CH 3

3 Biosynthesis of pectins
Debra Mohnen
3.1 Introduction
Pectin is the most structurally complicated polysaccharide in the plant cell wall.
Accordingly, the study of its synthesis is complex owing to the large number
of enzymes required. At least 12 activated sugar substrates are required for
pectin synthesis. The known activated sugars are nucleotide-sugars, although
the possibility that lipid-linked sugars may be involved cannot be ruled out.
The regulation of the synthesis of the activated sugar substrates is likely to be
important in the overall regulation of pectin synthesis. Based on the known
structure of pectin, at least 14 distinct enzyme activities are required to syn-
thesize the activated sugar substrates and 58 distinct glycosyl-, methyl- and
acetyltransferases are required to synthesize the complicated family of polymers
known as pectin. While progress has been made in characterizing some of the
pectin biosynthetic enzymes in crude or partially puried plant homogenates,
in no case has a single enzyme been completely characterized in regard to
enzyme structure, subcellular location, protein sequence, gene identity and
enzyme regulation. Several recent comprehensive reviews on pectin structure
(Mohnen, 1999; Ridley et al., 2001), synthesis (Mohnen, 1999; Ridley et al.,
2001) and function (Ridley et al., 2001; Willats et al., 2001a) and on nucleotide-
sugar interconversion pathways (Reiter and Vanzin, 2001) have been published
and should be consulted for detailed background information. In addition,
several general reviews on cell wall synthesis (Gibeaut, 2000; Reid, 2000;
Dhugga, 2001; Perrin et al., 2001) and on progress and strategies to identify
wall biosynthetic genes (Keegstra and Raikhel, 2001; Perrin et al., 2001) and
glycosyltransferases (Henrissat et al., 2001; Keegstra and Raikhel, 2001) have
recently been published. The goal of this review is to summarize our present
level of understanding of pectin synthesis.
The rst part of the review outlines our understanding of the subcellular
location of pectin synthesis. The nucleotide-sugars required for pectin synthesis
are then introduced and progress towards understanding the mechanism, site(s)
and regulation of their synthesis is reviewed. Finally, a list of the glycosyl-,
methyl- and acetyltransferases required for pectin synthesis is provided and
progress towards identifying and characterizing these enzymes is summarized.
It is hopedthat this reviewwill provide a foundationtofacilitate the identication
of the pectin biosynthetic genes and the development of better molecular tools
to study pectin biosynthesis.
BIOSYNTHESIS OF PECTINS 53
3.2 What is the structure of newly synthesized pectin?
One of the challenges of studying pectin synthesis is that we do not know the
structure of de novo synthesized pectin. For example, the detailed structural
characterization of pectin isolated from the wall (ONeill et al., 1990; Mohnen,
1999; Ridley et al., 2001) has led to the identication of the family of complex
polysaccharides known as homogalacturonan (HGA), rhamnogalacturonan I
(RG-I) and the substituted galacturonans such as the ubiquitous rhamnogalac-
turonan II (RG-II) (Ridley et al., 2001), and the less prevalent xylogalacturonan
(Schols et al., 1990, 1995; Yu and Mort, 1996), and apiogalacturonan (Hart
and Kindel, 1970; Watson and Orenstein, 1975). However, we do not yet know
whether these polysaccharides are synthesized as one polymer or whether they
are synthesized as individual polymers that become interconnected during or
following their insertion into the wall (see Figure 3.1). Furthermore, we do not
know whether the structural differences found in each of the polysaccharides
isolated from the wall, such as variations in the degree and pattern of methyl-
and acetyl-esterication of homogalacturonan (Willats et al., 2001b), arise in
the wall (i.e. in muro by wall-localized enzymes) or whether the differences
occur, at least in part, during synthesis. This uncertainty makes it challenging to
propose models for how pectin is synthesized and to predict which enzymes are
required intracellularly for synthesis, rather than being required extracellularly
for in muro modeling of pectin. For the purpose of this review, the working
hypothesis is that homogalacturonan can be synthesized as in independent
polymer. It is also proposed that substituted galacturonans such as ubiquitous
RG-II (Ridley et al., 2001) and the other less prevalent xylogalacturonan (Schols
et al., 1990, 1995; Yu and Mort, 1996) and apiogalacturonan (Ridley et al.,
2001) can be synthesized as modied versions of homogalacturonan. Finally,
it is hypothesized that RG-I is synthesized as an independent polymer that
may, or may not, be covalently linked to homogalacturonan or to substituted
galacturonans (see Figure 3.1). It must be stressed, however, that no unequivocal
evidence is available to support the hypothesized independence of RG-I and
HGA synthesis or of the dependence of RG-II synthesis on HGA. One of
the goals, or outcomes, of current research in pectin synthesis should be the
elucidation of how the three main pectic polymers, HGA, RG-I and RG-II, are
linked together during synthesis.
3.3 Subcellular location of pectin synthesis
Several lines of evidence indicate that pectin is synthesized in the Golgi and
transported to the wall via membrane-bound vesicles. The plant Golgi apparatus
is a dynamic series of membrane-bound vesicles and stacks that function in
the biosynthesis pectins and hemicellulose, in the glycosylation of proteins
F
i
g
u
r
e
3
.
1
M
o
d
e
l
s
f
o
r
d
i
f
f
e
r
e
n
t
p
o
s
s
i
b
l
e
p
o
l
y
m
e
r
i
c
o
r
g
a
n
i
z
a
t
i
o
n
s
o
f
t
h
e
p
e
c
t
i
c
p
o
l
y
s
a
c
c
h
a
r
i
d
e
s
.
W
h
e
n
p
e
c
t
i
n
i
s
i
s
o
l
a
t
e
d
f
r
o
m
t
h
e
w
a
l
l
i
t
o
f
t
e
n
a
p
p
e
a
r
s
a
s
i
f
t
h
e
t
h
r
e
e
p
e
c
t
i
c
p
o
l
y
s
a
c
c
h
a
r
i
d
e
s
H
G
A
,
R
G
-
I
a
n
d
R
G
-
I
I
a
r
e
c
o
v
a
l
e
n
t
l
y
l
i
n
k
e
d
t
o
e
a
c
h
o
t
h
e
r
.
T
h
i
s
i
s
b
a
s
e
d
o
n
t
h
e
o
b
s
e
r
v
a
t
i
o
n
t
h
a
t
t
r
e
a
t
m
e
n
t
o
f
i
s
o
l
a
t
e
d
c
e
l
l
w
a
l
l
s
w
i
t
h
e
n
d
o
p
o
l
y
g
a
l
a
c
t
u
r
o
n
a
s
e
,
w
h
i
c
h
c
l
e
a
v
e
s
t
h
e
1
,
4
-
G
a
l
A
l
i
n
k
a
g
e
s
i
n
H
G
A
,
r
e
l
e
a
s
e
s
R
G
-
I
,
R
G
-
I
I
a
n
d
f
r
a
g
m
e
n
t
s
o
f
H
G
A
(
I
s
h
i
i
a
n
d
M
a
t
s
u
n
a
g
a
,
2
0
0
1
)
.
W
h
a
t
i
s
n
o
t
c
l
e
a
r
i
s
w
h
e
t
h
e
r
t
h
e
s
e
p
o
l
y
m
e
r
s
a
r
e
s
y
n
t
h
e
s
i
z
e
d
a
s
o
n
e
l
a
r
g
e
p
o
l
y
m
e
r
(
f
)
t
h
a
t
m
a
y
a
l
s
o
i
n
c
l
u
d
e
o
t
h
e
r
m
o
d
i
c
a
t
i
o
n
s
s
u
c
h
a
s
x
y
l
o
g
a
l
a
c
t
u
r
o
n
a
n
(
Y
u
a
n
d
M
o
r
t
,
1
9
9
6
;
H
u
i
s
m
a
n
e
t
a
l
.
,
2
0
0
1
b
)
o
r
w
h
e
t
h
e
r
H
G
A
(
a
)
,
R
G
-
I
(
b
)
a
n
d
R
G
-
I
I
(
c
)
a
r
e
s
y
n
t
h
e
s
i
z
e
d
a
s
s
e
p
a
r
a
t
e
p
o
l
y
m
e
r
s
.
A
l
t
e
r
n
a
t
i
v
e
l
y
,
R
G
-
I
a
n
d
R
G
-
I
I
m
a
y
b
e
s
y
n
t
h
e
s
i
z
e
d
o
n
s
e
p
a
r
a
t
e
H
G
A
-
c
o
n
t
a
i
n
i
n
g
p
o
l
y
m
e
r
s
(
e
a
n
d
d
,
r
e
s
p
e
c
t
i
v
e
l
y
)
.
and in the synthesis of lipids (Staehelin and Moore, 1995; Nebenf uhr and
Staehelin, 2001). The Golgi vesicles move along actin laments via myosin
motors (Nebenf uhr et al., 1999) and it is believed that this movement targets
the transport of pectin and other macromolecules to the cell wall.
The autoradiographic identication of radiolabeled polysaccharides in Golgi
cisternae and their chase from the Golgi to the cell wall using non-radiolabeled
precursors (Northcote and Pickett-Heaps, 1966; Northcote, 1970) was among
the rst evidence that pectin is synthesized in the Golgi. For example, Golgi-
enriched fractions isolated from cells grown in the presence of radiolabeled
glucose contained radioactive galactose (Gal), arabinose (Ara) and galactosy-
luronic acid (GalA, galacturonic acid), the major glycosyl residues found in the
pectic polysaccharides (Stoddart and Northcote, 1967; Harris and Northcote,
1971).
The identication of pectin-specic carbohydrate epitopes in the Golgi, via
immunocytochemistry of thin cell sections using anti-pectin antibodies (Moore
et al., 1991; Staehelin and Moore, 1995; Willats et al., 2000), provides fur-
ther evidence that pectins are synthesized in the Golgi apparatus (Staehelin
and Moore, 1995) and suggests that specic pectic carbohydrate epitopes are
sublocalized in the Golgi. Studies using antibodies reactive to HGA-like and
the RG-I-like epitopes suggest that the syntheses of homogalacturonan (HGA)
and rhamnogalacturonan I (RG-I) begin in the cis-Golgi (Lynch and Staehelin,
1992; Zhang and Staehelin, 1992; Staehelin and Moore, 1995) and continue into
the medial Golgi (Moore et al., 1991; Zhang and Staehelin, 1992; Staehelin and
Moore, 1995) with more extensive branching taking place in the trans-Golgi
cisternae (Zhang and Staehelin, 1992; Staehelin and Moore, 1995). Further-
more, studies using antibodies reactive against relatively unesteried HGA
(JIM5 (VandenBosch et al., 1989; Knox et al., 1990); PGA/RG-I (Moore et al.,
1986; Moore and Staehelin, 1988; Lynch and Staehelin, 1992); 2F4 (Liners
et al., 1989)) and relatively esteried HGA (JIM7 (Knox et al., 1990)) sug-
gest that HGA becomes methyl-esteried in the medial and trans-Golgi (Vian
and Roland, 1991; Liners and Van Cutsem, 1992; Zhang and Staehelin, 1992;
Sherrier andVandenBosch, 1994; StaehelinandMoore, 1995), andis transported
to the plasma membrane in vesicles as a highly methyl-esteried polymer that
is inserted into the wall (Carpita and Gibeaut, 1993; Liners et al., 1994; Dolan
et al., 1997). The de-esterication of HGA by pectin methylesterases (Micheli,
2001) in the wall or at cell plate (Dolan et al., 1997) produces more acidic
HGA (Stoddart and Northcote, 1967; Shea et al., 1989; Liners and Van Cutsem,
1992; Li et al., 1994; Marty et al., 1995). Such a spatial partitioning of HGA
esterication and de-esterication is supported by the localization of esteried
HGA throughout the cell wall (Fujiki et al., 1982; Knox et al., 1990; Vian
and Roland, 1991; Liners and Van Cutsem, 1992; Liners et al., 1994; Sherrier
and VandenBosch, 1994; Marty et al., 1995; Dolan et al., 1997; Willats et al.,
2001a), the localization of relatively unesteried HGA in the middle lamella,
F
i
g
u
r
e
3
.
2
T
w
o
m
o
d
e
l
s
f
o
r
t
h
e
s
u
b
c
e
l
l
u
l
a
r
l
o
c
a
l
i
z
a
t
i
o
n
o
f
t
h
e
n
u
c
l
e
o
t
i
d
e
-
s
u
g
a
r
t
r
a
n
s
f
o
r
m
i
n
g
e
n
z
y
m
e
s
r
e
q
u
i
r
e
d
f
o
r
p
e
c
t
i
n
s
y
n
t
h
e
s
i
s
.
(
a
)
M
o
d
e
l
s
h
o
w
i
n
g
t
h
e
p
r
o
d
u
c
t
i
o
n
o
f
a
r
e
q
u
i
r
e
d
n
u
c
l
e
o
t
i
d
e
-
s
u
g
a
r
o
n
t
h
e
c
y
t
o
s
o
l
i
c
s
i
d
e
o
f
t
h
e
G
o
l
g
i
.
S
u
b
s
e
q
u
e
n
t
u
s
e
o
f
t
h
e
s
u
g
a
r
b
y
a
G
o
l
g
i
l
u
m
e
n
a
l
g
l
y
c
o
s
y
l
t
r
a
n
s
f
e
r
a
s
e
r
e
q
u
i
r
e
s
t
r
a
n
s
p
o
r
t
o
f
t
h
e
s
u
g
a
r
i
n
t
o
t
h
e
G
o
l
g
i
l
u
m
e
n
.
(
b
)
M
o
d
e
l
s
h
o
w
i
n
g
t
h
e
t
r
a
n
s
p
o
r
t
o
f
a
p
r
e
c
u
r
s
o
r
n
u
c
l
e
o
t
i
d
e
-
s
u
g
a
r
i
n
t
o
t
h
e
G
o
l
g
i
a
n
d
t
h
e
s
u
b
s
e
q
u
e
n
t
G
o
l
g
i
l
u
m
e
n
-
l
o
c
a
l
i
z
e
d
t
r
a
n
s
f
o
r
m
a
t
i
o
n
o
f
t
h
e
p
r
e
c
u
r
s
o
r
n
u
c
l
e
o
t
i
d
e
-
s
u
g
a
r
i
n
t
o
a
n
u
c
l
e
o
t
i
d
e
-
s
u
g
a
r
r
e
q
u
i
r
e
d
f
o
r
p
e
c
t
i
n
s
y
n
t
h
e
s
i
s
.
O
n
c
e
p
r
e
s
e
n
t
i
n
t
h
e
G
o
l
g
i
t
h
e
s
u
g
a
r
r
e
s
i
d
u
e
o
f
a
p
p
r
o
p
r
i
a
t
e
n
u
c
l
e
o
t
i
d
e
-
s
u
g
a
r
s
i
s
t
r
a
n
s
f
e
r
r
e
d
o
n
t
o
a
g
r
o
w
i
n
g
p
e
c
t
i
c
p
o
l
y
m
e
r
b
y
p
e
c
t
i
n
b
i
o
s
y
n
t
h
e
t
i
c
g
l
y
c
o
s
y
l
t
r
a
n
s
f
e
r
a
s
e
s
.
T
h
e
r
e
s
u
l
t
i
n
g
N
D
P
i
s
h
y
d
r
o
l
y
z
e
d
b
y
a
G
o
l
g
i
-
r
e
s
i
d
e
n
t
n
u
c
l
e
o
s
i
d
e
-
d
i
p
h
o
s
p
h
a
t
a
s
e
a
n
d
t
h
e
r
e
s
u
l
t
i
n
g
N
M
P
i
s
t
r
a
n
s
p
o
r
t
e
d
o
u
t
o
f
t
h
e
G
o
l
g
i
b
y
a
n
a
p
p
r
o
p
r
i
a
t
e
N
M
P
:
N
D
P
-
s
u
g
a
r
t
r
a
n
s
p
o
r
t
e
r
.
F
i
g
u
r
e
3
.
2
(
c
o
n
t
i
n
u
e
d
)
58 PECTINS AND THEIR MANIPULATION
and the frequently observed absence of unesteried HGA epitopes in the trans-
Golgi vesicles. In spite of such results, however, the fact that some cell types
show a different localization of unesteried HGA, such as melon callus cells
(Vian and Roland, 1991) that contain unesteried HGA in the trans-Golgi,
suggests that HGA can be inserted into the wall in a relatively unesteried
state in some cells. This calls into question the general belief that HGA is
necessarily synthesized in a highly esteried form (Knox et al., 1990; Casero
and Knox, 1995). It is important to realize that specic pectic epitopes localize
to different Golgi compartments in different cell types (Knox et al., 1990;
Lynch and Staehelin, 1992; Casero and Knox, 1995; Staehelin and Moore,
1995), suggesting that pectin synthesis may differ in different cell types, in
different species, at different points during development, or even at different
locations in the same wall (Stacey et al., 1995; Willats et al., 1999; McCartney
et al., 2000; Orla and Knox, 2000; Willats et al., 2000). However, since the
absence of a specic carbohydrate epitope may be due to masking of the epitope
rather than to a lack of the synthesis of the epitope, it is necessary to conrm
carbohydrate-epitope immunocytochemistry results with localization studies on
the biosynthetic enzymes themselves in order to make conclusions regarding
the mechanism of biosynthesis.
The rst enzymatic evidence for the location of a pectin biosynthetic glyco-
syltransferase was the localization of HGA-galacturonosyltransferase (GalAT)
to the Golgi and the demonstration that its catalytic site faces the Golgi lumen
(Sterling et al., 2001). Most of the GalAT activity in pea (Pisum sativum) co-
localizes in linear and discontinuous sucrose gradients with the Golgi marker
latent UDPase and is separated from the endoplasmic reticulum, mitochondria
and plasma membrane (Sterling et al., 2001). The catalytic site of GalAT was
shown to reside within the lumen of the Golgi, since GalATactivity was reduced
by treatment with proteinase K only if Golgi membranes were rst permeabi-
lized with detergent (Sterling et al., 2001). The enzymes that methyl-esterify
HGA have also been localized to the Golgi, further conrming the location of
pectin synthesis. Tobacco HGAmethyltransferase (HGA-MT) activity localizes
to the Golgi and the enzymes catalytic site was shown to face the Golgi lumen
(Goubet and Mohnen, 1999a). The localization of roughly half of the pectin
methyltransferase activity in ax to the Golgi (Vannier et al., 1992; Bourlard
et al., 1997b) provides further evidence that HGA methyl-esterication occurs
in the Golgi.
The location of pectin synthesis in the Golgi leads to the question of where the
nucleotide-sugar substrates are synthesizedandof howthe substrates gainaccess
tothe enzyme. It has beenproposedthat the nucleotide-sugars requiredfor pectin
synthesis are synthesized on the cytosolic side of the Golgi and transported
into the Golgi lumen by specic nucleotide-sugar:nucleoside monophosphate
antiporters (Sterlinget al., 2001) (see Figure 3.2a). While this model is consistent
with the topology of some nucleotide-sugar biosynthetic enzymes in animals
(Berninsone and Hirschberg, 2000) and plants (Schroeder and Hagiwara, 1989;
Munoz et al., 1996; Neckelmann and Orellana, 1998; Baldwin et al., 2001),
there are also indications that some nucleotide biosynthesis enzymes, such as
UDP-glucuronic acid decarboxylase (Hayashi et al., 1988; Kearns et al., 1993),
may actually reside in the Golgi (see Figure 3.2b). Thus, until the subcellular
location of the enzymes is conrmed experimentally, two models for the location
of the nucleotide-transforming enzymes must be considered (Figure 3.2). For
those nucleotide-sugars that are synthesized in the cytosol, it has been shown in
both animals (Capasso and Hirschberg, 1984) and plants (Munoz et al., 1996;
Wang et al., 1997; Neckelmann and Orellana, 1998; Baldwin et al., 2001) that
transport occurs via nucleotide-sugar:nucleoside monophosphate antiporters
that reside in the endoplasmic reticulum or Golgi membranes. As shown in
Figure 3.2a, nucleotide-sugars that are synthesized on the cytosolic side of the
Golgi are predictedtobe transportedintothe Golgi lumenbyspecic nucleotide-
sugar:nucleoside monophosphate antiporters. The channeling of nucleotide-
sugars from the cytosol into the Golgi may be facilitated by nucleotide-sugar
binding proteins (Faik et al., 2000). Once the nucleotide-sugar is transported
into the Golgi it is used as a substrate by a pectin biosynthetic glycosyltrans-
ferase that transfers the glycosyl residue onto a growing polymer. The released
nucleoside diphosphate (NDP) is hydrolyzed by a Golgi-localized nucleotide
-5
-diphosphatase (NDPase) (Orellana et al., 1997) into NMP and inorganic

phosphate. The nucleoside monophosphate is then transported out of the Golgi
by a nucleotide-sugar:nucleoside monophosphate antiporter.
3.4 Synthesis of the nucleotide-sugar substrates
required for pectin synthesis
It is commonly accepted that nucleotide-sugars are the immediate substrates for
the glycosyltransferases that drive pectin biosynthesis. Radiolabeled nucleotide-
sugars have been used in vitro as substrates to assay a number of pectin biosyn-
thetic glycosyltransferases including 1,4-galacturonosyltransferase (Sterling
et al., 2001; Takeuchi and Tsumuraya, 2001) and galactosyltransferase (Geshi
et al., 2000; Peugnet et al., 2001). Figure 3.3 shows a summary of the nucleotide-
sugar interconversion pathways that are believed or proposed to be the primary
pathways for the synthesis of the activated sugars required for pectin synthe-
sis (Feingold and Avigad, 1980; Feingold and Barber, 1990; Mohnen, 1999;
Gibeaut, 2000; Reiter andVanzin, 2001; Ridley et al., 2001). Although the inter-
conversion pathways are likely the main pathways for the synthesis of the
nucleotide-sugars, there is a set of alternative pathways for the synthesis of the
NDP-sugars knownas the salvage pathway. Inthe salvage pathwayl-Ara, d-Gal,
d-Man (mannose), d-GalA, d-GlcA, l-Rha, l-Fuc (fucose), d-Glc (glucose) and
d-Xyl (xylose) are recycled fromthe wall by conversion into sugar-1-phosphates
F
i
g
u
r
e
3
.
3
S
u
m
m
a
r
y
o
f
n
u
c
l
e
o
t
i
d
e
-
s
u
g
a
r
t
r
a
n
s
f
o
r
m
a
t
i
o
n
p
a
t
h
w
a
y
s
r
e
q
u
i
r
e
d
f
o
r
p
e
c
t
i
n
b
i
o
s
y
n
t
h
e
s
i
s
.
A
d
a
p
t
e
d
f
r
o
m
R
e
i
t
e
r
a
n
d
V
a
n
z
i
n
(
2
0
0
1
)
,
M
o
h
n
e
n
(
1
9
9
9
)
,
F
e
i
n
g
o
l
d
a
n
d
B
a
r
b
e
r
(
1
9
9
0
)
.
N
o
t
e
t
h
a
t
t
h
e
s
a
l
v
a
g
e
p
a
t
h
w
a
y
s
a
r
e
n
o
t
s
h
o
w
n
(
s
e
e
t
e
x
t
)
.
by the action of C-1-Kinases (Hassid et al., 1959; Hassid, 1967; Feingold and
Avigad, 1980; Feingold and Barber, 1990). The resulting sugar-1-phosphates
are transformed into nucleotide-sugars by pyrophosphorylases that transfer the
nucleoside monophosphate (NMP) from a nucleoside triphosphate (NTP) onto
the phosphate of the sugar-1-phosphate withthe release of pyrophosphate: sugar-
1-P + NTP NDP-sugar + PP
i
(Hassid et al., 1959). The so-called salvage
pathwayis thought tofunctioninthe re-utilizationof glycosyl residues following
turnover of the wall. The synthesis of UDP-GlcA from GlcA-1-P derived from
myoinositol (the myoinositol pathway) (Hassid, 1967; Feingold and Barber,
1990) also uses, in part, some of the enzymes from the salvage pathway. The
genes for some of the enzymes in the salvage pathway have been identied,
including galactokinase (AGK1 and GAL1) (Kaplan et al., 1997; Sherson et al.,
1999), arabinose kinase (ARA1) (Sherson et al., 1999), and GDP-d-mannose
pyrophosphorylase (also referred to mannose-1-phosphate guanylyltransferase)
(VTC1 and CYT1) (Keller et al., 1999; Lukowitz et al., 2001).
Although the amount of each type of glycosyl residue required for wall
synthesis will depend on the exact structure of pectin being synthesized in
that cell type at that specic point of development, a comparison of the glycosyl
residue composition of pectins isolated fromwalls of sycamore cell suspensions
(Eberhard et al., 1989), tobacco cell suspensions (Mohnen et al., 1996) and
Arabidopsis leaves (Zablackis et al., 1996) (see Table 3.1) gives an indication
of the relative proportion of the different glycosyl residues in pectin. The most
abundant glycosyl residue in pectin is d-GalA followed by, depending on the
species and cell type, l-Ara, l-Rha, d-Gal, d-GlcA and d-Xyl. The other
glycosyl residues required for pectin synthesis (i.e. l-Fuc, l-Gal, d-Apiose,
l-aceric acid, d-Kdo (ketodeoxymanno-octulopyranosylonic acid), and d-Dha
(deoxylyxo-heptulopyranosylaric acid)) represent 1% or less of normalized
mol% of pectin (Ridley et al., 2001).
The nucleotide-sugars involved in pectin synthesis (Mohnen, 1999; Ridley
et al., 2001) and in general cell wall synthesis (Feingold and Avigad, 1980;
Feingold and Barber, 1990; Gibeaut, 2000) and the molecular genetics of plant
nucleotide-sugar interconversion pathways (Reiter andVanzin, 2001) have been
reviewed. The reader is directed to these reviews for additional background
information. Here, only the salient features of the biosynthetic pathways will
be presented. Hexose phosphates made directly from carbohydrate products of
photosynthesis or from transported sucrose or stored starch are the precursors
for the nucleotide-sugars (see Figure 3.3). As mentioned above, while it has tra-
ditionally been held that the nucleotide-sugars are synthesized on the cytosolic
side of the Golgi, there is evidence that some nucleotide-sugars, such as UDP-
Xyl, may at least in part be synthesized in the Golgi lumen (Hayashi et al.,
1988). The nucleotide-sugars required for pectin synthesis will be described in
the order of the abundance of their respective glycosyl residues in pectin, as
based on the composition of the three different pectins shown in Table 3.1.
Table 3.1 Comparison of the glycosyl residue composition
a
of pectic polysaccharides
b
released from
tobacco, sycamore and Arabidopsis walls
Normalized mol%
Glycosyl residue
a
Sycamore suspension
c
Tobacco suspension
d
Arabidopsis leaves
e
Galacturonic acid 56.0 74.2 72.1
Arabinose 15.2 2.6 6.6
Rhamnose 7.4 6.4 9.8
Galactose 11.6 1.4 6.5
Glucuronic acid 5.0 7.5 0.3
Xylose 1.5 0.5 2.0
Fucose 1.2 1.0 0.7
Unknown
f
6.5
Mannose 1.8 0 0
a
Glycosyl residues <1% of total are not shown.
b
Pectic polysaccharides released from total walls by digestion with endopolygalacturonase.
c
Data from Eberhard et al. (1989).
d
Data from Mohnen et al. (1996).
e
Calculated from data in Zablackis et al. (1996).
f
An unidentied uronic acid.
3.4.1 Uridine diphosphate--d-galacturonic acid (UDP-GalA)
UDP-GalA is a substrate for the synthesis of all the pectic polymers (i.e.
HGA, RG-I, RG-II). The main route for synthesis of UDP-GalA in the plant is
the 4-epimerization of UDP-GlcA catalyzed by UDP-glucuronate 4-epimerase
(UDP-GlcA 4-epimerase) (EC 5.1.3.6) (Neufeld et al., 1958; Feingold et al.,
1960; Ankel and Tischer, 1969; Feingold and Avigad, 1980; Mitcham et al.,
1991; Liljebjelke et al., 1995; Eidson et al., 1996). The reaction is believed to
proceed through a 4-keto intermediate and may require a tightly bound NAD
+
coenzyme (Feingold andAvigad, 1980). UDP-glucuronate 4-epimerase activity
has been recovered from plant homogenates as both soluble and membrane-
bound enzyme (Feingold et al., 1960; Liljebjelke et al., 1995).
UDP-d-GlcA
UDP-GlcA 4-epimerase
UDP-d-GalA (1)
The biochemically best-characterized plant UDP-GlcA 4-epimerase is that
from the blue green alga Anabaena os-aquae (Gaunt et al., 1974). The
Anabaena UDP-GlcA 4-epimerase has a K
m
for UDP-GlcA of 37 M, a pH
optimum of 8.5, and an equilibrium constant of 2.6 in the direction of UDP-
GalA formation (Gaunt et al., 1974). Crude extracts from many different plant
species have been shown to have UDP-GlcA 4-epimerase activity and have
been used to make radiolabeled UDP-GalA via the 4-epimerization of UDP-
GlcA radiolabeled in the sugar (Neufeld et al., 1958; Feingold et al., 1960;
Mitcham et al., 1991; Liljebjelke et al., 1995) or in the nucleotide (Orellana
and Mohnen, 1999) moiety. UDP-[
14
C]GalA has also been synthesized for
in vitro use by the enzymatic oxidation of UDP-[
14
C]Gal to UDP-[
14
C]GalA
(Rao and Mendicino, 1976; Kelleher and Bhavanandan, 1986; Basu et al., 2000)
with a yield of >90% UDP-[
14
C]GalA using a simple polyethyleneimine (PEI-
cellulose) column chromatography purication step (Basu et al., 2000). We
nd it necessary to purify the UDP-[
14
C]GalA synthesized from UDP-[
14
C]Gal
by high-pressure liquid chromatography (HPLC) to remove contaminants that
inhibit 1,4-galacturonosyltransferase activity. With the HPLCpurication step
we obtain 27% yield of UDP-[
14
C]GalA (J. Sterling and D. Mohnen, unpub-
lished results). The UDP-GlcA 4-epimerase from plants has not been puried
to homogeneity, nor has its gene been cloned. However, a gene (Cap1J) for a
bacterial UDP-GlcA 4-epimerase from Streptococcus pneumoniae type 1 has
been identied (Mu nos et al., 1999). The bacterial enzyme has a K
m
for UDP-
GlcA of 240 M, a pH optimum of 7.5, an equilibrium constant of 1.3 in the
direction of UDP-GalA, and M
r
of 80,000. The bacterial enzyme appears to
require a tightly bound NAD
+
for activity (Mu nos et al., 1999). Reiter and
Vanzin (2001) report that BLASTsearches of the Arabidopsis genome yields six
predicted coding regions with high degrees of sequence similarity to the Cap1J
gene from Streptococcus pneumoniae. Denitive evidence that these putative
UDP-GlcA epimerase genes encode functional UDP-GlcA 4-epimerase has not
yet been reported.
3.4.2 Uridine diphosphate--l-arabinose (UDP-l-Ara)
UDP-l-Ara is a substrate for the synthesis of RG-I and RG-II. UDP-l-Ara
is formed by the 4-epimerization of UDP-Xyl catalyzed by UDP-arabinose 4-
epimerase (EC5.1.3.5) (Feingold et al., 1960; Fan and Feingold, 1970; Feingold
and Avigad, 1980; Robertson et al., 1995). UDP-arabinose 4-epimerase activ-
ity has been identied in particulate preparations from multiple plant species
(Feingold and Avigad, 1980).
UDP-d-Xyl
UDP-xylose 4-epimerase
UDP-l-Ara (2)
A partially puried UDP-xylose 4-epimerase from wheat germ (Fan and
Feingold, 1970) was shown to have a pH optimum of 8.0, an apparent K
m
of
1.5 mM for UDP-Xyl and 0.5 mM for UDP-l-Ara, and an equilibrium constant
of 0.8 in the direction of UDP-Ara (Fan and Feingold, 1970). A comparable
equilibrium constant of 1.0 has been reported for the UDP-xylose 4-epimerase
from mung bean (Feingold et al., 1960). If NAD
+
is required for the reaction,
it must be tightly bound to the enzyme (Feingold and Avigad, 1980). The
wheat germ UDP-xylose 4-epimerase (Fan and Feingold, 1970; Feingold and
Avigad, 1980) has been used to synthesize non-radiolabeled and radiolabeled
UDP--l-arabinopyranose (Pauly et al., 2000) for use for in vitro wall polymer
biosynthesis studies. The Ara in the synthesized UDP-Ara is in the pyranose
form, while the predominant form of arabinose is arabinofuranose in the wall
polysaccharides, proteoglycans, arabinans and arabinogalactan proteins (Hassid
et al., 1959; Feingold and Avigad, 1980; Carpita, 1996). It is not known when
the mutorotation occurs; however, it is believed to occur during polysaccha-
ride biosynthesis, presumably by arabinosyltransferase(s) that catalyze ring
rearrangement before formation of the glycosidic bond (Carpita, 1996). An
Arabidopsis mutant (mur4) has been identied that has reduced membrane-
bound UDP-xylose 4-epimerase activity in the leaves, cotyledons and owers
(Burget and Reiter, 1999).
3.4.3 Uridine diphosphate--l-rhamnose (UDP-l-Rha)
l-Rhamnose is a component of RG-I and RG-II. Plants such as tobacco (Barber,
1963), mung bean (Barber, 1962), Silene dioica (Kamsteeg et al., 1978),
pummelo (Citrus maxima) (Bar-Peled et al., 1991) and Chlorella pyrenoidasa
(Barber and Chang, 1967) can convert UDP-d-Glc to UDP-l-rhamnose in an
NADH-dependent reaction (reviewed in Feingold and Avigad, 1980; Feingold
and Barber, 1990). UDP-4-keto-6-deoxy-d-Glc is an intermediate in the con-
version (Barber, 1963; Barber and Chang, 1967; Kamsteeg et al., 1978). UDP-
Rha has been shown to be a substrate in plants for the rhamnosylation of
secondary metabolites such as avonoid-glycosides (Bar-Peled et al., 1993)
and it is assumed that UDP-l-Rha is the nucleotide-sugar substrate for the
synthesis of RG-I and RG-II. However, it should be noted that it has not yet
been experimentally conrmed that UDP-Rha is the substrate for RG-I and RG-
II synthesis. A biosynthetic scheme for the synthesis of UDP-Rha in plants has
been proposed (Kamsteeg et al., 1978; Feingold and Avigad, 1980; Mohnen,
1999) based on the rfb genes-encoded pathway for the synthesis of dTDP-
rhamnose fromdTDP-glucose in bacteria (Stevenson et al., 1994). The proposed
pathway is shown in equation 3.
UDP-d-Glc
UDP-glucose
4,6-dehydratase
[UDP-4-keto-6-deoxy-Glc]
UDP-4-keto-l-rhamnose
3,5-epimerase
[UDP-4-keto-6-deoxy-l-mannose]
UDP-4-ketorhamnose
reductase
UDP-l-Rha (3)
Basedonthe bacterial pathway, the proposedpathwayfor UDP-Rha synthesis
in plants begins with the conversion of UDP-d-Glc to UDP-4-keto-6-deoxy-Glc
catalyzed by UDP-glucose 4,6-dehydratase (EC 4.2.1.76). The UDP-4-keto-
6-deoxy-Glc is subsequently epimerized to UDP-4-keto-6-deoxy-l-mannose
by UDP-4-keto-l-rhamnose 3,5-epimerase. Finally, the UDP-4-keto-6-deoxy-
l-mannose is reduced to UDP-l-rhamnose by UDP-4-ketorhamnose reductase.
None of these enzymes has been puried to homogeneity or cloned in plants. It is
also not clear how many enzymes would encode the required enzyme activities.
Blast searches of the Arabidopsis genome database using E. coli dTDP-l-
rhamnose biosynthetic genes (E. coli has unique genes for each of the three
required enzymatic activities for dTDP-rhamnose synthesis) have identied
three Arabidopsis genes with signicant sequence similarity to dTDP-d-glucose
4,6-dehydratase (Reiter and Vanzin, 2001). Based on the primary sequence of
these genes, it has been proposed that they may each encode all three enzymatic
activities required for UDP-Rha synthesis (Reiter and Vanzin, 2001). Blast
searchers have also identied other Arabidopsis genes with sequence similarity
to individual E. coli genes that encode only one of the enzymatic activities
required for dTDP-Rha synthesis (see Reiter and Vanzin, 2001). However,
direct proof that any of these Arabidopsis putative UDP-Rha biosynthetic genes
actually encodes a UDP-Rha biosynthetic protein has not yet been reported.
3.4.4 Uridine diphosphate--d-galactose (UDP-Gal)
UDP-Gal is a substrate for the synthesis of RG-I and RG-II. UDP-Gal is
formed fromUDP-Glc by a 4-epimerization catalyzed by UDP-Glc 4-epimerase
(EC 5.1.3.2) (Fan and Feingold, 1969; Feingold and Avigad, 1980). The reac-
tion mechanism includes an enzyme-bound UDP-4-keto-hexose intermediate
(Maxwell, 1957; Maitra and Ankel, 1971; Wee and Frey, 2001) which binds
the enzyme approximately 100 times more tightly than UDP-Glc (Feingold and
Avigad, 1980; Wee and Frey, 2001).
UDP-Glc
UDP-Glc 4-epimerase
UDP-Gal (4)
The structure of the enzyme from E. coli has been determined by X-ray crys-
tallography (Bauer et al., 1992). The bacterial enzyme comprises two identical
39.5 kDa subunits (WilsonandHogness, 1969; Bauer et al., 1992), eachof which
binds a NAD
+
cofactor (Bauer et al., 1992). Each subunit folds into a distinct
N-terminal domain, primarily responsible for NAD
+
/NADH positioning, that
has a seven-stranded parallel -pleated sheet anked on either side by -helices.
The small C-terminal motif is responsible for binding the UDP-sugar (Thoden
et al., 1996a, 1996b; Thoden and Holden, 1998). The active site is located
between the two domains. The size of the enzyme varies in different species, as
does the tightness by which the enzyme binds NAD
+
. For example, the bovine
enzyme is a monomer of 40 kDa that requires exogenous NAD
+
for activity
while the UDP-d-Glc 4-epimerase from Candida pseudotropicalis is made up
of two identical 60 kDa subunits each of which contains one tightly bound
NAD
+
(Maxwell, 1957; Geren and Ebner, 1977; Feingold and Avigad, 1980).
The UDP-Glc 4-epimerase from leaves of Vicia faba is a soluble cytoplasmic
protein with a pH optimum of 8.8 and an apparent K
m
for UDP-Gal of 95 M
(K onigs and Heinz, 1974). A UDP-Glc 4-epimerase puried from wheat germ
extract has M
r
of 100 000 and requires NAD
+
for activity (Fan and Feingold,
1969; Feingold and Avigad, 1980). The tightness of binding of NAD
+
to plant
UDP-Glc 4-epimerase appears to be species-specic (Feingold and Avigad,
1980) and both soluble and membrane-bound activities have been recovered
in plants (Feingold and Avigad, 1980). An Arabidopsis gene for UDP-Glc 4-
epimerase (UGE1) has been cloned and expressed in E. coli (Dormann and
Benning, 1996). The Arabidopsis expressed protein encodes a 39 kDa protein
with a broad pH optimum from 7.0 to 9.55 and an apparent K
m
for UDP-Glc of
110 M (Dormann and Benning, 1996). A cDNA encoding a predicted 39 kDa
putative UDP-Glc 4-epimerase frompea (Pisumsativum) that has 92%sequence
homology to the Arabidopsis gene has also been cloned (Lake et al., 1998)
although no characteristics of the enzyme have been reported. Two cDNAs that
encode UDP-Glc epimerases of 39.3 and 38.4 kDa from developing seeds of
guar (Cyamopsis tetragonoloba) endosperm have also been reported (Joersbo
et al., 1999). Analysis of the Arabidopsis genome has led to the identication
of four coding regions with signicant sequence identity to UGE1 (Reiter and
Vanzin, 2001), two of which (UGE2 and UGE3) encode functional UDP-Glc
epimerases (Reiter and Vanzin, 2001).
3.4.5 Uridine diphosphate--d-glucuronic acid (UDP-GlcA)
UDP-d-Glucuronic acid is the likely substrate for the incorporation of GlcAinto
RG-II and into some side branches of RG-I. UDP-GlcA is produced either by
the oxidation of UDP-Glc catalyzed by UDP-Glc 6-dehydrogenase (Feingold
et al., 1960; Feingold and Avigad, 1980) or by the uridylation of Glc-1-P via
the myoinositol pathway (Feingold and Avigad, 1980).
UDP-d-Glc
UDP-glucose dehydrogenase
UDP-d-GlcA (5)
UDP-Glc 6-dehydrogenase (EC 1.1.1.22) catalyzes the 4-electron oxidation
of UDP-Glc at C-6 and the reduction of two moles of NAD
+
(Feingold and
Avigad, 1980; Feingold and Franzen, 1981). The reaction is ordered: beginning
with binding of UDP-Glc, followed by binding of NAD
+
(Feingold andAvigad,
1980; Hempel et al., 1994; Campbell et al., 2000), reduction of the rst bound
NAD
+
and release of the rst NADH. This is followed by binding of the second
NAD
+
, reduction and release of the second NADH and nally release of the
UDP-GlcA (Feingold and Avigad, 1980; Campbell et al., 1997). Bovine UDP-
Glc 6-dehydrogenase consists of six 52 kDa subunits (Zalitis and Feingold,
1969; Gainey et al., 1972; Franzen et al., 1978; Feingold and Avigad, 1980;
Jaenicke et al., 1986; Hempel et al., 1994) with one mole of substrate bound per
two moles of enzyme (i.e. half-of-the-site behavior) (Franzen et al., 1978;
Hempel et al., 1994). In contrast, UDP-Glc 6-dehydrogenase from E. coli
consists of two identical subunits of 50 kDa each (Schiller et al., 1976; Feingold
and Franzen, 1981). The X-ray crystal structure of UDP-glucose dehydroge-
nase from the bacteria Streptococcus pyogenes has been solved (Campbell
et al., 2000). The S. pyogenes UDP-Glc dehydrogenase appears to exist as
either a monomer or dimer in solution. Each monomer consists of two discrete
/ domains connected by a long -helix. Each domain consists of a core
-sheet sandwiched between -helices. The N-terminal domain contains a six-
stranded parallel -sheet that binds NAD
+
(Campbell et al., 2000). UDP-
Glc 6-dehydrogenases have been puried 1000-fold from pea ((Strominger
and Mapson, 1957), 12-fold from germinating lily (Lilium longiorum) pollen
(Davies and Dickinson, 1972), 62-fold from soybean nodules (Stewart and
Copeland, 1998), and 341-fold from elicitor-treated French bean (Phaseolus
vulgaris L) cell suspensions (Robertson et al., 1996). The apparent K
m
values
for UDP-Glc for the different enzymes were 70 M, 300 M, 50 M, and
5.5 mM, respectively, and the apparent K
m
values for NAD
+
were 115 M,
400 M, 120 M and 20 M. The soybean enzyme has a pH optimum of 8.4,
a native molecular mass of 272 kDa, and a subunit molecular mass of 47 kDa,
suggesting that the enzyme functions as a hexamer (Stewart and Copeland,
1998). All known eukaryotic UDP-Glc 6-dehydrogenases are cooperatively
inhibited by UDP-Xyl, suggesting a feedback inhibition of the enzyme by
UDP-Xyl (Feingold and Avigad, 1980; Campbell et al., 1997). A cDNA clone
for UDP-Glc 6-dehydrogenase from soybean that is highly homologous to
the cloned bovine UDP-GlcDH gene (Hempel et al., 1994) encodes a protein
with a predicted molecular mass of 52.9 kDa (Tenhaken and Thulke, 1996).
The soybean gene has a conserved NAD
+
-binding site motif and contains the
catalytic Cys residue (Hempel et al., 1994; Tenhaken and Thulke, 1996). An
Arabidopsis gene (UGD) encoding UDP-Glc dehydrogenase has been identied
and its gene expression has been studied using -glucuronidase and green uo-
rescent protein reporter constructs (Seitz et al., 2000). Three additional putative
UDP-Glc dehydrogenases have been identied in Arabidopsis via sequence
analysis (Reiter and Vanzin, 2001). The four genes share 8393% sequence
identity (Seitz et al., 2000). The protein with UDP-Glc 6-dehydrogenase activity
that was puried from French bean (Robertson et al., 1996) does not share
the characteristics of the cloned UDP-GlcDH from soybean (Strominger and
Mapson, 1957) or Arabidopsis (Seitz et al., 2000). The putative UDP-GlcDH
fromFrenchbean(Robertsonet al., 1996) has a molecular mass of 40 kDa, a high
apparent K
m
of 5.5 mM for UDP-Glc, co-puries with alcohol dehydrogenase
activity and is preferentially located in cells that make secondary walls. It is
unclear whether the 40 kDa protein from French bean represents a bona de
multifunctional UDP-GlcDH preferentially expressed during secondary wall
synthesis (Robertson et al., 1996), or whether it is an alcohol dehydrogenase
that can oxidize UDP-Glc in vitro but plays little or no role in the formation of
UDP-GlcA in planta.
3.4.6 Uridine diphosphate--d-xylose (UDP-Xyl)
UDP-Xyl is the expected substrate for the synthesis of xylogalacturonan and
RG-II. UDP-Xyl is produced by the decarboxylation of UDP-GlcA catalyzed
by UDP-GlcA carboxylase (EC 4.1.1.35) (Feingold et al., 1960; Feingold and
Avigad, 1980; Hayashi et al., 1988; Hannapel, 1991).
UDP-d-GlcA
UDP-GlcA decarboxylase
UDP-d-Xyl (6)
UDP-GlcA decarboxylase contains a tightly-bound NAD
+
and catalyzes a
reaction that proceeds via a UDP-4-keto-hexose intermediate (Feingold and
Avigad, 1980). Partially puried UDP-GlcA decarboxylase from wheat germ
(John et al., 1977) has a pH optimum of 7.0 and consists of two 210 kDa
isoenzymes that do not require exogenous NAD
+
for activity. Both isozymes are
activated by low (<100 M) concentrations of UDP-GlcA, indicating coopera-
tive allosteric regulation by UDP-GlcA (John et al., 1977). The apparent K
m
values of the fully activated wheat germisozymes for UDP-GlcAwere 0.18 mM,
and 0.53 mM, respectively. Both UDP-GlcA decarboxylase isozymes are allo-
sterically inhibited by UDP-Xyl (John et al., 1977). UDP-GlcAdecarboxylase is
recovered as both a soluble and membrane-bound enzyme (Hayashi et al., 1988).
In soybean, the membrane-bound UDP-GlcA carboxylase has a pH optimum
of 6.07.5 and an apparent K
m
of 240 M for UDP-GlcA (Hayashi et al.,
1988) while the soluble UDP-GlcA carboxylase has an apparent K
m
of 700 M
(Hayashi et al., 1988). At least some of the UDP-GlcA carboxylase activity has
been reported to reside within the lumen of the Golgi (Hayashi et al., 1988).
The rst gene for UDP-GlcA decarboxylase was identied in Cryptococcus
neoformans (Bar-Peled et al., 2001). The C. neoformans gene encodes a
46.5 kDa protein that catalyzes the production of UDP-Xyl from UDP-GlcA.
The enzyme has an apparent K
m
for UDP-GlcA of 700 M, a pH optimum
of 7.5 and is inhibited by NADH, UDP and UDP-Xyl. Three Arabidopsis
genes shown to encode functional UDP-d-glucuronate decarboxylases (acces-
sions AF387787, AF387788, AF387789) have been identied (M. Bar-Peled,
unpublished). The purication and partial sequencing of UDP-d-glucuronate
decarboxylases from pea has led to the identication of a pea gene (accession
BAB40967) that encodes a functional UDP-GlcAdecarboxylase (see Reiter and
Vanzin, 2001).
3.4.7 Uridine diphosphate--l-fucose (GDP-Fuc)
GDP-l-Fuc is the likely substrate for the synthesis of RG-II and for some of
the side branches of RG-I. Soluble enzyme preparations from various plant
species can convert GDP-d-Man to GDP-l-Fuc (Liao and Barber, 1971). The
synthesis of GDP-l-Fuc using enzyme preparations from Phaseolus vulgaris
requires NADPHor NADH, occurs at a pHoptimumof 6.97.8, has an apparent
K
m
for GDP-d-Man of 160 M (Liao and Barber, 1971) and an apparent
molecular mass of 120 kDa (Liao and Barber, 1972). The reaction proceeds
via the C-4 oxidation and C-6 reduction of GDP-Man catalyzed by GDP-
d-Man 4,6-dehydratase (EC 4.2.1.47) (Liao and Barber, 1971; Feingold and
Avigad, 1980). The product formed, GDP-4-keto-6-deoxy-d-mannose, appears
to tightly bind a GDP-4-keto-6-deoxy-d-Man 3,5-epimerase, which converts
it to a GDP-4-keto-6-deoxy-l-galactose intermediate (Feingold and Avigad,
1980; Bonin et al., 1997) that is then reduced by a GDP-4-keto-l-fucose reduc-
tase activity to yield GDP-l-fucose (Feingold and Avigad, 1980). Although it
was previously thought that the nal two enzyme activities might reside on
separate proteins, it has been shown in humans (Sullivan et al., 1998) and
in transgenic Saccharomyces cerevisiae harboring the E. coli genes (Mattila
et al., 2000) that a single enzyme, GDP-keto-6-deoxymannose 3,5-epimerase-
4-reductase, catalyzes both the epimerization and reduction steps. The rst
enzyme in the pathway, GDP-d-Man-4,6-dehydratase, is inhibited by GDP-
fucose (Sullivan et al., 1998; Kornfeld and Ginsburg, 1966), indicating feedback
inhibition.
GDP-d-Man
GDP-d-Man 4,6-dehydratase
GDP-4-keto-6-deoxy-d-mannose
GDP-keto-6-deoxymannose
3,5-epimerase-4-reductase
GDP-Fuc (7)
The Arabidopsis mutant mur1 is defective in the synthesis of l-fucose in
the aerial parts of the plant (Reiter et al., 1993). The MUR1 gene encodes
a 41.9 kDa GDP-d-mannose 4,6-dehydratase (Bonin et al., 1997). A second
Arabidopsis gene, GMD1, encodes a second GDP-d-mannose 4,6-dehydratase
that is highlyexpressedinroots (Boninet al., 1997). TheArabidopsis gene GER1
encodes a GDP-keto-6-deoxymannose 3,5-epimerase-4-reductase (Bonin and
Reiter, 2000). A second putative GDP-keto-6-deoxymannose 3,5-epimerase-4-
reductase gene, GER2, with 88% amino acid identity to GER1 has been identi-
ed by DNAsequence analysis (Reiter andVanzin, 2001). The dwarf phenotype
associated with mur1-1 and mur1-2 was shown to be due to a substitution of l-
galactose for the l-fucose and 2-O-methyl-l-galactose for 2-O-methyl-l-fucose
in RG-II. This change in RG-II structure results in a reduction in the amount
of borate crosslinked RG-II dimer (ONeill et al., 2001) that is present in the
walls and leads to the dwarsm. This result provides unequivocal proof that the
pectic polysaccharide RG-II is essential for normal plant growth (ONeill et al.,
2001).
3.4.8 Uridine diphosphate--d-apiose (UDP-apiose)
UDP-apiose is a substrate for the synthesis of the species-specic substituted
galacturonan apiogalacturonan that is found in some aquatic monocotyledonous
plants such as Spirodela polyrrhiza (Watson and Orenstein, 1975) and Lemna
minor (Hart and Kindel, 1970). Apiogalacturonan is a homogalacturonan in
which d-apiose or apiobiose (d-Apif-1,3-d-apiose) are attached to O-2 or O-
3 of HGA. UDP-apiose is also the likely substrate for the synthesis of RG-II
(ONeill et al., 2001; Ridley et al., 2001). UDP-apiose is formed by a decarboxy-
lation and rearrangement of UDP-GlcAcatalyzed by a NAD
+
-dependent UDP-
apiose/UDP-Xyl synthase (Wellmann and Grisebach, 1971; Baron et al., 1973;
Kindel and Watson, 1973; Watson and Orenstein, 1975; Matern and Grisebach,
1977; Feingold and Barber, 1990). UDP-Xyl is also a product of the in vitro
enzymatic reaction (Matern and Grisebach, 1977) with UDP-apiose:UDP-Xyl
ratios of 1.4 reported (Matern and Grisebach, 1977). The UDP-apiose synthase
and UDP-Xyl synthase activities could not be separated in a 1400-fold puried
protein preparation, leading to the suggestion that a single multifunctional
proteinis responsible for bothactivities (WellmannandGrisebach, 1971; Matern
and Grisebach, 1977). However, it has more recently been suggested that xylose
may be an articial product recovered in in vitro reactions (Gardiner et al., 1980)
and the name UDP-apiose synthase has been used for the enzyme (Gardiner
et al., 1980). It is believed that UDP-apiose synthesis occurs via the formation
of an l-threo-4-pentosulose intermediate, common to both UDP-apiose and
UDP-Xyl formation, followed by ring contraction and epimerization (Matern
and Grisebach, 1977).
UDP-d-GlcA
UDP-apiose synthase
UDP-d-apiose
UDP-Xyl (possible in vitro side product?) (8)
Partially puried UDP-apiose/UDP-Xyl synthase from Lemna minor has
optimum activity at 1 mM NAD
+
and a pH of 8.08.3 (Kindel et al., 1971).
Partially puried UDP-apiose/UDP-Xyl synthase from parsley (Matern and
Grisebach, 1977) is composed of an 86 kDa protein consisting of two identical
44 kDa subunits and a 65 kDa protein consisting of two identical 34 kDa subunits
(Matern and Grisebach, 1977). The 86 kDa protein contains all the enzyme
activity, binds 0.5 mol of UDP-GlcA per mol of protein and, in the presence
of UDP-GlcA, binds 0.5 mol NAD
+
per mol of catalytic protein (Matern and
Grisebach, 1977). The 65 kDa protein is enzymatically inactive but was reported
to be required for stability of the 86 kDa protein (Matern and Grisebach, 1977).
UDP-d-glucose and UDP-methyl-d-GlcA are competitive inhibitors of UDP-
apiose/UDP-Xyl synthase (Gebb et al., 1975).
3.4.9 Uridine diphosphate--l-galactose (GDP-Gal)
GDP-l-Gal is a possible substrate for the l-Gal in RG-II and for the l-Gal that
is substituted for the l-Fuc (Zablackis et al., 1996) in xyloglucan synthesized in
Arabidopsis mur1 mutants. Mur1 mutants have a mutation in GDP-d-mannose
4,6-dehydratase and, thus, have reduced amounts of l-Fuc in the aerial portions
of the plant that is replaced with l-Gal (Zablackis et al., 1996). It has been
proposed that GDP-d-Man is converted to GDP-l-Gal by GDP-d-mannose 3,5-
epimerase (Feingold and Avigad, 1980).
GDP-d-Man
GDP-d-mannose 3,5-epimerase
GDP-l-Gal (9)
The reversible 3,5-epimerization of GDP-Man has been reported using
extracts from Chlorella pyrenoidosa (Hebda et al., 1979; Hebda and Barber,
1978). The partially puried Chlorella GDP-d-mannose 3,5-epimerase had a
molecular mass of 100 kDa, a broad pH optimum centering at 8.1, an appar-
ent K
m
of 96 M for GDP-d-Man and 97 M for GDP-l-Gal, and an
equilibrium constant of 2.9 in the direction of GDP-Man (Hebda et al.,
1979).
3.4.10 XXX-Kdo, XXX-Dha and XXX-aceric acid
The identity and biosynthetic pathways for the activated glycosyl donors of the
Kdo, Dha and aceric acid in RG-II have not been experimentally established in
plants. In contrast, a great deal of information is available regarding the synthe-
sis in bacteria of cytidine 5
-monophosphate-3-deoxy-d-manno-octulosonate
(CMP-Kdo), the activated donor of the Kdo found in lipopolysaccharides and
other extracellular bacterial polysaccharides (Unger, 1981; Raetz, 1990; Pazzani
et al., 1993; Baasov and Kohen, 1995; Rosenow et al., 1995; Jelakovic et al.,
1996; Jelakovic and Schulz, 2001). Assuming that plants use CMP-Kdo to
synthesize RG-II, and that they synthesize CMP-Kdo using a similar pathway as
bacteria, the following pathway is proposed. d-Ribulose 5-phosphate is isomer-
ized to d-arabinose 5-phosphate by d-arabinose-5-phosphate isomerase (Unger,
1981). The d-arabinose 5-phosphate is condensed with phosphoenolpyruvate by
Kdo-8-phosphate synthetase (2-dehydro-3-deoxyphosphooctonate aldolase) to
form Kdo 8-phosphate (2-dehydro-3-deoxy-d-octonate 8-phosphate) (Unger,
1981; Doong et al., 1991). The 8-phosphate is removed from Kdo 8-phosphate
by Kdo-8-phosphate phosphatase to produce Kdo and inorganic phosphate
(Unger, 1981). Finally, CMP-Kdo and pyrophosphate are formed from cytidine
5
-triphosphate (CTP) and Kdo by CMP-Kdo synthetase (Unger, 1981). Kdo-

8-phosphate synthetase has been identied in multiple plant species and has
been partially puried from spinach (Doong et al., 1991). Kdo-8-phosphate
synthetase has a pH optimum of 6.2, an apparent K
m
of 270 M for arabinose
5-phosphate and an apparent K
m
of 35 M for phosphoenolpyruvate (Doong
et al., 1991). A cDNA from pea has been identied that encodes a 31.7 kDa
functional Kdo-8-phosphate synthetase whenexpressedinE. coli (Brabetz et al.,
2000). The expressed pea enzyme has a pH optimum of 6.1.
The identity and the biosynthetic pathway in plants for the activated glycosyl
donor for Dha (3-deoxy-d-lyxo-2-heptulosaric acid) is not known. However,
it has been proposed that 3-deoxy-d-arabino-heptulosonate 7-phosphate syn-
thase could catalyze the condensation of phosphoenolpyruvate with threose
to generate a precursor of Dha (Doong et al., 1992). A cytosolic form of 3-
deoxy-d-arabino-heptulosonate 7-phosphate synthase with a wide substrate
specicity has been identied in plants (Doong et al., 1992). cDNAs encoding
plastidic 3-deoxy-d-arabino-heptulosonate 7-phosphate synthases involved in
the shikimate pathway leading to aromatic secondary metabolism have been
identied from potato (Solanum tuberosum L.) (Dyer et al., 1990) and other
plant species (Herrmann, 1995). An alternative route for the synthesis of the
activated donor of Dha might be the interconversion of CMP-Kdo to CMP-
Dha through oxidation and decarboxylation reactions. There is no information
available regarding the nature or mode of synthesis of the activated donor for
aceric acid.
3.5 Glycosyltransferases involved in pectin biosynthesis
Elucidating the mechanism by which polysaccharides are synthesized is a chal-
lenging endeavor. Polymer synthesis involves at least three stages: initiation,
elongation and termination. While nothing is known about the initiation and
termination of pectin synthesis, considerable effort has been directed towards
identifying and characterizing glycosyltransferases that presumably catalyze the
elongation of pectin. Such glycosyltransferases transfer a glycosyl residue from
an activated sugar donor (e.g. a nucleotide-sugar) either onto endogenous pectic
acceptors in the Golgi or onto exogenous oligosaccharide or polysaccharide
pectic acceptors in in vitro reactions using permeabilized Golgi or microsomes
or detergent-solubilized enzymes. Although it has recently been proposed to be
useful to make a distinction between the terms glycosyltransferase and glycan
synthase (Perrin et al., 2001) when discussing polysaccharide synthesis, it
appears premature at this time to make this distinction for the pectin biosynthetic
enzymes. Glycan synthases have been dened as enzymes that synthesize the
backbone of a polysaccharide (i.e. the 1,4-linked d-galactosyluronic acid back
bone of HGA or the alternating [-d-GalA-1,2--l-Rha-1,4] backbone of
RG-I). It is often presumed that such enzymes should act processively, that
is, catalyze multiple rounds of catalysis before releasing their oligosaccha-
ride/polysaccharide acceptor. It is likely that such processivity, if it occurs
for pectin synthesis, could require protein complexes. Such complexes may,
or may not, remain intact in the homogenates and fractions used for studying
pectin synthesis. Since both RG-I and the substituted regions of HGA (e.g.
RG-II) are highly branched, it is not clear that it would be more favorable for
overall biosynthetic rates and structure delity for pectin synthesis to occur
processively or distributively. No evidence for in vitro processivity of pectin
synthesis has yet been reported. For these reasons all the pectin biosynthetic
enzyme activities identied to date are dened in this review as glycosyltrans-
ferases. If these glycosyltransferases are eventually shown to act processively
when associated with other polypeptides in complexes, then it is recommended
that the appropriate glycan synthase name be used for the holoenzyme complex.
Alternatively, if the glycosyltransferases identied to date are eventually shown
to act processively under specic reaction conditions (e.g. in the presence of
specic cofactors or substrates), then it is recommended that these enzymes
be named their corresponding glycan synthase, as put forward by Perrin et al.
(2001).
As mentioned above, it is not known whether all of the pectic polysaccharides
are synthesized as a single polysaccharide or whether separate populations of
polymers are synthesized. It is likely that the substituted galacturonan named
RG-II is synthesized in the same polymer that contains contiguous regions
of HGA, since RG-II isolated from cell walls is extended on both ends by
regions of HGA. It is also possible that some HGA is synthesized without
substituted HGAregions. It is less clear whether the syntheses of HGAand RG-I
occur on the same polysaccharide chain or whether they occur as independent
biosynthetic events. Work from the groups of Voragen (Schols et al., 1995)
and Mort (Yu and Mort, 1996), suggesting that RG-I is covalently attached to
xylogalacturonan-like regions, indicates that RG-I and HGA may exist as a
single polymer. In the following discussion, the enzymes activities required for
the synthesis of pectin will be divided into those required for HGA synthesis,
substituted galacturoronan synthesis (including RG-II and xylogalacturonan),
and RG-I synthesis.
3.5.1 Synthesis of homogalacturonan
Homogalacturonan (HGA) is a partially methyl-esteried and acetylated homo-
polymer of 1,4-linked d-galactosyluronic acid (Ridley et al., 2001). It is
unclear how much the degree of polymerization (DP) of HGA varies within
pectin; however, a DP range of 72100 has been reported (Thibault et al.,
1993). As shown in Table 3.2, the synthesis of HGA requires at least one
homogalacturonan 1,4-galacturonosyltransferase (HGA-GalAT) (also referred
as polygalacturonate: 1,4-galacturonosyltranferase), a homogalacturonan-
methyltransferase (HGA-MT) (also referred to as pectin methyltransferase),
and a homogalacturonan 3-O-acetyltransferase (HGA-AT).
Table 3.2 Glycosyltransferase activities required for HGA biosynthesis
a
Enzyme
b
Type of transferase Acceptor substrate Enzyme activity Reference for structure
Glycosyl-
d-GalAT

GalA1,4-GalA 1,4-GalAT ONeill et al. (1990)
Methyl-
HGA methyltransferase GalA1,4-GalA
(n)
HGA-MT Goubet et al. (1998);
(HGA-MT) Vannier et al. (1992);
Kauss et al. (1967)
Acetyl-
HGA: GalA 3-O- GalA1,4-GalA
(n)
HGA-AT Ishii (1997); De Vries et al.
acetyltransferase (1986); Ishii (1995);
(HGA-AT) Rombouts and Thibault (1986)
a
Adapted from Ridley et al. (2001).
b
All sugars are d sugars and have pyranose rings unless otherwise indicated. Glycosyltranferases add to
the glycosyl residue on the left
of the indicated acceptor.

3.5.1.1 Homogalacturonan galacturonosyltransferase (HGA-GalAT)
Membrane-bound 1,4-galacturonosyltransferase (GalAT) activity has been
identied and partially characterized in mung bean (Villemez et al., 1966;
Kauss and Swanson, 1969), tomato (Lin et al., 1966), turnip (Lin et al., 1966),
and sycamore (Bolwell et al., 1985), tobacco suspension (Doong et al., 1995),
radish roots (Mohnen et al., 1999), enriched Golgi from pea (Sterling et al.,
2001), Azuki bean (Vigna angularis) (Takeuchi and Tsumuraya, 2001) and
Arabidopsis (Sterling and Mohnen, unpublished results) (see Table 3.3). The
GalAT from pea has been localized to the Golgi (Sterling et al., 2001) with
its catalytic site facing the lumenal side of the Golgi, (Sterling et al., 2001).
These results provide the rst direct enzymatic evidence that the synthesis
of HGA occurs in the Golgi. In in vitro reactions, GalAT adds [
14
C]GalA
from UDP-[
14
C]GalA (Liljebjelke et al., 1995) onto endogenous acceptors in
microsomal membrane preparations to produce radiolabeled products of large
molecular mass (i.e. 105 kDa in tobacco microsomal membranes (Doong
et al., 1995) and 500 kDa in pea Golgi (Sterling et al., 2001)). The cleav-
age of up to 89% of the radiolabeled product into GalA, digalacturonic acid
(diGalA) and trigalacturonic acid (triGalA) following exhaustive hydrolysis
with a puried endopolygalacturonase conrmed that the product synthesized
by tobacco GalAT was largely HGA. The product produced in vitro in tobacco
microsomes is 50% esteried (Doong et al., 1995), while the product pro-
duced in pea Golgi did not appear to be esteried (Sterling et al., 2001).
These results suggest that the degree of methyl-esterication of newly synthe-
sized HGA may be species-specic and that methyl-esterication occurs after
the synthesis of at least a short stretch of HGA. GalAT activity in detergent-
permeabilized microsomes frometiolated azuki bean seedlings adds [
14
C]GalA
Table 3.3 Comparison of catalytic constants and pH optimum of HGA 1,4-galacturonosyl-
transferases
a,b
Apparent
K
m
for V
max
UDP-GalA pH (pmol mg
1
Enzyme
b
Plant source (M) optimum min
1
) Reference
GalAT
a
Mung bean 1.7 6.0 4700 Villemez et al. (1966)
GalAT Mung bean n.d. n.d. n.d. Crombie and Reid (2001)
GalAT Pea n.d.
e
6.0 n.d. Cumming and Brett (1986)
GalAT Pea n.d. n.d. n.d. Sterling et al. (2001)
GalAT Sycamore 770 n.d. ? Bolwell et al. (1985)
GalAT Tobacco 8.9 7.8 150 Doong et al. (1995)
GalAT (sol)
c
Tobacco 37 6.37.8 290 Doong and Mohnen (1998)
GalAT (per)
d
Azuki bean 140 6.87.8 2700 Takeuchi and Tsumuraya
(2001)
a
Adapted from Mohnen (1999).
b
Unless indicated, all enzymes are measured in particulate preparations.
c
(sol): detergent-solubilized enzyme.
d
(per): detergent-permeabilized enzyme.
e
n.d.: not determined.
from UDP-[
14
C]GalA onto acid-soluble polygalacturonate (PGA) exogenous
acceptors (Takeuchi and Tsumuraya, 2001). Treatment of the radiolabeled prod-
uct with a puried fungal endopolygalacturonase yielded GalA and diGalA,
conrming that the activity identied was GalAT. The azuki bean enzyme
has a broad pH range of 6.87.8 and a surprisingly high specic activity of
13002000 pmol mg
1
min
1
, especially considering the large amount (3.1
4.1 nmol mg
1
min
1
) of polygalacturonase activity that was also present in the
microsomal preparations. As with the product made by tobacco, no evidence
for the processive transfer of galactosyluronic acid residues onto the acceptor
was obtained (see below).
GalAT can be solubilized from membranes with detergent (Doong and
Mohnen, 1998). Solubilized GalAT adds GalA onto the nonreducing end
(Scheller et al., 1999) of exogenous HGA acceptors of degrees of polymeri-
zation 10 (Doong et al., 1995). The bulk of the HGA elongated in vitro by
solubilized GalAT from tobacco membranes (Doong and Mohnen, 1998) and
detergent-permeabilized Golgi from pea (Sterling et al., 2001) is elongated by
a single GalA residue. These results suggest that solubilized GalAT in vitro
acts nonprocessively, (i.e. distributively). The lack of in vitro processivity of
the solubilized GalAT may indicate that the enzyme does not synthesize HGA
in a processive manner in vivo, or it may be an artifact due to the dissociation
of a required biosynthetic complex or cofactor(s)/substrate(s) during solubi-
lization of the enzyme. Attempts to recover processive in vitro GalAT activity
by using alternative pectic acceptors or high concentrations (up to 10 mM) of
UDP-GalA were not successful (H.F. Quigley and D. Mohnen, unpublished
results; Ridley et al., 2001). There is also no evidence that the inclusion of the
methyl donor S-adenosylmethionine (Takeuchi and Tsumuraya, 2001; Kauss
and Swanson, 1969; Doong et al., 1995) and/or the acetyl donor acetyl-CoA
promotes the processivity of GalAT (H.F. Quigley and D. Mohnen, unpublished
results; Ridley et al., 2001). Thus, the question of whether or not GalAT in vivo
is processive remains to be resolved. The gene for HGA-GalAT has not yet
been identied, although efforts to purify the enzyme are on-going in several
laboratories.
3.5.1.2 HGA methyltransferase (HGA-MT)
The methyl-esterication of HGA at the C-6 carboxyl group is catalyzed by
HGA methyltransferase (HGA-MT). Although the enzyme has been referred
to as pectin methyltransferase, the term HGA-MT is preferred to distinguish
HGA-MT from the enzymes that methylate RG-I or RG-II. HGA-MT has
been identied in microsomal membranes from mung bean (Kauss et al., 1967,
1969; Crombie and Reid, 1998), ax (Vannier et al., 1992; Schaumann et al.,
1993), tobacco (Goubet et al., 1998), and soybean (Ishikawa et al., 2000) (see
Table 3.4).
Membrane-bound HGA-MTs from ax (Bruyant-Vannier et al., 1996;
Bourlard et al., 1997) and tobacco (Goubet and Mohnen, 1999b) have been sol-
ubilized using detergent. Two apparent HGA-MT isozymes, PMT5 and PMT7,
from ax have been reported with pH optima of 5.0 and 6.5, respectively
(Bourlard et al., 2001). Efforts to purify these apparent isozymes resulted
in the identication of a small additional polypeptide with HGA-MT activ-
ity designated PMT18. The isoelectric points and apparent molecular masses
of PMT5, PMT 7 and PMT18 were 5.86.5, 8.79.2 and 4.04.5, and 40,
110 and 18 kDa, respectively (Bourlard et al., 2001). It is proposed that the
18 kDa protein is a subunit of the 40 and 110 kDa proteins, based on the
appearance of the larger proteins when PMT18 is rechromatographed by size-
exclusion chromatography and by the appearance of an 18 kDa band when
PMT5 and PMT7 are separated by SDS-polyacrylamide gel electrophoresis
(Bourlard et al., 2001). Furthermore, photoafnity labeling of PMT5 and PMT7
with [
3
H]S-adenosylmethionine yielded a single labeled band at 18 kDa. No
information on the gene encoding HGA-MT is available.
HGA-MT is localized to the Golgi (Vannier et al., 1992; Bourlard et al.,
1997b; Baydoun et al., 1999; Goubet and Mohnen, 1999a) with its catalytic site
facing the Golgi lumen (Goubet and Mohnen, 1999a). Available biochemical
evidence suggests that at least a small stretch of HGA is synthesized prior
to its methylation by HGA-MT in the Golgi. This conclusion is based on
studies with intact membranes which showthat UDP-GalAstimulates HGA-MT
activity (Kauss and Swanson, 1969; Goubet et al., 1998) and from studies with
detergent-permeabilized membranes and solubilized HGA-MTwhich showthat
Table 3.4 Comparison of catalytic constants and pH optimum of HGA methyltransferases
a
Apparent Apparent
K
m
for molecular
Plant SAM
d
pH mass
Enzyme
b,c
source (M) optimum V
e
max
(kDa) Reference
HGA-MT
b
Mung 59 6.67.0 2.7
f
Kauss and Hassid (1967);
bean Kauss et al. (1969);
Kauss et al. (1967)
PMT Flax 1030 6.8 n.d.
h
Schaumann et al. (1993);
Vannier et al. (1992)
PMT (sol)
g
Flax 0.5 7.1
i
or n.d. Bourlard et al. (1997a,b);
5.5
j
Bruyant-Vannier et al.
(1996)
HGA-MT Tobacco 38 7.8 49 Goubet et al. (1998)
HGA-MT (sol) Tobacco 18 7.8 7.3 Goubet and Mohnen
(1999b)
PMT-MT Soybean 230 6.8 1360 Ishikawa et al. (2000)
PMT-MT5
k
Flax n.d. 5.0 n.d. 40 Bourlard et al. (2001)
PMT-MT7
k
Flax n.d. 6.5 n.d. 110 Bourlard et al. (2001)
PMT-MT18
k
Flax n.d. n.d. n.d. 18 Bourlard et al. (2001)
a
b
Unless indicated, all enzymes are measured in particulate preparations.
c
All enzymes are thought to be HGA-MT. However, since some authors use the previous name (pectin
methyltransferase) it is included for clarity.
d
Apparent K
m
for S-adenosylmethionine.
e
V
max
in pmol min
1
mg
1
protein.
f
V
max
is calculated from data in Kauss et al. (1969).
g
(sol): detergent-solubilized enzyme.
h
n.d.: not determined.
i
From Bruyant-Vannier et al. (1996).
j
From Bourlard et al. (1997a,b).
k
Puried enzymes.
polygalacturonic or pectin are acceptors for HGA-MT in vitro. Some of the
HGA-MTs in detergent-permeabilized membranes from ax and soybean show
a preference for partially esteried pectin (Bourlard et al., 1997b; Ishikawa
et al., 2000; Bourlard et al., 2001) over polygalacturonic acid. These results
suggest that multiple HGA-MTs may exist that differ in their specicity for
HGAof differing degrees of methylation. Such HGA-MTs may be preferentially
involved in the initial methylation of HGA or in the methylation of more highly
esteried HGA.
3.5.1.3 HGA acetyltransferase (HGA-AT)
The GalA residues in HGA may, depending upon the species, be partially O-
acetylated at C-2 or C-3 (Ishii, 1995; Ishii, 1997). Pectin O-acetyltransferase
activity has been identied in microsomes from suspension-cultured potato
cells (Pauly and Scheller, 2000). The incubation of potato microsomes with
[
14
C]acetyl-CoAyieldeda salt/ethanol precipitable product fromwhichapproxi-
mately 8%of the radioactivity could be solubilized by treatment with endopoly-
galacturonase and pectin methylesterase. These results suggest that 8% of the
radiolabeled acetate was transferred either onto HGA or onto solubilized RG-
II or RG-I fragments. It remains to be shown whether the described activ-
ity represents HGA-AT or an enzyme that acetylates one of the other pectic
polysaccharides that may be covalently linked to HGA.
3.5.2 Synthesis of substituted homogalacturonans
There have been no reports of a systematic effort to study the glycosyltrans-
ferases required for the synthesis of RG-II, the most structurally complicated
polysaccharide in the cell wall. As shown in Table 3.5, at least 24 transferase
activities are likely required to synthesize RG-II. It is possible that the apio-
syltransferase identied in the studies of apiogalacturonan synthesis in Lemna
is related to, or is the same apiosyltransferase(s) as that required for RG-II
synthesis (see below), however, this remains to be shown.
3.5.2.1 Rhamnogalacturonan-II methyltransferase (RG-II-MT)
The GalA in the HGA backbone of RG-II may be partially methyl-esteried. A
detergent-solubilized pectin methyltransferase activity from suspension-
cultured ax cells was identied that could transfer methyl groups from
S-adenosylmethionine onto RG-II isolated from wine (Bourlard et al., 1997a).
The addition of RG-II to enzyme reactions gave a 7-fold stimulation of methyl-
transferase activity above the level obtained in the absence of exogenous accep-
tor. The radiolabeled product had a size similar to RG-II monomers and RG-II
dimers (Bourlard et al., 1997; Ridley et al., 2001). It has not yet been shown
where in RG-II the methyl group was added. The identied methylation could
represent methyl-esterication of the HGA backbone of RG-II. Alternatively,
it could represent methyl-etherication of RG-II since RG-II contains methyl
groups on non-galacturonic glycosyl residues (e.g. 2-O-methylxylose and 2-O-
methylfucose (Darvill et al., 1978; ONeill et al., 1996)) of side chain residues.
More research is required to determine the exact location of the methylation
and of the identity of the potentially novel enzyme activity reported.
3.5.2.2 Apiogalacturonan apiosyltransferase
The substituted galacturonan known as apiogalacturonan is produced in some
aquatic monocotyledonous plants (Hart andKindel, 1970; WatsonandOrenstein,
1975). Apiogalacturonan has apiose or apiobiose (d-Apif-1,3-d-apiose)
attached to O-2 or O-3 of HGA (Hart and Kindel, 1970; Watson and Orenstein,
1975). Table 3.6 shows some of the transferase activities likely to be required
to synthesize apiogalacturonan. The anomeric conguration of the glycosidic
linkage of apiose to HGA may be in the conguration (Watson and Orenstein,
1975). It is not known how apiogalacturonan is related to RG-II, which has
Table 3.5 Glycosyltransferase activities likely to be required for RG-II biosynthesis
a,b
Enzyme
d
Type of RG-II
glycosyl side Parent Acceptor Enzyme Reference for
transferase chain
c
polymer substrate activity structure
d-GalAT HGA/RG-II
4
d-GalAT A RG-II l-Rha1,3
-Apif 1,2-GalAT ONeill et al. (1997);

Carpita and Gibeaut
(1993)
d-GalAT A RG-II l-Rha1,3
-Apif 1,3-GalAT ONeill et al. (1997);

Carpita and Gibeaut
(1993)
l-RhaT A,B RG-II Apif 1,2-GalA 1,3
-l-RhaT ONeill et al. (1997);

Carpita and Gibeaut
(1993)
l-RhaT C RG-II Kdo2,3-GalA 1,5-l-RhaT ONeill et al.
(2001, 1997);
Carpita and Gibeaut
(1993)
l-RhaT B RG-II l-Ara1,4-Gal 1,2-l-RhaT ONeill et al. (1997);
Carpita and Gibeaut
(1993)
l-RhaT B
2
RG-II l-Ara1,4-Gal 1,3-l-RhaT
l-GalT B RG-II GlcA1,4-Fuc 1,2-l-GalT ONeill et al. (1997);
Carpita and Gibeaut
(1993)
d-GalT B RG-II l-AcefA1,3-Rha 1,2-GalT Vidal et al. (2000);
ONeill et al. (1997);
Carpita and Gibeaut
(1993)
l-AraT D RG-II Dha2,3-GalA 1,5-l-Araf T ONeill et al. (2001,
1997); Carpita and
Gibeaut (1993)
l-AraT B RG-II Gal1,2-l-AcefA 1,4-l-ArapT Vidal et al. (2000,
1997); Carpita and
Gibeaut (1993)
l-AraT B
2
RG-II l-Rha1,2-l-Ara 1,2-l-Araf T ONeill et al. (1997);
Carpita and Gibeaut
(1993)
l-FucT A RG-II l-Rha1,3
-Apif 1,4-l-FucT ONeill et al. (1997);

Carpita and Gibeaut
(1993)
l-FucT B RG-II Gal1,2-l-AceAf 1,2-l-FucT Vidal et al. (2000);
Carpita and Gibeaut
(1993)
d-Apif T A,B RG-II GalA1,4-GalA 1,2-Apif T ONeill et al. (1997);
Carpita and Gibeaut
(1993)
Table 3.5 (continued)
Enzyme
d
Type of RG-II
glycosyl side Parent Acceptor Enzyme Reference for
transferase chain
c
polymer substrate activity structure
d-XylT A RG-II l-Fuc1,4-l-Rha 1,3-XylT ONeill et al. (1997);
Carpita and Gibeaut
(1993)
d-GlcAT A RG-II l-Fuc1,4-l-Rha 1,4-GlcAT ONeill et al. (1997);
Carpita and Gibeaut
(1993)
d-KdoT C RG-II GalA1,4-GalA 2,3-KdoT ONeill et al. (2001,
1997); Carpita and
Gibeaut (1993)
d-DhaT D RG-II GalA1,4-GalA 2,3-DhaT ONeill et al. (2001,
1997); Carpita and
Gibeaut (1993)
l-AcefA B RG-II l-Rha1,3
-Apif 1,3-AceAf T Vidal et al. (2000,

1997); Carpita and
Gibeaut (1993)
Methyl-RG-II: RG-II d-Xyl1,3-l-Fuc ONeill et al. (1997);
xylose 2-O- Carpita and Gibeaut
methyltransferase (1993)
RG-II:fucose 2-O- RG-II l-Fuc1,2-d-Gal ONeill et al. (1997);
methyltransferase Carpita and Gibeaut
(1993)
Acetyl- RG-II l-Fuc1,2-d-Gal ONeill et al. (1997);
RG-II:fucose Carpita and Gibeaut
acetyltransferase (1993)
RG-II:aceric RG-II l-AcefA1,3-l-Rha Vidal et al. (2000,
acid 3-O- 1997); Carpita and
acetyltransferase Gibeaut (1993)
a
b
This list of glycosyltransferase activites is based on the most extended structure of RG-II (Ridley et al.,
2001). Note that terminal Rhap3Arap- in side chain B and the terminal Araf2Rhap- in side
chain B are not present in all RG-II preparations (ONeill et al., 2001).
c
The side chain of RG-II that contains the specied glycosyl residue. See Ridley et al. (2001), ONeill
et al. (2001) for side chain structure.
d

two of its four side branches attached to an HGA backbone by a Apif linked
to the O-2 of HGA (Ridley et al., 2001). It is also not known whether the
apiosyltransferases that synthesize RG-II are the same as those involved in
apiogalacturonan synthesis since no studies specically directed at the 1,2-
apiosyltransferase involved in RG-II synthesis have been reported. The in vivo
Table 3.6 Some of the glycosyltransferase activities likely to be required for apiogalacturonan
biosynthesis
Enzyme
a,b
d-GalAT

d-Apif T GalA1,4-GalA 1,2-Apif T Hart and Kindel (1970);
Watson and Orenstein (1975)
d-Apif T GalA1,4-GalA 1,3-Apif T Hart and Kindel (1970);
d-Apif T Apif 1,2-GalA 1,3-Apif T Hart and Kindel (1970);
d-Apif T Apif 1,3-GalA 1,3-Apif T Hart and Kindel (1970);
a
All sugars are d sugars and have pyranose rings unless otherwise indicated.
b
Glycosyltranferases add to the glycosyl residue on the left

synthesis of apiogalacturonan, however, has been studied in vegetative fronds
of Spirodela polyrrhiza (Longland et al., 1989) and a d-apiosyltransferase has
been identied and characterized in cell-free particulate preparations fromduck-
weed (Lemna minor) (Pan and Kindel, 1977). The apiosyltransferase transferred
[
14
C]apiose from UDP-[
14
C]apiose onto endogenous acceptors in particulate
membrane preparations from Lemna. The enzyme has a pH optimum of 5.7 and
anapparent K
m
for UDP-apiose of 4.9 M(PanandKindel, 1977). Interestingly,
the rate of apiosyltransferase activity was increased twofold by the inclusion of
UDP-GalA in the reaction (Pan and Kindel, 1977) and the product synthesized
in the presence of UDP-GalA bound more tightly to anion exchange resin
than the product synthesized without UDP-GalA (Mascaro and Kindel, 1977).
These results suggest either that the apiosyltransferase transfers apiose onto
a growing HGA chain or that the amount of endogenous HGA substrate was
limiting. The product was solubilized in 1%ammoniumoxalate, as expected for
apiogalacturonans isolated from Lemna wall (Mascaro and Kindel, 1977) and
the product was fragmented by treatment with a fungal pectinase as expected
for apiogalacturonan. The fact that acid hydrolysis of the
14
C-labeled product
yielded [
14
C]apiose and [
14
C]apiobiose (Mascaro and Kindel, 1977) conrmed
that an apiogalacturonan apiosyltransferase was identied. There are no reports
of the purication of the enzyme.
3.5.2.3 Xylogalacturonan xylosyltransferase
Xylogalacturonan is a region of HGA in which some of the GalA residues
are substituted at O-3 with -d-xylose (see (Schols et al., 1995). Table 3.7
shows some of the transferases likely to be required for xylogalacturonan
synthesis. Xylosyltransferase activity was identied during studies of apio-
galacturonan synthesis (Mascaro and Kindel, 1977; Pan and Kindel, 1977).
Table 3.7 Glycosyltransferase activities likely required to synthesize the substituted galacturonan known
as xylogalacturonan
Enzyme
a
d-GalAT

d-XylT GalA1,4-GalA 1,3-XylT Aspinall (1980); Yu and
Mort (1996); Kikuchi et al.
(1996); Schols et al. (1995)
a

The product produced was not characterized in detail; however, at least some of
the radioactive xylose appeared to be incorporated into apiogalacturonan and/or
HGA. Thus, the enzyme may have been a xylogalacturonan xylosyltransferase.
There have been no reports of the identication of the 1,3-xylosyltransferase
that transfers xylose fromUDP-Xyl onto the l-Fuc1,4-l-rhamnosyl portion of
the side branch of RG-II (Ridley et al., 2001).
3.5.2.4 Other glycosyltransferases
There are no reports of targeted studies for the glycosyltransferases that insert
fucose, Kdo, Dha or aceric acid into pectins. However, success in identifying
fucosyltransferase genes involved in the synthesis of hemicellulose could be
useful in the study of pectin synthesis. Fucose is a component of RG-II and RG-I.
No fucosyltransferase involved in the synthesis of either of these pectic poly-
mers has knowingly been studied. However, an Arabidopsis gene for an 1,2-
fucosyltranferase that fucosylates a side branch in the hemicellulose xyloglucan
has been described (Perrin et al., 1999) that has 3573.8% amino acid sequence
identify to 10 other putative fucosyltransferase genes in Arabidopsis (Perrin
et al., 2001). The possibility that one or more of these genes may encode fuco-
syltransferase(s) involved in RG-I or RG-II synthesis remains to be investigated.
3.5.3 Synthesis of rhamnogalacturonan I (RG-I)
RG-I is a family of polysaccharides with an alternating [4)--d-GalpA-
(12)--l-Rhap-(1] backbone in which roughly 2080% of the rhamnoses
are substitutedbyarabinans, galactans or arabinogalactans (Carpita andGibeaut,
1993; Mohnen, 1999; Ridley et al., 2001). Table 3.8 shows a list of all the likely
enzyme activities required to synthesize all the known structures of RG-I, based
on the available structural information. There is considerable evidence from
immunocytochemistry studies of plant tissues using antibodies against specic
carbohydrate epitopes found in RG-I (Willats et al., 2001a) that supports the
viewthat the precise structure of the side chains of RG-I varies in a cell type- and
Table 3.8 Glycosyltransferases required for RG-I biosynthesis
a
Enzyme
b
Type of Parent Reference for
glycosyltransferase polymer Acceptor substrate Enzyme activity structure
d-GalAT RG-I

l-Rha1,4-GalA 1,2-GalAT ONeill et al. (1990);
Eda et al. (1986); Lau
et al. (1985)
d-GalAT RG-I/HGA
c
GalA1,2-l-Rha 1,4-GalAT
l-RhaT RG-I GalA1,2-l-Rha 1,4-l-RhaT ONeill et al. (1990);
Eda et al. (1986); Lau
et al. (1985)
l-RhaT HGA/RG-I
c
GalA1,4-GalA 1,4-l-RhaT
d-GalT RG-I l-Rha1,4-GalA 1,4-GalT ONeill et al. (1990);
Lau et al. (1987)
d-GalT RG-I Gal1,4-Rha 1,4-GalT ONeill et al. (1990);
Lau et al. (1987)
d-GalT RG-I Gal1,4-Gal 1,4-GalT Morita (1965a,b);
Aspinall et al. (1967);
Stephen (1983);
Aspinall (1980);
Lau et al. (1987)
d-GalT RG-I Gal1,4-Gal 1,6-GalT ONeill et al. (1990);
Lau et al. (1987)
d-GalT RG-I/AGP
d
Gal1,3-Gal 1,3-GalT Carpita and Gibeaut
(1993)
d-GalT RG-I/AGP
d
Gal1,3-Gal 1,6-GalT Carpita and Gibeaut
(1993)
d-GalT RG-I/AGP
d
Gal1,6-Gal1,3-Gal 1,6-GalT Carpita and Gibeaut
(1993)
d-GalT RG-I l-Araf-1,4-Gal 1,5-GalT Huisman et al. (2001a)
l-AraT RG-I Gal1,4-Rha 1,3-l-Araf T ONeill et al. (1990);
Lau et al. (1987)
l-AraT RG-I l-Araf1,3-Gal 1,2-l-Araf T ONeill et al. (1990);
Lau et al. (1987)
l-AraT RG-I l-Araf1,2-Ara 1,5-l-Araf T ONeill et al. (1990);
Lau et al. (1987)
l-AraT RG-I l-Rha1,4-GalA 1,4-Araf T Lau et al. (1987)
l-AraT RG-I l-Araf1,5-Ara 1,5-l-Araf T Carpita and Gibeaut
(1993)
(1993)
(1993)
(1993)
l-AraT RG-I Gal1,4-Gal 1,3-l-Araf T Morita (1965a,b);
Stephen (1983);
Aspinall (1980);
Table 3.8 (continued)
Enzyme
b
Type of Parent Reference for
glycosyltransferase polymer Acceptor substrate Enzyme activity structure
Carpita and Gibeaut
(1993)
l-AraT RG-I l-Araf-1,3-Gal 1,5-l-Araf T Morita (1965a,b);
Stephen (1983);
Aspinall (1980)
l-AraT RG-I/AGP
d
Gal1,6-Gal 1,3-l-Araf T Carpita and Gibeaut
(1993)
l-AraT RG-I/AGP
d
Gal1,6-Gal 1,6-l-Araf T Carpita and Gibeaut
(1993)
l-AraT RG-I Gal1,4-Gal 1,4-l-ArapT Huisman et al. (2001a)
l-FucT RG-I Gal1,4-Gal 1,2-l-Fucf T ONeill et al. (1990);
Lau et al. (1987)
d-GlcAT RG-I Gal. . . 1,6-GlcAT An et al. (1994)
d-GlcAT RG-I Gal. . . 1,4-GlcAT An et al. (1994)
Methyl-
RG-I:GlcA 4-O- RG-I GlcA1,6-Gal An et al. (1994)
methyltransferase
Acetyl- RG-I GalA1,2-l-Rha1,4
(n)
Lerouge et al. (1993);
RG-I:GalA Ishii (1997);
3-O/2-O-acetyl- ONeill et al. (1990);
transferase Bacic et al. (1988);
Komalavilas and
Mort (1989)
a
b

c
Enzyme that may be required to make an HGA/RG-I junction.
d
Enzyme activity would also be required to synthesize arabinogalactan proteins (AGPs) (see Gaspar
et al., 2001).
development-specic manner. Thus, it not expected that all of the biosynthetic
enzyme activities shown in Table 3.8 will necessarily be expressed in any given
cell type synthesizing a specic RG-I structure.
3.5.3.1 RG-I galactosyltransferase (GalT)
The synthesis of the family of polysaccharides known as RG-I requires at least
eight different galactosyltransferase (GalT) activities to synthesize the diverse
side chain linkages found in the RG-I structures identied to date (see Table 3.8).
Probable 1,4-GalT and 1,3-GalT activities were identied in early studies of
microsomal preparations frommung bean (McNab et al., 1968; Panayotatos and
Villemez, 1973). Amore recent study conrmed that a 1,4-galactosyltranferase
activity with a pH of optimum of 6.5 was present in mung bean microsomes
based on sensitivity of the product to digestion with endo-1,4-galactanase
(Brickell and Reid, 1996).
Galactosyltransferases have also been identied in particulate homogenates
(Goubet and Morvan, 1993; Goubet and Morvan, 1994) and solubilized enzyme
(Goubet, 1994) fromax (LinumusitatissimumL.). Flax GalTs solubilized from
microsomes with detergent transferred [
3
H]Gal fromUDP-[
3
H]Gal onto exoge-
nous RG-I-enriched and pectic 1,4-galactan acceptors (Peugnet et al., 2001)
to yield a radiolabeled product of high molecular mass. Interestingly, the pH
optimum for transfer onto lupin pectic 1,4-galactan (i.e. pH 6.5) was different
fromthe pHoptimumfor transfer of Gal onto an endopolygalacturonase-treated
RG-I-enriched fractions from ax (i.e. two optima: pH 6.5 and 8.0) (Peugnet
et al., 2001). Cleavage of the large radiolabeled product with either rhamno-
galacturonan I hydrolase (RGaseA) or with rhamnogalacturonan I lyase (RGase
B) resulted in a fragmentation of the radiolabeled product into lower molecular
mass material that, at least in part, eluted as expected for small oligomers (i.e.
DP 36) (Peugnet et al., 2001). This result conrmed that the GalTs added Gal
onto RG-I (Peugnet et al., 2001). The fragmentation of at least a portion of the
radiolabeled product with 1,4-endogalactanase further demonstrated that at
least some of the GalT activity represented 1,4-galactosyltransferase (Peugnet
et al., 2001). The GalT at pH 8.0 has an apparent K
m
of 460 M for UDP-Gal.
The characteristics of the GalT at pH 8.0 are consistent with an enzyme that
adds galactose onto short galactan side branches of RG-I.
An RG-I:1,4-galactosyltransferase was identied and partially character-
ized from potato suspension cultured cells (Geshi et al., 2000). The potato
GalT transfers [
14
C]Gal from UDP-[
14
C]Gal onto endogenous acceptor(s) in
the membranes to produce a >500 kDa product. The product can be fragmented
by endo-1,4-galactanase into [
14
C]Gal and [
14
C]galactobiose. Treatment of the
intact radiolabeled product with rhamnogalacturonase A, an endohydrolase that
cleaves the glycosidic linkage between the GalA and the Rha in the RG-I back-
bone (Azadi et al., 1995), yielded radiolabeled fragments between 50 kDa and
180 kDa in size (Geshi et al., 2000). The subsequent treatment of the [
14
C]Gal-
labeled fragments with endo-1,4-galactanase yielded [
14
C]Gal and slightly
larger fragments that themselves could be cleaved to [
14
C]Gal by a puried -
galactosidase. These results suggest that potato microsomal membranes contain
enzymes that both initiate and elongate 1,4-galactan side chains of RG-I. The
potato 1,4-GalT has a pH optimum of 6.06.5.
A fenugreek gene for an -d-1,6-galactosyltransferase involved in the syn-
thesis of the hemicellulose galactomannan (Edwards et al., 1999) has been
identied. Eight Arabidopsis genes withsequence similarityexist inArabidopsis
(Perrin et al., 2001) and the possibility that one or more of these putative
galactosyltransferases is involved in pectin synthesis remains to be explored.
d-Galactose is a component of both RG-II and RG-I, while l-galactose is found
only in RG-II. No information is available on the enzyme that adds l-Gal onto
RG-II.
3.5.3.2 RG-I arabinosyltransferase
RG-I andRG-II containl-arabinose inmultiple linkages (seeTables 3.5and3.8).
Most of the arabinose is in the furanose ring form, although it has recently been
reported that a terminal arabinose exists in the pyranose formin some RG-I side
chains (Huisman et al., 2001a). Arabinosyltransferase activity has been iden-
tied in microsomes from mung bean (Phaseolus aureus) shoots (Odzuck and
Kauss, 1972) and frombean (Phaseolus vulgaris) hypocotyl and callus (Bolwell
and Northcote, 1981). Denitive evidence that theseAraTactivities are involved
in pectin synthesis was not demonstrated (see Mohnen, 1999, for review).
The arabinosyltransferase activity in bean hypocotyl and callus was primarily
associated with enriched Golgi, and to a lesser extent with enriched endoplasmic
reticulum(Bolwell and Northcote, 1983). The difculty of studying arabinosyl-
transferases that specically synthesize pectin, as opposed to other hemicellu-
losic polysaccharides or arabinogalactan proteins has been discussed (Nunan
and Scheller, 2001). An approach that entails the use of detergent-solubilized
microsomes andspecic pectic oligo/polysaccharide acceptors has beenreported
to yield arabinosylation of pectic acceptors (Nunan and Scheller, 2001).
3.5.3.3 RG-I methyltransferase (RG-I-MT)
A detergent-solubilized pectin methyltransferase (PMT) from ax has been
reported to use an RG-I-enriched fraction as an exogenous acceptor (Bourlard
et al., 1997a). Specically, pectin methyltransferase activity was stimulated
in the presence of an enriched RG-I fraction 1.5-fold to 1.7-fold above levels
recovered using endogenous acceptor. The resulting radiolabeled product had a
size similar to RG-I. It was not shown, however, where in RG-I the methylation
occurred. Thus, it is not clear whether the methylation occurred on GalA in
the RG-I backbone, or whether it occurred on possible HGA tails that may
be covalently linked to RG-I. Also, it was not shown whether some of the
methylation may have occurred on a non-galacturonic substituent in RG-I such
as methylation at the 4-position of glucuronic acid in the side branches of RG-I
(An et al., 1994). The location on the polymer of the methylation in the RG-I-
enriched fraction and, thus, the identity of the potentially novel enzyme activity,
remains to be determined.
3.5.3.4 RG-I acetyltransferase (RG-I-AT)
The GalAresidues in the alternating [4)--d-GalpA-(12)--l-Rhap-(1]
backbone of RG-I may be acetylated on C-2 and/or C-3 (Komalavilas and
Mort, 1989). Microsomes from suspension-cultured potato cells (Pauly and
Scheller, 2000) contain an RG-I acetyltransferase that transfers [
14
C]acetate
from [
14
C]acetyl-CoA onto RG-I yielding a >500 kDa radiolabeled product
(Pauly and Scheller, 2000). The release of [
14
C]acetate following incubation of
the radiolabeled product with a puried rhamnogalacturonan O-acetyl esterase,
and the fragmentation of the product by rhamnogalacturonan lyase (RGase B)
conrmed that the enzyme was an RG-I acetyltransferase (Pauly and Scheller,
2000). The RG-I acetyltransferase has an apparent K
m
for acetyl-CoAof 35 M,
an apparent V
max
of 54 pmol min
1
mg
1
protein and a pHoptimumof 7.0, with
80% of activity recovered at pH 6.58.0.
3.6 Future directions and resources for studying pectin biosynthesis
The partial characterization and, in some cases, purication of selected pectin
biosynthetic genes has provided a core of biochemical information on the
enzymes. However, to signicantly increase our understanding of when, where
and how the enzymes interact to produce pectin, it is essential that the genes
for the biosynthetic enzymes be identied (see Henrissat et al., 2001; Keegstra
and Raikhel, 2001; Perrin et al., 2001; Reiter and Vanzin, 2001). The identi-
cation of the genes would provide primary structure information and allow the
generation of antibodies against the biosynthetic enzymes that could be used
for analysis of enzyme localization and provide a means to identify members of
the expected biosynthetic protein complexes. The genes could also be used to
express the enzymes for use in detailed kinetic, structure and function studies.
The manipulation of the genes in transgenic plants should allow hypotheses
regarding pectin structurefunction in the plant to be tested and would facilitate
the elucidation of howthe individual biosynthetic enzymes interact to synthesize
pectin.
The identicationof the pectinbiosynthetic genes will likelyoccur usingmul-
tiple strategies including enzyme purication, mutant identication and charac-
terization, and DNA sequence/motif similarity computer searches. In all cases,
however, the denitive identication of any pectin biosynthetic gene will require
proof of enzyme activity. Thus, continued progress in identifying and making
available the required nucleotide-sugar and oligo/polysaccharide substrates for
the biosynthetic enzymes is essential. The National Science Foundation-funded
Plant Cell Wall Biosynthesis Research Coordination Network, also referred to
as WallBioNet (http://xyloglucan.prl.msu.edu), serves as an information center
and resource for researchers studying plant cell wall biosynthesis. It provides
a central location for updated information on progress in, and available tools
for, studying wall biosynthesis research. It also partially supports the synthesis
of rare and needed substrates and acceptors for wall biosynthesis that are made
available to the scientic community.
Acknowledgments
I thank my colleagues at the CCRC for their helpful discussions. This effort
was supported in part by NSF grant No. MCB-0090281, NRI competitive
USDA award 2001-35318-11111 and DOE-funded center grant DE-FG05-93-
ER20097.
References
An, J., ONeill, M.A., Albersheim, P. and Darvill, A.G. (1994) Isolation and structural characterization
of -d-glucosyluronic acid and 4-O-methyl -d-glucosyluronic acid-containing oligosaccharides
from the cell-wall pectic polysaccharide, rhamnogalacturonan I. Carbohydr. Res., 252, 235243.
Ankel, H. andTischer, R. (1969) UDP-d-Glucuronate 4-epimerase in blue-green algae. Biochim. Biophys.
Acta, 178, 415419.
Aspinall, G.O. (1980) Chemistry of cell wall polysaccharides, in The Biochemistry of Plants, vol. 3 (ed.
J. Preiss), Academic Press, NewYork, pp. 473500.
Aspinall, G.O., Begbie, R., Hamilton, A. andWhyte, J.N.C. (1967) Polysaccharides of soy-beans. Part III.
Extraction and fractionation of polysaccharides from cotyledon meal. J. Chem. Soc., 10651070.
Azadi, P., ONeill, M.A., Bergmann, C., Darvill, A.G. and Albersheim, P. (1995) The backbone of the
pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase.
Glycobiology, 5, 783789.
Baasov, T. and Kohen, A. (1995) Synthesis, inhibition, and acid-catalyzed hydrolysis studies of model
compounds of the proposed intermediate in the Kdo8P-synthase-catalyzed reaction. J. Am. Chem.
Soc., 117, 61656174.
Bacic, A., Harris, P.J. and Stone, B.A. (1988) Structure and function of plant cell walls, in The
Biochemistry of Plants, vol. 14 (ed. J. Preiss), Academic Press, NewYork, pp. 297371.
Baldwin, T.C., Handford, M.G., Yuseff, M.-I., Orellana, A. and Dupree, P. (2001) Identication and
characterization of GONST1, a Golgi-localized GDP-mannose transporter in Arabidopsis. Plant
Cell, 13, 22832295.
Bar-Peled, M., Lewinsohn, E., Fluhr, R. and Gressel, J. (1991) UDP-rhamnose:avanone-7-O-
glucoside-2
-O-rhamnosyltransferase. Purication and characterization of an enzyme catalyzing

the production of bitter compounds in citrus. J. Biol. Chem., 266, 2095320959.
Bar-Peled, M., Fluhr, R. and Gressel, J. (1993) Juvenile-specic localization and accumulation of a
rhamnosyltransferase and its bitter avonoid in foliage, owers, and young citrus fruits. Plant
Physiol., 103, 13771384.
Bar-Peled, M., Grifth, C.L. and Doering, T.L. (2001) Functional cloning and characterization of a
UDP-glucuronic acid decarboxylase: the pathogenic fungus Cryptococcus neoformans elucidates
UDP-xylose synthesis. Proc. Natl. Acad. Sci. USA, 98, 1200312008.
Barber, G.A. (1962) The enzymatic synthesis of uridine diphosphate l-rhamnose. Biochem. Biophys.
Res. Commun., 8, 204208.
Barber, G.A. (1963) The formation of uridine diphosphate l-rhamnose by enzymes of the tobacco leaf.
Arch. Biochem. Biophys., 103, 276282.
Barber, G.A. and Chang, M.T.Y. (1967) Synthesis of uridine diphosphate l-rhamnose by enzymes of
Chlorella pyrenoidosa. Arch. Biochem. Biophys., 118, 659663.
Baron, D., Streitberger, U. and Grisebach, H. (1973) Improved method for purication of UDP-
apiose/UDP-xylose synthase from cell cultures of parsley. Biochim. Biophys. Acta, 293, 526533.
Basu, S.S., Dotson, G.D. and Raetz, C.R.H. (2000) A facile enzymatic synthesis of uridine diphospho-
[
14
C]galacturonic acid. Anal. Biochem., 280, 173177.
Bauer, A.J., Rayment, I., Frey, P.A. and Holden, H.M. (1992) The molecular structure of UDP-galactose
4-epimerase from Escherichia coli determined at 2.5

A resolution. Proteins, 12, 372381.
Baydoun, E.A.H., Rizk, S.E. and Brett, C.T. (1999) Localisation of methyltransferases involved in
glucuronoxylan and pectin methylation in the Golgi apparatus in etiolated pea epicotyls. J. Plant
Physiol., 155, 240244.
Berninsone, P.M. and Hirschberg, C.B. (2000) Nucleotide-sugar transporters of the Golgi apparatus.
Curr. Opin. Struct. Biol., 10, 542547.
Bolwell, G.P. and Northcote, D.H. (1981) Control of hemicellulose and pectin synthesis during
differentiation of vascular tissue in bean (Phaseolus vulgaris) callus and in bean hypocotyl. Planta,
152, 225233.
Bolwell, G.P. and Northcote, D.H. (1983) Arabinan synthase and xylan synthase activities of
Phaseolus vulgaris. Subcellular localization and possible mechanism of action. Biochem. J., 210,
497507.
Bolwell, G.P., Dalessandro, G. and Northcote, D.H. (1985) Decrease of polygalacturonic acid synthase
during xylem differentiation in sycamore. Phytochemistry, 24, 699702.
Bonin, C.P. and Reiter, W.-D. (2000) A bifunctional epimerase-reductase acts downstream of the MUR1
gene product and completes the de novo synthesis of GDP-l-fucose in Arabidopsis. Plant J., 21,
445454.
Bonin, C.P., Potter, I., Vanzin, G.F. and Reiter, W.-D. (1997) The MUR1 gene of Arabidopsis thaliana
encodes an isoform of GDP-d-mannose-4,6-dehydratase, catalyzing the rst step in the de novo
synthesis of GDP-l-fucose. Proc. Natl. Acad. Sci. USA, 94, 20852090.
Bourlard, T., Pellerin, P. and Morvan, C. (1997a) Rhamnogalacturonans I and II are pectic substrates for
ax-cell methyltransferases. Plant Physiol. Biochem., 35, 623629.
Bourlard, T., Schaumann-Gaudinet, A., Bruyant-Vannier, M.-P. and Morvan, C. (1997b) Various pectin
methyltransferase activities with afnity for lowand highly methylated pectins. Plant Cell Physiol.,
38, 259267.
Bourlard, T., Vannier, M.-P., Schaumann, A., Bruyant, P. and Morvan, C. (2001) Purication of several
pectin methyltransferases from cell suspension cultures of ax. C. R. Acad. Sci. Paris, Science de
la vie, 324, 335343.
Brabetz, W., Wolter, F.P. and Brade, H. (2000) A cDNA encoding 3-deoxy-d-manno-oct-2-ulosonate-
8-phosphate synthase of Pisum sativum L (pea) functionally complements a kdsA mutant of the
Gram-negative bacterium Salmonella enterica. Planta, 212, 136143.
Brickell, L.S. and Reid, J.S.G. (1996) Biosynthesis in vitro of pectic (1-4)--d-galactan, in Pectins and
Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science,
Amsterdam, pp. 127134.
Bruyant-Vannier, M.-P., Gaudinet-Schaumann, A., Bourlard, T. and Morvan, C. (1996) Solubilization
and partial characterization of pectin methyltransferase from ax cells. Plant Physiol. Biochem.,
34, 489499.
Burget, E.G. and Reiter, W.-D. (1999) The mur4 mutant of Arabidopsis is partially defective in the
de novo synthesis of uridine diphospho l-arabinose. Plant Physiol., 121, 283389.
Campbell, R.E., Sala, R.F., Van De Rijn, I. and Tanner, M.E. (1997) Properties and kinetic analysis of
UDP-glucose dehydrogenase from group A streptococci. J. Biol. Chem., 272, 34163422.
Campbell, R.E., Mosimann, S.C., Van De Rijn, I., Tanner, M.E. and Strynadka, N.C.J. (2000) The rst
structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold
oxidation. Biochemistry, 39, 70127023.
Capasso, J.M. and Hirschberg, C.B. (1984) Mechanisms of glycosylation and sulfation in the
Golgi apparatus: evidence for nucleotide-sugar/nucleoside monophosphate and nucleotide
sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane. Proc. Natl. Acad.
Sci. USA, 81, 70517055.
Carpita, N.C. (1996) Structure and biogenesis of the cell walls of grasses. Annu. Rev. Plant Physiol.
Plant Mol. Biol., 47, 445476.
Carpita, N.C. and Gibeaut, D.M. (1993) Structural models of primary cell walls in owering plants:
consistency of molecular structure with the physical properties of the walls during growth. Plant
J., 3, 130.
Casero, P.J. and Knox, J.P. (1995) The monoclonal antibody JIM5 indicates patterns of pectin deposition
in relation to pit elds at the plasma-membrane-face of tomato pericarp cell walls. Protoplasma,
188, 133137.
Crombie, H.J. and Reid, J.S.G. (1998) Pectin methyltransferase: activities in particulate and solubilised
preparations from mung bean (Vigna radiata) hypocotyls and tomato (Lycopersicon esculentum)
pericarp. Cell Walls 98: 8th International Cell Walls Meeting: John Innes Centre, Norwich,UK,
Abstract 1.39.
Crombie, H.J. and Reid, J.S.G. (2001) A homogalacturonan synthase from mung bean hypocotyls. Cell
Wall 01:9th International Cell Wall Meeting, Toulouse, France, Abstract, p. 131.
Cumming, C.M. and Brett, C.T. (1986) Agalacturonyltransferase involved in pectin biosynthesis, in Cell
Walls 86. Proceedings of the Fourth Cell Wall Meeting. Paris, Universit e Pierre et Marie Curie
Ecole Normale Sup erieure, Paris, pp. 360363.
Darvill, A., McNeil, M. and Albersheim, P. (1978) Structure of plant cell walls: VIII. A new pectic
polysaccharide. Plant Physiol., 62, 418422.
Davies, M.D. and Dickinson, D.B. (1972) Properties of uridine diphosphoglucose dehydrogenase from
pollen of Lilium longiorum. Arch. Biochem. Biophys., 152, 5361.
De Vries, J.A., Voragen, A.G.J., Rombouts, F.M. and Pilnik, W. (1986) Structural studies of apple pectins
with pectolytic enzymes. in Chemistry and Function of Pectins (eds M.L. Fishman and J.J. Jen),
American Chemical Society, Washington, DC, pp. 3848.
Dhugga, K.S. (2001) Building the wall: genes and enzymes complexes for polysaccharides synthases.
Curr. Opin. Plant Biol., 4, 488493.
Dolan, L., Linstead, P. and Roberts, K. (1997) Developmental regulation of pectic polysaccharides in
the root meristem of Arabidopsis. J. Expe. Botany, 48, 713720.
Doong, R.L. and Mohnen, D. (1998) Solubilization and characterization of a galacturono-
syltransferase that synthesizes the pectic polysaccharide homogalacturonan. Plant J., 13,
363374.
Doong, R.L., Ahmad, S. and Jensen, R.A. (1991) Higher plants express 3-deoxy-d-manno-octulosonate
8-phosphate synthase. Plant Cell Environ., 14, 113120.
Doong, R.L., Gander, J.E., Ganson, R.J. and Jensen, R.A. (1992) The cytosolic isoenzyme of 3-deoxy-d-
arabino-heptulosonate 7-phosphate synthase in Spinacia oleracea and other higher plants: extreme
substrate ambiguity and other properties. Physiol. Plant., 84, 351360.
Doong, R.L., Liljebjelke, K., Fralish, G., Kumar, A. and Mohnen, D. (1995) Cell free synthesis of pectin:
identication and partial characterization of polygalacturonate 4--galacturonosyltransferase and
its products from membrane preparations of tobacco (Nicotiana tabacum L. cv samsun) cell
suspension cultures. Plant Physiol., 109, 141152.
Dormann, P. and Benning, C. (1996) Functional expression of uridine 5
-diphosphoglucose 4-epimerase
(EC-5.1.3.2) from Arabidopsis-thaliana in Saccharomyces-cerevisiae and Escherichia-coli. Arch.
Biochem. Biophys., 327, 2734.
Dyer, W.E., Weaver, L.M., Zhao, J., Kuhn, D.N., Weller, S.C. and Herrmann, K.M. (1990) A cDNA
encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from Solanum tuberosum L.
J. Biol. Chem. 265, 16081614.
Eberhard, S., Doubrava, N., Marf` a, V., et al. (1989) Pectic cell wall fragments regulate tobacco thin-cell-
layer explant morphogenesis. Plant Cell, 1, 747755.
Eda, S., Miyabe, K., Akiyama, Y., Ohnishi, A. and Kato, K. (1986) A pectic polysaccharide from cell
walls of tobacco (Nicotiana tabacum) mesophyll. Carbohydr. Res., 158, 205216.
Edwards, M.E., Dickson, C.A., Chengappa, S., Sidebottom, C., Gidley, M.J. and Reid, J.S.G. (1999)
Molecular characterisation of a membrane-bound galactosyltransferase of plant cell wall matrix
polysaccharide biosynthesis. Plant J., 19, 691697.
Eidson, B.T., Chan, J., Vandersall, A.S., et al. (1996) Metabolism of UDP-galacturonic acid in tobacco
cell free membrane preparations. Plant Physiol., 111S, 101
Faik, A., Desveaux, D. and Maclachlan, G. (2000) Sugar-nucleotide-binding and autoglycosylating
polypeptides(s) from nasturtium fruit: biochemical capacities and potential functions. Biochem. J.,
347, 857864.
Fan D.-F. and Feingold, D.S. (1969) Nucleoside diphosphate-sugar 4-epimerases I. uridine diphosphate
glucose 4-epimerase of wheat germ. Plant Physiol., 44, 599604.
Fan, D.-F. and Feingold, D.S. (1970) Nucleoside diphosphate-sugar 4-epimerases. II. Uridine
diphosphate arabinose 4-epimerase of wheat germ. Plant Physiol., 46, 592595.
Feingold, D.S. and Avigad, G. (1980) Sugar nucleotide transformations in plants, in The Biochemistry
of Plants, vol. 3 (ed. J. Preiss), Academic Press, NewYork, pp. 101170.
Feingold, D.S. and Barber, G.A. (1990) Nucleotide sugars, in Methods in Plant Biochemistry, vol. 2,
Carbohydrates (ed. P.M. Dey), Academic Press, London, pp. 3978.
Feingold, D.S. and Franzen, J.S. (1981) Pyridine nucleotide-linked four-electron transfer
dehydrogenases. Trends Biochem. Sci., 6, 103105.
Feingold, D.S., Neufeld, E.F. and Hassid, W.Z. (1960) The 4-epimerization and decarboxylation of
uridine diphosphate d-glucuronic acid by extracts fromPhaseolus aureus seedlings. J. Biol. Chem.,
235, 910913.
Franzen, J.S., Marchetti, P., Ishman, R., Ashcom, J. and Feingold, D.S. (1978) Half-sites oxidation of
bovine liver UDP glucose dehydrogenase. Biochem. J., 173, 701704.
Fujiki, Y., Hubbard, A.L., Fowler, S. and Lazarow, P.B. (1982) Isolation of intracellular membranes
by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biol., 93,
97102.
Gainey, P.A., Pestell, T.C. and Phelps, C.F. (1972) Astudy of the subunit structure and the thiol reactivity
of bovine liver UDP-glucose dehydrogenase. Biochem. J., 129, 821830.
Gardiner, S.E., Schr oder, J., Matern, U., Hammer, D. and Hahlbrock, K. (1980) mRNA-dependent
regulation of UDP-apiose synthase activity in irradiated plant cells. J. Biol. Chem., 255,
1075210757.
Gaspar, Y., Johnson, K.L., McKenna, J.A., Bacic, A. and Schultz, C.J. (2001) The complex structures
of arabinogalactan-proteins and the journey towards understanding function. Plant Mol. Biol., 47,
161176.
Gaunt, M.A., Maitra, U.S. and Ankel, H. (1974) Uridine diphosphate galacturonate 4-epimerase from
the blue-green alga Anabaena os-aquae. J. Biol. Chem., 249, 23662372.
Gebb, C., Baron, D. and Grisebach, H. (1975) Spectroscopic evidence for the formation of a 4-keto
intermediate in the UDP-apiose/UDP-xylose synthase reaction. Eur. J. Biochem., 54, 493498.
Geren, C.R. and Ebner, K.E. (1977) Purication and characterization of UDP-galactose-4-epimerase
from bovine tissues. J. Biol. Chem., 252, 20822088.
Geshi, N., Jorgensen, B., Scheller, H.V. and Ulvskov, P. (2000) In vitro biosynthesis of 1,4--galactan
attached to rhamnogalacturonan I. Planta, 210, 622629.
Gibeaut, D.M. (2000) Nucleotide sugars and glycosyltransferases for synthesis of cell wall matrix
polysaccharides. Plant Physiol. Biochem., 38, 6980.
Goubet, F. (1994) Etude de la biosynthese de polysaccharides parietaux des bres cellulosiques au
cours du developpement du lin. PhD Universit e de Rouen, pp. 1176.
Goubet, F., Council, L.N. and Mohnen, D. (1998) Identication and partial characterization of the
pectin methyltransferase homogalacturonan-methyltransferase from membranes of tobacco cell
suspensions. Plant Physiol., 116, 337347.
Goubet, F. and Mohnen, D. (1999a) Subcellular localization and topology of homogalacturonan
methyltransferase in suspension-cultured Nicotiana tabacum cells. Planta, 209, 112117.
Goubet, F. and Mohnen, D. (1999b) Solubilization and partial characterization of homogalacturonan-
methyltransferase from microsomal membranes of suspension-cultured tobacco cells. Plant
Physiol., 121, 281290.
Goubet, F. and Morvan, C. (1993) Evidence for several galactan synthases in ax (Linum usitassimum
L.) suspension-cultured cells. Plant Cell Physiol., 34, 12971303.
Goubet, F. and Morvan, C. (1994) Synthesis of cell wall galactans from ax (Linum usitatissimum L.)
suspension-cultured cells. Plant Cell Physiol., 35, 719727.
Hannapel, D.J. (1991) Distribution of potato tuber proteins during development. Am. Potato J., 68,
179190.
Harris, P.J. and Northcote, D.H. (1971) Polysaccharide formation in plant Golgi bodies. Biochim.
Biophys. Acta, 237, 5664.
Hart, D.A. and Kindel, P.K. (1970) Isolation and partial characterization of apiogalacturonans from the
cell wall of Lemna minor. Biochem. J., 116, 569579.
Hassid, W.Z., Neufeld, E.F. and Feingold, D.S. (1959) Sugar nucleotides in the interconversion of
carbohydrates in higher plants. Proc. Natl. Acad. Sci. USA, 45, 905915.
Hassid, W.Z. (1967) Transformation of sugars in plants. Annu. Rev. Plant Physiol., 18, 253280.
Hayashi, T., Koyama, T. and Matsuda, K. (1988) Formation of UDP-xylose and xyloglucan in soybean.
Plant Physiol., 87, 341345.
Hebda, P.A. and Barber, G.A. (1978) Further studies of the guanosine 5
-diphosphate -d-mannose:
guanosine 5
-diphosphate -l-galactose epimerase of Chlorella pyrenoidosa. Fed. Proc. Fed. Am.

Soc. Exp. Biol., 37, 1774.
Hebda, P.A., Behrman, E.J. and Barber, G.A. (1979) The guanosine 5
-diphosphate d-mannose:
guanosine 5
-diphosphate l-galactose epimerase of Chlorella pyrenoidosa. Arch. Biochem.

Biophys., 194, 496502.
Hempel, J., Perozich, J., Romovacek, H., Hinich, A., Kuo, I. and Feingold, D.S. (1994) UDP-glucose
dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases.
Protein Sci., 3, 10741080.
Henrissat, B., Coutinho, P.M. and Davies, G.J. (2001) A census of carbohydrate-active enzymes in the
genome of Arabidopsis thaliana. Plant Mol. Biol., 47, 5572.
Herrmann, K.M. (1995) The shikimate pathway as an entry to aromatic secondary metabolism. Plant
Physiol., 107, 712.
Huisman, M.M.H., Br ull, L.P., Thomas-Oates, J.E., Haverkamp, J., Schols, H.A. and Voragen, A.G.J.
(2001a) The occurrence of internal (1-5)-linked arabinofuranose and arabinopyranose residues in
arabinogalactan side chains from soybean pectic substances. Carbohydr. Res., 330, 103114.
Huisman, M.M.H., Fransen, C.T.M., Kamerling, J.P., Vilegenthart, J.F.G., Schols, H.A. and Voragen,
A.G.J. (2001b) The CDTA-soluable pectic substances from soybean meal are composed of
rhamnogalacturonan and xylogalacturonan but not homogalacturonan. Biopolymers, 58, 279294.
Ishii, T. (1995) Pectic polysaccharides from bamboo shoot cell-walls. Mokuzai Gakkaishi, 41, 669676.
Ishii, T. (1997) O-Acetylated oligosaccharides from pectins of potato tuber cell walls. Plant Physiol.,
113, 12651272.
Ishii, T. and Matsunaga, T. (2001) Pectic polysaccharide rhanogalacturonan II is covalently liked to
homogalacturonan. Phytochemistry, 57, 969974.
Ishikawa, M., Kuroyama, H., Takeuchi, Y. and Tsumuraya, Y. (2000) Characterization of pectin
methyltransferase from soybean hypocotyls. Planta, 210, 782791.
Jaenicke, R., Rudolph, R. and Feingold, D.S. (1986) Dissociation and reconstitution of bovine liver
UDPGDH. Biochemistry, 25, 72837287.
Jelakovic, S. and Schulz, G.E. (2001) The structure of CMP:2-keto-3-deoxy-manno-octonic acid
synthetase and of its complexes with substrates and substrate analogs. J. Mol. Biol., 312, 143155.
Jelakovic, S., Jann, K. and Schulz, G.E. (1996) The 3-dimensional structure of capsule-specic CMP-
2-keto-3-deoxy-manno-octonic acid synthetase from Escherichia-coli. FEBS Lett., 391, 157161.
Joersbo, M., Pedersen, S.G., Nielsen, J.E., Marcussen, J. and Brunstedt, J. (1999) Isolation and expression
of two cDNA clones encoding UDP-galactose epimerase expressed in developing seeds of the
endospermous legume guar. Plant Sci., 142, 147154.
John, K.V., Schutzbach, J.S. and Ankel, H. (1977) Separation and allosteric properties of two forms of
UDP-glucuronate carboxy-lyase. J. Biol. Chem., 252, 80138017.
Kamsteeg, J., Van Brederode, J. and Van Nigtevecht, G. (1978) The formation of UDP-l-rhamnose
from UDP-d-glucose by an enzyme preparation of red campion (Silene dioica (L) clairv) leaves.
FEBS Lett., 91, 281284.
Kaplan, C.P., Tugal, H.B. and Baker, A. (1997) Isolation of a cDNA encoding an Arabidopsis
galactokinase by functional expression in yeast. Plant Mol. Biol., 34, 497506.
Kauss, H. and Hassid, W.Z. (1967) Enzymatic introduction of the methyl ester groups of pectin. J. Biol.
Chem., 242, 34493453.
Kauss, H. and Swanson, A.L. (1969) Cooperation of enzymes responsible for polymerization and
methylation in pectin biosynthesis. Z. Naturforsch., 24, 2833.
Kauss, H., Swanson, A.L. and Hassid, W.Z. (1967) Biosynthesis of the methyl ester groups of pectin by
transmethylation from S-adenosyl-l-methionine. Biochem. Biophys. Res. Commun., 26, 234240.
Kauss, H., Swanson, A.L., Arnold, R. and Odzuck, W. (1969) Biosynthesis of pectic substances.
Localization of enzymes and products in a lipid-membrane complex. Biochim. Biophys. Acta, 192,
5561.
Kearns, A.E., Vertel, B.M. and Schwartz, N.B. (1993) Topography of glycosylation and UDP-xylose
production. J. Biol. Chem., 268, 1109711104.
Keegstra, K. and Raikhel, N. (2001) Plant glycosyltransferases. Curr. Opin. Plant Biol., 2001, 219224.
Kelleher, F.M. and Bhavanandan, V.P. (1986) Re-examination of the products of the action of galactose
oxidase. Evidence for the conversion of rafnose to 6-carboxyrafnose. J. Biol. Chem., 261,
1104511048.
Keller, R., Renz, F.S. and Kossmann, J. (1999) Antisense inhibition of the GDP-mannose pyro-
phosphorylase reduces the ascorbate content in transgenic plants leading to developmental
changes during senescence. Plant J., 19, 131141.
Kikuchi, A., Edashige, Y., Ishii, T. and Satoh, S. (1996) A xylogalacturonan whose level is dependent
on the size of cell clusters is present in the pectin from cultured carrot cells. Planta, 200, 369372.
Kindel, P.K. and Watson, R.R. (1973) Synthesis, characterization and properties of uridine 5
(-d-apio-
d-furanosyl pyrophosphate). Biochem. J., 133, 227241.
Kindel, P.K., Gustine, D.L. and Watson, R.R. (1971) Purication and properties of UDP-d-glucuronic
acid cyclase from Lemna minor. Fed. Proc. Fed. Am. Soc. Exp. Biol., 30, 1117.
Knox, J.P., Linstead, P.J., King, J., Cooper, C. and Roberts, K. (1990) Pectin esterication is spatially reg-
ulated both within cell walls and between developing tissues of root apices. Planta, 181, 512521.
Komalavilas, P. and Mort, A.J. (1989) The acetylation at O-3 of galacturonic acid in the rhamnose-rich
portion of pectins. Carbohydr. Res., 189, 261272.
Kornfeld, R.H. and Ginsburg, V. (1966) Control of synthesis of guanosine 5
-diphosphate d-mannose
and guanosine 5
-diphosphate l-fucose in bacteria. Biochim. Biophys. Acta, 117, 7987.

K onigs, B. and Heinz, E. (1974) Investigation of some enzymatic activities contributing to the
biosynthesis of galactolipid precursors in Vicia faba. Planta, 118, 159169.
Lake, M.R., Williamson, C.L. and Slocum, R.D. (1998) Molecular cloning and characterization of a
UDP-glucose-4-epimerase gene (galE) and its expression in pea tissues. Plant Physiol. Biochem.,
36, 555562.
Lau, J.M., McNeil, M., Darvill, A.G. and Albersheim, P. (1985) Structure of the backbone of
rhamnogalacturonan I, a pectic polysaccharide in the primary cell walls of plants. Carbohydr.
Res., 137, 111125.
Lau, J.M., McNeil, M., Darvill, A.G. and Albersheim, P. (1987) Treatment of rhamnogalacturonan I
with lithium in ethylenediamine. Carbohydr. Res., 168, 245274.
Lerouge, P., ONeil, M.A., Darvill, A.G. and Albersheim, P. (1993) Structural characterization of
endo-glycanse-generated oligoglycosyl side chains of rhamnogalacturonan I. Carbohydr. Res.,
243, 359371.
Li, Y.Q., Chen, F., Linskens, H.F. and Cresti, M. (1994) Distribution of unesteried and esteried
pectins in cell walls of pollen tubes of owering plants. Sex Plant Reprod., 7, 145152.
Liao, T.H. and Barber, G.A. (1971) The synthesis of guanosine 5
-diphosphate l-fucose by enzymes of

a higher plant. Biochim. Biophys. Acta, 230, 6471.
Liao, T.H. and Barber, G.A. (1972) Purication of guanosine 5
-diphosphate d-mannose oxidoreductase

from Phaseolus vulgaris. Biochim. Biophys. Acta, 276, 8593.
Liljebjelke, K., Adolphson, R., Baker, K., Doong, R.L. and Mohnen, D. (1995) Enzymatic synthesis
and purication of uridine diphosphate [
14
C]galacturonic acid: a substrate for pectin biosynthesis.
Anal. Biochem., 225, 296304.
Lin, T.-Y., Elbein, A.D. and Su, J.C. (1966) Substrate specicity in pectin synthesis. Biochem. Biophys.
Res. Commun., 22, 650657.
Liners, F. and Van Cutsem, P. (1992) Distribution of pectic polysaccharides throughout wall of
suspension-cultured carrot cells. Protoplasma, 170, 1021.
Liners, F., Letesson, J.-J., Didembourg, C. and Van Cutsem, P. (1989) Monoclonal antibodies against
pectin. Recognition of a conformation induced by calcium. Plant Physiol., 91, 14191424.
Liners, F., Gaspar, T. and Van Cutsem, P. (1994) Acetyl- and methyl-esterication of pectins of friable
and compact sugar-beet calli: consequences for intercellular adhesion. Planta, 192, 545556.
Longland, J.M., Fry, S.C. and Trewavas, A.J. (1989) Developmental control of apiogalacturonan
biosynthesis and UDP-apiose production in a duckweed. Plant Physiol., 90, 972976.
Lukowitz, W., Nickle, T.C., Meinke, D.W., Last, R.L., Conklin, P.L. and Somerville, C.R. (2001)
Arabidopsis cyt1 mutants are decient in a mannose-1-phosphate guanylyltransferase and point
to a requirement of N-linked glycosylation for cellulose biosynthesis. Proc. Natl. Acad. Sci. USA,
27, 22622267.
Lynch, M.A. and Staehelin, L.A. (1992) Domain-specic and cell type-specic localization of two
types of cell wall matrix polysaccharides in the clover root tip. J. Cell Biol., 118, 467479.
Maitra, U.S. and Ankel, H. (1971) Uridine diphosphate-4-keto-glucose, an intermediate in the uridine
diphosphate-galactose-4-epimerase reaction. Proc. Natl. Acad. Sci. USA, 68, 26602663.
Marty, P., Goldberg, R., Liberman, M., Vian, B., Bertheau, Y. and Jouan, B. (1995) Composition and
localization of pectic polymers in the stems of two Solanum tuberosum genotypes. Plant Physiol.
Biochem., 33, 409417.
Mascaro, L.J., Jr. and Kindel, P.K. (1977) Characterization of [
14
C]apiogalacturonans synthesized in a
cell-free system from Lemna minor. Arch. Biochem. Biophys., 183, 139148.
Matern, U. and Grisebach, H. (1977) UDP-apiose/UDP-xylose synthase. Subunit composition and
binding studies. Eur. J. Biochem., 74, 303312.
Mattila, P., R abin a, J., Hortling, S., Helin, J. and Renkonen, R. (2000) Functional expression
of Escherichia coli enzymes synthesizing GDP-l-fucose from inherent GDP-d-mannose in
Saccharomyces cerevisiae. Glycobiology, 10, 10411047.
Maxwell, E.S. (1957) The enzymatic interconversion of uridine diphosphogalactose and uridine
diphosphoglucose. J. Biol. Chem., 229, 139151.
McCartney, L., Ormerod, A.P., Gidley, M.J. and Knox, J.P. (2000) Temporal and spatial regulation of
pectic (1-4)--d-galactan in cell walls of developing pea cotyledons: implications for mechanical
properties. Plant J., 22, 105113.
McNab, J.M., Villemez, C.L. and Albersheim, P. (1968) Biosynthesis of galactan by a particulate
preparation from Phaseolus aureus seedlings. Biochem. J., 106, 355360.
Micheli, F. (2001) Pectin methylesterases: cell wall enzymes with important roles in plant physiology.
Trends Plant Sci., 6, 414419.
Mitcham, E.J., Gross, K.C. and Wasserman, B.P. (1991) Synthesis of uridinediphospho-[U-
14
C]-d-
galacturonic acid by enzyme particulate fractions and purication via high performance liquid
chromatography. Phytochem. Anal., 2, 112115.
Mohnen, D. (1999) Biosynthesis of pectins and galactomannans, in Comprehensive Natural Products
Chemistry, vol. 3, Carbohydrates and Their Derivatives Including Tannins, Cellulose, and Related
Lignins (ed. B.M. Pinto), Elsevier, Oxford, pp. 497527.
Mohnen, D., Doong, R.L., Liljebjelke, K., Fralish, G. and Chan, J. (1996) Cell free synthesis of the
pectic polysaccharide homogalacturonan, in Pectins and Pectinases (eds J. Visser and A.G.J.
Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 109126.
Mohnen, D., Quigley, H.F., Adams, K.L., et al. (1999) A multi-enzyme approach to study pectin
biosynthesis. Annual Meeting of the American Society of Plant Physiology, Abstract No. 203, p. 65.
Moore, P.J. and Staehelin, L.A. (1988) Immunogold localization of the cell-wall-matrix polysaccharides
rhamnogalacturonan I and xyloglucan during cell expansion and cytokinesis in Trifolium pratense
L.; implication for secretory pathways. Planta, 174, 433445.
Moore, P.J., Darvill, A.G., Albersheim, P. and Staehelin, L.A. (1986) Immunogold localization of
xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.
Moore, P.J., Swords, K.M.M., Lynch, M.A. and Staehelin, L.A. (1991) Spatial organization of the
assembly pathways of glycoproteins and complex polysaccharides in the golgi apparatus of plants.
J. Cell Biol., 112, 589602.
Morita, M. (1965a) Polysaccharides of soybean seeds. Part I. Polysaccharide consituents of hot-water-
extract fractions of soybean seeds and an arabinogalactan as its major component. Agric. Biol.
Chem., 29, 564573.
Morita, M. (1965b) Polysaccharides of soybean seeds. Part II. A methylated arabinogalactan isolated
from methylated product of hot-water extract fraction of soybean seed polysaccharides. Agric.
Biol. Chem., 29, 626630.
Mu nos, R., L opex, R., de Frutos, M. and Garca, E. (1999) First molecular characterization of a
uridine diphosphate galacturonate 4-epimerase: an enzyme required for capsular biosynthesis in
Streptococcus pneumoniae type 1. Mol. Microbiol., 31, 703713.
Munoz, P., Norambuena, L. and Orellana, A. (1996) Evidence for a UDP-glucose transporter in Golgi
apparatus-derived vesicles from pea and its possible role in polysaccharide biosynthesis. Plant
Physiol., 112, 15851594.
Nebenf uhr, A. and Staehelin, L.A. (2001) Mobile factories: Golgi dynamics in plant cells. Trends Plant
Sci., 6, 160167.
Nebenf uhr, A., Gallagher, L.A., Dunahay, T.G., et al. (1999) Stop-and-go movements of plant Golgi
stacks are mediated by the acto-myosin system. Plant Physiol., 121, 11271141.
Neckelmann, G. and Orellana, A. (1998) Metabolism of uridine 5
-diphosphate-glucose in Golgi
vesicles from pea stems. Plant Physiol., 117, 10071014.
Neufeld, E.F., Feingold, D.S. and Hassid, W.Z. (1958) Enzymatic conversion of uridine diphosphate
d-glucuronic acid to uridine diphosphate galacturonic acid, uridine diphosphate xylose, and
uridine diphosphate arabinose. J. Am. Chem. Soc., 80, 4430.
Northcote, D.H. (1970) The Golgi apparatus. Endeavor, XXX, 2633.
Northcote, D.H. and Pickett-Heaps, J.D. (1966) A function of the Golgi Apparatus in polysaccharide
synthesis and transport in the root-cap cells of wheat. Biochem. J., 98, 159167.
Nunan, K.J. and Scheller, H.V. (2001) Biosynthesis of arabinan. Cell Walls 01:9th International Cell
Wall Meeting, Toulouse, France, p. 110 (Abstract)
ONeill, M.A., Warrenfeltz, D., Kates, K., et al. (1996) Rhamnogalacturonan-II, a pectic polysaccharide
in the walls of growing plant cell, forms a dimer that is covalently crosslinked by a borate ester
in vitro conditions for the formation and hydrolysis of the dimer. J. Biol. Chem., 271, 2292322930.
ONeill, M., Albersheim, P. and Darvill, A. (1990) The pectic polysaccharides of primary cell walls, in
Methods in Plant Biochemistry (ed. P.M. Dey), vol. 2, Academic Press, London, pp. 415441.
ONeill, M.A., Warrenfeltz, D., Kates, K., et al. (1997) Rhamnogalacturonan-II, a pectic polysaccharide
in the walls of growing plant cell, forms a dimer that is covalently crosslinked by a borate ester
in vitro conditions for the formation and hydrolysis of the dimer. J. Biol. Chem., 272, 3869
ONeill, M.A., Eberhard, S., Albersheim, P. and Darvill, A.G. (2001) Requirement of borate crosslinking
of cell wall rhamnogalacturonan II for Arabidopsis growth. Science, 294, 846849.
Odzuck, W. and Kauss, H. (1972) Biosynthesis of pure araban and xylan. Phytochemistry, 11, 24892494.
Orellana, A. and Mohnen, D. (1999) Enzymatic synthesis and purication of [
3
H]uridine diphosphate
galacturonic acid for use in studying Golgi-localized transporters. Analy. Biochem., 272,
224231.
Orellana, A., Neckelmann, G. and Norambuena, L. (1997) Topography and function of Golgi
uridine-5
-diphosphatase from pea stems. Plant Physiol., 114, 99107.

Orla, C. and Knox, J.P. (2000) Spatial regulation of pectic polysaccharides in relation to pit elds in
cell walls of tomato fruit pericarp. Plant Physiol., 122, 775781.
Pan, Y.-T. and Kindel, P.K. (1977) Characterization of particulate d-apiosyl- and d-xylosyltransferase
from Lemna minor. Arch. Biochem. Biophys., 183, 131138.
Panayotatos, N. and Villemez, C.L. (1973) The formation of a -(14)-d-galactan chain catalysed by
a Phaseolus aureus enzyme. Biochem. J., 133, 263271.
Pauly, M. and Scheller, H.V. (2000) O-Acetylation of plant cell wall polysaccharides: identication
and partial characterization of rhamnogalacturonan O-acetyl-transferase from potato suspension
cultured cells. Planta, 210, 659667.
Pauly, M., Porchia, A., Olsen, C.E., Nunan, K.J. and Scheller, H.V. (2000) Enzymatic synthesis
and purication of UDP--l-arabinopyranose, a substrate for the biosynthesis of a plant
polysaccharides. Anal. Biochem., 278, 6973.
Pazzani, C., Rosenow, C., Boulnois, G.J., Bronner, D., Jann, K. and Roberts, I.S. (1993) Molecular
analysis of region-1 of the Escherichia-coli K5 antigen gene-clustera region encoding proteins
involved in cell-surface expression of capsular polysaccharide. J. Bacteriol., 175, 59785983.
Perrin, R., Wilkerson, C. and Keegstra, K. (2001) Golgi enzymes that synthesize plant cell wall poly-
saccharides: nding and evaluating candidates in the genomic era. Plant Mol. Biol., 47, 115130.
Perrin, R.M., DeRocher, A.E., Bar-Peled, M., et al. (1999) Xyloglucan fucosyltransferase, an enzyme
involved in plant cell wall biosynthesis. Science, 284, 19761979.
Peugnet, I., Goubet, F., Bruyant-Vannier, M.-P. et al. (2001) Solubilization of rhamnogalacturonan I
galactosyltransferases from membranes of a ax cell suspension. Planta, 213, 435445.
Raetz, C.R.H. (1990) Biochemistry of endotoxins. Annu. Rev. Biochem., 59, 129170.
Rao, A.K. and Mendicino, J. (1976) Preparation of UDP-d-[U-
14
C]galacturonic acid and UDP-d-[6-
3
H]galactose with high specic activities. Analy. Biochem., 72, 400406.
Reid, J.S.G. (2000) Cementing the wall: cell wall polysaccharide synthesising enzymes. Curr. Opin.
Plant Biol., 3, 512516.
Reiter, W.-D. and Vanzin, G.F. (2001) Molecular genetics of nucleotide sugar interconversion pathways
in plants. Plant Mol. Biol., 47, 95113.
Reiter, W.-D., Chapple, C.C.S. and Somerville, C.R. (1993) Altered growth and cell walls in a
fucose-decient mutant of Arabidopsis. Science, 261, 10321035.
Ridley, B.L., ONeill, M.A. and Mohnen, D. (2001) Pectins: structure, biosynthesis, and oligogala-
cturonide-related signaling. Phytochemistry, 57, 929967.
Robertson, D., McCormack, B.A. and Bolwell, G.P. (1995) Cell-wall polysaccharide biosynthesis and
related metabolism in elicitor-stressed cells of French bean (Phaseolus vulgaris L.). Biochem. J.,
306, 745750.
Robertson, D., Smith, C. and Bolwell, G.P. (1996) Inducible UDP-glucose dehydrogenase from French
bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity.
Biochem. J., 313, 311317.
Rombouts, F.M. and Thibault, J.F. (1986) Sugar beet pectins: chemical structure and gelation through
oxidative coupling, in Chemistry and Function of Pectins (eds M.L. Fishman and J. J. Jen),
American Chemical Society, Washington, DC, pp. 4960.
Rosenow, C., Roberts, I.S. and Jann, K. (1995) Isolation from recombinant Escherichia-coli and
characterization of CMP-Kdo synthetase, involved in the expression of the capsular K5
polysaccharide (K-CKS). FEMS Microbiol. Lett., 125, 159164.
Schaumann, A., Bruyant-Vannier, M.-P., Goubet, F. and Morvan, C. (1993) Pectic metabolism in
suspension-cultured cells of ax, Linum usitatissimum. Plant Cell Physiol., 34, 891-897.
Scheller, H.V., Doong, R.L., Ridley, B.L. and Mohnen, D. (1999) Pectin biosynthesis: a solubilized
galacturonosyltransferase from tobacco catalyzes the transfer of galacturonic acid from UDP-
galacturonic acid onto the non-reducing end of homogalacturonan. Planta, 207, 512517.
Schiller, J.G., Lamy, F., Frazier, R. and Feingold, D.S. (1976) UDP-glucose dehydrogenase from
Esherichia coli purication and subnit structure. Biochim. Biophys. Acta, 453, 418425.
Schols, H.A., Posthumus, M.A. andVoragen, A.G.J. (1990) Structural features of hairy regions of pectins
isolated from apple juice produced by the liquefaction process. Carbohydr. Res., 206, 117129.
Schols, H.A., Bakx, E.J., Schipper, D. and Voragen, A.G.J. (1995) A xylogalacturonan subunit present
in the modied hairy regions of apple pectin. Carbohydr. Res., 279, 265279.
Schroeder, J.I. and Hagiwara, S. (1989) Cytosolic calcium regulates ion channels in the plasma
membrane of Vicia faba guard cells. Nature, 338, 427430.
Seitz, B., Klos, C., Wurm, M. and Tenhaken, R. (2000) Matrix polysaccharide precursors in Arabidopsis
cell walls are synthesized by alternate pathways with organ-specic expresssoin patterns. Plant
J., 21, 537546.
Shea, E.M., Gibeaut, D.M. and Carpita, N.C. (1989) Structural analysis of the cell walls regenerated by
carrot protoplasts. Planta, 179, 293308.
Sherrier, D.J. and VandenBosch, K.A. (1994) Secretion of cell wall polysaccharides in Vicia root hairs.
Plant J., 5, 185195.
Sherson, S., Gy, I., Medd, J., Schmidt, R., et al. (1999) The arabinose kinase, ARA1, gene of Arabidopsis
is a novel member of the galactose kinase gene family. Plant Mol. Biol., 39, 10031012.
Stacey, N.J., Roberts, K., Carpita, N.C., Wells, B. and McCann, M.C. (1995) Dynamic changes in
cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia
elegans into tracheary elements. Plant J., 8, 891906.
Staehelin, L.A. and Moore, I. (1995) The plant Golgi apparatus: structure, functional organization and
trafcking mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol., 46, 261288.
Stephen, A.M. (1983) Other plant polysaccharides, in The Polysaccharides, vol. 2 (ed. G.O. Aspinall),
Academic Press, NewYork, pp. 97-193.
Sterling, J., Quigley, H.F., Orellana, A. and Mohnen, D. (2001) The catalytic site of the pectin
biosynthetic enzyme -1,4-galacturonosyltransferase (GalAT) is located in the lumen of the Golgi.
Stevenson, G., Neal, B., Liu, D., et al. (1994) Structure of the O antigen of Escherichia coli K-12 and
the sequence of its rfb gene cluster. J. Bacteriol., 176, 41444156.
Stewart, D.C. and Copeland, L. (1998) Uridine 5
- diphosphate-glucose dehydrogenase from soybean

nodules. Plant Physiol., 116, 349355.
Stoddart, R.W. and Northcote, D.H. (1967) Metabolic relationships of the isolated fractions of the
pectic substances of actively growing sycamore cells. Biochem. J., 105, 4559.
Strominger, J.L. and Mapson, L.W. (1957) Uridine diphosphoglucose dehydrogenase of pea seedlings.
Biochem. J., 66, 567572.
Sullivan, F.X., Kumar, R., Kriz, R., et al. (1998) Molecular cloning of human GDP-mannose 4,6-
dehydratase and reconstitution of GDP-fucose biosynthesis in vitro. J. Biol. Chem., 273, 81938202
(Abstract).
Takeuchi, Y. and Tsumuraya, Y. (2001) In vitro biosynthesis of homogalacturonan by a membrane-
bound galacturonosyltransferase from epicotyls of azuki bean. Biosci. Biotechnol. Biochem., 65,
15191527.
Tenhaken, R. and Thulke, O. (1996) Cloning of an enzyme that synthesizes a key nucleotide-sugar
precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase. Plant
Physiol., 112, 11271124.
Thibault, J.-F., Renard, C.M.G.C., Axelos, M.A.V., Roger, P. and Crepeau, M.-J. (1993) Studies of the
length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydr. Res., 238, 271286.
Thoden, J.B. and Holden, H.M. (1998) Dramatic differences in the binding of UDP-galactose and UDP-
glucose to UDP-galactose 4-epimerase from Escherichia coli. Biochemistry, 37, 1146911477.
Thoden, J.B., Frey, P.A. and Holden, H.M. (1996a) Crystal structures of the oxidized and reduced
forms of UDP-galactose 4-epimerase isolated from Escherichia coli. Biochemistry, 35,
25572566.
Thoden, J.B., Frey, P.A. and Holden, H.M. (1996b) High-resolution X-ray structure of UDP-galactose
4-epimerase complexed with UDP-phenol. Protein Sci., 5, 21492161.
Unger, F.M. (1981) The chemistry and biological signicance of 3-deoxy-d-manno-2-octulosonic acid
(KDO). Adv. Carbohydr. Chem. Biochem., 38, 323388.
VandenBosch, K.A., Bradley, D.J., Knox, J.P., Perotto, S., Butcher, G.W. and Brewin, N.J. (1989)
Common components of the infection thread matrix and the intercellular space identied by
immunocytochemical analysis of pea nodules and uninfected roots. EMBO J., 8, 335342.
Vannier, M.P., Thoiron, B., Morvan, C. and Demarty, M. (1992) Localization of methyltransferase
activities throughout the endomembrane complex system of ax (Linum usitatissimum L)
hypocotyls. Biochem. J., 286, 863868.
Vian, B. and Roland, J.-C. (1991) Afnodetection of the sites of formation and of the further distribution
of polygalacturonans and native cellulose in growing plant cells. Biol. Cell, 71, 4355.
Vidal, S., Doco, T., Williams, P., et al. (2000) Structural characterization of the pectic polysaccharide
rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing
oligoglycosyl side chain. Carbohydr. Res., 326, 277294.
Villemez, C.L., Swanson, A.L. and Hassid, W.Z. (1966) Properties of a polygalacturonic acid-
synthesizing enzyme system from Phaseolus aureus seedlings. Arch. Biochem. Biophys., 116,
446452.
Wang, J., Dudareva, N., Bhakta, S., Raguso, R.A. and Pichersky, E. (1997) Floral scent production
in Clarkia breweri (Onagraceae) II. Localization and developmental modulation of the enzyme
S-adenosyl-l-methionine:(iso)eugenol O-methyltransferase and phenylpropanoid emission. Plant
Physiol., 114, 213221.
Watson, R.R. and Orenstein, N.S. (1975) Chemistry and biochemistry of apiose. Adv. Carbohydr. Chem.
Biochem., 31, 135184.
Wee, T.G. and Frey, P.A. (2001) Studies on the mechanism of action of uridine diphosphate galactose
4-epimerase. II. Substrate-dependent reduction by sodiumborohydride. J. Biol. Chem., 248, 3340.
Wellmann, E. and Grisebach, H. (1971) Purication and properties of an enzyme preparation fromLemna
minor L. catalyzing the synthesis of UDP-apiose and UDP-d-xylose from UDP-d-glucuronic
acid. Biochim. Biophys. Acta, 235, 389397.
Willats, W.G.T., Steele-King, C.G., Marcus, S.E. and Knox, J.P. (1999) Side chains of pectin polysac-
charides are regulated in relation to cell proliferation and cell differentiation. Plant J., 20, 619628.
Willats, W.G.T., Steele-king, C.G., McCartney, L., Orla, C., Marcus, S.E. and Knox, J.P. (2000)
Making and using antibody probes to study plant cell walls. Plant Physiol. Biochem., 38, 2736.
Willats, W.G.T., McCartney, L., Mackie, W. and Knox, J.P. (2001a) Pectin: cell biology and prospects
for functional analysis. Plant Mol. Biol., 47, 927.
Willats, W.G.T., Orla, C., Limberg, G., et al. (2001b) Modulation of the degree and pattern of methyl-
estericaton of pectic homogalacturonan in plant cell walls. J. Biol. Chem., 276, 1940419413.
Wilson, D.B. and Hogness, D.S. (1969) The enzymes of the galactose operon in Escherichia coli. II.
The subunits of uridine diphosphogalactose 4-epimerase. J. Biol. Chem., 244, 21322136.
Yu, L. and Mort, A.J. (1996) Partial characterization of xylogalacturonans from cell walls of ripe
watermelon fruit: inhibition of endopolygalacturonase activity by xylosylation, in Pectins and
Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science,
Amsterdam, pp. 7988.
Zablackis, E., York, W.S., Pauly, M., et al. (1996) Substitution of l-fucose by l-galactose in cell walls
of arabidopsis mur1. Science, 272, 18081810.
Zalitis, J. and Feingold, D.S. (1969) Purication and properties of UDPGDH from beef liver. Arch.
Biochem. Biophys., 132, 457465.
Zhang, G.F. and Staehelin, L.A. (1992) Functional compartmentation of the golgi apparatus of plant
cells; immunocytochemical analysis of high-pressure frozen- and freeze-substituted sycamore
maple suspension culture cells. Plant Physiol., 99, 10701083.

CH 3

Încărcat de

Informații document

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

CH 3

Încărcat de

Drepturi de autor:

Formate disponibile

3 Biosynthesis of pectins

-diphosphatase (NDPase) (Orellana et al., 1997) into NMP and inorganic

-triphosphate (CTP) and Kdo by CMP-Kdo synthetase (Unger, 1981). Kdo-

of the indicated acceptor.

-Apif 1,2-GalAT ONeill et al. (1997);

-Apif 1,3-GalAT ONeill et al. (1997);

-l-RhaT ONeill et al. (1997);

-Apif 1,4-l-FucT ONeill et al. (1997);

-Apif 1,3-AceAf T Vidal et al. (2000,

of the indicated acceptor.

of the indicated acceptor.

of the indicated acceptor.

of the indicated acceptor.

-O-rhamnosyltransferase. Purication and characterization of an enzyme catalyzing

-diphosphate -l-galactose epimerase of Chlorella pyrenoidosa. Fed. Proc. Fed. Am.

-diphosphate l-galactose epimerase of Chlorella pyrenoidosa. Arch. Biochem.

-diphosphate l-fucose in bacteria. Biochim. Biophys. Acta, 117, 7987.

-diphosphate l-fucose by enzymes of

-diphosphate d-mannose oxidoreductase

-diphosphatase from pea stems. Plant Physiol., 114, 99107.

- diphosphate-glucose dehydrogenase from soybean

S-ar putea să vă placă și