Novel rhamnogalacturonan I and arabinoxylan polysaccharides of flax seed mucilage

Abstract

The viscous seed mucilage of flax (Linum usitatissimum) is a mixture of rhamnogalacturonan I and arabinoxylan with novel side group substitutions. The rhamnogalacturonan I has numerous single nonreducing terminal residues of the rare sugar l-galactose attached at the O-3 position of the rhamnosyl residues instead of the typical O-4 position. The arabinoxylan is highly branched, primarily with double branches of nonreducing terminal l-arabinosyl units at the O-2 and O-3 positions along the xylan backbone. While a portion of each polysaccharide can be purified by anion-exchange chromatography, the side group structures of both polysaccharides are modified further in about one-third of the mucilage to form composites with enhanced viscosity. Our finding of the unusual side group structures for two well-known cell wall polysaccharides supports a hypothesis that plants make a selected few ubiquitous backbone polymers onto which a broad spectrum of side group substitutions are added to engender many possible functions. To this end, modification of one polymer may be accompanied by complementary modifications of others to impart functions to heterocomposites not present in either polymer alone.

Figure 1.

Micrographs of fresh samples of Arabidopsis and flax seeds in the presence or absence of 0.2% (w/v) ruthenium red (Sigma) in water containing 0.1% Tween 20 for 18 h. Samples without ruthenium red were then stained with the reagent 3 h before photography. A, Arabidopsis seeds placed directly in ruthenium red solution. B, Flax seeds placed directly in ruthenium red solution. C, Arabidopsis seeds incubated in water overnight, swirled gently, and stained with ruthenium red. D, Flax seeds incubated in water overnight, swirled gently, and stained with ruthenium red.

Figure 2.

Separation of partly methylated alditol acetates by gas chromatography reveals greater complexity of the flax mucilage. A, Arabidopsis mucilage. B, Flax mucilage. Partly methylated alditol acetates were made after carboxyl reduction with CMC and NaBD₄ to generate 6,6-dideutero-hexitols of Gal and Glc to differentiate the respective uronic acid from its neutral sugar. Peaks designated as GalA are 6,6-dideuterogalactosyl residues. Elution profiles and linkage structures were determined as described previously (Carpita and Shea, 1989).

Figure 3.

Electron-impact mass spectra confirm the 2,3-Rha (A) and 2,4-Rha (B) branch point residues. The anomeric carbons of all partly methylated sugars are reduced with NaBD₄ to tag all fragments containing the C-1. Cleavage on either side of the C-4 methoxylated carbon gives m/z 262 and m/z 131 as major primary fragments, unequivocally showing the C-2 and C-3 acetylations indicative of the 2,3-Rha branch point residue. In contrast, cleavage on either side of the C-3 methoxylated carbon gives m/z 190 and m/z 203 as major primary fragments, unequivocally showing the C-2 and C-4 acetylations indicative of the typical 2,4-Rha branch point residue. [See online article for color version of this figure.]

Figure 4.

Separation of three fractions of the flax mucilage by anion-exchange chromatography. The mucilage polysaccharides dissolved in 10 mL of 30 mm ammonium acetate, pH 5.2, were loaded on a 2.5-cm × 10-cm column of Sephadex A-25 (Sigma) equilibrated in the same buffer and, after a 5-mL elution in 30 mm running buffer, were separated in an 80-mL gradient of ammonium acetate, pH 5.2, from 30 mm to 1.2 m. Carbohydrate in 1-mL samples was determined by the phenol-sulfuric acid assay (Dubois et al., 1956). Three fractions were pooled as designated in gray, dialyzed against deionized water, and freeze dried for further analysis.

Figure 5.

l-Gal is a side group substituent of the mucilage RG I, and d-Gal is associated with the arabinoxylan. A, Arabinoxylan. B, RG I. Arabinoxylan and RG I were hydrolyzed to monosaccharide by TFA, and the Gal in dry residues dissolved in water was oxidized by l-Gal and d-Gal dehydrogenase. The resultant monosaccharides were separated by high-performance anion-exchange chromatography to show the relative amounts of each Gal moiety remaining. PAD, Pulsed-amperometric detection. [See online article for color version of this figure.]

Figure 6.

A tight association of the arabinoxylan and RG I enhances mucilage viscosity. About 100 μL of 1 mg mL⁻¹ solutions in water was assayed by spinning-disc viscometry. The arabinoxylan, RG I, and composite materials were collected after anion-exchange chromatography as described for Figure 4. Values (in millipascals per second at 20°C) are relative to water (1 mPa s⁻¹ at 20°C) and were independent of shear rate. The arrowhead represents the expected additive contributions of arabinoxylan (AX)-void and RG I in the in vitro mixture. [See online article for color version of this figure.]