Arabinose-rich polymers as an evolutionary strategy to plasticize

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DOI 10.1007/s00425-012-1785-9
ORIGINAL ARTICLE
Arabinose-rich polymers as an evolutionary strategy to plasticize
resurrection plant cell walls against desiccation
John P. Moore • Eric E. Nguema-Ona • Mäite Vicré-Gibouin • Iben Sørensen
William G.T. Willats • Azeddine Driouich • Jill M. Farrant
•
Received: 19 June 2012 / Accepted: 11 October 2012
Ó Springer-Verlag Berlin Heidelberg 2012
Abstract A variety of Southern African resurrection
plants were surveyed using high-throughput cell wall profiling tools. Species evaluated were the dicotyledons,
Myrothamnus flabellifolia and Craterostigma plantagineum; the monocotyledons, Xerophyta viscosa, Xerophyta
schlecterii, Xerophyta humilis and the resurrection grass
Eragrostis nindensis, as well as a pteridophyte, the resurrection fern, Mohria caffrorum. Comparisons were made
between hydrated and desiccated leaf and frond material,
with respect to cell wall composition and polymer abundance, using monosaccharide composition analysis, FT-IR
spectroscopy and comprehensive microarray polymer profiling in combination with multivariate data analysis. The
data obtained suggest that three main functional strategies
J. P. Moore (&) E. E. Nguema-Ona
Institute for Wine Biotechnology, Department of Viticulture
and Oenology, Faculty of AgriSciences, Stellenbosch University,
Matieland 7602, South Africa
e-mail: [email protected]
E. E. Nguema-Ona M. Vicré-Gibouin A. Driouich
Laboratoire ‘Glycobiologie et Matrice Extracellulaire Végétale’,
Glyco-MEV, IFRMP23-PRIMACEN IBiSA, Université de
Rouen, 76821 Mont-Saint-Aignan, France
I. Sørensen W. G.T.Willats
Department of Plant Biology and Biotechnology, Faculty of Life
Sciences, University of Copenhagen, 1001 Copenhagen,
Denmark
Present Address:
I. Sørensen
Department of Plant Biology, Cornell University,
Ithaca, NY 14853, USA
J. M. Farrant
Department of Molecular and Cell Biology,
University of Cape Town, Rondebosch 7701, South Africa
appear to have evolved to prepare plant cell walls for
desiccation. Arabinan-rich pectin and arabinogalactan
proteins are found in the resurrection fern M. caffrorum and
the basal angiosperm M. flabellifolia where they appear to
act as ‘pectic plasticizers’. Dicotyledons with pectin-rich
walls, such as C. plantagineum, seem to use inducible
mechanisms which consist of up-regulating wall proteins
and osmoprotectants. The hemicellulose-rich walls of the
grass-like Xerophyta spp. and the resurrection grass
E. nindensis were found to contain highly arabinosylated
xylans and arabinogalactan proteins. These data support a
general mechanism of ‘plasticising’ the cell walls of resurrection plants to desiccation and implicate arabinose-rich
polymers (pectin-arabinans, arabinogalactan proteins and
arabinoxylans) as the major contributors in ensuring flexibility is maintained and rehydration is facilitated in these
plants.
Keywords Arabinans Arabinogalactan proteins Arabinoxylans Cell wall profiling Resurrection plants
Abbreviations
AIR
Alcohol insoluble residue
CoMPP Comprehensive microarray polymer profiling
CBM
Carbohydrate binding module
mAb
Monoclonal antibody
FT-IR
Fourier transform-infrared spectroscopy
RG
Rhamnogalacturonan
HG
Homogalacturonan
XyG
Xyloglucan
AXyG
Arabinoxyloglucan
AX
Arabinoxylan
AGP
Arabinogalactan protein
XTH
Xyloglucan transhydrolase
XET
Xyloglucan endotransglycosylase
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Introduction
Resurrection plants possess the unique property of being
able to survive dehydration of their vegetative tissues to the
air dry state, corresponding to a tissue water concentration
of or below 0.1 g H2O g-1 dry mass and a water potential
of B-100 MPa, for extended periods and to recover full
metabolic competence upon rehydration (Gaff 1971). This
group of plants encompass species from disparate lineages
(Oliver et al. 2000), including members from a number of
angiosperm families (i.e. Scrophulariaceae, Myrothamnaceae and Velloziaceae), as well as lower plant groups
including the pteridophytes, an example being the resurrection fern, Mohria caffrorum (Farrant et al. 2009).
Observation of resurrection plants dehydrating inevitably
highlights the dramatic morphological changes that
accompany desiccation, which include substantial tissue
compaction and cell collapse (Webb and Arnott 1982;
Vicré et al. 1999; Vander Willigen et al. 2003). This leads
to the obvious question as to how these plants are able to
withstand such substantial water loss and particularly the
effect on the cell wall, the cell structure responsible for
shape and support of cells and tissues, and its structural/
compositional integrity. Investigations into the cell wallassociated changes that may occur in leaf tissues due to
desiccation have been performed in relatively few resurrection plant species. Early studies on Craterostigma wilmsii have highlighted xyloglucan remodeling and calcium
ion re-distribution (Vicré et al. 1999, 2004) as important
wall responses, while in the sister species, Craterostigma
plantagineum, expansin proteins have been implicated in
improving extensibility under desiccation (Jones and
McQueen-Mason 2004). A recent study has also implicated
dehydrin proteins in effecting cell wall protection of
Polypodium polypodioides during desiccation and subsequent rehydration (Layton et al. 2010). A study of the
leaf cell wall composition of the woody resurrection plant
Myrothamnus flabellifolia suggested that constitutive protection was afforded to the wall of this species through the
presence of significant amounts of highly flexible pectinassociated arabinans and arabinogalactan proteins (Moore
et al. 2006). Biophysical studies on Eragrostis spp. implicated wall extensibility as being correlated with desiccation
tolerance, but no investigation into the polymer system(s) responsible for this was conducted (Balsamo et al.
2005, 2006). Further evidence for wall protein involvement
was observed in Boea hygrometrica, where a glycine-rich
protein is found to be up-regulated and apoplastically targeted during desiccation (Wang et al. 2009). Recently, a
transcriptomic study on C. plantagineum provided further
support for cell wall remodeling under desiccation stress
showing transcripts associated with cell wall genes, such as
the XET/XTH gene family, being differentially regulated
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in response to water stress (Suarez Rodriguez et al. 2010).
These studies provide clear evidence for cell wall
involvement in the desiccation tolerance phenomenon of
resurrection plants; however, the nature of the changes that
have been observed thus far suggests a certain degree of
species specificity in the response to desiccation. Clearly,
as tolerance to desiccation re-evolved/re-activated from the
seed genetic programming available (Illing et al. 2005;
Farrant and Moore 2011) in each of the different angiosperm families over the course of evolution, so too
remodeling, that enhanced wall plasticity, must have
occurred in the specific wall systems of each species. It is
difficult to elucidate the functionally conserved approaches
to desiccation stress and their associated cell wall changes
without understanding the nature of the evolutionary
challenges each family/species encountered in the different
angiosperm resurrection plant lineages. To address this
question, a comparative survey was conducted on a variety
of Southern African resurrection plant species using highthroughput cell wall profiling methods (see Nguema-Ona
et al. 2012) coupled to multivariate data analysis techniques. Monosaccharide composition analysis, comprehensive microarray polymer profiling (CoMPP) analysis
and fourier transform infrared spectroscopy (FT-IR) were
used in combination with chemometrics to compare cell
wall composition between species as well as between
hydration states within the same species. It was decided
to profile M. flabellifolia Welw. (Moore et al. 2007),
C. plantagineum Hochst. (Bartels 2005), Xerophyta viscosa
Baker (Mowla et al. 2004), Xerophyta schlecterii Baker,
Xerophyta humilis Baker (Illing et al. 2005) and E. nindensis Ficalho and Hiern (Vander Willigen et al. 2003) as
these would provide a good coverage of the different resurrection plant ‘types’ that occur in the angiosperms (see
Fig. 1a–g for photographs of the various resurrection plants
surveyed). Profiles were also obtained from the resurrection fern M. caffrorum Christenh. (Farrant et al. 2009) as
this species provided a good out-group (a pteridophyte
member). This also resulted in the first cell wall analysis of
this fern species, which is of interest since limited information is available on the composition of non-angiosperm
resurrection plant cell walls.
Materials and methods
Plant material
All resurrection plants used in the study were collected
from their natural habitats within South Africa and subsequently maintained in environmentally controlled glasshouse conditions at the Department of Botany, University
of Cape Town, a greenhouse as previously described
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Fig. 1 A photographic panel
showing the South African
resurrection plants surveyed in
the study: Mohria caffrorum (a),
Eragrostis nindensis (b),
Xerophyta schlecterii (c),
Xerophyta viscosa (d),
Xerophyta humilis (e),
Craterostigma plantagineum
(f) and Myrothamnus
flabellifolia (g)
(Sherwin and Farrant 1996; vander Willigen et al. 2003)
until experimentation commenced. C. plantagineum
Hochst. and X. humilis were collected from Barakalalo
National Park (Limpopo Province, South Africa), M. flabellifolia and X. schlecterii from a private farm situated
in the Vaalwater area (northwest Limpopo Province),
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X. viscosa from the Cathedral Peak Nature Reserve
(Kwazulu Natal Province) and Eragrostis nindensis was
collected from the Gamsberg area (North western Cape
Province). Mohria caffrorum plants were collected during
the dry (summer) and rainy (winter) seasons from Table
Mountain Nature Reserve (Western Cape Province). Plants
were maintained fully hydrated by regular watering until
dehydration was initiated. Relative water contents (RWC)
of leaves (or fronds) from ten individual fully turgid plants
were determined as described previously (Sherwin and
Farrant 1996). Drying was commenced by withholding of
water until whole plants had reached an air dry state
(usually at water contents B10 % RWC) after which no
further change in water content occurred. Regular monitoring of RWC, as described above, was performed until
the plants had been maintained in air dry state for 1 week.
Leaf (or frond) tissues from four to six individual plants in
the fully hydrated state, and completely air dry state, were
harvested and pooled and frozen in liquid nitrogen for
lyophilisation and further analysis as described below.
Isolation and fractionation of cell wall material
Lyophilised leaf or frond material was ground to a fine
powder, under liquid nitrogen, using a pestle and mortar.
Powdered lyophilate was suspended in boiling 80 % (v/v)
aqueous ethanol for 15 min to deactivate the enzymes
present. A series of ethanol extractions were performed to
remove pigments, alkaloids, tannins, soluble sugars and
other low molecular weight metabolites from the cell wall
containing residues. Residues were extracted for 12 h at
room temperature twice with methanol-chloroform (1:1,
v/v), twice with methanol-acetone (1:1, v/v), and finally
with acetone–water (4:1, v/v). The residue was air dried
and then destarched at 60 °C in a 50 mM acetate, pH 5.4,
buffer using a thermostable a-amylase and amyloglucosidase (EC 3.2.1.1; Megazyme International, Wicklow, Ireland). After dialysis against distilled water, cell wall
residues (alcohol insoluble residues) were freeze dried and
stored at room temperature until further use.
Composition analysis of cell wall material
A gas liquid chromatography method (York et al. 1985)
was used to determine the monosaccharide content of cell
wall residues and fractions. Approximately, 5 mg of wall
residue or fractionated material was hydrolysed (2 M TFA
(trifluoroacetic acid), 110 °C, 2 h) and the liberated
monosaccharides converted to methoxy sugars using 1 M
methanolic HCl at 80 °C for 24 h. Silylation was performed at 80 °C (20 min) to produce trimethyl-silyl-glycosides which were dissolved in cyclohexane. The
derivatives were separated and analysed in a gas
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chromatograph (5890 series II; Hewlett Packard, Palo Alto,
CA, USA) coupled to a flame ionisation detector, using a
30 m 9 0.25 mm (i.d.) HPS-MS column. The oven temperature program was stabilised at 120 °C for 2 min,
ramped at 10 °C/min to 160 °C, then at 1.5 °C/min to
220 °C and finally at 20 °C/min to 280 °C. Myo-inositol
(0.5 lmol) was used as the internal standard. Derivatives
were identified based on their retention time and quantified
by determination of their peak areas. Monosaccharides
(from Sigma-Aldrich, St. Louis, MO, USA) were used as
standards to determine the retention time of the nine main
monosaccharides found in plant cell walls. The sugar
composition was expressed as mole percentage of each
monosaccharide. Error bars in the histograms in Fig. 2
represent the standard deviation (SD) of the mean of five
biological samples with two technical replicates per biological sample.
Infrared (IR) spectroscopy of cell wall fractions
A NEXUS 670 FTIR instrument (Thermo Electron, Waltham, MA, USA) containing a Golden Gate Diamond ATR
(Attenuated Total Reflectance) accessory with a type IIa
diamond crystal was used for ATR-FT-IR measurements.
The spectra were recorded between 4,000 and 650 cm-1
with a Geon-KBr beamsplitter and DTGS/Csl detector.
Spectral data (128 co-added scans per sample) were processed using UnscramblerTM (CamoÒ Inc., Oslo, Norway).
The spectral region (600–4,000 cm-1) displayed was limited to the wall protein, lipid and carbohydrate regions
(700–2,000 cm-1). Principal component analysis of the
spectral data was performed using UnscramblerTM (CamoÒ
Inc.) with data normalised, averaged and models verified
using cross-validation.
CoMPP analysis of cell wall material
AIR was prepared as described in the ‘Isolation and fractionation of cell wall material’ section and two extraction
steps were used. First, an aqueous cyclohexanediaminetetraacetic acid (CDTA) extraction was performed on the AIR
followed by a NaOH extraction. Approximately, 10 mg of
material was used to perform the CoMPP analyses as
described in Moller et al. (2007) and extractant volumes
were adjusted for weight. Samples were printed as three
technical replicates in three concentrations, giving a total of
nine spots per sample. The heatmap was produced using
the online tool (http://cgi.snafu.de/provart/user-cgi-bin/
heatmapper.pl). The numbers represent averaged values
from two independent sets of E. nindensis, X. schlecterii,
X. humilis, X. viscosa D and H samples and four M. caffrorum D and H samples. C. plantagenium and M. flabellifolia
are represented by one set of D and H samples each. The
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highest signal in the entire data set was set to 100 and all
other data adjusted accordingly. A cut off value of 5 was
imposed. Data were converted into UnscramblerTM (CamoÒ
Inc.) format and analysed using principal component analysis (PCA) and cluster analysis (average linkage clustering,
using a Squared Euclidean distance measure). Principal
component analysis of the CoMPP data was performed using
UnscramblerTM (CamoÒ Inc.) with data normalised and
averaged and models verified using cross-validation.
Statistical and multivariate tools
All statistical analyses were performed in consultation and
collaboration with Professor Martin Kidd of the Centre for
Statistical Consultation (Stellenbosch
University).
Descriptive statistical analyses and analysis of the variance
(one way ANOVA) were performed with the statistical
package of Microsoft Excel 2010 and Statistica 10 software. All the tests were performed at P = 0.05. FT-IR
spectral and CoMPP datasets were converted into
Unscrambler software process format using the built-in
software conversion algorithms. Spectral datasets were
baseline corrected, smoothed using a Savitsky–Golay filter,
processed for multiplicative scatter correction and averaged. Principal component analysis (PCA) and cluster
analysis (average linkage clustering, using a Squared
Euclidean distance measure) were performed using
UnscramblerTM (CamoÒ Inc.).
Results
To provide an overview of the general cell wall ‘type’ (e.g.
grass-like rich in xylans or herbaceous-like rich in pectins)
of each of the resurrection plants evaluated and to determine
if dehydration resulted in wall polymer changes, a total
monosaccharide composition was performed on AIR prepared from hydrated and desiccated leaf samples (Fig. 2a–
h). Monosaccharide compositional analysis of total AIR
sourced from M. flabellifolia produced a highly similar
profile to that obtained in an in-depth leaf cell wall study
performed on this species by Moore et al. (2006). The main
monosaccharides present (Fig. 2a), Ara at ca. 25 mol %,
Xyl at ca. 15 mol %, GalUA at ca. 10 mol % and Glc at ca.
7.5 mol %, correspond to the prior study where the leaves
were shown to be composed of xylan-rich vascular tissue
and arabinan-rich pectin polymers (Moore et al. 2006). No
differences were found between hydrated and desiccated
samples supporting the proposal (Moore et al. 2006) that
this species is constitutively protected against desiccation
through the ‘plasticising’ properties (Moore et al. 2008a) of
the pectin-associated arabinans and AGPs. In contrast,
inspection of the monosaccharide profile obtained from C.
plantagineum leaves (Fig. 2b) revealed the major sugars:
GalUA at ca. 40 mol %, Glc at ca. 20 mol %, Xyl at ca.
10 mol %, Ara at ca. 10 mol %, Gal at ca. 10 mol %,
which implies a pectin-rich cell wall. This correlates to a
study performed on the related species C. wilmsii which
showed that this herbaceous resurrection plant is pectin-rich
containing an abundance of GalUA (Vicré et al. 2004). No
difference in wall composition between hydrated and desiccated states is evident for C. plantagineum (Fig. 2b),
although fractionation of C. wilmsii leaves revealed modification of XyG composition and additionally ion analysis
showed calcium re-distribution due to dehydration stress
(Vicré et al. 1999, 2004), and an in-depth analysis of
C. plantagineum leaf cell walls may, therefore, show similar desiccation-induced wall changes.
To date, no cell wall analysis has been performed on
Xerophyta spp. many of which are resurrection plants,
although unlike the homoiochlorophyllous dicotyledonous
Myrothamnus and Craterostigma spp. are grass-like monocotyledons and poikilochlorophyllous. Analysis of AIR
sourced from X. viscosa, X. schlecterii and X. humilis
(Fig. 2c–e) reveals generally similar profiles with the main
wall sugars present: Xyl at ca. 20–30 mol %, GalUA at ca.
20–25 mol %, Ara at ca. 10–15 mol % and Glc at ca.
10–20 mol %. The high Xyl and GalUA content strongly
suggests that Xerophyta spp. contain mainly xylans and
pectin polymers in equivalent abundance in their leaf walls.
An interesting difference between X. schlecterii and the
other two Xerophyta species is the higher Man content of
ca. 10 mol % (Fig. 2d) compared to less than 5 mol %
(Fig. 2c, e). The variable nature of Glc content (Fig. 2c–e)
may be due to residual co-precipitated starch which is
known to show variation in resurrection plants between
hydrated and desiccated states. Starch contents are variable
amongst hydration states in resurrection plants, with many
species mobilizing starch into sucrose (as an osmoticum)
during dehydration. The datasets showing variable Glc
levels probably reflect insufficient de-starching and reflect
known physiological phenomena in resurrection plants. No
significant differences between hydrated and desiccated
leaf composition are found for X. viscosa and X. schlecterii
(Fig. 2c, d), suggesting that these walls may be constitutively protected from desiccation. In contrast, in X. humilis,
significant differences are observed for Ara: from ca.
10 mol % hydrated to ca. 25 mol % dehydrated, and Xyl:
from ca. 20 mol % to ca. 30 mol %, (Fig. 2e) which
appears to indicate that dehydration may cause an increase
in wall arabinoxylan content and/or arabinosylation of wall
xylans in this species. A similar profile was obtained from
E. nindensis (Fig. 2f), a monocotyledon resurrection grass,
which had a leaf wall almost exclusively composed of
arabinoxylans with a major sugar composition of Ara: at
ca. 15 mol % and Xyl: at ca. 40 mol %. The comparative
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a
b
30.0
45.0
40.0
25.0
35.0
30.0
mol %
mol %
20.0
15.0
10.0
25.0
20.0
15.0
10.0
5.0
5.0
0.0
0.0
Ara
Fuc
Xyl
Man
Gal GalUA Glc GlcUA
Ara
35.0
40.0
30.0
35.0
25.0
30.0
20.0
15.0
*
Gal GalUA Glc GlcUA
*
25.0
20.0
0.0
Ara
Rha
Fuc
Xyl
Man
Gal GalUA Glc GlcUA
Ara
f
*
40.0
35.0
Rha
Fuc
Xyl
Man
Gal GalUA Glc GlcUA
50.0
40.0
*
*
mol %
mol %
Man
5.0
0.0
25.0
Xyl
10.0
5.0
30.0
Fuc
15.0
10.0
e
Rha
d
40.0
mol %
mol %
c
Rha
20.0
15.0
10.0
*
30.0
20.0
10.0
5.0
0.0
0.0
Ara
g
Rha
Fuc
Xyl
Man
h
35
*
25
25
20
20
15
*
*
Rha
Fuc
Xyl
Man
Gal GalUA Glc GlcUA
35
30
mol %
mol %
30
*
*
*
15
10
10
5
5
0
0
Ara
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Ara
Gal GalUA Glc GlcUA
Rha
Fuc
Xyl
Man
Gal GalUA Glc GlcUA
Ara
Rha
Fuc
Xyl
Man
Gal GalUA Glc GlcUA
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b Fig. 2 Monosaccharide compositional analysis of total AIR sourced
from leaves of Myrothamnus flabellifolia (a), Craterostigma plantagineum (b), Xerophyta viscosa (c), Xerophyta schlecterii (d),
Xerophyta humilis (e), Eragrostis nindensis (f), and fronds of Mohria
caffrorum (sensitive form, g) and Mohria caffrorum (tolerant form,
h). White bars represent hydrated desiccated leaves and shaded bars
represent desiccated leaves. Monosaccharide codes are for arabinose
(Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl), mannose (Man),
galactose (Gal), galacturonic acid (GalUA), glucose (Glc) and
glucuronic acid (GlcUA). Error bars in the histograms represent
the standard deviation (SD) of the mean of five biological samples
with two technical replicates per biological sample. Statistically
significant differences, based on one-way ANOVA variance testing,
are indicated on the histograms as an asterisk
data suggest a mean difference in Ara (although not statistically significant) of almost 10 mol % between hydrated
and dehydrated states (Fig. 2f). Again variable Glc contents may be related to residual starch (Fig. 2f).
The non-angiosperm resurrection fern M. caffrorum is
unusual in that it is seasonally desiccation-tolerant, i.e. it
switches from a desiccation-sensitive state during the wet
winter months and produces desiccation-tolerant vegetative
tissue during the dry summer season (Farrant et al. 2009).
Hence, it is useful to profile both ‘sensitive’ and ‘tolerant’
tissue for changes in wall composition to identify wall
adaptations that assist this species in tolerating desiccation.
A profile of AIR sourced from desiccation-sensitive fronds
of M. caffrorum (Fig. 2g) displays Ara, Xyl, Man, Gal,
GalUA and Glc as the main monosaccharides at ca.
5–10 mol %, 5–10 mol %, 22 mol %, 10–20 mol %,
5–10 mol % and 15–30 mol %, respectively. In contrast, a
profile of AIR sourced from tolerant fronds (Fig. 2h)
yielded Ara, Xyl, Man, Gal, GalUA and Glc at sugar
abundances of ca. 20–25 mol %, 10–15 mol %, 15 mol %,
7.5 mol % and 10–20 mol %, respectively. The sugar
compositions of the cell walls of both sensitive and tolerant
forms of M. caffrorum are clearly complex and more difficult to interpret in terms of polymer sources. However,
what is clear is the dramatic increase in the abundance of
Ara in the tolerant frond cell walls, an increase of
15–20 mol % from sensitive tissue (Fig. 2g, h), indicating
an important role for polymers containing this sugar in
desiccation tolerance.
Fourier transform infrared (FT-IR) spectroscopy has
been used to profile plant cell walls both in vivo (with
specialised equipment) and on processed AIR material
(Chen et al. 1998; Alonso-Simón et al. 2004). The methodology offers a rapid non-invasive and non-destructive
method to obtain data and provide insight into the underlying chemical nature of the material analysed. Spectral
data are composed of a superposition of spectral signatures
from the various polymer systems present in the AIR, and
identification of the various chemical functional groups
(and polymers that contain them) is possible through
comparison with reference data generated from isolated
polymers. The methodology has been put to use most
effectively in screening strategies to identify cell wall
mutants in plant populations by comparison with wild-type
data (Chen et al. 1998), mostly facilitated using multivariate data analysis techniques (e.g. chemometric methods
commonly PCA). FT-IR spectroscopy coupled to PCA was
used to provide an alternative method to non-invasively
scan intact AIR samples (i.e. the entire cell wall network)
sourced from resurrection plants and to allow comparison
of the samples with each other and between material from
hydrated and dehydrated plants. An inspection of the PCA
plot generated from spectral data (averaged from three
replicates) (Fig. 3), accounting for 69 % of the total variation in the dataset with PC1 and PC2 accounting for 51
and 18 % of the variation, respectively, shows clear clustering of the different resurrection plant samples. The plot
showed that M. flabellifolia, M. caffrorum (tolerant form
only), X. schlecterii, X. viscosa and E. nindensis had distinct clusters (Fig. 3) and that no dramatic differences were
evident between hydrated and dehydrated plants for these
species. This is consistent with the observation that these
species do not show cell wall compositional changes upon
desiccation. It is also interesting to note that M. flabellifolia
and E. nindensis showed distinct clustering (Fig. 3), due to
their unique wall compositions, i.e. an arabinan-rich/AGPrich pectin wall in the former and an arabinoxylan-rich wall
in the latter case. The two species C. plantagineum and
X. humilis show major differences between hydrated and
desiccated leaves (Fig. 3). The nature of these differences
is difficult to interpret from the loading plots (data not
shown), as the spectral overlaps between the different
polymer systems suggest that multiple polymer networks
contribute to driving the variation observed in the score
plot (Fig. 3). Even though loading plots are not simple for
interpretation, the advantage of this approach is that overall
wall composition and architecture can be probed in a
holistic manner giving insight into the broad functional
chemistry present. Clearly, as most of the resurrection
plants do not show substantial wholesale changes in wall
structure and composition, although the nature of the differences found for C. plantagineum and X. humilis would
be interesting to discern, more subtle influences must be at
play during desiccation. From this analyses, it is clear that
more subtle differences are involved in preparing resurrection plant cell walls to desiccation, and so more specific
techniques are needed, such as those using molecular
probes (e.g. commercial antibodies to wall glycans) to
determine structural alterations.
To complement these chemical and spectroscopic analyses and results, and to provide additional detailed independent datasets, CoMPP analysis of the different
resurrection plant AIR samples was performed. CoMPP
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Fig. 3 Principal component analysis of FT-IR spectroscopic data
generated from total AIR sourced from leaves and fronds of hydrated
and desiccated resurrection plants. Codes indicate resurrection plant
names and hydration state: M flab H: Myrothamnus flabellifolia
hydrated, M flab D: Myrothamnus flabellifolia desiccated, C plant H:
Craterostigma plantagineum hydrated, C plant D: Craterostigma
plantagineum desiccated, X vis H: Xerophyta viscosa hydrated, X vis
D: Xerophyta viscosa desiccated, X sch H: Xerophyta schlecterii
hydrated, X sch D: Xerophyta schlecterii desiccated, X hum H:
Xerophyta humilis hydrated, X hum D: Xerophyta humilis desiccated,
E nind H: Eragrostis nindensis hydrated, E nind D: Eragrostis
nindensis desiccated, M caff H: Mohria caffrorum hydrated and M
caff D: Mohria caffrorum desiccated
analysis allows the profiling and the analysis of cell wall
material using sets of monoclonal antibodies (mAbs) and
carbohydrate binding modules (CBMs) with specificities
toward plant cell wall glycan epitopes (Moller et al. 2007).
CoMPP analysis provides information about the relative
abundance of epitopes in the extracted material rather than
fully quantitative data but in contrast to fully quantitative
biochemical techniques, it can provide information about
polysaccharide rather than monosaccharide occurrence.
CoMPP analysis was performed by extracting with CDTA
(to predominantly extract pectin-type material) and NaOH
(to predominantly extract hemicellulosic polymers) from
AIR prepared from hydrated and desiccated vegetative
tissues. To provide an overview of the CDTA and NaOH
datasets, a PCA analysis was performed on each of these
data matrices (Figs. 4, 5). Inspection of the CDTA score
plot (Fig. 4a) and corresponding loading plot (Fig. 4b)
reveals distinct clusters associated with specific epitopes
present in certain AIR samples. E. nindensis, X. viscosa, X.
schlecterii and X. humilis are clustered together (Fig. 4a)
and seem to possess higher abundances of epitopes
recognised by mAbs LM1, JIM20 (both anti-extensin) LM2
(anti-AGP) and LM5 (anti-galactan). M. caffrorum positioning (Fig. 4a) corresponded to higher relative amounts
of AGPs recognised by mAbs JIM8, JIM13, MAC207 and
JIM4, and XyG recognised by mAb LM15 (Fig. 4b).
Similarly, M. flabellifolia and C. plantagineum appeared to
separate from the rest of the data samples (Fig. 4a) based
on higher relative amounts of HG epitopes (mAbs JIM5,
JIM7) arabinan epitopes (mAb LM13), AGP (mAb JIM13)
and xylan/AX epitopes (mAb LM11) mAbs (Fig. 4b). No
major differences in clustering for desiccated and hydrated
samples are evident from inspecting the CDTA score plot
(Fig. 5a). Interestingly, PCA analysis of the NaOH dataset
displayed different clustering patterns to the CDTA analysis (Figs. 4, 5). X. viscosa, X. schlecterii, X. humilis
C. plantagineum and M. caffrorum clustered in the centreleft of the score plot (Fig. 5a) and, thus, represent the
average samples in the dataset, and appears to be due to the
high relative amounts of galactans (LM5), mannans
(BS400-4), AGPs (MAC207) and extensins (JIM20, LM1)
in these plants. M. flabellifolia samples distinctly separate
123
Planta
from the main cluster (Fig. 5a) correlating with higher
relative amounts of xylan (mAb LM10), xylan/AX (mAb
LM11), arabinan (mAbs LM13 and LM6) and AGP epitopes (mAbs JIM13, LM2 and JIM8) (Fig. 5a). E. nindensis samples also cluster separately from the main sample
cluster (Fig. 5a) and this appears to be the result of higher
abundances of (1,3)-b-D-glucan (mAb BS400-2), XyG
(mAb LM15), arabinan (mAb LM6), AGP (mAb LM2) and
xylan/AX epitopes (mAb LM11) (Fig. 5b). No major differences between hydrated and desiccated states were
evident.
Further inspection of the CoMPP datasets (Figs. 4c, 5c)
allows detailed comparison of polymers present between
species and also between hydration states of the same
species. For the woody resurrection plant M. flabellifolia,
the analysis is generally confirmatory of prior studies
insofar as the CDTA extracts containing HG epitopes
(mAbs JIM5 and JIM7), arabinans (mAbs LM6 and LM13)
and AGPs (mAbs LM2, JIM4, JIM8 and JIM13) were
detected (Fig. 4c) confirming the pectin-rich nature of the
wall ‘plasticised’ by the presence of arabinans and AGPs
(Moore et al. 2006). The NaOH treatment of M. flabellifolia AIR extracted further hemicellulosic polymers with
co-extracted pectin polymers evidenced (Fig. 5c) by the
presence of arabinans (mAbs LM6 and LM13), galactans
(mAb LM5) and AGPs (mAbs LM2, JIM4, JIM8 and
JIM13) found together with XyG (mAb LM15) and fucosylated XyG (mAb CCRC-M1), xylans (mAb LM10),
AXs (mAb LM11), mannan (BS400-4), extensins (mAbs
LM1 and JIM20), (1,3)-b-D-glucan (mAb BS400-2) and
cellulose (CBM3a). This confirms previous chemical
analysis of M. flabellifolia leaves inferring all these wall
components present and additionally the binding of the
CCRC-M1 mAb concurs with prior MS analysis showing
fucosylated XyG motifs occurring in the walls (Moore
et al. 2006). No significant differences appear between
hydration states in both datasets for M. flabellifolia.
In the case of C. plantagineum, the walls are rich in
pectin supported by the abundant HG epitopes (mAbs JIM5
and JIM7) and associated arabinans chains (mAb LM6) in
the CDTA extract, but are poor in both abundance and
diversity of AGPs (mAbs LM2 and JIM13 in the CDTA
extract and mAb JIM13 in the NaOH extract) (Fig. 4c).
Interestingly, extensin epitopes (mAbs LM1 and JIM20)
are detected in CDTA extracts from AIR material (Fig. 4c).
The walls are clearly pectin-rich as evidenced by HG (mAb
JIM5), galactan (mAb LM5) and arabinan (mAb LM6) coextracted with hemicellulose polymers: mainly XyGs
(mAb LM15), mannan (mAb BS400-4), extensins (mAbs
LM1 and JIM20), (1,3)-b-D-glucan (mAb BS400-2) and
cellulose (CBM3a) using NaOH extraction (Fig. 4c). The
herbaceous nature of this species is supported by the lack
of xylans (no LM10 or LM11 epitopes detected) implying
no significant vasculature/reinforced strands (e.g. sclerenchyma) present in the leaves. No obvious differences
between hydrated and desiccated samples appear present.
The Xerophyta spp. studied show similar CDTAextractable pectin components including HGs (mAbs JIM5
and JIM7), arabinan (mAb LM6), AGPs (mAbs LM2, JIM4
and MAC207) and extensins (mAbs LM1 and JIM20)
(Fig. 4c). In addition, the AGP epitopes recognised by
mAbs JIM8 and JIM13 were present in X. humilis and the
JIM13 epitope was present in X. schlecterii CDTAextractable material (Fig. 4c). NaOH extraction liberates a
combination of ‘tightly bound’ pectin-associated material
and hemicellulosic polymers from AIR prepared from
Xerophyta spp. The pectin components include arabinan
(mAb LM6), galactan (mAb LM5) and AGP (mAb LM2)
(Fig. 5c). In addition, AGP epitopes specific to X. schlecterii include those recognised by mAbs LM2, JIM8, JIM13
and MAC207; to X. viscosa include those recognised by
mAbs LM2, JIM4 and JIM8; and to X. humilis include
those recognised by mAbs LM2 and JIM13 (Fig. 5c). The
arabinan epitope recognised by LM13 is also found in
X. schlecterii and X. humilis. XyG epitopes present in all
Xerophyta spp. include that recognised by LM15 and also,
interestingly mAb CCRC-M1, indicating fucosylated XyG
motifs are found in these species. The cellulose (recognised
by CBM3a), (1,3)-b-D-glucan (recognised by mAb BS4002) and extensin (mAbs LM1 and JIM20) epitopes are
present in all three resurrection plants (Fig. 5c). Most
remarkably, the xylan (mAb LM10) and xylan/AX (mAb
LM11) epitopes are only found in X. humilis (Fig. 5c),
indicating the probable source of the previously characterised elevated levels of Ara and Xyl found in this species
compared to X. schlecterii and X. viscosa. Additionally, the
mannan epitope recognised by BS400-4 appears unique to
X. humilis AIR (Fig. 5c). No obvious differences between
hydrated and dehydrated tissues appear evident for both
CDTA and NaOH extracts.
Certainly, the monosaccharide composition analysis of
the AIR sourced from E. nindensis suggests a significantly
different wall structure than the other resurrection plants
studied. This is supported by the CoMPP analysis which
suggests a minimal pectin component with only epitopes to
HGs (mAbs JIM5 and JIM7), arabinan (mAb LM6) and
AGP (mAb LM2) recognised (Fig. 4c). Galactans, arabinans and AGPs recognised by mAb LM5, LM6 and LM2,
respectively, are also present in the NaOH extract (Fig. 5c).
In addition to cellulose (recognised by CBM3a) and (1,3)b-D-glucan (recognised by mAb BS400-2) polymers, evidence for XyG (mAb LM15), xylan (mAb LM10), xylan/
AX (mAb LM11) and mannan (mAb BS400-4) being
present (Fig. 5c) strongly supports previous compositional
analysis. No differential binding of Abs or CBMs to
E. nindensis samples prepared from hydrated versus
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Planta
Fig. 4 Principal component
analysis of CoMPP data
generated from CDTA
extraction of total AIR sourced
from leaves and fronds of
hydrated and desiccated
resurrection plants. A score plot
is indicated (a) and associated
codes indicate resurrection plant
names and hydration state: M
flab H: Myrothamnus
flabellifolia hydrated, M flab D:
Myrothamnus flabellifolia
desiccated, C plant H:
Craterostigma plantagineum
hydrated, C plant D:
Craterostigma plantagineum
desiccated, X vis H: Xerophyta
viscosa hydrated, X vis D:
Xerophyta viscosa desiccated, X
sch H: Xerophyta schlecterii
hydrated, X sch D: Xerophyta
schlecterii desiccated, X hum H:
Xerophyta humilis hydrated, X
hum D: Xerophyta humilis
desiccated, E nind H: Eragrostis
nindensis hydrated, E nind D:
Eragrostis nindensis desiccated,
M caff H: Mohria caffrorum
hydrated and M caff D: Mohria
caffrorum desiccated. A loading
plot is indicated (b).
Comprehensive microarray
polymer profiling (CoMPP)
analysis of resurrection plant
leaf and frond cell wall fractions
from CDTA-extractable
material (c). The heatmap
shows the relative abundance of
plant cell wall glycan-associated
epitopes present in AIR and
colour intensity is correlated to
mean spot signals. Sequential
extractions were carried out
with CDTA and the extracted
material spotted onto
nitrocellulose, which was
probed with sets of antibodies
and carbohydrate binding
modules. The values in the
heatmap are mean spot signals
from three experiments and the
highest signal in the entire data
set was set to 100 and all other
data adjusted accordingly. A cut
off value of 5 was imposed
123
a
b
c
Planta
Fig. 5 Principal component
analysis of CoMPP data
generated from NaOH
extraction of total AIR sourced
from leaves and fronds of
hydrated and desiccated
resurrection plants. A score plot
is indicated (a) and associated
codes indicate resurrection plant
names and hydration state: M
flab H: Myrothamnus
flabellifolia hydrated, M flab D:
Myrothamnus flabellifolia
desiccated, C plant H:
Craterostigma plantagineum
hydrated, C plant D:
Craterostigma plantagineum
desiccated, X vis H: Xerophyta
viscosa hydrated, X vis D:
Xerophyta viscosa desiccated, X
sch H: Xerophyta schlecterii
hydrated, X sch D: Xerophyta
schlecterii desiccated, X hum H:
Xerophyta humilis hydrated, X
hum D: Xerophyta humilis
desiccated, E nind H: Eragrostis
nindensis hydrated, E nind D:
Eragrostis nindensis desiccated,
M caff H: Mohria caffrorum
hydrated and M caff D: Mohria
caffrorum desiccated. A loading
plot is indicated (b).
Comprehensive microarray
polymer profiling (CoMPP)
analysis of resurrection plant
leaf and frond cell wall fractions
from NaOH-extractable
material (c). The heatmap
shows the relative abundance of
plant cell wall glycan-associated
epitopes present in AIR and
colour intensity is correlated to
mean spot signals. Sequential
extractions were carried out
with NaOH and the extracted
material spotted onto
nitrocellulose which was probed
with sets of antibodies and
carbohydrate binding modules.
The values in the heatmap are
mean spot signals from three
experiments and the highest
signal in the entire data set was
set to 100 and all other data
adjusted accordingly. A cut off
value of 5 was imposed
a
b
c
123
Planta
dehydrated samples was observed (Fig. 5c). The pectin
extract from M. caffrorum (tolerant form) was found to be
fairly simple in composition being constituted of HGs
(mAbs JIM5 and JIM7) and AGPs (mAb sJIM4, JIM8,
JIM13 and MAC207) (Fig. 4c). Further pectin components,
HGs (mAb JIM5), galactan (mAb LM5), arabinan (mAb
LM6) and AGPs (mAbs JIM4, JIM13 and MAC207), were
also found in the NaOH extract (Fig. 5c). Hemicellulosic
polymers present include XyG (mAb LM15), fucosylated
XyG (mAb CCRC-M1) and mannan (mAb BS400-4) as
well as (1,3)-b-D-glucan (mAb BS400-2) and cellulose
(CBM3a) (Fig. 5c) supporting the compositional data previously reported. No significant differences between
hydration states could be detected using CoMPP analysis
on M. caffrorum material (Figs. 4c, 5c).
Cluster analysis was performed on the CoMPP datasets
(Fig. 6) to determine which of the two factors: species
identity or the hydration state was of greater importance in
determining cell wall composition. Cluster analysis performed on the CDTA CoMPP dataset results in a dendrogram (Fig. 6a) showing that M. caffrorum, M. flabellifolia,
C. plantagineum and X. humilis clustered mainly according
to their species status, with hydration state not playing
a major role. The other three species: E. nindensis,
X. schlecteri and X. viscosa by contrast did not show as
clear species clustering. Cluster analysis of the NaOH
CoMPP dataset confirmed that, based on broad hemicellulose components, resurrection plants clustered based on
their species status and not hydration states (Fig. 6b). As
cluster analysis involves different algorithms, the results
are not comparable to PCA analysis nor are comparisons to
‘loading variables’ possible. From this analysis, it appears
pectin components show greater variation in relation to
environmental conditions rather than NaOH-extractable
components which are conserved structural ‘hemicellulosic’ components. This would tend to imply that constitutive
protection mechanisms predominate and that resurrection
plants possess cell walls pre-adapted to survive major
fluctuations in water content.
Discussion
Desiccation tolerance of vegetative tissues is clearly a
complex phenomenon and research over the past few
decades has reinforced the realisation that multiple factors
‘act in concert’ to effect protection (Moore et al. 2009). A
range of general properties are necessary to achieve tolerance, these include molecular signalling mechanisms and
water stress signal perception, alleviation of osmotic
stresses (e.g. producing osmo-protectants), activation of
antioxidant systems, modification of photosynthesis, limiting of mechanical/membrane damage and production of
123
desiccation-protectant proteins (e.g. LEAs) (see Bartels
and Hussein 2011; Cushman and Oliver 2011; Moore and
Farrant 2012 for current reviews concerning vegetative
desiccation tolerance mechanisms in resurrection plants).
Recent studies have revealed strong evidence that desiccation tolerance re-activated/re-evolved from orthodox
seed genetic programming, which remained ‘switched on’
during germination and growth (Farrant and Moore 2011).
In the context of the current study, the role of the cell wall
is only one aspect contributing to the much more extensive
phenomenon/property of desiccation tolerance (Moore
et al. 2008a). Nevertheless, acquiring comprehensive desiccation tolerance requires the protection of the cell wall in
parallel to activating other cellular/metabolic processes
(Moore et al. 2008b). The data presented here suggest that
resurrection plants from disparate plant lineages not only
possess unique species-specific cell walls, but have also
evolved ‘wall-specific’ solutions to desiccation stress.
To summarise the main findings of this study, a series of
schematic illustrations are provided (Fig. 7), which
encapsulates the main wall adaptations found in each of the
resurrection plant species ‘types’ surveyed. In M. flabellifolia, pectin-associated arabinans and AGPs provide constitutive protection (Moore et al. 2006, 2008a), while in
Craterostigma spp. a number of inducible-responses are
observed from xyloglucan remodelling and calcium ion
deposition to expansin production (Vicré et al. 1999, 2004;
Jones and McQueen-Mason 2004; Suarez Rodriguez et al.
2010). The arabinan ‘plasticising’ hypothesis is further
strengthened by the arabinan epitopes (recognised by mAbs
LM13 and LM6) detected in M. flabellifolia tissue. The
presence of high amounts of Ara-containing polymers
suggests an interesting manner by which desiccation tolerance may have evolved in this species. Arabinans have
been shown to be present in high abundances (40 % noncellulosic sugar component) in Arabidopsis seed tissue cell
walls and upon germination these arabinans are actively
metabolised as a fuel reserve (Gomez et al. 2009). It is
tempting to speculate that these arabinans are serving a
dual function protecting the seeds against desiccation when
dehydrated (Webb and Arnott 1982) and switching to a
germination energy store during imbibition. In the case of
M. flabellifolia seeds, this catabolic breakdown of Ara
polymers is inhibited ensuring that the seedlings possess
arabinan-rich cell walls, which are able to constitutively
resist desiccation allowing for the retention of seed-encoded vegetative desiccation tolerance in this species. The
Xerophyta spp. have a generally hemicellulose-rich wall;
however, a significant amount of pectin is also present. In
this study, the data suggest that arabinosylation of xylans is
important, possibly preventing irreversible crystallisation
of xylans in muro and/or the aggregation of other polymers
due to water loss, particularly in X. humilis which shows
Planta
a
Average linkage clustering using Squared Euclidian distance
M caff H
M caff D
M flab H
M flab D
C plant H
C plant D
E nin H
X hum H
X hum D
X sch D
E nin D
X vis D
X vis H
X sch H
Relative distance
b
Average linkage clustering using Squared Euclidian distance
M flab H
M flab D
C plant H
C plant D
X hum H
X hum D
X vis H
X vis D
X sch H
X sch D
M caff H
M caff D
E nin H
E nin D
Relative distance
Fig. 6 Cluster analysis of CoMPP datasets from CDTA (a) and
NaOH (b) extractable cell wall polymers prepared from resurrection
plant leaves (fronds). Codes indicate resurrection plant names and
hydration state: M flab H: Myrothamnus flabellifolia hydrated, M flab
D: Myrothamnus flabellifolia desiccated, C plant H: Craterostigma
plantagineum hydrated, C plant D: Craterostigma plantagineum
desiccated, X vis H: Xerophyta viscosa hydrated, X vis D: Xerophyta
viscosa desiccated, X sch H: Xerophyta schlecterii hydrated, X sch D:
Xerophyta schlecterii desiccated, X hum H: Xerophyta humilis
hydrated, X hum D: Xerophyta humilis desiccated, E nind H:
Eragrostis nindensis hydrated, E nind D: Eragrostis nindensis
desiccated, M caff H: Mohria caffrorum hydrated and M caff D:
Mohria caffrorum desiccated
increased arabinose incorporation into the leaf wall due to
desiccation. Different AGP epitopes are also found in the
three Xerophyta spp. and these may contribute to the protection of pectin components against dehydration. The cell
walls of the resurrection grass E. nindensis are markedly
hemicellulose-rich and almost exclusively composed of
xylans and cellulose. Desiccation stress causes this species
to change its Ara:Xyl ratio in the favour of Ara, thereby
providing more support to the role of highly arabinosylated
xylans in protecting the wall from water-deficit damage. A
recent study has implicated arabinosylation of xylans in
improving the hydration rate and capacity of polymer
mixtures (Ying et al. 2011). The resurrection fern M. caffrorum possesses a very different cell wall profile to the
123
Planta
a
b
EXPANSIN
Ca2+
AGP
Ca2+
Ca2+
Ca2+
AGP
XG modification
AGP
AGP
Ca2+
Ca2+
Ca2+
EXPANSIN
c
d
AGP
AGP
AGP
AGP
AGP
AGP
cellulose
side chain
(arabinan)
xyloglucan
side chain
(branched arabinan)
rhamnogalcturonan I
(Rha: grey, GalA: white)
expansin
EXPANSIN
xyloglucan structural
alteration (e.g. cleavage)
saccharide (sucrose)
arabinogalactan protein
XG modification
xylan
AGP
calcium ions
Ca2+
(galacto-)mannan
Fig. 7 Schematic illustrations summarising the main cell wall
adaptations documented in the various resurrection plants surveyed.
The different resurrection plant wall ‘types’ characterised are
Myrothamnus flabellifolia (a), Craterostigma wilmsii and Craterostigma plantagineum (b), Mohria caffrorum (tolerant form, c) and
Xerophyta spp. and Eragrostis nindensis (d). The data to support
some of these models are collated from a number of papers including:
Jones et al. (2003); Moore et al. (2006, 2007, 2008a, b); Vicré et al.
(1999, 2004). A key legend identifying the different schematic objects
present in the various cell wall illustrations is provided
angiosperm species surveyed. The cell walls of this species
appear to be composed of a mixture of pectins, XyGs,
mannans and galactomannans and interestingly seems to
utilise mainly AGPs as cell wall desiccation-protectants.
The remodelling of the wall between desiccation-tolerant
and -sensitive states reinforces the importance of AGPs and
arabinans in this species which appear altered as a function
of desiccation and seasonal life cycle phase. What is
common regarding these wall responses is that they appear
to involve improving plasticity, i.e. acting as pasticizers.
The chemical definition of plasticizers involves their use as
dispersants added to polymer blends to increase the plasticity and/or fluidity of a material. In the context of cell
walls the role of these substances (e.g. as loosening agents)
as key determinants in influencing plasticity (see Beckman
1971 and Wu et al. 1988 for useful reviews on plant cell
wall plasticity) during growth and development. The role
of AGPs, key constituents of cell walls, as pectic plasticizers is reviewed in Lamport et al. (2006), where they are
shown to play a role in response to osmotic stress (i.e. salt).
If AGPs were the first ‘pectic plasticizer’ as these data
imply, then it would be tempting to speculate that in
addition to a plasticizer (mechanical stabilising function)
these wall-membrane interface proteins also had a ‘mechanosensor’ function, involved in water loss signal perception. The involvement of GRPs in resurrection plant
desiccation phenomena (Wang et al. 2009), distantly related to AGPs, might point to a functional role in the cell
wall membrane interface, currently an area of active
research (Hamann 2012). It is also tempting to speculate on
an evolutionary framework for wall adaptations, with the
first ‘solution’ involving the recruitment of AGPs and
123
Planta
pectic-arabinan incorporation into the walls of pteridophytes (M. caffrorum) and basal angiosperms (M. flabellifolia) as a remedy to repeated desiccation/rehydration
cycles. The RG-1 backbone of potato pectin has been
shown experimentally to be dependent on arabinan side
chain substitution/abundance for efficient rehydration
(Larsen et al. 2011). The development of the standard
dicotyledon pectin-rich walls necessitated a combination of
inducible factors (e.g. expansins) to be employed as is
found in C. plantagineum and C. wilmsii. The evolution
of monocotyledon grass-like (Xerophyta spp.) and grass
(E. nindensis) walls required modification of the xylan
components of these walls (i.e. via arabinosylation) to
improve solubility and prevent desiccation-induced polymer aggregation in the resurrection plant species when
dehydrating/rehydrating.
A major limitation of the current study is that this profiling approach does not give any insight into the spatial/
structural determinants that may be important in resurrection plant cell walls. The high-throughput profiling methodology (Nguema-Ona et al. 2012) performed in this study
has, however, given useful insights into which areas (i.e.
wall networks and epitopes) might be productive to invest
in, in respect of more in-depth cell wall analyses. Certainly
epitopes associated with arabinans, AGPs and arabinoxylans would be useful areas to probe, using commercially
available wall glycan antibodies, in specific types of resurrection plants as a fraction of dehydration/rehydration. A
range of wall probes and immunomicroscopy-based
methodology are available (Lee et al. 2011), which will be
used in future studies to ‘follow-up’ on the leads obtained
from the current study. Nevertheless, a clear ‘general
mechanism’ is evident, in that resurrection plant cell walls
need to be maintained in a flexible state during desiccation
and be easily rehydrated when water becomes available to
the plant. The use of Ara-containing polymers and arabinosylation of existing polymers appear to be a repeated
evolutionary strategy employed to ‘plasticise’ the cell wall
and to ensure polymer aggregation due to water loss does
not occur in these species. The data presented here also
suggest a more universal role for Ara-rich polymers (e.g.
RGII, AGP and AX) in respect of cell wall function in
relation to hydration properties in general plant growth and
development processes.
Acknowledgments Our thanks go to Borakalalo National Park for
donation of X. humilis, John and Sandy Burrows and Elizabeth Parker
(Lydenberg district) for donation of C. wilmsii and X. viscosa, and
Rupert and Tanya Baber (Waterberg district) for donation of M. flabellifolia, Professor Martin Kidd (Centre for Statistical Consultation,
Stellenbosch University) is thanked for help and discussions on statistical data analysis. We would like to thank Jonatan Fangel (University of Copenhagen, Denmark) for help with the CoMPP figure
artwork. The work was supported by grants to Jill Farrant from the
Harry Oppenheimer Trust Foundation and University of Cape Town.
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