全 文 ：Received 20 Aug. 2003 Accepted 25 Sept. 2003
Supported by the State Key Basic Research and Development Plan of China (G199016002) and the National Natural Science Foundation of
China (30170056, 30070614).
* Author for correspondence. Tel: +86 (0)10 62757016; Fax: +86 (0)10 62751526; E-mail: .
http://www.chineseplantscience.com
Changes of Soluble Protein, Peroxidase Activity and Distribution
During Regeneration After Girdling in Eucommia ulmoides
HOU Hong-Wei1, Kalima-N’Koma MWANGE1, 2, WANG Ya-Qing1 , CUI Ke-Ming1*
(1. College of Life Sciences, Peking University, Beijing 100871, China;
2. High Commission of Atomic Energy/Regional Center of Nuclear Research, B. P. 868, Kinshasa 11, D. R. Congo)
Abstract: Peroxidases are known to play important roles in plant wound healing. Biochemical analysis
and histochemical localization techniques were used to assess changes and distribution of peroxidases in
the recovering bark after girdling in Eucommia ulmoides Oliv. Between 4 and 21 days after girdling (DAG),
peroxidases activity in the girdled trees significantly increased by 30-40 times over that in ungirdled trees.
During the whole bark recovery process (from 0 to 63 DAG), the peroxidase signal was not found in the
tissue regions subjected to intense cell division activity (regenerating cambial zone and phellogen). However,
high peroxidase activity was detected in the callus, cortex-like, mature phloem and xylem. Interestingly, it
was shown that, in maturing xylem and phloem cells, there was respectively an inward and outward
peroxidase activity gradient on both sides of the cambium zone. An isoelectric-focusing electrophoresis of
the extracted protein displayed two isozyme bands of peroxidase: PODⅠand PODⅡ. PODⅠwas only
detected in the xylem fraction and could play a role in xylem differentiation. PODⅡ was only identified in
the recovering bark portion and could be more engaged in bark regeneration process. A relationship
between IAA and peroxidase is also discussed.
Key words: peroxidase; cambium; Eucommia ulmoides ; girdling; regeneration; isozyme
Many woody and herbaceous dicotyls species can re-
generate new bark after girdling, provided that the peeling
treatment is suitably made and enough immature xylem cells
are left on the exposed stem (Li et al., 1981; 1982; 1983;
1988; Li and Cui, 1984; 1985; 1988; Zhao et al., 1984; Cui
and Li, 1986; Lu et al., 1987; Li and Xu, 1988; Liu and Li,
1990; Cui, 1992). As found out by Li et al. (1981; 1982), cells
in the inner layer of the immature xylem usually dedifferen-
tiate to produce a new cambium. Since cell dedifferentiation
and redifferentiation are important in plant morphological
reconstitution, many investigations have been fundamen-
tally conducted on anatomical and physiological changes
in plant tissues regeneration. Nowadays, biochemical
changes in this process are more and more attracting the
scientists’ attention.
Plant peroxidase (EC 1.11.1.7) is a family of enzymes. In
plant kingdom, they have been implicated in the lignifica-
tion of secondary cell walls by catalyzing the polymeriza-
tion of phenolics into lignin and in the formation of cova-
lent cross-links between ferulylated polysaccharides, hy-
droxyproline-rich glycoproteins and lignin (Fry, 1988). Per-
oxidases have also been involved in plant wound healing
(Roberts et al., 1988), periderm suberization (Espelie et al.,
1986) and auxin catabolism (Hinnman and Lang, 1965). It
was reported that the peroxidase isozymogram, changing in
different tissues and at different developmental stages of
plants, is also correlated with organogenesis and tissue dif-
ferentiation (Barcelo and Munoz, 1992; Cui et al., 1993).
Changes of peroxidase isozymogram normally occur earlier
than the eyeable morphological changes, which could re-
flect the development status. Thus, peroxidase is widely
considered as a suitable natural marker system for investi-
gating plants growth and development (Scandalios, 1974;
Robert and Aloni, 1988; Luo et al., 1999).
Even though valuable researches on peroxidases have
sufficiently been conducted on wound healing in many plant
species, some important questions on their specific action
still remain unanswered. What are, for instance, the spatial
and temporal patterns of peroxidases distribution in ringed
plant tissues at different stages of bark reconstitution?
Where does the peroxidases (or their isozymes) activity take
place in these tissues? What is the correlation between the
morphological change and the localization pattern of these
peroxidases?
The objective of this study was to establish the relation-
ship between peroxidases activity and tissue regeneration
occurring during bark reconstitution process in Eucommia
ulmoides, subsequent to a relatively severe girdling.
Acta Botanica Sinica
植 物 学 报 2004, 46 (2): 216-223
217
HOU Hong-Wei et al.: Changes of Soluble Protein, Peroxidase Activity and Distribution During Regeneration After Girdling in
Eucommia ulmoides
1 Materials and Methods
1.1 Plant materials and sampling
Twenty Eucommia ulmoides Oliv. trees were selected
at the tree plantation of Shunyi District, Beijing (China).
The trees were six-year-old, 5 to 6 m tall, with a mean diam-
eter of 12 cm measured at 1.3 m above the ground level.
Trees were girdled in early summer according to Li et al.
(1981) and wrapped in transparent plastic sheet to avoid
desiccation.
Samples for peroxidase and protein extraction were taken
at 0, 1, 2, 4, 7, 10, 14, 18, 21, 28, 35, 42 and 63 days after
girdling (DAG) as follows: 0.5 - 1.0 g of tissues was scraped
on the girdled area with a clean and sharp knife in a manner
to include immature xylem, cambium and the progressively
regenerating bark tissues. The scraped tissues were imme-
diately stored into liquid N2 for more than 1 h, then, pre-
served at -70 ℃ until further analyses. At each sampling
time, about 25 cm2 of barks were removed from ungirdled
trees, taken as controls, and both the inner layers of the
bark (comprising cambium and immature phloem cells, as
revealed under light microscope) and the outer layers of
the exposed xylem (including immature xylem) were har-
vested and treated as above.
For peroxidases localization in plant tissue, small blocks
(0.5 cm× 0.5 cm× 1.0 cm in size) were excised at each
sampling date on the girdled part (including some mature
and immature xylem cell layers as well as the regenerating
tissues) and immediately fixed for 2 h in 2% FAA in order to
adequately immobilize enzymes without diminishing the per-
oxidase activity.
1.2 Peroxidase localization
Peroxidase staining was performed on cryo-cross-sec-
tions (10 µm thick) of the above-mentioned tissue blocks
made using a LEICA cryomicrotome (LEICA CM 1850-
Cryostat, Germany). Sections were first suspended in phos-
phate buffer (PB, pH 7.2) for 5 min at 4 ℃. They were then
treated in 0.1% ammonium molybdate for 5 min at room
temperature, before being stained, for 1 min, with 0.1% ben-
zidine containing one drop of 30% H2O2. Once stained,
sections were rapidly observed and photographed under
light microscope. Control sections were heated for 5 min at
90 ℃ to inhibit peroxidase activity as advised by Micheal
(1975) and Gahan (1984), then submitted to the same stain-
ing procedure as above.
1.3 Proteins and peroxidases extraction and quantifica-
tion
About 0.5 g of the scraped plant tissues was finely
ground in liquid N2 and received 3 mL of the extracting
buffer (3 mL 0.1 mol/L Tris-HCl, pH 8.5) for one night at 4 ℃
under a continuous shaking. The supernatant obtained af-
ter centrifugation (6 000 r/min, 15 min, 4 ℃) was kept at 4 ℃
until later use.
Proteins were determined by using the protein-dye bind-
ing method developed by Bradford (1976) using bovine
serum albumin (BSA) as protein standard. Peroxidases ac-
tivity was assessed by diluting 0.1 mL of the supernatant
into 1 mL of the extracting solution. Immediately after an
addition of 2 mL of the reaction mixture (Phosphate buffer
(pH 6.0): H2O2 : guaiacol / 50 mL : 0.028 mL : 0.019 mL) to the
supernatant, the reaction progress was measured by re-
cording the resulted absorbance at 470 nm using a spectro-
photometer (Agilent 8453 UV-Vis, USA), and the straight
line zone was used as measurement of peroxidase activity
(OD/ g FW) (Kochba et al., 1977).
1.4 Electrophoresis
At each harvesting time within the 35 DAG, 100 mg of
samples were scraped on the girdled area and proteins ex-
tracted according to Cui et al. (1993). At 35 DAG, an extra-
sampling was done and consisted in splitting the immature
xylem and the recovered bark in two distinct samples. The
gel preparation and the isoelectric-focusing electrophore-
sis procedure were totally inspired from Cui et al. (1993).
After electrophoresis, the gel slabs were placed in the sub-
strate buffer until the isozymes bands were visible. The
substrate for peroxidase was a mixture (1:2:1) of 2.2% ben-
zidine (in 20% acetic acid), 0.35% o-dianisidine and 0.6%
H2O2. The gel slabs were, then, dried and photographed.
2 Results
2.1 Peroxidase localization in the recovering bark of E.
ulmoides
The peroxidase signal in the E. ulmoides tissue was
identified by an intense reddish-brown color (Fig.1B, D and
F) as compared to the control (heated sections) (Fig. 1A, C
and E). The signal appeared in part of sections containing
peroxidase after reacting with benzidine (used as substrate).
Sections in which peroxidases were denatured through
heating did not develop the above signal. Most peroxi-
dases were found in the cell wall and protoplasm of the
maturing xylem cells (Fig.1B), the callus (Fig.1D), and the
phloem parenchyma cells (Fig.1F). On the contrary, cam-
bial cells were not at all stained, but immature xylem cells
showed an inward increasing gradient of peroxidases
staining. Less peroxidase was detected in the radially en-
larging cells (Fig.1B).
Peroxidase localization in the recovering bark greatly
varied with time and observed tissues (Fig.1 G-O). At 0
Acta Botanica Sinica植物学报 Vol.46 No.2 2004218
DAG, there was very few staining in the section, mainly
localized in immature xylem cells (involving radially enlarg-
ing and maturing xylem cells) (Fig.1G). At 1 and 4 DAG,
peroxidases signal was mainly distributed in the callus cells
(Fig.1H-J). At 7 DAG, cell division occurs in the inner layer
of the immature xylem, where there was no obvious peroxi-
dases staining (Fig.1K). This region will later generate the
cambium and will stay almost unstained until the full formation
219
HOU Hong-Wei et al.: Changes of Soluble Protein, Peroxidase Activity and Distribution During Regeneration After Girdling in
Eucommia ulmoides
of the cambium (Fig.1L-O). About 14 DAG, the phellogen-
like starts to initiate at three to five layers beneath the sur-
face cells, which will develop into the regenerated periderm.
Apart from the phellogen-like, the initiating cambium and
the enlarging xylem cells, a high activity of peroxidase was
shown in the remaining callus and the differentiating xylem,
specially at 14, 21 and 35 DAG (Fig. 1L-N). This period
corresponded with the completion of the bark recovery pro-
cess and the effective start of the cambium activity, pro-
ducing xylem inwards and phloem outwards. At 63 DAG
(Fig. 1O), much more layers of cambium have differentiated
into a lot of xylem cells inward and phloem cells outward,
but the peroxidase staining in the cambium remained
imperceptible.
2.2 Soluble protein and peroxidase change pattern in the
recovering bark
Results on the soluble protein content and the peroxi-
dase activity found out in the reconstituting bark of E.
ulmoides after girdling are highlighted in Fig.2. Broadly,
the soluble protein content in ungirdled trees did not sen-
sibly vary; it remained below 3 mg/g FW (Fig. 2A). In girdled
trees, however, the protein content remarkably fluctuated.
It brutally increased from 2 DAG and remained significantly
different (P<0.01) from that in ungirdled trees. Low at 0 and
1 DAG, it reached the highest amount (about 13 mg/g FW)
at 14 DAG and slowly decreased later.
The peroxidase activity measured in the regenerating
tissue (Fig.2B) followed the general trend of the soluble
protein content as exposed above. But, its increase started
from 4 DAG and gradually followed the evolution of the
bark regeneration process, before reaching the highest peak
(about 110 OD/g FW) on 21 DAG. Compared to the situa-
tion in ungirdled trees, the peroxidases activity in girdled
plants has abruptly increased by approximately 30 to 40
times between 4 and 21 DAG.
The ratio between peroxidase and soluble protein con-
tent (Fig.2C), expressing the activity of peroxidase per gram
of protein, has significantly (P<0.05) fallen down in girdled
trees compared to the control at 2 and 4 DAG. It, then,
radically increased over the control (P<0.01) with time and
reached its maximum level (about 11 OD/mg protein) at 21-
28 DAG. This ratio drastically decreased with the complete
re-establishment of the bark (28-63 DAG).
2.3 Isoperoxidase detection
The isoelectric-focusing electrophoresis result is illus-
trated in Fig.3. Up to 2 DAG, the gel displayed, at pH 8.0,
only a band of isozyme (identified as PODⅠ in this study)
in the samples from both girdled and ungirdled trees (Fig.
3A-B). From 4 DAG (Fig.3C), another band (identified here
as PODⅡ) was detected at pH 9.0, with a gradually in-
creasing intensity with time, only in the extract from girdled
trees. These two bands remained visible up to 35 DAG (Fig.
Fig.1. Identification of peroxidase in Eucommia ulmoides tissues. A-F: longitudinal cryosections across the cambium region at 0 (A and
B), 7 (C and D), and 63 (E and F) DAG, respectively. Sections previously heated at 90 ℃ for 5 min (A, C and E) were not stained. The
non-heated cryo-sections displayed peroxidase in maturing xylem (B), callus (D) and phloem (E) cell layers. There was minor staining in
the radially enlarging cell and no staining in the cambium. G-O: peroxidases were localized in transverse cryosections of the progres-
sively recovering bark in E. ulmoides after girdling. From 0 to 2 (G-I) DAG, few peroxidase signals was detected in immature xylem cells
and the regenerated tissues, showing less peroxidase activity in the tissues at this period. At 4 (J) and 7(K) DAG, the growing callus was
intensely stained but not the dividing immature xylem cell layers. At 14 (L), 21 (M) , 35 (N) and 63 (O) DAG, the peroxidase was not
localized in the developing periderm and cambium regions, but invades all the remaining recovered tissues as well as the maturing xylem
cell layers. Abbreviations: c, cambium; ca, callus; cl, cork layer; co, cortex-like; ix, immature xylem; mgx, maturing xylem; mx, mature
xylem; ph, phloem; pr, phellogen region; re, radially enlarging cell layers. Bar = 200 mm.
←
Fig.2. Variation of soluble protein (A), peroxidase activity (B)
and peroxidase: protein ratio (C) in the regenerating bark of
Eucommia ulmoides (0 to 63 DAG).
Acta Botanica Sinica植物学报 Vol.46 No.2 2004220
3C-G). The gel electrophoresis of immature xylem (Fig. 3H,
lanes X) and recovered bark (Fig.3H, lanes B) from the 35
DAG samples also exhibited the above two bands.
However, PODⅠ was detected in the immature xylem
fraction, whereas PODⅡwas identified in the recovered
bark portion.
3 Discussion
A lot of investigations have demonstrated the impor-
tant roles played by peroxidases in plant development and
wound healing. This study suggests that peroxidases could
be greatly involved in the following three aspects during
tissue regenerating process after girdling.
3.1 “Repulsive” relationship between peroxidase and
indole-3-acetic acid (IAA) in bark regeneration and xylem
differentiation
It has been previously reported that peroxidases cata-
lyze the oxidative decarboxylation of IAA and play an im-
portant role in regulating the concentration of endogenous
IAA in plant tissues (Reineke and Bandurski, 1987; Fahn
and Werker, 1990; Siddiqu, 1991). In the other hand, IAA
was stated to inhibit peroxidase gene expression in tobacco
(Klotz and Lagrimini, 1996). This somewhat “repulsive” re-
lationship between peroxidase and IAA was once more
monitored by the results obtained in both this study on
peroxidases and that by Mwange et al. (2003) on IAA con-
tent and distribution in the bark regeneration process.
As a matter of fact, the high IAA content, measured in
the progressively recovering bark in the first 4 DAG, was
mainly localized in the radially enlarging cells of the imma-
ture xylem cells and callus and, later (7-63 d), most of the
IAA was detected in the cambium and phellogen zones
(Mwange et al., 2003). In this study, however, no peroxi-
dases were observed in tissue undergoing active cell divi-
sion and/or containing high IAA level. Thus, the few per-
oxidase traces were detected in the immature xylem cells (0
-7 d) and, later, in the forming (14 d) and formed (21-63 d)
cambial region and phellogen (Fig.1). This possible inter-
active effect between peroxidases and IAA was also illus-
trated by the decrease of peroxidase: protein ratio observed
between 2 and 4 d (Fig.2C), period during which a remark-
able increase of IAA content has been previously moni-
tored (Mwange et al., 2003). In this time, immature xylem
cells started dedifferentiating into callus cells and contained
high IAA.
As IAA has been often reported to play a key role in the
vascular tissue regeneration process (Aloni, 1995; Cui et
al., 1995; Wang and Cui, 1998), the importance of peroxi-
dases in bark regeneration could be the regulation of IAA
concentration. That is, peroxidases could catalyse the IAA
oxidation, resulting in its gradual diminution in differentiat-
ing cell layers (such as maturing and mature xylem and
phloem cells).
In addition, some investigations have previously indi-
cated that variations of isoperoxidase band patterns were
correlated with the periodicity of cambial activity and the
bark regeneration process, and assumed that some isozyme
activities contributed to the regulation of IAA concentra-
tion (Cui et al., 1993; 1995). The present study showed that
PODⅠ in the samples from both girdled and ungirdled
trees could be identical to PODⅢB reported by Luo et al.
(1999) （their isoelectric points were all identified at pH
8.0） and specific for xylem differentiation. This suggests
that the above isozyme would be more likely involved in
the normal xylem cell differentiation, from immature into
mature xylem. Figure 3H clearly showed that PODⅠ was
only present in the xylem fraction. On the other hand, POD
Ⅱ, which appeared in the gel from 4 DAG exclusively in
girdled E. ulmoides, could be more engaged in bark regen-
eration process. Indeed, this peroxidase isozyme was only
found in the regenerating bark fraction (Fig.3H) of the girdled
Fig.3. Peroxidase isozyme zymograms after an isoelectric-
focusing electrophoresis. Only PODⅠ isozymes were present
in the girdled trees at 1 (A) and 2 (B) days after girdling (DAG).
Both PODⅠ and PODⅡ were visible in the following stages of
wound healing (C-G). The intensity of PODⅡ was low at 4
DAG (C) and gradually increased with time at 7 (D), 14 (E), 21
(F) and 35 (G) DAG. PODⅠ could be involved in xylem differ-
entiation as it was detected only in the xylem fraction and POD
Ⅱ could be active in cell dedifferentiation as it was located in the
recovered bark fraction of 35 DAG zymogram (H). 1, 2, regener-
ating bark tissues; 3, 4, differentiating xylem tissues; X, xylem; B,
bark tissues.
221
HOU Hong-Wei et al.: Changes of Soluble Protein, Peroxidase Activity and Distribution During Regeneration After Girdling in
Eucommia ulmoides
trees, which will later give rise to several tissue cell layers
(phloem, cortex-like and periderm cells). Furthermore,
Mwange et al. (2003) observed an over-production of IAA
at 2 to 4 DAG and its decrease from 4 DAG. They stated
that this IAA increase was mainly involved in the callus
production and the immature xylem cell division, whereas
its decline coincided with the beginning of cell dedifferen-
tiation (phellogen, cambium) and transdifferentiation (Cao,
2003①) processes. The IAA reduction in these processes
could be attributed to peroxidases in their role of regulating
IAA content. PODⅡ could probably insure this IAA
diminution. This could explain why POD Ⅱ band density
started decreasing with cambium formation and activity
three weeks after girdling （Fig.3F, G）. Further studies
on isolating PODⅠ and PODⅡ and exploring their mo-
lecular composition as well as their gene expression will
be of great interest in elucidating the mechanisms of cell
division and differentiation.
3.2 Relationship between peroxidase and the formation of
the secondary cell wall
Other research works have described the peroxidase
involvement in the secondary cell wall lignification by cata-
lyzing the polymerization of phenolics into lignin and by
intervening in the formation of covalent cross-links between
ferulylated polysaccharides, hydroxyproline-rich glycopro-
teins and lignin (Fry, 1988; Roberts et al., 1988). In these
trials, peroxidases were located in the secondary wall thick-
ening xylem cells as shown in Fig. 1, which were undergo-
ing secondary cell wall formation and lignification in all
stages of bark regeneration.
Recently, Liszkay et al. (2003) have reported that per-
oxidases in cell wall produce hydroxyl radicals (· OH), which
mediate cell extension growth. These radicals could be ca-
pable of cleaving wall polymers, mediate cell wall loosen-
ing and, consequently, participate in the cell growth
process. Our studies support the above findings.
3.3 Synthesis of new proteins during the regeneration
after girdling
It was noticed that the soluble protein content appeared
earlier (2 d before) than that of peroxidase in the recovering
bark. This implies that the synthesis of some new proteins
was highly required for their traditional role in tissue
regeneration. The soluble protein content change found in
this study was consistent to that of IAA by Mwange et al.
(2003). This suggests that the release of free IAA from con-
jugated IAA could be enzyme-catalyzed. These enzymes
could participate in the increase of soluble proteins.
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