Modulation of the Degree and Pattern of Methyl-esterification ofPectic Homogalacturonan in Plant Cell WallsIMPLICATIONS FOR PECTIN METHYL ESTERASE ACTION, MATRIX PROPERTIES, AND CELL ADHESION*

Received for publication, December 13, 2000, and in revised form, February 28, 2001Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M011242200

William G. T. Willats‡, Caroline Orfila‡, Gerrit Limberg§¶, Hans Christian Buchholti,Gert-Jan W. M. van Alebeek**, Alphons G. J. Voragen**, Susan E. Marcus‡,Tove M. I. E. Christensen§, Jørn D. Mikkelsen§, Brent S. Murray‡‡, and J. Paul Knox‡ §§

From the ‡Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom, §Danisco Biotechnology,Langebrogade 1, DK 1001 Copenhagen K, Denmark, iDanisco 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

Homogalacturonan (HG) is a multifunctional pecticpolysaccharide of the primary cell wall matrix of allland plants. HG is thought to be deposited in cell walls ina highly methyl-esterified form but can be subsequentlyde-esterified by wall-based pectin methyl esterases(PMEs) that have the capacity to remove methyl estergroups from HG. Plant PMEs typically occur in multi-gene families/isoforms, but the precise details of thefunctions of PMEs are far from clear. Most are thoughtto act in a processive or blockwise fashion resulting indomains of contiguous de-esterified galacturonic acidresidues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and cancontribute to intercellular adhesion. We demonstratethat, in addition to blockwise de-esterification, HG witha non-blockwise distribution of methyl esters is also anabundant feature of HG in primary plant cell walls. Apartially methyl-esterified epitope of HG that is gener-ated in greatest abundance by non-blockwise de-esteri-fication is spatially regulated within the cell wall matrixand occurs at points of cell separation at intercellularspaces in parenchymatous tissues of pea and other an-giosperms. Analysis of the properties of calcium-medi-ated gels formed from pectins containing HG domainswith differing degrees and patterns of methyl-esterifica-tion indicated that HG with a non-blockwise pattern ofmethyl ester group distribution is likely to contributedistinct mechanical and porosity properties to the cellwall matrix. These findings have important implicationsfor our understanding of both the action of pectinmethyl esterases on matrix properties and mechanismsof intercellular adhesion and its loss in plants.

The load-bearing components of primary cell walls, princi-pally the cellulose and hemicellulose polysaccharide network,are embedded in a pectic matrix that is structurally complex

and heterogeneous. The pectic matrix contributes to both thephysical integrity and physiological status of cell walls, but thefunctional implications of the structural complexity of this ma-trix are poorly understood. Typically, heterogeneous popula-tions of pectic polymers are present in primary cell walls, andour present understanding is that all essentially consist ofgalacturonan backbones with or without various side chainadditions (1–3). Backbone domains consist of either contiguous1,4-linked a-D-galacturonic acid (homogalacturonan, HG)1 orrepeats of the disaccharide (34)-a-D-GalA-(132)-a-L-Rha-(13)(rhamnogalacturonan, RG). GalA residues in HG may be meth-yl-esterified, acetylated, and/or substituted with xylose or api-ose (2–4). Oligosaccharide side chains may be attached to bothRG and HG domains to form the branched domains known asRG-I and RG-II, respectively (2, 3, 5, 6).

The HG domain of the pectic network is implicated in influ-encing a range of cell wall properties that impact upon cellexpansion, cell development, intercellular adhesion, and de-fense mechanisms. Stretches of HG with un-esterified GalAresidues can associate by calcium cross-linking (7). Such asso-ciation promotes the formation of supramolecular pectic gels,which are important in controlling the porosity and mechanicalproperties of cell walls and contribute to the maintenance ofintercellular adhesion (8, 9). Plant cells are adhered by contactacross middle lamellae, which are HG-rich regions of the cellwall developed from cell plates formed at cytokinesis. In plants,cell adhesion is a default state and cell separation an activeprocess that is under developmental control (8, 10). In additionto the roles of the HG polysaccharide domain, HG-derivedoligogalacturonides generated by pectinolytic cleavage are in-volved in signaling processes during development and in de-fense responses to plant pathogens (11–13).

It is thought that HG is highly methyl-esterified when ex-ported into cell walls and is subsequently de-esterified by theaction of pectin methyl esterases (PMEs) in the cell wall (3, 14).PME genes occur in multigene families and encode isoformswith differing action patterns with respect to the removal of

* This work was supported by United Kingdom Biotechnology andBiological Sciences Research Council and the EU Framework IV and VInitiatives. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely 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.

1 The abbreviations: HG, homogalacturonan; IDA, immuno-dot assay;PME, pectin methyl esterase; RG, rhamnogalacturonan; N, newton(s);pPME, plant pectin methyl esterase; fPME, fungal pectin methyl ester-ase; 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-linkedimmunosorbent assay; ciELISA, competitive inhibition enzyme-linkedimmunosorbent assay; PIPES, 1,4-piperazinediethanesulfonic acid;CDTA, cyclohexanediamine N,N,N9,N9-tetraacetic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 22, Issue of June 1, pp. 19404–19413, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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methyl esters. However, the specific functions of PME popula-tions in the context of cell expansion and other processes arenot well understood (15–17). Methyl esters can be distributedin diverse patterns along HG chains and it is clear that theaction patterns of plant PMEs (pPMEs) can be influenced bylocal cell wall pH, the existing balance of methyl and freecarboxyl groups on HG substrates, and metal ion concentration(16, 18–22). In addition to plants, some fungi and bacteria alsoproduce PMEs, and it is significant that the action patterns ofplant and fungal PMEs are thought to be different. It is gen-erally proposed that pPMEs remove methyl esters in a proces-sive blockwise fashion (single chain mechanism), giving rise tolong contiguous stretches (blocks) of un-esterified GalA resi-dues in HG domains of pectin (23–25). In contrast, the action offungal PMEs (fPMEs) is generally regarded as random (ormultiple chain mechanism), resulting in the de-esterification ofsingle GalA residues per enzyme/substrate interaction (23–25).However, the precise nature of the action patterns of fPME andpPME are far from clear, and some pPMEs appear to have thecapacity to remove a limited number of methyl esters perreaction, giving rise to short un-esterified blocks (20). More-over, recently developed procedures for the enzymatic finger-printing of pectic fragments have indicated that the actionpatterns of fPMEs are non-blockwise, but probably not in factrandom (24). In this report, the action pattern of an orangepPME is referred to as blockwise, whereas the action pattern ofan Aspergillus fPME and the chemical process of base catalysisare referred to as non-blockwise.

Previous investigations of the action patterns of PMEs havefocused on the action of isolated PMEs on pectin substrates invitro (16). However, the use of anti-HG monoclonal antibodieswith appropriate binding specificities allows the products ofde-esterification processes to be analyzed in planta in the con-text of cell wall architecture and cell development. We haveused monoclonal antibodies with differing binding require-ments with respect to the distribution of methyl ester groups toinvestigate the spatial regulation of HG with blockwise andnon-blockwise methyl group distributions in pea. The mono-clonal antibody PAM1 (which binds specifically to longstretches of un-esterified HG produced most readily by block-wise de-esterification) and JIM5 (which binds to a range ofpartially methyl-esterified HG domains) have been describedpreviously (26–28). Non-blockwise de-esterification of HG hasbeen analyzed using a monoclonal antibody (LM7) described forthe first time in this report. In addition, we have investigatedthe functional implications of varying degrees and patterns ofmethyl-esterification of HG in the context of the physical andphysiological properties of calcium-HG gels. Taken together,the results indicate that wall-based pPMEs with a range ofaction patterns can produce HG with non-blockwise and block-wise distributions of methyl esters at discrete cell wall microdo-mains, resulting in distinct spatially regulated matrixproperties.

EXPERIMENTAL PROCEDURES

Monoclonal Antibodies—Monoclonal antibody LM7 was generatedusing hybridoma technology subsequent to the immunization of a groupof rats with lime pectin with a degree of methyl-esterification (DE) of22.9%, a degree of amidation of 27.3% and an average molecular massof 84 kDa. Immunization, hybridoma preparation, and cloning proce-dures were performed as described previously (29). LM7 was selected bydifferential immuno-dot assay (IDA) screens of a series of pectin sam-ples differing in DE with blockwise and non-blockwise patterns ofde-esterification (see below). Monoclonal antibodies JIM5 and PAM1were produced by hybridoma and phage display technologies, respec-tively (26–28).

Production of Lime Pectins with Defined DE—A series of lime pectinswith different patterns (blockwise and non-blockwise), and defined DEwere prepared by enzymatic and chemical treatments of a commercial

highly methyl-esterified (81%) lime pectin (E81, GRINDSTEDy PectinURS 1200) as described previously (24). Briefly, one series was pro-duced by blockwise de-esterification of E81 with a pPME isolated fromorange peel (P-series), while another series was produced by non-block-wise de-esterification of E81 with a fPME from Aspergillus niger (F-series). A further set of samples was also produced by non-blockwisede-esterification of E81 by base catalysis (B-series). A sample of com-pletely de-esterified pectin (E0) was prepared by treatment with fPMEfollowed by base catalyzed de-esterification.

Digestion of Pectin B34 with endo-Pectin Lyase (PL) and endo-Polyg-alacturonase II (PG II)—Pectin sample B34 (prepared by base catalysisand with a DE of 34%) was digested with PL (EC 3.2.1.15) or PG II (EC4.2.2.10), 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 5mg/ml by overnight rocking at room temperature. 0.1 unit of PL or 0.2unit of PG II was added to 1 ml of the above pectin solution and in bothcases incubated at room temperature for 20 h. The reaction was stoppedby boiling for 5 min.

Competitive Inhibition ELISAs (ciELISAs)—The effect of PL and PGII digestion of B34 on the binding of LM7 was assessed by ciELISAswith untreated B34 as the immobilized antigen. Untreated B34 (50mg/ml in Tris-buffered saline (TBS)) was coated (100 ml/well) overnightat 4 °C onto microtiter plates (Maxisorp, Nunc, Denmark). The coatingsolution was removed, and plates were blocked at room temperaturewith 3% bovine serum albumin in TBS (3%BSA/TBS) for 2 h (200ml/well). Following washing, competitor solutions (untreated B34, B34digested with PL, and B34 digested with PG II) were applied (100ml/well) as 5-fold serial dilutions in 3%BSA/TBS. All competitor solu-tions also contained LM7 at a final level of 1/100 dilution of hybridomasupernatant (corresponding to ;90% maximal binding on antibodycapture ELISAs). After 2 h of incubation, plates were washed andsecondary antibody (anti-rat IgG horseradish peroxidase conjugate,Sigma, Poole, United Kingdom) diluted 1/1000 in 3%BSA/TBS applied(100 ml/well) and incubated for 2 h at room temperature. After washing,plates were developed with a tetramethyl benzidine-based substrate(150 ml/well). After stopping the reaction with 2 M H2SO4 (35 ml/well),absorbances were read at 450 nm. Concentrations of competitors re-sulting in 50% inhibition (IC50) of antibody binding were determined.Values from controls with no competitor were taken as 0% inhibition ofantibody binding, and values from controls with no LM7 antibodyrepresented 100% inhibition of binding. In some cases, CaCl2 or MgCl2were added to competitor solutions to a maximum level of 1 mM.

Immuno-dot Assays—Pectins were dissolved in water to a concentra-tion of 5 mg/ml or 10 mg/ml and applied as 1-ml aliquots to nitrocellulose(Scheicher & Schuell) in a 5- or 10-fold dilution series. Nitrocellulosemembranes 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 for1 h in primary antibodies diluted in 5%M/PBS. JIM5 and LM7 wereused as 1/10 dilutions of hybridoma supernatants while PAM1 was usedat a concentration of ;1 3 1011 phage particles/ml (;1/100 dilution ofphage prepared by polyethylene glycol precipitation; Ref. 27). In allcases, membranes were incubated in primary antibodies for 1.5 h. Afterwashing, 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 substratesolution (25 ml of de-ionized water, 5 ml of MeOH containing 10 mg/ml4-chloro-1-naphthol, 30 ml 6% (v/v) H2O2). In some cases, immobilizedpectin samples were chemically de-esterified by incubation of mem-branes 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 sterilevermiculite, 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 mm. Sections were placed immediately in fixative consistingof 4% paraformaldehyde in 50 mM PIPES, 5 mM MgSO4, and 5 mM

EGTA. Following 30 min of fixation, sections were washed in the PIPESbuffer 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 hybridomasupernatants, respectively. PAM1 was used at a concentration of ;5 31011 phage particles/ml (; 1/20 dilution of phage prepared by polyeth-ylene glycol precipitation; Ref. 27). Sections were washed by gentlyrocking in PBS prior to incubation for 1 h in secondary antibody. Forvisualization of LM7 and JIM5 binding, the secondary antibody wasanti-rat IgG coupled to fluorescein isothiocyanate (Sigma). For visual-ization of PAM1 binding, a secondary antibody was prepared by conju-

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gating an anti-M13 antibody (Amersham Pharmacia Biotech) to fluo-rescein isothiocyanate using a protein conjugation kit (Sigma). Allsecondary antibodies were used at dilutions of 1/100 in 5%M/PBS. Afterwashing in PBS, sections were mounted in anti-fade agent (Citifluor,Agar Scientific) and examined on a microscope equipped with epifluo-rescence illumination (Olympus BH-2). In some cases, hand sectionswere chemically de-esterified by incubation in 0.1 M Na2CO3 for 1 hprior to processing.

In certain cases plant material was embedded in resin for electronmicroscopy. 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. Materialwas then post-fixed in 0.1% (w/v) osmium tetroxide in 0.1 M sodiumphosphate buffer, for 1 h at 4 °C, washed extensively with 0.1 M sodiumphosphate buffer, and then dehydrated in an ethanol series. Dehy-drated material was infiltrated with resin (LR White) (London Resin,Reading, United Kingdom), then placed in gelatin capsules containingresin and allowed to polymerize at 37 °C for 5 days. Material used fortransmission electron microscopy but not immunogold labeling, wasstained en bloc with 4% (w/v) uranyl acetate in distilled water overnightat 4 °C, then washed extensively with water before being dehydratedand embedded as described above.

For immunogold labeling, sections obtained from resin-embeddedmaterial (;0.1 mm thick) were incubated in 3%BSA/TBS for 30 min.Sections were then incubated in a solution containing JIM5 or LM7diluted 1/10 in 3%BSA/TBS for 1.5 h. The sections were washed with3%BSA/TBS, and then incubated in secondary antibody (anti-rat mono-clonal antibody conjugated to 10 nm colloidal gold (Sigma) diluted 1/40in 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 thenwashed extensively with distilled water. Sections were observed withan electron microscope (1200ex, JEOL, Tokyo, Japan). All incubationswere at room temperature.

Calcium-mediated Gelation of Lime Pectin Samples—A subset of theP-, F-, and B-series of lime pectins that contained the epitopes recog-nized by LM7 or PAM1 to various levels were selected for analysis of theeffects of the degree and pattern of methyl-esterification on the physicalproperties of calcium-mediated gels. Gels were prepared from E0, F11,F31, P41, F43, and B34, essentially as described elsewhere (30). Pectinsolutions (2% (w/v) in de-ionized water) were prepared with gentlerocking at 4 °C for at least 18 h. For casting gels, 900 ml of pectinsolution was transferred to a 2-ml syringe (with an internal diameter of8.6 mm) from which the nozzle end had been removed. 67 ml of 500 mM

CaCl2 was added as a layer to the top of the pectin solution (to give afinal CaCl2 concentration of 35 mM) and the cut end of the syringesealed 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 auniform height using a custom-made nylon cutting block with guideslots.

Rheological Testing of Calcium-mediated Pectin Gels—Gel samplesprepared as described above were subjected to compressive tests todetermine their elasticity under low strain and their yield points underhigh strain. Compression tests were performed using an in-house tex-ture measuring device, known as the “Ministron,” constructed in theUniversity of Leeds Department of Food Science instrument workshop.This device was used to analyze the controlled compression of gelsamples between two parallel stainless steel plates at a precisely de-fined speed. The lower plate, on which the sample rested, is mounted ona high precision load cell (Maywood Instruments, Basingstoke, UnitedKingdom). The gap between the plates and the force, F, on the load cellare electronically logged throughout the experiment at a suitable fre-quency. For all experiments, at least four samples from different gelpreparations were analyzed.

Gels were compressed by reducing the gap between the plates at arate of 0.1 mm s21. The elasticity, E, of the gels in the low strain regionwas calculated from Equation 1.

E 5 F/~A z s! (Eq. 1)

s is the strain, and A is sample cross-sectional area. The strain is givenby Equation 2.

s 5 Dh/h0 (Eq. 2)

h0 is the initial height of the sample, and Dh is the change in height dueto compression. The line of best fit to the plot of F versus s, in the region0.1 , s , 0.2, was used to calculate E. Below this range samples did notalways 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 5 0.2 the change in A was negligible forall samples, and for 0.1 , s , 0.2 the line of best fit to the data alwayshad a regression coefficient of at least 0.95.

Analysis of the Water Holding Capacity of Calcium-mediated PectinGels under Compression—It was observed that there were significantdifferences in the water expelled from gel samples during compression.The final percentage of water lost was calculated from the change in thevolume 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 sbecame negative. Yielding was sometimes also accompanied by visiblesplitting of the gel piece.

Determination of the Porosity of Calcium-mediated Pectin Gels—Theporosity of gels to protein was determined by incubating gels in asolution of BSA and measuring the incorporation of protein into gelsover time. For each time point, two gel blocks (prepared as describedpreviously) above were incubated with gentle rocking in 2 ml of BSAsolution (5 mg/ml in de-ionized water). Following incubation, the twogel blocks were washed briefly in de-ionized water, briefly blotted dry onfilter paper to remove surface liquid, and incubated with gentle rockingin 2 ml of 50 mM calcium chelator (CDTA) (pH 7) until gels werecompletely dissolved (;30 min). The protein concentration of the solu-tions were then analyzed using Bradford protein assays. For eachpectin gel sample, four replicates of the protocol described were ana-lyzed for each time point.

RESULTS

Monoclonal Antibody LM7 Recognizes an Epitope of HG Pro-duced by Non-blockwise De-esterification—Monoclonal anti-body LM7 was generated subsequent to immunization with alime pectin (containing 88.3% GalA) and selected by IDAscreening on the basis of its specific binding to a subset ofF-series and B-series pectins that have non-blockwise patternsof methyl-esterification as shown in Fig. 1a. The binding of thepreviously characterized anti-HG JIM5 (26, 28) and the phageantibody PAM1 (27) to the same pectin samples are shown for

FIG. 1. The monoclonal antibody LM7 binds to a subset ofF-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 withdefined degrees and different patterns of methyl-esterification. Theseries was produced by the de-esterification of a common high esterpectin sample with a DE of 81% by digestion with a pPME (P-series), anfPME (F-series), or by base catalysis (B-series). A completely de-ester-ified pectin sample (E0) was produced by digestion of E81 with fungalpectin methyl esterase followed by base catalysis. pPME removes con-tiguous methyl groups from relatively long stretches of HGHG, result-ing in a blockwise de-esterification, while treatment with fPME andbase results in non-blockwise de-esterification. All samples were ap-plied to nitrocellulose in dilution series as indicated.

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comparison (Fig. 1, b and c). Both the degree and the pattern ofde-esterification influence the capacity of these antibodies tobind to HG domains. LM7 did not bind at the highest leveltested (1 mg) to any of the P-series pectins or to F-series pectinswith DE of 76%, 69%, or 11% (Fig. 1a). LM7 did bind to F-seriespectins with DE from 58% to 31% with increasing avidity, andLM7 bound to F31 with a detection limit of ,0.2 mg (Fig. 1a).LM7 bound weakly, at the highest level tested (1 mg), to aB-series sample with a DE of 43% and bound to B-series pectinswith DEs of 34 and 15% with detection limits of ,0.2 mg. Thebinding of LM7 to all samples was abolished when blots werede-esterified by treatment with 0.1 M Na2CO3 prior to labeling(data not shown). The binding profile of LM7 indicates that it isspecific for a partially methyl-esterified domain of HG and thatits epitope is most readily produced by the non-blockwise de-esterification processes such as that produced by fPME actionand base catalysis. In contrast, PAM1 binds to long (.30 res-idues) contiguous stretches of de-esterified GalA residues pro-duced 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 alsoproduced by extensive de-esterification by fPME or base catal-ysis as indicated by binding to F11 and B15, respectively (Fig.1b). The optimal binding requirements of JIM5 are not fullydefined, and JIM5 has the capacity to bind to a wide range ofHG epitopes with varying degrees and patterns of methyl-ester-ification as shown in Fig. 1c and as discussed elsewhere (28).

The Partially Methyl-esterified Epitope Recognized by LM7 IsDegraded by the Action of Both Endo-polygalacturonase andPectin Lyase—In order to explore the structure of LM7 epitopefurther, its susceptibility to digestion by PG II and PL wasassessed. The products of enzymatic digestion were analyzedby ciELISAs using untreated B34 as the immobilized antigenas shown in Fig. 2. The use of ciELISAs allowed the binding ofLM7 to digest fragments to be analyzed in solution, rather thanin an immobilized state, as would be the case for IDAs. This isimportant because the binding of pectic fragments to nitrocel-lulose sheets is related to fragment size. For example, oligoga-lacturonides with degrees of polymerization less than 15 arenot immobilized effectively onto nitrocellulose sheets (data notshown).

When untreated B34 was used as a soluble competitor incompetitive inhibition ELISAs, 20 mg/ml was required toachieve a 50% inhibition (IC50) of LM7 binding. LM7 binding toB34 was abolished entirely following complete digestion withPG II and PL, and the digestion fragments failed to produceany significant inhibition of LM7 binding even at the highestlevel used (1 mg/ml) as shown in Fig. 2. The PG II used from A.niger has an absolute requirement for de-esterified GalA resi-dues to be present at both sides of the cleavage position (sub-sites 11 and 21). Moreover, optimal cleavage occurs wheresubsite 12 is de-esterified, whereas whether or not subsites 22and 23 are de-esterified appears to be less critical (24). Incontrast, PL cleaves optimally in regions of HG that are fullymethyl-esterified (24). The susceptibility of the epitope recog-nized by LM7 to both PGII and PL cleavage, and the profile ofLM7 binding in IDAs (Fig. 1), indicate that the LM7 epitopecontains both contiguous methyl-esterified GalA residues andcontiguous un-esterified GalA residues.

Confirmation of epitope structure can be obtained by dem-onstration of binding to oligosaccharides in ciELISAs, and thisgenerally indicates carbohydrate epitope sizes of 4 to 6 sugars(27, 28). Fully methyl-esterified and fully un-esterified oligoga-lacturonides with degrees of polymerization up to 8, preparedas described elsewhere (28), were not effective inhibitors ofLM7 binding in ciELISAs (data not shown). In order to char-acterize the LM7 epitope fully, a range of oligogalacturonideswith intermediate degrees and defined patterns of methyl-esterification would be required, and currently these are notavailable. Nonetheless, it is clear that LM7 binds to a HGdomain of pectin that contains both un-esterified and methyl-esterified GalA residues, that this structure is generated mostreadily by non-blockwise de-esterification processes, and thatthe epitope is distinct from any other previously characterizedHG epitope.

LM7 Binding Is Not Dependent on, nor Reduced by, Calcium-mediated Chain Association of HG Domains—The possible ef-fects of the formation of conformational HG structures on LM7binding were investigated by addition of divalent cations tociELISA assays. The presence of calcium or magnesium at upto 1 mM had no effect on LM7 binding to either immobilized orsoluble pectin sample F31, as shown in Fig. 3. Levels of calcium

FIG. 3. LM7 binding is not dependent on, nor abolished by,calcium-mediated HG chain association. The binding of LM7 toF31 was assessed in the presence of CaCl2 and MgCl2(LM71CaCl21F31 and LM71MgCl21F31, respectively). Control sam-ples (LM71CaCl2 and LM71MgCl2) without F31 in the solution wereincluded to assess the effects of calcium and magnesium ions on LM7binding to the immobilized antigen.

FIG. 2. The LM7 epitope is degraded by polygalacturonase andpectin lyase. Competitive inhibition ELISAs of the effects of pectino-lytic digestion on the binding in solution of LM7 to pectin sample B34.Untreated B34 was used as the immobilized antigen. The binding ofLM7 to B34 was assessed for untreated B34 (B34 NT), B34 completelydigested with endo-polygalacturonase II (B34 PG II) and for B34treated with pectin lyase (B34 PL).

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above 1 mM resulted in gel formation and disruption of theassay. Surface labeling of calcium pectate gel blocks (preparedas described previously) indicated that LM7 binding was re-tained when HG was cross-linked via calcium (data not shown).Taken together, these results indicate that LM7 binding isneither dependent on, nor abolished by, calcium-mediated HGchain association of HG domains.

The Partially Methyl-esterified Epitope Recognized by LM7Occurs in Discrete Micro-domains of Primary Cell Walls—Thepartially methyl-esterified epitope recognized by LM7 wasfound to be abundant in plant tissues. The epitope was mostreadily visualized in plant materials that had undergone prep-arations that maintained maximum antigenicity, i.e. hand-cutsections or cryosections of non-embedded material. The epitoperecognized by LM7 appears to be unstable in pectin prepara-tions and in plant material. The capacity of F31 and B34 pectinsamples to be recognized by LM7 was gradually lost when thesesamples were stored as frozen solutions (data not shown). Sim-ilarly, the epitope was lost from plant materials when they hadundergone extensive preparation, such as resin-embedding forimmunocytochemistry. As discussed, LM7 binding is criticallydependent on DE being with a certain range, and the instabil-ity of the epitope is probably due to de-esterification occurringduring freeze/thawing or processing.

The distribution of the LM7 epitope was examined mostextensively in pea seedlings (a system that is amenable tohand-sectioning) and immunofluorescent labeling of sectionsindicated that the LM7 epitope was restricted to discrete re-gions of cell walls in the roots, stems, and leaves of seedlings.The distribution of the LM7 epitope in pith parenchyma cells ina transverse section of a pea stem internode is shown in Fig. 4a.In this region, most cell junctions are expanded to some extentto form intercellular spaces. The LM7 epitope was restricted tothe region of the cell wall lining the intercellular spaces be-tween the parenchyma cells, and no LM7 epitope was detectedin other regions of the cell walls in this tissue. Fig. 4d shows ahigher magnification of an individual intercellular space andshows that the LM7 epitope was particularly abundant at thecorners of the intercellular space, i.e. the point between adher-ent and separated cell walls. For comparison, the labelingpatterns of JIM5 and PAM1 on equivalent sections are shownin Fig. 4 (panels b and e and panels c and f, respectively). JIM5

bound to all primary cell walls (Fig. 4b). At higher magnifica-tion (Fig. 4e), the increased abundance of the JIM5 epitope inthe region of the cell wall lining the intercellular spaces and inthe region of the wall closest to the plasma membrane wasevident. The blockwise de-esterified HG epitope recognized byPAM1 occurred in discrete cell wall domains (Fig. 4, c and f).Like LM7 and JIM5, PAM1 bound to material in the cell walllining intercellular spaces and, like JIM5, did not bind to thecentral regions of the cell walls (including the middle lamellae).In contrast to LM7, PAM1 also bound to the region of the wallclosest to the plasma membrane, but, unlike JIM5, PAM1labeling was absent from inner regions of the cell wall adjacentto the intercellular space (Fig. 4f). The reason for the appar-ently thicker cell walls and more diffuse nature of labelingobtained using PAM1 is due to the fact that PAM1 is a phagedisplay antibody and large (;800 nm long) intact phage parti-cles were used as the primary stage antibody.

The localization of the LM7 epitope at intercellular spaceswas consistent throughout all tissues in the pea stem, and therelationship between the occurrence of the LM7 epitope and theformation of intercellular space was examined in more detail byexamination of cortical parenchyma tissue. The immunolabel-ing 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 largerparenchyma cells toward the center of the stem are shown inFig. 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 junc-tions, LM7 bound to all of the developing space (which at thisstage is filled with expanded middle lamellae) but did not bindto any other regions of the cell wall (Fig. 5a). At larger, air-filled junctions, the LM7 epitope was most abundant at thecorners of the triangular intercellular spaces as viewed in thesections shown in Fig. 5 (b and c). The epitope recognized byLM7 is therefore most abundant in regions of the expandedmiddle lamellae at the point of separation of cell walls. Theultrastructure of intercellular spaces was investigated bytransmission electron microscopy. However, as discussedabove, the epitope recognized by LM7 is prone to instabilityand, when stem material was resin-embedded for immunoflu-orescent or immunogold detection, the LM7 epitope could not

FIG. 4. The HG epitope recognizedby LM7 occurs at cell junctions inplant tissues. Figure shows micrographsof the binding of LM7 (a and d), JIM5 (band e), and PAM1 (c and f) to the cell wallsof pea stem cortical cells as visualized byimmunofluorescent labeling. All sectionswere hand-cut transverse sections of peastem. Arrowheads and arrows in d–f indic-ate the corners and linings of intercellularspaces, respectively. The double arrowheadin f indicates a region of cell wall close tothe plasma membrane. Scale bars in a-c 5100 mm; scale bars in d–f 5 20 mm.

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be detected. However, the fine structures of comparable inter-cellular 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 thecorners of the junction (Fig. 5, e and f). The position of thismaterial appears to correspond to regions of the wall contain-ing the LM7 epitope as localized by immunofluorescent label-ing of non-embedded material (Fig. 5, b and c). Pectic materialtypically stains darkly in the transmission electron microscopystaining protocol used, and immunogold labeling with JIM5indicated the presence of HG at this position (Fig. 5f, inset).

The LM7 epitope was also found to occur in regions of thethickened outer cell walls of stem epidermal cells as shown inFig. 6a. The distribution of the LM7 epitope was restricted todiscrete regions of the outer epidermal cell wall that werealigned with radial cell walls between epidermal cells but wasabsent entirely from the radial cell walls themselves (Fig. 6a).In contrast, the JIM5 epitope occurred only sparsely in most ofthe thickness of the outer tangential epidermal cell walls butabundantly in radial epidermal cell walls (Fig. 6b).

In addition to its occurrence in pea stem tissue discussedabove, the LM7 epitope was detected at comparable locations inepidermal cell walls and intercellular spaces in all pea seedlingorgans examined. These included domains within the outer cellwall (just under the cuticle) of the leaf epidermis in line withradial cell walls as shown in Fig. 6c, leaf parenchyma near themidrib (Fig. 6d), and root cortical parenchyma (Fig. 6e). Fur-thermore, the LM7 epitope was found in equivalent regions ofdeveloping intercellular spaces and epidermal cell junctions ina range of angiosperms including carrot (Apiaceae), maize(Poaceae), Silene latifolia (Caryophyllaceae), Kalanchoe dai-gremontiana (Crassulaceae), and Nicotiana tabacum (So-lanaceae) (data not shown).

Functional Implications of Blockwise and Non-blockwise De-esterification of HG Domains of Pectin—A series of model pec-tin samples containing the epitopes recognized by PAM1 orLM7 were used in a series of in vitro assays to investigate theimplications for a range physical properties of blockwise ornon-blockwise distributions of methyl esters on HG domains.Calcium gels formed from pectins E0, F11, F31, P41, F43, andB34 all maintained a stable shape under gravity but differed intheir opacities, as shown in Fig. 7a. The different opacities ofthe gels suggested that the degree and pattern of de-esterifica-

tion of HG domains influenced the structure and pore size ofthe gel and that this was reflected in differences in the lightscattering properties.

The degree and pattern of methyl-esterification was found toeffect the elasticity of the gels and their response to compres-sive strain. There were significant differences in the extent andmanner of deformation of gels when compressed by a force of9.82 N or to yield point as shown in Fig. 7 (b and c). Compres-sion testing of gels indicated that both the degree and patternof methyl-esterification were important in determining theyield point and elasticity of gels, as shown in Figs. 7d and 8.The mean values of the yield strain, yield force, and elasticitiesare given in the tables of Figs. 7d and 8a, with standarddeviations given in parentheses. These values were obtainedfrom averages from at least two pairs of measurements, eachpair being made on a gel formed from a completely separatesolution. The plots of F versus s shown are those of the averagevalues for the samples.

F31 and F43 have the same distribution pattern of methylgroups and differ only in DE. Although F31 formed a strong gelthat did not yield at a force of 9.82 N, gels formed from F43were relatively weak under compression and yielded at a meanforce of 0.92 N (Fig. 7d). Samples F43 and P41 have differentdistribution patterns of methyl groups but differ in DE by only2%. However, there was nearly a 3-fold difference in the yieldpoint of gels formed from P41 and F43 (Fig. 7d). The outstand-ing feature of gels formed from P41 was the fact that, althoughthe gels did yield due to the development of fractures in the gelbelow F 5 9.82 N, on removal of the force the sample recoveredits original dimensions almost immediately, as seen in Fig. 7 (band c). In complete contrast, and as shown in Fig. 7 (b and c),gels formed from F43 showed practically no recovery on re-moval of the compressive force and behaved more like a plasticmaterial, irreversibly deformed by a stress above its yieldstress. Similarly, samples F31 and B34 have similar DE, butgels prepared from these samples differed greatly in their re-sponse to compression. The force at which gels formed from B34yielded was at least 10 times less than the yield force of gelsformed from F31. These results strongly suggest that, althoughmethyl groups on both F31 and B34 are distributed in a non-blockwise fashion in both cases, the distribution patterns arenot identical. This was also suggested by their differing capac-ities to be degraded by PL (24).

FIG. 5. The LM7 epitope is presentthroughout intercellular spaceformation in pea stem parenchyma.a– c, micrographs showing the duallabeling of the cell walls of pea stem cort-ical parenchyma cells with LM7 (immu-nofluorescent labeling, green) and thecellulose-binding fluorophor calcofluor(blue) showing the positions HG epitopesand cellulose in relation to intercellularspaces at different stages of formation.d–f, transmission electron micrographs ofapproximately equivalent cell junctions tothose in a–c. The junction shown in dconsists entirely of cell wall material,whereas junctions at positions near themiddle of the cortex are progressively sepa-rated resulting in the formation of interc-ellular spaces (e and f). The darkly stainedmaterial accumulated at the corners ofexpanding air spaces (e and f) contains HG,as indicated by immunogold labeling withJIM5 (inset to f). Scale bars: a–c 5 5 mm;d–f 5 1 mm, inset to f 5 500 nm.

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With respect to the elasticity of gels, decreasing DE wasbroadly 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 wasthe case for the yield force, both the degree and pattern ofmethyl-esterification appeared to be important in determining

FIG. 6. The LM7 epitope occurs atregions of outer epidermal cell wallsand at intercellular spaces through-out pea seedlings. Figure shows micro-graphs of the binding of LM7 (a and c–e)and JIM5 (b) to the cell walls of pea stemepidermal cells (a and b), leaf epidermaland parenchyma cells (c and d, respec-tively), and root parenchyma cells (e). Allsections were hand-cut transverse sec-tions. Arrowheads in a–c indicate the po-sition of a region of outer epidermal cellwall overlying cell junctions. Arrows in cindicate the limits of the thick outer epi-dermal wall. Scale bars: a and d 5 50 mm;b, c, e, and f 5 10 mm.

FIG. 7. The degree and pattern of methyl-esterification of HGdomains influences the response of calcium-pectin gels to com-pression. a, 2% (w/v) gels were formed from a subset of the P-, F-, andB-series pectins by the addition of calcium chloride to a final concen-tration of 35 mM and equilibration for at least 24 h. Gels were cast in8.6-mm diameter syringes and cut to a height of 4 mm. b and c, theappearance 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 versusstrain curves for pectin gels under compression. Gels were prepared asdescribed in a and compressed at a rate of 0.1 mm s21 to a maximumforce of 9.82 N.

FIG. 8. The degree and pattern of methyl-esterification of HGdomains influences the elasticity of calcium-pectin gels. Theelasticity of calcium-mediated pectin gels prepared described as in Fig.7a. a, elastic moduli were calculated from the gradients of force/straincurves at low strain. b, the graph shows the relationship betweenelasticity and degree of methyl-esterification for calcium-mediated pec-tin gels.

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the elasticity of gels, as not all the samples fell exactly on thesame curve.

The Effect of the Degree and Pattern of Methyl-esterificationon the Water Holding Capacity and Porosity of Calcium-medi-ated Pectin Gels—Gels formed from E0, F11, F31, P41, F43,and B34 differed significantly in their water holding capacitiesand porosity to protein as shown in Fig. 9. For the F-seriessamples, there was some correlation between DE and waterholding capacity as shown in Fig. 9a. However, for the samplesas a whole, the correlation between DE and water holdingcapacity was not strong. For example, there were significantdifferences in the water holding capacities of gels formed fromF31 and B34 and in the water holding capacities of gels formedfrom P41 and F43. It is worth noting that the pectin gels thatshowed the largest volume loss (E0 and F11) were also the onesthat appeared to collapse under compression with relativelylittle expansion, or increase in the lateral dimension. For thesesamples this implies a collapse of the gel structure as water islost, unlike F43 mentioned previously, which maintains a co-

herent structure, loses relatively little water, and exhibits thepronounced broadening of an incompressible, plastic material.

For all the samples, the rate of incorporation of BSA into gelsdecreased over time, but there were significant differences inthe amount of protein that had been incorporated into gelsafter 20 h as shown in Fig. 9b. Although the gels formed fromF31 and B34 had similar porosities, the gels formed from P41and F43 had the highest and lowest porosities, respectively.Additionally, the porosity of gels formed from F11 was moresimilar to that of gels formed from F43 than it was to gelsformed from F31. From these results, it appears that the pat-tern rather the degree of methyl-esterification of HG may bethe more important factor in determining both the water hold-ing capacity and the porosity of calcium-mediated pectin gels.

DISCUSSION

The formation of the pectic network in the primary cell wallmatrix is contingent upon polysaccharide synthesis in the Golgiapparatus, its deposition and assembly in the cell wall, andsubsequent modification by cell wall-based enzymes in re-sponse to functional requirements. The modulation of the de-gree and pattern of methyl-esterification of HG is one aspect ofthis functional fine tuning, and the work reported here indi-cates that HG domains with distinctive physical properties areproduced in discrete microdomains of primary cell walls. Thefact that LM7 bound to cell walls in a range of organs andspecies indicates that a non-blockwise pattern of methyl-ester-ification is a widespread aspect of HG modification in plants.

It is likely that the abundance and pattern of methyl estergroups varies along an HG chain, and, although some epitopestructures, such as that recognized by LM7, have distinct loca-tions within the intercellular matrix, they do not necessarilyoccur exclusively. For example, the epitopes recognized byLM7, JIM5, and PAM1 were all present at the lining of inter-cellular spaces of pea stem parenchyma. Therefore, discretemicrodomains of the cell wall matrix are likely to contain HGwith a mixture of HG methyl ester distribution patterns result-ing in complex combinations of physical properties. In vitroanalysis of calcium-mediated model pectin gels indicated thatthe compressive strength, elasticity, water holding capacity,and the porosity of gels was significantly influenced by both thepattern as well as the degree of methyl-esterification of HGdomains. Although it is possible that some variation of thedegree and pattern of methyl-esterification of HG may be gen-erated during synthesis, it is thought that HG is usually highlymethyl-esterified prior to insertion into cell walls (3, 14). It istherefore likely that the activity of PMEs with varying de-esterification action patterns is an important mechanism formodifying matrix properties in planta.

The results reported here demonstrate that blockwise andnon-blockwise distribution patterns of methyl groups on HGcan significantly influence the physical properties of calcium-mediated pectin gels. Details of PME action and its widerconsequences on the cell wall environment are far from clear.One aspect of PME action may be to generate extracellular pHgradients that in turn orchestrate cell wall loosening via pH-sensitive processes. For example, partial inhibition of the ex-pression of a pea PME gene (rcpme1) has been correlated withchanges in extracellular pH and in cell development (31). More-over, PME activity can itself modulated by pH. Kinetic analysisof PMEs from mung bean hypocotyl demonstrated the coexist-ence of three isoforms with different pH and ion sensitivitiesand different Km and Vmax (15). In vitro analysis of the mungbean PME isoforms indicated that different action patternscould be generated by different pH conditions and the DE of thesubstrate (16). These observations raise the possibility of feed-back mechanisms modulating HG structure and hence cell wall

FIG. 9. The degree and pattern of methyl-esterification of HGdomains affects the water holding capacity and porosity of cal-cium-pectin gels. a, the water holding capacity of gels was assessed bythe 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, andP41). The graph shows the relationships between volume loss undercompression and degree of methyl-esterification. b, the porosity of pec-tin gels to protein was assessed by the rate of incorporation of BSA intogels. Gels were incubated in a solution of BSA in de-ionized water (5mg/ml) and removed at selected time points (loading time). The amountof BSA that had entered the gels was determined by dissolving gels in50 mM CDTA and assaying the amount of protein in the resultingsolutions.

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matrix properties. An another aspect of possible feedback isthat HG backbones with differing methyl-esterification pat-terns are differentially susceptible to subsequent enzymaticcleavage. PME action on a highly esterified HG is requiredbefore polygalacturonases can cleave effectively. Plant polyga-lacturonases occur in large multigene families, and membersare likely to have a range of functions in plant growth anddevelopment although these are as yet uncertain (32, 33). Thegeneration of HG substrates susceptible to polygalacturonasecleavage is likely to be greatly influenced by PME activities.

The LM7 epitope is the first spatially regulated HG epitopeto be found to occur at a consistent location within cell wallsand intercellular matrices across a range of plant organs andspecies. This consistency suggests a specific functionality forthe LM7 epitope-rich pectin at these locations. The occurrenceof the LM7 epitope within the cell walls of epidermal celljunctions and intercellular spaces indicates that a pectic HGdomain with a non-blockwise distribution of methyl esters (andwith consequent distinctive properties) has a role associatedwith cell adhesion at cell junctions. Of the model pectin sam-ples tested, F31 and B34 contained the LM7 HG epitope at thehighest abundance. These samples have similar DE and areboth the products of (different) non-blockwise de-esterificationprocedures, but gels formed from the B34 and F31 differ sig-nificantly in their physical properties. This suggests that evensubtle differences in the distribution patterns of methyl groups(revealed by enzymatic fingerprinting in the case of B34 andF31; Ref. 24) may have profound effects on matrix properties.The occurrence of the LM7 epitope in plant material indicatedthe presence of HG with non-blockwise distributions of methylgroups, but not the precise distribution pattern. However, asmore PME genes are cloned and their products characterized indevelopmental contexts, these details may become clear.

The driving force for intercellular space formation appears tobe turgor pressure producing tensile forces in cell walls andinducing a tendency toward reduced volume and hence spher-ical cell shapes (34). Three-way cell junctions are thereforesubjected to forces tending toward cell separation (34). To ini-tiate space formation, a region of primary cell wall must first bedismantled to allow the middle lamellae to link up (8) and asshown schematically in Fig. 10. Intercellular space then resultsfrom controlled splitting at the middle lamellae. As space de-

velops, the stresses are greatest at regions of adhered wallsbordering the separated cell walls and the intercellular space.The LM7 epitope-rich pectin appears to be present from theearliest stages of intercellular space formation and to be mostabundantly maintained at the points of cell to cell contact.Pectin containing this epitope may have a direct role in main-taining cell wall to cell wall links at these points throughcalcium-mediated cross-linking. Discrete electron-dense re-gions within middle lamellae of pea cotyledon parenchymahave been proposed to be involved in limiting cell separation inthis tissue (35). In the outer thickened cell wall of epidermalcells, the LM7 epitope occurs in discrete regions that are asso-ciated with cell junctions and may be involved in maintainingthe integrity of the outer cell layer. An accumulation of calciumin equivalent regions of outer epidermal cell walls (togetherwith an accumulation at the corners of intercellular spaces) hasbeen reported in mung bean hypocotyl (36). The common aspectbetween the corners of an intercellular space and points ofouter epidermal cell wall at the plant surface is that they areboth points of contact between two cells, although in the formercase cell separation occurs to some extent. LM7 epitope-richpectin may provide an appropriate environment (porosity, ionicstatus, etc.) for processes that directly maintain cell to cellcontacts (or indeed for enzymes involved in dismantling suchcontacts, although this seems less likely as cell separation isnot generally a feature of epidermal cell junctions). An addi-tional possibility is that the LM7-binding pectin may have adefensive role at points of intercellular attachment. For exam-ple, a subtly altered pattern of HG methyl ester groups mayalter both the capacity to be degraded by microbial pectinasesand also the precise nature and properties of any oligogalactu-ronide products released.

In conclusion, the observations reported here demonstratethat modulations of the pattern and degree of methyl-esterifi-cation of pectic HG occur within discrete regions of primary cellwalls and, in particular, that a non-blockwise pattern of methylesters of HG is an abundant feature of HG. We also show thatthe pattern and degree of methyl group distribution signifi-cantly affect the mechanical and physiological properties ofcalcium-mediated pectin gels and are therefore likely to influ-ence the in vivo functionalities of pectic HG domains. In thisway, a highly methyl-esterified HG polysaccharide that is de-posited in the cell wall can potentially be modified in differentways to generate distinct functional properties. Understandingthe cell biological context of the products of PME action will becrucial for determining the functions of PME multigene familymembers.

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FIG. 10. Schematic diagram showing occurrence of the LM7epitope in relation to cell junctions and the formation of intercel-lular space. a, intercellular space forms at the junction between old (o)and new (n) cell walls and involves the linking up of middle lamellae. Thisrequires the dismantling of a region of the older cell wall (indicated by thedotted circle). b, a non-expanded intercellular junction with no air space.The gray triangle indicates the position of expanded middle lamella ma-terial that occupies the space completely and corresponds to the positionof LM7 labeling. c, an expanded cell junction with a large intercellularspace (is). The arrows indicate the forces generated by intracellular turgorpressure that drive cell separation. The gray triangles indicate the regionsof the cell that correspond to the position of LM7 labeling. In all cases, cindicates the interior of cells. In all cases, thick lines indicate the plasmamembrane face of cell walls and thin lines the position of middle lamellae.Figure was adapted from Jarvis (34).

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Methyl-esterification of Pectic HG in Plant Cell Walls 19413

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Jørn D. Mikkelsen, Brent S. Murray and J. Paul KnoxW. M. van Alebeek, Alphons G. J. Voragen, Susan E. Marcus, Tove M. I. E. Christensen, William G. T. Willats, Caroline Orfila, Gerrit Limberg, Hans Christian Buchholt, Gert-Jan

ESTERASE ACTION, MATRIX PROPERTIES, AND CELL ADHESIONHomogalacturonan in Plant Cell Walls: IMPLICATIONS FOR PECTIN METHYL

Modulation of the Degree and Pattern of Methyl-esterification of Pectic

doi: 10.1074/jbc.M011242200 originally published online March 6, 20012001, 276:19404-19413.J. Biol. Chem. 

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