J. S. Grant Reid

Structural aspects of the interaction of mannan-based polysaccharides with bacterial cellulose

Interactions between cellulose and glucomannan/galactomannans have been studied through molecular and ultrastructural analysis of composites formed by deposition of cellulose from Acetobacter aceti ssp. xylinum into solutions containing either glucomannan or galactomannans of varying Man:Gal ratio. 13C NMR suggests that unsubstituted mannan segments can bind to cellulose by undergoing a conformational transition to an extended 2-fold form. Konjac glucomannan induces a coalescence of cellulose fibrils and a dramatic reduction of crystallinity: low galactose galactomannans also show these trends. The inferred mannan/cellulose sandwich structure may underlie the densification processes which can accompany mannan-containing secondary cell wall formation. Medium galactose galactomannans additionally form cross- links of varying lengths between cellulose fibrils and show evidence for self-association. The presence of cellulose fibrils promotes network formation from galactomannan solutions under conditions where this would not normally occur. The principles of interaction between cellulose and mannan- based polymers at both molecular and ultrastructural levels are discussed in the context of plant cell wall design and assembly. Interactions between cellulose and glucomannan/galactomannans have been studied through molecular and ultrastructural analysis of composites formed by deposition of cellulose from Acetobacter aceti ssp. xylinum into solutions containing either glucomannan or galactomanannans of varying Man:Gal ratio. 13C NMR suggests that unsubstituted mannan segments can bind to cellulose by undergoing a conformational transition to an extended 2-fold form. Konjac glucomannan induces a coalescence of cellulose fibrils and a dramatic reduction of crystallinity: low galactose galactomannans also show these trends. The inferred mannan/cellulose sandwich structure may underlie the densification processes which can accompany mannan-containing secondary cell wall formation. Medium galactose galactomannans additionally form cross-links of varying lengths between cellulose fibrils and show evidence for self-association. The presence of cellulose fibrils promotes network formation from galactomannan solutions under conditions where this would not normally occur. The principles of interaction between cellulose and mannan-based polymers at both molecular and ultrastructural levels are discussed in the context of plant cell wall design and assembly.

Cell Wall Storage Carbohydrates in Seeds—Biochemistry of the Seed “Gums” and “Hemicelluloses”

Publisher Summary In recent years, there has been a reawakening of interest in the physiology and biochemistry of the cell wall storage carbohydrates of seeds. This chapter outlines the structures and occurrence of cell wall storage carbohydrates to give an account of current research on their metabolism and to explore their overall biological significance in the seeds which contain them. Seeds are generally treated with alkali to extract polysaccharides of the “hemicellulose” type or with water to extract “gum” polysaccharides. Consequently, the molecules with which this article is concerned are still widely classified as seed gums and hemicelluloses. The chapter indicates the principal types of carbohydrate molecules stored in the cell walls of seeds and their distribution.

Xyloglucan (amyloid) mobilisation in the cotyledons of Tropaeolum majus L. seeds following germination

The levels of cell-wall xyloglucan (amyloid) in nasturtium (Tropaeolum majus L.) cotyledons were monitored during a 28-d period covering seed imbibition, germination and early seedling development. The activities of the following enzymes capable of hydrolysing the glycosidic linkages in the xyloglucan were assayed in cotyledon extracts over the same period: endo-(1→4)-β-glucanase (EC 3.2.1.4), β-glucosidase (EC 3.2.1.21), α-xylosidase and β-galactosidase (EC 3.2.1.23). The endo-β-glucanase was assayed viscometrically using xyloglucan as substrate, and the three glycosidases using appropriate p-nitrophenylglycosides. Alpha xylosidase and β-galactosidase, the enzymes which would be expected to hydrolyse the side-chains from the xyloglucan molecule, were also assyed using xyloglucan as substrate. Under our culture conditions, xyloglucan levels remained constant at 30 mg per cotyledon pair for 7 d, that is until 3 d after germination: thereafter, the amount of xyloglucan diminished to zero in a 12-d period. The most rapid period of depletion was between days 9 and 13. The mobilisation of all reserve substances from the cotyledons resulted in a weight-loss of 92 mg: xyloglucan, therefore, is an important storage substance, representing 33% by weight of the seeds substrate reserves. It is a cell-wall storage polysaccharide. Xyloglucan mobilisation was accompanied by a 17-fold increase in endo-β-glucanase activity, a 7-fold increase in β-galactosidase and an 8-fold increase in α-xylosidase activities, all determined using xyloglucan as substrate. All three activities began to increase at day 5, peaked at days 12–14 when the most rapid phase of xyloglucan breakdown was over, and had declined to zero by days 22–25. The levels of theses enzymes have been shown to be consistent with their being responsible for xyloglucan hydrolysis in vivo. Nitrophenyl-β-galactosidase activity increased up to day 3, remained constant and then increased again 2.5-fold from day 5, peaking at day 11. Nitrophenyl-β-glucosidase remained relatively constant up to day 16 and then decreased to zero by day 25. Nitrophenyl-α-xylosidase activity was not detected.

Purification and properties of a novel β-galactosidase or exo-(1→4)-β-D-galactanase from the cotyledons of germinated Lupinus angustifolius L. seeds

The main polysaccharide component of the thickened cell walls in the storage parenchyma of Lupinus angustifolius L. cotyledons is a linear (1 → 4)-β-linked d-galactan, which is mobilised after germination (L.A. Crawshaw and J.S.G Reid, 1984, Planta 160, 449–454). The isolation from the germinated cotyledons of a β-d-galactosidase or exo-(1 → 4)-β-d-galactanase with a high specificity for the lupin galactan is described. The enzyme, purified using diethylaminoethyl-cellulose, carboxymethyl-cellulose and affinity chromatography on lactose-agarose, gave two bands (major 60 kDa, minor 45 kDa) on sodium dodecyl sulphate-gel electrophoresis, and two similar bands on isoelectric focusing (major, pI 7.0, minor pI 6.7, both apparently possessing enzyme activity). The minor component cross-reacted with an antiserum raised against, and affinity-purified on, the major band. Both components had a common N-terminal sequence. The minor component was probably a degradation product of the major one. The enzyme had limited β-galactosidase action, catalysing the hydrolysis of p-nitrophenyl-β-d-galactopyranoside and (1→ 4)- and (1 → 6)-β-linked galactobioses. Lactose [β-d-galactopyranosyl-(1 → 4)-d-glucose] was hydrolysed only very slowly and methyl-β-d-galactopyranoside not at all. Lupin galactan was hydrolysed rapidly and extensively to galactose, whereas other cell-wall polysaccharides (xyloglucan and arabinogalactan) with terminal non-reducing β-d-galactopyranosyl residues were not substrates. A linear (1 → 4)-β-linked galactopentaose was hydrolysed efficiently to the tetraose plus galactose, but further sequential removals of galactose to give the tetraose and lower homologues occurred more slowly. Galactose, γ-galactonolactone and Cu+2 were inhibitory. No endo-β-d-galactanase activity was detected in lupin cotyledonary extracts, whereas exo-galactanase activity varied pari passu with galactan mobilisation. Exo-galactanase protein was detected, by Western immunoblotting of cotyledon extracts, just before the activity could be assayed and then increased and decreased in step with the enzyme activity. The exo-galactanase is clearly a key enzyme in galactan mobilisation and may be the sole activity involved in depolymerising the dominant (1 → 4)-β-galactan component of the cell wall.

Effects of structural variation in xyloglucan polymers on interactions with bacterial cellulose

A cellulose/xyloglucan framework is considered to form the basis for the mechanical properties of primary plant cell walls and hence to have a major influence on the biomechanical properties of growing, fleshy plant tissues. In this study, structural variants of xyloglucan have been investigated as components of composites with bacterial cellulose as a simplified model for the cellulose/xyloglucan framework of primary plant cell walls. Evidence for molecular binding to cellulose with perturbation of cellulose crystallinity was found for all xyloglucan types. High molecular mass samples gave homogeneous centimeter-scale composites with extensive cross-linking of cellulose with xyloglucan. Lower molecular mass xyloglucans gave heterogeneous composites having a range of microscopic structures with little, if any, cross-linking. Xyloglucans with reduced levels of galactose substitution had evidence of self-association, competitive with cellulose binding. At comparable molecular mass, fucose substitution resulted in a modest promotion of microscopic features characteristic of primary cell walls. Taken together, the data are evidence that galactose substitution of the xyloglucan core structure is a major determinant of cellulose composite formation and properties, with additional fucose substitution acting as a secondary modulator. These conclusions are consistent with reported structural and mechanical properties of Arabidopsis mutants lacking specific fucose and/or galactose residues.

Control of mannose/galactose ratio during galactomannan formation in developing legume seeds

Galactomannan deposition was investigated in developing endosperms of three leguminous species representative of taxonomic groups which have galactomannans with high, medium and low galactose content. These were fenugreek (Trigonella foenum-graecum L.; mannose/galactose (Man/Gal) = 1.1), guar (Cyamopsis tetragonoloba (L.) Taub.; Man/Gal = 1.6) and Senna occidentalis (L.) Link. (Man/Gal = 3.3), respectively. Endosperms were analysed at different stages of seed development for galactomannan content and the levels, in cell-free extracts, of a mannosyltransferase and a galactosyltransferase which have been shown to catalyse galactomannan biosynthesis in vitro (M. Edwards et al., 1989, Planta 178, 41–51). There was a close correlation in each case between the levels of the biosynthetic mannosyl- and galactosyltransferases and the deposition of galactomannan. The relative in vitro activities of the mannosyl- and galactosyltransferases in fenugreek and guar were similar, and almost constant throughout the period of galactomannan deposition. In Senna the ratio mannosyltransferase/galactosyltransferase was always higher than in the other two species, and it increased substantially throughout the period of galactomannan deposition. In fenugreek and guar the galactomannans present in the endosperms of seeds at different stages of development had the Man/Gal ratios characteristic of the mature seeds. By contrast the galactomannan present in Senna endosperms at the earliest stages of deposition had a Man/Gal ratio of about 2.3. During late deposition this ratio increased rapidly, stabilising at about 3.3, the ratio characteristic of the mature seed. The levels of α-galactosidase in the developing endosperms of fenugreek and guar were low and remained fairly constant throughout the deposition of the galactomannan. In Senna, α-galactosidase activity in the endosperm was low during early galactomannan deposition, but increased subsequently, peaking during late galactomannan deposition. The developmental patterns of the α-galactosidase activity and of the increase in Man/Gal ratio of the Senna galactomannan were closely similar, indicating a cause-and-effect relationship. The endosperm α-galactosidase activity in Senna was capable, in vitro, of removing galactose from guar galactomannan without prior depolymerisation of the molecule. In fenugreek and in guar the genetic control of the Man/Gal ratio in galactomannan is not the result of a post-depositional modification, and must reside in the biosynthetic process. In Senna, the Man/Gal ratio of the primary biosynthetic galactomannan product is controlled by the biosynthetic process. Yet the final Man/Gal ratio of the galactomannan in the mature seed is, to an appreciable extent, the result of galactose removal from the primary biosynthetic product by an α-galactosidase activity which is present in the endosperm during late galactomannan deposition.

Tobacco transgenic lines that express fenugreek galactomannan galactosyltransferase constitutively have structurally altered galactomannans in their seed endosperm cell walls

Galactomannans [(1→6)-α-d-galactose (Gal)-substituted (1→4)-β-d-mannans] are major cell wall storage polysaccharides in the endosperms of some seeds, notably the legumes. Their biosynthesis in developing legume seeds involves the functional interaction of two membrane-bound glycosyltransferases, mannan synthase (MS) and galactomannan galactosyltransferase (GMGT). MS catalyzes the elongation of the mannan backbone, whereas GMGT action determines the distribution and amount of Gal substitution. Fenugreek (Trigonella foenum-graecum) forms a galactomannan with a very high degree of Gal substitution (Man/Gal = 1.1), and its GMGT has been characterized. We now report that the endosperm cell walls of the tobacco (Nicotiana tabacum) seed are rich in a galactomannan with a very low degree of Gal substitution (Man/Gal about 20) and that its depositional time course is closely correlated with membrane-bound MS and GMGT activities. Furthermore, we demonstrate that seeds from transgenic tobacco lines that express fenugreek GMGT constitutively in membrane-bound form have endosperm galactomannans with increased average degrees of Gal substitution (Man/Gal about 10 in T1 generation seeds and about 7.5 in T2generation seeds). Membrane-bound enzyme systems from transgenic seed endosperms form galactomannans in vitro that are more highly Gal substituted than those formed by controls under identical conditions. To our knowledge, this is the first report of structural manipulation of a plant cell wall polysaccharide in transgenic plants via a biosynthetic membrane-bound glycosyltransferase.

A new polysaccharide from a traditional Nigerian plant food: Detarium senegalense Gmelin☆

The seed flour of an African leguminous plant, Detarium senegalense Gmelin, is used traditionally in Nigeria as a thickening agent in foods. Recent studies have shown that the detarium seed contains a large amount of water-soluble, non-starch polysaccharide (s-NSP), which suggests it has important nutritional properties. The aims of the present study were to characterise the structure and solution properties of purified s-NSP. The main monosaccharide residues of the extracted s-NSP were glucose, xylose, and galactose in the ratio of 1.39:1.00:0.52, suggesting structural similarity to the xyloglucan group of cell wall storage polysaccharides. This was confirmed by comparing the oligosaccharides released on endo-(1 --> 4)-beta-D-glucanase digestion with those obtained from tamarind seed xyloglucan. The intrinsic viscosity [eta] of a sample of the detarium polysaccharide was found to be 8.9 dl/g, indicating that the sample was of high molecular weight, a result confirmed by light scattering. Histochemical examination of detarium seed using bright field and epifluorescence microscopy showed the presence of xyloglucan in highly thickened cell walls, which were particularly prominent at the cell junctions.

A xyloglucan-oligosaccharide-specific ?-d-xylosidase or exo-oligoxyloglucan-?-xylohydrolase from germinated nasturtium (Tropaeolum majus L.) seeds: Purification, properties and its interaction with a xyloglucan-specific eneto-(1?4)-?-d-glucanase and other hydrolases during storage-xyloglucan mobilisation

The α-xylosidase which is involved in the postgerminative mobilisation of xyloglucan in nasturtium seed cotyledons has now been purified to apparent homogeneity by a facile procedure involving lectin affinity chromatography. The purified enzyme, a glycoprotein, moved as a single band (apparent molecular weight 85000) on sodium dodecyl sulphate-gel electrophoresis, whilst isoelectric focusing gave a number of enzymatically active protein bands spanning the range pI = 5.0 to 7.1 (maximum activity at pI = 6.1). The enzyme did not hydrolyse the simple α-xylosides p-nitrophenyl-α-d-xylopyranoside and woprimeverose (α-d-Xyl(1→6)-d-Glc), or polymeric tamarind-seed xyloglucan. It released xylose from a complex mixture of oligosaccharides produced by exhaustive hydrolysis of tamarind seed xyloglucan using the xyloglucan-specific endo-(1→4)-β-d-glucanase from germinated nasturtium seeds (M. Edwards et al. 1986, J. Biol. Chem., 261. 9489–9494). The three xyloglucan oligosaccharides of lowest molecular size were purified from this mixture and were shown by 1H-nuclear magnetic resonance (1H-NMR) and enzymatic analysis to have the structures:

The Seeds of Lotus japonicus Lines Transformed with Sense, Antisense, and Sense/Antisense Galactomannan Galactosyltransferase Constructs Have Structurally Altered Galactomannans in Their Endosperm Cell Walls

Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1→6)-α-galactose (Gal) substitution of the (1→4)-β-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense (“hairpin loop”) constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T1 generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T1 generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T2 generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.