Asparagine biosynthesis in soybean nodules

Two auxin-induced endo-1,4-β-glucanases (EC 3.2.1.4) were purified from pea (Pisum sativum L. var. Alaska) epicotyls and used to degrade purified pea xyloglucan. Hydrolysis yielded nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and heptasaccharide (glucose/xylose, 4:3) as the products. The progress of hydrolysis, as monitored viscometrically (with amyloid xyloglucan) and by determination of residual xyloglucan-iodine complex (pea) confirmed that both pea glucanases acted as endohydrolases versus xyloglucan. K_m values for amyloid and pea xyloglucans were approximately the same as those for cellulose derivatives, but V_max values were lower for the xyloglucans. Auxin treatment of epicotyls in vivo resulted in increases in net deposits of xyloglucan and cellulose in spite of a great increase (induction) of endogenous 1,4-β-glucanase activity. However, the average degree of polymerization of the resulting xyloglucan was much lower than in controls, and the amount of soluble xyloglucan increased. When macromolecular complexes of xyloglucan and cellulose (cell wall ghosts) were treated in vitro with pea 1,4-β-glucanase, the xyloglucan component was preferentially hydrolyzed and solubilized. It is concluded that xyloglucan is the main cell wall substrate for pea endo-1,4-β-glucanase in growing tissue.     

Structure of Hemicellulose Isolated from Rice Endosperm Cell Wall : Mode of Linkages and Sequences in Xyloglucan, β-Glucan and Arabinoxylan

A hemicellulosic polysaccharide, which was homogeneous on sedimentation analysis and also on electrophoresis, was isolated from the rice endosperm cell walls by the combination of alkaline extraction, ion exchange chromatography and iodine complex formation. It is composed of arabinose, xylose and glucose (molar ratio, 1.0: 2.0: 5.7) together with a small amount of galactose and rhamnose. Methylation analysis, Smith degradation and fragmentation with cellulase showed that this polysaccharide is composed of three distinct polysaccharide moieties i.e., xyloglucan, β-glucan and arabinoxylan. The xyloglucan consists of β-(1→4)-linked glucan back bone and short side chains of single xylose units or galactosylxylose both attached to C-6 of the glucose residues. The β-glucan contains both (1 →3)-and (1→4)-linkages similarly to the other cereal β-glucans, but differ from them in containing the blocks of (1→3)-linked glucose residues in the chain. The arabinoxylan has a highly branched structure, in which 78% of (1→4)-linked xylose residues have short side chains of arabinose at C-3 position.

On the basis of these findings, the interconnection of these polysaccharide moieties is discussed.     

Isolation and structural characterization of a polysaccharide FCAP1 from the fruit of Cornus officinalis

A water-soluble polysaccharide, FCAP1, was isolated from an alkaline extract from the fruits of Cornus officinalis. Its molecular weight was 34.5 kDa. Monosaccharide composition analysis revealed that it was composed of fucose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.29:0.19:1.74:1:3.30:1.10. On the basis of partial acid hydrolysis and methylation analysis, FCAP1 was shown to be a highly branched polysaccharide with a backbone of β-(1→4)-linked-glucose partially substituted at the O-6 position with xylopyranose residues. The branches were composed of (1→3)-linked-Ara, (1→4)-linked-Man, (1→4,6)-linked-Man, (1→4)-linked-Glc, and (1→2)-linked-Gal. Arabinose, fucose, and galactose were located at the terminal of the branches. The structure was further elucidated by a specific enzymatic degradation with an endo-β-(1→4)-glucanase and MALDI-TOF-MS analysis. Oligosaccharides generated from FCAP1 indicated that FCAP1 contained XXXG-type and XXG-type xyloglucan fragments.     

Pea Xyloglucan and Cellulose: VI. Xyloglucan-Cellulose Interactions in Vitro and in Vivo

Since xyloglucan is believed to bind to cellulose microfibrils in the primary cell walls of higher plants and, when isolated from the walls, can also bind to cellulose in vitro, the binding mechanism of xyloglucan to cellulose was further investigated using radioiodinated pea xyloglucan. A time course for the binding showed that the radioiodinated xyloglucan continued to be bound for at least 4 hours at 40°C. Binding was inhibited above pH 6. Binding capacity was shown to vary for celluloses of different origin and was directly related to the relative surface area of the microfibrils. The binding of xyloglucan to cellulose was very specific and was not affected by the presence of a 10-fold excess of (1→2)-β-glucan, (1→3)-β-glucan, (1→6)-β-glucan, (1→3, 1→4)-β-glucan, arabinogalactan, or pectin. When xyloglucan (0.1%) was added to a cellulose-forming culture of Acetobacter xylinum, cellulose ribbon structure was partially disrupted indicating an association of xyloglucan with cellulose at the time of synthesis. Such a result suggests that the small size of primary wall microfibrils in higher plants may well be due to the binding of xyloglucan to cellulose during synthesis which prevents fasciation of small fibrils into larger bundles. Fluorescent xyloglucan was used to stain pea cell wall ghosts prepared to contain only the native xyloglucan:cellulose network or only cellulose. Ghosts containing only cellulose showed strong fluorescence when prepared before or after elongation; as predicted, the presence of native xyloglucan in the ghosts repressed binding of added fluorescent xyloglucan. Such ghosts, prepared after elongation when the ratio of native xyloglucan:cellulose is substantially reduced, still showed only faint fluorescence, indicating that microfibrils continue to be coated with xyloglucan throughout the growth period.     

Cooperative action of β-glucan synthetase and UDP-xylose xylosyl transferase of Golgi membranes in the synthesis of xyloglucan-like polysaccharide

Golgi membranes of pea seedling tissue contain a UDP xylose:polysaccharide xylosyl transferase, the action of which is stimulated by UDP glucose. In the presence of both nucleotide-sugars a heteropolysaccharide containing both xylose and glucose (xyloglucan) is produced. Transfer of xylose and glucose units is presumed to be due to separate enzymes, because their properties differ in a number of respects. Xylosyl units appear to be transferred to a glucan core polysaccharide that is produced from UDP glucose by β-1,4-glucan synthetase. This, rather than cellulose biosynthesis, is inferred to be the in vivo role of Golgi membrane β-1,4-glucan synthetase.     

An Examination of Centrifugation as a Method of Extracting an Extracellular Solution from Peas, and Its Use for the Study of Indoleacetic Acid-induced Growth

A technique of centrifuging pea epicotyl sections which extracts water-soluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests for protein, hexose, pentose, and malate dehydrogenase indicate that significant damage to the cells occurs above 3,000g. Below this force, there is little damage, as evidenced by the similar growth rates of centrifuged and noncentrifuged sections. Centrifugation at 1,000g extracts polysaccharides containing rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose. An increase in xylose and glucose, presumably xyloglucan, is induced by treating sections with indoleacetic acid. Much of the alcohol-insoluble, water-soluble polysaccharide within the wall is extractable by centrifugation, since nearly as much arabinose and xylose are extractable by centrifugation as by homogenization. The utility of this method for the study of cell wall metabolism is discussed.     

Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells

Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-[¹⁴C] fucose and UDP-[¹⁴C]xylose to xyloglucan occurred mainly in dictyosomeenriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with [³H]fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.     

Characterization of the adsorption of Xyloglucan to Cellulose

The binding of xyloglucan- and cello-oligosaccharides to cellulosescan be expressed by Langmuir adsorption isotherms, in whichthe levels of adsorption maxima are all similar but very low.In the present study, although the adsorption constant increasedwith increases in the degree of polymerization (DP) of the 1,4-rß-glucosylresidues of xyloglucan- and cello-oligosaccharides, the adsorptionconstant of cellopentitol to cellulose was similar to that ofhendecosanosaccharide (glucose/xylose, 12 : 9), demonstratingless extensive binding in the case of xyloglucan oligosaccharidesin spite of longer chains of 1,4-rß-glucosyl residues.The binding to cellulose of xyloglucans from pea and Tamarindusindica can also be expressed as Langmuir adsorption isotherms.The adsorption constant for pea xyloglucan with a DP for 1,4-rß-glucosylresidues of 150 was obviously higher than that for Tamarindusxyloglucan with a DP of 3,000. The adsorption maximum and adsorptionconstant of Tamarindus xyloglucan decreased gradually as theDP of 1,4-rß-glucosyl residues decreased from 3,000to 64. This result demonstrates that fucosylated pea xyloglucanhas a higher adsorption constant for cellulose than non-fucosylatedTamarindus xyloglucan when the DP of 1,4-rß-glucosylresidues is identical. These findings indicate that xyloglucanbinds to cellulose as a mono-layer and fucosyl residues contributeto the increase in adsorption affinity. (Received June 4, 1994; Accepted September 10, 1994)     

Pea Xyloglucan and Cellulose : IV. Assembly of beta-Glucans by Pea Protoplasts

The synthesis and assembly of xyloglucan were examined during early stages of wall regeneration by protoplasts isolated from growing regions of etiolated peas. During early stages of cultivation, fluorescence microscopy showed that the protoplast surface bound Calcofluor and ammonium salt of 8-anilino-1-naphthalene sulfonic acid and, in time, it also bound fluorescent fucose-binding lectin. Based on chemical analysis, 1,3-β-glucan was the main polysaccharide formed by protoplasts and xyloglucan and cellulose were minor wall components. Binding between cellulose and xyloglucan was not as strong as that in tissues of intact pea plants, i.e. mild alkali could dissolve most xyloglucan from the protoplast. However, the addition of exogenous pea xyloglucan into the culture medium stimulated the deposition of new polysaccharides into the protoplast wall and enhanced the close association of newly formed xyloglucan with cellulose.     

Presence of a xyloglucan in the cell wall of Phaseolus aureus hypocotyls

The non-cellulosic ß-glucan₁ in the cell wall of Phaseolusaureus hypocotyb was studied. Evidence that xyloglucan is presentin a hemicellulose fraction was obtained by its isolation fromcell wall preparations. This polysaccharide was homogeneouson zone electrophoresis and ultracentrifugation. On acid hydrolysis,it gave glucose, xylose, galactose, and fucose in the approximatemolar ratio of 10 : 7 : 2.5 : 1. Its solution gave a reddishviolet color with iodine-staining solution. The results of partialacid hydrolysis and cellulase treatment suggest a structurein which xylose, galactose, and fucose attached as side chainsto a sequenceof ß-l,4-linked glucose. The xyloglucanisolated accounted for 13.9% of the total non-cellulosic fractions. (Received May 10, 1976; )     

The Oligosaccharide Units of the Xyloglucans in the Cell Walls of Bulbs of Onion, Garlic and their Hybrid

Xyloglucans were isolated from the 24% KOH-soluble fractionof the cell walls of bulbs of onion (Allium cepa), garlic (Alliumsativum) and their hybrid. The polysaccharides yielded singlepeaks upon gel filtration with average moleular weights of 65,000for onion, 55,000 for garlic and 82,000 for the hybrid. Compositionalanalysis of the oligosaccharide units after digestion with anendo-1,4-ß-glucanase from Streptomyces indicated thatthe polysaccharides were constructed of four kinds of repeatingoligosaccharide unit, namely, a decasaccharide (glucose/xylose/galactose/fucose,4 : 3 : 2 : 1), a nonasaccharide (glucose/xylose/galactose/fucose,4 : 3 : 1 : 1), an octasaccharide (glucose/xylose/galactose,4 : 3 : 1 ) , and a heptasaccharide (glucose/xylose, 4 : 3).The xyloglucan from the hybrid contained highly fucosylatedunits that resembled those from onion rather than from garlic.The analysis also revealed that the xyloglucans from Alliumspecies contain highly substituted xylosyl residues with fucosyl-galactosylresidues, suggesting that these monocotyledonous plants resembledicotyledons in the structural features of their xyloglucans. (Received November 1, 1993; Accepted June 16, 1994)     

Effects of the Degree of Polymerization on the Binding of Xyloglucans to Cellulose

Xyloglucan oligosaccharides were isolated with various degreesof polymerization (DP) and reduced with tritiated sodium borohydride.The ³H-oligosaccharides were tested for their ability to bindto amorphous and microcrystalline celluloses and to cellulosefilter paper. The time course of binding indicated that theradiolabeled oligosaccharides continued to be bound for at least1 h after heating at 120°C. The binding probably requiredthe organization of the oligosaccharides and celluloses by gradualannealing after heating. Although neither pentasaccharide (glucose:xylose, 3 : 2), heptasaccharide (glucose: xylose, 4 : 3) andnonasaccharide (glucose : xylose : galactose : fucose, 4 : 3: 1 : 1) failed to bind to the celluloses, binding occurredwith oligosaccharides with DP equivalent to more than four consecutive1,4-ß-glucosyl residues. The extent of binding tothe celluloses increased gradually from octasaccharide (glucose:xylose, 5 : 3) to hendecosanosaccharide (glucose/xylose, 12: 9), with the increase in the DP of 1,4-ß-glucosylresidues. The binding of reduced cello-dextrins to celluloserequired at least 4 consecutive 1,4-ß-glucosyl residues.The extent of binding of cellopentitol or cellohexitol to cellulosewas similar to that of hendecosanosaccharide, showing lowerbinding for xyloglucan oligosaccharides in spite of longer chainsof 1,4-ß-glucosyl residues. These findings suggestthat the mode of binding to cellulose of xyloglucan oligosaccharidesis different from that of cello-oligosaccharides. (Received February 18, 1994; Accepted June 1, 1994)     

Effect of IAA on Growth and Soluble Cell Wall Polysaccharides Centrifuged from Pine Hypocotyl Sections

Auxin-induced elongation and cell wall polysaccharide metabolism were studied in excised hypocotyl sections of ponderosa pine (Pinus ponderosa) seedlings. Sections excised from hypocotyls of ponderosa pine elongate in response to the addition of auxin. The neutral sugar composition of the extracellular solution removed from hypocotyl sections by centrifugation was examined. In cell wall solution from freshly excised sections, glucose, galactose, xylose, and arabinose make up more than 90% of the neutral sugars, while rhamnose, fucose, and mannose are relatively minor components. The neutral sugar composition of the polysaccharides of the pine cell wall solution is both qualitatively and quantitatively similar to that of pea. Following auxin treatment of pine hypocotyls, the neutral sugar composition of the cell wall changes; glucose, xylose, rhamnose, and fucose increase by nearly 2-fold relative to controls in buffer without auxin. These changes in neutral sugars in response to auxin treatment are similar to those found in pea, with the exception that in pea, rhamnose levels decline in response to auxin treatment.     

Localization of Xyloglucan in the Macromolecular Complex Composed of Xyloglucan and Cellulose in Pea Stems

A macromolecular complex composed of xyloglucan and cellulosewas isolated from elongating regions of stems of etiolated pea(Pisum sativum L. var Alaska) seedlings and binding of a xyloglucan-specificantibody was examined after treatment of the complex with endo-1,4-ß-glucanaseor 24% KOH. The antibody bound to the complex but the extentof binding was reduced after treatment of the complex with endo-1,4-ß-glucanaseand was hardly detectable after treatment with 24% KOH. Themolecular weight of the xyloglucan that remained (5%) in theß-glucanase-treated complexes was less than 9,200.Pea xyloglucan was allowed to bind to enzymeand alkali-treatedcomplexes to generaly reconstituted complexes. The amount ofthe antibody that bound to each type of reconstituted complexwas similar but was much lower than that bound to the nativecomplex. Immunogold labeling indicated that most of the antigenwas widely distributed between microfibrils in the native complex,whereas the antigen appeared to be confined to the microfibrilsin the reconstituted complexes. These findings suggest thata part of each xyloglucan molecule is strongly associated withcellulose microfibrils while the rest is free of the microfibrilsin the native complex. ¹This work was supported in part by a grant from the YamadaScience Foundation.     

Effect of cellulose synthesis inhibition on growth and the integration of xyloglucan into pea internode cell walls

2,6-Dichlorobenzonitrile (DCB, 100 μm) inhibited by 80 to 85% the incorporation of [³H]glucose into cellulose in stem segments of etiolated pea (Pisum sativum) seedlings. The inhibition lasted for at least 24 h. In the period 1 to 4 h after the excision of the segments, DCB did not influence elongation in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). However, during the period 1 to 24 h after excision, DCB enhanced endogenous and 2,4-D-stimulated elongation by 65 and 34%, respectively. DCB did not affect the incorporation of ³H from [³H]arabinose into xyloglucan, and did not change the ability of the [³H]xyloglucan formed in vivo to bind strongly to the cell wall. Therefore, at least 80 to 85% of newly synthesized cellulose was excess to the requirements for tight wall binding of newly synthesized xyloglucan. This conflicts with the hypothesis that xyloglucan is held in the cell wall solely by direct hydrogen bonding to the surfaces of cellulosic microfibrils.     

Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a beta-1,4-linked d-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.     

Cell wall assembly in fucus zygotes: I. Characterization of the polysaccharide components

Fertilization triggers the assembly of a cell wall around the egg cell of three brown algae, Fucus vesiculosus, F. distichus, and F. inflatus. New polysaccharide polymers are continually being added to the cell wall during the first 24 hours of synchronous embryo development. This wall assembly involves the extracellular deposition of fibrillar material by cytoplasmic vesicles fusing with the plasma membrane. One hour after fertilization a fragmented wall can be isolated free of cytoplasm and contains equal amounts of cellulose and alginic acid with no fucose-containing polymers (fucans) present. Birefringence of the wall caused by oriented cellulose microfibrils is not detected in all zygotes until 4 hours, at which time intact cell walls can be isolated that retain the shape of the zygote. These walls have a relatively low ratio of fucose to xylose and little sulfate when compared to walls from older embryos. When extracts of walls from 4-hour zygotes are subjected to cellulose acetate electrophoresis at pH 7, a single fucan (F₁) can be detected. By 12 hours, purified cell walls are composed of fucans containing a relatively high ratio of fucose to xylose and high levels of sulfate, and contain a second fucan (F₂) which is electrophoretically distinct from F₁. F₂ appears to be deposited in only a localized region of the wall, that which elongates to form the rhizoid cell. Throughout wall assembly, the polyuronide block co-polymer alginic acid did not significantly vary its mannuronic (M) to guluronic (G) acid ratio (0.33-0.55) or its block distribution (MG, 54%; GG, 30%; MM, 16%). From 6 to 24 hours of embryo development, the proportion of the major polysaccharide components found in purified walls is stable. Alginic acid is the major polymer and comprises about 60% of the total wall, while cellulose and the fucans each make-up about 20% of the remainder. During the extracellular assembly of this wall, the intracellular levels of the storage glucan laminaran decreases. A membrane-bound β-1, 3-exoglucanase is found in young zygotes which degrades laminaran to glucose. It is postulated that hydrolysis of laminaran by this glucanase accounts, at least in part, for glucose availability for wall biosynthesis and the increase in respiration triggered by fertilization. The properties and function of alginic acid, the fucans, and cellulose are discussed in relation to changes in wall structure and function during development.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

Xyloglucan (amyloid) formation in the cotyledons of Tropaeolum majus L. seeds

Mature seeds of Tropaeolum majus L. contain the cell wall polysaccharide xyloglucan (amyloid), protein and lipid as storage substances. The transitory occurrence of starch during the process of seed development could be substantiated.[U-¹⁴C]-labelled xylose, glucose and glucuronic acid were fed to ripening seeds and the incorporation of radioactivity into xyloglucan, starch and the sugar nucleotide fraction of the cotyledons was determined. The results indicate that exogenous supplied xylose is not incorporated directly into xyloglucan, but is transformed to glucose before incorporation into xyloglucan and starch. Radioactivity from glucuronic acid was predominantly found in the xylose moiety of xyloglucan. Incubation of seeds with [6-¹⁴C]-labelled glucose resulted in an incorporation of labelled hexoses into amyloid and starch, whereas xylose residues of amyloid remained unlabelled.Abbreviations p.a. post anthesis - UDP uridine 5-diphosphate - GDP guanosine 5-diphosphate - TLC thin layer chromatography - HPLC high pressure liquid chromatography     

Biosynthesis of the fucose-containing xyloglucan nonasaccharide by pea microsomal membranes

Pea microsomal membranes catalyze the transfer of [¹⁴C]fucose (Fuc) from GDP-[U-¹⁴C]fucose, with or without added unlabeled UDP-glucose (Glc), UDP-xylose (Xyl) or UDP-galactose (Gal), to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, [¹⁴C] fucose residues occur exclusively in a fragment corresponding in size to the xyloglucan nonasaccharide, Glc₄ Xyl₃ Gal Fuc. This fragment contains a single labeled fucose residue per oligomer, α-linked in a terminal nonreducing position. By comparison, in incubations where GDP-[¹⁴C] fucose is absent and replaced by UDP-[³H]xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products is an octasaccharide. In the presence of both GDP-[¹⁴C]fucose and UDP-[³H]xylose, a nonasaccharide containing the two labels is produced. Fucose and xylose residues are transferred within a few minutes to acceptor molecules of molecular weight up to 300,000. Such products do not elongate detectably over 60 minutes of incubation. The data support the conclusion that the nonasaccharide subunit of xyloglucan may be generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis is dependent on the inclusion of exogenous GDP-fucose.