ULTRASTRUCTURAL CHANGES IN THE CELL WALLS OF CAMBIAL DERIVATIVES DURING WOOD FORMATION IN INDIAN ELM (HOLOPTELEA INTEGRIFOLIA) - Brill
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
IAWA Journal, Vol. 33 (4), 2012: 403– 416
ULTRASTRUCTURAL CHANGES IN THE CELL WALLS OF
CAMBIAL DERIVATIVES DURING WOOD FORMATION IN
INDIAN ELM (HOLOPTELEA INTEGRIFOLIA)
Karumanchi S. Rao1,*, Yoon Soo Kim2 and Pramod Sivan1
Summary
Sequential changes occurring in cell walls during expansion, secondary
wall (SW) deposition and lignification have been studied in the differ-
entiating xylem elements of Holoptelea integrifolia using transmission
electron microscopy. The PATAg staining revealed that loosening of the
cell wall starts at the cell corner middle lamella (CCML) and spreads to ra-
dial and tangential walls in the zone of cell expansion (EZ). Lignification
started at the CCML region between vessels and associated parenchyma
during the final stages of S2 layer formation. The S2 layer in the vessel
appeared as two sublayers,an inner one and outer one.The contact ray
cells showed SW deposition soon after axial paratracheal parenchyma had
completed it, whereas noncontact ray cells underwent SW deposition and
lignification following apotracheal parenchyma cells. The paratracheal
and apotracheal parenchyma cells differed noticeably in terms of propor-
tion of SW layers and lignin distribution pattern. Fibres were found to
be the last xylem elements to complete SW deposition and lignification
with differential polymerization of cell wall polysaccharides. It appears
that the SW deposition started much earlier in the middle region of the
fibres while their tips were still undergoing elongation. In homogeneous
lignin distribution was noticed in the CCML region of fibres.
Key words: Cambial derivatives, Holoptelea integrifolia, lignification,
secondary wall deposition, wood formation.
Introduction
During secondary xylem differentiation, thin-walled precursor cambial cells undergo
dramatic transformation including cell expansion, secondary wall (SW) deposition,
bordered pit formation, lignification and programmed cell death (Savidge 1996).
There is considerable variation in the structure and chemical composition between the
primary and SW of xylem elements. The primary wall is mainly composed of cellulose,
hemicelluloses and pectic polysaccharides whereas the SW is composed of cellulose,
hemicellulose and lignin. Therefore, SWs of woody plants are considered composite
1) Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat,
India.
2) Department of Forest Products and Technology, Chonnam National University, Gwangju
500-757, South Korea.
*) Corresponding author [E-mail: kayesrao@yahoo.com].
Downloaded from Brill.com12/07/2021 12:24:59PM
via free access404 IAWA Journal, Vol. 33 (4), 2012
materials consisting of a tight mixture of polysaccharides and polyphenols organized
around the crystalline and amorphous cellulose microfibrillar (CMFs) network. In this
composite, cellulose represents 40–50% of dry weight which provides the precise and
specific anisotropic organization of the thick SW because of its supra-molecular
microfibril architecture (Ruel et al. 2006). Despite overall similarities there are also
substantial differences in structure and composition of cell walls between different
trees growing in temperate and tropical climates. Considerable work has been car-
ried out on ultrastructural aspects of wood formation in softwood species (Donaldson
2001; Putoczki et al. 2008). The process of cell differentiation during wood formation
is not fully understood particularly in hardwood species which have a complex wood
structure, in particular in tropical species. Therefore, in the present study we selected
the Indian elm tree (Holoptelea integrifolia), a tropical, deciduous, diffuse porous tree
with storied cambium. Holoptelea integrifolia belongs to the family Ulmaceae, having
15 genera and about 200 species, distributed over tropical and temperate regions of
the Northern Hemisphere including the Indian peninsula to Indo China and Srilanka
(Sharma & Singh 2012). The Indian elm is a large deciduous tree, distributed throughout
the greater part of India up to an altitude of 2,000 feet. The anatomical structure and
tissue composition of wood in Holoptelea integrifolia has been reported by Pearson and
Brown (1932). The wood is considered to be of commercial importance and is used for
indoor building purposes, low cost furnitures, cabinet works, plywood, packing cases,
match boxes and for paper pulp. It is also a good fuel wood with high calorific value.
Rao et al. (2011) reported the development of derivatives from the storied cambium
in Holoptelea. This study describes the sequential ultrastructural changes occurring in
the cell wall during cell expansion, SW deposition and lignification in differentiating
xylem elements.
Materials and Methods
The material used for this study was collected from the main trunk of three Holoptelea
integrifolia trees growing in the Jambughoda forest of Gujarat State, India during Sep-
tember 2004 when the cambium was active. After chiseling away the 3 × 5 cm wide
strips of bark with outer cambial cell layers, ice-cold Karnovsky’s fixative solution in
Sorensen’s buffer at pH 7 (Karnovsky 1965) was applied to the exposed inner cambial
zone cell layers and the soft differentiating xylem tissue on the main trunk. Tangential
strips (10 × 5 mm, 1–2 mm thick) of xylem containing both differentiating layer and
mature tissue were cut using sharp double-edge blades from the exposed surface of
the trunk. About 8 such stripes were collected from the trunk surface and immediately
dropped into ice-cold fixative. After reaching the laboratory, the samples were diced
into 1–2-mm pieces with a sharp blade and put into fresh fixative solution followed by
secondary fixation in 2% osmium tetroxide each for overnight duration at 4 °C. After
a cold buffer wash, the tissues were dehydrated in graded series of cold acetone and
embedded in Spurr’s epoxy resin (Spurr 1969).
Semi-thin sections were cut on RMC Powertome X ultramicrotome using a glass
knife and stained with toluidine blue and photographed with Image Pro-Plus. For
transmission electron microscopy (TEM), ultra-thin transverse sections were taken
Downloaded from Brill.com12/07/2021 12:24:59PM
via free accessRao et al. — Ultrastructure of developing cell walls 405
using a diamond knife, placed on nickel and gold grids and subjected to periodic acid-
theocarbohydrazide-silver proteinate (PATAg, Thiery 1967) and potassium perman-
ganate (Donaldson 1992) staining for visualization of cell wall polysaccharides and
lignin, respectively. Sections were examined in a Jeol 1212 TEM at 80 kV.
Immuno-gold labeling was carried out according to Joseleau and Ruel (1997) using
polyclonal antibodies raised in rabbit against non-condensed syringyl/guaiacyl lignin
(S/G 5/1DHP). Ultra-thin sections, mounted on uncoated nickel grids (300 mesh),
were incubated in the antiserum (primary antibody) diluted in PBS buffer containing
0.05% tween. Sections were then labelled with goat-anti-rabbit antiserum (secondary
antibody) conjugated to colloidal gold (10 nm particle size). Sections were examined
with a Jeol 1212 TEM at 80 kV after counterstaining with 4% uranyl acetate. Control
samples were treated as described above, except that either the primary antibody was
omitted from the incubation solution or pre-immune rabbit serum was used instead of
the primary antibody.
Results
The sequential developmental stages of xylogenesis from cambial derivatives towards
mature xylem were identified from transverse semi-thin sections of xylem stained
with toluidine blue (Fig. 1A). The zone of cell expansion (EZ) that started from the
centripetal end of the cambial zone (CZ) was identified by the presence of randomly
arranged larger cells distinct from the radially seriated cambial cells. The differentiating
cells initially undergo radial expansion followed by tangential expansion as they are
moved away from the cambial zone.The radial seriation found in the cambial zone is
lost among cambial derivatives as they undergo tangential expansion. Cell specializa-
tion started well before the deposition of SW material. Among all differentiating cells,
vessels in particular appear largest in transverse diameter followed by axial parenchyma
and fibres. The cells in the EZ showed thin primary walls, often appearing wavy due
to the pressure caused during trimming of the samples. The cell walls of EZ appeared
similar in electron density to those of the cambial zone following PATAg staining.
These cells showed a distinct thin primary wall layer while cell corners were apparently
loosely organized and electron translucent (Fig. 1B). The loosely organized CMFs were
more prominent in the cells differentiating into fibres undergoing intrusive elongation.
Fibre initials were also recognized by their narrow lumen area, distinct from the sur-
rounding cells and representing transversely cut portions of the intrusively growing cell
ends (Fig. 1B). The primary walls in the cells of EZ revealed electron translucent areas
throughout the middle lamellae region following PATAg reaction indicating loosely
organized middle lamellae surrounded by primary walls (Fig. 1C). Such loose organi-
zation of cell walls continues even during the initial stage of SW deposition (Fig. 1D).
However, the primary walls soon became compact and appear uniformly dense when the
SW deposition is in progress, while the CCML showed electron translucent areas.
Among the differentiating xylem elements, vessel member initials are the first cells
to deposit SW layers that appear as loosely organized CMFs against the primary wall
(Fig. 1E). Interestingly, the initial deposition of SW layers starts on the centripetal end
(towards xylem) of the vessel primary wall and appears to spread gradually towards
Downloaded from Brill.com12/07/2021 12:24:59PM
via free access406 IAWA Journal, Vol. 33 (4), 2012
➤
Figure 1. Light (A) and transmission electron micrographs (B–I) of transverse sections of dif-
ferentiating wood from the main trunk of Holoptelea integrifolia. – A: Differentiating xylem
close to cambial zone (CZ) showing zones of expansion (thick vertical bar) and maturation
(thin vertical bar). Toluidine blue staining. – B: Group of xylem elements from EZ with pri-
mary walls. Note the electron translucent CCML (arrows) close to smaller cells representing
fibre tips (asterisks). PATAg staining. – C: Expanding xylem elements with their primary walls
of adjacent cells separated by electron translucent (arrows) middle lamella. PATAg staining. –
D: Differentiating fibre showing initiation of SW deposition (arrowhead). Note the adjacent fibre
tip (asterisk) showing no sign of SW deposition. Arrows indicate electron translucent areas in
the primary wall. PATAg staining. – E: Portion of primary wall of differentiating vessel showing
initial stages of SW deposition (horizontal line). Note the vessel associated parenchyma cells
Downloaded from Brill.com12/07/2021 12:24:59PM
via free accessRao et al. — Ultrastructure of developing cell walls 407
the centrifugal end (towards cambium). The early deposited CMFs of the SW appear
more compactly arranged than the recently deposited ones (Fig. 1F). The vessel as-
sociated parenchyma did not show SW deposition at the time when the SW deposi-
tion started in the vessels. The completely deposited SW appeared three-layered with
the S2 layer having two sublayers, an inner and outer one, followed by a thin S3 layer
(Fig. 1G). Lignin deposition began in vessels at the time of S2 layer deposition. The
KMnO4 staining indicated that traces of lignin deposition first occurred in the middle
lamella region between the vessel and associated parenchyma cells (Fig. 1H). The
CCML and primary wall region of the vessel wall adjacent to the ray parenchyma also
showed electron dense areas. With progress of SW deposition, lignin spreads radi-
ally among wall layers from the middle lamella towards the cell lumen. Lower lignin
levels were noticed in the S1 layer whereas the S2 layer showed distinct differences in
lignification pattern between its sublayers. The inner sublayer was characterized by
more lignin than the outer one (Fig. 1I). The thin S3 layer also showed more lignin
than the other SW layers.
Vessel associated parenchyma cells were the second xylem element type that un-
derwent SW deposition. The SW deposited in these cells was thinner than those of the
adjacent vessels except near the cell corners (Fig. 1I). The KMnO4 staining revealed
electron dense areas in the middle lamella region towards the vessel wall while the
wall shared with the adjacent fibre undergoing SW deposition was electron translu-
cent. Among SW layers, S1 and S2 layers of paratracheal parenchyma showed more or
less similar thickness while lignin proportion was higher in the S2 and thin S3 layer
(Fig. 2A). Randomly distributed electron translucent regions appeared in the CCML
region between vessels and associated parenchyma indicating in homogeneous lignin
distribution.
The contact rays showed SW deposition and subsequent lignification after the
completion of cell wall lignification in the adjacent vessel associated parenchyma cells
(Fig. 1G), whereas the SW deposition in non-contact rays occurred later, and these
cells were still without SW when the adjacent fibres were undergoing the final stages of
S2 layer formation. Due to the elongation of developing fibres, the SW deposition starts
in the middle region of the cell and then spreads to the cell ends. The process of SW
deposition and lignification in fibres ends only after ray and apotracheal parenchyma
←
(P) with primary walls. PATAg staining. – F: Differentiating vessel (V) showing SW deposition
(horizontal line). Arrow indicates recently deposited, loosely organized CMFs compared to the
compactly packed microfibrils close to the primary wall. Note the deposition of SW material in
the associated parenchyma cells (P). PATAg staining. – G: Vessel and associated parenchyma
cells (P) after completion of SW deposition. Short lines indicate the sublayers in the S2 layer of
SW. Note the initiation of SW deposition (arrows) in the adjacent ray cells (R). PATAg staining. –
H: Portion of vessel wall (V) showing early stages of lignin deposition in the form of electron
dense small bodies in the middle lamellae (arrows). KMnO4 staining. – I: Walls of vessel (V) and
associated parenchyma cells (P) showing complete lignification. Note the outer (white asterisk)
and inner sublayers (black asterisk) of the S2 layer in the vessel SW. KMnO4 staining. — Scale
bars: A = 50 µm; B, G, I = 2 µm; C, D, E, H = 1 µm; F = 0.5 µm.
Downloaded from Brill.com12/07/2021 12:24:59PM
via free access408 IAWA Journal, Vol. 33 (4), 2012
Figure 2. Transmission electron micrographs of transverse sections of differentiating xylem in
Holoptelea integrifolia. – A: Inhomogeneous distribution of lignin (arrow) in the CCML be-
tween vessel associated parenchyma (P) and fibre. Note the fibre (F) with developing SW.
KMnO4 staining. – B: Apotracheal parenchyma cells (P) showing completion of SW deposition.
Note the fibre (F) with incomplete SW deposition. Arrows indicate simple pits on the adjacent
parenchyma cells. PATAg staining. – C: Apotracheal parenchyma (P) with adjacent differentiating
fibres (F). Note the parenchyma with completely lignified SW while the fibres are without S3
layer. KMnO4 staining. – D: Ray cell (R) showing SW deposition (arrow) in the wall adjacent to
another ray cell while there is still no SW deposition in the wall adjacent to a fibre (arrowhead).
Note the fibres with S1 and S2 layers. KMnO4 staining. – E: Ray cells adjacent to fibres showing
SW deposition. Note the lower ray cells with completely formed SW while the upper one shows
incompletely formed SW (arrow). The arrowhead indicates the portion of the tangential wall with
plasmodesmatal connections. PATAg staining. – F: Ray cell (R) with completely lignified radial
walls (arrow). Note the SW of adjacent fibre (F) with no S3 layer. KMnO4 staining. — Scale
bars: A, C–F = 1 µm; B = 2 µm.
→
Figure 3.Transmission electron micrographs of transverse sections of differentiating xylem
in the Holoptelea integrifolia. – A: Portion of the cell wall between parenchyma cell (P) and
fibres (F). Note the fibre with S1, S2 layers while parenchyma cells show no sign of SW. PATAg
staining. – B: CCML between fibres (arrow) showing electron translucent areas indicating
loosely organized CMFs. Note the incompletely formed S2 layer showing small angle CMFs in
Downloaded from Brill.com12/07/2021 12:24:59PM
via free accessRao et al. — Ultrastructure of developing cell walls 409
comparison to S1 layer. Bars represent the extent of S1 and S2 layer. PATAg staining. – C: Fibre
wall showing electron dense CCML (arrow). Note the S2 layer in the final stages of deposition.
PATAg staining. – D: Lignin deposition in the cell walls of fibres (F) and adjacent apotracheal
parenchyma (P). Note the lignin distribution (arrow) close to parenchyma wall. KMnO4 stain-
ing. – E: Group of fibres showing lignin distribution in the CML and outer region of S2 layer
(arrows). Note the absence of S3 layer in the fibre. KMnO4 staining. – F: Fibre adjacent to ray
cell showing lignified SW layers. Note the intense lignin deposition in the S3 layer (arrow).
KMnO4 staining. – G: Vessel (V) and associated parenchyma (P) showing the presence of
condensed GS type lignin epitopes in the middle lamella and SW layers. – H: Vessel wall (V)
showing heavy labeling of inner S2 layer (vertical line); P = parenchyma cell. — Scale bars:
A, D–H = 1 µm; B = 0.5 µm; C = 2 µm.
Downloaded from Brill.com12/07/2021 12:24:59PM
via free access410 IAWA Journal, Vol. 33 (4), 2012
cells have developed lignified SWs. The SW of apotracheal parenchyma appears rela-
tively thin and uniform with simple pits (Fig. 2B). No apparent difference was found
in the lignin distribution between S1 and S2 wall layers of parenchyma cells while a
thin S3 layer was characterized by high lignin deposition (Fig. 2C).
In non-contact ray cells, the SW layer may be laid down first on the radial walls com-
mon between the adjacent ray cells (Fig. 2D). Similar to vessels, ray cells also showed
SW deposition extending radially from xylem towards cambium (Fig. 2E). The area
of a ray cell’s tangential wall with plasmodesmatal connections and no SW deposition
may develop into a pit membrane of a simple pit (Fig. 2E). The fibres remain with
an incomplete SW even after lignification of SW layers in the adjacent ray cells. The
radial wall between adjacent ray cells was thicker than the wall shared with adjacent
fibres (Fig. 2F). The SW layering was not apparent in ray cells.
The deposition of SW in fibres started prior to that of ray and apotracheal parenchyma
cells (Fig. 3A). It appears that the deposition of the thick S2 layer in elongated fibres
takes longer than that of other cells. Hence fibres are found to be the last elements in
the xylem to complete the process of SW deposition and lignification in Holoptelea.
Fibre cells which were 15–20 cells radially away from the cambial zone showed the
initiation of SW deposition. In transverse view, larger diameter cells representing
middle portion of the differentiating fibres showed more SW material. The adjacent
narrow diameter cells representing intrusively growing terminal portions of fibres had
relatively less SW. The CCML region of fibres remained loosely organized until the
partial deposition of the S2 layer (Fig. 3 B) and became condensed following completion
of S2 layer deposition (Fig. 3 C). The newly formed S1 and S2 layers of SW had loosely
organized CMFs, gradually changing in orientation. After the complete deposition of
SW layers, the S1 layer had CMFs running at a right angle to the cell axis while S2 was
characterized by helicoidal arrangement of CMFs (Fig. 3B). The lignification of fibres
started at the CCML region when the adjacent apotracheal parenchyma had completed
wall deposition and lignification (Fig. 3D). The CCML region showed both electron
translucent and opaque regions indicating heterogeneous lignin distribution (Fig. 3D).
Subsequently, lignin deposition spread towards the inner wall layers (Fig. 3E). The
SW layers showed distinct differences in lignin distribution pattern between its three
layers, the S1 layer presents more electron translucent regions than the other regions
of SW indicating that this layer had a far lower lignin concentration (Fig. 3F). The last
formed thin S3 layer in fibres showed more lignin compared to the S2 layer (Fig. 3F).
Electron microscopic analysis of sections incubated with lignin SG type antibodies
revealed that the cell walls of apotracheal parenchyma showed relatively weak labeling
whereas heavy labeling was noticed in the SW of vessels (Fig. 3G). The distribution
of epitopes were mostly confined to the CCML and inner regions of the S2 layer while
S1 and S3 layers were weakly labeled (Fig. 3H).
Discussion
The pattern of SW deposition and lignification has been followed in both the axial and
radial elements of Holoptelea using PATAg and KMnO4 staining methods under TEM.
Downloaded from Brill.com12/07/2021 12:24:59PM
via free accessRao et al. — Ultrastructure of developing cell walls 411
The first visual change in the cell wall structure in the zone of expansion (EZ) is the
loosening of wall materials at the cell corners and its extension to radial and tangential
walls. Similarly, the middle lamella region of cells in EZ becomes completely electron
translucent following PATAg reaction indicating structural disorganization of wall
polysaccharides. The primary walls of cambial derivatives have a high content of cel-
lulose and methylated pectins (Catesson 1990; Catesson et al. 1994) and the changes in
the amount and composition of wall polysaccharides plays an important role in cell ex-
pansion (Catesson & Roland 1981; Barnett 1992). The presence of substantial amounts
of acidic pectin arising from de-esterification of methylated pectins is a characteristic
feature of cambial xylem derivatives and allows greater radial growth (Catesson 1994).
Therefore, loosely organized middle lamellas in the expanding xylem derivatives of
Holoptelea can be due to the accumulation of acidic polysaccharides associated with
cell wall expansion and elongation growth.
In Holoptelea, though all the elements in the axial system show heterogeneity in tim-
ing and proportion of SW layers, no apparent difference was found in the pattern of SW
deposition. Samuels et al. (2006) proposed that a dramatic shift occurs at the transition
between primary and SW production.The present study shows that the SW deposition
starts in the vessel elements followed by associated parenchyma. A similar pattern has
been reported in poplar (Murakami et al. 1999) and in Japanese oak (Yoshinaga et al.
1997). The initial deposition of CMFs begins from the wall facing mature xylem and
spreads towards the opposite wall of the vessel. The CMFs deposition is believed to
be controlled by cortical microtubules. During the final stage of the radial expansion
phase, cortical microtubules assume a helical array, wrapping transversely around the
late radially expanding cells in a pattern that strongly correlates with the microfibril
deposition of first SW layers (Funada et al. 2000; Chaffey & Burlow 2002; Samuels
et al. 2006). The variation found in the orientation of CMFs is in agreement with
previous reports. The early deposited CMFs of SW appear more compactly arranged
than the recently deposited ones. The cellulose chains are less tightly packed on the
surface than in the interior of CMFs (Newman 1994) and, therefore, the inner structure
of the wood cellulose microfibril differs from the structure of its outer surface layer
(Xu et al. 2007).
TEM study reveals that the SW deposition initiates at the middle region of fibre
walls even while the tip is undergoing an elongation process. In addition to symplastic
growth, fibres have intrusive growth, with local cell wall biosynthesis in the fibre tips
(Dejardin et al. 2010). There are different opinions regarding the timing of SW deposi-
tion in xylem elements. Theoretically, the SW is deposited after the primary wall ceases
to grow in surface (Esau & Charvet 1978). In developing fibres of bamboo, the SW
formation occurs only after fibres have stopped elongating (He et al. 2000). In contrast,
SW deposition has been found in elongating cotton fibres (Schubert et al. 1973) and in
Dendrocalamus asper (Gritsch & Murphy 2005). These authors postulated that elonga-
tion of primary walls and SW deposition are not separate events and a substantial amount
of SW is deposited before elongation stops. A similar pattern of early SW deposition
prior to cessation of radial expansion has also been reported in conifer tracheids (Abe
et al. 1997). The SW deposition in elongating fibres of Holoptelea also suggests that
Downloaded from Brill.com12/07/2021 12:24:59PM
via free access412 IAWA Journal, Vol. 33 (4), 2012
cell expansion and SW deposition are not independent events, rather they can occur
simultaneously. Another conspicuous variation in pattern of SW deposition found in the
fibres of Holoptelea is that, although it begins earlier than in parenchyma cells, the S3
layer deposition proceeds only after the completion of SW deposition and lignification
of rays and all other axial elements. This differential polymerization of wall material
might be due to the longer duration of elongation growth and to the thicker SW layers
in fibres, and hence they are the last xylem elements to complete SW deposition and
lignification.
A clearly distinguishable difference has been noticed in the PATAg staining reactivity
between compound middle lamella (CML) and SW layers of the differentiating xylem
of Holoptelea. On the basis of a number of studies CMFs orientation in a variety of
taxa such as Arabidopsis, poplar and loblolly pine Samuels et al. (2006) support a view
that cellulose synthesizing enzymes for the SW formation are different from cellulose
synthases (CesAs) used in primary wall formation. Therefore, the heterogeneous nature
of interaction between CMFs and other wall polysaccharides could result in differential
staining intensity in primary and SW following PATAg staining.
Remarkable differences have been found in the proportion of SW layers in axial
elements of xylem. In vessels, the thickest S2 layer has two sublayers deposited in
more or less equal proportion. The paratracheal parenchyma cells show overall reduc-
tion in SW deposition; however, they have a distinct, thick S2 layer and thin S1 and S3
layers. On the other hand, fibre SW was characterized by a greater S2 wall thickening
and relatively thin S3 layer. This variation in thickness of SW among different xylem
elements might be related to the speed and extent of the cell differentiation process. In
Holoptelea, the vessel elements and axial parenchyma are storied as they are derived
from the storied cambium. The vessels undergo significant expansion both in radial and
tangential directions before SW formation and lignification. On the other hand, fibre
initials show limited lateral expansion but elongate vertically to attain lengths several
times longer than fusiform cambial cells (Rao et al. 2011).
In vessels, when the S2 layer formation progresses, lignin deposition starts at the
CCML region between the vessel and paratracheal cells and contact ray cells. On the
other hand, axial parenchyma and fibres show lignification at a later stage. In Magnolia
and Populus lignification was first observed in the vessel walls particularly at cell corners
and middle lamellae (Terashima et al. 1986a, b). Murakami et al. (1999) also noticed
that the lignification starts in vessel elements and contact rays while fibres lignify later.
It has been suggested that non-lignifying xylem parenchyma is the source of peroxidase
necessary for the polymerization of cinnamoyl alcohols in the secondary cell walls of
dying/dead xylem vessels (Barcelo 2005). In Holoptelea, the pattern of lignification
is similar to that reported in other species. Lignin starts depositing at the CCML and
spreads to the primary wall after the start of SW formation, while lignification of the
SW begins when SW formation is complete as judged by the presence of a S3 layer
(Donaldson 1992; Ruel et al. 2006). Intensive peroxidase labeling at the cell corner
regions of young xylem elements before SW deposition has been reported in poplar
which implies that peroxidases are incorporated into the developing primary cell wall
(Kim et al. 2002; Grunwald et al. 2002). These authors suggested that peroxidase may
Downloaded from Brill.com12/07/2021 12:24:59PM
via free accessRao et al. — Ultrastructure of developing cell walls 413
be involved in the preferential onset of lignification in these wall areas soon after the
deposition of first SW polysaccharides. Lignification is more in evidence in the cell
corners and CML region. According to Fujino and Itoh (1998) the highly porous carbo-
hydrate matrix in the middle lamellae and primary wall allows greater lignin deposition
by space filling than the densely packed secondary cell wall. Rausel and Lim (1995)
suggested that the more ordered CMFs and tighter space in the middle lamellae favour
the linkage with condensed lignin and further endwise polymerization leading to a
non-condensed type of lignin. Our immunolabelling study also shows the presence of
a non-condensed GS type lignin in the inner SW layer of the vessel elements. In poplar,
condensed lignin subunits are localized in cell corners of xylem derivatives prior to
S1 layer formation while non-condensed lignin units are labeled in the inner wall layers
(Grunward et al. 2002). According to Ruel et al. (2006) the first deposited condensed
lignin is more likely to interact with hemicelluloses coating the CMF surface and these
could be the site of lignin-carbohydrate complexes which serve as anchors for lignin
polymerization.
According to Higuchi (1997) and Terashima (2000), the developmental sequence
of lignin deposition in relation to pectin/polysaccharide matrix in softwood tracheids
and hardwood fibres is principally the same. In Holoptelea, the lignification shows a
progressive advance towards the cell lumen with the corresponding progress in the
deposition of S2 and S3 layers. The S1 layers show less lignin compared to the S3 layer.
In the tracheid SW of Pinus, the lignin distribution in the S1 region is reported to have
a distinct variation in its concentration at outer and lower regions (Maurer & Fengel
1991; Donaldson 1992). It is obviously difficult to visualize cell wall changes occur-
ring in a large population of different elements during xylogenesis, particularly when
cell division and differentiation are at their peak level. One of the common features
found in the SW of fibres and vessels is the low lignin level in the S1 layer and the
highly lignified S3 layer. However, vessels are characterized by the presence of two
sublayers in the S2, i.e. inner and outer layers. Singh and Donaldson (1999) reported
a similar kind of sublayering of the S2 wall layer in compression wood tracheids of
pine. In the case of the vessel SW of Holoptelea, the inner layer shows a higher lignin
concentration than the outer one. The lignin concentration in vessel walls is reported
to be higher than that of fibres and ray parenchyma (Saka 1991). The higher lignin
content in the vessel wall might be associated with the relatively high proportion of the
highly lignified S2 inner layer. In the case of fibres, the thick S2 layer has no apparent
variation in overall lignin distribution.
Compared to axial xylem elements, little information is available on the SW deposi-
tion and lignification pattern of ray cells which varies greatly among species. The present
study shows that SW deposition and lignification follow different timing patterns for
contact and non-contact ray cells. Contact ray cells undergo SW deposition and lignifi-
cation immediately after completion of vessel differentiation while these events occur
in non-contact rays at a later stage. Interestingly, the radial walls between adjacent ray
cells are thicker than those adjacent to axial elements. However, the lignin distribution
was higher in the wall adjacent to fibres. Eriksson et al. (1988) suggested that ray cells
have a higher lignin concentration than fibre SWs. On the other hand, distinguishable
Downloaded from Brill.com12/07/2021 12:24:59PM
via free access414 IAWA Journal, Vol. 33 (4), 2012
heterogeneity has also been found in the SW thickness and lignification pattern between
paratracheal and apotracheal parenchyma cells. The former shows more lignin content
in CML, S2 and S3 layers while apotracheal cells show more lignin in the CML and S3
layer. The difference in wall thickness was more apparent in the cell corner region of
vessels and associated parenchyma cells. In oak wood, SW deposition and lignification
occur first in the vessel elements and vessel-associated cells (Yoshinaga et al. 1997).
However, the difference in lignification pattern between apotracheal and paratracheal
parenchyma cells is far from clear. We assume that the heterogeneity in the proportion
of SW and pattern of lignification in the two types of axial parenchyma might be as-
sociated with their function. The paratracheal parenchyma may have adapted to resist
the compressive stresses developed during the formation of adjacent large thick-walled
vessels, therefore developing a thick, highly lignified SW. On the other hand, thinner
walls and less lignification in apotracheal parenchyma might be related to storage
function. However, more information from different hardwood species is required to
differentiate the cell wall structure and function between apotracheal and paratracheal
parenchyma.
In conclusion, the present study shows that during wood formation, the cell specializa-
tion starts much before the deposition of SW material. SW deposition spreads in a radial
direction among the population of cells and within the same cell from xylem towards
the cambium in vessel elements and ray parenchyma. The pattern of cell expansion,
SW deposition and lignification in fibres indicates that these events occur sequentially
but independently commencing before the completion of the preceding step. Fibres
and vessel elements are the first elements to begin differentiation among cambial de-
rivatives. However, fibres are the last cells to complete the differentiation process due
to their elongation growth. Distinct variation was also noticed in the thickness of SW
and lignification pattern between apotracheal and paratracheal parenchyma cells which
may be related to their function. The ray cells are characterized by the absence of a
distinctly layered SW, a feature perhaps related to radial arrangement of cells. Lignin
deposition first begins in vessel elements and then spreads to the associated cells while
its distribution is heterogeneous within the wall layers of xylem elements.
Acknowledgements
KRS is grateful to the Korean Federation of Science and Technology Societies for the Brain Pool
Scientist award. The authors are thankful to Dr. Katia Ruel for providing lignin antibodies and Prof.
A.M. Catesson for her helpful suggestions during the preparation of this manuscript.
References
Abe, H., R. Funada, J. Ohtani & K. Fukazawa. 1997. Changes in the arrangement of cellulose
microfibrils associated with the cessation of cell expansion in tracheids. Trees Structure and
Function 11: 328–332.
Barcelo, A.R. 2005. Xylem parenchyma cells deliver the H2O2 necessary for lignification in
differentiating xylem vessels. Planta 220: 747–756.
Barnett, J.R. 1992. Reactivation of cambium in Aesculus hippocastanum L.: A transmission
electron microscope study. Ann. Bot. 70: 169–177.
Downloaded from Brill.com12/07/2021 12:24:59PM
via free accessRao et al. — Ultrastructure of developing cell walls 415
Catesson, A.M. 1990. Cambial cytology and biochemistry. In: M. Iqbal (ed.), The vascular
cambium: 63–112. Research Studies Press, Tauton, Somerset.
Catesson, A.M. 1994. Cambial ultrastructure and biochemistry: changes in relation to vascular
tissue differentiation and the seasonal cycle. Internat. J. Plant Sci. 155: 251–261.
Catesson, A. M., R. Funada, D. Robert-Baby, M. Quinet-Szély, J. Chu-Bâ & R. Goldberg. 1994.
Biochemical and cytochemical cell wall changes across the cambial zone. IAWA J. 15:
91–101
Catesson, A.M & J.C. Roland. 1981. Sequential changes associated with cell wall formation and
fusion in the vascular cambium. IAWA Bull. n.s. 2: 151–162.
Chaffey, N. & P. Barlow. 2002. Myosin, microtubules and microfilaments: co-operation between
cytoskeletal components during cambial cell division and secondary wall differentiation in
trees. Planta 214: 526–536.
Dejardin, A., F. Laurans, D. Arnaud, C. Breton, G. Pilate & J-C. Leple. 2010. Wood formation
in angiosperms. Comptes Rendus Biologies 333: 325–334.
Donaldson, L.A. 1992. Lignin distribution during latewood formation in Pinus radiata D. Don.
IAWA Bull. n.s. 13: 381–387.
Donaldson, L.A. 2001. Lignification and lignin topochemistry – an ultrastructural view. Phy-
tochemistry 57: 859–873.
Eriksson, I., O. Lidbrandt & U.Westermark. 1988. Lignin distribution in Birch (Betula verru-
cosa) as determined by mercurization with SEM-TEM-EDXA. Wood Sci. & Technol. 22:
251–257.
Esau, K. & I. Charvat. 1978. On vessel member differentiation in the bean (Phaseolus vulgaris
L.). Ann. Bot. 42: 665–677.
Fujino, T. & T. Itoh. 1998. Changes in the three-dimensional architecture of cell walls during
lignification of xylem cells in Eucalyptus tereticornis. Holzforschung 52: 111–116.
Funada, R., O. Furusawa, M. Shibagaki, H. Miura, T. Miura, H. Abe & J. Ohtani. 2000. The
role of cytoskeleton in secondary xylem differentiation in conifers. In: R.A. Savidge, J.R.
Barnett & R. Napier (eds.), Cell and molecular biology of wood formation: 255–264. Bios
Scientific Publishers, Oxford, UK.
Gritsch, C.S. & R. J. Murphy. 2005. Ultrastructure of fibre and parenchyma cell walls during
early stages of culm development in Dendrocalamus asper. Ann. Bot. 95: 619–629.
Grünwald, C., K. Ruel, Y.S. Kim & U. Schmitt. 2002. On the cytochemistry of cell wall forma-
tion in poplar trees. Plant Biol. 4: 13–21.
He, X.Q., Y.Q. Wang, Y.X. Hu & J.X. Lin. 2000. Ultrastructural study of secondary wall for-
mation in stem fibre of Phyllostachys pubescens. Acta Bot. Sinica 42: 1003–1008.
Higuchi, T. 1997. Biochemistry and molecular biology of wood. Springer Verlag, Berlin, Heidel-
berg, New York.
Joseleau, J.P. & K. Ruel. 1997. Study of lignification by non-invasive technique in growing
maize internodes. An investigation by FTIR, CP/MAS 13C NMR and immunocytochemical
transmission electron microscopy. Plant Physiol. 114: 1123–1133.
Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in
electron microscopy. J. Cell Biol. 27: 137–138.
Kim, Y.S., S.G. Wi, C. Grünwald & U. Schmitt. 2002. Immuno-electron microscopic localization
of peroxidases in the differentiating xylem of Populus spp. Holzforschung 56: 355–359.
Maurer, A. & D. Fengel. 1991. Electron microscopic representation of structural details in soft-
wood cell walls by very thin ultramicrotome sections. Holz-Roh-Werkstoff 49: 53–56.
Murakami, Y., R. Funada, Y. Sano & J. Ohtani. 1999. The differentiation of contact cells and isola-
tion cells in the xylem ray parenchyma of Populus maximowiczii. Ann. Bot. 84: 429–435.
Downloaded from Brill.com12/07/2021 12:24:59PM
via free access416 IAWA Journal, Vol. 33 (4), 2012
Newman, R.H. 1994. Crystalline forms of cellulose in softwoods and hardwoods. J. Wood Chem.
& Technol. 14: 451–466.
Pearson, R.S. & H.P. Brown. 1932. Commercial timbers of India. Their distribution, suppliers,
anatomical structure, physical and mechanical properties and uses. Vol. 2: 548. Government
of India. Central Publication Branch, Calcutta.
Putoczki, T.L., J.A. Gerrard, B.G. Butterfield & S.L. Jackson. 2008. The distribution of un-
esterified and methyl-esterified pectic polysaccharides in Pinus radiata. IAWA J. 29:
115–127.
Rao, K.S., J.S. Kim & Y.S. Kim. 2011. Early changes in the radial walls of storied fusiform
cambial cells during fibre differentiation. IAWA J. 32: 333–340.
Raussel, M.R. & C. Lim. 1995. Dynamic model lignin growing in restricted spaces. Macromol-
ecules 28: 370–376.
Ruel, K., V. Chevalier-Billosta, F. Guillemin, J.B. Sierra & J.-P. Joseleau. 2006. The wood cell
wall at the ultrastructural scale – formation and topochemical organization. Maderas Ciencia
Tecnologia 8: 107–116.
Saka, S. 1991. Chemical composition and distribution. In: D.N.S. Hon & N. Shiraishi (eds.),
Wood and cellulosic chemistry: 59–88. Marcel Dekker Inc., New York.
Samuels, A.L., K. Kaneda & K.H. Rensing. 2006. The cell biology of wood formation: from
cambial division to mature secondary xylem. Can. J. Bot. 84: 631–639.
Savidge, R.A. 1996. Xylogenesis. IAWA J. 17: 269–310.
Schubert, A.M., C.R. Benedict, J.D. Berlin & R.J. Kohel. 1973. Cotton fiber development-kinetics
of cell elongation and secondary wall thickening. Crop Sci. 13: 704–709.
Sharma, J. & V. Singh. 2012. Holoptelea integrifolia. An overview. Europ. J. Applied Sci. 4:
42– 46.
Singh, A.P. & L.A. Donaldson. 1999. Ultrastructure of tracheid cell walls in radiata pine (Pinus
radiata) mild compression wood. Can. J. Bot. 77: 32–40.
Spurr, A.R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy.
J. Ultrastruct. Res. 26: 31–45.
Terashima, N. 2000. Formation and ultrastructure of lignified plant cell walls. In: Y.S. Kim (ed.),
New horizons in wood anatomy: 169–180. Chonnam National University Press, Gwangju.
Terashima, N., K. Fukushima & K. Takabe. 1986a. Heterogeneity in formation of lignin-XI. An
autoradiographic study on the formation of guaiacyl and syringyl lignin in Magnolia kobus
DC. Holzforschung 40: 101–105.
Terashima, N., K. Fukushima & S. Tsuchiya. 1986b. Heterogeneity in formation of lignin-XII. An
autoradiographic study on the formation of guaiacyl and syringyl lignin in poplar. J. Wood
Chem. & Technol. 6: 495–504.
Thiery, J.P. 1967. Mise en evidence des polysaccharides sur coupes fines en microscopie élec-
tronique. J. Microscopy 6: 987–1017.
Xu, P., L.A. Donaldson, Z.R. Gergely & L.A. Staehelin. 2007. Dual-axis tomography: a new
approach for investigating the spatial organization of wood cellulose microfibrils. Wood
Sci. & Technol. 41: 101–116.
Yoshinaga, A., M. Fujita & H. Saiki. 1997. Cellular distribution of guaiacyl and syringyl lignins
within an annual ring in oak wood. Mokuzai Gakkaishi 43: 384–390.
Downloaded from Brill.com12/07/2021 12:24:59PM
via free accessYou can also read
