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Annals of Botany 84 : 429–435, 1999
Article No. anbo.1999.0931, available online at http:\\www.idealibrary.com on
The Differentiation of Contact Cells and Isolation Cells in the Xylem Ray
Parenchyma of Populus maximowiczii
Y. M U R A K A M I, R. F U N A DA*, Y. S A N O and J. O H T A N I
Department of Forest Science, Faculty of Agriculture, Hokkaido UniŠersity, Kita 9, Nishi 9, Kita-ku,
Sapporo 060-8589, Japan
Received : 24 March 1999
Returned for revision : 27 April 1999
Accepted : 11 June 1999
A histochemical analysis was made of the differentiation of contact cells and isolation cells in the xylem ray
parenchyma of Populus maximowiczii. The contact cells formed secondary walls at approximately the same time as
adjoining vessel elements. The lignification of the cell walls of contact cells and vessel elements began earlier than that
of wood fibres and isolation cells. Thus, the formation of the secondary wall, including lignification, of the contact
cells might occur at the same time as that of the vessel elements to which they are directly connected. By contrast,
the isolation cells began to form secondary walls later than the vessel elements and wood fibres in the vicinity of the
isolation cells. After the deposition of the secondary wall, a protective layer was formed in contact cells but no
isotropic layer was observed in isolation cells. The results suggest the importance of vessel elements in the
determination of the differentiation of adjoining ray parenchyma cells.
# 1999 Annals of Botany Company
Key words : Contact cell, isolation cell, vessel element, xylem differentiation, Populus maximowiczii Henry.
INTRODUCTION
Ray parenchyma cells play an important role in the storage,
conductive and secretory systems of woody plants. Mostly,
they maintain radial continuity between the phloem and
xylem, thereby facilitating the radial and lateral transport of
materials (Larson, 1994).
The ray parenchyma cells of xylem are of two types,
namely contact cells and isolation cells, and they differ with
respect to their contacts, through pits, with vessel elements.
Contact cells are ray cells that make direct connections with
adjacent vessel elements through half-bordered pit pairs,
and the isolation cells are ray cells that make no pitmediated direct connections with adjacent vessel elements
(Braun, 1967). Sauter and Kloth (1986) proposed that
isolation cells might be more specialized for radial translocation than contact cells. By contrast, tyloses develop
from contact cells and not from isolation cells. Therefore,
contact cells might function to protect living ray parenchyma
cells from hydrolases released by dead vessel elements
(Schmid, 1965 ; O’Brien, 1970). In contrast, Chafe (1974)
proposed that contact cells might function as ‘ transfer cells ’
because both types of cell have a similar structure. It
appears that contact cells and isolation cells differ in terms
of both form and function, even though both types of cell
are derived from the same cambial ray cells (Braun, 1970).
Thus, it seems possible that these two types of cell might
be formed via different processes of differentiation from
cambial ray cells.
* For correspondence. Fax j81 117361791, e-mail funada!for.
agr.hokudai.ac.jp
0305-7364\99\100429j07 $30.00\0
Ray parenchyma cells generally form an additional layer
on the inner side of the secondary wall (Schmid, 1965). This
layer contains high levels of polysaccharides (Czaninski,
1973). Chafe (1974) reported that, in Populus tremuloides,
such an additional layer in contact cells was unlignified
during the first growing season (this layer was referred to as
the protective layer), while that of isolation cells was
lignified within one growing season (this layer was referred
to as the isotropic layer). The protective layer was finally
lignified when the formation of heartwood began. Using
histochemical techniques, Fujii, Harada and Saiki (1981)
found that there was little difference between the protective
layer and the isotropic layer in terms of their ultrastructure
and chemical compositions prior to lignification. Thus, they
concluded that these two layers might have the same origin
and that lignification might begin at different times in the
two layers. These observations indicate that the formation
of the secondary wall, including lignification, differs between
the two types of ray parenchyma cell. Such differences might
be dependent on the disposition of ray parenchyma cells
with respect to the vessel elements.
The purpose of the present study was to monitor the
differentiation of contact cells and isolation cells in Populus
maximowiczii by histochemical staining. The rays of Populus
have a simple composition, being homogeneous and
uniseriate (Panshin and de Zeeuw, 1980). Therefore, it is
easy to determine, by observation, whether or not ray
parenchyma cells make direct contact with adjacent vessel
elements. We postulated that the deposition and lignification
of cell walls of the two types of ray parenchyma cell would
provide a suitable model for clarification of the mechanism
of differentiation of secondary xylem cells derived from
cambial cells.
# 1999 Annals of Botany Company
430
Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
F. 1. A differentiating vessel element (V) and other elements around it, at a distance of approximately 500 µm from the cambium, in radial
section. Arrows indicate transverse walls of contact cells with birefringence. Arrowheads indicate ray-vessel pits of contact cells. No birefringence
was visible in cell walls of isolation cells (asterisks). The left side of each micrograph corresponds to the outer side of the tree. Light micrograph
(A) and polarizing light micrograph (B). Bar l 50 µm.
Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
431
F. 2. Electron micrographs showing tangential sections of differentiating ray parenchyma cells at a similar stage to that shown in Fig. 1. Contact
cell with a secondary wall (A) and an isolation cell without a secondary wall (B). V, Vessel element ; F, wood fibre ; CC, contact cell ; IC, isolation
cell. Bars l 2 µm.
MATERIALS AND METHODS
Samples from a straight, healthy tree (approx. 30 years old)
Populus maximowiczii Henry (height, 18 m ; diameter at
breast height, 30 cm) growing in the Tomakomai Experimental Forest of Hokkaido University, were used. Small
blocks containing phloem, cambial zone and differentiating
xylem were taken from the stem at breast height in July
1995, when cambial activity was very high. At that time, the
successive stages of differentiation of ray parenchyma cells
could be observed within a single radial section.
All sample blocks were immediately placed in a 4 %
solution of glutaraldehyde in 0n1  phosphate buffer (pH 7n2)
and fixed overnight at room temperature. After washing
with the same buffer, some blocks were postfixed in 1 %
osmium tetroxide in the same buffer for 2 h at room
temperature. Other sample blocks for ultraviolet microscopy
were not postfixed in 1 % osmium tetroxide. Samples were
then washed with buffer and dehydrated through a graded
ethanol series. They were then embedded in epoxy resin.
For standard light microscopy and polarizing light
microscopy, radial and tangential sections (1 µm thick)
were cut with a glass knife on an ultramicrotome (Ultracut
J ; Reichert, Austria). Sections were stained with a 1 %
aqueous solution of safranin or a 1 % aqueous solution of
gentian violet. They were observed with a light microscope
(BHS-2 ; Olympus, Japan) and a polarizing microscope
(BHS-751P ; Olympus).
For determination of the extent of lignification, radial
and tangential sections (1 µm thick) were cut with a glass
knife, placed on quartz slides and mounted in glycerine with
quartz coverslips. Photographs were taken under an
ultraviolet (UV) microscope (MPM-800 ; Carl Zeiss, Germany) at a wavelength of 280 nm with a bandwidth of 5 nm
(Fukazawa, 1992 ; Sano and Nakada, 1998).
For transmission electron microscopy, ultra-thin tangential sections (70 nm thick) were cut with a diamond knife
and stained with uranyl acetate and lead citrate. The ultrathin sections were then observed with a transmission electron
microscope (JEM-100C ; JEOL, Japan) operated at an
accelerating voltage of 100 kV.
432
Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
RESULTS
Figure 1 shows a differentiating vessel element, which was
located approx. 500 µm from the cambium, and the other
elements around it. In this region, the birefringence of the
cell walls of the axial xylem cells was detectable with a
polarizing light microscope (Fig. 1 B). This birefringence
indicated that the deposition of secondary walls had started
in these cells (Abe et al., 1997). Ray parenchyma cells, which
were oriented transversely, were also observed. The parenchyma cells with ray-vessel pits (arrowheads in Fig. 1 A
and B) were considered to be contact cells. The birefringence
of transverse walls of contact cells was clearly visible
(arrows in Fig. 1 B). Several thin-walled ray parenchyma
cells (asterisks in Fig. 1 A and B) were observed between
contact cells. These cells were considered to be isolation
cells because they had no ray-vessel pits. Serial, radial, semithin sections revealed that isolation cells were longer than
contact cells and that there were many intercellular spaces
between isolation cells (data not shown). The walls of
isolation cells were not birefringent under the polarizing
light microscope, and indication that the deposition of
secondary walls had not yet started in these cells.
Figure 2 shows the ultrastructure of cell walls in
differentiating ray parenchyma cells in tangential section
of a sample at a similar stage to that shown in Fig. 1. The
contact cells formed ray-vessel pits (Fig. 2 A). The thick
secondary walls of contact cells and vessel elements had
already formed. The contact cells were present at the upper
and lower margins of the ray. By contrast, the isolation cells
had thin cell walls (Fig. 2 B), and the thickness was similar
to that of the cell walls of cambial cells.
The UV absorption of cell walls at 280 nm was apparent
on radial sections of a region similar to that shown in Fig. 1.
F. 4. UV photomicrograph showing a tangential section of a
differentiating vessel element and other elements around it at a similar
stage to that shown in Fig. 3. Arrows indicate cell walls of contact cells
and vessel elements that absorbed UV light. Arrowheads indicate cell
walls of isolation cells (asterisks) that did not absorb UV light. V,
Vessel element ; F, wood fibre. Bar l 10 µm.
F. 3. UV photomicrograph showing a radial section of a
differentiating vessel element (V) and other elements around it at a
similar stage to that shown in Fig. 1. UV absorption of cell walls is
visible in vessel elements (large arrows) and contact cells (small
arrows). The cell walls of wood fibres absorbed UV light very weakly
(arrowheads). The left side of this micrograph corresponds to the outer
side of the tree. Bar l 50 µm.
The UV absorption of cell walls was visible first in vessel
elements (large arrows in Fig. 3). This absorption indicated
that the lignification of these cell walls had begun. UV
absorption by the secondary walls of contact cells was as
strong as that by cell walls of vessel elements (small arrows
in Fig. 3). By contrast, the cell walls of wood fibres absorbed
UV light only very weakly (arrowheads in Fig. 3).
Figure 4 shows differentiating axial elements and ray
parenchyma cells at a similar stage to that shown in Fig. 3
on a tangential section. The UV absorption of cell walls of
vessel elements and contact cells was stronger than that of
wood fibres in the vicinity of vessel elements (arrows in Fig.
4). Strong UV absorption was observed at the middle
lamella in contact cells, but no UV absorption was detected
Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
433
F. 5. Electron micrographs showing tangential sections of differentiating ray parenchyma cells at distances from the cambium that ranged from
approx. 1500–2000 µm. Contact cell with a protective layer (arrows) (A) and an isolation cell without an isotropic layer (B). V, Vessel element ;
CC, contact cell ; IC, isolation cell. Bars l 2 µm.
at pit membranes of ray-vessel pits. Moreover, the cell walls
(arrowheads in Fig. 4) of isolation cells (asterisks in Fig. 4)
did not absorb UV light.
Various terms have been used for the additional layers of
parenchyma cells. Schmid (1965) names these layers the
‘ protective layer ’. Later Chafe (1974) and Chafe and
Chauret (1974) used the term ‘ protective layer ’ in the case
of vessel-associated ray parenchyma cells and the term
‘ isotropic layer ’ in the case of axial parenchyma cells and
non-vessel-associated ray parenchyma cells. By contrast,
Fujii, Harada and Saiki (1980, 1981) proposed both layers
be called the ‘ amorphous layer ’ because there appeared to
be little difference between them in their original chemical
composition and fine structure. In this report, we use
separate designations for the protective layer and the
isotropic layer because these layers appear to have different
functions. Figure 5 shows ray parenchyma cells at distances
from the cambium that range from approx. 1500–2000 µm.
The protective layer was formed (arrows in Fig. 5 A) in the
contact cells after the deposition of the secondary wall. In
agreement with the observations reported by Chafe (1974),
this layer was not limited only to the side where vessel
elements were located, but was also present on the opposite
side. However, the protective layer on the opposite side was
much thinner than that on the side on which vessel elements
were located. Czaninski (1973) and Chafe (1974) reported
that the deposition of the protective layer began at the site
of secondary wall thickening. Figure 6 shows ray parenchyma cells at a similar stage to that shown in Fig. 5 in
tangential section. The protective layer of contact cells
(arrows in Fig. 6) and pit membranes of ray-vessel pits
(arrowheads in Fig. 6) did not absorb UV light. The
434
Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
F. 6. UV photomicrograph showing a tangential section of
differentiating ray parenchyma cells at a similar stage to that shown in
Fig. 5. No UV absorption by the protective layer (arrows) and pit
membranes of ray-vessel pits (arrowheads) is evident in the contact
cells. UV absorption of cell walls is evident in the isolation cells
(asterisks). V, Vessel element ; F, wood fibre. Bar l 10 µm.
secondary walls of isolation cells absorbed UV light
(asterisks in Fig. 6), but no isotropic layer was apparent in
isolation cells (Fig. 5 B).
DISCUSSION
The cell walls of isolation cells in the regions shown in Figs
1 and 2 were primary walls because they were thin and
exhibited no birefringence under polarized light. By contrast,
contact cells formed secondary walls at approximately the
same time as vessel elements. Chafe (1974) observed that the
initiation of the deposition of the secondary wall in vessel
elements occurred slightly earlier than in contact cells. At
this stage, in our samples, UV absorption of cell walls of
vessel elements and contact cells appeared to be stronger
than that of the wood fibres in the vicinity of vessel elements
(Figs 3 and 4). By contrast, no UV absorption of cell walls
of isolation cells was detected (arrowheads in Fig. 4). These
results indicated that the lignification of the cell walls of
contact cells and vessel elements began earlier than that of
wood fibres and isolation cells. Thus, the timing of the
formation of the secondary walls, including lignification, of
the contact cells was similar to that of the vessel elements
with which they were directly connected. The similarity in
timing of differentiation between contact cells and vessel
elements suggests that differentiation of contact cells might
be influenced by vessel elements through ray-vessel pits. By
contrast, the differentiation of isolation cells, which make
no direct pit-mediated connections with adjacent vessel
elements, might not be directly affected by the differentiation
of vessel elements.
Our results indicate that contact cells and vessel elements
began to form lignified secondary walls earlier than the
wood fibres in the vicinity of the vessel elements. By
contrast, the pit membranes of ray-vessel pits and the
protective layers in contact cells were not lignified soon after
deposition of secondary walls (Fig. 6). Barnett, Cooper and
Bonner (1993) suggested that maintenance of an apoplastic
pathway around a lignified cell with living protoplast was
the primary function of the protective layer. The contact
cells might specialize in the translocation of water and
materials between ray and vessel. In contrast, the differentiating isolation cells with unlignified tangential walls might
become pathways for temporary radial continuity between
the phloem and xylem. In this way, easy radial translocation
is maintained through the rows of isolation cells.
We observed no isotropic layer in isolation cells after
deposition of secondary walls (Fig. 5 B). Chafe (1974)
reported that isolation cells of Populus tremuloides formed
secondary walls and isotropic layers, all of which became
lignified, within a single growth season. By contrast, Fujii,
Harada and Saiki (1979) reported that the cell wall in
differentiated isolation cells of Populus maximowiczii consisted of only a cellulosic secondary wall. The reason for the
discrepancy among species that belong to the same genus is
that the isotropic layer might be only partially deposited in
the isolation cells and, thus, the occurrence of an isotropic
layer in ultra-thin sections would depend on the place in
which the isolation cells were sectioned. In addition, there is
the possibility that isolation cells had not yet begun to form
the isotropic layer in the present study because our specimens
were collected in July. Chafe (1974) reported that deposition
of the protective layer began after completion of the
deposition of the cellulosic secondary wall. However, the
isotropic layer might not be fully developed soon after
completion of the deposition of the cellulosic secondary wall
in isolation cells.
Our results indicate that the structure of the ray
parenchyma cells of the secondary xylem differs considerably
between contact cells and isolation cells, which are derived
from the same cambial ray cells. Therefore, these ray
parenchyma cells are formed by different processes of
differentiation, depending on whether or not they make
direct connections, through pits, with adjoining vessel
elements. Our observations indicate the importance of
Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
neighbouring cells in controlling xylem differentiation.
Expanding vessels might send some signals to adjoining
cells through the plasma membrane and primary wall.
These signals determine the direction of differentiation of
ray parenchyma cells. Although the nature of these putative
signals remains to be characterized, some specific proteins
or polysaccharides of the cell wall, which trigger cell
differentiation, might be secreted and reach future contact
cells from differentiating vessel elements. In contrast, future
isolation cells might not receive such signals. Therefore,
future contact cells and isolation cells might differ originally
in both structure and the nature of the primary wall and
plasma membrane. They might have different numbers and
distributions of plasmodesmata with different structures
since it has been proposed that plasmodesmata play an
important role in the fate of xylem cells (see Gunning, 1978 ;
Barnett, 1981, 1982). More detailed information about
plasmodesmata in cambial ray cells is needed to clarify the
mechanisms of differentiation of xylem ray cells.
Cambial cells and their derivatives have been proposed to
serve as a model system for studies of cytodifferentiation in
secondary tissues in situ because it is possible to follow the
process of differentiation in a radial direction (Abe et al.,
1995 ; Chaffey, Barlow and Barnett, 1997 ; Chaffey, Barnett
and Barlow, 1997 ; Funada et al., 1997). The successive
differentiation of contact cells and isolation cells, both of
which are derived from cambial ray cells, is also a useful
model system for studies of the mechanism of control of the
formation and lignification of secondary walls in secondary
xylem.
A C K N O W L E D G E M E N TS
This work was supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education, Science
and Culture, Japan (Nos 08760157 and 09760152) and from
the Japan Society for the Promotion of Science (no. JSPSRFTF 96L00605).
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