Surface architecture of the plant cell: Biogenesis of the cell wall,with special emphasis on the role of the plasma membrane in cellulose biosynthesis

Cell wall structure and biogenesis in the unicellular green alga, Oocystis apiculata, is described. The wall consists of an outer amourphous primary layer and an inner secondary layer of highly organized cellulosic microfibrils. The primary wall is deposited immediately after cytokinesis. Golgi-derived products contribute to this layer. Cortical microtubules underlie the plasma membrane immediately before and during primary wall formation. They function in maintaining the elliptical cell shape. Following primary wall synthesis, Golgi-derived materials accumulate on the cell surface to form the periplasmic layer. This layer functions in the deposition of coating and cross-linking substances which associate with cellulosic microfibrils of the incipient secondary wall. Secondary wall microfibrils are assembled in association with the plasma membrane. Freeze-etch preparations of untreated, living cells reveal linear terminal complexes in association with growing cellulosic microfibrils. These complexes are embedded in the EF fracture face of the plasma membrane. The newly synthesized microfibril lies in a groove of the outer leaflet of the plasma membrane. The groove is decorated on the EF fracture face by perpendicular structures termed “ridges.” The ridges interlink with definitive rows of particles associated with the PF fracture face of the inner leaflet of the plasma membrane. These particles are termed “granule bands,” and they function in the orientation of the newly synthesized microfibrils. Microfibril development in relation to a coordinated multienzyme complex is discussed. The process of cell wall biogenesis in Oocystis is compared to that in higher plants.     

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.     

Further evidence from sectioned material in support of the existence of a linear terminal complex in cellulose synthesis

Summary Transmembrane linear terminal complexes considered to be involved in the synthesis of cellulose microfibrils have been described in the plasma membrane ofBoergesenia forbesii. Evidence for the existence of these structures has been obtained almost exlusively using the freeze etching technique. In the present study an attempt has been made to complete these studies using conventional fixation, staining, and sectioning procedures. In developing cells ofBoergesenia forbesii, strongly stained structures traversing the plasma membrane and averaging 598.9 nm ± 171.3 nm in length, 28.7 nm ± 4.2 nm in width, and 35.2 nm ± 6.6 nm in depth have been demonstrated. These structures are considered to be linear terminal complexes. At their distal (cell wall) surface, they appear to be closely associated with cellulose microfibrils. At the proximal (cytoplasmic) surface, they are associated with microtubules and polysomes. A model of the possible interrelation of the terminal complexes and microtubules leading to the generation of cell wall microfibrils is proposed.     

Arrays of plasma-membrane “rosettes” involved in cellulose microfibril formation of Spirogyra

The cell-wall structure and plasma-membrane particle arrangement during cell wall formation of the filamentous chlorophycean alga Spirogyra sp. was investigated with the freeze-fracture technique. The cell wall consists of a thick outer slime layer and a multilayered inner wall with ribbon-like microfibrils. This inner wall shows three differing orientations of microfibrils: random orientation on its outside, followed by axial bundles of parallel microfibrils, and several internal layers of bands of mostly five to six parallel associated microfibrils with transverse to oblique orientation. The extraplasmatic fracture face of the plasma membrane shows microfibril imprints, relatively few particles, and “terminal complexes” arranged in a hexagonal package at the end of the imprint of a microfibril band. The plasmatic fracture face of the plasma membrane is rich in particles. In places, it reveals hexagonal arrays of “rosettes”. These rosettes are best demonstrable with the double-replica technique. These findings on rosette arrays of the zygnematacean alga Spirogyra are compared in detail with the published data on the desmidiacean algae Micrasterias and Closterium.     

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)     

THE SITES OF CELLULOSE SYNTHESIS IN ALGAE: DIVERSITY AND EVOLUTION OF CELLULOSE-SYNTHESIZING ENZYME COMPLEXES

Information on the sites of cellulose synthesis and the diversity and evolution of cellulose-synthesizing enzyme complexes (terminal complexes) in algae is reviewed. There is now ample evidence that cellulose synthesis occurs at the plasma membrane-bound cellulose synthase, with the exception of some algae that produce cellulosic scales in the Golgi apparatus. Freeze-fracture studies of the supramolecular organization of the plasma membrane support the view that the rosettes (a six-subunit complex) in higher plants and both the rosettes and the linear terminal complexes (TCs) in algae are the structures that synthesize cellulose and secrete cellulose microfibrils. In the Zygnemataceae, each single rosette forms a 5-nm or 3-nm single “elementary” microfibril (primary wall), whereas rosettes arranged in rows of hexagonal arrays synthesize criss-crossed bands of parallel cellulose microfibrils (secondary wall). In Spirogyra, it is proposed that each of the six subunits of a rosette might synthesize six β-1,4-glucan chains that cocrystallize into a 36-glucan chain “elementary” microfibril, as is the case in higher plants. One typical feature of the linear terminal complexes in red algae is the periodic arrangement of the particle rows transverse to the longitudinal axis of the TCs. In bangiophyte red algae and in Vaucheria hamata, cellulose microfibrils are thin, ribbon-shaped structures, 1–1.5 nm thick and 5–70 nm wide; details of their synthesis are reviewed. Terminal complexes appear to be made in the endoplasmic reticulum and are transferred to Golgi cisternae, where the cellulose synthases are activated and may be transported to the plasma membrane. In algae with linear TCs, deposition follows a precise pattern directed by the movement and the orientation of the TCs (membrane flow). A principal underlying theme is that the architecture of cellulose microfibrils (size, shape, crystallinity, and intramicrofibrillar associations) is directly related to the geometry of TCs. The effects of inhibitors on the structure of cellulose-synthetizing complexes and the relationship between the deposition of the cellulose microfibrils with cortical microtubules and with the membrane-embedded TCs is reviewed In Porphyra yezoensis, the frequency and distribution of TCs reflect polar tip growth in the apical shoot cell.The evolution of TCs in algae is reviewed. The evidence gathered to date illustrates the utility of terminal complex organization in addressing plant phylogenetic relationships.     

The cytoplasmic domain of the cellulose-synthesizing complex in vascular plants

The cytoplasmic domain of the rosette terminal complex has been imaged in situ in patches of plasma membrane isolated from tobacco BY-2 protoplasts. By partially extracting the plasma membrane lipids, cellulose microfibrils were observed through the plasma membrane. Rosette terminal complexes were identified on the basis of their association with the ends of these cellulose microfibrils. The cytoplasmic domain of the rosette terminal complex has been shown to be hexagonal in shape and has been measured to be 45-50 nm in diameter and 30-35 nm tall. These findings demonstrate that the terminal complex does indeed have a substantial cytoplasmic component, and that the hexagonal array observed in the lipid bilayer by freeze fracture is actually only a small part of the overall complex. These findings will allow better modeling of the terminal complex and may facilitate predictions of how many proteins are associated with the rosette terminal complex in vivo.     

Cell wall biogenesis in Oocystis: experimental alteration of microfibril assembly and orientation.

Cell wall biogenesis in the unicellular green alga Oocystis apiculata has been studied. Under normal growth conditions, a cell wall with ordered microfibrils is synthesized. In each layer there are rows of parallel microfibrils. Layers are nearly perpendicular to each other. Terminal linear synthesizing complexes are located in the plasma membrane, and they are capable of bidirectional synthesis of cellulose microfibrils. Granule bands associated with the inner leaflet of the plasma membrane appear to control the orientation of newly synthesized microfibrils. Subcortical microtubules also are present during wall synthesis. Patterns of cell wall synthesis were studied after treatment with EDTA and EGTA as well as divalent cations (MgSO4, CaSO4, Cacl2). 0.1 M EDTA treatment for 15 min results in the disassociation of the terminal complexes from the ends of microfibrils. EDTA-treated cells followed by 15 min treatment with MgSO4 results in reaggregation of the linear complexes into a paired state, remote from the original ends to which they were associated. After 90 min treatment with MgSO4, normal synthesis resumes. EGTA and calcium salts do not affect the linear complexes or microfibril orientation. Treatments with colchicine and vinblastine sulphate do not depolymerize the microtubles, but the wall microfibril orientation is altered. With colchicine or vinblastine, the change in orientation from layer to layer is inhibited. The process is reversible upon removal of the drugs. Lumicolchicine has no effect upon microfibril orientation, but granule bands are disorganized. Treatment with coumarin, a known inhibitor of cellulose synthesis, causes the loss of visualization of subunits of the terminal complexes. The possibility of the existence of a membrane-associated colchicine-sensitive orientation protein for cellulose microfibrils is discussed. Transmembrane modulation of microfibril synthesis and orientation is presented.     

Sucrose synthase localizes to cellulose synthesis sites in tracheary elements.

The synthesis of crystalline cellulose microfibrils in plants is a highly coordinated process that occurs at the interface of the cortex, plasma membrane, and cell wall. There is evidence that cellulose biogenesis is facilitated by the interaction of several proteins, but the details are just beginning to be understood. In particular, sucrose synthase, microtubules, and actin have been proposed to possibly associate with cellulose synthases (microfibril terminal complexes) in the plasma membrane. Differentiating tracheary elements of Zinnia elegans L. were used as a model system to determine the localization of sucrose synthase and actin in relation to the plasma membrane and its underlying microtubules during the deposition of patterned, cellulose-rich secondary walls. Cortical actin occurs with similar density both between and under secondary wall thickenings. In contrast, sucrose synthase is highly enriched near the plasma membrane and the microtubules under the secondary wall thickenings. Both actin and sucrose synthase lie closer to the plasma membrane than the microtubules. These results show that the preferential localization of sucrose synthase at sites of high-rate cellulose synthesis can be generalized beyond cotton fibers, and they establish a spatial context for further work on a multi-protein complex that may facilitate secondary wall cellulose synthesis.     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

Visualization of particle complexes in the plasma membrane of Micrasterias denticulata associated with the formation of cellulose fibrils in primary and secondary cell walls

Highly ordered arrays of intramembrane particles are observed in freeze- fractured plasma membranes of the green alga Micrasterias denticulata during the synthesis of the secondary cell wall. The observable architecture of the complex consists primarily of a precise hexagonal array of from 3 to 175 rosettes, consisting of 6 particles each, which fracture with the P-face. The complexes are observed at the ends of impressions of cellulose fibrils. The distance between rows of rosettes is equal to the center-to-center distance between parallel cellulose fibrils of the secondary wall. Correlation of the structure of the complex with the pattern of deposition indicates that the size of a given fibril is proportional to the number of rosettes engaged in its formation. Vesicles containing hexagonal arrays of rosettes are found in the cytoplasm and can be observed in the process of fusing with the plasma membrane, suggesting that the complexes are first assembled in the cytoplasm and then incorporated into the plasma membrane, where they become active in fibril formation. Single rosettes appear to be responsible for the synthesis of microfibrils during primary wall growth. Similar rosettes have now been detected in a green alga, in fern protonemata, and in higher plant cells. This structure, therefore, probably represents a significant component of the cellulose synthesizing mechanism in a large variety of plant cells.     

Evidence for an intramembrane component associated with a cellulose microfibril-synthesizing complex in higher plants

Freeze-fracture of rapidly frozen, untreated plant cells reveals terminal complexes on E-fracture faces and intramembrane particle rosettes on P-fracture faces. Terminal complexes and rosettes are associated with the ends of individual microfibril impressions on the plasma membrane. In addition, terminal complexes and rosettes are associated with the impressions of new orientations of microfibrils. These structures are sparse within pit fields where few microfibril impressions are observed, but are abundant over adjacent impressions of microfibrils. It is proposed that intramembrane rosettes function in association with terminal complexes to synthesize microfibrils. The presence of a cellulosic microfibril system in Zea mays root segments is confirmed by degradation experiments with Trichoderma cellulase.     

Plasma membrane "rosettes" in carrot and sycamore suspension culture cells

Suspension culture cells of carrot, Daucus carota L., and sycamore, Acer pseudoplatanus L., were freeze-fractured after ultrarapid freezing without fixation or cryoprotection in a propane-jet freezer. Infrequently, rosettes (ca. 24 nm diameter) of six (occasionally five) subunits (ca. 8 nm diameter) were observed in P-face views of the plasma membrane of both taxa. When present, rosette density was approximately 1/micron 2. Generally, rosettes were less frequently seen on plasma membranes exhibiting numerous vesicle fusion figures. Due to the high quality of the freezing, cellulose microfibril impressions were rarely seen on either PF or EF views of the plasma membrane, thus precluding correlations between microfibrils on the one hand and rosettes (and terminal globules) on the other. The presence of rosettes in suspension culture cells of these two species supports the putative role of rosettes in cellulose biosynthesis in higher plants.     

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL-SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm^?2, whereas that of the exoplasmic fracture face was 325 μm^?2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm^?2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm² of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.     

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 ASSOCIATION OF ROSETTE AND GLOBULE TERMINAL COMPLEXES WITH CELLULOSE MICROFIBRIL ASSEMBLY IN NITELLA TRANSLUCENS VAR. AXILLARIS (CHAROPHYCEAE)1,2

A freeze-fracture investigation of the putative cellulose synthesizing complex (terminal complex) morphology in Nitella translucens var. axillaris (A. Br.) R.D.W. internodal cells revealed single solitary EF globules and PF rosettes on the plasma membrane. The average density of rosettes in elongating internodal cells was 5.6 μm^?2 with slight spatial variation observed. In only three other algal genera (all zygnematalean) have rosette / globule terminal complexes been observed, while this characteristic is common to all vascular plants and one moss thus far investigated. This evidence strongly suggests that the rosette type of terminal complex morphology is an additional characteristic of charophycean algae and lends further support to the hypothesis that this group of algae represents the evolutionary line that gave rise to vascular plants. Observations were also made from the freeze-fracture of Nitella internodal cells concerning the orientation of cell wall microfibrils and cytoskeletal elements near the plasma membrane. The pattern of microfibril orientation in growing internodal cells is initially transverse to the cell long axis, becoming progressively axial presumably due to the strain of elongation. In mature internodal cells, the pattern of microfibril orientation is helicoidal. Microtubules appressed to the inner surface of the plasma membrane are oriented parallel to the most recently formed microfibrils in elongating and mature internodal cells.     

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     

Winding threads around plant cells: a geometrical model for microfibril deposition

In this paper, a geometrical model is put forward to account for the deposition orientation of plant cell wall microfibrils (CMFs). The model presupposes the insertion in the plasma membrane of CMF initiation complexes, which, once inserted, are moved through the fluid plane of the plasma membrane by the kinetic force of CMF synthesis, leaving CMFs in their wake. Deposition occurs in a limited space and the CMFs are linked to wall matrix molecules. CMF orientation is governed by the laws of geometry and, taking space-limiting conditions into account, therefore depends on (1) cell geometry, (2) the other wall molecules linked to the CMFs, and (3) the number of CMF initiation complexes inserted into the plasma membrane. The model does not exclude the idea that cortical microtubules may determine initial CMF orientation after cell division by determining the cell elongation direction.     

The assembly of cellulose microfibrils in Valonia macrophysa Kütz

The assembly of cellulose microfibrils was investigated in artificially induced protoplasts of the alga, Valonia macrophysa (Siphonocladales). Primary-wall microfibrills, formed within 72 h of protoplast induction, are randomly oriented. Secondary-wall lamellae, which are produced within 96 h after protoplast induction, have more than three orientations of highly ordered microfibrils. The innermost, recently deposited micofibrils are not parallel with the cortical microtubules, thus indicating a more indirect role of microtubules in the orientation of microfibrils. Fine filamentous structures with a periodicity of 5.0–5.5 nm and the dimensions of actin were observed adjacent to the plasma membrane. Linear cellulose-terminal synthesizing complexes (TCs) consisting of three rows, each with 30–40 particles, were observed not only on the E fracture (EF) but also on P fracture (PF) faces of the plasma membrane. The TC appears to span both faces of the bimolecular leaflet. The average length of the TC is 350 nm, and the number of TCs per unit area during primary-wall synthesis is 1 per m². Neither paired TCs nor granule bands characteristic of Oocystis were observed. Changes in TC structure and distribution during the conversion from primary- to secondary-wall formation have been described. Cellulose microfibril assembly in Valonia is discussed in relation to the process among other eukaryotic systems.Abbreviations TC terminal complex - EF E (outer leaflet) fracture face of the plasma membrane - PF P (inner leaflet) fracture face of the plasma membrane - MT microtubule - PS protoplasmic surface of the membrane     

The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts of Mougeotia

Protoplasts of the filamentous green alga Mougeotia sp. are spherical when isolated and revert to their normal cylindrical cell shape during regeneration of a cell wall. Sections of protoplasts show that cortical microtubules are present at all times but examination of osmotically ruptured protoplasts by negative staining shows that the microtubules are initially free and become progressively cross-bridged to the plasma membrane during the first 3 h of protoplast culture. Cell-wall microfibrils areoobserved within 60 min when protoplasts are returned to growth medium; deposition of microfibrils that is predominantly transverse to the future axis of elongation is detectable after about 6 h of culture. When regenerating protoplasts are treated with either colchicine or isopropyl-N-phenyl carbamate, drugs which interfere with microtubule polymerization, they remain spherical and develop cell walls in which the microfibrils are randomly oriented.