The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

Patterns of cortical and perinuclear microtubule organization in meristematic root cells ofAdiantum capillus veneris

Summary The interphase meristematic root cells ofAdiantum capillus venerispossess a well developed cytoskeleton of cortical microtubules (Mts), which disappear at prophase. The preprophase-prophase cells display a well organized preprophase microtubule band (PMB) and a perinuclear Mt system. The observations favour the suggestion that the cell edges included in the PMB cortical zone possess a Mt organizing capacity and thus play an important role in PMB formation. The perinuclear Mts are probably organized on the nuclear surface. The preprophase-prophase nuclei often form protrusions towards the PMB cortical zone and the spindle poles, assuming a conical or rhomboid shape. Mts may be involved in this nuclear shaping.Reinstallation of cortical Mts in dividing cells begins about the middle of cytokinesis with the reappearance of short Mts on the cell surface. When cytokinesis terminates, numerous Mts line the postcytokinetic daughter wall. Many of them converge or form clusters in the cytoplasm occupying the junctions of the new and the old walls. In the examined fern, the cortical Mt arrays seem to be initiated in the cortex of post-cytokinetic root cells. A transitory radial perinuclear Mt array, comparable to that found in post-telophase root cells of flowering plants, was not observed inA. capillus veneris.     

Microtubule and actin filament organization during stomatal morphogenesis in the fernAsplenium nidus

Summary The newly-formed guard cell mother cells (GMCs) ofAsplenium nidus are small, lens-shaped and are formed by one or two asymmetrical divisions. Their growth axis is parallel to the plane of their future division, a process during which the internal periclinal wall (IPW) is detached from the partner wall of the underlying cell(s). This oriented GMC expansion occurs transversely to a microfibril bundle, which is deposited externally to a U-like microtubule (Mt) bundle and a co-localized actin filament (Af) bundle. They line the IPW and the major part of the anticlinal walls. The deposition of the microfibril bundle is followed by the slight constriction of the internal part of the GMCs and the broadening of the substomatal cavity. The IPW forms a distinct bulging distal to the neighbouring leaf margin, as well as a less defined proximal one. During the IPW bulging, the Mts and Afs under the external periclinal wall (EPW) attain a radial organization. This is followed by thinning of the central EPW region, which becomes impregnated with a callose-like glucan. The rest of the EPW becomes unequally thickened. The disintegration of the U-like Mt bundle is succeeded by the organization of radial Mt and Af arrays under the IPW. The radial Mt systems, controlling the alignment of the newly-deposited microfibrils, allow the GMC to assume a round paradermal profile. The GMCs form a preprophase Mt band (PPB) perpendicular to the interphase U-like Mt bundle. The anticlinal PPB portions appear first and those lining the periclinal walls later. The cytoplasm adjacent to the latter walls retain the radial Mt systems during early preprophase, simultaneously with the anticlinal PPB portions. The observations suggest that the GMCs of the fernA. nidus obtain a unique form, as a result of a particular polarity established in the cortical cytoplasm of the periclinal walls, in which Mts and Afs appear involved. This polarity persists in cell division and is inherited to guard cells (GCs). It provides primary morphogenetic information not only to GMCs but also to GCs.Abbreviations Af actin filament - EPW external periclinal wall - GC guard cell - GMC guard cell mother cell - IPW internal periclinal wall - Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band     

Microtubule organization and morphogenesis of stomata in caffeine-affected seedlings ofZea mays

Summary Treatment ofZea mays seedlings with a 5 mM caffeine solution inhibits cytokinesis in guard cell mother cells (GMCs), producing unicellular, binucleate aberrant stomata (a-stomata). Ventral wall (VW) strips of limited length, which usually meet the wall portions of GMCs adjoining the cortical zone of the preprophase microtubule band (PMB), are laid down in many a-stomata.In a-stomata with or without VW-strips, the periclinal walls are lined by numerous microtubules (Mts) converging on their mid-region, where local wall thickenings are deposited. When the VW-strips reach the mid-region of the periclinal walls, thickenings lined by numerous Mts rise at their free margins. In certain a-stomata an anticlinal wall column, surrounded by a dense Mt bundle, grows centripetally from either or both of the periclinal wall thickenings. In wall thickenings, the cellulose microfibrils are co-aligned with the adjacent Mts. Pore formation is initiated in all a-stomata. Deposition of an electron dense intra-wall material followed by lysis precedes pore opening. This process is closely related to the a-stornata morphogenesis. These observations show that the primary morphogenetic phenomenon in a-stomata is the establishment of an intense and stable polarity in the cytoplasm abutting on the mid-region of the periclinal walls and/or the adjacent plasmalemma area. Prime morphogenetic factor(s), including microtubule organizing centres (MTOCs), seem to function in these sites. Morphogenesis in a-stomata is a Mt-dependent process that is carried out as in normal stomata but in the absence of a VW.Abbreviations a-stomata unicellular binucleate aberrant stomata - CIPC chlorisopropyl-N-phenyl carbamate - GC guard cell - GMC guard cell mother cell - Mt microtubule - MTOC microtubule organizing centre - PMB preprophase microtubule band - VW ventral wall     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

Photocontrol of the orientation of cell division inAdiantum

The rate of transition from one- to two-dimensional growth of fernAdiantum gametophytes under white light depends on the age of gametophyte cultured under red light. When gametophytes were cultured for longer period under red light, the rate of transition decreased and the number of abnormal gametophytes increased. Although the first step of the transition was the first longitudinal cell division following the two transverse ones (Wada and Furuya, 1970), the time-lapse-video study revealed that the apical cell of protonemata became flattened in the plane perpendicular to the incident ray of white light before the first longitudinal cell division. Analytical study of growing part of the apical cell with grains of activated charcoal as markers revealed that the apical cell flattening occurred evenly throughout the equatorial circumference of the cell even in the shaded side of the protonemata as well as in the side irradiated with white light.     

Effects of colchicine and amiprophos-methyl on microfibril arrangement and cell shape inAdiantum protonemal cells

Summary 5 mM colchicine and 1 g/ml amiprophos-methyl, known antimicrotubule agents, were applied to fernAdiantum protonemata under red light. Both drugs caused microtubule disruption and subsequent apical swelling of protonemal cells after certain lag periods. While the lag periods for the onset of microtubule disruption after application of the two drugs were different (within 15 minutes in amiprophos-methyl, 1 hour in colchicine), the lag periods of apical swelling after microtubule disruption were nearly the same (approx. 70 minutes). The results suggest that the apical swelling is a consequence of microtubule disruption.In cells examined 1 hour after microtubule disruption by either drug, the microfibril arrangement of the innermost layer of the cell wall was random at the tip, transverse in the subapical region, and roughly longitudinal in the cylindrical region. This pattern of microfibrils was similar to that of untreated cells in which the microtubules show a similar arrangement (Murata and Wada 1989). Surprisingly, even after approx. 4 hours of microtubule disruption, when apical swelling had occurred in most cells, the pattern of microfibril deposition was not altered. The role of microtubules in oriented microfibril deposition and the mechanism of control of cell shape are discussed.Abbreviations APM amiprophos-methyl - DMSO dimethylsulfoxide - MT(s) microtubule(s) - PBS phosphate buffered saline     

Microtubules and cell shaping in the mesophyll ofNigella damascena L.

Summary Cell shaping in the mesophyll ofNigella damascena was investigated with the aim of determining the origin of the arm-like protrusions, which are characteristic of, e.g., arm-palisade cells. It was found that hoops of cell wall were deposited during the early stages of cell expansion. The hoops were interconnected, thus embracing the cells with a wide-meshed net of local wall reinforcement. The pattern of wall deposition in the extra-cellular matrix correlated with a pattern of bands of microtubules in the cortical cytoplasm of the cells. During lateral expansion bulges were forced through the comparatively thin walls of spaces between the meshes, giving rise to the arm-like protrusions. After establishing the cell shape the bands of microtubules disintegrated and cell wall was uniformly deposited. The results are discussed in the context of the mode of cell shaping observed in the mesophyll of other systems and of a previous, classical hypothesis on the origin of arms in mesophyll cells.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - PME phosphate-magnesium-EGTA-buffer     

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.     

Microtubules and epithem-cell morphogenesis in hydathodes of Pilea cadierei

When cell divisions have ceased, the epithem of the hydathodes of Pilea cadierei Gagnep. et Guill. consists of small polyhedral cells exhibiting a meristematic appearance, and completely lacks intercellular spaces. The cortical microtubules in epithem cells exhibit a unique organization: they are not scattered along the whole wall surface but form groups lying at some distance from each other. In sections, from two to eight groups of microtubules can be observed, each lining a wall region averaging between 0.5 and 1.5 m in length. These groups represent sections of microtubule bundles girdling a major part or the whole of the cell periphery. They are connected to one another by anastomoses, forming a microtubular reticulum. The assembly of microtubule bundles is followed by the appearance of distinct local thickenings in the adjacent wall areas. The cellulose microfibrils in the thickenings are deposited in parallel to the underlying microtubules. Gradually, the vacuolating epithem cells undergo swelling, except for the areas bounded by the wall thickenings. Since the latter, and actually their constituent bundles of cellulose microfibrils, cannot extend in length the differential cell growth results in schizogenous formation of intercellular spaces between contiguous cell walls at their thickened regions. The spaces then broaden and merge to become an extensive intercellular space system. As a result of the above processes, the epithem cells become constricted and finally deeply lobed. The observations show that (i) the cortical microtubules are intimately involved in the morphogenesis of the epithem cells and (ii) the initiation and development of the epithem intercellular spaces is a phenomenon directly related to cell morphogenesis and therefore to the cortical microtubule cytoskeleton. The sites of initiation of these spaces are highly predictable.     

Live imaging of microtubule organization,cell expansion,and intercellular space formation in Arabidopsis leaf spongy mesophyll cells

Leaf spongy mesophyll cells form an interconnected network of branched cells and intercellular spaces to maximize the surface area available for light capture and photosynthetic gas exchange. To investigate the morphogenetic events leading to cell separation and branching in Arabidopsis thaliana, we used mesophyll-specific promoters to facilitate imaging of mesophyll cell shape and microtubule (MT) organization over multiple spatiotemporal scales without interference from the overlying epidermal cells. We show that cells enlarge by selective expansion of cell wall regions in contact with intercellular spaces. Cell–cell contacts remain relatively fixed in size, forming the termini of interconnecting branches. Surprisingly, classic schizogeny (de-adhesion of neighboring cells) is relatively infrequent, being related to the local topology of cell junctions during early expansion. Intercellular spaces cue the position of stable MT bundles, which in turn promote efficient dilation of intercellular spaces and cell branching. Our data provide insights into mesophyll morphogenesis and MT organization and lay the groundwork for future investigations.

Live imaging reveals a positive feedback loop between cell expansion and microtubule organization that facilitates efficient cell branching and intercellular space dilation in spongy mesophyll cells.     

Three-dimensional study of the intercellular gas space inVigna radiata hypocotyl

Summary Infiltration of the intercellular gas space is often used in physiological experimentation either to reach internal targets with various biochemical probes or to extract molecules from the apoplast. Such investigations require a good understanding of the organization and structure of the gas space system. This system was studied in the mung bean hypocotyl using different approaches, in particular internal microcasting of seedlings. Results show the presence of two continuous, more or less independent, networks, one in the cortical parenchyma, the other in the pith. The narrow tubules of one or the other network connect all cells within a tissue. Infiltration with physiological solutions does not significantly disturb living cells, at least in the short term, but opens a large field for experimental applications.     

Growth,cell wall formation and differentiation in the protonema of the moss,Funaria hygrometrica: Effects of plasmolysis on the developmental program and its expression

Summary Cultivation ofFunaria protonemata under plasmolytic or slightly subplasmolytic conditions initially causes a cessation of growth which is accompanied by a transient disappearance (or strong reduction in frequency, respectively) of putative cellulose synthesizing particle rosettes in the plasma membrane. Simultaneously, the formation and exocytosis of cell wall materialsecreting Golgi vesicles is slowed down. The latter process does not become apparent for several hours, though the reduction in activity can be proved indirectly. As a consequence of the imbalance between exocytosis, cell wall material accumulates in the plasmolytic space, generally at the cell tip. This indicates that the pattern of local, polar deposition of cell wall formation and cell elongation, membrane debris as well as wall material is maintained for some time. Later, however, the whole protoplast may become covered by new wall layers. Potentially growing filament tips and the distal region of nontip cells increase in diameter after longer cultivation in subplasmolytic conditions. It is suggested that normal wall growth results from a softening of the existing wall, its stretching and simultaneous stabilization by the apposition of new wall layers. We believe that the swelling is caused by a change in the equilibrium between the obviously less affected softening process and the imperfect stabilization by new wall layers because the wall layers which are formed at reduced turgor pressure are looser than normal and may have a changed composition.Kinetin-induced buds do not develop under plasmolytic conditions. Instead, spiral filaments are formed which readily give rise to buds when the osmotic value of the (kinetin-containing) medium is normalized. The results show that plasmolysis affects the expression of the developmental program rather than its initiation or maintenance.     

Cell shaping and microtubules in developing mesophyll of wheat (Triticum aestivum L.)

Summary Differentiated mesophyll cells ofTriticum aestivum (cv. Star) exhibit a lobed outline resembling tube-shaped balloons with almost regularly spaced constrictions. It was shown that these constrictions are probably the result of hoops of wall reinforcements laid down during early stages of cell expansion. It appears that these hoops prevent expansion in the corresponding regions and thus give rise to the peculiar cell shape. The comparatively thin cell walls of the bulges are uniformly reinforced after the lobed shape is established.By using immunofluorescence techniques a change in the pattern of cortical microtubule arrangement was observed which corresponded to the pattern of cell wall deposition. Discrete bands of microtubules were found beneath the sites of hoop reinforcement. These bands disintegrated during late stages of cell expansion with microtubules fanning out into the almost empty regions of the bulges.Abbreviations DMSO dimethyl sulfoxid - EGTA ethylene glycol bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanat - MSB microtubule stabilizing buffer - PBS phosphate buffered saline - PIPES 1,4-piperazine diethanesulfonic acid - PMSF phenylmethyl sulfonylfluoride     

Cell division and morphogenesis in angiosperm embryogenesis

Angiosperm embryogenesis generates the basic body organization of flowering plants. The underlying processes of pattern formation, which establishes the diversity of position-dependent cell fates, and morphogenesis, which brings about the shape of the embryo, may not only involve intercellular communication and controlled cell expansion but also non-random cell divisions. Genetic analysis ofArabidopsisembryogenesis which displays a large invariant pattern of cell divisions suggests that unequal cell divisions segregate cell fates and are thus involved in pattern formation whereas other oriented cell divisions and differential mitotic rates reflect patterning and rather play a role in morphogenesis.     

Microtubule and actin filament organization during acentral divisions in potato suspension culture cells

Summary Microtubule and filamentous(F)-actin organization in the potato suspension culture line HH260 was studied by fluorescence microscopy in double-labelled cells. During interphase, microtubules and F-actin were randomly arrayed in isodiametric cells but were aligned transversely to the direction of growth in elongated cells. Microtubules and F-actin coaligned in preprophase bands which were, however, comparatively rare and diffuse. Interestingly, more than half of the cells in telophase contained phragmoplasts that were either horseshoe-shaped or straight, instead of being round. We traced the cause of this difference to preprophase, where misplaced nuclear localization away from the central axis of cells may give rise to acentrally placed spindles and, subsequently, to acentrally placed phragmoplasts and cell plates. Further, we hypothesize that it is the uneven fusion of the expanding cell plates with the parent plasma membrane, and the accompanying depolymerization of those parts of the phragmoplasts, that gives the incomplete phragmoplasts observed.Abbreviations DAPI 4,6-diamidino-2-phenylindole - MBS 3-maleimidobenzoyl-N-hydroxy-succinimidester - PMSF phenyl-methylsulfonyl fluoride - SB stabilization buffer     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator     

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