Download Evidence for land plant cell wall biosynthetic mechanisms in

Annals of Botany 114: 1217–1236, 2014
doi:10.1093/aob/mcu171, available online at www.aob.oxfordjournals.org
RESEARCH IN CONTEXT: PART OF A SPECIAL ISSUE ON PLANT CELL WALLS
Evidence for land plant cell wall biosynthetic mechanisms in charophyte
green algae
Maria D. Mikkelsen1,*, Jesper Harholt1, Peter Ulvskov1, Ida E. Johansen1, Jonatan U. Fangel1,
Monika S. Doblin2, Antony Bacic2 and William G. T. Willats1
1
Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg, Denmark
and ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Victoria 3010, Australia
* For correspondence. E-mail [email protected]
2
Received: 4 December 2013 Returned for revision: 29 January 2014 Accepted: 8 July 2014 Published electronically: 9 September 2014
† Background and Aims The charophyte green algae (CGA) are thought to be the closest living relatives to the land
plants, and ancestral CGA were unique in giving rise to the land plant lineage. The cell wall has been suggested to be a
defining structure that enabled the green algal ancestor to colonize land. These cell walls provide support and protection, are a source of signalling molecules, and provide developmental cues for cell differentiation and elongation. The
cell wall of land plants is a highly complex fibre composite, characterized by cellulose cross-linked by non-cellulosic
polysaccharides, such as xyloglucan, embedded in a matrix of pectic polysaccharides. How the land plant cell wall
evolved is currently unknown: early-divergent chlorophyte and prasinophyte algae genomes contain a low number of
glycosyl transferases (GTs), while land plants contain hundreds. The number of GTs in CGA is currently unknown, as
no genomes are available, so this study sought to give insight into the evolution of the biosynthetic machinery of CGA
through an analysis of available transcriptomes.
† Methods Available CGA transcriptomes were mined for cell wall biosynthesis GTs and compared with GTs
characterized in land plants. In addition, gene cloning was employed in two cases to answer important evolutionary
questions.
† Key Results Genetic evidence was obtained indicating that many of the most important core cell wall polysaccharides have their evolutionary origins in the CGA, including cellulose, mannan, xyloglucan, xylan and pectin, as well as
arabino-galactan protein. Moreover, two putative cellulose synthase-like D family genes (CSLDs) from the CGA
species Coleochaete orbicularis and a fragment of a putative CSLA/K-like sequence from a CGA Spirogyra
species were cloned, providing the first evidence that all the cellulose synthase/-like genes present in early-divergent
land plants were already present in CGA.
† Conclusions The results provide new insights into the evolution of cell walls and support the notion that the CGA
were pre-adapted to life on land by virtue of the their cell wall biosynthetic capacity. These findings are highly significant for understanding plant cell wall evolution as they imply that some features of land plant cell walls evolved
prior to the transition to land, rather than having evolved as a result of selection pressures inherent in this transition.
Key words: Plant cell wall, charophyte green algae, polysaccharides, glycosyl transferases, transcriptomes,
evolution, CAZy, cellulose synthase-like, CSLA, CSLK, CSLD, Coleochaete orbicularis, Spirogyra.
IN T RO DU C T IO N
The colonization of land by the ancestral green algae was one of
the most important events in the history of life. This transition
and the subsequent explosive radiation of land plants triggered
the development of diverse ecosystems that support other life
forms and led to significant changes in atmospheric conditions.
Land colonization is thought to have occurred around 470
million years ago (Kenrick and Crane, 1997; Waters, 2003;
Niklas and Kutschera, 2010) and is believed to have occurred
only once, giving rise to a vast diversity of land plant species
(Graham, 1993; Karol et al., 2001; Lewis and McCourt, 2004;
McCourt et al., 2004; Becker and Marin, 2009).
The charophyte green algae (CGA) are considered the closest
living relatives of the land plants. The CGA include six monophyletic classes: the Mesostigmatophyceae, Chlorokybophyceae,
Klebsormidiaceae, Charophyceae, Coleochaetophyceae and
Zygnematophyceae. Mesostigmatophyceae and Chlorokybophyceae are together thought to represent the early divergent
Streptophyta (Lemieux et al., 2007; Rodriguez-Ezpeleta et al.,
2007; Timme et al., 2012). The Mesostigmatophyceae is represented by a single scaly biflaggelate species, Mesostigma
viride (Karol et al., 2001; Lemieux et al., 2007), while the
Chlorokybophyceae is represented by a single sarcinoid (nonmotile cells occurring in packages of four) species (Chlorokybus
atmophyticus) (Lemieux et al., 2007; Rodriguez-Ezpeleta et al.,
2007). The Klebsormidiaceae comprises three genera and
approx. 45 species and is believed to be the earliest divergent
class after the Mesostigmatophyceae and Chlorokybophyceae
(Timme et al., 2012). While the earliest emerging branches of
the CGA are considered phylogenetically well resolved, the
closest living relative to the land plants, thought to be in either
the Zygnematophyceae (6000 species in 50 genera), the
Coleochaetophyceae (20 species in three genera) or the
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Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
Charophyceae (81 species in six genera), has not yet been conclusively determined (Turmel et al., 2006, 2007; Becker and
Marin, 2009; Wodniok et al., 2011; Timme et al., 2012; Turmel
et al., 2013; Zhong et al., 2013). However, although no CGA
genomes have been published to date, chloroplast genome analysis as well as recent evidence from phylogenetic analysis of transcriptomic data has suggested that the Zygnematophyceae is the
closest living relative of land plants (Turmel et al., 2006, 2007;
Wodniok et al., 2011; Timme et al., 2012; Zhong et al., 2013).
The morphology of the CGAvaries from unicellular species to
species with complex multicellular body plans as in the
Charophyceae (Lewis and McCourt, 2004). Certain ancestral
CGA are thought to have possessed particular properties that
enabled only them to colonize land, although these characteristics are not necessarily in ‘plant-like’ body plans (Stebbins and
Hill, 1980; Wodniok et al., 2011). Particular cell wall architectural designs are one feature of the ancestral CGA that are
thought to have been important in conferring a pre-adaptation
to life on land. Cell walls are crucial for protection against
biotic and abiotic stress and play key roles in cell differentiation
and in the ability for upright growth of many land plants (Carpita
and Gibeaut, 1993; Graham et al., 2000; Cosgrove, 2005; Harris,
2005; Sørensen et al., 2010). Moreover, cell wall compositions
are commonly used characters informing the phylogenetic classification of algae (Stebbins, 1992; Buckeridge et al., 1999;
Graham and Wilcox, 1999; Xue and Fry, 2012).
As subtle modifications in the cell wall polymers can have profound effects on cell wall function (Niklas, 2004), the adaptations necessary for life on land probably required changes in
the chemical composition and overall architectural arrangement
of the cell walls (Tsekos 1999; Popper and Fry, 2003, 2004;
Niklas, 2004; Carafa et al., 2005; Van Sandt et al., 2007; Fry
et al., 2008a, b; Sørensen et al., 2008, 2010).
To better understand the complexities and diversity of green
plant cell walls we must understand the origins of individual
components, including the constituent polymers and the
enzyme-encoding genes responsible for their synthesis.
Considerable research has focused on the investigation of cell
wall structure and composition of terrestrial plants, in particular
flowering plants. These studies underpin our current understanding that cell walls are fibre composite materials based on a
load-bearing network of cellulose microfibrils cross-linked by
non-cellulosic polysaccharides, including xyloglucan, xylans,
mixed linkage (1 – 3), (1,4)-b-D-glucans (Bacic et al., 1988;
Fry, 2004; Scheller and Ulvskov, 2010), and probably also
pectins (Zykwinska et al., 2007). The primary cell wall of
most land plant species also contains pectins, a highly diverse
group of polysaccharides (Ridley et al., 2001). In addition to
the polysaccharides, the land plant cell wall also contains glycoproteins and in some cell types also the phenylpropanoid
polymer lignin (Boerjan et al., 2003).
The cell walls of the earliest divergent land plants to the
flowering plants all consist of similar groups of polysaccharides,
although their fine structures have undergone extensive modifications (Peña et al., 2008; Popper and Tuohy, 2010; Sørensen
et al., 2010; Popper et al., 2011). Recent work has shown that
several of the extant CGA share many cell wall components
with land plants (Popper and Fry, 2003; Domozych et al.,
2007a; Eder et al., 2008; Popper, 2008; Eder and Lütz-Meindl,
2009; Sørensen et al., 2010, 2011).
The biosynthesis of highly complex cell wall polysaccharides
and glycoproteins requires a wide array of glycosyltransferases
(GTs). To some extent these have been characterized, although
many catalytic activities and functions remain to be elucidated.
Analysis of the available moss and lycopod genomes suggests
that the families of GT-encoding genes in flowering plants are
mostly represented, although the number of GTs is usually considerably smaller (Harholt et al., 2012). Consistent with this, the
prasinophyte and chlorophyte algae, representing the earliest divergent green plants, contain a significantly lower number of
GTs in their genomes than any land plants (Ulvskov et al., 2013).
Due to the lack of a sequenced CGA genome, only a few GTs in
the CGA have been cloned or otherwise described. Here, we
provide genetic evidence of the evolution of cell wall biosynthesis in the CGA through analysis of available CGA transcriptomic data and newly cloned sequences. We show that the
CGA contain many GT sequences related to land plant genes
known to be involved in the synthesis of the highly specialized
land plant cell wall, including cellulose, mannans, xyloglucan,
xylans, pectins, arabinonogalactan proteins (AGPs) and extensins. Furthermore, we present the full-length sequences of two
cellulose synthase-like D (CSLD) proteins from Coleochaete
orbicularis and a partial sequence of a CSLA/K-like sequence
from Spirogyra sp., both presenting particularly important evolutionary steps in the cell wall biosynthesis machinery.
Together, these data suggest that many GTs involved in the biosynthesis of cell wall polysaccharides and glycoproteins of the
land plant cell wall evolved before terrestrialization of the
extant algal ancestor.
M AT E R I A L A N D M E T HO D S
Proteomes and database creation
Translated expressed sequence tag (EST) sequences were
screened for putative GT-encoding hits according to Ulvskov
et al. (2013) but with some minor modifications to allow for
quality control of relatively short ESTs. Most notably, the
CCD (NCBI’s conserved domain database), which is very
useful for quality control of full-length sequence hits, is not guaranteed to work for short sequences. The revised procedure used
was as follows. All sequences in the CAZy-database as of
September 2013 were downloaded except those that were annotated in CAZy as fragments or partial sequences. Also eliminated
were sequences regarded as outdated by NCBI. An NCBI
BLAST + database was prepared from the sequences, and used
with BLASTX at an E-value cut-off of 10 – 10. Control for false positives was a three-step procedure. The same translated ESTs were
blasted against an Arabidopsis thaliana protein blast database
(TAIR10 from www.arabidopsis.org) from which all known
GTs were subtracted. Translated ESTs giving better hits to the
GT-depleted database were eliminated. Both the top hit and the
second best hit were examined from the blast against CAZy.
‘Close in E-value’ is defined as less than 30 orders of magnitude
difference for E-values better than 10 – 100, and less than 20, 10
and 5 orders of magnitude for E-values better than 10 – 50, 10 – 20
and 10 – 10, respectively. The third level of quality control is
manual inspection of alignments of the translated EST, the bait
that pulled out the hit and selected members of the particular
clade in the CAZy-family in question. It must also be taken
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
into consideration that some impurities from other organisms can
give false positive results, although these were minimized in
many cases by more thorough investigations.
Phylogenetic analysis
Phylogenetic analysis was performed via http://www.phylo
geny.fr (Dereeper et al., 2008, 2010). The sequences were aligned using Muscle v. 3.7 with default settings. The positions with
gaps were removed and the curated sequences were used for
building maximum-likelihood phylogenetic trees using phyML
with default settings, including the WAG substitution matrix.
The phylogenetic trees were statistically supported by approximate
likelihood-ratio tests using default settings and values between
0 and 1 were obtained, as with bootstrap values. Approximate
likelihood-ratio-test (aLRT) values are included for described
clades and when values are under 0.7 where CGA sequences
are present. Additionally, aLRT values are also included in
Figure 3 and Supplementary Data Fig. S3 for low supported clustering of chlorophyte sequences.
In two instances aLRT values are not included for a clade
(Fig. 5, branch point before the E and F clade; Supplementary
Data Fig. S7, branch point before the A clade), due to very
short branches annotated with a 0.0 score that the algorithm
inserts, as it can only make strictly bifurcating trees.
For clarification, cosmetic rearrangement of the trees was
made using Adobe Illustrator. When constructing trees, CGA
sequences were chosen that span the same part of the land
plant sequences in the alignment and sequences long enough
to not change the clade structure of the land plant sequences,
while all CGA sequences are included and divided into GT families in Supplementary Data File S1. CGA sequences probably
originating from the same gene were not excluded in our analysis,
and hence one gene might be represented by more than one accession in the phylogenetic trees. Only CGA sequences long enough
to not disturb the clustering of the land plant sequences were used
to generate the trees, and alignments are included in Supplementary Data File S2.
Cloning of CSLD orthologues from C. orbicularis
The C. orbicularis culture was obtained from David
S. Domozych, Skidmore College, and was grown for 10– 14 d
in 250-mL Falcon 353136 flasks with 0.2-mm vented plugs in a
Sanyo Versatile Environment Test Chamber at 23 8C with 16 h
illumination per day from Philips master TL-D 36W/840 lamps.
The medium containing 80 % Bold Basal Medium (BBM)
(Sigma, B5282, 500 mL), 5.88 mM NaNO3, 100 mL soil extract,
1 mL vitamin mix (Sigma G1019, 50 mL), pH 6.5– 6.8, was sterilized by autoclaving. Soil extract was prepared from 200 mL
unfertilized garden soil mixed with 700 mL deionized water.
The mixture was heated to near boiling point and incubated
at 90 8C in a waterbath for 2 h and left to cool overnight. The
heat treatment was repeated and, after cooling overnight, the
extract was left for 24 h to run through a filter paper.
Cells were harvested by centrifugation and resuspended in
four volumes of BBM soil medium with 2 mg mL – 1 Drisilase
(Sigma D9515-G), 20 mM MES, pH5.5, 85 mg mL – 1 mannitol
and incubated for 30 min at room temperature. Cells were harvested by centrifugation at 4000 g for 5 min, homogenized in
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liquid nitrogen and RNAwas extracted from approx. 100 mg protoplasts using the Spectrum Plant Total RNA Kit (SigmaAldrich, St Louis, MO, USA). cDNA was synthesized using
the SuperScript Reverse Transcriptase kit and OligodT primers
according to the manufacturer’s protocol (Life Technologies,
Carlsbad, CA, USA).
Based on an alignment of CSLD sequences from A. thaliana,
Physcomitrella patens and Selaginella moellendorffii, the degenerate primers, forward: 5′ -GGIWSICAYTGGCCIGGIAC
NTGG-3′ and reverse: 5′ -GTSTTGTCYTCATACCAGCTGC-3′
were used to PCR-amplify a central 985-bp fragment of
CoCSLD. Two sequences identified among the PCR products
were named CoCSLD1 and CoCSLD2. The 5′ sequence of
CoCSLD1 and CoCSLD2 were obtained by 5′ -RACE
(Clontech SMARTER RACE, Palo Alto, CA, USA) using GSP
primers, forward: 5′ -GAGGGAGACGGGTGTCGACATCCGT
G-3′ and reverse 5′ -GCGGCAATCGCGTGTCCACCTCACTC3′ , respectively. Among C. orbicularis ESTs, GW592522
(758 bp) and GW593916 (694 bp) were identified as CSLDlike. Amplifi-cation and sequencing of the predicted open
reading frames showed that the 3′ sequence of CoCSLD1 and
CoCSLD2 corresponded to GW593916 and GW592522, respectively. The sequence of 5′ untranslated regions and complete
open reading frames were assigned GenBank accession numbers
KF928161 and KF928162, respectively.
Cloning of Spirogyra sp. CSLA/K EST
The Spirogyra sp. culture was obtained from David
S. Domozych, Skidmore College, and was grown for 10– 14 d
in Petri dishes in a Sanyo Versatile Environmental Test
Chamber at 18 8C with 16 h illumination per day from Philips
master TL-D 36W/840 lamps. The medium containing 800 mL
BBM, 5.88 mM NaNO3 and 1 mL vitamin mix (Sigma G1019,
50 mL), pH 6.8 – 7.2, was sterilized by autoclaving.
Cells were harvested and washed over a 5-mm nylon mesh
(Streno, Farum, Denmark) with sterile water and RNA extracted
from approx. 100 mg material as described above. cDNA
was also synthesized as previously described. Alignments of
translated land plant CSLA sequences were used to design degenerate primers 5′ -GTICARYTICCIATGTAYAAYGAR-3′ and
5′ -AMNGCCATRTCCATRTCYTC-3′ . A 573-bp fragment
was amplified (KF928160) from Spirogyra sp. cDNA.
R E S U LT S AN D D I S C U S S I O N
To investigate the evolutionary origins of the land plant cell wall,
available CGA transcriptomes were searched for genes related to
land plant cell wall biosynthesis (GT) genes. The available CGA
transcriptomes analysed are from different classes of CGA,
covering species from the early divergent Mesostigmatophyceae (Mesostigma viride), Chlorokybophyceae (Chlorokybus
atmophyticus) and Klebsormidiophyceae (Klebsormidium
flaccidum and Klebsormidium subtile) to species of the later divergent Charaphyceae (Nitella mirabilis, Nitella hyalina and
Chara vulgaris), Coleochaetophyceae (Chaetosphaeridium
globosum, Coleochaete orbicularis and Coleochaete scutata)
and Zygnematophyceae (Closterium peracerosum, Penium
margaritaceum and Spirogyra pratensis) (Table 1). We recognize the limitations of these data: transcriptomes reflect the
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Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
TA B L E 1. Number of GTs found in the analysed CGA transcriptomes
Class
Order
Family
Species
Reference
No. of
ESTs
No. of
GT
hits*
GTs/
ESTs
(%)
Mesostigmatophyceae
Mesostigmatales
Mesostigmataceae
Mesostigma viride
15 972
19
0.12
Chlorokybophyceae
Chlorokybales
Chlorokybaceae
12 496
158
1.3
Klebsomidiophyceae
Klebsormidiales
Klebsormidiaceae
Timme et al. (2012)
24 913
282
1.1
Klebsomidiophyceae
Charaphyceae
Klebsormidiales
Charales
Klebsormidiaceae
Characeae
Chlorokybus
atmophyticus
Klebsormidium
flaccidum
Klebsormidium subtile
Nitella mirabilis
**EC: Nedelcu et al.
(2006); **DN: Simon et al.
(2006)
Timme et al. (2012)
4827
83 522
9
419
0.19
0.5
Charaphyceae
Charaphyceae
Coleochaetophyceae
Charales
Charales
Chaetosphaeridiales
Characeae
Characeae
Chaetosphaeridiaceae
Wodniok et al. (2011)
J. H. Thierer et al.
(unpublished)
Timme et al. (2012)
Wodniok et al. (2011)
Timme et al. (2012)
40 615
13 615
24 200
176
17
165
0.4
0.12
0.7
Coleochaetophyceae
Coleochaetales
Colechaetaceae
18 386
188
1
Coleochaetophyceae
Zygnematophyceae
Coleochaetales
Desmidiales
Colechaetaceae
Closteriaceae
5346
3236
25
12
0.5
0.4
Zygnematophyceae
Desmidiales
Peniaceae
29 220
345
1.2
Zygnematophyceae
Zygnematales
Zygnemataceae
9587
168
1.8
Nitella hyalina
Chara vulgaris
Chaetosphaeridium
globosum
Coleochaete
orbicularis
Coleochaete scutata
Closterium
peracerosum
Penium
margaritaceum
Spirogyra pratensis
Timme and Delwiche
(2010)
Wodniok et al. (2011)
**AU: Sekimoto et al.
(2003); **BW: Sekimoto
et al. (2006)
Timme et al. (2012)
Timme and Delwiche
(2010)
* The number of GTs determined in this study may include false positive sequences.
** Accession number prefix.
expression profiles of the different CGA in one or at best multiple
but not all developmental stages when the RNA was extracted
and is influenced by the quality of the extraction and subsequent
sequencing. All these factors contribute to the total number of
ESTs and also the number and type of GTs identified.
Nevertheless, it was assumed that if land plant-type GT genes
were present in the CGA genomes, there would be a reasonable
chance that they would be represented in at least one of the
sampled transcriptomes.
The translated CGA transcriptomes were blasted against an
in-house database constructed from GTs extracted from the
CAZy database (www.cazy.org; Lombard et al., 2013) to identify putative candidate GTs. False positive hits were minimized,
but probably not completely eliminated, as some transcriptomic
sequences were very short. The analysed transcriptomes are presented in Table 1, including the number of sequences and GT hits
in each transcriptome, while strains and cell types are presented
in Supplementary Data Table S1. Generally, a higher number of
GT hits was found in the transcriptomes with a greater total
number of ESTs. However, the percentage of GTs still varies
considerably, from 0.12 to 1.3, i.e. over 10-fold difference, probably indicative of large differences in the data-sets, rather than
true evolutionary differences (Table 1).
The presence of a sequence with significant similarity to a land
plant GT in one or more CGA provides evidence that the evolution of the GT sequence, and hence the biosynthesis of the implicated cell wall polymer, is likely to have occurred prior to the
transition to land. The present study focuses on CGA sequences
orthologous to known land plant genes and genes identified in
selected prasinophyte algae (Ostreococcus tauri, Ostreococcus
lucimarinus and Micromonas sp. RCC299). The cell wall-related
GT hits from CGA have been divided into families in Fig. 1,
while other GT families are listed in Supplementary Data
Table S2. Only the cell wall-related GT families are discussed
in this paper and are presented in the section relevant to the
polymer they may be involved in producing.
Cellulose and cellulose synthase-like Ds
Cellulose synthases. Cellulose is the most abundant polymer in
nature and has been found not only in plants and algae but also in
bacteria, cyanobacteria and tunicates (Hess et al., 1928; Naylor
and Russell-Wells, 1934; Brown, 1985; Kimura and Itoh, 1995;
Nobels et al., 2001; Roberts et al., 2002). While the chlorophyte
algae synthesize cellulose in linear terminal complexes (TCs),
land plants and also CGA primarily synthesize cellulose in
rosette TCs, an ability probably derived from their CGA ancestor
(Tsekos, 1999; Baldan et al., 2001; Roberts et al., 2002, Roberts
and Roberts, 2007). Both K. flaccidum and C. atmophyticus have
been shown to produce only small amounts of cellulose, suggesting
that the later divergent CGA more closely resemble land plants with
respect to usage of cellulose as a major polysaccharide in the cell
wall (Sørensen et al., 2011).
In embryophytes, cellulose derived from rosettes is produced
by cellulose synthases (CESAs), members of the GT2 family.
Other distantly related GT2 CESA sequences are present in bacteria, cyanobacteria, ascomycetes, some red algae and early
evolved land plants [these are often referred to as linear
CESAs (Fangel et al., 2012; Ulvskov et al., 2013)]. In addition
to various chlorophytes, several bacteria, stramenopiles,
0
6
GT2
5
4
4
2
12
27
2
37
GT8
0
0
0
3
7
18
0
20
GT14
0
0
0
0
0
1
0
9
GT24
1
0
1
0
1
1
0
GT31
0
0
0
0
1
13
0
GT34
2
1
1
0
2
2
0
20
7
0
2
8
0
0
GT37
0
0
0
0
1
2
0
12
3
0
1
2
2
2
GT43
0
0
0
0
0
1
0
4
1
0
0
2
2
0
4
2
GT47
1
3
3
1
6
18
0
13
5
0
6
20
0
0
50
13
GT48
1
0
0
0
2
2
0
3
1
0
2
4
2
0
4
4
GT61
0
0
0
0
0
14
0
5
0
0
1
2
0
0
0
GT64
1
2
0
0
0
2
0
3
0
0
0
2
0
0
4
GT75
0
0
0
0
3
2
0
1
1
0
0
3
0
1
GT77
6
4
9
0
7
6
0
22
6
1
6
7
0
0
GT92/DUF23
0
0
0
0
1
0
0
2
0
0
0
3
0
0
19
Kobito
0
0
0
0
1
0
0
1
2
0
0
0
1
0
0
0
2
2
5
6
0
0
0
0
0
0
0
11
1
0
0
0
0
0
0
0
1
5
0
0
15 972
12 496
24 913
4827
83 522
40 615
13 615
24 200
18 386
5346
3236
29 220
9587
Bacterial‐type
CESAs
Total
9
20
5
19
22
1
3
28
14
24
10
47
42
0
1
9
8
23
14
39
42
0
0
0
1
5
2
12
11
0
0
0
1
0
1
1
1
1
8
0
0
8
5
20
16
40
33
2
2
18
8
6
8
2
9
6
6
18
10
5
2
10
4
45
27
35
39
12
7
11
13
5
3
5
25
8
1
1
5
3
3
4
2
6
4
3
5
41
4
7
4
16
19
18
13
9
8
12
C. orbicularis
10
C. globosum
0
C. vulgaris
0
N. hyalina
A. thaliana
3
O. sativa
N. mirabilis
0
S. moellendorffii
K. subtile
0
P. patens
K. flaccidum
1
S. pratensis
C. atmophyticus
0
P. margaritaceum
M. viride
0
C. peracerosum
Micromonas sp. RCC299
DUF266
C. scutata
O. lucimarinus
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O. tauri
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
1
1
4
3
18
4
29
12
10
0
9
10
0
0
3
1
3
3
0
1
14
7
0
2
F I G . 1. GT family members found in CGA transcriptomes, selected prasinophyte algae and sequenced land plants. The numbers depict how many sequences were
found in the different species and are also illustrated by the intensity of the colours. Full gene sequences from sequenced genomes are shown in yellow– orange, while
transcriptome partial sequences are shown in blue. (Only GTs discussed in the text are shown; the other GT families are shown in Supplementary Data Table S2.)
rhodophytes, glaucophytes, slime moulds, dinoflagellates and
tunicates have been found to produce cellulose using linear
TCs (Brown et al., 1976; Tsekos and Reiss, 1994; Kimura and
Itoh, 1995; Tsekos, 1999; Blanton et al., 2000; Nobles et al.,
2001; Sekida et al., 2004; Okuda and Sekida, 2007; Robert and
Robert, 2009). The bacterial-type CESAs lack domains characteristic of the rosette-forming TC CESAs including a
Zn-binding domain, a plant conserved region (P-CR) and a
hypervariable region (HVR; Pear et al., 1996; Roberts et al.,
2002; Gu and Somerville, 2010). Several lines of evidence
have been provided to suggest that these domains may be
involved in forming the rosette TC structure (Arioli et al.,
1998; Delmer, 1999; Kurek et al., 2002).
While no bacterial-type CESAs has yet been found in the few
available sequenced genomes of chlorophytes, P. patens and
S. moellendorffii possess both bacterial- and rosette-type CESAs
(Harholt et al., 2012; Ulvskov et al., 2013). Only rosette TCs
have been found in P. patens, which are presumed to produce
cellulose (Roberts et al., 2012). The bacterial-type CESAs in
S. moellendorffii and P. patens may participate in cellulose synthesis but evidence for this remains lacking. The intermediate state of
having both bacterial- and vascular plant-type CESAs is nonetheless very interesting and analysing the transcriptomes of the CGAs
shows that this transition in CESA types may date further back in
evolutionary history. The CGA transcriptomes also contain both
types of CESAs (Fig. 2, Supplementary Data Fig. S1). This is in
agreement with the previously cloned and predicted rosette
TC-forming McCESA1 (AAM83096.1) from the later divergent
CGA Mesotaenium caldariorum, showing 59 % identity to land
plant CESAs (Roberts et al., 2002).
Several bacterial-type CESA sequences were found in the
CGA transcriptomes of N. mirabilis and a low scoring sequence
in N. hyalina (JO290135; Supplementary Data Table S1).
The sequences found in N. mirabilis (JV751589, JV751588,
JV741555, JV812725, JV746415, JV762468, JV762467,
JV774960, JV774959) have low E-values (down to 6.00E-84
1222
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
CESA
Sp
0_
8
29
18
A1
JOMcCES
02_Pm
JO2057
0·7
JO257201_Kf
0·6
JO201441_Ca
JV767035_Nm,
JV767034_Nm
0·16
m
0_N m
740 2_N
6
7
4
JV
93
76
JV
CS
Co
C
LD
1
LD
S
oC
2
0·98
5
LD
S
AtC
CSLD
0·1
F I G . 2. Phylogenetic tree of the GT2 rosette-forming CESAs and CSLDs. Full-length C. orbicularis CoCSLD1 (KF928161) and CoCSLD2 (KF928162) sequences
cluster closely to the land plant CSLDs. The C. atmophyticus sequence lies ancestral to both the land plant clades CESA (green) and CSLD (brown, including
AtCSLD5), while the later divergent C. orbicularis and S. pratensis, M. caldariorum and P. margaritaceum sequences are more closely related to the land plant
sequences. CGA sequences are indicated by accession numbers following a two-letter code for the species: P. margaritaceum (Pm), S. pratensis (Sp), K. flaccidum
(Kf ), C. atmophyticus (Ca), N. mirabilis (Nm). Land plant sequences are from A. thaliana, O. sativa, P. patens and S. moellendorffi. aLRT values are shown for
major clades and for CGA containing branches when under 0.7. The scale bar is an indicator of genetic distance based on branch length.
over a maximum of 606 amino acid residues) compared with
S. moellendorffi and P. patens sequences, suggesting that the
bacterial-type CESA is present in some if not all CGA.
The rosette-type CESA sequences found in the later divergent
CGA (examples: JO182980, JO205702) share high identity with
the land plant genes, as evidenced by the cloned CESA gene
from M. caldariorum (Fig. 2, Supplementary Data Fig. S1).
Interestingly, a 162 amino acid-long sequence was found in the
earlier divergent CGA C. atmophyticus (JO201441) showing as
much similarity to land plant CESAs as to the closely related
clade of land plant CESA-like proteins, the CSLDs (Fig. 2,
Supplementary Data Fig. S1). This might indicate that the
protein is a very close relative of the ancestral CESA/CSLD
protein before it evolved into the two separate CESA and CSLD
proteins. The C. atmophyticus sequence is closely related to the
CESA/CSLDs and does not resemble the bacterial-type CESAs,
suggesting that the divergence from bacterial-type CESAs
may have occurred before the divergence of the later CGA
(Supplementary Data Fig. S1). Several similarly intermediate
CESA/CSLD sequences were found in the CGA, perhaps reflecting the evolution of the CESA/CSLD clades. The C. atmophyticus
sequence represents the earliest divergent CGA example, while a
sequence from K. flaccidum (JO257201) clusters nearer the land
plant CESAs, although more distantly than the later divergent
P. margaritaceum and S. pratensis sequences (JO205702,
JO182980), the latter closely related to the McCESA1.
CSLDs. Several land plant CSLDs have been investigated and
they have been characterized as either b-1,4-glucan synthases
(Manfield et al., 2004; Park et al., 2011) or b-1,4-mannan
synthases (Yin et al., 2011). The CSLDs have been shown to
be implicated in early cell differentiation and tip growth
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
(Bernal et al., 2007; Yin et al., 2011) and they have been found in
all land plants investigated dating back to the early divergent
mosses and lycopods (Harholt et al., 2012). Evidence has recently been provided for the likely existence of a CSLD in CGA
with three CSLD-type ESTs being identified from Coleochaete
nitellarum (Sørensen et al., 2011).
The CGA transcriptomes were also found to contain a few
sequences resembling land plant CSLDs (JO249957, JO249127,
JG446158, JO276353, JO271158, JO253400) but could not be
included in the tree due to their sequence location (Supplementary Data File S1). As the existence of CSLDs in CGA
could prove an important evolutionary step in cell wall biosynthesis, we decided to clone the full-length gene from the CGA
C. orbicularis using degenerate primers designed towards
JO249957 and JO249127. To our surprise, C. orbicularis does
not only contain one CSLD sequence, it contains at least two distinctly different genes. The encoding proteins were named
CoCSLD1 and CoCSLD2 (GenBank accession numbers
KF928161 and KF928162, respectively) and are 1310 and
1312 amino acids long with 51.6 and 50.8 % amino acid identity
to AtCSLD5 (NP_171773.1), respectively. The CoCSLDs
contain all the rosette TC CESA-specific domains, including
the Zn-binding domain, P-CR, HVR, as well as the conserved
sub-regions U1 – 4 containing the D-D-D-QXXRW motif
(Supplementary Data Fig. S2). This finding further supports
the observation by Roberts et al. (2002) that these regions are
not embryophyte-specific but rather streptophyte-specific.
The CoCSLDs cluster together, somewhat separated from the
land plant proteins, comparable to the distance the McCESA
clusters from the land plant CESAs (Fig. 2). This means that
not only did the CGAs have CESAs before land colonization,
they also possessed CSLDs (Fig. 2). Together with the sequence
found in C. atmophyticus, the evolution of the CESA/CSLD
branch of the GT2 family seems to have been somewhat resolved,
probably starting with one ancestral gene sharing resemblance to
the C. atmophyticus sequence that evolved into two distinctly different protein clades, CESA and CSLD, present in the later divergent CGA and land plants.
Mannan and xyloglucan backbone
In higher plants, the backbone of mannans and xyloglucan
(XyG) is synthesized by the CSLAs and CSLCs of the GT2
family, respectively (Dhugga et al., 2004; Liepman et al., 2005;
Cocuron et al., 2007; Goubet et al., 2009). The mannan backbone
consists of b-1,4-linked mannose that can be interspaced with
b-1,4-linked glucose in glucomannan, while the backbone of
XyG consists of b-1,4-linked glucose. Mannans have been
found throughout Viridiplantae from prasinophyte algae to flowering plants, including CGA (Morrison et al., 1993; Pettolino
et al., 2001; Dunn et al., 2007; Domozych et al., 2009b; Estevez
et al., 2009; Ordaz-Ortiz et al., 2009; Sørensen et al., 2011).
While mannan is produced by CSLAs in terrestrial plants, in
the prasinophyte and chlorophyte algae, only one ancestral
CSLK gene has been identified, which has been suggested to
encode a protein that produces mannan in these algae (Yin
et al., 2009; Fangel et al., 2012; Ulvskov et al., 2013). The
CSLKs cluster between the CSLAs and the CSLCs. The
CSLKs seem to have evolved into CSLAs and CSLCs before
the divergence of land plants from the chlorophyte algae, as
1223
P. patens and S. moellendorffii have both CSLA and CSLC but
no CSLK-like proteins (Harholt et al., 2012). As the early divergent land plants contain CSLAs and CSLCs with high sequence
identity to their counterparts in flowering plants, the split is likely
to have occurred in the CGA.
In searches of the CGA transcriptomes, some CSLC-like
sequences were identified including a sequence from
S. pratensis (JO191557) that clusters with the land plant
sequences and one full-length CSLC from Chara globularis
is available online (AY995817, Fig. 3, Supplementary Data
Fig. S3). This suggests strongly that although somewhat distantly
related to the land plants, CSLC proteins seem to have been
present in the later divergent CGA. This could imply that the
split from CSLK to CSLC and CSLA families occurred in the ancestral CGA. No CSLA-like sequences were identified in the
CGA transcriptomes, however, which counters this hypothesis.
To address the issue of whether CSLA-type sequences were
either missing from the CGA transcriptomes or are truly absent
from their genomes, we sought to clone a CSLA-like sequence
from a CGA species. We cloned a sequence encoding a 191
amino acid-long polypeptide from Spirogyra sp. (KF928160),
covering approximately one-third of the predicted full-length sequence of land plant CSLAs. The sequence was verified by comparisons to as yet unpublished CGA sequence contigs from the
1KP dataset (http://www.onekp.com/). Interestingly, the translated Spirogyra sp. sequence resembled CSLKs more than
CSLAs in an unresolved branch with a low aLRT test value of
only 0.16, and we therefore call it CSLA/K-like (Fig. 3). This
low value is due to the sequence length and position of the
CSLA/CSLK-like sequence compared with the land plant
sequences and this also results in a low resolution of the CSLK
clade. The CSLK clustering is better resolved in Supplementary
Data Fig. S3, where the analysed CGA transcriptome sequence
is around half of the land plant sequence length.
The S. pratensis transcriptome contains a sequence clustering
into the land plant CSLCs, strongly suggesting that Spirogyra
contains both a CSLC and a CSLA/K-like sequence (Supplementary Data Fig. S3). Spirogyra therefore can be considered
to represent a snapshot of evolution in a transition stage of the
CGA CSLA/K-like gene just before its evolution into a CSLA
gene. CSLCs seem to have diverged from the CSLKs faster
than the CSLAs, implying that there must have been a greater
evolutionary pressure towards the development of the CSLCs
compared with the CSLAs.
Together, these findings suggest that the CESA and CSL proteins present in the earlier divergent land plants P. patens and
S. moellendorffii were already present in CGA, although the CGA
CSLA/K-like protein is still not completely diverged towards
the CSLAs. This could further imply that the cell wall polymers
the land plant-encoding proteins produce, mannans and xyloglucan, might already have been produced to some extent by
orthologous proteins in CGA before the transition to land.
Galactomannan
The mannan backbone in galactomannan can be substituted
with galactose. A GalT from Trigonella foenum-graecum
(CAB52246.1) found in the GT34B clade has been characterized
as a galactomannan galactosyltransferase (Edwards et al., 1999)
and orthologues of this protein seem to be present in all the
1224
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
CSLC
CSLA
0·68
0·26
0·16
7_
81
5
99
AY
Cg
0·16
0·13
OlCSLK
CS
LK
CrCSLK
OtCSLK
Vc
MprC
MpCSLK SLK
pC
Ss
SLK
CsC
LK
CvCS
SL
A/
K-
lik
e
0·17
CSLK
0·1
F I G . 3. Phylogenetic tree of the GT2 CSLA, CSLC and CSLK clades. The Spirogyra sp. CSLA/K-like sequence (KF928160) clusters distantly in the CSLK clade
(brown) in an unresolved branch, while the C. globosum sequence (AAX98242) clusters ancestral to the land plant CSLC clade (orange). Land plant CSLAs are
depicted in red. Land plant sequences are from A. thaliana, O. sativa, P. patens and S. moellendorffi. Chlorophyte and prasinophyte sequences are VcCSLK:
Volvox carteri XP_002945948.1, CrCSLK: Chlamydomonas reinhardtii XP_001696519.1, MpCSLK: Micromonas pusilla XP_003064171.1, MprCSLK:
Micromonas sp. RCC299 XP_002508565.1, OtCSLK: O. tauri XP_003083844.1, OlCSLK: O. lucimarinus XP_001421850.1, CsCSLK: Coccomyxa subellipsoidea
XP_005648248.1, CvCSLK: Chlorella variabilis XP_005844039.1. aLRT values are shown for major clades and for CGA containing branches when below 0.7. The
scale bar is an indicator of genetic distance based on branch length.
terrestrial plants sequenced, although no orthologous sequences
were found in the CGA transcriptomes (Fig. 4). While it cannot
be ruled out that a gene will be revealed when a CGA genome is
fully sequenced, the lack of an orthologous GalT sequence in the
analysed CGA transcriptomes might suggest that CGA make
mannan without galactose branching. In-depth analyses of
CGA cell walls will be required to confirm this hypothesis.
Xyloglucan
XyG is a major hemicellulose in land plants and possesses a
complex side-chain arrangement that differs between species
and between tissues of the same species (Penã et al., 2008,
2012). It seems that XyG biosynthesis has undergone elaborate
diversification after the transition to land, as evidenced by the
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
_Cg
5474
JO16
1225
8_Nm
JV78867
A clade
18
JO
C clade
32
80
_S
0·
0· 68
04
p
D clade
0·75
0·62
JO
18
0·22
59
54
_S
p
m
6_N
400
4
JV7
_Nh
554
m
_N
86
8
10
m
8
N
5_
00
4
74 m
JV 1_N
45
89
7
V
JV
J
TfGalT
83
JO2
B clade
0·1
F I G . 4. Phylogenetic tree of the GT34 family. An S. pratensis sequence clusters together with land plant GT34A clade (blue), while most other CGA sequences included
cluster ancestral to the GT34B clade (brown), which includes GalT from Trigonella foenum-graecum. No CGA sequences are found in the GT34C clade (green) or the
GT34D clade (orange). Land plant sequences are from A. thaliana, O. sativa, P. patens and S. moellendorffi. aLRT values are shown for major clades and for CGA containing branches when below 0.7. Accession numbers and a two-letter code are noted on each translated CGA EST: S. pratensis (Sp), C. globosum (Cg), N. mirabilis (Nm),
N. hyalina (Nh). For layout purposes long branches are bent. The scale bar is an indicator of genetic distance based on branch length.
wide variety of side-chains identified in numerous species
(Hoffman et al., 2005; Penã et al., 2008; Hsieh and Harris,
2009; Hsieh et al., 2009).
XyG in CGA has not been detected using conventional
biochemical methods using hydrolytic enzymes and it has therefore been suggested that XyG was an embryophyte invention
(Popper and Fry, 2003). However, XyG has been detected in
many CGA species including Netrium digitus, Chara corallina,
C. nitellarum, Cosmarium turpini and Spirogyra sp. using
XyG-specific monoclonal antibodies (Ikegaya et al., 2008;
Domozych et al., 2009b; Sørensen et al., 2011). Characteristic
glucan and xylosic linkages, 4,6-Glcp, 1,4-Glcp and terminal
xylose have also been detected through methylation analysis in
Spirogyra sp. (Sørensen et al., 2011). Further evidence for
the presence of XyG in CGA has come from biochemical
activity and sequence analysis of endotransglucosylases and
endotransglucosylases/hydrolases, including an XTH EST from
C. nitellarum (HO204633.1; Van Sandt et al., 2007; Fry et al.,
2008a, b; Del Bem and Vincentz, 2010; Sørensen et al., 2011).
In addition to an ancestral CSLC in CGA, other proteins seem
to be present that in land plants are involved in the biosynthesis of
the XyG side-chains. GT family 34 contains land plant xylosyltransferases shown to add a side-chain xylose to the glucan backbone of XyG, all of which cluster in the GT34A clade (Cavalier
et al., 2008). Oryza sativa and S. moellendorffii have one and two
proteins in this clade, respectively, while P. patens has none
(Harholt et al., 2012). P. patens proteins instead cluster in the
GT34D clade, which have also been suggested to have XyG xylosyltransferase activity (Zabotina et al., 2008).
The S. pratensis transcriptome contains one sequence
(JO183280) that covers 58 % of A. thaliana xyloglucan xylosyltransferase 5 with an E-value of 1E-148 (Supplementary Data
File S1). This sequence is surprisingly not ancestral to the land
plant clade, strongly suggesting that the XyG biosynthetic machinery is present in some if not all CGA (Fig. 4).
In most land plants, the XyG side-chain can be further galactosylated and three GT47 proteins have been proposed to be involved
in this process in A. thaliana, including MUR3 and XLT2
1226
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
m
606_P m
JO213
_P
20
JO
66
30
C clade (XGD)
m
0_N
0204
JV8
m
41_N
8020
_Kf
2416 Nm
_
69
AtX
0·
69
0·4
1
JV789
JV775117_Nm +
JV775118_Nm
h
114_N
JO294
0·
89 JV At
AR
50 7
63_ _N 875 AD
46
Nh m
1
_N
m
87_Nm
122
o
_C
74
1
1 m
25 _N
de
m
JO 68
cla
_N
7
9
1
F
2
6
80 806
m Ca
a
a
JV JV8815_N 70_
0
9
_C
5_C
8
a
JV
92
50 9814 402_C
1
7
7 JO1 194
JO
9
JO 0_Sp
1
JO
344
JO18 _Pm
0
5
_Co
7
2
JO21
8171
JO23 5_Co
JO24051
JO2 JO185
97
130
54_ 2_Sp
Pm
27
13
73
JO26
80
JV
JV
9
0·93
0·7
JO3
GD
1
+ JV
B clade (ARAD)
0·87
0·9
E clade
0·4
JO212341_Pm + JO220287_Pm
e
tIRX10-lik
_Sp m
2798
JO18 4701_P
1
JO2
T1
D clade (GUT)
6_Kf
f
_K
556
254
JO
AtXU
JO25960
1·0
A clade (MUR)
0
X1
/IR
T2 Kf
GU 50_
f
40 10_K
25
6
JO O254 _Kf
J 918
RA8
53
AtIRX7/AtF
JO2
AtGUT1/A
3
UR
JO2
147
J
Pm
JO217969_P
m
1
O2
41_
9
53
JO
21
32
_
41
m
75
_P
22
90
JO
73
21
26_Pm
JO2125
m
P
1_
JO
AtXL
T2
M
At
04
_P
m
Pm
1·0
F I G . 5. Phylogenetic tree of the GT47 family. Translated CGA ESTs fall in all land plant clades in GT47, including GT47B (AtARAD1, blue), GT47A (AtMUR3,
orange), GT47D (AtGUT1 and 2, brown), GT47C (AtXGD1, red), GT47E ( purple), and GT47F (green). Accession numbers and a two-letter code are noted on each
CGA sequence: P. margaritaceum (Pm), S. pratensis (Sp), K. flaccidum (Kf ), C. atmophyticus (Ca), N. mirabilis (Nm), N. hyalina (Nh), C. orbicularis (Co). Land plant
sequences are from A. thaliana, O. sativa, P. patens and S. moellendorffi. aLRT values are shown for major clades and for CGA containing branches when below 0.7.
The scale bar is an indicator of genetic distance based on branch length.
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
S. moellendorffii, while no fucosylated XyG has been found in
P. patens, nor any CGA tested to date (Puhlmann et al., 1994;
Moller et al., 2007; Sørensen et al., 2011; Harholt et al., 2012).
Fucosylated XyG has therefore been suggested to have evolved
after the divergence of mosses and hornworts (Peña et al.,
2008; Sørensen et al., 2011).
AtFUT1 (Q9SWH5.2) from GT37 is involved in fucosylation
of XyG in A. thaliana, while other fucosyltransferases from the
GT37 clade are implicated in fucosylation of AGPs (Sarria
et al., 2001; Wu et al., 2010a). Several GT37-type encoding
sequences were found amongst the CGA ESTs [JO291477 (Nh),
JO274235 (Kf), JO190875 (Sp), JO184025 (Sp), JO183715
(Sp), JV812705 (Nm)] and these cluster together with P. patens
JV
78
68
30
_N
m
JV
80
07
45
_N
m
(NP_179627.2, NP_201028.1; Madson et al., 2003; Li et al., 2004;
Jensen et al., 2012). MUR3 and XLT2 are found in the A clade of
GT47 along with P. patens, S. moellendorffii and O. sativa
sequences (Harholt et al., 2012). XyG in A. thaliana roots can
also be branched with galactoronic acid, an activity performed
by XUT1, which also clusters into the A clade (NP_176534.2;
Peña et al., 2012). Sequences ancestral to the land plant
MUR3 clade were found in the CGA P. margaritaceum and
K. flaccidum (Fig. 5), suggesting that the machinery for the next
level of XyG side-chain biosynthesis, at least in a land plant
polymer-directed sense, could be present in CGA.
Fucosylated XyG has been shown in several seed plants including A. thaliana as well as in the seedless vascular plant
1227
AtFUT
1
0·69
0·06
JV729965_Nm
m
_N m
89 1_N
8
89 989
79
JV
V7 78
J
/
m /JV
_N Nm
8
88 0_
89 989
7
8
JV V7
J
Nm
h
_N
_K
f
77
JO
274
235
14
JO
29
05_Nm
7
JV812
0·1
p
JO183715_S
JO184025_Sp
JO1
908
75_
Sp
3_
432
S. moellendorffii
P. patens
F I G . 6. Phylogenetic tree of the GT37 family. All translated CGA ESTs fall ancestral to the FUT clade (AtFUT1, grey) and cluster together with or ancestral to
sequences from early divergent land plants (arch). Accession numbers and a two-letter codes are noted on each CGA sequence: S. pratensis (Sp), K. flaccidum
(Kf ), N. mirabilis (Nm), N. hyalina (Nh). Land plant sequences are from A. thaliana, O. sativa, P. patens and S. moellendorffi. aLRT values are shown for major
clades and for CGA containing branches when below 0.7. The scale bar is an indicator of genetic distance based on branch length.
1228
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
and S. moellendorffii sequences in another clade separate to the
O. sativa and A. thaliana sequences (Fig. 6). This might indicate
an alternative function of these divergent proteins. Such a suggestion has already been made by Harholt et al. (2012) who hypothesized them to be xylosyltransferases, although biochemical
evidence is still lacking (Moller et al., 2007; Harholt et al., 2012).
Together, these data suggest that although XyG has not yet
been detected in CGA by conventional biochemical procedures
(Popper and Fry, 2003), the CGA are likely to at least have the
genetic machinery for making xylosylated and perhaps even
galactosylated XyG comparable to terrestrial plants.
(1,3 –1,4)-b-D-Glucan
Mixed-linkage (1,3 – 1,4)-b-D-glucan (MLG) has been found
in grasses, the horsetail Equisetum arvense (Buckeridge et al.,
2004; Fry et al., 2008b; Sørensen et al., 2008), the CGA
Micrasterias denticulata (Eder et al., 2008) and several other
CGA species (Eder et al., 2008; Sørensen et al., 2011). In
grasses, MLG is synthesized by two clades of the GT2 family,
the CSLFs and CSLHs (Burton et al., 2006, 2011; Doblin
et al., 2009). None of these genes has been found in any of the
other MLG-producing plants and it has therefore been postulated
that other genes, evolved through convergent evolution, are responsible for producing MLG in these plants (Harholt et al., 2012).
Consistent with this, CGA transcriptomes lack CSLF and CSLH
sequences and like the earlier divergent land plants P. patens and
S. moellendorffii, only contain members of the CESA, CSLD,
CSLA and CSLC clades of GT2 (Harholt et al., 2012). It cannot,
however, be ruled out that complete genome sequencing of a
CGA might reveal one or more genes from other GT2 clades.
Xylan
Xylans are a major and diverse group of cell wall polysaccharides and xylan backbone biosynthesis has in several land
plant species been shown to involve a complex, reducing endoligosaccharide. This oligosaccharide might function as either
a biosynthesis primer or a terminator (York and O’Neill, 2008).
Immuno-glycan microarray analysis has indicated the presence of 4-linked xylose in several CGA, including species in
the Charophyceae, Coleochaetophyceae and Zygnematophyceae,
which was verified by methylation analysis (Morrison et al.,
1993; Domozych et al., 2009b; Sørensen et al., 2011).
Xylan biosynthesis requires many GTs, involving at least
seven different GT activities and several GT families, including
GT8, GT43 and GT47. In A. thaliana IRX8/GAUT12
(Q9FH36.1, GT8), PARVUS (Q9LN68.1; GATL clade of
GT8) and IRX7/FRA8 (Q9ZUV3.1; GT47) have all been suggested to be involved in the synthesis of the complex reducing
end of xylan (Brown et al., 2005, 2007; Lee et al., 2007; Peña
et al., 2007; York and O’Neill, 2008; Cantarel et al., 2009).
Orthologous genes have been found in the earlier divergent
land plants P. patens and S. moellendorffii, suggesting that
their origins might date back to before land colonization
(Harholt et al., 2012). In the CGA transcriptomes, there are
many sequences sharing similarities to all GAUT clades in
GT8 (Fig. 7) including several sequences related to AtIRX8/
AtGAUT12 (JO271878, JV740401, JV799913, JO283871).
The latter suggests that synthesis of the complex reducing end
of xylan might pre-date colonization of land. In addition, a
couple of distantly related GATL-encoding ESTs (JO257712,
JV745132) are also present in CGA (data not shown). The
GATL sequences in CGA seem more distantly related to the
flowering plant proteins; however, this is not surprising, as this
is also the case for the GATL-related S. moellendorffii sequence
(Harholt et al., 2012).
Elongation of the xylan backbone has been suggested to be
performed by the IRX9 (Q9ZQC6.1, GT43), IRX14 (Q8L707.1,
GT43), GUT2/IRX10 (ABF58973.1, GT47) and GUT1/IRX10like (Q940Q8.1, GT47) proteins in A. thaliana (Brown et al.,
2005, 2007, 2009; Lee et al., 2007; Penã et al., 2007; York
and O’Neill, 2008; Cantarel et al., 2009; Wu et al., 2009,
2010b). Orthologous genes are also present in P. patens and
S. moellendorffii, except for IRX9, where S. moellendorffii
only has an IRX9-like protein (Harholt et al., 2012).
A C. orbicularis sequence (JO238317) falls nicely into the
GT43B clade with the land plant IRX14 and IRX14-like
sequences (Supplementary Data Fig. S4). A sequence from
S. pratensis (JO191580) clusters near the IRX9-like land plant
sequences, although a longer sequence is necessary to make
any conclusions (139 amino acids compared with 394 amino
acids of IRX9H (Q9SXC4.2) from A. thaliana). In CGA, the
GT47D clade, including the IRX7/FRA8 branch, the GUT1/
IRX10-like and GUT2/IRX10 branch, is represented by long
sequences with high identity to the land plant sequences
(Fig. 5). In the GUT clade, as in some of the other phylogenetic
trees, a branch point has a low aLRT value, indicating that the
CGA sequences cluster ancestral to the land plant sequences
but that the internal phylogeny between the CGA sequences
cannot be fully resolved. Glucuronic and methyl-glucuronic
acid are added to xylan by the GT8 GUX-type glucuronosyltransferases (Mortimer et al., 2010; Oikawa et al., 2010). Several
sequences from CGA cluster at the base of the GUX clade,
as in the P. patens and S. moellendorffii GUX-like proteins
(Supplementary Data Fig. S5). As the GUX clade also contains
starch initiation proteins (PGSIP6, which are not included in the
tree; Lao et al., 2003; Chatterjee et al., 2005), the CGA-encoded
proteins may together with the P. patens and S. moellendorffii
proteins be involved in starch initiation and not xylan biosynthesis,
although this needs further investigation.
In the GT61 family, the GT61C clade has been shown to be
involved in side-chain addition to xylan (Anders et al., 2011;
Chiniquy et al., 2012). While the earlier divergent land plants
species under investigation in this study do not contain
members of the GT61C clade, K. flaccidum contains a sequence
(JO255459) that lies ancestral to both GT61A and GT61C, but it
is not of sufficient length to determine the phylogenetic relationship between JO255459 and GT61A and GT61C (data not
shown). Only full-length or near to full-length sequences will
enable us to further analyse whether a true GT61C clade
protein exists in CGA. In S. pratensis, JO186429 shows high sequence identity (4.00E-38 over 115 amino acids) to the land plant
GT61A clade, although no function has yet been determined for
members of this clade (Supplementary Data Files S1, S2).
Furthermore, several of the CGA species analysed contain
sequences similar to those in the GT61B clade that are involved
in N-glycosylation in land plants (Strasser et al., 2000).
In summary, parts of or perhaps the whole xylan biosynthesis
machinery was already present in CGA, suggesting that xylan
21
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Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
0·1
F I G . 7. Phylogenetic tree of GAUT from the GT8 family. Translated CGA ESTs fall inside land plant GAUT-B (AtGAUT8, red) and GAUT-C (AtGAUT12, brown)
clades, while other CGA sequences lie ancestral to the GAUT-A clade (AtGAUT1 and AtGAUT7, blue). Accession numbers and a two-letter code are noted on each
CGA EST: S. pratensis (Sp), K. flaccidum (Kf), N. mirabilis (Nm), N. hyalina (Nh), C.orbicularis (Co), C. globosum. Land plant sequences are from A. thaliana,
O. sativa, P. patens and S. moellendorffi. aLRT values are shown for major clades and for CGA containing branches when below 0.7. The scale bar is an indicator
of genetic distance based on branch length.
biosynthesis in its complexity may also very well pre-date the
transition to land.
Pectin
Pectins are matrix polysaccharides that play important roles
such as controlling cell wall porosity and calcium complexations
in land plant cell walls. Pectins consist of three domains, homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II). The biosynthesis of pectin is not well
understood, with only four different types of verified GTs and
one putative activity identified to date (Atmodjo et al., 2013).
Complex formation and GTs with apparent lack of catalytic activity also complicate elucidation of pectin biosynthesis
(Atmodjo et al., 2011; Harholt et al., 2012). Despite these difficulties, activities or putative activities have been found for all the
major pectin components except the RG-I backbone.
In higher plants, the most abundant pectin polymer is HG. HG
has been found in the later divergent CGA orders Charophyceae,
Coleochaetophyceae and Zygnematophyceae (Domozych et al.,
2009a; Sørensen et al., 2011) and is abundant in species such as
P. margaritaceum and C. corallina (Proseus and Boyer, 2006;
Domozych et al., 2007b; Sørensen et al., 2011), where it forms
complexes with calcium, as in land plants (Sørensen et al.,
2011). The degree of methyl esterification has been found to
vary in P. margaritaceum and N. digitus (Popper and Fry,
2003; Domozych et al., 2007b; Eder and Lütz-Meindl, 2009;
Sørensen et al., 2011).
The GAUT family members of GT8 have been shown to synthesize HG in land plants (Sterling et al., 2006). A. thaliana has
15 GAUT homologues with single mutants displaying varying
phenotypes, but all are believed to be galacturonosyltransferases
with the majority involved in HG biosynthesis (Atmodjo et al.,
2013).
A total of 37 CGA sequences orthologous to GAUT were
found, including 17 in the phylogenetic tree shown in Fig. 7,
the earliest occurrence being observed in K. flaccidum
(JO253621, JO254590, JO271878). Not all CGA sequences
1230
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
were of sufficient quality and length to be included in the phylogenetic tree but enough are present to obtain meaningful
information.
The most thoroughly characterized GAUT activity is encoded
by GAUT1 (Q9LE59.1; GAUT-A clade) from A. thaliana
(Sterling et al., 2006), orthologues of which were identified
from several CGA including C. orbicularis, C. globosum and
K. flaccidum (JO248621, JO160886, JO253621); this is in agreement with an already released GAUT1 orthologous EST from
C. nitellarum (Sørensen et al., 2011). GAUT1 functions in a
complex with GAUT7 (Q9ZVI7.2, GAUT-A clade) in A. thaliana
(Atmodjo et al., 2011). Only very ancestral GAUT7 orthologues
were identified in CGA, supporting the suggestion by Harholt
et al. (2012) that the anchoring function of GAUT7 is specific
for later divergent land plants. No clear orthologues of
GAUT2– GAUT7 (GAUT-A) were found in CGA, but some ancestral sequences cluster at the base of these sequences
(JO191147, JV741764, JO292792). CGA sequences group in
the remaining clades, including QUA1/GAUT8 (Q9LSG3.1,
GAUT-B clade) and GAUT12 (GAUT-C) (Bouton et al.,
2002; Orfila et al., 2004; Persson et al., 2007), consistent with
previous findings of a GAUT8 EST in C. nitellarum (Sørensen
et al., 2011).
HG can be xylosylated to produce xylogalacturonan. This
xylosylation is catalysed by XGD1 (Q94AA9.2) in A. thaliana
and which is found in a specific part of clade GT47C (Jensen
et al., 2008). No clear orthologues of this specific part of
GT47C were identified in the CGA transcriptomes (Fig. 5) and
as neither S. moellendorffii nor P. patens has more than ancestral
members of GT47C, xylosylation of HG, or at least the proliferation of members of GT47C, appears to be a late embryophyte
invention (Harholt et al., 2012).
RG-I in terrestrial plants can have various side-chains, most
notably arabinans and galactans. The existence of arabinans
and galactans in earlier divergent taxa of the green plant
lineage has not been thoroughly investigated. Short linear
stretches of arabinosyl residues have, however, been shown to
be present in C. corallina cells through immunolabelling with
an arabinan-specific antibody (Domozych et al., 2009b). The
protein synthesizing the RG-I backbone has not yet been identified, while ARAD1 (NP_850241.1) and GalS1 (AAP68307.1)
from A. thaliana have been suggested to be arabinan and galactan
side-chain synthases, respectively (Harholt et al., 2006; Liwanag
et al., 2012). ARAD1 is found in clade GT47B and similar CGA
sequences were found to be present in the analysed transcriptomes from K. flaccidum and N. mirabilis (JO262416,
JV801369, respectively) (Fig. 5). ARAD1 is the only characterized GT47B member and some CGA sequences cluster in the
other sub-clades of GT47B (Fig. 5). In addition, two CGA
sequences from N. mirabilis lie ancestral to the GT47B clade
(JV802040, JV802041).
The galactan synthase GalS1 is located in clade GT92/DUF23.
We cannot find a clear distinction between GT92 and DUF23 and
have therefore collapsed these two families into one. In this screen,
we found CGA sequences that are ancestral to the higher plant
GalS sequences, clustering close to one S. moellendorfii and two
P. patens sequences (Supplementary Data Fig. S6).
The GT65 protein ECTOPICALLY PARTING CELLS 1
(EPC1; NP_191142.1) from A. thaliana has also been suggested
to be involved in galactan synthesis because knock-out mutants
show decreased galactan content (Singh et al., 2005; Bown et al.,
2007). However, EPC1 orthologues have been found in
Galdieria sulphuraria, a red alga that contains no galactan in
its wall. It therefore seems unlikely that EPC1 is directly involved
in galactan biosynthesis at least in red algae (Ulvskov et al.,
2013). EPC1-like sequences are present in the analysed CGA
transcriptomes (data not shown) but a function cannot be suggested for them as yet.
The distinct structure of RG-II shows a great degree of conservation between embryophyte species, despite the recent finding
of minor differences in size and methylation patterning (Pabst
et al., 2013). RG-II is believed to be a land plant feature, emerging at least partly in mosses and has not been detected in
green algae (Domozych et al., 1980; Becker et al., 1994, 1998;
Matsunaga et al., 2004; Sørensen et al., 2011). Moss RG-II has
not been purified and structurally characterized, but some diagnostic sugars have been identified (Matsunagu et al., 2004).
Some unusual sugars of RG-II are present in algae, including
2-keto-3-deoxyoctonate (KDO) (York et al., 1985) and
3-deoxy-2-heptulosaric acid (DHA) (Becker et al., 1994, 1998;
Domozych et al., 1991, 1992), the former found in the scales
or theca of prasinophyte species and the latter in the scales of
M. viride (York et al., 1985; Becker et al., 1991; Domozych
et al., 1991). Furthermore, low levels of 3,4-linked GalA, an
RG-II-specific sugar linkage, has been found in C. nitellarum
(Sørensen et al., 2011).
One RG-II biosynthetic activity has been identified in
A. thaliana to date, the xylosylation reaction performed by
RGXT from the GT77B clade (Egelund et al., 2006, 2008).
While three or four RGXT homologues are found in
A. thaliana, only a single RGXT orthologue is found in
O. sativa, P. patens and S. moellendorffii (Egelund et al., 2006,
2008; Harholt et al., 2012). Surprisingly, we found 34 RGXT
CGA sequences in total (at least 14 N. mirabilis sequences lie ancestral to GT77B), including two in the earlier divergent
K. flaccidum (JO269496, JO255157; Fig. 8). This finding
could point towards a gradual evolution of RG-II, starting in
CGA and not in mosses as previously thought. The high
number of CGA sequences found could, however, indicate that
the RGXTs in CGA are not involved in RG-II synthesis, as
only a quantitatively minute amount of RG-II is produced in
mosses (Fry, 2011). We propose that RGXT was recruited
from the CGA ancestor for making RG-II in embryophytes but
whether its role in the CGA is making a polysaccharide unrelated
to RG-II, or what might be termed an evolutionary precursor of
RG-II, needs further investigation.
Extensins and AGPs
The family of hydroxyproline (Hyp)-rich glycoproteins comprises several sub-classes of structural and chimeric proteins
(Showalter et al., 2010). The latter kind includes, for example,
the plasma membrane-localized extensin-like receptor kinases
that may be involved in cell wall signalling (Bai et al., 2012).
These are not dealt with here; only the glycosylation enzymes
of the two overlapping sub-classes, extensins and AGPs, were
analysed. Both extensins and AGPs are non-enzymatic cell
wall proteins with characteristic patterns of glycosylation.
Extensins feature single Gal residues a-linked to Ser or Thr
and short arabinan side-chains linked in the b-configuration to
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
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F I G . 8. Phylogenetic tree of the GT77 family. Translated CGA ESTs fall inside the land plant GT77A (blue) and GT77D (green) clades, while other CGA sequences
lie more ancestral to the GT77B (brown) and GT77C (AtXEG113, orange) clades and the uncharacterized last land plant clade. Accession numbers and a two-letter
code are noted on each CGA sequence: P. margaritaceum (Pm), S. pratensis (Sp), K. flaccidum (Kf), C. atmophyticus (Ca), N. mirabilis (Nm), N. hyalina (Nh),
C. orbicularis (Co). Land plant sequences are from A. thaliana, O. sativa, P. patens and S. moellendorffi. aLRT values are indicated for major clades and for branches
with CGA sequences when below 0.7. The scale bar is an indicator of genetic distance based on branch length.
hydroxyproline (Kieliszewski et al., 2011). The glycans of AGPs
are quite complex and no AGP-glycan structure has yet been
fully elucidated. Two glycan models have been proposed, one
quite compact and suggestive of a repeating building block
(Tan et al., 2012) and one more extended model without repeating motifs (Tryfona et al., 2012). Both feature a b-1,3-galactan
linked to Hyp, but the latter model provides evidence for
extended b-1,6-linked side chains while the b-1,6-links are
merely single residue kinks in the former. The galactans are
decorated with rhamnose, arabinofuranose (Araf ) and methylglucuronic acid residues.
Whether Hyps are glycosylated by AGP or extensin-like
glycan structures is determined by the context in which the
Hyp is situated in the amino acid sequence of the protein.
Single Hyps carry AGP-type glycans and contiguous Hyps are
preferentially arabinosylated (Tan et al., 2003). Extensins quite
1232
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
similar to vascular plant extensins, albeit with a somewhat richer
set of glycan structures, are found as the major constituent of the
cell wall of the chlorophyte alga Chlamydomonas reinhardtii
(Bollig et al., 2007). This finding and the presence of orthologous
extensin biosynthetic genes in the prasinophytes indicate that
extensins are ancestral to Viridiplantae (Ulvskov et al., 2013).
The biosynthesis of extensins is rather well understood in
A. thaliana, where SGT1 encoded by At3g01720 galactosylates
Ser or Thr residues (Saito et al., 2014). The GT that adds the innermost Ara is unknown, but the next b-1,2-linked Arafs are
believed to be transferred by XEG113 (NP_850250.1) from the
C clade of GT77 and the third Araf by the REDUCED
RESIDUAL ARABINOSE proteins (RRAs) of GT77 clade A
(Egelund et al., 2007; Gille et al., 2009; Velasquez et al.,
2011; Fig. 8). The GT that adds the fourth Araf residue, which
is linked in a-1,3 is presently unknown. CGA sequences
similar to the three known GTs that glycosylate extensin were
identified [JV747861 (Nm), JV77 4710 (Nm), JO197897 (Ca),
JO210666 (Pm), JO158654 (Cg), JO192987 (Ca), JO255878
(Kf ); Fig. 8]. This is in good agreement with the omnipresence
of extensins throughout Viridiplantae.
Glycosylation that results in the arabinogalactan structures of
AGPs is far from being elucidated. It is generally believed to
involve GTs from families GT31, GT14 and GT14-like. The
plant members of the GT14-like family were formerly known
as DUF266 s and we find this discrimination useful for functional annotation and thus retain the term DUF266.
GT31 enzymes all appear to transfer hexosyl monosaccharides, but linkage and acceptor vary (Narimatsu, 2006; Strasser
et al., 2007; Egelund et al., 2011; Basu et al., 2013; Geshi
et al., 2013). At least two A. thaliana GT31 proteins are involved
in AGP biosynthesis, namely GALT2 (NP_193838.2) and
GALT31A (NP_174569.1), adding the first galactose residue
to Hyp and extending the 1,6-galactose side-chains, respectively
(Basu et al., 2013; Geshi et al., 2013). As GALT1 (Q8L7F9.1) is
involved in N-glycosylation, it cannot be concluded that all
Viridiplantae GT31 are involved in AGP biosynthesis and
therefore only orthologues to the two known AGP activities are
discussed.
GALT2 is found in GT31 clade B (Supplementary Data Fig.
S7) and two short CGA sequences (JO246062, JO214276, 136
and 149 amino acids, respectively) with good sequence similarity to this cluster of higher plant sequences were identified, but
which are too short to include in phylogenetic analyses. The
second known activity, GALT31A, is located in GT31 clade A
and two long CGA sequences cluster closely in the same clade
(Fig. S7, JO249593, JO185579, 323 and 355 amino acids, respectively).
GT14 is involved in AGP biosynthesis via the glucuronosyltransferase activity of GlcAT14 (NP_198815.1, Knoch et al.,
2013). No clear orthologues to GlcAT14 were found in the
CGA transcriptomes, although some ancestral GT14 sequences
were identified (Supplementary Data Fig. S8), suggesting that
this family of proteins is present in CGA. Based on mutant
studies in O. sativa, BC10 (ABN72585.1) was identified as a putative AGP biosynthetic GT (Zhou et al., 2009). BC10 features a
DUF266 domain which is closely related to GT14 enzymes
(Hansen et al., 2012). At least one sequence with good similarity
(with 42 % identity and an E-value of 4e-78) to BC10 can be
identified in C. globosum (JO162810; Supplementary Data
Fig. S9), indicating that BC10, as opposed to GlcAT14, has an
evolutionary origin in CGA or earlier.
CON CLU DI NG REMA RKS
CGA occupy an especially significant position in the tree of life
between basal green algae and terrestrial plants. Our transcriptomic analysis of cell wall biosynthetic genes traces evolution
in three distinct ways: (1) genes encoding GTs involved in extensin biosynthesis form an unbroken sequence of homologous
genes at the base of Viridiplantae and are also found in the
most divergent chlorophyte algae; (2) bacterial-type CESA
genes are probably inherited from prokaryotes, yet the evolutionary lineage seems broken as it is absent from sequenced prasinophytes, chlorophytes and glaucophytes but appears in N. mirabilis,
a later divergent CGA, as well as in earlier divergent land plants;
and finally (3) we observe an explosion of polysaccharide biosynthetic genes in the CGAs representing the genetic complement
required to synthesize the major polysaccharide classes found in
walls of vascular plants.
These findings are highly significant for understanding plant
cell wall evolution as they imply that some features of land
plant cell walls evolved prior to the transition to land, rather
than having evolved as a result of selection pressures inherent
in this transition. Indeed, it is possible that the ability to synthesize such walls was an aspect of the pre-adaptation that could
explain in part why the ancestors of the CGA and not other
algae gave rise to the land plant lineage.
S U P P L E M E N TARY D ATA
Supplementary data are available online at www.aob.oxford
journals.org and consist of the following. Table S1: description
of trancriptomes analysed, including strains and growth stages.
Table S2: GT family members found in CGA transcriptomes,
selected prasinophyte algae and sequenced land plants. Fig.
S1: phylogenetic tree of GT2 rosette-forming CESA, CSLD
and bacterial-type CESAs. Fig. S2: alignment of CoCSLDs,
CESAs, CSLDs and bacterial-type CESAs. Fig. S3: phylogenetic tree of GT2 CSLA, CSLC and CSLK clades. Fig. S4: phylogenetic tree of GT43. Fig. S5: phylogenetic tree of the GUX
clade of GT8. Fig. S6: phylogenetic tree of GT92/DUF23. Fig.
S7: phylogenetic tree of GT31. Fig. S8: phylogenetic tree of
GT14. Fig. S9: phylogenetic tree of DUF266. Data file S1:
GTs found in the analysed CGA transcriptomes. Data file S2:
sequence alignments from the presented phylogenetic trees.
ACK N OW L E DG E M E N T S
Special thanks to Professor David S. Domozych for the algal starter
cultures used for cloning the full-length Coleochaete orbicularis
CoCSLD sequences and the Spirogyra sp. CSLA/K-like fragment.
We thank Karin Olesen for expertise and technical assistance with
growing the algal cultures. We also thank the 1KP initiative (http://
www.onekp.com/) for allowing us to verify the cloned Spirogyra
sp. CSLA/K-like sequence in their large transcriptomic data
sets. M.S.D. and A.B wish to acknowledge the support of
the Australian Research Council for funding to the ARC
Centre of Excellence in Plant Cell Walls (CE11000010007). J.H.
was supported by the Villum Foundation’s Young Investigator
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
Programme. M. D. M. was supported by The Danish Council for
Strategic Research (101597).
LIT E RAT URE CITED
Anders N, Wilkinson MD, Lovegrove A, et al. 2011. Glycosyl transferases in
family 61 mediate arabinofuranosyl transfer onto xylan in grasses.
Proceedings of the National Academy of Sciences of the USA. 109:
989– 993.
Arioli T, Peng L, Betzner AS, et al. 1998. Molecular analysis of cellulose biosynthesis in Arabidopsis. Science 279: 717– 720.
Atmodjo MA, Sakuragi Y, Zhu X, et al. 2011. Galacturonosyltransferase
(GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic
homogalacturonan:galacturonosyltransferase complex. Proceedings of the
National Academy of Sciences of the United States of America 13:
20225–20230.
Atmodjo MA, Hao Z, Mohnen D. 2013. Evolving views of pectin biosynthesis.
Annual Review of Plant Biology 64: 747– 779.
Bacic A, Harris PJ, Stone BA. 1988. Structure and function of plant cell walls. In
Preiss J, ed. The biochemistry of plants. New York: Academic Press,
297– 371.
Bai L, Zhang GZ, Zhou Y, et al. 2012. Plasma membrane-associated
proline-rich extensin-like receptor kinase 4 a novel regulator of Ca2+ signalling is required for abscisic acid responses in Arabidopsis thaliana. Plant
Journal 60: 314 –327.
Baldan B, Andolfo P, Navazio L, Tolomio A, Mariani P. 2001. Cellulose in
algal cell wall: an ‘in situ’ localization. European Journal of
Histochemistry 45: 51– 56.
Basu D, Liang Y, Liu X, et al. 2013. Functional identification of a
hydroxyproline-o-galactosyltransferase specific for arabinogalactan
protein biosynthesis in Arabidopsis. Journal of Biological Chemistry 288:
10132–10143.
Becker B, Marin B. 2009. Streptophyte algae and the origin of embryophytes.
Annals of Botany 103: 999–1004.
Becker B, Becker D, Kamerling JP, Melkonian M. 1991. 2-Keto-sugar acids in
green flagellates: a chemical marker for prasinophycean scales. Journal of
Phycology 27: 498–504.
Becker B, Marin B, Melkonian M. 1994. Structure, composition, and biogenesis of prasinophyte cell coverings. Protoplasma 181: 233–244.
Becker B, Melkonian M, Kamerling JP. 1998. The cell wall (theca) of
Tetraselmis striata (Chlorophyta): macromolecular composition and structural elements of the complex polysaccharides. Journal of Phycology 34:
779– 787.
Bernal AJ, Jensen JK, Harholt J, et al. 2007. Disruption of ATCSLD5 results in
reduced growth, reduced xylan and homogalacturonan synthase activity and
altered xylan occurrence in Arabidopsis. Plant Journal 52: 791– 802.
Blanton RL, Fuller D, Iranfar N, Grimson MJ, Loomis WF. 2000. The cellulose synthase gene of Dictyostelium. Proceedings of the National Academy
of Sciences of the USA 97: 2391–2396.
Boerjan W, Ralph J, Baucher M. 2003. Lignin biosynthesis. Annual Review of
Plant Biology 54: 519–546.
Bollig K, Lamshoeft M, Schweirner K, Marner FJ, Budzikiewicz H. 2007.
Structural analysis of linear hydroxyproline-bound O-glycans of
Chlamydomonas reinhardtii – conservation of the inner core in
Chlamydomonas and land plants. Carbohydrate Research 342: 2557– 2566.
Bouton S, Leboeuf E, Mouille G, et al. 2002. QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis. Plant Cell 14: 2577–2590.
Bown L, Kusaba S, Goubet F, et al. 2007. The ectopically parting cells 1–2
(epc1–2) mutant exhibits an exaggerated response to abscisic acid.
Journal of Experimental Botany 58: 1813–1823.
Brown RM Jr. 1985. Cellulose microfibril assembly and orientation: recent
developments. Journal of Cell Science Supplement 2: 13– 32.
Brown RM Jr, Willison MJH, Richardson CL. 1976. Cellulose biosynthesis in
Acetobacter xylinum: visualization of the site of synthesis and direct measurement of the in vivo process. Proceedings of the National Academy of
Sciences of the USA 73: 4565– 4569.
Brown DM, Zeef LAH, Ellis J, Goodacre R, Turner SR. 2005. Identification of
novel genes in Arabidopsis involved in secondary cell wall formation using
expression profiling and reverse genetics. Plant Cell 17: 2281–2295.
1233
Brown DM, Goubet F, Wong VW, et al. 2007. Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis.
Plant Journal 52: 1154– 1168.
Brown DM, Zhang ZN, Stephens E, Dupree P, Turner SR. 2009.
Characterization of IRX10 and IRX10-like reveals an essential role in glucuronoxylan biosynthesis in Arabidopsis. Plant Journal 57: 732–746.
Buckeridge MS, Vergara CE, Carpita NC. 1999. The mechanism of synthesis
of a mixed-linkage (13),(14)b-D-glucan in maize. Evidence for multiple sites of glucosyl transfer in the synthase complex. Plant Physiology
120: 1105– 1116.
Buckeridge MS, Rayon C, Urbanowicz B, Tine MAS, Carpita NC. 2004.
Mixed linkage (13), (14)-b-D-glucans of grasses. Cereal Chemistry
81: 115–127.
Burton RA, Wilson SM, Hrmova M, et al. 2006. Cellulose synthase-like CslF
genes mediate the synthesis of cell wall (1,3;1,4)-beta-D-glucans. Science
311: 1940– 1942.
Burton RA, Collins HM, Kibble NAJ, et al. 2011. Over-expression of specific
HvCslF cellulose synthase-like genes in transgenic barley increases the
levels of cell wall (1,3;1,4)-b-D-glucans and alters their fine structure.
Plant Biotechnology Journal 9: 117– 135.
Carafa A, Duckett JG, Knox JP, Ligrone R. 2005. Distribution of cell-wall
xylans in bryophytes and tracheophytes: new insights into basal interrelationships in land plants. New Phytologist 168: 231–240.
Carpita NC, Gibeaut DM. 1993. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties
of the walls during growth. Plant Journal 3: 1 –30.
Cavalier DM, Lerouxel O, Neumetzler L, et al. 2008. Disrupting two
Arabidopsis thaliana xylosyltransferase genes results in plants deficient in
xyloglucan, a major primary cell wall component. Plant Cell 20:
1519– 1537.
Chatterjee M, Berbezy P, Vyas D, Coates S, Barsby T. 2005. Reduced expression of a protein homologous to glycogenin leads to reduction of starch
content in Arabidopsis leaves. Plant Science 168: 501–509.
Chiniquy D, Sharma V, Schultink A, et al. 2012. XAX1 from glycosyltransferase family 61 mediates xylosyltransfer to rice xylan. Proceedings of the
National Academy of Sciences of the USA 109: 17117– 17122.
Cocuron JC, Lerouxel O, Drakakaki G, et al. 2007. A gene from the cellulose
synthase-like C family encodes a beta-1,4 glucan synthase. Proceedings of
the National Academy of Sciences of the USA 104: 8550– 8555.
Cosgrove D. 2005. Growth of the plant cell wall. Nature Reviews. Molecular Cell
Biology 6: 850– 861.
Del Bem LE, Vincentz MG. 2010. Evolution of xyloglucan-related genes in
green plants. BMC Evolutionary Biology 10: 341–388.
Delmer DP. 1999. Cellulose biosynthesis: exciting times for a difficult field of
study. Annual Review of Plant Physiology and Plant Molecular Biology
50: 245–276.
Dereeper A, Guignon V, Blanc G, et al. 2008. Phylogeny.fr: robust phylogenetic
analysis for the non-specialist. Nucleic Acids Research 36: W465– W469.
Dereeper A, Audic S, Claverie JM, Blanc G. 2010. BLAST-EXPLORER
helps you building datasets for phylogenetic analysis. BMC Evolutionary
Biology 10: 8.
Dhugga KS, Barreiro R, Whitten B, et al. 2004. Guar seed b-mannan synthase
is a member of the cellulase synthase super gene family. Science 303:
363–366.
Doblin MS, Pettolino FA, Wilson SM, et al. 2009. A barley cellulose synthaselike CSLH gene mediates (1,3;1,4)-beta-D-glucan synthesis in transgenic
Arabidopsis. Proceedings of the National Academy of Sciences of the
United States of America 106: 5996–6001.
Domozych DS, Stewart KD, Mattox KR. 1980. The comparative aspects of cell
wall chemistry in the green algae (Chlorophyta). Journal of Molecular
Evolution 15: 1–12.
Domozych DS, Wells B, Shaw PJ. 1991. Basket scales of the green alga
Mesostigma viride: chemistry and ultrastructure. Journal of Cell Science
100: 397–407.
Domozych DS, Wells B, Shaw PJ. 1992. Scale biogenesis in the green alga,
Mesostigma viride. Protoplasma 167: 19–32.
Domozych DS, Elliott L, Kiemle SN, Gretz MR. 2007a. Pleurotaenium trabecula, a desmid of wetland biofilms: the extracellular matrix and adhesion
mechanisms. Journal of Phycology 43: 1022–1038.
Domozych DS, Serfis A, Kiemle SN, Gretz MR. 2007b. The structure and biochemistry of charophycean cell walls: I. Pectins of Penium margaritaceum.
Protoplasma 230: 99–115.
1234
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
Domozych DS, Lambiasse L, Kiemle SN, Gretz MR. 2009a. Cell wall development and bipolar growth in the desmid Penium margaritaceum
(Zygnematophyceae, Streptophyta). Asymmetry in a symmetric world.
Journal of Phycology 45: 879– 893.
Domozych DS, Sørensen I, Willats WGT. 2009b. The distribution of cell wall
polymers during antheridium development and spermatogenesisin the
Charophycean green alga, Chara corallina. Annals of Botany 104:
1045–1056.
Dunn EK, Shoue DA, Huang X, et al. 2007. Spectroscopic and biochemical
analysis of regions of the cell wall of the unicellular ‘mannan weed,’
Acetabularia acetabulum. Plant and Cell Physiology 48: 122–133.
Eder M, Lütz-Meindl U. 2009. Analyses and localization of pectin-like carbohydrates in cell wall and mucilage of the green alga Netrium digitus.
Protoplasma doi/10.1007/s00709– 009–0040–0.
Eder M, Tenhaken R, Driouich A, Lütz-Meindl U. 2008. Occurrence and characterisation of arabinogalactan-like proteins and hemicelluloses in
Micrasterias (Streptophyta). Journal of Phycology 44: 1221–1234.
Edwards ME, Dickson CA, Chengappa S, Sidebottom C, Gidley MJ, Reid JS.
1999. Molecular characterisation of a membrane-bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis. Plant Journal 191:
691–697.
Egelund J, Petersen BL, Motawia MS, et al. 2006. Arabidopsis thaliana
RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II. Plant
Cell 18: 2593– 2607.
Egelund J, Obel N, Ulvskov P, et al. 2007. Molecular characterization of two
Arabidopsis thaliana glycosyltransferase mutants, rra1 and rra2, which
have a reduced residual arabinose content in a polymer tightly associated
with the cellulosic wall residue. Plant Molecular Biology 64: 439–451.
Egelund J, Damager I, Faber K, Ulvskov P, Petersen BL. 2008. Functional
characterisation of a putative rhamnogalacturonan II specific xylosyltransferase. FEBS Letters 582: 3217–3222.
Egelund J, Ellis M, Doblin M, Qu Y, Bacic A. 2011. Genes and enzymes of the
GT31 family: towards unravelling the function(s) of the plant glycosyltransferase family members. Annual Plant Reviews 41: 213 –234.
Estevez JM, Fernández PV, Kasulin L, Dupree P, Ciancia M. 2009. Chemical
and in situ characterization of macromolecular components of the cell walls
from the green seaweed Codium fragile. Glycobiology 19: 212–228.
Fangel JU, Ulvskov P, Knox JP, et al. 2012. Cell wall evolution and diversity.
Frontiers in Plant Science 3: 152.
Fry SC. 2004. Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells. New Phytologist 161: 641–675.
Fry SC. 2011. Cell wall polysaccharide composition and covalent crosslinking.
In Ulvskov P, ed. Plant polysaccharides, biosynthesis and bioengineering.
Oxford: Blackwell, 1 –42.
Fry SC, Mohler KE, Nesselrode BHWA, Franková L. 2008a. Mixed-linkage
b-glucan: xyloglucan endotransglucosylase, a novel wall-remodeling
enzyme from Equisetum (horsetails) and charophytic algae. Plant Journal
55: 240– 252.
Fry SC, Nesselrode BH, Miller JG, Mewburn BR. 2008b. Mixed-linkage
(13,14)-b-D-glucan is a major hemicellulose of Equisetum (horsetail)
cell walls. New Phytologist 179: 104– 115.
Geshi N, Johansen JN, Dilokpimol A, et al. 2013. Galactosyltransferase acting
on arabinogalactan protein glycans is essential for embryo development in
Arabidopsis. Plant Journal 76: 128– 137.
Gille S, Hänsel U, Ziemann M, Pauly M. 2009. Identification of plant cell wall
mutants by means of a forward chemical genetic approach using hydrolases.
Proceedings of the National Academy of Sciences of the USA 106:
14699– 14704.
Goubet F, Barton CJ, Mortimer JC, et al. 2009. Cell wall glucomannan in
Arabidopsis is synthesised by CSLA glycosyltransferases, and influences
the progression of embryogenesis. Plant Journal 60: 527–538.
Graham LE. 1993. Origin of land plants. New York: Wiley.
Graham LE, Wilcox L. 1999. Algae. San Francisco: Benjamin Cummings.
Graham LE, Cook ME, Busse JS. 2000. The origin of plants: body plan changes
contributing to a major evolutionary radiation. Proceedings of the National
Academy of Sciences of the USA 97: 4535–4540.
Gu Y, Somerville C. 2010. Cellulose synthase interacting protein A new factor in
cellulose synthesis. Plant Signaling and Behavior 5: 1571–1574.
Hansen SF, Harholt J, Oikawa A, Scheller HV. 2012. Plant glycosyltransferases beyond CAZy: a perspective on DUF families. Frontiers in Plant
Science 3: 59.
Harris PJ. 2005. Diversity in plant cell walls. In Henry RJ, ed. Plant diversity and
evolution: genotypic and phenotypic variation in higher plants.
Wallingford, UK: CAB International Publishing, 201– 227.
Harholt J, Jensen JK, Sørensen SO, Orfila C, Pauly M, et al. 2006.
ARABINAN DEFICIENT 1 is a putative arabinosyltransferase involved
in biosynthesis of pectic arabinan in Arabidopsis. Plant Physiology 140:
49–58.
Harholt J, Sørensen I, Fangel J, et al. 2012. The glycosyltransferase repertoire
of the spikemoss Selaginella moellendorffii and a comparative study of its
cell wall. PLOS ONE 7: e35846.
Hess K, Haller R, Katz JR. 1928. Die Chemie der Zellulose und Ihrer Begleiter.
Leipzig: Akademische Verlagsgesellschaft.
Hoffman M, Jia Z, Penã MJ, et al. 2005. Structural analysis of xyloglucans in
the primary cell walls of plants in the subclass Asteridae. Carbohydrate
Research 115: 1826–1840.
Hsieh YS, Harris PJ. 2009. Xyloglucans of monocotyledons have diverse structures. Molecular Plant 2: 943– 965.
Hsieh YSY, Paxton M, Ade CP, Harris PJ. 2009. Structural diversity, functions
and biosynthesis of xyloglucans in angiosperm cell walls. NZ Journal of
Forest Science 39: 187– 196.
Ikegaya H, Hayashi T, Kaku T, Iwata K, Sonobe S, Shimmen T. 2008.
Presence of xyloglucan-like polysaccharide in Spirogyra and possible involvement in cell–cell attachment. Phycological Research 56: 216– 222.
Jensen JK, Sørensen SO, Harholt J, Geshi N, Sakuragi Y, et al. 2008.
Identification of a xylogalacturonan xylosyltransferase involved in pectin
biosynthesis in Arabidopsis. Plant Cell 20: 1289–1302.
Jensen JK, Schultink A, Keegstra K, Wilkerson CG, Pauly M. 2012.
RNA-Seq analysis of developing nasturtium seeds (Tropaeolum majus):
identification and characterization of an additional galactosyltransferase
involved in xyloglucan biosynthesis. Molecular Plant 5: 984.
Karol KG, McCourt RM, Cimino MT, Delwiche CF. 2001. The closest living
relatives of land plants. Science 294: 2351– 2353.
Kenrick P, Crane PR. 1997. The origin and early evolution of plants on land.
Nature 389: 33–39.
Kieliszewski MJ, Lamport DTA, Tan L, Cannon MC. 2011.
Hydroxyproline-rich glycoproteins: form and function. In Ulvskov P, ed.
Plant polysaccharides, biosynthesis and bioengineering. Oxford:
Blackwell 321–342.
Kimura S, Itoh T. 1995. Evidence for the role of the glomerulocyte in cellulose
synthesis in the tunicate Metandrocarpa uedai. Protoplasma 186: 24–33.
Knoch E, Dilokpimol A, Tryfona T, et al. 2013. A b-glucuronosyltransferase
from Arabidopsis thaliana involved in biosynthesis of type II arabinogalactan has a role in cell elongation during seedling growth. Plant Journal
doi:10.1111/tpj.12353.
Kurek I, Kawagoe Y, Jacob-Wilk D, Doblin M, Delmer DP. 2002.
Dimerization of cotton fiber cellulose synthase catalytic subunits occurs
via oxidation of the zinc-binding domains. Proceedings of the National
Academy of Sciences of the USA 99: 11109–11114.
Lao NT, Long D, Kiang S, et al. 2003. Mutation of a family 8 glycosyltransferase
gene alters cell wall carbohydrate composition and causes a humiditysensitive semi-sterile dwarf phenotype in Arabidopsis. Plant Molecular
Biology 53: 647– 661.
Lee C, O’Neill MA, Tsumuraya Y, Darvill AG, Ye ZH. 2007. The irregular
xylem9 mutant is deficient in xylanxylosyltransferase activity. Plant and
Cell Physiology 48: 1624– 1634.
Lewis LA, McCourt RM. 2004. Green algae and the origin of land plants.
American Journal of Botany 91: 1535– 1556.
Li XM, Cordero I, Caplan J, Molhoj M, Reiter WD. 2004. Molecular analysis
of 10 coding regions from Arabidopsis that are homologous to the MUR3
xyloglucan galactosyltransferase. Plant Physiology 134: 940– 950.
Lemieux C, Otis C, Turmel M. 2007. A clade uniting the green algae
Mesostigma viride and Chlorokybus atmophyticus represents the deepest
branch of the Streptophyta in chloroplast genome-based phylogenies.
BMC Biology 5: 2.
Liepman AH, Wilkerson CG, Keegstra K. 2005. Expression of cellulose
synthase-like (Csl) genes in insect cells reveals that CslA family members
encode mannan synthases. Proceedings of the National Academy of
Sciences of the USA 102: 2221–2226.
Liwanag AJM, Ebert B, Verhertbruggen Y, et al. 2012. Pectin biosynthesis:
GALS1 in Arabidopsis thaliana is a b-1,4-galactan b-1,4-galactosyl
transferase. Plant Cell 24: 5024– 5036.
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B.
2013. The Carbohydrate-active enzymes database (CAZy) in 2013.
Nucleic Acids Research 42: D490–D495.
Madson M, Dunand C, Li X, et al. 2003. The MUR3 gene of Arabidopsis
encodes a xyloglucan galactosyltransferase that is evolutionarily related to
animal exostosins. Plant Cell 15: 1662– 1670.
Manfield IW, Orfila C, McCartney L, et al. 2004. Novel cell wall architecture of
isoxaben-habituated Arabidopsis suspension-cultured cells: global transcript profiling and cellular analysis. Plant Journal 40: 260– 275.
Matsunaga T, Ishii T, Matsumoto S, et al. 2004. Occurrence of the primary cell
wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes,
and bryophytes. Implications for the evolution of vascular plants. Plant
Physiology 134: 339–351.
McCourt RM, Delwiche CF, Karol KG. 2004. Charophyte algae and land plant
origins. Trends in Ecology and Evolution 19: 661 –666.
Moller I, Sørensen I, Bernal AJ, et al. 2007. High throughput mapping of cell
wall polymers within and between plants using novel microarrays. Plant
Journal 50: 1118–1128.
Morrison JC, Greve LC, Richmond PA. 1993. Cell wall synthesis during
growth and maturation of Nitella internodal cells. Planta 189: 321–328.
Mortimer JC, Miles GP, Brown DM, et al. 2010. Absence of branches from
xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass. Proceedings of the National Academy of Sciences of
the USA 107: 17409–17414.
Narimatsu H. 2006. Human glycogene cloning: focus on beta
3-glycosyltransferase and beta 4-glycosyltransferase families. Current
Opinion in Structural Biology 16: 567– 575.
Naylor G, Russell-Wells B. 1934. On the presence of cellulose and its distribution in the cell walls of brown and red algae. Annals of Botany (London) 48:
635– 641.
Nedelcu AM, Borza T, Lee RW. 2006. A land plant– specific multigene family
in the unicellular Mesostigma argues for its close relationship to
Streptophyta. Molecular Biology and Evolution 23: 1011–1015.
Niklas KJ. 2004. The cell walls that bind the tree of life. BioScience 54: 831– 841.
Niklas KJ, Kutschera U. 2010. The evolution of the land plant life cycle. New
Phytology 185: 27– 41.
Nobles DR, Romanovicz DK, Brown RM. 2001. Cellulose in cyanobacteria.
Origin of vascular plant cellulose synthase? Plant Physiology 127:
529– 542.
Oikawa A, Joshi HJ, Rennie EA, et al. 2010. An integrative approach to the
identification of Arabidopsis and rice genes involved in xylan and secondary
wall development. PLOS ONE 5.
Okuda K, Sekida S. 2007. Cellulose: molecular and structural biology.
Brown M Jr, Saxena IM, eds. New York: Springer.
Orfila C, Sørensen SO, Harholt J, Geshi N, Crombie H, et al. 2004.
QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects homogalacturonan and xylan biosynthesis.
Planta 222: 613– 622.
Ordaz-Ortiz JJ, Marcus SE, Knox JP. 2009. Cell wall microstructure analysis
implicates hemicellulose polysaccharides in cell adhesion in tomato fruit
pericarp parenchyma. Molecular Plant 2: 910–921.
Park S, Szumlanski AL, Gu F, Guo F, Nielsen E. 2011. A role for CSLD3 during
cell-wall synthesis in apical plasma membranes of tip-growing root-hair
cells. Nature Cell Biology 13: 973– 980.
Pear JR, Kawagoe Y, Schreckengost WE, Delmer DP, Stalker DM. 1996.
Higher plants contain homologs of the bacterial celA genes encoding the
catalytic subunit of cellulose synthase. Proceedings of the National
Academy of Sciences of the United States of America 93: 12637–12642.
Peña MJ, Zhong R, Zhou GK, et al. 2007. Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis. Plant Cell 19: 549–563.
Peña MJ, Darvill AG, Eberhard S, York WS, O’Neill MA. 2008. Moss and
liverwort xyloglucans contain galacturonic acid and are structurally distinct
from the xyloglucans synthesized by hornworts and vascular plants.
Glycobiology 18: 891–904.
Peña MJ, Kong Y, York WS, O’Neill MA. 2012. A galacturonic acid–containing xyloglucan is involved in arabidopsis root hair tip growth. Plant Cell 24:
4511–4524.
Persson S, Paredez A, Carroll A, et al. 2007. Genetic evidence for three unique
components in primary cell-wall cellulose synthase complexes in
Arabidopsis. Proceedings of the National Academy of Sciences of the
United States of America 104: 15566– 15571.
1235
Pettolino F, Hoogenraad NJ, Ferguson C, et al. 2001. A (1–
4)-b-mannan-specific monoclonal antibody and its use in the immunocytochemical location of galactomannans. Planta 214: 235– 242.
Popper ZA. 2008. Evolution and diversity of green plant cell walls. Current
Opinion in Plant Biology 11: 286–292.
Popper ZA, Fry SC. 2003. Primary cell wall composition of bryophytes and
charophytes. Annals of Botany 91: 1 –12.
Popper ZA, Fry SC. 2004. Primary cell wall composition of the pteridophytes
and spermatophytes. New Phytologist 164: 165–174.
Popper ZA, Tuohy MG. 2010. Beyond the green: understanding the evolutionary puzzle of plant and algal cell walls. Plant Physiology 153: 373– 383.
Popper Z, Michel G, Hervé C, et al. 2011. Evolution and diversity of plant
cell walls: from algae to flowering plants. Annual Review of Plant Biology
62: 567–590.
Proseus TE, Boyer JS. 2006. Periplasm turgor pressure controls wall deposition
and assembly in growing Chara corallina cells. Annals of Botany 98:
93–105.
Puhlmann J, Bucheli E, Swain MJ, et al. 1994. Generation of monoclonal antibodies against plant cell wall polysaccharides. I. Characterization of a
monoclonal antibody to a terminal a-(1,2)-linked fucosyl-containing
epitope. Plant Physiology 104: 699– 710.
Ridley BL, O’Neill MA, Mohnen D. 2001. Pectins: structure, biosynthesis, and
oligogalacturonide-related signaling. Phytochemistry 57: 929– 967.
Roberts AW, Roberts E. 2007. Evolution of the cellulose synthase (CesA) gene
family: insights from green algae and seedless plants. In Brown RM,
Saxena IM, eds. Cellulose: molecular and structural biology. Dordrecht:
Springer, 17– 34.
Roberts EM, Roberts AW. 2009. A cellulose synthase (CESA) gene from the red
alga Porphyra yezoensis (Rhodophyta). Journal of Phycology 45: 203– 212.
Roberts AW, Roberts EM, Delmer DP. 2002. Cellulose synthase (CesA) genes
in the green alga Mesotaenium caldariorum. Eukaryotic Cell 1: 847–855.
Roberts AW, Roberts EM, Haigler CH. 2012. Moss cell walls: structure and
biosynthesis. Frontiers in Plant Science 3: 166.
Rodriguez-Ezpeleta N, Philippe H, Brinkmann H, Becker B, Melkonian M.
2007. Phylogenetic analyses of nuclear, mitochondrial, and plastid multigene data sets support the placement of Mesostigma in the Streptophyta.
Molecular Biology and Evolution 24: 723– 731.
Saito F, Suyama A, Oka T, Yoko-O T, Matsuoka K, Jigami Y, Shimma YI.
2014. Identification of novel peptidyl serine a-galactosyltransferase gene
family in plants. Journal of Biological Chemistry 289: 20405– 20420.
Sarria R, Wagner TA, O’Neill MA, et al. 2001. Characterization of a family of
Arabidopsis genes related to xyloglucan fucosyltransferase. Plant
Physiology 127: 1595–1606.
Scheller HV, Ulvskov P. 2010. Hemicelluloses. Annual Review of Plant Biology
61: 263–289.
Sekida S, Horiguchi T, Okuda K. 2004. Development of thecal plates and pellicle in the dinoflagellate Scrippsiella hexapraecingula (Peridiniales,
Dinophyceae) elucidated by changes in stainability of the associated membranes. European Journal of Phycology 39: 105–114.
Sekimoto H, Tanabe Y, Takizawa M, Ito N, Fukumoto RH, Ito M. 2003.
Expressed sequence tags from the Closterium peracerosum-strigosumlittorale complex, a unicellular charophycean alga, in the sexual reproduction process. DNA Research 31: 147 –153.
Sekimoto H, Tanabe Y, Tsuchikane Y, et al. 2006. Gene expression profiling
using cDNA microarray analysis of the sexual reproduction stage of the unicellular Charophycean alga Closterium peracerosum-strigosum-littorale
complex. Plant Physiology 141: 271 –279.
Showalter AM, Keppler B, Lichtenberg J, Gu DZ, Welch LR. 2010. A bioinformatics approach to the identification, classification, and analysis of
hydroxyproline-rich glycoproteins. Plant Physiology 153: 485– 513.
Simon A, Glöckner G, Felder M, Melkonian M, Becker B. 2006. EST analysis
of the scaly green flagellate Mesostigma viride (Streptophyta): implications
for the evolution of green plants (Viridiplantae). BMC Plant Biology 6: 2.
Singh SK, Eland C, Harholt J, Scheller HV, Marchant A. 2005. Cell adhesion
in Arabidopsis thaliana is mediated by ECTOPICALLY PARTING CELLS
1 – a glycosyltransferase (GT64) related to the animal exostosins. Plant
Journal 43: 384– 397.
Stebbins GL. 1992. Comparative aspects of plant morphogenesis: a cellular, molecular and evolutionary approach. American Journal of Botany 79:
589–598.
Stebbins GL, Hill GJC. 1980. Did multicellular plants invade the land.
American Naturalist 115: 342 –353.
1236
Mikkelsen et al. — Biosynthetic mechanisms in cell walls of charophyte green algae
Sterling JD, Atmodjo MA, Inwood SE, et al. 2006. Functional identification of
an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase. Proceedings of the National Academy of Sciences of the United States
of America 103: 5236–5241.
Strasser R, Mucha J, Mach L, et al. 2000. Molecular cloning and functional expression of b 1,2-xylosyltransferase cDNA from Arabidopsis thaliana.
FEBS Letters 472: 105 –108.
Strasser R, Bondili JS, Vavra U, et al. 2007. A unique beta
1,3-galactosyltransferase is indispensable for the biosynthesis of
N-glycans containing lewis a structures in Arabidopsis thaliana. Plant
Cell 19: 2278– 2292.
Sørensen I, Pettolino FA, Wilson SM, et al. 2008. Mixed-linkage
(13),(14)-b-D-glucan is not unique to the Poales but is an abundant
component of equisetum arvense cell walls. Plant Journal 54: 510– 521.
Sørensen I, Domozych D, Willats WGT. 2010. How have plant cell walls
evolved? Plant Physiology 153: 366 –372.
Sørensen I, Pettolino FA, Bacic A, et al. 2011. The charophycean green algae
provide insights into the early origins of plant cell walls. Plant Journal
68: 201– 211.
Tan L, Leykam JF, Kieliszewski MJ. 2003. Glycosylation motifs that direct arabinogalactan addition to arabinogalactan-proteins. Plant Physiology 132:
1362–1369.
Tan L, Varnai P, Lamport DTA, et al. 2012. Plant O-hydroxyproline arabinogalactans are composed of repeating trigalactosyl subunits with short bifurcated side chains. Journal of Biological Chemistry 285: 24575– 22458.
Tryfona T, Liang HC, Kotake T, Tsumuraya Y, Stephens E, Dupree P. 2012.
Structural characterization of Arabidopsis leaf arabinogalactan polysaccharides. Plant Physiology 160: 653–666.
Timme R, Delwiche C. 2010. Uncovering the evolutionary origin of plant molecular processes: comparison of Coleochaete (Coleochaetales) and
Spirogyra (Zygnematales) transcriptomes. BMC Plant Biology 10: 96.
Timme RE, Bachvaroff TR, Delwiche CF. 2012. Broad phylogenomic sampling and the sister lineage of land plants. PLOS ONE 7: e29696.
Tsekos I. 1999. The sites of cellulose synthesis in algae: diversity and evolution
of cellulose-synthesising enzyme complexes. Journal of Phycology 35:
635–655.
Tsekos I, Reiss HD. 1994. Tip growth and the frequency and distribution of cellulose microfibril-synthesizing complexes in the plasma membrane of
apical shoot cells of the red alga Porphyra yezoensis. Journal of
Phycology 30: 300– 310.
Turmel M, Otis C, Lemieux C. 2006. The chloroplast genome sequence of
Chara vulgaris sheds new light into the closest green algal relatives of
land plants. Molecular Biology and Evolution 23: 1324–1338.
Turmel M, Pombert JF, Charlebois P, Otis C, Lemieux C. 2007. The green
algal ancestry of land plants as revealed by the chloroplast genome.
International Journal of Plant Sciences 168: 679– 689.
Turmel M, Otis C, Lemieux C. 2013. Tracing the evolution of streptophyte
algae and their mitochondrial genome. Genome Biology and Evolution 5:
1817–1835.
Ulvskov P, Paiva DS, Domozych D, Harholt J. 2013. Classification, naming and
evolutionary history of glycosyltransferases from sequenced Green and Red
algal genomes. PLOS ONE 8: e76511.
Van Sandt VST, Stiperaere H, Guisez Y, Verbelen JP, Vissenberg K. 2007.
XET activity is found near sites of growth and cell elongation in bryophytes
and some green algae: new insights into the evolution of primary cell wall
elongation. Annals of Botany 99: 39– 51.
Velasquez SM, Ricardi MM, Dorosz JG, et al. 2011. O-glycosylated cell wall
proteins are essential in root hair growth. Science 332: 1401– 1403.
Waters ER. 2003. Molecular adaptation and the origin of land plants. Molecular
Phylogenetics and Evolution 29: 456–463.
Wodniok S, Brinkmann H, Glöckner G, et al. 2011. Origin of land plants: do
conjugating green algae hold the key? BMC Evolutionary Biology 11: 104.
Wu AM, Rihouey C, Seveno M, et al. 2009. The Arabidopsis IRX10 and
IRX10-LIKE glycosyltransferases are critical for glucuronoxylan biosynthesis during secondary cell wall formation. Plant Journal 57: 718–731.
Wu YY, Williams M, Bernard S, et al. 2010a. Functional identification of two
nonredundant Arabidopsis alpha(1,2)fucosyltransferases specific to arabinogalactan proteins. The Journal of Biological Chemistry 285:
13638–13645.
Wu AM, Hornblad E, Voxeur A, et al. 2010b. Analysis of the Arabidopsis
IRX9/IRX9-L and IRX14/IRX14-L pairs of glycosyltransferase genes
reveals critical contributions to biosynthesis of the hemicellulose glucuronoxylan. Plant Physiology 153: 542 –554.
Xue X, Fry SC. 2012. Evolution of mixed-linkage (1_3, 1_4)-b-D-glucan (MLG)
and xyloglucan in Equisetum (horsetails) and other monilophytes. Annals of
Botany 109: 873–886.
Yin L, Verhertbruggen Y, Oikawa A, et al. 2011. The cooperative activities of
CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development. M Plant doi:10.1093/mp/ssr026.
Yin YB, Huang JL, Xu Y. 2009. The cellulose synthase superfamily in fully
sequenced plants and algae. BMC Plant Biology 9: 99.
York WS, O’Neill MA. 2008. Biochemical control of xylan biosynthesis 2
which end is up? Current Opinion in Plant Biology 11: 258– 265.
York WS, Darvill AG, McNeil M, Albersheim P. 1985.
3-deoxy-Dmanno-2-octulosonic acid (Kdo) is a component of rhamnogalacturonan II, a pectic polysaccharide in the primary cell walls of plants.
Carbohydrate Research 138: 109–126.
Zabotina OA, van de Ven WTG, Freshour G, et al. 2008. The Arabidopsis XT5
protein encodes a putative a-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis. Plant Journal 56: 101–115.
Zhong B, Liu L, Yan Z, Penny D. 2013. Origin of land plants using the multispecies coalescent model. Trends in Plant Science 18: 492– 495.
Zhou Y, Li S, Qian Q, et al. 2009. BC10, a DUF266-containing and
Golgi-located type II membrane protein, is required for cell-wall biosynthesis in rice (Oryza sativa L.). Plant Journal 57: 446–462.
Zykwinska A, Thibault JF, Ralet MC. 2007. Organization of pectic arabinan
and galactan side chains in association with cellulose microfibrils in
primary cell walls and related models envisaged. Journal of Experimental
Botany 58: 1795–1802.