A helicoidal cell wall texture in root hairs ofLimnobium stoloniferum

Summary The cell wall of root hairs ofLimnobium stoloniferum is composed of two fibrillar layers: an outer layer with a dispersed texture and an inner layer with a helicoidal texture. In stained oblique sections the helicoidal layer appears as a series of bow-shaped structures. In sections which were shadow-casted after the embedding medium was removed, the following properties of the helicoidal layer can be directly observed. (1) It is build up of superimposed lamellae. (2) Each lamella consists of parallel oriented microfibrils. (3) Going into the helicoidal layer, there is a counter-clockwise discontinuous rotation of the microfibril orientation in successive lamellae. (4) Between adjacent lamellae the average angular displacement of the microfibril orientation is about 23 degrees. The dispersed outer layer is also polylamellated, but with randomly arranged microfibrils in each lamella. Both layers are present in the lateral wall as well as in the apical wall of the root hairs. Observations indicate that in the cell wall of the tip the parallel oriented microfibrils of the outermost helicoidal lamellae become distorted towards a dispersed arrangement. The suggestion is made that the dispersed outer layer is derived from the helicoidal layer.     

Microtubules do not control microfibril orientation in a helicoidal cell wall

Summary The secondary cell wall layer of the young root hair ofEquisetum hyemale (L) has a helicoidal texture. The cortical microtubules in these hairs maintain an axial alignment while microfibrils are being deposited with a different orientation in each subsequent layer. The role of cortical microtubules in microfibril orientation is disputed.I gratefully acknowledge the support of Professor Dr. M. M. A.Sassen and the technical assistance of M.Wolters-Arts.     

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     

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.     

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.     

Helicoidal cell-wall texture in root hairs

It is shown that root hairs of most aquatic plants have a helicoidal cell-wall texture. Cell walls of root hairs of the aquatic/marshland plant Ranunculus lingua, however, have an axial microfibril alignment. The occurrence of a helicoidal wall texture is not limited to root hairs of aquatic plants: the terrestrial plant Zebrina purpusii has a helicoidal root-hair wall texture, too. With the exception of the grasses, the occurrence of root hairs with helicoidal cell walls pertains to species with predetermined root-hair-forming cells, trichoblasts. The rotation mode of the helicoid is species-specific. The average angle between fibrils of adjacent lamellae varies from 23° to 40°. In Hydrocharis morsus-ranae, cortical microtubules have a net-axial orientation and thus do not parallel nascent microfibrils. The deposition of the helicoidal cell wall is discussed.In honour of Prof. Dr. H.F Linskens (Nijmegen) on the occasion of his 65th birthday     

LIGHT AND ELECTRON MICROSCOPE STUDIES OF THE CELL WALL STRUCTURE OF THE ROOT HAIRS OF RAPHANUS SATIVUS

Dawes , Clinton J., and Edwin Bowler . (U. of California, Los Angeles.) Light and electron microscope studies of the cell wall structure of the root hairs of Raphanus sativus. Amer. Jour. Bot. 46(8): 561–565. Illus. 1959.—The structure and development of the cell wall of the root hair of Raphanus sativus were studied under the light and electron microscopes. The outer layer of the root hair consists of mucilage which covers the entire hair and forms a thick cap at the tip. Beneath the mucilage a thin cuticle covers the inner layers of the cell wall. These layers consist of cellulose microfibrils, varying in pattern, in a granular matrix, presumably pectic in nature. The microfibrils of the outer layer, apparently laid down at the tip, are reticulate in arrangement. In mature regions of the root hair, the wall is thickened by an inner layer of parallel and longitudinally orientated microfibrils. Pores in the cellulose wall are evident and increase in number and size near the base of the hair.     

The plasma membrane of growing root hairs is composed of zones of local differentiation

Growing root hairs of cress (Lepidium sativum L.) were investigated using freeze-fracture and electron-microscopic techniques. Three zones of differentiation could be detected: the tip zone, the zone of vacuolation and the foot zone. Corresponding to these zones, the plasmatic fracture face of the plasma membrane showed areas of pronounced differentiation with respect to the distribution and frequency of intramembranous particles (IMPs). The tip zone was characterized by an irregular fracture plane caused by a large number of blisters which were more or less free of IMPs. These blisters coincided in size and shape with Golgi vesicles accumulated in the ground cytoplasm near the very tip. Outside these blisters, IMPs were randomly distributed. The surrounding cell wall was very thin and mainly composed of amorphous material. The plasma membrane of the vacuolation zone often revealed areas of hexagonally ordered particles (HOPS). Such patterns of particles were observed in chemically fixed and unfixed root hairs with a maximum surface density of 1200 HOPS per area. Mostly, however, 15–50 HOPS per area were found. The number of such areas increased with increasing distance from the tip up to five areas per m². Additionally, imprints of large cellulose microfibrils could be detected in unfixed material; they were mainly parallel to the root-hair axis and sometimes ended in areas of HOPS. However, HOPS were observed only in approximately 60% of the root hairs. Otherwise, large areas free of IMPs were interspersed between areas of randomly distributed IMPs. The particle frequency was relatively low and varied greatly in the tip as well as in the vacuolation zone, that is, from 1200 to 2000 IMPs m^-2. Finally, the plasma membrane of the foot zone showed a very constant number of approx. 2000 IMPs m^-2. These particles were mainly distinct and randomly distributed. In this zone, HOPS were never observed in spite of the fact that the cell wall was composed of numerous parallel-running cellulose microfibrils. Since membrane material is mainly incorporated in the tip zone where IMPs are statistically distributed, the results indicate that the plasma membrane of the outgrowing part of the root-hair cells is characterized by a high lateral mobility of its components. Furthermore, they indicate that specifically arranged particles are involved in the synthesis of cellulose microfibrils. These areas of HOPS seem to be locally restricted and — or limited with respect to their lifetime.Abbreviations cmf(s) cellulose microfibril(s) - EF extraplasmatic fracture face - HOPS hexagonally ordered particles - IMP intramembranous particle - PF plasmatic fracture face - pm plasma membrane Dedicated to Professor Dr. Kurt Mühlethaler, Zürich, on the occasion of his 65th birthday     

Cellulose-synthesizing terminal complexes and microfibril structure in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)*

The brown alga Sphacelaria rigidula Kützing synthesizes cellulose microfibrils as determined by CBH I-gold labeling. The cellulose microfibrils are thin, ribbon-like structures with a uniform thickness of about 2.6 nm and a variable width in the range of 2.6-30 nm. Some striations appear along the longitudinal axis of the microfibrils. The developed cell wall in Sphacelaria is composed of three to four layers, and cellulose micro-fibrils are deposited in the third layer from the outside of the wall. A freeze fracture investigation of this alga revealed cellulose-synthesizing terminal complexes (TCs), which are associated with the tip of microfibril impressions in the plasmatic fracture face of the plasma membrane. The TCs consist of subunits arranged in a single linear row. The average diameter of the sub-units is about 6 nm, and the intervals between the neighboring subunits, about 9 nm, are relatively constant. The number of subunits constituting the TC varies between 10 and 100, so that the length of the whole TC varies widely. A model that has been proposed for the assembly of thin, ribbon-like microfibrils was applied to microfibril assembly in Sphacelaria.     

CYTOPLASMIC MICROTUBULE AND WALL MICROFIBRIL ORIENTATION IN ROOT HAIRS OF RADISH

The fine structure of young root hairs of radish was studied, with special attention to cytoplasm-wall relationships. Hairs up to 130 µ in length were examined after fixation of root tips in glutaraldehyde followed by osmium tetroxide. Microtubules occur axially aligned in the cytoplasm just beneath the plasmalemma, and extend from the base of the hair to within 2 to 3 µ of the tip. Poststaining with uranyl acetate and lead citrate clearly reveals in thin sections the presence of the two layers of cellulose microfibrils known from studies on shadowed wall preparations: an outer layer of randomly arranged microfibrils arising at the tip, and a layer of axially oriented microfibrils deposited on the inside of this layer along the sides. The youngest microfibrils of the inner, oriented layer first appear at a distance of about 25 µ from the tip. Although the microfibrils of the inner layer and the adjacent microtubules are similarly oriented, the oriented microtubules also extend through the 20- to 25-µ zone near the tip where the wall structure consists of random microfibrils. This suggests that the role of microtubules in wall deposition or orientation may be indirect.     

Tracing cellulose microfibril orientation in inner primary cell walls

Summary By quantitative analysis of cellulose microfibril orientation at different levels in the primary cell wall of a number of cell types, the development of wall texture was studied. Meristematic, isodiametric and cylindrical parenchyma cells and cells of a suspension culture were used. Within the newly deposited microfibril population, various orientations were recognized on the micrographs. Within subpopulations the orientation of undercrossing and overcrossing microfibrils were measured. These measurements showed a gradual shift in cellulose microfibril orientation in the different levels. Microfibrils showed predominant orientations at particular levels but microfibrils of intermediate orientation also occurred, although at a much lower density. As cellulose microfibrils of intermediate orientation were not closely packed, lamellae were not formed. Interwoven microfibrils were occasionally present, indicating that differently orientated microfibrils are occasionally deposited simultaneously. Also gradual changes in orientation over the entire inner cell wall surface were observed. From these observations it was inferred that microfibril deposition occurs with a small but regular and progressive change in orientation, the rotational motion, related to that of a helicoidal system.Dedicated to Professor Dr. M. M. A. Sassen on the occasion of his 65th birthday     

Effects of colchicine on cell shape and on microfibril arrangement in the cell wall of Closterium acerosum

Cell morphogenesis in Closterium acerosum (Schrank) Ehrenberg was greatly influenced by colchicine. Addition of colchicine to the medium led to production of tadpole-shaped cells, by decreasing the length and increasing the thickness of the new semicells. Transversely oriented wall microtubules and microfibrils, characteristic of normally elongating semicells, were not observed in colchicine-treated semicells, randomly oriented microfibrils being present instead. About 3.5 h after septum formation, the randomly oriented microfibrils began to be overlaid by bundles of microfibrils as seen in normal semicells at the later stage of elongation. When colchicine treatment was terminated 1 h after septum formation, cell elongation was partially restored and microfibrils were deposited parallel to each other and transversely to the cell axis, indicating that the effect of colchicine on microfibril arrangement in growing semicells is reversible.     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

A model for the pattern of deposition of microfibrils in the cell wall of Glaucocystis

Freeze-fracturing of Glaucocystis nostochinearum Itzigsohn cells during cell-wall microfibril deposition indicates that unidirectionally polarized microfibril ends are localized in a zone of synthesis covering about 30% of the sarface area of the plasma membrane. Within this zone there are about 6 microfibril ends/m² cell surface. It is proposed that microfibrils are generated by the passage of their tips over the cell surface and that the pattern of microfibril organization at the poles of the cells, in which microfibrils of alternate layers are interconnected at 3 rotation centres, results directly from the pattern of this translation of microfibril tips. In a model of the deposition pattern it is proposed that the zone of synthesis may split into 3 sub-zones as the poles are approached, each sub-zone being responsible for the generation of one rotation centre. It is demonstrated that the microfibrillar component of the entire wall could be generated by the steady translation of the microfibril tips (at which synthesis is presumed to occur) over the cell surface at a rate of 0.25–0.5 m min^-1. Microcinematography indicates that the protoplast rotates during cell-wall deposition, and it is proposed that this rotation may play a role in the generation of the microfibril deposition pattern.     

Deposition and reorientation of cellulose microfibrils in elongating cells of Petunia stylar tissue

According to Roelofsen and Houwink's (1953, Acta Bot. Neerl. 2, 218–225) multinet growth hypothesis, microfibrils originally deposited transversely in the cell wall become gradually reoriented towards more axial orientations during cell elongation. To establish the extent of reorientation, microfibrils were studied during their deposition and elongation, using stylar parenchyma and transmitting tissue cells of Petunia hybrida L. At the inner surface of very young cells, microfibrils were deposited in alternating Z- and S-helical orientations. The following sequence in deposition, from the exterior to the interior side of the wall, could be inferred: Axial: 150°–180° (Z-helical), 0°–30° (S-helical); oblique: 110°–150° (Z-helical), 30°–70° (S-helical); transverse: 90°–110° (Z-helical), 70°–90° (S-helical). With the increasing pitch, the density of the deposited microfibrils increased as well, giving rise to an alternating helical texture. During elongation, only transversely S- and Z-helically oriented microfibrils were deposited and all microfibrils underwent a certain reorientation as described in the multinet growth hypothesis. The texture resembled that of young cells and the wall maintained its thickness. The extent of passive reorientation was in agreement with the theoretical calculations made by Preston.Dedicated to Professor Dr. A.B. Wardrop, Melbourne, on the occasion of his 70th birthday     

Ultrastructure,differentiation and cell wall texture of trichoblasts and root hairs of ceratopteris thalictroides (L.) brongn. (Parkeriaceae)

Trichoblasts and root hairs of Ceratopteris thalictroides (L.) Brongn. were studied by different techniques to survey their morphological features. Trichoblasts could be identified at an early stage by an intra-vacuolar precipitate appearing during fixation. Special attention was paid to root-hair initiation. No structures or changes were observed that play a role in the initiation of papilla formation. When the papilla is formed, vesicles and periplasmic membranes can be observed which may play a role in the weakening of the cell wall during the papilla outgrowth.During root-hair growth, the nucleus of the trichoblast moves from the trichoblast to a subapical position in the root hair. The nuclei of all root cells contain 2 types of nuclear inclusions, one of which is proteinaceous.The cell wall of the Ceratopteris root hair has a helicoidal texture and because the cortical microtubules run longitudinally in the root hair, no correlation can be made between the directions of microtubules and microfibrils in these root hairs.     

Microtubules and morphogenesis in ordinary epidermal cells ofVigna sinensis leaves

Summary Undifferentiated ordinary epidermal cells (ECs) ofVigna sinensis leaves possess straight anticlinal walls and cortical microtubules (Mts) scattered along them. At an early stage of EC differentiation cortical Mts adjacent to the above walls form bundles normal to the leaf plane, loosely interconnected through the cortical cytoplasm of the internal periclinal wall. At the upper ends of the Mt bundles, Mts fan out towards the external periclinal wall and form radial arrays. Mt bundles and radial arrays exhibit strict alternate disposition between neighbouring ECs. An identical reticulum of cellulose microfibril (CM) bundles is deposited outside the Mt bundles. Local wall pads rise at the junctions of anticlinal walls with the external periclinal one, where the CM bundles terminate. They display radial CMs fanning towards the external periclinal wall. The CM bundles and radial CM systems prevent local cell bulging, but allow it in the intervening wall areas. In particular, the radial CM systems dictate the pattern of EC waviness by favouring local tangential expansion of external periclinal wall. As a result, ECs obtain an undulate appearance. Constrictions in one EC correspond with protrusions of adjacent ECs. ECs affected by colchicine entirely lose their Mts and do not develop wavy walls, an observation substantiating the role of cortical Mts in EC morphogenesis.Abbreviations CM cellulose microfibril - DTT dithiothreitol - EC epidermal cell - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

Orientation of Wall Microfibril Deposition in Root Cells of Pisum sativum L. var Alaska

The arrangement of wall microfibrils on the inner surface ofcortical cells in pea roots was observed by a replica method.In the elongating region, microfibrils were deposited transverselyto the root axis. After cell elongation stopped, the orientationof microfibril deposition changed discontinuously from a transverseto an oblique one. The change occurred at 6–7 mm fromthe root tip. The oblique orientation seemed to change discontinuouslyto another oblique one as time passed. (Received January 24, 1986; Accepted May 15, 1986)     

Colchicine and microfibril orientation

Summary Investigations on the mechanism of orientation of the cellulose microfibrils of the green algaOocystis solitaria have been carried out. This organism demonstrates easily observable and highly ordered microfibrils in its wall, which are arranged parallel to one another and regularly alternate at 90 from layer to layer of which there are approximately 30. During the entire wall development, and always parallel to one of the microfibril directions, are microtubules lying in the cortical cytoplasm. In the presence of 10^–2 M colchicine, microtubules are no longer detected and the typical cell wall pattern is not developed. The possible role of microtubules in the orientation of cellulose microfibrils is briefly discussed.     

Dynamic changes in the arrangement of cortical microtubules in conifer tracheids during differentiation.

The arrangement of cortical microtubules (MTs) in differentiating tracheids of Abies sachalinensis Masters was examined by confocal laser scanning microscopy after immunofluorescent staining. The arrays of MTs in the tracheids during formation of the primary wall were not well ordered and the predominant orientation changed from longitudinal to transverse. During formation of the secondary wall, the arrays of MTs were well ordered and their orientation changed progressively from a flat S-helix to a steep Z-helix and then to a flat S-helix as the differentiation of tracheids proceeded. The orientation of cellulose microfibrils (MFs) on the innermost surface of cell walls changed in a similar manner to that of the MTs. These results provide strong evidence for the co-alignment of MTs and MFs during the formation of the semi-helicoidal texture of the cell wall in conifer tracheids.Abbreviations MT cortical microtubule - MF cellulose microfibril - S1, S2 and S3 the outer, middle and inner layers of the secondary wall The authors thank Mr. T. Itoh of the Electron Microscope Laboratory, Faculty of Agriculture, Hokkaido University, for his technical assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (no. 06404013).