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.)

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

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

Tomato: a crop species amenable to improvement by cellular and molecular methods

Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.

Redox activities and ROS, NO and phenylpropanoids production by axenically cultured intact olive seedling roots after interaction with a mycorrhizal or a pathogenic fungus

Las raíces de las plántulas de olivo, en cultivo axénico, fueron colocadas alternativamente en contacto con Rhizophagus irregulares (micorrícicos) o con hongos Verticillim dahliae (patógenos). También se incluyeron tratamientos MeJA. Las raíces intactas (generación de anión superóxido, superóxido dismutasa y actividades de peroxidasa) se midieron en las actividades in vivo del apoplasto. Todos nuestros resultados mostraron que las actividades redox apoplásticas de raíces de las plántulas intactas en contacto con el hongo micorriza compatible fueron claramente atenuados en comparación con el hongo patógeno o tratado con MeJA, incluso en las primeras etapas usadas en el tratamiento. Los fenoles totales, flavonoides y glucósidos fenilpropanoides, también fueron cuantificados. Las raíces en contacto con el hongo micorriza no mejoraron la biosíntesis de compuestos fenólicos con respecto a los controles, mientras que los de contacto con el patógeno mejoraron de forma significativa la biosíntesis de todas las fracciones fenólicas medidas. Las especies reactivas del oxígeno y la acumulación de óxido nítrico en las raíces fueron examinadas por microscopía de fluorescencia. Todos ellas presentaron una acumulación mucho mayor en las raíces en contacto con el patógeno que con el hongo micorriza. En total, estos resultados indican que las raíces de las plántulas intactas de olivo, claramente diferenciadas entre micorrizas y hongos patógenos, atenuan las reacciones de defensa contra la primera para facilitar su creación, mientras que induce una reacción de defensa fuerte y sostenida contra el segundo. Ambas especies reactivas de oxígeno y nitrógeno parecían estar involucrados en estas respuestas desde los primeros momentos de contacto. Sin embargo, se necesitan más investigaciones para aclarar la diafonía propuesta entre ellos y sus respectivas funciones en estas respuestas ya que las imágenes de fluorescencia de las raíces revelaron que las especies reactivas del oxígeno se acumulan principalmente en el apoplasto (congruente con las actividades redox medidas en este compartimento), mientras el óxido nítrico se almacena principalmente en el citosol.Roots of intact olive seedlings, axenically cultured, were alternatively placed in contact with Rhizophagus irregularis (mycorrhizal) or Verticillim dahliae (pathogenic) fungi. MeJA treatments were also included. In vivo redox activities in the apoplast of the intact roots (anion superoxide generation, superoxide dismutase and peroxidase activities) were measured. All our results showed that apoplastic redox activities of intact seedling roots in contact with the compatible mycorrhizal fungus were clearly attenuated in comparison with the pathogenic fungus or treated with MeJA, even at the early stages of treatment used. Total phenolics, flavonoids and phenylpropanoid glycosides were also quantified. Roots in contact with the mycorrhizal fungus did not enhance the biosynthesis of phenolic compounds with respect to controls, while those in contact with the pathogenic one significantly enhanced the biosynthesis of all phenolic fractions measured. Reactive oxygen species and nitric oxid accumulation in roots were examined by fluorescence microscopy. All of them presented much higher accumulation in roots in contact with the pathogenic than with the mycorrhizal fungus. Altogether these results indicate that intact olive seedling roots clearly differentiated between mycorrhizal and pathogenic fungi, attenuating defense reactions against the first to facilitate its establishment, while inducing a strong and sustained defense reaction against the second. Both reactive oxygen and nitrogen species seemed to be involved in these responses from the first moments of contact. However, further investigations are required to clarify the proposed crosstalk between them and their respective roles in these responses since fluorescence images of roots revealed that reactive oxygen species were mainly accumulated in the apoplast (congruently with the measured redox activities in this compartment) while nitric oxid was mainly stored in the cytosol.-- Ministerio de Ciencia e Innovación. Proyecto CGL2009-12406 -- Junta de Extremadura. Proyecto PRI09A023peerReviewe

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

Exome-wide somatic mutation characterization of small bowel adenocarcinoma

Small bowel adenocarcinoma (SBA) is an aggressive disease with limited treatment options. Despite previous studies, its molecular genetic background has remained somewhat elusive. To comprehensively characterize the mutational landscape of this tumor type, and to identify possible targets of treatment, we conducted the first large exome sequencing study on a population-based set of SBA samples from all three small bowel segments. Archival tissue from 106 primary tumors with appropriate clinical information were available for exome sequencing from a patient series consisting of a majority of confirmed SBA cases diagnosed in Finland between the years 2003-2011. Paired-end exome sequencing was performed using Illumina HiSeq 4000, and OncodriveFML was used to identify driver genes from the exome data. We also defined frequently affected cancer signalling pathways and performed the first extensive allelic imbalance (Al) analysis in SBA. Exome data analysis revealed significantly mutated genes previously linked to SBA (TP53, KRAS, APC, SMAD4, and BRAF), recently reported potential driver genes (SOX9, ATM, and ARID2), as well as novel candidate driver genes, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We also identified clear mutation hotspot patterns in ERBB2 and BRAF. No BRAF V600E mutations were observed. Additionally, we present a comprehensive mutation signature analysis of SBA, highlighting established signatures 1A, 6, and 17, as well as U2 which is a previously unvalidated signature. Finally, comparison of the three small bowel segments revealed differences in tumor characteristics. This comprehensive work unveils the mutational landscape and most frequently affected genes and pathways in SBA, providing potential therapeutic targets, and novel and more thorough insights into the genetic background of this tumor type.Peer reviewe

Auxin-jasmonate crosstalk in Oryza sativa L. root system formation after cadmium and/or arsenic exposure

Soil pollutants may affect root growth through interactions among phytohormones like auxin and jasmonates. Rice is frequently grown in paddy fields contaminated by cadmium and arsenic, but the effects of these pollutants on jasmonates/auxin crosstalk during adventitious and lateral roots formation are widely unknown. Therefore, seedlings of Oryza saliva cv. Nihonmasari and of the jasmonate-biosynthetic mutant coleoptile photomorphogenesis2 were exposed to cadmium and/or arsenic, and/or jasmonic acid methyl ester, and then analysed through morphological, histochemical, biochemical and molecular approaches. In both genotypes, arsenic and cadmium accumulated in roots more than shoots. In the roots, arsenic levels were more than twice higher than cadmium levels, either when arsenic was applied alone, or combined with cadmium. Pollutants reduced lateral root density in the wild-type in every treatment condition, but jasmonic acid methyl ester increased it when combined with each pollutant. Interestingly, exposure to cadmium and/or arsenic did not change lateral root density in the mutant. The transcript levels of OsASA2 and OsYUCCA2, auxin biosynthetic genes, increased in the wild-type and mutant roots when pollutants and jasmonic acid methyl ester were applied alone. Auxin (indole-3-acetic acid) levels transiently increased in the roots with cadmium and/or arsenic in the wild-type more than in the mutant. Arsenic and cadmium, when applied alone, induced fluctuations in bioactive jasmonate contents in wild-type roots, but not in the mutant. Auxin distribution was evaluated in roots of OsDR5::GUS seedlings exposed or not to jasmonic acid methyl ester added or not with cadmium and/or arsenic. The DR5::GUS signal in lateral roots was reduced by arsenic, cadmium, and jasmonic acid methyl ester. Lipid peroxidation, evaluated as malondialdehyde levels, was higher in the mutant than in the wild-type, and increased particularly in As presence, in both genotypes. Altogether, the results show that an auxin/jasmonate interaction affects rice root system development in the presence of cadmium and/or arsenic, even if exogenous jasmonic acid methyl ester only slightly mitigates pollutants toxicity