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.     

Orientation of cortical microtubules correlates with cell shape and division direction

Summary Cortical microtubules in the epidermis of regeneratingGraptopetalum plants were examined by in situ immunofluorescence. Paradermal slices of tissue were prepared by a method that preserves microtubule arrays and also maintains cell junctions. To test the hypothesis that cortical microtubule arrays align perpendicular to the direction of organ growth, arrays were visualized and their orientation quantified. A majority of microtubules are in transverse orientation with respect to the organ axis early in shoot development when the growth habit is uniform. Later in development, when growth habit is non-uniform and the tissue is contoured, cortical microtubules are increasingly longitudinal and oblique in orientation. Microtubules show only a minor change in orientation at the site of greatest curvature, the transition zone of a developing leaf. To assess the role of the division plane on orientation of arrays, the pattern of microtubules was examined in individual cells of common shape. Cells derived from transverse divisions have predominately transverse cortical arrays, whereas cells derived from oblique and longitudinal divisions have non-transverse arrays. The results show that, regardless of the stage of development, microtubules orient with respect to cell shape and plane of division. The results suggest that cytoskeletal function is best considered in small domains of growth within an organ.Abbrevations DMSO dimethylsulfoxide - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetra acetic acid - FITC fluorescein isothiocyanate - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.     

The effects of microtubule and microfilament disrupting agents on cytoskeletal arrays and wall deposition in developing cotton fibers

Summary The effects of various cytoskeletal disrupting agents (cholchicine, oryzalin, trifluralin, taxol, cytochalasins B and D) on microtubules, microfilaments and wall microfibril deposition were monitored in developing cotton fibers, using immunocytochemical and fluorescence techniques. Treatment with 10^–4 M colchicine, 10^–6 M trifluralin or 10^–6 M oryzalin resulted in a reduction in the number of microtubules, however, the drug-stable microtubules still appear to influence wall deposition. Treatment with 10^–5 M taxol increased the numbers of microtubules present within 15 minutes of application. New microtubules were aligned parallel to the existing ones, however, some evidence of random arrays was observed. Microtubules stabilized with taxol appeared to function in wall organization but do not undergo normal re-orientations during development. Microtubule disrupting agent had no detectable affect on the microfilament population. Exposure to either 4×10^–5 M cytochalasin B or 2×10^–6M cytochalasin D resulted in a disruption of microfilaments and a re-organization of microtubule arrays. Treatment with either cytochalasin caused a premature shift in the orientation of microtubules in young fibers, whereas in older fibers the microtubule arrays became randomly organized. These observations indicate that microtubule populations during interphase are heterogeneous, differing at least in their susceptibility to disruption by depolymerizing agents. Changes in microtubule orientation (induced by cytochalasin) indicate that microfilaments may be involved in regulating microtubule orientation during development.     

Cell proliferation,cell shape,and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.     

Microtubule patterning in apical epidermal cells ofVinca minor preceding leaf emergence

Summary Serial thin sectioning for electron microscopy was carried out on the cortical cytoplasm of surface cells of the apical dome ofVinca minor. The cellulose reinforcement pattern in the outer epidermal walls forming this surface is known to correlate well with the decussate phyllotaxis pattern. The purpose of this study was to determine the location of microtubules immediately under these epidermal walls as a first step toward finding out how the cellulose pattern arises. First, correspondence between the patterns of microtubules and cellulose was checked. Second, the role of potential orienting cues for the alignment of microtubule arrays in specific cells was evaluated.Microtubule arrays which were well or moderately ordered (70% of the total interphase cells) generally had alignment parallel to the adjacent leaf base, as has been seen for cellulose. The aligned features or cues potentially correlating with a given array were: (1) orientation and length of the previous anticlinal cross-wall, (2) alignment of microtubules in adjacent cells, and (3) direction of inferred stretch, parallel to the nearby leaf bases. All three features were found to agree with the microtubule alignment in 17 of 34 cells with ordered arrays. At least two features agreed in 33 of the 34 cases. All 34 cells with ordered arrays had at least one feature parallel to the array. Random association between microtubule orientation and these features would lead to such correlations less than 0.01% of the time. Thirty percent of the interphase cells showed no obvious order. Most of these cells were located in the central linear corridor region of the apex. The unordered cells were more likely than the ordered cells to have more than one orientation specified by the potential cues; i.e., no single orientation parallel to all of the cues existed. This indicates that uniformity of the orientation cues may be as important as their direction.     

Microtubule arrays in regeneratingMougeotia protoplasts may be oriented by electric fields

Summary Initially non-polar protoplasts of the green algaMougeotia will regenerate to re-establish their original cylindrical cell shape. The orientation of the growth axis of regenerating protoplasts held in agarose was independent of both the direction of incident white light and gravity. Protoplasts elongated parallel to applied DC electric fields of approx. 0.2 Vcm^–1 (1 mV/protoplast) and greater, with an increasing percentage oriented with increasing field strength. At the maximum field strength used (10 mV/cell), 53% of protoplasts were oriented within +- 10° of the 0/180° axis of the field. In untreated controls, the orientation of elongation was random. Protoplast survival was unaffected by field treatment. Some protoplasts (up to 37% in 10 mV/cell fields) formed outgrowths towards the cathode and occasionally towards the anode. Regenerating protoplasts in fields displayed the normal sequence of microtubule reorganization. This means that the positioning of the ordered symmetrical array of microtubules centred on two foci that appears within 3 to 4 h, and the subsequent organization of microtubules by 8 to 12 h into a band that intersects both foci and which is transverse to the axis of elongation (Galway and Hardham 1986), may be controlled by externally applied electric fields. In the region of this microtubule band, the applied field causes the plasma membrane to be stretched parallel to the field (Bryant and Wolfe 1987). We suggest that microtubules may become oriented perpendicular to the direction of field-induced membrane stretching, and that membrane stretching may be one of the orienting mechanisms for membrane-linked microtubules in elongating plant cells.Abbreviations PBS phosphate buffered saline - PMM protoplast maintenance medium - DMM dilute maintenance medium - MES 2(N-morpholino)ethanesulfonic acid - TRIS tris(hydroxymethyl)aminomethane - ANOVA analysis of variance     

Microtubule rearrangements accompanying dedifferentiation in mesophyll cultures ofZinnia elegans L.

Summary To determine the orientation of cortical microtubule arrays in mesophyll cells ofZinnia, a new technique designed to increase the rate of fixation of excised leaf tissue and subsequent permeabilization of mesophyll cell walls was developed. This procedure resulted in immunolabeling of high percentages of mesophyll cells, making it possible to quantify cells with different types of cortical microtubule arrays. When developing palisade mesophyll cells were fixed in situ, most of the cells had cortical microtubules organized in parallel arrays oriented transverse to the long axis. Delay in the transfer of leaf tissue to fixative resulted in increased numbers of cells with random cortical microtubule orientations, indicating that arrays may become reoriented rapidly during leaf excision and cell isolation procedures. The role of wound-induced microtubule reorientation in mesophyll dedifferentiation and tracheary element development is discussed.Abbreviations BSA bovine serum albumin - CMT cortical microtubule - TE tracheary element - TBS tris-buffered saline     

Microtubules and coated vesicles in guard-cell protoplasts ofAllium cepa L.

Protoplasts were prepared from the guard cells ofA. cepa. Epidermal peels taken from expanding green leaves and largely free of mesophyll were treated with Cellulysin, and protoplasts were harvested after 18 h of digestion. That the protoplasts were derived from guard cells was ascertained from their characteristic vacuolar autofluorescence and from observations showing that all other epidermal cells are killed in the peeling procedure. The protoplasts proved to be a good system with which to view the cell cortex and inner surface of the plasmalemma. The lysis of cells adhering to polylysine-treated, Formvar-coated grids, followed by negative staining in uranyl acetate, showed that many microtubules normally present in ordered arrays in situ remain closely applied to the inner surface of the plasmalemma in protoplasts. In addition, numerous vesiculate elements including coated vesicles and/or pits are present amongst the microtubules. Similar vesicles are evident in thin sections of fixed, embedded guard cells and protoplasts. The significance of these structures in the cell cortex is discussed.     

Tobacco protoplasts differentiate into elongate cells without net microtubule depolymerization

Summary Microtubules (MTs) are important for plant cell morphogenesis because they influence the deposition of cell plate and wall components. It has been observed that tobacco protoplasts contain a disordered MT array in the cortex. Following several days in culture, these protoplasts become elongate cells with an orderly cortical MT array. The transformation of the MT array may occur by net depolymerization of the disordered MTs and repolymerization of MTs into an ordered array, or by movement of the array as an integral unit. To experimentally distinguish between these two possibilities, the drug taxol was used to stabilize MTs. Protoplasts derived from suspension cultured tobacco,Nicotiana tabacum, were grown in a medium containing the two plant hormones -naphthaleneacetic acid and benzyladenine, in the presence or absence of 10M taxol. Changes in cell size and shape were quantified using a video image analysis system. Cell elongation had begun within 48h of protoplast conversion, in both treatments, and continued for 7 days. Immunolocalization of tubulin showed that, in the majority of cells, MTs were disorganized immediately following protoplast conversion. After elongation, the MT arrays were observed to have reoriented to an ordered state. Taxol-treated protoplasts were found to elongate faster and to a greater extent than the non-treated controls. Additionally, the cortical array of taxol-treated protoplasts reorganized more quickly. These data indicate that the net depolymerization of disordered cortical MTs is not necessarily required for the differentiation of a protoplast into an elongate cell.Abbreviations APM amiprophosmethyl - BSA bovine serum albumin - DIC differential interference contrast - DTT dithiothreitol - EGTA ethylenegrycol-bis-(-aminoethyl ether)N,N,N,N-tetra-acetic acid - ELISA enzyme-linked immunosorbent assay - FMS Fukuda, Murashige, and Skoog - MS Murashige and Skoog - MT(s) microtubule(s) - PBS phosphate buffered saline - PIPES piperazine-N,N-bis (2-ethanesulfonic acid, 1.5 sodium) - PM plasma membrane - Tris Tris(hydroxymethyl)amino-methane     

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     

Ultrastructure of the cell wall regeneration of isolated protoplasts of Skimmia japonica thunb

Isolated protoplasts obtained from leaves and from stem callus cultures of Skimmia japonica were cultivated for 72 h to regenerate a new cell wall. During this process the structural changes in the protoplasts and at the surface of the plasmalemma were studied in ultrathin sections and after freeze-fracturing and deep-etching.The cultured protoplasts show an apparent increase in cell organelles compared to the freshly isolated protoplasts. In particular, mitochondria, endoplasmic reticulum, and ribosomes, many of them appear as polysomes, become numerous. Moreover, special connections between the ER and the plasmalemma are visible. Most important are the fracture faces of the plasmalemma with two different arrangements of membrane-bound particles: (1) particles in hexagonal arrays and (2) rows of ca. 14 particles. Their orientation usually conforms with that of the regenerated microfibrils of the cell wall. According to these results the following model for microfibril synthesis and orientation in higher plants is proposed: While the cytoplasmic activity is involved in the production of cellulose precursors and enzymes, the hexagonal arrays may respresent specialized regions for the outward passage of these cellulose precursors. The rows of membrane-associated particles may function as a linear enzyme complex (matrix) for microfibril biosynthesis and orientation.Abbreviations ER endoplasmic reticulum - IAA -indolylacetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid     

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     

Effect of ultraviolet radiation on cell division and microtubule organization inPetunia hybrida protoplasts

Summary Mesophyll protoplasts isolated fromPetunia hybrida were subjected to UV radiation (280–360 nm) in an attempt to assess whether (a) UV radiation has an effect on cortical microtubule organization, (b) UV radiation affects the progression of protoplasts through the cell cycle, and (c) there is a connection between the effect of UV radiation on cell division and the polymerization state of the microtubules. The proto plasts were irradiated with the following UV doses: 4, 8, 12, and 24mmol photons/m², 30 min after isolation. Cell cycle analysis and immuno-localization of microtubules were carried out 0, 24, 48, and 72 h after irradiation. The length of cortical microtubules was determined after irradiation and in corresponding controls. We found that UV radiation induced breaks in cortical microtubules resulting in shorter fragments with increasing dose. Also, the protoplasts were delayed in their progression through the cell cycle, with G1 and G2 phases being affected as well as the S phase. The commencement of DNA synthesis in the irradiated protoplasts followed the re-establishment of a microtubule network. At 48 h after irradiation the protoplasts in all treatments, except for the 24 mmol/m², had cortical microtubules of similar length, and at 72 h after irradiation only the protoplasts that had received 24 mmol photons/m² had not started dividing.Abbreviations BSA bovine serum albumin - DMSO dimethyl sulfoxide - FDA fluorescein diacetate - MT microtubules - MTSB microtubule stabilizing buffer - PAR photosynthetically active radiation (400–700 nm) - PBS phosphate buffered saline - UV ultraviolet     

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells

Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine. Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized. Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre- prophase bands, the mitotic spindle, and phragmoplast microtubules. The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.     

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

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

Establishing and maintaining axial growth: wall mechanical properties and the cytoskeleton

Organ morphology depends on cell placement and directional cell expansion. Microtubules are involved in both of these processes so genetic approaches to understand the role microtubules play in organ expansion are not straightforward. Our use of the temperature-sensitive mor1-1 mutants led to the surprising discovery that Arabidopsis thaliana (L.) Heynh. root cells can establish and maintain transverse cellulose texture without well organized microtubule arrays. This work also demonstrated that cells can lose the ability to expand anisotropically without losing transversely oriented cellulose microfibrils. We suggest that microtubule disruption affects the cells ability to generate long cellulose microfibrils, which may be essential for achieving growth anisotropy. Thus organ shape may depend not only on the orientation but also on the relative length of cellulose microfibrils during axis establishment and growth. More recent work has shown an important correlation between microtubule organization and the deposition patterns of the glycosylphosphatidylinositol (GPI)-anchored wall protein COBRA. Loss of microtubule organization is associated with the dissipation of transverse banding patterns of COBRA, suggesting that COBRAs function in maintaining anisotropic expansion may be microtubule-dependent.     

Is the recovery of microtubule orientation in pea roots dependent on the cell wall?

This study tested several aspects of a model proposed by Williamson (1990, 1991) in which stresses in plant cell walls, detected by stress-receptive portions of inelastic cellulose microfibrils, orient microtubules via interactions with cell wall-linked transmembrane proteins. Young expanding cells of pea root tips have highly ordered transverse arrays of microtubules oriented perpendicular to the direction of cell expansion. The recovery of these ordered MT arrays after depolymerisation with oryzalin was assessed. It was shown that treating roots with disruptors of microfibril synthesis (2,6-dichlorobenzonitrile and calcofluor white) or the disruption of Arg-Gly-Asp (RGD)-mediated wall-membrane links did not affect the orientation of recovering microtubule arrays. Furthermore, cell wall stresses themselves appeared unnecessary for regeneration of transverse arrays. The relevance of these findings to Williamson's hypothesis is discussed.     

Microtubules in statocytes from roots of cress (Lepidium sativum L.)

Summary Statocytes in root caps ofLepidium sativum L. were examined by means of ultrathin serial sections to evaluate the amount and distribution of cortical microtubules. The microtubules encircle the cell, oriented normal to the root length axis. In the distal cell edges, microtubules form a network, separating the distal complex of endoplasmic reticulum from the plasmalemma. Preprophase bands in meristem cells are observable rarely, structures which can be regarded as nucleating sites for microtubules are lacking. During ageing of the root cap cells, the number of microtubules increases in combination with a decrease of microtubule length. Development of the roots on a horizontal clinostat preserves a younger developmental stage of the microtubule system regarding amount and length of the individual microtubules. Evidence for an involvement of microtubules in graviperception is low, whereas their role in orienting cellulose microfibrils cannot be ruled out. Compression of the distal network of microtubules after centrifugation of the roots indicates that microtubules in statocytes ofLepidium sativum L. roots might function in stabilizing the distal complex of endoplasmic reticulum.