From the Centre for Plant Sciences, University of
Leeds, Leeds LS2 9JT, United Kingdom, § Danisco
Biotechnology, Langebrogade 1, DK 1001 Copenhagen K, Denmark,
Danisco Cultor, Edwin Rahrs Vej 38, DK-8220 Brabrand, Denmark,
the ** Department of Agrotechnology and Food Sciences, Laboratory of
Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD Wageningen,
The Netherlands, and the
Procter Department
of Food Science, University of Leeds,
Leeds LS2 9JT, United Kingdom
Received for publication, December 13, 2000, and in revised form, February 28, 2001
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ABSTRACT |
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Homogalacturonan (HG) is a multifunctional pectic
polysaccharide of the primary cell wall matrix of all land plants. HG
is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl
esterases (PMEs) that have the capacity to remove methyl ester groups
from HG. Plant PMEs typically occur in multigene families/isoforms, but
the precise details of the functions of PMEs are far from clear. Most
are thought to act in a processive or blockwise fashion resulting in
domains of contiguous de-esterified galacturonic acid residues. Such
de-esterified blocks of HG can be cross-linked by calcium resulting in
gel formation and can contribute to intercellular adhesion. We
demonstrate that, in addition to blockwise de-esterification, HG with a
non-blockwise distribution of methyl esters is also an abundant feature
of HG in primary plant cell walls. A partially methyl-esterified
epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix
and occurs at points of cell separation at intercellular spaces in
parenchymatous tissues of pea and other angiosperms. Analysis of the
properties of calcium-mediated gels formed from pectins containing HG
domains with differing degrees and patterns of methyl-esterification
indicated that HG with a non-blockwise pattern of methyl ester group
distribution is likely to contribute distinct mechanical and porosity
properties to the cell wall matrix. These findings have important
implications for our understanding of both the action of pectin methyl
esterases on matrix properties and mechanisms of intercellular adhesion
and its loss in plants.
The load-bearing components of primary cell walls, principally the
cellulose and hemicellulose polysaccharide network, are embedded in a
pectic matrix that is structurally complex and heterogeneous. The
pectic matrix contributes to both the physical integrity and physiological status of cell walls, but the functional implications of
the structural complexity of this matrix are poorly understood. Typically, heterogeneous populations of pectic polymers are present in
primary cell walls, and our present understanding is that all essentially consist of galacturonan backbones with or without various
side chain additions (1-3). Backbone domains consist of either
contiguous 1,4-linked The HG domain of the pectic network is implicated in influencing a
range of cell wall properties that impact upon cell expansion, cell
development, intercellular adhesion, and defense mechanisms. Stretches
of HG with un-esterified GalA residues can associate by calcium
cross-linking (7). Such association promotes the formation of
supramolecular pectic gels, which are important in controlling the
porosity and mechanical properties of cell walls and contribute to the
maintenance of intercellular adhesion (8, 9). Plant cells are adhered
by contact across middle lamellae, which are HG-rich regions of the
cell wall developed from cell plates formed at cytokinesis. In plants,
cell adhesion is a default state and cell separation an active process
that is under developmental control (8, 10). In addition to the roles
of the HG polysaccharide domain, HG-derived oligogalacturonides
generated by pectinolytic cleavage are involved in signaling processes
during development and in defense responses to plant pathogens
(11-13).
It is thought that HG is highly methyl-esterified when exported into
cell walls and is subsequently de-esterified by the action of pectin
methyl esterases (PMEs) in the cell wall (3, 14). PME genes occur in
multigene families and encode isoforms with differing action patterns
with respect to the removal of methyl esters. However, the specific
functions of PME populations in the context of cell expansion and other
processes are not well understood (15-17). Methyl esters can be
distributed in diverse patterns along HG chains and it is clear that
the action patterns of plant PMEs (pPMEs) can be influenced by local
cell wall pH, the existing balance of methyl and free carboxyl groups
on HG substrates, and metal ion concentration (16, 18-22). In addition to plants, some fungi and bacteria also produce PMEs, and it is significant that the action patterns of plant and fungal PMEs are
thought to be different. It is generally proposed that pPMEs remove
methyl esters in a processive blockwise fashion (single chain
mechanism), giving rise to long contiguous stretches (blocks) of
un-esterified GalA residues in HG domains of pectin (23-25). In
contrast, the action of fungal PMEs (fPMEs) is generally regarded as
random (or multiple chain mechanism), resulting in the
de-esterification of single GalA residues per enzyme/substrate
interaction (23-25). However, the precise nature of the action
patterns of fPME and pPME are far from clear, and some pPMEs appear to
have the capacity to remove a limited number of methyl esters per
reaction, giving rise to short un-esterified blocks (20). Moreover,
recently developed procedures for the enzymatic fingerprinting of
pectic fragments have indicated that the action patterns of fPMEs are non-blockwise, but probably not in fact random (24). In this report,
the action pattern of an orange pPME is referred to as blockwise,
whereas the action pattern of an Aspergillus fPME and the
chemical process of base catalysis are referred to as
non-blockwise.
Previous investigations of the action patterns of PMEs have focused on
the action of isolated PMEs on pectin substrates in vitro
(16). However, the use of anti-HG monoclonal antibodies with
appropriate binding specificities allows the products of de-esterification processes to be analyzed in planta in the
context of cell wall architecture and cell development. We have used
monoclonal antibodies with differing binding requirements with respect
to the distribution of methyl ester groups to investigate the spatial regulation of HG with blockwise and non-blockwise methyl group distributions in pea. The monoclonal antibody PAM1 (which binds specifically to long stretches of un-esterified HG produced most readily by blockwise de-esterification) and JIM5 (which binds to a
range of partially methyl-esterified HG domains) have been described
previously (26-28). Non-blockwise de-esterification of HG has been
analyzed using a monoclonal antibody (LM7) described for the first time
in this report. In addition, we have investigated the functional
implications of varying degrees and patterns of methyl-esterification
of HG in the context of the physical and physiological properties of
calcium-HG gels. Taken together, the results indicate that wall-based
pPMEs with a range of action patterns can produce HG with non-blockwise
and blockwise distributions of methyl esters at discrete cell wall
microdomains, resulting in distinct spatially regulated matrix properties.
Monoclonal Antibodies--
Monoclonal antibody LM7 was generated
using hybridoma technology subsequent to the immunization of a group of
rats with lime pectin with a degree of methyl-esterification (DE) of
22.9%, a degree of amidation of 27.3% and an average molecular mass
of 84 kDa. Immunization, hybridoma preparation, and cloning procedures were performed as described previously (29). LM7 was selected by
differential immuno-dot assay (IDA) screens of a series of pectin
samples differing in DE with blockwise and non-blockwise patterns of
de-esterification (see below). Monoclonal antibodies JIM5 and PAM1 were
produced by hybridoma and phage display technologies, respectively
(26-28).
Production of Lime Pectins with Defined DE--
A series of lime
pectins with different patterns (blockwise and non-blockwise), and
defined DE were prepared by enzymatic and chemical treatments of a
commercial highly methyl-esterified (81%) lime pectin (E81,
GRINDSTEDTM Pectin URS 1200) as described previously (24). Briefly,
one series was produced by blockwise de-esterification of E81 with a
pPME isolated from orange peel (P-series), while another series was
produced by non-blockwise de-esterification of E81 with a fPME from
Aspergillus niger (F-series). A further set of samples was
also produced by non-blockwise de-esterification of E81 by base
catalysis (B-series). A sample of completely de-esterified pectin (E0)
was prepared by treatment with fPME followed by base catalyzed
de-esterification.
Digestion of Pectin B34 with endo-Pectin Lyase (PL) and
endo-Polygalacturonase II (PG II)--
Pectin sample B34 (prepared by
base catalysis and with a DE of 34%) was digested with PL (EC
3.2.1.15) or PG II (EC 4.2.2.10), both from Aspergillus
niger. B34 was dissolved in 50 mM NaOAc (pH 5.0 for PL
and pH 4.2 for PG II) at a concentration of 5 mg/ml by overnight
rocking at room temperature. 0.1 unit of PL or 0.2 unit of PG II was
added to 1 ml of the above pectin solution and in both cases incubated
at room temperature for 20 h. The reaction was stopped by boiling
for 5 min.
Competitive Inhibition ELISAs (ciELISAs)--
The effect of PL
and PG II digestion of B34 on the binding of LM7 was assessed by
ciELISAs with untreated B34 as the immobilized antigen. Untreated B34
(50 µg/ml in Tris-buffered saline (TBS)) was coated (100 µl/well)
overnight at 4 °C onto microtiter plates (Maxisorp, Nunc, Denmark).
The coating solution was removed, and plates were blocked at room
temperature with 3% bovine serum albumin in TBS (3%BSA/TBS) for
2 h (200 µl/well). Following washing, competitor solutions
(untreated B34, B34 digested with PL, and B34 digested with PG II) were
applied (100 µl/well) as 5-fold serial dilutions in 3%BSA/TBS. All
competitor solutions also contained LM7 at a final level of 1/100
dilution of hybridoma supernatant (corresponding to ~90% maximal
binding on antibody capture ELISAs). After 2 h of incubation,
plates were washed and secondary antibody (anti-rat IgG horseradish
peroxidase conjugate, Sigma, Poole, United Kingdom) diluted 1/1000 in
3%BSA/TBS applied (100 µl/well) and incubated for 2 h at room
temperature. After washing, plates were developed with a tetramethyl
benzidine-based substrate (150 µl/well). After stopping the reaction
with 2 M H2SO4 (35 µl/well),
absorbances were read at 450 nm. Concentrations of competitors
resulting in 50% inhibition (IC50) of antibody binding
were determined. Values from controls with no competitor were taken as
0% inhibition of antibody binding, and values from controls with no
LM7 antibody represented 100% inhibition of binding. In some cases,
CaCl2 or MgCl2 were added to competitor
solutions to a maximum level of 1 mM.
Immuno-dot Assays--
Pectins were dissolved in water to a
concentration of 5 mg/ml or 10 mg/ml and applied as 1-µl aliquots to
nitrocellulose (Scheicher & Schuell) in a 5- or 10-fold dilution
series. Nitrocellulose membranes were air-dried at room temperature for
at least 30 min. After blocking for 1 h in phosphate-buffered
saline (PBS) containing 5% (w/v) fat-free milk powder (5%M/PBS),
membranes were incubated for 1 h in primary antibodies diluted in
5%M/PBS. JIM5 and LM7 were used as 1/10 dilutions of hybridoma
supernatants while PAM1 was used at a concentration of ~1 × 1011 phage particles/ml (~1/100 dilution of phage
prepared by polyethylene glycol precipitation; Ref. 27). In all cases,
membranes were incubated in primary antibodies for 1.5 h. After
washing, membranes were incubated for 1.5 h in secondary antibody
(anti-rat horseradish peroxidase conjugate (for JIM5 and LM7) (Sigma,
Poole, United Kingdom) or anti-M13 horseradish peroxidase conjugate
(for PAM1) (Amersham Pharmacia Biotech) diluted 1/1000 in 5%M/PBS.
Membranes were briefly washed prior to development in substrate
solution (25 ml of de-ionized water, 5 ml of MeOH containing 10 mg/ml
4-chloro-1-naphthol, 30 µl 6% (v/v) H2O2).
In some cases, immobilized pectin samples were chemically de-esterified
by incubation of membranes in 0.1 M
Na2CO3 for 1 h prior to processing.
Immunolabeling of Plant Material--
Pea (Pisum
sativum L. cv. Avola) seeds were imbibed overnight in tap water,
sown in sterile vermiculite, and grown for 7-15 days. Regions (0.5 cm
long) of stem, petiole, or root were excised and sectioned by hand to a
thickness of ~100-300 µm. Sections were placed immediately in
fixative consisting of 4% paraformaldehyde in 50 mM PIPES,
5 mM MgSO4, and 5 mM EGTA.
Following 30 min of fixation, sections were washed in the PIPES buffer
and then incubated for 1 h in primary antibody diluted in
5%M/PBS. JIM5 and LM7 were used as 10- and 3-fold dilutions of
hybridoma supernatants, respectively. PAM1 was used at a concentration
of ~5 × 1011 phage particles/ml (~ 1/20 dilution
of phage prepared by polyethylene glycol precipitation; Ref. 27).
Sections were washed by gently rocking in PBS prior to incubation for
1 h in secondary antibody. For visualization of LM7 and JIM5
binding, the secondary antibody was anti-rat IgG coupled to fluorescein
isothiocyanate (Sigma). For visualization of PAM1 binding, a secondary
antibody was prepared by conjugating an anti-M13 antibody (Amersham
Pharmacia Biotech) to fluorescein isothiocyanate using a protein
conjugation kit (Sigma). All secondary antibodies were used at
dilutions of 1/100 in 5%M/PBS. After washing in PBS, sections were
mounted in anti-fade agent (Citifluor, Agar Scientific) and examined on
a microscope equipped with epifluorescence illumination (Olympus BH-2).
In some cases, hand sections were chemically de-esterified by
incubation in 0.1 M Na2CO3 for
1 h prior to processing.
In certain cases plant material was embedded in resin for electron
microscopy. Regions (2 mm long) of stem were fixed in 2.5% (w/v)
glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 2 h at 4 °C, then washed extensively in 0.1 M
sodium phosphate buffer. Material was then post-fixed in 0.1% (w/v)
osmium tetroxide in 0.1 M sodium phosphate buffer, for
1 h at 4 °C, washed extensively with 0.1 M sodium
phosphate buffer, and then dehydrated in an ethanol series. Dehydrated
material was infiltrated with resin (LR White) (London Resin, Reading,
United Kingdom), then placed in gelatin capsules containing resin and
allowed to polymerize at 37 °C for 5 days. Material used for
transmission electron microscopy but not immunogold labeling, was
stained en bloc with 4% (w/v) uranyl acetate in distilled
water overnight at 4 °C, then washed extensively with water before
being dehydrated and embedded as described above.
For immunogold labeling, sections obtained from resin-embedded material
(~0.1 µm thick) were incubated in 3%BSA/TBS for 30 min. Sections
were then incubated in a solution containing JIM5 or LM7 diluted 1/10
in 3%BSA/TBS for 1.5 h. The sections were washed with 3%BSA/TBS,
and then incubated in secondary antibody (anti-rat monoclonal antibody
conjugated to 10 nm colloidal gold (Sigma) diluted 1/40 in 3%BSA/TBS
for 1.5 h. Section were washed with TBS and post-stained with 4%
uranyl acetate in distilled water for 15 min, and then washed
extensively with distilled water. Sections were observed with an
electron microscope (1200ex, JEOL, Tokyo, Japan). All incubations were
at room temperature.
Calcium-mediated Gelation of Lime Pectin Samples--
A subset
of the P-, F-, and B-series of lime pectins that contained the epitopes
recognized by LM7 or PAM1 to various levels were selected for analysis
of the effects of the degree and pattern of methyl-esterification on
the physical properties of calcium-mediated gels. Gels were prepared
from E0, F11, F31, P41, F43, and B34, essentially as described
elsewhere (30). Pectin solutions (2% (w/v) in de-ionized water) were
prepared with gentle rocking at 4 °C for at least 18 h. For
casting gels, 900 µl of pectin solution was transferred to a 2-ml
syringe (with an internal diameter of 8.6 mm) from which the nozzle end
had been removed. 67 µl of 500 mM CaCl2 was
added as a layer to the top of the pectin solution (to give a final
CaCl2 concentration of 35 mM) and the cut end of the syringe sealed with tape. Gels were left to equilibrate for
24-48 h at 4 °C. Polymerized gels were removed using the syringe plunger and cut to a uniform height using a custom-made nylon cutting
block with guide slots.
Rheological Testing of Calcium-mediated Pectin Gels--
Gel
samples prepared as described above were subjected to compressive tests
to determine their elasticity under low strain and their yield points
under high strain. Compression tests were performed using an in-house
texture measuring device, known as the "Ministron," constructed in
the University of Leeds Department of Food Science instrument workshop.
This device was used to analyze the controlled compression of gel
samples between two parallel stainless steel plates at a precisely
defined speed. The lower plate, on which the sample rested, is mounted
on a high precision load cell (Maywood Instruments, Basingstoke, United
Kingdom). The gap between the plates and the force, F, on
the load cell are electronically logged throughout the experiment at a
suitable frequency. For all experiments, at least four samples from
different gel preparations were analyzed.
Gels were compressed by reducing the gap between the plates at a rate
of 0.1 mm s Analysis of the Water Holding Capacity of Calcium-mediated Pectin
Gels under Compression--
It was observed that there were
significant differences in the water expelled from gel samples during
compression. The final percentage of water lost was calculated from the
change in the volume of the gels after compression up to a force of
9.82 N (E0, F11, and F31) or by the force that resulted in yielding
(P41, F43, and B34). The yield point was taken as the point when the gradient of F versus s became
negative. Yielding was sometimes also accompanied by visible splitting
of the gel piece.
Determination of the Porosity of Calcium-mediated Pectin
Gels--
The porosity of gels to protein was determined by incubating
gels in a solution of BSA and measuring the incorporation of protein
into gels over time. For each time point, two gel blocks (prepared as
described previously) above were incubated with gentle rocking in 2 ml
of BSA solution (5 mg/ml in de-ionized water). Following incubation,
the two gel blocks were washed briefly in de-ionized water, briefly
blotted dry on filter paper to remove surface liquid, and incubated
with gentle rocking in 2 ml of 50 mM calcium chelator
(CDTA) (pH 7) until gels were completely dissolved (~30 min). The
protein concentration of the solutions were then analyzed using
Bradford protein assays. For each pectin gel sample, four replicates of
the protocol described were analyzed for each time point.
Monoclonal Antibody LM7 Recognizes an Epitope of HG Produced by
Non-blockwise De-esterification--
Monoclonal antibody LM7 was
generated subsequent to immunization with a lime pectin (containing
88.3% GalA) and selected by IDA screening on the basis of its specific
binding to a subset of F-series and B-series pectins that have
non-blockwise patterns of methyl-esterification as shown in Fig.
1a. The binding of the previously characterized anti-HG JIM5 (26, 28) and the phage antibody
PAM1 (27) to the same pectin samples are shown for comparison (Fig. 1,
b and c). Both the degree and the pattern of
de-esterification influence the capacity of these antibodies to bind to
HG domains. LM7 did not bind at the highest level tested (1 µg) to
any of the P-series pectins or to F-series pectins with DE of 76%,
69%, or 11% (Fig. 1a). LM7 did bind to F-series pectins with DE from 58% to 31% with increasing avidity, and LM7 bound to F31
with a detection limit of <0.2 µg (Fig. 1a). LM7 bound weakly, at the highest level tested (1 µg), to a B-series sample with
a DE of 43% and bound to B-series pectins with DEs of 34 and 15% with
detection limits of <0.2 µg. The binding of LM7 to all samples was
abolished when blots were de-esterified by treatment with 0.1 M Na2CO3 prior to labeling (data
not shown). The binding profile of LM7 indicates that it is specific
for a partially methyl-esterified domain of HG and that its epitope is
most readily produced by the non-blockwise de-esterification processes
such as that produced by fPME action and base catalysis. In contrast,
PAM1 binds to long (>30 residues) contiguous stretches of
de-esterified GalA residues produced by the blockwise action of pPME as
shown in Fig. 1b. However, un-esterified blocks are also
produced if enough non-blockwise de-esterification occurs and the PAM1
epitope is also produced by extensive de-esterification by fPME or base
catalysis as indicated by binding to F11 and B15, respectively (Fig.
1b). The optimal binding requirements of JIM5 are not fully
defined, and JIM5 has the capacity to bind to a wide range of HG
epitopes with varying degrees and patterns of methyl-esterification as shown in Fig. 1c and as discussed elsewhere (28).
The Partially Methyl-esterified Epitope Recognized by LM7 Is
Degraded by the Action of Both Endo-polygalacturonase and Pectin
Lyase--
In order to explore the structure of LM7 epitope further,
its susceptibility to digestion by PG II and PL was assessed. The products of enzymatic digestion were analyzed by ciELISAs using untreated B34 as the immobilized antigen as shown in Fig.
2. The use of ciELISAs allowed the
binding of LM7 to digest fragments to be analyzed in solution, rather
than in an immobilized state, as would be the case for IDAs. This is
important because the binding of pectic fragments to nitrocellulose
sheets is related to fragment size. For example, oligogalacturonides
with degrees of polymerization less than 15 are not immobilized
effectively onto nitrocellulose sheets (data not shown).
When untreated B34 was used as a soluble competitor in competitive
inhibition ELISAs, 20 µg/ml was required to achieve a 50% inhibition
(IC50) of LM7 binding. LM7 binding to B34 was abolished entirely following complete digestion with PG II and PL, and the digestion fragments failed to produce any significant inhibition of LM7
binding even at the highest level used (1 mg/ml) as shown in Fig. 2.
The PG II used from A. niger has an absolute requirement for
de-esterified GalA residues to be present at both sides of the cleavage
position (subsites +1 and
Confirmation of epitope structure can be obtained by demonstration of
binding to oligosaccharides in ciELISAs, and this generally indicates
carbohydrate epitope sizes of 4 to 6 sugars (27, 28). Fully
methyl-esterified and fully un-esterified oligogalacturonides with
degrees of polymerization up to 8, prepared as described elsewhere
(28), were not effective inhibitors of LM7 binding in ciELISAs (data
not shown). In order to characterize the LM7 epitope fully, a range of
oligogalacturonides with intermediate degrees and defined patterns of
methyl-esterification would be required, and currently these are not
available. Nonetheless, it is clear that LM7 binds to a HG domain of
pectin that contains both un-esterified and methyl-esterified GalA
residues, that this structure is generated most readily by
non-blockwise de-esterification processes, and that the epitope is
distinct from any other previously characterized HG epitope.
LM7 Binding Is Not Dependent on, nor Reduced by, Calcium-mediated
Chain Association of HG Domains--
The possible effects of the
formation of conformational HG structures on LM7 binding were
investigated by addition of divalent cations to ciELISA assays. The
presence of calcium or magnesium at up to 1 mM had no
effect on LM7 binding to either immobilized or soluble pectin sample
F31, as shown in Fig. 3. Levels of
calcium above 1 mM resulted in gel formation and disruption
of the assay. Surface labeling of calcium pectate gel blocks (prepared
as described previously) indicated that LM7 binding was retained when
HG was cross-linked via calcium (data not shown). Taken together, these results indicate that LM7 binding is neither dependent on, nor abolished by, calcium-mediated HG chain association of HG domains.
The Partially Methyl-esterified Epitope Recognized by LM7 Occurs in
Discrete Micro-domains of Primary Cell Walls--
The partially
methyl-esterified epitope recognized by LM7 was found to be abundant in
plant tissues. The epitope was most readily visualized in plant
materials that had undergone preparations that maintained maximum
antigenicity, i.e. hand-cut sections or cryosections of
non-embedded material. The epitope recognized by LM7 appears to be
unstable in pectin preparations and in plant material. The capacity of
F31 and B34 pectin samples to be recognized by LM7 was gradually lost
when these samples were stored as frozen solutions (data not shown).
Similarly, the epitope was lost from plant materials when they had
undergone extensive preparation, such as resin-embedding for
immunocytochemistry. As discussed, LM7 binding is critically dependent
on DE being with a certain range, and the instability of the epitope is
probably due to de-esterification occurring during freeze/thawing or processing.
The distribution of the LM7 epitope was examined most extensively in
pea seedlings (a system that is amenable to hand-sectioning) and
immunofluorescent labeling of sections indicated that the LM7 epitope
was restricted to discrete regions of cell walls in the roots, stems,
and leaves of seedlings. The distribution of the LM7 epitope in pith
parenchyma cells in a transverse section of a pea stem internode is
shown in Fig. 4a. In this
region, most cell junctions are expanded to some extent to form
intercellular spaces. The LM7 epitope was restricted to the region of
the cell wall lining the intercellular spaces between the parenchyma
cells, and no LM7 epitope was detected in other regions of the cell
walls in this tissue. Fig. 4d shows a higher magnification
of an individual intercellular space and shows that the LM7 epitope was
particularly abundant at the corners of the intercellular space,
i.e. the point between adherent and separated cell walls.
For comparison, the labeling patterns of JIM5 and PAM1 on equivalent
sections are shown in Fig. 4 (panels b and
e and panels c and f,
respectively). JIM5 bound to all primary cell walls (Fig.
4b). At higher magnification (Fig. 4e), the
increased abundance of the JIM5 epitope in the region of the cell wall
lining the intercellular spaces and in the region of the wall closest
to the plasma membrane was evident. The blockwise de-esterified HG
epitope recognized by PAM1 occurred in discrete cell wall domains (Fig.
4, c and f). Like LM7 and JIM5, PAM1 bound to
material in the cell wall lining intercellular spaces and, like JIM5,
did not bind to the central regions of the cell walls (including the
middle lamellae). In contrast to LM7, PAM1 also bound to the region of
the wall closest to the plasma membrane, but, unlike JIM5, PAM1
labeling was absent from inner regions of the cell wall adjacent to the
intercellular space (Fig. 4f). The reason for the apparently
thicker cell walls and more diffuse nature of labeling obtained using
PAM1 is due to the fact that PAM1 is a phage display antibody and large
(~800 nm long) intact phage particles were used as the primary stage antibody.
The localization of the LM7 epitope at intercellular spaces was
consistent throughout all tissues in the pea stem, and the relationship
between the occurrence of the LM7 epitope and the formation of
intercellular space was examined in more detail by examination of
cortical parenchyma tissue. The immunolabeling of a small non-expanded
intercellular space without air, occurring in the cortical parenchyma
closer to the epidermis, and larger intercellular air spaces that occur
between larger parenchyma cells toward the center of the stem are shown
in Fig. 5 (panel a
and panels b and c respectively. Fig.
5 (a-c) shows dual labeling of sections with LM7 and the
cellulose-binding fluorescent probe, calcofluor. At non-expanded
junctions, LM7 bound to all of the developing space (which at this
stage is filled with expanded middle lamellae) but did not bind to any
other regions of the cell wall (Fig. 5a). At larger,
air-filled junctions, the LM7 epitope was most abundant at the corners
of the triangular intercellular spaces as viewed in the sections shown
in Fig. 5 (b and c). The epitope recognized by
LM7 is therefore most abundant in regions of the expanded middle
lamellae at the point of separation of cell walls. The ultrastructure
of intercellular spaces was investigated by transmission electron
microscopy. However, as discussed above, the epitope recognized by LM7
is prone to instability and, when stem material was resin-embedded for
immunofluorescent or immunogold detection, the LM7 epitope could not be
detected. However, the fine structures of comparable intercellular
junctions between parenchyma cells are shown in Fig. 5
(d-f). These microgaphs indicate that, as air spaces form,
there is an accumulation of darkly staining material at the corners of
the junction (Fig. 5, e and f). The position of
this material appears to correspond to regions of the wall containing
the LM7 epitope as localized by immunofluorescent labeling of
non-embedded material (Fig. 5, b and c). Pectic
material typically stains darkly in the transmission electron
microscopy staining protocol used, and immunogold labeling with JIM5
indicated the presence of HG at this position (Fig. 5f,
inset).
The LM7 epitope was also found to occur in regions of the thickened
outer cell walls of stem epidermal cells as shown in Fig. 6a. The distribution of the
LM7 epitope was restricted to discrete regions of the outer epidermal
cell wall that were aligned with radial cell walls between epidermal
cells but was absent entirely from the radial cell walls themselves
(Fig. 6a). In contrast, the JIM5 epitope occurred only
sparsely in most of the thickness of the outer tangential epidermal
cell walls but abundantly in radial epidermal cell walls (Fig.
6b).
In addition to its occurrence in pea stem tissue discussed above, the
LM7 epitope was detected at comparable locations in epidermal cell
walls and intercellular spaces in all pea seedling organs examined.
These included domains within the outer cell wall (just under the
cuticle) of the leaf epidermis in line with radial cell walls as shown
in Fig. 6c, leaf parenchyma near the midrib (Fig.
6d), and root cortical parenchyma (Fig. 6e).
Furthermore, the LM7 epitope was found in equivalent regions of
developing intercellular spaces and epidermal cell junctions in a range
of angiosperms including carrot (Apiaceae), maize (Poaceae),
Silene latifolia (Caryophyllaceae), Kalanchoe
daigremontiana (Crassulaceae), and Nicotiana tabacum
(Solanaceae) (data not shown).
Functional Implications of Blockwise and Non-blockwise
De-esterification of HG Domains of Pectin--
A series of model
pectin samples containing the epitopes recognized by PAM1 or LM7 were
used in a series of in vitro assays to investigate the
implications for a range physical properties of blockwise or
non-blockwise distributions of methyl esters on HG domains. Calcium
gels formed from pectins E0, F11, F31, P41, F43, and B34 all maintained
a stable shape under gravity but differed in their opacities, as shown
in Fig. 7a. The different
opacities of the gels suggested that the degree and pattern of
de-esterification of HG domains influenced the structure and pore size
of the gel and that this was reflected in differences in the light
scattering properties.
The degree and pattern of methyl-esterification was found to effect the
elasticity of the gels and their response to compressive strain. There
were significant differences in the extent and manner of deformation of
gels when compressed by a force of 9.82 N or to yield point as shown in
Fig. 7 (b and c). Compression testing of gels
indicated that both the degree and pattern of methyl-esterification were important in determining the yield point and elasticity of gels,
as shown in Figs. 7d and 8.
The mean values of the yield strain, yield force, and elasticities are
given in the tables of Figs. 7d and 8a, with
standard deviations given in parentheses. These values were
obtained from averages from at least two pairs of measurements, each
pair being made on a gel formed from a completely separate solution.
The plots of F versus s shown are
those of the average values for the samples.
F31 and F43 have the same distribution pattern of methyl groups and
differ only in DE. Although F31 formed a strong gel that did not yield
at a force of 9.82 N, gels formed from F43 were relatively weak under
compression and yielded at a mean force of 0.92 N (Fig. 7d).
Samples F43 and P41 have different distribution patterns of methyl
groups but differ in DE by only 2%. However, there was nearly a 3-fold
difference in the yield point of gels formed from P41 and F43 (Fig.
7d). The outstanding feature of gels formed from P41 was the
fact that, although the gels did yield due to the development of
fractures in the gel below F = 9.82 N, on removal of
the force the sample recovered its original dimensions almost
immediately, as seen in Fig. 7 (b and c). In complete contrast, and as shown in Fig. 7 (b and
c), gels formed from F43 showed practically no recovery on
removal of the compressive force and behaved more like a plastic
material, irreversibly deformed by a stress above its yield stress.
Similarly, samples F31 and B34 have similar DE, but gels prepared from
these samples differed greatly in their response to compression. The
force at which gels formed from B34 yielded was at least 10 times less
than the yield force of gels formed from F31. These results strongly
suggest that, although methyl groups on both F31 and B34 are
distributed in a non-blockwise fashion in both cases, the distribution
patterns are not identical. This was also suggested by their differing capacities to be degraded by PL (24).
With respect to the elasticity of gels, decreasing DE was broadly
correlated with increasing elasticity as shown in Fig. 8 (a
and b), as would be expected if free carboxyl groups are
required for calcium cross-linking to occur. However, as was the case
for the yield force, both the degree and pattern of
methyl-esterification appeared to be important in determining the
elasticity of gels, as not all the samples fell exactly on the same curve.
The Effect of the Degree and Pattern of Methyl-esterification on
the Water Holding Capacity and Porosity of Calcium-mediated Pectin
Gels--
Gels formed from E0, F11, F31, P41, F43, and B34 differed
significantly in their water holding capacities and porosity to protein
as shown in Fig. 9. For the F-series
samples, there was some correlation between DE and water holding
capacity as shown in Fig. 9a. However, for the samples as a
whole, the correlation between DE and water holding capacity was not
strong. For example, there were significant differences in the water
holding capacities of gels formed from F31 and B34 and in the water
holding capacities of gels formed from P41 and F43. It is worth noting
that the pectin gels that showed the largest volume loss (E0 and F11)
were also the ones that appeared to collapse under compression with
relatively little expansion, or increase in the lateral dimension. For
these samples this implies a collapse of the gel structure as water is
lost, unlike F43 mentioned previously, which maintains a coherent structure, loses relatively little water, and exhibits the pronounced broadening of an incompressible, plastic material.
For all the samples, the rate of incorporation of BSA into gels
decreased over time, but there were significant differences in the
amount of protein that had been incorporated into gels after 20 h
as shown in Fig. 9b. Although the gels formed from F31 and
B34 had similar porosities, the gels formed from P41 and F43 had the
highest and lowest porosities, respectively. Additionally, the porosity
of gels formed from F11 was more similar to that of gels formed from
F43 than it was to gels formed from F31. From these results, it appears
that the pattern rather the degree of methyl-esterification of HG may
be the more important factor in determining both the water holding
capacity and the porosity of calcium-mediated pectin gels.
The formation of the pectic network in the primary cell wall
matrix is contingent upon polysaccharide synthesis in the Golgi apparatus, its deposition and assembly in the cell wall, and subsequent modification by cell wall-based enzymes in response to functional requirements. The modulation of the degree and pattern of
methyl-esterification of HG is one aspect of this functional fine
tuning, and the work reported here indicates that HG domains with
distinctive physical properties are produced in discrete microdomains
of primary cell walls. The fact that LM7 bound to cell walls in a range
of organs and species indicates that a non-blockwise pattern of
methyl-esterification is a widespread aspect of HG modification in plants.
It is likely that the abundance and pattern of methyl ester groups
varies along an HG chain, and, although some epitope structures, such
as that recognized by LM7, have distinct locations within the
intercellular matrix, they do not necessarily occur exclusively. For
example, the epitopes recognized by LM7, JIM5, and PAM1 were all
present at the lining of intercellular spaces of pea stem parenchyma.
Therefore, discrete microdomains of the cell wall matrix are likely to
contain HG with a mixture of HG methyl ester distribution patterns
resulting in complex combinations of physical properties. In
vitro analysis of calcium-mediated model pectin gels indicated
that the compressive strength, elasticity, water holding capacity, and
the porosity of gels was significantly influenced by both the pattern
as well as the degree of methyl-esterification of HG domains. Although
it is possible that some variation of the degree and pattern of
methyl-esterification of HG may be generated during synthesis, it is
thought that HG is usually highly methyl-esterified prior to insertion
into cell walls (3, 14). It is therefore likely that the activity of
PMEs with varying de-esterification action patterns is an important
mechanism for modifying matrix properties in planta.
The results reported here demonstrate that blockwise and non-blockwise
distribution patterns of methyl groups on HG can significantly influence the physical properties of calcium-mediated pectin gels. Details of PME action and its wider consequences on the cell wall environment are far from clear. One aspect of PME action may be to
generate extracellular pH gradients that in turn orchestrate cell wall
loosening via pH-sensitive processes. For example, partial inhibition
of the expression of a pea PME gene (rcpme1) has been correlated with changes in extracellular pH and in cell development (31). Moreover, PME activity can itself modulated by pH. Kinetic analysis of PMEs from mung bean hypocotyl demonstrated the coexistence of three isoforms with different pH and ion sensitivities and different
Km and Vmax (15).
In vitro analysis of the mung bean PME isoforms indicated
that different action patterns could be generated by different pH
conditions and the DE of the substrate (16). These observations raise
the possibility of feedback mechanisms modulating HG structure and
hence cell wall matrix properties. An another aspect of possible
feedback is that HG backbones with differing methyl-esterification
patterns are differentially susceptible to subsequent enzymatic
cleavage. PME action on a highly esterified HG is required before
polygalacturonases can cleave effectively. Plant polygalacturonases
occur in large multigene families, and members are likely to have a
range of functions in plant growth and development although these are
as yet uncertain (32, 33). The generation of HG substrates susceptible
to polygalacturonase cleavage is likely to be greatly influenced by PME activities.
The LM7 epitope is the first spatially regulated HG epitope to be found
to occur at a consistent location within cell walls and intercellular
matrices across a range of plant organs and species. This consistency
suggests a specific functionality for the LM7 epitope-rich pectin at
these locations. The occurrence of the LM7 epitope within the cell
walls of epidermal cell junctions and intercellular spaces indicates
that a pectic HG domain with a non-blockwise distribution of methyl
esters (and with consequent distinctive properties) has a role
associated with cell adhesion at cell junctions. Of the model pectin
samples tested, F31 and B34 contained the LM7 HG epitope at the highest
abundance. These samples have similar DE and are both the products of
(different) non-blockwise de-esterification procedures, but gels formed
from the B34 and F31 differ significantly in their physical properties. This suggests that even subtle differences in the distribution patterns
of methyl groups (revealed by enzymatic fingerprinting in the case of
B34 and F31; Ref. 24) may have profound effects on matrix properties.
The occurrence of the LM7 epitope in plant material indicated the
presence of HG with non-blockwise distributions of methyl groups, but
not the precise distribution pattern. However, as more PME genes are
cloned and their products characterized in developmental contexts,
these details may become clear.
The driving force for intercellular space formation appears to be
turgor pressure producing tensile forces in cell walls and inducing a
tendency toward reduced volume and hence spherical cell shapes (34).
Three-way cell junctions are therefore subjected to forces tending
toward cell separation (34). To initiate space formation, a region of
primary cell wall must first be dismantled to allow the middle lamellae
to link up (8) and as shown schematically in Fig.
10. Intercellular space then results
from controlled splitting at the middle lamellae. As space develops,
the stresses are greatest at regions of adhered walls bordering the
separated cell walls and the intercellular space. The LM7 epitope-rich
pectin appears to be present from the earliest stages of intercellular
space formation and to be most abundantly maintained at the points of cell to cell contact. Pectin containing this epitope may have a direct
role in maintaining cell wall to cell wall links at these points
through calcium-mediated cross-linking. Discrete electron-dense regions
within middle lamellae of pea cotyledon parenchyma have been proposed
to be involved in limiting cell separation in this tissue (35). In the
outer thickened cell wall of epidermal cells, the LM7 epitope occurs in
discrete regions that are associated with cell junctions and may be
involved in maintaining the integrity of the outer cell layer. An
accumulation of calcium in equivalent regions of outer epidermal cell
walls (together with an accumulation at the corners of intercellular
spaces) has been reported in mung bean hypocotyl (36). The common
aspect between the corners of an intercellular space and points of
outer epidermal cell wall at the plant surface is that they are both
points of contact between two cells, although in the former case cell
separation occurs to some extent. LM7 epitope-rich pectin may provide
an appropriate environment (porosity, ionic status, etc.) for processes
that directly maintain cell to cell contacts (or indeed for enzymes involved in dismantling such contacts, although this seems less likely
as cell separation is not generally a feature of epidermal cell
junctions). An additional possibility is that the LM7-binding pectin
may have a defensive role at points of intercellular attachment. For
example, a subtly altered pattern of HG methyl ester groups may alter
both the capacity to be degraded by microbial pectinases and also the
precise nature and properties of any oligogalacturonide products
released.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galacturonic acid
(homogalacturonan, HG)1 or
repeats of the disaccharide
(
4)-
-D-GalA-(1
2)-
-L-Rha-(1
) (rhamnogalacturonan, RG). GalA residues in HG may be methyl-esterified, acetylated, and/or substituted with xylose or apiose (2-4).
Oligosaccharide side chains may be attached to both RG and HG domains
to form the branched domains known as RG-I and RG-II, respectively (2, 3, 5, 6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. The elasticity, E,
of the gels in the low strain region was calculated from Equation 1.
s is the strain, and A is sample
cross-sectional area. The strain is given by Equation 2.
(Eq. 1)
h0 is the initial height of the sample,
and
(Eq. 2)
h is the change in height due to compression. The
line of best fit to the plot of F versus
s, in the region 0.1 < s < 0.2, was
used to calculate E. Below this range samples did not always
compress uniformly, due to not having exactly parallel sides, whereas
at higher strains there were significant increases in cross-sectional
area and/or water loss from the sample, contributing to non-linear
F versus s plots. Up to
s = 0.2 the change in A was negligible for
all samples, and for 0.1 < s < 0.2 the line of
best fit to the data always had a regression coefficient of at least
0.95.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The monoclonal antibody LM7 binds to a subset
of F-series and B-series model pectins. Figure shows IDA of LM7
(a), PAM1 (b), and JIM5 (c) binding to
a series of lime pectin samples with defined degrees and different
patterns of methyl-esterification. The series was produced by the
de-esterification of a common high ester pectin sample with a DE of
81% by digestion with a pPME (P-series), an fPME (F-series), or by
base catalysis (B-series). A completely de-esterified pectin sample
(E0) was produced by digestion of E81 with fungal pectin methyl
esterase followed by base catalysis. pPME removes contiguous methyl
groups from relatively long stretches of HGHG, resulting in a blockwise
de-esterification, while treatment with fPME and base results in
non-blockwise de-esterification. All samples were applied to
nitrocellulose in dilution series as indicated.
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Fig. 2.
The LM7 epitope is degraded by
polygalacturonase and pectin lyase. Competitive inhibition ELISAs
of the effects of pectinolytic digestion on the binding in solution of
LM7 to pectin sample B34. Untreated B34 was used as the immobilized
antigen. The binding of LM7 to B34 was assessed for untreated B34
(B34 NT), B34 completely digested with
endo-polygalacturonase II (B34 PG II) and for B34 treated
with pectin lyase (B34 PL).
1). Moreover, optimal cleavage occurs where
subsite +2 is de-esterified, whereas whether or not subsites
2 and
3 are de-esterified appears to be less critical (24). In contrast, PL
cleaves optimally in regions of HG that are fully methyl-esterified
(24). The susceptibility of the epitope recognized by LM7 to both PGII
and PL cleavage, and the profile of LM7 binding in IDAs (Fig. 1),
indicate that the LM7 epitope contains both contiguous
methyl-esterified GalA residues and contiguous un-esterified GalA residues.
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Fig. 3.
LM7 binding is not dependent on, nor
abolished by, calcium-mediated HG chain association. The binding
of LM7 to F31 was assessed in the presence of CaCl2 and
MgCl2 (LM7+CaCl2+F31 and
LM7+MgCl2+F31, respectively). Control samples
(LM7+CaCl2 and LM7+MgCl2)
without F31 in the solution were included to assess the effects of
calcium and magnesium ions on LM7 binding to the immobilized
antigen.
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Fig. 4.
The HG epitope recognized by LM7
occurs at cell junctions in plant tissues. Figure shows
micrographs of the binding of LM7 (a and d), JIM5
(b and e), and PAM1 (c and
f) to the cell walls of pea stem cortical cells as
visualized by immunofluorescent labeling. All sections were hand-cut
transverse sections of pea stem. Arrowheads and
arrows in d-f indicate the corners and linings
of intercellular spaces, respectively. The double
arrowhead in f indicates a region of cell wall
close to the plasma membrane. Scale bars in
a-c = 100 µm; scale bars in
d-f = 20 µm.
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Fig. 5.
The LM7 epitope is present throughout
intercellular space formation in pea stem parenchyma.
a-c, micrographs showing the dual labeling of the
cell walls of pea stem cortical parenchyma cells with LM7
(immunofluorescent labeling, green) and the
cellulose-binding fluorophor calcofluor (blue) showing the
positions HG epitopes and cellulose in relation to intercellular spaces
at different stages of formation. d-f,
transmission electron micrographs of approximately equivalent cell
junctions to those in a-c. The junction shown in
d consists entirely of cell wall material, whereas
junctions at positions near the middle of the cortex are progressively
separated resulting in the formation of intercellular spaces
(e and f). The darkly stained material
accumulated at the corners of expanding air spaces (e and
f) contains HG, as indicated by immunogold labeling with
JIM5 (inset to f). Scale
bars: a-c = 5 µm; d-f = 1 µm, inset to f = 500 nm.
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Fig. 6.
The LM7 epitope occurs at regions of outer
epidermal cell walls and at intercellular spaces throughout pea
seedlings. Figure shows micrographs of the binding of LM7
(a and c-e) and JIM5 (b) to the cell
walls of pea stem epidermal cells (a and b), leaf
epidermal and parenchyma cells (c and d,
respectively), and root parenchyma cells (e). All sections
were hand-cut transverse sections. Arrowheads in
a-c indicate the position of a region of outer epidermal
cell wall overlying cell junctions. Arrows in c
indicate the limits of the thick outer epidermal wall.
Scale bars: a and
d = 50 µm; b, c, e,
and f = 10 µm.
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Fig. 7.
The degree and pattern of
methyl-esterification of HG domains influences the response of
calcium-pectin gels to compression. a, 2% (w/v) gels
were formed from a subset of the P-, F-, and B-series pectins by the
addition of calcium chloride to a final concentration of 35 mM and equilibration for at least 24 h. Gels were cast
in 8.6-mm diameter syringes and cut to a height of 4 mm. b
and c, the appearance of calcium-mediated pectin gels
(prepared as described in a) following compression by the
application of a linearly increasing force. Compression was stopped
when a force of 9.82 N was reached (E0, F11, and F31), or when gels
yielded (F43, B34, and P41). d, force versus
strain curves for pectin gels under compression. Gels were prepared as
described in a and compressed at a rate of 0.1 mm
s 1 to a maximum force of 9.82 N.
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Fig. 8.
The degree and pattern of
methyl-esterification of HG domains influences the elasticity of
calcium-pectin gels. The elasticity of calcium-mediated pectin
gels prepared described as in Fig. 7a. a, elastic
moduli were calculated from the gradients of force/strain curves at low
strain. b, the graph shows the relationship between
elasticity and degree of methyl-esterification for calcium-mediated
pectin gels.
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Fig. 9.
The degree and pattern of
methyl-esterification of HG domains affects the water holding capacity
and porosity of calcium-pectin gels. a, the water
holding capacity of gels was assessed by the change in volume of gels
following compression by a force of 9.82 N (E0, F11, and F31) or by a
force at which gels yielded (F43, B34, and P41). The graph
shows the relationships between volume loss under compression and
degree of methyl-esterification. b, the porosity of pectin
gels to protein was assessed by the rate of incorporation of BSA into
gels. Gels were incubated in a solution of BSA in de-ionized water (5 mg/ml) and removed at selected time points (loading time). The amount
of BSA that had entered the gels was determined by dissolving gels in
50 mM CDTA and assaying the amount of protein in the
resulting solutions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
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Fig. 10.
Schematic diagram showing occurrence
of the LM7 epitope in relation to cell junctions and the formation of
intercellular space. a, intercellular space forms at
the junction between old (o) and new (n) cell
walls and involves the linking up of middle lamellae. This requires the
dismantling of a region of the older cell wall (indicated by the
dotted circle). b, a non-expanded
intercellular junction with no air space. The gray
triangle indicates the position of expanded middle lamella
material that occupies the space completely and corresponds to the
position of LM7 labeling. c, an expanded cell junction with
a large intercellular space (is). The arrows
indicate the forces generated by intracellular turgor pressure that
drive cell separation. The gray triangles
indicate the regions of the cell that correspond to the position of LM7
labeling. In all cases, c indicates the interior of cells.
In all cases, thick lines indicate the plasma
membrane face of cell walls and thin lines the
position of middle lamellae. Figure was adapted from Jarvis (34).
In conclusion, the observations reported here demonstrate that
modulations of the pattern and degree of methyl-esterification of
pectic HG occur within discrete regions of primary cell walls and, in
particular, that a non-blockwise pattern of methyl esters of HG is an
abundant feature of HG. We also show that the pattern and degree of
methyl group distribution significantly affect the mechanical and
physiological properties of calcium-mediated pectin gels and are
therefore likely to influence the in vivo functionalities of
pectic HG domains. In this way, a highly methyl-esterified HG
polysaccharide that is deposited in the cell wall can potentially be
modified in different ways to generate distinct functional properties.
Understanding the cell biological context of the products of PME action
will be crucial for determining the functions of PME multigene family members.
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FOOTNOTES |
---|
* This work was supported by United Kingdom Biotechnology and Biological Sciences Research Council and the EU Framework IV and V Initiatives.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Alexander von Humboldt Stiftung, D-53173 Bonn, Germany.
§§ To whom correspondence should be addressed. Fax: 44-113-2333144; E-mail: j.p.knox@leeds.ac.uk.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M011242200
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ABBREVIATIONS |
---|
The abbreviations used are: HG, homogalacturonan; IDA, immuno-dot assay; PME, pectin methyl esterase; RG, rhamnogalacturonan; N, newton(s); pPME, plant pectin methyl esterase; fPME, fungal pectin methyl esterase; DE, degree of methyl-esterification; TBS, Tris-buffered saline; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PL, endo-pectin lyase; PG II, endo-polygalacturonase II; ELISA, enzyme-linked immunosorbent assay; ciELISA, competitive inhibition enzyme-linked immunosorbent assay; PIPES, 1,4-piperazinediethanesulfonic acid; CDTA, cyclohexanediamine N,N,N',N'-tetraacetic acid.
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