Sugar Beet Cell Walls Research Articles

Assessment of In Vitro Binding of Isolated Pectic Domains to Cellulose by Adsorption Isotherms, Electron Microscopy, and X-ray Diffraction Methods

Isolated pectic domains representative of the pectic backbone and the neutral sugar side chains were tested for their ability to interact with cellulose in comparison to the well-known binding of xyloglucan. Pectic side chains displayed a significant in vitro binding capacity to cellulose, whereas pectic backbone domains exhibited only slight adsorption to cellulose microfibrils. To support the binding results, electron microscopy and X-ray diffraction were applied. Celluloses from bacteria and sugar beet cell walls were used as substrates for the precipitation of isolated pectic domains or xyloglucan by acetone vapor diffusion. Pectic side chains grew attached to the cellulose surfaces, whereas pectic backbone domains were observed separately from cellulose microfibrils. Xyloglucan seeded with cellulose provoked a decrease of microfibrils entanglement, but no clear cross-links between neighboring microfibrils were observed. These results led to the elucidation of the pectic domains responsible for binding with cellulose microfibrils.

Sugar beet ( Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall

Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after 18O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or α-1 → 5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (β-1 → 4-linked-dimer), Ara (α-1 → 5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.

Isolation of diferulic bridges ester-linked to arabinan in sugar beet cell walls

After degradation of sugar beet cell walls with Driselase® and fractionation of the solubilised products by hydrophobic interaction chromatography, a dehydrodiferuloylated oligoarabinan was isolated. Its structure was assigned to two dimers of (1 → 5)-linked arabinose units esterified by a central 8-O-4′ ferulic dimer. These results provide the first direct evidence that pectic arabinans in sugar beet cell walls may be covalently cross-linked through dehydrodiferulates.

Isolation from sugar beet cell walls of arabinan oligosaccharides esterified by two ferulic acid monomers.

Side chains of sugar beet (Beta vulgaris) pectins, which are mainly composed of arabinose (Ara) and galactose (Gal) residues, are esterified by ferulic acid units. Enzymatic hydrolysis of beet cell walls yielded several feruloylated oligosaccharides, which were separated by hydrophobic interaction chromatography. Two new oligomers were isolated in the fraction eluted by 25:75 (v/v) ethanol:water. An arabinotriose and an arabinotetraose esterified by two ferulic acid residues were obtained, and their structure was elucidated by mass spectrometry. It is shown that feruloyl groups are linked to O-5 of Ara residues, in addition to the known O-2 position. This work establishes for the first time, to our knowledge, that two neighboring Ara units may be esterified by two ferulic acid units. This close proximity may have important biochemical implications.

Extraction of pectic polysaccharides from sugar-beet cell walls

Previous methods of extracting pectin from sugar-beet have used pulp as the starting material. As the temperature and pressure of the pulping process may modify the architecture of the cell wall, we have adapted a relatively non-disruptive method to characterise cell wall material (CWM) isolated directly from the sugar-beet. Cell walls from mature sugar-beets (Beta vulgaris L Aztec) were sequentially extracted four times with imidazole and twice with sodium carbonate to produce six heterogeneous pectic polysaccharide extracts, and with KOH to produce a hemicellulosic extract which was predominantly xylans. Heterogeneity of the extracted pectins was indicated by differences in FTIR spectra, uronic acid content, % methyl esterification, % feruloylation, % acetylation, molecular weight distribution and neutral sugar composition. The highest proportion of feruloyl esters was found in polysaccharides solubilised by the second sodium carbonate extraction. Anion exchange chromatography of these polysaccharides gave three fractions, one of which contained most of the feruloyl ester. These results indicate that feruloyl esters are not randomly distributed among the different pectic polysaccharides in the sugar-beet cell wall, and that esterification is likely to be dependent on the local sugar sequence or conformation. © 2000 Society of Chemical Industry

Structure and properties of apple and sugar-beet pectins extracted by chelating agents

Pectins were extracted from apple or sugar-beet cell walls (alcohol-insoluble solids) at pH 4.5 and 20°C and at pH 6.5 and 80°C in acetate and phosphate buffer solutions, respectively, and in the same buffers containing cyclohexanediaminetetraacetic acid or ethylenediaminetetraacetic acid. The yields were very different between the two pH and temperature conditions, but variations with the nature of the extractant were small at pH 4.5 and 20°C, and no differences were observed at pH 6.5 and 80°C. The pectins extracted at pH 6.5 and 80°C were richer in neutral sugars and acetic acid. Degradation of the pectins extracted at high pH and temperature was shown by the decrease of the intrinsic viscosity and by the shift of the uronic acid peak toward the total volume in gel-filtration chromatography. These pectins contained a high molecular weight population rich in neutral sugars and a low molecular weight acid population, in contrast to the pectins extracted at low temperature and pH, for which neutral and acidic sugars had similar elution profiles. At pH 6.5 and 80°C, rather extensive degradation of the pectins occurred. Calcium-binding experiments (potentiometry with specific electrode and conductimetry) indicated a statistical distribution of free carboxyl groups. These results do not support the hypothesis that chelating-agent extractable pectins are held in the cell wall by calcium cross-links involving the formation of “egg-boxes”.

A pectin Lyase Inhibitor Protein from Cell Walls of Sugar Beet

A protein from sugar beet roots inhibited pectin lyase (PNL) that was isolated from Rhizoctonia solani. The PNL inhibitor protein (PNLIP) was extracted from healthy sugar beet cell walls and partially purified by ammonium sulfate fractionation, cation exchange, and gel permeation chromatography. Electrophoresis in polyacrylamide with sodium dodecyl sulfate showed a single major band of 57.5 kDa. Isoelectric focusing gave a major band at pI 9.9. Kinetic analysis indicated that the inhibition was uncompetitive (coupling). Pectin lyase inhibitory activity was greater in cell wall extracts from rotted and adjacent tissue than in healthy tissue []