Primary cell wall composition of pteridophytes and spermatophytes


  • Zoë A. Popper,

    1. The Edinburgh Cell Wall Group, Institute of Cell and Molecular Biology, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, UK
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  • Stephen C. Fry

    Corresponding author
    1. The Edinburgh Cell Wall Group, Institute of Cell and Molecular Biology, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, UK
      Author for correspondence: S. C. Fry l. +44 131650 5320 Fax: +44 131650 5392 Email: S.Fry@Ed.Ac.UK
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Author for correspondence: S. C. Fry l. +44 131650 5320 Fax: +44 131650 5392 Email: S.Fry@Ed.Ac.UK


  • • Primary cell walls (PCWs) of major vascular plant taxa were analysed as a contribution towards understanding wall evolution.
  • • Alcohol-insoluble residues from immature shoots were acid- or enzyme-hydrolysed and the products analysed chromatographically and electrophoretically.
  • • There were phylogenetic differences in abundance of mannose, galacturonate and glucuronate residues, mixed-linkage glucan (MLG) and tannins. Eusporangiate pteridophytes (lycopodiophytes, a psilotophyte, an equisetophyte and a eusporangiate fern) were richer in mannose than leptosporangiate ferns, gymnosperms and angiosperms. Galacturonate was always the most abundant uronate; glucuronate was not abundant in PCWs of vascular plants except angiosperms (especially monocots and some magnoliids). MLG was detected in the Poaceae and Flagellariaceae, but no other vascular plants. Proanthocyanidins were associated with PCWs from leptosporangiate ferns, gymnosperms and some angiosperms, but not eusporangiate pteridophytes. Xyloglucan was present in all vascular plants tested.
  • • The results imply that major evolutionary changes in the PCW occurred not only during the charophyte–bryophyte and bryophyte–lycopodiophyte transitions but also after plants attained the vascular condition and upright growth habit, particularly during the eusporangiate–leptosporangiate transition.


Early cells evolved in an aqueous environment and the primary cell wall (PCW) evolved as a strategy for coping with the associated osmotic problems (Gerhart & Kirschner, 1997). The PCW has become one of the defining characteristics of plants, many of which now live in a terrestrial environment. The PCW is fundamentally involved in many plant processes, including tissue cohesion, defence (e.g. against microbes), ion-exchange, the production of oligosaccharins and the regulation of cell expansion (Goldberg et al., 1994; Brett & Waldron, 1996; Cassab, 1998; Dumville & Fry, 1999). Demands on the PCW may have changed during terrestrial plant evolution, thereby influencing its optimal composition.

Angiosperm crop species are still the most extensively studied and the PCW composition of all higher plants is assumed to be comparable though not identical, the major monosaccharide residues being d-glucose (Glc), d-galactose (Gal), d-mannose (Man), d-xylose (Xyl), l-arabinose (Ara), l-fucose (Fuc), l-rhamnose (Rha) and d-galacturonate (GalA) (Albersheim, 1976; McNeil et al., 1984; Fry, 2000). The PCW has been found to differ between vascular plants at both the monosaccharide and polysaccharide level. Gramineous monocot PCWs contain the same monosaccharide residues as the rest of the angiosperms (both non-gramineous monocots and dicots) but usually have more Xyl and less Gal and Fuc (Burke et al., 1974; Carpita, 1996). Gymnosperm PCWs are similar in composition to those of dicotyledonous angiosperms but contain more Man residues (Edashige & Ishii, 1996). At the polysaccharide level, mixed-linkage glucan (MLG) appears to occur uniquely in gramineous monocots and closely related members of the Poales (Smith & Harris, 1999).

Extant pteridophytes (= non-seed vascular plants, including ferns, horsetails and club-mosses) are the relatively few surviving progeny of a great diversity of extinct taxa that formerly were ecologically dominant (Raven, 1993; Kenrick & Crane, 1997). Few studies exist of the PCW composition of pteridophytes, although studies by Vissenberg et al. (2003) show the PCWs of lycopodiophytes (early diverging pteridophytes) to contain xyloglucan endotransglucosylase (XET), an enzyme activity capable of transglycosylating xyloglucan (a cell wall polysaccharide). Additionally, we previously reported (Popper et al., 2001) that lycopodiophytes, the earliest diverging extant vascular plants (Raubeson & Jansen, 1992; Manhart, 1994, 1995; Pryer et al., 1995; Wolf, 1997; Duff & Nickrent, 1999) uniquely contain high concentrations of 3-O-methyl-d-galactose (MeGal) in their PCWs. Homosporous lycopodiophytes, bryophytes and charophytes also contain 3-O-methyl-rhamnose (MeRha) in their PCWs (Popper et al., 2004). MeRha has been shown to occur as a modification of the non-reducing Rha residue present on the aceric acid-containing side chain of rhamnogalacturonan II (RG-II); the primary structure of RG-II appears otherwise to be conserved (Matsunaga et al., 2004). Matsunaga et al. (2004) also report that the sporophyte PCWs of lycopodiophytes, equisetophytes, psilotophytes and ferns contain amounts of RG-II comparable with those in angiosperm PCWs. By contrast, PCW material from the gametophyte generation of all bryophytes analysed contained only 1% of the amounts of an RG-II-like polysaccharide present in angiosperm PCWs (Matsunaga et al., 2004).

We are therefore documenting the evolution of the PCW composition of vascular plants. Vascular plants (tracheophytes) form a well-supported monophyletic group which originated around 420 million years ago (Judd et al., 1999). It is of interest to identify any changes in cell wall composition that have accompanied steps in their subsequent diversification. In the present paper we report that PCW composition varies within the tracheophytes as well as between vascular plants and bryophytes.

Materials and Methods

Source of plant material

Sources of vascular plant material are listed in Table 1. All material collected was of immature leaf and/or stem tissues which were still expanding. Seeds of Secale cereale L., Triticum aestivum L., Zea mays L. and Hordeum vulgare L. were obtained commercially (PBI, Hauxton, UK) and grown in the dark, and alcohol-insoluble residue (AIR) was prepared from the coleoptiles as described by Popper et al. (2001).

Table 1.  Composition of alcohol-insoluble residues from young growing shoot tissues of diverse vascular plant taxa (arrangement of taxa after Judd et al., 1999)
  • *

    Accession numbers followed by (E) refer to material from the Royal Botanic Garden, Edinburgh (RBGE); DRB, glass houses of the Daniel Rutherford Building, University of Edinburgh; PBI, Plant Breeding International, Cambridge, UK; Johnson's, W.W. Johnson & Son Ltd, UK. Other material was collected by Z.A.P. Collection sites were: Cairngorms Hills (57°05–10′ N, 3°35–45′ W) and Roslin Glen (55°51′-N, 3°10′-W).

  • – Denotes compound not detected; blank denotes compound not tested for presence/absence in particular plant.

∼ HomosporousLycopodium pinifolium Blume19835037(E)++ +  
Huperzia selago (L.) Bernh. ex. Schrank and Mart.Cairngorms++ +  
Diphasiastrum alpinum (L.) Holub.Cairngorms++  
∼ HeterosporousSelaginella apoda (L.) Spring.19677705(E)++ +  
Selaginella erythropus Spring.19715473(E)++ + + +
Selaginella pallescens (C. Presl.) Spring.19697710(E)++ ++ +
EquisetophyteEquisetum debile Roxb. ex. Vaucher19731694(E)++ + +  
PsilotophytePsilotum nudum (L.) P. Beauv.DRB++ + ++ +
∼ Eusporangiate fernMarattia fraxinea Sm.19697183(E)++ ++ +
∼ Leptosporangiate fernsOsmunda regalis L.19578631(E)+±++ +
Todea barbara (L.) T. Moore19652792(E)+±++ +
Dryopteris crispifolia Rasbach et al. 19920813(E)+±++ +
Asplenium australassium (J.Sm.) Hook.19933661(E)+±++ +
Nephrolepis lauterbachii H. Christ.19933715(E)+±++ +
Onoclea sensiblis L.19662802(E)+±++ +
Phyllitis scolopendrum L.19731529(E)+±++ +
Blechnum spicant (L.) RothCairngorms+±+   
Salvinia auriculatea Aubl.19830813(E)+±++ +
Platycerium bifurcatum (Car.) C. Chr.19734554(E)+ +   
∼ GymnospermsEncephalartos altensteinii Lehm.19754185(E)++ + + +
Pinus sylvestris L.Cairngorms++ + +
Gnetum gnemon L.19902511(E)+± + +
Gnetum indicum Merr.19550226(E)+± + +
Gnetum montana Markgr.19791010(E)+± + +
∼ Angiosperms:
∼∼ Non-monocot paleoherbNymphaea colorata Peter19972169(E)++ + +
∼∼ Magnoliid complexAustrobaileya scandens C.T. White19973060(E)++ + + +
Hernandia cordigera Vieill.19696964(E)+± + +
Drimys lanceolata (Poir.) Baill.19531038(E)+± + +
Calycanthus floridus Makino19696408(E)+± ++ +
Schizandra rubiflora Rehder and E.H. Wilson19091003(E)+± ++ +
Illicium verum Hook. f.19741584(E)+± + +
∼∼ Monocots:
∼∼∼ AcoralesAcorus calamus L.DRB+    
∼∼∼ AlismatalesLemna sp.DRB+    
Vallisneria spiralis L.19697868(E)+    
∼∼∼ ZingiberalesCalathea zebrina (Sims.) Lindl.19696326(E)+± ±+
∼∼∼ CommelinalesCallisia repens L.19831987(E)+± ±+ +
Cyanotis longifolia Wight19672908(E)+±±++ +
Dichorisandra thyrsifolia J.C. Mikan19644295(E)+± ++ +
Geogenthus undatus (K.Koch and Linden) Mildbr. and Strauss19696892(E)+±   
Pallisota albertii L.Gentil19140061(E)+± ±+
Siderasis fuscata (Lodd.) H.E. Moore19633223(E)+± ±+ +
∼∼∼ JuncalesJuncus effusus L.DRB+± ++ +
Cyperus esculentus L.19960902(E)+± ++ +
Cyperus papyrus L.20000863(E)+± ++
∼∼∼ Poales:
∼∼∼∼ PoaceaeSecale cereale L.PBI+    +
Triticum aestivum L.PBI+    +
Zea mays L.PBI+    +
Hordeum vulgare L.PBI+    +
Avena sativa L.PBI+    +
∼∼∼∼ RestionaceaeElegia capensis (Burm. f.) Schelpe19860037(E)+± ±+ +
∼∼∼∼ FlagellariaceaeFlagellaria guineensis Schum.19720171(E)+± ±+ ++
∼∼ EudicotsHelleborus argutifolius Viv.19780406(E)+± + +
Spinacia oleracea (L.) T. MooreJohnson's+    
Fallopia japonica (Houtt.) Ronse DeraeneRoslin Glen+± + +
Phaseolus aureus L.Seeds, Sainsbury's+    

Paper chromatography (PC) and thin-layer chromatography (TLC)

Whatman no. 1 paper was used for one-dimensional analytical PC by the descending method. Solvent systems used were (i) butan-1-ol : acetic acid : water (12 : 3 : 5 by volume) for 16 h; (ii) as for system 1 followed in the same dimension, by ethyl acetate : pyridine : water (8 : 2 : 1 by volume) for 18 h; (iii) ethyl acetate : pyridine : water (8 : 2: 1 by volume) for 18–72 h; (iv) ethyl acetate : pyridine : water (10 : 4 : 3 by volume) for 24 h. Paper chromatograms were stained with aniline hydrogen-phthalate; faint spots were most readily visible by their fluorescence when viewed under a 366-nm UV lamp (Fry, 2000). TLC was on Merck silica gel or cellulose (VWR International, Poole, UK). The solvent system used for silica gel TLC was (v) butan-1-ol : acetic acid : water (3 : 1 : 1 by volume) and that for cellulose TLC was (vi) as for solvent system (v) followed by ethyl acetate : pyridine : water (10 : 4 : 3 by volume). Silica gel TLC plates were stained with thymol–sulphuric acid (Jork et al., 1994); cellulose TLC plates were stained with aniline hydrogen-phthalate.

Paper electrophoresis (PE)

PE was on Whatman no. 1 paper. Samples were loaded 12 cm from the cathode end. The buffer was 200 ml 0.1 m boric acid, 113.5 ml 0.1 m NaOH, pH 9.4. Typical running conditions for paper of width 38 cm were 3.0 kV (approx. 100 mA) for 1 h. Electrophoretograms were stained with silver nitrate (Trevelyan et al., 1950) (the alkali step was modified by the addition of 4% pentaerythritol).

High-pressure liquid chromatography (HPLC)

A Dionex HPLC (Camberley, UK) was used with a CarboPac PA1 anion-exchange column (4 mm internal diameter, 250 mm long) and a pulsed amperometric detector with gold electrode. The flow rate was 1.0 ml min−1 at room temperature, and 20-µl samples were injected. Before injection, samples were filtered (Millex-HV4 4-mm syringe filters, acetate membrane, pore size 0.45 µm; Millipore, Bedford, MA, USA). Mono- and disaccharides were separated by the following eluents (gradients were linear between all specified time points): 0–1.80 min, 0.02 m NaOH; 1.80–1.81 min, 0.02→0 m NaOH; 1.81–30 min, H2O; 30–40 min, 0→0.03 m NaOH; 40–76 min, 0.03→0.80 m NaOH; 76–81 min, 0.8 m NaOH; 81–82 min, 0.80→0.02 m NaOH; 82–90 min, 0.02 m NaOH.

Ion-exchange chromatography

Before ion-exchange samples were de-lactonized by adjusting to pH 13 (0.1 m NaOH) and incubating for 8 s. Samples were neutralized with 0.1 m formic acid and made up to 1 ml before loading onto columns (1.5 ml bed volume) of Dowex 1 × 4–200 strongly basic anion-exchanger (Sigma, Poole, UK). The resin was pretreated by washing (1 h in each wash) in (i) 0.5 m NaOH; (ii) 0.5 m formic acid (twice); and (iii) 2 m sodium formate. The resin was finally washed in buffer A (10 mm pyridinium formate, pH 5.5). Neutral sugars were eluted in 4 ml buffer A. The acidic fraction was then eluted with 4 ml buffer B (pyridine : formic acid : water; 1 : 1 : 23, pH 4.8). Neutral and acidic fractions were dried and re-dissolved in 100 µl water and the neutral fraction was de-salted using cation-exchange columns of bed-volume 1.5 ml. The cation-exchange resin (Dowex 50 W 8100–200, H+ form from Sigma) was pretreated by shaking 1 h in 1 m HCl then rinsing in water until the filtrate was neutral. The neutral sugars were eluted from the columns in 1.5 ml water then dried in vacuo and re-dissolved in water ready for analysis.

Licheninase digestion

MLG was detected by licheninase digestion as described by Popper & Fry (2003).

Hemicellulose extraction, Driselase digestion and acid hydrolysis

Hemicelluloses were extracted from AIR and their component di- and monosaccharides analysed as described by Popper & Fry (2003). The presence of the disaccharide isoprimeverose after Driselase digestion is indicative of xyloglucan. Acid hydrolysis (2 m TFA, 120°C, 1 h) was used to essentially release all the monosaccharides present in the PCW matrix as described by Popper & Fry (2003).

Sodium borohydride reduction

Sodium borohydride reduction followed by acid hydrolysis can be used to identify the reducing terminus of a disaccharide by conversion to an alditol. About 0.1 mg Psilotum nudum disaccharide was dissolved in 0.2 ml of 0.5 m NaBH4 containing 1 m ammonia and incubated for 16 h at 25°C. Excess NaBH4 was destroyed by the addition of 30 µl glacial acetic acid. Ammonium and Na+ were removed on a cation-exchange column. The sample was dried and redissolved in 0.1 ml methanol : acetic acid (10 : 1 by volume) six times to remove borate then subjected to acid hydrolysis. The products were separated by cellulose TLC; sugars were stained with aniline hydrogen-phthalate.

Detection of tannins

Condensed tannins (proanthocyanidins) were identified by their ability to form a red coloration in hot acid (Fry, 2000).


Mannose-containing polysaccharides

The neutral fraction of TFA hydrolysates of PCW-rich material was analysed by PC. The concentration of Man residues was higher in lycopodiophytes, psilotophytes, equisetophytes and a eusporangiate fern than it was in all leptosporangiate ferns tested and most gymnosperms and angiosperms (Fig. 1 and similar results, not shown; Table 1). Psilotum AIR was particularly rich in Man residues (Fig. 1).

Figure 1.

Paper chromatogram of the acid hydrolysis products of vascular plant alcohol-insoluble residues (AIRs). The PC was developed in solvent system 3 and stained with aniline hydrogen-phthalate. The ‘marker mix’ contained lactose, Gal, Man, Ara and Xyl.

In an investigation of the origin of the Man in Psilotum, the neutral fraction of a Driselase digest from Psilotum AIR gave a major spot (intensity similar to that of Glc) that stained brown with aniline hydrogen-phthalate and had a similar RGlc (the distance moved by a compound relative to the distance moved by Glc in the same system) to cellobiose in solvent system 1. Cellobiose stains brown with aniline hydrogen-phthalate, as do all reducing disaccharides of Glc; but cellobiose is usually completely digested by Driselase to Glc. The Psilotum disaccharide was purified by preparative PC and run alongside Glc disaccharide markers; in all PC and PE systems tested the disaccharide had similar RGlc values to cellobiose or kojibiose (Dumville & Fry, 2003). TFA hydrolysis of the disaccharide released Man and Glc (RGlc values in solvent system three distinguished these from all six other neutral aldohexoses, including allose). After prolonged sodium borohydride reduction of the disaccharide, acid hydrolysis still gave Glc and Man, suggesting the presence of two disaccharides, one with Man at the reducing terminus and the other with Glc. Enzymic hydrolysis of a glucomannan with endo-β-d-glucanase is much less selective than digestion with endo-β-d-mannanase and gives mixtures of oligosaccharides that contain both Man and Glc at the reducing terminus (Goldberg et al., 1991). No other plant polysaccharides than glucomannans are known that could yield a disaccharide composed of Glc and Man. Our results suggest that two disaccharides originating from endo-β-d-glucanase digestion of a glucomannan were present in our Driselase digested Psilotum AIR.


On TFA hydrolysis of PCW-rich material from all leptosporangiate ferns tested, the suspension became bright red (Fig. 2). This suggested the presence of condensed tannins (proanthocyanidins), which however, were not detectable in equisetophytes, psilotophytes, lycopodiophytes or a eusporangiate fern (Fig. 2). The butanol/HCl test showed the presence of high concentrations of proanthocyanidins in leptosporangiate ferns and lower levels in gymnosperms and angiosperms (data not shown). Our results suggest that proanthocyanidins are present in high concentration in leptosporangiate fern AIRs whereas they are undetectable in the more primitive vascular plants (lycopodiophytes, equisetophytes, a psilotophyte and a eusporangiate fern) and present at low concentrations in more recently evolved vascular plants (gymnosperms and angiosperms).

Figure 2.

Coloration of alcohol-insoluble residues (AIRs) from various vascular plants on acid hydrolysis. AIRs were of (a) Selaginella pallescens (Selaginellaceae); (b) Equisetum debile (Equisetaceae); (c) Huperzia selago (Lycopodiaceae); (d) Selaginella apoda (Selaginellaceae); (e) Marattia fraxinea (eusporangiate fern); (f) Psilotum nudum (Psilotaceae); (g) Lycopodium pinifolium (Lycopodiaceae); (h) Diphasiastrum alpinum (Lycopodiaceae); and the following leptosporangiate ferns: (i) Nephrolepis lauterbachii; (j) Onoclea sensiblis; (k) Dryopteris crispifolia; (l) Todea barbara; (m) Phyllitis scolopendrum; (n) Osmunda regalis; (o) Salvinia auriculatea; (q) Platycerium bifurcatum; (r) Blechnum spicant and (s) Asplenium australassium. (Sample (P) was from Cyanotis longifolia (a monocot).)

Uronic acids

The major uronic acid present in all land plants tested was GalA. Among vascular plants, monocots, some members of the magnoliid complex and non-monocot paleoherbs had a higher GlcA residue concentration than the eudicots and monocots (Table 1).

Mixed-linkage glucan

We detected the presence of MLG in gramineous monocots (Poaceae) and Flagellaria guineensis (Flagellariaceae) within the Poales. MLG was not detected in any other vascular plants studied including Acorus calamus, thought to be one of the earliest diverging members of the monocots, and Elegia capensis (Restionaceae), one of the early diverging members of the Poales (Fig. 3).

Figure 3.

Licheninase digestion of alcohol-insoluble residues (AIRs) from three species of monocot. Digestion products were loaded on silica gel TLC, developed in solvent system 5 and stained with thymol-H2SO4. MLG3, MLG4, MLG5 and MLG6 are tri-, tetra-, penta- and hexasaccharide repeat units of barley mixed-linkage glucan. Samples were (a) Acorus calamus undigested; (b) Acorus calamus digested; (c) Flagellaria guineensis undigested; (d) Flagellaria guineensis digested; (e) Siderasis fuscata undigested; (f) Siderasis fuscata digested; and (g) digested authentic mixed-linkage glucan from barley. Markers were (h) MLG3 and MLG4 and (i) Glc.


Xyloglucan was found in all vascular plants tested (Table 1; Fig. 4). The concentration of isoprimeverose (the diagnostic disaccharide of xyloglucan) relative to weight of AIR digested appeared to be similar between the most primitive extant vascular plants, lycopodiophytes and all non-gramineous angiosperms (Fig. 4).

Figure 4.

HPLC of Driselase digestion-products from lycopodiophytes (top three traces) and early diverging angiosperms (bottom six traces).


High XET action in all vascular plants tested, from lycopodiophytes to gramineous monocots (Vissenberg et al., 2003), correlates well with our finding that its substrate, the polysaccharide xyloglucan, is also present in all vascular plants tested. Xyloglucan has been characterized from dicot PCWs where it is the major hemicellulose that hydrogen-bonds to, and probably forms tethers between, adjacent cellulose microfibrils (Fry, 1989; Carpita & Gibeaut, 1993). XET activity can cut and rejoin tethering xyloglucan chains, thus allowing cell wall loosening and cell expansion (Fry, 1989, 1992, 1995). The importance of xyloglucan is suggested by the universality of its occurrence in vascular plants. Xyloglucan derived from angiosperms has been reported to vary in side-chain composition. Some storage xyloglucans contain no Fuc (Kooiman, 1961); those extracted from the Solanaceae have α-l-Ara linked to Xyl at position 2, contain little Fuc and are less substituted with Xyl (Eda & Kato, 1978; Akiyama & Kato, 1982; Ring & Selvendran, 1981). Xyloglucan has been found to occur at a lower concentration in the PCWs of bryophytes (Popper & Fry, 2003), where it may differ in composition. Bryophyte xyloglucan may lack the Fuc side-chain since xyloglucan was not detected in bryophyte water-conducting cells by CCRC-M1, an antibody which recognizes an epitope containing a terminal α-Fuc residue (1→2)-linked to a β-Gal residue (Ligrone et al., 2002). XET activity has been detected in sporophyte and gametophyte tissues from the liverwort Marchantia and gametophyte tissue from a moss, Mnium (Fry et al., 1992).

Edashige & Ishii (1996) reported glucomannan to be a major component (10.6% of total PCW, w/w) in suspension-cultured cells of the gymnosperm Cryptomeria japonica. This is consistent with our finding of a high concentration of Man residues in the acid hydrolysate of AIR from young tissue of some gymnosperms. Among the gymnosperms, we studied a cycad (Encephalartos alteinsteinii), a conifer (Pinus sylvestris) and three gnetophytes (Gnetum gnemon, Gnetum indicum, Gnetum montana). The cycad and conifer had similar, high concentrations of Man in their AIRs. Gnetophyte AIR contained a lower concentration of Man residues than the other gymnosperms. Molecular evidence provided by developmental genes suggested that the gnetophytes are more closely related to conifers than to flowering plants (Winter et al., 1999) but our Man data suggest that the gnetophytes are more similar to angiosperms than to the rest of the gymnosperms.

The mannan content revealed a pronounced segregation between eusporangiate and leptosporangiate pteridophytes. A eusporangium is one that originates from several initial cells, has a sporangial wall more than one cell thick and tends to produce numerous sporocytes; a leptosporangium originates from a single initial cell, has a one-cell-thick wall and produces few sporocytes (Foster & Gifford, 1959). Among the non-seed plants, the PCWs of leptosporangiate ferns (Table 1) contained far less Man than those of the bryophytes (Popper & Fry, 2003), the lycopodiophytes, an equisetophyte, a psilotophyte and a eusporangiate fern (Marattia fraxinea) (Table 1). Lycopodiophytes, psilotophytes and equisetophytes, like M. fraxinea, exhibit the eusporangiate condition; and gene sequencing studies have shown that the eusporangiate ferns are more closely related to the equisetophytes and psilotophytes than to the leptosporangiate ferns (Pryer et al., 2001). Leptosporangiate ferns appear to have diversified in an environment dominated by seed plants (Schneider et al., 2004) and may therefore have faced selective pressures similar to those experienced by the seed plants themselves. This circumstance could explain why the PCWs of leptosporangiate ferns, including Osmunda, one of the earliest diverging extant leptosporangiate ferns (Pryer et al., 2001; Schneider et al., 2004), are more similar to those of seed plants than to those of eusporangiate pteridophytes. It is possible that the PCW composition of the vegetative tissues of bryophytes, lycopodiophytes, equisetophytes, psilotophytes and eusporangiate ferns is similar to that of the secondary cell wall of leptosporangiate ferns, gymnosperms and angiosperms. The neoteny theory of plant evolution postulates that angiosperms arose from their ancestors by a modified and extended juvenile phase (Takhtajan, 1976).

Lycopodiophytes are a well-supported monophyletic group which diverged early during vascular plant evolution. Within the lycopodiophytes there are two distinct groups: homosporous and the more advanced heterosporous. All lycopodiophytes tested contain MeGal in their PCWs (Popper et al., 2001) but the homosporous genera (Lycopodium, Huperzia and Diphasiastrum) are the only lycopodiophytes that resemble bryophytes in having high concentrations of MeRha in their PCWs (Popper et al., 2004). Matsunaga et al. (2004) also isolated MeRha from PCW material of a homosporous lycopodiophyte and a psilotophyte and showed the MeRha to be a component of RG-II (an important structural pectic polysaccharide) in these taxa. Bryophyte and charophyte PCWs also contain MeRha (Popper et al., 2004). These results suggest that the earliest-diverging extant vascular plants share some PCW characters with bryophytes and that during vascular plant evolution there was a reduction in hexose methylation.

Tannins may not be strictly a PCW component but are often associated with PCW-rich material and some tannins may be deposited within the PCW. There are condensed and non-condensed tannins. Condensed tannins (proanthocyanidins) are specifically detectable by the butanol/HCl test (Fry, 2000). They are an important class of secondary plant metabolites; in many cases they are the active principles of medicinal plants (De Bruyne et al., 1999). However, it is likely they evolved as a deterrent to herbivory as they can have negative effects on feed digestibility (Schofield et al., 2001).

Proanthocyanidins are present in AIR of leptosporangiate ferns and absent from that of the lycopodiophytes, a psilotophyte, an equisetophyte and the eusporangiate fern M. fraxinea. This chemical demarcation between leptosporangiate and eusporangiate pteridophytes coincides with that found for mannan (see Table 1) and supports the molecular evidence that the eusporangiate ferns are more closely related to the equisetophytes and psilotophytes than to the leptosporangiate ferns (Pryer et al., 2001). Bate-Smith & Learner (1954) reported the presence of a ‘moderate[ly]’ strong positive reaction for the presence of proanthocyanidin in M. fraxinea. Vascular plants (tracheophytes) form a well-supported, monophyletic group including the lycopodiophytes, psilotophytes, equisetophytes and eusporangiate ferns as well as the leptosporangiate ferns, gymnosperms and angiosperms (Judd et al., 1999). Leptosporangiate ferns are the earliest diverging plants in which proanthocyanidins start to predominate over flavonols (De Bruyne et al., 1999). Proanthocyanidins remain important in early diverging angiosperms but synthesis decreases in more advanced orders (De Bruyne et al., 1999). It seems likely that the production of proanthocyanidins evolved at the same time as the leptosporangiate condition rather than the vascular condition as proposed by Bate-Smith (1977).

GalA was the most abundant PCW uronic acid in all land plants (including all bryophytes) tested. However, the GlcA : GalA ratio was relatively high in early diverging bryophytes (Popper & Fry, 2003; Popper et al., 2003). We did not find GlcA in high concentration in any vascular plants tested but it was present in higher concentration in earlier diverging angiosperms than the more recently diverged ones. Jarvis et al. (1988) also found the Commelinanae, more recently evolved monocots, to have a greatly reduced uronic acid residue concentration. 4-O-Methylglucuronic acid has been reported to be present in high concentration in some dicot secondary cell walls, but is present at a lower concentration of about 0.2–0.8% of the d. wt. in all gramineous monocot PCWs tested (Darvill et al., 1980; Harris et al., 1997).

Our results confirm the report by Smith & Harris (1999) that MLG is present in the AIR of Flagellaria (Poales, Flagellariaceae). We did not detect MLG in Elegia capensis AIR (Poales, Restionaceae). However, Smith & Harris (1999) found concentrations of MLG in various species of Restionaceae to vary from undetectable to 0.1% (w/w, MLG/total cell wall composition). The super-order Poanae, as described by Takhtajan (1996), appears to be well defined as all families (Flagellariaceae, Joinvilleaceae, Restionaceae, Anarthriaceae, Ecdeiocolaceae, Centrolepidaceae, Poaceae) contain MLG.

Our results suggest that throughout vascular plant evolution the PCW and associated tannins have been adapted and modified. Stebbins (1992) thought that alterations in cell wall composition may have played a leading role in the evolution of vascular plants. Within and between the three monophyletic groups of extant vascular plants: (1) lycopodiophytes; (2) equisetophytes; psilotophytes, eusporangiate and leptosporangiate ferns; and (3) seed plants (Pryer et al., 2001) there are alterations in PCW composition. In particular, there is a pronounced chemical demarcation between the eusporangiate pteridophytes (high mannan, low tannin) and the leptosporangiate pteridophytes (low mannan, high tannin). However, the major monosaccharide residue composition appears to be relatively stable and all vascular plants contain a cellulose–xyloglucan network. Therefore it seems likely that all vascular plants accomplish cell expansion in the same way despite differences in PCW composition.


We thank Mrs J. Miller for technical assistance. We are very grateful to the horticultural staff at the RBGE, especially Mr Philip O. Ashby, for the supply of specimens. Z.A.P. thanks the BBSRC for a research studentship.