全 文 ：Received 21 Jul. 2003 Accepted 15 Sept. 2003
Supported by the National Natural Science Foundation of China (30270083).
* Author for correspondence. Tel: +86 (0)20 85288393; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 443-450
Cytochemical Localization of Pectinase: the Cytochemical Evidence for Resin
Ducts Formed by Schizogeny in Pinus massoniana
LI Ai-Min1, 2, WANG Yu-Rong1, WU Hong1*
(1. College of Life Sciences, South China Agricultural University, Guangzhou 510642, China;
2. Department of Bioengineering, Huaihua College, Huaihua 418008, China)
Abstract : Initiation and development of resin ducts were studied in cortex of stem of Pinus massoniana
Lamb. by thin section. It is shown that the development of resin ducts can be divided into four stages: the
initial stage, the intercellular space forming stage, the lumen expanding stage and the mature stage. To
further investigate the original mode of resin ducts, cytochemical localizat ion of pectinase with a
transmission electron microscope (TEM) was conducted. Results showed that the pectinase was active
during resin duct development. At the initial stage, reaction products of pectinase first appeared in the
corner of swollen cell wall among the future epithelial cells and later along the walls. At the intercellular
space forming stage, reaction products occurred in the cell wall/intercellular space interface and its
density decreased while the intercellular space enlarged. At the mature stage, no reaction products were
found in the walls of the epithelial cells. These results indicate that pectinase catalyzes the degradation of
the middle lamella of the epithelial cells during the resin ducts development. The cytochemical evidence
supports that the resin ducts are formed by schizogeny. This report represents the first direct evidence
for the involvement of pectinase in resin ducts formation .
Key words : initiation and development; cytochemical localization; pectinase; resin ducts; Pinus massoniana
Resin ducts are a common pro tect ive st ructure in
conifers. The oleoresin, a mixture of roughly equal amounts
of turpentine and rosin (Steele et al., 1998), is synthesized
and accumulated in resin ducts. Its toxic and sticky proper-
ties repel invading ins ects and inhibit pathogen ic fungi
(Nagy et al., 2000). So, the resin ducts in conifers are con-
sidered as the first line of defense against insects and patho-
gens (Berryman,1972; Boucher et al., 2001). In addition,
res in ducts are an important taxonomic characteristic in
conifers (Wu and Hu, 1997; Lin et al., 2001; Lin et al., 2002).
Conifer resins are important renewable resources and pro-
vide a range of commercially useful products for chemicals
and pharmaceuticals (McGarvey and Croteau, 1995).
There are many papers on resin ducts in Pinaceae, mostly
focusing on their structure (Wooding and Northcote, 1965;
Fahn and Benayoun, 1976; Fahn , 1979; Wu and Müller,
1999), distribution (Bannan, 1936; Reid and Watson, 1966;
Wu and Hu, 1997; Lin et al., 2002), initiat ion and develop-
ment (Chat taway, 1950; Werker and Fahn, 1969; Wu and
Hu, 1994), histochemical changes during their development
(Wu and Hu, 1995; Li and Wu, 2003) and systematic signifi-
cance (Hanes, 1927; Wu and Hu, 1997; Lin et al., 2000; Lin
et al., 2001; Lin et al., 2002). The rate of oleoresin flow from
freshly made wounds is determined in part by the number
and size of radial resin ducts (Mergen and Echols, 1955)
and vertical duct density (Blanche et al., 1992). So, under-
standing the initiation and development of resin ducts can
help to regulate the number and size of resin ducts and,
consequently, to increase the oleoresin production.
About the initiation and development of resin ducts,
previous authors considered that the res in ducts were
formed by schizogeny, based on characteristics of cell mor-
phology during development of resin ducts employing light
microscopy (Esau, 1965; Werker and Fahn, 1969; Fahn, 1979).
Since different kinds of fixatives, and embedding media, as
well as dehydrating, staining and paraffine sect ion p ro-
ces ses would in fluence the observation and analysis of
results (Hu and Yu, 1993), ultrastructural changes of initia-
t ion o f res in ducts canno t be obs erved us ing ligh t
microscopy. With the development of electron microscopy
techniques, electron microscopy was immediately employed
to study the initiation and development of resin ducts. Fahn
and Benayoun (1976) first observed multivesicular struc-
ture in the region of the middle lamella and in invaginations
of the plasmalemma outside the cytoplasm at initial stages
of resin duct development in Pinus halepensis. In the
cytoplasm, precisely in the vicin ity of the multivesicular
structures, Golgi bodies and similar vesicles were often
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004444
obs erved . Therefore, these authors thought it could be
possible that vesicles developing from Golgi bodies carry
lytic enzyme fo r the dissolution of the middle lamella. Wu
and Hu (1994) drew the same conclusion based on the de-
velopment of resin ducts in P. tabulaeformis under the
transmis sion electron microscope (TEM). These resu lts
further support the viewpoint that resin ducts are formed
by s chizogeny. However, there is no direct evidence re-
vealing the biochemical and physiological changes in cell
wall during initiation and development of resin ducts.
Schizogenous resin ducts are formed by separation of
the middle lamella of neighboring cells (Fahn, 1979). Pecti-
nase hydrolyzes pectin in the cell wall and middle lamella of
plant cells, resulting in cell separation (Hadfield and Bennett,
1998; Hong et al., 2000; A tkinson et al., 2002). We used
thin section and cytochemistry for TEM to study initiation,
development and changes of pectinas e of cortical resin
ducts in P. massoniana, so as to provide cytochemical evi-
dence for original mode of resin ducts formation.
1 Materials and Methods
1.1 Plant materials
Vegetative shoot ap ices o f Pinus massoniana Lamb.
(about 20 years old) were collected from cultivated popula-
t ions on the campus of South China Agricu ltural
University，from November 2001 to April 2002.
1.2 Methods
1.2.1 Thin sections for light microscopy The materials
were cut into 0.5 mm× 0.5 mm×1 mm blocks and fixed in
5% (V/V) glu taraldehyde in 0.1 mol/L sodium phosphate
buffer (pH 7.2) for 4 h at 4 ℃, then washed in three changes
of 0.1 mol/L sodium phosphate buffer (pH 7.2), postfixed in
1% (W/V) aqueous osmium tetroxide for 2 h at room tem-
perature and washed in th ree changes o f distilled water.
The samples were then dehydrated through alcohol series
and embedded in Epon812. Sections were cut to a th ick-
ness of 1 µm using g las s knives on an ult ramicrotome
(Reichert-Jung, Austria). Sections were stained with PAS
and toluidine blue O (Xu and Hu, 1986), examined and pho-
tographed using Leica DMLB light microscopy.
1.2.2 Cytochemical localization of pectinas e for TEM
Tissue samples were fixed in Karnovsky’s (1965) fixative in
0.05 mol/L sodium phosphate buffer (pH 7.2) for 2-4 h at 4
℃, washed in 20 changes of 0.05 mol/L sodium phosphate
buffer (pH 7.2) and stored in the same buffer overnight at 0
℃. They were incubated in 0.5% pectin in 0.1 mol/L sodium
acetate buffer (pH 5.0) for 20 min at room temperature, then
transferred to hot Benedict’s reagent and incubated for 10
min at 80-90 ℃. Control 1 was incubated in 0.1 mol/L
sod ium acetate buffer without pectin for 20 min at room
temperature, then transferred to hot Benedict’s reagent and
incubated for 10 min at 80-90 ℃. Control 2 was incubated
in Benedict’s reagent fo r 10 min at 80-90 ℃,then trans-
ferred to pectin solut ion for 20 min at room temperature.
Control 3 was incubated in pect in so lution for 20 min at
room temperature, then trans ferred to 0.05 mol/L sodium
phosphate buffer (pH 7.2) for 10 min at 80-90 ℃. All above
treated samples were allowed to coo l, rinsed with 0.05
mol/L sodium cacodylate buffer (pH 7.2), and then postfixed
for 2 h in 1% osmium tetroxide in the same buffer at room
temperature. The samples were washed with distilled water
and dehydrated through a graded ethanol series (Allen and
Nessler, 1984), then infiltrated and embedded in Epon812.
Ultra-thin sections were cut with a diamond knife on a Leica
UCT ultramicrotome, and then stained with uranyl acetate
and lead citrate. The s ections were examined and photo-
graphed under Philip FEI-TECHNAI 12 transmission elec-
tron microscope at 80 kV.
2 Results
The mature cortical resin ducts of P. massoniana were
found to range into a circle in cross sections of stem. They
are elongated structures throughout their length of epithe-
lial cells surrounding an intercellular space. During the resin
ducts formation a series of changes in structure took place.
Four different stages were identified: initial stage, intercel-
lular space forming stage, lumen expanding stage and ma-
ture stage.
2.1 Initial stage
Under light micros cope, a group of four to s ix cells
formed a rosette at about 360 µm from the stem apex in the
cortex. These are the initial cells of resin ducts (Fig.1). The
duct initial cells, which arranged closely and dyed densely,
are easily recognized from the neighboring cells. Under
TEM, a few of electron opaque crys talline depos its ap-
peared in middle lamella among the initial duct cells (Fig.7).
In addition, some high electron density granules were scat-
tered in walls (Fig.8). These deposits and granules should
be the products formed by the reaction of Benedict’s solu-
tion with reducing s ugars liberated during hydrolysis of
pectin in the incubation medium by pectinase. Their posi-
tions indicated reaction localization of this hydrolysis. Re-
action products distributing in middle lamella implied that
pectinase have been transported to extracellular space from
endocellular space and accumulated in middle lamella at
initial stage. During the development, the wall in the corner
between the future epithelial cells began swelling, and re-
action products accumulated continuously in middle lamella


LI Ai-Min et al.: Cytochemical Localization of Pectinase: the Cytochemical Evidence for Resin Ducts Formed by Schizogeny
in Pinus massoniana 447
of wall and became patches. Microfibrils near the reaction
products started loosening (Fig.9). No any reaction prod-
ucts were found in the swollen walls in corner in controls
(Fig.15 show the result of con trol 3, results of others con-
trols were not shown).
2.2 Intercellular space forming stage
At this stage, intercellular space started to form among
the further epithelial cells as seen using light microscopy
(Fig.2). However, the observation with TEM revealed that
the middle lamella with patches of reaction products started
to degrade when the reaction products became much more
dense, and the intercellular space occurred (Figs.10,12). The
intercellular space first appeared in the corner among the
forthcoming epithelial cells and then gradually enlarged
(Fig.10). Patches of reaction products were not observed in
middle lamella of walls in con trols , bu t there were s ome
vesicles and electron-dense granules scattered in cell walls
(Fig.16) or in intercellular space (Fig.17). Since tissues were
boiled during specimen preparation to allow the Benedict’s
reaction occurrence, some cellular disruption was visible.
These vesicles could be lysosome vesicles and the gran-
ules might be some structure proteins of wall or broken wall
remnants.
2.3 Lumen expending stage
After the intercellular space formed, the middle lamella
among neighboring cells continuously kept dissolving so
that the intercellular space was further expanded along walls
and the duct of 6-7 cells surrounding a lumen was formed
(Fig.3). The cells surrounding the lumen evolved in epithe-
lial cells in the future, and the cells enclosing epithelial cells
were sheath cells, which usually consist of one to two cell
layers. Afterwards, a number of ep ithelial cells separated
from radial wall, then some sheath cells penetrated between
the radial walls of epithelial cells and became epithelial cells.
The number of epithelial cells increased (Fig.4). At the same
time, the epithelial cells underwent tangential prolongation
(Fig.5) and radial division for fu rther developing lumen.
While observed with TEM, the duct lumen lacks dis tinct
reaction products at the lumen expanding stages, only hav-
ing some residual substances and vesicles (Fig.13). Some
residual substances were also found in lumen of controls
(Fig.18).
2.4 Mature stage
With the increasing number of epithelial cells and en-
larg ing vo lume of duct lumen , the resin ducts came into
mature stage. At this stage, the tangentially elongated epi-
thelial cells consisted of 12-30 cells with thin walls, and
their volumes were obviously smaller than those of sheath
cells (Fig .6). Some of them were filled with po lyphenols.
The duct lumen appeared transparent under TEM (Fig.14).
3 Discussion
The middle lamella of plan t cells are most ly made of
pect in, which are shown to play an important role in cell
adhesion. In addition, pectin exists in the primary cell wall.
Pectin is a family of polysaccharides that contain 1,4-linked
a-galactosyluronic residues (Ridley et al., 2001). Pectinase
(EC 3.2.1.15) is an enzyme that randomly hydro lyzes the
a-1,4 linkages of pectin and then degrades pectin (Allen
and Nessler, 1984; Hadfield and Bennett, 1998). Increasing
of pectinase expression during a number of developmental
processes is thought to be involved in cell wall breakdown.
These include fruit ripening (Crookes and Grierson, 1983;
Atkinson et al., 2002), leaf abscission (Morre, 1968; Taylor
et al., 1990), pollen tube growth (Pressey and Reger, 1989;
Pressey, 1991), and nonarticulated laticifers intrusive growth
(Wilson et al., 1976). To our knowledge, endogenous secre-
to ry st ructu res with lumina are fo rmed in th ree ways,
schizogenesis, (1) separation of walls of neighboring cells;
lysigenesis, (2) the disintegration of cells in the area where
the space develops, and schizo-lysigenesis, (3) combina-
tion of schizogenesis with lysigenesis (Esau, 1965; Fahn,
1979). In the present paper we have directly revealed the
changes of cell walls during resin duct development of P.
massoniana by cytochemistry localization of pectinase. We
have confirmed that resin ducts were fo rmed sch izog-
enously and the pectinase degraded the middle lamella re-
gion of the future epithelial cell walls during the resin duct
development.
As observed using light microscopy, the development
of resin ducts in P. masson iana (p res ent paper), in P.
tabulaeformis (Wu, 1990) and P. halepensis (Werker and
Fahn, 1969; Fahn and Benayoun , 1976), shows a similar
pattern. The development course is that the middle lamella
of cell wall swells in the rosette initial cells, then the middle
lamella disintegrates to form intercellular space and the in-
tercellular space further expands to form a mature resin duct.
During the res in duct development, we found that pecti-
nase in midd le lamella displayed regular changes. At the
initial stage, the cell walls showed no distinct changes in
light microscopy, but under TEM, the middle lamella in cor-
ners among the future epithelial cells swelled and appeared
clump deposits . Whereas, the unswollen walls had a few
or no deposits. Since pectin is the most abundant element
in the middle lamella of the walls and the primary walls
contained very little, pectinases have been eliminated from
cytoplasm, accumulating in middle lamella and then gradu-
ally degrading pect ins in swollen walls , releasing more
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004448
reducing sugars , that resulted in the increase of reaction
products with Benedict’s reagent. Sutherland et al. (1999)
thought that changes to pectins during ripening involve at
least three processes: solubilization of pectin, depolymer-
ization o f pectin, and los s o f galactos e s ide chains .
Furthermore, the wall swelling occurred as a result of
changes to the viscoelastic properties of the cell wall dur-
ing pect in so lubilisation (Redgwell et al., 1997). So, the
deposits first appeared in swollen middle lamella. While the
intercellular space was forming, pectin in middle lamella
gradually decreased and pectinase activity diminished so
that the deposits declined. Deposits with low density only
dist ributed in intercellular space and completely cleared
away while the lumen was being formed. The deposits gradu-
ally increased in number, then decreased . The phenom-
enon of deposits quant ity changes indicated that pecti-
nase activity is low or absent at initial stage, gradually rises
to a maximum at the intercellular space forming stage, then
declines. These changes are s imilar to those no ticed dur-
ing leaf abscission (Morre, 1968).
Fahn and Benayoun (1976) observed electron dense lines
in the region of the middle lamella at the early s tages of
res in duct development in P. halepensis us ing elect ron
micros copy. Thes e lines were accompanied by small and
very dark globules. These globu les, as well as electron-
translucent vesicles, occurred in very small developing in-
tercellu lar space. In addition to multivesicular structures,
many of those ves icles, filled with electron-dense granu-
lated material, were observed in the invaginations of the
plasmalemma outside the cytoplasm. In the cytoplasm, near
the multives icular structures Golgi bodies and similar
vesicles were often seen. The above researchers suggested
that vesicles, developing from the Golgi bodies, could carry
lytic enzymes for the dissolution of the middle lamella. We
found that the products , res ulted from the reaction of
Bened ict’s solution with reducing sugars liberated by
pectinases, first appeared in the corners among the neigh-
boring initial cells and, later, along the walls. The phenom-
enon agrees with the results from morphological observa-
tion by Fahn and Benayoun (1976) and Wu and Hu (1994).
Besides these, we observed some plasmalemma invagina-
tions and a little s mall granule outside the plasmalemma
(Fig.12). It proved that the vesicles, found out by Fahn and
Benayoun (1976), undoubtedly carry pectinase. Moreover,
s ome ves icles were seen in cell walls in the cont ro l
treatments, but the number was low and the delimitation
unclear, compared with the results provided by Fahn and
Benayoun (1976). It is poss ib le that during specimen
preparat ion s ome cells were d is rupted res u lt ing in
the occurrence of these vesicles.
Although remarkable advances have been achieved
about pect inase at molecular level (Taylor, 1990; Hadfield
and Bennett, 1998; Hong et al., 2000; Wang et al., 2000;
Atkinson et al., 2002), the pectinase biosynthetic pathways
and the elimination patterns have not been fully character-
ized at cytological level. Modified method des cribed by
Allen and Nes sler (1984) enhances the s ens it iv ity of
pectinase. Pectinase biosynthetic pathways and elimina-
tion patterns at cytological level can be elucidated. On the
other hand, cytochemical ev idence of formation pathway
of secreto ry st ructu re and o ther plan t development
proces ses, involving pecto lysis, can be supp lied . In
addition, genes modulating resin duct formation have not
been reported. Further studies on in itiation and develop-
ment of resin ducts at cytochemistry and immunocytochem-
istry level should p rovide more abundant in formation to
reveal molecu lar mechanism in morphogenesis of res in
ducts.
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