Annals of Botany 116: 225–236, 2015
doi:10.1093/aob/mcv089, available online at www.aob.oxfordjournals.org
Sugar composition of the pectic polysaccharides of charophytes, the closest algal
relatives of land-plants: presence of 3-O-methyl-D-galactose residues
Christina O’Rourke,1,y Timothy Gregson,1,z Lorna Murray,2 Ian H. Sadler2 and Stephen C. Fry1*
1
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford
Building, The King’s Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK and 2EastChem School of Chemistry, The
University of Edinburgh, The King’s Buildings, Edinburgh EH9 3JJ, UK
*For correspondence. E-mail [email protected]
†
Present address: Piramal Healthcare, Earl’s Road, Grangemouth FK3 8XG, UK.
‡
Present address: Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK.
Received: 31 March 2015 Returned for revision: 13 April 2015 Accepted: 28 April 2015 Published electronically: 25 June 2015
Background and Aims During evolution, plants have acquired and/or lost diverse sugar residues as cell-wall constituents. Of particular interest are primordial cell-wall features that existed, and in some cases abruptly changed,
during the momentous step whereby land-plants arose from charophytic algal ancestors.
Methods Polysaccharides were extracted from four charophyte orders [Chlorokybales (Chlorokybus atmophyticus), Klebsormidiales (Klebsormidium fluitans, K. subtile), Charales (Chara vulgaris, Nitella flexilis),
Coleochaetales (Coleochaete scutata)] and an early-diverging land-plant (Anthoceros agrestis). ‘Pectins’ and
‘hemicelluloses’, operationally defined as extractable in oxalate (100 C) and 6 M NaOH (37 C), respectively, were
acid- or Driselase-hydrolysed, and the monosaccharides analysed chromatographically. One unusual monosaccharide, ‘U’, was characterized by 1H/13C-nuclear magnetic resonance spectroscopy and also enzymically.
Key Results ‘U’ was identified as 3-O-methyl-D-galactose (3-MeGal). All pectins, except in Klebsormidium, contained acid- and Driselase-releasable galacturonate, suggesting homogalacturonan. All pectins, without exception,
released rhamnose and galactose on acid hydrolysis; however, only in ‘higher’ charophytes (Charales,
Coleochaetales) and Anthoceros were these sugars also efficiently released by Driselase, suggesting rhamnogalacturonan-I. Pectins of ‘higher’ charophytes, especially Chara, contained little arabinose, instead possessing
3-MeGal. Anthoceros hemicelluloses were rich in glucose, xylose, galactose and arabinose (suggesting xyloglucan
and arabinoxylan), none of which was consistently present in charophyte hemicelluloses.
Conclusions Homogalacturonan is an ancient streptophyte feature, albeit secondarily lost in Klebsormidium.
When conquering the land, the first embryophytes already possessed rhamnogalacturonan-I. In contrast, charophyte
and land-plant hemicelluloses differ substantially, indicating major changes during terrestrialization. The presence
of 3-MeGal in charophytes and lycophytes but not in the ‘intervening’ bryophytes confirms that cell-wall chemistry
changed drastically between major phylogenetic grades.
Key words: Charophytic algae, charophytes, Embryophyta, Streptophyta, Chlorokybus, Klebsormidium, Chara,
Coleochaete, plant cell-wall evolution, pectin, pectic polysaccharides, rhamnogalacturonan-I, 3-O-methylD-galactose.
INTRODUCTION
All land-plants, from liverworts to angiosperms, comprise a single taxon, the Embryophyta, believed to have evolved from a
single aquatic green-algal ancestor approximately 460 Mya
(Leliaert et al., 2012). That ancient aquatic is presumably extinct, but phylogenetic studies imply that its closest living relatives are freshwater algae of the division Charophyta. The
charophytes plus the embryophytes together constitute the
Streptophyta. Nevertheless, a major evolutionary chasm separates the earliest-diverging land-plants from their closest charophytic relatives. It remains uncertain which of three extant
charophytic orders (Charales, Coleochaetales or Zygnematales)
is closest related to the land-plants (Karol et al., 2001;
Wodniok et al., 2011; Zhong et al., 2013); two earlier-diverging
orders of charophytes (Klebsormidiales and Chlorokybales) are
clearly less closely related to them.
Adapting to life on land must have necessitated numerous
changes; among the most important, enabling an upright growth
habit in a windy atmosphere that lacks buoyancy, and
conferring resistance to the desiccation and new herbivores and
pathogens encountered on land, would have involved cell walls.
We are interested in the primordial wall components that were
seconded into new roles when charophytic algae adapted to terrestrial life.
The chemistry of charophyte cell walls has been relatively
little explored. At least some charophytes are known to contain
cellulose (Hotchkiss and Brown, 1987) and a-D-galacturonaterich polysaccharides (Cherno et al., 1976; Proseus and Boyer,
C The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.
V
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O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
226
2006; Domozych et al., 2014), but few charophytic polysaccharides have been described in detail. Recent studies of charophyte cell walls have relied heavily on immunochemistry
(Domozych et al., 2009, 2014; Ikegaya et al., 2008). The
immunochemical approach depends on assumptions about the
specificity of the antibodies used; however, it is difficult to predict the ability of an antibody to recognize a postulated carbohydrate epitope that is not available in pure form for testing.
Thus, as an alternative, we have adopted the chemical strategy
of comparing the monosaccharide residues that comprise the
wall polysaccharides of charophytes and embryophytes, aiming
to discover primordial features of land-plant cell walls by characterizing the walls of their closest living algal relatives. New
data on primordial wall polysaccharides may also offer new insights into how angiosperm cell walls function, e.g. in their
growth-controlling roles. Thus, evolutionary knowledge of
charophyte polysaccharides could inform and re-focus agriculturally relevant research on the mechanisms and control of
crop-plant growth.
Land-plant primary walls consist mainly of polysaccharides –
categorized into cellulose, pectins and hemicelluloses – whose
chemistry has been studied in some detail (Albersheim et al.,
2011; Fry, 2011). Primary cell-wall polysaccharides are often assumed to be built of a common set of major monosaccharide residues. However, distinct taxonomically defined differences in
wall chemistry are emerging (Popper and Fry, 2003, 2004;
Nothnagel and Nothnagel, 2007; Fry et al., 2008; Brennan and
Harris, 2011; Sørensen et al., 2011). Here we have extended this
knowledge, focusing on charophyte ‘pectic’ polysaccharides.
In land-plants, pectins may play structural, hydrating, lubricating and porosity-defining roles in the primary cell wall.
Pectins can be chemically defined as polysaccharides rich in
4-linked a-D-galacturonic acid residues (often partially methyland/or acetyl-esterified); they are often also rich in a-L-rhamnose, b-D-galactose and a-L-arabinose residues (Albersheim
et al., 2011; Fry, 2011; Peaucelle et al., 2012). An alternative
(operational) definition would characterize pectins as the polysaccharide fraction that can be solubilized from the plant cell
wall by hot solutions of chelating agents, e.g. oxalate at pH4
or EDTA at pH65. This operational definition will be
employed here, avoiding unwarranted assumptions about the
chemical nature of charophyte ‘pectins’. The present paper
explores the monosaccharide composition of the operationally
defined ‘pectins’ of charophytes.
MATERIALS AND METHODS
Source of plant material
Chara vulgaris L. was collected from Blackford Pond,
Edinburgh, UK, and Nitella flexilis (L.) C. Agardh from an
unnamed moorland pond near Heriot, Scottish Borders, UK.
Both were manually freed of fragments of angiosperm pondweeds and any extraneous material such as snails’ eggs.
Cultures of Coleochaete scutata Brébisson, Klebsormidium
subtile (Kütz.) Tracanna ex Tell, K. fluitans (F. Gay) Lokhorst
and Chlorokybus atmophyticus Geitler were purchased from the
Culture Collection of Algae and Protozoa (CCAP),
Dunstaffnage, UK, and maintained on 3N-BBMþV medium
(http://www.ccap.ac.uk/media/documents/3N_BBM_V.pdf);
these cultures were not axenic, but the named alga was the
only photosynthetic organism present. Axenic cell-suspension cultures of the hornwort Anthoceros agrestis Paton
(Vogelsang et al., 2006) were a generous gift of Dr Maike
Petersen, University of Marburg, Germany. Lycopodium
clavatum L. was from Harehope Hill, near Peebles, UK.
All plant and cell samples were was washed extensively in
water, removing any free mucilage, then stirred in 70–77 % (v/
v) ethanol (acidified with 1 % formic acid) at 20 C for 16 h,
and centrifuged at 5000 g for 10 min. The resulting cell-wallrich alcohol-insoluble residue (AIR) was washed several times
in 70 % ethanol, then acetone, and finally dried.
For the experiment described in Fig. 1, the AIR was freed of
non-covalently bound proteins by stirring in phenol/acetic acid/
water (UAW; 2:1:1, w/v/v) at 70 C for 1 h, followed by rinsing
in ethanol.
For the experiments described in Figs 2–4 and 6, the samples
were de-starched as follows. The AIR was suspended at
10 mg mL–1 in 40 mM lutidine (OAc–) buffer, pH 67, in 025 %
(w/v) chlorobutanol, stirred at 100 C for 15 min (gelatinizing
any starch), and cooled to 60 C. Next, 01 volumes of a solution of heat-stable a-amylase (Bacillus amyloliquifaciens;
Sigma A7595 (Sigma-Aldrich, Poole, Dorset, UK); 10 mL of
the commercial solution dialysed against water then diluted to
45 mL with the lutidine buffer) was added, and incubation was
continued at 60 C for 72 h. Ethanol and ammonium formate
were then added to give final concentrations of 70 % (v/v) and
1 % (w/v), respectively, and the suspension was incubated at
20 C for 16 h, precipitating any water-soluble polysaccharides
among the cell walls, which were thoroughly rinsed with 70 %
ethanol and dried.
Source of enzymes and authentic carbohydrates
a-Amylase from Bacillus licheniformis (specific activity
>500 U (mg protein)–1; Sigma-Aldrich) was used at a final concentration of 4 U mL–1, where one unit liberates 1 mg of maltose from starch in 3 min at pH 69 and 20 C. D-Galactose
oxidase from Dactylium dendroides (specific activity 500
U mg–1; Sigma-Aldrich) was used at a final concentration
of 4 U mL–1, where 1 U produces a DA425 of 1 min–1 (in an otolidine–peroxidase system) at pH 60 and 25 C. Driselase, a
mixture of hydrolytic enzymes from the basidiomycete Irpex
lacteus, was freed of carbohydrates as described (Fry, 2000).
Tamarind xyloglucan was a generous gift from Dainippon
Pharmaceutical Co., Osaka, Japan; konjac glucomannan, beet
arabinan, wheat arabinoxylan and barley mixed-linkage glucan
were from Megazyme, Bray, Ireland. Yeast a-mannan was isolated in our laboratory. Methylglucuronoxylan, homogalacturonan (‘polygalacturonic acid’), citrus pectin, laminarin and
monosaccharides were from Sigma-Aldrich. Authentic 3-Omethyl-D-galactose (3-MeGal) was obtained from Lycopodium
clavatum AIR as described (Popper et al., 2001).
Extraction of polysaccharides
‘Pectin’ was solubilized from AIR with 02 M ammonium oxalate (pH 40–43), at 100 C, for 2 h followed by a further 16 h.
The 2-h and 16-h pectic extracts (P1 and P2) were dialysed and
freeze-dried.
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
Cell walls
(AIR)
α-Cellulose
100
% of total AIR weight
Wash ‘W’
80
227
Hemicellulose b
Oxalate pH 4·3,
Hemicellulose a
2 h at 100 °C
Pectin 1
Pectin 2
Pectin 1
60
Oxalate pH 4·3,
Pectin 2
16 h at 100 °C
40
6 M NaOH,
20
Wash
‘W’
pH 4,
Hemicelluloses
72 h at 37 °C
neutralize,
dialyse
20 °C
ro
s
ce
te
ae
ch
eo
C
ol
α-Cellulose
An
th
o
lla
C
ha
ra
N
ite
C
Kl
hl
or
eb
ok
so
yb
rm
us
id
i
um
Kl
eb
flu
so
ita
rm
ns
id
iu
m
su
bt
ile
0
Insoluble:
Hemicellulose a
Soluble:
Hemicellulose b
FIG. 1. Fractionation of charophyte and hornwort cell walls into broad polymer classes. The bar chart shows the yield of each polymer class, after fractionation by
the method shown in the scheme.
Xyl
F
Fuc
Xyl
Xyl
Ara
Ara
Man
Man
Glc
Glc
Gal
Gal
GalA
GalA
Xyl
‘U’
Ara
Ara
Man
Man
Glc
Gl
Glc
Gal
Gal
GlcN
GlcA
GalA
GalA
P1 P2 Hb P1 P2 W Ha Hb P1 P2 W Ha Hb MM MM
P1 P2 W Hb P1 P2 W Hb P1 Hb P1 P2 Hb MM
Origin
MMs
Chlorokybus
Klebsormidium
fluitans
Klebsormidium
subtile
Nitella
Chara
Coleochaete
Anthoceros
FIG. 2. Paper chromatography of monosaccharide constituents of matrix polysaccharide fractions from charophytes and a hornwort. Chromatography was on
Whatman No. 20 paper in BAW (12:3:5) followed by EPW (8:2:1). Stain: AgNO3. Polysaccharide fractions are ‘pectins’ (P1, P2), hemicelluloses a and b (Ha, Hb),
and the wash after alkali extraction (W). MM, monosaccharide marker mixture; GlcN, glucosamine; ‘U’, unfamiliar monosaccharide in Chara hydrolysate.
Rhamnose was allowed to run off the end of the paper, thus improving the resolution of the slower-migrating monosaccharides.
In some experiments, hemicellulose was then extracted from
the oxalate-insoluble residue in 6 M NaOH, at 37 C for 72 h.
The extract was neutralized with acetic acid and dialysed
against water; material that precipitated during these operations
(termed hemicellulose a; Ha) was sedimented by centrifugation
at 5000 g for 10 min, rinsed in water and freeze-dried.
The remaining solution (hemicellulose b; Hb) was also freezedried. The NaOH-inextractable material was rinsed several
times with pH 4 buffer, and the pooled washings (wash ‘W’)
were dialysed and freeze-dried. The final residue (‘a-cellulose’)
was rinsed in water and freeze-dried.
Polysaccharide hydrolysis and chromatography of sugars
AIR or polysaccharide fractions (5 mg) were hydrolysed
with 1 mL of 2 M trifluoroacetic acid (TFA) at 120 C for 1 h.
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
228
MeRha
MeXyl
MeXyl
MeXyl
Rha
Rha
Rib
Xyl Fuc
Rib Fuc
Xyl
Xyl,Fuc
Ara
Man
Glc
Gal
GlcN
GlcA
GalA
Rha
Xyl,Fuc
‘U’
Ara
Ara
Man
Glc
Man
Glc
Gal
Gal
GlcN
GlcA
GalA
GlcN
GlcA
GalA
MMs
P1 P2 Hb P1 P2 W Ha Hb P1 P2 W Ha Hb
Chlorokybus
Klebsormidium
fluitans
MMs
Klebsormidium
subtile
P1 P2 W Hb P1 P2 W Hb P1 Hb P1 P2 Hb MMs
Nitella
Chara
Coleochaete
Anthoceros
Fig. 3. Thin-layer chromatography of monosaccharide constituents of matrix polysaccharide fractions from charophytes and a hornwort. TLC was performed on silica-gel in ethyl acetate/pyridine/acetic acid/H2O (6 : 3 : 1 : 1). Stain: thymol/H2SO4. Polysaccharide fractions are ‘pectins’ (P1, P2), hemicelluloses a and b (Ha, Hb),
and the wash after alkali extraction (W). MM, monosaccharide marker mixture; GlcN, glucosamine; ‘U’, unfamiliar monosaccharide in Chara pectin hydrolysates.
The hydrolysate was dried, re-dissolved in water and chromatographed. Analytical paper chromatography was performed on
Whatman No. 1 or No. 20, typically in butan-1-ol/acetic acid/
water (BAW; 12:3:5, v/v/v) for 16 h and/or in ethyl acetate/pyridine/water (EPW; 8:2:1, v/v/v) for 16–24 h; sugars were
stained with aniline hydrogen-phthalate or AgNO3 (Fry, 2000).
Thin-layer chromatography (TLC) was on Merck silica-gel
plates, usually in BAW (4:1:1) or ethyl acetate/pyridine/acetic
acid/water (EPAW; 6:3:1:1); sugars were stained with thymol/
H2SO4 (Jork et al., 1994).
Other samples of AIR were digested in 1 % Driselase (purified) in pyridine/acetic acid/water (PyAW; 1:1:98, pH 47)
containing 05 % chlorobutanol at 37 C for 48 h. Ethanol (three
volumes) was then added, and the digest was incubated at 80 C
for 30 min. After centrifugation at 13000 g for 5 min, the supernatant was dried in vacuo and redissolved in water for chromatographic analysis. Driselase digests were analysed by highpressure liquid chromatography (HPLC) on a Dionex CarboPac
PA1 column in a NaOH/H2O gradient at 1 mL min–1: 0–2 min,
20 mM NaOH; 2–40 min, water; 40–75 min, water ! 800 mM
NaOH (linear gradient); 75–82 min, 800 mM NaOH; 82–90 min,
20 mM NaOH.
Purification and HPLC of an unfamiliar sugar (‘U’) from Chara
Hydrolysate from Chara vulgaris AIR (25 mg) was applied
as a 20-cm streak on Whatman No. 3 paper, and the unknown
(‘U’) was partially purified by chromatography in, sequentially,
BAW (12:3:5) for 40 h, phenol/water (UW; 4:1, w/w) for 24 h,
and EPW (8:2:1) for 40 h. After each preparative run, only the
fringes were stained with aniline hydrogen-phthalate, then the
unstained majority of the ‘U’ zone was eluted according to
Eshdat and Mirelman (1972).
Partially purified ‘U’ (100 mg) from the third chromatogram was freed of traces of soluble, paper-derived
polysaccharides on a 100-mL Bio-Gel P-2 column in PyAW
(1:1:98 by vol., pH 47), and 10 % of each 2-mL fraction was
analysed by TLC in BAW (4:1:1). Authentic 3-MeGal was
used as a marker.
Partially purified ‘U’, with and without a spike of
authentic 3-MeGal (50 mg), was subjected to analytical
HPLC on a Dionex CarboPac PA1 column in a NaOH/H2O
gradient at 1 mL min–1: 0–01 min, 20 mM NaOH; 01–50 min,
20 mM NaOH – 2 mM NaOH, linear gradient; 5–45 min,
2 mM NaOH; 45–75 min, 2 mM NaOH – 800 mM NaOH, concave gradient; 75–81 min, 800 mM NaOH; 81–82 min, 800 mM
NaOH – 20 mM NaOH, linear gradient; 82–90 min, 20 mM
NaOH.
Nuclear magnetic resonance (NMR) methods
The approach used was essentially that previously
reported by Popper et al. (2001). A 4 -mg sample of ‘U’ in D2O
was examined on a Bruker AVANCE III 800 MHz
spectrometer operating at 79972 MHz for protons and
20110 MHz for 13C nuclei. The composition of the ‘U’ preparation was determined by a series of one-dimensional (1D) and
2D NMR spectroscopy experiments as described in the Results
section.
Enzymic determination of enantiomerism of 3-MeGal
Samples of authentic D-galactose, L-galactose, methyl b-Dgalactopyranoside and ‘U’ (each at 025 mg mL–1) were incubated with D-galactose oxidase (4 U mL–1) in 03 % collidine
(OAc–) buffer, pH 60, for up to 96 h. At intervals, 16 mL of the
reaction mixture (: 4 mg substrate) was added to 10 mL of
50 % formic acid, dried and analysed by TLC in BAW (4:1:1).
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
Klebsormidium
fluitans
Chlorokybus
Pectin
1
Pectin
2
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
+
–
–
–
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
+
–
±
–
±
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
HemiGlc
cellulose
Man
a
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Hemi- Gal
cellulose Glc
Man
b
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
+
+
–
+
–
–
–
±
–
–
+
±
–
–
±
+
–
–
Klebsormidium
subtile
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
±
+
–
±
±
–
–
±
±
±
±
+
–
–
Nitella
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
Chara
+
±
±
+
+
+
+
+
+
+
±
±
+
±
±
+
–
–
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
229
Coleochaete
+
+
±
+
+
+
+
+
±
+
±
+
±
±
–
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
Anthoceros
+
+
+
+
+
+
+
+
+
+
+
+
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
+
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
+
–
+
+
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
GalA
GlcA
Gal
Glc
Man
Ara
Xyl
Fuc
Rha
+
+
+
–
+
+
+
+
–
+
+
+
+
–
+
FIG. 4. Semi-quantification of monosaccharide residues of matrix polysaccharide fractions from charophytes and a hornwort, as revealed by acid hydrolysis.
Polysaccharide fractions are ‘pectins’ (P1, P2) and hemicelluloses a and b (Ha, Hb). The coloured horizontal bars indicate staining intensity of each monosaccharide
on an arbitrary scale. The þ, 6 and – symbols to the right of each bar indicate release, partial release or no release, respectively, of the corresponding monosaccharide upon Driselase digestion (cf. Fig. 6). In some cases (especially mannose), the Driselase yield was difficult to assess owing to the presence of a trace of the monosaccharide (or a co-eluting peak) in the Driselase-only blanks, so no symbol is shown. In other cases, acid hydrolysis results were not available, but Driselase
digestions were performed and a high (þ) or low (6) yield of the monosaccharide was recorded.
RESULTS
Yield of operationally defined polysaccharide fractions from
diverse charophytes and Anthoceros
AIR samples from several charophytes and a cell-culture of the
hornwort Anthoceros were fractionated into polysaccharide
classes (Fig. 1). In each case, a substantial proportion
(35–63 %) of the AIR was extractable in hot oxalate, and thus
operationally defined as ‘pectic’. Of the total ‘pectin’, a high
proportion was solubilized quickly (as fraction P1) in
Anthoceros and the ‘higher’ charophytes (Chara, Nitella and
Coleochaete). In the ‘lower’ charophytes (Klebsormidium and
Chlorokybus), most of the pectin was extracted only by prolonged heating.
All organisms tested yielded hemicelluloses (extractable in 6
M NaOH at 37 C), the great majority of which was Hb (i.e. remained soluble when the NaOH was removed). An appreciable
amount of material (up to 15 % of the AIR) was subsequently
solubilized from the residual (NaOH-inextractable) wall by a
mildly acidic buffer. A final residue (‘a-cellulose’) was found
in all cases, although this was a highly variable proportion of
the AIR, ranging from <4 % in Chlorokybus to 20–30 % in
Klebsormidium, Chara and Nitella.
Monosaccharide residue composition of ‘pectins’
The polysaccharide fractions reported in Fig. 1 were
acid-hydrolysed under conditions which hydrolyse matrix polysaccharides but not cellulose, and the monosaccharide products
were resolved by paper chromatography (Fig. 2) and TLC
(Fig. 3). The spots were semi-quantified by staining intensity
(Fig. 4). The following description collates information from
both paper chromatography and TLC.
Classically, the pectic polysaccharides of land-plants are
rich in GalA, Rha, Gal and Ara, with a trace of 2-Omethylxylose (2-MeXyl) and several other minor sugars – a
pattern confirmed in our representative land-plant,
Anthoceros. The pectins of the ‘higher’ charophytes generally
also followed this trend, except for a notably low yield of Ara
and 2-MeXyl residues. The co-occurrence of GalA, Rha and
Gal supports the possible presence in higher charophytes of
rhamnogalacturonans in addition to homogalacturonan. The
pectins of higher charophytes contained little or no Ara and 2MeXyl, although Coleochaete did produce a spot identified as
3-O-methylrhamnose (3-MeRha; acofriose), which has been
observed before in charophytes and lower land-plants (Popper
and Fry, 2004).
230
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
Among the lower charophytes, Klebsormidium ‘pectin’ was
rich in Xyl and Gal, and also contained Man, Ara and Rha; it
thus resembled land-plant pectin in certain respects (Gal, Ara,
Rha), but the presence of Man and the absence of GalA singled
out Klebsormidium ‘pectin’ as differing fundamentally from
that of land-plants. Klebsormidium fluitans and K. subtile resembled each other closely in ‘pectin’ composition. The ‘pectin’ of the other lower charophyte tested, Chlorokybus,
resembled land-plant pectin surprisingly closely in some
respects, containing GalA, Rha, Gal, Ara and 2-MeXyl, but
differed in also possessing GlcA, Glc and a trace of Man.
Unexpectedly, Xyl was also a major component of the ‘pectins’ of Anthoceros, Chara, Nitella and Klebsormidium. A difference in the colour of staining distinguishes Fuc (reddish)
from Xyl (purple/blue) on TLC (Fig. 3). Very little Xyl was
present in Coleochaete (as noted before; Popper and Fry, 2003)
and in Chlorokybus.
Chara pectin consistently yielded an unfamiliar monosaccharide, indicated on Figs 2 and 3 as ‘U’, which was not present in
the other plants tested. ‘U’ was obtained from the AIR of
Chara but not from that of the majority of land-plants, including Equisetum (a eusporangiate fern-ally), Zea (a poalean
monocot) and Vinca (a dicot); in a hydrolysate of total Chara
AIR, ‘U’ exceeded Ara (Fig. 5). Its colour of staining suggested
that ‘U’ was a neutral hexose.
Xyl
Xyl
Ara
‘U’
Ara
Man
Man
Glc
Glc
Gal
Gal
Monosaccharide residue composition of ‘hemicelluloses’
The majority of the alkali-extractable hemicellulose remained soluble after neutralization (and was thus Hb). As expected, the Hb of the land-plant (Anthoceros) was rich in Glc,
Xyl, Gal and Ara, compatible with the presence of xyloglucan
and arabinoxylan. None of these four characteristic residues
was consistently present in all charophyte Hb samples. Glc was
abundant in them all except Klebsormidium; Xyl was abundant in all except Coleochaete and Chlorokybus; Gal was
abundant only in Coleochaete and Klebsormidium; and Ara
was abundant only in Coleochaete. The Hb of Chlorokybus was
almost pure glucan, whose linkage remains to be elucidated: it
is clearly not a (1!4)-b-glucan since it was water-soluble, and
it was not a mixed-linkage (1!3), (1!4)-b-glucan since it was
not digestible by lichenase. Very little Man was present in
Anthoceros Hb, indicating negligible mannan; Chara and
Nitella were the only charophytes with Man-rich Hb.
In the case of Klebsormidium, the Ha fraction was also analysed. The two Klebsormidium spp. differed greatly, their Ha
compositions being K. fluitans, Glc >> Rha Xyl; and K.
subtile, Gal Man > Ara Xyl >> Rha. Thus, surprisingly,
the major component of K. fluitans Ha (Glc) was almost undetectable in the Ha of K. subtile.
Many charophyte polysaccharides are resistant to Driselase
digestion
The polysaccharide fractions investigated by acid hydrolysis
were also treated with ‘Driselase’, a mixture of hydrolytic enzymes from the basidiomycete Irpex lacteus. Driselase typically
gives almost complete digestion of angiosperm primary cell
walls to yield mono- and disaccharides (Fry, 2000), and the
GalA
GalA
MM
H
NH
Chara
H
NH
Equisetum
H
NH
Zea
H
NH
MM
Vinca
Fig. 5. Paper chromatography of monosaccharide constituents of matrix polysaccharides from a charophyte, a eusporangiate fern, a poalean monocot and a eudicot. AIR from the young (growing) shoot tissue of each plant was hydrolysed
(H) in trifluoroacetic acid (TFA) at 120 C and the products were resolved by
paper chromatography in EPW (8 : 2 : 1) and stained with aniline hydrogenphthalate. Staining colours are: uronic acids, orange; neutral pentoses, red; neutral hexoses (including O-methylhexoses), brown. NH, non-hydrolysed control
treated with TFA at 20 C instead of 120 C; MM, monosaccharide marker mixture; ‘U’, unfamiliar brown-staining monosaccharide in Chara hydrolysate.
effectiveness of the present batch was verified on several authentic polysaccharides from land-plants (Fig. 6B–G). [Note
that incubation of Driselase alone yields traces of autolysis
products (cyan curves in Fig. 6), including Man, which must be
taken into account in any evaluation of the polysaccharide products.] However, HPLC showed that, unlike land-plant polysaccharides, many of the charophyte samples gave only low yields
of monosaccharides (Fig. 6H–N).
As a generalization, the ‘higher’ charophyte pectins were
well digested to monosaccharides by Driselase, suggesting that
they resembled land-plant pectins. In contrast, the pectins of the
‘lower’ charophytes were poorly digested to monosaccharides
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
GalA
Xyl
Man
Glc
Gal
Fuc
Rha
Ara
150
100
GalA
H
15
Chlorokybus
Hb
P2
P1
10
50
5
0
0
80
60
40
20
Bk
Klebsormidium fluitans
Gal
Glc
I
15
Ara
10
Glc
Xyloglucan
Ara
B
Gal
15
Glc
80
60
40
20
Monosaccharides
Gal
A
GlcA
200
231
Hb
Ha
P2
P1
10
5
5
Bk
Klebsormidium subtile
15
20
Glc
Man
J
Gal
β-Glucomannan,
α-Mannan
C
Glc
30
Ara
0
80
60
40
20
0
Hb
H
Ha
P2
P1
10
E
Laminarin,
MLG
Glc
40
GalA
Glc
Xyl
Gal
Bk
0
80
60
40
20
L
Chara
Hb
Ha
P2
P1
Bk
10
20
15
Glc
M
Coleochaete
Gal
0
80
60
40
20
Rha
Ara
Arabinan
Gal
Rha
20
GalA
Ara
F
Fuc
0
GalA
5
10
Hb
P2
10
10
P1
Bk
GalA
80
40
N
GalA
0
80
60
40
20
15
P2
P1
Bk
5
20
Anthoceros
Hb
10
G
Gal
R
Rha
Ara
A
60
Homogalacturonan,
Pectin
Glc
G
Gal
5
Rha
Ara
0
100
5
15
30
30
Hb
a
Ha
P2
P1
Fuc
50
Nitella
10
GalA
0
15
Glc
*
5
K
Gal
*
10
Bk
Rha
Ara
15
0
80
60
40
20
Rha
Methylglucuronoxylan,
Arabinoxylan
20
PAD response (nC per pulse)
D
Xyl
0
25
5
Ara
PAD response (nC per pulse)
10
0
0
5
10
15
20
25
30
75
80
Retention time (min)
5
10
15
20
25
30
75
80
Retention time (min)
FIG. 6. HPLC of Driselase digests of charophyte polysaccharide fractions and standard polysaccharides. Authentic polysaccharides (A–G) and various charophyte
and hornwort fractions (H–N) were digested with Driselase and the products analysed by HPLC on a Dionex PA1 column; cyan curve (Bk) ¼ blank showing
Driselase autolysis products. In D, the acidic oligosaccharides indicated by asterisks are probably MeGlcA-Xyl2 (black) and GlcA-Xyl2 (red); Driselase does not release free GlcA or MeGlcA from heteroxylans. The 779-min peak in several charophyte samples represents an unidentified acidic disaccharide. Polysaccharide
fractions analysed are ‘pectins’ (P1, P2) and hemicelluloses a and b (Ha, Hb), obtained as in Fig. 1. Curves have been arbitrarily slid up or down the y-axis so that
samples can be distinguished easily. The horizontal dashed line indicates a change in the y-axis scale.
by Driselase, indicating that they differed fundamentally from
land-plant pectins (e.g. in linkage, enantiomerism or anomerism), despite having some sugar residues in common.
Exceptions to this generalization were the GalA of
Chlorokybus, suggesting that this structurally simple and earlydiverging charophyte possesses homogalacturonan; and the Ara
of Klebsormidium ‘pectin’, suggesting that it may be present as
non-reducing terminal a-L-Ara residues, which are targeted by
232
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
Driselase in many land-plant polysaccharides (arabinoxylans
and pectic arabinogalactans).
The major sugars of Hb in Coleochaete (Gal, Glc, Ara) were
well digested to monosaccharides by Driselase, suggesting linkages reminiscent of those found in land-plant hemicelluloses. In
contrast, the major Hb residues in all other charophytes, including Chara and Nitella, were not well released as monosaccharides by Driselase – Ara again being an exception. Curiously,
the abundant Glc found in the Ha of K. fluitans, Nitella and
Chara and in Hb of Chlorokybus (and Coleochaete) was very
well released as the monosaccharide by Driselase. However,
the abundant Glc in the Hb of Chara and Nitella was
surprisingly resistant to Driselase. We conclude that the hemicelluloses of most charophytes differ starkly from those of
land-plants, Coleochaete being closest to the land-plants in this
respect.
Purification of ‘U’ from Chara AIR
The unfamiliar monosaccharide ‘U’, found in acid hydrolysates of Chara and Coleochaete AIR, migrated slightly faster
than Ara on paper chromatography in EPW (8:2:1; Fig. 5). The
yield of ‘U’ in Chara (10 mg (mg AIR)–1, estimated by paper
chromatography) approximately equalled that of fucose and exceeded that of arabinose. It stained brown with aniline hydrogen-phthalate, and, when viewed under 366-nm UV light, the
stained spot gave a bluish-white fluorescence – properties suggesting a neutral, reducing hexose, deoxyhexose or
O-methylhexose.
‘U’ was partially purified by preparative paper chromatography in three successive solvents (Supplementary Data Fig. S1)
followed by gel-permeation chromatography (GPC) on Bio-Gel
P-2 (Fig. S2). TLC confirmed an acceptable purity (Fig. S2).
On TLC, ‘U’ co-migrated with authentic 3-MeGal. In addition, purified ‘U’ was run by HPLC alone and after spiking
with authentic 3-MeGal obtained from Lycopodium. Under
these conditions, ‘U’ co-eluted with 3-MeGal, suggesting that
this was its identity (Fig. S3).
NMR spectroscopy results
The structure of ‘U’ was investigated by NMR. Signals in
the 44–54 d region of the pre-saturation and SHAKA 1D proton NMR spectra (Fig. 7) showed the presence of a- and banomers of two monosaccharides. In addition, a strong singlet
at 345 d indicated the presence of an O-methyl group. The 2D
proton–proton chemical shift spectrum (COSY) showed many
signals which corresponded to those obtained (Popper et al.,
2001) from a sample of 3-O-methyl-D-galactose. The presence
of this compound in the ‘U’ sample was confirmed as described
below (we arbitrarily assume the D-enantiomer, which is confirmed in the following section).
1. A series of selective 1D TOCSY experiments in which the
signal corresponding to H-1 of the b-anomer and in which
the mixing time was successively increased showed the
successive appearance of the signals from H-2, H-3, H-4
and H-5 (Fig. 8). These signals occurred at the expected chemical shifts for 3-O-methyl-b-D-galactose.
The coupling constants were also in agreement with those
for MeGal, thus confirming the configuration of the ring
protons and excluding the possibility of a glucose derivative (Supplementary Data Table S1).
2. A 1D-NOESY experiment in which the signal corresponding to the O-methyl protons was irradiated showed enhancement of only the H-3 and H-4 protons on the ring,
demonstrating them to be spatially close to the protons of
the O-methyl group (Fig. 9). If the O-methyl group were
on O-4 or O-6, H-3 would not be enhanced and if the Omethyl group were on O-2, H-4 would not be enhanced.
3. A 2D proton–carbon (HSQC) chemical shift correlation
spectrum gave signals in agreement with those obtained by
Popper et al. (2001) for 3-MeGal.
The identity of the other monosaccharide, present as a contaminant in ‘U’, was confirmed as arabinose from signals in the
COSY spectrum by comparison with those from an authentic
sample.
3-MeGal (‘U’) from Chara is the D-enantiomer
Both the D- and the L-enantiomers of 3-MeGal occur in polysaccharides of a red alga (Jania rubens; Navarro and Stortz,
2008), and we therefore investigated the optical isomerism of
Chara ‘U’. D-Galactose oxidase oxidizes D- but not L-Gal derivatives, including 3-MeGal. After 8 h of incubation with D-Gal
oxidase both D-Gal and methyl b-D-galactoside were oxidized
to products including galactose dialdehyde or its methyl glycoside, whereas L-Gal was unchanged after 24 h (Fig. 10). Chara
‘U’ was partially oxidized within 24 h and completely within
96 h (Fig. 10). Its susceptibility to D-galactose oxidase showed
that ‘U’ is a D-Gal derivative.
DISCUSSION
This study shows that the three ‘higher’ charophytes examined
(Coleochaete, Chara and Nitella) all possess pectic polysaccharides with sugar residue compositions broadly resembling those
of land-plant pectins, except that the Ara content is distinctly
lower in the charophytes. In contrast, the oxalate-extractable
polymers (operationally ‘pectins’) of lower charophytes are
more variable: those of the two Klebsormidium spp. studied
lack detectable GalA, and thus do not include appreciable
amounts of pectin in the generally accepted (chemical) sense of
the term. In contrast, the oxalate-extractable ‘pectins’ of
Chlorokybus, which is thought to be an extremely early-diverging charophyte, do possess GalA. Furthermore, Chlorokybus
pectin releases free GalA on digestion with Driselase, suggesting the same linkage as in land-plant pectins [4-linked a-DGalpA]. We conclude that a-D-GalpA-rich pectin is an ancient
feature of the Streptophyta, albeit secondarily lost from one order: the Klebsormidiales. Although we have not tested the
Zygnematales, an order of charophytes that may have a
particularly close affinity to the bryophytes, it may therefore be
assumed that when conquering the land 460 Mya, the first embryophytes already possessed a-D-GalpA-rich pectins – which
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
233
O-Me protons
PRESAT
Gal H-1β
Ara H-1β
Gal H-1α
Ara H-1α
SHAKA
5·0
4·5
4·0
3·5
Proton chemical shift (ppm)
FIG. 7. Pre-saturation and SHAKA proton NMR spectra of a sample containing ‘U’. These experiments were to suppress the residual water signal. Signals from
anomeric protons and from an O-methyl group are clearly visible.
H2O
1H
4H
5H
2H
3H
20 ms
40 ms
80 ms
120 ms
160 ms
400 ms
4·5
4·0
3·5
Proton chemical shift (ppm)
FIG. 8. Selective total correlation spectrum (TOCSY) of a sample containing ‘U’, showing signals from the O-methyl b-galactose spin system only. Spectra obtained
by selective excitation of the H-1 signal of the b-anomer at 459. The signals from the ring protons appear in sequence as the mixing time is increased from 20 to
400 ms.
have been retained in the primary walls of all subsequently
evolving land-plants.
Pectins of the higher charophytes, like those of land-plants
such as Anthoceros, also possess Driselase-releasable Rha, suggesting that rhamnogalacturonan-I (RG-I) may be present.
[RG-I, but not RG-II, is digestible by Driselase.] It remains to
be seen whether the charophytic RG closely resembles the
well-characterized land-plant RG-I. Speaking in favour of such
a resemblance, Driselase-releasable neutral Gal residues were
also present in the higher-charophyte pectins. The Rha and Gal
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
234
OH
OH
6
4
5
H
H3CO
2
3
H2O
H O
1
H
OH
OH
H
H
400 ms
4H
O-Me
3H
O-Me irradiated NOESY
4·5
4·0
3·5
Proton chemical shift (ppm)
FIG. 9. 1D Nuclear Overhauser spectrum (NOESY) of a sample containing ‘U’, with irradiation of the signal from the O-methyl protons. Only proton signals which
are enhanced appear in the spectrum. The signal from the irradiated protons appears strongly negative. The 400-ms 1D TOCSY spectrum showing the signals from
protons H1–H5 in 3-O-methyl b-galactose is shown for comparison.
Oxidation
product
of ‘U’
Oxidation
products
Chara ‘U’
D-
& L-Gal &
Me β-D-Gal
D-Gal
L-Gal
Methyl β-D-Gal
96 h
24 h
0h
24 h
8h
0h
24 h
8h
0h
24 h
8h
0h
Oxidation
products
Chara ‘U’
FIG. 10. Enzymic evidence that Chara U is the D-enantiomer. Samples of authentic D-galactose, L-galactose, methyl b-D-galactopyranoside and the Chara unknown
(‘U’) (4 mg of each) were incubated with D-galactose oxidase for up to 96 h. At the indicated intervals, a portion of the reaction mixture was analysed by TLC in
BAW (4 : 1 : 1). [In this TLC system, galactose does not resolve from methyl b-D-galactoside; 3-MeGal runs slightly faster.]
O’Rourke et al. — 3-O-methyl-D-galactose in charophyte pectins
residues of both lower charophytes (Klebsormidium and
Chlorokybus), in contrast, were not efficiently released by
Driselase, indicating that they were not linked through landplant RG-I-like glycosidic bonds.
We found that the pectins of the higher charophytes, especially Chara, also contained 3-MeGal residues – potentially
replacing the diminished arabinose. 3-MeGal is undetectable in
the cell walls of most land-plant taxa, a significant exception
being that it is a major wall component of lycophytes (Popper
et al., 2001), where it is thus an automorphy. 3-MeGal residues
were not detected in bryophytes (Popper et al., 2001), although
they are found taxonomically scattered across various kingdoms. For example, 3-MeGal has been found in a neutral polysaccharide extracted from the green alga Chlorella vulgaris
(Ogawa et al., 1994; 40 mg (mg hydrolysate)–1), although it
does not appear to have been reported from any other genera of
the Chlorophyta. Among the non-lycopod land-plants, 3-MeGal
has been found as a component of specific extraprotoplasmic
polymers in two lamialean dicots including an arabinogalactanprotein of sage (Salvia sp.) (Capek, 2008) and a hot-water-extracted neutral (1!4)-galactan from Acanthus ebracteatus (a
mangrove plant) which contained 26–33 mol % 3-MeGal
(Hokputsa et al., 2004) but is otherwise absent. Among basidiomycete fungi, 3-MeGal residues occur in a soluble extracellular
(1!4)-a-D-galactan from the oyster mushroom Pleurotus
ostreatoroseus (Rosado et al., 2002) and in a hot-water-extractable polysaccharide from the bracket fungus Phellinus igniarius
(Yang et al., 2009); both these fungal polysaccharides contained 33 mol % 3-MeGal. The corallinalean red seaweed
Jania rubens possesses a hot-water-extractable sulphated xylogalactan, with a repeat unit [ . . . !3)-b-D-Gal-(1!4)-a-L-Gal(1! . . . ] in which both D-Gal and L-Gal residues are partially
3-O-methylated (Navarro and Stortz, 2008). The freshwater red
alga Porphyridium also possesses small amounts of 3-MeGal in
a soluble extracellular polysaccharide (Percival and Foyle,
1979). Paulsen et al. (1992) also found an extracellular polysaccharide of a cryptophycean soil alga (Cryptomonas sp.) to possess 15 mol % 3-MeGal residues. Among animals, 3-MeGal
occurs in the haemocyanin (an N-glycosylated glycoprotein) of
certain molluscs (Staudacher, 2012); however, it does not appear to have been reported from vertebrates or from bacteria
(Staudacher, 2012).
The distribution of 3-MeGal among charophyte taxa (present
work) indicates that many of the higher charophytes possess
this sugar residue, and that it was thus quite probably present in
the pectin of the immediate charophytic ancestors of the first
land-plants. However, 3-MeGal appears to have been quickly
lost as a major wall component by the land-plants, specifically
the bryophytes (as judged by its absence from all modern liverworts, hornworts and mosses; Popper and Fry, 2003, 2004),
though retained (or reintroduced) by the lycophytes (Popper
et al., 2001). It is not known whether the 3-MeGal of lycophytes is a pectic component and thus whether it may occupy
the same molecular niche as in the charophytes. There is little
obvious physiological or anatomical resemblance between
Chara and Lycopodium, precluding any simple interpretation of
the functional significance of 3-MeGal. Indeed, the roles of 3MeGal and other O-methyl sugars in the diverse polymers of
the diverse organisms in which they occur remain a mystery
(Staudacher, 2012).
235
Hemicelluloses were extractable from all six charophytes examined, but none of these charophytes contained high concentrations of all four monosaccharide residues that typify the
major land-plant hemicelluloses (xyloglucans, heteroxylans) –
Glc, Xyl, Gal and Ara. Although further work is required to
characterize the charophytic hemicelluloses in detail, it appears
likely that abrupt changes in hemicellulose chemistry occurred
during terrestrialization.
CONCLUSIONS
Our survey shows that an acid- and Driselase-digestible a-DGalA-rich polysaccharide, presumably homogalacturonan, was
an ancient feature of the Streptophyta, albeit secondarily lost
from the Klebsormidiales. Only the ‘higher’ charophytes and
the embryophytes also possess pectic material from which
Driselase is able to release the monomers rhamnose and galactose – presumably RG-I. Thus, when conquering the land, the
first embryophytes probably already possessed homogalacturonan and RG-I. In contrast, none of the modern charophytes investigated possesses hemicelluloses that contain all four major
residues (Glc, Xyl, Gal, Ara) characteristic of xyloglucan and
arabinoxylan, indicating that major changes in the principal
hemicelluloses occurred during terrestrialization. The discovery
of 3-MeGal in higher charophyte pectins confirms that during
their long evolutionary history the Streptophyta have
experimented with numerous diverse sugar residues for the construction of their cell-wall polysaccharides. The occurrence of
3-MeGal in charophytes and lycophytes but not in a large
‘intervening’ grade of plants – the bryophytes – supports the
hypothesis that major steps in plant evolution were accompanied by notable changes in cell-wall chemistry.
SUPPLEMENTARY DATA
Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Table S1: 1H and 13C
chemical shifts and coupling constants for 3-O-methyl-a-D-galactose. Figure S1: preparative paper chromatography of sugar
‘U’ from Chara. Figure S2: removal of polysaccharide contaminants from ‘U’ as isolated by preparative paper chromatography. Figure S3: Chara ‘U’ co-elutes with authentic 3-MeGal on
HPLC.
ACKNOWLEDGEMENTS
We thank Dr Lenka Franková for valuable discussions and
Mrs Janice Miller for technical assistance. We also thank the
Leverhulme Foundation (sponsor reference F00158/CI) for
funding this work.
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