Swelling Behavior of the Tomato Cell Wall Network - American

The swelling of tomato pectin and isolated tomato pericarp cell wall material was investigated in .... leads to an excess of counterion within the gel...
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Biomacromolecules 2001, 2, 450-455

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Swelling Behavior of the Tomato Cell Wall Network Alistair J. MacDougall,* Neil M. Rigby, Peter Ryden, C. William Tibbits,† and Stephen G. Ring Food Biopolymer Section, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. Received November 3, 2000; Revised Manuscript Received January 8, 2001

The swelling of tomato pectin and isolated tomato pericarp cell wall material was investigated in aqueous media under different ionic conditions, pH, and external osmotic stress. Conditions were chosen to include those that would be encountered in vivo. Swelling in these systems was strongly influenced by the polyelectrolyte nature of the polymer and the extent of cross-linking with divalent counterions. Introduction The plant cell wall is a complex macromolecular assembly. Current models of the primary cell wall of dicotyledonous plants show cellulose microfibrils, cross-linked by xyloglucans, dispersed in a predominantly pectin matrix. Typically the isolated cell wall contains 50% pectin, while the middle lamella region, which lies between adjoining cell walls, is almost entirely composed of pectin. For polymer networks generally, there is a strong effect of polymer concentration on material characteristics including transport properties, mechanical behavior, and ionic relationships. Rather surprisingly, there is relatively little information available on the physicochemical and biochemical factors which influence plant cell wall hydration. It is this aspect which is the focus of this study, more particularly the role of the pectin. The pectic polysaccharides are perhaps structurally the most complex polymers within the wall.1,2 They consist of a backbone of (1 f 4) R-D-galacturonosyl residues interrupted with typically a 10% substitution of (1 f 2) R-Lrhamnopyranosyl residues. A portion of the rhamnosyl residues are branch points for neutral sugar side chains which contain L-arabinose and D-galactose. The rhamnosyl substitution is thought to cluster in “hairy” regions leaving “smooth” sequences of the galacturonan backbone. The backbone may be partially acetylated and may be further substituted with terminal xylose. A portion of the galacturonosyl residues of extracted pectins are methyl esterified. The complexity of pectin structures means that a range of interactions can potentially affect their behavior and that of the cell wall. Of particular importance are the charge interactions of the free uronic acids, which can either cross-link the network, through association with polyvalent counterions, or contribute to network swelling through a Donnan effect. The interaction of counterions with polyelectrolytes is dependent on ionic strength and charge density along the * Corresponding author: Telephone: 44-1603-255000. Fax: 44-1603507723. E-mail: [email protected]. † Current address: Institute of Cell and Molecular Biology, The University of Edinburgh, Darwin Building, King’s Buildings, Edinburgh, EH9 3JR.

polymer backbone.3-5 At low ionic strength the macroion has a relatively high affinity for the counterion. On increasing ionic strength the electrostatic field of the macroion becomes progressively screened and the affinity for the counterion is reduced. This reduction in affinity with increasing counterion concentration is anticooperative and is the generally observed behavior of polyelectrolytes and also for pectins, more particularly at low ionic strengths and charge densities.6 In addition to this simple screening of Coulomb interactions, for sufficiently strongly charged polyelectrolytes, it becomes favorable for a fraction of the counterions to “condense” on the polymer backbone.4 To estimate whether counterion condensation is likely to be observed, a dimensionless parameter, ξ, can be calculated from ξ)

q2 04πkTb

(1)

where  is the dielectric constant of the medium, 0 the dielectric permittivity of free space, q the elementary charge, k Boltzmann’s constant, and b the spacing between charges.5 For many polyelectrolytes, including pectin,7 counterion condensation has been observed above a threshold value of ξ ) 1. There is also the possibility of specific effects, as in the strong interaction of Ca2+ with oligogalacturonate sequences.8 For pectins, this interaction is dependent both on the degree of methyl esterification and on the distribution of unesterified galacturonic acid residues along the backbone.9 The strength of the interaction increases with increasing charge density and increasing localization of that charge along the backbone.6 Crystallographic and NMR evidence suggests that Ca2+ ions can induce conformational changes and promote chain association of oligogalacturonates.10,11 For pectins containing oligogalacturonate sequences, the addition of Ca2+ ions to concentrated solutions can cause gelation. A model for the cross-link in these gels is the “eggbox”, in which oligogalacturonate segments enclose Ca2+ ions.12,13 A consequence of the Ca2+-induced conformational change is that the binding of one Ca2+ ion can facilitate the binding of others, leading to a cooperative binding behavior. In experi-

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mental studies, both anticooperative (at relatively low concentrations of supporting electrolyte, e.g., 0.01 M NaCl) and cooperative behaviors (at higher ionic strengths) are observed.6 In addition to their role in cross-linking, the charged pectin residues also have a potential role in influencing swelling. For neutral polymer networks, swelling can be characterized in terms of an interaction parameter describing the affinity of polymer and solvent.14 For lattice model representations of the entropy of mixing of a polymer and a diluent, this interaction parameter is a measure of energetic interaction between a polymer repeating unit and diluent and also includes any local entropic effects.15 For aqueous solutions of polysaccharides, these local effects can be significant. At high concentrations of neutral polymer, the osmotic pressure generated by the network can be large. Additionally, for polyelectrolyte gels, the requirement for electrical neutrality leads to an excess of counterion within the gel compared to the external medium.14 This excess generates an osmotic pressure difference between the gel and external medium, which increases with decreasing ionic strength. The excess osmotic pressure leads to the swelling of the gel until a balance is achieved between the osmotic pressure which drives swelling and the restorative force arising from the deformation of the cross-linked network.14 At intermediate salt concentrations an estimate of the contribution to osmotic pressure, π, due to a polyelectrolyte may be obtained from3,14,16-18 π≈

RTc2 A(c + 4Acs)

(2)

for univalent electrolytes, where c and cs are the molar concentrations of polymer segment and salt and A is the number of monomers between effective charges. The greater the charge on the polymer, and the lower the ionic strength, the greater the osmotic pressure generated. However at high charge densities, the phenomenon of counterion condensation can reduce the counterion fraction which can contribute to this Donnan effect. In this article, we compare the swelling behavior of a pectin isolated from tomato fruit cell wall, and the isolated cell wall material (CWM) as a function of ionic strength and osmotic stress. Experimental Methods Reagents. Fluorescein isothiocynate (FITC)-labeled dextran, dextran, poly(ethylene glycol) (PEG), and cyclohexanediaminetetraacetic acid (CDTA) (>99%) were obtained from Sigma. Osmotic Pressure Experiments. The concentration dependence of osmotic pressure for dextran and PEG were calculated from log π ) a + b(wt %)c, using published values of the constants.19,20 The suitability of this reference data was confirmed by experiment.21 Preparation and Characterization of CWM. Pericarp tissue was taken from mature green tomatoes (var. Solairo). Fruit harvested in late autumn (which had a low content of

starch grains as assessed by iodine/KI staining) were peeled to remove the outer cuticle, and the pericarp was dissected and immersed in liquid nitrogen. The frozen pericarp was shattered in a Waring blender run for 1 min at low speed and placed in phenol saturated with buffer (Tris HCl, pH 8.2) at 20 °C. Upon standing, the CWM accumulated at the bottom of the aqueous phase, was recovered by suction, and was extracted a second time with buffered phenol. After being washed with 50 mM KCl (50 volumes), the cell wall suspension was recovered by centrifugation and adjusted to pH 4.5 with acetic acid. Phenol was then removed from the CWM by repeated washing with 50 mM KCl (5 × 50 volumes) followed by centrifugation. Intact cells were removed on nylon cloth (300 µm mesh). Tomato pectin was extracted from ball-milled CWM with the chelating agent CDTA as described previously.22 The neutral monosaccharide content of the CWM was determined after hydrolysis and separation of the sugars as their alditol acetates.23 The uronic acid content was determined by colorimetric analysis.24 The degree of methyl esterification was determined by headspace analysis after release of methanol with alkali. Assays were carried out in a total of 2 mL, containing pectin or CWM, 1 µmol propanol as an internal standard, and NaOH at a concentration of 1 N. After standing at room temperature for 2 h the headspace was analyzed on a Perkin-Elmer Autosystem XL GC (Perkin-Elmer, Cambridge, U.K.) equipped with a Perkin-Elmer HS 40 XL autosampler. Vials were incubated at 95 °C for 15 min, pressurized to 100 kPa and an injection of 0.05 min made on the GC. The GC column was a 25 m × 0.32 mm ID, 4.0 µm film BP5 column (SGE, Ringwood, Australia), operated at 50 °C. Detection was by FID. Standards containing up to 15 µmol of methanol were tested and gave a linear response. The calcium content of the CWM was determined after ashing by atomic absorption spectroscopy. As the drying of polysaccharides is known to encourage their further association, the CWM and the isolated pectin were stored frozen. Swelling of Pectin Films. Films of the K+ form of chelator-extracted tomato pectin were prepared by air-drying. The swelling as a function of osmotic stress and ionic strength was determined from the change in dimension of 6 mm diameter disks which were bisected with a razor. The length of the straight edge was measured with a traveling microscope to 10 µm.21 The dimensions of unswollen films were determined after drying over P2O5 in vacuo at 60 °C. For a set of experiments, films were preequilibrated in an aqueous solution (of the required ionic composition) of PEG 8000 with an osmotic pressure of 4 MPa before being transferred to a PEG solution of the required osmotic pressure and ionic composition. Preliminary measurements on the change in size of the disk and its thickness confirmed that the swelling was isotropic, and reversible under the conditions used. From the change in linear dimension the volume change was calculated. Each data point in the figures is the average measurement from three disks. Measurement of Cell Wall Swelling. The swelling of the cell wall as a function of salt concentration (KCl) was determined from the exclusion of a macromolecular probe (FITC-labeled dextran Mw 260 000). Preliminary experiments

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established that the FITC-dextran did not bind to the CWM under the conditions used. The KCl concentration of the solution in which the CWM was suspended was varied from the initial level of 50 mM by washing with distilled water. For swelling measurements, 1.5 mL of 0.2% w/w FITCdextran solution was added to a known weight of hydrated sediment containing ∼25 mg CWM and mixed. After equilibration at 25 °C (16 h) the suspension was centrifuged and the concentration of FITC-dextran in the supernatant determined from its absorbance at 450 nm. The final KCl concentration was estimated by conductivity. The lowest conductivity observed was 10 µS (equivalent to 0.05 mM KCl). The pH of the supernatant was also measured. From the dilution of the FITC-dextran, the volume and mass of the solution accessible to the macromolecular probe was determined. The hydrated mass of CWM was determined from the difference between the total mass of suspension and the mass of solution accessible to the probe. Measurements of the variation of cell wall hydration with pH in the range 3-5.5 were made by equilibrating the CWM in 20 mM Na acetate buffer. Cell wall swelling was also measured as a function of the osmotic stress provided by a concentrated dextran solution (Mw 480 000), with the unlabeled dextran acting as the macromolecular probe for determining swelling. In this case the mass fraction of dextran in the supernatant was determined by freeze-drying (making an allowance for the salt content). The standard deviation obtained from replicated swelling measurements was less than 5% of the mean value. Results and Discussion Cell Wall Isolation. In vitro studies of cell wall swelling require the isolation of a CWM, with a minimum of physical, chemical, and enzymic modification and minimal contamination with cytoplasmic components. For these reasons, a buffered phenol extraction was used. The carbohydrate composition of the isolated CWM was as follows: Dgalactose, 12.5; L-arabinose, 3.2; D-xylose, 2.8; L-rhamnose, 0.4; D-glucose, 38.5; D-galacturonic acid, 22.0. All were expressed as % w/w of the CWM. The purified CWM was starch-free (as indicated by the absence of blue/red color on staining with iodine/potassium iodide solution). From the known composition of the noncellulosic polysaccharides of tomato cell wall, the pectic polysaccharide content was estimated at ∼55% w/w of the total carbohydrate present. The degree of methyl esterification of the pectic polysaccharides in the CWM, obtained from the total uronic acid content and determination of the methanol released in alkali, was 61%. The calcium content of the cell walls was 0.05% w/w. This is low compared to previous estimates25 and may reflect the growing conditions of the plants, or differences in the method of preparation of CWM. The buffered phenol extraction that we have used is very effective in removing cytoplasmic proteins to leave a CWM which is >90% w/w carbohydrate. In addition, we have also equilibrated the CWM in 50 mM KCl to remove readily exchangeable counterions. The carbohydrate content of the chelator-extracted pectin was as follows: D-galactose, 10.1; L-arabinose, 2.9; D-xylose,

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Figure 1. Salt dependence of the swelling of tomato pectin at an osmotic pressure of 0.5 MPa at 20 °C. Key: closed squares, experimental data; open squares, predicted behavior.

0.1; L-rhamnose, 0.7; D-glucose, 0.5; D-galacturonic acid. 71.0. All were expressed as % w/w, with the degree of methyl esterification of the uronic acid being 68%. From the methyl ester content of the pectic polysaccharides, it is possible to calculate an average charge spacing between uronic acid residues along the rhamnogalacturonan backbone of the pectic polysaccharides. These values are 1.12 nm for the pectin in the cell wall and 1.5 nm for the extracted pectin For water at 25 °C, ξ ≈ 0.71/b (if b is expressed in nmseq 1), with values of 0.63 and 0.47 for the cell wall and extracted pectin, respectively. These data suggest that counterion condensation should not have a major effect on the polyelectrolyte behavior of these materials. Swelling of Tomato Pectin Films. The swelling of tomato pectin was examined as a function of ionic strength and osmotic stress, at 20 °C and pH 5.5. The K+ form of the dried pectin swells rapidly in water at room temperature and eventually dissolves. Exposure of the film to a constant osmotic stress of 0.5 MPa maintained film integrity permitting the study of swelling behavior as a function of monovalent salt concentration (KCl). In the region 0.1-1 M, the observed swelling is ∼1.5 v/v with little dependence on concentration (Figure 1). Reduction of the KCl concentration below 0.1 M results in a dramatic increase in observed swelling, reaching 19.5 v/v at 5 mM, and is consistent with a polyelectrolyte contribution to swelling. The observed behavior was compared with that predicted from eq 2. Qualitative agreement was obtained although the predicted swelling was substantially underestimated at the lowest ionic strength, and at intermediate ionic strengths the swelling was overestimated. A potential origin of the latter effect is that, at intermediate KCl concentrations, some counterion condensation may occur or, alternatively, some counterionmediated association of pectin chains may cross-link this system, reducing the observed swelling. The swelling of the pectin, as a function of osmotic stress, is shown in Figure 2 at a constant KCl concentration of 50 mM. On reducing osmotic stress below 1.0 MPa, a marked increase in swelling is observed. The predicted concentration of polymer, expressed as v/v, needed to generate the balancing osmotic pressure from solely a polyelectrolyte effect (eq 2) is shown

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Figure 2. Relationship between applied osmotic pressure and swelling of tomato pectin films in 50 mM KCl at 20 °C. Key: closed squares, experimental data; open squares, predicted behavior. Figure 4. Dependence of the swelling of tomato CWM on KCl concentration.

Figure 3. Dependence of the swelling of tomato pectin films as a function of osmotic pressure in the presence of (a) 1 M KCl and (b) 6 mM CaCl at 20 °C.

for comparison. Again, the similarity in the forms of the curves suggests that pectin swelling under these conditions derives mainly from a polyelectrolyte effect, although the predicted swelling is underestimated at the lowest applied osmotic stress. In Figure 3, the swelling of the pectin film as a function of osmotic stress in 1 M KCl and 6 mM CaCl2 is compared. In 1 M KCl the predicted Donnan contribution to swelling is substantially reduced (cf. Figure 1). The data presented in Figure 3 show that swelling is suppressed over the range of osmotic stress applied. Changing the counterion to Ca2+ at a concentration of 6 mM has a similar effect in reducing

swelling. Even after allowance is made for the change from monovalent to divalent counterion, swelling is much less than might be expected at this ionic strength from a Donnan effect. Ca2+ is a known cross-linking agent for these pectic polysaccharides, even at semidilute concentrations of polymer;22 therefore, it is expected that it should act as a crosslinker in these more concentrated systems. The stiffness of the cross-linked network counteracts the swelling pressure. The reason for the choice of 6 mM Ca2+ is that this is the concentration found in apoplastic sap (the solution in which the cell wall is bathed in vivo).26,27 Also found in the sap is Mg2+. At a 50 mM concentration of counterion and an osmotic stress of 0.5 MPa we found that the effectiveness of both Mg2+ and Ca2+ in reducing swelling was similar, with swelling ratios of 1.7 and 1.6 being observed, respectively. As swelling is much less than might be predicted from a Donnan effect, this suggests that Mg2+ may also function as a cross-linker under these conditions. Cell Wall Swelling. The swelling of the CWM as a function of aqueous KCl concentration was determined from the exclusion of a macromolecular probe from the cell wall network. The macromolecular probe chosen for this study was a FITC-labeled dextran with a reported Mw of 250 000 and a radius of gyration of ≈250 Å.28 The macromolecular probe will therefore be excluded from networks with smaller pore sizes. The swelling behavior, determined as the mass of hydrated cell wall/dry mass, of the purified CWM is shown in Figure 4. The volumetric swelling behavior was estimated using a value for the density of pectin at 20 °C of 1600 kg m-3. At salt concentrations >50 mM, the observed swelling was ∼10 g/g (∼15 v/v). At lower salt concentrations, there was some increase in swelling to ∼14 g/g (∼ 22 v/v) in water saturated with atmospheric CO2. This effect was not as marked as for the isolated polysaccharide, although still consistent with a polyelectrolyte contribution to swelling. In vivo, the plant cell wall is exposed to the ionic environment of the apoplastic sap and the osmotic stress of the cell contents. There is a developing interest in how the osmotic stress in biological systems affects the assembly and

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Figure 5. Effect of applied osmotic pressure on the swelling of tomato CWM in 50 mM KCl.

interactions of cellular components.29 The osmotic stress being exerted on the cell wall by solutes contained within plant cells can be determined from microprobe measurements of the hydrostatic (turgor) pressure in individual cells.30 Reported values for the expanding cells of higher plants are generally in the range 0.1-1 MPa. For mature green tomato fruit pericarp cells, the turgor pressure has been estimated at 0.14 MPa, falling to 0.03 MPa as the fruits ripen31sthe decrease being associated with failure of the cell membranes to continue to act as an effective barrier to the movement of solutes. The swelling behavior of CWM as a function of osmotic stress was therefore examined within the range 0.01-0.5 MPa in the presence of 30 mM KCl (Figure 5). In this experiment, an unlabeled high molecular weight dextran was used to provide the osmotic stress and to act as the macromolecular probe for determining cell wall swelling. The swelling decreases from ∼7.5 to ∼3 g/g (∼11 to 4 v/v) as the osmotic pressure is increased to 0.16 MPa, with little further dependence of swelling on osmotic pressure up to 0.5 MPa (data not shown). The calculated difference in osmotic pressure, arising from a polyelectrolyte effect, generated by changing the cell wall concentration from 7.5 to 3 g/g is 0.16 MPa. While exactly equivalent to the applied osmotic stress, this agreement should be treated with some caution. First, it assumes ionization of all the unesterified galacturonic acid residues and does not take into account the effect of specific ion binding on observed behavior. This estimate also does not include the contribution of the neutral polysaccharide component to the observed swelling behavior. The demonstration that there is a marked dependence of cell wall hydration on osmotic stress in vitro indicates that physiological variations in the osmotic stress exerted on the cell wall, by the cell contents, have the potential to affect the concentration of cell wall polymers and therefore cell wall properties. The in vivo environment of the plant cell wall is also subject to variations in pH. In Figure 6 is shown the pH dependence of cell wall swelling in the pH range 6.0-1.2 at a concentration of acetate buffer of 0.02 M. This

MacDougall et al.

Figure 6. pH dependence of the swelling of tomato CWM.

experiment covers the known variation in pH observed in the apoplast of ripening tomato fruit cells. In this tissue the pH of the extruded apoplastic sap has been found to decrease from 5 to 4 as ripening proceeds.27 Over this interval there is a marked increase in the swelling of tomato CWM from ∼8 g/g (∼12 v/v) at pH 5 to 12 g/g (∼19 v/v) at pH 4. A dependence of swelling on pH is a typical feature of the swelling of covalently cross-linked polyelectrolytes. For weakly charged polyelectrolytes, a change in pH which reduces the dissociation of the ionizable groups will reduce the charge on the polymer, and consequently reduce the magnitude of the Donnan effect and lead to reduced swelling. As the pKa for the ionization of the carboxyl group of the anhydrogalacturonic acid of pectin is ∼3.2532 a fall in pH from 5 to 4 would reduce the charge on the polymer (to a small extent) and hence the magnitude of the Donnan contribution to cell wall swelling. The observed increase in swelling is therefore the reverse of what might be expected. Experiments on the gelation of isolated pectins, and the results presented above on tomato pectin films, demonstrate that calcium mediated interactions can play a major role in the cross-linking of pectin networks. In contrast to covalent cross-linking, this cross-linking is expected to show a pH dependence. Treatment of tomato CWM at acid pH reduced the calcium level from 0.05% w/w at pH 5 to 0.042% at pH 3.25 and 0.0025% at pH 2.1. One explanation for the pH dependent increase in swelling on changing the pH from 5 to 4 is that the change in calcium content results in reduced cross-linking, a reduced restorative force opposing swelling, and consequently an observed increase in swelling. Support for this suggestion comes from experiments on calcium crosslinked tomato pectin gels which showed an increase in swelling and a decrease in gel stiffnesssindicative of a decrease in cross-linkingsas the pH was reduced from 5 to 3.25. In this case further reduction in pH resulted in a decreased swelling32 which was not observed for the CWM. The current data indicate that small changes in pH can have a marked effect on the swelling of CWM in vitro and such behavior is potentially relevant to the functioning of the cell wall network in vivo.

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Conclusions Concentrated networks of tomato pectin exhibit a swelling behavior in aqueous solution which is characteristic of weak polyelectrolytes in that increased swelling is observed as the concentration of monovalent counterion is reduced. Both Ca2+ and Mg2+ can cross-link these concentrated networks and reduce swelling. The isolated tomato cell wall displays qualitatively similar behavior. Cell wall swelling showed a strong dependence on osmotic stress over the range of osmotic stress to which the cell wall would be exposed in vivo. Cell wall swelling also showed a significant pH dependence in the range over which tomato pericarp cell wall pH is reported to vary. These observations suggest that cell wall behavior in vivo will be modified by changes in the ionic composition of the apoplast and by variation in the osmotic stress exerted on the cell wall by the cell contents. Acknowledgment. The authors wish to thank the core strategic grant of the BBSRC and Zeneca for participating in a BBSRC CASE award for C. W. Tibbits. References and Notes (1) Schols, H. A.; Voragen, A. G. J. Carbohydr. Res. 1994, 256, 8395. (2) Schols, H. A.; Voragen, A. G. J.; Colquhoun, I. J. Carbohydr. Res. 1994, 256, 97-111. (3) Barrat, J.-L.; Joanny, J.-F. AdV. Chem. Phys. 1996, 94, 1-67. (4) Manning, G. S. Ber. Bunsen Ges. Phys. Chem. 1996, 100, 923928. (5) Manning, G. S.; Ray, J. J. Biomol. Struct. Dynamics 1998, 16, 461476. (6) Garnier, C.; Axelos, M. A. V.; Thibault, J.-F. Carbohydr. Res. 1994, 256, 71-81. (7) Ghagare, S.; Savitsky, G. B.; Spencer, H. G. Polymer 1992, 33, 4725-4727. (8) Kohn, R. Pure Appl. Chem. 1975, 42, 371-99. (9) Thibault, J. F.; Rinaudo, M. Biopolymers 1985, 24, 2131-2143. (10) Walkinshaw, M. D.; Arnott, S. J. Mol. Biol. 1981, 153, 1055-1073.

Biomacromolecules, Vol. 2, No. 2, 2001 455 (11) Jarvis, M. C.; Apperley, D. C. Carbohydr. Res. 1995, 275, 131145. (12) Grant, G. T.; Morris, E. R.; Rees, D. A.; Smith, P. J. C.; Thom, D. FEBS Lett. 1973, 32, 195-8. (13) Morris, E. R.; Powell, D. A.; Gidley, M. J.; Rees, D. A. J. Mol. Biol. 1982, 155, 507-516. (14) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953. (15) Moates, G. K.; Noel, T. R.; Parker, R.; Ring, S. G. Carbohydr. Res. 1997, 298, 327-333. (16) Dobrynin, A. V.; Colby, R. H.; Rubinstein, M. Macromolecules 1995, 28, 1859-1871. (17) Rubinstein, M.; Colby, R. H.; Dobrynin, A. V.; Joanny, J.-F. Macromolecules 1996, 29, 398-406. (18) Skouri, R.; Schosseler, F.; Munch, J. P.; Candau, S. J. Macromolecules 1995, 28, 197-210. (19) Parsegian, V. A.; Rand, R. P.; Fuller, N. L.; Rau, D. C. Methods Enzymol. 1986, 127, 400-416. (20) Parsegian, V. A. http://dir/nichd.nih.gov/Lpsb/docs/osmdata/osmdata.html. (21) Ryden, P.; MacDougall, A. J.; Tibbits, W.; Ring, S. G. Biopolymers 2000, 54, 398-405. (22) MacDougall, A. J.; Needs, P. W.; Rigby, N. M.; Ring, S. G. Carbohydr. Res. 1996, 293, 235-249. (23) Blakeney, A. B.; Harris, P. J.; Henry, R. J.; Stone, B. A. Carbohydr. Res. 1983, 113, 291-299. (24) Blumenkrantz, N.; Asboe-Hansen, G. Anal. Biochem. 1973, 54, 484489. (25) Huber, D. J. Phytochemistry 1991, 30, 2523-2527. (26) MacDougall, A. J.; Parker, R.; Selvendran, R. R. Plant Physiol. 1995, 108, 1679-1689. (27) Almeida, D. P. F.; Huber, D. J. Physiol. Plant. 1999, 105, 506512. (28) Seksek, O.; Biwersi, J.; Verkman, A. S. J. Cell Biol. 1997, 138, 131142. (29) Leikin, S.; Parsegian, V. A.; Rau, D. C.; Rand, R. P. Annu. ReV. Phys. Chem. 1993, 369-395. (30) Tomos, A. D.; Leigh, R. A. Annu. ReV. Plant Physiol. Plant Mol. Biol. 1999, 50, 447-472. (31) Shackel, K. A.; Greve, C.; Labavitch, J. M.; Ahmadi, H. Plant Physiol. 1991, 97, 814-816. (32) Tibbits, C. W.; MacDougall, A. J.; Ring, S. G. Carbohydr. Res. 1998, 310, 101-107.

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