Download genes and enzyme complexes for polysaccharide synthases

488
Building the wall: genes and enzyme complexes for
polysaccharide synthases
Kanwarpal S Dhugga
The complete sequence of the Arabidopsis genome has
revealed a total of 40 cellulose synthase (CesA) and cellulose
synthase-like (Csl) genes. Recent studies suggest that each
CESA polypeptide contains only one catalytic center, and that
two or more polypeptides from different genes might be
needed to form a functional cellulose synthase complex.
Addresses
Agronomic Traits, Traits and Technology Development, Pioneer Hi-Bred
International, Inc., Johnston, Iowa 50131, USA;
e-mail: [email protected]
Current Opinion in Plant Biology 2001, 4:488–493
1369-5266/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
CesA
cellulose synthase gene
Csl
cellulose synthase-like
EST
expressed sequence tag
IRX1
IRREGULAR XYLEM1
RSW1 RADIAL SWELLING1
Introduction
Cell walls, in addition to providing strength, define the
shape and size of a plant cell. Secondary wall in the form of
wood and fiber is a major source of renewable biomass. In
the seeds of some plant species, such as endospermic
legumes, the endosperm wall acts as a storage compartment
for Golgi-synthesized, hemicellulosic polysaccharides [1].
These polysaccharides have a role similar to that of starch
in the seed cotyledons of non-endospermic legumes.
Polysaccharides make up the majority of the cell wall.
Cellulose, a paracrystalline form of H-bonded β-1,4-glucan
chains, is the primary determinant of strength. Although
cellulose forms only about one third of an expanding
primary wall, it is the dominant constituent of the walls of
most mature cells.
Whereas cellulose is deposited directly from the plasma
membrane, the rest of the matrix polysaccharides are first
made in the Golgi and then exported to the wall by exocytosis. Structural proteins constitute a small proportion of
the growing cell wall, and their precise function remains to
be determined. Enzymes such as glucanases, expansins,
and transglycosylases are believed to affect the wall properties by altering the size of hemicellulosic polymers or by
dissociating hydrogen bonds. Progress in understanding the
roles of structural proteins and enzymes in the cell wall, in
understanding cellulose synthesis, and in identifying
oligoglycosyltransferases that are involved in polysaccharide
synthesis has been addressed in recent reviews [2–4,5•,6,7].
This review summarizes the literature published during
the past year on genes encoding polysaccharide synthases
and their functional significance.
Genes for polysaccharide synthases
The biochemical approach to identifying plant polysaccharide synthases has achieved little success over the past
several decades despite the fact that detergent-solubilized
callose synthase can be purified [8,9]. A gene for bacterial
cellulose synthase (CesA) was, however, isolated following
enzyme purification and peptide sequencing [10]. The
breakthrough in isolating the plant CesA gene came via the
genomics approach when Pear et al. [11] isolated two
cDNA clones from developing cotton fibers by screening a
few hundred expressed sequence tags (ESTs). The proteins predicted by these cDNA clones contained stretches
of amino acids that were highly similar to those of the
bacterial CESA [10,12]. The main reason for success was
the choice of tissue, that is, developing cotton fibers
during the peak secondary wall formation period when
cellulose is synthesized at a rapid rate. Usually, the frequency
of CesA-related sequences in the public and proprietary
(DuPont) EST databases is approximately 5 × 10–4 [5•]
(Figure 1; KS Dhugga, unpublished data).
Genes with sequence similarity to cotton CesA have been
isolated from many plant species; and evidence for the
involvement of CesA genes in cellulose synthesis has come
from studies employing mutational genetics [13,14,15••,16].
Another class of sequences, which shows little similarity to
the CesA genes and is termed cellulose synthase-like (Csl),
was uncovered from the Arabidopsis EST database [5•,17].
The CSL proteins differ from the CESA proteins in that they
lack an amino-terminal Zn-finger domain [5•].
Recently, a gene has been isolated from several different
plant species that has similarity to FKS (FK506-hypersensitive locus), a fungal β-1,3-glucan synthase gene [18,19,20•].
This gene encodes a protein with 16 transmembrane
domains, and its central soluble domain shows some
similarity to the fungal enzyme. The predicted plant
polypeptide contains neither the conserved D residues nor
the QXXRW motif that are found in other polymerizing
β-glycosyltransferases [21]. These motifs are also absent
from the fungal FKS polypeptide.
A gene for curdlan (β-1,3-glucan) synthase isolated from
Agrobacterium is similar to CesA and contains the conserved
motifs of a β-glycosyltransferase [22]. Furthermore, upon
loss of its CesA gene, Dictyostelium also loses β-1,3-glucan
synthase activity [23]. These findings are consistent with
earlier suggestions that cellulose synthase makes cellulose
as well as callose, the latter upon wounding of the plant
Genes and enzyme complexes for polysaccharide synthases Dhugga
489
Figure 1
(a) Seedling
(b) Root
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sA esA esA esA esA esA esA esA
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(d) Stalk
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(c) Leaf
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(h) Embryo
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(j) Pollen
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Expression of maize CesA genes in different
tissues as studied by massively parallel
signature sequencing [32,33]. With this
technique, each cDNA is attached to the
surface of a unique microbead. A highly
expressed mRNA is represented on a
proportionately large number of microbeads.
Signature sequences of 16–20 nucleotides
are then obtained from these microbeads by
iterative cycles of restriction with a type IIs
endonuclease, adaptor ligation, and
hybridization with encoded probes. For each
tissue, the data were averaged across the
following numbers of libraries, each derived
from the in-bred line B73: ear shoot, 3;
embryo, 3; endosperm, 4; kernel, 2; leaf, 2;
pollen, 1; root, 6; seedling, 2; stalk, 2; and
tassel, 1. The number of signatures in each
library ranged from 1.2 × 106 to 2.2 × 106.
The level of expression of a gene is
determined by the abundance of its signature
in the total pool. None of the genes were
found to be expressed in pollen.
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sA sA sA sA sA sA sA sA
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Current Opinion in Plant Biology
490
Cell biology
tissue [24]. The FKS homologs might make callose in
intact cells or cell parts, such as the cell plate, plasmodesmatal channels, pollen mother cells, the pollen tube, and
the sieve plates. CESA might start to make callose in
response to tissue wounding.
of even the highly purified callose synthase fractions and
has been difficult to identify by peptide sequencing [8,28]
(KS Dhugga, unpublished data). Sophisticated and highly
sensitive proteomics technologies combined with the sizable public and proprietary EST databases should prove
useful in overcoming these problems.
Gene families
Completion of the Arabidopsis genome sequence has
allowed the compilation of genes encoding glycosyltransferase activities, some of which are involved in
polysaccharide synthesis. On the basis of similar aminoacid sequences, and in some cases of hydrophobic clusters,
Henrissat and colleagues have assigned all available plant
and non-plant glycosyltransferases to 52 families (as of
July 2, 2001; URL http://afmb.cnrs-mrs.fr/~pedro/CAZY/
db.html). A total of 353 sequences for glycosyltransferases
that fall into 26 different families have been identified in
Arabidopsis [25••]. Family 2, to which plant CesA and Csl
genes belong, contains 40 sequences, of which ten are CesA
genes and the remainder are Csl genes [4,25••]. The 30 Csl
genes can be grouped into six families [5•]. Family 48, to
which the fungal FKS homologs have been assigned,
contains 12 genes [18,20•,25••].
Although the functions of about half of the CesA genes have
been determined by mutational genetics, progress in
determining the exact role of the Csl genes in cell-wall
synthesis has been slow [4,13,14,15••,26,27]. Only one of
the 30 Csl genes, CslD3, has been assigned a role, that of
root-hair formation [26,27]. The mutant cslD3 gene affects
only the development of root hairs although it is also
expressed in other tissues [27]. As root hair cells lack the
physical support of other cells around them, alteration in
wall composition of these cells could readily lead to a visible
mutant phenotype. Such a phenotype could be further
explained by the lack of expression of other, compensating
Csl genes in these cells. An indication as to what type of
polysaccharide CslD3 might synthesize comes from the work
of Doblin et al. [20•] who suggested that a similar gene in
tobacco might be involved in making cellulose in tip-growing
cells, such as those in the pollen tube and root hairs. This
tobacco gene is highly expressed in the growing pollen tube,
and its deduced polypeptide, unlike those encoded by the
Arabidopsis Csl genes, contains an amino-terminal Zn-finger
domain. As the CslD family is closest to CesA family and
appears to be the most ancient of the Csl families, it is
possible that its members do indeed make cellulose [5•,20•].
Biochemical activity has been difficult to associate with any
of the products of the CesA or Csl genes because polysaccharides synthases, particularly CESA, are rather labile. Golgi
enzymes involved in the synthesis of wall matrix polysaccharides, such as xylan, mixed-linked glucan, xyloglucan
and polygalacturonan, are amenable to biochemical assays.
These enzymes might, therefore, prove to be useful in
relating the polypeptides from partially purified enzymes
to specific CesA or Csl genes. Paradoxically, the catalytic
polypeptide appears to constitute only a very small proportion
Functional significance
On the basis of amino-acid sequence similarity alone, it
appears that functional specificity of CESAs might be
conserved across species [29••]. Maize CESA1, for example,
groups with RADIAL SWELLING1 (RSW1), which is
known to be involved in primary wall formation [13].
Another derived maize polypeptide, CESA8, is closely
related to CESAs from other species that are expressed in
secondary-wall-forming tissues such as cotton fiber and
pine xylem. Expression patterns of CesA genes, as studied
by in situ hybridization, support the groupings made on the
basis of sequence comparisons [29••]. These findings
suggest that the CesA gene families had formed before the
separation of monocots and dicots.
Recent studies indicate that coordinate expression of two
or more genes in the same cell might be needed for the
formation of a functional CESA [15••,30••]. Two distinct
genes involved in the formation of secondary wall in
Arabidopsis, IRREGULAR XYLEM1 (IRX1) and IRX3, are
coordinately expressed in the same cell types [30••]. A similar
phenotype results when either of these genes is mutated.
Similarly, PROCUSTE1 (PRC1) and RSW1, the genes
involved in primary wall formation, are expressed in
elongating cells [15••]. Mutation of either of these genes
leads to a similar phenotype, suggesting that both genes
must be expressed coordinately to form a functional CESA
complex [15••]. Inhibition of crystalline cellulose synthesis
by a herbicide resulted in the association of the CESA
polypeptides with the noncrystalline cellulose in cotton
fibers [31••]. Presence of a mixture of CESA1 and CESA2
polypeptides in the product-associated fraction suggested
once again that both of these genes are expressed together
in developing cotton fibers.
We have studied the expression of maize CesA genes using
massively parallel signature sequencing technology [32,33].
On average, 1.4 million signatures were obtained from
each library (Figure 1). The number of unique signatures
ranged from about 11 500 for the pollen library to approximately 87 000 for a root library; multiple signatures are
possible from each gene. The expression profiles of eight
maize CesA genes, as found in 26 libraries that were derived
from ten different tissues, are shown in Figure 1. CesA9 is
left out of the figure because it is nearly identical to CesA4,
and the only signature identified from these genes is common to both genes. None of the genes was detected in the
pollen library. The correlation coefficients for the expression levels between different CesA gene family members
across all of the libraries are: CesA1 versus CesA2, 0.78;
CesA1 vs CesA6, 0.7; CesA1 vs CesA7, 0.76; CesA1 vs CesA8,
Genes and enzyme complexes for polysaccharide synthases Dhugga
0.66; CesA2 vs CesA7, 0.87; CesA2 vs CesA8, 0.91; and CesA7
vs CesA8, 0.74. CesA4 was expressed only in a few tissues at
a very low level. The expression patterns of CesA3 and
CesA5 did not correlate with those of any of the other
genes. Consistent with previous reports, these data suggest
that genes CesA1, CesA2, CesA7, and CesA8 might be coordinately expressed in similar tissues, and that polypeptides
encoded by any two or more of these genes could assemble to form a functional CESA unit [15••,30••,31••]. CESA1
and CESA2 form a clade with the primary-wall-forming
enzymes, and CESA7 and CESA8 with the secondarywall-forming ones [29••]. As whole tissues were used for
library construction, it is difficult to judge whether these
genes were expressed in different cell types from the same
tissue in different combinations; for example, CesA1 and
CesA2 in the young, expanding cells and CesA7 and CesA8 in
the vascular bundles.
The CesA5 gene stands out by being highly expressed in the
endosperm tissue (Figure 1). From DuPont’s genome database for maize, consisting of approximately 500 000 ESTs
derived from more than 200 tissue libraries, CesA5 appears to
be preferentially expressed in elongating tissues, including
the coleoptiles (KS Dhugga, unpublished data). Endosperm
walls are rich in mixed-linked glucan. This polysaccharide is
also abundantly deposited in the walls of coleoptile and
other elongating cells, although it is turned over once the
cell stops expanding [34]. The expression pattern of CesA5
suggests that it might encode a Golgi enzyme involved in
the synthesis of mixed-linked glucan. In the absence of any
information on the molecular nature of Golgi polysaccharide
polymerases, however, it is difficult to judge at this stage
whether CESA5 is indeed a Golgi enzyme.
Mechanism of chain elongation
A two-site model for the formation of dextran was proposed by Robyt et al. [35]. Subsequently, this model was
extended to explain cellulose synthesis in bacteria [36].
Han and Robyt suggested that a cellulose chain grows
toward the reducing end, but others have reported growth
in the reverse direction [36,37]. In either case, a two-site
model involving two different CESA polypeptides could
explain the anomaly created by the report that SpsA, a
bacterial glycosyltransferase belonging to family 2, has
only one catalytic center in each polypeptide and the
previous proposals that two sites are needed to form a
molecule such as cellulose [21,38,39]. Two sites juxtaposed to each other could supply alternate residues for the
elongating cellulose chain, solving the problem of requiring
the chain to rotate after the addition of each residue if the
same polypeptide were to contain both the catalytic centers.
The three sites proposed for the formation of mixed-linked
glucan in maize, for example, could be supplied by a
homotrimer of CESA5 [40].
The picture becomes somewhat complicated by the recent
finding that cellulose chains are tightly attached to the
cotton CESA1 and CESA2 polypeptides [31••]. The high
491
molecular mass glucan-polysaccharide complex, obtained
from cultured cotton fibers after treatment with a herbicide,
was subjected to cellulase treatment to release the CESA
polypeptides. A tryptic fragment from the same region of
both the CESA genes had 2–6 glucosyl residues attached to
it. Although the exact residue to which the oligosaccharides were attached was not identified, the peptide itself
was the same as that previously reported to match the
amino-acid sequence around the glucose-binding arginyl
residue of the reversibly glycosylated polypeptide [41,42].
These findings support Robyt’s model of the cellulose
chain growing toward the reducing end and the chain
being covalently linked to the CESA polypeptide at all
times until released. Formation of a second covalent
intermediate would, however, seem necessary to form a
β-linkage in the resulting cellulose chain. The possibility
remains, however, that the oligosaccharides are covalently
attached to CESA only upon herbicide treatment and that
under normal conditions this enzyme does not form a
covalent intermediate.
Conclusions
The exact roles of only a few of the 40 CesA and Csl genes
from Arabidopsis have been determined, in each case by
mutational genetics. The number of catalytic centers in each
CESA polypeptide and the mechanism of chain elongation
are currently hot topics. Determination of three-dimensional
structure of the soluble, catalytic portion of the CESA is
necessary to answer this question unequivocally.
Enzymes that can be assayed in vitro could prove useful in
determining the role of the remaining polysaccharide
synthase genes now that protein identification by highly
sensitive proteomics techniques is becoming a routine.
Reverse genetics, gene silencing [43,44], and biochemical
approaches will help to unravel the exact function in cell
wall polysaccharide synthesis of the remaining genes.
Determining whether substrate supply or CESA/CSL
enzyme levels limit cell wall formation will have commercial implications in improving biomass productivity [45]. It
is not yet known to what extent the strength of a tissue is
determined by the properties of a particular CESA enzyme.
This type of information might be useful in improving the
mechanical strength of tissues in commercial crops, thereby
overcoming abiotic problems such as lodging.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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Cosgrove DJ: New genes and new biological roles for expansins.
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Campbell P, Bram J: Xyloglucan endotransglycosylase: diversity of
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492
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5. Richmond TA, Somerville CR: The cellulose synthase superfamily.
•
Plant Physiol 2000, 124:495-498.
A thorough discussion on different CesA and Csl gene families and their
members from Arabidopsis. The genomic structures of representative
members from each family are also presented. (See also the website URL
http://cellwall.stanford.edu/)
6.
Saxena IM, Brown RM Jr: Cellulose synthases and related
enzymes. Curr Opin Plant Biol 2000, 3:523-531.
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Keegstra K, Raikhel N: Plant glycosyltransferases. Curr Opin Plant
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8.
Dhugga KS, Ray PM: Purification of 1,3-β-glucan synthase
activity from pea tissue: two polypeptides of 55 kDa and 70 kDa
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Meikle PJ, Ng KF, Johnson E, Hoogenraad NJ, Stone BA: The
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11. Pear JR, Kawagoe Y, Schreckengost WE, Delmer DP, Stalker DM:
Higher plants contain homologs of the bacterial celA genes
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12. Wong HG, Fear AL, Calhoon RD, Eichinger GH, Mayer R, Amikam D,
Benziman M, Gelfand DH, Meade JH, Emerick AW et al.:
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Hofte H, Plazinski J, Birch R et al.: Molecular analysis of cellulose
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irregular xylem3 locus of Arabidopsis encodes a cellulose
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15. Fagard M, Desnos T, Desprez T, Goubet F, Refregier G, Mouille G,
•• McCann M, Rayon C, Vernhettes S, Hofte H: PROCUSTE1 encodes
a cellulose synthase required for normal cell elongation
specifically in roots and dark-grown hypocotyls of Arabidopsis.
Plant Cell 2000, 12:2409-2423.
The authors report the map-based cloning of the Arabidopsis CesA6
(PRC1) gene, which is expressed in elongating cells. Coordinate expression
of PRC1 and RSW1 (CesA1) in different tissues is shown, and the authors
suggest that the expression of at least two different CesA genes in the same
cell is needed to form a functional enzyme complex.
16. Turner SR, Somerville CT: Collapsed xylem phenotype of
Arabidopsis identifies mutants deficient in cellulose deposition in
the secondary cell wall. Plant Cell 1997, 9:689-701.
17.
Cutler S, Somerville C: Cellulose synthesis: cloning in silico. Curr
Biol 1997, 7:R108-R111.
22. Stasinopoulos SJ, Fisher PR, Stone BA, Stanisich VA: Detection of
two loci involved in (1fwdarw3)-β-glucan (curdlan) biosynthesis
by Agrobacterium sp. ATCC31749, a comparative sequence
analysis of the putative curdlan synthase gene. Glycobiol 1999,
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23. Blanton RL, Fuller D, Iranfar N, Grimson MJ, Loomis WF: The
cellulose synthase gene of Dictyostelium. Proc Natl Acad Sci USA
2000, 97:2391-2396.
24. Delmer D: Cellulose biosynthesis: exciting times for a difficult
field of study. Annu Rev Plant Physiol Plant Mol Biol 1999,
50:245-276.
25. Henrissat B, Cautinho PM, Davies GJ: A census of carbohydrate
•• active enzymes in the genome of Arabidopsis thaliana. Plant Mol
Biol 2001, 47:55-57.
Extensive tables of different glycosyltransferases identified from the completely sequenced Arabidopsis genome are presented. A total of 353
glycosyltransferases spread over 26 different families are shown. Information
on these and other carbohydrate-active enzymes is regularly updated at the
internet site: URL http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html
26. Favery B, Ryan E, Foreman J, Linstead P, Boudonck K, Steer M,
Shaw P, Dolan L: KOJAK encodes a cellulose synthase-like protein
required for root hair cell morphogenesis in Arabidopsis. Genes
Dev 2001, 15:79-89.
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Wang X, Cnops G, Vanderhaeghen R, De Block S, Van Montagu M,
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28. Wu A, Wasserman BP: Limited proteolysis of (1,3)-β-glucan
(callose) synthase from Beta vulgaris L.: topology of proteasesensitive sites and polypeptide identification using Pronase E.
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•• Delmer DP: A comparative analysis of the plant cellulose synthase
(CesA) gene family. Plant Physiol 2000, 123:1313-1323.
CESA polypeptides involved in primary or secondary wall formation are
shown to group into different clades across monocot and dicot species. This
grouping is supported by evidence obtained by in situ analysis of genes from
different clades. Nine CesA genes from maize, their chromosomal map
locations, and their expression patterns in different tissues are presented.
30. Taylor NG, Laurie S, Turner SR: Multiple cellulose synthase
•• catalytic subunits are required for cellulose synthesis in
Arabidopsis. Plant Cell 2000, 12:2529-2539.
The map-based cloning of AtCesA8 (IRX1) is reported, as is its coordinate
expression with AtCesA7 (IRX3) in the same tissues. Transformation of the
irx3 mutant with his-tagged IRX3 restored the wild-type phenotype. When
detergent-solubilized membranes were adsorbed onto a Ni-column, IRX1
was also adsorbed, indicating that IRX1 and IRX3 are associated with each
other in the membranes.
31. Peng L, Xian F, Roberts E, Kawagoe Y, Greve LC, Kreuz K,
•• Delmer DP: The experimental herbicide CGA 325’615 inhibits
synthesis of crystalline cellulose and causes accumulation of
non-crystalline β-1,4-glucan associated with CesA protein. Plant
Physiol 2001, 126:981-992.
Data presented in this paper suggest that the cellulose chain is covalently
attached to the CESA polypeptide. The site of attachment is probably the
same as that previously reported for the reversibly glycosylated polypeptide
that has been implicated in Golgi polysaccharide synthesis [41,42].
18. Hong Z, Delauney AJ, Verman DPS: A cell plate-specific callose
synthase and its interaction with phragmoplastin. Plant Cell 2001,
13:755-768.
32. Brenner S, Williams SR, Vermaas EH, Storck T, Moon K, McCollum C,
Mao JI, Luo S, Kirchner JJ, Eletr S et al.: In vitro cloning of complex
mixtures of DNA on microbeads: physical separation of
differentially expressed cDNAs. Proc Natl Acad Sci USA 2000,
97:1665-1670.
19. Cui X, Shin H, Song C, Laosinchai W, Amano Y, Brown RM:
A putative plant homolog of the yeast β-1,3-glucan synthase
subunit FKS1 from cotton (Gossypium hirsutum L.) fibers. Planta
2001, 213:223-230.
33. Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D,
Luo S, McCurdy S, Foy M, Ewan M et al.: Gene expression analysis
by massively parallel signature sequencing (MPSS) on microbead
arrays. Nat Biotechnol 2000, 18:630-634.
20. Doblin MS, De Melis L, Newbigin E, Bacic A, Read SM: Pollen tubes
•
of Nicotiana alata express two genes from different β-glucan
synthase families. Plant Physiol 2001, 125:2040-2052.
A gene that appears to be a chimera between the CesA and CslD genes is
expressed in the pollen and pollen tube, and is suggested to encode a
cellulose synthase.
34. Vergara CE, Carpita NC: β-D-glycan synthesis and the CesA
gene family: lessons to be learned from the mixed-linked
(1,3),(1,4)β-D-glucan synthase. Plant Mol Biol 2001, 47:145-160.
21. Saxena IM, Brown RM, Fevre M, Geremia RA, Henrissat B:
Multidomain architecture of β-glycosyl transferases:
implications for mechanism of action. J Bacteriol 1995,
177:1419-1424.
36. Han NS, Robyt JF: The mechanism of Acetobacter xylinum
cellulose biosynthesis: direction of chain elongation and the role
of lipid pyrophosphate intermediates in the cell membrane.
Carbohydr Res 1998, 313:125-133.
35. Robyt JF, Kimble BK, Walseth TF: The mechanism of
dextransucrase action. Arch Biochem Biophys 1974, 165:634-640.
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