A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

Floral structure of Emmotum (Icacinaceae sensu stricto or Emmotaceae), a phylogenetically isolated genus of lamiids with a unique pseudotrimerous gynoecium,bitegmic ovules and monosporangiate thecae

Background and Aims

Icacinaceae sensu stricto consist of a group of early branching lineages of lamiids whose relationships are not yet resolved and whose detailed floral morphology is poorly known. The most bizarre flowers occur in Emmotum: the gynoecium has three locules on one side and none on the other. It has been interpreted as consisting of three fertile and two sterile carpels or of one fertile carpel with two longitudinal septa and two sterile carpels. This study focused primarily on the outer and inner morphology of the gynoecium to resolve its disputed structure, and ovule structure was also studied. In addition, the perianth and androecium were investigated.

Methods

Flowers and floral buds of two Emmotum species, E. harleyi and E. nitens, were collected and fixed in the field, and then studied by scanning electron microscopy. Microtome section series were used to reconstruct their morphology.

Key Results

The gynoecium in Emmotum was confirmed as pentamerous, consisting of three fertile and two sterile carpels. Each of the three locules behaves as the single locule in other Icacinaceae, with the placenta of the two ovules being identical, which shows that three fertile carpels are present. In addition, it was found that the ovules are bitegmic, which is almost unique in lamiids, and that the stamens have monosporangiate thecae, which also occurs in the closely related family Oncothecaceae, but is not known from any other Icacinaceae sensu lato so far.

Conclusions

The flowers of Emmotum have unique characters at different evolutionary levels: the pseudotrimerous gynoecium at angiosperm level, the bitegmic ovules at lamiid level and the monosporangiate thecae at family or family group level. However, in general, the floral morphology of Emmotum fits well in Icacinaceae. More comparative research on flower structure is necessary in Icacinaceae and other early branching lineages of lamiids to better understand the initial evolution of this large lineage of asterids.     

Holoparasitic Rafflesiaceae possess the most reduced endophytes and yet give rise to the world's largest flowers

Background and Aims

Species in the holoparasitic plant family Rafflesiaceae exhibit one of the most highly modified vegetative bodies in flowering plants. Apart from the flower shoot and associated bracts, the parasite is a mycelium-like endophyte living inside their grapevine hosts. This study provides a comprehensive treatment of the endophytic vegetative body for all three genera of Rafflesiaceae (Rafflesia, Rhizanthes and Sapria), and reports on the cytology and development of the endophyte, including its structural connection to the host, shedding light on the poorly understood nature of this symbiosis.

Methods

Serial sectioning and staining with non-specific dyes, periodic–Schiff''s reagent and aniline blue were employed in order to characterize the structure of the endophyte across a phylogenetically diverse sampling.

Key Results

A previously identified difference in the nuclear size between Rafflesiaceae endophytes and their hosts was used to investigate the morphology and development of the endophytic body. The endophytes generally comprise uniseriate filaments oriented radially within the host root. The emergence of the parasite from the host during floral development is arrested in some cases by an apparent host response, but otherwise vegetative growth does not appear to elicit suppression by the host.

Conclusions

Rafflesiaceae produce greatly reduced and modified vegetative bodies even when compared with the other holoparasitic angiosperms once grouped with Rafflesiaceae, which possess some vegetative differentiation. Based on previous studies of seeds together with these findings, it is concluded that the endophyte probably develops directly from a proembryo, and not from an embryo proper. Similarly, the flowering shoot arises directly from the undifferentiated endophyte. These filaments produce a protocorm in which a shoot apex originates endogenously by formation of a secondary morphological surface. This degree of modification to the vegetative body is exceptional within angiosperms and warrants additional investigation. Furthermore, the study highlights a mechanical isolation mechanism by which the host may defend itself from the parasite.     

Reconstructing the ancestral angiosperm flower and its initial specializations

Increasingly robust understanding of angiosperm phylogeny allows more secure reconstruction of the flower in the most recent common ancestor of extant angiosperms and its early evolution. The surprising emergence of several extant and fossil taxa with simple flowers near the base of the angiosperms-Chloranthaceae, Ceratophyllum, Hydatellaceae, and the Early Cretaceous fossil Archaefructus (the last three are water plants)-has brought a new twist to this problem. We evaluate early floral evolution in angiosperms by parsimony optimization of morphological characters on phylogenetic trees derived from morphological and molecular data. Our analyses imply that Ceratophyllum may be related to Chloranthaceae, and Archaefructus to either Hydatellaceae or Ceratophyllum. Inferred ancestral features include more than two whorls (or series) of tepals and stamens, stamens with protruding adaxial or lateral pollen sacs, several free, ascidiate carpels closed by secretion, extended stigma, extragynoecial compitum, and one or several ventral pendent ovule(s). The ancestral state in other characters is equivocal: e.g., bisexual vs. unisexual flowers, whorled vs. spiral floral phyllotaxis, presence vs. absence of tepal differentiation, anatropous vs. orthotropous ovules. Our results indicate that the simple flowers of the newly recognized basal groups are reduced rather than primitively simple.     

The Immense Diversity of Floral Monosymmetry and Asymmetry Across Angiosperms

Floral monosymmetry and asymmetry are traced through the angiosperm orders and families. Both are diverse and widespread in angiosperms. The systematic distribution of the different forms of monosymmetry and asymmetry indicates that both evolved numerous times. Elaborate forms occur in highly synorganized flowers. Less elaborate forms occur by curvature of organs and by simplicity with minimal organ numbers. Elaborate forms of asymmetry evolved from elaborate monosymmetry. Less elaborate form come about by curvature or torsion of organs, by imbricate aestivation of perianth organs, or also by simplicity. Floral monosymmetry appears to be a key innovation in some groups (e.g., Orchidaceae, Fabaceae, Lamiales), but not in others. Floral asymmetry appears as a key innovation in Phaseoleae (Fabaceae). Simple patterns of monosymmetry appear easily “reverted” to polysymmetry, whereas elaborate monosymmetry is difficult to lose without disruption of floral function (e.g., Orchidaceae). Monosymmetry and asymmetry can be expressed at different stages of floral (and fruit) development and may be transient in some taxa. The two symmetries are most common in bee-pollinated flowers, and appear to be especially prone to develop in some specialized biological situations: monosymmetry, e.g., with buzz-pollinated flowers or with oil flowers, and asymmetry also with buzz-pollinated flowers, both based on the particular collection mechanisms by the pollinating bees. Floral monosymmetry has developed into a model trait in evo-devo studies, whereas floral asymmetry to date has not been tackled in molecular genetic studies.     

7th SOSORT consensus paper: conservative treatment of idiopathic & Scheuermann's kyphosis

Abstract

Thoracic hyperkyphosis is a frequent problem and can impact greatly on patient's quality of life during adolescence. This condition can be idiopathic or secondary to Scheuermann disease, a disease disturbing vertebral growth. To date, there is no sound scientific data available on the management of this condition. Some studies discuss the effects of bracing, however no guidelines, protocols or indication's of treatment for this condition were found. The aim of this paper was to develop and verify the consensus on managing thoracic hyperkyphosis patients treated with braces and/or physiotherapy.

Methods

The Delphi process was utilised in four steps gradually modified according to the results of a set of recommendations: we involved the SOSORT Board twice, then all SOSORT members twice, with a Pre-Meeting Questionnaire (PMQ), and during a Consensus Session at the SOSORT Lyon Meeting with a Meeting Questionnaire (MQ).

Results

There was an unanimous agreement on the general efficacy of bracing and physiotherapy for this condition. Most experts suggested the use of 4-5 point bracing systems, however there was some controversy with regards to physiotherapeutic aims and modalities.

Conclusion

The SOSORT panel of experts suggest the use of rigid braces and physiotherapy to correct thoracic hyperkyphosis during adolescence. The evaluation of specific braces and physiotherapy techniques has been recommended.     

Synorganisation without organ fusion in the flowers of Geranium robertianum (Geraniaceae) and its not so trivial obdiplostemony

Background and Aims

Synorganisation of floral organs, an important means in angiosperm flower evolution, is mostly realized by congenital or post-genital organ fusion. Intimate synorganisation of many floral organs without fusion, as present in Geranium robertianum, is poorly known and needs to be studied. Obdiplostemony, the seemingly reversed position of two stamen whorls, widely distributed in core eudicots, has been the subject of much attention, but there is confusion in the literature. Obdiplostemony occurs in Geranium and whether and how it is involved in this synorganisation is explored here.

Methods

Floral development and architecture were studied with light microscopy based on microtome section series and with scanning electron microscopy.

Key Results

Intimate synorganisation of floral organs is effected by the formation of five separate nectar canals for the proboscis of pollinators. Each nectar canal is formed by six adjacent organs from four organ whorls. In addition, the sepals are hooked together by the formation of longitudinal ribs and grooves, and provide a firm scaffold for the canals. Obdiplostemony provides a guide rail within each canal formed by the flanks of the antepetalous stamen filaments.

Conclusions

Intimate synorganisation in flowers can be realized without any fusion, and obdiplostemony may play a role in this synorganisation.