Discovery of organellar calcium signaling components in Arabidopsis thaliana

In dieser Dissertation wird die Rolle von Organellen in der Kalzium-abhängigen Signaltransduktion in Pflanzen untersucht. Pflanzen und andere eukaryotische Organismen nutzen Kalzium als sekundären Botenstoff („secondary messenger“), um sich schnell an veränderte Umweltbedingungen anzupassen, oder um ihre Entwicklungsprozesse zu steuern. Die Bedeutung von Kalzium-abhängiger Signaltransduktion („calcium signaling“) in der Pflanzenphysiologie ist heute gut etabliert, ist aber bisher hauptsächlich auf zytosolische Prozesse fokussiert. Um weitere Einblicke in „calcium signaling“ in pflanzlichen Organellen zu bekommen, habe ich den Einfluss von Kalzium auf Plastiden, Mitochondrien und Peroxisomen untersucht, und zum Teil auch den Einfluss dieser Organellen auf das „calcium signaling“ im Rest der Zelle. Ich habe meine Arbeit mit einer Suche nach Kalzium-bindenden Proteinen in Chloroplasten in einem gerichteten proteomischen Ansatz und intensiven Recherchen in Datenbanken begonnen. Am Ende waren die Resultate dieser Arbeit nicht nur auf Plastiden beschränkt, da ich auch neuartige Komponenten des „calcium signaling“ in Mitochondrien und Peroxisomen entdeckt habe. Die Proteomischen Ansätze führten zur Identifizierung von zwei bestätigten Kalzium-bindenden Proteinen im Chloroplasten: LENA (Little E-enriched protein A), welches vermutlich eine Komponente des lange gesuchten Kalziumspeichers im Plastiden darstellt, und SAMTL (S adenosyl methionine transporter-like), einem Mitglied der Mitochondrial Carrier Familie (MCF), welches vermutlich in einer Kalzium-abhängigen Weise SAM in Chloroplasten transportiert. Zusätzlich wurden drei weitere Kalzium-abhängige MCF Proteine identifiziert, die ATP/Phosphat Carrier APC1, 2 und 3. Phylogenetische Analysen und funktionelle Komplementation von Hefemutanten legen nahe, dass APC1-3 in Mitochondrien lokalisiert sind und eine Rolle unter Sauerstoffmangelbedingungen spielen könnten. Außerdem wurde ein organellenlokalisiertes EFHand Protein mit einer Masse von 18 kDa (OEF18) identifiziert, welches dual im äußeren Chloroplasten Envelope und in Peroxisomen lokalisiert ist und in den Prozess der Organellenteilung involviert sein lönnte. Zusätzlich zu meinen Ansätzen, neuartige organellenlokalisierte Kalzium-bindende Proteine zu identifizieren, habe ich auch einen möglichen „cross-talk“ zwischen „calcium signaling“ und Proteinphosphorylierung im Chloroplasten untersucht. Dabei konnte eindeutig eine Kalziumabhängige Phosphorylierung der drei Chloroplastenproteine, PsaN (Photosystem I Untereinheit N), der FtsH Protease VAR1 (Variegated 1) und CAS (Calcium Sensor Protein) gezeigt werden. Über die funktionelle Bedeutung dieser neuen Regulation kann soweit nur spekuliert werden, aber diese Ergebnisse bilden jetzt eine stabile Basis für weitere Untersuchungen über die Rolle der Kalziumabhängigen Protein Phosphorylierung in Chloroplasten.My thesis is focused on the role of organelles in plant calcium signaling. Plants and other eukaryotic organisms use calcium ions as secondary messengers in order to acclimate rapidly to changes of the environment or to assist in developmental programs. The importance of calcium signaling on plant physiology is well established today, but has so far focused mainly on calcium signaling in the cytosol. To get insights into plant organellar calcium signaling, I have studied the impact of calcium on plastids, mitochondria and peroxisomes, and to a minor extent also the influence of these organelles on calcium signaling in the rest of the cell. Initially, I started with a search for calcium binding proteins in the chloroplast by targeted proteomics and data mining. However, the results of this work were in the end not only restricted to plastids as I discovered novel calcium signaling components of the mitochondria and peroxisomes. The proteomic approaches resulted in the identification of two confirmed chloroplast calcium binding proteins. LENA (Little E-enriched protein A) is predicted to be a protein component of the long-hypothesized calcium storage of the plastid. SAMTL (S-adenosyl methionine transporter-like) is a member of the mitochondrial carrier family and predicted to transport SAM into chloroplasts in a calcium-dependent manner. The three calcium-dependent mitochondrial carrier family (MCF) proteins, APC1, 2 and 3 (ATP/phosphate carriers 1, 2 and 3) localize to mitochondria. Phylogenetic analysis and complementation of yeast mutants suggested APC1, 2 and 3 to be mitochondrial ATP importers, which could be needed during periods of anoxia. Furthermore an organellar EF-hand protein of 18 kDa (OEF18) was discovered in the course of a study on the impact of protein N-acylation for organellar targeting. This protein was identified by data mining as well as in the proteomic approach. Subsequent work showed that OEF18 is dually targeted to the plastid outer envelope and peroxisomal membrane and might be involved in the process of organellar division. In addition to my approaches to identify novel organellar Ca2+ binding proteins, I have studied a potential crosstalk between calcium signaling and protein phosphorylation in chloroplasts. Three chloroplast proteins, PsaN (subunit N of Photosystem I), the FstH protease VAR1 (Variegated 1) and CAS (Calcium sensing protein) were conclusively found to be phosphorylated in a calcium dependent manner. The implications of this novel regulation are speculative, but the presented findings provide a stable base to further investigate the impact of calcium dependent protein phosphorylation on chloroplast function

Plant proteases and programmed cell death

Proteolysis affects many processes in plant development and during stress responses, as well as being crucial in cellular protein homeostasis and recycling of resources. Beyond bulk degradation, proteases can have important signaling functions or affect cellular pathways by precise cleavage of signaling proteins. This special issue covers key research themes in the diverse but increasingly interconnected fields of programmed cell death (PCD) and plant protease activity. Future trends are also highlighted, such as accelerated substrate discovery facilitated by large-scale deposition of N-terminomic data to easily accessible databases, or better profiling using genetically encoded protease activity reporters

Actos de disposición de bienes sociales por un solo cónyuge

En nuestro sistema jurídico hay diversos problemas que resolver, lo que se ha tratado en esta investigación es uno de ellos, se basó en las disposiciones que hace un cónyuge sobre bienes sociales sin la intervención del otro cónyuge, pues bien, se ha partido cuando en un matrimonio rige la sociedad de gananciales y por ende los cónyuges han debido de actuar de manera conjunta para disponer o gravar bienes sociales, pero ellos omitieron seguir esta regla. Esta situación ha traído conflictos en la jurisprudencia nacional, ya que el acto arbitrario de disposición unilateral de un bien social no tiene una sanción específica, habiendo optado algunos magistrados por sancionar estos actos con la nulidad y otros con la ineficacia. Para obtener este resultado hemos recopilado sentencias en casación del año 2012 al año 2014, y se han seleccionado las que aporten al desarrollo de la investigación, también se han realizado entrevistas a conocedores del Derecho con experiencia en este ámbito. Habiéndose llegado a las conclusiones que existen diferentes criterios para la interpretación de la inobservancia al Art. 315 del Código Civil, y que no hay una sanción uniforme la cual deban seguir los magistrados generando incertidumbre para las partes en un proceso así como también para los abogados defensores

The ROS wheel: refining ROS transcriptional footprints

In the last decade, microarray studies have delivered extensive inventories of transcriptome-wide changes in messenger RNA levels provoked by various types of oxidative stress in Arabidopsis (Arabidopsis thaliana). Previous cross-study comparisons indicated how different types of reactive oxygen species (ROS) and their subcellular accumulation sites are able to reshape the transcriptome in specific manners. However, these analyses often employed simplistic statistical frameworks that are not compatible with large-scale analyses. Here, we reanalyzed a total of 79 Affymetrix ATH1 microarray studies of redox homeostasis perturbation experiments. To create hierarchy in such a high number of transcriptomic data sets, all transcriptional profiles were clustered on the overlap extent of their differentially expressed transcripts. Subsequently, meta-analysis determined a single magnitude of differential expression across studies and identified common transcriptional footprints per cluster. The resulting transcriptional footprints revealed the regulation of various metabolic pathways and gene families. The RESPIRATORY BURST OXIDASE HOMOLOG F-mediated respiratory burst had a major impact and was a converging point among several studies. Conversely, the timing of the oxidative stress response was a determining factor in shaping different transcriptome footprints. Our study emphasizes the need to interpret transcriptomic data sets in a systematic context, where initial, specific stress triggers can converge to common, aspecific transcriptional changes. We believe that these refined transcriptional footprints provide a valuable resource for assessing the involvement of ROS in biological processes in plants

N-terminal proteomics assisted profiling of the unexplored translation initiation landscape in Arabidopsis thaliana

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well-and poorly-annotated genomes

Diverse biological effects of glycosyltransferase genes from Tartary buckwheat

Background: Tartary buckwheat (Fagopyrum tataricum) is an edible cereal crop whose sprouts have been marketed and commercialized for their higher levels of anti-oxidants, including rutin and anthocyanin. UDP-glucose flavonoid glycosyltransferases (UFGTs) play an important role in the biosynthesis of flavonoids in plants. So far, few studies are available on UFGT genes that may play a role in tartary buckwheat flavonoids biosynthesis. Here, we report on the identification and functional characterization of seven UFGTs from tartary buckwheat that are potentially involved in flavonoid biosynthesis (and have varying effects on plant growth and development when overexpressed in Arabidopsis thaliana.) Results: Phylogenetic analysis indicated that the potential function of the seven FtUFGT proteins, FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, FtUFGT15, FtUFGT40, and FtUFGT41, could be divided into three Arabidopsis thaliana functional subgroups that are involved in flavonoid biosynthesis of and anthocyanin accumulation. A significant positive correlation between FtUFGT8 and FtUFGT15 expression and anthocyanin accumulation capacity was observed in the tartary buckwheat seedlings after cold stress. Overexpression in Arabidopsis thaliana showed that FtUFGT8, FtUFGT15, and FtUFGT41 significantly increased the anthocyanin content in transgenic plants. Unexpectedly, overexpression of FtUFGT6, while not leading to enhanced anthocyanin accumulation, significantly enhanced the growth yield of transgenic plants. When wild-type plants have only cotyledons, most of the transgenic plants of FtUFGT6 had grown true leaves. Moreover, the growth speed of the oxFtUFGT6 transgenic plant root was also significantly faster than that of the wild type. At later growth, FtUFGT6 transgenic plants showed larger leaves, earlier twitching times and more tillers than wild type, whereas FtUFGT15 showed opposite results. Conclusions: Seven FtUFGTs were isolated from tartary buckwheat. FtUFGT8, FtUFGT15, and FtUFGT41 can significantly increase the accumulation of total anthocyanins in transgenic plants. Furthermore, overexpression of FtUFGT6 increased the overall yield of Arabidopsis transgenic plants at all growth stages. However, FtUFGT15 shows the opposite trend at later growth stage and delays the growth speed of plants. These results suggested that the biological function of FtUFGT genes in tartary buckwheat is diverse

Extracellular peptide Kratos restricts cell death during vascular development and stress in Arabidopsis

During plant vascular development, xylem tracheary elements (TEs) form water-conducting, empty pipes by genetically regulated cell death. Cell death is prevented from spreading to non-TEs by unidentified intercellular mechanisms, downstream of METACASPASE9 (MC9)-mediated regulation of autophagy in TEs. Here, we identified differentially abundant extracellular peptides in vascular-differentiating wild-type and MC9-down-regulated Arabidopsis cell suspensions. A peptide named Kratos rescued the abnormally high ectopic non-TE death resulting from either MC9 knockout or TE-specific overexpression of the ATG5 autophagy protein during experimentally induced vascular differentiation in Arabidopsis cotyledons. Kratos also reduced cell death following mechanical damage and extracellular ROS production in Arabidopsis leaves. Stress-induced but not vascular non-TE cell death was enhanced by another identified peptide, named Bia. Bia is therefore reminiscent of several known plant cell death-inducing peptides acting as damage-associated molecular patterns. In contrast, Kratos plays a novel extracellular cell survival role in the context of development and during stress response.Peer reviewe

Pars Pro Toto: Every Single Cell Matters

Compared to other species, plants stand out by their unparalleled self-repair capacities. Being the loss of a single cell or an entire tissue, most plant species are able to efficiently repair the inflicted damage. Although this self-repair process is commonly referred to as “regeneration,” depending on the type of damage and organ being affected, subtle to dramatic differences in the modus operandi can be observed. Recent publications have focused on these different types of tissue damage and their associated response in initiating the regeneration process. Here, we review the regeneration response following loss of a single cell to a complete organ, emphasizing key molecular players and hormonal cues involved in the model species Arabidopsis thaliana. In addition, we highlight the agricultural applications and techniques that make use of these regenerative responses in different crop and tree species.</jats:p