Annals of Botany 104: 1045– 1056, 2009
doi:10.1093/aob/mcp193, available online at www.aob.oxfordjournals.org
The distribution of cell wall polymers during antheridium development and
spermatogenesis in the Charophycean green alga, Chara corallina
David S. Domozych1,*, Iben Sørensen2 and William G. T. Willats2
1
Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College, 815 North Broadway, Saratoga Springs,
NY 12866, USA and 2Department of Biology, University of Copenhagen, Copenhagen Biocentre, Ole Maaløes Vej 5,
2200 Copenhagen, Denmark
Received: 24 April 2009 Returned for revision: 27 May 2009 Accepted: 8 July 2009 Published electronically: 19 August 2009
† Background and Aims The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The
processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics
in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive
analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.
† Methods Microarrays of cell wall components and immunocytochemical methods were employed in order to
analyse cell wall macromolecules during antheridium development.
† Key Results Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the
developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of
sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers
and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the
sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the
probes employed were found on the scale-covered sperm cells.
† Conclusions Antheridium development in C. corallina is a rapid event that includes the production of cell walls
that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in
the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific
zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules
from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.
Key words: Chara corallina, antheridium, cell wall, spermatogenesis, pectins.
IN T RO DU C T IO N
The structure and development of plant tissues and organs is a
manifestation of the coordinated activities of their inclusive
populations of cells. The synchronization of the expression
of selected gene sets, cell cycle activities and signal transduction pathways all contribute to the construction of the final
functioning morphological unit. The inclusive subcompartmentalized units of cells often display considerable
structural and physiological diversity but ultimately contribute
to the functional competency of the total unit. One fundamental example of this developmental process in multicellular
green plants is the production of gametangia during the
sexual reproductive phase of their life cycles. In primitive nonvascular plants (e.g. bryophytes) or in the gametophyte phase
of primitive vascular plants (e.g. pteridophytes), specific
‘pre-gametangial’ cells undergo multiple cell divisions and
their derivative units differentiate into specialized structures
such as the antheridium or archegonium (Bell and Hemsley,
2000; Renzaglia et al., 2000). These units are further delineated into specific sub-compartments such as the jacket and
a developing mass of sperm cells in the antheridium, or in
the archegonium, the neck, venter and the egg. In gymnosperms and angiosperms, gametogenesis yields significantly
* For correspondence. E-mail [email protected]
reduced gametangial units such as pollen and archegonia or
egg sacs embedded within a protective ovule. All of these
gametogenesis-based developmental events and the resulting,
functional morphological units optimize strategies for
gamete production, protection, transfer and the ultimate fertilization event.
In plants, the production of the cell wall is central to morphological development. This entails the synthesis, secretion
and post-secretory remodelling of multiple cell wall polymers
that are guided by complex gene expression patterns and multiple signal transduction cascades responding to environmental
cues (Lerouxel et al., 2006; Geisler et al., 2008). For most
plant cells, this includes the establishment of a network of cellulose microfibrils tethered by non-cellulosic cross-linking
polymers (commonly referred to as hemicelluloses) and
embedded in a matrix of pectins, various proteoglycans and,
in some specialized cells, phenolic (e.g lignins) or phenylpropanoid polymers (e.g. sporopollenin) (Carpita and McCann,
2000). The most widely studied examples of cell wall structure, biochemistry and development in plant gametogenesis
are from angiosperms and gymnosperms, especially those
relating to the unique anisotropic growth mechanism exhibited
during post-pollination pollen tube development (Hepler et al.,
2001; Cardenas et al., 2008; Lee and Yang, 2008). Here, coordinated modes of cell wall macromolecule production in the
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
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Domozych et al. — Cell wall macromolecules of the antheridium of Chara
tube yield distinct polymer gradations and microloci (including oscillations) that result in pronounced unipolar growth
toward the egg. The egg itself is the product of a complex
differentiation process and it is embedded in its own unique
gametophyte tissue that is surrounded by protective sporophyte
tissue. However, there is a virtual absence of information
concerning cell wall dynamics during gametogenesis in earlydivergent plants including bryophytes and their algal ancestors, the Charophycean green algae or CGA (Streptophyta,
Virideplantae; Lewis and McCourt, 2004). Advanced clades
of the CGA such as the Charales and Coleochaetales display
complex thallus morphology including the production of
multicellular gametangia that, in some cases, exhibit notable
similarity to those found in primitive land plants. This lack
of information creates a significant gap in our basic understanding of the evolution of developmental processes in multicellular plants as gametangia represent one of the first,
specialized multicellular units derived from a vegetative
thallus in green plants. Likewise, the absence of data severely
limits our understanding of the evolutionary origins of, and
adaptations associated with, sexual reproductive strategies in
green plants. Elucidation of cell wall dynamics during gametangium development in early-divergent plants would
provide important insight into the structural and functional
roles of specific cell wall polymers during the evolution of
green plants and in their complex life cycles.
Detailed analysis of cell walls during gametangium development in the CGA and early-divergent land plants is technically challenging because of the short period of time during
the year that male and female gametangia are available for
study, and their small size makes it hard to obtain enough
material for traditional biochemical studies. However, a large
panel of molecular probes, monoclonal antibodies (mAbs)
and carbohydrate-binding modules (CBMs) has been developed that enables cell wall components to be studied in
muro (Willats et al., 2000; Willats and Knox, 2003).
Moreover, a recently developed technique (comprehensive
microarray polymer profiling or CoMPP) that combines the
specificity of mAbs and CBMs with the high throughput
capacity of microarrays enables the rapid analysis of cell
wall polysaccharide composition in diverse samples (Moller
et al., 2007; Sørensen et al., 2008). In this study, which
forms part of a long-term study of the cell walls of the
CGA, both immunocytochemical and CoMPP analyses were
utilized to undertake a detailed survey of cell wall polymer distribution during antheridium development and spermatogenesis in Chara corallina.
M AT E R IA L S A ND M E T HO DS
General
Chara corallina was collected from a freshwater wetland in
Porter Corners, NY (USA) and was subsequently cultured in
aquaria in the Greenhouse facility of Skidmore College.
Thalli with antheridia were obtained during the month of
May when water temperature reached 21 8C and the photoperiod was 14 h light/10 h dark. Antheridium-laden thalli
were excised 10 cm from the apical tip and placed in sterile
well water till further use.
Antheridium excision for CoMPP
Thalli were washed gently with deionized water and then
placed on the stage of a Wild M36 stereo microscope (Wild,
Heerbrugg, Switzerland). Individual antheridia were excised
by hand and placed in ice-cold (4 8C) 80 % ethanol. After
90 min the antheridia were spun down at 500 g on an
International Clinical Centrifuge (Needham, MA, USA) and
the ethanol was removed. The antheridia were resuspended
in 10 ml of 80 % ethanol at 4 8C for 90 min. This process
was repeated twice more. The antheridia were then washed
three times with acetone and air dried in a fume hood. The
resultant material was collected and stored at 220 8C until
further use.
CoMPP
CoMPP was carried out essentially as described in Sørensen
et al. (2008). Starting material was 10 mg of alcohol-insoluble
residue (AIR). Cell wall polymers were sequentially
extracted with 50 mM trans-1,2-diaminocyclohexane-N,N,
N0 ,N0 -tetraacetic acid (CDTA), pH 7.5 and 4 M NaOH with
0.1 % (v/v) NaBH4, and extractions printed in three dilutions
and six replicates, giving a total of 18 spots per sample. The
entire experiment was repeated three times and the data
represent an average of these. The data were converted into a
heatmap format using the online BAR heatmapper tool
(http://bar.utoronto.ca/ntools/cgi-bin/ntools_heatmapper.cgi).
Binding specificity of each mAb/CBM is provided in Table 1.
Light microscopy (LM)
An Olympus SZX12 stereo microscope equipped with a
DP70 camera (Olympus, Melville, NY, USA) was used for
obtaining overview images of antheridial position on the
thallus. For cytochemical work, thalli containing one nodal
zone containing lateral branches with antheridia were excised
with a scalpel and fixed for 1 h at 4 8C in 1 % paraformaldehyde (EMS, Fort Washington, PA, USA) in 0.05 M cacodylate
buffer with 2 mM CaCl2 ( pH 7.4). The antheridia were washed
in 0.05 M cacodylate buffer with 2 mM CaCl2, three times for
10 min each. The antheridia were then slowly dehydrated
over 6 h in a series of ethanol solutions and then placed in a
1 : 1 ratio of ethanol– London resin (LR; EMS) overnight.
The antheridia were then infiltrated with 100 % LR for 2 d at
4 8C (three times) and then placed in cold LR in flat-bottomed
Beem capsules. The antheridia were then UV polymerized.
Sections of the antheridia (0.5 mm) were cut with a diamond
knife on a Reichert Ultracut ultramicrotome (MOC, Valley
Cottage, NY, USA). Sections were collected in wells of an
immunoslide (EMS) coated with 1 % silane (Sigma
Chemical, St Louis, MO, USA). Immunolabelling of the sections followed previously described protocols (Domozych
et al., 2007) with tetramethylrhodamine isothiocyanate
(TRITC)-conjugated anti-rat secondary antibodies. Antibodies
were obtained from Plant Probes (Leeds, UK) or BioSupplies
(Parkville, Australia). In order to determine if xylan, xyloglucan (XyG) or mannan epitopes were being masked by pectins
in the cell wall, some sections were first incubated in a
100 mg mL21 solution of pectolyase (Sigma Chemical; in a
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
1047
TA B L E 1. Summary of antibodies employed and results of immunocytochemical labeling
mAb/CBM/dye
Specificity
Shield cells
Sperm cell initials
References
Cellulose
Calcofluor
CBM3a
b-Glucans, cellulose
Crystalline cellulose
11
11
11
11
Hughes and McCully (1975)
Blake et al. (2006)
Pectins
JIM5
JIM7
LM7
LM5
LM6
LM13
LM8
Low DE HG
High DE HG
Partial DE, non-blockwise
(1– 4)-Galactan
(1– 5)-Arabinan
Arabinan
Xylogalacturonan
11
1
2
2
2
2
2
11
1
2
2
1
1
2
Clausen et al. (2003)
Clausen et al. (2003)
Willats et al. (1998)
Jones et al. (2003)
Freshour et al. (2003)
Verhertbruggen et al. (2009)
Willats et al. (2004)
Xyloglucan
CCRC-1
LM15
Fucosylated XyG
Non-fucosylated XyG
2
2
2
1 capitulum
Marcus et al. (2008)
Mannan
BS-400-4
b(1 –4)-Mannan/galacto-b(1–4)-mannan
2
2
Pettolino et al. (2001)
Xylan
LM10
LM11
(1– 4)-Xylan
b(1 –4)-Xylan/arabinoxylan
2
2
2
2
McCartney et al. (2005)
McCartney et al. (2005)
b1– 3 glucan
BS-400– 2
b(1 –3)-Glucan
1
2
Tormo et al. (1996)
AGP
LM2
JIM13
JIM8
AGP
AGP
AGP
2
11
11
2
2
2
Smallwood et al. (1996)
Knox (1991)
Extensin
LM1
JIM19
JIM20
Extensin
Extensin
Extensin
2
2
2
2
2
2
McCabe et al. (1997)
Smallwood et al. (1994)
Smallwood et al. (1994)
Key: þþ, intense labelling; þ, labeling; 2, no label; DE, degree of methyl esterification.
pH 4, CAPSO buffer, Herve et al., 2009) for 2 h at room temperature or in 50 mM CDTA ( pH 6.9) for 2 h. at room temperature. The treated sections were washed five times in deionized
H2O and then labelled with mAbs as described above. Control
samples excluded primary mAb incubation. CBM3a labelling
was performed essentially as described in Blake et al.
(2006). For general labelling of b-glucans, sections were
treated with 0.1 mg mL21 Calcofluor (Sigma) for 2 min and
then repeatedly washed with deionized H2O. LM and fluorescence light microscopy (FLM) imaging employed an
Olympus BX-60 light microscope (Olympus, USA) equipped
with fluorescence optics and a DP-70 camera.
Transmission electron microscopy (TEM) cytochemistry
Excised antheridia were fixed with 0.5 % glutaraldehyde at
4 8C for 1 h in cacodylate buffer (see above). After 30 min,
the antheridia were washed with cacodylate and then lightly
fixed for 1 h in 0.5 % OsO4/0.05 M cacodylate buffer. After
washing with cacodylate buffer three times (10 min each),
the antheridia were dehydrated in acetone, infiltrated in an
acetone/Spurrs low viscosity medium (EMS) and then
embedded in flat-bottomed Beem capsules using heat polymerization (60 8C, 9 h). Sections of 60– 80 nm were cut on the
ultramicrotome and collected on gold or nickel, formvarcoated grids. Immunogold labelling followed previously
described protocols (Domozych, 2007) and used goat anti-rat
antibody conjugated with 15 nm gold particles. For determination of potential pectin masking, sections on grids were
treated with pectolyase or CDTA as described above before
immunogold labelling. For control experiments, the primary
antibody incubation was excluded. TEM imaging took place
on a JEOL 1010 TEM at 80 kV (JEOL, Peabody, MA, USA).
R E S ULT S
Antheridia were present on thalli from May to August when
water temperature exceeded 21 8C. The appearance of antheridia typically preceded oogonia by 1 – 2 d and full antheridial
development was completed within 3 – 4 d. Antheridia arose
from the axillary regions of lateral branches emerging from
the first 2 – 3 nodes of apical portions of thalli. Antheridia
were juxtaposed to oogonia on the lateral branches (Fig. 1A)
and were easily recognizable by the bright orange pigmentation of the epidermal-like shield cells (Fig. 1B).
Mature antheridia were excised from lateral branches and
used for CoMPP and microscopy-based studies. CoMPP provides semi-quantitative information about the relative abundance of epitopes occurring on cell wall components, and
the data are presented as a heatmap (Fig. 2). In the heatmap,
mean spot signals from the CoMPP arrays are correlated to
colour intensity, and the highest value in the entire data pool
1048
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
A
B
Oogonium
Shield cells
Antheridium
Extensin (mAb LM1)
Extensin (mAb JIM19)
Extensin (mAb JIM20)
AGP (mAb JIM13)
0
0
AGP (mAb JIM8)
0
0
AGP (mAb LM2)
0 5·5 0 36·6 37·1 55·5 0
21 19·9 25·5 14·6 32·1 49 0
b(1–3)-glucan (mAb BS 400-2)
0
0
Cellulose (CBM3a)
b(1–4)-xylan/arabinoxylan (mAb LM11)
0 11·4 0
0 7·7 13
b(1–4)-xylan (mAb LM10)
b(1–4)-mannan (mAb BS 400-4)
XG (mAb LM15)
XG (+fuc., mAb CCRC-M1)
Xylogalacturonan (mAB LM8)
0 11·1 0 0
0 26·8 14·8 0
Arabinan (mAb LM13)
0
0
a(1–5)-arabinan (mAb LM6)
b(1–4)-galactan (mAb LM5)
CDTA 100 0
NaOH 10·6 0
HG (Partially DE, non-blockwise, mAb LM7)
HG (High DE, mAb JIM7)
HG (Low DE, mAb JIM5)
F I G . 1. The gametangia of C. corallina. (A) An overview of antheridial positioning on a lateral branch. Each antheridium is closely situated near an oogonium.
Scale bar ¼ 400 mm. (B) A magnified view of an antheridium and highlighting the brightly coloured shield cells of the epidermis. Scale bar ¼ 90 mm.
F I G . 2. CoMPP profile of the antheridium. The printed microarrays were probed with a panel of monoclonal antibodies and a CBM listed above the heatmap.
CDTA and NaOH refer to the signal values from the two different extractions. BS, Biosupplies.
was set to 100 and all other values adjusted accordingly. The
heatmap shows the signals obtained for cell wall components
extracted with CDTA and NaOH, two solvents that in land
plants extract predominantly pectic and hemicellulosic polysaccharides, respectively. CoMPP indicated that antheridia
contained the pectic homogalacturonan (HG) epitope with a
low degree of methyl esterification that is recognized by
mAb JIM5, but not the partially methyl-esterified HG epitopes
recognized by mAbs LM7 and JIM7. b(1 – 4)-galactan and
a(1 – 5)-arabinan often occur as side chains on land plant
pectins and whilst b(1– 4)-galactan was not detected in antheridia, a(1 –5)-arabinan (recognized by mAb LM6) was.
Interestingly, LM6 binding was strongest in the NaOH rather
than the CDTA fraction, suggesting that in antheridia this
epitope may be associated primarily with hemicellulosic
rather than pectic polymers. The presence of cellulose and
callose was indicated by the binding of CBM3a and mAb
BS 400-2, respectively, and three hemicellulosic epitopes
were also detected; a mannan epitope recognized by mAb
BS 400-4, a xylan-containing epitope recognized by mAb
LM11 and a XyG epitope recognized by mAb LM15. It is
noteworthy that the fucosylated XyG epitope recognized by
mAb CCRC-M1 was not detected and also that, in contrast
to most studies on land plant cell walls, LM15 bound more
strongly to CDTA- rather than to NaOH-extracted material.
This suggests that XyG is less tightly held in the cell wall
than is typical in land plants, and may be associated with the
pectic matrix in antheridia. Three arabinogalactan protein
(AGP) epitopes (recognized by mAbs LM2, JIM8 and
JIM13) were detected in both CDTA and NaOH extractions,
but three anti-extensin mAbs (LM1, JIM19 and JIM20) did
not produce signals above background.
For immunofluorescence studies, 500 nm sections of fixed
and LR-embedded antheridia were obtained. Table 1 summarizes the immunocytochemistry-based results of this study. The
cell walls of all regions of the antheridium were labelled with
Calcofluor and CBM3a (Fig. 3A– C), which label b-glucans
and crystalline cellulose, respectively. Both the end walls
and cross-walls of developing sperm cell initials, organized
in filaments, were labelled as well. The cell walls of all
areas of the antheridium including the sperm cell initials
were also labelled with the anti-HG mAb JIM5 (Fig. 3D, E).
JIM7, an mAb which labels relatively highly esterified HG,
also labelled the cell walls of all components of the antheridium including those of the sperm cell initials (Fig. 3F). The
anti-a(1 ! 5)-arabinan mAb LM6 labelled only the cell
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
Calc
CBM3a
1049
CBM3a
S
S
SC
A
JIM5
B
C
JIM5
JIM7
D
E
F
JIM13
JIM8
I
J
LM6
G JIM13
H
F I G . 3. Immunofluorescence and Calcofluor images of the antheridium and developing sperm cell initials. (A) The cell walls of all cell types of the antheridium
are labelled with Calcofluor including the shield cells (S), sperm cell initials (SCI), the capitulum (C) and pedicel (P). Scale bar ¼ 35 mm. (B) A CBM3a-labelled
antheridium. The cell walls of all component cells are labelled including the sperm cell initials (SCI) and shield cells (S). Scale bar ¼ 45 mm. (C) A magnified
view of the sperm cell initials labelled with CBM3a (arrow). Note that both the thick end walls and thin cross-walls are labelled. Scale bar ¼ 9 mm. (D) An
antheridium labelled with JIM5. Note that the cell walls of all cell types are labelled. Scale bar ¼ 35 mm. (E) A magnified image of sperm cell initials labelled
with JIM5. Note that both end and cross-walls label. Scale bar ¼ 21 mm. (F) A view of the sperm cell initials labelled with JIM7. Scale bar ¼ 20 mm. (G) A
magnified view of the sperm cell initials (arrow) labelled with LM6. These are the only antheridial cells that label with this mAb. Scale bar ¼ 20 mm. (H)
An antheridium labelled with JIM13. The shield cells (arrow) are the only cells that are labelled with this mAb. Scale bar ¼ 35 mm. (I) A magnified view of
the shield cell epidermis labelled with JIM13. Only the shield cells are labelledf Scale bar ¼ 20 mm. (J) A JIM8-labelled antheridium demonstrating that
only the shield cells are labelled (arrow). Scale bar ¼ 15 mm.
walls of the sperm cell initials (Fig. 3G), whilst the anti-AGP
mAb JIM13 labelled just the cell walls of the outer epidermal
layer of shield cells (Fig. 3H, I). This was also the case for the
anti-AGP, JIM8, as its binding was restricted to just the outer
cell wall of the epidermal cells (Fig. 3J). Binding of the
anti-XyG mAb, LM15, was highly restricted and was confined
to certain cell walls of the capitulum and its immediate derivatives (Fig. 4A). Control preparations that included elimination
of primary antibody incubation did not reveal any labelling
(Fig. 4B). Treatment of sections with pectolyase or CDTA
prior to labelling did not result in enhanced labelling of
LM15 (Fig. 4C, D) or labelling with LM11 or BS 400-4
(data not shown).
TEM-based ultrastructural and immunogold analyses of
60– 80-nm sections demonstrated the microarchitectural
characteristics and refined polymer mapping of the cell walls
of the various regions of the antheridium. The most distinct
region of the antheridium was the sperm filament that included
as many as 25– 40 sperm cell initials attached end to end
(Fig. 5A). Each cell was surrounded by a cell wall, and the
cross-walls between the sperm cell initials were perforated
by plasmodesmata (Fig. 5B, C). The cross-walls of the
sperm cell initials were formed by a cell plate during cytokinesis (Fig. 5B; also see Cook et al., 1998). The side walls of the
sperm cell initials were thin and the junctions between the side
and cross-walls were distinctively V-shaped (Fig. 5D). During
latter stages of development, the sperm cell initial protoplast
condensed and retracted from the cell wall (Fig. 5C). A noticeable, moderately osmiophilic substance filled the spaces
between sperm filaments and was never found to label with
any mAb, Calcofluor or CBM3a in either LM or TEM preparations. During the late stages of development when sperm
1050
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
LM15
Control
SCI
C
P
A
B
CDTA/LM15
Pectolyase/LM15
C
D
F I G . 4. Immunofluorescence profiles. (A) An antheridium labelled with LM15. Only the cell walls of the immediate cell products of the capitulum (C; arrows)
are labelled. The sperm cell initials (SCI) and pedicel (P) are also apparent. Scale bar ¼ 15 mm. (B) A control preparation whereby the primary antibody was
eliminated from the labelling process. Scale bar ¼15 mm. (C) A preparation whereby sections were treated with pectolyase before LM15 labelling. No new labelling is apparent. Scale bar ¼ 15 mm. (D) A preparation whereby sections were treated with CDTA before LM15 labelling. No new labelling is apparent. Scale
bar ¼ 15 mm.
cells started to retract from the cell walls, no antibody labelling
was ever found upon sperm cell membranes where scales were
located. The epidermis of the antheridium consists of thickwalled shield cells. The outermost region of the cell wall of
the shield cells was thick and fibrous with a higher density
of fibres situated on the outer loci (Fig. 6A). The side walls
were considerably thinner (Figs 5B and 6B) and in many
cases ended in distinct protuberances that only partially separated adjacent cells (Fig. 6C). This created large expanses of
cytoplasmic continuity between shield cells. Chloroplasts
lined the outer cytoplasmic strata and possessed distinct
accumulations of pigment globules (Fig. 5A; see also
Pickett-Heaps 1975). These globules most probably represented the carotenoid depositions that produced the distinct
orange coloration of the antheridium.
JIM5 labelled all portions of the shield cell cell wall
(Fig. 6D) including the thick protuberances and the cross-walls
(Fig. 6E, F). JIM7 (Fig. 6G) and JIM13 (Fig. 6H) also labelled
all of the various regions of the shield cell cell walls. The cell
walls of sperm cell initials were also labelled with JIM5
(Fig. 7A), especially at the V-shaped junctions where cell
walls of individual cells met (Fig. 7B). JIM7 also labelled
the sperm cell initial cell walls, with higher labelling in the
cross-wall region and lesser labelling in the V-shaped junction
(Fig. 7C, D). JIM13 did not label any region of the cell wall of
sperm cell initials (Fig. 7E). LM6 labelled only the sperm cell
initial cell walls including the cross-wall region (Fig. 7F).
However, unlike other antibody labelling, it appeared as if
the label was localized on the plasma membrane of the crosswall region. The electron-dense material found between the
sperm cell filaments was not labelled with any of the antibodies. Control labelling included elimination of primary antibody incubation (Fig. 7G). During the final stage of
spermatogenesis, the sperm cell protoplasts retract from the
cell wall, condense, produce flagella (Fig. 8A) and become
coated with small scales (Fig. 8B). At this time, no part of
the plasma membrane or flagellar membrane surfaces or
material found between the cell and old cell wall was labelled
with any of the antibodies.
D IS C US S IO N
This study represents the first detailed study of the cell wall
components in C. corallina antheridia cell walls using an
extensive panel of cell wall probes. The CoMPP technique
enabled a large number of epitopes to be surveyed using a
small amount of material and also provided information
about the extractabilities and possible inter-relationships of
cell wall components. The LM and TEM immunolocalization
studies provided insights into the cellular locations of the epitopes. In general, there was a close agreement between the
observations from the CoMPP and immunolabelling studies.
However, for certain epitopes this was not the case. For
example, JIM7 bound strongly to sections through antheridia
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
*
1051
SCI
SCI
A
*
B
SCI
*
SCI
SCI
SCI
C
*
D
F I G . 5. Ultrastructural features of spermatogenesis. (A) A longitudinal section through an antheridial filament (arrow). Each filament contains 20–30 sperm cell
initials (SC) and each cell is surrounded by a cell wall. The sperm cell initials sit is a moderately electron-dense material (*) within the antheridium. Scale bar ¼
4 mm. (B) Cell plate formation in the antheridial filament. The plate (large arrows) develops in centripetal fashion between the daughter sperm cell initials
(SCI) and entails the formation of plasmodesmata (small arrows). Scale bar ¼ 375 nm. (C) A magnified view of sperm cell initials (SCI) during a later stage
of development. Note that the protoplast is beginning to condense and retract from the end wall (arrowhead). The cross-walls (small arrows) remain intact.
V-shaped junctions are present where the cross-walls meet with the end wall (large arrows). Scale bar ¼ 1 mm. (D) A magnified view of the V-shaped cell
wall junctions (arrowhead) of developing sperm cell initials (SCI). These are the zones where the end walls (large arrows) and cross-walls (small arrows)
merge. Scale bar ¼ 250 nm.
cell wall, but did not produce a signal above background in
CoMPP. One explanation for this could be that the JIM7
epitope was not extracted by the CDTA extraction used –
possibly because the polysaccharide bearing the JIM7
epitope was very firmly held in the cell wall by association
with other polymers. Conversely, the b(1 –4) xylan/arabinoxylan and b(1 – 4) mannan epitopes recognized by LM11
and BS 400-2 were detected by CoMPP, but not in the immunolabelling studies, and one reason for this might be masking
of these epitopes by other cell wall components. It has been
shown in land plants that hemicellulosic epitopes can be
masked in immunolabelling studies by pectin and can be
unmasked by pectinolytic enzymes (Herve et al., 2009).
However, treatment of LM and TEM antheridia sections
with pectolyase and CDTA before labelling failed to demonstrate any new or enhanced labelling with these mAbs, and it
is possible that these epitopes may be masked by cell wall
epitopes other than pectins. The results obtained in this
study generally corresponded well with previous biochemical
studies of total thallus cell wall chemistry of Chara (Gillet
et al., 1992, 1998; Gillet and Liners, 1995; Popper and Fry,
2003; Proseus and Boyer, 2006; Popper, 2008) in that
pectins and cellulose were determined to be abundant cell
wall polymers. However, previous screening for XyG in
Chara has yielded inconclusive results (Popper and Fry,
2003; Van Sandt et al., 2005; Popper, 2008). The disaccharide, isoprimeverose, is produced by the digestion of XyG (but
no other polymers) by the enzyme driselase, and this effect is
generally assumed to be diagnostic for XyG. A study by
Popper and Fry failed to detect isoprimeverose upon driselase
digestion of C. corallina cell wall material. The material used
for this study did not contain antheridia (S. Fry, University of
Edinburgh, UK, pers. comm.). Labelling with LM15 indicated a highly restricted location of the LM15 epitope and
it is possible that the material selected for driselase treatment
simply did not include XyG-containing cell walls. It is also
possible that XyG in C. corallina has a somewhat different
structure from that of land plant XyGs and is not driselase
digestible. The LM15 mAb was generated using a XXXG
heptasaccharide (i.e. an oligosaccharide consisting of four
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Domozych et al. — Cell wall macromolecules of the antheridium of Chara
OL
CW
*
A
B
JIM5
*
P
C
D
JIM5
JIM5
E
F
JIM7
G
JIM13
H
F I G . 6. Ultrastructure and immunogold labelling of the shield cells of the antheridium. (A) An image of the thick cell wall (CW) on the outside of the shield
cells. The cell wall is fibrillar with a gradient of thick fibrillar density on the outer layer (OL) and less density on the inside. Scale bar ¼ 500 nm. (B) The crosswall (CW) between shield cells. This cell wall is thinner than that of the outer cell wall and is adjacent to thin peripheral layers of cytoplasm carrying the globulecontaining (arrow) plastids (*). Scale bar ¼ 420 nm. (C) A cell wall protuberance (P) characteristic of many of the shield cells. The terminus of the protuberance
ends in the middle of the cell. Note the plastid (*) in the peripheral cytoplasm adjacent to the terminus. Scale bar ¼ 750 nm. (D) The outer wall region of a shield
cell labelled with JIM5. Note the abundance of label (arrows) throughout the cell wall. Scale bar ¼ 520 nm. (E) A JIM5-labelled protuberance showing the dense
labelling in the protuberance swelling (arrow). Scale bar ¼ 550 nm. (F) A JIM5-labelled region (arrows) of the cross-wall of a shield cell. Scale bar ¼ 430 nm.
(G) A view of the outer region of the shield cell labelled with JIM7. Note the label throughout the cell wall (arrow). Scale bar ¼ 575 nm. (H) The outer wall of a
shield cell labelled with JIM13. The label is also found throughout the cell wall (arrow). Scale bar ¼ 560 nm.
contiguous 1,4-linked glucose resides, three of which bear
1,6-linked xylose residues; Herve et al., 2009); however,
LM15 also binds to XXXG that is further substituted with
galactose. The possibility cannot be excluded that
C. corallina XyG is substituted such that it is detectable by
LM15 but not digestible by driselase. Although LM15 has
been shown not to cross-react with a wide range of other
cell wall polymers, it is also formally possible that the
LM15 labelling observed represents LM15 binding to a
non-XyG cell wall component.
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
JIM5
1053
JIM5
SCI
*
SCI
A
B
JIM7
JIM7
C
*
D
JIM13
LM6
Control
SCI
*
*
SC
E
F
G
F I G . 7. Immunogold labelling of sperm cell initials. (A) A sperm cell initial (SCI) labelled with JIM5. The end and cross-walls are all labelled with the mAb
(arrows). Scale bar ¼ 1 mm. (B) A magnified view of the V-shaped junction and end walls of sperm cell initials labelled with JIM5. Note that there is no labelling
in the material outside of the walls (*) Scale bar ¼ 400 nm. (C) A view through a cross-wall of a sperm cell initial labelled with JIM7. The label is found throughout the cell wall (arrows) that is interrupted by plasmodesmata. Scale bar ¼ 150 nm. (D) A view of the V-shaped junction labelled with JIM7 (arrow). Note that
there is no labelling in the material in the space outside of the wall (*). Scale bar ¼ 200 nm. (E) The JIM13-labelled interface between the inner wall of a shield
cell (large arrow) and the end wall of a sperm cell initial (SCI). Note that the shield cell wall is labelled with the mAb but not the cell wall of the sperm cell initial.
Scale bar ¼ 200 nm. (F) The cross-wall region of sperm cell initials labelled with LM6. Note the labelling at this zone, including the plasma membrane of this
region (arrows). (G) A control preparation where the primary antibody was eliminated during labelling. No labelling is found in the cell walls (arrow) or the
material outside of the cell walls (*). Scale bar ¼ 400 nm.
The formation of an antheridium in C. corallina represents a
rapid developmental phenomenon originating with an adaxial
cell positioned on the lateral branches near the thallus apex.
These cells are located at a nodal region, i.e. a central location
for the initiation of many morphogenetic events in the alga,
and undergo multiple cell divisions to yield an octant of undifferentiated cells (Pickett-Heaps, 1975). From this octant,
further cell divisions and subsequent differentiation events
yield three layers of cells, the outer epidermis, the medial manubrium and the internal capitulum. Further divisions and
differentiation yield the mature components of the functioning
antheridium. These developmental events are regulated by
environmental cues including long photoperiods and warm
water temperatures, as well as chemical signals including the
gibberellin, GA3 (Kazmierczak, 1999; Kazmierczak et al.,
1999; Kazmierczak and Stepinski, 2005). The production of
antheridia is also temporally orchestrated with the formation
of oogonia and, ultimately, both gametangia are geographically positioned in closely set pairs. The proximity of the
male and female gametangia most probably increases the
rate of success of sperm transfer to the egg and subsequent fertilization. The mature antheridium is a highly distinctive structure and includes: (a) a pedicel or stalk that connects the
antheridium to the vegetative branch; (b) an epidermis or
1054
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
SC
SC
A
B
F I G . 8. Ultrastructure of the last stages of spermatogenesis. (A) An overview of sperm cell initials transforming into sperm cells. Note that the protoplasts condense and retract from the cell walls and flagella emerge (arrows). Scale bar ¼ 1 mm. (B) A JIM5-labelled sperm cell initial during sperm cell development. Not
that the mAb labels the cell wall (large arrows) but not the scales upon the flagellar surface (small arrow) or material (*) between the sperm cell flagellum and cell
wall. Scale bar ¼ 205 nm.
jacket consisting of a layer of shield cells; (c) the manubrium
that sits internal to the jacket and yields the capitulum; and (d)
the capitulum (i.e. both primary and secondary capitula, sensu
Pickett-Heaps, 1969) whose cells ultimately give rise to sperm
cells. The epidermal shield cells are brightly coloured due to
the accumulation of carotenoid pigment globules in their
chloroplasts, a characteristic also found in the antheridia of
some bryophytes (Shaw and Goffinet, 2000). The shield cells
often possess incomplete cross-walls that terminate in distinct
swollen protuberances. These protuberances are labelled with
Calcofluor, CBM3a, JIM5 and JIM7, but not with LM5 or
LM6. These results indicate the presence of cellulose and
HGs with varying degrees of esterification but not rhamnogalactouran I (RG-I)-like macromolecules. Pectin-containing cell
wall protuberances like those found here are not commonly
found in plant cells but have been described in some taxa of
most of the major groups of land plants (Leroux et al.,
2007). Many of these protuberances have been found in cells
that are associated with specific physical stress stimuli including wounding and grafting (Davies and Lewis, 1981;
Donaldson and Singh, 1984). Likewise, cell wall thickenings
of strategically placed cells have been implicated in the forceful dissemination of gametes and spores of bryophytes (Shaw
and Goffinet, 2000). The functional role of the protuberances
in the Chara’s shield cells has yet to be definitively described
but it is possible that they contribute to the dehiscence mechanism exhibited by the mature antheridia. Immediately prior to
sperm release, the antheridial epidermis ruptures and forcefully unravels in a rosette pattern (Pickett-Heaps, 1975) that,
in turn, facilitates the spread of the sperm. The alternation of
the thick protuberances with the more uniform outer and
inner wall regions of the shield cells may contribute to the generation of a controlled, i.e., organized, centripetal action that
displaces the sperm outward from the antheridium. Further
work will be needed to elucidate both the functional role of
the protuberances and the specific role of its pectin constituents in Chara. This includes analysis of rapid remodelling of
HG chemistry that might allow for dramatic changes in cell
wall charge and shield cell osmotic conditions that ultimately
lead to rupture.
One of the more unique and spectacular events during
antheridial development in Chara is the production of sperm
cells. These gametes are derived from cells that arise in distinct, elongate filaments known as antheridial or spermatogenous filaments. In the early stages of spermatogenesis, the
sperm cell initials are covered with thin cell walls and are
produced by a cytokinetic mechanism that includes a
phragmoplast and tubulo-vesicular cell plate. Fields of plasmodesmata in the cross-walls between sperm cell initials are
created during the cytokinetic process (Pickett-Heaps, 1975;
Cook et al., 1998, 1997). The cell walls contain both cellulose
and HGs with varying degrees of esterification. The cell walls
of sperm cell initials also labelled with LM6, which is specific
for a(1 – 5)-arabinan side chains of RG-I and sometimes considered a marker of AGP glycans (Lee et al., 2006).
Interestingly, immunogold labelling with LM6 was found
both on the plasma membrane and cell walls of the sperm
cell initials. JIM13 and JIM8, which label a glycan component
of AGPs, did not label the cell walls of sperm initial cells but
did label the shield cell cell walls. These results suggest that
AGPs are components of the antheridum’s epidermis but are
not involved in spermatogenesis. The LM6 labelling may
simply be recognizing RG-I epitopes involved in early sperm
cell initial formation. XyG localization with LM15 was
limited to the capitular cells and early sperm cell initials but
not in cells of the maturing filaments. This demonstrates a
role for XyGs during the key, early transformation of capitulum cells into young sperm filaments and their inclusive
sperm cell initials but not in later stages of sperm cell initial
multiplication. All of these results demonstrate that antheridial
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
development and spermatogenesis employ cell wall polymers
similar to those found in the vegetative thallus of Chara or
in the primary cell walls of land plants. Further biochemical
analyses will be required in order to refine the identity of
these polymers and to identify specific moieties unique to
the cell walls of C. corallina and specifically its antheridium.
The development of the spermatogenous filaments in Chara
entails many distinct transformational events (Kwiatkowska,
2003). When a sperm cell initial transforms into a functioning
sperm cell, the protoplast retracts from the cell wall, becomes
helical, produces two flagella and, most importantly, becomes
covered with small scales. The scales cover both the sperm
cell’s plasma membrane and flagellar membranes. In the
immunocytochemical part of this study, we found no antibody
labelling of the sperm cell surface once scales appeared upon
the retracted protoplast. This demonstrates a fundamental
switch in extracellular matrix polymer production during the
final stage of sperm development, i.e. the conversion of a
cell-wall-covered cell to a scale-covered cell. A scale covering
is a condition that is commonly seen in the motile cells of the
primitive green algal group, the Prasinophyceae, and the basal
taxon of the CGA, Mesostigma viride (Domozych et al.,
1991). Scales consist of polymers that contain 2-keto sugars
and are very different from those found in the cell walls of
green plants (Becker et al., 1991, 1994; Domozych et al.,
1991). The functional significance of scales in primitive green
algae and on motile sperm cells of advanced CGA remains
unknown. However, the production of scales during the last
phase of spermatogenesis demonstrates that gene expression
controlling scale synthesis occurs quickly, precisely and in a
coordinated fashion amongst sperm cells. Also, the secretory
apparatus (e.g. Golgi apparatus and secretory vesicle network)
must switch over rapidly from processing cell wall macromolecules to scales. Conversely, the production of scales occurs
only in the motile phase of the Chara lifecycle (e.g. sperm)
while all other cells are covered with cell wall. These observations lend support to the idea that cell wall polymers most
probably arose in non-motile phases (Mattox and Stewart,
1984). Further analyses will be needed to determine if cell
wall macromolecules similar to those found in land plants are
found in the non-motile phases of prasinophytes or Mesostigma.
Approximately 470 million years ago, land plants evolved
from a group of green algal ancestors that are currently represented by the CGA. Gametangium development during
sexual reproduction phases of the life cycle represents a critical
phenomenon that is necessary for gene transfer and survival.
Cell wall development is an integral part of this morphogenesis.
The green algal ancestors of land plants, the CGA, evolved
mechanisms that employed many cell wall polymers also
found in land plants to yield complex gametangia like the antheridium. Further studies will be needed to investigate the cell wall
dynamics of other structures including the oogonia and zygotes
in the CGA in order to elucidate more fully the evolution of the
cell wall and its roles in sexual reproduction.
ACK N OW L E DG E M E N T S
We thank Amy Snyder for her efforts in isolating antheridia,
and Catherine R. Domozych for her help in reviewing this
manuscript. We thank J. Paul Knox of the University of
1055
Leeds for many of the antibodies used in this study. Part of
this work was supported by a grant from National Science
Foundation (DBI-0419131; to D.S.D.).
L I T E R AT U R E CI T E D
Becker B, Becker D, Kamerling JP, Melkonian M. 1991. 2-Keto-sugar acids
in green flagellates: a chemical marker for prasinophycean scales. Journal
of Phycology 27: 498 –504.
Becker B, Marin B, Melkonian M. 1994. Structure, composition and biogenesis of prasinophyte cell coverings. Protoplasma 181: 233 –244.
Bell PR, Hemsley AR. 2000. Green plants, 2nd edn. Cambridge: Cambridge
University Press.
Blake AW, McCartney L, Flint JE, et al. 2006. Understanding the biological
rationale for the diversity of cellulose-directed carbohydrate-binding
modules in prokaryotic enzymes. Journal of Biological Chemistry 281:
29321–29329.
Cardenas L, Lovy-Wheeler A, Kunkel JG, Hepler PK. 2008. Pollen tube
growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization. Plant Physiology 146: 1611– 1621.
Carpita N, McCann M. 2000. The cell wall. In: Buchanan BB, Wilhelm G,
Jones RL. eds. Biochemistry and molecular biology of plants. Bethesda,
MD: American Society of Plant Physiologists, 52– 108.
Clausen MH, Willats WGT, Knox JP. 2003. Synthetic methyl hexagalacturonate hapten inhibitors of anti-homogalacturonan monoclonal antibodies.
Carbohydrate Research 338: 1797–1800.
Cook ME, Graham LE, Botha CEJ, Lavin CA. 1997. Comparative ultrastructure of plasmodesmata of Chara and selected bryophytes: towards
an elucidation of the evolutionary origin of plant plasmodesmata.
American Journal of Botany 84: 1169–1178.
Cook ME, Graham LE, Lavin CA. 1998. Cytokinesis and nodal anatomy in
the charophycean green alga Chara zeylanica. Protoplasma 203: 65– 74.
Davies WP, Lewis BG. 1981. Development of pectic projections on the
surface of wound callus cells of Daucus carota. L. Annals of Botany
47: 409– 413.
Domozych DS, Wells B, Shaw PJ. 1991. Basket scales of the green alga,
Mesostigma viride, chemistry and ultrastructure. Journal of Cell Science
100: 397 –407.
Domozych DS, Serfis A, Keimle S, Gretz MR. 2007. The structure and biochemistry of the homogalacturonans of the cell wall of the desmid,
Penium margaritaceum. Protoplasma 230: 99–115.
Donaldson LA, Singh AP. 1984. Cell wall protuberances in the resin-pocket
callus of Pinus nigra. Canadian Journal of Botany 62: 570–574.
Freshour G, Bonin CP, Reiter D, Albersheim P, Darvill AG, Hahn MG.
2003. Distribution of fucose-containing xyloglucans in cell walls of the
Mur1 mutant of Arabidopsis. Plant Physiology 131: 1602–1612.
Geisler DA, Sampathkumar A, Mutwil M, Persson S. 2008. Laying down
the bricks: logistic aspects of cell wall biosynthesis. Current Opinion in
Plant Biology 11: 647– 652.
Gillet C, Liners F. 1995. Changes in distribution of short pectic polysaccharides induced by monovalent ions in the Nitella cell wall. Canadian
Journal of Botany 74: 26–30.
Gillet C, Cambier P, Liner F. 1992. Release of small polyuronides from
Nitella cell walls during ionic exchange. Plant Physiology 100: 846– 852.
Gillet C, Voue M, Cambier P. 1998. Site-specific counterion binding and
pectic chains conformational transitions in the Nitella cell wall. Journal
of Experimental Botany 49: 797–805.
Hepler PK, Vidali L, Cheung AY. 2001. Polarized cell growth in higher
plants. Annual Review of Cell and Developmental Biology 17: 159– 187.
Herve C, Rogowski A, Gilbert HJ, Knox JP. 2009. Enzymatic treatments
reveal differential capacities for xylan recognition and degradation in
primary and secondary cell walls. The Plant Journal 58: 413–422.
Hughes J, McCully ME. 1975. The use of an optical brightener in the study of
plant structure. Stain Technology 50: 319– 329.
Jones L, Seymour GB, Knox JP. 1997. Localization of pectin galactan
in tomato cell walls using monoclonal antibody specific to
(1– 4)b-D-galactan. Plant Physiology 113: 1405–1412.
Kazmierczak A. 1999. Determination of GA3 in Chara vulgaris by capillary
electrophoresis system. Acta Physiologia Plantarum 21: 344– 348.
Kazmierczak A, Stepinski D. 2005. GA3 content in young and mature antheridia of Chara tomentosa estimated by capillary electrophoresis. Folia
Histochemica et Cytochemica 43: 65–67.
1056
Domozych et al. — Cell wall macromolecules of the antheridium of Chara
Kazmierczak A, Kwiatkowska M, Poplonska K. 1999. GA3 content in
antheridia of Chara vulgaris at the proliferative stage and in spermiogenesis estimated by capillary electrophoresis. Folia Histochemica
et Cytobiologica 37: 49– 52.
Knox JP, Linstead PJ, Peart J, Cooper C, Roberts K. 1991.
Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation. The Plant
Journal 1: 317–326.
Kwiatkowska M. 2003. Plasmodesmal changes are related to different developmental stage of antheridia of Chara species. Protoplasma 222: 1 –11.
Lee YJ, Yang Z. 2008. Tip growth: signaling in the apical zone. Current
Opinion in Plant Biology 11: 662– 671.
Lee KJD, Sakata Y, Mau S-L, et al. 2005. Arabinogalactan-proteins are
required for apical cell extension in the moss Physcomitrella patens.
The Plant Cell 17: 3051–3065.
Lerouxel O, Cavalier DM, Liepman AH, Keegstra K. 2006. Biosynthesis of
plant cell wall polysaccharides – a complex process. Current Opinion in
Plant Biology 9: 621– 630.
Leroux O, Knox JP, Leroux F, et al. 2007. Intercellular pectic protuberances
in Asplenium: new data on their composition and origin. Annals of Botany
100: 1165–1173.
Lewis LA, McCourt RM. 2004. Green algae and the origin of land plants.
American Journal of Botany 36: 1535– 1556.
Marcus SE, Verhertbruggen Y, Hervé C, et al. 2008. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls. BMC
Plant Biology 8: 60. doi:10.1186/1471-2229-8-60.
Mattox KR, Stewart KD. 1984. Classification of the green algae: a concept
based on comparative cytology. In: Irvine DEG, John DM. eds.
Systematics of the green algae. London: Academic Press, 29–72.
McCabe PF, Valentine TA, Forsberg LS, Pennell RI. 1997. Soluble signals
from cells identified at the cell wall establish a developmental pathway in
carrot. The Plant Cell 9: 2225– 2241.
McCartney L, Marcus SE, Knox JP. 2005. Monoclonal antibodies to plant
cell wall xylans and arabinoxylan. Journal of Histochemistry and
Cytochemistry 53: 543–546.
Moestrup O. 1970. The fine structure of mature spermatozoids of Chara
corallina, with special reference to microtubules and scales. Planta 93:
295–308.
Moller I, Sørensen I, Bernal AJ, et al. 2007. High-throughput mapping of
cell wall polymers within and between plants using novel microarrays.
The Plant Journal 50: 1118–1128
Pettolino FA, Hoogenraad NJ, Ferguson C, Bacic A, Johnson E, Stone BA.
2001. A (1 –4)-b-mannan-specific monoclonal antibody and its use in the
immunocytochemical location of galactomannans. Planta 214: 235–242.
Pickett-Heaps JD. 1969. Ultrastructure and differentiation in Chara (Fibrosa).
IV. Spermatogenesis. Australian Journal of Biological Sciences 21:
655–690.
Pickett-Heaps JD. 1975. Green algae. Sunderland, MA: Sinauer Associates.
Popper ZA. 2008. Evolution and diversity of green plant cell walls. Current
Opinion in Plant Biology 11: 286–292.
Popper ZA, Fry SC. 2003. Primary cell wall composition of bryophytes and
charophytes. Annals of Botany 91: 1 –12.
Proseus TE, Boyer JS. 2006. Calcium pectate chemistry controls growth rate
of Chara corallina. Journal of Experimental Botany 57: 3989– 4002.
Renzaglia KS, Duff RJ, Nickrent DL, Garbary DJ. 2000. Vegetative and
reproductive innovations of early land plants: implications for a unified
phylogeny. Philosophical Transactions of the Royal Society B:
Biological Science 355: 769– 793.
Shaw AJ, Goffinet B. 2000. Bryophyte biology. Cambridge: Cambridge
University Press.
Smallwood M, Beven A, Donovan N, et al. 1994. Localization of cell wall
proteins in relation to the developmental anatomy of the carrot root
apex. The Plant Journal 5: 237–246.
Smallwood M, Yates EA, Willats WGT, Martin H, Knox JP. 1996.
Immunochemical comparison of membrane-associated and secreted
arabinogalactan-proteins in rice and carrot. Planta 198: 452– 459.
Sørensen I, Pettolino FA, Wilson SM, et al. 2008. Mixed linkage (1 ! 3),
(1 ! 4)-b-D-glucan is not unique to the Poales and is an abundant
component of Equisetum arvense cell walls. The Plant Journal 54:
510– 521
Tormo J, Lamed R, Chirino AJ, et al. 1996. Crystal structure of a bacterial
family-III cellulose-binding domain: a general mechanism for attachment
to cellulose. EMBO Journal 15: 5739–5751.
Van Sandt VST, Stieperaere H, Guisez Y, Verbelen J-P, Vissenberg K.
2007. XET activity is found near sites of growth and cell elongation in
bryophytes and some green algae: new insights into the evolution of
primary cell wall elongation. Annals of Botany 99: 39–51.
Verhertbruggen Y, Marcus SE, Haeger A, Ordaz-Ortiz JJ, Knox JP. 2009.
An extended set of monoclonal antibodies to pectic homogalacturonan.
Carbohydrate Research. In press. doi:10.1016/j.carres.2008.11.010.
Willats WGT, Knox JP. 2003. Molecules in context: probes for cell wall
analysis: In: Rose JKC. ed. The plant. Boca Raton, FL: Blackwell
Publishing/CRC Press, 92– 110.
Willats WGT, Marcus SE, Knox JP. 1998. Generation of a monoclonal
antibody specific to (1–5)-a-arabinan. Carbohydrate Research 308:
149– 152.
Willats WGT, Steele-King CG, McCartney L, Orfila C, Marcus SE, Knox
JP. 2000. Making and using antibody probes to study plant cell walls.
Plant Physiology and Biochemistry 38: 27– 36.
Willats WG, McCartney L, Steele-King CG, et al. 2004. A xylogalacturonan
epitope is specifically associated with plant cell detachment. Planta 218:
673– 681.