Cellulose Microfibril Assembly Research Articles

Role of monolignol glucosides in supramolecular assembly of cell wall components in ginkgo xylem formation

Abstract The physical, chemical and biological properties of wood depend on the supramolecular assembly of cellulose microfibrils (CMFs), hemicelluloses (HCs) and lignin in the growing cell walls. Based on the 13C-tracer studies of ginkgo xylem formation, a hypothetical scenario for the role of monolignol glucosides (MLGs) in the assembly is proposed as follows: (1) Both moieties, aglycone monolignols and glycone d-glucose (d-Glc), play essential roles in a cooperative manner in delivery of hydrophobic and highly reactive p-hydroxycinnamyl- (H), coniferyl- (G) alcohols to the hydrophilic site of lignin deposition. (2) The d-Glc liberated at lignification site is converted into essential HCs mainly via Golgi apparatus under the influence of diurnally changing turgor pressure, and partly converted in the apoplast. (3) At cell corner middle lamella, a pressure-resistant layer of HG-lignin-HCs-CMFs is formed, and allows expansion of new cells in cambium region by elevation of turgor pressure. The deformable G-lignin-HCs-CMFs layer at secondary wall shrinks by dehydration of the swollen gel of HCs-CMFs during differentiation, and contributes posture control of standing tree. On-demand quick supply of a large amount of monolignols and HCs can be achieved by the large storage and delivery of MLGs in the growing ginkgo xylem.

BcsAB synthesized cellulose on nickel surface: polymerization of monolignols during cellulose synthesis alters cellulose morphology

The bacterial BcsA-B cellulose synthase synthesizes cellulose II when immobilized on nickel surfaces. To explore how lignin interacts with cellulose, lignin was polymerized either during or after cellulose formation from surface immobilized BcsA-B, leading to significant alterations in cellulose and lignin morphology. To facilitate lignin detection, polymerized coniferyl alcohol monolignols were detected by lignin autofluorescence. This compound was previously demonstrated as a suitable lignin monolignol exhibiting autofluorescence that enables lignin detection even at low concentrations. Cellulose and lignin coated nickel surfaces were studied under confocal microscopy, as well as by atomic force microscopy and X-ray diffractometry (XRD). As a consequence of simultaneous lignin and cellulose deposition on the surface, cellulose microfibril morphology was altered. XRD data indicated that cellulose crystal size was significantly decreased by polymerized lignin incorporation, suggesting that simultaneous lignin polymerization prevents the proper assembly of cellulose microfibrils. In contrast, polymerizing lignin after cellulose synthesis yielded a cellulose that was very similar in character to control (lignin-free) samples in terms of crystal morphology.

Inhomogeneity of Cellulose Microfibril Assembly in Plant Cell Walls Revealed with Sum Frequency Generation Microscopy.

Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.

From nano- to micrometer scale: the role of microwave-assisted acid and alkali pretreatments in the sugarcane biomass structure

BackgroundTo date, great strides have been made in elucidating the role of thermochemical pretreatments in the chemical and structural features of plant cell walls; however, there is no clear picture of the plant recalcitrance and its relationship to deconstruction. Previous studies precluded full answers due to the challenge of multiscale features of plant cell wall organization. Complementing the previous efforts, we undertook a systematic, multiscale, and integrated approach to track the effect of microwave-assisted H2SO4 and NaOH treatments on the hierarchical structure of plants, i.e., from a nano- to micrometer scale. We focused on the investigation of the highly recalcitrant sclerenchyma cell walls from sugarcane bagasse.ResultsThrough atomic force microscopy and X-ray diffraction analyses, remarkable details of the assembly of cellulose microfibrils not previously seen were revealed. Following the H2SO4 treatment, we observed that cellulose microfibrils were almost double the width of the alkali pretreated sample at the temperature of 160 °C. Such enlargement led to a greater contact between cellulose chains, with a subsequent molecule alignment, as indicated by the X-ray diffraction (XRD) results with the conspicuous expansion of the average crystallite size. The delignification process had little effect on the local nanometer-sized arrangement of cellulose molecules. However, the rigidity and parallel alignment of cellulose microfibrils were partially degraded. The XRD analysis also agrees with these findings as evidenced by large momentum transfer vectors (q > 20 nm−1), interpreted as indicators of the long-range order of cell wall components, which were similar for all the studied samples except with application of the NaOH treatment at 160 °C. These changes were followed by the eventual swelling of the fiber cell walls.ConclusionsBased on an integrated approach, we presented multidimensional architectural models of cell wall deconstruction resulting from microwave-assisted pretreatments. We provided direct evidence supporting the idea that hemicellulose is the main barrier for the swelling of cellulose microfibrils, whereas lignin adds rigidity to cell walls. Our findings shed light on the design of more efficient strategies, not only for the conversion of biomass to fuels but also for the production of nanocellulose, which has great potential for several applications such as composites, rheology modifiers, and pharmaceuticals.

Impact of plant matrix polysaccharides on cellulose produced by surface-tethered cellulose synthases

Surface immobilized BcsA-B cellulose synthases synthesize crystalline cellulose II under in vitro conditions and were used to explore the interaction between cellulose and hemicelluloses and pectin. The morphology of the cellulose microfibrils changed in the presence of xyloglucan and glucomannan, while pectin did not significantly impact morphology. X-ray diffractometry and FT-IR spectroscopy indicated that crystal size and crystallinity were significantly affected by xyloglucan and glucomannan but not altered by pectin. Glucomannan had the most significant impact on the structure of cellulose and inhibits crystallization. The presence of xyloglucan and glucomannan prevents the proper assembly of cellulose microfibrils and changes the crystalline properties of cellulose II in in vitro conditions, but did not have any impact on cellulose allomorph.

Vibrational sum-frequency-generation (SFG) spectroscopy study of the structural assembly of cellulose microfibrils in reaction woods

The cellulose microfibril assemblies in secondary cell walls of tension wood and compression wood were studied with vibrational sum frequency generation (SFG) spectroscopy. The tension wood contains the gelatinous layer with highly-crystalline and highly-aligned cellulose microfibrils. The SFG spectral features of tension wood changed depending on the azimuth angle between the polarization of the incident IR beam and the preferential alignment axis of the cellulose microfibrils. The SFG spectra of the compression wood did not show any dependence on the azimuth angle, implying that the overall orientation of cellulose microfibrils in compression wood is not highly aligned. Instead, the decrease of cellulose content in compression wood brought about larger separation between cellulose microfibrils, which was manifested as changes in CH2/OH intensity ratio in SFG spectra. These results implied that SFG spectral features are sensitive to cellulose microfibril alignments and inter-fibrillar separations.

Probing crystal structure and mesoscale assembly of cellulose microfibrils in plant cell walls, tunicate tests, and bacterial films using vibrational sum frequency generation (SFG) spectroscopy.

This study reports that the noncentrosymmetry and phase synchronization requirements of the sum frequency generation (SFG) process can be used to distinguish the three-dimensional organization of crystalline cellulose distributed in amorphous matrices. Crystalline cellulose is produced as microfibrils with a few nanometer diameters by plants, tunicates, and bacteria. Crystalline cellulose microfibrils are embedded in wall matrix polymers and assembled into hierarchical structures that are precisely designed for specific biological and mechanical functions. The cellulose microfibril assemblies inside cell walls are extremely difficult to probe. The comparison of vibrational SFG spectra of uniaxially-aligned and disordered films of cellulose Iβ nanocrystals revealed that the spectral features cannot be fully explained with the crystallographic unit structure of cellulose. The overall SFG intensity, the alkyl peak shape, and the alkyl/hydroxyl intensity ratio are sensitive to the lateral packing and net directionality of the cellulose microfibrils within the SFG coherence length scale. It was also found that the OH SFG stretch peaks could be deconvoluted to find the polymorphic crystal structures of cellulose (Iα and Iβ). These findings were used to investigate the cellulose crystal structure and mesoscale cellulose microfibril packing in intact plant cell walls, tunicate tests, and bacterial films.

Evolution and impact of cellulose architecture during enzymatic hydrolysis by fungal cellulases

The enzymatic hydrolysis of cellulose is still considered as a main limiting step of the biological production of biofuels from ligno-cellulosic biomass. Glycoside hydrolases from Trichoderma reesei are currently used to produce fermentable glucose units from degradation of cellulose packed in a complex assembly of cellulose microfibrils. The present work describes the structural evolution of two prototypical samples of cellulose (a micro-crystalline cellulose and a bleached sulfite pulp) over 5 length scale orders of magnitude. The results were obtained through wide angle, small angle and ultra-small angles synchrotron X-ray scattering, completed by Small Angle Neutron Scattering and particle size analyzers. These structural evolutions were followed as a function of enzymatic conversion. The results show that whereas there is no change at the nanometer scale, drastic changes occur at micron. The observed decrease of the size of the cellulose particles is accompanied by a smoothing of the crystalline surfaces that can be explained by a two-step mechanism of the enzymatic hydrolysis.

Building and degradation of secondary cell walls: are there common patterns of lamellar assembly of cellulose microfibrils and cell wall delamination?

During cell wall formation and degradation, it is possible to detect cellulose microfibrils assembled into thicker and thinner lamellar structures, respectively, following inverse parallel patterns. The aim of this study was to analyse such patterns of microfibril aggregation and cell wall delamination. The thickness of microfibrils and lamellae was measured on digital images of both growing and degrading cell walls viewed by means of transmission electron microscopy. To objectively detect, measure and classify microfibrils and lamellae into thickness classes, a method based on the application of computerized image analysis combined with graphical and statistical methods was developed. The method allowed common classes of microfibrils and lamellae in cell walls to be identified from different origins. During both the formation and degradation of cell walls, a preferential formation of structures with specific thickness was evidenced. The results obtained with the developed method allowed objective analysis of patterns of microfibril aggregation and evidenced a trend of doubling/halving lamellar structures, during cell wall formation/degradation in materials from different origin and which have undergone different treatments.

Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana

A cinnamoyl-CoA reductase 1 knockout mutant in Arabidopsis thaliana was investigated for the consequences of lignin synthesis perturbation on the assembly of the cell walls. The mutant displayed a dwarf phenotype and a strong collapse of its xylem vessels corresponding to lower lignin content and a loss of lignin units of the noncondensed type. Transmission electron microscopy revealed that the transformation considerably impaired the capacity of interfascicular fibers and vascular bundles to complete the assembly of cellulose microfibrils in the S(2) layer, the S(1) layer remaining unaltered. Such disorder in cellulose was correlated with X-ray diffraction showing altered organization. Semi-quantitative immunolabeling of lignins showed that the patterns of distribution were differentially affected in interfascicular fibers and vascular bundles, pointing to the importance of noncondensed lignin structures for the assembly of a coherent secondary wall. The use of laser capture microdissection combined with the microanalysis of lignins and polysaccharides allowed these polymers to be characterized into specific cell types. Wild-type A. thaliana displayed a two-fold higher syringyl to guaiacyl ratio in interfascicular fibers compared with vascular bundles, whereas this difference was less marked in the cinnamoyl-CoA reductase 1 knockout mutant.

Cellulose Biosynthesis: Current Views and Evolving Concepts

To outline the current state of knowledge and discuss the evolution of various viewpoints put forth to explain the mechanism of cellulose biosynthesis. * Understanding the mechanism of cellulose biosynthesis is one of the major challenges in plant biology. The simplicity in the chemical structure of cellulose belies the complexities that are associated with the synthesis and assembly of this polysaccharide. Assembly of cellulose microfibrils in most organisms is visualized as a multi-step process involving a number of proteins with the key protein being the cellulose synthase catalytic sub-unit. Although genes encoding this protein have been identified in almost all cellulose synthesizing organisms, it has been a challenge in general, and more specifically in vascular plants, to demonstrate cellulose synthase activity in vitro. The assembly of glucan chains into cellulose microfibrils of specific dimensions, viewed as a spontaneous process, necessitates the assembly of synthesizing sites unique to most groups of organisms. The steps of polymerization (requiring the specific arrangement and activity of the cellulose synthase catalytic sub-units) and crystallization (directed self-assembly of glucan chains) are certainly interlinked in the formation of cellulose microfibrils. Mutants affected in cellulose biosynthesis have been identified in vascular plants. Studies on these mutants and herbicide-treated plants suggest an interesting link between the steps of polymerization and crystallization during cellulose biosynthesis. * With the identification of a large number of genes encoding cellulose synthases and cellulose synthase-like proteins in vascular plants and the supposed role of a number of other proteins in cellulose biosynthesis, a complete understanding of this process will necessitate a wider variety of research tools and approaches than was thought to be required a few years back.

Roles of microtubules and cellulose microfibril assembly in the localization of secondary-cell-wall deposition in developing tracheary elements

The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.

The mechanism and regulation of cellulose synthesis in primary walls: lessons from cellulose-deficient Arabidopsis mutants

Cellulose-deficient Arabidopsis mutants were identified using FT-IR microspectroscopy. The study of these mutants not only led to the identification of actors in cellulose synthesis, but also provided insights in the organization of the hexameric terminal complex from CESA mutants and identified unsuspected accessory proteins with so far unknown roles in the synthesis and/or assembly of cellulose microfibrils. Finally, mutant analysis established a role for protein glycosylation in cellulose synthesis and provided new perspectives on the developmental regulation of cell wall synthesis and the role that cellulose synthesis plays in the control of cell elongation.

Cellulose microfibril assembly and orientation in some bangiophyte red algae: relationship between synthesizing terminal complexes and microfibril structure, shape, and dimensions

As shown by freeze-fracture electron microscopy, the linear terminal complexes (TCs) in Erythrocladia subintegra Rosenvinge, Erythrotrichia carnea (Dillwyn) J. Agardh, Porphyra leucosticta Thuret, and Porphyra yezoensis Ueda typically have regularly spaced rows of particles aligned transverse to the longitudinal axis of the TCs. The spacing between neighboring transverse particle rows is almost constant and ranges from 10.5 to 13.5 nm among the four species. The TC subunits appear to be interconnected by filament-like structures in transverse as well as longitudinal directions along the long axis of the TC. In the investigated species, both linear TCs and microfibrils are randomly distributed and the microfibrils show a flat, ribbon-like morphology. Isolated cellulose microfibrils in negatively stained preparations from Erythrocladia and Porphyra appear as thin, ribbon-shaped structures, 1 to 1.5 nm thick (constant) and 5 to 70 nm wide (variable). It is proposed that each protein particle subunit in an Erythrocladia TC synthesizes three glucan chains that are then assembled into a minisheet. Each transverse row of four TC subunits contributes to a fibrillar (minicrystal) component containing 12 glucan chains, with dimensions of 1.20 × 1.59 nm. The number of fibrillar (minicrystal) components assembled by a given TC and then laterally associated equals the number of transverse particle rows. The laterally associated minicrystal components contribute to the variation in microfibrillar width. The arrangement and consolidation of polymerases (cellulose synthase) and variation in their number in a single linear TC (Erythrocladia 32 to 140 subunits, Erythrotrichia 30 to 110, Porphyra yezoensis 11 to 25, Porphyra leucosticta 6 to 24) are responsible for the structure and flat, ribbon-like morphology of the microfibrils and for the variation in the width/diameter of the cellulose microfibrils. In red algae, cellulose occurs as highly oriented crystalline microfibrils.

Cellulose synthesized by Acetobacter xylinum in the presence of acetyl glucomannan

Acetobacter xylinum was cultured in Hestrin-Schramm medium (control medium) and Hestrin-Schramm medium containing acetyl glucomannan (mannan medium). Loose bundles of the cellulose microfibrils are formed in the mannan medium in contrast to the normal ribbons being produced in the control medium. Rapid-freeze and substitution method followed by metal-shadowing revealed the droplet-like structures around the microfibril synthesized in the mannan medium. The cellulose synthesized in the mannan medium was stained heavily by the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) method, while the cellulose synthesized in the control medium was not stained. X-ray diffractometry and FT-IR spectroscopy indicated that the addition of mannan induced a change in the crystal structure from the algal-bacterial type to the cotton-ramie type. Thus the presence of acetyl glucomannan in the medium prevents the assembly of cellulose microfibrils and changes the crystal structure of cellulose.

New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicateMetandrocarpa uedai

Cellulose synthesizing enzyme complexes (terminal complexes, TCs) have been found in the plasma membrane of epidermal cells in the tunicateMetandrocarpa uedai by using freeze-fracture replication techniques for electron microscopy. Assembly of cellulose microfibrils by TCs is a universal phenomenon in the biological kingdoms. The TCs are locally distributed in the plasma membrane of the epidermal cells facing the tunic, and no TCs are observed on the lateral membranes bordered by tight junctions. The TCs consist of two types of membrane subunits: large particles (14.5 nm in diameter) on the periphery and small subunit particles (7.2 nm) filling the center; the latter are hypothesized to be involved in cellulose synthesis. The TCs are the linear type (ca. 195 nm in length and 78 nm in width). Direct connections of TCs with the termini of microfibrils were observed. Amorphous regions, which were hypothesized the nascent microfibrils, were associated with the depressions of the TCs. The distortion of microfibrils on their terminus indicates that the crystallization may occur at the margin of TCs from which the microfibrils are discharged. This report provides evidence that: (1) The outer cell membrane of epidermis is the site for the assembly of cellulose microfibrils in the tunic; (2) a new type of TC is involved in the biosynthesis of cellulose microfibrils in the tunicates; (3) disorganized glucan chains may be synthesized in the depression of TCs and crystallized outside the E-surface of the epidermal cell membrane.

Cellulose microfibril assembly inErythrocladia subintegra Rosenv.: An ideal system for understanding the relationship between synthesizing complexes (TCs) and microfibril crystallization

The marine red algaErythrocladia subintegra synthesizes cellulose microfibrils as determined by CBH I-gold labelling, X-ray and electron diffraction analyses. The cellulose microfibrils are quite thin, ribbon-like structures, 1–1.5 nm in thickness (constant), and 10–33 nm in width (variable). Several laterally associated minicrystal components contribute to the variation in microfibrillar width. Electron diffraction analysis suggested a uniplanar orientation of the microfibrils with their (101) lattice planes parallel to the plasma membrane surface of the cell. The linear particle arrays bound in the plasma membrane and associated with microfibril impressions recently demonstrated inErythrocladia have been shown in this study to be the cellulose-synthesizing terminal complexes (TCs). The TCs appear to be organized by a repetition of transverse rows consisting of four TC subunits, rather than by four rows of longitudinallyarranged TC subunits. The number of transverse rows varied between 8–26, corresponding with variation in the length of the TCs and the width of the microfibrils. The spacings between the neighboring transverse rows are almost constant being 10.5–11.5 nm. Based on the knowledge thatAcetobacter, Vaucheria, andErythrocladia synthesize similar thin, ribbon-like cellulose microfibrils, the structural characteristics common to the organization of distinctive TCs occurring in these three organisms has been discussed, so that the mode of cellulose microfibril assembly patterns may be deciphered.

Effects of 2,6-dichlorobenzonitrile and Tinopal LPW on the structure of the cellulose synthesizing complexes ofVaucheria hamata

The effects of 2,6-dichlorobenzonitrile (DCB, a known inhibitor of cellulose synthesis) and Tinopal LPW (TPL, an agent which disrupts glucan crystallization) on the structure of cellulose synthesizing complexes (terminal complexes, TCs) in the xanthophycean algaVaucheria hamata were investigated. DCB (10 μM) inhibits nascent fibril formation from the TC subunit (based on the absence of impressions) although it does not alter the overall shape of the rectangular TC during the short treatment of 20 min. With a prolonged treatment (60 min), the arrangement of TC subunits becomes disordered, and particles generally exhibited as doublets of subunits are released from each other. DCB also interferes with the formation of the overall shape of the TC although it does not disturb the conversion into TC rows of the subunits (the zymogenic precursor of the TC) packed in the globules. A 15 min treatment with TPL (1 mM) destroys the TC integrity by reducing the subunits into small fragments or particulate aggregates. The particulate rows of the TC are interrupted at many points, and fragments and particulate aggregates are dispersed by prolonged treatment (45 min) with TPL. Unlike DCB, TPL inhibits the conversion of globule subunits into TC rows. New insights on the structural characteristics necessary for cellulose microfibril assembly and possible mechanisms for the biogenesis of theVaucheria TC come from these data.

Cellulose synthesizing complexes in some giant marine algae

ABSTRACT The structure of putative cellulose synthesizing complexes (TCs) has been studied in giant marine algae and is discussed in relation to the assembly of cellulose microfibrils. Including previous work, 14 species belonging to nine genera in the Siphonocladales and two species in the Cladophorales are known to have linear TCs on both E- and P-fracture faces of the plasma membrane. Species studied in the present paper included Boodlea composita, Dictyosphaeria cavernosa, Ernodesmis verticillata, Siphonocladus tropicus, Struvea elegans, Valoniopsis pachynema and Chaetomorpha aerea. Contrary to their fairly consistent width (30–36 nm), TCs have a wide distribution of length among indIvidual species and at various stages of development in the same species. Most of the TCs have a random arrangement of subunits, but sometimes they are arranged in three rows. The mean TC length is greater during secondary wall synthesis than in primary wall synthesis in all of the following species: Boodlea composita, Dictyosphaeria cavernosa, Siphonocladus tropicus, Valonia macrophysa, Valonia ventricosa and Chaetomorpha aerea. These results support previous results suggesting that the linear TCs increase their length during cell wall development. The size of TC subunits, ranging from 7.3 to 8.9 nm, was smaller than the structural membrane particles on the plasma membrane in all of the species examined. It is suggested that the spacing between individual glucan chains will be reduced to half after crystallization of cellulose microfibrils, on the basis of evidence that the width of microfibrils is as wide as that of TC. The width of microfibrils ranged from 11.2 to 23.6 nm, while most of the species had microfibrils with a width in the range 14 to 16 nm. The width of microfibrils in Boergesenia was the largest among the giant marine algae. The formation of TCs from subunits, which are transmembrane particles, is characteristic of Siphonocladales in spite of their varying cell morphology.

Xyloglucan in the cell walls of cotton fiber

Xyloglucans occur widely in the primary walls of higher plant cells where they are bound in close association with cellulose microfibrils. Evidence was recently presented that the polysaccharide functions in the organization of cellulose microfibril assembly by preventing the fasciation of the fibrils and could aid in spreading the fiber network over the cell surface’. In pea stems, auxin treatment induces the development of endo-(1+4)-/3-D-glucanase activities and these are responsible for xyloglucan turnover 2J. The integrity of xyloglucan could then control the ability of microfibrils to loosen, and of cells to elongate and expand during growth. Cotton fibers are highly elongated cells similar in shape to pollen tubes and root hairs. However, unlike these latter cell types, cotton fibers elongate not by tip growth, but by deposition of wall components and elongation throughout the length of the fiber”‘. During elongation, the fibers deposit a primary wall similar in composition to that of many other dicot species 5 that includes xyloglucan as one component6,7. We recently showed that xyloglucan deposition is restricted to the elongation phase of fiber development ‘. The mechanism by which cell shape is determined in cotton fibers is not understood, but the interaction of xyloglucan with cellulose microfibrils and the orientation of such tibrils might be expected to play some role. In this report, we have examined the fine structure of cotton fiber xyloglucan and compared its structure to that found in cell walls of other dicotyledonous plants. Cotton fiber cell wall polysaccharides were sequentially fractionated into hot water-soluble and 24% potassium hydroxide, and an insoluble fraction. Each fraction was analyzed by methylation analysis8 and the iodine-sodium sulfate method9 in order to determine the xyloglucan content. The results indicated that 95% of total xyloglucan was concentrated in the 24% potassium hydroxide extract.