al.,
998), Nicotiana sylvestris (Criqui et al., 1992), and
Zantedeschia aethiopica L. (Lino-Neto et al., 1998).
1
2
.2. Oxidation of lunularin (1) with the peroxidase
To investigate whether lunularin (1) is a substrate of
the puri®ed 37-kDa peroxidase, oxidation of 1 with the
peroxidase in the presence of hydrogen peroxide was
examined. Negative FAB-mass spectrum of the reaction
products showed the peaks at m/z 213, 425, 637, and
849, corresponding to lunularin (1) (MW 214), lunularin
Fig. 2. SDS±PAGE of the secreted proteins from Marchantia poly-
morpha. SDS±PAGE: lane 1, ammonium sulfate precipitation stained
by Coomassie Blue; lane 2, puri®ed peroxidase stained by Ag (Silver
Stain II kit, Wako). Molecular mass values are shown in the left margin.
Table 1
Puri®cation of the peroxidase from suspension culture of M. polymorpha
Step
Total actitivy
units)
Total protein
(mg)
Speci®c activity
(units/mg)
Yield
(%)
Puri®cation
(fold)
(
(
NH
4
)
2
SO
4
Fr.
77600
14300
7710
2480
72.8
22.6
1.8
32.3
196
341
(100)
(1.0)
6.1
11
Butyl Toyopearl
DEAE Toyopearl
ConA Sepharose
18.4
9.9
5.3
4130
2240
69

2
00
T. Hirata et al. / Phytochemistry 55 (2000) 197±202
considered that the dimer (2) obtained in the coupling
reaction of lunuralin (1) catalyzed by the 37-kDa per-
oxidase may be an intermediate for the biosynthesis of
the cyclic bis(bibenzyl) compounds such as marchantin
C (3) in M. polymorpha (Friederich et al., 1999a,b), the
presence of 3 could not be detected in the enzymatic
oxidation. Recently, it has been reported that the speci®c
cytochrome P-450 enzymes from M. polymorpha cata-
lyzed the biosynthesis of marchantins A and C from
phenylalanine via lunularic acid (4), but not lunularin
(
1) (Friederich et al., 1999a,b).
The puri®ed peroxidase from M. polymorpha may
allow us to shed some light on the biogenetic pathway
that leads to oligomers from phenolic compounds and
presumed cell wall constituents as a defense reaction
against chemical stress. Further work is necessary to
clarify the physiological functions of the secreted per-
oxidase in M. polymorpha.
3
. Experimental
3
.1. Materials
The cultured suspension cells of M. polymorpha
liverwort) were prepared by the culture of M. poly-
(
morpha cells in MSK-II medium with continuous shaking
for two weeks (Chen et al., 1996).
dimer (MW 426), trimer (MW 638), and tetramer (MW
50), respectively, as shown in Fig. 3. This indicates that
8
Lunularin (1; 4-[2-(3-hydroxyphenyl)ethyl]phenol)
1
0 0
[ H NMR (CDCl ) ꢀ 2.83 (4H, s 1 -H and 2 -H ), 6.64
3 2 2
lunularin (1) was converted into a dimer, a trimer and a
tetramer, and some presumed polymers were also pro-
duced. One of the dimeric compounds was con®rmed to
0
0
00
(1H, s, 2 -H), 6.65 (1H, d, J=7.8 Hz, 4 -H), 6.75 (1H, d,
J=7.6 Hz, 6 -H), 6.76 (2H, d, J=8.3 Hz, 2-H and 6-H),
7.07 (2H, d, J=8.3 Hz, 3-H and 5-H), and 7.14 (1H, t,
0
0
1
be perrottetin E (2) (Asakawa, 1995) by H NMR ana-
00
lysis of its trimethyl ether. This observation indicates
that the 37-kDa peroxidase has an activity for oxidative
phenol-coupling of lunularin (1). Asakawa et al. (1987,
J=7.8 Hz, 5 -H)] was synthesized from p-methoxybenzyl
0
chloride and triphenyl phosphine via 3,4 -dimethoxystil-
bene. Concanavalin-A Sepharose and low range mole-
cular weight protein standard were obtained from
Amersham Pharmacia Biotech. All other chemicals were
from Wako Pure Chemical Ind. and Sigma.
1990; Asakawa, 1995) have reported that cyclic bis(bi-
benzyl) compounds such as marchantin C (3; MW 424)
were isolated from M. polymorpha. Although it was
Fig. 3. Negative FAB-mass spectrum of the reaction products in the oxidation of lunularin (1) with the 37-kDa peroxidase.

T. Hirata et al. / Phytochemistry 55 (2000) 197±202
201
3
.2. Enzyme and protein assay
for peroxidase activity and with Coomassie Brilliant
Blue R-250 for total protein concentration. Periodate/
Schi€ staining was used for detection of glycosidic residues.
The molecular mass of the peroxidase was estimated by
SDS±PAGE and native PAGE with the low range
molecular weight standards, and by gel ®ltration using a
Tosoh G3000SW HPLC column.
The activity of the enzyme during the puri®cation was
monitored by spectrophotometric measurements of the
oxidation products of guaiacol (o-methoxyphenol) at
470 nm, according to the reported methods (Hendriks et
al., 1991; Miller et al., 1992). The reaction was per-
ꢀ
formed at 25 C with a reaction mixture containing
3
1 mM H O , 12.5 mM guaiacol and 200 ml of enzyme
3.5. N-Terminal amino acid sequencing
2
2
preparation in 3 ml of 3.1 mM sodium phosphate bu€er
pH 7.0). One unit of peroxidase activity is de®ned as
the amount of enzyme that catalyzed the oxidation of
(
Puri®ed peroxidase on a 12.5% SDS±PAGE gel was
degraded partially by V8 endopeptidase and the peptide
fragment on the SDS±PAGE gel was blotted onto a
polyvinylidene di¯uoride (PVDF) membrane (Immobi-
lon-PSQ, Millipore). The Coomassie stained peptide
band was excised. Sequence analysis was performed
with an online phenylthiohydantoin amino acids analy-
zer (Applied Biosystems Model 473A pulsed liquid
sequencer).
1.0 mmol of guaiacol per min.
Protein concentration was determined by the method
of Bradford, with bovine serum albumin as a standard
protein (Bradford, 1976). The protein concentration
during the enzyme puri®cation process was determined
by measurement of the absorbance at 280 nm.
3.3. Puri®cation steps
3
.6. Thermal stability
The suspension cells (1380 g) of M. polymorpha were
cultured for two weeks in 5.5 l of fresh MSK-II medium
The 37-kDa peroxidase was incubated in 2 ml of
ꢀ
ꢀ
(
Ono et al., 1979) at 25 C. Bornyl acetate (6.9 mmol)
10 mM sodium phosphate bu€er (pH 7.0) at 60 C for
20 h. An aliquot (0.2 ml) was taken out at 0.5, 1, 2, 6,
12, and 20 h, and assayed for peroxidase activity with
guaiacol as substrate.
was added to the culture and it was allowed to grow for
a further four days. The culture medium was collected by
®ltration and then fractionated with 40±80% ammonium
sulfate. The precipitated proteins were resuspended in buf-
fer A (10 mM sodium phosphate bu€er, pH 7.0) and
dialyzed against water. The solution was loaded onto a
butyl-Toyopearl 650 M column equilibrated with bu€er A
containing 1.5 M ammonium sulfate and eluted with the
ammonium sulfate gradient decreasing from 1.5 to 0 M in
bu€er A. The peroxidase was released from the column at
an ammonium sulfate concentration of about 0.5 M.
After the active fraction was dialyzed against bu€er B
3.7. Oxidation of lunularin (1) with the peroxidase
To a solution (50 ml) of the 37-kDa peroxidase (150
units) in 50 mM sodium phosphate bu€er (pH 5.0),
10 mg of lunularin (1) and 0.3 ml of 10% hydrogen
peroxide were added and the mixture was incubated at
ꢀ
37 C for 10 min. After the incubation, the reaction
mixture was extracted with EtOAc to give a crude pro-
duct (8 mg). The crude products were analyzed by
negative FAB-mass spectroscopy, which was recorded
by the negative ion mode with a glycerol matrix on a
JEOL SX-102A mass spectrometer equipped with a
Xenon FAB gun (Fig. 3). The negative FAB-mass
spectrum showed the molecular ion peaks at m/z 213,
425, 637, and 849, corresponding to lunularin (1) (MW
214), lunularin dimer (MW 426), trimer (MW 638), and
tetramer (MW 850), respectively. However, a control
experiment using a thermally denatured enzyme pre-
paration did not result in formation of any oligomeric
products.
(
5 mM Tris±HCl, pH 8.0), the fraction was loaded onto
a DEAE-Toyopearl 650 M column equilibrated with
bu€er B and eluted with a NaCl gradient from 0 to
1.5 M in bu€er B. A cationic peroxidase fraction was
eluted from the column, and then anionic peroxidases
were released at about 1 M NaCl concentration. The
cationic peroxidase fraction was further loaded onto a
concanavalin-A Sepharose column equilibrated with
bu€er C (100 mM acetate bu€er, pH 6.0 containing 1 M
NaCl, 1 mM MnCl and 1 mM MgCl ) and eluted with
2
2
a methyl d-glucopyranoside gradient from 0 to 100 mM
in bu€er C. A cationic peroxidase was eluted from the
column at 60 mM methyl d-glucopyranoside.
Furthermore, the crude products for the enzymatic
reaction were subjected to reversed-phase high perfor-
mance liquid chromatography (HPLC) on an ODS col-
umn (Inertsil ODS-2, 4.6Â250 mm, GL Sciences Inc.),
eluted with H O±CH CN±2-propanol (4:5:1, v/v/v) at a
3.4. Electrophoresis and molecular mass assay
SDS±PAGE was performed with a 12.5% gel. Iso-
2
3
electric focusing was performed with an Ampholine
PAG plate range between 3.5 and 9.5. These gels were
stained with guaiacol in sodium acetate bu€er (pH 5)
¯ow rate of 0.3 ml/min with monitoring at 210 nm.
HPLC analysis showed three peaks at retention time
(Rt) 16.4 (85%), 25.0 (11%), and 33.9 (3%) min.

2
02
T. Hirata et al. / Phytochemistry 55 (2000) 197±202
Products at Rt 16.4 and 25.0 were isolated by HPLC.
The negative FAB-mass spectra of the isolated products
at Rt 16.4 and 25.0 exhibited a molecular ion peak at m/
z 213 and 425, respectively, which indicated lunularin
acterization of tomato cDNAs encoding glutathione peroxidase-like
proteins. European Journal of Biochemistry 253, 445±451.
Friederich, S., Maier, U.H., Deus-Neumann, B., Asakawa, Y., Zenk,
M.H., 1999a. Biosynthesis of cyclic bis(bibenzyls) in Marchantia
polymorpha. Phytochemistry 50, 589±598.
(
1) and lunularin dimer (2). The product at Rt 25.0 was
Friederich, S., Rue€er, M., Asakawa, Y., Zenk, M.H., 1999b. Cyto-
chromes P-450 catalyze the formation of marchantins A and C in
Marchantia polymorpha. Phytochemistry 50, 589±598.
further methylated with diazomethane and puri®ed by
thin layer chromatography on silica gel with hexane±
EtOAc (4:1, v/v) to give a trimethyl ether: MS (EI) m/z
Hendriks, T., Wijsman, H.J., van Loon, L.C., 1991. Petunia perox-
idase a: isolation, puri®cation and characteristics. European Journal
of Biochemistry 199, 139±146.
+
1
4
68 (M ); H NMR (CDCl ) d2.82 and 2.87 (8H, ±CH ±
3
2
), 3.76 (3H, s, ±OMe), 3.77 (3H, s, ±OMe), 3.82 (3H, s, ±
OMe), and 6.7±7.2 (m, 14H, phenyl H), identical to that
of authentic perrottetin E trimethyl ether.
Ishida, A., Ono, K., Matsusaka, T., 1985. Cell wall-associated perox-
idase in cultured cells of liverwort, Marchantia polymorpha L.
changes of peroxidase level and its localization in the cell wall. Plant
Cell Reports 4, 54±57.
Ishida, A., Ookubo, K., Ono, K., 1987. Formation of hydrogen per-
oxide by NAD(P)H oxidation with isolated cell wall-associated
peroxidase from cultured liverwort cells, Marchantia polymorpha L.
Plant Cell Physiology 28, 723±726.
Acknowledgements
Izumi, S., Yamamoto, Y., Hirata, T., 1995. Secretion of an esterase
from the cultured suspension cells of Marchantia polymorpha. Phy-
tochemistry 38, 831±833.
The authors thank Professor Yoshinori Asakawa of
Tokushima Bunri University, for a gift of authentic
sample of lunularin and the H NMR spectrum of per-
1
Lino-Neto, T., Tavares, R.M., Palme, K., Pais, M.S.S., 1998. Nucleo-
tide sequence of a cDNA encoding a glutathione peroxidase
(Accession No. AF053311) from Zantedeschia aethiopica (L.).
Spreng. Plant Physiology 118, 1102.
rottetin E trimethyl ether, and to the Instrument Center
for Chemical Analysis of Hiroshima University, for
measurement of FAB-mass spectra. This work was
supported in part by a Grant-in-Aid for Scienti®c
Research (No. 09480142) from the Ministry of Education,
Science, Sports and Culture, Japan and a grant of the UK-
Japan Collaboration Research Project (1997±1998) of
Japanese Society for the Promotion of Science (JSPS).
Martin-Alonso, J.M., Ghosh, S., Coca-Prados, M., 1993. Cloning of
the bovine plasma selenium-dependent glutathione peroxidase (GP)
cDNA from the ocular ciliary epithelium: expression of the plasma
and cellular forms within the mammalian eye. Journal of Biochem-
istry 114, 284±291.
Miller, V.P., DePillis, G.D., Ferrer, J.C., Mauk, A.G., Ortiz de Mon-
tellano, P.R., 1992. Monooxygenase activity of cytochrome c per-
oxidase. Journal of Biological Chemistry 267, 8936±8942.
Mullineaux, P.M., Karpinski, S., Jimenez, A., Cleary, S.P., Robinson,
C., Creissen, G.P., 1998. Identi®cation of cDNAs encoding plastid-
targeted glutathione peroxidase. Plant Journal 13, 375±379.
Nishizaki, A., Satake, A., Chen, L., Izumi, S., Hirata, T., 1996.
Secretion of a 112 kDa phosphatase from the cultured suspension
cells of liverworts. Chemical Letters 1996, 807±808.
References
Asakawa, Y., 1995. In: Herz, W., Kirby, G.W., Moore, R.E., Steglich,
W., Tamm, C. (Eds.), Progress in the Chemistry of Organic Natural
Products, vol. 65, p. 1. Springer, Vienna.
Asakawa, Y., Tori, M., Takikawa, K., Krishnamurty, H.G., Kar,
S.K., 1987. Cyclic bis(bibenzyls) and related compounds from the
liverworts Marchantia polymorpha and Marchantia palmata. Phy-
tochemistry 26, 1811±1816.
O'Donnell, J.P., Wan, L., van Huystee, R.B., 1992. Characterization
of two forms of cationic peroxidase from cultured peanut cells.
Biochemical Cell Biology 70, 166±169.
Asakawa, Y., Tori, M., Masuya, T., Frahm, J.-P., 1990. ent-Sesqui-
terpenoids and cyclic bis(bibenzyls) from the German liverwort
Marchantia polymorpha. Phytochemistry 29, 1577±1584.
Bartling, D., Radzio, R., Steiner, U., Weiler, E.W., 1993. A glu-
tathione S-transferase with glutathione-peroxidase activity from
Arabidopsis thaliana. Molecular cloning and functional character-
ization. European Journal of Biochemistry 216, 579±586.
Bradford, M., 1976. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein-
dye binding. Analytical Biochemistry 72, 248±254.
Ono, K., Ohyama, K., Gamborg, O.L., 1979. Regeneration of the
liverwort Marchantia polymorpha L. from protoplasts isolated from
cell suspension culture. Plant Science Letters 14, 225±229.
Roeckel-Drevet, P., Gagne, G., Tourvielle de Labrouhe, D., Dufaure,
J.P., Nicolas, P., Drevet, J.R., 1998. Molecular characterization,
organ distribution and stress-mediated induction of two glutathione
peroxidase-encoding mRNAs in sun¯ower (Helianthus annuus).
Physiology Plantarum 103, 385±394.
Sugimoto, M., Sakamoto, W., 1997. Putative phospholipid hydro-
peroxide glutathione peroxidase gene from Arabidopsis thaliana
induced by oxidative stress. Genes & Genetic Systems 72, 311±
316.
Chen, L., Izumi, S., Ito, D.I., Iwaeda, T., Utsumi, R., Hirata, T., 1996.
Secretion of allelochemicals from the cultured suspension cells of
Marchantia polymorpha. Chemical Letters 1996, 205±206.
Chibbar, R.N., Cella, R., Albani, D., Huystee, R.B.V., 1984. The
growth of and peroxidase synthesis by two carrot cell lines. Journal
of Experimental Botany 35, 1846±1852.
Sugimoto, M., Furui, S., Suzuki, Y., 1997. Molecular cloning and
characterization of a cDNA encoding putative phospholipid hydro-
peroxide glutathione peroxidase from spinach. Bioscience Bio-
technology and Biochemistry 61, 1379±1381.
Criqui, M.C., Jamet, E., Parmentier, Y., Marbach, J., Durr, A., Fleck,
J., 1992. Isolation and characterization of a plant cDNA showing
homology to animal glutathione peroxidases. Plant Molecular Biol-
ogy 18, 623±627.
Wan, L., van Huystee, R.B., 1993. A study on glycosylation of catio-
nic peanut peroxidase. Biochemical & Biophysical Research Com-
munications 194, 1398±1405.
Yang, B.Y., Gray, J.S.S., Montgomery, R., 1996. The glycans of
horseradish peroxide. Carbohydrate Res. 287, 203±212.
Depege, N., Drevet, J., Boyer, N., 1998. Molecular cloning and char-