Induction of embryogenesis in [isolated] microspores and pollen of Brassica napus L. cv. Topas

Artificial systems to produce plant embryos are important tools for basic research as well as for plant breeding. It is possible to produce large amounts of embryos by methods like somatic embryogenesis or embryogenic microspore cultures. Such high amounts of embryos, which are easier to handle than zygotic embryos, are the prerequisite for biochemical and molecular genetic investigations on the one hand, and for biotechnological use on the other hand. Moreover, embryos derived from microspores or pollen represent a very efficient basis for the production of plant hybrids: Because of their haploid origin, microspore-derived embryos are after diploidization dihaploid, and thus, homozygous.The induction of embryogenesis in microspores and pollen of Brassica napus is realized by their cultivation under heat shock conditions (32 °C for at least 24 h). This dissertation presents results from our investigations on the induction phase of embryogenesis. Cellular changes during the first 24 h of cultivation of microspores and pollen were analysed to discern cytological differences between this induction phase of embryogenesis and the normal pattern of pollen development in planta. Combining light as well as electron-microscopy for the analysis of the cytoskeleton (microtubules and microfilaments), it was shown that the development of microspores and pollen in B. napus to a mature, tricellular microgametophyte is comparable to other angiosperms. Only the disappearance of the central vacuole before microspore mitosis is different from the common pathway (Chapter 2). Stage specific developmental patterns of microtubules and microfilaments could be detected in the microspores and pollen.Symmetrical divisions are a prerequisite for the embryogenic development of cultivated microspores and pollen, and three pathways for the induction of such divisions were identified (Chapter 3). (i) In vacuolated microspores cultivation under embryogenic conditions causes a migration of the nucleus to the centre of the cell where the division takes place. This phenomenon is accompanied by the disruption or altered formation of the microtubules. (ii) In late microspores the embryogenic conditions cause a turn of the mitotic spindle up to 90°. Both events (i and ii) result in symmetrically divided cells forming a bicellular proembryo. (iii) The developmental switch from young, bicellular pollen to the formation of proembryos is caused by a disrupted pollen development (arrest of the generative cell at the pollen wall) followed by division of the vegetative cell. In this case, microtubules, normally detectable in the vegetative cell perpendicular to the generative cell, are disrupted. In summary, all three pathways can lead to the formation of proembryos, and the microtubular cytoskeleton seems to be involved in these developmental changes. Although changes were also visible concerning the microfilaments under embryogenic conditions, their role in the induction of embryogenesis could not be confirmed.Because of the obvious changes in the cell cycle of microspores and pollen cultivated under embryogenic conditions, the synthesis of DNA was investigated in vivo and in vitro (Chapter 4). The incorporation and detection of bromodeoxyuridine as well as the determination of the ploidy level of the nuclei by microspectrophotometry were used for these investigations. DNA replication could be shown in vivo within the nucleus of the late microspore and also within the generative nucleus of the late bicellular pollen. In normal development the vegetative nucleus remains in the G1- phase. Under embryogenic conditions, the pattern of replication in microspores remained the same, but the vegetative nucleus of the young bicellular pollen re-entered the cell cycle and exhibited DNA synthesis.Changes in phosphorylation patterns were analysed using the monoclonal antibody MPM-2 (Chapter 5). The antibody MPM-2, raised against mitotic proteins of HeLa-cells, recognizes phosphorylated, mitosis-specific proteins in animal and plant cells. In developing microspores and pollen of B. napus, MPM-2 bound to proteins of all developmental stages, especially to proteins in the nuclei. Moreover, there were no differences in phosphorylated epitopes between microspores and pollen cultivated under embryogenic and non-embryogenic conditions. This might be caused by the fact that this antibody recognizes phosphorylated epitopes of various proteins.Because of the heat shock conditions used for the induction of embryogenesis, the subcellular localisation of heat shock proteins (HSPs) was performed (Chapter 6). Western blot analysis of proteins separated by two-dimensional gel electrophoresis revealed a strong signal at 70 kDa. Immunocytochemical investigations using an antibody raised against HSP70 showed a distinct stage- specific subcellular localization of HSP70 in vivo as well as in vitro. The embryogenic cultivation caused an altered localization of HSP70, which became detectable within the nucleus of the vegetative cell. Its localization could therefore be correlated with the initiation of DNA replication. Possible relations between HSP70 and replication were discussed.Chapter 7 describes the localisation of a specific mRNA within developing microspores and pollen of B. napus and Arabidopsis thaliana. Using freeze sectioned material and in situ- hybridization with a digoxygenin labelled probe, specific gene expression was demonstrated for the generative cell of both species.Finally, the expression of polarity during the development of microspore-derived and zygotic embryos was compared (Chapter 8). Investigations by scanning electron microscopic techniques showed that the embryo formation between them are similar from the globular stage onwards. The distribution of calcium ions, calmodulin and starch was used to find early signs of polarity. However, the accumulation of starch and the position of a residual pollen wall were the only hints for a predisposition of the radial axis of the developing embryo.In chapter 9 the embryogenesis in isolated microspores and pollen is considered as a biphasic process. The induction phase of embryogenesis represents the dedifferentiation of a developing organism followed by differentiation to a real plant embryo. Our results are combined with results of other groups to create a general scheme on induction of embryogenesis in microspores and pollen of B. napus.</em

Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane

This is the final version of the article. Available from Elsevier via the DOI in this record.The 30 kDa N-BAR domain of the human Bin1 protein is essential for the generation of skeletal muscle T-tubules. By electron cryo-microscopy and electron cryo-tomography with a direct electron detector, we found that Bin1-N-BAR domains assemble into scaffolds of low long-range order that form flexible membrane tubules. The diameter of the tubules closely matches the curved shape of the N-BAR domain, which depends on the composition of the target membrane. These insights are fundamental to our understanding of T-tubule formation and function in human skeletal muscle.This work was supported by grants from the Deutsche Forschungsgemeinschaft (GRK 1026, SFB610) (A.A., T.G., J.B.), the BMBF ZIK program (A.M., J.B.), the European Regional Development Fund of the European Commission (A.M., T.G.: EFRE 1241 12 0001), and the state Sachsen-Anhalt (A.M., T.G., J.B.)

Mechanostimulation of Medicago truncatula leads to enhanced levels of jasmonic acid

Wounding of plants leads to endogenous rise of jasmonic acid (JA) accompanied with the expression of a distinct set of genes. Among them are those coding for the allene oxide cyclase (AOC) that catalyses a regulatory step in JA biosynthesis, and for 1-deoxy-D-xylulose 5-phosphate synthase 2 (DXS2), an enzyme involved in isoprenoid biosynthesis. To address the question how roots and shoots of Medicago truncatula respond to mechanostimulation and wounding, M. truncatula plants were analysed in respect to JA levels as well as MtAOC1 and MtDXS2-1 transcript accumulation. Harvest-caused mechanostimulation resulted in a strong, but transient increase in JA level in roots and shoots followed by a transient increase in MtAOC1 transcript accumulation. Additional wounding of either shoots or roots led to further increased JA and MtAOC1 transcript levels in shoots, but not in roots. In situ hybridization revealed a cell-specific transcript accumulation of MtAOC1 after mechanostimulation in companion cells of the vascular tissue of the stem. AOC protein, however, was found to occur constitutively in vascular bundles. Further, transcript accumulation of MtDXS2-1 was similar to that of MtAOC1 in shoots, but its transcript levels were not enhanced in roots. Repeated touching of shoots increased MtAOC1 transcript levels and led to significantly shorter shoots and increased biomass. In conclusion, M. truncatula plants respond very sensitively to mechanostimulation with enhanced JA levels and altered transcript accumulation, which might contribute to the altered phenotype after repeated touching of plants

Detection of influenza C virus but not influenza D virus in Scottish respiratory samples

AbstractBackgroundA newly proposed genus of influenza virus (influenza D) is associated with respiratory disease in pigs and cattle. The novel virus is most closely related to human influenza C virus and can infect ferrets but infection has not been reported in humans.ObjectivesTo ascertain if influenza D virus can be detected retrospectively in patient respiratory samples.Study design3300 human respiratory samples from Edinburgh, Scotland, covering the period 2006–2008, were screened in pools of 10 by RT-PCR using primers capable of detecting both influenza C and D viruses.ResultsInfluenza D was not detected in any sample. Influenza C was present in 6 samples (0.2%), compared with frequencies of 3.3% and 0.9% for influenza A and B viruses from RT-PCR testing of respiratory samples over the same period. Influenza C virus was detected in samples from individuals <2 years or >45 years old, with cases occurring throughout the year. Phylogenetic analysis of nearly complete sequences of all seven segments revealed the presence of multiple, reassortant lineages.ConclusionWe were unable to detect viruses related to influenza D virus in human respiratory samples. Influenza C virus was less prevalent than influenza A and B viruses, was associated with mild disease in the young (<2 years) and old (>45 years) and comprised multiple, reassortant lineages. Inclusion of influenza C virus as part of a diagnostic testing panel for respiratory infections would be of limited additional value

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

Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis

Background: Adventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored. Results: MeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression. Conclusions: The JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species

Does mycorrhization influence herbivore-induced volatile emission in Medicago truncatula?

Symbiosis with mycorrhizal fungi substantially impacts secondary metabolism and defensive traits of colonised plants. In the present study, we investigated the influence of mycorrhization (Glomus intraradices) on inducible indirect defences against herbivores using the model legume Medicago truncatula. Volatile emission by mycorrhizal and non-mycorrhizal plants was measured in reaction to damage inflicted by Spodoptera spp. and compared to the basal levels of volatile emission by plants of two different cultivars. Emitted volatiles were recorded using closed-loop stripping and gas chromatography/mass spectrometry. The documented volatile patterns were evaluated using multidimensional scaling to visualise patterns and stepwise linear discriminant analysis to distinguish volatile blends of plants with distinct physiological status and genetic background. Volatile blends emitted by different cultivars of M. truncatula prove to be clearly distinct, whereas mycorrhization only slightly influenced herbivore-induced volatile emissions. Still, the observed differences were sufficient to create classification rules to distinguish mycorrhizal and non-mycorrhizal plants by the volatiles emitted. Moreover, the effect of mycorrhization turned out to be opposed in the two cultivars examined. Root symbionts thus seem to alter indirect inducible defences of M. truncatula against insect herbivores. The impact of this effect strongly depends on the genetic background of the plant and, hence, in part explains the highly contradictory results on tripartite interactions gathered to date

Effect of plyometric jump training on skeletal muscle hypertrophy in healthy individuals: A systematic review with multilevel meta-analysis

Objective: To examine the effect of plyometric jump training on skeletal muscle hypertrophy in healthy individuals.Methods: A systematic literature search was conducted in the databases PubMed, SPORTDiscus, Web of Science, and Cochrane Library up to September 2021.Results: Fifteen studies met the inclusion criteria. The main overall finding (44 effect sizes across 15 clusters median = 2, range = 1–15 effects per cluster) indicated that plyometric jump training had small to moderate effects [standardised mean difference (SMD) = 0.47 (95% CIs = 0.23–0.71); p &amp;lt; 0.001] on skeletal muscle hypertrophy. Subgroup analyses for training experience revealed trivial to large effects in non-athletes [SMD = 0.55 (95% CIs = 0.18–0.93); p = 0.007] and trivial to moderate effects in athletes [SMD = 0.33 (95% CIs = 0.16–0.51); p = 0.001]. Regarding muscle groups, results showed moderate effects for the knee extensors [SMD = 0.72 (95% CIs = 0.66–0.78), p &amp;lt; 0.001] and equivocal effects for the plantar flexors [SMD = 0.65 (95% CIs = −0.25–1.55); p = 0.143]. As to the assessment methods of skeletal muscle hypertrophy, findings indicated trivial to small effects for prediction equations [SMD = 0.29 (95% CIs = 0.16–0.42); p &amp;lt; 0.001] and moderate-to-large effects for ultrasound imaging [SMD = 0.74 (95% CIs = 0.59–0.89); p &amp;lt; 0.001]. Meta-regression analysis indicated that the weekly session frequency moderates the effect of plyometric jump training on skeletal muscle hypertrophy, with a higher weekly session frequency inducing larger hypertrophic gains [β = 0.3233 (95% CIs = 0.2041–0.4425); p &amp;lt; 0.001]. We found no clear evidence that age, sex, total training period, single session duration, or the number of jumps per week moderate the effect of plyometric jump training on skeletal muscle hypertrophy [β = −0.0133 to 0.0433 (95% CIs = −0.0387 to 0.1215); p = 0.101–0.751].Conclusion: Plyometric jump training can induce skeletal muscle hypertrophy, regardless of age and sex. There is evidence for relatively larger effects in non-athletes compared with athletes. Further, the weekly session frequency seems to moderate the effect of plyometric jump training on skeletal muscle hypertrophy, whereby more frequent weekly plyometric jump training sessions elicit larger hypertrophic adaptations.</jats:p