Abstract

Background and Aims

Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii ‘C-Fern’, a widely used model system for ferns.

Methods

Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of ‘C-Fern’ sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes.

Key Results

While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns.

Conclusions

The differences and similarities in glycan cell-wall composition between ‘C-Fern’ gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte.

INTRODUCTION

Plant cell walls are carbohydrate-based extracellular matrices involved in many essential biological processes that regulate or impact upon plant growth and development, morphology, biomechanics and cellular responses to environmental factors (Albersheim et al., 2010). As a result of these multiple functions, cell walls display a considerable degree of structural and compositional diversity. Many innovations that facilitated the diversification of embryophytes also led to increasingly complex plant body plans. The most prominent structural innovations are those that are required for the acquisition, retention and transport of water and solutes, as well as providing increased support to accommodate a trend towards taller stems for improved spore dispersal or more efficient light capture (Bateman et al., 1998). As cell walls determine most of the fundamental features of specialized plant tissues it is safe to state that they have played a central role in the evolution of land plants, either through (functional or structural) elaboration of ancestral polymers or through the acquisition of new components.

While our knowledge of the structural complexity of plant cell-wall components is well established, our understanding of how this reflects evolution remains incomplete (Niklas, 2004; Popper and Tuohy, 2010; Sørensen et al., 2010; Fangel et al., 2012). Recent publications highlighted that the presence and relative proportions of cell-wall components may vary between representatives of different plant lineages (Harris, 2005; Popper, 2008; Fry, 2011; Fangel et al., 2012). For example, fern primary cell walls were reported to contain relatively high proportions of mannose-rich polymers and a lower concentration of xyloglucans, leading Silva et al. (2011) to describe a new (primary) cell-wall type (type III) typical of ferns.

To gain a more complete understanding of plant cell-wall evolution it will be necessary to place the known diversity of cell-wall polymers in spatio-temporal and taxonomic contexts. Preferably, such studies require comparative investigations at different taxonomic levels and different levels of anatomical organization. Moreover, while vegetative innovations may have had the greatest visual impact, early land plant evolution was also characterized by successive transformations of the reproductive system and the life cycle. Unlike in all other land plants, the gametophyte is the dominant stage in bryophytes, with the sporophyte being fully dependent on the gametophyte for survival. Within the vascular plants, ferns sensu lato (s.l.) (Pteridophyta sensu stricto, thus excluding Lycopodiophyta but including Equisetum, or monilophytes) are the largest group of plants that alternate between independent gametophyte and sporophyte generations. These generations show several morphological and physiological differences. While gametophytes are small and flattened organisms, sporophytes are large and initiate vascular and mechanical tissues. To the best of our knowledge, there is no published account comparing cell-wall composition between both generations.

Although the ferns s.l. currently lack a representative with a fully sequenced genome, a cultivar of Ceratopteris richardii, referred to as ‘C-Fern’, was introduced in the late 1980s as a fern model system (Hickok et al., 1987; Leroux et al., 2013a). Traditionally, this homosporous leptosporangiate fern was either included in a family of its own, Parkeriaceae (Hooker, 1825; Copeland, 1947; Pichi-Sermolli, 1977), or ascribed to Pteridaceae (Hooker, 1858; Copeland, 1947; Tryon et al., 1990), or to the large and diverse pteridioid clade (Schuettpelz and Pryer, 2008).

The goal of this paper was to explore the level of variation in glycan epitope presence and distribution between tissues, cell types and structures in different organs and generations of C. richardii ‘C-Fern’. Are glycan epitopes that are associated with complex tissues in the sporophyte also present in the morphologically less complex gametophyte? Are primary and secondary cell walls of different organs and tissues similar in glycan epitope composition? We adopted a two-level antibody-based strategy: first screening for specific cell-wall components by probing glycan microarrays with monoclonal antibodies, followed by detailed in situ immunocytochemical analyses.

MATERIALS AND METHODS

Plant material

Ceratopteris richardii ‘C-Fern’ spores, purchased from Carolina Biological Supply Company (Burlington, USA), were sterilized and cultured as described in the ‘C-Fern’ Web Manual (www.c-fern.org). The spores were grown on agar plates in a growth cabinet at 28 °C under continuous light (80 μmol m–2 s–1). Young sporophytes, which emerged after fertilization, were planted in potting soil and kept in a plastic container with a lid that was placed in a growth cabinet under the same conditions as mentioned above.

Embedding of plant material

Segments of roots, petioles, laminae and whole gametophytes were fixed in 4 % (v/v) paraformaldehyde in PEM buffer [100 mm PIPES, 10 mm MgSO4 and 10 mm ethylene glycol tetraacetic acid (EGTA), pH 6·9] at room temperature for 2 h. After thoroughly washing in phosphate-buffered saline (PBS), samples were dehydrated in an ethanol gradient (30, 50, 70, 94 and 100 % ethanol) before gradually infiltrating with LR-White resin (medium grade, London Resin Company, London, UK). Infiltrated specimens were sealed in flat embedding moulds using Aclar film (Electron Microscopy Sciences, Hatfield, PA, USA) and cured in an oven at 58 °C for 24 h. Transverse sections of 0·5 μm, cut using an ultramicrotome (Reichert-Jung Ultracut E) equipped with glass knives, were mounted on Vectabond-treated slides (Vector Labs, Peterborough, UK). For anatomical observations, sections were stained with 0·05 % (w/v) toluidine blue O (Merck, Darmstadt, Germany) in 0·1 % (w/v) Na2B4O7. Micrographs were taken using an Olympus XC10 digital camera mounted on an Olympus BX51 epifluorescence microscope. The drawings shown in Fig. 1 were made by hand.

Fig. 1.

‘C-Fern’ anatomy. (A) Hermaphroditic gametophyte with photosynthetic tissue (pt) bearing antheridia (an), archegonia (ar) and rhizoids (rh). Note remainders of the spore walls (sp). (B) Transverse section of root showing central cylinder (cc) surrounded by an endodermis, aerenchyma (ae) and epidermis (ep). (C) Transverse section of petiole showing stomata (st) in the epidermis (ep), as well as two vascular bundles in aerenchyma (ae). (D) Detail of the large concentric amphicribral vascular bundle in C, consisting of xylem (x), phloem (ph) and pericycle (pe) surrounded by an endodermis (en) and sclerenchyma sheath (ss). (E) Transverse section through the lamina showing vascular bundles with xylem tracheids (x), mesophyll tissue (me) with large intercellular spaces and epidermal tissues (ep) with stomata (st).

Fig. 1.

‘C-Fern’ anatomy. (A) Hermaphroditic gametophyte with photosynthetic tissue (pt) bearing antheridia (an), archegonia (ar) and rhizoids (rh). Note remainders of the spore walls (sp). (B) Transverse section of root showing central cylinder (cc) surrounded by an endodermis, aerenchyma (ae) and epidermis (ep). (C) Transverse section of petiole showing stomata (st) in the epidermis (ep), as well as two vascular bundles in aerenchyma (ae). (D) Detail of the large concentric amphicribral vascular bundle in C, consisting of xylem (x), phloem (ph) and pericycle (pe) surrounded by an endodermis (en) and sclerenchyma sheath (ss). (E) Transverse section through the lamina showing vascular bundles with xylem tracheids (x), mesophyll tissue (me) with large intercellular spaces and epidermal tissues (ep) with stomata (st).

Alcohol insoluble residue (AIR)

Fresh plant tissue was collected from different organs or structures including roots, petioles, fertile laminae, spores and gametophytes. After homogenization in liquid nitrogen using a mortar and pestle, a series of 70 % (v/v) ethanol extractions were performed to remove pigments, alkaloids, tannins and soluble sugars from the cell-wall-containing residues. A final 5-min wash with 100 % acetone was performed prior to air-drying the pellets overnight.

Glycan microarray analysis

For each sample three (extraction) replicates of approximately 10 mg AIR were processed separately. We sequentially extracted pectins and non-cellulosic polysaccharides using 50 mm CDTA (diamino-cyclohexane-tetraacetic acid, pH 7·5) and 4 mm NaOH, respectively. Analysis was performed as described by Moller et al. (2007) and extractant volumes were adjusted according to weight. All extracts were serially diluted (1 : 1 and 1 : 5) and two technical replicates were printed on nitrocellulose membranes, giving a total of 18 spots for each sample. Details of the probes used in this study are shown in Table 1. The labelled arrays were scanned and processed using ImaGene 6·0 microarray analysis software (Biodiscovery, http://www.biodiscovery.com). The highest mean spot signal in the data set was assigned a value of 100 % and all other signals were adjusted accordingly. The resulting heatmap was generated in Excel (Microsoft, Redmond, WA, USA) and is shown in Fig. 2. The resulting values represent averages from the technical replicates. A minimum of 5 % was imposed, and values below this are represented by ‘0’.

Table 1.

List of monoclonal antibodies used in this study

 mAb Specificity Reference(s) 
Pectin-related LM18 low-esterified HG Verhertbruggen et al. (2009a) 
LM19 low-esterified HG Verhertbruggen et al. (2009a) 
LM20 high-esterified HG Verhertbruggen et al. (2009a) 
LM7 non-blockwise partially methyl-esterified HG Willats et al. (2001), Clausen et al. (2003) 
LM8 xylogalacturonan Willats et al. (2004) 
LM5 (1→4)-β-galactan Jones et al. (1997) 
LM6 (1→5)-β-arabinan Willats et al. (1998) 
LM13 linearized (1→5)-α-l-arabinan Verhertbruggen et al. (2009b) 
LM16 processed arabinan Verhertbruggen et al. (2009b) 
Hemicellulose-related LM10 (1→4)-xylan McCartney et al. (2005) 
LM11 (1→4)-β-xylan/arabinoxylan McCartney et al. (2005) 
LM15 XXXG-motif of xyloglucan Marcus et al. (2008) 
LM25 XXXG/galactosylated xyloglucan Pedersen et al. (2012) 
LM21 heteromannan Marcus et al. (2010) 
LM22 heteromannan Marcus et al. (2010) 
BS-400–2 (1→3)-β-glucan (callose) Meikle et al. (1991) 
AGP-related LM2 β-linked glucuronic acid Smallwood et al. (1996), Yates et al. (1996) 
JIM8 AGP glycan Pennell et al. (1991, 1992
JIM13 AGP glycan Knox et al. (1991), Yates et al. (1996) 
 mAb Specificity Reference(s) 
Pectin-related LM18 low-esterified HG Verhertbruggen et al. (2009a) 
LM19 low-esterified HG Verhertbruggen et al. (2009a) 
LM20 high-esterified HG Verhertbruggen et al. (2009a) 
LM7 non-blockwise partially methyl-esterified HG Willats et al. (2001), Clausen et al. (2003) 
LM8 xylogalacturonan Willats et al. (2004) 
LM5 (1→4)-β-galactan Jones et al. (1997) 
LM6 (1→5)-β-arabinan Willats et al. (1998) 
LM13 linearized (1→5)-α-l-arabinan Verhertbruggen et al. (2009b) 
LM16 processed arabinan Verhertbruggen et al. (2009b) 
Hemicellulose-related LM10 (1→4)-xylan McCartney et al. (2005) 
LM11 (1→4)-β-xylan/arabinoxylan McCartney et al. (2005) 
LM15 XXXG-motif of xyloglucan Marcus et al. (2008) 
LM25 XXXG/galactosylated xyloglucan Pedersen et al. (2012) 
LM21 heteromannan Marcus et al. (2010) 
LM22 heteromannan Marcus et al. (2010) 
BS-400–2 (1→3)-β-glucan (callose) Meikle et al. (1991) 
AGP-related LM2 β-linked glucuronic acid Smallwood et al. (1996), Yates et al. (1996) 
JIM8 AGP glycan Pennell et al. (1991, 1992
JIM13 AGP glycan Knox et al. (1991), Yates et al. (1996) 

HG, homogalacturonan; AGP, arabinogalactan protein.

Fig. 2.

Heatmap of the glycan microarray analysis indicating the relative abundance of plant cell-wall-associated glycan epitopes in sequential CDTA and NaOH extractions of different organs and structures.

Fig. 2.

Heatmap of the glycan microarray analysis indicating the relative abundance of plant cell-wall-associated glycan epitopes in sequential CDTA and NaOH extractions of different organs and structures.

Indirect immunofluorescence imaging

Immunolocalization was carried out on sections obtained from the same LR-White blocks that were prepared for anatomical observations. The monoclonal antibodies used in this study were purchased from Plantprobes (http://www.plantprobes.net) or Biosupplies (http://www.biosupplies.com.au), and are listed in Table 1. A pectate lyase pretreatment was performed to remove pectic homogalacturonan (HG) as these polymers have been reported to mask hemicellulose-related epitopes such as xyloglucan (Marcus et al., 2008) or mannan (Marcus et al., 2010). This treatment consists of sequential incubation with 0·1 m sodium carbonate (pH 11·4) for 2 h at room temperature and 10 μg mL–1 pectate lyase obtained from Cellvibrio japonicus (Megazyme, E-PLYCJ) in CAPS buffer (50 mmN-cyclohexyl-3-aminopropanesulfonic acid, 2 mm CaCl2) for 2 h at room temperature. Sections were blocked in 3 % (w/v) non-fat milk protein in PBS (prepared from a 10× stock solution: 80 g NaCl, 28·6 g Na2HPO4.12H2O and 2 g KH2PO4 in 1 litre de-ionized H2O, pH 7·2) (MP/PBS) for 5 min and incubated in primary monoclonal antibody (mAb) in MP/PBS for 1·5 h at room temperature. Rat mAbs were used in a 10-fold dilution and the mouse mAb, BS-400–2, was used in a 50-fold dilution. After thoroughly washing with PBS, sections were incubated with a 100-fold dilution of anti-rat or anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody in MP/PBS for 1·5 h at room temperature. Control of background fluorescence was carried out by omitting primary antibodies. Slides were washed extensively with PBS, stained with Calcofluor White (fluorescent brightener 28, Sigma, St Louis, MO, USA; 0·25 μg mL–1 in dH2O) and mounted in anti-fade agent (Citifluor AF2, Agar Scientific, Stanstead, UK). The slides were examined using an epifluorescence microscope (Olympus BX51) and images captured with an Olympus XC10 camera.

Confocal microscopy

For the whole mount surface labelling, entire gametophytes were fixed in PEM buffer containing 16 % (v/v) paraformaldehyde and washed in PBS. After pectate lyase pretreatment the gametophytes were immunolabelled and mounted as described above. The gametophytes were examined with an Olympus Fluoview 300 confocal microscope using 488-nm Ar laser excitation and collecting the emission signal through FITC-specific filters.

RESULTS

Anatomy of ‘C-Fern’

As ‘C-Fern’ is not yet a widely studied model plant we first provide a brief overview of its anatomy (Fig. 1). ‘C-Fern’ spores develop into elongate male or heart-shaped hermaphroditic gametophytes (Fig. 1); only the latter produce both sperm-bearing antheridia and egg-containing archegonia (Fig. 1A). Rhizoids anchor the gametophytes to the soil and share morphological features with sporophyte root hairs. The sporophyte consists of a rhizome from which roots and leaves emerge (Hou and Hill, 2002; Hou and Blancaflor, 2009). Roots have an aerenchymatous ground tissue and are surrounded by an epidermis with root hairs, which are prominent in young roots (Fig. 1B). A pericycle and an endodermis surround the central vascular tissue; a sclerenchyma sheath is absent. The number and arrangement of the petiole vascular bundles, each surrounded by a sclerenchyma sheath, are variable depending on the age and size of the leaves (Fig. 1C, D). The vascular bundles are concentric, with xylem occupying the centre and being completely surrounded by phloem, a pericycle and a single-layered endodermis with Casparian strips (Fig. 1D). The lamina consists of vascular bundles embedded in mesophyll tissue with large intercellular spaces (Fig. 1E). Numerous stomata are present in the lower and upper cutinized epidermal tissues.

Glycan microarray analysis

The heatmap of the glycan microarray analysis (Fig. 2), which largely directed the layout of our in situ experiments, illustrates that the relative abundance of glycan epitopes varies between different generations and organs. One way of making sense of this variation is to match differences in anatomy with differences in (relative) epitope abundance. For instance, the petiole contains a large proportion of sclerenchyma compared with the root and the high relative amount of xylan epitopes in the petiole sample might indicate that the latter tissue has xylan-rich walls.

Gametophytes showed the highest relative abundance of arabinogalactan protein (AGP) epitopes recognized by the mAbs LM2, JIM8 and JIM13. All glycan epitopes, except for JIM13, were found in relatively low amounts in the spores. Some epitopes were not detected in levels above background signal in any of the samples analysed and include the pectic HG LM7, xylogalacturonan LM8 and pectic arabinan LM16 epitopes. Note that the representative nature of the glycan array results depends greatly on the extractability of cell-wall components from alcohol-insoluble residues. It cannot be ruled out that components such as lignins, suberins and sporopollenins may have hindered full extraction of non-cellulosic glycan polymers in the samples. To interpret the variation observed in the heatmap, we performed detailed in situ immunolabelling experiments using the same antibodies. The results, presented from the perspective of primary and secondary cell walls, are summarized in Table 2. To show the full extent of cell walls we stained sections with Calcofluor White, which stains β-glucans such as cellulose and xyloglucans (Figs 3A, 4A, 5A, O, J and 6A, H, N). A control in which the primary antibody was omitted enabled the distinction of antibody binding from autofluorescence (Figs 3B, 4B, 5B, K and 6B, I, O).

Table 2.

Results of immunocytochemical labelling (all slides, excluding those utilized for LM19 and LM20 labelling, were pretreated with pectate lyase)

   Pectin-related antibodies
 
Hemicellulose-related antibodies
 
AGP-related antibodies
 
LM19 LM20 LM5 LM6* LM15 LM25 LM11 LM21 BS- 400–2 LM2 JIM13 
Primary cell walls 
Sporophyte petiole epidermis ++1 ++ – – – – +4 – – – 
aerenchyma ++1,2 ++ – – – – – – – 
endodermis ++1 ++ – – – – – – – 
sclerenchyma sheath +3 +3 – – – – – – – 
pericycle +3 ++ – – – – – – – 
phloem ++ – ++ – – – – 
xylem +3 ++ – – – – – – – – – 
root epidermis +3 – – – – – – – – 
root hair – – ++ – – NA ++ ++ 
aerenchyma +3 – – – – +/– – – – 
endodermis – – – – – +/– – – – 
pericycle – – – – – +/– – – – 
phloem – – – +/– – – – 
xylem – – – – – – – – – 
lamina epidermis ++ – – – ++4 – – – 
mesophyll +3 ++ – – – – ++ – – – 
vascular bundle – 5 +/–6 – +/–6 – ++ – – – 
sporangium  +6 – – – +/– – ++ – – – 
Gametophyte   rhizoids – – ++ ++ ++ – – ++ ++ 
photosynthetic tissue – – – +/– – +/– – – – 
archegonia ++ – – ++ ++ – – – – 
antheridia ++ – – – – ++ – – 
Secondary cell walls 
Sporophyte petiole sclerenchyma sheath – – – – – ++ ++ ++ – – 
xylem tracheids – – – – – ++ ++ – – – 
root xylem tracheids – – – – – ++ ++ – – – 
lamina xylem tracheids – – – – – ++ ++ – – – 
sporangium thickened annulus walls of the sporangia +7 – – – – – – – – – – 
   Pectin-related antibodies
 
Hemicellulose-related antibodies
 
AGP-related antibodies
 
LM19 LM20 LM5 LM6* LM15 LM25 LM11 LM21 BS- 400–2 LM2 JIM13 
Primary cell walls 
Sporophyte petiole epidermis ++1 ++ – – – – +4 – – – 
aerenchyma ++1,2 ++ – – – – – – – 
endodermis ++1 ++ – – – – – – – 
sclerenchyma sheath +3 +3 – – – – – – – 
pericycle +3 ++ – – – – – – – 
phloem ++ – ++ – – – – 
xylem +3 ++ – – – – – – – – – 
root epidermis +3 – – – – – – – – 
root hair – – ++ – – NA ++ ++ 
aerenchyma +3 – – – – +/– – – – 
endodermis – – – – – +/– – – – 
pericycle – – – – – +/– – – – 
phloem – – – +/– – – – 
xylem – – – – – – – – – 
lamina epidermis ++ – – – ++4 – – – 
mesophyll +3 ++ – – – – ++ – – – 
vascular bundle – 5 +/–6 – +/–6 – ++ – – – 
sporangium  +6 – – – +/– – ++ – – – 
Gametophyte   rhizoids – – ++ ++ ++ – – ++ ++ 
photosynthetic tissue – – – +/– – +/– – – – 
archegonia ++ – – ++ ++ – – – – 
antheridia ++ – – – – ++ – – 
Secondary cell walls 
Sporophyte petiole sclerenchyma sheath – – – – – ++ ++ ++ – – 
xylem tracheids – – – – – ++ ++ – – – 
root xylem tracheids – – – – – ++ ++ – – – 
lamina xylem tracheids – – – – – ++ ++ – – – 
sporangium thickened annulus walls of the sporangia +7 – – – – – – – – – – 

Key: +, labelling; –, no labelling; +/–, weak labelling; ++, intense labelling; 1, including middle lamellae; 2, only primary cell walls close to the epidermis were labelled; 3, restricted to middle lamellae; 4, except for guard cell walls; 5, only endodermis and pericycle; 6, restricted to phloem; 7, restricted to the inner cell-wall layer; NA, data not available; *also binds to AGPs.

In situ cell-wall analysis of the petiole and the root

For the detection of pectic HG in the petiole we used two mAbs, LM19 and LM20, directed against HG with low and high levels of methyl-esterification, respectively. While the LM19 epitope was largely confined to the middle lamellae and cell-wall junctions (Fig. 3C), the LM20 epitope was more abundant in primary cell walls (Fig. 3E). Closer to the epidermis, LM19 also bound to the primary cell walls (Fig. 3C). As pectic HG is known to mask hemicellulosic epitopes (Marcus et al., 2008, 2010) we pretreated sections with pectate lyase, which resulted in removal of the LM19 epitope (Fig. 3D). We used antibodies that recognize either galactan (LM5) or arabinan (LM6), which generally occur as side chains on pectic rhamnogalacturonan-I (RG-I); however, the LM6 epitope has also been attributed to arabinogalactan proteins in the moss Physcomitrella patens (Lee et al., 2005). While LM5 weakly bound to phloem tissue (Fig. 3F), the LM6 epitope was not detected (Fig. 3G). Heteroxylans were immunoprobed using the LM11 mAb, which bound to the secondary cell walls of the xylem tracheids and the sclerenchyma sheath (Fig. 3H). Pectate lyase pretreatment resulted in enhanced labelling of the phloem cell walls with the anti-xyloglucan mAb LM15 (Fig. 3I). LM25, an anti-xyloglucan mAb sharing the XXXG-specificity with LM15, but also recognizing galactosylated xyloglucan, bound to most primary cell walls in the petiole, including those of the sclerenchyma sheath (Fig. 3J). Binding to the phloem was stronger compared with all other cell or tissue types (Fig. 3J). While LM21, directed against heteromannan, bound to all primary cell walls, it labelled only the secondary cell walls of the tracheids, but not those of the sclerenchyma sheath (Fig. 3K). Pectate lyase pretreatment also resulted in enhanced labelling (Fig. 3K). Moreover, binding to walls facing large intercellular spaces was considerably stronger, both before and after pectate lyase pretreatment (Fig. 3K, inset). We also probed slides with a mAb directed against callose (Biosupplies BS-400–2), which bound only to the secondary cell walls of the sclerenchyma sheath (Fig. 3L).

Fig. 3.

In situ localization of cell-wall glycan epitopes in equivalent transverse sections of a ‘C-Fern’ petiole. (A) Calcofluor White staining all cell walls. (B) No primary antibody control. (C) Detection of LM19 HG epitope (low levels of methyl-esterification) in all middle lamellae and aerenchyma primary cell walls close to epidermis (location of epidermis indicated with arrow). (D) Pectate lyase removed all pectic HG epitopes. (E) LM20 anti-HG (high levels of methyl-esterification) binds to all primary cell walls. (F) The LM5 pectic galactan epitope restricted to phloem. (G) AGP/pectic arabinan epitope LM6 not detected. (H) LM11 (anti-arabinoxylan) strongly labels secondary cell walls of xylem tracheids and sclerenchyma sheath. (I) Anti-xyloglucan mAb LM15 binding to phloem cell walls. (J) LM25 anti-xyloglucan labels primary cell walls of aerenchyma and phloem. (K) LM21 anti-heteromannan binds to all primary cell walls and tracheid secondary walls. Inset shows that LM21 strongly labels cell walls facing air cavities. (L) Callose immunodetected in sclerenchyma sheath secondary cell walls. ae, aerenchyma; ss, sclerenchyma sheath; ph, phloem; x, xylem; PL−, no pectate lyase pretreatment; PL+, pectate lyase pretreatment.

Fig. 3.

In situ localization of cell-wall glycan epitopes in equivalent transverse sections of a ‘C-Fern’ petiole. (A) Calcofluor White staining all cell walls. (B) No primary antibody control. (C) Detection of LM19 HG epitope (low levels of methyl-esterification) in all middle lamellae and aerenchyma primary cell walls close to epidermis (location of epidermis indicated with arrow). (D) Pectate lyase removed all pectic HG epitopes. (E) LM20 anti-HG (high levels of methyl-esterification) binds to all primary cell walls. (F) The LM5 pectic galactan epitope restricted to phloem. (G) AGP/pectic arabinan epitope LM6 not detected. (H) LM11 (anti-arabinoxylan) strongly labels secondary cell walls of xylem tracheids and sclerenchyma sheath. (I) Anti-xyloglucan mAb LM15 binding to phloem cell walls. (J) LM25 anti-xyloglucan labels primary cell walls of aerenchyma and phloem. (K) LM21 anti-heteromannan binds to all primary cell walls and tracheid secondary walls. Inset shows that LM21 strongly labels cell walls facing air cavities. (L) Callose immunodetected in sclerenchyma sheath secondary cell walls. ae, aerenchyma; ss, sclerenchyma sheath; ph, phloem; x, xylem; PL−, no pectate lyase pretreatment; PL+, pectate lyase pretreatment.

With few exceptions we found similar distribution patterns of glycan epitopes in roots (Fig. 4) and petioles (Fig. 3). LM19 and LM20 generally bound to middle lamellae and primary cell walls, respectively (Fig. 4C–E). The LM5 epitope was detected in the phloem tissue in the central cylinder (Fig. 4F). Although relatively high amounts of the LM6 arabinan epitope were detected in the glycan array, the antibody only weakly labelled cell membranes of the aerenchyma and the parenchymatous cell types of the vascular bundle (Fig. 4G). However, we processed complete root systems, including younger and/or lateral roots with root hairs for the preparation of AIR. Surface labelling of young roots showed strong binding of LM6 to the surface of the root hairs (Fig. 4G, inset). As a sclerenchyma sheath is absent, LM11 binding was restricted to the xylem tracheids (Fig. 4H). LM15 and LM25 also showed similar binding patterns (Fig. 4I, J), although the signal intensity of LM25 binding was lower (at similar exposure times) compared with that in petioles (Figs 3J and 4J). Likewise, LM21 binding in the cortex parenchyma was weaker, and not altered after pectate lyase pretreatment (Fig. 4K). The anti-callose mAb did not label the root sections (Fig. 4L).

Fig. 4.

In situ localization of cell-wall glycan epitopes in equivalent transverse sections of a ‘C-Fern’ root. (A) Calcofluor White staining all cell walls. (B) No primary antibody control. (C) Detection of LM19 HG epitope (low levels of methyl-esterification) in all middle lamellae. (D) Pectate lyase removed all pectic HG epitopes. (E) LM20 anti-HG (high levels of methyl-esterification) mAb binds to all primary cell walls. (F) LM5 pectic galactan epitope restricted to phloem. (G) Weak binding of the AGP/pectic arabinan LM6 antibody to cell membranes of aerenchyma and parenchymatous cell types of the central cylinder. Labelling of whole root tips reveals abundance of LM6 epitope in root hair cell walls (inset). (H) LM11 (anti-arabinoxylan) binds strongly to xylem tracheid secondary cell walls. (I) Anti-xyloglucan epitope LM15 weakly labels phloem cell walls. (J) Detection of the LM25 xyloglucan epitope in the primary cell walls of aerenchyma and phloem. (K) LM21 anti-heteromannan binds strongly to tracheid and xylem parenchyma cell walls and weakly labels all remaining primary cell walls. (L) Callose not immunodetected. ae, aerenchyma; ph, phloem; x, xylem; PL−, no pectate lyase pretreatment; PL+, pectate lyase pretreatment.

Fig. 4.

In situ localization of cell-wall glycan epitopes in equivalent transverse sections of a ‘C-Fern’ root. (A) Calcofluor White staining all cell walls. (B) No primary antibody control. (C) Detection of LM19 HG epitope (low levels of methyl-esterification) in all middle lamellae. (D) Pectate lyase removed all pectic HG epitopes. (E) LM20 anti-HG (high levels of methyl-esterification) mAb binds to all primary cell walls. (F) LM5 pectic galactan epitope restricted to phloem. (G) Weak binding of the AGP/pectic arabinan LM6 antibody to cell membranes of aerenchyma and parenchymatous cell types of the central cylinder. Labelling of whole root tips reveals abundance of LM6 epitope in root hair cell walls (inset). (H) LM11 (anti-arabinoxylan) binds strongly to xylem tracheid secondary cell walls. (I) Anti-xyloglucan epitope LM15 weakly labels phloem cell walls. (J) Detection of the LM25 xyloglucan epitope in the primary cell walls of aerenchyma and phloem. (K) LM21 anti-heteromannan binds strongly to tracheid and xylem parenchyma cell walls and weakly labels all remaining primary cell walls. (L) Callose not immunodetected. ae, aerenchyma; ph, phloem; x, xylem; PL−, no pectate lyase pretreatment; PL+, pectate lyase pretreatment.

In situ cell-wall analysis of the lamina and sporangia

The prevalence of the LM20 over the LM19 HG epitope was also apparent in the lamina (Fig. 5C, D). LM5 weakly labelled the phloem tissue in the vascular bundles (data not shown). The LM6 arabinan epitope was not detected in the lamina, which includes the guard cell walls (Fig. 5E). As expected, the anti-xylan mAb LM11 labelled the tracheids in the xylem tissue (Fig. 5F). While the anti-xyloglucan mAb LM15 only weakly bound to phloem cells (data not shown), LM25 labelled most primary cell walls, especially those close to the outer surface, including the epidermal cells and the guard cell walls, as well as primary cell walls in the vascular bundles (Fig. 5G). LM21, which recognizes heteromannan, showed a similar binding pattern, except for the guard cell walls, which were not labelled, and the tracheid walls, which were weakly labelled (Fig. 5H). The BS-400–2 callose epitope was not detected (Fig. 5I). As our lamina sample also contained sporangia, we immunolabelled transverse sections through sporangia. Cell walls of the annulus resembled those of the petiole sclerenchyma sheath in terms of glycan epitope abundance. While LM19 (Fig. 5L), LM25 (data not shown) and LM21 (Fig. 5N) only labelled primary walls, the LM11 xylan (Fig. 5M) and BS-400–2 callose (Fig. 5P) epitopes were detected in secondary cell walls. The spores in the sporangia were not labelled with any of the antibodies used. Pectate lyase treatment did not alter binding patterns of the antibodies.

Fig. 5.

In situ localization of cell-wall glycan epitopes in equivalent transverse sections of a ‘C-Fern’ lamina (A–I) and a mature sporangium (J–P). (A) Calcofluor White staining all cell walls, including guard cell walls (inset). (B) No primary antibody control. (C) Detection of LM19 HG epitope (low levels of methyl-esterification) in all middle lamellae and in outer periclinal walls of the epidermis and guard cell walls (inset). (D) The LM20 anti-HG (high levels of methyl-esterification) mAb binds to all primary cell walls. (E) The AGP/pectic arabinan epitope LM6 is not detected in the lamina. (F) LM11 (anti-arabinoxylan) strongly labels xylem tracheid secondary cell walls. (G) The LM25 xyloglucan epitope is found in all primary cell walls, including those of guard cells (inset). (H) LM21 anti-heteromannan binds to all cell walls, except to those of guard cells (inset). (I) Callose is not immunodetected. (J) Calcofluor White staining all cell walls. (K) No primary antibody control showing weak autofluorescence of exospore walls. (L) The anti-pectic HG antibody LM19 binds weakly to primary cell walls and to inner layers of secondary cell walls of the annulus. (M) LM11 (anti-arabinoxylan) strongly labels secondary cell walls of the annulus. (N) The LM21 heteromannan epitope is immunodetected in all primary cell walls. (O, P) The BS-400–2 callose antibody binds weakly to secondary cell walls of the annulus. ep, epidermis; gc, guard cell wall; me, mesophyll; x, xylem; ann, annulus with secondary cell walls; sp, spore; PL−, no pectate lyase pretreatment; PL+, pectate lyase pretreatment.

Fig. 5.

In situ localization of cell-wall glycan epitopes in equivalent transverse sections of a ‘C-Fern’ lamina (A–I) and a mature sporangium (J–P). (A) Calcofluor White staining all cell walls, including guard cell walls (inset). (B) No primary antibody control. (C) Detection of LM19 HG epitope (low levels of methyl-esterification) in all middle lamellae and in outer periclinal walls of the epidermis and guard cell walls (inset). (D) The LM20 anti-HG (high levels of methyl-esterification) mAb binds to all primary cell walls. (E) The AGP/pectic arabinan epitope LM6 is not detected in the lamina. (F) LM11 (anti-arabinoxylan) strongly labels xylem tracheid secondary cell walls. (G) The LM25 xyloglucan epitope is found in all primary cell walls, including those of guard cells (inset). (H) LM21 anti-heteromannan binds to all cell walls, except to those of guard cells (inset). (I) Callose is not immunodetected. (J) Calcofluor White staining all cell walls. (K) No primary antibody control showing weak autofluorescence of exospore walls. (L) The anti-pectic HG antibody LM19 binds weakly to primary cell walls and to inner layers of secondary cell walls of the annulus. (M) LM11 (anti-arabinoxylan) strongly labels secondary cell walls of the annulus. (N) The LM21 heteromannan epitope is immunodetected in all primary cell walls. (O, P) The BS-400–2 callose antibody binds weakly to secondary cell walls of the annulus. ep, epidermis; gc, guard cell wall; me, mesophyll; x, xylem; ann, annulus with secondary cell walls; sp, spore; PL−, no pectate lyase pretreatment; PL+, pectate lyase pretreatment.

In situ cell-wall analysis of gametophytic walls

Both sections and whole-mount material of hermaphroditic gametophytes were immunoprobed (Fig. 6). Resin-embedded gametophytes were sectioned such that both vegetative tissues and reproductive structures were exposed for labelling. LM19 bound weakly to the cell walls of the photosynthetic tissue and strongly to the rhizoids (Fig. 6C, F, P). LM20, by contrast, strongly labelled the photosynthetic tissue but appeared to be absent in the rhizoids (Fig. 6D, G, Q). LM6 did not bind to sections of gametophytic tissues (Fig. 6J). The LM25 xyloglucan epitope was detected in all tissues, and appeared to be more abundant in the rhizoids (Fig. 6K). LM21, binding to heteromannan, weakly labelled all primary cell walls except those of the rhizoids and strongly labelled the archegonial, but not antheridial, cell walls (Fig. 6I). The anti-callose mAb BS-400–2 bound to some of the rhizoids and to cell-wall material between spermatocytes in the antheridia (Fig. 6M). We also incubated sections with an anti-AGP mAb, LM2, but no labelling was observed (data not shown). Labelling of whole-mount samples appeared to be successful for investigating the in situ cell-wall composition of rhizoids. In most cases we were able to confirm the results obtained in labelled sections. However, both LM6 (arabinan/AGP-related epitope) (Fig. 6R) and LM2 (AGP-related epitope) (Fig. 6T), which were not detected in sections, strongly bound to rhizoids in whole-mount-labelled gametophytes. In fact, of all those used, we obtained the brightest fluorescence with these two antibodies. The rhizoids were also labelled intensively with the anti-xyloglucan probes LM15 (Fig. 6S) and LM25 (data not shown). Pectate lyase pretreatment did not alter binding patterns of the antibodies.

Fig. 6.

In situ localization of cell-wall glycan epitopes in equivalent sections through a ‘C-Fern’ gametophyte and in rhizoids. (A) Calcofluor White staining all cell walls. (B) No primary antibody control. (C) The LM19 HG epitope (low levels of methyl-esterification) only detected in photosynthetic tissue at the base of the gametophyte, near rhizoids. (D) The LM20 anti-HG (high levels of methyl-esterification) mAb binds to all primary cell walls of the photosynthetic tissue except to zones where LM19 is abundant. (E–G) Details showing the localization of pectic HG epitopes in transverse sections through the photosynthetic tissue. Note that LM20 binds strongly to the outer cell walls. (H) Calcofluor White staining all cell walls of a larger section that includes antheridia (double arrow) and archegonia (arrows). (I) No primary antibody control. (J) The AGP/pectic arabinan epitope LM6 is not detected. (K) LM25 xyloglucan epitope labels all primary cell walls. Note strongly labelled rhizoid cell walls. (L) LM21 anti-heteromannan binds weakly to primary cell walls but strongly labels archegonial cell walls (detail in inset). (M) Anti-callose antibody BS-400–2 binds strongly to antheridia (detail in inset). (N) Calcofluor White staining all rhizoidal walls. (O) No primary antibody control. (P) LM19 anti-HG (low levels of methyl-esterification) binds weakly to the rhizoid cell walls. (Q) LM20 anti-HG (high levels of methyl-esterification) not detected. (R–T) AGP/pectic arabinan mAb LM6, xyloglucan mAb LM15 and AGP-related mAB LM2 strongly label rhizoids. rh, rhizoids; pt, photosynthetic tissue.

Fig. 6.

In situ localization of cell-wall glycan epitopes in equivalent sections through a ‘C-Fern’ gametophyte and in rhizoids. (A) Calcofluor White staining all cell walls. (B) No primary antibody control. (C) The LM19 HG epitope (low levels of methyl-esterification) only detected in photosynthetic tissue at the base of the gametophyte, near rhizoids. (D) The LM20 anti-HG (high levels of methyl-esterification) mAb binds to all primary cell walls of the photosynthetic tissue except to zones where LM19 is abundant. (E–G) Details showing the localization of pectic HG epitopes in transverse sections through the photosynthetic tissue. Note that LM20 binds strongly to the outer cell walls. (H) Calcofluor White staining all cell walls of a larger section that includes antheridia (double arrow) and archegonia (arrows). (I) No primary antibody control. (J) The AGP/pectic arabinan epitope LM6 is not detected. (K) LM25 xyloglucan epitope labels all primary cell walls. Note strongly labelled rhizoid cell walls. (L) LM21 anti-heteromannan binds weakly to primary cell walls but strongly labels archegonial cell walls (detail in inset). (M) Anti-callose antibody BS-400–2 binds strongly to antheridia (detail in inset). (N) Calcofluor White staining all rhizoidal walls. (O) No primary antibody control. (P) LM19 anti-HG (low levels of methyl-esterification) binds weakly to the rhizoid cell walls. (Q) LM20 anti-HG (high levels of methyl-esterification) not detected. (R–T) AGP/pectic arabinan mAb LM6, xyloglucan mAb LM15 and AGP-related mAB LM2 strongly label rhizoids. rh, rhizoids; pt, photosynthetic tissue.

DISCUSSION

This paper provides the first account of cell-wall glycan-epitope distribution in sporophytes and gametophytes of the same fern species. The mAbs used in this study have been developed to characterize angiosperm cell-wall composition but are clearly applicable to the analysis of fern cell walls, indicating that at least some of the cell-wall structures present in angiosperm cell-wall polysaccharides are conserved. However, we cannot exclude the possibility that fern cell walls may contain components that are not present in angiosperm walls and are therefore not detectable with the current set of commercially available glycan-directed probes.

‘C-Fern’ gametophyte and sporophyte walls were rich in pectic HG. We specifically immunolocalized pectic HG with low levels of esterification in the middle lamellae and cell-wall junctions, locations where calcium-mediated HG gels may contribute to cell-to-cell cohesion. While the glycan array results showed a higher relative abundance of the LM19 epitope compared with the LM20 epitope, LM19 labelling in sections was generally weaker. It is possible that some LM19 epitopes were lost during sample processing or that they are not fully detectable in resin sections.

We used two antibodies recognizing galactan (LM5) and arabinan (LM6), which commonly occur as side-chains of pectic RG-I in angiosperms (Albersheim et al., 2010). The LM5 epitope was only detected in phloem tissue in the root, petiole and lamina of the sporophyte, which suggests that galactans may be involved in controlling differentiation and/or function of phloem tissue. The lack of LM5 epitope detection in the gametophyte and the absence of any published account on the presence of galactans in fern gametophytes suggest that these polymers might play an important role in the elaboration of plant body plans and the differentiation of complex tissues or cell-wall architectures. It is of interest that the LM5 galactan epitope was (variably) detected in the cortical parenchyma and water-conducting cells of moss and liverwort gametophytes, which, compared with fern gametophytes, show a relatively higher degree of anatomical complexity (Ligrone et al., 2002).

While arabinan has been immunolocalized in guard cell walls of angiosperms (e.g. Jones et al., 2003) and ferns s.l., including Equisetum arvense (Verhertbruggen et al., 2009b) and Adiantum raddianum (Leroux et al., 2013b), we did not detect it in ‘C-Fern’ guard cell walls. Pectate lyase pretreatment or labelling with undiluted hybridoma supernatant did not result in binding of LM6 to guard cell walls. This suggests that either arabinans are not detectable or may not be required for guard cell functioning in ‘C-Fern’. It is of interest that the ‘C-Fern’ plants used in this study were grown in high relative humidity, which may have obviated the functional need for arabinan in guard cell walls. Moore et al. (2013) hypothesized that in resurrection plants, arabinose-rich polymers, which include pectic arabinans, facilitate cell-wall flexibility and rehydration, properties that are important to the functioning of guard cell walls, especially in dryer environments. While appearing to be absent in sporophytic guard cell walls, we did detect the LM6 epitope in gametophytic rhizoids and sporophytic root hairs, but only after whole-mount surface labelling. Lee et al. (2005) reported that LM6, in addition to pectic arabinan, also binds to arabinogalactan proteins. Several observations suggest that in ‘C-Fern’ rhizoids and root hairs, the LM6 epitope may be associated with AGPs rather than pectic arabinan. First, we localized LM2 and JIM13 AGP-related epitopes in rhizoids, but as for LM6, these epitopes were also lost during sample processing but were readily detected in whole-mount labelled gametophytes. Secondly, AGPs are often associated with cell membranes and we weakly immunodetected the LM6 epitope in cell membranes of ‘C-Fern’ root tissues. The detection of AGP glycan epitopes in ‘C-Fern’ rhizoids and root hairs in this study, as well as in pollen tubes (Nguema-Ona et al., 2012) and Physcomitrella patens protonema (Lee et al., 2005), suggests that AGPs may play a key role in tip growth and control cell expansion in elongating cells. Moreover, the observed similarities in epitope abundance, particularly AGP and xyloglucan epitopes, in sporophyte root hairs and gametophyte rhizoids of ‘C-Fern’ strongly suggest that a similar gene regulatory network may control their development. As AGP epitopes were detected in the glycan array but nearly absent in resin sections, they were probably solubilized during sample processing. Therefore, we cannot draw any firm conclusions on AGP epitope distribution in ‘C-Fern’ organs and tissues.

Antheridial protoplasts of differentiating spermatocytes appeared to be embedded in a callose-rich matrix, an observation previously made by Cave and Bell (1973) on the basis of Periodic acid–Schiff and aniline blue staining. Similar patterns of callose deposition were reported in flowering plants during microsporogenesis (Nguema-Ona et al., 2012). Such callose matrices may function as a barrier isolating the differentiating protoplasts from gametophytic tissues, or as a temporary wall that, after dissolution, results in an antheridium with free sperm cells. Whilst we detected a high relative amount of the callose epitope in the root NaOH extraction, the antibody did not label sections through this organ and pectate lyase treatments did not result in new or enhanced binding. However, callose generally occurs in phloem sieve plates and such structures are not commonly observed in transverse sections, and might explain why we did not detect the BS-400–2 callose epitope. We did, however, detect callose in the secondary cell walls of the sclerenchyma sheath and sporangium annulus in the petiole and lamina, respectively, suggesting that this polymer might be involved in secondary cell-wall formation of non-vascular secondary cell walls. The callose found in the annulus secondary walls may have accounted for the callose detected in the NaOH extraction of laminae in our glycan array analysis.

We used two antibodies that recognize different substructures of xyloglucan. LM15, which is directed against the XXXG-motif, labelled phloem cell walls. This epitope was previously specifically detected in phloem tissue of Equisetum ramosissimum (Leroux et al., 2011) and Adiantum raddianum (Leroux et al., 2013b). A related anti-xyloglucan probe, LM25, directed towards a galactosylated xyloglucan epitope, bound more extensively, and labelled the primary cell walls of the phloem and the aerenchyma. These observations suggest that fine structural modification of xyloglucan polymers may have played a role in the evolution of specialized tissues in tracheophytes.

Without exception, all secondary cell walls of the sporophyte, including those of the sclerenchyma sheath, xylem and sporangial annuli, were xylan-rich. In contrast, we did not detect the LM11 xylan epitope in gametophytes, suggesting that xylan polymers might be restricted to sporophytes. This is consistent with the results of Carafa et al. (2005), who found that xylan epitopes were specifically detected in mechanical and vascular tissues in tracheophytes, but also in specific cell-wall layers in pseudoelaters and spores in hornwort sporophytes. Kulkarni et al. (2012) reported the occurrence of xylan epitopes in the moss Physcomitrella patens, and observed that xylans were detected in walls that contained relatively small amounts of xyloglucan. It is of interest to note that the anti-xyloglucan probe LM25 bound extensively to all gametophytic cell walls. Further analysis is necessary to provide conclusive evidence on the absence of xylans in fern gametophytes. We detected a mannan epitope in tracheid cell walls but not in the secondary cell walls of the sclerenchyma sheath nor in sporangial annuli. This suggests that sclerenchyma secondary walls in ‘C-Fern’ are xylan- rather than mannan-rich, an apparent exception among ferns as the mannan epitope was previously found in secondary walls of the strengthening tissue of Equisetum ramosissimum (Leroux et al., 2011) and many leptosporangiate ferns (O. Leroux, unpubl. res.). It is known from the literature that mannans and/or xylans are the most abundant hemicelluloses in secondary cell walls of vascular plants, with their relative abundance depending on plant lineage and tissue or cell type (Harris, 2005; Donaldson and Knox, 2012; Kim and Daniel, 2012a, b). It would be of interest to determine the relative timing of xylan, mannan and lignin deposition in fern tracheid and sclerenchyma cell walls. While we showed the differential occurrence of the LM21 mannan epitope in secondary cell walls, they were detected in nearly all primary cell walls of C-Fern sporophytes and gametophytes, supporting reports that fern primary cell walls are mannan-rich (e.g. Popper and Fry, 2004; Marcus et al., 2010; Silva et al., 2011). Several observations suggest a mechanical role for mannans in primary cells walls of ‘C-Fern’. Guard cell walls were the only epidermal walls in which no mannans where detected, and, in aerenchyma tissue of the petiole, LM21 binding was considerably stronger in the walls delineating the air cavities.

CONCLUSIONS

Comparison of the abundance and distribution of glycan epitopes between ‘C-Fern’ gametophytes and sporophytes indicated that functional specialization is largely reflected in the composition of cell walls. This was demonstrated by the restricted detection of xylan (LM11) and galactan (LM5) epitopes in specialized sporophyte tissues, as well as by a clear similarity in glycan epitope abundance between sporophytic and gametophytic tissues that perform similar functions (photosynthetic tissues and rhizoids/root hairs). These observations emphasize that gaining insight in tissue- and cell-type-specific cell-wall architectures is essential for evaluating taxonomic variation in cell-wall composition and is therefore key to a better understanding of cell-wall evolution.

While many aspects are in favour of selecting ‘C-Fern’ as a model system, we need to keep in consideration that, as in all major plant lineages, great morpho-anatomical diversity also exists within ferns, and therefore results obtained in ‘C-Fern’ cannot be generalized and are not necessarily transferable to other fern taxa. However, when genetic data emerge, with Ceratopteris currently being a good candidate for whole-genome sequencing, detailed information of tissue-specific distribution of cell-wall components will contribute to a more integrated understanding of cell-wall evolution by filling the ‘plant lineage gap’ between seed plants and lycophytes and allowing comparison of cell-wall features of genome sequenced taxa across the (land) plant kingdom.

ACKNOWLEDGEMENTS

We thank the Electron Microscopy Facility at the Centre for Microscopy and Imaging (Anatomy, School of Medicine, NUI Galway), Julia Schuckel (University of Copenhagen) for her help with the glycan microarray analysis, Myriam Claeys for assistance with semi-thin sectioning and Mie Degraeve for providing the anatomical drawings. This research was supported financially by the Research Foundation – Flanders, Belgium (FWO-Vlaanderen) (K2.090.12N and K2.136.13N). O.L. was supported by an IRCSET EMPOWER award (PD/2011/2326). This work was partly done during a research stay funded by the Erasmus staff mobility/staff training programme.

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Author notes

These authors contributed equally to the manuscript.

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