Transcriptional landscapes of floral meristems in barley.

Organ development in plants predominantly occurs postembryonically through combinatorial activity of meristems; therefore, meristem and organ fate are intimately connected. Inflorescence morphogenesis in grasses (Poaceae) is complex and relies on a specialized floral meristem, called spikelet meristem, that gives rise to all other floral organs and ultimately the grain. The fate of the spikelet determines reproductive success and contributes toward yield-related traits in cereal crops. Here, we examined the transcriptional landscapes of floral meristems in the temperate crop barley (Hordeum vulgare L.) using RNA-seq of laser capture microdissected tissues from immature, developing floral structures. Our unbiased, high-resolution approach revealed fundamental regulatory networks, previously unknown pathways, and key regulators of barley floral fate and will equally be indispensable for comparative transcriptional studies of grass meristems.

Hybrid dwarfness in crosses between wheat (Triticum aestivum L.) and rye (Secale cereale L.): a new look at an old phenomenon.

The existence of hybrid dwarfs from intraspecific crosses in wheat (Triticum aestivum) was described 100 years ago, and the genetics underlying hybrid dwarfness are well understood. In this study, we report a dwarf phenotype in interspecific hybrids between wheat and rye (Secale cereale). We identified two rye lines that produce hybrid dwarfs with wheat and have none of the hitherto known hybrid dwarfing genes. Genetic analyses revealed that both rye lines carry a single allelic gene responsible for the dwarf phenotype. This gene was designated Hdw-R1 (Hybrid dwarf-R1). Application of gibberellic acid (GA3 ) to both intraspecific (wheat-wheat) and interspecific (wheat-rye) hybrids showed that hybrid dwarfness cannot be overcome by treatment with this phytohormone. Histological analysis of shoot apices showed that wheat-rye hybrids with the dwarf phenotype at 21 and 45 days after germination failed to develop further. Shoot apices of dwarf plants did not elongate, did not form new primordia and had a dome-shaped appearance in the seed. The possible relationship between hybrid dwarfness and the genes responsible for the transition from vegetative to generative growth stage is discussed.

Chromatin alterations during pollen development in Hordeum vulgare.

The dynamics of posttranslational histone modifications in relation to nuclear architecture has been analyzed during pollen development in Hordeum vulgare L. cv. Igri. Notwithstanding the asymmetry of cytokinesis associated with pollen mitosis I, immunolabeling revealed that the vegetative and generative nuclei initially display identical chromatin modification patterns. Yet, differential chromatin modification patterns between vegetative and generative nuclei emerge with the development of conspicuous differences in nuclear morphology as visualized by 4',6-diamidino-2-phenylindole staining. The temporal and spatial distribution of most histone modifications observed is in agreement with reduced gene activity in the generative nucleus and increased expression in the vegetative nucleus as indicated by immunolabeling of active RNA polymerase II. Signals of trimethylation of histone H3 lysine 27 proved to be particularly enriched in euchromatic domains of subtelomeric regions. In the context of nuclear differentiation in bicellular pollen, this modification became restricted to the vegetative nucleus, indicating a role in activating rather than suppressing gene expression. The presence of acetylated histone H3 at lysine 9 in the cytoplasm of the generative cell is indicative of a more complex, still unknown function of this particular modification.

Time-lapse imaging of the initiation of pollen embryogenesis in barley (Hordeum vulgare L.).

Pollen embryogenesis provides exciting opportunities in the areas of breeding and biotechnology as well as representing a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular. A cell culture system was devised in which immature barley pollen could be cultured as a monolayer trapped between the bottom glass-cover slip of a live-cell chamber and a diaphanous PTFE membrane within a liquid medium over a period of up to 28 d, allowing the process of embryogenesis to be tracked in individual pollen. Z-stacks of images were automatically captured every 3min, starting from the unicellular pollen stage up to the development of multicellular, embryogenic structures. The method should prove useful for the elucidation of ultrastructural features and molecular processes associated with pollen embryogenesis.

Ultrastructural changes associated with cryopreservation of potato (Solanum tuberosum l.) shoot tips.

The ultrastructure of cells within shoot tips of S. tuberosum 'Désirée' was studied after different steps of the DMSO droplet cryopreservation method. After 2 h of DMSO treatment, cells contained numerous small vesicles, while at the same time mitochondria and chloroplasts had increased in size and vacuoles had assumed an irregular shape. After rapid cooling in liquid nitrogen, subsequent rewarming, and 1 h incubation there were no apparent changes in the ultrastructural organization of the cells, suggesting that they might be still intact. However, two days after rewarming, the meristematic dome area and part of the epidermis showed signs of extensive damage. Rupture of plasmalemma, plasmolysis and destruction of cell organelles as well as strong heterochromatisation of nuclei were observed. Survival and regeneration of cells were found mainly in leaf primordial regions. Here cells were very active, containing many mitochondria and intact or regenerating chloroplasts. Alternating temperature preculture of donor plants before shoot tip isolation improved the cryopreservation results (plant regeneration 46.5 percent) as compared to constantly warm precultured shoot tips (plant regeneration 20.0 percent), which showed slightly stronger damage after rewarming from liquid nitrogen.

Elimination of chromosomes in Hordeum vulgare x H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatinization.

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the development of Hordeum vulgare x H. bulbosum hybrid embryos that is restricted to an early stage of development. In almost all embryos most of the H. bulbosum chromatin undergoes a fast rate of elimination within nine days after pollination. There are differences in the mitotic behaviour between the parental chromosomes, with H. bulbosum chromatids segregating asymmetrically at anaphase. We provide evidence for a chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei differs and heterochromatinization and disintegration of the nuclear envelope of micronuclei are the final steps of chromosome elimination.

A fast, high spatial resolution optical tomographic scanner for measurement of absorption in gel dosimetry.

A fast tomographic optical density measurement system has been constructed and evaluated for application in Fricke 3D gel dosimetry. Although the potential for full three-dimensional radiation dosimetry with Fricke gel dosimeters has been extensively reported, its application has been limited due to a lack of fast optical density measurement systems. In this work, the emphasis of the design has been to achieve a short scan time through the use of precision optics and minimal moving parts. The system has been demonstrated in the laboratory to be able to achieve better than 1mm resolution and a scanning time per tomographic slice of 2.4 seconds. Full volumetric sampling of a 10 cm diameter by 7cm long cylinder can be achieved in 3 minutes. When applied with a Fricke based gel dosimeter a linear response between reconstructed CT number and absolute dose was better than 3%.

Novel phosphorylation of histone H3 at threonine 11 that temporally correlates with condensation of mitotic and meiotic chromosomes in plant cells.

A novel mitosis-specific phosphorylation site in histone H3 at threonine 11 has been described for mammalian cells. This modification is restricted to the centromeric region while phosphorylation at the classical H3 sites, Ser10 and Ser28 occurs along the entire chromosomal arms. Using phosphorylation state-specific antibodies we found that phosphorylation at threonine 11 occurs also in plant cells, during mitosis as well as meiosis. However, in contrast to animal cells, ph(Thr11)H3 was distributed along the entire length of condensed chromosomes, whereas H3 phosphorylated at Ser10 and Ser28 appeared to be restricted to centromeric/pericentromeric chromatin. Phosphorylation at Thr11 started in prophase and ended in telophase, it correlated with the condensation of mitotic and meiotic chromosomes and was independent of the distribution of late replicating heterochromatin and Giemsa-banding positive regions. Interestingly, treatment of cells with the phosphatase inhibitor cantharidin revealed a high level of Thr11 phosphorylation in interphase cells, in this case particularly in pericentromeric regions. These data show that histone modifications are highly dynamic. Moreover, animal and plant organisms may have evolved individual histone codes.

Discovery of an extended bundle sheath in Ricinus communis L. and its role as a temporal storage compartment for the iron chelator nicotianamine.

The extended bundle sheath (EBS) is a specialized layer of cells that enhances the lateral transport of photoassimilates within the leaf. This little-known tissue is often considered to be legume-specific. We identified an EBS in cotyledons and leaves of the non-legume Ricinus communis L. By means of cytological and immunological studies and using the localization of the iron-chelator nicotianamine as an established indicator for mass transport, we confirmed its role as a transport tissue and a temporal sink. Observations on cotyledons of Ricinus seedlings further proved that the EBS carries out these tasks from a very early stage of development onwards. This is the first time that information has been obtained on the physiological role of an EBS in a non-legume. Our results support the idea of its widespread occurrence among higher plants.

Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions.

BACKGROUND: The dsDNA bacteriophage PRD1 has a membrane inside its icosahedral capsid. While its large size (66 MDa) hinders the study of the complete virion at atomic resolution, a 1.65-A crystallographic structure of its major coat protein, P3, is available. Cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction have shown the capsid at 20-28 A resolution. Striking architectural similarities between PRD1 and the mammalian adenovirus indicate a common ancestor. RESULTS: The P3 atomic structure has been fitted into improved cryo-EM reconstructions for three types of PRD1 particles: the wild-type virion, a packaging mutant without DNA, and a P3-shell lacking the membrane and the vertices. Establishing the absolute EM scale was crucial for an accurate match. The resulting "quasi-atomic" models of the capsid define the residues involved in the major P3 interactions, within the quasi-equivalent interfaces and with the membrane, and show how these are altered upon DNA packaging. CONCLUSIONS: The new cryo-EM reconstructions reveal the structure of the PRD1 vertex and the concentric packing of DNA. The capsid is essentially unchanged upon DNA packaging, with alterations limited to those P3 residues involved in membrane contacts. These are restricted to a few of the N termini along the icosahedral edges in the empty particle; DNA packaging leads to a 4-fold increase in the number of contacts, including almost all copies of the N terminus and the loop between the two beta barrels. Analysis of the P3 residues in each quasi-equivalent interface suggests two sites for minor proteins in the capsid edges, analogous to those in adenovirus.

Organization of immature human immunodeficiency virus type 1.

Immature retrovirus particles contain radially arranged Gag polyproteins in which the N termini lie at the membrane and the C termini extend toward the particle's center. We related image features to the polyprotein domain structure by combining mutagenesis with cryoelectron microscopy and image analysis. The matrix (MA) domain appears as a thin layer tightly associated with the inner face of the viral membrane, separated from the capsid (CA) layer by a low-density region corresponding to its C terminus. Deletion of the entire p6 domain has no effect on the width or spacing of the density layers, suggesting that p6 is not ordered in immature human immunodeficiency virus type 1 (HIV-1). In vitro assembly of a recombinant Gag polyprotein containing only capsid (CA) and nucleocapsid (NC) domains results in the formation of nonenveloped spherical particles which display two layers with density matching that of the CA-NC portion of immature HIV-1 Gag particles. Authentic, immature HIV-1 displays additional surface features and an increased density between the lipid bilayers which reflect the presence of gp41. The other internal features match those of virus-like particles.

Cryo-electron microscopy reveals the functional organization of an enveloped virus, Semliki Forest virus.

Semliki Forest virus serves as a paradigm for membrane fusion and assembly. Our icosahedral reconstruction combined 5276 particle images from 48 cryo-electron micrographs and determined the virion structure to 9 A resolution. The improved resolution of this map reveals an N-terminal arm linking capsid subunits and defines the spike-capsid interaction sites. It illustrates the paired helical nature of the transmembrane segments and the elongated structures connecting them to the spike projecting domains. A 10 A diameter density in the fusion protein lines the cavity at the center of the spike. These clearly visible features combine with the variation in order between the layers to provide a framework for understanding the structural changes during the life cycle of an enveloped virus.

The intact retroviral Env glycoprotein of human foamy virus is a trimer.

Electron microscopy of negatively stained human foamy virus particles provides direct evidence for the trimeric nature of intact Env surface glycoproteins. Three-dimensional image reconstruction reveals that the Env trimer is a tapering spike 14 nm in length. The spikes were often arranged in hexagonal rings which shared adjacent Env trimers.

Bacteriophage PRD1 contains a labile receptor-binding structure at each vertex.

Bacteriophage PRD1 is a membrane-containing virus with an unexpected similarity to adenovirus. We mutagenized unassigned PRD1 genes to identify minor capsid proteins that could be structural or functional analogs to adenovirus proteins. We report here the identification of an amber mutant, sus525, in an essential PRD1 gene XXXI. The gene was cloned and the gene product was overexpressed and purified to near homogeneity. Analytical ultracentrifugation and gel filtration showed that P31 is a homopentamer of about 70 kDa. The protein was shown to be accessible on the virion surface and its absence in the sus525 particles led to the deficiency of two other viral coat proteins, protein P5 and the adsorption protein P2. Cryo-electron microscopy and image reconstruction of the sus525 particles indicate that these proteins are located on the capsid vertices, because in these particles the entire vertex structure was missing along with the peripentonal major capsid protein P3 trimers. Sus525 particles package DNA effectively but loose it upon purification. All of the PRD1 vertex structures are labile and potentially capable of mediating DNA delivery; this is in contrast to other dsDNA phages which employ a single vertex for packaging and delivery. We propose that this arises from a symmetry mismatch between protein P2 and the pentameric P31 in analogy to that between the adenovirus penton base and the receptor-binding spike.

The carboxy-terminal p3Gag domain of the human foamy virus Gag precursor is required for efficient virus infectivity.

Proteolytic processing of foamy virus Gag proteins appears to be different from that of other retroviruses. A single carboxy-terminal cleavage site is consistently detectable in human foamy virus (HFV) Gag precursor protein p74Gag derived from infected cells and/or purified virus particles. Using a recombinant HFV protease, we have determined the p74Gag cleavage site that results in p70Gag and the carboxy-terminal p3Gag (Pfrepper et al., 1997, Biochem. Biophys. Res. Commun. 237, 548-553). To study the biological functions of p3Gag, proviral DNA clones were constructed coding for a carboxy-terminally truncated p70Gag lacking the entire p3Gag protein. Removal of p3Gag resulted in an about 100-fold lower virus titer. The expression of other HFV proteins and the processing of Pol proteins were indistinguishable from those of wild-type-transfected cells. The defect in viral infectivity of the p3 mutants was partially restored by coexpressing the full-length p74Gag protein in trans. The deletion of p3Gag resulted in particle assembly with wild-type virion morphology and encapsidation of Pol proteins. Our data show that the carboxy-terminal p3Gag protein has an important function for viral infectivity but is not required for preassembly of capsids, virus morphogenesis, and incorporation of Pol proteins into virions.

A 60-kDa plant microtubule-associated protein promotes the growth and stabilization of neurotubules in vitro.

The search for microtubule-associated proteins (MAPs) in plants is relatively recent. In particular, the "classical MAPs," which stimulate the polymerization and stabilization of microtubules, have only been examined in heterogeneous fractions. As a first step in dissecting the role of individual MAPs, we have chromatographically purified a single 60-kDa protein from a carrot MAP fraction and analyzed its effects on tubulin assembly. MAP60 promoted the formation of long, morphologically regular brain microtubules in vitro, an effect inhibited by preincubation of the MAP with affinity-purified antibodies against this protein. MAP60 also increased the stability of microtubules to dilution and significantly enhanced cold stability to the normally cold-sensitive neurotubules. These in vitro properties are consistent with a role for MAP60 in regulating the turnover/assembly of dynamic plant microtubules in vivo.

Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus.

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.

Brefeldin A effects on tobacco pollen tubes.

Pollen germination and pollen tube growth were clearly affected by the drug brefeldin A: both processes could be stopped completely. Notable thickening of the cell wall was not observed in non-growing pollen tubes, and plasma streaming remained vigorous in the presence of brefeldin A. Over time, the tip region became filled with a dense cytoplasm that lacked large organelles. Ultrastructural observation showed the rapid dissociation and disappearance of Golgi bodies. The concomitant appearance of vesicle-like structures attached to the endoplasmic reticulum (ER) suggests a fusion of the Golgi with the ER similar to that which happens in animal cells. Secretory vesicles disappeared from drug-treated tubes, and the tip region became filled with tubular ER. As the actions of the drug seemed to be reversible, brefeldin A may contribute substantially to research on Golgi dynamics and tip growth in pollen tubes.