Isolation of xyloglucans from etiolated Glycine max and Vigna sesquipedalis hypocotyls

Xyloglucans isolated from cell walls of etiolated Glycine maxand Vigna sesquipedalis hypocotyls were subjected to fragmentationanalysis with cellulase for structural comparison with thosederived from Phaseolus aureus hypocotyls. The xyloglucans fromG. max and V. sesquipedalis had glucose, xylose, galactose andfucose in the approximate molar ratio of 10:6:4:1 and 10:7:3:1,respectively. However, the results of cellulase fragmentationanalysis of xyloglucans from the three species suggested thatthe basic structure of the xyloglucans in the cell walls ofthese bean-hypocotyls is almost the same; the structure is basedon two repeating oligosaccharide units, one of which consistsof glucose and xylose and the other of glucose, xylose, galactoseand fucose. ¹ Present address: Toppan Printing Co., Ltd., Okaji, Sendai980, Japan. (Received February 3, 1977; )     

Auxin-Induced Changes in the Cell Wall Xyloglucans: Effects of Auxin on the Two Different Subtractions of Xyloglucans in the Epicotyl Cell Wall of Vigna angularis

In order to study the IAA-induced modifications of the cellwall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara)epicotyl segments, the xyloglucans were subfractionated intotwo components, i.e., 4K-U and 24K xyloglucans, which were obtainedby extraction with 4% KOH solution containing 8 M urea and 24%KOH solution, respectively. The weight-average molecular weightsof 4K-U and 24K xyloglucans were estimated to be 40 x 10⁴ and106 x 10⁴, respectively. Complete acid hydrolysis of 4K-U and24K xyloglucans gave glucose, xylose, galactose and fucose inmole % 48.3 : 33.5 : 13.8 : 4.4 and 45.3 : 30.9 : 19.6 : 4.3,respectively. Treatment of epicotyl segments with IAA (0.1 mM) caused a decreasein the amount of 24K xyloglucans and an increase in 4K-U xyloglucans,whereas the total amount of the two xyloglucans remained constant.Furthermore, IAA treatment caused a decrease in the molecularweight of 24K xyloglucans from 106 x 10⁴ to 78 x 10⁴ withoutcausing changes in their sugar compositions. With 4K-U xyloglucans,IAA caused an increase in the mole % of xylose and a decreasein the mole % of galactose and fucose. ¹ This paper is dedicated to the late Professor Joji Ashida. (Received November 26, 1982; Accepted February 7, 1983)     

Inhibition of the Breakdown of Xyloglucans in Azuki Bean Epicotyls by Concanavalin A

Indole-3-acetic acid at 10 µM caused a 30% decrease inthe weight-average molecular mass of xyloglucans extracted with24% KOH from the cell walls of epicotyl segments of azuki bean(Vigna angularis Ohwi et Ohashi cv. Takara). Concanavalin A(Con A) at 2 g liter^–1 completely inhibited the IAA-inducedchange in the molecular mass of the xyloglucans. Con A alsosuppressed the autolysis of pectin-depleted cell walls, as wellas the breakdown of xyloglucans by a protein fraction that hadbeen extracted with 1 M NaCl from the cell walls of azuki beanepicotyls. These results indicate that Con A is a potent inhibitorof the breakdown of xyloglucans both in vivo and in vitro. Mostof the activity responsible for the decrease in staining byiodine and the increase in reducing power of solution of xyloglucansin the protein fraction from cell walls bound to a column ofCon A-Sepharose and was eluted by the specific hapten, methyl     

Unchanged Molecular-Weight Distribution of Xyloglucans in Outer Tissue Cell Walls along Intact Growing Hypocotyls of Squash (Cucurbita maxima Duch.) Seedlings

The changes in the mechanical properties and compositions ofcell walls in outer and inner tissues were investigated alongthe hypocotyls of squash (Cucurbita maxima Duch.) seedlings.The endogenous growth capacity decreased and the minimum stress-relaxationtime (T_O) of cell walls in outer tissues increased from theapical to the basal region of hypocotyls. A high correlationwas observed between values of To in outer tissues and endogenousgrowth (r=–0.99). The values of T_O in inner tissues didnot change from the apical to the basal region of hypocotyls. In outer tissues, the levels of neutral sugars in pectin decreasedconsiderably from the apical to the basal region of hypocotyls.However, relative amounts of hemicellulose showed little differencealong the hypocotyls. Levels and molecular weights of hemicellulosicxyloglucans in outer tissues were about 2-3 times greater thanthose in inner tissues. The amount of xyloglucans in outer tissuesincreased in the middle region of hypocotyls, and xyloglucansin upper and basal regions had similar molecular weights. Bycontrast, in inner tissues, amounts of cell-wall material decreasedtoward the basal region. Amounts and molecular weights of hemicellulosicxyloglucans also decreased along the hypocotyls. These results clearly show that cell-wall metabolism duringaging of intact growing stem tissues differs markedly betweenouter and inner tissues, and the absence of a simple relationship between the molecular weights of xyloglucans and the mechanicalproperties of the cell walls in outer tissues indicates thatthe changes in the mechanical properties of the cell walls inintact growing tissues cannot be explained only by the molecularweights of xyloglucans. Thus, the regulation of the mechanicalproperties of cell walls in intact growing stems may be somewhatdifferent from that in auxin-treated stem sections, in whichauxin promotes the depolymerization of xyloglucan molecules. (Received November 28, 1991; Accepted November 16, 1992)     

Variation in the Content and Composition of Sterols in Alfalfa Seedlings Treated with Compactin (ML-236B) and Mevalonic Acid

Compactin (ML-236B), a specific inhibitor of 3-hydroxy-3-methylglutarylCoA reductase, inhibited the elongation of roots and hypocotylsof Medicago sativa seedlings when it was applied to the roots.Addition of mevalonic acid, the direct product of the enzyme,together with compactin relieved the growth inhibition of roots. The contents and compositions of sterols were studied in threeparts of M. sativa seedlings—roots, hypocotyls and cotyledons.Compactin (20 µM) decreased the sterol contents of rootsand hypocotyls by about a half but did not affect that of cotyledons.On the other hand, mevalonic acid (2 mM) increased the sterolcontent of roots more than threefold the nontreated controllevel but not the contents of hypocotyls and cotyledons. Mevalonicacid added in combination with compactin had a similar effecton the sterol content of roots as when it was added alone. The major sterol in all three parts was stigmasterol whetheror not compactin or mevalonic acid was present. However, thevariation of the sterol composition in the roots was distinct;mevalonic acid-treated roots markedly accumulated ⁷-sitosterol,24-methylenecycloartanol and squalene. (Received October 16, 1986; Accepted April 2, 1987)     

Changes in Wall Polysaccharides of Squash (Cucurbita maxima Duch.) Hypocotyls under Water Stress Condition: II. Composition of Pectic and Hemicellulosic Polysaccharides

Hypocotyl growth of dark-grown squash (Cucurbita maxima Duch.)seedlings was greatly reduced by the addition of polyethyleneglycol (60 mM) to the hydroponic solution through inhibitionof cell elongation. Measurement of the mechanical propertiesof the cell walls revealed that the cell wall of stressed hypocotylswas loosened as much as that of the unstressed hypocotyls, suggestingthat the stressed hypocotyl could not elongate even though thecell wall loosened. Galactose and arabinose in the pectic fraction,which are probably attached to high mol wt rhamnogalacturonans,increased under stressed as well as under unstressed condition.Other polysaccharides including pectic low mol wt galacturonans,hemicellulosic xyloglucans, galactoglucomannans, arabinans,and glucuronoarabinoxylans increased more under unstressed condition.The mol wt of xyloglucans in the hemicellulosic fraction increasedunder unstressed but not under stressed condition. These results suggest that changes in wall structure, such asincreases in high mol wt rhamnogaracturonans rich in arabinoseand galactose residues, and the suppression of polymerizationof xyloglucans are involved in the process of cell wall loosening. (Received December 15, 1986; Accepted June 8, 1987)     

Investigation of the heterogeneity of xyloglucans from the cell walls of apple

Cell-wall material from parenchymatous tissues of apple was sequentially extracted with 50mm NaOH at 1°, m KOH at 1° and 20°, and 4m KOH at 20°, to leave a residue of α-cellulose. From the 4m KOH-soluble fraction, a crude xyloglucan was isolated by anion-exchange chromatography, and further resolved into seven xyloglucans by borate anion-exchange chromatography. The relative amounts of the xyloglucans, in order of elution, were 2.7:1.3:29.7:1.0:3.2:1.2:10.3. The structural features of five of the xyloglucans were determined by methylation analysis. These results show that apple xyloglucans exhibit heterogeneity.     

Structural features of cell-wall polymers of the apple

Cell-wall material was isolated from ripe-apple cortical tissues by sequential extraction with aqueous 1.5% sodium dodecyl sulphate and aqueous 90% methyl sulphoxide. The wall material, which contained ～1% of protein, with proline and hydroxyproline as the preponderant amino acids, was sequentially extracted with water at 80°, oxalate at 80°, m KOH at 1°, and m and 4m KOH at 20°, to leave a residue of α-cellulose, which was associated with an appreciable amount of arabinose-rich pectic material. The depectinated material was also extracted with 6m guanidinium thiocyanate at 20° to solubilise preferentially polysaccharides rich in mannose. The hot-water-soluble pectic substances were richer in arabinose compared with the oxalate-soluble ones and were resolved into five fractions by anion-exchange chromatography. The bulk of the hemicelluloses, which were xyloglucans, were solubilised by 4m KOH. The alkali-soluble hemicellulose polymers were resolved by anion-exchange chromatography into polysaccharides, mainly xyloglucans, arabinoxylan-pectic-xyloglucan, and arabinoxylan-pectic complexes. Small amounts of polysaccharide-protein-polyphenol complexes (where the polysaccharide moieties were arabinoxylans), pectic substances, and xyloglucans were also present. The glycosidic linkages of the above polymers were determined by methylation analysis. The general structural features of the cell-wall polymers are discussed.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Purification of Xyloglucan Hydrolase/Endotransferase from Cell Walls of Azuki Bean Epicotyls

An enzyme involved in the breakdown of xyloglucans was purifiedfrom an extract of cell walls of azuki bean epicotyls obtainedwith 1 M NaCl and purified by column chromatography on severaldifferent resins. The purified enzyme gave a single band ofa protein with a molecular mass of about 32 kDa after SDS-PAGE.The enzyme hydrolyzed the xyloglucans of high molecular massfrom azuki cell walls to yield fragments of about 50 kDa withoutproduction of any oligo- or monosaccharides. Moreover, the enzymehad hardly any effect on xyloglucans of less than 60 kDa. Theenzyme also hydrolyzed xyloglucans from tamarind, but it didnot react with cellulose derivatives. In the presence of pyridylamino-labeledxyloglucan oligosac-charides as acceptor substrates, the enzymecatalyzed the transfer of 50-kDa products to the oligosaccharides.The K_m value of the enzyme for xyloglucans of 540 kDa was similarin the presence and in the absence of xyloglucan oligosaccharidesas acceptors: 1.0 mg ml^–1. These results suggest thatthe enzyme was an endotransferase but had unusual acceptor specificity,preferring smaller acceptors such as water. (Received September 9, 1996; Accepted March 16, 1997)     

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)     

Differential Effect of Auxin on Molecular Weight Distributions of Xyloglucans in Cell Walls of Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls

Effects of indole-3-acetic acid (IAA) on the mechanical properties of cell walls and structures of cell wall polysaccharides in outer and inner tissues of segments of dark grown squash (Cucurbita maxima Duch.) hypocotyls were investigated. IAA induced the elongation of unpeeled, intact segments, but had no effect on the elongation of peeled segments. IAA induced the cell wall loosening in outer tissues as studied by the stress-relaxation analysis but not in inner tissues. IAA-induced changes in the net sugar content of cell wall fractions in outer and inner tissues were very small. Extracted hemicellulosic xyloglucans derived from outer tissues had a molecular weight about two times as large as in inner tissues, and the molecular weight of xyloglucans in both outer and inner tissues decreased during incubation. IAA substantially accelerated the depolymerization of xyloglucans in outer tissues, while it prevented that in inner tissues. These results suggest that IAA-induced growth in intact segments is due to the cell wall loosening in outer tissues, and that IAA-accelerated depolymerization of hemicellulosic xyloglucans in outer tissues is involved in the cell wall loosening processes.     

Dimethipin (2,3-Dihydro-5,6-dimethyl-1,4-dithiin 1,1,4,4-tetraoxide) Effects on Soybean Seedling Growth and Metabolism

Three-day-old dark-grown soybean [Glycine max (L.) Merr.] seedlingswere transferred to 2 mM CaSO₄ or 10^–5 M dimethipin in2 nM CaSO₄ and root-fed via liquid culture. Plants were placedin continuous darkness or in continuous white light (200 µE.m^–2?s^–11,PAR) at 25?C. Dimethipin inhibited root and shoot elongationin dark-grown plants after 24 h and 48 h, respectively. In thelight, root elongation was inhibited also after 24 h, but hypocotylelongation was not significantly affected. Extractable phenylalanineammonia-lyase (PAL) activity per axis in dimethipin-treateddark-grown axes was not generally affected but, in the lightdimethipin caused a significant decrease in PAL activity (24to 96 h). Total soluble hydroxyphenolics in axes were not affectedby dimethipin in light- or dark-grown plants. Anthocyanin andchlorophyll levels were lowered in hypocotyls of dimethipin-treatedplants after 48 to 96 h. Soluble protein in hypocotyls of light-or dark-grown seedlings was not substantially affected by dimethipin.Nitrate reductase (NR) activity (per organ) was generally notaffected by dimethipin in light-grown cotyledons, but in theroots of these seedlings, NR activity was significantly decreased.Proteolytic enzyme activity using three substrates (leucine-p-nitroanilide,LPNA; proline-p-nitroanilide, PPNA; and benzoylarginine-p-nitroanilide,BAPA) indicated little effect on enzyme activities per organin roots and hypocotyls. These data suggest that dimethipinat low concentrations can cause significant growth inhibitionin soybean seedlings grown in either light or darkness and thatfurthermore, extractable activities of some enzymes associatedwith nitrogen metabolism and secondary metabolism are alteredby this chemical. Light also plays a role in the activity ofthis chemical. (Received November 29, 1983; Accepted January 25, 1984)     

Properties of Two N-Linked Glycoproteins Associated with the Nuclear Envelope in Mung Bean Hypocotyls

Nuclei from mung bean (Vigna radiata) hypocotyls contained twoglycoproteins of 50 and 49 kDa, respectively, that reacted withconcanavalin A. The glycoproteins were released from the nuclearenvelope by treatment with 2 M KCl but not with nucleases. Theglycoproteins, tentatively named gp50 and gp49, were isolatedand characterized. Gel-permeation chromatography suggested thatgp50 and gp49 seem to exist as a complex with other components.The glycoproteins could be detected only in the nuclear fractionby immunoblot analysis with specific antibodies, and they werenot detected in endoplasmic reticulum, plasma membrane, vacuolarmembrane or mitochondria. Agglutinin I from Ulex europeaus,peanut agglutinin, soybean agglutinin and wheat germ agglutininall failed to bind to the glycoproteins. Treatment with glycopeptidaseF removed all oligosaccharides from the glycoproteins and decreasedtheir molecular masses by about one thousand daltons each. Theseresults suggest that the glycoproteins contained N-linked, high-mannose-typeoligosaccharides with six or seven hexose residues. gp50 andgp49 seemed to be isoforms of a single glycoprotein becausethe two proteins had some common properties. Nuclear fractionsfrom azuki bean (Phaseolus angularis) and soybean (Glycine max)contained proteins that were immunologically similar to gp50and gp49. (Received March 18, 1995; Accepted May 24, 1995)     

QUANTITATIVE SEPARATION OF INORGANIC POLYPHOSPHATES IN CHLORELLA CELLS

Using unicellular green alga, Chlorella ellipsoidea, which hadbeen labeled with ³²P-phosphate, re-examination was performedon our routine method for the quantitative separation of inorganicpolyphosphates (poly-Pi). Confirming the previous results, itwas demonstrated that poly-Pi's in Chlorella cells are clearlyseparated from each other by successive extractions with cold8% TCA (poly-Pi "A"), with cold KOH at pH 9 (poly-Pi "B"), andwith 2 N KOH (poly-Pi's "C" and "D"; the former precipitateson neutralization with PCA, leaving the latter in solution).Use of 2N KOH was found to be most suitable for the purposeof fractionation of poly-Pi's "C" and "D." (Received February 25, 1965; )     

PHYSIOLOGICAL ACTIVITIES OF HELMINTHOSPOROL AND HELMINTHOSPORIC ACID: I. EFFECTS ON GROWTH OF INTACT PLANTS

Helminthosporol (H-ol) and helminthosporic acid (H-acid) wereeffective in promoting elongation of leaf sheaths of rice, Japanesebarnyard grass and dwarf maize (d-2 and d-5) and of hypocotylsof taisai (Brassica chinensis), but inactive in leaf sheathsof oat and wheat, hypocotyls of sesame and morning glory (Pharbitisnil) and epicotyls of Pharbitis and dwarf and tall peas. Onthe elongation of the leaf sheath of maize d-1, H-ol was promotivebut the activity of H-acid was doubtful. On hypocotyls of lettuceand daikon (Raphanus sativus), only H-acid was active. Multiplicationrate and size of fronds of Lemna perpusila were not affectedby either of the substances. Compared with gibberellic acid for the effect on the shoot growth,H-ol and H-acid were weak in activity and narrower in the scopeof plants that responded. H-ol and H-acid characteristicallypromoted the elongation of the primary root. Comparative effectivenessof H-ol and H-acid varied with plant species or parts examined. ¹ This study was supported in part by grant-in aid of the Ministryof Education (No. 0417). The results reported here were presentedat the Annual Meeting of the Botanical Society of Japan at Kanazawain 1964 (S).     

Stress relaxation properties of the cell wall of growing intact plants

The cell wall of dark-grown Avena coleoptiles and the epidermisof light-grown mungbean hypocotyls was subjected to stress-relaxationanalysis and the following results were obtained. 1. Actively growing apical regions of the organs, either coleoptilesor hypocotyls, had certain threshold values of minimum stress-relaxationtime, T_O, 0.04 sec for coleoptile cell wall and 0.03 sec forthe epidermal cell wall of hypocotyls. The cell wall of thebasal region of the organs, which were mature and not growing,had a higher value of To. 2. When the apical regions of the organs, either coleoptilesor hypocotyls, ceased to grow, their cell walls showed T_O valuesabove these thresholds. 3. The relaxation rate, b, was small in the cell wall of activelygrowing regions of the organs, compared with that of non-growingregions. 4. The maximum relaxation time, T_m, was variable and no significantrelationship with growth capacity was found. 5. The extensibility, mm/gr, was large not only in activelygrowing regions of the organs but also in fully grown regions,suggesting that the value represents complex properties of thecell wall including the history of cell wall extension. From these results, we concluded that biochemical modificationsoccur in the cell wall matrix of actively growing organs ofeither monocots or dicots, and these are the bases of the capacityof the cell wall to extend and are represented chiefly by T_oand possibly by b. (Received August 12, 1974; )     

Glucans in the Cell Walls Regenerated from Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere cultured for 5 days. The cell walls regenerated from theprotoplasts were mainly composed of glucans having 1,3- and1,4-linkages. To investigate the molecular species, these glucanswere separated into four fractions: EDTA (50 mM, pH 4.5)-soluble(fraction E), KOH (24%)- soluble but not precipitatable by neutralizationwith acetic acid (fraction K-S), KOH (24%)-soluble and precipitatableby neutralization with acetic acid (fraction K-P), and KOH (24%)-insoluble(fraction C). By means of sugar composition analysis, methylationanalysis, periodate oxidation and enzymatic digestion, the molecularspecies of the glucans contained in the regenerated cell wallswere deduced to be ß-1,4-glucan (cellulose) and ß-1,3-glucan.Fraction C was mainly composed of ß-1,4-glucan; ß-1,3-glucanwas mainly recovered in fraction K-P. The ß-l,3-glucanwas soluble in dilute alkali solution, but was only slightlysoluble in water. The ß-1,3-glucan had an essentiallyunbranched structure, and its weight average molecular weightestimated by gel permeation chromatography was 4.5–5.0x 10⁴. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan (Received May 21, 1981; Accepted October 13, 1981)     

Hemicellulose fractions and associated protein of lupin hypocotyl cell walls

The alkali extraction of polysaccharide fractions from depectinated primary cell walls of lupin hypocotyls was studied using sequential extractions at 0° and 18–22°. Aqueous 10% KOH at 0° removed hemicellulose-A (95%) heteroglycan-B (80%) and linear 1–4 linked hemicellulose-B (60%). Arabinose accounts for 88% of the monosaccharides of the linear 1–4 linked hemicellulose-B extracted between 2 and 5 h at 18–22°. Extraction of the 0° and 18–22° alkali-soluble fractions by denaturants, was also examined. 6M guanidine thiocyanate removed about 60% of the 0° 10% KOH soluble polysaccharide but little of the 18–22° soluble material. Although rapidly extracted by 10% KOH at 0° the hemicellulose-A is not extracted by this reagent. Analyses of cell walls and extracted fractions showed that there is little change in amino acid composition and little extraction of wall protein upon removal of about 60% of total wall hemicellulose with 10% KOH at 0°. It is therefore not bound to the wall through galactosylserine links. 10% KOH at 18–22° caused a marked change in composition and extracted most of the wall protein. An alkali resistant fraction high in hydroxyproline and low in serine was not extracted by 24% KOH at 18–22° in 24 hr.     

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.