J. Phycol. 44, 1257–1268 (2008)
2008 Phycological Society of America
DOI: 10.1111/j.1529-8817.2008.00568.x
CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM
BHARUCHAE F. MINOR (OEDOGONIALES, CHLOROPHYTA) 1
Jose´ M. Estevez2,3
Carnegie Institution, Plant Biology, Stanford University, Stanford, California 94305, USA
Patricia I. Leonardi
Departamento de Biologı́a, Bioquı́mica y Farmacia, Universidad Nacional del Sur, CERZOS-CONICET, 8000 Bahı́a Blanca, Argentina
and Josefina S. Alberghina
Departamento de Biodiversidad y Biologı́a Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria – Pabellón 2, 1428 Buenos Aires, Argentina
Cell wall changes in vegetative and suffultory cells
(SCs) and in oogonial structures from Oedogonium
bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several
carbohydrate epitopes. Vegetative cells and SCs
develop only a primary cell wall (PCW), whereas
mature oogonial cells secrete a second wall, the
oogonium cell wall (OCW). Based on histochemical
and immunolabeling results, (1 fi 4)-b-glucans in
the form of crystalline cellulose together with a variable degree of Me-esterified homogalacturonans
(HGs)
and
hydroxyproline-rich
glycoprotein
(HRGP) epitopes were detected in the PCW. The
OCW showed arabinosides of the extensin type and
low levels of arabinogalactan-protein (AGP) glycans
but lacked cellulose, at least in its crystalline form.
Surprisingly, strong colabeling in the cytoplasm of
mature oogonia cells with three different antibodies
(LM-5, LM-6, and CCRC-M2) was found, suggesting
the presence of rhamnogalacturonan I (RG-I)–like
structures. Our results are discussed relating the
possible functions of these cell wall epitopes with
polysaccharides and O-glycoproteins during oogonium differentiation. This study represents the first
attempt to characterize these two types of cell walls
in O. bharuchae, comparing their similarities and differences with those from other green algae and land
plants. This work represents a contribution to the
understanding of how cell walls have evolved from
simple few-celled to complex multicelled organisms.
Abbreviations: AGP, arabinogalactan protein; BF,
basic fuchsine; CW, calcofluor white; HG, homogalacturonan; HRGP, hydroxyproline-rich glycoprotein;
LSCM,
laser
scanning
confocal
microscopy; MB, methylene blue; OCW, oogonium cell wall; PCW, primary cell wall; RG-I,
rhamnogalacturonan I; RR, ruthenium red; SC,
suffultory cell, TBO, toluidine blue ortochromatic; XG, xyloglucan
The order Oedogoniales is a monophyletic group
within the Chlorophyceae lineage made up of green
algae with an unusual form of cytokinesis and cell
elongation by ring formation, stephanokont type of
zooids, and a complex oogamous sexual reproduction (Pickett-Heaps 1975, Graham and Wilcox 2000,
Alberghina et al. 2006). Oogenesis begins with a
highly asymmetrical division of the oogonial initial
cell (Ohashi 1930, Coss and Pickett-Heaps 1974,
Pickett-Heaps 1975). Following mitosis, cytokinesis
cuts off the oogonial initial cell into the small basal
SC and an apical daughter cell, which becomes the
swollen oogonium. The SC may undergo further
divisions to form more oogonial structures.
Cell wall changes during oogonial development
are crucial for the fertilization process (PickettHeaps 1975). During the expansion of the cell
wall, a period of active biosynthesis of polysaccharides and glycoproteins takes place in the wall as
the cell increases in volume several times (Ohashi
1930, Pickett-Heaps and Fowke 1970, Pickett-Heaps
1975). In addition, the cell wall in the already
formed oogonium is weakened to permit the
opening of the fertilization pore or circumcision
(Hoffman 1971, Coss and Pickett-Heaps 1974,
Mrozinska 1985). To study changes in cell wall epitopes from vegetative to reproductive states during
cell differentiation, we have chosen Oedogonium
because it is an adequate model organism due to
its simple thallus structure composed of a single
row of cells.
Key index words: cell wall; cellulose; green algae;
HRGP; Oedogonium bharuchae f. minor; oogonium
development; pectins
1
Received 16 May 2007. Accepted 14 March 2008.
Author for correspondence: e-mail [email protected] and
[email protected].
3
Present address: Departamento de Fisiologı́a, Biologı́a Molecular
y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Ciudad Universitaria – Pabellón 2, 1428 Buenos Aires,
Argentina.
2
1257
1258
J O S É M . E S T E V E Z E T A L .
The PCW of embryophytes comprises highly complex macromolecules, such as cellulose microfibrils,
matrix polysaccharides, and structural glycoproteins.
Moreover, the wall plays essential biological roles,
including mediation of ion exchange, tissue cohesion, defense against pathogens, and regulation of
cell expansion (Carpita and Gibeaut 1993, Fry 2000,
Somerville et al. 2004, Cosgrove 2005). Both the
chemical composition and macromolecular organization of the different cell wall components are
highly dynamic during development and differentiation of cells and tissues (Carpita et al. 2001). In the
past few years, it has become evident that the cell
wall composition shows considerable similarities and
also many divergences between different embryophyte plant groups. The analysis of PCWs has shown
that xyloglucans (XGs) are major polysaccharides
with a role in forming bridges between cellulosic microfibrils in embryophytes, except for commelinid
monocots (Carpita and Gibeaut 1993). XGs also
occur in pteridophytes, mosses, and liverworts but
are absent in charophycean green algae, which are
the closest living algal relatives to land plants
(Popper and Fry 2003, 2004). Unfortunately, the cell
wall composition has been poorly studied in nonvascular plants and even less in green algae with the
exception of well-known volvocacean cell wall (Percival and McDowell 1981, Takeda 1996, Edelmann
et al. 1998, Popper and Fry 2003, Domozych et al.
2007a,b), and practically no information is available
on the nature of the cell wall for the Oedogoniales.
The aim of this study was to characterize the
changes that take place in the cell wall during the
differentiation from SC to oogonium structures in
the O. bharuchae f. minor using histochemical and
immunochemical probes. A series of monoclonal
antibodies was used to determine the presence and
location of diverse cell wall carbohydrate epitopes
comprising nonesterified HGs, HGs with different
degrees and patterns of methyl-esterification, RG-I
associated structures, and finally, O. glycans present
in AGP and extensins.
MATERIALS AND METHODS
Algal cultures. The algal material used in this study was
composed of O. bharuchae f. minor $ (BAFC-FyCE N 49) and
O. bharuchae f. minor # (BAFC-FyCE N50). O. edogonium bharuchae f. minor is a macrandrous and dioecious species in which the
ellipsoidal oogonia are located in the female filament in series of
one to five, and the oogonial aperture occurs by means of a
circumcision in supreme position (Vélez 1995). The culture
medium used for induction of the sexual reproduction was that
reported by Hill (1980), but with less than 95% of nitrogen
content. Cultures were deposited in the culture collection of the
Phycology and Experimental Microalgae Culture Laboratory
(BAFC-FyCE), at the Departamento de Biodiversidad y Biologı́a
Experimental, Universidad de Buenos Aires (UBA). Reproductive filaments with oogonia were fixed at room temperature in
1.5% glutaraldehyde in culture medium, postfixed in 1% OsO4
in sodium cacodylate buffer (0.05 M), dehydrated through a
graded acetone series, and embedded with Spurr’s low viscosity
resin. For TEM, thin sections were cut with a diamond knife and
stained with uranyl acetate and lead citrate. Later, they were
examined using a JEOL 100 CX-II electron microscope (Jeol,
Akishima, Tokyo, Japan) at the Centro de Investigaciones
Básicas y Aplicadas de Bahı́a Blanca (CRIBABB).
Cell wall histochemistry. The staining procedures used in the
LM histochemical characterization as described in Krishnamurthy (1999) were performed on thin sections as follows:
(i) Toluidine Blue O (TBO) at pH 6.8 for labeling anionic
polysaccharides and at pH = 1.0 for staining only the sulfated
polysaccharides (red-purple, c metachromasia). (ii) Ruthenium Red in aqueous solution for acidic polysaccharides (red).
(iii) Basic Fuchsine (BF; 4% w ⁄ v) in NaOH 0.1 M (pH 12.5)
methylene blue (2% w ⁄ v) for neutral (pink) and acidic
polysaccharides (red–purple), respectively (Robinson et al.
1987). (iv) Calcofluor White M2R (CW) in aqueous solution
for b-linked glycans including (1 fi 4)-, (1 fi 3)-, and (1 fi 3,
1 fi 4)-linked b-glucans (Wood et al. 1983). In addition, CW
could react with other polysaccharides, such as chitin and XGs.
(v) Aniline blue fluorochrome (Sirofluor) 0.1% in 0.1 M
K3PO4 (pH 12) for detecting callose (Stone et al. 1984). As a
control for propidium iodide (PI), CW, and callose probes,
unstained sections were exposed to UV light under the same
conditions used to detect autofluorescence from chloroplast
chl, nucleolar DNA, and ⁄ or cell walls. Phloroglucinol-HCl 2%
(w ⁄ w) in 95% (v ⁄ v) aqueous ethanol was used for detecting
lignin. This stain specifically reacts with p-hydroxycinnamaldehyde end groups (particularly 5- and ⁄ or 4-O-linked coniferyl
and sinapyl aldehydes). Reference and specificity for each
histochemical probe used in this study are included in Table 1.
All the histochemical reagents were purchase from Sigma (St.
Louis, MO, USA) unless otherwise indicated.
In situ carbohydrate immunolabeling. Thin sections were
collected on microscope slides (ProbeOn; Fisher Pittsburgh,
PA, USA) and blocked in PBS containing 10% (w ⁄ v) BSA
(PBS ⁄ BSA) for 60 min. Rat monoclonal antibodies (dil 1:50–
1:500) specific for carbohydrate epitopes (see Table 1 for
specificity) were incubated overnight at 4C and washed with
PBS ⁄ BSA (2x). For detecting antibody binding, a secondary
antibody anti-rat-IgG coupled with Alexa Fluor 488 (AF;
Molecular Probes, Carlsbad, CA, USA) (1:250) in PBS ⁄ BSA
was added for 1–2 h at room temperature. After washing with
PBS for 5 min (2·), the samples were observed in a laser
scanning confocal microscope (see below). Primary antibodies
were omitted for labeling procedure to serve as controls.
Additionally, specific controls were performed for the different
groups of primary antibodies: anti-AGP, antiextensins, and
antipectin epitopes (see Table 1). After removal of Spurr resin
with a solution containing 1.5 g NaOH in 100 mL EtOH,
sequential enzymatic digestions were performed to remove: (i)
soluble AGP and extensins (HRGP), and (ii) pectins. Cell wall
polysaccharides and glycoproteins (HRGP) were extracted as
described elsewhere (Fry 2000, Estevez et al. 2006, Domozych
et al. 2007a,b). Briefly, they were sequentially extracted using
(i) 50 mM Tris–Cl, pH 8, 10 mM EDTA, 1% Triton X-100, 0.1%
b-mercaptoethanol for soluble AGP and extensins; (ii) pectins
were extracted with 50 mM diamino-hexane-tetra-acetic acid
(CDTA) at pH 7.5. In addition, after removing the CDTA
solvent, two enzymes were added to remove all the remaining
pectins: a polygalacturonase (Sigma P-3429 EC.3.2.1.15) and a
rhamnogalacturonase (AN2528.2) isolated from Aspergillus
nidulans and expressed on Pichia pastoris (Bauer et al. 2006).
Both enzymes were incubated using 50 mM acetate buffer pH
5.5 at 37C. Finally, cellulose was digested with a cellulase from
Aspergillus niger (Sigma C-1184, EC.3.2.1.4) using 50 mM
acetate buffer at pH 5.0 at 37C (Okada 1988). Then, extracted
and ⁄ or enzymatic digested thin sections and nonextracted and
undigested sections were incubated using the same immunolabeling probes as described above to corroborate the primary
antibody specificity (Fig. S1, a–h, in the supplementary
Okada (1988)
Bauer et al. (2006)
Bauer et al. (2006)
Bolam et al. (1998)
Smallwood et al. (1996)
Yates et al. (1996)
Knox et al. (1991)
Smallwood et al. (1994)
Smallwood et al. (1994)
Smallwood et al. (1994)
Clausen et al. (2003)
Clausen et al. (2003)
Liners et al. (1989)
Jones et al. (1997)
Willats et al. (1998)
Puhlmann et al. (1994)
Steffan et al. (1995)
Puhlmann et al. (1994)
(1 fi 4)-, and (1 fi 3, 1 fi 4)-linked b-glucans
HG
RG-I
Crystalline cellulose
b-d-GlcAp in the AGP glycan
AGP glycan [b-d-GlcAp-(1 fi 3)-a-d-GlcAp-(1 fi 2)-l-Rha]
AGP glycan
a-Araf-(1 fi 3)-b-l-Araf-(1[ fi 2)-b-l-Araf-(1 fi )]1–-2 or [(1 fi 2)-b-l-Araf]1–2
a-Araf-(1 fi 3)-b-l-Araf-(1[ fi 2)-b-l-Araf-(1 fi )]1–2 or [(1 fi 2)-b-l-Araf]1–2
a-Araf-(1 fi 3)-b-l-Araf-(1[ fi 2)-b-l-Araf-(1 fi )]1–2 or [(1 fi 2)-b-l-Araf]1–2
Partially low methyl-esterified HG epitope: unesterified residues adjacent to
or flanked by residues with methyl-ester groups
Partially methyl-esterified HG epitope: methyl-esterified residues with adjacent
or flanking unesterified GlcAp ⁄ alternating methyl-esterified GlcAp residues
Nonblockwise partially Me-HG
HG, Ca+2-linked
[(1 fi 4)-b-d-Galp]4
[(1 fi 5)-a-l-Araf]4-5
RG-I, specific epitope is unknown
Arabinosylated [(1 fi 6)-b-d-Galp]
(1 fi 2)-linked a-l-Fucp in XG and ⁄ or in RG-I backbone
Clausen et al. (2003)
Krishnamurthy (1999)
Krishnamurthy (1999)
Robinson et al. (1987); Krishnamurthy (1999)
Wood et al. (1983)
Robinson et al. (1987); Krishnamurthy (1999)
Krishnamurthy (1999)
Stone et al. (1984); Krishnamurthy (1999)
Reference
Polyanions (at pH = 6.5), sulfated polysaccharides (at pH = 1)
Anionic polysaccharides
Anionic polysaccharides
(1 fi 3)-, (1 fi 4)-, and (1 fi 3, 1 fi 4)-linked b-glucans
Neutral polysaccharides
Lignin, specifically p-hydroxycinnamaldehyde end groups
b-(1 fi 3)-glucan (callose)
Specificity
AGP, arabinogalactan protein; HG, homogalacturonan; RG-I, rhamnogalacturonan; XG, xyloglucan.
*This antibody could react with glycans in AGP.
LM-7
2F4
LM-5
LM-6
CCRC-M2
CCRC-M7*
XGs ⁄ RG epitope
CCRC-M1
JIM-7
Dyes
Toluidine Blue O (TBO)
Ruthenium Red (RR)
Methylene Blue (CB)
Calcofluor White (CW)
Basic Fuchsine (BF)
Phloroglucinol-HCl
Siroflour (Aniline Blue)
Enzymes
Cellulase
Polygalacturonase
Rhamnogalacturonase
CBM
CBM2a
Monoclonal antibodies
AGP epitopes
LM-2
MAC-207
JIM-16
Extensin epitopes
JIM-11
JIM-20
LM-1
Pectin epitopes
JIM-5
Probe
Table 1. Probes used for cell wall characterization of the green alga Oedogonium bharuchae f. minor.
C E L L W A L L E P I T O P E S I N T H E G R E E N A L G A O E D OG O N I U M
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J O S É M . E S T E V E Z E T A L .
material). To label crystalline cellulose, sections were incubated with 2.5 lg Æ mL)1 of CBM2a in PBS containing 5%
(w ⁄ v) milk protein (MP ⁄ PBS) for 1.5 h. Samples were washed
in PBS at least three times and then incubated with a 100-fold
dilution of rabbit anti-His polyclonal antibody (Sigma) in
MP ⁄ PBS for 1.5 h. Then, a secondary antibody anti-rabbit-IgG
coupled with Alexa Fluor 488 (AF; Molecular Probes, Eugene,
OR, USA) (1:250) in MP ⁄ PBS was added for 1–2 h at room
temperature. After washing with PBS for 5 min (2·), the
samples were observed in a laser scanning confocal microscope
(see below). As a control for this labeling, the rabbit anti-His
polyclonal (used as primary antibody) or the CBM2a were
omitted during labeling procedure. The antibodies CCRC-M1,
CCRC-M2, and CCRC-M7 were obtained from the Complex
Carbohydrate Research Center (Athens, GA, USA), and the
antibodies 2F4, JIM-5, JIM-7, JIM-11, JIM-16, JIM-20, LM-1,
LM-2, LM-5, LM-6, LM-7, and MAC 206 from Plant Probes
(Leeds, UK). In addition, CMB2a was provided by Paul Knox
(University of Leeds, UK).
Laser scanning confocal microscopy (LSCM). Imaging was
performed using an MRC 1024 laser scanning confocal head
(Bio-Rad, Hercules, CA, USA) mounted on a Diaphot 200
inverted microscope (Nikon, Tokyo, Japan), a Zeiss 510 laser
scanning confocal microscope (Thornwood, NY, USA) and a
Leica TCS SP2 AOBS (Wetzlar, Germany). The objectives used
were a 60· Nikon PlanApo water immersion (WI) 1.2 NA
numerical aperture (Technical Instruments, San Francisco, CA,
USA), a 40X Nikon PlanApo WI 0.9 NA, and a HCX PL APO
63X ⁄ 1.2 W Corr ⁄ 0.17 Lbd. Bl. The samples were excited with
two lasers (Ar ⁄ Kr and He ⁄ Cd) with the following wavelengths:
568 nm for propidium iodide (PI) or chl fluorescence (CF)
and 488 nm for Alexa Fluor secondary antibody (AF). Thin
sections were mounted on coverslips in glycerol:water (1:1).
Three-dimensional reconstructions (Z-projections) of image
stacks (n = 30 slices) were performed using Image J version
1.39 software (http://rsb.info.nih.gov/ij/) at maximum intensity. All images were processed with Adobe Photoshop 6.0
and assembled with Adobe Illustrator 10.0 software (Adobe
Systems, Mountain View, CA, USA).
RESULTS AND DISCUSSION
In O. bharuchae f. minor, the vegetative cells and
SCs (Fig. 1a) showed only a single wall layer, designated here as PCW. At least two different cell wall
stages were recognized (stages I–II; Fig. 1, c–m) during oogonium development (Fig. 1b). Immature
oogonia (IO) only showed PCW (stage I; Fig. 1, c, f,
and j), and female gametangia close to their maturation phase had a new wall layer beneath PCW.
This new layer, designated here as OCW, was
located inside the PCW (stages IIa–c; Fig. 1, d–e).
In addition, an outermost electron-dense layer was
detected in the PCW of the mature oogonia (Fig. 1,
f–m), possibly indicating the presence of a nonpolysaccharide component, such as putative wall (glyco)proteins (see also Fig. 5). Although both PCW
and OCW had a multilayer structure at TEM level,
OCW was much more electron-dense than PCW,
Fig. 1. Cell wall morphology during oogonium development in Oedogonium bharuchae f. minor. (a–b) Light micrographs of vegetative
and oogonial cells. (a) General aspect of vegetative cells. (b) General view of an oogonium. (c–m) Transmission electron micrographs of
cell walls in oogonial cells. (c–e) General views of oogonium developmental stages (St., stages I–II). Scale bars = 5 lm. (c) Immature oogonium in the beginning of its differentiation (stage I). (d) Already expanded oogonium (stage IIa). (e) Complete and mature oogonium
(stage IIc). (f–i) Oogonium cell wall (OCW) development. The OCW region shown in detail is indicated in Fig. 1b (rectangle). Scale
bars = 2 lm. (f) Beginning of the cell wall expansion where only the primary cell wall (PCW) is observed. (g) Fully expanded oogonium
with the incipient development of the oogonium cell wall (OCW) (stage IIa). (h–i) Complete development of the OCW (stages IIb-c).
Mature oogonia, possibly before (stage IIb) and after being fertilized (stage IIc). (j–m) Details of oogonium cell wall layers through the
developmental stages from the beginning of the undifferentiated oogonia (j), immature oogonium (k), and mature oogonium possibly
before (l) and after being fertilized (m). Scale bars = 1 lm. PCW (white arrowheads), OCW (asterisks), outer cell wall layer of the PCW
(white-black arrows) and the new layer synthesized below the OCW (black arrows) are indicated in the figure. ID = immature oogonium;
MO = mature oogonium.
C E L L W A L L E P I T O P E S I N T H E G R E E N A L G A O E D OG O N I U M
suggesting a different chemical composition, as
shown by the histochemical analysis (Fig. 2) and
epitope labeling (see Figs. 3–5).
Cell wall histochemistry. PCW and OCW layers were
characterized using general histochemical probes
(Table 1, Fig. 2). PCW was strongly reactive to BF
dye (Fig. 2, a–c), but very weak reaction or no reaction was observed for TBO dye, both at pH = 1 (not
shown) and pH = 6.8 (Fig. 2d), and for RR probe
(Fig. 2e). In addition, strong labeling was detected
with CW (Fig. 2, f and j) suggesting the presence of
(1 fi 4)- and ⁄ or (1 fi 3, 1 fi 4)-linked b-glucans
[the negative Aniline Blue fluorescence eliminated
(1 fi 3)-linked b-glucans]. Immature oogonia
showed similar PCW features, but their thickness
was 40 % lower (n = 20) than that in the undifferentiated vegetative cells and SCs (Fig. 2b), due to
the cell expansion process during the first steps of
oogonial development.
The OCW was clearly reactive to the cationic dyes
such as RR (Fig. 2, g, n, and p), Methylene Blue
(MB, Fig. 2, h, k, and q), and TBO at pH = 6.8
(Fig. 2, i and l), but it was essentially unreactive to
the acidic TBO (at pH = 1.0), specific for sulfated
macromolecules (Fig. 2m; Krishnamurthy 1999).
Strong labeling was observed for anionic molecules in the cytoplasm of the oogonia (Fig. 2, h, k,
q, and m). No reaction in PCW and OCW was
observed with phloroglucinol–HCl or with Aniline
Blue fluorochrome (results not shown), suggesting
that neither p-hydroxycinnamaldehyde end groups
usually found in lignin nor callose [(1 fi 3)-bglucans)] is present in either the PCW or the OCW
of O. bharuchae (Table 1).
To test if b-glycans stained with CW were in fact
cellulose, sections were labeled with CBM 2a that
specifically binds crystalline cellulose (Bolam et al.
1998). A strong reaction was observed only in the
PCW, but no signal was found in the OCW (Fig. 2,
r–u) as shown before for CW staining (Fig. 2, r–s).
In addition, negative results were obtained in the
cellulase-treated sections with CBM2a and CW
(results not shown). All these results together indicate that crystalline cellulose is present in the PCW
of O. bharuchae f. minor. On the other hand, from
these results, we cannot eliminate the possibility
that amorphous cellulose and ⁄ or (1 fi 3, 1 fi 4)linked b-glucans are also present in the PCW.
Based on the histochemical stainings, PCW and
OCW differ in composition. To obtain further information on the differences in cell wall polymers
between these two wall layers, a set of monoclonal
antibodies was used against well-characterized carbohydrate epitopes (Table 1; Figs. 3–5). For primary
mAb specificity, sequential extractions with Tris–Cl
buffer and CDTA solvents were used as controls to
remove soluble HRGP (AGP and extensins) and
pectins, respectively (Fry 2000, Estevez et al. 2006,
Domozych et al. 2007a,b) from thin Oedogonium sections before performing the labeling. In addition, a
1261
mixture of polygalacturonase and rhamnogalacturonase was added after CDTA extraction to remove
the putative remaining pectins (see Materials and
Methods for details). Then, preextracted and enzymatically digested thin sections were incubated with
each primary antibody (Table 1) to test for the presence of AGP and extensin epitopes, non- and partially methyl-esterified HG, and RG-I epitopes. The
comparison between nontreated and preextracted,
digested thin sections provides a necessary control
to test the epitope specificity of the primary mAbs
(Figs. 3–5 and Fig. S1, a–h).
Pectin-associated epitopes. Nonesterified HGs crosslinked by Ca+2 were present in the PCW from oogonial cells and absent in the SCs (Fig. 3, a and b). A
similar pattern of labeling was observed with the
low-esterified Me-HG epitope recognized by JIM-5
mAb (Fig. 3, c and d). JIM-5 mAb labels HG molecules with nonesterified residues located adjacent to
or flanked by residues with methyl-ester groups
(Clausen et al. 2003). The high-Me esterified-HG
(JIM-7 mAb) was located in the PCW from the oogonial cells and SCs (Fig. 3, e and f). JIM-7 reacted to
partially Me-esterified HG epitope only when the
methyl-esterified residues were adjacent or flanked
by unesterified GalAp, or alternated with methylesterified GalAp units. In addition, nonblockwise
de-esterified HG was more strongly labeled with
LM-7 mAb in the PCW from SCs than from oogonial cells (Fig. 3, e and f; Clausen et al. 2003).
Similarly, arabinosylated (1 fi 6)-b-d-Galp structures,
possibly associated with type-II arabinogalactans
(II-AGs, Steffan et al. 1995), were detected with
CCRC-M7 mAb in the PCW from SCs but not from
the expanded oogonium (Fig. 3, g and h).
It is well known that HG forms stiff gels through
Ca2+-mediated crosslinking, and that growing plant
cells usually synthesize HG in which most of the carboxyl groups are methyl esterified (Cosgrove 2005).
Highly esterified HG usually does not form stiff gels,
and their secretion might help the expanding wall
to remain pliant. Carboxyl-based crosslinking sites
are subsequently unmasked, as cells stop growing,
by action of pectin methyl esterases. This reaction
leaves free the carboxyl group for Ca 2+ crosslink
and gel formation (Li et al. 2002, Cosgrove 2005).
It was proposed, at least for rapidly growing cells
like pollen tubes, that highly esterified pectins are
secreted at the tip. These are subsequently deesterified by methyl estereases (Li et al. 2002). This
hypothesis is compatible with immunocitochemical
results obtained from SCs and oogonial cells. The
presence of HG with a variable degree of nonblockwise methyl esterification was observed only in the
PCW, which would be regularly esterified (JIM-7
and LM-7 mAbs) in the vegetative cells and SCs
(Fig. 3, a, b, e, and f). The putative blocks of GalAp
units, possibly flanked by methyl-esterified units
(detected with JIM-5) or nonesterifed HG (shown
by 2F4 mAb), were present mainly in the oogonial
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J O S É M . E S T E V E Z E T A L .
Fig. 2. Cell wall histochemistry light micrographs of the suffultory cell (SC) and oogonia of Oedogonium bharuchae f. minor using LM
and laser scanning confocal microscopy (LSCM). (a–f) Histochemistry of the cell wall polysaccharides. Primary cell wall (PCW) is indicated
with arrowheads. (a–c) Methylene Blue (anionic polysaccharides)-Basic Fuchsine (neutral polysaccharides) (MB-BF) staining. (b) Detail of
the PCW in the SC (top) and in the immature oogonia (bottom). (c) Junction zone between the oogonium and SC. (d) Toluidine Blue
O staining for anionic polysaccharides (mainly carboxylated groups) (TBO, pH = 6.8). (e) Ruthenium Red for anionic polysaccharides
(RR). (f) Calcofluor White staining (CW), specific for (1 fi 3)-, (1 fi 4)-, and (1 fi 3, 1 fi 4)-linked b-glucans. (g–q) General aspect of
the oogonia (top panels) and details of the apical and basal zones (bottom panels). (g–i) Localization of the anionic polysaccharides.
Presence (white arrowheads) and absence (asterisks) of oogonium cell wall (OCW) together with the PCW (black arrowheads) are indicated in the figures. (g) RR staining for anionic polysaccharides. (h) MB-BF staining. (i) TBO (pH = 6.8) for carboxylated polysaccharides.
(j) CW staining. Absence of (1 fi 3)-, (1 fi 4)-, and (1 fi 3, 1 fi 4)-linked b-glucans in the OCW. (k–n) Detail of the PCW and OCW on
the boundary between SC and mature oogonium. (k) MB-BF staining. (l) TBO staining of anionic (mainly carboxylated) polysaccharides
(pH = 6.8). (m) TBO staining only for sulfated polymers (pH = 1). (n) RR staining. (o–q) Details of PCW and OCW in the mature oogonia. (o) Unstained section. (p) Detail of the cell walls in the oogonia with RR staining. (q) MB-BF staining. (r–u) Strong labeling with
CBM2a that binds to crystalline cellulose in the PCW of SC (r–s) and in the oogonial PCW (t–u). Note that there is no labeling in the
OCW layer. SC observed under LM (r) and labeled cellulose using CBM2a in the LSCM (s). (t–u) Mature oogonium observed using LM
as a reference (t) and CBM2a labeling in the LSCM (u). CBM2a is shown in pseudogreen (Alexa fluor 488). Scale bars = 5 lm. Absence
of labeling is shown in the OCW (**) layer. IO = immature oogonism; MO = mature oogonium.
C E L L W A L L E P I T O P E S I N T H E G R E E N A L G A O E D OG O N I U M
PCW (Clausen et al. 2003). Deesterification of HG
has been reported in cell walls of higher plants and
unicellular green algae (Zhang and Staehelin 1992,
Luetz-Meindl and Brosch-Salomon 2000, Domozych
et al. 2007a,b). In the case of Oedogonium, it would
clearly take place after the expansion of the PCW in
the already developed oogonia. Further time-lapse
experiments are needed to determine the beginning of the deesterification process of HG.
Similar labeling patterns in the cytoplasm of
oogonial cells were observed for the following antibodies: LM-5 (Fig. 4, a and b), which recognizes the
epitope (1 fi 4)-b-d-galactan (Jones et al. 1997);
LM-6 (Fig. 4, c and d), specific for a (1 fi 5)-a-larabinan-structure (Willats et al. 1998); and CCRCM2 (Fig. 4, e and f), which reacts to RG-I polymers
(although its structure is unknown, Puhlmann et al.
1994). All of them showed the same pattern of
strong intracellular labeling. In addition, similar
cytoplasmatic staining was observed with MB and
TBO dyes (Fig. 2, h and i, respectively), most likely
due to the presence of negatively charged units
(e.g., GalAp). Consequently, this colabeling with the
three mAbs suggests that O. baruchae is able to synthesize pectins with RGI-like structures only at a
mature stage of oogonial development. However,
further chemical characterization is needed to confirm these results. In the green algae Penium, 6-linked
Gal units have been detected, possibly arising as
component of type II-AG side chains and also as a
positive indicator of RG-I like pectins (Domozych
et al. 2007a). In addition, in Sphagnum leaf pectins,
linkage analysis revealed 4-GalAp, 2-Rha, and 2,4Rha, providing some evidence of RG-I like molecules (Kremer and Pettolino 2004). So far, there is
no clear evidence whether the RG-I molecules present in vascular plants are also synthesized by simpler
organisms, such as green algae and bryophytes
(Kremer and Pettolino 2004, Domozych et al.
2007a). On the contrary, based on the biochemical
data available, pectins of the HG type seem to be
present in ancient organisms like green algae that
preceded the emergence of green plants onto land
(Zhang and Staehelin 1992, Luetz-Meindl and
Brosch-Salomon 2000, Ligrone et al. 2002, Popper
and Fry 2003, Hoffman et al. 2005, Domozych et al.
2007a).
Most of the carbohydrate epitopes in O. bharuchae
seem to be regulated during oogonial development,
as described for many cell wall epitopes from different plant tissues (Knox et al. 1991, Smallwood et al.
1994, Knox 1997, Willats et al. 1998). It was proposed that RG-I with (1 fi 4)-b-d-galactan side
chains increases the firmness of the cell wall in pea
cotyledons (McCartney et al. 2000), which may also
explain its presence in the cambial zone of the
xylem cells (Ermel et al. 2000). The (1 fi 4)-b-dgalactan structure has been associated with water
absorption in the peripheral cells of flax roots
(Vicré et al. 1998) and PCWs of elaters in the horn-
1263
wort Megaceros (Kremer and Pettolino 2004). The
presence of high amounts of this type of galactan
and arabinan epitopes in the cytoplasm of the oogonia could be related with the physiological event in
which the oogonia increase the turgor force to
break the cell wall by water uptake. A layer between
PCW and OCW, already described as the ‘‘thin layer’’
in other species of Oedogonium and Bulbochaete,
has been related to the wall weakening during the
fertilization process. However, its composition still
remains unknown (Pickett-Heaps and Fowke 1970,
Hoffman 1971, 1973, Retallack and Butler 1973,
Pickett-Heaps 1975).
HRGP epitopes. The presence of arabinogalactanprotein (AGP) epitopes in the PCW from SCs and
oogonium cells was based on LM2 labeling, which
is specific for b-d-GlcAp units in the AGP-glycan
type (Fig. 5, a and b; Smallwood et al. 1996) and
also on the MAC 207 probe (Fig. 5, c and d).
These AGP epitopes were detected in the outer
layer of the PCW (MAC-207) or on the PCW surface (LM2) of the oogonial cells. In contrast, the
inner layers of the OCW and PCW yielded positive
signals to JIM-20 (Fig. 5e), indicating that arabinosides of the extensin type were present in the oogonial wall and absent in the vegetative-SC cell walls
(Fig. 5f; Smallwood et al. 1994). JIM-11 (Fig. 5, g
and h), which also recognizes an extensin epitope,
showed a very low reaction in the oogonium walls
(Fig. 5g) and much higher labeling in the PCW of
SCs (Fig. 5h).
AGP and extensins, bearing short arabinoside
substituents, are members of a large group of
proteoglycans (glycoproteins) known as HRGPs
(Gaspar et al. 2001, Showalter 2001). Although the
specific functions for most of the individual HRGPs
remain elusive, extensins in vascular plants and
volvocacean algae have been involved in tensile cell
wall strength, in cell plate formation, and in defense
responses by forming interpenetrating cross-linked
networks in the wall (Fry 1982, Brady et al. 1998,
Held et al. 2004, Cannon et al. 2008). Several studies have implicated AGP in many aspects of plant
growth and development (Gaspar et al. 2001, Showalter 2001, Motose et al. 2004). In O. bharuchae f.
minor, arabinosides of the extensin type were found
in the already expanded PCW, in the inner face of
OCW (Fig. 5e), and in the PCW of SCs (Fig. 4g).
On the basis of the different pattern of immunolabeling obtained with JIM-20 in comparison to
JIM-11 (Fig. 5, e–h), we suggest that these two antibodies recognize different structures of the extensin
type, probably involving distinct populations of
O-glycosylated HRGP. Unfortunately, the precise
extensin epitope fine structure recognized by these
two antibodies remains unknown (Smallwood et al.
1994).
The HRGP epitopes in O. bharuchae walls appear
to be common cell wall components usually found
in many other green algal groups, according to
1264
J O S É M . E S T E V E Z E T A L .
Fig. 3. Immunolocalization by laser scanning confocal microscopy (LSCM) of the homogalacturonan (HG) epitopes present in the
suffultory cell (SC) and mature oogonium cell walls (OCWs). (a–b) Labeling of nonesterified HG with the 2F4 in the primary cell wall (PCW)
from oogonial cell (a) but not in the SC (b). (c–d) Labeling of putative nonesterified and ⁄ or low-esterified homogalacturonan (HG) with
the JIM-5 antibody in both oogonium (c) and SC walls (d). (e–f) Labeling with JIM-7 antibody against low and ⁄ or high-esterified HG in the
oogonia (e) and SC (f). (g–h) Labeling with LM-7 that reacts with nonblockwise partially Me-HG epitope less strongly in the oogonium (g) in
comparison with SC (h). (i–j) Labeling with CCRC-M7, which recognizes the arabinosylated [(1 fi 6)-b-d-Galp] epitope, only in the SC (j).
This epitope could be present also in the AGP glycans. In all cases, propidium iodide (PI, in pseudored color) was used for referencing the
PCW, whereas the monoclonal antibody labeling (in pseudogreen, Alexa fluor 488, AF) and the merge of both channels (merge) are shown
on the left panels. Chl fluorescence (CF) is also detected in the pseudored channel. Scale bars = 5 lm. Absence of labeling is shown in the
PCW (*) and OCW (**), whereas the positive labeling on the cell walls is indicated with an arrowhead. See Table 1 for antibody specificity.
the following evidence: (i) Several HRGPs of the
extensin type and vegetative serine-proline repeat
proteins (VSP) were reported to be the main com-
ponents of the extracellular matrix in volvocaceans
(Lamport and Miller 1971, Roberts 1974, Woessner
et al. 1994, Ender et al. 2002, Hallmann 2006).
C E L L W A L L E P I T O P E S I N T H E G R E E N A L G A O E D OG O N I U M
(ii) At least two expressed sequenced tags (ESTs)
from a cDNA library obtained in the green alga
Ulva linza (Ulvophyceae; ESTs AJ891903 and
AJ892864) have been assigned to HRGP genes
(Stanley et al. 2005). (iii) Hydroxyproline residues
have been detected in the cell wall proteins of
many different green algal groups, including many
species of chlorophyceans, ulvophyceans, and charophyceans with the exception of Charales (Gotelli
1265
and Cleland 1968, Kieliszewski and Lamport
1994). (iv) HRGP epitopes (AGP and extensins)
have been partially characterized and immunolocated in the cell wall of the siphonous seaweed
Codium (Caulerpales, Ulvophyceae; J. M. Estevez,
P. V. Fernández, L. Kasulin, P. Dupree, and M.
Ciancia, unpublished results). (v) AGP epitopes
have been detected on the cell walls of the green
algae Micrasterias (Charophyceae) and Pleurotaenium (Conjugatophyceae, Streptophyta) (Luetz-Meindl and Brosch-Salomon 2000, Domozych et al.
2007b). These observations support the idea, first
proposed by Kieliszewski and Lamport (1994), that
diverse groups of HRGPs, including extensins,
AGP, gum arabic glycoprotein (GAGP), nodulins,
and other proteins have evolved from a small set
of archetypal polypeptides having a common
ancestor, possibly belonging to an extinct group
within the green algae.
Fucosylated epitope. The absence of labeling with
CCRC-M1 antibody on both cell wall types in
O. bharuchae (Fig. S1, i and j), which recognizes terminal nonreducing (1 fi 2)-linked a-l-Fucp units
usually present on the xyloglucan backbones (XG)
and in RG-I polymer (Puhlmann et al. 1994), suggests the complete deficiency of fucosylated XG or
fucosylated RG-I, at least with the usual glycan
structures occurring in vascular plants (except for
several well-characterized species of Poaceae, Lamiales, Solanales, and Asteridae, where low- or nonfucosylated XGs were reported; Hoffman et al.
2005). In addition, XGs are not part of the cell walls
of the Charophyceae algae (Klebsormidium, Coleochaete, and Chara; Popper and Fry 2003). On this basis,
the ability to synthesize fucosylated XG or RG-I with
structures similar to those in current vascular plants
seems to have started after green algae diversification, at late stages of the aquatic-to-land transition
(Ligrone et al. 2002, Popper and Fry 2003, Hoffman
et al. 2005).
Fig. 4. Immunolocalization by laser scanning confocal microscopy (LSCM) of the rhamnogalacturonan (RG-I like) epitopes.
(a–f) Labeling of the (1 fi 4)-b-d-galactan epitope, which is recognized by LM5 antibody only in the cytoplasm (Cy) of the
mature oogonium (a) but not in the suffultory cell (SC) (b).
(c–d) Labeling of the LM-6 indicates the presence of (1 fi 5)
-a-l-arabinan epitopes not only in the cytoplasm of the oogonium
(c), but also in lower amounts in the primary cell wall (PCW) in
the SC (d). (e–f) Labeling of the CCRC-M2 mAb that reacts with
RG-I polysaccharides. The precise structure of the epitope is still
unknown. High labeling was observed only in the cytoplasm (Cy)
of the mature oogonium (e), but very weak reaction in the SC
(f). In all cases, propidium iodide (PI, in pseudored color) was
used for referencing the cellulose in the PCW, whereas the
monoclonal antibody labeling (in pseudogreen, Alexa fluor 488,
AF) and the merge of both channels (merge) are shown on the
left panels. Chl fluorescence (CF) is also detected in the pseudored channel. Scale bars = 5 lm. Absence of labeling is shown
in the PCW (*) and OCW (**), while the positive labeling on the
cell walls is indicated with an arrowhead. See Table 1 for antibody
specificity.
1266
J O S É M . E S T E V E Z E T A L .
Fig. 5. Immunolocalization by laser scanning confocal microscopy (LSCM) of hydroxyproline-rich glycoprotein (HRGP) epitopes present in the suffultory cell (SC) and mature oogonium cell walls. (a–b) Labeling with LM-2 antibody that recognizes b-d-GlcAp-units in the
AGP-glycan moieties in the oogonium (a) and SC (b). (c–d) Labeling of AGP glycans, which is recognized by MAC207 antibody in the primary cell wall (PCW) from both oogonium (c) and SC (d). (e–h) Immunolabeling with anti-extensin antibodies JIM-20 (e–f) and JIM-11
(g–h). (e–f) Labeling with JIM-20, which recognizes arabinosides of the extensin type, only in the oogonium cell wall (OCW) (e), but not
in the SC cell walls (f). (g–h) Labeling with JIM-11, with much lower reaction in OCW (g) than in SC walls (h). In all cases, propidium
iodide (PI, in pseudored color) was used for referencing the PCW, whereas the monoclonal antibody labeling (in pseudogreen, Alexa
fluor 488, AF) and the merge of both channels (merge) are shown on the left panels. Chl fluorescence (CF) is also detected in the pseudored channel. Scale bars = 5 lm. Absence of labeling is shown in the PCW (*) and OCW (**), while the positive labeling on the cell
walls is indicated with an arrowhead. See Table 1 for antibody specificity.
CONCLUSION
Based on our results, the PCW in the vegetativeSCs and oogonial cells of O. bharuchae f. minor is
composed of (1 fi 4)-b-glucans in the form of crystalline cellulose, non- and variable Me-esterified
HG, extensins, and AGP epitopes. In addition, arabinosides of the extensin type and low amounts of
AGP epitopes were found in the OCW. Putative
RG-I like molecules including (1 fi 4)-b-d-galactan
and (1 fi 5)-a-l-arabinan side chains were detected
as epitopes in the cytoplasm of mature oogonia.
Additional chemical data would be necessary to
reveal the fine chemical structures of HRGP- (AGP
and extensins) and pectin-related epitopes present
in Oedogonium walls. A comparison between the cell
walls present in green algae, including chlorophyceans and charophyceans, would provide a deeper
understanding of how cell walls have evolved from
simple organisms (unicellular, filamentous, etc.)
with one to few different cell types to much more
complex ones with >20–30 cell types (e.g., in current land plant groups).
The authors thank C. Somerville from the Carnegie Institution of Washington (Stanford University) for his valuable
C E L L W A L L E P I T O P E S I N T H E G R E E N A L G A O E D OG O N I U M
help throughout the study and for providing the antibodies
and enzymes used. We also thank P. Knox (University of
Leeds, UK) for providing the CMB2a and C. G. Vélez for the
specimens of Oedogonium bharuchae f. minor. J. M. E. was
responsible for designing, performing, interpreting most of
the experiments, and writing the manuscript. P. I. L.
obtained thin sections, supervised and performed TEM
experiments, and discussed the results. J. S. A. fixed the algal
material, performed the cultures and sexual inductions, performed TEM experiments, and discussed the results. We are
also indebted to S. Pietrocovsky for helpful comments on the
manuscript. J. M. E. supported by AGENCIA (PICT 2007-963).
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Supplementary Material
The following supplementary material is available for this article:
Fig. S1. Immunolocalization by laser scanning
confocal microscopy (LSCM) of cell wall epitopes
present in the suffultory cell (SC) and mature
oogonium cell walls (OCW).
This material is available as part of the online
article from: http://www.blackwell-synergy.com/
doi/abs/10.1111/j.1529-8817.2008.00568.x.
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