Genetic modulation of ABA mediating stress response in the genus populus

Genetische Modulation der ABA-vermittelten Stressantwort in der Gattung Populus Das Phytohormon Abscisinsäure (ABA) spielt eine entscheidende Rolle bei der Stressantwort auf Trockenheit und Salinität. Über die Regulation der ABA-Biosynthese im Modellorganismus für Bäume, der Pappel, ist bisher kaum etwas bekannt. Neben der NCED3 wird dem Molybdoenzym Aldehydoxidase (AO) hier eine wichtige Rolle zugesprochen. Die Untersuchung der Regulation der ABA-Synthese durch die AO steht im Fokus dieser Arbeit. Es wird vermutet, dass die ABA-Biosynthese nicht nur in verschiedenen Pflanzenorganen und Entwicklungsstadien unterschiedlich reguliert wird, sondern auch in verschiedenen Spezies. Deshalb trägt diese Arbeit dazu bei, die Regulation der ABA-Synthese im Besonderen für holzige Arten zu klären. Für die Untersuchungen wurden erstmalig transgene P. x canescens-Pflanzen erzeugt, die eine genetische Modulation im letzten Schritt der ABA-Biosynthese besitzen. Es konnten für die AO und ihren Aktivator ABA3 jeweils knock down-Pflanzen mittels RNAi und Überexprimierer erzeugt werden. Auch ein ABA3-Promoterkonstrukt, das GUS als Reportergen enthält, wurde transformiert und regeneriert. Die knock down-Pflanzen und Überexprimierer zeigten morphologisch keine Unterschiede zum Wildtyp. Die AO-Aktivität war in beiden Typen von RNAi-Pflanzen drastisch reduziert, die XDH-Aktivität zusätzlich in den ABA3-RNAi-Linien. Im unbehandelten Zustand war keine Auswirkung auf den ABA-Gehalt der Transformanten zu beobachten. Obwohl sich die AO-Aktivitäten in den Salz- und Trockenstressexperimenten nicht signifikant veränderten, war ein drastischer Anstieg des ABA-Gehaltes, auch in den RNAi-Pflanzen, zu beobachten. Der ABA3-Promoter zeigte keine Induktion unter Salz- und Trockenstress. Diese Ergebnisse indizieren eine geringe Bedeutung der AO bei der ABA-Biosynthese bei Pappel. Möglicherweise wird die Biosynthese in erster Linie durch die NCED kontrolliert oder ein alternativer Pathway über die Reduktion AB-Aldehyd zu AB-Alkohol und anschließende Oxidation zu ABA spielt eine Rolle.Genetic modulation of ABA mediating stress response in the genus populus The phytohormone abscisic acid (ABA) plays an essential role in stress response to drought and salinity. In the model tree poplar there is hardly anything known about the regulation of ABA-biosynthesis. Besides NCED3, the molybdo-enzyme aldehyde oxidase (AO) is ascribed an important role here. The analysis of regulation of ABA-synthesis via AO is the focus of this work. ABA-synthesis is assumed to be differently regulated in diverse plant organs and states of development, but also in various species. Therefore this work contributes to decipher the regulation of ABA-biosynthesis in woody species. For the first time transgenic P. x canescens plants with a genetic modulation in the last step of ABA-synthesis were generated. For AO and its activator ABA3 knock down plants via RNAi and over-expression plants were produced. An ABA3 promoter construct was also transformed and plants were regenerated. The knock down and over-expression plants showed no morphological differences in comparison to the wild type. AO activity was reduced dramatically in both types of RNAi-plants, XDH activity additionally in the ABA3-RNAi lines. Under non-treated conditions there was no effect on ABA content in the transgenic plants. Although the AO activities in salt and drought stress experiments did not change significantly, a strong increase of ABA content, even in the RNAi plants, was observed. The ABA3 promoter showed no induction through salt and drought stress. The results indicate a minor relevance of AO in ABA-biosynthesis in poplar. Maybe the biosynthesis is primarily controlled via NCED or an alternative pathway through reduction of AB-aldehyde to AB-alcohol and subsequent oxidation to ABA plays a role

RNAi-mediated suppression of isoprene emission in poplar transiently impacts phenolic metabolism under high temperature and high light intensities: a transcriptomic and metabolomic analysis

In plants, isoprene plays a dual role: (a) as thermo-protective agent proposed to prevent degradation of enzymes/membrane structures involved in photosynthesis, and (b) as reactive molecule reducing abiotic oxidative stress. The present work addresses the question whether suppression of isoprene emission interferes with genome wide transcription rates and metabolite fluxes in grey poplar (Populusxcanescens) throughout the growing season. Gene expression and metabolite profiles of isoprene emitting wild type plants and RNAi-mediated non-isoprene emitting poplars were compared by using poplar Affymetrix microarrays and non-targeted FT-ICR-MS (Fourier transform ion cyclotron resonance mass spectrometry). We observed a transcriptional down-regulation of genes encoding enzymes of phenylpropanoid regulatory and biosynthetic pathways, as well as distinct metabolic down-regulation of condensed tannins and anthocyanins, in non-isoprene emitting genotypes during July, when high temperature and light intensities possibly caused transient drought stress, as indicated by stomatal closure. Under these conditions leaves of non-isoprene emitting plants accumulated hydrogen peroxide (H2O2), a signaling molecule in stress response and negative regulator of anthocyanin biosynthesis. The absence of isoprene emission under high temperature and light stress resulted transiently in a new chemo(pheno)type with suppressed production of phenolic compounds. This may compromise inducible defenses and may render non-isoprene emitting poplars more susceptible to environmental stress

Numerical modelling of mining subsidence, upsidence and valley closure using UDEC

Ground subsidence due to mining has been the subject of intensive research for several decades, and it remains to be an important topic confronting the mining industry today. In the Southern Coalfield of New South Wales, Australia, there is particular concern about subsidence impacts on incised river valleys – valley closure, upsidence, and the resulting localised loss of surface water under low flow conditions. Most of the reported cases have occurred when the river valley is directly undermined. More importantly, there are a number of cases where closure and upsidence have been reported above unmined coal. These latter events are especially significant as they influence decisions regarding stand-off distances and hence mine layouts and reserve recovery. The deformation of a valley indicates the onset of locally compressive stress conditions concentrated at the base of the valley. Compressive conditions are anticipated when the surface deforms in a sagging mode, for example directly above the longwall extraction; but they are not expected when the surface deforms in a hogging mode at the edge of the extraction as that area is typically in tension. To date, explanations for valley closure under the hogging mode have considered undefined compressive stress redistributions in the horizontal plane, or lateral block movements and displacement along discontinuities generated in the sagging mode. This research is investigating the possibilities of the block movement model and its role in generating compressive stresses at the base of valleys, in the tensile portion of the subsidence profile. The numerical modelling in this research project has demonstrated that the block movement proposal is feasible provided that the curvatures developed are sufficient to allow lateral block movement. Valley closure and the onset of valley base yield are able to be quantified with the possibility of using analytical solutions. To achieve this, a methodology of subsidence prediction using the Distinct Element code UDEC has been developed as an alternative for subsidence modelling and prediction for isolated longwall panels. The numerical models have been validated by comparison with empirical results, observed caving behaviour and analytical solutions, all of which are in good agreement. The techniques developed in the subsidence prediction UDEC models have then been used to develop the conceptual block movement model. The outcomes of this research have vast implications. Firstly, it is shown that valley closure and upsidence is primarily a function of ground curvature. Since the magnitude of curvature is directly related to the magnitude of vertical subsidence there is an opportunity to consider changes in the mine layout as a strategy to reduce valley closure. Secondly, with further research there is the possibility that mining companies can assess potential damage to river valleys based on how close longwall panels approach the river valley in question. This has the added advantage of optimising the required stand off distances to river valley and increasing coal recovery

El Diario de Pontevedra : periódico liberal: Ano XXIV Número 7067 - 1907 novembro 8

Quantification of total fatty acids in complemented A. thaliana lines. GC measurements of seeds and seedlings for Fig. 8c are shown. (XLSX 127 kb

Additional file 6: of Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa

Hypocotyl measurements used for calculating the effect of transgenes on postgerminative growth. Individual hypocotyl lengths are listed along with calculations for Figs. 6 and 8. EVC = Empty vector control. (XLSX 76 kb

DAG and TAG molecular species as well as acyl-CoA pool under replete and N-deplete conditions.

<p>The amounts of the molecular species of diacylglycerol (DAG) und triacylglycerol (TAG) are displayed with a threshold of 0.1 μmol/g in (A). These data were produced by neutral loss scanning which allows the determination of the sum (number of carbon atoms and of double bonds) of two (for DAG) or three FAs (for TAG) but not always the elucidation of the distinct fatty acid composition of the molecular species. Therefore, the possible fatty acid composition of the different molecular species are given in (B). (C): The diagrams show the acyl-CoA pool as comparison of day 0 (beige) and the last day of the growth kinetic of growth under replete conditions (blue), N-depletion with normal light (red) and N-depletion with high light (green). Data are mean values of 3 biological replicates, for the comprised time point 0d, 9 biological replicates were used. Error bars indicate standard deviation.</p

Growth parameters and total fatty acid composition of <i>Phaeodactylum tricornutum</i> control cultures under normal light (blue) or with N-depletion under normal light (red) or high light (green).

<p>In (A) the dry weight content [mg/ml] is depicted and in (B) the chlorophyll <i>a</i> amount in relation to the dry weight [mg/g]. Samples were taken at days 0, 2, 5 and 7 for N-replete growth and at days 0, 2, 3 and 6 for N-depleted growth. These samples were used for determination of growth parameters as well as for lipid and metabolite analysis. The total FA composition in μmol/g is displayed in (C) showing day 0 and the last time point of each condition. Day 0 comprises the mean of all conditions (beige). Day 7 of replete conditions is shown in blue, day 6 of N-deplete with normal light in red and day 6 of N-deplete with high light in green. Data are mean values of 3 biological replicates, for the comprised time point 0d, 4 biological replicates were used. Error bars indicate standard deviation.</p

Profiling analysis of the central metabolites of <i>Phaeodactylum tricornutum</i> under replete and N-deplete conditions.

<p><i>Phaeodactylum tricornutum</i> cultures were analyzed by metabolite profiling. Bar plots show mean values with standard deviations of 3 biological replicates for prominent metabolite markers of the selected clusters. The first 4 bar plots (light to dark blue) show the relative amounts of the compounds for replete conditions (0, 3, 5, 7d), while the next bar plots show the data for N-deplete conditions (0, 3, 6d) under normal light (light to dark red) or high light (light to dark green). The identity of the indicated compounds was confirmed by high resolution MS² experiments. Pathway visualization was applied using VANTED 2.1 software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164673#pone.0164673.ref039" target="_blank">39</a>].</p

Metabolite fingerprinting analysis of <i>Phaeodactylum tricornutum</i> under replete and N-deplete conditions.

<p><i>Phaeodactylum tricornutum</i> cultures grown under replete condition and normal light (NL), or grown under N-depleted conditions (-N) and normal light (NL) or high light (HL) were harvested at the indicated time points and extracted by two phase partitioning. The fingerprint of metabolites of the polar extraction phase was generated by UPLC-TOF-MS analysis. A subset of 935 high-quality features (FDR < 10⁻⁵) derived from the positive as well as the negative ionization mode were used for clustering and visualization by means of one-dimensional self-organizing map (1D-SOM, <a href="http://marvis.gobics.de/" target="_blank">http://marvis.gobics.de</a>). Prototype 2 (blue frame) represents features with reduced relative amounts under N-depleted conditions, while features combined in prototype 4–6 (red frame) are enriched under N-depletion. Horizontal and vertical dimensions correspond to prototypes and experimental conditions, respectively. The heat map colors represent average intensity values according to the color map on the right-hand side. The width of each prototype column is proportional to the number of marker candidates assigned to this prototype. Bar plots show mean values with standard deviations of 3 biological replicates for prominent metabolite markers of the selected clusters. The first 4 bar plots (light to dark blue) show the relative amounts of the compounds for replete conditions (0, 3, 5, 7d), while the next bar plots show the data for N-deplete conditions (0, 3, 6d) under normal light (light to dark red) or high light (light to dark green). The identity of the indicated compounds was confirmed by high resolution MS² experiments. Visualization was applied using VANTED 2.1 software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164673#pone.0164673.ref039" target="_blank">39</a>].</p

Acyl chain fragments for MRM and NL scanning.

<p>Acyl chain fragments for MRM and NL scanning.</p