Celery (Apium graveolens L.) parenchyma cell walls examined by atomic force microscopy: effect of dehydration on cellulose microfibrils

Atomic force microscopy (AFM) was used to image celery (Apium graveolens L.) parenchyma cell walls in situ. Cellulose microfibrils could clearly be distinguished in topographic images of the cell wall. The microfibrils of the hydrated walls appeared smaller, more uniformly distributed, and less enmeshed than those of dried peels. In material that was kept hydrated at all times and imaged under water, the microfibril diameter was mainly in the range 6–25 nm. The cellulose microfibril diameters were highly dependent on the water content of the specimen. As the water content was decreased, by mixing ethanol with the bathing solution, the microfibril diameters increased. Upon complete dehydration of the specimen we observed a significant increase in microfibril diameter. The procedure used to dehydrate the parenchyma cells also influenced the size of cellulose microfibrils with freeze-dried material having larger diameters than air-dried material. Received: 16 November 1999 / Accepted: 7 March 2000     

Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy

We used atomic force microscopy (AFM), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis – a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5‐nm wide cellulose microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high‐resolution AFM images of the newly deposited wall surface showed that single microfibrils merge into and out of short regions of microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose microfibrils when imaged by FESEM, but not by AFM. The AFM thus preferentially detects cellulose microfibrils by probing through the soft matrix in these hydrated walls. AFM‐based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near‐native state, with implications for microfibrils motions in different lamellae during uniaxial and biaxial extensions.     

Cellulose microfibril alignment recovers from DCB-induced disruption despite microtubule disorganization

Cellulose microfibril deposition patterns define the direction of plant cell expansion. To better understand how microfibril alignment is controlled, we examined microfibril orientation during cortical microtubule disruption using the temperature-sensitive mutant of Arabidopsis thaliana, mor1-1. In a previous study, it was shown that at restrictive temperature for mor1-1, cortical microtubules lose transverse orientation and cells lose growth anisotropy without any change in the parallel arrangement of cellulose microfibrils. In this study, we investigated whether a pre-existing template of well-ordered microfibrils or the presence of well-organized cortical microtubules was essential for the cell to resume deposition of parallel microfibrils. We first transiently disrupted the parallel order of microfibrils in mor1-1 using a brief treatment with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). We then analysed the alignment of recently deposited cellulose microfibrils (by field emission scanning electron microscopy) as cellulose synthesis recovered and microtubules remained disrupted at the mor1-1 mutant's non-permissive culture temperature. Despite the disordered cortical microtubules and an initially randomized wall texture, new cellulose microfibrils were deposited with parallel, transverse orientation. These results show that transverse cellulose microfibril deposition requires neither accurately transverse cortical microtubules nor a pre-existing template of well-ordered microfibrils. We also demonstrated that DCB treatments reduced the ability of cortical microtubules to form transverse arrays, supporting a role for cellulose microfibrils in influencing cortical microtubule organization.     

Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein

It has long been hypothesized that cortical microtubules (MTs) control the orientation of cellulose microfibril deposition, but no mutants with alterations of MT orientation have been shown to affect this process. We have shown previously that in Arabidopsis, the fra2 mutation causes aberrant cortical MT orientation and reduced cell elongation, and the gene responsible for the fra2 mutation encodes a katanin-like protein. In this study, using field emission scanning electron microscopy, we found that the fra2 mutation altered the normal orientation of cellulose microfibrils in walls of expanding cells. Although cellulose microfibrils in walls of wild-type cells were oriented transversely along the elongation axis, cellulose microfibrils in walls of fra2 cells often formed bands and ran in different directions. The fra2 mutation also caused aberrant deposition of cellulose microfibrils in secondary walls of fiber cells. The aberrant orientation of cellulose microfibrils was shown to be correlated with disorganized cortical MTs in several cell types examined. In addition, the thickness of both primary and secondary cell walls was reduced significantly in the fra2 mutant. These results indicate that the katanin-like protein is essential for oriented cellulose microfibril deposition and normal cell wall biosynthesis. We further demonstrated that the Arabidopsis katanin-like protein possessed MT-severing activity in vitro; thus, it is an ortholog of animal katanin. We propose that the aberrant MT orientation caused by the mutation of katanin results in the distorted deposition of cellulose microfibrils, which in turn leads to a defect in cell elongation. These findings strongly support the hypothesis that cortical MTs regulate the oriented deposition of cellulose microfibrils that determines the direction of cell elongation.     

Molecular Rigidity in Dry and Hydrated Onion Cell Walls

Solid-state nuclear magnetic resonance relaxation experiments can provide information on the rigidity of individual molecules within a complex structure such as a cell wall, and thus show how each polymer can potentially contribute to the rigidity of the whole structure. We measured the proton magnetic relaxation parameters T2 (spin-spin) and T1p (spin-lattice) through the 13C-nuclear magnetic resonance spectra of dry and hydrated cell walls from onion (Allium cepa L.) bulbs. Dry cell walls behaved as rigid solids. The form of their T2 decay curves varied on a continuum between Gaussian, as in crystalline solids, and exponential, as in more mobile materials. The degree of molecular mobility that could be inferred from the T2 and T1p decay patterns was consistent with a crystalline state for cellulose and a glassy state for dry pectins. The theory of composite materials may be applied to explain the rigidity of dry onion cell walls in terms of their components. Hydration made little difference to the rigidity of cellulose and most of the xyloglucan shared this rigidity, but the pectic fraction became much more mobile. Therefore, the cellulose/xyloglucan microfibrils behaved as solid rods, and the most significant physical distinction within the hydrated cell wall was between the microfibrils and the predominantly pectic matrix. A minor xyloglucan fraction was much more mobile than the microfibrils and probably corresponded to cross-links between them. Away from the microfibrils, pectins expanded upon hydration into a nonhomogeneous, but much softer, almost-liquid gel. These data are consistent with a model for the stress-bearing hydrated cell wall in which pectins provide limited stiffness across the thickness of the wall, whereas the cross-linked microfibril network provides much greater rigidity in other directions.     

Xyloglucan in the primary cell wall: assessment by FESEM,selective enzyme digestions and nanogold affinity tags

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load‐bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high‐resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase‐digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan‐specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold‐conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan‐depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.     

Formation of macromolecular lignin in ginkgo xylem cell walls as observed by field emission scanning electron microscopy

Formation of macromolecular lignin in ginkgo cell walls. In the lignifying process of xylem cell walls, macromolecular lignin is formed by polymerization of monolignols on the pectic substances, hemicellulose and cellulose microfibrils that have deposited prior to the start of lignification. Observation of lignifying secondary cell walls of ginkgo tracheids by field emission scanning electron microscopy suggested that lignin-hemicellulose complexes are formed as tubular bead-like modules surrounding the cellulose microfibrils (CMFs), and that the complexes finally fill up the space between CMFs. The size of one tubular bead-like module in the middle layer of the secondary wall (S2) was tentatively estimated to be about 16+/-2 nm in length, about 25+/-1 nm in outer diameter, with a wall thickness of 4+/-2 nm; the size of the modules in the outer layer of the secondary wall (S1) was larger and they were thicker-walled than that in the middle layer (S2). Aggregates of large globular modules were observed in the cell corner and compound middle lamella. It was suggested that the structure of non-cellulosic polysaccharides and mode of their association with CMFs may be important factors controlling the module formation and lignin concentration in the different morphological regions of the cell wall.     

Solid-state 13C-NMR spectroscopy shows that the xyloglucans in the primary cell walls of mung bean (Vigna radiata L.) occur in different domains: a new model for xyloglucan-cellulose interactions in the cell wall

Xyloglucans (XG) with different mobilities were identified in the primary cell walls of mung beans (Vigna radiata L.) by solid-state 13C-NMR spectroscopy. To improve the signal:noise ratios compared with unlabelled controls, Glc labelled at either C-1 or C-4 with 13C-isotope was incorporated into the cell-wall polysaccharides of mung bean hypocotyls. Using cell walls from seedlings labelled with d-[1-13C]glucose and, by exploiting the differences in rotating-frame and spin-spin proton relaxation, a small signal was detected which was assigned to Xyl of XGs with rigid glucan backbones. After labelling seedlings with d-[4-13C]glucose and using a novel combination of spin-echo spectroscopy with proton spin relaxation-editing, signals were detected that had 13C-spin relaxations and chemical shifts which were assigned to partly-rigid XGs surrounded by mobile non-cellulosic polysaccharides. Although quantification of these two mobility types of XG was difficult, the results indicated that the partly-rigid XGs were predominant in the cell walls. The results lend support to the postulated new cell-wall models in which only a small proportion of the total surface area of the cellulose microfibrils has XG adsorbed on to it. In these new models, the partly-rigid XGs form cross-links between adjacent cellulose microfibrils and/or between cellulose microfibrils and other non-cellulosic polysaccharides, such as pectic polysaccharides.     

Localization of cell wall polysaccharides in normal and compression wood of radiata pine: relationships with lignification and microfibril orientation

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.     

Crystalline Cellulose in Hydrated Primary Cell Walls of Three Monocotyledons and One Dicotyledon

The molecular ordering of cellulose, including its crystallinity,in the unlignified primary cell walls of three monocotyledons(Italian ryegrass, pineapple, and onion) and one dicotyledon(cabbage) was characterized by solid-state ¹³C NMR spectroscopy.These species were chosen because their primary cell walls havedifferent non-cellulosic polysaccharides and this may affectthe molecular ordering of cellulose. Values of the proton rotating-framerelaxation [T_1p(H)] and spin-spin relaxation [T₂(H)] time constantsshowed that the cellulose in the cell walls of all four specieswas in a crystalline rather than an amorphous state. Furthermore,a resolution enhancement procedure showed that the triclinic(I) and the monoclinic (I_rß) crystal forms of cellulosewere present in similar proportions in these cell walls. However,the calculated cross-sectional dimensions of the cellulose crystallitesvaried among the cell walls (in the range 2–3 nm): thelargest were in the Italian ryegrass, the smallest were in theonion and cabbage, and those of intermediate size were in thepineapple. The crystallite dimensions may thus be affected bythe non-cellulosic polysaccha-ride compositions of the cellwalls. ⁴Present address: Food Science Postgraduate Programme, Departmentof Chemistry, The University of Auckland, Private Bag 92019,Auckland, New Zealand.     

Plant cell wall architecture is revealed by rapid-freezing and deep-etching

Summary This paper reports on preliminary investigations into the structure of cell walls of varying complexity as revealed by the rapidfreeze deep-etch technique. Three cell types from different species were examined in order to compare the three-dimensional arrangement of random, polylamellate and helicoidal walls. Each cell type displayed a distinctive level of organisation with respect to the cellulose microfibrils and the matrix material. In polylamellated walls, the microfibrils within each layer were linked to each other by 16–20 nm long side chains regularly spaced along the length of the microfibril. In helicoidal walls, the shifting of the microfibrils could cleary be seen, yet no recognisable structures were observed which could mediate this movement.     

Structure and association of wall fibrils produced by regenerating tobacco protoplasts

A study has been made of the wall fibrils produced by tobacco protoplasts, using scanning electron microscopy in conjunction with negative staining. It has been shown that the fibres seen in scanning electron microscopy correspond to aggregates of microfibrils. These aggregates are only visible where they are lifted clear of the protoplast surface. Negative staining of fixed protoplasts shows that the aggregation of microfibrils into the fibres visible in scanning electron microscopy is probably produced by air-drying. Gentle disruption of microfibrils produces both random broken fragments and bundles of short pieces of fibrillar material about 60 nm in length. This material is present in undisrupted young walls, but not in undisrupted older walls. The microfibrils in young walls seem much more fragile and liable to breakage than those in older walls. These results are discussed in terms of the interpretation of scanning electron microscope images and the mechanism of cellulose microfibril formation by higher plants.Abbreviations SEM Scanning electron microscopy     

Celery (Apium graveolens) parenchyma cell walls: cell walls with minimal xyloglucan

The primary walls of celery ( Apium graveolens L.) parenchyma cells were isolated and their polysaccharide components characterized by glycosyl linkage analysis, cross-polarization magic-angle spinning solid-state ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR) and X-ray diffraction. Glycosyl linkage analysis showed that the cell walls consisted of mainly cellulose (43 mol%) and pectic polysaccharides (51 mol%), comprising rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). The amounts of xyloglucan (2 mol%) and xylan (2 mol%) detected in the cell walls were strikingly low. The small amount of xyloglucan present means that it cannot coat the cellulose microfibrils. Solid-state ¹³C NMR signals were consistent with the constituents identified by glycosyl linkage analysis and allowed the walls to be divided into three domains, based on the rigidity of the polymers. Cellulose (rigid) and rhamnogalacturonan (semi-mobile) polymers responded to the CP/MAS ¹³C NMR pulse sequence and were distinguished by differences in proton spin relaxation time constants. The arabinans, the most mobile polymers, responded to single-pulse excitation (SPE), but not CP/MAS ¹³C NMR. From solid-state ¹³C NMR of the cell walls the diameter of the crystalline cellulose microfibrils was determined to be approximately 3 nm while X-ray diffraction of the cell walls gave a value for the diameter of approximately 2 nm.     

Role of the putative membrane-bound endo-1,4-beta-glucanase KORRIGAN in cell elongation and cellulose synthesis in Arabidopsis thaliana

A temperature-sensitive, elongation-deficient mutant of Arabidopsis thaliana was isolated. At the non-permissive temperature of 31 degrees C, the mutation impaired tissue elongation; otherwise, tissue development was normal. Hypocotyl cells that had established cell walls at 21 degrees C under light-dark cycles ceased elongation and swelled when the mutant was shifted to 31 degrees C and darkness, indicating that the affected gene is essential for cell elongation. Analysis of the cell walls of mutant plants grown at 31 degrees C revealed that the cellulose content was reduced to 40% and the pectin content was increased to 162% of the corresponding values for the wild type grown at the same temperature. The increased amounts of pectin in the mutant were bound tightly to cellulose microfibrils. No change in the content of hemicellulose was apparent in the 31 degrees C-adapted mutant. Field emission-scanning electron microscopy suggested that the structure of cellulose bundles was affected by the mutation; X-ray diffraction, however, revealed no change in the crystallite size of cellulose microfibrils. The regeneration of cellulose microfibrils from naked mutant protoplasts was substantially delayed at 31 degrees C. The recessive mutation was mapped to chromosome V, and map-based cloning identified it as a single G-->A transition (resulting in a Gly(429)-->Arg substitution) in KORRIGAN, which encodes a putative membrane-bound endo-1,4-beta-glucanase. These results demonstrate that the product of this gene is required for cellulose synthesis.     

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.     

Cellulose orientation determines mechanical anisotropy in onion epidermis cell walls

The role of cellulose microfibril orientation in determining cell wall mechanical anisotropy and in the control of the wall plastic versus elastic properties was studied in the adaxial epidermis of onion bulb scales using the constant-load (creep) test. The mean or net cellulose orientation in the outer periclinal wall of the epidermis was parallel to the long axis of the cells. In vitro cell wall extensibility was 30-90% higher in the direction perpendicular to the net microfibril orientation than parallel to it. This was the case for the size of the initial deformation occurring just after the load application and for the rate of time-dependent creep. Loading/unloading experiments confirmed the presence of a real irreversible component in cell wall extension. The plastic component of the time-dependent deformation was higher perpendicular to the net cellulose orientation than parallel to it. An acid buffer (pH 4.5) increased the creep rate by 25-30% but this response was not related to cellulose orientation. The present data provide direct evidence that the net orientation of cellulose microfibrils confers mechanical anisotropy to the walls of seed plants, a characteristic that may be relevant to understanding anisotropic cell growth.     

How do expansins control plant growth? A model for cell wall loosening via defect migration in cellulose microfibrils

Expansins are plant cell wall-loosening proteins that promote cell growth and are essential for many critical developmental processes and stress responses. The molecular basis for expansin action is uncertain. Recently, it has been proposed that expansins loosen the wall by means of the generation of mobile conformational defects at the surface of cellulose microfibrils. The present work addresses this hypothesis by elaborating three assumptions: (1) microfibril–matrix interfaces cause steep stress gradients on the microfibril surface, (2) stress gradients drive the motion of conformational defects along the microfibril surface toward the microfibril–matrix interfaces, and (3) the approach of the defects to the microfibril–matrix interfaces facilitates the dissociation of matrix polysaccharides from cellulose microfibrils.     

Microfibrillar structure of growing cell wall in a coenocytic green alga,Boergesenia forbesii

A fine structure of cell wall lamellae in a coenocytic green algaBoergesenia forbesii was examined by electron microscopy. The wall has a polylamellate structure containing cellulose microfibrils 25 to 30 nm in diameter. The outer surface of the cell was covered by a thin structureless lamella, underneath which existed a lamella containing randomly-oriented microfibrils. The major part of the wall consisted of two types of lamellae, multifibrillar lamella and a transitional, matrix-rich one. In the former, microfibrils were densely arranged more or less parallel with each other. In the transitional lamella, existing between the multifibrillar ones, the microfibril orientation shifted about 30° within the layer. The fibril orientation also shifted 30° between adjacent transitional and multifibrillar layers, and consequently the microfibril orientation in the neighboring multifibrillar layers shifted 90°. It was concluded that the orientation rotated counterclockwise when observed from inside the cell. Each lamella in the thallus wall become thinner with cell expansion, but no reorientation of microfibrils in the outer old layers was observed. In the rhizoid, the outer lamellae sloughed off with the tip growth.     

The control of cellulose microfibril deposition in the cell wall of higher plants

In maize (Zea mays L.) and pine (Pinus taeda L.) seedlings, cellulose microfibril impressions are present on freeze-fractured plasma membranes. It has been proposed that impressions of newly synthesized microfibrils are a record of the movement of terminal synthesizing complexes through the plasma membrane (Mueller and Brown, 1980, J. Cell Biol. 84, 315–326). The association of terminal complexes with the ends of microfibril impressions or with the ends of microfibrils torn through the membrane indicates the orientation of microfibril tips. Unidirectionally-oriented microfibril tips (all pointing in the same direction) are associated with the organized deposition of parallel arrays of microfibrils. Multidirectionally-oriented microfibril tips were observed in a cell in which microfibril deposition was unusually disorganized. Microfibril patterns around pit fields are asymmetric and resemble flow patterns. Unidirectionally-oriented tears are associated with these microfibrils. Although microfibril orientations are deflected around pit fields, the main axis of microfibril orientation is maintained across the surface of the cell. The hypothesis is proposed that the interaction of a flowing plasma membrane with microfibril synthesizing complexes in the plane of the membrane may result in unidirectional deposition and asymmetric microfibril impressions around pit fields.Some of this work has been published in preliminary form (Brown 1979)     

Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro

The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca2+ and 8 mM Mg2+. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 μm in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.