Evidence for In Vitro Binding of Pectin Side Chains to Cellulose

Abstract

Pectins of varying structures were tested for their ability to interact with cellulose in comparison to the well-known adsorption of xyloglucan. Our results reveal that sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectins, which are rich in neutral sugar side chains, can bind in vitro to cellulose. The extent of binding varies with respect to the nature and structure of the side chains. Additionally, branched arabinans (Br-Arabinans) or debranched arabinans (Deb-Arabinans; isolated from sugar beet) and galactans (isolated from potato) were shown bind to cellulose microfibrils. The adsorption of Br-Arabinan and galactan was lower than that of Deb-Arabinan. The maximum adsorption affinity of Deb-Arabinan to cellulose was comparable to that of xyloglucan. The study of sugar beet and potato alkali-treated cell walls supports the hypothesis of pectin-cellulose interaction. Natural composites enriched in arabinans or galactans and cellulose were recovered. The binding of pectins to cellulose microfibrils may be of considerable significance in the modeling of primary cell walls of plants as well as in the process of cell wall assembly.

The well-known model of primary cell walls (PCWs) of dicotyledons emphasizes noncovalent interactions between cell wall polymers and suggests two independent, but interacting, networks where the cellulose-xyloglucan network is embedded in a matrix of pectic polysaccharides (Carpita and Gibeaut, 1993; Somerville et al., 2004).

Cellulose, the primary structural element of the cell wall, is a homopolymer composed of (1→4)-linked β-d-Glcp residues. The linear chains of parallel alignment are tightly linked by hydrogen bonds to form microfibrils. Xyloglucan, the most abundant hemicellulosic polysaccharide in the PCWs of dicotyledons, is composed of a cellulose-like backbone consisting of (1→4)-linked β-d-Glcp residues, branched at O-6 by α-d-Xylp residues, which can be further substituted at O-2 by β-d-Galp residues (Fry, 1989). Some of the Galp residues may be substituted at O-6 by α-d-Fucp. Pectins are major components of dicotyledon PCWs and of the middle lamella. Their backbone is composed of smooth homogalacturonan (HG) and hairy rhamnogalacturonan (RG) I regions (O'Neill et al., 1990). HG, a linear chain composed of (1→4)-linked α-d-GalUAp units, can be methyl esterified at O-6 of carboxyl groups and acetyl esterified at O-2 and/or O-3 of secondary hydroxyl groups (Ralet et al., 2001). Some HGs might be substituted to form RG II or xylogalacturonan. RG II is a complex polysaccharide composed of GalUAp, Rhap, Galp, and some unusual sugars. Dimers of RG II were found to be cross-linked by two diester bonds through a boron atom (Fleischer et al., 1999). Xylogalacturonan contains β-d-Xylp residues attached to O-3 of the HG backbone (Le Goff et al., 2001). RG I contains a backbone of the repeating disaccharide unit: (1→2)-α-L-Rhap-(1→4)-α-d-GalUAp (Renard et al., 1995). They are predominantly substituted at O-4 of Rhap residues by neutral sugar side chains (Schols and Voragen, 1994). The proportion of branched Rhap residues depends on the plant source (Voragen et al., 1995). Neutral sugar side chains of arabinan, galactan, and arabinogalactan (AG) I and II may be present. Arabinan side chains are composed of (1→5)-α-l-Araf residues, which can be more or less branched by α-l-Araf units attached at O-2 and/or O-3. Arabinan-rich pectins have been isolated from apple (Malus domestica), sugar beet (Beta vulgaris), carrot (Daucus carota), and onion (Allium cepa; Voragen et al., 1995). Galactan side chains contain (1→4)-linked β-d-Galp units and are commonly found in potato (Solanum tuberosum), soybean (Glycine soja), and lupin (Lupinus albus; Voragen et al., 1995). In the Amaranthaceae family, to which spinach (Spinacia oleracea) and sugar beet belong, arabinan and galactan side chains are esterified by ferulic acid (Ishii and Tobita, 1993; Ralet et al., 1994). AG I is composed of a (1→4)-linked β-d-Galp backbone, which is branched mostly with terminal Araf units at the O-3 of Galp residues (McNeil et al., 1984). AG II constitutes a group of short (1→3)- and (1→6)-β-d-Galp chains, containing side chains of (1→6)-α-L-Araf-[(1→6)-β-d-Galp]_n units where n = 1, 2, or 3 (Ridley et al., 2001).

Xyloglucan is known to interact with cellulose presumably through hydrogen bonds (Hayashi et al., 1987; Vincken et al., 1995). It is believed that xyloglucan can either bind to the surface of cellulose microfibrils or cross-link the adjacent microfibrils. This network is embedded in a matrix in which various types of noncovalent cross-links between pectins have been claimed. Indeed, the chains of HG may be cross-linked by calcium ions, hydrogen bonds, and hydrophobic interactions (Voragen et al., 1995). The degree of methylation (DM) and degree of acetylation (DA) of HG, as well as the distribution of methyl and acetyl groups, play an important role in these interactions (Thibault and Rinaudo, 1986; Ralet et al., 2003). The oxidative coupling of arabinan and galactan side chains containing ferulic acid (Saulnier and Thibault, 1999) may form another chemical cross-link leading to a covalent network in which the cellulose-xyloglucan complex may be embedded.

Although Chanliaud and Gidley (1999) ruled out in vitro molecular interactions between cellulose microfibrils from Acetobacter xylinum and citrus or apple pectins, it has been recently proposed that pectic neutral sugar side chains could bind to cellulose (Iwai et al., 2001; Oechslin et al., 2003; Vignon et al., 2004). Indeed, an arabinan-cellulose composite was isolated from the spine fibers of the cactus Opuntia ficus-indica after successive alkali extractions (Vignon et al., 2004). Oechslin et al. (2003) suggested interactions between cellulose and pectic galactan side chains in apple cell walls. In the cell walls of Nicotiana plumbaginifolia, Iwai et al. (2001) showed that Ara-rich pectins are strongly associated with cellulose-hemicellulose complexes.

In this work, the in vitro adsorption of pectins of varying structures to Avicel microcrystalline cellulose was studied and compared to the well-known adsorption of xyloglucan. Some polymers that exhibited a clear adsorption to Avicel cellulose were also tested for their ability to bind to PCW cellulose. We provide evidence for molecular interactions between cellulose microfibrils and pectins, presumably through the arabinan and/or galactan side chains. Alkali treatment of sugar beet and potato cell wall materials (CWM) were performed with the aim of providing further evidence of potential pectin-cellulose interactions.

RESULTS

Some Pectins and Pectic Subunits Are Able to Bind to Cellulose Microfibrils

Xyloglucan, commercial and laboratory-extracted pectins, and commercial pectic neutral sugar side chains were tested for their ability to bind to Avicel microcrystalline cellulose. The chemical and physicochemical characteristics of these polysaccharides are presented in Table I. Solutions of the various polysaccharides were prepared at different concentrations and aliquots were mixed with a known quantity of cellulose. Polysaccharide solutions and polysaccharide-cellulose blends were kept under continuous head-over-tail mixing at 40°C for 6 h, centrifuged, and the supernatants were tested for their GalUA or total neutral sugar content by colorimetric assays. The amount of adsorbed matter was calculated from the difference in sugar content measured for polysaccharide solutions and polysaccharide-cellulose blend supernatants. The binding capacity of the different polymers to cellulose was then expressed by binding isotherms where the mass of bound material per mass of cellulose (q_e) was plotted versus the concentration of free material remaining in solution at equilibrium concentration (C_e).

Table I.

Sugar composition and macromolecular characteristics of Avicel microcrystalline cellulose, PCW cellulose, xyloglucan, arabinan-rich sugar beet pectin, galactan-rich potato pectin, C-30, C-70, Br-Arabinan, Deb-Arabinan, and galactan

M_W, Weight-average molar mass; M_n, number-average molar mass; I, polydispersity index; nd, not determined.

	Neutral Sugars and GalUA								Degrees of Esterification		Physicochemical Parameters
	Rha	Fuc	Ara	Xyl	Man	Gal	Glc	GalUA	DM	DA	Mw	I
	mg/g										kD	Mw/Mn
Avicel cellulose	0	0	3	13	15	0	912	29	nd	nd	nd	nd
PCW cellulose	0	0	2	26	18	4	925	17	nd	nd	nd	nd
Xyloglucan	0	0	16	266	0	152	457	nd	nd	nd	763	1.4
Sugar beet pectin	26	2	201	2	1	55	4	589	16	19	98	5.4
Potato pectin	26	4	70	3	2	463	6	293	0	0	nd	nd
C-30	19	0	0	3	0	85	6	820	28	0	111	8.9
C-70	16	0	25	1	0	43	0	770	70	2	104	6.3
Br-Arabinan	37	6	681	2	4	138	4	98	0	19	43	1.8
Deb-Arabinan	45	2	518	0	0	181	0	114	0	0	26	2.2
Galactan	24	5	80	3	0	608	3	182	0	0	nd	nd

The amount of xyloglucan from Tamarindus indica adsorbed to cellulose (Fig. 1) is in good agreement with values reported by Vincken et al. (1995). In the range of concentrations from 2.5 μg/mL to 10 μg/mL, xyloglucan bound completely to cellulose, as illustrated by the steep part of the isotherm, suggesting the presence of specific interactions between xyloglucan and cellulose. The extent of binding increased with increasing concentration of xyloglucan and apparently reached a plateau value of approximately 13 μg of bound material per milligram of cellulose, above 750 μg/mL of xyloglucan. This indicates that all potential binding sites of the cellulose surface are occupied. The estimated weight-average molar mass of xyloglucan was very high (763 kD). The index of polydispersity close to 1 (Table I) indicates the presence of a homogeneous population of xyloglucan.

Figure 1.

Adsorption isotherms (μg/mg) of xyloglucan from Tamarindus indica (□), arabinan-rich sugar beet pectin (♦), galactan-rich potato pectin (▾), citrus pectin (C-30; ○), and citrus pectin (C-70; ▵) to Avicel microcrystalline cellulose.

The same experiment was performed with commercial citrus pectins of DM 30 (C-30) and 70 (C-70), composed mainly of GalUA (820 mg/g and 770 mg/g for C-30 and C-70, respectively; Table I). These pectins do not bind to cellulose (Fig. 1), in agreement with results reported by Chanliaud and Gidley (1999), who used pectins similarly rich in GalUA. All these pectins were of commercial origin and contained very limited amounts of neutral sugar side chains as a consequence of their extraction in harsh acidic conditions. However, cell walls contain pectins rich in neutral sugar side chains (Voragen et al., 1995). We have therefore extracted arabinan-rich and galactan-rich pectins in conditions that allow preservation of their side chains. Pectins containing GalUA (589 mg/g) and Ara (201 mg/g) were obtained from sugar beet CWM (sugar beet pectin), while pectins containing GalUA (293 mg/g) and Gal (463 mg/g) were isolated from potato CWM (potato pectin; Table I). The fractionation of these pectins by anion-exchange chromatography (data not shown) revealed that they were totally bound to the column, indicating that the arabinan and galactan chains comprise the hairy regions of both pectins and are not present as free macromolecules. In contrast to the commercial pectins tested, these samples were found to bind to cellulose. The binding of the arabinan-rich pectin was slightly higher than that of the galactan-rich pectin (Fig. 1), but, in both cases, the observed binding was lower than that of xyloglucan. The amount of pectins adsorbed to cellulose increased with increasing concentration and a plateau value of approximately 4 μg and approximately 2.5 μg/mg of cellulose for the arabinan- and galactan-rich pectins, respectively, was reached above 300 μg/mL of pectin. The absence of binding of citrus pectins poor in neutral sugar side chains and the binding of arabinan- and galactan-rich pectins suggest that pectins may bind to cellulose through their side chains and not through their main backbone. The arabinan-rich pectin appeared highly heterogeneous with respect to molar mass. Several pectic populations exhibiting different binding capacities to cellulose are likely to be present. The weight-average molar mass of galactan-rich pectin was difficult to assess due to very high heterogeneity of this pectin.

Xyloglucan and the arabinan-rich pectin, the two polysaccharides that exhibited the higher adsorption to Avicel cellulose, were tested for their ability to bind to PCW cellulose (Fig. 2). A very important binding capacity of xyloglucan to PCW cellulose was observed. A maximum binding capacity of xyloglucan to PCW cellulose of approximately 33 μg/mg of cellulose was reached above 750 μg/mL of xyloglucan, showing that PCW cellulose can adsorb about 3 times more xyloglucan than Avicel cellulose. Moreover, the steep part of the isotherm, suggesting the presence of specific interactions between xyloglucan and cellulose, was shown for a wider range of concentrations (up to 50 μg/mL and up to 10 μg/mL for PCW cellulose and Avicel cellulose, respectively; Figs. 1 and 2). A higher binding capacity to PCW cellulose was also shown for the arabinan-rich pectin (Fig. 2). The amount of pectin adsorbed to cellulose increased with increasing concentration and a plateau value of approximately 8 μg/mg of cellulose was reached above 500 μg/mL of pectin. The PCW cellulose appeared able to adsorb about 2 times more arabinan-rich pectin than the Avicel cellulose. These findings emphasize the influence of cellulose origin in binding processes, in agreement with results reported by Hayashi et al. (1987) and Vincken et al. (1995). These authors suggested that the binding of xyloglucan to cellulose is a function of the cellulose surface available and could also depend on the index of cellulose crystallinity.

Figure 2.

Adsorption isotherms (μg/mg) of xyloglucan from Tamarindus indica (□) and arabinan-rich sugar beet pectin (♦) to PCW cellulose.

Commercial side chains of branched arabinans (Br-Arabinans) and debranched arabinans (Deb-Arabinans) isolated from sugar beet, and commercial galactan side chains isolated from potato, were used in binding experiments with Avicel cellulose in order to confirm the assumption that binding to cellulose could possibly take place through the pectic side chains. Arabinans were composed mainly of Ara (681 mg/g and 518 mg/g for Br-Arabinan and Deb-Arabinan, respectively) with minor amounts of Gal, Rha, and GalUA (Table I). Galactan contained essentially Gal (608 mg/g), with residual amounts of Ara, Rha, and GalUA (Table I). The binding capacity of these polysaccharides to cellulose varied with respect to their nature and structure, as shown in Figure 3. Galactan and Br-Arabinan displayed binding capacities to cellulose comparable to that of arabinan- and galactan-rich pectins. The amount of each polysaccharide adsorbed to cellulose increased with increasing concentrations and the saturation effect of the cellulose surface was observed between 750 μg/mL and 1,000 μg/mL of polysaccharide, where the maximum of material adsorbed was approximately 5 μg/mg of cellulose. The most significant binding was measured for Deb-Arabinan (Fig. 3). For concentrations above 750 μg/mL, the amount of bound Deb-Arabinan reached a plateau value of approximately 11 μg/mg of cellulose. The maximum binding capacity of Deb-Arabinan to cellulose was close to that of xyloglucan. Significant differences were observed, however, at the beginning of the isotherm where the initial amount of Deb-Arabinan adsorbed to cellulose was lower than that of xyloglucan. Mishima et al. (1998) demonstrated that polymers presenting an affinity for cellulose were β-d-(1→4)-linked glucans and suggested interaction mechanisms based on surface complementarity. Deb-Arabinans were recently shown to adopt a 2-fold helix conformation with a pitch of 0.868 nm (Janaswamy and Chandrasekaran, 2005). This conformation, although compatible with potential binding to cellulose (1.036-nm pitch; Sugiyama et al., 1991), probably does not allow surface complementarities as good as those presumed between xyloglucan and cellulose.

Figure 3.

Adsorption isotherms (μg/mg) of isolated pectic side chains: Deb-Arabinan (▴), Br-Arabinan (•), and galactan (▪) to Avicel microcrystalline cellulose.

Binding Strength of Xyloglucan, Pectins, and Pectic Side Chains to Avicel Microcrystalline Cellulose

The reversibility of the composites obtained by the in vitro adsorption of xyloglucan-, arabinan-, and galactan-rich pectins, and isolated neutral sugar side chains to Avicel cellulose, was appraised. The incubation of the xyloglucan-Avicel cellulose composite with sodium acetate buffer revealed that the adsorption of xyloglucan to cellulose is irreversible in such conditions because no xyloglucan could be removed. The adsorption of pectins to Avicel cellulose was slightly weaker because approximately 5% of arabinan-rich and approximately 8% of galactan-rich pectins bound to Avicel cellulose were pulled from their surfaces after incubation with sodium acetate buffer. The binding of the Br-Arabinans and Deb-Arabinans and the galactan side chains to cellulose appeared quite strong because only approximately 2% of bound material was removed under the conditions used. The adsorption of xyloglucan to cellulose, however, appears stronger than that of pectins or pectic domains to cellulose. The better surface complementarity between xyloglucan and cellulose suggested above could explain the observed irreversibility of the xyloglucan-cellulose composite.

Kinetics of Binding of Xyloglucan and Deb-Arabinan to Avicel Microcrystalline Cellulose

The kinetics of binding to cellulose was studied for a low concentration (50 μg/mL) of xyloglucan and Deb-Arabinan. Two kinetic behaviors were observed: (1) Xyloglucan binding increased sharply for the first 15 min to reach a value of approximately 2.25 μg of xyloglucan bound per milligram of cellulose (Fig. 4); and (2) Deb-Arabinan binding occurred almost instantaneously and a binding capacity of approximately 0.7 μg of Deb-Arabinan per milligram of cellulose was reached within 5 min (Fig. 4). These differences in the initial binding rates of xyloglucan and Deb-Arabinan to cellulose are probably due to the huge difference with respect to weight-average molar mass for these two polysaccharides (Table I). Indeed, the best binding conformation is reached more slowly by a large macromolecule like xyloglucan (763 kD) than by a small one like Deb-Arabinan (26 kD). Once this conformation reached above 5 min and 15 min for Deb-Arabinan and xyloglucan, respectively, binding increases smoothly for both polymers and reaches an apparent plateau value at 4 to 6 h (Fig. 4) in agreement with the findings of Hayashi et al. (1987).

Figure 4.

Adsorption kinetics of xyloglucan (□) and Deb-Arabinan (▴) at 50 μg/mL to microcrystalline Avicel cellulose.

Isolation of Natural Composites

Interactions between pectins and cellulose were also studied in sugar beet and potato cell walls. The CWMs were treated in various alkaline conditions and the chemical composition of the residues was determined in order to look for the presence of pectic populations differing in their binding capacity to cellulose.

Ara (225 mg/g), GalUA (225 mg/g), and Glc (237 mg/g) are the three main sugars detected in the untreated sugar beet CWM (Table II). This composition suggests that the major polysaccharides in these cell walls are arabinan-rich pectins and cellulose. The total amount of noncellulosic Glc, Xyl, and Fuc (34 mg/g) indicates a very low xyloglucan content, as previously reported by Renard and Jarvis (1999). The treatment of sugar beet CWM by mild alkali (0.05 n NaOH, 4°C) led to the extraction of the arabinan-rich pectin, which was the one used in the binding assays. However, about 86% of the cell wall remained insoluble after this treatment (Table II). This residue still contained high amounts of Ara (227 mg/g, which corresponds to 87% of the Ara initially present in CWM), GalUA (167 mg/g, which corresponds to 64% of the GalUA initially present in CWM), and cellulosic Glc (275 mg/g, which corresponds to 99% of the cellulosic Glc initially present in CWM). In order to peel off pectic and hemicellulosic polysaccharides from cellulose, NaOH treatments of varying severity (0.05 n, 0.275 n, 0.5 n; 40°C, 65°C, 90°C) were applied to sugar beet CWM (Table II). Treatments at 40°C led only to a limited sugar solubilization, close to that observed at 4°C (Table II). A more important sugar solubilization was observed at 65°C for 0.05, 0.275, and 0.5 n NaOH. The remaining cellulose-enriched residues were still rich in Ara (from 38% to 60% of the Ara initially present in CWM). The treatment solubilized more GalUA as only 39% to 49% of the GalUA initially present in the CWM remained in the residues. After treatment in severe conditions (0.05 n, 0.275 n, or 0.5 n NaOH at 90°C), cellulose-enriched residues still containing some pectic and hemicellulosic material were recovered. It is noteworthy that 55% to 88% of the noncellulosic Glc and 13% to 49% of the Xyl, but only 2% to 8% of the Ara and 1% to 7% of the GalUA initially present in CWM, were still present in those residues, revealing that the potential pectin-cellulose interaction is certainly weaker than the xyloglucan-cellulose interaction.

Table II.

Sugar composition (mg/g) of sugar beet CWM and the sugar beet residues obtained after alkaline extractions with NaOH

R-SB, Sugar beet residues.

	Yield	GalUA	Rha	Fuc	Ara	Xyl	Man	Gal	Glc
									Cellulosic	Noncellulosic
	%
Sugar beet CWM	100	225	16	2	225	19	14	54	237	13
R-SB (0.05 n, 4°C)	86	167	14	2	227	22	16	54	275	13
R-SB (0.05 n, 40°C)	80	207	14	2	220	21	20	64	253	13
R-SB (0.275 n, 40°C)	79	229	17	2	224	20	18	63	248	19
R-SB (0.5 n, 40°C)	75	230	15	2	209	17	19	56	238	19
R-SB (0.05 n, 65°C)	64	171	17	2	212	21	17	59	280	22
R-SB (0.275 n, 65°C)	52	201	19	2	225	25	20	65	272	23
R-SB (0.5 n, 65°C)	42	193	14	2	205	23	15	57	347	24
R-SB (0.05 n, 90°C)	28	58	5	2	66	33	22	24	523	41
R-SB (0.275 n, 90°C)	14	22	0	2	24	35	18	0	675	65
R-SB (0.5 n, 90°C)	11	18	0	2	31	23	23	0	693	65

The same treatments were applied to potato CWM and comparable results were obtained. The untreated potato CWM was mainly composed of galactan-rich pectins and cellulose (Table III). The total amount of noncellulosic Glc, Xyl, and Fuc (62 mg/g) indicates a low xyloglucan content. About 11% of the potato CWM was extracted by weak alkali conditions (0.05 n NaOH at 4°C) to give a galactan-rich pectin, which was shown to form weak associations with cellulose under in vitro conditions (compare with binding assays). The potato CWM, however, was still rich in pectins and cellulose with some hemicelluloses (Table III). After alkaline treatments of increasing severity, remaining CWMs were progressively enriched in cellulosic Glc, pectic and hemicellulosic polymers being more and more washed off. High amounts of hemicelluloses (56% to 70% of the noncellulosic Glc and 45% to 59% of the Xyl initially present in CWM) and some remaining pectic substances (2% to 8% of the Gal and 2% to 6% of the GalUA initially present in CWM) were still present in those residues. As previously suggested for sugar beet CWM, the potential pectin-cellulose interaction in potato CWM is most likely weaker than the xyloglucan-cellulose interaction.

Table III.

Sugar composition (mg/g) of potato CWM and the potato residues obtained after alkaline extractions with NaOH

R-P, Potato residues.

	Yield	GalUA	Rha	Fuc	Ara	Xyl	Man	Gal	Glc
									Cellulosic	Noncellulosic
	%
Potato CWM	100	162	13	2	52	26	10	234	281	34
R-P (0.05 n, 4°C)	89	146	11	2	50	29	17	205	315	34
R-P (0.05 n, 40°C)	79	156	13	2	55	35	19	229	355	36
R-P (0.275 n, 40°C)	74	143	10	2	51	30	14	206	360	36
R-P (0.5 n, 40°C)	70	159	10	2	50	27	14	222	372	36
R-P (0.05 n, 65°C)	72	140	10	2	49	33	13	181	368	39
R-P (0.275 n, 65°C)	57	127	10	2	43	38	17	161	472	39
R-P (0.5 n, 65°C)	48	130	10	2	34	40	18	119	539	47
R-P (0.05 n, 90°C)	34	28	0	1	24	45	18	53	634	70
R-P (0.275 n, 90°C)	26	15	0	0	15	45	12	17	746	73
R-P (0.5 n, 90°C)	26	15	0	0	15	47	13	14	826	73

DISCUSSION

In this work, the binding ability of pectins or pectic domains of varying structures to cellulose was studied and compared to the well-known adsorption of xyloglucan to cellulose. It is shown that pectins can bind in vitro to cellulose microfibrils, probably through their neutral sugar side chains. The arabinan- and galactan-rich pectins bind to cellulose microfibrils, in contrast to commercial pectins, which are almost free of side chains. The extent of binding varies with respect to the nature and structure of the side chains, the adsorption level of Deb-Arabinan being comparable to that of xyloglucan and higher than that of Br-Arabinan and galactan. Our findings also emphasize that binding is modulated by the cellulose origin. PCW-derived cellulose appeared capable of binding much higher amounts of xyloglucan- and arabinan-rich pectin than Avicel microcrystalline cellulose. These differences in binding ability are most likely due to differences both in cellulose surface availability and crystallinity index (Hayashi et al., 1987; Vincken et al., 1995).

The weight ratios of xyloglucan adsorbed in vitro to cellulose (1.3% and 3.3% [w/w] for Avicel cellulose and PCW cellulose, respectively) are in good agreement with the results of Hayashi et al. (1987), who found that between 1% and 5% of pea (Pisum sativum) xyloglucan bound in vitro to cellulose of various origins, and of Vincken et al., (1995), who found that 0.84% of tamarind xyloglucan bound in vitro to Avicel cellulose. These reconstituted complexes, however, are not as tightly organized as in the cell walls. Indeed, it was demonstrated that mild alkaline conditions are sufficient to remove xyloglucan adsorbed in vitro to cellulose while only very severe alkaline conditions effectively solubilize xyloglucan from pea cell walls (Hayashi and Maclachlan, 1984). Moreover, when Acetobacter synthesizes cellulose in the presence of xyloglucan, the amount of xyloglucan bound to cellulose is very high (approximately 38% [w/w]; Whitney et al., 1995), probably because xyloglucan binds not only to the surface of microfibrils but also is entrapped within the microfibrils. Our results also show strong in vivo xyloglucan-cellulose interactions in sugar beet or potato CWM with more than 50% of the xyloglucan initially present that remained associated with cellulosic material after alkali treatments at 90°C. These results are in good agreement with the findings of Pauly et al. (1999) on etiolated pea stems. The interaction of xyloglucan to cellulose backbone is highly specific and believed to be mediated by hydrogen bonds. Xyloglucan backbone is likely to adopt a cellulose-like conformation and a change from twisted to flat ribbon conformation seems to be required for efficient bonding (Levy et al., 1991). It is noteworthy that, in vivo, only some xyloglucan domains interact strongly with cellulose surfaces, while others appear quite mobile as observed by solid-state NMR (Bootten et al., 2004). The latter could correspond to molecular domains that constitute the cross-links between cellulose microfibrils and the tails and loops that extend away from the microfibril surface (Pauly et al., 1999).

In contrast, there is very little information about the way in which pectic polysaccharides could interact with cellulose. Our results reveal that side chain-rich pectins and isolated arabinan and galactan side chains can bind to cellulose under in vitro conditions. It is likely that interactions between pectic side chains and cellulose are also mediated by hydrogen bonds, as suggested for xyloglucan. The branching of arabinan and galactan chains seems to be a limiting factor in the binding capacity, probably because of the steric hindrance of the substituants. Deb-Arabinan chains led to an increased binding capacity that could be due to a higher degree of alignment of arabinan backbone with cellulose microfibrils. The possibility of multilayer formation by self-association of Deb-Arabinan must also be taken into account, especially because steps were observed in the binding isotherm. As shown for xyloglucan, arabinans and galactans could be more tightly bound to cellulose in vivo. Alkali treatments applied to sugar beet and potato CWM revealed that several pectic populations could exist. Pectins that can be easily solubilized for smooth alkaline conditions could constitute a putative pectic population weakly associated with cellulose microfibrils. These pectins were indeed shown to interact only slightly with cellulose microfibrils under in vitro conditions. Another putative pectic population seems to be more tightly bound to cellulose microfibrils because it withstands extraction in more severe alkaline conditions. These findings are in good agreement with data from the literature. Indeed, Oechslin et al. (2003) found considerable amounts of Ara and Gal that could not be extracted by 1 and 4 n NaOH and remained associated with the cellulosic residue of apple CWM. An arabinan-cellulose composite (1:1, w/w) was recovered after alkaline treatment and chlorite bleaching of prickly pear (Pyrus communis) spines from O. ficus-indica (Vignon et al., 2004). Solid-state NMR relaxation experiments showed that around 15% of arabinan interacts strongly with cellulose and exhibits solid-like behavior, whereas 25% undergoes hindered motions and 60% is in a liquid-like state. In sugar beet cell walls, Renard and Jarvis (1999) ruled out a close association of galacturonan or arabinan with cellulose microfibrils. However, a clear signal at 108 ppm on ¹³C CP-MAS NMR spectra for short contact times (1 and 3 ms) was observed. The presence of this specific signal was recently claimed to prove that part of the arabinan is in strong interaction with the cellulose (Vignon et al., 2004). By analogy with xyloglucan, it is likely that only limited or specific domains of arabinan and galactan chains are bound to cellulose microfibrils, while others are highly mobile. Indeed, arabinan molecular motions are enhanced in the presence of water and Vignon et al. (2004) proposed that cellulose microfibrils interact strongly with a swollen, but stiff, arabinan gel. Sugar beet and potato cell walls contain very little xyloglucan (around one-fifth to one-sixth of the mass of the cellulose), which is clearly insufficient to provide a complete coating of the cellulose microfibril surface (Renard and Jarvis, 1999). On the contrary, arabinan and galactan chains are present in large amounts, similar to that of cellulose. Although arabinan and galactan chains most probably do not bind to cellulose as strongly as xyloglucan, it is very likely that they can fulfill a coating function and provide a continuum between the cellulose and pectic network. This hypothesis, taking into account the galacturonan-side chain-xyloglucan ratio, is illustrated in Figure 5.

Figure 5.

Schematic model of sugar beet or potato cell walls showing the hypothetical connections between pectic molecules and cellulose microfibrils. Shaded solid bands, Cellulose microfibril; jagged line, RG; dotted line, HG; thin solid line, neutral sugar side chain; thick solid line, xyloglucan. 1, Side chain binding one single pectic molecule onto the surface of one cellulose microfibril. 2, Calcium-mediated cross-links between HG regions. 3, Side chains belonging to one single pectic molecule cross-linking two cellulose microfibrils.

Pectic populations, and particularly their arabinan or galactan side chains, that are highly mobile are likely to contribute to the cell wall porosity as plasticizers and water-binding agents. Jones et al. (2003) investigated the effects of modifying specific guard cell wall polymers on the ability of stomata to open or close. The enzymatic digestion of cell walls revealed that only pectic-degrading enzymes had profound effects on stomatal function, especially arabinanase, after which the stomatal pore failed to open. Jones et al. (2003) suggested that arabinans could maintain flexibility in guard cell walls by providing steric hindrance.

Pectic side chains are also thought to be involved in cell and tissue development. Distinct locations in relation to cell development of arabinan and galactan have been observed at the carrot root apex and in suspension-cultured cells (Willats et al., 1999). Arabinans occurred in the wall of dividing cells, whereas galactans were present in cell walls following induction, before any visible elongation. The disassembly of PCWs was observed during ripening. The modification of structure was correlated with solubilization and depolymerization of the constituent polysaccharides, especially the pectic side chains (Gross and Sams, 1984). In melon (Cucumis melo) fruit, the changes in pectic solubility coincided with a loss of Gal from tightly bound pectins (Rose et al., 1998).

Very little information is available on the biosynthesis of pectins and their side chains. Immunochemistry, using antibodies directed against cell wall polysaccharide epitopes (Moore et al., 1991), showed that HG and RG I-like epitopes are present in both the cis- and medial Golgi. Extensive branching of pectin is supposed to occur in the trans-Golgi cisternae. Assembly of pectins, which remain soluble during synthesis, may occur during the transport to the plasma membrane, where pectins are inserted into the wall. The interaction between xyloglucan and cellulose was claimed to occur when the xyloglucan molecules, assembled in the Golgi apparatus, are secreted into the cell wall in a soluble form and integrated with the newly synthesized cellulose microfibrils (Moore et al., 1991). By analogy with the xyloglucan hypothesis, binding of pectins via their neutral side chains to cellulose may also take place during this process.

The observed binding of pectins to cellulose microfibrils may be of considerable significance in the modeling of PCWs of plants and in the process of cell wall assembly. The pectic network could interact with the cellulose-xyloglucan network through pectic neutral sugar side chains. The role of the fine structure of arabinan and galactan will be further studied in order to elucidate the properties of these pectin-cellulose complexes.

MATERIALS AND METHODS

CWM, Cellulose, and Noncellulosic Polysaccharides

Sugar beet (Beta vulgaris) CWM was prepared from fresh sugar beet pulp (approximately 2 kg; sugar factory in Cagny, France) by boiling in 5 L of 75% (w/v) ethanol for 20 min. This operation was carried out three times. The slurry was filtered through nylon cloth and the insoluble material was left for 12 h with 75% (w/v) ethanol (5 L) and filtered again. This step was repeated until the filtrate gave a negative reaction to the phenol sulfuric acid test (Dubois et al., 1956). The residue was then dried by solvent exchange (ethanol, acetone) and left overnight at 40°C. Potato (Solanum tuberosum) CWM was prepared from potato pulp (Roquette, France). A suspension of 100 g of pulp in 2 L of water at 40°C was adjusted to pH 7 with 1 n NaOH. The temperature was raised to 100°C and 120 L Termamyl were added. The suspension was maintained for 1 h at 100°C and filtered through a G3 sintered glass. The residue was washed with boiling water, dried by solvent exchange (ethanol, acetone), and left overnight at 40°C.

The PCW cellulose was prepared from sugar beet CWM as described by Heux et al. (1999). A suspension of 50 g of sugar beet CWM in 1.5 L of 0.1 n HCl was maintained for 30 min at 85°C and filtered through a nylon cloth. The extraction was carried out three times. The residue was washed abundantly with distilled water and subjected to an alkaline extraction (0.5 n NaOH, 1.5 L for 30 min at 80°C). This operation was performed three times. The final residue was washed abundantly with distilled water, dried by solvent exchange (ethanol, acetone), and left overnight at 40°C. The PCW cellulose, at 0.6% in water, was treated for 15 min at 25°C in a Waring blender (22,400 rpm). The sample was then homogenized by 10 passes through an APV Gaulin homogenizer operating from 500 to 1,800 bars. The obtained cellulose suspension was freeze dried.

Avicel microcrystalline cellulose PH-101 was purchased from Fluka.

Xyloglucan was extracted from a powder of tamarind seeds (Dainippon Pharmaceutical). The powder was boiled with citric acid (2 g/L) for 40 min, centrifuged, and the clean supernatant was concentrated under vacuum at 40°C. Xyloglucan was obtained by precipitation with 1 volume of 95% (w/v) ethanol. After 1 night at 4°C, the precipitate was treated by solvent exchange (ethanol, acetone) and dried overnight at 40°C.

Commercial C-30 and C-70 citrus pectins were from Danisco. The arabinan- and galactan-rich pectins were isolated from sugar beet and potato CWM, respectively. The sugar beet (5 g) and potato (4 g) CWMs were stirred with 150 mL of 0.05 n NaOH at 4°C for 30 min. The extractions were carried out three times. After filtration through G3 sintered glasses, supernatants were pooled, adjusted to pH 5 with 1 n HCl, concentrated under vacuum at 40°C, and precipitated with 4 volumes of 95% (w/v) ethanol. After 1 night at 4°C, precipitates were carefully rinsed with 70% (w/v) ethanol and solubilized in water. Solutions were concentrated under vacuum and freeze dried. Br-Arabinan and Deb-Arabinans (from sugar beet) and galactan (from potato) were purchased from Megazyme.

Alkali Treatment of CWM

The sugar beet (5 g) and potato (4 g) CWMs were stirred with 150 mL of 0.05 n, 0.275 n, or 0.5 n NaOH at 40°C, 65°C, or 90°C for 1 h. The extraction was carried out three times. The final residues were recovered after filtration through G3 sintered glass, abundantly washed with distilled water, dried by solvent exchange (ethanol, acetone), and left overnight at 40°C.

Binding Assays

Binding assays were performed in 20 mm sodium acetate buffer (pH 5.8) at 40°C. Solutions of the different polysaccharides were prepared at 1 mg/mL, eventually heated to give perfectly cleared solutions, and diluted to give a range of concentration from approximately 2.5 μg/mL to approximately 1 mg/mL. After centrifugation (15 min at 4,000g), supernatants were recovered and aliquots (1.5 mL) were added to cellulose samples (approximately 7.5 mg). Polysaccharide solutions and polysaccharide-cellulose blends were incubated for 6 h at 40°C (head-over-tail mixing), then centrifuged for 10 min at 9,000g, and supernatants (1,250 μL) were removed for analysis. GalUA and/or neutral sugar content were quantified in the polysaccharide solutions and in the polysaccharide-cellulose blend supernatants using the automated colorimetric m-hydroxybiphenyl and/or orcinol methods, respectively (Thibault, 1979; Tollier and Robin, 1979). The amount of adsorbed matter was calculated from the difference in sugar content measured for polysaccharide solutions and polysaccharide-cellulose blends, taking into account the amount of sugars released by a cellulose blank (approximately 7.5 mg of cellulose in 1.5 mL sodium acetate buffer). Binding assays were performed in triplicate. The average and the corresponding error measurements were then calculated.

Desorption Process

Polysaccharide-cellulose blends obtained after incubation, centrifugation, and removal of 1,250 μL of supernatant were suspended in 1,250 μL of sodium acetate buffer (pH 5.8). After incubation (24 h at 40°C, head-over-tail mixing), samples were centrifuged for 10 min at 9,000g and supernatants were analyzed for their sugar content as described above. The amount of desorbed polymer was calculated, taking into account the quantity of soluble polymer that was still present in the pellet.

Adsorption Kinetics

Kinetic experiments were performed for xyloglucan and Deb-Arabinan for a concentration of 50 μg/mL. Polysaccharide-cellulose blends were prepared as described above (binding assays) and incubated for 5 to 360 min at 40°C with head-over-tail mixing. Samples were either centrifuged for 10 min at 9,000g or, for short incubation times (5, 10, and 15 min), immediately filtered through a Maxi clean IC-4 (Altech) resin. The amount of polymer adsorbed was calculated colorimetrically as described above.

Analysis

Uronic acid (as GalUA) and total neutral sugar (as Ara or Gal) content were determined colorimetrically by the automated m-hydroxybiphenyl and orcinol methods, respectively (Thibault, 1979; Tollier and Robin, 1979).

The individual neutral sugars were analyzed as their alditol acetate derivatives by gas chromatography after hydrolysis by 4 n H₂SO₄ at 100°C for 2 h for arabinans and galactans, 3 h for xyloglucan, and 6 h for pectic samples. Longer hydrolysis times were applied to pectic samples in order to provide a good estimation of Rha content. Avicel microcrystalline cellulose, sugar beet CWM, potato CWM, and potato and sugar beet residues were hydrolyzed by 4 n H₂SO₄ at 100°C for 2 h. Cellulosic Glc was measured as the difference in Glc content with and without prehydrolysis by 72% (w/v) H₂SO₄ for 90 min at 25°C. Inositol was used as an internal standard.

Methanol and acetic acid were released by alkaline de-esterification in the presence of CuSO₄ and quantified by HPLC on a C18 column as previously described (Levigne et al., 2002). Isopropanol was used as an internal standard. DM and DA were calculated as the molar ratio of methanol and acetic acid to GalUA, respectively.

Anion-exchange chromatography was performed on a DEAE Sepharose CL-6B column (30×2.6 cm; Pharmacia). Samples were applied onto the column equilibrated with 50 mm sodium succinate buffer (pH 4.5) at a flow rate of 1.5 mL/min. The column was first eluted with 370 mL of 50 mm sodium succinate buffer (pH 4.5). NaCl gradient (0–0.6 m NaCl) was then applied. Fractions of 12 mL were collected and analyzed colorimetrically for their content of total neutral sugars and GalUA.

The molar mass distribution and polydispersity index were determined by viscometric or light-scattering detection after high-performance size exclusion chromatography. The system used was composed of one Shodex SB-G precolumn followed by two Shodex OH-pak SB HQ 805 and 804 columns in series with a multiangle laser light-scattering detector (MALLS, mini Dawn; Wyatt), a differential refractometer (ERC 7517 A), and a differential viscometer (T-50A; Viscotek). Elution was performed with 50 mm NaNO₃ containing 0.02% NaN₃ at a flow rate of 0.7 mL/min at room temperature. The system was calibrated using pullulan standards. The molar mass determined by viscometric detection was calculated through universal calibration.