The Synthesis of Cellulose Microfibrils by Particulate Fractions Released from Acetobacter xylinum by Washing

Washing A. xylinum whole cells in 0.01 m phosphate-0.003 m citrate,pH 6.2, leads to the release of particulate fractions whichretain the ability to synthesize cellulose microfibrils fromglucose and from UDPG. These fractions contain both outer andcytoplasmic membrane fragments. Frccze-etching shows the microfibrilsto arise from outer membrane vesicles. A scheme to describethe synthesis of cellulose microfibrils by A. xylinum is proposed.     

Changes in the Arrangement of Microtubules and Microfibrils in Differentiating Conifer Tracheids During the Expansion of Cells

The arrangements of microtubules and the cellulose microfibrilsof radial walls in tracheids of Abies sachalinensis Mastersduring the expansion of cells were examined by immunofluorescenceand field-emission scanning electron microscopy. The radialdiameter of tracheids increased to three to four times thatof cambial initial cells. Microfibrils on the innermost surfaceof primary walls of conifer tracheids at early stages were notwell ordered and most of the microfibrils were oriented longitudinally.As each cell expanded, microfibrils in the process of depositionwere still not well ordered but their orientation changed fromlongitudinal to transverse. When cell expansion ceased, microfibrilswere well ordered and oriented transversely. Cortical microtubulesshowed a change in orientation similar to that of the microfibrils.These results indicate that the orientation of cortical microtubulesis correlated with that of microfibrils as they are being laiddown and with cell morphogenesis in conifer tracheids.Copyright1995, 1999 Academic Press Microfibril, microtubule, tracheid, cell expansion, Abies sachalinensis Masters, field-emission scanning electron microscopy, immunofluorescence microscopy     

Electron Microscopy of a Non-pellicle-forming Strain of Acetobacter xylinum

A non-pellicle-forming culture of Acetobacter xylinum, isolatedfrom a pellicle-forming culture by repeated transfer of thelatter through agitated medium, has been examined. The cellsof the non-pellicle-producing culture are often greatly elongatedand possess a profusion of intra-cytoplasmic membranes. Freeze-fracturedcells reveal extensive face views of both the outer and cytoplasmicmembranes and show a cell coat composed of a large number ofshort fibrils arising from the outer membrane. X-ray diffractionof the cells in some cases shows the cellulose-I pattern butshadow-contrasted preparations do not show long microfibrils.It is suggested that the non-pellicle formers produce celluloseas only very short microfibrils which are synthesised all overthe cell surface and that there are changes in the outer membranecompared to pellicle-producing cells. The possible significanceof these differences between pellicle-forming and non-pellicle-formingA. xylinum strains is discussed.     

Microfibrillar Structure, Cortical Microtubule Arrangement and the Effect of Amiprophos-methyl on Microfibril Orientation in the Thallus Cells of the Filamentous Green Alga, Chamaedoris orientalis

Microfibrillar structure, cortical microtubule orientation andthe effect of amiprophos-methyl (APM) on the arrangement ofthe most recently deposited cellulose microfibrils were investigatedin the marine filamentous green alga, Chamaedoris orientalis.The thallus cells of Chamaedoris showed typical tip growth.The orientation of microfibrils in the thick cell wall showedorderly change in longitudinal, transverse and oblique directionsin a polar dependent manner. Microtubules run parallel to thelongitudinally arranged microfibrils in the innermost layerof the wall but they are never parallel to either transverseor obliquely arranged microfibrils. The ordered change in microfibrilorientation is altered by the disruption of the microtubuleswith APM. The walls, deposited in the absence of the microtubules,showed typical helicoidal pattern. However, the original crossedpolylamellate pattern was restored by the removal of APM. Thissuggests that cortical microtubules in this alga do not controlthe direction of microfibril orientation but control the orderedchange of microfibril orientation. Amiprophos-methyl, Chamaedoris orientalis, coenocytic green alga, cortical microtubule, microfibrillar structure, tip growth     

The Net Orientation of Wall Microfibrils in the Outer Periclinal Epidermal Walls of Seedling Leaves of Wheat

Use of the dichroic stain chlor-zinc-iodine revealed that thenet orientation of cellulose wall microfibrils in the outerparadermal wall of the epidermis of seedling wheat leaves isprincipally transverse in the extension zone. The net orientationof microfibrils changes abruptly to principally longitudinalat the end of cell elongation. The net angle of orientationof microfibrils in the extension zone was not a function ofRht-dosage (number of dwarfing alleles), and neither leaf extensionrate nor estimated maximum relative elemental rate of elongationwere functions of microfibril orientation. The results indicatethat elongation rates are not regulated by the net angle oforientation of microfibrils and support the concept that leafextension rate is regulated by the length of the extension zone.Copyright1995, 1999 Academic Press Cellulose wall microfibrils, extension zone, elongation, Rht, wheat, Triticum aestivum L     

Modification in Cell Shape Unrelated to Cellulose Microfibril Orientation in Growing Thallus Cells of Chaetomorpha moniligera

Cellulose microfibril orientation patterns in thallus cellsof Chaetomorpha moniligera were studied, and the relationshipbetween the microfibril and the peripheral microtubule arrangementsduring cell-shape modification by colchicine was examined. Inthe cuttings from growing thalli, linearly arranged cylindricalcells developed into cask-shaped cells during 4–6 daysof culture at 27?C. In the cylindrical cells, microfibrils formingthe innermost portion of the wall were arranged alternatelyin longitudinal and transverse directions, but peripheral microtubuleswere always arranged only in a longitudinal direction. Thesefeatures were also noted in the cask-shaped cells. Colchicineat 10^–3M and 3?10^–3M accelerated both cell expansionand wall thickening with matrix deposition, but the directionsin which both microfibrils and microtubules were arranged werethe same as those of the cylindrical cells. These results indicatethat (1) the microfibril and microtubule arrangements of Chaetomorphaare not necessarily correlated, (2) changes in cell shape ofChaetomorpha are not necessarily accompanied by changes in thearrangement of cell-wall microfibrils, and (3) colchicine playsa role in the loosening and thickening of cell walls by enhancingmatrix deposition. (Received June 2, 1986; Accepted February 13, 1987)     

Submicroscopic Structure of the Cereal Starch Grain

The submicroscopic structure of the starch grains of Zea maysand Triticum sativum was studied electron-optically from replicasmade of internal, fracture surfaces. In corn starch, long cylindricalmicrofibrils were found, arranged in a radial direction andimbedded in an amorphous matrix. Their diameter was uniformlyabout 200 Å. Microfibrils were also indicated in wheatstarch because of the prominence of their ends in surface view.Many microfibrils in corn starch appeared to be helically coiled.The more ordered starch substance was presumed to be localizedprincipally in the microfibrils.     

Direct Visualization of Lignifying Secondary Wall Thickenings in Zinnia elegansCells in Culture

The lignifying secondary wall thickenings of tracheary elementsthat were differentiating from Zinnia mesophyll cells in suspensionculture were examined by a freeze-etch replica technique. Cellulosemicrofibrils in primary and secondary wall thickenings differedin terms of both width and arrangement. The primary wall wasobserved as a randomly arranged network of microfibrils. Bycontrast to microfibrils in the secondary wall thickenings werehighly organized, with many pores and spaces between them. Numerousfilamentous and granular cross-links were observed in both primarywalls and secondary wall thickenings. As lignifica-tion proceeded,the cellulose microfibrils in secondary wall thickenings becameobscure as a result of the deposition of large numbers of sphericalbodies around and between the microfibrils. This material hadcompletely covered the fibrous matrix by the end of lignification.It might have been composed of the products of the dehydrogenationof monolignols. We also noted that the microfibrils appearedto be slightly irregular or wavy just after the start of lignificationbut were straighter and appeared to be more rigid when lignificationwas complete. (Received July 25, 1996; Accepted April 24, 1997)     

Orientation of Microtubules during Regeneration of Cell Wall in Selected Giant Marine Algae

The microtubules in highly synchronized aplanospores of twogiant marine algae, Boergesenia forbesii and Valonia ventricosa,were examined by immunofluorescence microscopy throughout theregeneration of the cell wall. Microtubule orientation was alwaysrandom up to 20 h after wounding, although the orientation ofcellulose microfibrils changed from random to parallel withinthat time period. When the rhizoid cells were in the stage ofelongation at 7 to 10 days after wounding, highly ordered microtubuleswere always observed along the longitudinal cell axis exceptat the very tip of the cells where random ones were found. Incontrast, the microfibrils in the innermost lamellae of newlysynthesized cell walls showed three different orientations,that is, transverse, longitudinal and oblique to the longitudinalcell axis. These observations suggest that microtubules maycontrol cell shape, but not the orientation of microfibrils.The mechanism of cell wall construction in these algae is discussedin relation to the self-assembly mechanism thought to operatein the construction of helicoidal cell walls. ³ Present address: Polymer Research Laboratory, Mitsui ToatsuChemicals, Inc., Yokohama, Kanagawa 244, Japan. (Received November 18, 1987; Accepted April 11, 1988)     

Changes in Cell Wall Architecture of Differentiating Tracheids of Pinus thunbergii during Lignification

The cell wall architecture, before and after lignification,of differentiating tracheids in Pinus thunbergii has been examinedusing a rapid-freeze deep-etching technique combined with transmissionelectron microscopy. Replicas of cells from the cambial zoneshowed that the unlignified primary cell wall was highly porouswith microfibrils extensively interconnected by crosslinks.The unlignified secondary cell wall has unidirectional microfibrils,more or less associated in bundles, forming a wavy pattern aroundpores of characteristic slit-like shape with narrowing ends.As the lignification progresses, the cell wall structure becomesdense, with no detectable pores. Delignification of wood samplesleads to the reappearance of crosslinks, individual microfibrilsand pores in the secondary cell wall, although in a somewhataltered shape. In addition, cellulose-synthesizing enzyme complexes(rosettes) have for the first time been detected on the plasmamembrane of differentiating xylem cells of softwood. (Received August 28, 1998; Accepted March 10, 1999)     

Electron Microscopic Investigations of the Walls of Green Algae: I. A PRELIMINARY ACCOUNT OF WALL LAMELLATION AND DEPOSITION IN VALONIA VENTRICOSA

A survey of the structure of wall lamellae in the green algaValonia ventricosa has revealed the following details. The microfibrilsare not so constant in width as had previously been suggested;they vary in this dimension from about 100 A to about 350 A.The length of the shadow cast by separated microfibrils shadowedwith palladium-gold or with uranium suggests that the microfibrilsare flattish ribbons rather than circular cylinders. In theintermediate lamellae of the wall they are in intimate contact,probably with an interfibrillar cementing material, but in outerlamellae they become more or less widely separated. This separationis assumed to occur as a result of adjustment to size increasein the vesicle. Observation of the innermost lamella shows thatthe microfibrils here are arranged at random, in marked contrastto the condition in other lamellae. Here and there granulardeposits on this lamella appear to be connected with the cytoplasm,and it is suggested that these may represent points in the cytoplasmassociated with microfibril production. Occasional twistingof microfibrils over and around each other in most of the walllamellae suggests most strongly that the wall and the cytoplasmare not so clearly separated during deposition as has been thought.     

Increase in the Amount of celA1 Protein in Tobacco BY-2 Cells by a Cellulose Biosynthesis Inhibitor, 2,6-dichlorobenzonitrile

The biochemical analysis of cellulose biosynthesis by plantshas been a difficult problem due to the lack of a reliable assayprocedure for cellulose synthase activity. Recently, the celAlgene was cloned from cotton fiber, and this gene was identifiedfrom the rsw1 mutant of Arabidopsis as a catalytic subunit ofcellulose synthase (Arioli et al. 1998). The cloning of thesegenes enables us to obtain specific antibodies against cellulosesynthase. A highly specific antibody against celAl protein wasprepared and used to detect the protein from microsomal fractionof tobacco BY-2 cells. The quantity of celAl protein in microsomalfraction of normal BY-2 cells was under the detection limit,although they contained a large quantity of cellulose. In contrast,cells habituated to 1 µM DCB (a specific inhibitor ofcellulose biosynthesis) produced 1/10 of cellulose content ofthe normal cells, but had much more celAl protein than the normalcells. The amount of polysaccharides in the EDTA-soluble fractionwas relatively increased in habituated cells. The results suggestthat celAl protein is stabilized upon DCB binding and that thecrystallization of cellulose microfibrils is inhibited simultaneously. (Received January 28, 1998; Accepted May 7, 1998)     

Cystolith Development and Structure in Pilea cadierei (Urticaceae)

Cystolith formation, structure and composition have been investigatedin leaves and stem internodes of Pilea cadierei (Urticaceae)using a variety of techniques at the light and electron microscopelevels. The development of lithocysts from epidermal cells hasbeen followed. These cells are cytoplasmically similar to otherepidermal cells but possess a much more active Golgi apparatusand more numerous mitochondria. The cystolith is a spindle-shapedbody composed of concentric layers of longitudinally orientatedcellulose microfibrils associated with pectins and other cellwall polysaccharides. At maturity it is heavily impregnatedwith calcium carbonate. Some cystoliths also contain siliconand are covered in a sheath of siliceous material. Cystolithformation occurs at the tip of a peg that grows in from thelithocyst wall. Evidence from ultrastructure suggests that thelithocytst cytoplasm transports carbohydrates to the cystolithvia Golgi vesicles, and organizes the deposition of cystolithcellulose microfibrils via a system of microtubules lying beneaththe plasma membrane that envelopes the growing cystolith. Thepeg is composed of heavily staining amorphous material likethat of an apoplastically sealed cell wall. It is incapableof supporting the migration of lanthanum ions into the cystolith.We conclude that cystoliths are isolated volumes of apoplastthat act as repositories for inorganic salts, principally calciumcarbonate. We propose that calcium ions move into the lithocystprotoplast from surrounding cells and are then transported acrossthe plasma membrane boundary into the cystolith. This proposalconflicts with previous suggestions that calcium enters by diffusionthrough the peg. Cystolith, lithocyst, cell wall, calcium, silicon, cytochemistry, electron probe analysis, Pilea cadierei     

Localization of the Xyloglucan in Cell Walls in a Suspension Culture of Tobacco by Rapid-Freezing and Deep-Etching Techniques Coupled with Immunogold Labelling

Localization of xyloglucan in cell walls regenerated from tobaccoprotoplasts (Nicotiana tabacum L.; cv. BY-2) is visualized byrapid-freezing and deep-etching (RFDE) electron microscopy coupledwith immunogold electron microscopy. Xyloglucan was alreadydeposited in the cell wall 3 h after culture initiation. Xyloglucanwas mainly localized along microfibrils with a lesser amountin intersections between two crossed microfibrils in 120-hour-oldcells. These data support the previous hypothesis of Keegstraet al. (1973) that propose an interconnection between xyloglucanand cellulose. (Received May 22, 1998; Accepted July 13, 1998)     

Changes in microfibril arrangement on the inner surface of the epidermal cell walls in the epicotyl of Vigna angularis ohwi et ohashi during cell growth

Throughout the entire period of cell growth, the microfibrils on the inner surface of the outer tangential walls of the epidermal cells of Vigna angularis epicotyls are running parallel to one another and their orientation differs from cell to cell. Although transverse, oblique and longitudinal microfibrils can be observed irrespective of cell age, the frequency distribution of microfibril orientation changes with age. In young cells, transversely oriented microfibrils predominate. In cells of medium age, which are still undergoing elongation, transverse, oblique and longitudinal microfibrils are present in quite similar frequencies. In old, non-growing cells, longitudinally oriented microfibrils are predominent. A decrease in the relative frequency of transversely oriented microfibrils with cell age was also observed in the radial epidermal walls.     

The Microfibrillar Component of the Pollen Intine Some Structural Features

The microfibrillar polysaccharide component of the pollen intinecan be isolated by progressive chemical digestion of the exineand the cellular contents and the extraction of the matrix materials.The resulting intine ‘ghosts’ reveal various characteristicstructural features. The microfibrils have apparent individualdiameters in the range of 5–15 nm, but they are commonlyassociated laterally to form ribbons, or aggregated in strandsor cables of dimensions great enough to be resolved with theoptical microscope. These often show preferred orientations,which can be associated with pollen grain shape and with thedisposition-of the germination apertures. The apertural intinemay be structurally complex, as in Abutilon hybridum, where,after the removal of the exine, the polysaccharide caps whichoverlie the protein storage sites of the pollen grain wall retainthe elaborate patterning of the original cytoplasmic evaginationsfrom the vegetative cell. Secale cereale, Narcissus pseudonarcissus, Abutilon hybridum, Crocus vernus, pollen grain, intine, exine, wall pattern, germination apertures, polysaccharide microfibrils, fluorescence microscopy     

The Effects of Light Irradiation on the Orientation of Microtubules in Seedlings of Avena sativa L. and Pisum sativum L.

The effects of light irradiation on the arrangement of corticalmicrotubules (MTs) were examined in etiolated A vena mesocotylsand coleoptiles, and in Pisum epicotyls. Elongation of A venamesocotyls ceased as a result of irradiation with white lightwithin 1 h. The predominant orientation of MTs became more longitudinalwithin 1 h in epidermal cells and changed from transverse tooblique, after the elongation ceased, in parenchymal cells.Irradiation with red and with blue light also caused cessationof cell elongation and the same changes in the orientation ofMTs. Elongation of Avena coleoptiles ceased as a result of irradiationwith white light within 24 h. The predominant orientation ofMTs became more longitudinal in epidermal cells and changedfrom transverse to oblique in parenchymal cells. The changein orientation of MTs in epidermal cells preceded that in parenchymalcells. In Pisum epicotyls, elongation ceased as a result ofirradiation with white light within 1 h. Although the orientationsof MTs in epidermal cells did not show any remarkable change,those in parenchymal cells changed from transverse to obliqueafter cell elongation ceased. The change in orientation of MTs and the cessation of cell elongationof A vena mesocotyls induced by white-light irradiation wereboth significantly retarded by treatment with IAA. This resultsuggests that IAA is involved in maintaining the transverseorientation of MTs in Avena mesocotyls. (Received February 22, 1989; Accepted August 2, 1989)     

Arrangement of Cellulose Microfibrils in Walls of Elongating Parenchyma Cells

The arrangement of cellulose microfibrils in walls of elongating parenchyma cells of Avena coleoptiles, onion roots, and celery petioles was studied in polarizing and electron microscopes by examining whole cell walls and sections. Walls of these cells consist firstly of regions containing the primary pit fields and composed of microfibrils oriented predominantly transversely. The transverse microfibrils show a progressive disorientation from the inside to the outside of the wall which is consistent with the multinet model of wall growth. Between the pit-field regions and running the length of the cells are ribs composed of longitudinally oriented microfibrils. Two types of rib have been found at all stages of cell elongation. In some regions, the wall appears to consist entirely of longitudinal microfibrils so that the rib forms an integral part of the wall. At the edges of such ribs the microfibrils can be seen to change direction from longitudinal in the rib to transverse in the pit-field region. Often, however, the rib appears to consist of an extra separate layer of longitudinal microfibrils outside a continuous wall of transverse microfibrils. These ribs are quite distinct from secondary wall, which consists of longitudinal microfibrils deposited within the primary wall after elongation has ceased. It is evident that the arrangement of cellulose microfibrils in a primary wall can be complex and is probably an expression of specific cellular differentiation.     

Observations on the Fine Structure of Plant Cell Walls II. The Microfibrillar Framework of the Parenchymatous Cell Wall in Cucurbita

The microfibrillar framework of parenchymatous walls in Cucurbitawas observed in petioles treated so as to remove various non-cellulosiccell wall components. Such extraction typically results in separationof the microfibrillar components into concentric lamellae. Thenumber and thickness of these lamellae vary according to theage and type of cell wall. The microfibrils appear to be orientatedwithin the plane of their lamellae but the orientation may varyin successive lamellae, and in many walls the crossed polylamellatecondition was detected. The collenchyma—and the outerepidermal cell walls show an alternation of lamellae with almostvertical microfibrils with those with a practically transverseorientation. In ordinary parenchymatous walls the alternationis not so extreme and is revealed only by the occasional presenceof the ‘herring bone pattern’ in non-radial sections.As a rule the lamellae are continuous around the circumferenceof a cell though individual lamellae may vary in thickness andsometimes appear to ‘fade out’. The present observationssuggest that growth of the primary wall occurs by depositionof microfibrils in successive lamellae thus confirming the basicpremise of the multinet theory of growth.     

Observations on the Fine Structure of Plant Cell Walls III. The Sieve Tube Wall in Cucurbita

The sieve tube wall in Cucurbita was examined in ultra-thinsections of petioles treated in different ways for the removalof non-cellulosic wall components. The sections were stainedwith permanganate. The microfibrillar components of the wallare arranged in concentric lamellae. The earliest (outermost)part of the wall is similar to that of ordinary parenchyma inhaving its lamellae composed of thinly-distributed microfibrilsreadily separated from one another by certain treatments suchas pectinase extraction. In the characteristically-thickenedinner (nacreous) layer the microfibrils are very densely packedand the lamellae do not separate readily. The microfibrils inthis layer of the wall are very close to transverse and the‘crossed fibrillar’ orientation is not easily discernible.