Download Primary cell wall composition of pteridophytes and spermatophytes

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Primary cell wall composition of pteridophytes and
spermatophytes
Blackwell Publishing, Ltd.
Zoë A. Popper and Stephen C. Fry
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
Summary
Author for correspondence:
S. C. Fry
Tel. +44 131650 5320
Fax: +44 131650 5392
Email: [email protected]
Received: 25 March 2004
Accepted: 6 May 2004
• 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.
Key words: angiosperms, cell walls (primary), evolution, gymnosperms, polysaccharides, pteridophytes, tannins, vascular plants.
New Phytologist (2004) 164: 165–174
© New Phytologist (2004) doi: 10.1111/j.1469-8137.2004.01146.x
Introduction
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), ionexchange, 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.
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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 -glucose (Glc), -galactose (Gal), -mannose
(Man), -xylose (Xyl), -arabinose (Ara), -fucose (Fuc), rhamnose (Rha) and -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
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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--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).
New Phytologist (2004) 164: 165– 174
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 hydrogenphthalate; 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  boric acid,
113.5 ml 0.1  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  NaOH; 1.80–1.81 min, 0.02→0  NaOH; 1.81–
30 min, H2O; 30–40 min, 0→0.03  NaOH; 40–76 min,
0.03→0.80  NaOH; 76–81 min, 0.8  NaOH; 81–82 min,
0.80→0.02  NaOH; 82–90 min, 0.02  NaOH.
Ion-exchange chromatography
Before ion-exchange samples were de-lactonized by adjusting
to pH 13 (0.1  NaOH) and incubating for 8 s. Samples
were neutralized with 0.1  formic acid and made up to 1 ml
before loading onto columns (1.5 ml bed volume) of Dowex
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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)
Classification
Lycopodiophytes:
∼ Homosporous
∼ Heterosporous
Equisetophyte
Psilotophyte
Filicophytes:
∼ Eusporangiate fern
∼ Leptosporangiate ferns
Spermatophytes:
∼ Gymnosperms
Species
Source*
Xyloglucan Mannans Tannins GlcA GalA MLG
Lycopodium pinifolium Blume
Huperzia selago (L.) Bernh. ex. Schrank
and Mart.
Diphasiastrum alpinum (L.) Holub.
Selaginella apoda (L.) Spring.
Selaginella erythropus Spring.
Selaginella pallescens (C. Presl.) Spring.
Equisetum debile Roxb. ex. Vaucher
Psilotum nudum (L.) P. Beauv.
19835037(E)
Cairngorms
+
+
++
++
–
–
–
–
Cairngorms
19677705(E)
19715473(E)
19697710(E)
19731694(E)
DRB
+
+
+
+
+
+
+
++
++
++
+++
+++
–
–
Marattia fraxinea Sm.
Osmunda regalis L.
Todea barbara (L.) T. Moore
Dryopteris crispifolia Rasbach et al.
Asplenium australassium (J.Sm.) Hook.
Nephrolepis lauterbachii H. Christ.
Onoclea sensiblis L.
Phyllitis scolopendrum L.
Blechnum spicant (L.) Roth
Salvinia auriculatea Aubl.
Platycerium bifurcatum (Car.) C. Chr.
19697183(E)
19578631(E)
19652792(E)
19920813(E)
19933661(E)
19933715(E)
19662802(E)
19731529(E)
Cairngorms
19830813(E)
19734554(E)
+
+
+
+
+
+
+
+
+
+
+
++
±
±
±
±
±
±
±
±
±
Encephalartos altensteinii Lehm.
Pinus sylvestris L.
Gnetum gnemon L.
Gnetum indicum Merr.
Gnetum montana Markgr.
19754185(E)
Cairngorms
19902511(E)
19550226(E)
19791010(E)
+
+
+
+
+
19972169(E)
19973060(E)
19696964(E)
19531038(E)
19696408(E)
19091003(E)
∼ Angiosperms:
∼ ∼ Non-monocot paleoherb Nymphaea colorata Peter
∼ ∼ Magnoliid complex
Austrobaileya scandens C.T. White
Hernandia cordigera Vieill.
Drimys lanceolata (Poir.) Baill.
Calycanthus floridus Makino
Schizandra rubiflora Rehder and
E.H. Wilson
Illicium verum Hook. f.
∼ ∼ Monocots:
∼ ∼ ∼ Acorales
Acorus calamus L.
∼ ∼ ∼ Alismatales
Lemna sp.
Vallisneria spiralis L.
∼ ∼ ∼ Zingiberales
Calathea zebrina (Sims.) Lindl.
∼ ∼ ∼ Commelinales
Callisia repens L.
Cyanotis longifolia Wight
Dichorisandra thyrsifolia J.C. Mikan
Geogenthus undatus (K.Koch and
Linden) Mildbr. and Strauss
Pallisota albertii L.Gentil
Siderasis fuscata (Lodd.) H.E. Moore
∼ ∼ ∼ Juncales
Juncus effusus L.
Cyperus esculentus L.
Cyperus papyrus L.
∼ ∼ ∼ Poales:
∼ ∼ ∼ ∼ Poaceae
Secale cereale L.
Triticum aestivum L.
Zea mays L.
Hordeum vulgare L.
Avena sativa L.
∼ ∼ ∼ ∼ Restionaceae
Elegia capensis (Burm. f.) Schelpe
© New Phytologist (2004) www.newphytologist.org
–
–
++
++
–
++
–
–
–
–
–
–
–
–
–
–
–
–
–
–
++
++
++
++
++
++
++
++
–
–
–
–
–
–
–
–
–
++
–
++
++
±
±
±
–
–
–
–
–
++
+
++
++
++
–
–
–
–
–
+
+
+
+
+
+
+
++
±
±
±
±
–
–
–
–
+
+
++
++
++
++
++
++
–
–
–
–
–
–
19741584(E)
+
±
–
++
–
DRB
DRB
19697868(E)
19696326(E)
19831987(E)
19672908(E)
19644295(E)
19696892(E)
+
+
+
+
+
+
+
+
±
±
±
±
±
19140061(E)
19633223(E)
DRB
19960902(E)
20000863(E)
+
+
+
+
+
±
±
±
±
±
PBI
PBI
PBI
PBI
PBI
19860037(E)
+
+
+
+
+
+
±
–
–
–
–
+
+
+
+
+
+
+
+
+
+
±
–
–
–
–
–
–
–
–
±
±
+
+
+
++
++
++
±
±
+
+
+
+
++
++
++
+
–
–
–
–
–
++
+
+
+
+
+
–
±
New Phytologist (2004) 164: 165–174
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Table 1 continued
Classification
Species
Source*
Xyloglucan Mannans Tannins GlcA GalA MLG
∼ ∼ ∼ ∼ Flagellariaceae
∼ ∼ Eudicots
Flagellaria guineensis Schum.
Helleborus argutifolius Viv.
Spinacia oleracea (L.) T. Moore
Fallopia japonica (Houtt.)
Ronse Deraene
Phaseolus aureus L.
19720171(E)
19780406(E)
Johnson’s
Roslin Glen
+
+
+
+
±
±
±
–
++
++
±
–
++
Seeds, Sainsbury’s +
+
–
–
–
–
*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.
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  NaOH; (ii) 0.5  formic acid (twice); and
(iii) 2  sodium formate. The resin was finally washed in buffer
A (10 m 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 redissolved in 100 µl water and the neutral fraction was desalted 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  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.
Excess NaBH4 was destroyed by the addition of 30 µl glacial
acetic acid. Ammonium and Na+ were removed on a cationexchange 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).
Results
Mannose-containing polysaccharides
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  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  NaBH4
containing 1  ammonia and incubated for 16 h at 25°C.
New Phytologist (2004) 164: 165– 174
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).
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 hydrogenphthalate, 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,
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Fig. 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.
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--glucanase is
much less selective than digestion with endo-β--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--glucanase digestion of a glucomannan
were present in our Driselase digested Psilotum AIR.
Tannins
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
© New Phytologist (2004) www.newphytologist.org
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).
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).
New Phytologist (2004) 164: 165–174
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Fig. 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).)
Xyloglucan
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).
Discussion
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
New Phytologist (2004) 164: 165– 174
Fig. 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 thymolH2SO4. 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.
storage xyloglucans contain no Fuc (Kooiman, 1961); those
extracted from the Solanaceae have α--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 CCRCM1, 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 suspensioncultured cells of the gymnosperm Cryptomeria japonica. This
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Research
Fig. 4 HPLC of Driselase digestion-products
from lycopodiophytes (top three traces) and
early diverging angiosperms (bottom six
traces).
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
© New Phytologist (2004) www.newphytologist.org
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
New Phytologist (2004) 164: 165–174
171
172 Research
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).
New Phytologist (2004) 164: 165– 174
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;
www.newphytologist.org © New Phytologist (2004)
Research
(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.
Acknowledgements
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.
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