Microtubules in epidermal and cortical root cells of Brassica rapa during clinorotation

Using confocal microscopy the organization of tubulin cytoskeleton including endoplasmic and cortical microtubules (CMTs) has been studied in epidermal and cortical cells of the different growth zones of main root of Brassica rapa L. 6-days-old seedlings in control conditions and under clinorotation. It was shown that changes in CMTs orientation occured only in the distal elongation zone (DEZ). In the control, CMT arrays oriented transversely to the root long axis. Under clinorotation appearance of the shorter randomly organized CMTs was observed. Simultaneously, a significant decrease in the cell length in the central elongation zone (CEZ) under clinorotation was detected. It is suggested that the decline of anisotropic growth typical for CEZ cells is connected with CMTs disorientation under clinorotation.     

Microtubules spatial alterations in root cells of Brassica rapa under clinorotation

Organization of tubulin cytoskeleton in epidermis and cortex cells in different root growth zones in Brassica rapa L. 6-day-old seedlings under clinorotation has been investigated. It was shown that changes in cortical microtubules orientation occur only in the distal elongation zone. In control, cortical microtubule arrays oriented transversely to the root long axis. Whereas under clinorotation an appearance of shorter randomly organized cortical microtubules was observed. Simultaneously, a significant decrease in a cell length in the central elongation zone under clinorotation was revealed. It is suggested that the decline of anisotropic growth, typical for central elongation zone cells, is connected with cortical microtubules disorientation under clinorotation.     

Histological changes during maturation in male and female cones of the cycad Zamia furfuracea and their significance in relation to pollination biology

The cycad Zamia furfuracea L.Fil. Is pollinated by a curculionid beetle, Rhopalotria mollis Sharp which completes its life cycle in male cones of the cycad, and effectively pollinates female coneS. Idioblasts within parenchyma in both male and female cones appear to contain toxic compounds, including at least one neurotoxin, 2-amino-3-(methylamino) propanoic acid (BMAA), and a toxic glycoside, methylazoxymethanol-/!-primeveroside (macrozamin). Idioblasts appear structurally unmodified in male cones throughout the period of pollen maturation, and feeding weevils consume much of the starch-rich microsporophyll parenchyma tissue, including idioblastS. During this activity no appreciable change in morphology or staining reactions of male-cone idioblasts is detectable. Prior to pollen receptivity, female-cone idioblasts resemble those of male cones. Thereafter, many female-cone idioblasts show marked changes in morphology and content not caused by the weevils themselveS. Idioblast changes in female cones are probably associated with the defence of female-cone resources against predation by animals, including pollinating weevils, and may relate to mobilization of toxinS. Absence of similar morphological changes in male-cone idioblasts is correlated with toxin sequestration, enabling the pollinator to breed and feed without intoxication.     

Aberrant microtubule organization in dividing root cells of p60-katanin mutants

Aberrant microtubule organization has been recently recorded in dividing root cells of fra2 and lue1 p60-katanin Arabidopsis thaliana mutants. Here, we report similar defects in the bot1 and ktn1-2 mutants of the same plant, proposing that they constitute a consistent phenotype of p60-katanin mutants. In addition, we show that the Targeting Protein for Xklp2 (TPX2) protein co-localizes with microtubules on the surface of prophase nuclei of the mutants, probably participating in multipolar spindle assembly. As microtubule organization defects are not observed in metaphase/anaphase spindles and initiating phragmoplasts, we also discuss the putative association of the observed aberrations with the nuclear envelope and we emphasize on the mechanism of bipolar metaphase spindle organization in the mutants. It seems that chromosome-mediated spindle assembly, probably minimally dependent on microtubule severing by p60-katanin, dominates after nuclear envelope breakdown, restoring bipolarity.     

Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair cells.     

The relation between cell size,chromosome length and the orientation of chromosomes in dividing root cortex cells

Cells in the root meristem are organised in longitudinal files. Repeated transverse cell divisions in these files are the prime cause of root growth. Because of the orientation of the cell divisions, we expected to find mitoses with an spindle axis parallel to the file axis. However, we observed in the root cortex ofVicia faba large number of oblique chromosome orientations. From metaphase to telophase there was a dramatic increase of the rotation of the spindle axis. Measurements of both the size of the cortex cells and the chromosome configurations indicated that most cells were too small for an orientation of the spindle parallel to the file axis. Space limitation force the spindle into an oblique position. Despite this spindle axis rotation, most daughter cells remained within the original cell file. Only in extremely flat cells did the position of the daughter nuclei forced the cell to set a plane of division parallel to the file axis, which result in side-by-side orientation of the daughter cells. Telophase spindle axis rotations are also observed inCrepis capillaris andPetunia hybrida.. These species have respectively medium and small sized chromosomes compared toVicia. Since space limitation, which causes the rotation, depends both on cell and chromosome size, the frequency and extent of the phenomenon in former two species is comparatively low.     

Microtubules self-repair in living cells

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Sesquiterpenes from the conifer root rot pathogen Heterobasidion occidentale

Investigation of the production of secondary metabolites of Heterobasidion occidentale led to the isolation and identification of six sesquiterpenes (illudolone A and B, illudolactone A and B, deoxyfomannosin A and B) along with the well-known sesquiterpene fomannosin and the previously described benzohydrofuran fomannoxin. The structures and relative configurations of the compounds were determined by 1D and 2D NMR spectroscopic analysis as well as by HRMS. Their absolute configuration and biosynthesis were suggested and discussed in relation to fomannosin. Four compounds showed growth inhibiting activity against several basidiomycetes, Phlebiopsis gigantea, Phanerochaete chrysosporium and H. occidentale, and toxicity towards the moss Physcomitrella patens. In addition, one compound displayed activity against the bacterium Variovorax paradoxus as well as against the ascomycete Fusarium oxysporum.     

Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-m-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations APM Amiprophosmethyl - FITC Fluorescein isothiocyanate - LATB Latrunculin B     

Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana

The polarized growth of cells as diverse as fungal hyphae, pollen tubes, algal rhizoids and root hairs is characterized by a highly localized regulation of cell expansion confined to the growing tip. In apically growing plant cells, a tip-focused [Ca2+]c gradient and the cytoskeleton have been associated with growth. Although actin has been established to be essential for the maintenance of elongation, the role of microtubules remains unclear. To address whether the microtubule cytoskeleton is involved in root hair growth and orientation, we applied microtubule antagonists to root hairs of Arabidopsis. In this report, we show that depolymerizing or stabilizing the microtubule cytoskeleton of these apically growing root hairs led to a loss of directionality of growth and the formation of multiple, independent growth points in a single root hair. Each growing point contained a tip-focused gradient of [Ca2+]c. Experimental generation of a new [Ca2+]c gradient in root hairs pre-treated with microtubule antagonists, using the caged-calcium ionophore Br-A23187, was capable of inducing the formation of a new growth point at the site of elevated calcium influx. These data indicate a role for microtubules in regulating the directionality and stability of apical growth in root hairs. In addition, these results suggest that the action of the microtubules may be mediated through interactions with the cellular machinery that maintains the [Ca2+]c gradient at the tip.     

Gene technologies in Pinus radiata and Picea abies: tools for conifer biotechnology in the 21st century

Conifer biotechnology is reviewed, covering transformation, molecular analysis of transgenic material, functional analysis of genes and promoters in conifers and model systems, and the development of early screening technologies for new introduced traits. Technologies and concepts that promise to be of significant advantage to conifer biotechnology in the new century are discussed.     

Microtubules in the microspikes and cortical cytoplasm of isolated cells

In thin sections through microspikes extending from the surface of isolated cells, a core has been seen which may contain microtubular elements. The differences between these and microtubules seen elsewhere in the cytoplasm are attributed to their rapid growth and exposed location which make them especially vulnerable to injury by preparative treatment. In support of this view it is shown that cytoplasmic microtubules may be altered or even destroyed by exposing the cells to changes in osmotic pressure. Associated with these straight microtubules in the cytoplasm were also found solid microfilaments. The form of these components and their location and alignment in portions of cells which are under tension or in motion suggest that they function in the structural support of the cell and its microspikes and in the transmission of tension in the cytoplasm. A second type of microtubule, smaller in diameter and tortuous in form, was also seen in certain cells and is presumed, from its shape, to have little to do with cytoplasmic support.     

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion (Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek (Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the -oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.Abbreviations BDM 2,3-butanedione monoxime - DAPI 4,6-diamidino-2-phenylindole - ER endoplasmic reticulum - GFP green fluorescent protein     

Cataphylls of the Middle Triassic cycad Antarcticycas schopfii and new insights into cycad evolution

Cataphylls associated with the Middle Triassic stem genus Antarcticycas are described, and their impact on understanding cycad evolution is discussed. The cataphylls of Antarcticycas are triangular in outline and flattened adaxially with lateral flanges. The outer surfaces are covered with a ramentum of filamentous hairs, the epidermis is a single cell layer thick, and the ground tissue is parenchymatous with mucilage canals and sclereids. Vascular bundles form a distinct inverted omega-shaped pattern characteristic of the Cycadales observed in petioles of extant species. The structures in Antarcticycas are interpreted as cataphylls based on overall morphology, presence of straight vascular strands in the cortex of the associated stem, and lack of fascicular cambia in the vascular bundles. Because much of the overall diversity of Cycadales is represented by fossils, integrating fossil taxa into explicit phylogenetic hypotheses is important for understanding cycad evolution. Therefore, character and minimum age mapping were performed on a phylogeny of extant and fossil taxa including Antarcticycas. The results suggest that major extant lineages of Cycadales had diverged by the Permian to Triassic and that certain synapomorphies for Cycadales had evolved by the Permian. Evidence of insect feeding on Antarcticycas suggests that associations between cycads and insects are ancient.     

Electron microscopy of dividing cells

During intranuclear mitosis in plasmodia of Physarum polycephalum the primordium of spindle microtubules which is a somewhat electron opaque, amorphous structure with fibrous or granular elements, occurs in the center of early prophase, 20 to 30 minutes before metaphase. Then, the primordium seems to divide into two parts. Spindle microtubules develop radially from the primordia of spindle microtubules. These spindle microtubules increase in number and length during prophase. Spindle microtubules are completed in about five minutes before metaphase. The nuclear envelope remains intact during prophase, but after metaphase it breaks at the polar regions. The nuclear envelope of the daughter nucleus is re-formed from the original nuclear envelope.The authors wish to express their thanks Professor K. Ueda for valuable advice. They would also like to thank Professor N. Kamiya of Osaka University who kindly supplied the material (Physarum polycephalum) used in the present study.     

Microtubules and actin microfilaments in the amphibian bladder granular cells

Microtubules and microfilaments were localized by an immunocytochemical method in the granular cells of the frog bladder after fixation and isolation. An extensive array of microtubules was observed in the granular cells with an orientation towards the luminal plasma membrane in the supranuclear zone. Actin filaments formed a continuous bundle that underlined the cellular membrane. After incubation in the presence of colchicine, nocodazole, or tubulozole, the microtubular network appeared fragmented but did not disappear completely. These observations are related to the role of the cytoskeleton in the permeability response of the frog bladder epithelium to vasopressin.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Evolutionary history of the conifer root rot fungus Heterobasidion annosum sensu lato

We investigated two hypotheses for the origin of the root rot fungus Heterobasidion annosum species complex: (i) that geology has been an important factor for the speciation (ii) that co-evolutionary processes with the hosts drove the divergence of the pathogen species. The H. annosum species complex consists of five species: three occur in Europe, H. annosum s.s., Heterobasidion parviporum and Heterobasidion abietinum, and two in North America, Heterobasidion irregulare and Heterobasidion occidentale; all with different but partially overlapping host preferences. The evolution of the H. annosum species complex was studied using six partially sequenced genes, between 10 and 30 individuals of each species were analysed. Neighbour-joining trees were constructed for each gene, and a Bayesian tree was built for the combined data set. In addition, haplotype networks were constructed to illustrate the species relationships. For three of the genes, H. parviporum and H. abietinum share haplotypes supporting recent divergence and/or possible gene flow. We propose that the H. annosum species complex originated in Laurasia and that the H. annosum s.s./H. irregulare and H. parviporum/H. abietinum/H. occidentale ancestral species emerged between 45 and 60 Ma in the Palaearctic, well after the radiation of the host genera. Our data imply that H. irregulare and H. occidentale were colonizing North America via different routes. In conclusion, plate tectonics are likely to have been the main factor influencing Heterobasidion speciation and biogeography.     

Cell wall pectins and xyloglucans are internalized into dividing root cells and accumulate within cell plates during cytokinesis

Recently, we have reported that cell wall pectins are internalized into apical meristem root cells. In cells exposed to the fungal metabolite brefeldin A, all secretory pathways were inhibited, while endocytic pathways remained intact, resulting in accumulation of internalized cell wall pectins within brefeldin A-induced compartments. Here we report that, in addition to the already published cell wall epitopes, rhamnogalacturonan I and xyloglucans also undergo large-scale internalization into dividing root cells. Interestingly, multilamellar endosomes were identified as compartments internalizing arabinan cell wall pectins reactive to the 6D7 antibody, while large vacuole-like endosomes internalized homogalacturonans reactive to the 2F4 antibody. As all endosomes belong topographically to the exocellular space, cell wall pectins deposited in these "cell wall islands", enclosed by the plasma-membrane-derived membrane, are ideally suited to act as temporary stores for rapid formation of cell wall and generation of new plasma membrane. In accordance with this notion, we report that all cell wall pectins and xyloglucans that internalize into endosomes are highly enriched within cytokinetic cell plates and accumulate within brefeldin A compartments. On the other hand, only small amounts of the pectins reactive to the JIM7 antibody, which are produced in the Golgi apparatus, localize to cell plates and they do not accumulate within brefeldin A compartments. In conclusion, meristematic root cells have developed pathways for internalization and recycling of cell wall molecules which are relevant for plant-specific cytokinesis.