Claudine Morvan

Structural features of galactans from flax fibres

Abstract Successive extraction of mechanically-isolated flax fibres with ethylene-diaminetetraacetic acid partially neutralized with NAOH EDTA-Na 2 and NaOH solubilized about 25% of the mass of fibres. The cation exchange capacity (CEC) of the fibres decreased from 0.100 ± 0.025 meq g −1 to 0.035 ± 0.015 meq g −1 after EDTA-Na2 extraction and then was reduced to virtually nil by extraction with NaOH. This final reduction in CEC by alkali was accompanied by the extraction of small charge molecules, notably phenolic acids. These may influence the binding selectivity of the fibre towards monovalent cations. Polymers solubilized by this extraction procedure included two main types of galactans; the first type consisted of β-1→4 galactans attached to a rhamnogalacturonan I-like polymer and the second type was β-1→3, β-1→6 linked galactans attached to proteins in some unknown manner. In addition, some β-1→3 glucan, β-1→4 glucan and glucomannan-like polymers were detected.

Partial purification of Flax cell wall pectin methylesterase.

Abstract Cell walls of Linum usitatissimum calli exhibited ca 1 μkat g−1 of pectin methylesterase activity (EC 3.1.1.11). Ion exchange and size exclusion chromatography gave three active fractions. The major one, representing 20% of the initial activity, with a chromatographic Mr of 100 000, and a SDS-PAGE one of 43 000 consisted of three isoforms with pIs of 5.5, 7.3 and 7.8, respectively. The second fraction, 3.5% of the initial activity, had a chromatographic Mr of 110 000, and was separated into five isoforms with pIs of 5.5, 7, 7.3, 7.8 and 8.8. The minor active fraction, 1 % of the initial activity, revealed one isoform (pI close to 10) but was contaminated with non-active proteins. The major active fraction had a Km of 0.147 mg ml−1 for a specific activity of 3.3 mkat g−1 and a complex behaviour in the presence of NaCl.

Control of Mung Bean Pectinmethylesterase Isoform Activities INFLUENCE OF pH AND CARBOXYL GROUP DISTRIBUTION ALONG THE PECTIC CHAINS

Well-characterized pectin samples with a wide range of degrees of esterification (39–74%) were incubated with the solubilized pure α and γ isoforms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata). Enzyme activity was determined at regular intervals along the deesterification pathway at pH 5.6 and pH 7.6. It has been demonstrated that the distribution of the carboxyl units along the pectin backbone controls the activity of the cell wall pectinmethylesterases to a much greater extent than the methylation degree, with a random distribution leading to the strongest activity. Polygalacturonic acid was shown to be a competitive inhibitor of the α isoform activity at pH 5.6 and to inhibit the γ isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in enzyme activity was shown to be correlated to the formation of deesterified blocks of 19 ± 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem.273, 33150–33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the substrate had been totally deesterified. Comparison of α and γ isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed.

Structural features of CDTA-soluble pectins from flax hypocotyls

Polymers extracted with CDTA-Na2 (trans-diaminocyclohexane-a-tetra-acetic acid partially neutralized with NaOH) after a boiling water treatment from cell walls of 3-day-old seedlings of flax, germinated on water and in darkness, contained three main components: (1) rhamnogalacturonan I (RG-I)-like polymers with ratios of rhamnose to galacturonic acid which increased with their molecular mass; (2) neutral polysaccharides including galactans as well as arabinans; and (3) galacturonic-acid enriched polymers. n nHigh degrees of acetylation were estimated for most of the rhamnogalacturonans but a minor RG-I-like fraction with a ratio of rhamnose to galacturonic acid of unity was acetylated (29%) as well as methylesterified (40%). n nHowever, the calcium activity coefficients of the fractionated polymers measured in solution were quite high which implies that these polyelectrolytes did not behave like linear ones. These polymers which reacted in solution as well as in situ not only with ruthenium red but also with ferric hydroxylamine, and which contained large amounts of acetylated RG-I-like blocks might be considered as early markers of the differentiation of the cellulosic fibre cells in flax.

Degradation of flax polysaccharides with purified endo-polygalacturonase

Abstract An endo-polygalacturonase was purified from a commercial preparation of Aspergillus by cation exchange and size exclusion chromatographies. It degraded a polysaccharide extracted from under-retted flax into three general size classes. The first product was a polysaccharide of high molecular weight (close to 100 000), enriched in galactose and characterized by a ratio of galacturonic acid:rhamnose 2:1. The second product was an acidic polysaccharide (the molecular weight of which was close to 10 000) enriched in glucose and rhamnose, which reacted little with the usual colorimetric methods for uronic acids and gave a negative circular dicroism bond. The last and major products were oligomers rich in galacturonic acids.

Characterization of mucilage polysaccharides, arabinogalactanproteins and cell-wall hemicellulosic polysaccharides isolated from flax seed meal: A wealth of structural moieties.

The present study aimed at analyzing the structural features of seed mucilage and cell-wall polysaccharides which accounted for 41% of the mass of flax meal (FM). A combination of high molar-mass mucilage-like polysaccharides (rhamnogalacturonan and arabinoxylan) was released from FM in water, together with arabinogalactan proteins and glucans. About half of FM homogalacturonans was extracted using a calcium chelator and boiling water. Hemicellulosic xyloglucans and xylans were further extracted with 1M KOH, in ∼13% FM-sugars yield. Structural characterization of the xyloglucan using specific enzyme hydrolysis, ion exchange chromatography (HPAEC) and matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectroscopy showed the presence of XXXG type xyloglucan, but also that of XXGG-structure, possibly characteristic of flax seeds. Hydrolysis of xylans with endo-(1→4)-β-D-xylanase, and analysis of the neutral and acidic oligosaccharides by MALDI-TOF-MS showed that xylan consisted of β-(1→4)-linked-D-xylopyranose backbone with some zones (DP 5-7) substituted with 4-O-MeGlcAGlcAGlc residues.

Structural features of water-soluble pectins from mung bean hypocotyls

Abstract Physicochemical and enzymic approaches were used to characterize highly methylated pectins isolated from mung bean hypocotyl cell walls. Young cell walls were particularly rich in rhamnogalacturonan-1-like polysaccharides, the galacturonic units of which might be fully methylated. Short, smooth, homogalacturonan blocks separated these hairy regions. The homogalacturonans included acidic and highly methylated blocks, the structures of which have been investigated through computer simulations. Such complex carbohydrates were also detected in the older parts of the hypocotyls but to a lesser extent. Mature cells were characterized by the presence of pure, sparsely methylated homogalacturonan with randomly distributed methyl groups.

Cell Wall Composition and Morphogenic Response in Callus Derived from Protoplasts of two Fibre flax (Linum usitatissimum L.) Genotypes

Summary Cotyledonary protoplasts of two French fibre flax ( Linum usitatissimum ) cultivars (Ariane and Viking) were isolated and cultured. The optimal conditions for high yield were from 4-5 day old seedlings, where 6-6.5x10 6 and 6.7-7.4x10 6 protoplasts were released from cv. Ariane and cv. Viking respectively. Culture in glutamine (20mM) containing medium led to first divisions after 2 days with an efficiency of 19% or 21.9% (cv. Ariane) and 49.3% or 44% (cv. Viking) of divided cells, when plating densities were 10 5 versus 5 x 10 4 protoplasts per mL. Rhizogenesis occurred on protoplast derived calli at high frequency 4 weeks after transfer onto solid medium supplemented with 0.5 μM 2.4 dichlorophenoxyacetic acid. Variations of cell wall sugar composition during culture on inducing and non-inducing media expressed some significant differences. The percentage of galacturonic acid increased on non-inducing medium, but remained constant on root-inducing medium. Glucose presented a peak of accumulation concomitant of the emergence of the root meristems 4 weeks after transfer onto 2,4-D containing medium. On the contrary galactose and arabinose variations were not characteristic of the rhizogenic process.