Abstract Previously we reported that purified Cell

Wall Peroxidase-Cationic (CWPO-C) from poplar callus

(Populus alba L.) oxidizes sinapyl alcohol and polymeric

substrate unlike other plant peroxidases and proposed

that this isoenzyme is a conceivable lignification specific

peroxidase. In this study, we cloned full-length cDNA of

CWPO-C and investigated the transcription of CWPO-C

gene in various organs and the localization of CWPO-C

protein in the differentiating xylem of poplar stem.Real-

time PCR analyses indicated that CWPO-C gene is

constitutively expressed in the developing xylem, leaf,

and shoot but not affected by many stress treatments.

Immunohistochemical analysis showed that CWPO-C

locates in the middle lamellae, cell corners, and second-

ary cell walls of the fiber cells during the lignification. The

intensity of the CWPO-C labeling increased gradually

from thecell wall thickeningstage tomaturestage of fiber

cells, which is very consistent with the increase of lignin

content in the developing xylem. These results strongly

support that CWPO-C is responsible for the lignification

of the secondary xylem. Interestingly, immuno-labeling

of CWPO-C was also observed inside of the ray paren-

chyma cells instead no signals were detected within the

developing fiber cells. This suggests that CWPO-C is

biosynthesized in the parenchyma cells and provided to

the middle lamellae, the cell corners, and the cell walls to

achieve lignin polymerization.

Keywords Lignification Æ Peroxidase Æ Poplar ÆImmunolocalization Æ Secondary xylem Æ Cell wall

Abbreviations

CWPO-C Cell wall peroxidase-cationic

2,4-D 2,4-Dichlorophenoxyacetic acid

Tris 2-Amino-2-hydroxymethylpropane-1,3-

diol

SDS-PAGE Sodium dodecyl sulfate

polyacrylamidegel-electrophoresis

PBS Phosphate buffered saline

Tween Poly(oxyethylene) sorbitan monolaulate

IgG Immune globulin G

PVDF Polyvinylidene difluoride

PIPES Piperazine-N,N¢-bis(2-ethanesulfonic

acid)

BSA Albumin from bovine serum

CTAB Cetyltrimethyl-aminonium bromide

cDNA Complementary DNA

PCR Polymerase chain reaction

DHP Dehydrogenative polymer

PAL Phenylalanine ammonialyase

4CL 4-Coumarate:coenzyme A ligase

CAD Cinnamyl alcohol dehydrogenase

CCoAOMT Caffeoyl-CoA 3-O-methyltransferase

CBB Coomassie brilliant blue R-250

RACE Rapid amplification of cDNA ends

S. Sasaki Æ Y. Tsutsumi (&) Æ R. KondoDepartment of Forest and Forest Products Sciences, KyushuUniversity, 6-10-1 Hakozaki, Higashiku,Fukuoka 812-8581, Japane-mail: y-tsutsu@agr.kyushu-u.ac.jp

K. BabaResearch Institute for Sustainable Humanosphere,Kyoto University, Gokasho, Uji 611-0011, Japan

T. NishidaDepartment of Forest Resource Science, ShizuokaUniversity, 836 Ohya, Shizuoka 422-8529, Japan

Plant Mol Biol (2006) 62:797–807

DOI 10.1007/s11103-006-9057-3

123

The cationic cell-wall-peroxidase having oxidation ability forpolymeric substrate participates in the late stage of lignificationof Populus alba L

Shinya Sasaki Æ Kei’ichi Baba Æ Tomoaki Nishida ÆYuji Tsutsumi Æ Ryuichiro Kondo

Received: 19 January 2006 / Accepted: 13 July 2006 / Published online: 27 September 2006� Springer Science+Business Media B.V. 2006

Introduction

Lignin biosynthesis is composed of monolignol synthesis

and polymerization. Recent works on the phenylprop-

anoid metabolism have proposed new biosynthetic

pathways; especially on the pathway for the hydroxyl-

ation and following methylation of 3- and 5-positions of

aromatic ring (Humphreys et al. 1999; Osakabe et al.

1999; Li et al. 2000; Schoch et al. 2001; Parvathi et al.

2001; Franke et al. 2002a, b). More recently, it is also

suggested that ferulic acid and sinapic acid would be

derived through the oxidation of coniferyl aldehyde and

sinapyl aldehyde (Nair et al. 2004).

On the other hand, the mechanism of lignin dehy-

drogenative polymerization is not still understood. So

far, many studies have focused on the identification

isoenzyme involved in lignin dehydrogenative poly-

merization. In these studies, peroxidase isoenzymes

were often classified into subgroups based on their

isoelectrophoretic point, anionic, neutral, and cationic.

Attempts have been made in tobacco and poplar to

identify anionic peroxidase isoenzymes specifically in-

volved in lignification (Lagrimini et al. 1987; Osakabe

et al. 1995; Christensen et al. 1998, 2001; Li et al.

2003). The syringaldazine oxidizing PXP 3-4 was ex-

pressed in the stem and in the root xylem. The

researchers discussed that the peroxidase functions in

secondary wall formation (Christensen et al. 2001). In

contrast, still other studies implicated wall bound cat-

ionic peroxidase isoenzymes as lignification peroxid-

ases (Sato et al. 1995; Blee et al. 2003).

Lagrimini et al. used transgenic plants in which the

anionic peroxidase gene was either over-expressed or

suppressed (Lagrimini et al. 1997). However, this

approach has failed to determine whether the anionic

peroxidase gene and lignification are correlated. On

the other hand, a down-regulation of the tobacco

cationic peroxidase isoenzyme TP-60 in tobacco using

an antisense strategy afforded transformants with

lignin reductions up to 40–50% of wild type plants

(Blee et al. 2003). In the case of poplar anionic

peroxidase isoenzyme (prxA3a), antisense transfor-

mants with lignin reductions up to 10–20% of control

plants were also obtained (Li et al. 2003). One of the

reasons for such a discrepancy to identify the ligni-

fication specific peroxidase isoenzyme is that plant

peroxidases have multiple molecular forms in indi-

vidual plant species. Another possible reason is that

lignification of the secondary xylem may be accom-

plished by the coordination of several peroxidase

isoenzymes.

Lignin polymerization is thought to result from the

oxidative coupling of a monolignol to a growing poly-

mer (Sarkanen 1971). Polymerization would be con-

tinued if a phenolic group on the lignin polymer is

oxidized to its radical (Hatfield and Vermerris 2001).

Therefore, a peroxidase isoenzyme that can oxidize a

lignin polymer efficiently is a candidate for lignification

peroxidases (Sasaki et al. 2004). Previously, we

reported that a peroxidase izoenzyme (Cell Wall Per-

oxidase-Cationic, CWPO-C) from poplar callus pref-

erentially uses sinapyl alcohol and syringaldazine as

substrates unlike other plant peroxidases. Further-

more, CWPO-C oxidizes polymer substrates synthetic

of lignin polymer and ferrocytochrome c (Sasaki et al.

2004; Aoyama et al. 2002; Tsutsumi et al. 1998).

Therefore, the kinetic study suggests that CWPO-C is a

specific peroxidase isoenzyme responsible for the lig-

nification of plant cell walls.

In this paper, in order to identify the role of CWPO-

C in lignification of poplar in poplar plant, we analyzed

the transcription of the CWPO-C gene in organs and in

stress response, and the localization of CWPO-C

protein in the secondary xylem in Populus alba L.

Materials and methods

Plant materials

We induced poplar (Populus alba L.) callus developed

on Murashige and Skoog basal medium supplemented

with 3% sucrose, 1.0 ppm 2,4-D, 0.5 ppm kinetin, and

0.8% agar. The callus was maintained on the medium

at 25�C in the dark. Peroxidase isoenzyme (CWPO-C)

was purified from poplar callus as described by Aoy-

ama et al. (Aoyama et al. 2002). The poplar tree

(P. alba) was grown in Kyushu University and used for

experiments of CWPO-C cloning, transcript, and

localization analysis. In the preparation of developing

xylem, bark was removed from the stem and thin flakes

(thickness was less than 1 mm) were whittled from the

surface of xylem using feather knife.

Amino acid sequence

Purified CWPO-C protein was dissolved in digestion

buffer (125 mM Tris–HCl pH 9.5, 1 mM EDTA, 0.1%

SDS), and heated at 94�C for 3 min. Lysil endprotease

was added to the solution, then the mixture was incu-

bated at 37�C for 8 h.

Purified CWPO-C protein was dissolved in diges-

tion buffer (100 mM Ammonium hydrogen carbonate

pH 7.8, 1 mM EDTA, 0.1% SDS), and heated at

94�C for 3 min. Endprotease Glu-C was added to

798 Plant Mol Biol (2006) 62:797–807

123

peroxidase solution, then the mixture was incubated

at 37�C for 28 h.

The samples treated with Lysil endprotease or

Endprotease Glu-C were separated on SDS-PAGE

and electrotransferred to PVDF membrane. The

membrane was stained with CBB reagent. The frag-

ment protein bands were cut out and subjected to an

automated Edman degradation sequencer (PPSQ-10

Shimadzu).

Cloning of CWPO-C cDNA

Total RNA was isolated from the developing xylem

of P. alba by the CTAB method (Murray and

Thompson 1980). Reverse transcriptase PCR and 3¢-RACE-PCR were performed by RNA PCR Kit

AMV Ver 2.1 (TaKaRa, Japan). The first-strand

cDNA was synthesized with an oligo (dT)-adapter

primer containing an M13 primer M4 sequence

according to the manufacturer’s protocol. For RT-

PCR, S1 (5¢-GARGCIWSICCIGGIGTRGT-3¢) and

A1 (5¢-TTYAAACGICGIAARCCIRAITTRCG-3¢)were used as sense primer and antisense primer

respectively. These primers were designed according

to the internal amino acid sequence of CWPO-C.

The PCR procedure started with 1 min of denatur-

ation at 94�C and was carried on the 45 cycles of

30 s of denaturation at 94�C, 30 s of annealing

at 55�C, and 2 min of extension at 72�C. For

3¢-RACE-PCR, M13 primer M4 was used as an

antisense primer (5¢-GTTTTCCCAGTCACACGAC-3¢).The sense primer (S2) was designed according to the

nucleotide sequence of CWPO-C (5¢-GTTTCATTG-

GGCATCCGATACATCT-3¢). The PCR procedure

started with 5 min of denaturation at 94�C and was

carried on the 40 cycles of 30 s of denaturation at 94�C,

30 s of annealing at 52�C, and 90 s of extension at

72�C. 5¢-Reverse transcriptase PCR was performed by

5¢ Full RACE CORE set (TaKaRa, Japan). The first-

strand cDNA was synthesized with a 5¢-phospholilated

antisense primer (5¢-GCAAAGAAAGATGAGTC-

AAAACT-3¢) according to the manufacturer’s

protocol. PCR amplification was accomplished with a

specific sense primer (S3; 5¢-AAGTTTGCAGCATTT-

GGTCTCAACGC-3¢) and a specific antisense primer

(A3; 5¢-GCGAGGGCTAGAATGTCTGC-3¢). The

PCR procedure started with 5 min of denaturation at

94�C and was carried on the 35 cycles of 30 s of

denaturation at 94�C, 30 s of annealing at 62�C, and

90 s of extension at 72�C.

The PCR products were subcloned into pXcmkn12

vector, and the resulting ligation product was trans-

formed into Escherichia coli strain DH5a according to

the manufacturer’s protocol. The clones were se-

quenced by a dideoxy method (Thermo Sequence

Cycle Sequence Kit, Amersham Bioscience) with a

sequencer (LIC-4000, Aloka, Japan).

Cloning of CWPO-C genomic DNA

Genome DNA was isolated from the developing xylem

of P. alba by the CTAB method (Murray and

Thompson 1980). For Genomic-PCR, S4 (5¢-AT-

GAGCCAAAAAGTAGTTTTAATG-3¢) and A4 (5¢-GAACTTTACCGCATCTTGTCG-3¢) were used as

sense primer and antisense primer respectively

(Fig. 1). The PCR procedure started with 1 min of

denaturation at 94�C and was carried on the 35 cycles

of 30 s of denaturation at 94�C, 30 s of annealing at

60�C, and 2 min of extension at 72�C.

Sequence alignment

The alignments of the peroxidase sequences have been

done using the CLASTAL W program. The region

representing the signal peptide of peroxidase homolog

was predicted with a primary structure analysis pro-

gram, SignalP (http://www.cbs.dtu.dk/services/SignalP/

). The name of each sequence corresponds to the

deduced protein of the following DNA accession

numbers: CWPO-C: AB210901, HRPC: J05552,

ATPA2: X99952, HRPA2: P80679

Stress treatment

Leaflets were cut off and subjected to wounding,

drought, and H2O2 treatments.

For wounding treatment, fully expanded leaves of

healthy poplar plants were detached and immedi-

ately cut into pieces with a razor blade. After

removing the midrib, six leaf pieces from six dif-

ferent leaves were treated, respectively. All samples

were put in petri dishes with a wet paper towel and

incubated under 25�C in darkness (Sasaki et al.

2002). For drought treatment, leaves were placed in

petri dishes with a dry paper towel and incubated

under 25�C in darkness. For H2O2 treatment, leaves

were soaked in 50 ml of aqueous 200 mM H2O2

under 25�C in darkness (Nanjo et al. 2004). All

stress treatments were conducted for 1, 2, 5, and

10 h, respectively, and then frozen in liquid nitro-

gen. For non-stress treatment, leaflets, shoots and

xylems were harvested and then immediately frozen

in liquid nitrogen.

Plant Mol Biol (2006) 62:797–807 799

123

Real-time PCR analysis

Total RNAs from xylem, shoot, leaf, and stress-treated

leaves were prepared by the CTAB method (Murray

and Thompson 1980).

Complementary DNA was synthesized in a final

volume of 20 ll that included 1 lg of total RNA

(4–5 ll of 0.2–0.3 lg/ll total RNA), 1 lM oligo-dT

18-mer primer, 10 unit Rnase inhibitor, and 10 units

of AMV Reverse Transcriptase (Takara, Japan)

according to the manufacturer’s instructions. After

reverse transcription for 60 min at 45�C, the samples

were heated for 5 min at 95�C to terminate the

reaction. Real-time PCR was performed in a final

volume of 10 ll with a Line Gene (Bio flux cor-

poration, Japan). The SYBR Premix Ex Taq kit

10 20 30 40 50 60 70 80 90 ATGAGCCAAAAAGTAGTTTTAATGTTTCTTTTGGTGGCCATGGCTGGCACCGCCACGGTGCAAGGCCAAGGCACTCGTGTTGGGTTTTAT M S Q K V V L M F L L V A M A G T A T V Q G Q G T R V G F Y

100 110 120 130 140 150 160 170 180GCAACGACGTGCCGTAGGGCTGAATCCATTGTTAGGGCAACAGTCCAGTCTCATTTCACTTCTGATTCCTCCATTGCCCCTGGGCTGCTC A T T C R R A E S I V R A T V Q S H F T S D S S I A P G L L

190 200 210 220 230 240 250 260 270AGGATGCATTTCCATGATTGCTTTGTGAATGGTTGTGATGCTTCCATCCTCATTGATGGCGCTAATACTGAAAAAACTGCGGGGCCAAAC R M H F H D C F V N G C D A S I L I D G A N T E K T A G P N

280 290 300 310 320 330 340 350 360CTTCTGTTGAGAGGATATGATGTCATTGCTGATGCCAAGACTCAGCTTGAAGCTGAGTGCCCTGGCGTTGTCTCATGTGCAGACATTCTA L L L R G Y D V I A D A K T Q L E A E C P G V V S C A D I L

370 380 390 400 410 420 430 440 450GCCCTCGCTGCTCGTGATTCTGTTGTTTTGACAAAGGGACTCACTTGGCCAGTGCCCACCGGACGGAGAGACGGTAGGGTTTCATTGGCA A L A A R D S V V L T K G L T W P V P T G R R D G R V S L A

460 470 480 490 500 510 520 530 540TCCGATACATCTAATTTGCCAGGTTTCACCGACTCCGTTGACGTGCAGAAACAGAAGTTTGCAGCATTTGGTCTCAACGCTCAAGATCTT S D T S N L P G F T D S V D V Q K Q K F A A F G L N A Q D L

550 560 570 580 590 600 610 620 630GTTACCCTTGTTGGAGGACACACCATAGGAACCACTGCTTGTCAATTCTTCAGGTACAGACTGTACAATTTCACGACAACAGGAAACGGT V T L V G G H T I G T T A C Q F F R Y R L Y N F T T T G N G

640 650 660 670 680 690 700 710 720GCGGACCCATCCATCAACCCTTCATTTGTCTCTCAACTACAGACACTCTGTCCACAGAACGGTGATGGGTCAAGGCGTATTGCTCTAGAC A D P S I N P S F V S Q L Q T L C P Q N G D G S R R I A L D

730 740 750 760 770 780 790 800 810ACCGGTAGCCAAAATAGTTTTGACTCATCTTTCTTTGCAAATTTGAGAAGTGGTCAAGGAATACTTGAATCTGATCAAAAGTTATGGACT T G S Q N S F D S S F F A N L R S G Q G I L E S D Q K L W T

820 830 840 850 860 870 880 890 900GATGCAACCACAAGAACTTTTGTCCAGCGCTTCCTTGGTGTCAGAGGCCTGGCTGGGCTCACGTTTGGTGTGGAGTTTGGCAGGTCCATG D A T T R T F V Q R F L G V R G L A G L T F G V E F G R S M

910 920 930 940 950 960 970 980 990GTCAAGATGAGTAACATCGGTGTGAAAACCGGCACTACTGGTGAAATTCGAAGAGTGTGTTCTGCTATAAATTGAACATTCTTTTCGATT V K M S N I G V K T G T T G E I R R V C S A I N *

1000 1010 1020 1030 1040 1050 1060 1070 1080TGTCTTAATGTATCGTTTTTTTAATCTTAATCTTTCCATTTTTCTGTTTACCCGACAAGATGCGGTAAAGTTCATAATATTATGTATCCT

1090 1100 1110 1120 1130 1140 1150 1160 1170ATTGCAAGAATGGGACACAAATCAAAAGGTCAAGAATTAATTGGACAAGGTTGATTGCTTTTGAACTCTCGTGTAATTGTAAAGCTTATC

1180 1190 1200 1210 1220ATTTGAACCAATAGATTGGGTCATTACATTTGTATTAGTAAAAAAAAAAAAAAAA

S4

a

b

c

S1

A1S2

A3

S3

A4

*

*

*

**

* *

*

Fig. 1 Complementary DNA and deduced amino acid se-quences of Populus alba L. CWPO-C. The amino acidsequences of the three fragments after lisylensopeptitase orendprotease Glu-C digestion are shown by underline, a, b, andc. All PCR primers are indicated by allows (refer to Material

and methods for detail). Putative signal peptide (22 aminoacids) is enclosed with the light-blue square. Two intron sitesare enclosed with the red square. Distal active site andproximal active site are shown by double-underline. Eight Cysresidues are shown by asterisks

800 Plant Mol Biol (2006) 62:797–807

123

(Takara, Japan) was used according to the manu-

facturer’s instructions with a final concentration of

0.2 lM for each primer. PCR amplification was

performed as follows: (i) an initial denaturation at

95�C for 1 min, (ii) 45 cycles, with 1 cycle consisting

of denaturation 95�C for 15 s, annealing at 60�C at

15 s, and elongation at 72�C for 30 s. Amplicon

specifity was verified by melting-curve analysis con-

ducted at 65–95�C with stepwise fluorescence

acquisition and by 2% agarose gel electrophoresis

staining with ethidium bromide. No fluorescence was

detected from real-time PCR amplification without a

template. Each primer sequence of CWPO-C

(Accession number: AB210901) is 5¢-ACACACCT-

AGGAACCACTG-3¢ (forward) and 5¢-AATACGC-

CTTGACCCATC-3¢ (reverse), respectively. Each

primer sequence of Populus alba Peroxidase 1 (PaPO1)

(Accession number: AB206042) as a positive control is

5¢-CCAAGGCCTGCTTCAGACG-3¢ (forward) and

5¢-TGCTATCTGGCCCAGCCAAG-3¢ (reverse).

Actin is used as a reference gene (Accession number:

AB025795). Each primer sequence is 5¢-GCCCAGAG

GTCCTCTTCCAA-3¢ (forward) and 5¢-GGGGCTA

GTGCTGAGATTT- CCTTGC-3¢ (reverse), respec-

tively. The ratio of gene specific expression was

defined as relative expression to the actin expression.

The data was accumulated for three individual

runs ± SD.

Preparation of anti-CWPO-C antiserum

Purified CWPO-C from poplar callus in PBS

(0.3 mg/500 ll) was thoroughly emulsified with an

equal volume of Freund’s adjuvant. Freund’s com-

plete adjuvant was used for the first injection into a

rat, and Freund’s incomplete adjuvant was used for

subsequent injections (three times). Each immuni-

zation was given in a total volume of 1 ml. A blood

sample was collected after the 3rd subsequent

injection.

Western-blot analysis for developing xylem

of P. alba

The homogenized developing xylem was extracted

three times with 50 mM Tris–HCl buffer (pH 7.5)

to ensure complete extraction of the soluble pro-

teins fraction (SP). The cell wall residue was incu-

bated with the same buffer plus 0.6 M NaCl to

extract the proteins ionically bound to the cell walls

(ionically bound proteins fraction, IBP). An equal

amount of protein of each fraction was separated by

SDS-PAGE. The separated proteins were trans-

ferred to a PVDF membrane. Blots were blocked

with PBS containing 5% (w/v) nonfat milk and

0.05% (w/v) Tween-20 for 1 h at room temperature.

After washing three times with PBS containing

0.05% (w/v) Tween-20, blots were incubated over-

night at room temperature with a rat polyclonal

antiserum against CWPO-C (1:1,000 dilution) in

above PBS. After washing three times, the blots

were incubated with horseradish peroxidase conju-

gated anti-rat goat IgG (1:10,000 dilution, Serotec

Ltd.) in above PBS for 1 h at room temperature,

then washed three times as described above. The

immunoreactive protein was visualized by 1 mM 3,3-

diaminobenzidine and 3 mM hydrogen peroxide in

50 mM Tris–HCl buffer (pH 7.5). Blots were incu-

bated with the solution for a few minutes at room

temperature.

Immunolocalization, hisrochemical lignin analysis,

and microscopy

Fresh hand-cut sections (5 mm thickness) from the

stem of one-year-old P. alba were immediately

submerged in freshly prepared 5% paraformalde-

hyde (w/v) in 20 mM PIPES buffer (pH 7.5)

and incubated at 4�C overnight. After rinsing with

phosphate buffer, they were dehydrated through

ethanol series, then embedded in LR White Resin

(hard, London Resin Company). Sections (2 lm

thickness) were prepared by a microtome with a

grass knife and mounted on slides. The sections

were firstly blocked with blocking buffer [1% BSA

(w/v) and 0.05% (w/v) Tween-20 in 10 mM Sodium

Phosphate 150 mM NaCl, pH 7.2 (PBS)] for 1 h at

room temperature. After washing five times with

PBS containing 0.05% (w/v) Tween-20, sections

were then incubated with CWPO-C antiserum

diluted 1:10–1:50 in the blocking buffer for 1 h at

room temperature. After washing five times as

described above, the sections were incubated with

Anti-rat goat IgG (H + L) Gold 5 nm (dilution 1:40,

British BioCell International, Ltd.) in the blocking

buffer for 1 h at room temperature. After washing

five times, the 5 nm gold particles were further

enhanced using a silver enhancing kit (Amersham

Bioscience). As a negative control, the same

sequential treatments of the sections, without the

CWPO-C antiserum, was omitted. The sections then

were mounted with 50% glycerol and observed with

a microscope, and images were taken using a digital

photo camera.

Plant Mol Biol (2006) 62:797–807 801

123

Results

Cloning of CWPO-C cDNA

To investigate the unique characteristics of CWPO-C,

we have attempted to obtain a CWPO-C cDNA

sequence. The digested peptide fragments of CWPO-C

by Lysil endprotease or Endprotease Glu-C were

subjected to amino acid sequencing. Subsequently,

three partial amino acid sequences [a: SIVRTAVQ, b:

AE(S or X)PGVVS, c: FAAFGLNAQDLV] were

determined (Fig. 1). A sense primer (S1) and an anti-

sense primer (A1) were designed based on the partial

amino acid sequences b and c, respectively, and used

for reverse transcriptase (RT)-PCR. A generated a

213 bp cDNA clone contained a plant peroxidase

consensus sequence (helix D region). To obtain the

full-length cDNA sequence, we used the 3¢-RACE-

PCR procedure, with a specific primer S2 (Fig. 1) and

M13 primer M4. The resulting 790 bp cDNA exhibited

90 bp overlap with the above mentioned 213 bp

sequence without any discrepancy. With the 5¢-RACE-

PCR procedure, the resulting 595 bp exhibited 248 bp

overlap with the above obtained 907 bp without any

sequence discrepancy. Finally, we obtained the

1,225 bp of a full-length CWPO-C cDNA clone.

The nucleotide sequence of CWPO-C predicted a

324-amino acid sequence containing a putative signal

peptide (22 amino acids) at the N-terminus (Fig. 1).

The Mw and pI of the deduced amino acid sequence of

the mature protein were calculated as to be 32.3 kDa

and 8.06, respectively. Previously, the molecular weight

of the purified CWPO-C protein was determined to be

approximately 32 kDa by SDS-PAGE (Tsutsumi et al.

1998) and Western-blot analysis (Fig. 4). Thus the

observed molecular weight is consistent with the cal-

culated mass. Additionally, we produced recombinant

CWPO-C protein with pQE-30 expression system

(Qiagen). The synthesized His-tag protein was purified

by Ni-NTA column, then subjected to Western-blot

analysis. The anti-CWPO-C antibody recognized a

single protein band with molecular mass of ca. 32 kDa

(data not shown). The result clearly indicated that

obtained cDNA encodes the CWPO-C protein. Cur-

rently the recombinant CWPO-C was expressed as an

inclusion body and the enzyme activity of the protein

has not been obtained yet.

The PSORT program (http://www.psort.nibb.ac.jp/)

predicts that the CWPO-C protein will localize outside of

the cell. CWPO-C conserved eight Cys residues for

putative disulfide bridge, as well as other plant peroxid-

ases (Fig. 1). The deduced amino acid sequence of

CWPO-C 1 -------MSQKVVLMFLLVAMAGTATVQGQGTRVGFYATTCRRAESIVRATVQSHFTSDS 53ATPA2 1 MAVTNLPTCDGLFIISLIVIVSSIFGTSSAQLNATFYSGTCPNASAIVRSTIQQALQSDT 60HRPC 1 -----------------------------MQLTPTFYDNSCPNVSNIVRDTIVNELRSDP 31TP-60 1 -----MGFKVFFFFAILFFSAVSAFAEDNSGLVMDYYKDSCPQAEDIIREQVKLLYKRHK 55

. . .* *. . *.* . ..

CWPO-C 53 SIAPGLLRMHFHDCFVNGCDASILIDGANT---EKTAGPNLL-LRGYDVIADAKTQLEAE 109ATPA2 60 RIGASLIRLHFHDCFVNGCDASILLDDTGSIQSEKNAGPNVNSARGFNVVDNIKTALENA 120HRPC 31 RIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFPVIDRMKAAVESA 91TP-60 55 NTAFSWLRNIFHDCFVESCDASLLLDSTRRMLSEKETDRSFG-MRNFRYIETIKEAVERE 114

.. . .* .******..****.*.* . ** . . *.. .. * . *

CWPO-C 109 CPGVVSCADILALAARDSVVLTKGLTWPVPTGRRDG-RVSLASDTSNLPGFTDSVDVQKQ 168ATPA2 120 CPGVVSCSDVLALASEASVSLAGGPSWTVLLGRRDSLTANLAGANSSIPSPIESLSNITF 180HRPC 91 CPRTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKD 151TP-60 114 CPGVVSCADILVLSGRDGIVALGGPYVPLKTGRRDGRKSRADILEQHLPDHNESMSVVLE 174

**..***.* * .. .. . .*. . . **** . .* .

CWPO-C 168 KFAAFGLN-AQDLVTLVGGHTIGTTACQFFRYRLYNFTTTGNGADPSINPSFVSQLQTLC 227ATPA2 180 KFSAVGLN-TNDLVALSGAHTFGRARCGVFNNRLFNFSGTGN-PDPTLNSTLLSTLQQLC 238HRPC 151 SFRNVGLNRSSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGL-PDPTLNTTYLQTLRGLC 210TP-60 174 RFANVGIN-APGVVALLGAHSVGRTHCVKLVHRLY----PEV--DPQLNPDHVPHMLKKC 227

* .*.* ..*.* * *. * * **... .. ** .* . .*

CWPO-C 227 PQNGDGSRRIAL---DTGSQNSFDSSFFANLRSGQGILESDQKLWTDATTRTFVQRFLGV 284ATPA2 238 PQNGSASTITNL---DLSTPDAFDNNYFANLQSNDGLLQSDQELFSTTGSS-TIAIVTSF 294HRPC 210 PLNGNLSALVDF---DLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPNATDTIPLVRSF 267TP-60 227 PDPIPDPKAVQYVRNDRGTPMKLDNNYYRNILENKGLMLVDHQLATDKRTK---PYVKKM 284

* .. . * .. .*. . *. *. .*. * .

CWPO-C 284 RGLAGLTFGVEFGRSMVKMSNIGVKTGTTGEIRRVCSAIN--- 324ATPA2 294 ASNQTLFFQA-FAQSMINMGNISPLTGSNGEIRLDCKKVNGS- 335HRPC 267 ANSTQTFFNA-FVEAMDRMGNITPLTGTQGQIRLNCRVVNSNS 309TP-60 284 AKSQDYFFKE-FARAITILTENNPLTGTKGEIRKQCNLANKLH 326

. .* * . . .. ..**. *.** * *

Fig. 2 Alignment of plantperoxidases includingATPA2, HRPA2, HRPC, andCWPO-C. Red lettersindicate active site residues.Green letters indicate thedistal Ca binding site. Purpleletters indicate the proximalCa binding site

802 Plant Mol Biol (2006) 62:797–807

123

CWPO-C showed similarities to the Gossypium hirsutum

cationic peroxidase [AAL93154] (83%), Capsicum ann-

uum cationic peroxidase [AAL35364] (77%) and Nico-

tiana tabacum cationic peroxidase [BAA82307] (72%).

Homology with Arabidopsis thaliana ATP A2 [X99952]

showed fewer similarities (45%). The alignment analysis

showed that the catalytic site of CWPO-C, including Arg-

39, His-43, Pro-135, and His-165 is identical to those of

ATP A2, HRP C, and other plant peroxidases (Fig. 2).

Real-time PCR analysis of CWPO-C transcript

We investigated the CWPO-C transcript in P. alba

with Real-time PCR analysis. CWPO-C was tran-

scribed in the developing xylem, shoot, and leaf

(Fig. 3). The difference was not seen in the amount of

expression of the gene (Fig. 3). CWPO-C transcript

was not changed by the wound stress, dry stress, and

hydrogen peroxide stress treatments (Fig. 3). On the

other hand, PaPO1 as a positive control, was clearly

was up-regulated by the wound stress treatment.

These results indicated that the CWPO-C gene is

constitutively expressed gene in various organs, but

not affected by many stresses.

Western blot analysis with specific antiserum for

poplar developing xylem

We tested the specificity of CWPO-C antiserum by

Western blot analysis toward the SP and the cell wall

bound proteins fraction (IBP) prepared from the

developing xylem of a poplar plant (Fig. 4). The anti-

CWPO-C antibody recognized a single protein band

with molecular mass of ca. 32 kDa only in cell wall

bound fractions, and the observed molecular weight is

identical to purified CWPO-C.

Localization analysis of CWPO-C protein on the

secondary xylem in P. alba

The immuno-labeling of CWPO-C was mainly ob-

served on the area surrounding the fiber cells in the

secondary wall formation (cell wall thickened and

matured stage) (Fig. 5A). The labeling was also ob-

served on parenchyma cells in the latter of secondary

wall formation (Figs. 5A, 6A). However the labeling

was less prevalent in the early stage of secondary

wall formation and not observed in the cambium

(Fig. 5A). In the magnified views, signals were

0

1

2

3

4

5

6

xyle

msh

oot

leaf 1h 2h 5h 10h 1h 2h 5h 10h 1h 2h 5h 10h

leaf 1h 2h 5h 10h

Per

oxid

ase/

Act

in

Wound Dry H2O2 Wound

CWPO-C PaPO1

Fig. 3 Relative abundance of CWPO-C and PaPO1 genestranscript in Populus alba L. determined by the Real-timePCR analysis. The ratio of CWPO-C or PaPO1 specificexpression to actin expression was defined as relative expressionand is the mean of three individual reactions ± SD. Legends:Dry: dry stress, H2O2: hydrogen peroxide stress, Wound: woundstress

CWPO-C SP IBP

xylem

94

67

43

30

20

(KDa)

Fig. 4 Western-blot analysis of CWPO-C protein in poplardeveloping xylem. The aliquot of each sample was resolved bySDS-PAGE (10% acrylamide gel). CWPO-C; purified CWPO-C(0.2 lg) from poplar callus, SP; Soluble proteins fraction (20 lg)from poplar developing xylem, IBP; Ionically bound proteinsfraction (20 lg) from the poplar

Plant Mol Biol (2006) 62:797–807 803

123

stronger on the intercellular layers and the cell cor-

ners (Fig. 6C). Also, signals were not observed in the

fiber cells but in the parenchyma cells (Fig. 6A). For

the control sample, the antibody signal is not seen

without CWPO-C antiserum treatment (Figs. 5B,

6B).

We observed immuno-localization of the CWPO-C

protein in the cross section of the poplar (Populus alba L.)

developing xylem (Fig. 7A). We also observed the

same section with the UV-fluorescence microscopic

technique that can estimate the lignin deposition pro-

cess in the secondary xylem (Fig. 7B). The blue fluo-

rescence is mainly due to lignin, and its intensity

depends on the amount of lignin. Thus the synchronous

observation of both pictures allows us to determine the

relation between lignification and CWPO-C. In the

cambium, the fluorescence was very weak or almost

invisible. The blue fluorescence became gradually

stronger along with the radial distance from the cam-

bium to the center of the stem (from left to right in

Fig. 7B). The blue fluorescence gradation typifies that

lignification is processing in the developing xylem. As

seen in the comparison of panels A and B in Fig. 7, it is

apparent that an increase of the intensities of the

CWPO-C labeling and lignin-derived fluorescence are

spatially temporally synchronized. This strongly sup-

ports that CWPO-C is associated with lignification.

Discussion

Angiosperm lignin is composed of two cinnamyl alco-

hols: coniferyl and sinapyl alcohols. In typical angio-

sperm woody plants, lignin in the secondary cell walls

of the fiber cells is mainly composed of syringyl units,

whereas lignin in middle lamellae and in vessel cell

Fig. 5 Immunohistochemicalanalysis of CWPO-C proteinin the secondary xylem ofone-year old poplar stem.Legend: (A) secondary wallformation stage labeled withCWPO-C antiserum, (B)without CWPO-C antiserum,Ca; cambium, Ct; cell wallthickening stage, Ma; maturestage, PC; parenchyma cells,FC; fiber cells, V; vessels,scale bar = 50 lm

Fig. 6 Magnified view of theimmunohistochemicalanalysis of CWPO-C proteinin the secondary xylem in 1-year old poplar stem. Thesignals were observed in theparenchyma cells (panel A).The signals were mainlyobserved on the middlelamella and the cell corners(panel C). Panel B; a serialsection without CWPO-Cantiserum. PC; parenchymacells, FC; fiber cells, V;vessels, CC; cell corners, SW;secondary walls, ML; middlelamellae, scale bar = 50 lm

804 Plant Mol Biol (2006) 62:797–807

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walls consists primarily of guaiacyl units (Saka and

Goring 1985). Therefore, lignification requires the

peroxidase isoenzyme that oxidizes both the coniferyl

and sinapyl alcohols. However, sinapyl alcohol is not a

good substrate for general plant peroxidases because

their substrate channels are too narrow for sinapyl

alcohol which possesses the additional methoxy group

at the 5-position of coniferyl alcohol (Ostergaard et al.

2000; Nielsen et al. 2001). In addition to the above fact,

lignin polymerization is thought to derive from the

oxidative coupling of a monolignol and the growing

polymer (Sarkanen 1971). Hence the peroxidase

responsible for the lignification should oxidize lignin

polymers to generate their radicals. In this regard, we

previously reported that a peroxidase isoenzyme,

CWPO-C, oxidizes both monolignols and synthetic

high molecular weight lignin. Its enzymatic character-

istics strongly support that CWPO-C is a lignification

specific peroxidase (Sasaki et al. 2004; Aoyama et al.

2002).

In order to investigate the relationship of CWPO-C

and lignification, in this study we analyzed the tran-

script of CWPO-C in poplar. Real-time PCR analysis

indicated that CWPO-C transcript was not up-regu-

lated by the examined all stress treatments, and it was

expressed in all examined tissues. These results indi-

cate that the CWPO-C gene is not a stress response

gene but a constitutively expressed gene in developing

xylem, leaf, and shoot.

Localization analysis indicated that the CWPO-C

protein is mainly located at the cell corners and in the

intercellular layers of the fiber cell during the second-

ary wall formation. The increase of immuno-labeling

was well consistent with the increase of lignin content

in the secondary xylem. Furthermore, the lignin con-

tents of the intercellular layers and cell corners are

much higher than the cell walls (Fergus and Goring

1970). Thus, the high intensity of the CWPO-C

immuno-labeling in the intercellular layers and cell

corners is in good agreement with the lignin localiza-

tions. These results also support that CWPO-C is

responsible for the lignification.

Takeuchi et al. investigated the immunolocalization

of anionic peroxiase prxA3a (Takeuchi et al. 2001).

PrxA3a was isolated from the Populus kitakaminsis

and was expressed in younger part of stem and shoot

tip (Osakabe et al. 1995). The prxA3a was found to be

localized in the developing fibers and vessels during

secondary wall formation. Under the electron micro-

scope, the labeling is observed on the plasma mem-

brane of developing vessels and fibers (Takeuchi et al.

2001; Takabe et al. 2001). On the other hand, the lignin

polymer oxidizing and cationic peroxidase, CWPO-C,

is localized on the middle lamella and the cell corners.

The localization of CWPO-C is similar to a cationic

peroxidase in the French bean (Smith et al. 1994).

Down regulation of another cationic peroxidase TP-60

crucially reduced the lignin content in tobacco plant

(Blee et al. 2003). Thus, lignin polymerization would

be achieved by the coordination of several peroxidase

isoenzymes, which are spatially and functionally dif-

ferent.

Immunohistochemical observations in this study

showed that the CWPO-C labeling was not observed

in the fiber cells. Thus, the fiber cells do not syn-

thesize the CWPO-C protein and supply it to the

cell walls, middle lamellae, and cell corners. Alter-

natively, immuno-detection revealed that the

CWPO-C protein locates inside of the parenchyma

cells. Thus the preferable explanation for the above

observation: lignin content and amount of CWPO-C

protein in the cell corners, middle lamellae, and

secondary cell walls keep increasing in the second-

ary wall formation, is that the ray parenchyma cells

provide the CWPO-C protein and monolignols to

these regions to complete lignification of the sec-

ondary xylem. It was reported that the localization

profile of other lignin biosynthetic enzymes was

similar to that of CWPO-C. Hosokawa et al. pos-

tulated that monolignols are supplied from not only

the tracheary elements themselves but also the sur-

rounding xylem parenchyma like cells (Hosokawa

et al. 2001). Feuillet et al. made observations of

CAD2 promoter activity (Feuillet et al. 1995). The

activity was not found in lignifying cells (vessels and

fibers) but was instead active in the adjacent

parenchyma cells (Feuillet et al. 1995). In the liter-

ature, some reports have described that the enzymes

involved in monolignol biosynthesis, PAL, 4CL,

CAD, and CCoAOMT, are located in the

Fig. 7 Immunohistochemical analysis of CWPO-C protein andultraviolet-fluorescence microscopic observation on the second-ary xylem in 1-year-old poplar. (A) Immunohistochemicalanalysis of CWPO-C protein on the secondary xylem, (B)ultraviolet-fluorescence microscopic observation of secondaryxylem. The blue fluorescence depends on the amount of lignin inthe cell walls (panel B). Legend: Ca: cambium zone, Ct: cell wallthickening stage, Ma; mature stage, FC; fiber cells, V; vessels,scale bar = 50 lm

Plant Mol Biol (2006) 62:797–807 805

123

parenchyma cells (Bevan et al. 1989; Feuillet et al.

1995; Hawkins et al. 1997; Zhong et al. 2000). Some

of them suggested that lignification occurred through

a process of ‘‘cell cooperation.’’ In this process, the

monolignols necessary for lignification are not pro-

duced in the lignifiying fiber or vessel cells, but in

associated parenchyma cells, and then exported to

the cells undergoing lignification (Feuillet et al.

1995). Therefore, it is also feasible that lignification

of the fiber cells of the secondary xylem in P. alba

progresses by the supply of CWPO-C from sur-

rounding parenchyma cells in vivo. The details of

the diffusion mechanism of CWPO-C protein to the

cell corners and intercellular layers are unknown,

however, the localization of a cationic peroxidase in

these regions has already been reported (Smith

et al. 1994). Further analysis of extracellular trans-

portation of CWPO-C and monolignols in vivo

would bring us understanding of lignin biosynthesis.

Acknowledgements We are grateful to Dr. Kazutoshi Sayamaand Mr. Naoto Ogawa, Shizuoka University for technical adviceon the preparation of CWPO-C antiserum. We also thank Dr.Mitsuhiro Furuse and Dr. Hironori Ando, Kyushu University fortechnical support regarding Real-time PCR analysis using LineGene. This work was supported by a grant-in-aid for scientificresearch fund from the Ministry of Education, Science andCulture of Japan (15380121).

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