Adapting INTACT to analyse cell-type-specific transcriptomes and nucleocytoplasmic mRNA dynamics in the Arabidopsis embryo

In the early embryo of vascular plants, the different cell types and stem cells of the seedling are specified as the embryo develops from a zygote towards maturity. How the key steps in cell and tissue specification are instructed by genome-wide transcriptional activity is poorly understood. Progress in defining transcriptional regulation at the genome-wide level in plant embryos has been hampered by difficulties associated with capturing cell-type-specific transcriptomes in this small and inaccessible structure. We recently adapted a two-component genetic nucleus labelling system called INTACT to isolate nuclei from distinct cell types at different stages of Arabidopsis thaliana embryogenesis. We have used these to generate a transcriptomic atlas of embryo development following microarray-based expression profiling. Here, we present a general description of the adapted INTACT procedure, including the two-component labelling system, seed isolation, nuclei preparation and purification, as well as transcriptomic profiling. We also compare nuclear and cellular transcriptomes from the early Arabidopsis embryo to assess nucleocytoplasmic differences and discuss how these differences can be used to infer regulation of gene activity

Tissue and Organ Initiation in the Plant Embryo : A First Time for Everything

Land plants can grow to tremendous body sizes, yet even the most complex architectures are the result of iterations of the same developmental processes: organ initiation, growth, and pattern formation. A central question in plant biology is how these processes are regulated and coordinated to allow for the formation of ordered, 3D structures. All these elementary processes first occur in early embryogenesis, during which, from a fertilized egg cell, precursors for all major tissues and stem cells are initiated, followed by tissue growth and patterning. Here we discuss recent progress in our understanding of this phase of plant life. We consider the cellular basis for multicellular development in 3D and focus on the genetic regulatory mechanisms that direct specific steps during early embryogenesis.</p

Synthesis and characterization of nanohybrid materials based on block copolymers and clays

Η ανάμειξη ανόργανων υλικών με πολυμερή οδηγεί στην παρασκευή σύνθετων υλικών τα οποία παρουσιάζουν βελτιωμένες ιδιότητες σε σχέση με τα αρχικά συστατικά. Μια σημαντική κατηγορία τέτοιων σύνθετων υλικών αποτελούν τα νανοσύνθετα υλικά στα οποία το ανόργανο πρόσθετο έχει τουλάχιστον μια διάσταση της τάξης του νανομέτρου. Σε αυτήν την κατηγορία ανήκουν τα υβρίδια που προκύπτουν από την ανάμειξη πολυμερών με πολυστρωματικούς πυριτιούχους πηλούς. Τα υλικά αυτά εμφανίζουν ανάλογα με τη δομή του σύνθετου υλικού υψηλότερη θερμική αντοχή και καλύτερες μηχανικές ιδιότητες ενώ συγχρόνως διατηρούν πλεονεκτήματα των αρχικών πολυμερών, όπως η διαφάνεια ή η ευκολία στην κατεργασία . Αντικείμενο της παρούσας εργασίας είναι η σύνθεση και η μελέτη των αλληλεπιδράσεων και των ιδιοτήτων νανοσύνθετων υλικών που αποτελούνται από πολυστρωματικούς πυριτιούχους πηλούς και δισυσταδικά συμπολυμερή πολυαιθυλενοξειδίου-πολυστυρενίου (PEO-b-PS). Ο πηλός που χρησιμοποιήθηκε ήταν ο φυσικός υδρόφιλος μοντμοριλλονίτης νατρίου (Na+) ενώ τα συμπολυμερή συντέθηκαν στο εργαστήριο με τη χρήση ελεγχόμενου ριζικού πολυμερισμού. Από την βιβλιογραφία είναι γνωστό ότι το ομοπολυμερές του PEO έχει την δυνατότητα παρεμβολής ανάμεσα στα στρώματα του μοντμοριλλονίτη Νa+καθώς είναι υδρόφιλο μόριο και επομένως οι αλληλεπιδράσεις του με την επιφάνεια του πηλού είναι ευνοϊκές. Αντίθετα το ομοπολυμερές του PS είναι υδρόφοβο με αποτέλεσμα οι αλληλεπιδράσεις του με τον πηλό να είναι μη ευνοϊκές και επομένως δεν δίνει δομές παρεμβολής αλλά φασικά διαχωρισμένες δομές. Σκοπός της εργασίας αυτής είναι να μελετήσει τι συμβαίνει στην περίπτωση των αμφίφιλων συμπολυμερών PEO-b-PS. Ποιες είναι οι συνολικές αλληλεπιδράσεις των συμπολυμερών με τον φυσικό υδρόφιλο μοντμοριλλονίτη και ποια η δομή των νανοσύνθετων υλικών που προκύπτουν. Ειδικότερα εξετάστηκε η επίδραση του μοριακού βάρους και της σύστασης των συμπολυμερών στην τελική δομή των νανοσύνθετων υλικών. Για τον σκοπό αυτό αρχικά συντέθηκαν επτά συμπολυμερή PEO-b-PS με ελεγχόμενο ριζικό πολυμερισμό μεταφοράς ατόμου (Atom Transfer Radical Polymerization, ATRP) και στην συνέχεια τα πολυμερή χαρακτηρίσθηκαν ως προς το μοριακό βάρος, την κατανομή μοριακών βαρών και την σύστασή τους. Μελετήθηκαν επίσης οι θερμικές ιδιότητες των πολυμερών και η θερμοδυναμική τους κατάσταση. Για την παρασκευή των νανοσύνθετων υλικών χρησιμοποιήθηκε η μέθοδος παρεμβολής τήγματος πολυμερούς. Στην συνέχεια χρησιμοποιήθηκε περίθλαση ακτίνων Χ για την μελέτη της δομής τους και για την εύρεση της διαστρωματικής τους απόστασης. Βρέθηκε ότι η δομή που εμφανίζουν τα νανοϋβρίδια είναι δομή παρεμβολής με διαστρωματικές αποστάσεις που μοιάζουν με αυτές που εμφανίζει το ομοπολυμερές PEO όταν αναμειγνύεται με το ίδιο ανόργανο υλικό και φαίνεται ότι ο λόγος που καθορίζει την τελική δομή σε όλες τις περιπτώσεις είναι η σύσταση PEO ως προς Na+. Για να ερευνηθεί ο ρόλος που παίζει η συστάδα του PS σε όλη την διαδικασία μελετήθηκαν οι αλληλεπιδράσεις που αναπτύσσονται ανάμεσα στις συστάδες του συμπολυμερούς και της ανόργανης επιφάνειας με την βοήθεια φασματοσκοπίας υπερύθρου. Τέλος, μελετήθηκε ο φθορισμός της συστάδας του πολυστυρενίου και η επίδραση που έχει σε αυτόν η θέση του πολυστυρενίου στο σύστημα. Η τεχνική που χρησιμοποιήθηκε για τον σκοπό αυτό είναι η φθορισμομετρία η οποία έδειξε ότι ο εγγενής φθορισμός του πολυστυρενίου χάνεται όταν αυτό βρίσκεται περιορισμένο μεταξύ των στρωμάτων του ανόργανου υλικού. Στην περίπτωση των νανοσύνθετων υλικών Na+/PEO-b-PS ο φθορισμός του PS είναι μετρήσιμος, γεγονός που αποδεικνύει ότι η συστάδα του PS βρίσκεται έξω από τις γαλαρίες.Mixing polymers with inorganic materials leads to the synthesis of composites, which have improved properties with respect to the original materials. An important category among them, are the nanohybrids in which the inorganic component has at least one dimension at the nanometer scale. This category includes hybrids that are formed by mixing polymers with layered silicate clays. Such materials exhibit (depending on their structure) high thermal resistance and improved mechanical properties, whereas they retain the advantages of the initial polymers, such as the transparency or the easy processability. The purpose of the present project is the synthesis and the study of the interactions and the properties of nanocomposite materials which consist of layered silicate clays and poly(ethylene oxide)-b-poly(styrene) (PEO-b-PS) diblock copolymers. The clay used is a natural hydrophilic sodium montmorillonite (Na+) whereas the copolymers were synthesized in the lab, in the framework of this work, utilizing controlled radical polymerization. It is well known from the literature that the PEO homopolymer has the ability to intercalate between the layers of the clay, as it is hydrophilic molecule, therefore has favorable interactions with the surface of the clay. On the other hand, the PS homopolymer is hydrophobic molecule and has unfavorable interactions with the clay, leading to phase separated structures. This study is focalized in the case of PEO-b-PS amphiphilic copolymers. The total interactions of the copolymers with the natural montmorillonite as well as the structure of the resulted nanocomposite materials are investigated. Especially, the influence of the molecular weight and the concentration of the copolymers to the final structure of the nanocomposite materials is examined in detail. Seven PEO-b-PS copolymers with varying molecular weight and composition were synthesized utilizing controlled Atom Transfer Radical Polymerization (ATRP). Their molecular weight and their polydispersity were evaluated with Size Exclusion Chromatography (SEC) whereas their composition was measured with 1H Nuclear Magnetic Resonance (1H NMR). The thermal properties and the thermodynamic condition of the polymers were also studied with Differential Scanning Calorimetry (DSC) and Small Angle X-ray Scattering (SAXS) respectively. For the preparation of the nanocomposite materials, the polymer melt intercalation method was used and hybrids with composition that varied from pure polymer to pure clay were synthesized. Following the synthesis, X-ray diffraction (XRD) was utilized to study the structure and to determine the interlayer distance. It was found that all nanohybrids have intercalated structure with interlayer distances that are similar to the ones that correspond to the respective of PEO homopolymer when it is mixed with the same inorganic material. There is, thus, a strong indication that in every case the final structure is determined by just the Na+/PEO ratio in the copolymer hybrids. In order to investigate the role of the PS block in the system, the interactions between the blocks of the copolymer and the inorganic surface were studied by infrared spectroscopy (FTIR). Finally, the fluorescence of the PS block as well as the way it is affected by whether the polystyrene is intercalated or not was studied. It was proved that the intrinsic fluorescence of PS is vanished when it is intercalated between the inorganic layers. In the case of the Na+/PEO-b-PS nanocomposites the PS fluorescence is measurable, evidencing that the PS block is outside of the inorganic galleries and only the PEO block of the copolymer intercalates

Publisher Correction : Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo

In the version of this Resource originally published, the author information was incorrect. Jos R. Wendrich should have had a present address: Department of Plant Biotechnology and Bioinformatics and VIB Center for Plant Systems Biology, Ghent University, Technologiepark 927, 9052 Ghent, Belgium. Mark Boekschoten and Guido J. Hooiveld should have been affiliated to the Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, 6708 WE Wageningen, The Netherlands. In addition, the version of Supplementary Table 5 originally published with this Resource was not the intended final version and included inaccurate citations to the display items of the Resource, and the file format and extension did not match. These errors have now been corrected in all versions of the Resource

Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo

During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered-in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell-Type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with a focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-Temporal manner, probably signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue-Snd cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development

Publisher Correction : Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo

In the version of this Resource originally published, the author information was incorrect. Jos R. Wendrich should have had a present address: Department of Plant Biotechnology and Bioinformatics and VIB Center for Plant Systems Biology, Ghent University, Technologiepark 927, 9052 Ghent, Belgium. Mark Boekschoten and Guido J. Hooiveld should have been affiliated to the Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, 6708 WE Wageningen, The Netherlands. In addition, the version of Supplementary Table 5 originally published with this Resource was not the intended final version and included inaccurate citations to the display items of the Resource, and the file format and extension did not match. These errors have now been corrected in all versions of the Resource

Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo

During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered-in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell-Type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with a focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-Temporal manner, probably signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue-Snd cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development

Prokaryotic Responses to Ammonium and Organic Carbon Reveal Alternative CO2 Fixation Pathways and Importance of Alkaline Phosphatase in the Mesopelagic North Atlantic

To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC) fixation, community composition (16S rRNA sequencing) and community gene expression (metatranscriptomics) in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e., pyruvate plus acetate) were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates—assumed to be related to autotrophic metabolisms—were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention