H. Kieft

Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth

The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.

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.

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.

Arabidopsis VILLIN2 and VILLIN3 are required for the generation of thick actin filament bundles and for directional organ growth

In plant cells, actin filament bundles serve as tracks for myosin-dependent organelle movement and play a role in the organization of the cytoplasm. Although virtually all plant cells contain actin filament bundles, the role of the different actin-bundling proteins remains largely unknown. In this study, we investigated the role of the actin-bundling protein villin in Arabidopsis (Arabidopsis thaliana). We used Arabidopsis T-DNA insertion lines to generate a double mutant in which VILLIN2 (VLN2) and VLN3 transcripts are truncated. Leaves, stems, siliques, and roots of vln2 vln3 double mutant plants are twisted, which is caused by local differences in cell length. Microscopy analysis of the actin cytoskeleton showed that in these double mutant plants, thin actin filament bundles are more abundant while thick actin filament bundles are virtually absent. In contrast to full-length VLN3, truncated VLN3 lacking the headpiece region does not rescue the phenotype of the vln2 vln3 double mutant. Our results show that villin is involved in the generation of thick actin filament bundles in several cell types and suggest that these bundles are involved in the regulation of coordinated cell expansion.

Winding threads around plant cells. Applications of the geometrical model for microfibril deposition.

SummaryBased on precise information about the orientations of cellulose microfibrils (CMFs) in the secondary cell wall of theEquisetum hyemale root hair, a geometrical model was recently put forward to account for the deposition orientation of CMFs. The model supposes that synthases spin out the CMFs and that geometrical laws dictate their movement. Taking space-limiting conditions into account, CMF orientation is dependent on cell morphology, the amount of other wall molecules adhering to the CMFs, and the number and distribution pattern of synthases. In the present paper this geometrical model for CMF deposition is further applied to nontip-growing angular cells with varying diameters, cells with tapering morphology, various distribution patterns of synthases, various matrix/fibril ratios, and intercalarily elongating cells. The model can accurately predict the actual wall textures in a great variety of cell walls. In the proposed model for CMF orientation, microtubules are not required as cellular guiding structures for the CMFs, not even in elongating walls. They are supposed to be involved in cell elongation, possibly by delivering wall material including CMF synthases.

Depth of dormancy in tomato (Lycopersicon esculentum Mill.) seeds is related to the progression of the cell cycle prior to the induction of dormancy

Cell cycle activities are initiated following imbibition of non-dormant seeds. However, it is not known whether cell cycle related events other than DNA replication also remain suppressed in imbibed dormant seeds. The objective of this study was to demonstrate that the transitions between the non-dormant and dormant (both primary and secondary) states are reflected in cell cycle events, such as DNA replication and the changing patterns of the microtubular cytoskeleton involved in the processes of growth and development. The present studies were conducted on seeds from tomato (Lycopersicon esculentum cv. Moneymaker) that possessed primary dormancy or were manipulated to attain secondary dormancy. In addition, a non-dormant abscisic acid (ABA)-deficient mutant, sit w , was used. DNA replication, as measured by flow cytometry, and � tubulin accumulation, analysed by immunoblotting, were compared with immunocytological studies of active DNA synthesis and microtubular cytoskeleton formation. It is shown that the depth of dormancy, which distinguishes primary and secondary dormancy, may depend on the progression of the cell cycle prior to the induction of dormancy.

Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.

Structure and development of somatic embryos formed in Arabidopsis thaliana pt mutant callus cultures derived from seedlings

SummarySeeds of theArabidopsis thaliana mutant primordia timing (pt) were germinated in 2,4-dichlorophenoxyacetic acidcontaining liquid medium. The seedlings formed somatic embryos and nonembryogenic and embryogenic callus in vitro in a time period of approximately two to three weeks. Embryogenesis and callus formation were monitored with respect to origin, structure, and development. Ten days after germination globular structures appeared in close vicinity of and on the shoot apical meristem (SAM). Somatic embryos formed either directly on the SAM region of the seedling or indirectly on embryogenic callus that developed at the SAM zone. Globular structures developed along the vascular tissue of the cotyledons as well, but only incidentally they formed embryos. Upon deterioration, the cotyledons formed callus. Regular subculture of the embryogenic callus gave rise to high numbers of somatic embryos. Such primary somatic embryos, grown on callus, originated from meristematic cell clusters located under the surface of the callus. Embryos at the globular and heart-shape stage were mostly hidden within the callus. Embryos at torpedo stage appeared at the surface of the callus because their axis elongated. Secondary somatic embryos frequently formed directly on primary ones. They preferentially emerged from the SAM region of the primary somatic embryos, from the edge of the cotyledons, and from the hypocotyl. We conclude that the strong regeneration capacity of thept mutant is based on both recurrent and indirect embryogenesis.