Oriented structure of pectic polysaccharides in pea epidermal cell walls

Polarized infrared absorption spectra of film specimens of theepidermal cell wall of the third internode of pea stems wererecorded before and after treatment with endopolygalacturonase(endo-PG) and endo-pectin lyase (endo-PL). The spectra showedthat the pectic polysaccharides solubilized with endo-PG wereessentially the same as those solubilized with endo-PL. Thedegree of esterification of the pectic polysaccharides was about20%, and their major sugar components were uronic acids (32.8%),arabinose (48.1%) and galactose (19.2%). The polarized infraredspectra showed that pectic polysaccharides have an orientedstructure in cell walls with their molecular chains orientedpreferentially parallel to the direction of cell elongation. ¹Present address: Research and Development, Kanzaki Paper Mfg.Co., Ltd., Amagasaki, Hyogo 660, Japan. ²Present address: Wakayama Research Laboratories, Kao Soap Co.,Ltd., Wakayama 640-91, Japan. (Received June 28, 1980; )     

Mixed-linkage (1-->3,1-->4)-beta-D-glucan is a major hemicellulose of Equisetum (horsetail) cell walls

Mixed-linkage (1-->3,1-->4)-beta-d-glucan (MLG) is a hemicellulose reputedly confined to certain Poales. Here, the taxonomic distribution of MLG, and xyloglucan, especially in early-diverging pteridophytes, has been re-investigated. Polysaccharides were digested with lichenase and xyloglucan endoglucanase (XEG), which specifically hydrolyse MLG and xyloglucan, respectively. The oligosaccharides produced were analysed by thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and alkaline peeling. Lichenase yielded oligo-beta-glucans from all Equisetum species tested (Equisetum arvense, Equisetum fluviatile, Equisetum scirpoides, Equisetum sylvaticum and Equisetum xtrachyodon). The major product was the tetrasaccharide beta-glucosyl-(1-->4)-beta-glucosyl-(1-->4)-beta-glucosyl-(1-->3)-glucose (G4G4G3G), which was converted to cellotriose by alkali, confirming its structure. Minor products included G3G, G4G3G and a nonasaccharide. By contrast, poalean MLGs yielded G4G3G > G4G4G3G > nonasaccharide > dodecasaccharide. No other pteridophytes tested contained MLG, including Psilotum and eusporangiate ferns. No MLG was found in lycopodiophytes, bryophytes, Chara or Nitella. XEG digestion showed that Equisetum xyloglucan has unusual repeat units. Equisetum, an exceedingly isolated genus whose closest living relatives diverged > 380 million years ago, has evolved MLG independently of the Poales. Equisetum and poalean MLGs share basic structural motifs but also exhibit clear-cut differences. Equisetum MLG is firmly wall-bound, and may tether neighbouring microfibrils. It is also suggested that MLG acts as a template for silica deposition, characteristic of grasses and horsetails.     

An enzymatically produced novel cyclomaltopentaose cyclized from amylose by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}

A bacterial strain AM7, isolated from soil and identified as Bacillus circulans, produced two kinds of novel cyclic oligosaccharides. The cyclic oligosaccharides were produced from amylose using a culture supernatant of the strain as the enzyme preparation. The major product was a cyclomaltopentaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. The other minor product was cyclomaltohexaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one alpha-(1-->6)-linkage. ICG5 was digested by alpha-amylase derived from Aspergillus oryzae, cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus, and maltogenic alpha-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans, and maltogenic alpha-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an alpha-(1-->6)-linkage in their molecules.     

Cell wall pectic (1-->4)-beta-d-galactan marks the acceleration of cell elongation in the Arabidopsis seedling root meristem

Here we demonstrate that the pectic rhamnogalacturonan-I-associated LM5 (1-->4)-beta-d-galactan epitope occurs in a restricted manner at the root surface of intact Arabidopsis seedlings. The root surface occurrence of (1-->4)-beta-d-galactan marks the transition zone at or near the onset of rapid cell elongation and the epitope is similarly restricted in occurrence in epidermal, cortical and endodermal cell walls. The extent of surface (1-->4)-beta-d-galactan occurrence is reduced in response to genetic mutations (stp-1, ctr-1) and hormone applications that reduce root cell elongation. In contrast, the application of the arabinogalactan-protein (AGP) binding beta-glucosyl Yariv reagent (betaGlcY) that disrupts cell elongation results in the persistence of (1-->4)-beta-d-galactan at the root surface and in epidermal, cortical and endodermal cell walls. This latter observation indicates that modulation of pectic (1-->4)-beta-d-galactan may be an event downstream of AGP function during cell expansion in the Arabidopsis seedling root.     

Mixed-linkage (1-->3),(1-->4)-beta-D-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls

Mixed-linkage (1-->3),(1-->4)-beta-D-glucan (MLG) is widely considered to be a defining feature of the cell walls of plants in the Poales order. However, we conducted an extensive survey of cell-wall composition in diverse land plants and discovered that MLG is also abundant in the walls of the horsetail Equisetum arvense. MALDI-TOF MS and monosaccharide linkage analysis revealed that MLG in E. arvense is an unbranched homopolymer that consists of short blocks of contiguous 1,4-beta-linked glucose residues joined by 1,3-beta linkages. However, in contrast to Poaceae species, MLG in E. arvense consists mostly of cellotetraose rather than cellotetriose, and lacks long 1,4-beta-linked glucan blocks. Monosaccharide linkage analyses and immunochemical profiling indicated that, in E. arvense, MLG is a component of cell walls that have a novel architecture that differs significantly from that of the generally recognized type I and II cell walls. Unlike in type II walls, MLG in E. arvense does not appear to be co-extensive with glucuroarabinoxylans but occurs in walls that are rich in pectin. Immunofluorescence and immunogold localization showed that MLG occurs in both young and old regions of E. arvense stems, and is present in most cell types apart from cells in the vascular tissues. These findings have important implications for our understanding of cell-wall evolution, and also demonstrate that plant cell walls can be constructed in a way not previously envisaged.     

An enzymatically produced novel cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->} (cyclic maltosyl-(1-->6)-maltose), from starch

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1-->4)- and alpha-(1-->6)-linkages. The cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages.     

Synthesis of methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside and methyl alpha-D-xylo-hex-4-ulopyranosyl-(1-->4)-alpha-D-galactopyranoside

The syntheses of methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside (1) and methyl alpha-D-xylo-hex-4-ulopyranosyl-(1-->4)-alpha-D-galactopyranoside (4) are reported. The keto-disaccharide 4 is of interest in our design, synthesis, and study of pectate lyase inhibitors. The key step in the syntheses was the high-yielding, stereospecific formation of methyl 4,6-O-benzylidene-2',3'-di-O-benzyl-alpha-D-glucopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (15), which was accomplished by reacting 2,3-di-O-benzyl-4,6-O-benzylidene-D-glucopyranosyl trichloroacetimidate (10) with methyl 2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (14) in the presence of a catalytic amount of tert-butyldimethylsilyl trifluoromethane sulfonate (TMSOTF). Compound 15 was either hydrogenolyzed to yield disaccharide 1 or treated with NaBH3CN-HCl in 1:1 tetrahydrofuran-ether to yield methyl 2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-alpha-D-galactopyranoside (2). The free 4'-OH of compound 2 was oxidized to a carbonyl group by a Swern oxidation, and the protecting groups were removed by hydrogenolysis to yield keto-disaccharide 4. These synthetic pathways were simple, yet high yielding.     

Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant QUASIMODO 1 suspension-cultured cells: implications for cell adhesion

Mutation in the Arabidopsis thaliana QUASIMODO 1 gene (QUA1), which encodes a putative glycosyltransferase, reduces cell wall pectin content and cell adhesion. Suspension-cultured calli were generated from roots of wild-type (wt) and qua1-1 A. thaliana plants. The altered cell adhesion phenotype of the qua1-1 plant was also found with its suspension-cultured calli. Cell walls of both wt and qua1-1 calli were analysed by chemical, enzymatic and immunohistochemical techniques in order to assess the role of pectic polysaccharides in the mutant phenotype. Compared with the wt, qua1-1 calli cell walls contained more arabinose (23.6 versus 21.6 mol%), rhamnose (3.1 versus 2.7 mol%), and fucose (1.4 versus 1.2 mol%) and less uronic acid (24.2 versus 27.6 mol%), and they were less methyl-esterified (DM: 22.9% versus 30.3%). When sequential pectin extraction of calli cell walls was performed, qua1-1 water-soluble and chelator-soluble extracts contained more arabinose and less uronic acid than wt. Water-soluble pectins were less methyl-esterified in qua1-1 than in wt. Chelator-soluble pectins were more acetyl-esterified in qua1-1. Differences in the cell wall chemistry of wt and mutant calli were supported by a reduction in JIM7 labelling (methyl-esterified homogalacturonan) of the whole wall in small cells and particularly by a reduced labelling with 2F4 (calcium-associated homogalacturonan) in the middle lamella at tricellular junctions of large qua1-1 cells. Differences in the oligosaccharide profile obtained after endopolygalacturonase degradation of alkali extracts from qua1-1 and wt calli indicated variations in the structure of covalently bonded homogalacturonan. About 29% more extracellular polymers rich in pectins were recovered from the calli culture medium of qua1-1 compared with wt. These results show that perturbation of QUASIMODO 1-1 gene expression in calli resulted in alterations of homogalacturonan content and cell wall location. The consequences of these structural variations are discussed with regard to plant cell adhesion.     

Multi‐scale spatial heterogeneity of pectic rhamnogalacturonan I (RG–I) structural features in tobacco seed endosperm cell walls

Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG–I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG–I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG–I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG–I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG–I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG–I glycan complexity in the context of cell‐wall architectures and in relation to cell‐wall functions in cell and tissue development.     

Synthesis and biological activities of octyl 2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-beta-L-fucopyranoside

Octyl 2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-beta-L-fucopyranoside, a fucosyl pentasaccharide with a regular structure resembling the repeating unit of a natural sulfated fucan, was chemically synthesized using a convergent '2+3' strategy. Regioselective 3-O-silylation of beta-thiofucopyranoside and AgOTf-catalyzed glycosylation of the protected glycosyl trichloroacetimidate facilitated a one-pot trisaccharide synthesis. The synthesized target compound showed good antitumor activity in vivo, and promising anticoagulant activity in vitro.     

Synthesis of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->3)]-beta-D-GlcpOLauryl, an oligosaccharide with anti-tumor activity

A concise and effective synthesis of lauryl heptasaccharide 17 was achieved from the key intermediates lauryl 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4-di-O-benzoyl-beta-D-glucopyranoside (10) and isopropyl 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-1-thio-beta-D-glucopyranoside (15). The key trisaccharide glycosyl acceptor 10 was constructed by coupling 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (3) with lauryl 6-O-acetyl-2,4-di-O-benzoyl-beta-D-glucopyranoside (9), followed by deacetylation. The thioglycoside donor 15 was obtained by condensation of 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (12), followed by debenzylidenation and acetylation. A bioassay of the inhibition of S180 noumenal tumors showed that lauryl heptasaccharide 17 could be employed as a potential agent for cancer treatment.     

Gram-scale syntheses of the (1-->3)-linked and (1-->4)-linked hyaluronan disaccharides

The first gram-scale syntheses of two hyaluronan disaccharides are described. Construction of the (1-->4)-linked disaccharide 12 was achieved in 12% overall yield using 2,3-bis-dimethyl acetal protection in combination with chlorosilane-induced carbamate cleavage methodologies. The uronic acid functionality was installed using TEMPO oxidation with NaOCl as the hypochlorite source. The (1-->3)-linked disaccharide 18 was achieved in 7% overall yield utilizing acetonide protection in addition to the chlorosilane-induced carbamate cleavage methodology and the TEMPO oxidation.     

A concise and practical synthesis of antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc

A concise and practical synthesis of the antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc (13), was achieved by coupling of a monosaccharide donor, 3-O-allyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-galactopyranosyl trichloroacetimidate (4) with a disaccharide acceptor, p-methoxyphenyl 2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (8), followed by deprotection. In spite of the existence of a C-2-ester substituent capable of neighboring-group participation in the donor, the coupling gave exclusively the alpha-linkage in satisfactory yield. The acceptor 8 was readily obtained from selective 3-O-benzoylation of the galactosyl ring of p-methoxyphenyl 2,6-di-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (7), which was prepared from p-methoxyphenyl beta-D-lactoside (5) via isopropylidenation, benzoylation, and deisopropylidenation. Donor 4 was obtained from p-methoxylphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside (1) via selective 4,6-di-O-debenzoylation, oxidative removal of 1-O-MP, benzylidenation, and trichloroacetimidate formation.     

Molecular cohesion in plant cell walls. Methylation analysis of pectic polysaccharides from the cotyledons of white mustard

Methylation analysis was used to characterize the pectic polysaccharides from mustard cotyledons, a tissue with potential for rapid biological change involving the walls. The methylated sugars were identified by g.l.c. and paper chromatography after conversion of uronic acid derivatives into [(3)H]hexoses, and confirmed by the formation of crystalline derivatives of most of the main products, which were: 2,3-di-O-methyl-d-[6-(3)H]galactose, 2-O-methyl-d-[6-(3)H]galactose, 3,4-di-O-methylrhamnose, 3-O-methylrhamnose, 2,3,5-tri-O-methyl-l-arabinose, 2,3-di-O-methyl-l-arabinose, 2-O-methyl-l-arabinose, 2,3,4-tri-O-methyl-d-xylose and 2,3,4,6-tetra-O-methyl-d-galactose in the molar proportions 1.00:1.14:0.54:0.74:2.86:2.50:2.24:1.88:0.32. The structural units present are similar to those in wellknown polysaccharides from mature tissues, but their proportions are strikingly different. Uninterrupted and unbranched galacturonan segments can therefore contribute little cohesion to these walls, and it is suggested that this correlates with a function of the wall matrix to hydrate and permit readjustment, during germination, of structural elements or wall surfaces or both.     

Synthesis of divalent beta-(1-->6)-branched (1-->3)-glucohexaose and trivalent beta-(1-->6)-branched (1-->3)-glucotriose

Hexaose, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp, based dimers were synthesized by twofold glycosidation of the hexaosyl trichloroacetimidate with hexylene 1,6-diol, diethylene glycol and triethylene glycol, respectively. Meanwhile, a triose, beta-1D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp, based trimer was obtained by glycosidation of the triosyl trichloroacetimidate with a glycerol-derived triol scaffold.     

Chromosomal Location of Genes Encoding Barley (1-->3, 1-->4)-beta-Glucan 4-Glucanohydrolases

Preparations of DNA from wheat (Triticum aestivum, cv Chinese Spring), barley (Hordeum vulgare, cv Betzes) and six euplasmic wheat-barley addition lines were digested to completion with restriction endonucleases and the products probed by Southern blot analysis using a cDNA-encoding barley (1→3, 1→4)-β-glucanase isoenzyme II. It is shown that one of the barley (1→3, 1→4)-β-glucanase genes is located on chromosome 1.     

Synthesis of a chitosan tetramer derivative, beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->4)-D-Glc N through a partial N-acetylation reaction by chitin deacetylase

We have synthesized beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->4)-D-GlcN (2) through a partial N-acetylation reaction of chitosan tetramer 1 by a chitin deacetylase from Colletotrichum lindemuthianum ATCC 56676. The compound was purified from the mixture of acetylation products of 1 using cation-exchange column chromatography and amine-adsorption column chromatography, and its structure was estimated by 1H NMR and FABMS analyses. The enzymatic reaction allows a regioselectivity that is hard to achieve by chemical N-acetylation.     

Glutamate synthase in developing pea cotyledons: Occurrence of NADH-dependent and ferredoxin-dependent enzymes

NADH-dependent and ferredoxin-dependent glutamate synthasesfrom developing pea cotyledons were separated by gel filtrationon a Sephadex G-200 column. The substrate requirements, molecularweights and effect of some inhibitors on both glutamate synthaseenzymes were investigated. ¹ Department of Agricultural Chemistry, Kyoto University, Kyoto606, Japan. ² To whom inquiries should be addressed. (Received August 9, 1979; )     

Regulation of pectic polysaccharide domains in relation to cell development and cell properties in the pea testa

The occurrence of pectic polysaccharide epitopes in cells and tissues of the pea testa during late stages of seed development have been examined in relation to anatomy and cell properties. Homogalacturonan, in a highly methyl-esterified form, was present throughout late development in all pea testa cell walls, including the thickened cell walls of the outer macrosclereid layer. Two epitopes, characteristic of the side-chains of the rhamnogalacturonan-I domain of pectic polysaccharides, occurred in restricted and separate cell layers of the pea testa. A (1-->4)-beta-D-galactan epitope was restricted to regions of the outer cell wall of the testa and to inner regions of the macrosclereid layer at 20 DAA and was absent from the osteosclereid and parenchyma cell walls. By 25 DAA the (1-->4)-beta-D-galactan epitope occurred only in the outer epidermal cell walls. A (1-->5)-alpha-L-arabinan epitope was also dependent on the developmental stage of the seed and was found with greatest abundance in the walls of the inner parenchyma cells. Cell separation studies indicated that, although calcium cross-links were involved in the maintenance of the link between the macrosclereid layer and proximal cell layers, most cell-to-cell adhesion in the testa was not due to calcium- or ester-based bonds.     

Identification of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-d-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->)

A structural characterization of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was carried out by NMR spectroscopy. H-1', 2'-OH, H-3', and 4'-OH of the 3-O-glycosylated residue and H-1 of the 6-O-glycosylated residue were found to cross-relax with protons of bound waters using the double-pulsed field-gradient spin-echo ROESY experiment. In the crystal structure, one water molecule is located in the center of the plate, and its temperature factor is very low, indicating that this water molecule is an intrinsic component.