Re-formation of microtubules in Closterium ehrenbergii Meneghini after cold-induced depolymerization

Immunofluorescence microscopy was used to examine the re-formation of microtubules (MT), after cold-induced depolymerization, in Closterium ehrenbergii. The C. ehrenbergii cells undergo cell division followed by semicell expansion in the dark period of daily light-dark cycles. Five types of MTs, namely the MT ring, hair-like MTs around the nuclei, spindle MTs, radially arranged MTs and transverse wall MTs, appeared and disappeared sequentially during and following cell division. The wall MTs were distributed transversely only in the expanding new semicells. When cells were chilled in ice water, wall MTs in expanding cells were fragmented, and then disappeared as did the other types of MTs, within 5 min. When cells were warmed at 20°C after 2 h chilling, wall MTs and the other types of MTs re-formed. At the early stage of wall-MT re-formation in expanding cells, small, star-like MTs were formed, and then randomly oriented MTs developed in both the expanding new and the old semicells. The MT ring was also re-formed at the boundary between the new and old semicells. There were no obvious MT-organizing centers in the random arrangement. As time passed, the randomly oriented wall MTs in the old semicells disappeared and those in the expanding new semicells gradually assumed a transverse orientation. These results indicate that wall MTs can be rearranged transversely after they have been re-formed and that nucleation of wall MTs is separable from the mechanism for ordering them.Abbreviations MT(s) microtubule(s) - MTOC(s) microtubule-organizing center(s)     

Immunofluorescence Microscopy of Microtubule Arrangement in Root Cells of Pisum sativum L. var Alaska

The arrangement of wall microtubules (MTs) in Pisum sativumroots was viewed immunofluorescently using cryosectioning. Mostcells in the tip region of pea roots (0–2 mm from tip)had wall MTs arranged transversely to the root axis. In theregion elongating at a higher rate (2–4 mm), wall MTsof epidermal, cortical and stelar cells were all transverselyarranged. In the region of about 5 mm from the tip, in whichcell elongation had already ceased, wall MTs in cortical cellschanged from a transverse to an oblique arrangement in relationto the root axis. Some cells had a crossed arrangement of wallMTs, which was interpreted as representing two sets of unidirectional,oblique wall MTs in opposite cell cortices of a single cell.This change was completed within a region of 1-mm width. Sinceroots elongated at a rate of 0.6 mm h^–1, it means thatthe arrangement of wall MTs changed within 2 h. An oblique arrangementof wall MTs was also observed in stelar cells. As the cellsaged, the oblique arrangement tended to change to a steeperor even a longitudinal one. (Received January 24, 1986; Accepted May 15, 1986)     

Exposure of Leaves of Cucumis sativus L. to Low Temperatures in the Light Causes Uncoupling of Thylakoids I. Studies with Isolated Thylakoids

Chilling of leaves of cucumber (Cucumis sativus L.) at 5?C inmoderate light for 5 h caused almost complete suppression ofphotosynthetic oxygen evolution in the leaves. Comparison ofelectron transport activities determined in the presence andabsence of an uncoupler, methylamine, indicated that thylakoidsprepared from such treated leaves were uncoupled without anysignificant changes in the capacity for electron transport.Immunoblotting revealed that the amount of coupling factor 1(CF₁) associated with membranes was reduced by the chillingtreatment in the light. Thylakoids prepared from leaves thathad been chilled in moderate light for 5 h and then re-warmedfor 1 h were coupled and capable of synthesising ATP. However,the capacity of leaf photosynthesis was not restored by therewarming. These results indicate that the thylakoids are uncoupledby the dissociation of some CF₁ complexes from the thylakoidmembranes during the chilling treatment of leaves in the lightand that thylakoids are recoupled by reassociation of CF₁ duringthe subsequent rewarming of the treated leaves at 25?C. It alsoappears that chilling in the light causes irreversible damageto reaction(s) other than those involved in electron transportand photophosphorylation. ¹ Present address: Department of Biological Science, Facultyof Science, Himeji Institute of Technology, Kamigori, Ako-gun,Hyogo, 678-12 Japan (Received July 1, 1991; Accepted October 4, 1991)     

The Effects of Light Irradiation on the Orientation of Microtubules in Seedlings of Avena sativa L. and Pisum sativum L.

The effects of light irradiation on the arrangement of corticalmicrotubules (MTs) were examined in etiolated A vena mesocotylsand coleoptiles, and in Pisum epicotyls. Elongation of A venamesocotyls ceased as a result of irradiation with white lightwithin 1 h. The predominant orientation of MTs became more longitudinalwithin 1 h in epidermal cells and changed from transverse tooblique, after the elongation ceased, in parenchymal cells.Irradiation with red and with blue light also caused cessationof cell elongation and the same changes in the orientation ofMTs. Elongation of Avena coleoptiles ceased as a result of irradiationwith white light within 24 h. The predominant orientation ofMTs became more longitudinal in epidermal cells and changedfrom transverse to oblique in parenchymal cells. The changein orientation of MTs in epidermal cells preceded that in parenchymalcells. In Pisum epicotyls, elongation ceased as a result ofirradiation with white light within 1 h. Although the orientationsof MTs in epidermal cells did not show any remarkable change,those in parenchymal cells changed from transverse to obliqueafter cell elongation ceased. The change in orientation of MTs and the cessation of cell elongationof A vena mesocotyls induced by white-light irradiation wereboth significantly retarded by treatment with IAA. This resultsuggests that IAA is involved in maintaining the transverseorientation of MTs in Avena mesocotyls. (Received February 22, 1989; Accepted August 2, 1989)     

Immunofluorescence Microscopical Observation of Cortical Microtubule Arrangement as Affected by Gibberellin in d5 Mutant of Zea mays L.

The relationship between the dwarf habit of growth and corticalmicrotubule (MT) orientation as affected by gibberellin wasexamined using a gibberellin responding dwarf mutant of Zeamays L. (d₅). The 4 mm portion of the mesocotyl below the coleoptilarnode of dark-grown seedlings was divided into four 1 mm segments.MTs were observed by means of immunofluorescence microscopyon the tangential surface of the epidermal cells (EP-t), theradial surfaces of epidermal (EP-r) and cortex cells (C-r) inboth normal and d₅ seedlings. MT arrangement in EP-t was transverse,oblique and/or longitudinal. In lower regions, cells with transverselyoriented MTs decreased, while those with obliquely/longitudinallyoriented MTs increased. The frequency of the occurrence of transverselyoriented MTs was much higher in normal than in d₅. In EP-r almostall the cells of the four regions had transversely orientedMTs. In d₅ seedlings only a few percent of cells had obliquelyoriented MTs. In C-r all the cells of the four regions of normalseedlings had transversely oriented MTs, while in d₅, 20% ofthe uppermost region cells showed oblique/longitudinal orientationand more in lower regions. By GA₃ (100µM) treatment for24 h, the growth of d₅ mesocotyls was stimulated, and the orientationof MTs in EP-t, EP-r and C-r of any regions became transverse. ¹Present address: Department of Forestry, Faculty of Agriculture,the University of Tokyo, Yayoi-1-Chome, Bunkyo-ku, Tokyo, 113Japan (Received August 16, 1990; Accepted January 22, 1991)     

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.     

Re-Formation and Ordering of Wall Microtubules in Spirogyra Cells

Processes of re-formation and ordering of wall microtubules(wall MTs) in Spirogyra cells were examined using immunofluorescencemicroscopy. Wall MTs were usually arranged nearly transverselyto the cell axis at all stages of the cell cycle. Two-hour treatmentwith amiprophosmethyl (APM) completely disrupted wall MTs, butremoval of the APM led to re-formation of randomly orientedwall MTs at many sites over the cell. The re-formed wall MTsgradually assumed a transversely oriented order without anyspecific MT-ordering centers, indicating that initiation andordering of MTs are different processes. Removal of APM after24-h treatment caused reformation of randomly oriented wallMTs, followed in some cells by gradual ordering to obliquelyoriented instead of transversely oriented wall MTs. This orderingoccurred with the same sign of obliquity as that of chloroplastspirals. When cells were centrifuged along the cell axis, chloroplastssedimented on the cross wall, but the transverse wall MTs didnot. In centrifuged cells, wall MTs were re-formed and orderedtransversely after MT depolymerization by APM for 2 h as innon-centrifuged cells. When cells were centrifuged for the final30 min in 2-h treatment with APM, wall MTs that re-formed afterremoval of APM were sometimes ordered transversely over thatpart of the cell which contained sedimented chloroplasts, butremained at random over the other part, as though the MT-orderingfactor was sedimented by the centrifugation. The mechanism determiningthe wall MT orientation is discussed. (Received January 12, 1987; Accepted April 27, 1987)     

Reinstatement of Microtubule Arrays from Cortical Nucleating Sites in Stomatal Complexes of Lolium rigidum Following Depolymerization of Microtubules by Oryzalin and High Pressure

Immunofluorescence visualization of microtubule (MT) arraysin stomatal complexes of Lolium rigidum shows that disassemblyof the arrays can be successfully achieved using oryzalin orhigh pressure treatments. Under conditions allowing for MT recovery,MTs reappear within an hour after oryzalin or within 5 min afterhigh pressure treatment. During recovery guard mother cells(GMCs) nucleate MTs at sites distributed randomly in the cellcortex. Even after 22 h of recovery the MTs are not arrangedinto any configuration found in untreated tissue. This inabilityto reorganize their MTs after treatment makes GMCs more sensitiveto the loss of MTs than are other cells of the leaf. In guardcells (GCs) MTs reappear around the pore at the junction ofthe periclinal and ventral walls. They subsequently appear throughoutmost of the cell cortex and the majority of stomatal complexesrecover fully organized MT arrays indistinguishable from thosein untreated cells. The results support and extend ultrastructuraland immunofluorescence observations that suggest that MTs inGCs of developing stomata are nucleated in the cell cortex. ²Present address: Department of Biology, The University of SouthwesternLouisiana, Lafayette, Louisiana 70504-2451, U.S.A. (Received April 24, 1990; Accepted July 7, 1990)     

Microtubule detachment from the microtubule-organizing center as a key event in the complete turnover of microtubules in cells

Microtubule (MT) response to different steady state temperatures and to rapid shifts in temperature was studied quantitatively in large, thin cells (LT-cells) from the goldfish scale. MT number and total tubulin concentration per cell were found to be fairly constant in cells from the same fish, regardless of cell size but between fish, could differ by a factor of two. The total tubulin concentration was similar to that found in mammalian tissue culture cells and the proportion in MT form increased with increasing steady state temperature. Total MT length quickly and exponentially decreased when cells were rapidly chilled to approximately -3 degrees C. In contrast, the average length of the MTs bound to the MT organizing center (MTOC) did not significantly change. Free MTs were generated during chilling and had an average length roughly half that of bound MTs. These observations suggest that 1) there is a functional block to rapid depolymerization at the unattached end of the MTOC bound MTs and 2) depolymerization of the MT occurs from the originally bound end only after its release from the MTOC. The presence of free MTs in a wide variety of cells suggests that these two features may be characteristic of steady state MTs in other cells. When the temperature of the LT-cells was abruptly raised, the number of MTs initiated on the MTOC rapidly increased and reached a brief steady state long before the MTs completely elongated. Many MTs then apparently detached from the MTOC and depolymerized before a final steady state was reached. When cells containing newly polymerized MTs were chilled to approximately -3 degrees C, the MTs detached from the MTOC more rapidly than those starting from steady state. Furthermore, the block to depolymerization at the unattached end was not complete. These observations suggest that newly formed, non-steady state MTs are different from the older, steady state MTs.     

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. II. Transition between the pre-prophase band and the mitotic spindle

Summary Changing patterns of tubulin immunofluorescence as onion root meristematic cells progress from a mature pre-prophase band (PPB) stage into mitosis are reported here. The PPB reaches its narrowest profile at maturity and then remains the same width throughout the rest of the transition. Concomitant with continuation of chromatin condensation and nucleolar breakdown, both initiated earlier in pre-prophase, alignment of fluorescent fibers along the nuclear envelope (NE) changes. Perinuclear microtubules (MTs), which were parallel to the PPB or randomly arranged when first seen at earlier stages of pre-prophase, assume the orientation of spindle MTS at late preprophase. They lie close to the NE and follow the nuclear contour, ultimately converging upon two focal points directly at the NE surface. MTs also can be seen traversing the cytoplasm between nucleus and cell periphery.As spindle initiation proceeds, PPB fluorescence intensity decreases and eventually is exceeded by the NE-associated fluorescence. PPB and spindle arrays co-exist briefly in the transition period, with spindle MTs typically aligned perpendicular to both the axis of the PPB and its constituent MTs. Total disappearance of the PPB occurs only after chromosome condensation is complete and the nucleus is contained within a spherical or ellipsoid cage of fluorescent fibers comprised of two non-overlapping half-spindles. Like the fully formed prophase spindle which follows, the incipient spindle is neither barrel-shaped nor fusiform, but rather displays MTs radiating from the poles in a smooth arc.     

Actomyosin is Involved in the Organization of the Microtubule Preprophase Band in Arabidopsis Suspension Cultured Cells

The microtubule preprophase bands （PPBs） participate in the sequence of events to position cell plates in most plants. However, the mechanism of PPB formation remains to be clarified. In the present study, the organization of PPBs in Arabidopsis suspension cultured cells was investigated by confocal laser scanning microscopy combined with pharmacological treatments of reagents specific for the cytoskeleton elements. Double staining of F-actin and microtubules （MTs） showed that actin filaments were arranged randomly and no colocalization with cortical MTs was observed in the interphase cells. However, cortical actin filaments showed colocalization with MTs during the formation of PPBs. A broad actin band formed with the broad MT band in the initiation of PPB and narrowed down together with the MT band to form the PPB. Nevertheless, broad MT bands were formed but failed to narrow down in cells treated with the F-actin disruptor latrunculin A. In contrast, in the presence of the F-actin stabilizer phalloidin, PPB formation did not exhibit any abnormality. Therefore, the integrity, but not the dynamics, of the actin cytoskeleton is necessary for the formation of normal PPBs. Treatment with 2, 3-butanedine monoxime, a myosin inhibitor, also resulted in the formation of broad MT bands, indicating that actomyosin may be involved in the rearrangement of MTs to form the PPBs. Double staining of MTs and myosin revealed that myosin concentrated on the PPB region during PPB formation. It is suggested that the actin cytoskeleton at the PPB site may serve as a rack to transport cortical MTs by using myosin when the broad MT band narrows down to form the PPB.     

Lipid changes in mature-green bell pepper fruit during chilling at 2°C and after transfer to 20°C subsequent to chilling

Lipid composition and pigment content in bell pepper ( Capsicum annuum L. cv. Bell Tower) fruit that were freshly harvested, chilled 14 days at 2° C. or chilled and then transferred to 20 °C for 3 days ("rewarmed") were determined. There was slight to moderate loss of membrane glycerolipids during chilling, with much greater losses after chilled fruit was rewarmed. Galactolipid (GL) loss exceeded that of phospholipid (PL). The ratio of monogalactosyl -to digalactosyl-diacylglycerol did not change in chilled or in rewarmed fruit, and there was no chlorophyll loss, but the amount of neutral carotenes declined during chilling and dropped further alter rewarming. Only minor changes in total membrane sterols (TMS = free sterols + steryl glycosides + acylated steryl glycosides) were noted in chilled and in rewarmed fruit (a small increase followed by a small decrease), but major changes occurred in sterol glycosylation and esterification. The ratio of stigmasterol to sitosterol increased during chilling and rose further after rewarming. Due to PL loss, the ratios of TMS and free sterols to PL increased in rewarmed fruit. The ratio of linolenate (18:3) to linoleate (18:2) rose during chilling and after rewarming in all fatty-acyl lipids (GL. PL. and acylated steryl glycosides), but the unsaturation index increased only in GL. These results indicate that most membrane damage occurs after rewarming of chilled fruit and that the chloroplasts are especially chilling sensitive.     

Experimental Studies on the Stability of the Cortical Microtubule Cytoskeleton in Relation to Polarity and Cell Elongation in the Coenocytic Green Alga, Chaetomorpha moniligera

The stability and ordered assembly of cytoskeletal microtubules(MTs) and the relationship between cell growth and MT cytoskeletonin the coenocytic green alga, Chaetomorpha moniligera Kjellmanwere examined. The cytoplasm of cylindrical growing cells ofChaetomorpha is covered with dense arrays of longitudinallyarranged cortical MTs which constitute the MT cytoskeleton.Seventy-five percent of MTs of the cytoskeleton disappearedwithin 4 h, with 25% remaining after 20 h following cold treatment.On terminating MT assembly with amiprophos-methyl (APM), thenumber of MTs decreased by 75% within 4 h. The remaining MTsdisappeared gradually within 24 h. The MT cytoskeleton of Chaetomorphawould thus appear to be composed of at least two kinds of MTsdiffering in stability. The MT cytoskeleton returned to normalafter treatment with APM for less than 48 h. However, this didnot occur after treatment with APM for more than 48 h, and theMT arrays became random. Cell elongation ceased completely within24 h after treatment with APM for less than 48 h but was restoredwithin 24 h after removing APM. The restoration of cell elongationwas no longer evident after removaI of APM for more than 48h. The results indicate that assembly of MTs into ordered arraysdepends on cell polarity and that in turn cell elongation isdependent on the polar-dependent arrays of MTs.Copyright 1994,1999 Academic Press Cell polarity, Chaetomorpha moniligera, coenocytic green alga, cold treatment, immunofluorescence, microtubule     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells

When metaphase PtK₁ cells are cooled to 6–8 ° C for 4–6 h the free, polar, and astral spindle microtubules (MTs) disassemble while the MTs of each kinetochore fiber cluster together and persist as bundles of cold-stable MTs. These cold-stable kinetochore fibers are similar to untreated kinetochore fibers in both their length (i.e., 5–6 m) and in the number of kinetochore-associated MTs (i.e., 20–45) of which they are comprised. Quantitative information concerning the lengths of MTs within these fibers was obtained by tracking individual MTs between serial transverse sections. Approximately 1/2 of the kinetochore MTs in each fiber were found to run uninterrupted into the polar region of the spindle. It can be inferred from this and other data that a substantial number of MTs run uninterrupted between the kinetochore and the polar region in untreated metaphase PtK₁ cells.     

Chilling Injury in Cucumber Leaves

When cucumber plants are chilled at 5 ?C and 85 per cent, r.h.the leaves wilt rapidly and lose water. Chlorophyll is lostin the light, but not in the dark. If chilled leaves are placedin water, electrolytes leak out rapidly, the amount dependingon the duration of chilling. There are marked reductions inthe rates of respiration and photosynthesis; Q₁₀ for respirationis 5.0 between 15 and 5 ?C. Phospholipid levels decline, butmore slowly than the rate of water loss. Leaves recover theirinitial fresh weight, rate of electrolyte leakage, and respirationand photosynthesis rates if the plants are returned to warmconditions within 1 to 2 d. When leaves are chilled at 100 per cent r.h. they do not losewater or phospholipids; electrolyte leakage is little more thanin controls, but respiration and photosynthesis are reducedas at 85 per cent r.h. It is envisaged that the leaf cell membranes suffer a physicalchange at 5 ?C which reduces the rates of respiration and photosynthesis,but allows the passage of water and electrolytes out of thecells; the water evaporates away but electrolytes become concentratedin the walls and can be leached out of the leaf. As water islost from the leaf symptoms of dehydration appear. Phospholipidloss may be associated with enzyme action following the lossof compart-mentation.     

The in vitro biosynthesis and secretion of glycerol by larval fat bodies of chilled Ostrinia nubilalis

Fat bodies from diapausing fifth-instar larvae of Ostrinia nubilalis were incubated in vitro at 5 or 23°C in Grace's medium and the glycerol contents of the organ and incubation medium determined. Fat bodies from diapausing larvae chilled 3 weeks at 5°C secreted glycerol into the medium at 5°C at a net rate of approx. 0.75 nmol/mg fat body dry wt/h for at least 96 h while the tissue levels remained essentially constant. Depending upon the experiment, from 6 to 15 times more glycerol was produced in 24 h at 5°C by these fat bodies than by those taken from diapausing unchilled larvae and incubated at either 5 or 23°C. A minimal chilling period of 10–12 days was recognized as necessary for chilled larval fat bodies to demonstrate rates of glycerol synthesis greater than those of unchilled larvae and the lag showed a temporal correlation with changes in haemolymph glycerol concentrations. These results suggest that this response to chilling by O. nubilalis is relatively slow. While incubation, at 23°C, of fat bodies from previously chilled larvae did not result in cessation of glycerol secretion, the rate of its appearance in the culture medium decreased during the 24-h incubation period. Although the ability of chilled fifth-instar larvae to accumulate glycerol is not dependent upon the diapause state results show that clearance of glycerol from the haemolymph by rewarmed O. nubilalis is related to diapause intensity.     

Nuclear-Cycle Dependence of the Development of the Preprophase Band in Tobacco BY-2 Cells

When tobacco BY-2 cells that had been treated with aphidicolinfor 24 h were cultured in the absence of aphidicolin, DNA synthesiswas initiated within 1 h. DNA synthesis was completed within4 h and then the preprophase band of microtubules (PPB) developed.However, when cells that had been treated with aphidicolin werecultured in the absence of aphidicolin for 1 h and then againin its presence, DNA synthesis, which was initiated during thehour in the absence of aphidicolin, was not completed withina further 10 h in the presence of aphidicolin. Moreover, thePPB did not develop even after the PPB had appeared and disappearedin cells that were cultured contemporaneously in the continuedabsence of aphidicolin. The development of the PPB seems to be causally associated withthe nuclear cycle of cell division in tobacco BY-2 cells. Thisprocess seems to require the completion of the replication ofall, or almost all, of the nuclear DNA. (Received July 25, 1992; Accepted November 24, 1992)     

Formation of a phragmosome-like structure in centrifuged protonemal cells of Adiantum capillus-veneris L.

A phragmosome (PS) is a transvacuolar aggregation of cytoplasm that develops in the plane of future cytokinesis and is found specifically in highly vacuolated cells. Although protonemal cells of Adiantum capillus-veneris L. usually do not form a PS, a PS-like structure developed at the site of a preprophase band (PPB) of microtubules (MTs) when the nucleus and endoplasm were displaced from the division site by centrifugation, leaving a PPB in the cortical cytoplasm. The PS-like structure contained endoplasmic MTs, F-actin, oil droplets and mitochondria. The structure did not develop when the cells were centrifuged before the formation of a PPB. Application of amiprophos-methyl (APM) before development of the PPB strongly inhibited the formation of the PS-like structure after centrifugation. The PS-like structure was dispersed after cytokinesis which occurred in the region of the displaced nucleus. Treatment with APM after the formation of the PS-like structure arrested the cell cycle at the M phase and inhibited the degradation of this structure. These results suggest that development of a PS-like structure is associated both with the formation of a PPB and with the stage of the cell cycle. Received: 9 July 1996 / Accepted: 12 September 1996