Plant Physiol. (1999) 119: 1315-1322

A Cross-Polarization, Magic-Angle-Spinning, ¹³C-Nuclear-Magnetic-Resonance Study of Polysaccharides in Sugar Beet Cell Walls¹

Catherine M.G.C. Renard^{2, *} and Michael C. Jarvis

Chemistry Department, University of Glasgow, Glasgow G12 8QQ, Scotland

	ABSTRACT

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Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T₁ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.

	INTRODUCTION

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The term "hemicellulose" has a long history (Schulze, 1891), and its survival into current usage is perhaps surprising. The polysaccharides that it describesxylans, xyloglucans, and glucomannanshave no common primary structure, but because they share the ability to adopt chain conformations similar to cellulose, they are all able to bind onto cellulose fibrils in a manner similar to the interchain bonding of cellulose itself. This has led to the supposition that they have common functions within the architecture of the plant cell wall, coating the microfibrils and cross-linking them, preventing their coalescence, or both (Hayashi, 1989; Carpita and Gibeaut, 1993).

To our knowledge, experimental evidence for these functions has thus far been restricted to xyloglucans. The cell walls of pea epicotyls contain at least enough xyloglucan to give a complete coating on the surface of the 3-nm microfibrils (Hayashi et al., 1993). High-resolution transmission electron microscopic images of onion and carrot cell walls revealed cross-links between microfibrils; these cross-links disappeared and the microfibrils aggregated when xyloglucans were removed with alkali (McCann et al., 1990). Extrusion of Acetobacter xylinum cellulose into xyloglucan solution gave a cross-linked network of fine microfibrils (Atalla et al., 1993). What would happen to the structure of a plant cell wall if there were not enough xyloglucan to coat the microfibril surface? Could other hemicelluloses or polysaccharides not normally classified as hemicelluloses fulfill the same functions and allow a normal cell wall to be formed? We have examined sugar beet (Beta vulgaris) cell walls, a good example of such a system, and discuss their functional architecture.

The swollen roots of sugar beets contain reserve parenchyma, but their cell walls differ in a number of key points from the primary cell walls of other dicots. Their pectins are substituted by ferulic acid on the arabinogalactan side chains, which seems to be a characteristic of the Chenopodiaceae, and by acetyl groups on the galacturonate backbone (Rombouts and Thibault, 1986a, 1986b). The cell walls appear to be almost devoid of xyloglucans, mannans, or xylans (Renard and Thibault, 1993). In spite of these differences, beet cell walls perform the same functions as cell walls whose cellulose fibrils are coated by xyloglucans. The question is, are there any other molecules coating the cellulose in beet cell walls?

Solid state ¹³C-NMR with CP and MAS is particularly useful for the investigation of mobility and domain size in polymer systems. The rate of relaxation (dissipation) of proton magnetization, which can be measured through the ¹³C spectrum by CP, is controlled by thermal motion and can therefore be used to probe the mobility of individual polymers within a complex structure (Schaefer et al., 1977; Kenwright and Say, 1993; Stejskal and Memory, 1994). In cell walls the proton-rotating, frame-spin-lattice relaxation time (¹H-T₁) is the most suitable indicator of mobility for rigid components such as cellulose, and the spin-spin relaxation time (¹H-T₂) is appropriate for more mobile polymers (Tekely and Vignon 1987; Newman et al., 1994). The ¹H-T₁ decreases and the ¹H-T₂ increases with increasing molecular motion in materials of this type. Extremely mobile, hydrated polymers can also be identified, because the rate of CP from ¹H to ¹³C is related to the ¹H-T₂ and becomes slow at very high levels of mobility (Ha et al., 1996). Proton T₁ relaxation and CP occur simultaneously when the CP contact time is varied, but can be separated experimentally by inserting a variable delay with proton-spin locking before a constant CP contact time.

If two solid components are close together, proton magnetization within them is averaged by spin diffusion. Since the rate of proton spin diffusion is a constant (dependent inversely on the ¹H-T₂), the time required for this spatial averaging is a measure of the distance between the two components (Newman, 1992). This principle can be used to derive spatial information from NMR-relaxation experiments. Averaging of the expected ¹H-T₁ values of two polymer components known to differ in mobility, e.g. cellulose and hemicellulose, implies that they are at most 2 to 3 nm apart, or somewhat less if both are relatively mobile (Ha et al., 1998). Shorter spatial separations of approximately 1 nm or less are implied between components whose magnetization is averaged during the contact time of a ¹H-T₂ experiment, or those that show similar CP kinetics because the proton pools contributing to their CP have merged.

In hydrated onion (Ha et al., 1996, 1997) and citrus cell walls (Jarvis et al., 1996), the microfibril components were characteristically immobile, whereas the matrix polysaccharides were of greater mobility. The most mobile components were pectins, especially methylated segments of the backbone and, in onions, galactan side chains (Foster et al., 1996; Ha et al., 1996). The majority of the onion xyloglucans, identified as coating the cellulose in the microfibrils, had ¹H-T₁ and ¹H-T₂ relaxation rates similar to those of cellulose (Ha et al., 1997), indicating a close spatial association. A minor xyloglucan component showed considerably faster ¹H-T₁ and slower ¹H-T₂ relaxation rates than cellulose, and was identified with cross-linking segments of the xyloglucan chains (Ha et al., 1997).

Our aim was to identify any polymer in beet cell walls that was closely associated with cellulose in the manner expected of hemicelluloses. We have used both ¹H-T₁ relaxation and CP kinetics from protons to ¹³C nuclei and DP-MAS ¹³C experiments for the highly mobile component.

	MATERIALS AND METHODS

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Plants

Cell Walls

Cellulose was isolated from de-esterified beet pulp after controlled-acid hydrolysis (0.1 M HCl for 72 h at 80°C; Renard et al., 1998), followed by neutralization to and extensive washing at pH 7, and was dried by solvent exchange (ethanol followed by acetone). Beet arabinan was from British Sugar (Norwich, UK).

Analysis

NMR

1

	RESULTS

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Chemical Characterization

This cell wall preparation was deficient in sugars (Xyl, Man, and Fuc) that commonly indicate the presence of hemicelluloses. Only about 12 mg/g Glc could be liberated from the cell walls without prehydrolysis (noncellulosic Glc), a further indication of very low xyloglucan content. In total, the Xyl, Fuc, Man, and noncellulosic Glc accounted for approximately 42 mg/g, whereas there was approximately 250 mg/g cellulose.

After controlled-acid hydrolysis (Renard et al., 1998), repeated washings of the beet cell walls at pH 7 removed most of the homogalacturonans. However, at that point the solid residue was very fine and gave stable suspensions. It became very difficult to isolate, and no corresponding mass balance could be calculated. This depectinated residue contained more than 80% cellulose, and was enriched in Xyl and Man, with residual GalUA (Table I). Man, Xyl, and noncellulosic Glc accounted for only 90 mg/g.

NMR Spectroscopy

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View larger version (18K): [in this window] [in a new window]   Figure 1. 13C CP-MAS NMR spectra of sugar beet cell walls at 1.0:0.5 ratio of cell wall to water. These spectra are not normalized and show the observed intensities in the varying experiments.

The beet spectrum showed one characteristic peak at 108 ppm that was not observed in onion or citrus cell walls, which was tentatively identified as the C-1 signal of sugar beet arabinans. To confirm this identification, a short-recycle, ¹³C DP-MAS spectrum was recorded (Fig. 2B). This spectrum showed substantial motional line narrowing and chemical shifts close to those observed for beet arabinan in aqueous solution (Fig. 2A). The CP-MAS spectrum of the depectinated residue (Fig. 2C) showed only the cellulose signals: C-1 (105 ppm), C-4 (89 and 85 ppm), and C-6 (65 and 62 ppm).

View larger version (20K): [in this window] [in a new window]   Figure 2. Spectra of beet cell wall components. A, 13C-solution NMR spectrum of sugar beet arabinan. B, 13C DP-MAS NMR spectrum of sugar beet cell walls. C, 13C CP-MAS NMR of depectinated cell walls with 1-ms contact time.

T₁ Decay

1

Subtraction of these delayed-contact spectra from the 1-ms spectrum (same CP time but no T₁ delay) after normalization to give equal intensity in the cellulose C-4 region (84-91 ppm) gave subspectra (Fig. 3) corresponding to components of high and intermediate mobility. Approximate T₁ was calculated on resolved peaks assuming a monocomponent exponential decay during the 14-ms T₁ delay period (Table II); although it is an oversimplification, this gives the relative decay rate to a first approximation. The signals displayed in the mobile subspectra correspond to components with T₁ < 10 ms, compared with 12 to 17 ms for the cellulose signals. These subspectra corresponded to pectic galacturonans (carboxy carbons at approximately 175-172 ppm, wide C-1 peak at 101-99 ppm, C-4 at approximately 80 ppm, and C-2, C-3, and C-5 at approximately 69 ppm) with both methoxy (54 ppm) and acetyl signals (21 ppm), plus some sharp signals at 108 (arabinan C-1), 77, and 62 ppm. The approximate T₁ calculated for the depectinated cell wall residue was 16 to 19 ms for all signals; spectra acquired with different delays differed only in their overall intensity, and no subspectra could be extracted.

View larger version (31K): [in this window] [in a new window]   Figure 3. Difference subspectra corresponding to the mobile elements and derived from rapid T1 decay (top spectrum), slow CP (middle spectrum), or both (bottom two spectra). Each point in the subspectrum corresponds to the difference in signal intensity (after normalization of the cellulose C-4 signals) between the corresponding points in the respective spectra.

View this table: [in this window] [in a new window]   Table II. 13C resonance assignments and approximate T1 (from delayed contact experiments) for hydrated sugar beet cell walls

CP Kinetics

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View larger version (20K): [in this window] [in a new window]   Figure 4. Variation in the intensity of arabinan C-1 (108 ppm), cellulose C-1 (105 ppm), galacturonan C-1 (100 ppm), and general carbohydrate signal (72 ppm) as a function of the CP contact time relative to their intensity after 1 ms of CP.

An alternative approach for obtaining spectral information on components of varying mobility is to compare spectra at different contact times. Using this approach the effects of CP kinetics and T₁ delays are superimposed. Difference spectra for (long-short) contact times are shown in Figure 3. They all contain positive peaks assignable to arabinans and pectic methoxy and acetyl groups, which are linked to their slow CP behavior. They also show negative peaks assigned to GalUA because, on average, their signal intensities are more influenced by rapid T₁ decay.

Crystallite Size within Cellulose Microfibrils

	DISCUSSION

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Although the plants used in the present study were young and had just begun to form thickened roots, the yield and composition of their cell walls were very similar to those found for mature beet (Renard and Thibault, 1993), with the characteristic high Ara content, the high degree of acetylation of the pectins, and the presence of ferulic acid (Thibault et al., 1994). The final beet cell walls contained enough calcium to neutralize about two-thirds of the free carboxyl groups of pectins. A procedure developed for pectin fractionation (Renard et al., 1998) made it possible to obtain a residue containing more than 80% of cellulose while avoiding concentrated alkali extractions that might lead to mercerization. A controlled-acid hydrolysis first solubilized the "hairy regions" of pectins as oligomers, leaving a solid consisting essentially of Glc and GalUA (Renard et al., 1998). The residual GalUA was present as homogalacturonans, which were insoluble under acidic conditions but became readily soluble upon careful neutralization. During this procedure there was only limited loss of Xyl (during the acid treatment) and almost no loss of Man relative to cellulose. The composition of the washed residue may be compared with that of the parenchymatous cellulose preparation from beets (Dinand et al., 1996), which contained 88% cellulose after treatment with 2% (0.5 M) NaOH and NaClO₃ bleaching, and likewise formed stable suspensions.

The sugars denoting cross-linking polysaccharides (i.e. Fuc, Xyl, Man, and noncellulosic Glc) amounted to only one-sixth of the mass of the cellulose in the beet cell walls and one-ninth of that in the depectinated cell walls. There is convincing evidence for the absence of a large amount of typical hemicelluloses in beet cell walls. Oosterveld et al. (1996) extracted only 19% of the beet cell walls in 4 M NaOH, and this extract contained mainly pectic rhamnogalacturonans, galacturonans, and arabinans, with a quantitatively minor (but normal) fucogalactoxyloglucan (less than 10%, as estimated from the DEAE fractionation by Oosterveld [1997]) and traces of (gluco)mannans.

Although cellulose, pectic galacturonan, and pectic arabinan were present in similar amounts in the beet cell walls, the cellulose signals in the spectrum were far more intense. The total intensity of the arabinan signals was considerably less than that predicted from the arabinan content of the cell walls, as would be expected since their T₁ decay was rapid and occurred simultaneously with particularly slow CP. Compared with hydrated citrus cell walls, the lower galacturonan content resulted in a decrease of the pectic galacturonan C-1 peak compared with the cellulose C-1 peak, and of the pectin C-4, C-2, and C-3 signals at approximately 80 and 69 ppm compared with the general C-2, C-3, and C-5 signals at 72 to 75 ppm.

Use of the Waltz-16 proton-decoupling sequence and a very short recycle time made it possible to record a DP-MAS ¹³C spectrum consisting exclusively of arabinan signals. Their chemical shifts were close to those of arabinans in solution, suggesting that the distribution of chain conformations was similar. The high degree of branching of beet arabinans and the abundance of nonreducing ends (Thibault and Rouau, 1990) explains the intensity of the C-5 and C-4 signals for terminal arabinofuranose residues, whereas the high degree of branching on C-3 led to a characteristic signal at 80.1 ppm. Cell walls investigated previously (Foster et al., 1996; Jarvis et al., 1996; Ha et al., 1997) did not have such high Ara contents, and no conclusions were drawn regarding the mobility of arabinan side chains. However, arabinans are known to be highly mobile in solution, because the glycosidic bonds in their backbone, Araf(15), are through the primary alcohol group. The spectrum of the depectinated cell wall was that of a type-I cellulose (Attalla et al., 1980), with a high proportion of crystallite surface chains (Newman et al., 1996).

The proton T₁ data made it possible to distinguish between cellulose, which is rigid and crystalline and thus has a longer T₁, and more mobile arabinans and galacturonans with shorter decay times. CP kinetics further separated the highly mobile arabinans from the galacturonans. The intensities of the arabinans were more influenced by their CP behavior, whereas those of galacturonans were more influenced by rapid T₁ decay. The particularly slow CP of the arabinan component (C-1 at 108 ppm) indicated a high degree of mobility, as did the DP-MAS spectrum. The absence of signals associated with galacturonan or arabinans in delayed-contact experiments implied that these polymers were not associated closely enough with the cellulose microfibril to be made more rigid by the association, as occurs for xyloglucan (Ha et al., 1997), or to have their observed proton T₁ significantly increased by averaging through proton spin diffusion. The minimum spatial separation between the mobile components themselves need not be large (minimum <1 nm) because mobility reduces the rate of spin diffusion.

In beet cell walls the total amount of hemicelluloses available to coat the cellulose surface was only one-sixth of the mass of the cellulose, and no other polysaccharide present in significant quantity fulfills a coating function. Beet cellulose may have a particularly low surface area, allowing it to be coated completely by a relatively small amount of hemicellulose. The surface area of cellulose fibrils depends on the fibril diameter. The percentage of interior cellulose chains was only 42% in the beet cell walls, compared with 48% for the depectinated beet residue. This corresponds to a crystallite size slightly greater than that in apple (38%; Newman et al., 1994) or Arabidopsis (40%; Newman et al., 1996). The data for beet indicate crystallites 2 to 3 nm wide in muro, the exact width depending on their shape and the packing density of the surface chains. After controlled-acid hydrolysis and washing there was no marked aggregation, although the cellulose content was >80%. Fibrils 2 to 4 nm in diameter have been isolated from beet cell walls by hydrolytic removal of the noncellulosic polysaccharides, thus avoiding highly alkali concentrations (Dinand et al., 1996). A slight tendency to aggregation where the fibrils contacted one another was observed by Dinand et al. (1996).

Assuming that a xyloglucan chain in the "flat" conformation described by Levy et al. (1991) is 1 nm wide and has a mean cross-sectional area of 0.7 nm² (compared with 0.32 nm² for cellulose), a 3-nm cellulose fibril will require an approximately equal mass of xyloglucan to provide a complete coating over its surface (Hayashi et al., 1993). We do not exclude the possibility that the crystallites of beet cellulose are aggregated into larger microfibrils and that these are disrupted upon removal of the noncellulosic polysaccharides. There is evidence (Ha et al., 1998) that this is the case in the onion cell wall, where the crystallite diameter is similar but the microfibril size (including the xyloglucan present) is 8 to 10 nm (McCann et al., 1990). However, 8- to 10-nm microfibrils would still require a xyloglucan:cellulose ratio of 1:2 by mass to provide a monolayer of xyloglucan at the surface.

Current cell wall models assume that hemicelluloses are effective only if they coat the cellulose microfibrils, but the data presented here show that beet microfibrils do not need such a coating to keep them separated. The aggregation observed after conventional extraction sequences that include strong alkali (McCann et al., 1990) may be due to the formation of cellulose II by mercerization rather than to the removal of hemicelluloses. The small amount of xyloglucan present in beet cell walls may be sufficient to cross-link the microfibrils, as the amounts that form the actual cross-links are minimal (McCann et al., 1990).

Although nature uses only a few building blocks for cell walls in terrestrial plants, we still do not understand the precise role of each of these blocks, nor how much redundancy or versatility there might be. Very few walls have actually been studied in detail, and we should pay more attention to variability in their function and design. Beet cell walls in particular are those of a storage tissue that is not undergoing fast growth or bearing weight.

	CONCLUSIONS

The polymers of the beet cell walls can be divided in three classes of mobility: (a) cellulose, which is highly rigid; (b) pectic galacturonans of intermediate mobility; and (c) highly mobile arabinans. We have not located any quantitatively significant fraction of the beet galacturonan or arabinan that would display the close association with cellulose microfibrils characteristic of hemicellulose. Indeed, the high mobility of arabinans in muro confirms that the pectic side chains fill the pores of the cell wall network rather than play a cross-linking role. Their structural role could be as plasticizers and water-binding agents.

The beet cell wall contains so little xyloglucan, xylan, and mannan that in the absence of any other coating polysaccharide most of the surface area of the cellulose must be bare. The 2- to 3-nm crystallites appear to have considerable stability in the absence of noncellulosic polymers. It follows that a molecular mechanism that does not involve coating of microfibrils must be responsible for limiting the aggregation of cellulose within the beet cell wall.

	FOOTNOTES

   Received July 9, 1998; accepted December 5, 1998.

	ABBREVIATIONS

Abbreviations: CP, cross-polarization. DP, direct polarization. MAS, magic-angle-spinning.

	ACKNOWLEDGMENTS

The authors thank Dr. Brett and Dr. Wende (Department of Botany, University of Glasgow) for the sugar beets. NMR spectra were recorded at the Engineering and Physical Sciences Research Council solid-state NMR service in Durham (UK).

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