Marc Lahaye

QUASIMODO1 Encodes a Putative Membrane-Bound Glycosyltransferase Required for Normal Pectin Synthesis and Cell Adhesion in Arabidopsis

Pectins are a highly complex family of cell wall polysaccharides. As a result of a lack of specific mutants, it has been difficult to study the biosynthesis of pectins and their role in vivo. We have isolated two allelic mutants, named quasimodo1 (qua1-1 and qua1-2), that are dwarfed and show reduced cell adhesion. Mutant cell walls showed a 25% reduction in galacturonic acid levels compared with the wild type, indicating reduced pectin content, whereas neutral sugars remained unchanged. Immersion immunofluorescence with the JIM5 and JIM7 monoclonal antibodies that recognize homogalacturonan epitopes revealed less labeling of mutant roots compared with the wild type. Both mutants carry a T-DNA insertion in a gene (QUA1) that encodes a putative membrane-bound glycosyltransferase of family 8. We present evidence for the possible involvement of a glycosyltransferase of this family in the synthesis of pectic polysaccharides, suggesting that other members of this large multigene family in Arabidopsis also may be important for pectin biosynthesis. The mutant phenotype is consistent with a central role for pectins in cell adhesion.

Isolation and structural determination of two 5,5′-diferuloyl oligosaccharides indicate that maize heteroxylans are covalently cross-linked by oxidatively coupled ferulates

Large amounts of ferulic acid (∼2.6% w/w) and dehydrodiferulic acids (∼1.3% total, w/w) were detected in saponified extracts of maize bran. After treatment of maize bran by mild acid hydrolysis and fractionation of the solubilised products by liquid chromatography, two dehydrodiferuloylated oligosaccharides (F8 and F9) were isolated. The compositions of F8 and F9 were shown to be 1:2:1 5,5′-diferulic acid (5,5′-diFA)–arabinose–xylose and 1:2 5,5′-diFA–arabinose, respectively. Their structure was determined using chromatography and LCMS and confirmed by 1H and 13C NMR spectroscopy. Fraction F9 was composed of 5,5′-diFA esterified at both carboxylic groups by a 5-O-l-arabinofuranoside moiety, while F8 was similar but with one of the arabinofuranosyl residues further substituted with a (1→2)-linked xylopyranosyl residue. These results provide direct evidence that the heteroxylans in maize cell walls are covalently cross-linked through dehydrodiferulates.

GDP‐d‐mannose 3,5‐epimerase (GME) plays a key role at the intersection of ascorbate and non‐cellulosic cell‐wall biosynthesis in tomato

The GDP-D-mannose 3,5-epimerase (GME, EC 5.1.3.18), which converts GDP-d-mannose to GDP-l-galactose, is generally considered to be a central enzyme of the major ascorbate biosynthesis pathway in higher plants, but experimental evidence for its role in planta is lacking. Using transgenic tomato lines that were RNAi-silenced for GME, we confirmed that GME does indeed play a key role in the regulation of ascorbate biosynthesis in plants. In addition, the transgenic tomato lines exhibited growth defects affecting both cell division and cell expansion. A further remarkable feature of the transgenic plants was their fragility and loss of fruit firmness. Analysis of the cell-wall composition of leaves and developing fruit revealed that the cell-wall monosaccharide content was altered in the transgenic lines, especially those directly linked to GME activity, such as mannose and galactose. In agreement with this, immunocytochemical analyses showed an increase of mannan labelling in stem and fruit walls and of rhamnogalacturonan labelling in the stem alone. The results of MALDI-TOF fingerprinting of mannanase cleavage products of the cell wall suggested synthesis of specific mannan structures with modified degrees of substitution by acetate in the transgenic lines. When considered together, these findings indicate an intimate linkage between ascorbate and non-cellulosic cell-wall polysaccharide biosynthesis in plants, a fact that helps to explain the common factors in seemingly unrelated traits such as fruit firmness and ascorbate content.

CHEMICAL STRUCTURE AND PHYSICO-CHEMICAL PROPERTIES OF AGAR

Advances in the chemistry and physico-chemical properties of agar since the review of Araki at the Fifth International Seaweed Symposium in 1965 are discussed. These advances are essentially the result of better separation techniques of the heterogeneous family of polysaccharides known as agar, the use of nuclear magnetic resonance spectroscopy, the use of agarases and, particularly, the use of combinations of the three approaches. Although physico-chemical methods have evolved, particularly molecular-weight determinations, X-ray diffraction data and molecular modelling of agar, correlations between chemical and functional properties of agar and agarose and their gelation mechanisms remain to be studied.

Average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides

Abstract A procedure to determine the absolute weight-average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides by aqueous size-exclusion chromatography coupled with low-angle laser light scattering is described. The molecular weights of the majority of the commercial samples investigated were between 80 000 and 140 000 with a polydispersity lower than 1·7. In contrast, most of the laboratory-extracted agarose-type polysaccharides had lower molecular weights.

Cell-wall polysaccharides from the marine green alga Ulva “rigida” (ulvales, chlorophyta). Extraction and chemical composition☆

Abstract Cell-wall polysaccharides from the green seaweed Ulva “rigida” were extracted sequentially with oxalate, 1 and 4 M KOH, sodium chlorite and 4 M KOH again. The chemical composition of the soluble and insoluble extracts and of the DEAE-Sepharose CL-6B fractions of the soluble polysaccharide were determined. Three main types of polysaccharide families were solubilized. The major family was composed of sulfated glucuronorhamnoxyloglycans (ulvan) and was essentially extracted with oxalate. Two hemicellulosic fractions were also isolated and consisted of glucuronans and glucoxylans. Minor fractions consisting of sulfated polysaccharides containing glucose, xylose, mannose and protein were also isolated.

Chemical and physical-chemical characteristics of dietary fibres from Ulva lactuca (L.) Thuret and Enteromorpha compressa (L.) Grev.

Dietary fibres from Ulva lactuca (L.) Thuret (sea lettuce) and Enteromorpha compressa (L.) Grev. (A.O. nori) were measured according to a ‘standard’ method and a ‘physiological’ protocol simulating the gastric and intestinal environments. U. lactuca contained 15.8–8.0% soluble and 24.2–32.6% insoluble fibres according to the ‘standard’ and ‘physiological’ methods, respectively. For E. compressa, these values were 14.9–15.9 and 21.6–28.7%, respectively. For both algae, the composition suggests that the soluble fibres were xylorhamnoglycuronans sulphates and insoluble fibres were essentially composed of glucans. No marked chemical compositional variation was observed between soluble fractions extracted under the simulated gastric and intestinal conditions. Fibres in both algae are hydrophilic but the water holding capacities were higher after extraction of soluble fibres (5.5–9.5 g g−1 for the dry algae; 14.0–16.0 g g−1 for the standard insoluble fibres). Water soluble fibres demonstrated low intrinsic viscosities at 37 °C in buffers, particularly those from E. compressa (36.0–36.5 ml g−1), and was affected by pH for those of U. lactuca (147.5 ml g−1 at pH 3.0 and 175.0 ml g−1 at pH 7.3).

Sugar determination in ulvans by a chemical-enzymatic method coupled to high performance anion exchange chromatography

The sugar determination of ulvans, the water-soluble polysaccharides from Ulva sp. and Enteromorpha sp., was optimized by combining partial acid prehydrolysis (2 mol L-1 trifluoroacetic acid, 120°C) with enzymic hydrolysis (with β-D-glucuronidase). The different constitutive sugars (rhamnose, galactose, glucose, xylose, glucuronic acid), released after hydrolysis, were separated by high performance anion-exchange chromatography and determined by pulsed amperometric detection. The ulvanobiouronic acid, β-D-GlcA-(1,4)-L-Rha, which is the main constituent of ulvans was always present after 3 h of trifluoroacetic acid hydrolysis (approx. 2% D.M.) when acid hydrolysis was performed alone but the xylose amount fell to 75% of its maximum value at this time. The optimal duration of 2 mol L−1 trifluoroacetic acid hydrolysis of ulvans (i.e. without any degradation of xylose, rhamnose and glucuronic acid) was 45 min. Additionnal treatment of the partial acid hydrolysate by purified β-D-glucuronidase allowed the hydrolysis of the residual ulvanobiouronic acid in rhamnose and glucuronic acid. High performance anion exchange chromatography coupled to this chemical-enzymic hydrolysis revealed to be a high resolution chromatographic technique for monitoring the hydrolysis of the aldobiouronic acid by β-D-glucuronidase. This method allowed the simultaneous quantitative determination of neutral and acidic sugars and revealed the presence of iduronic acid inulvans.

Developments on gelling algal galactans, their structure and physico-chemistry

The wide uses of the algal galactans agar, agarose and carrageenans arebased on their unique properties to form strong gels in aqueous solutions. These gels result from peculiar regular chemical structures, specific orderedmolecular conformations and aggregations. In recent years, newmethodologies and instruments have provided a more accurate view of therelationships between the chemical structure and the gelling characteristicsof these complex hybrid and heterogeneous polysaccharides. Methanolysisand reductive acid hydrolysis procedures coupled to differentchromatographic separations allowing the quantitative determination of allthe constituent sugars including the acid labile 3,6-anhydyrogalactose areparticularly emphasised. Means of determining sugar linkages, substitutionsand sequences using chemical, enzymatic and spectroscopic methods arealso presented. Developments in multi- and low-angle laser-light diffusiondetectors coupled to high performance size exclusion chromatography nowrender the determination of molecular weight and molecular weightdistribution of these galactans more accessible. Such techniques also yieldnew information on the aggregate formation of these sulphatedpolysaccharides. These and other data question the existence of thegenerally assumed intertwined double helical conformations of thesegalactans during gel formation.

In vitro fermentation by human faecal bacteria of total and purified dietary fibres from brown seaweeds

The in vitro degradation of dietary fibre from three brown seaweeds (Himanthalia elongata, Laminaria digitata and Undaria pinnatifida) was studied, using human faecal flora. Two sets of fibre were tested: (1) total algal fibres extracted from the whole algae, mainly composed of alginates, and (2) purified fibres (sulphated fucans, Na-alginates and laminarans) representative of those contained in the whole brown algae. Mannuronate, one algal component, was also investigated. Substrate disappearance and short-chain fatty acid (SCFA) production were monitored after 6, 12 and 24 h fermentation. Gas production was followed hourly during the first 9 h and then at 12 and 24 h. Sugarbeet fibre was used as a fermentation reference substrate. According to the fermentative indices used, most of each of the total algal fibres disappeared after 24 h (range 60-76%) but, unlike the reference substrate, they were not completely metabolized to SCFA (range 47-62%). Among the purified algal fibres, disappearance of laminarans was approximately 90% and metabolism to SCFA was approximately 85% in close agreement with the fermentation pattern of reference fibres. Sulphated fucans were not degraded. Na-alginates exhibited a fermentation pattern quite similar to those of the whole algal fibres with a more pronounced discrepancy between disappearance and production of SCFA: disappearance was approximately 83% but metabolism was only approximately 57%. Mannuronate was slowly fermented but its metabolism corresponded to its disappearance from the fermentative medium. Thus, the characteristic fermentation pattern of the total fibres from the three brown algae investigated was attributed to the peculiar fermentation of alginates, and mannuronate was shown not to be directly involved.