Transverse Microtubules Research Articles

Fate of nascent microtubules organized at the M/G1 interface, as visualized by synchronized tobacco BY-2 cells stably expressing GFP-tubulin: time-sequence observations of the reorganization of cortical microtubules in living plant cells.

Transgenic BY-2 cells stably expressing a GFP (green fluorescent protein)-tubulin fusion protein (BY-GT16) were subcultured in a modified Linsmaier and Skoog medium. The BY-GT16 cells could be synchronized by aphidicolin and the dynamics of their microtubules (MTs) were monitored by the confocal laser scanning microscopy (CLSM). We have succeeded in investigating the mode of reorganization of cortical MTs at the M/G1 interface. The cortical MTs were initially organized in the perinuclear regions and then they elongated to reach the cell cortex, forming the bright spots there. Subsequently, the first cortical MTs rapidly elongated from the spots and they were oriented parallel to the long axis towards the distal end of the cells. Around the time when the tips of the parallel MTs reached the distal end, the formation of transverse cortical MTs followed in the cortex near the division site, as we had previously suggested [Hasezawa and Nagata (1991) Bot. Acta 104: 206, Nagata et al. (1994) Planta 193: 567]. It was confirmed in independent observations that the appearance of the parallel MTs was followed by the appearance of the transverse MTs in each cell. We found that the transverse MTs spread through the whole cell cortex within about 20-30 min, while the parallel MTs disappeared. The significance of these observations on the mode of cortical MT organization is discussed.

Effects of phosphoprotein phosphatase inhibitors (phenylarsine oxide and cantharidin) on Tetrahymena.

The effects of phenylarsine oxide (PAO) (phosphotyrosine phosphatase inhibitor) and cantharidin (serine/threonine phosphatase [PP2A] inhibitor) treatments were analysed on the synthesis of phospholipids and glycolipids, and on the cytoskeletal elements (F-actin and tubulin containing structures) of Tetrahymena pyriformis. Both phosphatase inhibitors reduced the amount of incorporated 32P of the whole phospholipid content, but the ratio of phosphatidylserine (PS) and phosphatidylcholine (PC) to the total phospholipid content increased. Both treatments influenced the phosphatidylinositol (PI) system. These inhibitors also influenced the incorporation of palmitic acid into the phospholipids: in general PAO decreased, whereas cantharidin increased the amount of incorporated palmitic acid; 1 microM cantharidin significantly increased the labelling of PE and PA. The incorporation of mannose and glucosamine was influenced differently by PAO and cantharidin treatments: the latter elevated, while PAO decreased the labelling of glycolipids with these sugars. The effects of these treatments were visible also in the case of confocal scanning laser microscopic (CSLM) images: after treatments with both inhibitors, the F-actin containing cortical elements were destroyed, but the tubulin containing ones (longitudinal and transversal microtubules, oral apparatus and deep fibres) did not display significant alterations. The different effects of phosphatase inhibitors were visible also on the scanning electron microscopic (sEM) images: cantharidin treatments (1 microM) decreased the amount of dissolved membrane lipids after chemical dehydration of the cells with 2, 2-dimethoxy propane (DMP), but in the case of treatments with 10 microM, the surface pattern of cells was similar to the controls. On the other hand, after PAO treatments the surface pattern of Tetrahymena showed significant alterations. Both phosphatase inhibitors inhibited the phagocytotic activity of the cells. On the basis of present experiments we suppose that these treatments are able to influence signalling systems (e.g. PI) of Tetrahymena, and also the structure of the cytoskeleton and the functions (e.g. phagocytosis) which are connected with skeletal elements.

Growth dynamics and cytoskeleton organization during stem maturation and gravity-induced stem bending in Zea mays L.

Characterization of gravitropic bending in the maize stem pulvinus, a tissue that functions specifically in gravity responses, demonstrates that the pulvinus is an ideal system for studying gravitropism. Gravistimulation during the second of three developmental phases of the pulvinus induces a gradient of cell elongation across the non-growing cells of the pulvinus, with the most elongation occurring on the lower side. This cell elongation is spatially and temporally separated from normal internodal cell elongation. The three characterized growth phases in the pulvinus correspond closely to a specialized developmental sequence in which structural features typical of cells not fully matured are retained while cell maturation occurs in surrounding internodal and nodal tissue. For example, the lignification of supporting tissue and rearrangement of transverse microtubules to oblique that occur in the internode when cell elongation ceases are delayed for up to 10 d in the adjacent cells of the pulvinus, and only occurs as a pulvinus loses its capacity to respond to gravistimulation. Gravistimulation does not modify this developmental sequence. Neither wall lignification nor rearrangement of transverse microtubules occurs in the rapidly elongating lower side or non-responsive upper side of the pulvinus until the pulvinus loses the capacity to bend further. Gravistimulation does, however, lead to the formation of putative pit fields within the expanding cells of the pulvinus.

The cyclic reorientation of cortical microtubules in epidermal cells of azuki bean epicotyls: the role of actin filaments in the progression of the cycle.

The orientation of microtubules (MTs) was examined in epidermal cells of azuki bean (Vigna angular is Ohwi et Ohashi) epicotyls. The orientation of MTs adjacent to the outer tangential wall of the cells, which has a crossed polylamellate structure with lamellae of longitudinal cellulose microfibrils alternating with lamellae of transverse cellulose microfibrils, differed from one cell to another. Treatment with an auxin-free solution caused the accumulation of cells with longitudinal MTs and subsequent treatment with a solution that contained auxin resulted in the accumulation of cells with transverse MTs, showing that sequential treatments with auxin-free and auxin-containing solutions can synchronize the reorientation of MTs. The MTs, once reoriented from longitudinal to transverse, returned to longitudinal and then back to transverse once again, the duration of the cycle being about 6h. Gibberellic acid, known to increase the percentage of cells with transverse MTs, promoted reorientation of MTs from longitudinal to transverse and inhibited that from transverse to longitudinal. Cytochalasin D, an agent that disrupts actin filaments, speeded up the reorientation from transverse to longitudinal and slowed down that from longitudinal to transverse. It caused an increase in the percentage of cells with MTs in mixed orientation, and the percentage of such cells was highest when the percentage of cells with longitudinal MTs was decreasing and that of cells with transverse MTs was increasing.

Separation of Photolabile-Phytochrome and Photostable-Phytochrome Actions on Growth and Microtubule Orientation in Maize Coleoptiles (A Physiological Approach).

For separating the physiological actions of photolabile (phy-l) and photostable phytochromes, we compared the effects of red (R) and far-red (FR) light on elongation growth and microtubule reorientation in segments of maize (Zea mays L.) coleoptiles raised either in darkness (phy-l present) or preirradiated with R (phy-l eliminated). In 4.5-d-old dark-grown seedlings R first promoted growth and induced a transverse microtubule orientation. In continuous R the phytochrome action responsible for these responses was replaced by an opposite phytochrome action that produced a stable growth inhibition and longitudinal microtubule orientation. In R-preirradiated segments only the second type of phytochrome action could be observed. Reversion experiments with FR light pulses demonstrated that both types of phytochrome action were dependent on the FR-absorbing form of phytochrome and mirrored the actual phytochrome state after 1 h. We conclude from these and related results that growth promotion and transverse microtubule orientation are mediated by phy-l, whereas growth inhibition and longitudinal microtubule orientation are mediated by photostable phytochrome. The opposite actions of the two phytochromes can be separated by preirradiating the seedlings with R. Photoresponsiveness ascribed to phy-l disappeared after 5 d. phy-l appears to play a distinct but transitory role in coleoptile development.

The Ultrastructure of Zosterodasys agamalievi (Ciliophora: Synhymeniida)

ABSTRACT. The cell surface of the synhymeniid ciliate, Zosterodasys agamalievi, consists of shallow kinetal grooves separated by low cortical ridges. Numerous electron‐opaque bodies are located in the cortical ridges, inside the kinetal grooves, and are distributed in parallel rows between adjacent kineties. Well‐developed alveoli are present beneath the cell surface membrane. Zosterodasys agamalievi has a single micronucleus and a homomerous macronucleus. The infraciliature of the somatic monokinetid consists of an anteriorly‐directed kinetodesmal fiber, a well‐developed divergent postciliary microtubular ribbon, radially‐oriented transverse microtubules, and a short striated rootlet, which extends anteriorly from the base of the kinetosome into the cell. Zosterodasys agamalievi has a perioral band of paired cilia, the synhymenium, that winds obliquely across the ventral surface of the body, just posterior to the cytostome. The infraciliature of the anterior kinetosome of the synhymenium consists of two postciliary microtubules; a well‐developed, divergent post‐ciliary ribbon of microtubules and a short kinetodesmal fiber are associated with the posterior kinetosome. The cytopharynx is supported by 14‐16 nematodesmata which are capped distally by a capitulum. The cytopharynx is bound proximally by a fibrous sheath and is lined by radially‐arranged microtubular ribbons. No obvious oral ciliature is present.

Interaction of auxin, light, and mechanical stress in orienting microtubules in relation to tropic curvature in the epidermis of maize coleoptiles

Changes in the orientation of cortical microtubules (longitudinal vs. transverse with respect to the long cell axis) at the outer epidermal wall of maize coleoptile segments were induced by auxin, red or blue light, and mechanical stresses (cell extension or compression produced by bending). Immunofluorescent techniques were used for the quantitative determination of frequency distributions of microtubule orientation. Detailed kinetic studies showed that microtubule reorientations are temporally correlated with the simultaneously measured changes in growth rate elicited by auxin, red light, or blue light. Growth inhibition induced by depletion of endogenous auxin produces a longitudinal microtubule pattern that can be changed into a transverse pattern in a dose-dependent manner by applying exogenous auxin. A mid-point pattern with equal frequencies of longitudinal and transverse microtubules was adjusted at 2 μmol/1 auxin. Bending stress applied under these conditions adjusts permanent, maximally longitudinal and transverse microtubule orientations at the compressed and extended segment sides, respectively, quantitatively mimicking the responses to differential flank growth during phototropic and gravitropic curvature. During tropic curvature the changes in microtubule pattern reflect the distribution of growth rather than the distribution of auxin. The microtubule pattern responds to auxin-dependent growth changes and mechanical stress in a synergistic manner, confirming the functional equivalence of these factors in affecting microtubule orientation. Similar results were obtained when segment growth was altered by blue or red light instead of auxin in the presence or absence of mechanical stress. It is concluded from these results that growth changes, elicited by auxin, light, etc., and mechanical stress affect microtubule orientation through a common signal perception and transduction chain.

The surface pattern of Paramecium tetraurelia in interphase: an electron microscopic study of basal body variability, connections with associated ribbons and their epiplasmic environment

Summary In Paramecium tetraurelia, thousands of basal bodies with their three associated rootlets are anchored into the submembranous skeleton, the epiplasm, which is itself segmented into cortical units. In this study, we redescribe more precisely (after Pitelka [31], Hufnagel [19] and Allen [1]) the architecture of these structures after detergent treatment and tannic acid contrast reinforcement. Although this technique leads to loss of membranes and of some labile cytoskeletal elements, it allows to better underline how the three rootlets are linked together and to the basal body. The postciliary microtubules and ciliary rootlets (Kd fibers) are bound together by a “fingered node”, and the transverse microtubules to the previous fibers by dense links. The anterior basal body of the pairs is linked to the ciliary rootlet by a newly identified architecture in this species, the “bone-like node” [1]. A specific ciliary domain can be defined in the epiplasm of each cortical unit: epiplasmic rings encircling the basal bodies are closely apposed on the thicker part of the epiplasm and can include some apertures along the basal bodies towards the cilia. The thickness of the epiplasm seems to vary according to the presumptive elongation direction of the cortical units. Each basal body associated rootlet is specifically linked to the epiplasm itself. Because of their unexpected set of linkage organization, these rootlets may ensure the ciliary cohesiveness in the interphase cell as well as the transmission of the ciliary polarity during division as it was previously discussed [19]. We also show for the first time that there is a variability of the basal body architecture at the cellular level: its height, and the number of cartwheel elements change depending on its location on the cortex and its morphogenetical history; the longest basal bodies with 5 cartwheel elements are found in territories which result from hyperduplication and become invariant, i.e. do not undergo duplication during the next divisions, as is the case along the sutures and in the left oral and cortical areas [22]. These results are discussed in terms of morphogenetical behavior.

The cyclic reorientation of cortical microtubules on walls with a crossed polylamellate structure: effects of plant hormones and an inhibitor of protein kinases on the progression of the cycle

The outer tangential wall (OTW) of epidermal cells of azuki bean epicotyls has a crossed polylamellate structure, in which lamellae of longitudinal cellulose microfibrils alternate with lamellae of transverse cellulose microfibrils. This implies that the cyclic reorientation of cortical microtubules (MTs) from longitudinal to transverse and from transverse to longitudinal occurs on the OTW. Treatment with a solution that contained no auxin caused the accumulation of cells with longitudinal MTs, suggesting that auxin is required for the reorientation of MTs from longitudinal to transverse during the reorientation cycle. Treatment with 6-dimethylaminopurine (DMAP), an inhibitor of protein kinases that promoted the reorientation of MTs from transverse to longitudinal, resulted in the accumulation of cells with longitudinal MTs. Subsequent treatment with auxin caused a marked increase in the percentage of cells with transverse MTs and then a decrease in the percentage, indicating that the reorientation of MTs from longitudinal to transverse and then from transverse to longitudinal occurred during treatment with auxin. The percentage of cells with transverse MTs decreased more slowly in segments that had been pretreated with gibberellin A3 (GA) than in segments that had been pretreated without GA, suggesting that GA, in cooperation with auxin, caused the suppression of the reorientation of MTs from transverse to longitudinal.

Comparative Ultrastructure of the Cytoplasm in Species of the Genus Tracheloraphis (Ciliophora, Karyorelictida). III. Buccal Apparatus

Summary The structure of the apical mouth has been studied in Tracheloraphis dogieli, Tr. phoenicopterus, Tr. totevi and Tr. caudatus . There is no permanent cytostome in any of them. The apical surface is covered by only the plasma membrane with many extrusomes of the type of subpellicular bodies (pigmentocysts) beneath it. In Tr. dogieli , the apical area is flat and surrounded with a circular “lip”. In Tr. phoenicopterus , the apical surface is dome-shaped, and the cytoplasm of the body “head” is filled with refringent oval electron-lucent granules up to 2 μm in size, clearly different from the extrusomes. The peribuccal ciliature of Tracheloraphis includes a specialized circumoral kinety independent of the meridional somatic kineties. In most species, this kinety consists of paired kinetosomes, one ciliated and one barren. However, in Tr. dogieli the circumoral ciliature consists of a series of short radial rows of kinetosomes, at least four kinetosomes in each row (2 ciliated and 2 barren). The innermost kinetosome, which is barren, sends a ribbon of transverse microtubules towards the apical area. This kinetosomal array is immediately outside the circular “lip”. The cilia of these kinetosomes are directed forward and form the apical ciliary “crown”. Nemadesmata start from the kinetosomes of the “crown”, both ciliated and barren, and are directed obliquely backwards. The somatic kineties, formed by dikinetids, begin well behind this “crown” and are not connected with it. They bear no nemadesmata.

Formation of novel endoplasmic and cortical microtubular arrays inAdiantum protonemal cells induced by blue light irradiation and acropetal centrifugation

Protonemal cells ofAdiantum capillus-veneris were grown under red light conditions over 6 days and exposed to blue light for 8 hr (and then dim green light for 1 hr for technical reasons), before they were centrifuged acropetally over at least 1 hr at 2,000×g. After this treatment, an arrangement of endoplasmic microtubules (MTs) that resembled the shape of a tadpole could be detected some distance below the nucleus in about 40% of the cells. The percentage of protonemata bearing this Mtstructure was dependent on centrifugation time as well as the time of blue light irradiation. The size of the structure was constant at any time of its existence. Additionally, a wide belt of transversally oriented cortical MTs in the upper part of the protonemata was detected in many cells after blue light irradiation and acropetal centrifugation. Its formation rate seemed to be also dependent on the period of blue light irradiation and centrifugation time. None of the endoplasmic and few of the cortical transverse MT patterns could be seen without blue light irradiation. A strict coincidence in the formation of both MT patterns was not seen. Further, a few tadpole-shaped MT arrays remained during mitosis, whereas the cortical transverse MT pattern was found in stages other than metaphase and anaphase.

Somatic Cortical and Endoplasmic Fine Structure of Remanella granulosa Kahl (Ciliophora, Karyorelictida)

Summary Remanella granulosa Kahl (Family Loxodidae) is a flattened lancet-shaped ciliate with 10 kineties on its right (physiologically ventral) body side, and two kineties along the margins of the otherwise barren left body side. In the right side kineties, the kinetosomes are paired, both being ciliated. In each pair, the rear kinetosome gives rise to a postciliary ribbon of microtubules which are stacked to form prominent lamellated postciliodesmata. The rear kinetosome has also a medium-long striated kinetodesmal filament which contacts the postciliary ribbon of the preceding pair. The antherior kinetosome has a short ribbon of transverse microtubules. There are numerous mitochondria in the interkinetal ridges, but no obvious myonemes. In the two left-side kineties, the kinetosomes are also paired, the anterior being ciliated, the posterior barren. Some of the latter are connected with the inner bodies of the Muller's vesicles, likely to be gravity receptors. The endoplasm contains many skeletal spicules, especially near the right body side, and many food vacuoles, frequently with diatoms inside. There are also envacuolated bacteria likely to be symbiotic. Some endoplasmic mitochondria are conspicuously surrounded by membrane vesicles and lack definite cristae. Curious tiny bottle-shaped organelles, likely to be extrusomes, occur in groups between some kineties on the ciliated body side. On both sides of the body, the plasma membrane is underlain by microtubules and provided with envacuolated subpellicular pigment granules.

Reorientation of microtubules at the outer epidermal wall of maize coleoptiles by phytochrome, blue-light photoreceptor, and auxin

The effects of red and blue light on the orientation of cortical microtubules (MTs) underneath the outer epidermal wall of maize (Zea mays L.) coleoptiles were investigated with immunofluorescent techniques. The epidermal cells of dark-grown coleoptiles demonstrated an irregular pattern of regions of parallel MTs with a random distribution of orientations. This pattern could be changed into a uniformly transverse MT alignment with respect to the long cell axis by 1 h of irradiation with red light. This response was transient as the MTs spontaneously shifted into a longitudinal orientation after 1–2 h of continued irradiation. Induction/reversion experiments with short red and far-red light pulses demonstrated the involvement of phytochrome in this response. In contrast to red light, irradiation with blue light induced a stable longitudinal MT alignment which was established within 10 min. The blue-light response could not be affected by subsequent irradiations with red or far-red light indicating the involvement of a separate blue-light photoreceptor which antagonizes the effect of phytochrome. In mixed light treatments with red and blue light, the blue-light photoreceptor always dominated over phytochrome which exhibited an apparently less stable influence on MT orientation. Long-term irradiations with red or blue light up to 6 h did not reveal any rhythmic changes of MT orientation that could be related to the rhythmicity of helicoidal cell-wall structure. Subapical segments isolated from dark-grown coleoptiles maintained a longitudinal MT arrangement even in red light indicating that the responsiveness to phytochrome was lost upon isolation. Conversely auxin induced a transverse MT arrangement in isolated segments even in blue light, indicating that the responsiveness to blue-light photoreceptor was eliminated by the hormone. These complex interactions are discussed in the context of current hypotheses on the functional significance of MT reorientations for cell development.

Ultrastructure of Prorodon (Ciliophora, Prostomatida) Ii. Oral Cortex and Phylogenetic Conclusions

ABSTRACT. The ultrastructure of the oral region and the ultrastructural architecture of the basket of Prorodon aklitolophon and Prorodon teres are described. the oral region of Prorodon consists of: 1) A circumoral kinety at the anterior pole of the cell surrounding the typically slit‐shaped cytostomial funnel. This kinety is composed of inversely oriented dikinetids in which both kinetosomes are ciliated and are associated with a very short postciliary microtubular ribbon and a few transverse microtubules; 2) Three brush rows aligned in parallel and extended meridionally in the anterior part of the cell. the individual brush rows consist of dikinetids, but in contrast to the dikinetids around the cytopharynx they are not inverted and only the anterior kinetosomes bear specialized short brush cilia and are associated with a divergent‐tangential transverse microtubular ribbon. the posterior kinetosome is non‐ciliated and bears a prominent convergent postciliary microtubular ribbon. Schematized dikinetid patterns of both oral regions of Prorodon are provided. In addition, a three‐dimensional reconstruction of the basket of the genus Prorodon based on serial thin sections is presented. A phylogenetic tree, mainly based on stomatogenic data, is given to show the phylogenetic relationships of some prostomatid genera as well as the hypothesized sistergroup relationship of colpodid and prostomatid ciliates.

Structural and ultrastructural observations of the ciliate Phacodinium metchnicoffi certes, 1891 (Heterotrichea, Phacodiniida)

This work points out several specific ultrastructural characteristics of the ciliate Phacodinium metchnicoffi. The somatic infraciliature consists of several segments of aciliferous or partially ciliferous monokinetids or alignments of kinetosomes with a particular structuration. All alignments of kinetosomes in double rows are associated with striated kinetodesmal fibers and to a ribbon of postciliary microtubules. The transverse fibers are observed only from monokinetids showing also kinetosomes associated with a 1-2 ribbon of transverse microtubules. However, only the anterior kinetosome of the monokinetid presents a ribbon of transverse fibers and an isolated microtubule next to triplet 5. The somatic kinetosomes located in the zones surrounding the buccal area are clearly associated with a noded microfibrillar network and with nemadesmata on the remaining part of the cortex. The kinetosomes of adorai organelles show 1-2 transverse microtubules. Each organelle is provided with 4 rows of kinetosomes and on each organelle, the kinetosomes of the anterior row bear postciliary microtubules directed towards the front part of the membranelle. The kinetosomes of the left row are associated with 1 or 2 transverse microtubules (T2) as are the kinetosomes of the other rows. The paroral organelle consists of an internal row of kinetosomes accompanied by a succession of parallel small rows each consisting most often of 3 kinetosomes. Only the kinetosomes of the internal row bear postciliary microtubules and prepostciliary microtubules and also show a noded microfibrillar network connecting their proximal zones. Lastly, the taxonomic relationships between Transitella and Phacodinium are discussed.

Cortical Microtubule Organization and Internodal Cell Maturation in Chara corallina

SummaryThe arrangement of cortical microtubules, as documented by immunofluorescence and immunogold‐silver enhancement, changes during the growth and maturation of giant internodal cells of Chara corallina, a process taking approximately 45 days. Transverse microtubules are found throughout growth along with a subset of distinctly non‐transverse microtubules. During the second half of the growing period, when relative growth rates are diminishing, these non‐transverse microtubules become more abundant but a few days prior to growth cessation, they are mostly absent. At about the time of growth cessation the microtubules, while retaining their locally parallel alignment, begin to show increasing deviation from the transverse axis. Eventually, a mosaic of locally parallel yet variably oriented fields of microtubules forms. Many days after growth stops, microtubules become shorter and less numerous and lose parallel alignment, leading to the formation of a random MT pattern.

Observations sur la structure et l’utrastructure du cilié Myxophthirus anomalocardiae gen.nov., sp.nov. (Scuticociliatida, Thigmophryidae), parasite du bivalve Anomalocardia brasiliana Gmelin, 1791

We describe a new genus of Thigmophryidae which is characterized by the cytoskeleton of the thigmotactic area, the median position of the buccal cavity with paroral and adorai organelles. On the locomotive cortex, the kinetosomes are in pairs and ciliated with a median parasomal sac. With the posterior kinetosome are associated: a) a kinetodesmal fibre, b) a ribbon of postciliary fibres, c) a ribbon of transverse microtubules, d) a transverse tractus, e) a microfibrillar tractus. Two or three microtubules run along each kinety. On the thigmotactic cortex, each posterior kinetosome is associated with a kinetodesmal fibre, a postciliary ribbon, a ribbon of transverse fibres and a transverse tractus. Moreover, a skeletal fibre with periodic structure, at the proximal end of the kinetosomes, runs along the left of each kinety. The endoplasm is full of host cells or organisms of the palleal cavity. The ultrastructure of the ciliate is typical of Scuticociliates, just as the buccal organization, with presence of a scuticus. The paroral membrane is a row of dyads with a development of a microfibrillar network as at the level of the single adoral organelle the kinetosomes of which have postciliary fibres.

Effects of Gibberllin and Abscisic Acid on the Cortical Microtubule Orientation in Hypocotyl Cells of Light-Grown Cucumber Seedlings

Effects of gibberellin (GA₄) and abscisic acid (ABA) on the orientation of cortical microtubules (MTs) in the epidermal and cortex cells of light-grown cucumber (Cucumis sativus L. cv Aonagajibai) hypocotyls were examined by immunofluorescence microscopy. MTs were observed in the 0-1 mm region (region A) and 3-4 mm region (region D) below the cotyledonary node. GA₄ promoted and ABA inhibited hypocotyl elongation. In the control, the predominant pattern of MT arrangement in the tangential surface of epidermal cells (Ep-t) was oblique in region A and oblique/longitudinal in region D, while in the radial surface of epidermal cells (Ep-r), the MT arrangement was mostly transverse in both regions. The MT arrangement in the radial surface of cortex cells (C-r) was all transverse in both regions. GA₄ increased transversely oriented MTs and decreased obliquely and longitudinally oriented MTs, whereas ABA increased obliquely and longitudinally oriented MTs and decreased transversely oriented MTs in Ep-t and Ep-r in both regions. ABA counteracted GA₄-induced effects on MT orientation as well as elongation. The frequency of the occurrence of transverse MTs in Ep-t but not in Ep-r and C-r was found to be highly correlated to the growth rate of the hypocotyl, suggesting that the epidermis is the essential tissue that limits the growth of the hypocotyl.

Mode of action of natural growth inhibitors in radish hypocotyl elongation – influence of raphanusanin on auxin‐mediated microtubule orientation

Raphanusanin is a plant growth‐inhibiting substance which plays an important role in light growth inhibition and phototropism of radish hypocotyls. We investigated the effect of raphanusanin on indole‐3‐acetic acid (IAA)‐mediated orientation of microtubules (MT) in the outer epidermal cells of radish hypocotyl segments using immunofluorescence microscopy. IAA‐mediated MT reorientation preceded cell elongation induced by IAA. A change of IAA‐mediated MT orientation from longitudinal to transverse started within less than 15 min after IAA treatment, while significant growth promotion induced by IAA was found within about 30 min. The IAA‐mediated transverse MT orientations were significantly inhibited by simultaneously added raphanusanin. We also investigated the effect of raphanusanin on the MT orientation of the segments pretreated with IAA. The change of MT orientation induced by raphanusanin preceded growth inhibition of the segments. Within about 60 min after its application, raphanusanin initiated inhibition of the steady‐state elongation pre‐induced by IAA, while IAA‐mediated transverse MT orientations started to change into longitudinal orientations within less than 30 min after application of raphanusanin. Based on these results, it is suggested that raphanusanin induces growth inhibition through interference with the auxin‐mediated MT orientations.

A Reexamination of the Ultrastructure of Didinium nasutum and a Reanalysis of the Phylogeny of the Haptorid Ciliates

ABSTRACT. The cilia of Didinium nasutum are restricted to two girdles encircling the cell. Each row of cilia in both girdles is made up of two to three anterior pairs of kinetosomes followed by several single kinetosomes. Each single kinetosome has two sets of transverse microtubules, an overlapping postciliary microtubular ribbon, and a laterally directed kinetodesmal fiber. The pairs of kinetosomes are homologous to the oral dikinetids of other haptorians: the nonciliated kinetosome of the pair has a transverse microtubular ribbon that extends to line the membrane of the proboscis, a single short postciliary microtubule, and a nematodesma; the ciliated kinetosome has a ribbon of postciliary microtubules and two sets of transverse microtubules. The presence of these characters in Didinium invalidates Leipe & Hausmann's conclusion that the Didiniidae should be removed from the subclass that contains the other haptorians (Leipe, D. D. & Hausumann, K. 1989. Somatic infraciliature of the haptorid ciliate Homalozoon vermiculare (Kinetofragminophora, Gymnostomata) Ditransversalia n. subcl. and phylogenetic implications. J. Protozool., 36:280–289). In light of this, the justification for a subclass Ditransversalia is challenged and shown to be unnecessary.