Hydrodynamic flow in the cytoplasm of plant cells.

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007) shows that active transport of organelles gives rise to a drag in the cytosol, setting up a hydrodynamic flow, which contributes to a fast distribution of proteins and nutrients in plant cells. Here, we show experimentally that actively transported organelles produce hydrodynamic flow that significantly contributes to the movement of the molecules in the cytosol. We have used fluorescence recovery after photobleaching and show that in tobacco Bright Yellow 2 (BY-2) suspension cells constitutively expressing cytoplasmic green fluorescent protein (GFP), free GFP molecules move faster in cells with active transport of organelles than in cells where this transport has been inhibited with the general myosin inhibitor BDM (2,3-butanedione monoxime). Furthermore, we show that the direction of the GFP movement in the cells with active transport is the same as that of the organelle movement and that the speed of the GFP in the cytosol is proportional to the speed of the organelle movement. In large BY-2 cells with fast cytoplasmic streaming, a GFP molecule reaches the other side of the cell approximately in the similar time frame (about 16 s) as in small BY-2 cells that have slow cytoplasmic streaming. With this, we suggest that hydrodynamic flow is important for efficient transport of cytosolic molecules in large cells. Hydrodynamic flow might also contribute to the movement of larger structures than molecules in the cytoplasm. We show that synthetic lipid (DOPG) vesicles and 'stealth' vesicles with PEG phospholipids moved in the cytoplasm.

Intrusive growth of flax phloem fibers is of intercalary type.

Flax (Linum usitatissimum L.) phloem fibers elongate considerably during their development and intrude between existing cells. We questioned whether fiber elongation is caused by cell tip growth or intercalary growth. Cells with tip growth are characterized by having two specific zones of cytoplasm in the cell tip, one with vesicles and no large organelles at the very tip and one with various organelles amongst others longitudinally arranged cortical microtubules in the subapex. Such zones were not observed in elongating flax fibers. Instead, organelles moved into the very tip region, and cortical microtubules showed transversal and helical configurations as known for cells growing in intercalary way. In addition, pulse-chase experiments with Calcofluor White resulted in a spotted fluorescence in the cell wall all over the length of the fiber. Therefore, it is concluded that fiber elongation is not achieved by tip growth but by intercalary growth. The intrusively growing fiber is a coenocytic cell that has no plasmodesmata, making the fibers a symplastically isolated domain within the stem.

Primmorphs from seven marine sponges: formation and structure.

Primmorphs were obtained from seven different marine sponges: Stylissa massa, Suberites domuncula, Pseudosuberites aff. andrewsi, Geodia cydonium, Axinella polypoides, Halichondria panicea and Haliclona oculata. The formation process and the ultra structure of primmorphs were studied. A positive correlation was found between the initial sponge-cell concentration and the size of the primmorphs. By scanning electron microscopy (SEM) it was observed that the primmorphs are very densely packed sphere-shaped aggregates with a continuous pinacoderm (skin cell layer) covered by a smooth, cuticle-like structure. In the presence of amphotericin, or a cocktail of antibiotics (kanamycin, gentamycin, tylosin and tetracyclin), no primmorphs were formed, while gentamycin or a mixture of penicillin and streptomycin did not influence the formation of primmorphs. The addition of penicillin and streptomycin was, in most cases, sufficient to prevent bacterial contamination, while fungal growth was unaffected.

Temporal and tissue-specific expression of the tobacco ntf4 MAP kinase.

The large number of mitogen-activated protein (MAP) kinase genes identified to date in plants suggests that their encoded proteins have a wide array of functions in development and physiological responses, as has been indicated by studies on the factors which lead to the activation of these kinases. Signalling pathways involving members of a multigene family employ a variety of mechanisms to ensure response specificity, one of which is via differential gene expression. We have performed detailed analyses of the expression of the tobacco ntf4 MAP kinase gene using a variety of approaches. The ntf4 gene promoter region was isolated and a chimeric ntf4 promoter-GUS fusion construct was introduced into plants. GUS expression was detected in pollen, in developing and mature embryos, and shortly after seed germination, but not in other floral tissues and tissues such as leaf, root, or stem. This expression pattern was confirmed by northern and western analyses. In situ hybridization and immunolocalization studies showed that the expression of the ntf4 gene and its encoded protein p45Ntf4 occurred in embryos at least from the globular embryo stage until the mature seed, as well as in the seed endosperm. Taken together, the results show that the p45Ntf4 MAP kinase has a very restricted expression pattern, being found only in pollen and seeds. These findings should be important when considering MAP kinase function in plants.

Arabidopsis thaliana SHAGGY-related protein kinases (AtSK11 and 12) function in perianth and gynoecium development.

In higher plants, the correct patterning of the floral meristem in terms of organ type, number and form is the result of a concerted expression of a network of genes. We describe phenotypes of flower patterning, resulting from a reduction of transcript levels of the Arabidopsis SHAGGY-related protein kinase genes AtSK11(ASKalpha) and AtSK12(ASKgamma). The AtSK genes are plant homologues of the Drosophila shaggy (SGG) gene and the mammalian Glycogen-Synthase Kinase-3 (GSK-3). The SGG protein kinase is a key component of the wingless signalling pathway and is required for the establishment of tissue patterning and cell fate determination. The expression patterns of the AtSK11(ASKalpha) and AtSK12(ASKgamma) genes during wild-type Arabidopsis inflorescence development, detected by in situ hybridisation, have been shown to be consistent with a possible role in floral meristem patterning. AtSK11(ASKalpha) and AtSK12(ASKgamma) transcripts were detected at the periphery of the inflorescence meristem and in the floral meristem. At later stages the expression of the AtSK genes became localised in specific regions of developing flower organ primordia. Furthermore, we have obtained and analysed transgenic plants containing AtSK11(ASKalpha) and AtSK12(ASKgamma) gene specific antisense constructs. These plants developed flowers showing a higher number of perianth organs and an alteration of the apical-basal patterning of the gynoecium.

Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not.

We studied cell cycle events in embryos of tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds during imbibition in water and during osmoconditioning ("priming") using both quantitative and cytological analysis of DNA synthesis and beta-tubulin accumulation. Most embryonic nuclei of dry, untreated control seeds were arrested in the G(1) phase of the cell cycle. This indicated the absence of DNA synthesis (the S-phase), as confirmed by the absence of bromodeoxyuridine incorporation. In addition, beta-tubulin was not detected on western blots and microtubules were not present. During imbibition in water, DNA synthesis was activated in the radicle tip and then spread toward the cotyledons, resulting in an increase in the number of nuclei in G(2). Concomitantly, beta-tubulin accumulated and was assembled into microtubular cytoskeleton networks. Both of these cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The activation of DNA synthesis and the formation of microtubular cytoskeleton networks were also observed throughout the embryo when seeds were osmoconditioned. However, this pre-activation of the cell cycle appeared to become arrested in the G(2) phase since no mitosis was observed. The pre-activation of cell cycle events in osmoconditioned seeds appeared to be correlated with enhanced germination performance during re-imbibition in water.

Dynamics of artificially immobilized Nitrosomonas europaea: Effect of biomass decay.

The dynamics of growth and death of immobilized Nitrosomonas europaea were studied. For this, the death rate of suspended cells was determined in the absence of ammonium or oxygen by following the loss of respiration activity and by fluorescein-diacetate (FDA)/lissamine-green staining techniques. The death rates obtained (1.06 x 10(-6) s(-1) or 4.97 x 10(-6) s(-1) in the absence of oxygen or ammonium, respectively) were incorporated in a dynamic growth model and the effects on the performance of the immobilized-cell process illustrated by model simulations.These model simulations and experimental validation show that if decay of biomass occurs the biomass concentration in the center of the bead decreases. As a result, the systems react slower to changes in substrate concentrations than if all cells remain viable.To show that cells in the center of the bead died, the FDA and lissamine-green staining techniques were adapted for immobilized cells. It was shown that biomass decay occurred, especially in the center of the bead; the amount of cells decreased there, and the remaining cells were all stained with lissamine green indicating cell death. After the substrate availability was decreased, also cells near the surface of the bead lost their viability. The number of viable cells increased again after increasing the substrate concentration as the result of cell multiplication. At low substrate concentrations and low hydraulic retention times, as for example in the treatment of domestic wastewater, the death rate of cells is thus an important parameter for the performance of the immobilized-cell system. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 630-641, 1997.

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.

Bacteriological composition and structure of granular sludge adapted to different substrates.

The bacteriological composition and ultrastructure of mesophilic granular methanogenic sludge from a large-scale Upflow Anaerobic Sludge Blanket reactor treating wastewater from a sugar plant and of sludge granules adapted to ethanol and propionate were studied by counting different bacterial groups and by immunocytochemical methods. Propionate-grown granular sludge consisted of two types of clusters, those of a rod-shaped bacterium immunologically related to Methanothrix soehngenii and those consisting of two different types of bacteria with a specific spatial orientation. One of these bacteria reacted with antiserum against Methanobrevibacter arboriphilus AZ, whereas the other is most likely a propionate-oxidizing bacterium immunologically unrelated to Syntrophobacter wolinii. Sludge granules obtained from the large-scale Upflow Anaerobic Sludge Blanket reactor and granules cultivated on ethanol did not show the typical spatial orientation of bacteria. Examination of the bacterial composition of the three types of granules by light and electron microscopy, the most-probable-number method, and by isolations showed that M. arboriphilus and M. soehngenii were the most abundant hydrogenotrophic and acetoclastic methanogens in propionate-grown sludge. Methanospirillum hungatei and Methanosarcina barkeri predominated in ethanol-grown granules, whereas many morphotypes of methanogens were abundant in granules from the full-scale reactor.

The relationship between nodulin gene expression and the Rhizobium nod genes in Vicia sativa root nodule development.

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.

Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine-phalloidin.

The three-dimensional organization of the microfilamental cytoskeleton of developingGasteria pollen was investigated by light microscopy using whole cells and fluorescently labelled phalloidin. Cells were not fixed chemically but their walls were permeabilized with dimethylsulphoxide and Nonidet P-40 at premicrospore stages or with dimethylsulphoxide, Nonidet P-40 and 4-methylmorpholinoxide-monohydrate at free-microspore and pollen stages to dissolve the intine.Four strikingly different microfilamentous configurations were distinguished. (i) Actin filaments were observed in the central cytoplasm throughout the successive stages of pollen development. The network was commonly composed of thin bundles ramifying throughout the cytoplasm at interphase stages but as thick bundles encaging the nucleus prior to the first and second meiotic division. (ii) In released microspores and pollen, F-actin filaments formed remarkably parallel arrays in the peripheral cytoplasm. (iii) In the first and second meiotic spindles there was an apparent localization of massive arrays of phalloidin-reactive material. Fluorescently labelled F-actin was present in kinetochore fibers and pole-to-pole fibers during metaphase and anaphase. (iv) At telophase, microfilaments radiated from the nuclear envelopes and after karyokinesis in the second meiotic division, F-actin was observed in phragmoplasts.We did not observe rhodamine-phalloidin-labelled filaments in the cytoplasm after cytochalasin-B treatment whereas F-actin persisted in the spindle. Incubation at 4° C did not influence the existence of cytoplasmic microfilaments whereas spindle filaments disappeared. This points to a close interdependence of spindle microfilaments and spindle tubules.Based on present data and earlier observations on the configuration of microtubules during pollen development in the same species (Van Lammeren et al., 1985, Planta165, 1-11) there appear to be apparent codistributions of F-actin and microtubules during various stages of male meiosis inGasteria verrucosa.

Structure and function of the microtubular cytoskeleton during pollen development in Gasteria verrucosa (Mill.) H. Duval.

In a study of pollen development in Gasteria verrucosa, the changes in the spatial organization of microtubules were related to the processes of cell division, nuclear movement and cytomorphogenesis. Sections of polyethylene-glycol-embedded anthers of G. verrucosa were processed immunocytochemically to record the structure and succession of fluorescently labeled microtubular configurations. Using microspectrophotometric measurements the relative quantity of tubulin in microtubules per unit of cytoplasm was determined. Cell dimensions and nuclear positions were measured to relate changes in cell shape and nuclear movements to microtubular configurations. Microtubules were detected in the different cells during microsporogenesis and microgametogenesis. In microspore mother cells which are approximately isodiametric at interphase, microtubules were predominantly arranged in a criss-cross pattern. The microtubules probably function as a flexible cytoskeleton which sustains the integrity of the cytoplasm. Bundles of microtubules were observed in the microspores, in the generative cells and during nuclear division, where they functioned in establishing and maintaining cell and spindle shapes. Microtubules radiating from nuclear membranes appeared to fix the nucleus in position. In prophase of meiosis and after microspore mitosis, periods a high fluorescence intensity were distinguished indicating a variation in the quantity of microtubules.