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     

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.     

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.     

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.     

Cellulose synthase (CesA) genes in the green alga Mesotaenium caldariorum

Cellulose, a microfibrillar polysaccharide consisting of bundles of beta-1,4-glucan chains, is a major component of plant and most algal cell walls and is also synthesized by some prokaryotes. Seed plants and bacteria differ in the structures of their membrane terminal complexes that make cellulose and, in turn, control the dimensions of the microfibrils produced. They also differ in the domain structures of their CesA gene products (the catalytic subunit of cellulose synthase), which have been localized to terminal complexes and appear to help maintain terminal complex structure. Terminal complex structures in algae range from rosettes (plant-like) to linear forms (bacterium-like). Thus, algal CesA genes may reveal domains that control terminal complex assembly and microfibril structure. The CesA genes from the alga Mesotaenium caldariorum, a member of the order Zygnematales, which have rosette terminal complexes, are remarkably similar to seed plant CesAs, with deduced amino acid sequence identities of up to 59%. In addition to the putative transmembrane helices and the D-D-D-QXXRW motif shared by all known CesA gene products, M. caldariorum and seed plant CesAs share a region conserved among plants, an N-terminal zinc-binding domain, and a variable or class-specific region. This indicates that the domains that characterize seed plant CesAs arose prior to the evolution of land plants and may play a role in maintaining the structures of rosette terminal complexes. The CesA genes identified in M. caldariorum are the first reported for any eukaryotic alga and will provide a basis for analyzing the CesA genes of algae with different types of terminal complexes.     

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     

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)     

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     

COBRA, an Arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation

The orientation of cell expansion is a process at the heart of plant morphogenesis. Cellulose microfibrils are the primary anisotropic material in the cell wall and thus are likely to be the main determinant of the orientation of cell expansion. COBRA (COB) has been identified previously as a potential regulator of cellulose biogenesis. In this study, characterization of a null allele, cob-4, establishes the key role of COB in controlling anisotropic expansion in most developing organs. Quantitative polarized-light and field-emission scanning electron microscopy reveal that loss of anisotropic expansion in cob mutants is accompanied by disorganization of the orientation of cellulose microfibrils and subsequent reduction of crystalline cellulose. Analyses of the conditional cob-1 allele suggested that COB is primarily implicated in microfibril deposition during rapid elongation. Immunodetection analysis in elongating root cells revealed that, in agreement with its substitution by a glycosylphosphatidylinositol anchor, COB was polarly targeted to both the plasma membrane and the longitudinal cell walls and was distributed in a banding pattern perpendicular to the longitudinal axis via a microtubule-dependent mechanism. Our observations suggest that COB, through its involvement in cellulose microfibril orientation, is an essential factor in highly anisotropic expansion during plant morphogenesis.     

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.     

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.     

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.     

The plasma membrane of young Chara internodal cells revealed by rapid freezing

Young elongating internodal cells of Chara globularis var. capillacea (Thuill.) Zanev. were rapidly frozen and freze-fractured in order to observed transient events occurring within the plasma membrane. Several structures have been observed. Relatively small depressions, varying in depth, are prolific and scattered at random over the plasma membrane. Charasomes and clusters of particle rosettes are common. Arrays of intramembrane particle lines are a characteristic feature of the internodal cell plasma membrane. The charasomes and the arrays of particle lines occupy a considerable proportion of the plasma membrane. In these young cells, substantial movement must take place across this membrane and its basic structure must fluctuate accordingly. The innumerable small depressions may represent pinocytotic and secretory processes. The array of intramembrane particle lines may represent stages in fusion between the membranes of vesicles within the cytoplasm and the plasma membrane. The technique of ultra-rapid freezing allows these events and their intermediate stages to be visualised; some features of the membrane may only be seen by this method.     

Winding threads around plant cells Applications of the geometrical model for microfibril deposition

Summary Based on precise information about the orientations of cellulose microfibrils (CMFs) in the secondary cell wall of theEquisetum hyemale root hair, a geometrical model was recently put forward to account for the deposition orientation of CMFs. The model supposes that synthases spin out the CMFs and that geometrical laws dictate their movement. Taking space-limiting conditions into account, CMF orientation is dependent on cell morphology, the amount of other wall molecules adhering to the CMFs, and the number and distribution pattern of synthases. In the present paper this geometrical model for CMF deposition is further applied to nontip-growing angular cells with varying diameters, cells with tapering morphology, various distribution patterns of synthases, various matrix/fibril ratios, and intercalarily elongating cells. The model can accurately predict the actual wall textures in a great variety of cell walls. In the proposed model for CMF orientation, microtubules are not required as cellular guiding structures for the CMFs, not even in elongating walls. They are supposed to be involved in cell elongation, possibly by delivering wall material including CMF synthases.Abbreviation CMF cellulose microfibril     

Cell walls,plasma membranes,and dictyosomes during zygote maturation ofClosterium ehrenbergii

Summary The cell wall formation and its correlation with the plasma membrane and dictyosome were investigated by an electron microscope in the zygote cells ofClosterium ehrenbergii. During zygote maturation, six wall layers were formed outside the plasma membrane. Wall layer III was the thickest layer and consisted of microfibril bundles. Dictyosomes produced flat vesicles during formation of wall layer III. Hexagonal arrays of rosette particles appeared in the plasma membrane in this period, thus confirming the simultaneous occurrence of flat vesicles and hexagonal particle arrays in the formation of microfibril bundles even at different stages of the life cycle. Wall layer VI was second in thickness and consisted of single microfibrils. Neither flat vesicles nor hexagonal particle arrays were observed during formation of this layer.     

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

Summary The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.     

Distribution of plasma membrane rosettes and kinetics of cellulose formation in xylem development of higher plants

Summary Germ roots of several higher plants—maize (Zea mays), mung bean (Vigna radiata) and cress (Lepidium sativum)—were freeze-fractured without cryoprotection in order to confirm and extend the informations on frequency and distribution of plasma membrane particle complexes with respect to cellulose formation. In all three objects the PF of developing xylem elements showed rosette accumulations in the regions of wall thickenings. The rosette-distribution pattern ranges from random in a young stage, to more grouped in a probable intermediate stage to strictly localized in later stages. The frequency of rosettes increases from stage to stage.In all three objects the EF of developing xylem elements is relatively poor in particles. Observations of terminal globules were rare and undistinct. This leads to the assumption that rosettes on the PF and terminal globules on the EF are not part of the same complex.A comparison of the number and distribution of microtubules underlying the xylem wall thickenings with rosette frequency and distribution leads to the conclusion that there seem to be no direct connections between these two structures. Microtubules may be involved in grouping of rosettes, thus indirectly orienting microfibril deposition. Calculations based on the observed rosette frequencies and the amount of wall material formed indicate that in xylem development 1,000 nm elementary fibril per rosette per minute may be formed and that the active phase of one rosette may be about 10 minutes.Abbreviations EF exoplasmic fracture face - PF protoplasmic fracture face     

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)     

Cell-wall formation in Pelvetia embryos. A freeze-fracture study

The cell-wall formation in the egg of Pelvetia fastigiata (J.G. Agardh) DeToni (Fucaceae) was studied with freeze-fracture. 1. The wall is lamellated with microfibrils approximately parallel in each lamella. The average orientation of microfibrils turns about 35° in each subsequent lamella. This slow turn gives rise to bow-shaped arcs when the wall is obliquely cross fractured. 2. The organization of the fibrils in the innermost lamellae is visualized by their imprints on the plasma membrane. These imprints are the result of both turgor pressure and adhesion of fibrils to the membrane. 3. Strings of membrane particles appear on the plasma membrane shortly after fertilization. They seem to be formed by a fertilization-induced aggregation of isolated membrane particles. Later each string comes to lie under a fibril and along its imprint. Peculiar lateral rips indicate that some strings are tightly bound to a fibril and may be involved in its orientation. 4. Wall formation in Pelvetia is marked by pronounced secretory activities. Following fertilization, the fusion of cortical vesicles and other vesicles make numerous loci in the plasma membrane. In older embryos, fibril-free patches in the plasma membrane mark the position of microfibril elongation centers in the wall matrix. Prior to germination, these elongation centers and their corresponding membrane patches reach a high density at the presumptive rhizoid end.We wish dedicate this paper to R.D. Preston