A comprehensive analysis was carried out of the composition of seed coat mucilage from Arabidopsis thaliana using the Columbia-0 accession. Pectinaceous mucilage is released from myxospermous seeds upon imbibition, and in Arabidopsis consists of a water-soluble, outer layer and an adherent, inner layer. Analysis of monosaccharide composition in conjunction with digestion with pectolytic enzymes conclusively demonstrated that the principal pectic domain of both layers was rhamnogalacturonan I, and that in the outer layer this was unbranched. The macromolecular characteristics of the water-soluble mucilage indicated that the rhamnogalacturonan molecules in the outer layer were in a slightly expanded random-coil conformation. The inner, adherent layer remained attached to the seed, even after extraction with acid and alkali, suggesting that its integrity was maintained by covalent bonds. Confocal microscopy and monosaccharide composition analyses showed that the inner layer can be separated into two domains. The internal domain contained cellulose microfibrils, which could form a matrix with RGI and bind it to the seed. In effect, in the mum5-1 mutant where most of the inner and outer mucilage layers were water soluble, cellulose remained attached to the seed coat. Immunolabeling with anti-pectin antibodies indicated the presence of galactan and arabinan in the inner layer, with the latter only present in the non-cellulose-containing external domain. In addition, JIM5 and JIM7 antibodies labeled different domains of the inner layer, suggesting the presence of stretches of homogalacturonan with different levels of methyl esterification.

SummaryPectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions.

Pectins of varying structures were tested for their ability to interact with cellulose in comparison to the well-known adsorption of xyloglucan. Our results reveal that sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectins, which are rich in neutral sugar side chains, can bind in vitro to cellulose. The extent of binding varies with respect to the nature and structure of the side chains. Additionally, branched arabinans (Br-Arabinans) or debranched arabinans (Deb-Arabinans; isolated from sugar beet) and galactans (isolated from potato) were shown bind to cellulose microfibrils. The adsorption of Br-Arabinan and galactan was lower than that of Deb-Arabinan. The maximum adsorption affinity of Deb-Arabinan to cellulose was comparable to that of xyloglucan. The study of sugar beet and potato alkali-treated cell walls supports the hypothesis of pectin-cellulose interaction. Natural composites enriched in arabinans or galactans and cellulose were recovered. The binding of pectins to cellulose microfibrils may be of considerable significance in the modeling of primary cell walls of plants as well as in the process of cell wall assembly.The well-known model of primary cell walls (PCWs) of dicotyledons emphasizes noncovalent interactions between cell wall polymers and suggests two independent, but interacting, networks where the cellulose-xyloglucan network is embedded in a matrix of pectic polysaccharides (Carpita and Gibeaut, 1993;Somerville et al., 2004).Cellulose, the primary structural element of the cell wall, is a homopolymer composed of (1/4)-linked b-D-Glcp residues. The linear chains of parallel alignment are tightly linked by hydrogen bonds to form microfibrils. Xyloglucan, the most abundant hemicellulosic polysaccharide in the PCWs of dicotyledons, is composed of a cellulose-like backbone consisting of (1/4)-linked b-D-Glcp residues, branched at O-6 by a-D-Xylp residues, which can be further substituted at O-2 by b-D-Galp residues (Fry, 1989). Some of the Galp residues may be substituted at O-6 by a-D-Fucp. Pectins are major components of dicotyledon PCWs and of the middle lamella. Their backbone is composed of smooth homogalacturonan (HG) and hairy rhamnogalacturonan (RG) I regions (O'Neill et al., 1990). HG, a linear chain composed of (1/4)-linked a-D-GalUAp units, can be methyl esterified at O-6 of carboxyl groups and acetyl esterified at O-2 and/or O-3 of secondary hydroxyl groups (Ralet et al., 2001). Some HGs might be substituted to form RG II or xylogalacturonan. RG II is a complex polysaccharide composed of GalUAp, Rhap, Galp, and some unusual sugars.Dimers of RG II were found to be cross-linked by two diester bonds through a boron atom (Fleischer et al., 1999). Xylogalacturonan contains b-D-Xylp residues attached to O-3 of the HG backbone (Le Goff et al., 2001). RG I contains a backbone of the repeating di- Renard et al., 1995). They are predominantly substituted at O-4 of Rhap residues by neutral sugar side chains (Schols and Voragen, 1994). The proportion of branched Rhap residues depen...

Imbibed Arabidopsis (Arabidopsis thaliana) seeds are encapsulated by mucilage that is formed of hydrated polysaccharides released from seed coat epidermal cells. The mucilage is structured with water-soluble and adherent layers, with cellulose present uniquely in an inner domain of the latter. Using a reverse-genetic approach to identify the cellulose synthases (CESAs) that produce mucilage cellulose, cesa5 mutants were shown to be required for the correct formation of these layers. Expression of CESA5 in the seed coat was specific to epidermal cells and coincided with the accumulation of mucilage polysaccharides in their apoplast. Analysis of sugar composition showed that although total sugar composition or amounts were unchanged, their partition between layers was different in the mutant, with redistribution from adherent to water-soluble mucilage. The macromolecular characteristics of the water-soluble mucilage were also modified. In accordance with a role for CESA5 in mucilage cellulose synthesis, crystalline cellulose contents were reduced in mutant seeds and birefringent microfibrils were absent from adherent mucilage. Although the mucilage-modified5 mutant showed similar defects to cesa5 in the distribution of sugar components between water-soluble and adherent mucilage, labeling of residual adherent mucilage indicated that cesa5 contained less cellulose and less pectin methyl esterification. Together, the results demonstrate that CESA5 plays a major and essential role in cellulose production in seed mucilage, which is critical for the establishment of mucilage structured in layers and domains.