JXB Advance Access originally published online on March 23, 2007
Journal of Experimental Botany 2007 58(7):1795-1802; doi:10.1093/jxb/erm037
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged
UR 1268 Biopolymères, Interactions, Assemblages, INRA, F-44300 Nantes, France
* To whom correspondence should be addressed. E-mail: ralet{at}nantes.inra.fr
Received 6 December 2006; Revised 8 February 2007 Accepted 8 February 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called ‘natural’ composites), rich in pectic neutral sugar side chains and cellulose, as well as ‘artificial’ composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.
Key words:
Arabinan, galactan, enzymes, 
-L-arabinofuranosidase, endo-arabinanase, ß-galactosidase, endo-galactanase
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The plant cell wall constitutes a highly complex and dynamic entity of extreme importance in plant growth and development. The growing cell wall can be considered as a fibre composite (Carpita and Gibeaut, 1993), where cellulose microfibrils are embedded in a matrix of complex polysaccharides (hemicelluloses and pectins) and proteins.
Cellulose is synthesized as a linear chain of ß-(1
4)-linked Glcp residues. Parallel glucan chains associate by hydrogen bonds to form microfibrils, highly crystalline structures that are resistant to enzymatic attack. Xyloglucan, an abundant hemicellulose in primary cell walls, possesses a cellulose-like backbone branched at O-6 by Xylp residues. Some Xylp residues can be substituted at O-2 by Galp residues (Fry, 1989), which can be further branched at O-2 by Fucp residues. Pectins, a highly complex and heterogeneous group of polysaccharides, are composed of distinctive domains, which are believed to be covalently linked one to another. The pectin backbone is composed of two main structural domains: homogalacturonan (HG) and type I rhamnogalacturonan (RG I). HG is a homopolymer composed of -(14)-linked GalAp units that are often highly methyl-esterified at O-6 and sometimes acetyl-esterified at O-2 or O-3 (Ralet et al., 2001). RG I contains a backbone of the repeating disaccharide unit: (12)--L-Rhap-(14)--D-GalAp (Renard et al., 1995), predominantly substituted at O-4 of Rhap residues by neutral sugar side chains. In the primary cell wall, pectins are characterized by a high quantity of neutral sugar side chains, among which arabinan and galactan are the most abundant (Schols and Voragen, 1994). Arabinan side chains are composed of -(15)-linked Araf residues, which can be further branched by -L-Araf units at O-2 and/or O-3, whereas galactan side chains are constituted of (14)-linked ß-D-Galp units. A type II rhamnogalacturonan (RG II), a complex domain composed of GalA, Rha, Gal, and some unusual sugars, may also constitute part of the pectic molecule (Ishii and Matsunaga, 2001). RG II, although present in minor amounts, is believed to play a significant role in the cell wall architecture (O'Neill et al., 2004).
When the matrix polysaccharides are secreted into the wall, they are associated with newly synthesized cellulose microfibrils to form a strong and extensible network. Network formation implies the setting up of interactions between the wall polysaccharides. It was hypothesized that cellulose microfibrils can be not only coated but also tethered through non-covalent interactions with matrix polysaccharides. Xyloglucans are known to be involved in the primary cell wall assembly (Hayashi et al., 1987) and it has recently been demonstrated that pectins rich in neutral sugar side chains are also able to bind to cellulose microfibrils under in vitro conditions (Zykwinska et al., 2005). The hypothesis of non-covalent interactions between pectins and cellulose, mediated by the arabinan and galactan side chains, was therefore raised. Indeed, the use of isolated structural domains of pectins allowed the demonstration that only the neutral sugar side chains are responsible for the interaction with cellulose (Zykwinska et al., 2007). Moreover, the possibility of interactions between neutral sugar side chains rich in pectins and cellulose was investigated in muro (Zykwinska et al., 2006). Alkaline extractions of increasing severity performed on sugar beet and potato cell walls revealed the presence of pectic populations that can be more or less associated with cellulose microfibrils, which confirmed this study's hypotheses based on in vitro approaches (Zykwinska et al., 2005).
In the present work, the organization of pectic arabinan and galactan domains in association with cellulose microfibrils was studied. Arabinan and galactan chains may be present as loops, tails, cross-links, and/or ‘trains’ (Fig. 1). Enzymes and alkali were used in order to quantify these domains. -L-Arabinofuranosidase and ß-galactosidase, which degrade, respectively, the arabinan and galactan side chains from their non-reducing ends, were applied in order to estimate the amount of non-reducing tails that extend away from the microfibril surface. The presence of cross-links, loops, and some tails not yet degraded was determined by the use of the endo-enzymes: endo-arabinanase and endo-galactanase, which hydrolyse the -(15)- and ß-(14)-linkages of arabinan and galactan side chains, respectively. In addition, fragments of polymers not liberated by the enzymes are supposed to be aligned with cellulose microfibrils. These fragments, called ‘trains’, can be recovered after extraction by hot alkali. The above enzymatic and alkali method was applied to ‘natural’ composites that were obtained after alkali extraction of sugar beet and potato cell walls (Zykwinska et al., 2006). The alkali conditions were used in order to preserve RG I regions including neutral sugar side chains and to induce an important degradation of HG regions. ‘Artificial’ composites, created by in vitro assembly of isolated debranched arabinan and galactan side chains with cellulose microfibrils, were used as simplified models of cell walls that may facilitate the study of potential pectin/cellulose interactions.
|
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Material
Substrates
Sugar beet cell wall material (SB-CWM) and potato cell wall material (P-CWM) were prepared, respectively, from fresh sugar beet pulp (sugar factory in Cagny, France) and potato pulp (Roquette, France) by ethanol washings (Zykwinska et al., 2005).
Cellulose was prepared from SB-CWM, as described by Heux et al. (1999) and Zykwinska et al. (2005). Briefly, SB-CWM was sequentially treated with hot dilute acid (0.1 M HCl, 85 °C, 3x30 min) and hot dilute alkali (0.5 M NaOH, 80 °C, 3x30 min) in order to solubilize pectins and hemicelluloses. The primary cell wall (PCW) cellulose thereby obtained was suspended in distilled water, mixed in a Warring Blender and homogenized by 10 passes through an APV Gaulin homogenizer operating at 1000 bars.
Debranched arabinan (from sugar beet) and galactan (from potato) were purchased from Megazyme (Ireland).
‘Natural’ composites
SB-CWM (5 g) and P-CWM (4 g) were stirred with 150 ml of 0.275 M NaOH at 65 °C for 1 h. The extraction was performed three times. The sugar beet (SB) and potato (P) ‘natural’ composites 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.
‘Artificial’ composites
The ‘artificial’ composites were obtained as described elsewhere (Zykwinska et al., 2005). Briefly, solutions of debranched arabinan and galactan side chains were prepared at 1 mg ml–1 in 20 mM Na-acetate buffer (pH 5.8), and heated to give a perfectly clear solution eventually. After centrifugation for 15 min at 4000 g, aliquots (1.5 ml) were mixed with the cellulose (
5 mg). Polysaccharide solutions and polysaccharide/cellulose blends were incubated overnight at 40 °C (head-over-tail mixing), centrifuged for 15 min at 9000 g, and supernatants (1250 µl) were removed for analysis. The ‘artificial’ composites were recovered and directly treated with enzymes.
Enzymes
Endo-arabinanase (EC 3.2.1.99
[EC]
; 8.4 U ml–1, 1 U being the amount of enzyme that releases 1 µmol of reducing ends min–1) and endo-galactanase (EC 3.2.1.89
[EC]
; 0.5 U ml–1) were provided by Novozyme (Copenhagen, Denmark). They were purified from Aspergillus niger as described previously (Bonnin et al., 2002). -L-Arabinofuranosidase (250 U ml–1; Megazyme, Ireland) was from Aspergillus niger, whereas ß-galactosidase (EC 3.2.1.23
[EC]
; 4.4 U ml–1) provided by Sigma (Germany) was from Aspergillus oryzae.
Degradation procedures
The ‘natural’ and ‘artificial’ composites were sequentially degraded by enzymes. It was verified that, in the conditions used, the model substrates (arabinans, galactans) were completely degraded by the enzymes used in the present study.
‘Natural’ composites
The general scheme of degradation experiments performed on sugar beet (SB) and potato (P) ‘natural’ composites is presented in Fig. 2A and B, respectively. SB-composite (500 mg) was suspended in 40 ml of 50 mM Na-acetate buffer (pH 4.5) and -L-arabinofuranosidase (approximately 2.5 U) was added. After incubation for 24 h at 40 °C (head-over-tail mixing), the suspension was filtered through G4 sintered glass and washed three times with 20 ml of 50 mM Na-acetate buffer (pH 4.5). Supernatants were pooled for further analysis. The pellet was suspended in 25 ml of Na-acetate buffer (pH 4.5) and endo-arabinanase (approximately 2 U) was added. The suspension was incubated for 24 h at 40 °C (head-over-tail mixing) and filtered through G4 sintered glass. The composite was washed three times with water and the supernatants were pooled for analysis. The composite was then dried by solvent exchange (ethanol, acetone) and left overnight at 40 °C. One part of the dried composite was analysed for sugar content (SB-enz-composite), whereas the other part was suspended in 1 M NaOH for 1 h at 90 °C. The extraction was carried out twice. The final SB-OH-composite was filtered through G4 sintered glass, washed five times with water, dried by solvent exchange (ethanol, acetone), and left overnight at 40 °C.
|
P-composite (
500 mg) was sequentially treated with three enzymes: -L-arabinofuranosidase (approximately 2.5 U), ß-galactosidase (approximately 2 U), and endo-galactanase (approximately 1 U), as described above. The final P-OH-composite was obtained after two alkaline extractions with 1 M NaOH for 1 h at 90 °C. In parallel, controls were obtained as described above, without adding the enzymes.
Enzymatic and NaOH degradations were performed twice. The average and the corresponding error of measurement were then calculated.
‘Artificial’ composites
The general scheme of degradation experiments applied to debranched arabinan/cellulose and galactan/cellulose ‘artificial’ composites is presented in Fig. 3A and B. A 1250 µl aliquot of Na-acetate buffer (pH 5.8) containing -L-arabinofuranosidase (0.25 U) and ß-galactosidase (0.2 U) was added, respectively, to debranched arabinan/cellulose and galactan/cellulose blends. After incubation for 2 h at 40 °C (head-over-tail mixing), blends were centrifuged for 15 min at 9 000 g and supernatants (1250 µl) were removed for analysis. A 1250 µl aliquot of Na-acetate buffer (pH 5.8) containing endo-arabinanase (0.8 U) and endo-galactanase (0.5 U) was then added, respectively, to debranched arabinan/cellulose and galactan/cellulose blends. Incubation with enzyme was carried out for 2 h at 40 °C. Supernatants (1250 µl) left after centrifugation (15 min at 9000 g) were analysed. Polysaccharide/cellulose blends were finally suspended in 1 M NaOH for 30 min at 90 °C. The final residues were then centrifuged and washed three times with water, dried by solvent exchange, and left overnight at 40 °C. The same treatment as described above was applied to cellulose blanks (cellulose in Na-acetate buffer pH 5.8). Enzymatic and NaOH degradations were performed in triplicate. The average and the corresponding error of measurement were then calculated.
|
Analytical
The individual neutral sugars were analysed as their alditol acetate derivatives by gas–liquid chromatography (Blakeney et al., 1983). The composites before and after enzymatic and alkali treatments were firstly pre-hydrolysed by 72% (w/v) H2SO4 for 1.5 h at 25 °C. Samples were then hydrolysed by 2 M H2SO4 at 100 °C for 2 h. Soluble fractions removed after enzymatic and alkali extractions of ‘natural’ and ‘artificial’ composites were hydrolysed by 2 M TFA at 120 °C for 2 h. Inositol was added as an internal standard.
Uronic acid (as GalA) was determined colorimetrically by the automated m-hydroxybiphenyl method (Thibault, 1979).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Composition of sugar beet (SB) and potato (P) ‘natural’ composites
The ‘natural’ composites used in this study were recovered after alkaline extractions of sugar beet and potato cell walls (Zykwinska et al., 2006). Sugar composition of the SB-composite recovered revealed the presence of three main sugars, cellulosic Glc, Ara, and GalA (Table 1). Some Rha and Gal were also detected. This composition shows that the major polysaccharides present in this cell wall are arabinan-rich pectins and cellulose. The alkali treatment applied to P-CWM yielded a P-composite enriched in galactan-rich pectin and cellulose (Table 1). Some minor amounts of Rha and Ara were also detected. The low amount of hemicellulosic sugars (Xyl, Man, Fuc) in SB- and P-composites indicates minor amounts of xyloglucan and/or xylan.
|
Enzymatic and alkali degradation of ‘natural’ composites
SB-composite was sequentially degraded with
-L-arabinofuranosidase, endo-arabinanase, and NaOH (Fig. 2A). The amount of Ara residues (expressed as a percentage of the Ara initially present in the SB-composite) hydrolysed by enzymes and after NaOH treatment was quantified in each soluble fraction (Table 2). In parallel, the different residues obtained after enzymatic and NaOH treatments were analysed for their neutral sugar and galacturonic acid contents, and the values (expressed as mg g–1) were presented in Table 1. Incubation of SB-composite with -L-arabinofuranosidase released mainly Ara residues with some rare GalA, Rha, and Gal residues (data not shown). After taking into account the amount of Ara residues freed in the blank [SB-composite incubated with buffer only (6±1%)] it turned out that 46% of the Ara initially present in the SB-composite was released by the -L-arabinofuranosidase action (Table 2). In the next step, an endo-arabinanase, that hydrolyses the -(15)-linkage between two Ara residues, was added. This enzyme freed mostly Ara residues, and only some rare GalA, Rha, and Gal were quantified (data not shown). After subtraction of the blank (5±1%), it can be calculated that 35% of the Ara initially present in the SB-composite was released by endo-arabinanase (Table 2). The residue recovered after enzymatic treatment (SB-enz-composite) represented 700 mg g–1 of the SB-composite and contained only 25 mg of Ara/g, corresponding to 8% of the Ara initially present in the SB-composite (Table 1). Other pectic and hemicellulosic sugars were still present in high amounts. The Ara content quantified in the SB-enz-composite is in agreement with the one that was estimated in soluble fractions after enzymatic treatments (8% of the Ara not degraded by enzymes; Table 2). Finally, the SB-enz-composite recovered after enzymatic degradation was treated with hot alkali (1 M NaOH at 90 °C) that solubilized 7% of the Ara initially present in the SB-residue (Table 2) and most of the other pectic sugars (GalA, Rha, Gal; data not shown). The final SB-OH-composite was enriched in cellulosic Glc and almost devoid of arabinan-rich pectin, as only 8 mg of Ara g–1, corresponding to 1% of the Ara initially present in the SB-residue, were quantified (Table 1). The amount of hemicellulosic sugars remaining in the SB-OH-composite shows that the alkaline treatment did not extract all hemicelluloses present, as 31 mg of Xyl g–1 and 17 mg of Man g–1, corresponding, respectively, to 36% and 22% of the Xyl and the Man initially present in the SB-composite, were still quantified (Table 1).
|
A similar procedure to that described above was applied to the P-composite (Fig. 2B). The first step of the enzymatic sequential degradation was initiated with
-L-arabinofuranosidase that freed 9% of the Ara and 5% of the Gal initially present in the P-composite (Table 3). Although P-composite was first pre-treated with -L-arabinofuranosidase, ß-galactosidase was not very active, as only 2% of the Gal initially present in the P-composite was released, after subtraction of Gal residues released in the blank (3±1%; Table 3). Some minor GalA, Rha, and Ara were also freed during the treatment (data not shown). The endo-galactanase digestion of the remaining composite, hydrolysing the ß-(14)-linkage between two Gal residues, hydrolysed 29% of the Gal initially present in the P-composite, after taking into account the amount of Gal released in the blank (6±1%; Table 3). The P-enz-composite recovered after the three sequential enzymatic treatments represented 800 mg g–1 of the initial P-composite (Table 1). It was still rich in Gal, as about 106 mg of Gal g–1 was quantified, which corresponded to 55% of the Gal initially present in the P-composite. Other pectic and hemicellulosic sugars were also present (Table 1). The Gal content quantified in P-enz-composite was in agreement with the one quantified in soluble fractions (55% of the Gal not degraded by enzymes; Table 3). The P-enz-composite was finally treated with 1 M NaOH at 90 °C, which extracted 48% of the Gal initially present in the P-composite (Table 3). Indeed, galactan side chains that were not accessible for enzymes were almost completely extracted by hot alkali, and the remaining P-OH-composite contained only 3% of the Gal initially present in the P-composite. Small amounts of other pectic sugars, for example GalA and Ara, were also quantified (Table 1). Besides pectins, hemicelluloses present in limited amounts in the P-composite were not completely extracted by the alkaline treatment in severe conditions, as 34 mg of Xyl g–1 and 10 mg of Man g–1, corresponding, respectively, to 33% of the Xyl and 22% of the Man initially present in the P-composite, were still detected in P-OH-composite (Table 1).
|
Enzymatic and alkali degradation of ‘artificial’ composites
The structure of ‘artificial’ composites, obtained after in vitro association of debranched arabinan and galactan side chains with cellulose, was also studied using an enzymatic and alkali degradation approach (Fig. 3A). Incubation of debranched arabinan with cellulose resulted in the adsorption of 98±15 µg of debranched arabinan onto the cellulose surface. This ‘artificial’ composite was first degraded by
-L-arabinofuranosidase, which hydrolysed 19% of the Ara initially present in the debranched arabinan/cellulose composite (Table 2). The enzymatic digestion was then followed by endo-arabinanase, which liberated, in addition, 9% of the Ara initially present in this composite. Finally, the remaining debranched arabinan not degraded by both enzymes (72% of the Ara initially present) was completely extracted by 1 M NaOH at 90 °C (Table 2).
Galactan side chains incubated with cellulose led to the adsorption of 63±12 µg of galactan onto the cellulose surface. The galactan/cellulose ‘artificial’ composite obtained was first treated with ß-galactosidase (Fig. 3B) that hydrolysed 28% of the Gal initially present in this composite (Table 3). Secondly, an endo-galactanase was applied and freed 16% of the Gal initially present in the galactan/cellulose composite. The remaining galactan present in the ‘artificial’ composite after treatment with both enzymes (56% of the Gal initially present) was completely removed by NaOH treatment (Table 3).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In the present work, the macromolecular assembly of pectins within the primary cell walls was studied using an enzymatic and alkali degradation approach performed on sugar beet and potato cell wall ‘natural’ composites, and on ‘artificial’ ones created by the in vitro adsorption of isolated debranched arabinan and galactan side chains onto cellulose microfibrils. The ability of pectins to bind to cellulose microfibrils might be of great significance, especially in cell walls poor in xyloglucan (sugar beet, potato, celery, onion, carrot). In sugar beet and celery, the low xyloglucan content was claimed to be insufficient to provide either complete coating or tethering of cellulose microfibrils (Renard and Jarvis, 1999; Thimm et al., 2002). Therefore, pectins rich in arabinan and galactan side chains, that are particularly abundant in those cell walls, could be the sole polymers able to maintain the cell wall structure by interacting with cellulose microfibrils.
The sequential incubation of SB-composite with -L-arabinofuranosidase and endo-arabinanase released 92% of the total Ara present in this residue, whereas 45% of the total Gal present in the P-composite was released by the sequential -L-arabinofuranosidase, ß-galactosidase, and endo-galactanase treatment. The arabinan fraction that was not removed during degradation represented 8% of the total Ara in the SB-composite and may constitute the fraction closely associated with cellulose, representing fragments called ‘trains’. This arabinan fraction may correspond to the one of limited mobility quantified by solid-state NMR spectroscopy, which represented 20% of the total Ara present in the SB-composite (Zykwinska et al., 2006). The results obtained by these two different approaches are in agreement with the findings of Vignon et al. (2004), who reported that 15% of the arabinan quantified by NMR relaxation measurements interacted strongly with cellulose in an arabinan/cellulose ‘composite’ isolated from the spine fibres of the cactus Opuntia ficus-indica. The galactan fraction (in the P-composite) that was not hydrolysed by the enzymes constituted 55% of the total Gal present in the residue. This fraction was considerably higher than the one quantified by solid-state NMR (30%; Zykwinska et al., 2006). It is likely that the environment and/or the presence of short galactan side chains hindered the action of the enzymes. Long galactan side chains, on the one hand, and short galacto-oligomers, on the other hand, are supposed to be present in apple cell walls (Oechslin et al., 2003). Their presence was recently demonstrated in flax (Gur'janov et al., 2007). In addition, Gur'janov et al. (2007) showed that galactanase needs at least three linear Gal residues linked to the RG backbone to perform its activity.
The organization of the xyloglucans/cellulose network within pea stem cell walls was also studied using enzymatic and alkali approaches (Pauly et al., 1999). The treatment of the cell walls with xyloglucan-endoglucanase (XEG) released 36% of all xyloglucans present. The xyloglucan fraction liberated by the XEG action may constitute xyloglucan domains that correspond to cross-links and any tails or loops that could extend away from the cellulose surface. The KOH treatment, that solubilized 45% of all xyloglucan present, indicated xyloglucan domains closely associated with the cellulose surface. Finally, the remaining cell walls were treated with the cellulase that freed 19% of all xyloglucans present. This fraction most probably corresponds to xyloglucans entrapped within cellulose microfibrils during their biosynthesis (Pauly et al., 1999). The results reported by Pauly et al. (1999) indicate that 64% of all xyloglucans are strongly associated with cellulose. These findings showed that only a limited pectin fraction (8% of the arabinan-rich pectins in sugar beet cell wall residue) might be tightly associated with cellulose. Moreover, most of the remaining arabinans or galactans in the enzymatically treated sugar beet and potato cell wall residues were extracted in harsh NaOH conditions, whereas KOH treatment was not sufficient to extract all xyloglucans from pea cell walls (Pauly et al., 1999). Therefore, it appears unlikely that pectic side chains can be entrapped within cellulose microfibrils, probably because of their limited length (100 Ara residues in sugar beet pulp; Oosterveld et al., 2000). Pectin/cellulose associations, limited only to the cellulose surface, may explain why pectins are more easily removed from the cell walls than xyloglucans. It is most likely that the minimum length of arabinan chains required for interaction with cellulose is between 10 and 100 Ara residues. Indeed, it has been demonstrated that arabinan oligomers, with the degree of polymerization ranging from 1 to 10, could not bind to cellulose (data not shown).
In order to simplify the understanding of cell wall architecture, ‘artificial’ composites were created under in vitro conditions. The use of -L-arabinofuranosidase and ß-galactosidase allowed the non-reducing tails that represent 19% of the debranched arabinan and 28% of the galactan side chains adsorbed onto the cellulose surface to be quantified. A second domain corresponding to cross-links between cellulose microfibrils, loops, and some tails that were not degraded in the first step, was estimated by the use of the endo-enzymes (endo-arabinanase and endo-galactanase). This domain represented 9% of the debranched arabinan and 16% of the galactan adsorbed onto cellulose. The remaining arabinan and galactan side chains, not accessible for the enzymes, are most probably in close interaction with the cellulose surfaces. These ‘trains’ represent 72% of the debranched arabinan and 56% of the galactan adsorbed onto the cellulose surface.
The organization of xyloglucans/cellulose ‘artificial’ composites was also investigated by an enzymatic and alkali approach (Pauly et al., 1999). A first xyloglucan domain corresponding to cross-links, loops, and/or tails, and representing 15% of bound xyloglucan, was shown by XEG action. A cellulase action also released 34% of bound xyloglucans. Finally, the KOH treatment removed all of the remaining bound xyloglucan (51%). This domain most probably corresponds to xyloglucan ‘trains’, closely associated with the cellulose surfaces (Pauly et al., 1999).
The results reported by Pauly et al. (1999) on ‘artificial’ composites suggest that a small fraction of xyloglucans can tether the cellulose microfibrils (15%). Indeed, xyloglucan length was claimed to be sufficient to cross-link adjacent microfibrils (McCann et al., 1990). On the other hand, pectic arabinan and galactan side chains are most probably too short to tether the cellulose microfibrils. Moreover, in cell walls, pectic side chains are attached to RG I regions. Therefore, it can be envisaged that cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule (Fig. 4A). In addition, when taking into account the high density of pectins in cell walls, it may be thought that the same pectic molecule can be associated, on the one hand, via the side chains with cellulose microfibrils and, on the other hand, via calcium bridges for example (Ralet et al., 2003) with another pectic molecule. This pectic molecule can then interact with another one (via calcium bridges) or with cellulosic microfibrils. Therefore, tethering of microfibrils can be envisaged not only through one pectic molecule but also through a pectic network composed of several molecules (Fig. 4B).
|
In the present study, the hypothesis of tethering of cellulose microfibrils through one pectic molecule and/or a network of pectic molecules was raised. The proposed organization of polysaccharides within the primary cell walls completes the one envisaged by Talbott and Ray (1992). It seems very likely that cellulose microfibrils are held together by cohesive forces between laterally associated pectins and xyloglucans (Talbott and Ray, 1992). It appeared that pectins, together with xyloglucans, are able to stick to cellulose microfibrils and thus prevent their lateral associations into large bundles. Moreover, it emerged from the present work that cellulose microfibrils can be directly tethered by pectin molecules and this possibility should now be taken into account. Besides the presence of xyloglucan/cellulose and pectin/cellulose networks, it can be envisaged that pectins and xyloglucans are also linked together, as suggested by Thompson and Fry (2000), and Popper and Fry (2005). It was proposed that xyloglucan and pectic side chains of RG I are covalently linked in suspension-cultured cells of both dicotyledons and monocotyledons (spinach, tomato, rose, sycamore, Arabidopsis, maize, barley) (Popper and Fry, 2005). However, further studies are required to elucidate if these three different networks (xyloglucan/cellulose, pectin/cellulose, pectin/xyloglucan) co-exist in the primary cell walls of dicotyledons.
|
Acknowledgements |
|---|
The authors wish to thank Estelle Bonnin for helpful discussions, and Sylviane Daniel for enzyme purification. We are grateful to Roquette and Cagny sugar factories for providing the samples of potato and sugar beet pulps, respectively.
|
Abbreviations |
|---|
CWM, cell wall material; DebAra/cellulose, debranched arabinan/cellulose ‘artificial’ composite; Gal/cellulose, galactan/cellulose ‘artificial’ composite; HG, homogalacturonan; P-CWM, potato cell wall material; P-composite, potato ‘natural’ composite; PCW-cellulose, primary cell wall cellulose; RG I, rhamnogalacturonan I; RG II, rhamnogalacturonan II; SB-CWM, sugar beet cell wall material; SB-composite, sugar beet ‘natural’ composite.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Blakeney AB, Harris PJ, Henry RJ, Stone BA. A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydrate Research (1983) 113:291–299.[CrossRef][Web of Science]
Bonnin E, Dolo E, Le Goff A, Thibault JF. Characterization of pectin subunits released by an optimized combination of enzymes. Carbohydrate Research (2002) 337:1687–1696.[CrossRef][Web of Science][Medline]
Carpita NC, Gibeaut DM. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of walls during growth. The Plant Journal (1993) 3:1–30.[CrossRef][Web of Science][Medline]
Fry SC. The structure and functions of xyloglucan. Journal of Experimental Botany (1989) 40:1–11.
Gur'janov OP, Gorshkova TA, Kabel M, Schols HA, van Dam JEG. MALDI-TOF MS evidence for the linking of flax bast fibre galactan to rhamnogalacturonan backbone. Carbohydrate Polymers (2007) 67:86–96.[CrossRef][Web of Science]
Hayashi T, Marsden MPF, Delmar DP. Pea xyloglucan and cellulose. V. Xyloglucan–cellulose interactions in vitro and in vivo. Plant Physiology (1987) 83:384–389.
Heux L, Dinand E, Vignon MR. Structural aspects in ultrathin cellulose microfibrils followed by 13C CP-MAS NMR. Carbohydrate Polymers (1999) 40:115–124.[CrossRef][Web of Science]
Ishii T, Matsunaga T. Pectic polysaccharide rhamnogalacturonan II is covalently linked to homogalacturonan. Phytochemistry (2001) 57:969–974.[CrossRef][Web of Science][Medline]
McCann MC, Wells B, Roberts K. Direct visualization of cross-links in the primary plant cell wall. Journal of Cell Science (1990) 96:323–334.
Oechslin R, Lutz MV, Amado R. Pectic substances isolated from apple cellulosic residue: structural characterization of a new type of rhamnogalacturonan I. Carbohydrate Polymers (2003) 51:301–310.[CrossRef][Web of Science]
Oosterveld A, Beldman G, Schols HA, Voragen AGJ. Characterization of arabinose and ferulic acid rich pectic polysaccharides and hemicelluloses from sugar beet pulp. Carbohydrate Research (2000) 328:185–197.[CrossRef][Web of Science][Medline]
O'Neill MA, Ishii T, Albersheim P, Darvill AG. Rhamnogalacturonan II: structure and function of a borate cross-linked cell wall pectic polysaccharide. Annual Review of Plant Biology (2004) 55:109–139.[CrossRef][Medline]
Pauly M, Albersheim P, Darvill AG, York WS. Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants. The Plant Journal (1999) 20:629–639.[CrossRef][Web of Science][Medline]
Popper ZA, Fry SC. Widespread occurrence of a covalent linkage between xyloglucan and acidic polysaccharides in suspension-cultured angiosperm cells. Annals of Botany (2005) 96:91–99.
Ralet MC, Bonnin E, Thibault JF. Pectins. In: Biopolymers—Vandamme E, ed. (2001) 6. Weinhein: Wiley-VCH Verlag. 345–380.
Ralet MC, Crépeau MJ, Buchhlot HC, Thibault JF. Polyelectrolyte behavior and calcium binding properties of sugar beet pectins differing in their degrees of methylation and acetylation. Biochemical Engineering Journal (2003) 16:191–201.[CrossRef]
Renard CMGC, Crépeau MJ, Thibault JF. Structure of the repeating units in the rhamnogalacturonic backbone of apple, beet and citrus pectins. Carbohydrate Research (1995) 275:155–165.[CrossRef][Web of Science]
Renard CMGC, Jarvis MC. A cross-polarization, magic-angle spinning, 13C-nuclear-magnetic-resonance study of polysaccharides in sugar beet cell walls. Plant Physiology (1999) 119:1315–1322.
Schols HA, Voragen AGJ. Occurrence of pectic hairy regions in various plant cell wall materials and their degradability by rhamnogalacturonase. Carbohydrate Research (1994) 256:83–95.[CrossRef][Web of Science]
Talbott LD, Ray PM. Molecular size and separability features of pea cell wall polysaccharides. Plant Physiology (1992) 98:357–368.
Thibault JF. Automatization du dosage des substances pectiques par la méthode au métahydroxydiphényle. Lebensmittel Wissenschaft und Technologie (1979) 12:247–251.
Thimm JC, Burritt DJ, Sims IM, Newman RH, Ducker WA, Melton LD. Celery (Apium graveolens) parenchyma cell walls: cell walls with minimal xyloglucan. Physiologia Plantarum (2002) 116:164–171.[CrossRef][Medline]
Thompson JE, Fry SC. Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells. Planta (2000) 211:275–286.[CrossRef][Web of Science][Medline]
Vignon MR, Heux L, Malainine ME, Mahrouz M. Arabinan-cellulose composite in Opuntia ficus-indica prickly pear spines. Carbohydrate Research (2004) 339:123–131.[CrossRef][Web of Science][Medline]
Zykwinska A, Gaillard C, Buléon A, Pontoire B, Garnier C, Thibault JF, Ralet MC. Assessment of in vitro binding of isolated pectic domains to cellulose by adsorption isotherms, electron microscopy and X-ray diffraction methods. Biomacromolecules (2007) 8:223–232.[CrossRef][Web of Science][Medline]
Zykwinska A, Ralet MC, Garnier C, Thibault JF. Evidence for in vitro binding of pectic side chains to cellulose. Plant Physiology (2005) 139:397–407.
Zykwinska A, Rondeau-Mouro C, Garnier C, Thibault JF, Ralet MC. Alkaline extractability of pectic arabinan and galactan, and their mobility in sugar beet and potato cell walls. Carbohydrate Polymers (2006) 65:510–520.[CrossRef][Web of Science]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. A. Arsovski, T. M. Popma, G. W. Haughn, N. C. Carpita, M. C. McCann, and T. L. Western AtBXL1 Encodes a Bifunctional {beta}-D-Xylosidase/{alpha}-L-Arabinofuranosidase Required for Pectic Arabinan Modification in Arabidopsis Mucilage Secretory Cells Plant Physiology, July 1, 2009; 150(3): 1219 - 1234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Alonso-Simon, P. Garcia-Angulo, A. Encina, J. L. Acebes, and J. Alvarez Habituation of Bean (Phaseolus vulgaris) Cell Cultures to Quinclorac and Analysis of the Subsequent Cell Wall Modifications Ann. Bot., June 1, 2008; 101(9): 1329 - 1339. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||