STABILITY AND COLCHICINE EFFECT OF WALL MICROFIBRILLAR ALIGNMENT IN THE NITELLA RHIZOID

The cell wall of the Nitella rhizoid was stripped to make wedges of various thicknesses. Polarizing and interference microscopes were used to examine the post-deposition orientation of wall microfibrils. The fibrils appeared to maintain alignment after they were deposited. Since during growth the rhizoid wall elements are static in the cylindrical part or extend isotropically in the dome (Chen, 1973), these observations provide indirect evidence that the fibrillar reorientation observed in the Nitella internode is due to a passive reorientation during the predominant longitudinal cell elongation (Gertel and Green, 1977). The static microfibrils of the secondary wall of rhizoid, however, reoriented under the influence of colchicine, the alignment becoming almost random after 48 hrs. The disturbance of alignment started in the region adjacent to the plasma membrane, increasing in thickness with prolonged treatment.     

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.     

Cell wall architecture: normal development and environmental modification of guard cells of the Cyperaceae and related species*

Abstract. The development and cell wall architecture of guard cells in the Cyperaceae were studied with light and electron microscopy. Development occurs along parallel files and results in a stomatal complex that consists of two guard cells each flanked by a subsidiary cell. The developmental pattern and general morphology are thus similar to that in the Gramineae. Several key differences, however, were observed. Wall synthesis in the Cyperaceae, as observed in the polarization and fluorescence microscope, occurs suddenly, within three to four complexes along a file, but is more gradual in the Gramineae. Mature cell walls in the Cyperaceae predominantly contain microfibrils oriented radially relative to the pore, while those in the Gramineae contain axial microfibrils. This difference was demonstrated in numerous species using freshly-collected as well as preserved material. In Cyperus esculentus, however, the alignment of microfibrils appears to be subject to environmental modification. Plants grown in the greenhouse contain guard cells with axial microfibrils, compared to the radial arrangement found in those grown in the field. In the former, wall is deposited gradually, as in the Gramineae. On return to more stressful conditions, radially micellated guard cells again develop. In each case, the cortical cytoplasm adjacent to areas where the wall is to thicken contains microtubules oriented parallel to the microfibril alignment characteristic of that treatment. These results are discussed in terms of the role of varied wall architecture in stomatal mechanics, the regulation of cell wall biosynthesis, and the evolutionary relationship of the Cyperaceae, Gramineae, and other taxa.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

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.     

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     

Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots. II. Role of Cortical Microtubules and Cellulose Microfibrils

We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101–111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized.     

On the alignment of cellulose microfibrils by cortical microtubules: A review and a model

Summary The hypothesis that microtubules align microfibrils, termed the alignment hypothesis, states that there is a causal link between the orientation of cortical microtubules and the orientation of nascent microfibrils. I have assessed the generality of this hypothesis by reviewing what is known about the relation between microtubules and microfibrils in a wide group of examples: in algae of the family Characeae,Closterium acerosum, Oocystis solitaria, and certain genera of green coenocytes and in land plant tip-growing cells, xylem, diffusely growing cells, and protoplasts. The salient features about microfibril alignment to emerge are as follows. Cellulose microfibrils can be aligned by cortical microtubules, thus supporting the alignment hypothesis. Alignment of microfibrils can occur independently of microtubules, showing that an alternative to the alignment hypothesis must exist. Microfibril organization is often random, suggesting that self-assembly is insufficient. Microfibril organization differs on different faces of the same cell, suggesting that microfibrils are aligned locally, not with respect to the entire cell. Nascent microfibrils appear to associate tightly with the plasma membrane. To account for these observations, I present a model that posits alignment to be mediated through binding the nascent microfibril. The model, termed templated incorporation, postulates that the nascent microfibril is incorporated into the cell wall by binding to a scaffold that is oriented; further, the scaffold is built and oriented around either already incorporated microfibrils or plasma membrane proteins, or both. The role of cortical microtubules is to bind and orient components of the scaffold at the plasma membrane. In this way, spatial information to align the microfibrils may come from either the cell wall or the cell interior, and microfibril alignment with and without microtubules are subsets of a single mechanism.Dedicated to Professor Brian E. S. Gunning 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.     

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)     

WALL STRUCTURE AND LATERAL FORMATION IN THE ALGA BRYOPSIS

Green , Paul B. (U. Pennsylvania, Philadelphia.) Wall structure and lateral formation in the alga Bryopsis. Amer. Jour. Bot. 47(6) : 476–481. Illus. 1960.—The large multinucleate cells of Bryopsis pennata regularly produce laterals at the apical tip. Cells were freed of protoplasm and the remaining cell wall was flattened and studied in polarized light and interference microscopes. Both the cylindrical main axis and the cylindrical laterals are negatively birefringent and thus apparently have generally a transverse arrangement of microfibrils. Lateral formation is first seen in the formation of round fields of concentric microfibrils on the main axis which persist as lateral-bases. The center of a field protrudes, making a small out-pouching of the main axis. The thin-walled protrusion increases first mostly in diameter and then in length to make a lateral. There is a definite correlation between wall structure (optical anisotropy) and cell shape in Bryopsis.     

Microtubules and angiosperm bordered pit formation

Summary Microtubules have been shown to run around the perimeter of the pit aperture of developing bordered pits of Salix fragilis, L. These microtubules are parallel to the microfibrils being produced and do not converge with them. Although microtubules may be parallel to microfibrils in differentiating vessel elements as well as in fibres, many cases have been found where microtubules are not so orientated. There is no direct evidence for the involvement of microtubules in the orientation of microfibrils in the secondary wall, although their position during wall synthesis suggests some ancillary role.     

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.     

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.     

Cell wall synthesis during growth and maturation of Nitella internodal cells

An improved ¹³C-density-labeling method was used to study cell wall synthesis in rapidly expanding, slowly expanding and recently mature internodes of Nitella translucens var axillaris (A.Br.) R.D.W. As cells matured, the rate of wall synthesis slowed and the deposition of cellulose microfibrils changed from a predominantly transverse direction in the primary wall of rapidly expanding internodes to a helicoidal array in the secondary wall of mature internodes. The secondary wall was characterized by relatively higher rates of cellulose synthesis and lower rates of pectin synthesis than the primary wall. The synthesis of xyloglucan also decreased markedly at the transition to secondary wall synthesis, while the synthesis of mannose-rich hemicellulose increased. Even though structural differences were striking between the primary and secondary walls of Nitella, compositional differences between the two types of wall were quantitative rather than qualitative. The authors appreciate the assistance of Martin Yousef with the electron microscopy.     

Cortical microtubules and microfibril deposition in the cell wall of root hairs ofEquisetum hyemale

Summary The cell wall of root hairs ofEquisetum hyemale is shown to be composed of three different cell wall textures. The growing cell wall at the tip of the hair is composed of a dispersed texture of microfibrils, which continues along the outside of the whole hair. With increasing distance from the tip an increasing number of helicoidally arranged lamellae is deposited. These findings correspond with the observed isotropism of young hairs in polarized light.Hairs of approximately 4 days old become positive birefringent, indicating that longitudinally oriented layers prevail over layers with a transverse direction. This phenomenon starts at the base of the hair. Full-grown hairs are positive birefringent up to the tip and concordantly show a thick additional inner cell wall layer which forms a helical pattern the length of the hair, with a mean microfibril angle of 25 with the cell axis.Cortical microtubules, subjacent to the dispersed, the helicoidal and the helical wall texture are axially aligned and, thus, not in coalignment with the last deposited microfibrils.Coated and smooth vesicles are present in the cortical cytoplasm of both growing and full-grown hairs. Electron-dense profiles (20 nm in diameter), surrounded by a halo (of 50 nm) were observed on the wall-plasmalemma interface in full-grown hairs only. A relation of these structures with microfibril deposition could not be demonstrated. They might represent channels transporting material to the wall, which, in full-grown hairs, is heavily impregnated with a tawny brown substance.The general hypothesis that cortical microtubule orientation directs microfibril deposition is disputed.     

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     

The Cellulose System in the Cell Wall ofMicrasterias

The cellulose system of the cell wall ofMicrasterias denticulataandMicrasterias rotatawas analyzed by diffraction contrast transmission electron microscopy, electron diffraction, and X-ray analysis. The studies, achieved on disencrusted cell ghosts, confirmed that the cellulose microfibrils occurred in crisscrossed bands consisting of a number of parallel ribbon-like microfibrils. The individual microfibrils had thicknesses of 5 nm for a width of around 20 nm, but in some instances, two or three microfibrils merged into one another to yield larger monocrystalline domains reaching up to 60 nm in lateral size. The orientation of the cellulose ofMicrasteriasis very unusual, as it was found that in the cell wall, the equatorial crystallographic planes of cellulose having ad-spacing of 0.60 nm [(110) in the Iβ cellulose unit cell defined by Sugiyamaet al.,1991,Macromolecules24, 4168–4175] were oriented perpendicular to the cell wall surface. Up to now, such orientation has been found only inSpirogyra,another member of the Zygnemataceae group. The unusual structure of the secondary wall cellulose ofMicrasteriasmay be tentatively correlated with the unique organization of the terminal complexes, which in this alga occur as hexagonal arrays of rosettes.     

Helicoidal orientation of cellulose microfibrils in Nitella opaca internode cells: ultrastructure and computed theoretical effects of strain reorientation during wall growth

The ultrastructure of the mature internode cell wall of Nitella opaca is described. It is interpreted in terms of a helicoidal array of cellulose microfibrils set in a matrix. A helicoid is a multiple plywood made up of layers of parallel microfibrils. There is a progressive change in direction from ply to ply, giving rise to characteristic arced patterns in oblique sections. A critical tilting test, using an electron microscope fitted with a goniometric stage, showed the expected reversal of direction of the arced pattern. Nitella cell wall is thus more regularly structured than previous studies have shown. From a survey of the cell-wall literature, we show that such arced patterns are common. This indicates that the helicoidal structure may be more widespread than is generally realised, although numerous other cell walls show no signs of it. Nevertheless, there are examples in most major plant taxa, and in several types of cells, including wood tracheids. Most of the examples, however, need confirmation by tilting evidence. There are possible implications for wall morphogenesis. Helicoidal cell walls might arise by selfassembly via a liquid crystalline phase, since it is known that the cholesteric state is itself helicoidal. A computer graphics programme has been developed to plot the expected effects of growth strain on the patterns in oblique sections of helicoids with various original angles between consecutive layers. Herringbone patterns typical of crossed polylamellate texture can be generated in this way, indicating a possible mode of their formation.