TY - JOUR A1 - Zhang, Yunming A1 - Ramming, Anna A1 - Heinke, Lisa A1 - Altschmied, Lothar A1 - Slotkin, R. Keith A1 - Becker, Jörg D. A1 - Kappel, Christian A1 - Lenhard, Michael T1 - The poly(A) polymerase PAPS1 interacts with the RNA-directed DNA-methylation pathway in sporophyte and pollen development JF - The plant journal N2 - RNA-based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre-mRNAs into mature mRNAs ready for translation and RNA-based silencing processes, such as RNA-directed DNA methylation (RdDM). Polyadenylation of pre-mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence-labelling strategy, we have characterized this defect in more detail using RNA and small-RNA sequencing. In addition to global defects in the expression of pollen-differentiation genes, paps1 null-mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21- and particularly 24-nucleotide-long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double-mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double-mutant of paps1 and rna-dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast. KW - poly(A) polymerase KW - RNA-directed DNA methylation KW - pollen development KW - siRNAs KW - transposable elements KW - gynoecium development KW - Arabidopsis thaliana Y1 - 2019 U6 - https://doi.org/10.1111/tpj.14348 SN - 0960-7412 SN - 1365-313X VL - 99 IS - 4 SP - 655 EP - 672 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Wang, Meng A1 - Li, Panpan A1 - Ma, Yao A1 - Nie, Xiang A1 - Grebe, Markus A1 - Men, Shuzhen T1 - Membrane sterol composition in Arabidopsis thaliana affects root elongation via auxin biosynthesis JF - International journal of molecular sciences N2 - Plant membrane sterol composition has been reported to affect growth and gravitropism via polar auxin transport and auxin signaling. However, as to whether sterols influence auxin biosynthesis has received little attention. Here, by using the sterol biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) and sterol application, we reveal that cycloeucalenol, a CPI1 substrate, and sitosterol, an end-product of sterol biosynthesis, antagonistically affect auxin biosynthesis. The short root phenotype of cpi1-1 was associated with a markedly enhanced auxin response in the root tip. Both were neither suppressed by mutations in polar auxin transport (PAT) proteins nor by treatment with a PAT inhibitor and responded to an auxin signaling inhibitor. However, expression of several auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) was upregulated in cpi1-1. Functionally, TAA1 mutation reduced the auxin response in cpi1-1 and partially rescued its short root phenotype. In support of this genetic evidence, application of cycloeucalenol upregulated expression of the auxin responsive reporter DR5:GUS (beta-glucuronidase) and of several auxin biosynthesis genes, while sitosterol repressed their expression. Hence, our combined genetic, pharmacological, and sterol application studies reveal a hitherto unexplored sterol-dependent modulation of auxin biosynthesis during Arabidopsis root elongation. KW - Arabidopsis thaliana KW - auxin KW - auxin biosynthesis KW - cycloeucalenol KW - CPI1 KW - sitosterol KW - sterol Y1 - 2021 U6 - https://doi.org/10.3390/ijms22010437 SN - 1422-0067 VL - 22 IS - 1 PB - MDPI CY - Basel ER - TY - THES A1 - Vyse, Kora T1 - Elucidating molecular determinants of the loss of freezing tolerance during deacclimation after cold priming and low temperature memory after triggering N2 - Während ihrer Entwicklung müssen sich Pflanzen an Temperaturschwankungen anpassen. Niedrige Temperaturen über dem Gefrierpunkt induzieren in Pflanzen eine Kälteakklimatisierung und höhere Frosttoleranz, die sich bei wärmeren Temperaturen durch Deakklimatisierung wieder zurückbildet. Der Wechsel zwischen diesen beiden Prozessen ist für Pflanzen unerlässlich, um als Reaktion auf unterschiedliche Temperaturbedingungen eine optimale Fitness zu erreichen. Die Kälteakklimatisierung ist umfassend untersucht worden,über die Regulierung der Deakklimatisierung ist jedoch wenig bekannt. In dieser Arbeit wird der Prozess der Deakklimatisierung auf physiologischer und molekularer Ebene in Arabidopsis thaliana untersucht. Messungen des Elektrolytverlustes während der Kälteakklimatisierung und bis zu vier Tagen nach Deakklimatisierung ermöglichten die Identifizierung von vier Knockout-Mutanten (hra1, lbd41, mbf1c und jub1), die im Vergleich zum Wildtyp eine langsamere Deakklimatisierungsrate aufwiesen. Eine transkriptomische Studie mit Hilfe von RNA-Sequenzierung von A. thaliana Col-0, jub1 und mbf1c zeigte die Bedeutung der Hemmung von stressreaktiven und Jasmonat-ZIM-Domänen-Genen sowie die Regulierung von Zellwandmodifikationen während der Deakklimatisierung. Darüber hinaus zeigten Messungen der Alkoholdehydrogenase Aktivität und der Genexpressionsänderungen von Hypoxiemarkern während der ersten vier Tagen der Deakklimatisierung, dass eine Hypoxie-Reaktion während der Deakklimatisierung aktiviert wird. Es wurde gezeigt, dass die epigenetische Regulierung während der Kälteakklimatisierung und der 24-stündigen Deakklimatisierung in A. thaliana eine große Rolle spielt. Darüber hinaus zeigten beide Deakklimatisierungsstudien, dass die frühere Hypothese, dass Hitzestress eine Rolle bei der frühen Deakklimatisierung spielen könnte, unwahrscheinlich ist. Eine Reihe von DNA- und Histondemethylasen sowie Histonvarianten wurden während der Deakklimatisierung hochreguliert, was auf eine Rolle im pflanzlichen Gedächtnis schließen lässt. In jüngster Zeit haben mehrere Studien gezeigt, dass Pflanzen in der Lage sind, die Erinnerung an einen vorangegangenen Kältestress auch nach einer Woche Deakklimatisierung zu bewahren. In dieser Arbeit ergaben Transkriptom- und Metabolomanalysen von Arabidopsis während 24 Stunden Priming (Kälteakklimatisierung) und Triggering (wiederkehrender Kältestress nach Deakklimatisierung) eine unikale signifikante und vorübergehende Induktion der Transkriptionsfaktoren DREB1D, DREB1E und DREB1F während des Triggerings, die zur Feinabstimmung der zweiten Kältestressreaktion beiträgt. Darüber hinaus wurden Gene, die für Late Embryogenesis Abundant (LEA) und Frostschutzproteine kodieren, sowie Proteine, die reaktive Sauerstoffspezies entgiften, während des späten Triggerings (24 Stunden) stärker induziert als nach dem ersten Kälteimpuls, während Xyloglucan- Endotransglucosylase/Hydrolase Gene, deren Produkte für eine Restrukturierung der Zellwand verantwortlich sind, früh auf das Triggering reagierten. Die starke Induktion dieser Gene, sowohl bei der Deakklimatisierung als auch beim Triggering, lässt vermuten, dass sie eine wesentliche Rolle bei der Stabilisierung der Zellen während des Wachstums und bei der Reaktion auf wiederkehrende Stressbedingungen spielen. Zusammenfassend gibt diese Arbeit neue Einblicke in die Regulierung der Deakklimatisierung und des Kältestress-Gedächtnisses in A. thaliana und eröffnet neue Möglichkeiten für künftige, gezielte Studien von essentiellen Genen in diesem Prozess. N2 - Throughout their lifetime plants need to adapt to temperature changes. Plants adapt to nonfreezing cold temperatures in a process called cold priming (cold acclimation) and lose the acquired freezing tolerance during warmer temperatures through deacclimation. The alternation of both processes is essential for plants to achieve optimal fitness in response to different temperature conditions. Cold acclimation has been extensively studied, however, little is known about the regulation of deacclimation. This thesis elucidates the process of deacclimation on a physiological and molecular level in Arabidopsis thaliana. Electrolyte leakage measurements during cold acclimation and up to four days of deacclimation enabled the identification of four knockout mutants (hra1, lbd41, mbf1c and jub1) with a slower rate of deacclimation compared to the wild type. A transcriptomic study using RNA-Sequencing in A. thaliana Col-0, jub1 and mbf1c identified the importance of the inhibition of stress responsive and Jasmonate-ZIM-domain genes as well as the regulation of cell wall modifications during deacclimation. Moreover, measurements of alcohol dehydrogenase activity and gene expression changes of hypoxia markers during the first four days of deacclimation evidently showed that a hypoxia response is activated during deacclimation. Epigenetic regulation was observed to be extensively involved during cold acclimation and 24 h of deacclimation in A. thaliana. Further, both deacclimation studies showed that the previous hypothesis that heat stress might play a role in early deacclimation, is not likely. A number of DNA- and histone demethylases as well as histone variants were upregulated during deacclimation suggesting a role in plant memory. Recently, multiple studies have shown that plants are able to retain memory of a previous cold stress even after a week of deacclimation. In this work, transcriptomic and metabolomic analyses of Arabidopsis during 24 h of priming (cold acclimation) and triggering (recurring cold stress after deacclimation) revealed a uniquely significant and transient induction of DREB1D, DREB1E and DREB1F transcription factors during triggering contributing to fine-tuning of the second cold stress response. Furthermore, genes encoding Late Embryogenesis Abundant (LEA) and antifreeze proteins and proteins detoxifying reactive oxygen species were higher induced during late triggering (24 h) compared to primed samples, while cell wall remodelers of the class xyloglucan endotransglucosylase/hydrolase were early responders of triggering. The high induction of cell wall remodelers during deacclimation as well as triggering proposes that these proteins play an essential role in the stabilization of the cells during growth as well as the response to recurring stresses. Collectively this work gives new insights on the regulation of deacclimation and cold stress memory in A. thaliana and opens the door to future targeted studies of essential genes in this process. KW - cold stress KW - deacclimation KW - Arabidopsis thaliana KW - epigenetics KW - co-expression network analysis KW - WGCNA KW - RNA-sequencing KW - differential gene expression KW - hypoxia KW - transcription factors KW - Kältestress KW - Deakklimatisierung KW - Epigenetik KW - Koexpression Netzwerk Analysen KW - RNA-Sequenzierung KW - Differenzielle Genexpression KW - Hypoxie KW - Transkriptionsfaktoren Y1 - 2022 ER - TY - THES A1 - von Bismarck, Thekla T1 - The influence of long-term light acclimation on photosynthesis in dynamic light N2 - Photosynthesis converts light into metabolic energy which fuels plant growth. In nature, many factors influence light availability for photosynthesis on different time scales, from shading by leaves within seconds up to seasonal changes over months. Variability of light energy supply for photosynthesis can limit a plant´s biomass accumulation. Plants have evolved multiple strategies to cope with strongly fluctuation light (FL). These range from long-term optimization of leaf morphology and physiology and levels of pigments and proteins in a process called light acclimation, to rapid changes in protein activity within seconds. Therefore, uncovering how plants deal with FL on different time scales may provide key ideas for improving crop yield. Photosynthesis is not an isolated process but tightly integrates with metabolism through mutual regulatory interactions. We thus require mechanistic understanding of how long-term light acclimation shapes both, dynamic photosynthesis and its interactions with downstream metabolism. To approach this, we analyzed the influence of growth light on i) the function of known rapid photosynthesis regulators KEA3 and VCCN1 in dynamic photosynthesis (Chapter 2-3) and ii) the interconnection of photosynthesis with photorespiration (PR; Chapter 4). We approached topic (i) by quantifying the effect of different growth light regimes on photosynthesis and photoprotection by using kea3 and vccn1 mutants. Firstly, we found that, besides photosynthetic capacity, the activities of VCCN1 and KEA3 during a sudden high light phase also correlated with growth light intensity. This finding suggests regulation of both proteins by the capacity of downstream metabolism. Secondly, we showed that KEA3 accelerated photoprotective non-photochemical quenching (NPQ) kinetics in two ways: Directly via downregulating the lumen proton concentration and thereby de-activating pH-dependent NPQ, and indirectly via suppressing accumulation of the photoprotective pigment zeaxanthin. For topic (ii), we analyzed the role of PR, a process which recycles a toxic byproduct of the carbon fixation reactions, in metabolic flexibility in a dynamically changing light environment. For this we employed the mutants hpr1 and ggt1 with a partial block in PR. We characterized the function of PR during light acclimation by tracking molecular and physiological changes of the two mutants. Our data, in contrast to previous reports, disprove a generally stronger physiological relevance of PR under dynamic light conditions. Additionally, the two different mutants showed pronounced and distinct metabolic changes during acclimation to a condition inducing higher photosynthetic activity. This underlines that PR cannot be regarded purely as a cyclic detoxification pathway for 2PG. Instead, PR is highly interconnected with plant metabolism, with GGT1 and HPR1 representing distinct metabolic modulators. In summary, the presented work provides further insight into how energetic and metabolic flexibility is ensured by short-term regulators and PR during long-term light acclimation. N2 - Photosynthese wandelt Lichtenergie in metabolische Energie um, welche das Pflanzenwachstum antreibt. In der Natur wird die Verfügbarkeit von Licht von vielerlei Faktoren auf unterschiedlichen Zeitskalen beeinflusst, z. B. von der Beschattung durch Blätter innerhalb von Sekunden bis hin zu jahreszeitlichen Veränderungen über Monate. Fluktuationen in der Lichtenergieverfügbarkeit in der Natur kann die Biomasseakkumulation der Pflanzen limitieren. Pflanzen haben verschiedene Strategien entwickelt, um stark fluktuierendes Licht nutzen zu können. Diese reichen von der langfristigen Optimierung der Blattmorphologie und Physiologie und des Gehalts an Pigmenten und Proteinen in dem Prozess der Lichtakklimatisierung bis hin zu schnellen Veränderungen der Proteinaktivität innerhalb von Sekunden. Daher kann die Aufdeckung der Art und Weise, wie Pflanzen mit FL auf verschiedenen Zeitskalen umgehen, wichtige Ideen zur Verbesserung der Ernteerträge liefern. Die Photosynthese ist kein isolierter Prozess, sondern steht in enger Interaktion mit den nachgeschalteten Stoffwechselwegen. Daher benötigen wir mechanistisches Verständnis, wie Lichtakklimatisierung die dynamische Photosynthese als auch deren Interaktion mit Downstream-Metabolismus moduliert. Dafür haben wir den Einfluss von Lichtakklimatisierung auf i) die Funktion der schnellen Photosyntheseregulatoren KEA3 und VCCN1 in der dynamischen Photosynthese und ii) die flexible Interaktion von Photorespiration mit Photosynthese analysiert. Im ersten Themenkomplex (i) wurden die Auswirkungen verschiedener Wachstumslicht-bedingungen auf Photosynthese und Photoprotektion anhand von kea3- und vccn1-Mutanten quantifiziert. Zum einen konnten wir zeigen, dass neben der photosynthetischen Kapazität auch die Aktivitäten von VCCN1 und KEA3 während eines Hochlichtpulses mit der Wachstumslichtintensität korrelierten. Dies deutet auf eine Regulierung beider Proteine durch die Kapazität des Downstream-Metabolismus hin. Zum anderen beschleunigte KEA3 die Kinetik des photoprotektiven nicht-photochemischen Quenchings (NPQ) auf zweifache Weise: Direkt über die Herabregulierung der lumenalen Protonenkonzentration, was den pH-abhängigen NPQ deaktivierte, und indirekt über die Unterdrückung der Akkumulation des photoprotektiven Pigments Zeaxanthin. Für das zweite Thema (ii) untersuchten wir die Rolle des photorespiratorischen Metabolismus (PR), welcher ein toxisches Nebenprodukt der Kohlenstofffixierungsreaktionen recycelt, in der metabolischen Flexibilität in einer sich dynamisch verändernden Lichtumgebung. Dazu verwendeten wir die Mutanten hpr1 und ggt1 mit teilweise blockiertem PR Flux. Unsere Daten widerlegen, im Gegensatz zu früheren Berichten, eine allgemein größere physiologische Bedeutung von PR unter dynamischen Lichtbedingungen. Die beiden Mutanten zeigten ausgeprägte und distinkte metabolische Veränderungen während der Akklimatisierung an eine Bedingung mit höherer photosynthetischer Aktivität. Dies zeigt, dass PR nicht ausschließlich als zyklischer Entgiftungsweg für 2PG angesehen werden kann. Vielmehr ist PR tief in den pflanzlichen Stoffwechsel eingebettet, wobei GGT1 und HPR1 als distinkte Stellschrauben des Downstream-Metabolismus agieren. Zusammenfassend liefert die vorliegende Arbeit weitere Erkenntnisse darüber, wie die energetische und metabolische Flexibilität durch kurzfristige Regulatoren und den photorespiratorischen Metabolismus während der langfristigen Lichtakklimatisierung gewährleistet wird. KW - photosynthesis KW - fluctuating light KW - Arabidopsis thaliana KW - Photosynthese KW - fluktuierendes Licht Y1 - 2023 ER - TY - JOUR A1 - Ud-Din, Aziz A1 - Rauf, Mamoona A1 - Ghafoor, S. A1 - Khattak, M. N. K. A1 - Hameed, M. W. A1 - Shah, H. A1 - Jan, S. A1 - Muhammad, K. A1 - Rehman, A. A1 - Inamullah, T1 - Efficient use of artificial micro-RNA to downregulate the expression of genes at the post-transcriptional level in Arabidopsis thaliana JF - Genetics and molecular research N2 - Micro-RNAs are cellular components regulating gene expression at the post-transcription level. In the present study, artificial micro-RNAs were used to decrease the transcript level of two genes, AtExpA8 (encoding an expansin) and AHL25 (encoding an AT-hook motif nuclear localized protein) in Arabidopsis thaliana. The backbone of the Arabidopsis endogenous MIR319a micro-RNA was used in a site-directed mutagenesis approach for the generation of artificial micro-RNAs targeting two genes. The recombinant cassettes were expressed under the control of the CaMV 35S promoter in individual A. thaliana plants. Transgenic lines of the third generation were tested by isolating total RNA and by subsequent cDNA synthesis using oligo-dT18 primers and mRNAs as templates. The expression of the two target genes was checked through quantitative realtime polymerase chain reaction to confirm reduced transcript levels for AtExpA8 and AHL25. Downregulation of AtExpA8 resulted in the formation of short hypocotyls compared with those of the wild-type control in response to low pH and high salt concentration. This technology could be used to prevent the expression of exogenous and invading genes posing a threat to the normal cellular physiology of the host plant. KW - Artificial micro-RNA KW - Arabidopsis thaliana KW - qRT-PCR KW - AtExpA8 KW - AHL25 Y1 - 2016 U6 - https://doi.org/10.4238/gmr.15027439 SN - 1676-5680 VL - 15 PB - FUNPEC CY - Ribeirao Preto ER - TY - JOUR A1 - Thirumalaikumar, Venkatesh P. A1 - Gorka, Michal A1 - Schulz, Karina A1 - Masclaux-Daubresse, Celine A1 - Sampathkumar, Arun A1 - Skirycz, Aleksandra A1 - Vierstra, Richard D. A1 - Balazadeh, Salma T1 - Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90.1 and ROF1 JF - Autophagy N2 - In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS inArabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of annbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression ofHSPgenes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation ofNBR1resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS. KW - Arabidopsis thaliana KW - heat stress KW - HSFA2 KW - HSP90.1 KW - NBR1 KW - ROF1 KW - selective autophagy KW - stress memory KW - stress recovery Y1 - 2020 U6 - https://doi.org/10.1080/15548627.2020.1820778 SN - 1554-8635 SN - 1554-8627 VL - 17 IS - 9 SP - 2184 EP - 2199 PB - Taylor & Francis CY - Abingdon ER - TY - JOUR A1 - Tejos, Ricardo A1 - Rodriguez-Furlan, Cecilia A1 - Adamowski, Maciej A1 - Sauer, Michael A1 - Norambuena, Lorena A1 - Friml, Jiri T1 - PATELLINS are regulators of auxin-mediated PIN1 relocation and plant development in Arabidopsis thaliana JF - Journal of cell science N2 - Coordinated cell polarization in developing tissues is a recurrent theme in multicellular organisms. In plants, a directional distribution of the plant hormone auxin is at the core of many developmental programs. A feedback regulation of auxin on the polarized localization of PIN auxin transporters in individual cells has been proposed as a self-organizing mechanism for coordinated tissue polarization, but the molecular mechanisms linking auxin signalling to PIN-dependent auxin transport remain unknown. We used a microarray-based approach to find regulators of the auxin-induced PIN relocation in Arabidopsis thaliana root, and identified a subset of a family of phosphatidylinositol transfer proteins (PITPs), the PATELLINs (PATLs). Here, we show that PATLs are expressed in partially overlapping cell types in different tissues going through mitosis or initiating differentiation programs. PATLs are plasma membrane-associated proteins accumulated in Arabidopsis embryos, primary roots, lateral root primordia and developing stomata. Higher order patl mutants display reduced PIN1 repolarization in response to auxin, shorter root apical meristem, and drastic defects in embryo and seedling development. This suggests that PATLs play a redundant and crucial role in polarity and patterning in Arabidopsis. KW - PATELLIN KW - Auxin KW - Arabidopsis thaliana KW - Auxin transport KW - Canalization Y1 - 2018 U6 - https://doi.org/10.1242/jcs.204198 SN - 0021-9533 SN - 1477-9137 VL - 131 IS - 2 PB - Company of Biologists Limited CY - Cambridge ER - TY - JOUR A1 - Smirnova, Julia A1 - Fernie, Alisdair R. A1 - Spahn, Christian M. T. A1 - Steup, Martin T1 - Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants JF - Analytical biochemistry : methods in the biological sciences N2 - Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (alpha- and beta-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify beta-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels. (C) 2017 Published by Elsevier Inc. KW - Arabidopsis thaliana KW - beta-amylase assay KW - Disproportionating isozyme 2 (DPE2) dpe2-deficient plants KW - Maltose assay KW - Leaf maltose content Y1 - 2017 U6 - https://doi.org/10.1016/j.ab.2017.05.026 SN - 0003-2697 SN - 1096-0309 VL - 532 SP - 72 EP - 82 PB - Elsevier CY - San Diego ER - TY - THES A1 - Skirycz, Aleksandra T1 - Functional analysis of selected DOF transcription factors in the model plant Arabidopsis thaliana T1 - Funktionsanalyse ausgewählter DOF-Transkriptionsfaktoren bei der Modellpflanze Arabidopsis thaliana N2 - Transcription factors (TFs) are global regulators of gene expression playing essential roles in almost all biological processes, and are therefore of great scientific and biotechnological interest. This project focused on functional characterisation of three DNA-binding-with-one-zinc-finger (DOF) TFs from the genetic model plant Arabidopsis thaliana, namely OBP1, OBP2 and AtDOF4;2. These genes were selected due to severe growth phenotypes conferred upon their constitutive over-expression. To identify biological processes regulated by OBP1, OBP2 and AtDOF4;2 in detail molecular and physiological characterization of transgenic plants with modified levels of OBP1, OBP2 and AtDOF4;2 expression (constitutive and inducible over-expression, RNAi) was performed using both targeted and profiling technologies. Additionally expression patterns of studied TFs and their target genes were analyzed using promoter-GUS lines and publicly available microarray data. Finally selected target genes were confirmed by chromatin immuno-precipitation and electrophoretic-mobility shift assays. This combinatorial approach revealed distinct biological functions of OBP1, OBP2 and AtDOF4;2. Specifically OBP2 controls indole glucosinolate / auxin homeostasis by directly regulating the enzyme at the branch of these pathways; CYP83B1 (Skirycz et al., 2006). Glucosinolates are secondary compounds important for defence against herbivores and pathogens in the plants order Caparales (e.g. Arabidopsis, canola and broccoli) whilst auxin is an essential plant hormone. Hence OBP2 is important for both response to biotic stress and plant growth. Similarly to OBP2 also AtDOF4;2 is involved in the regulation of plant secondary metabolism and affects production of various phenylpropanoid compounds in a tissue and environmental specific manner. It was found that under certain stress conditions AtDOF4;2 negatively regulates flavonoid biosynthetic genes whilst in certain tissues it activates hydroxycinnamic acid production. It was hypothesized that this dual function is most likely related to specific interactions with other proteins; perhaps other TFs (Skirycz et al., 2007). Finally OBP1 regulates both cell proliferation and cell expansion. It was shown that OBP1 controls cell cycle activity by directly targeting the expression of core cell cycle genes (CYCD3;3 and KRP7), other TFs and components of the replication machinery. Evidence for OBP1 mediated activation of cell cycle during embryogenesis and germination will be presented. Additionally and independently on its effects on cell proliferation OBP1 negatively affects cell expansion via reduced expression of cell wall loosening enzymes. Summing up this work provides an important input into our knowledge on DOF TFs function. Future work will concentrate on establishing exact regulatory networks of OBP1, OBP2 and AtDOF4;2 and their possible biotechnological applications. N2 - Biologische Prozesse, wie beispielsweise das Wachstum von Organen und ganzen Organismen oder die Reaktion von Lebewesen auf ungünstige Umweltbedingungen, unterliegen zahlreichen Regulationsmechanismen. Besonders wichtige Regulatoren sind die sogenannten Transkriptionsfaktoren. Dabei handelt es sich um Proteine, die die Aktivität von Erbeinheiten, den Genen, beeinflussen. In Pflanzen gibt es etwa 2000 solcher Regulatoren. Da sie wichtige Kontrollelemente darstellen, sind sie von großem wissenschaftlichen und biotechnologischen Interesse. Im Rahmen der Doktorarbeit sollte die Funktion von drei Transkriptionsfaktoren, genannt OBP1, OBP2 und AtDOF4;2, untersucht werden. Sie wurden bei der Suche nach neuen Wachstumsregulatoren identifiziert. Als Untersuchungsobjekt diente die in der Öffentlichkeit kaum bekannte Pflanze Ackerschmalwand, lateinisch als Arabidopsis thaliana bezeichnet. Um die Funktion der Regulatoren zu entschlüsseln, wurden an der Modellpflanze genetische Veränderungen durchgeführt und die Pflanzen dann mit molekularbiologischen und physiologischen Methoden analysiert. Es zeigte sich, dass OBP1 an der Regulation der Zellteilung beteiligt ist. Alle Lebewesen sind aus Zellen aufgebaut. Gelingt es, die Zellteilung gezielt zu steuern, kann damit beispielsweise die Produktion von pflanzlicher Biomasse verbessert werden. Das OBP1-Protein übt auch einen Einfluss auf die Zellstreckung aus und beeinflusst auch auf diesem Wege das pflanzliche Wachstum. Die beiden anderen Proteine steuern Prozesse, die im Zusammenhang mit der Bildung von Pflanzeninhaltsstoffen stehen. OBP2 ist Teil eines zellulären Netzwerkes, dass die Synthese von sogenannten Glucosinolaten steuert. Glucosinolate kommen unter anderem in Broccoli und Kohl vor. Sie fungieren als Abwehrstoffe gegen Fraßinsekten. Einigen Glucosinolaten wird auch gesundheitsfördernde Wirkung zugesprochen. Das Protein AtDOF4;2 ist Komponente eines anderen Netzwerkes, dass die Bildung von Phenylpropanoiden steuert. Diese Substanzen haben strukturelle Funktion und spielen darüber hinaus eine Rolle bei der pflanzlichen Toleranz gegenüber tiefen Temperaturen. Mit der Doktorarbeit konnte das Wissen über die Transkriptionsfaktoren erheblich erweitert und die Grundlage für interessante zukünftige Arbeiten gelegt werden. Von großer Bedeutung wird es dabei sein, die Netzwerke, in die die Transkriptionsfaktoren eingebunden sind, noch besser zu verstehen. Dann wird es möglich sein, auch Teilnetzwerke gezielt zu beeinflussen, was für biotechnologische Anwendungen, beispielsweise bei der Präzisionszüchtung von nachwachsenden Rohstoffen, von zentraler Bedeutung ist. KW - Transkriptionsfaktoren KW - Arabidopsis thaliana KW - transcription factors KW - Arabidopsis thaliana KW - cell cycle KW - secondary metabolism Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16987 ER - TY - JOUR A1 - Sharma, Tripti A1 - Dreyer, Ingo A1 - Riedelsberger, Janin T1 - The role of K+ channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana JF - Frontiers in plant science N2 - Potassium (K+) is inevitable for plant growth and development. It plays a crucial role in the regulation of enzyme activities, in adjusting the electrical membrane potential and the cellular turgor, in regulating cellular homeostasis and in the stabilization of protein synthesis. Uptake of K+ from the soil and its transport to growing organs is essential for a healthy plant development. Uptake and allocation of K+ are performed by K+ channels and transporters belonging to different protein families. In this review we summarize the knowledge on the versatile physiological roles of plant K+ channels and their behavior under stress conditions in the model plant Arabidopsis thaliana. KW - plant potassium channel KW - Shaker KW - TPK KW - K-ir-like KW - Arabidopsis thaliana KW - voltage-dependent KW - voltage-independent Y1 - 2013 U6 - https://doi.org/10.3389/fpls.2013.00224 SN - 1664-462X VL - 4 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Schwarte, Sandra A1 - Wegner, Fanny A1 - Havenstein, Katja A1 - Groth, Detlef A1 - Steup, Martin A1 - Tiedemann, Ralph T1 - Sequence variation, differential expression, and divergent evolution in starch-related genes among accessions of Arabidopsis thaliana JF - Plant molecular biology : an international journal of fundamental research and genetic engineering N2 - Transitory starch metabolism is a nonlinear and highly regulated process. It originated very early in the evolution of chloroplast-containing cells and is largely based on a mosaic of genes derived from either the eukaryotic host cell or the prokaryotic endosymbiont. Initially located in the cytoplasm, starch metabolism was rewired into plastids in Chloroplastida. Relocation was accompanied by gene duplications that occurred in most starch-related gene families and resulted in subfunctionalization of the respective gene products. Starch-related isozymes were then evolutionary conserved by constraints such as internal starch structure, posttranslational protein import into plastids and interactions with other starch-related proteins. 25 starch-related genes in 26 accessions of Arabidopsis thaliana were sequenced to assess intraspecific diversity, phylogenetic relationships, and modes of selection. Furthermore, sequences derived from additional 80 accessions that are publicly available were analyzed. Diversity varies significantly among the starch-related genes. Starch synthases and phosphorylases exhibit highest nucleotide diversities, while pyrophosphatases and debranching enzymes are most conserved. The gene trees are most compatible with a scenario of extensive recombination, perhaps in a Pleistocene refugium. Most genes are under purifying selection, but disruptive selection was inferred for a few genes/substitutiones. To study transcript levels, leaves were harvested throughout the light period. By quantifying the transcript levels and by analyzing the sequence of the respective accessions, we were able to estimate whether transcript levels are mainly determined by genetic (i.e., accession dependent) or physiological (i.e., time dependent) parameters. We also identified polymorphic sites that putatively affect pattern or the level of transcripts. KW - Arabidopsis thaliana KW - Divergent evolution KW - Intraspecific genetic variation KW - Positive selection KW - Starch metabolizing enzymes KW - Transcript levels Y1 - 2015 U6 - https://doi.org/10.1007/s11103-015-0293-2 SN - 0167-4412 SN - 1573-5028 VL - 87 IS - 4-5 SP - 489 EP - 519 PB - Springer CY - Dordrecht ER - TY - JOUR A1 - Schwarte, Sandra A1 - Tiedemann, Ralph T1 - A Gene Duplication/Loss Event in the Ribulose-1,5-Bisphosphate-Carboxylase/Oxygenase (Rubisco) Small Subunit Gene Family among Accessions of Arabidopsis thaliana JF - Molecular biology and evolution N2 - Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated. KW - Arabidopsis thaliana KW - Arabidopsis lyrata KW - Rubisco KW - gene duplication KW - positive selection Y1 - 2011 U6 - https://doi.org/10.1093/molbev/msr008 SN - 0737-4038 VL - 28 IS - 6 SP - 1861 EP - 1876 PB - Oxford Univ. Press CY - Oxford ER - TY - GEN A1 - Schwarte, Sandra A1 - Brust, Henrike A1 - Steup, Martin A1 - Tiedemann, Ralph T1 - Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana T2 - BMC Research Notes N2 - Background Natural accessions of Arabidopsis thaliana are a well-known system to measure levels of intraspecific genetic variation. Leaf starch content correlates negatively with biomass. Starch is synthesized by the coordinated action of many (iso)enzymes. Quantitatively dominant is the repetitive transfer of glucosyl residues to the non-reducing ends of α-glucans as mediated by starch synthases. In the genome of A. thaliana, there are five classes of starch synthases, designated as soluble starch synthases (SSI, SSII, SSIII, and SSIV) and granule-bound synthase (GBSS). Each class is represented by a single gene. The five genes are homologous in functional domains due to their common origin, but have evolved individual features as well. Here, we analyze the extent of genetic variation in these fundamental protein classes as well as possible functional implications on transcript and protein levels. Findings Intraspecific sequence variation of the five starch synthases was determined by sequencing the entire loci including promoter regions from 30 worldwide distributed accessions of A. thaliana. In all genes, a considerable number of nucleotide polymorphisms was observed, both in non-coding and coding regions, and several amino acid substitutions were identified in functional domains. Furthermore, promoters possess numerous polymorphisms in potentially regulatory cis-acting regions. By realtime experiments performed with selected accessions, we demonstrate that DNA sequence divergence correlates with significant differences in transcript levels. Conclusions Except for AtSSII, all starch synthase classes clustered into two or three groups of haplotypes, respectively. Significant difference in transcript levels among haplotype clusters in AtSSIV provides evidence for cis-regulation. By contrast, no such correlation was found for AtSSI, AtSSII, AtSSIII, and AtGBSS, suggesting trans-regulation. The expression data presented here point to a regulation by common trans-regulatory transcription factors which ensures a coordinated action of the products of these four genes during starch granule biosynthesis. The apparent cis-regulation of AtSSIV might be related to its role in the initiation of de novo biosynthesis of granules. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 400 KW - Arabidopsis thaliana KW - starch synthases KW - genetic variation KW - transcript level Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-401128 ER - TY - THES A1 - Schaarschmidt, Stephanie T1 - Evaluation and application of omics approaches to characterize molecular responses to abiotic stresses in plants T1 - Evaluierung und Anwendung von Omics-Methoden zur Charakterisierung von abiotischem Stress in Pflanzen auf molekularer Ebene N2 - Aufgrund des globalen Klimawandels ist die Gewährleistung der Ernährungssicherheit für eine wachsende Weltbevölkerung eine große Herausforderung. Insbesondere abiotische Stressoren wirken sich negativ auf Ernteerträge aus. Um klimaangepasste Nutzpflanzen zu entwickeln, ist ein umfassendes Verständnis molekularer Veränderungen in der Reaktion auf unterschiedlich starke Umweltbelastungen erforderlich. Hochdurchsatz- oder "Omics"-Technologien können dazu beitragen, Schlüsselregulatoren und Wege abiotischer Stressreaktionen zu identifizieren. Zusätzlich zur Gewinnung von Omics-Daten müssen auch Programme und statistische Analysen entwickelt und evaluiert werden, um zuverlässige biologische Ergebnisse zu erhalten. Ich habe diese Problemstellung in drei verschiedenen Studien behandelt und dafür zwei Omics-Technologien benutzt. In der ersten Studie wurden Transkript-Daten von den beiden polymorphen Arabidopsis thaliana Akzessionen Col-0 und N14 verwendet, um sieben Programme hinsichtlich ihrer Fähigkeit zur Positionierung und Quantifizierung von Illumina RNA Sequenz-Fragmenten („Reads“) zu evaluieren. Zwischen 92% und 99% der Reads konnten an die Referenzsequenz positioniert werden und die ermittelten Verteilungen waren hoch korreliert für alle Programme. Bei der Durchführung einer differentiellen Genexpressionsanalyse zwischen Pflanzen, die bei 20 °C oder 4 °C (Kälteakklimatisierung) exponiert wurden, ergab sich eine große paarweise Überlappung zwischen den Programmen. In der zweiten Studie habe ich die Transkriptome von zehn verschiedenen Oryza sativa (Reis) Kultivaren sequenziert. Dafür wurde die PacBio Isoform Sequenzierungstechnologie benutzt. Die de novo Referenztranskriptome hatten zwischen 38.900 bis 54.500 hoch qualitative Isoformen pro Sorte. Die Isoformen wurden kollabiert, um die Sequenzredundanz zu verringern und danach evaluiert z.B. hinsichtlich des Vollständigkeitsgrades (BUSCO), der Transkriptlänge und der Anzahl einzigartiger Transkripte pro Genloci. Für die hitze- und trockenheitstolerante Sorte N22 wurden ca. 650 einzigartige und neue Transkripte identifiziert, von denen 56 signifikant unterschiedlich in sich entwickelnden Samen unter kombiniertem Trocken- und Hitzestress exprimiert wurden. In der letzten Studie habe ich die Veränderungen in Metabolitprofilen von acht Reissorten gemessen und analysiert, die dem Stress hoher Nachttemperaturen (HNT) ausgesetzt waren und während der Trocken- und Regenzeit im Feld auf den Philippinen angebaut wurden. Es wurden jahreszeitlich bedingte Veränderungen im Metabolitspiegel sowie für agronomische Parameter identifiziert und mögliche Stoffwechselwege, die einen Ertragsrückgang unter HNT-Bedingungen verursachen, vorgeschlagen. Zusammenfassend konnte ich zeigen, dass der Vergleich der RNA-seq Programme den Pflanzenwissenschaftler*innen helfen kann, sich für das richtige Werkzeug für ihre Daten zu entscheiden. Die de novo Transkriptom-Rekonstruktion von Reissorten ohne Genomsequenz bietet einen gezielten, kosteneffizienten Ansatz zur Identifizierung neuer Gene, die durch verschiedene Stressbedingungen reguliert werden unabhängig vom Organismus. Mit dem Metabolomik-Ansatz für HNT-Stress in Reis habe ich stress- und jahreszeitenspezifische Metabolite identifiziert, die in Zukunft als molekulare Marker für die Verbesserung von Nutzpflanzen verwendet werden könnten. N2 - Due to global climate change providing food security for an increasing world population is a big challenge. Especially abiotic stressors have a strong negative effect on crop yield. To develop climate-adapted crops a comprehensive understanding of molecular alterations in the response of varying levels of environmental stresses is required. High throughput or ‘omics’ technologies can help to identify key-regulators and pathways of abiotic stress responses. In addition to obtain omics data also tools and statistical analyses need to be designed and evaluated to get reliable biological results. To address these issues, I have conducted three different studies covering two omics technologies. In the first study, I used transcriptomic data from the two polymorphic Arabidopsis thaliana accessions, namely Col-0 and N14, to evaluate seven computational tools for their ability to map and quantify Illumina single-end reads. Between 92% and 99% of the reads were mapped against the reference sequence. The raw count distributions obtained from the different tools were highly correlated. Performing a differential gene expression analysis between plants exposed to 20 °C or 4°C (cold acclimation), a large pairwise overlap between the mappers was obtained. In the second study, I obtained transcript data from ten different Oryza sativa (rice) cultivars by PacBio Isoform sequencing that can capture full-length transcripts. De novo reference transcriptomes were reconstructed resulting in 38,900 to 54,500 high-quality isoforms per cultivar. Isoforms were collapsed to reduce sequence redundancy and evaluated, e.g. for protein completeness level (BUSCO), transcript length, and number of unique transcripts per gene loci. For the heat and drought tolerant aus cultivar N22, I identified around 650 unique and novel transcripts of which 56 were significantly differentially expressed in developing seeds during combined drought and heat stress. In the last study, I measured and analyzed the changes in metabolite profiles of eight rice cultivars exposed to high night temperature (HNT) stress and grown during the dry and wet season on the field in the Philippines. Season-specific changes in metabolite levels, as well as for agronomic parameters, were identified and metabolic pathways causing a yield decline at HNT conditions suggested. In conclusion, the comparison of mapper performances can help plant scientists to decide on the right tool for their data. The de novo reconstruction of rice cultivars without a genome sequence provides a targeted, cost-efficient approach to identify novel genes responding to stress conditions for any organism. With the metabolomics approach for HNT stress in rice, I identified stress and season-specific metabolites which might be used as molecular markers for crop improvement in the future. KW - Arabidopsis thaliana KW - Oryza sativa KW - RNA-seq KW - PacBio IsoSeq KW - metabolomics KW - high night temperature KW - combined heat and drought stress KW - natural genetic variation KW - differential gene expression KW - Arabidopsis thaliana KW - Oryza sativa KW - PacBio IsoSeq KW - RNA-seq KW - kombinierter Hitze- und Trockenstress KW - erhöhte Nachttemperaturen KW - Differenzielle Genexpression KW - Metabolomik KW - natürliche genetische Variation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-509630 ER - TY - JOUR A1 - Sakuraba, Yasuhito A1 - Bülbül, Selin A1 - Piao, Weilan A1 - Choi, Giltsu A1 - Paek, Nam-Chon T1 - Arabidopsis EARLY FLOWERING3 increases salt tolerance by suppressing salt stress response pathways JF - The plant journal KW - Arabidopsis thaliana KW - salt stress response KW - EARLY FLOWERING3 (ELF3) KW - reactive oxygen species KW - PHYTOCHROME INTERACTING FACTOR4 (PIF4) KW - JUNGBRUNNEN1 (JUB1/ANAC042) KW - ORESARA1 (ORE1/ANAC092) KW - SAG29 Y1 - 2017 U6 - https://doi.org/10.1111/tpj.13747 SN - 0960-7412 SN - 1365-313X VL - 92 SP - 1106 EP - 1120 PB - Wiley CY - Hoboken ER - TY - GEN A1 - Riano-Pachon, Diego Mauricio A1 - Nagel, Axel A1 - Neigenfind, Jost A1 - Wagner, Robert A1 - Basekow, Rico A1 - Weber, Elke A1 - Müller-Röber, Bernd A1 - Diehl, Svenja A1 - Kersten, Birgit T1 - GabiPD : the GABI primary database - a plant integrative "omics" database N2 - The GABI Primary Database, GabiPD (http:// www.gabipd.org/), was established in the frame of the German initiative for Genome Analysis of the Plant Biological System (GABI). The goal of GabiPD is to collect, integrate, analyze and visualize primary information from GABI projects. GabiPD constitutes a repository and analysis platform for a wide array of heterogeneous data from high-throughput experiments in several plant species. Data from different ‘omics’ fronts are incorporated (i.e. genomics, transcriptomics, proteomics and metabolomics), originating from 14 different model or crop species. We have developed the concept of GreenCards for textbased retrieval of all data types in GabiPD (e.g. clones, genes, mutant lines). All data types point to a central Gene GreenCard, where gene information is integrated from genome projects or NCBI UniGene sets. The centralized Gene GreenCard allows visualizing ESTs aligned to annotated transcripts as well as displaying identified protein domains and gene structure. Moreover, GabiPD makes available interactive genetic maps from potato and barley, and protein 2DE gels from Arabidopsis thaliana and Brassica napus. Gene expression and metabolic-profiling data can be visualized through MapManWeb. By the integration of complex data in a framework of existing knowledge, GabiPD provides new insights and allows for new interpretations of the data. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 137 KW - Phosphorylation sites KW - Arabidopsis thaliana KW - Information KW - Proteins KW - Families Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45075 ER - TY - JOUR A1 - Ralevski, Alexandra A1 - Apelt, Federico A1 - Olas, Justyna Jadwiga A1 - Müller-Röber, Bernd A1 - Rugarli, Elena I. A1 - Kragler, Friedrich A1 - Horvath, Tamas L. T1 - Plant mitochondrial FMT and its mammalian homolog CLUH controls development and behavior in Arabidopsis and locomotion in mice JF - Cellular and molecular life sciences N2 - Mitochondria in animals are associated with development, as well as physiological and pathological behaviors. Several conserved mitochondrial genes exist between plants and higher eukaryotes. Yet, the similarities in mitochondrial function between plant and animal species is poorly understood. Here, we show that FMT (FRIENDLY MITOCHONDRIA) from Arabidopsis thaliana, a highly conserved homolog of the mammalian CLUH (CLUSTERED MITOCHONDRIA) gene family encoding mitochondrial proteins associated with developmental alterations and adult physiological and pathological behaviors, affects whole plant morphology and development under both stressed and normal growth conditions. FMT was found to regulate mitochondrial morphology and dynamics, germination, and flowering time. It also affects leaf expansion growth, salt stress responses and hyponastic behavior, including changes in speed of hyponastic movements. Strikingly, Cluh(+/-) heterozygous knockout mice also displayed altered locomotive movements, traveling for shorter distances and had slower average and maximum speeds in the open field test. These observations indicate that homologous mitochondrial genes may play similar roles and affect homologous functions in both plants and animals. KW - Arabidopsis thaliana KW - Mitochondria KW - FMT KW - Hyponasty KW - Mice KW - CLUH; KW - Locomotion Y1 - 2022 U6 - https://doi.org/10.1007/s00018-022-04382-3 SN - 1420-682X SN - 1420-9071 VL - 79 IS - 6 PB - Springer International Publishing AG CY - Cham (ZG) ER - TY - JOUR A1 - Poxson, David J. A1 - Karady, Michal A1 - Gabrielsson, Roger A1 - Alkattan, Aziz Y. A1 - Gustavsson, Anna A1 - Doyle, Siamsa M. A1 - Robert, Stephanie A1 - Ljung, Karin A1 - Grebe, Markus A1 - Simon, Daniel T. A1 - Berggren, Magnus T1 - Regulating plant physiology with organic electronics JF - Proceedings of the National Academy of Sciences of the United States of America N2 - The organic electronic ion pump (OEIP) provides flow-free and accurate delivery of small signaling compounds at high spatio-temporal resolution. To date, the application of OEIPs has been limited to delivery of nonaromatic molecules to mammalian systems, particularly for neuroscience applications. However, many long-standing questions in plant biology remain unanswered due to a lack of technology that precisely delivers plant hormones, based on cyclic alkanes or aromatic structures, to regulate plant physiology. Here, we report the employment of OEIPs for the delivery of the plant hormone auxin to induce differential concentration gradients and modulate plant physiology. We fabricated OEIP devices based on a synthesized dendritic polyelectrolyte that enables electrophoretic transport of aromatic substances. Delivery of auxin to transgenic Arabidopsis thaliana seedlings in vivo was monitored in real time via dynamic fluorescent auxin-response reporters and induced physiological responses in roots. Our results provide a starting point for technologies enabling direct, rapid, and dynamic electronic interaction with the biochemical regulation systems of plants. KW - auxin KW - Arabidopsis thaliana KW - dendritic polymer KW - bioelectronics KW - polyelectrolyte Y1 - 2017 U6 - https://doi.org/10.1073/pnas.1617758114 SN - 0027-8424 VL - 114 SP - 4597 EP - 4602 PB - National Acad. of Sciences CY - Washington ER - TY - JOUR A1 - Parlitz, Steffi A1 - Kunze, Reinhard A1 - Müller-Röber, Bernd A1 - Balazadeh, Salma T1 - Regulation of photosynthesis and transcription factor expression by leaf shading and re-illumination in Arabidopsis thaliana leaves JF - Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants N2 - Leaf senescence of annual plants is a genetically programmed developmental phase. The onset of leaf senescence is however not exclusively determined by tissue age but is modulated by various environmental factors. Shading of individual attached leaves evokes dark-induced senescence. The initiation and progression of dark-induced senescence depend on the plant and the age of the affected leaf, however. In several plant species dark-induced senescence is fully reversible upon re-illumination and the leaves can regreen, but the regreening ability depends on the duration of dark incubation. We studied the ability of Arabidopsis thaliana leaves to regreen after dark-incubation with the aim to identify transcription factors (TFs) that are involved in the regulation of early dark-induced senescence and regreening. Two days shading of individual attached leaves triggers the transition into a pre-senescence state from which the leaves can largely recover. Longer periods of darkness result in irreversible senescence. Large scale qRT-PCR analysis of 1872 TF genes revealed that 649 of them are regulated in leaves during normal development, upon shading or re-illumination. Leaf shading triggered upregulation of 150 TF genes, some of which are involved in controlling senescence. Of those, 39 TF genes were upregulated after two days in the dark and regained pre-shading expression level after two days of re-illumination. Furthermore, a larger number of 422 TF genes were down regulated upon shading. In TF gene clusters with different expression patterns certain TF families are over-represented. KW - Arabidopsis thaliana KW - Dark-induced senescence KW - Expression profiling KW - Regreening KW - Transcription factor Y1 - 2011 U6 - https://doi.org/10.1016/j.jplph.2011.02.001 SN - 0176-1617 VL - 168 IS - 12 SP - 1311 EP - 1319 PB - Elsevier CY - Jena ER - TY - JOUR A1 - Pandey, Prashant K. A1 - Yu, Jing A1 - Omranian, Nooshin A1 - Alseekh, Saleh A1 - Vaid, Neha A1 - Fernie, Alisdair R. A1 - Nikoloski, Zoran A1 - Laitinen, Roosa A. E. T1 - Plasticity in metabolism underpins local responses to nitrogen in Arabidopsis thaliana populations JF - Plant Direct N2 - Nitrogen (N) is central for plant growth, and metabolic plasticity can provide a strategy to respond to changing N availability. We showed that two local A. thaliana populations exhibited differential plasticity in the compounds of photorespiratory and starch degradation pathways in response to three N conditions. Association of metabolite levels with growth-related and fitness traits indicated that controlled plasticity in these pathways could contribute to local adaptation and play a role in plant evolution. KW - Arabidopsis thaliana KW - natural variation KW - nitrogen availability KW - photorespiration KW - plasticity Y1 - 2019 U6 - https://doi.org/10.1002/pld3.186 SN - 2475-4455 VL - 3 IS - 11 PB - John Wiley & sonst LTD CY - Chichester ER - TY - JOUR A1 - Omidbakhshfard, Mohammad Amin A1 - Winck, Flavia Vischi A1 - Arvidsson, Samuel Janne A1 - Riano-Pachon, Diego M. A1 - Müller-Röber, Bernd T1 - A step-by-step protocol for formaldehyde-assisted isolation of regulatory elements from Arabidopsis thaliana JF - Journal of integrative plant biology N2 - The control of gene expression by transcriptional regulators and other types of functionally relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in all organisms. DNA transactions require the controlled interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successfully employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol will be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species. KW - Arabidopsis thaliana KW - chromatin KW - cis-regulatory elements KW - epigenomics KW - FAIRE-qPCR KW - FAIRE-seq KW - gene expression KW - gene regulatory network KW - transcription factor Y1 - 2014 U6 - https://doi.org/10.1111/jipb.12151 SN - 1672-9072 SN - 1744-7909 VL - 56 IS - 6 SP - 527 EP - 538 PB - Wiley-Blackwell CY - Hoboken ER - TY - THES A1 - Oberkofler, Vicky T1 - Molecular basis of HS memory in Arabidopsis thaliana T1 - Die molekulare Basis des Hitzestress-Gedächtnisses in Arabidopsis thaliana N2 - Plants can be primed to survive the exposure to a severe heat stress (HS) by prior exposure to a mild HS. The information about the priming stimulus is maintained by the plant for several days. This maintenance of acquired thermotolerance, or HS memory, is genetically separable from the acquisition of thermotolerance itself and several specific regulatory factors have been identified in recent years. On the molecular level, HS memory correlates with two types of transcriptional memory, type I and type II, that characterize a partially overlapping subset of HS-inducible genes. Type I transcriptional memory or sustained induction refers to the sustained transcriptional induction above non-stressed expression levels of a gene for a prolonged time period after the end of the stress exposure. Type II transcriptional memory refers to an altered transcriptional response of a gene after repeated exposure to a stress of similar duration and intensity. In particular, enhanced re-induction refers to a transcriptional pattern in which a gene is induced to a significantly higher degree after the second stress exposure than after the first. This thesis describes the functional characterization of a novel positive transcriptional regulator of type I transcriptional memory, the heat shock transcription factor HSFA3, and compares it to HSFA2, a known positive regulator of type I and type II transcriptional memory. It investigates type I transcriptional memory and its dependence on HSFA2 and HSFA3 for the first time on a genome-wide level, and gives insight on the formation of heteromeric HSF complexes in response to HS. This thesis confirms the tight correlation between transcriptional memory and H3K4 hyper-methylation, reported here in a case study that aimed to reduce H3K4 hyper-methylation of the type II transcriptional memory gene APX2 by CRISPR/dCas9-mediated epigenome editing. Finally, this thesis gives insight into the requirements for a heat shock transcription factor to function as a positive regulator of transcriptional memory, both in terms of its expression profile and protein abundance after HS and the contribution of individual functional domains. In summary, this thesis contributes to a more detailed understanding of the molecular processes underlying transcriptional memory and therefore HS memory, in Arabidopsis thaliana. N2 - Pflanzen können darauf vorbereitet werden, einen schweren Hitzestress (HS) zu überleben, indem sie zuvor einem leichten HS ausgesetzt werden. Die Information über den Priming-Stimulus wird von der Pflanze mehrere Tage lang aufrechterhalten. Diese Aufrechterhaltung der erworbenen Thermotoleranz, das so genannte HS-Gedächtnis, ist genetisch vom Erwerb der Thermotoleranz selbst trennbar, und in den letzten Jahren wurden mehrere spezifische Regulierungsfaktoren identifiziert. Auf molekularer Ebene korreliert das HS-Gedächtnis mit zwei Arten von Transkriptionsgedächtnis, Typ I und Typ II, die eine sich teilweise überschneidende Untergruppe von HS-induzierbaren Genen charakterisieren. Das Transkriptionsgedächtnis vom Typ I oder die anhaltende Induktion bezieht sich auf die anhaltende Transkriptionsinduktion eines Gens über das Niveau der Expression im ungestressten Zustand hinaus über einen längeren Zeitraum nach dem Ende der Stressbelastung. Das Transkriptionsgedächtnis des Typs II bezieht sich auf eine veränderte Transkriptionsreaktion eines Gens nach wiederholter Exposition gegenüber einem Hitzestress von ähnlicher Dauer und Intensität. Insbesondere bezieht sich dabei die verstärkte Re-Induktion auf ein Transkriptionsmuster, bei dem ein Gen nach der zweiten Stressexposition in deutlich höherem Maße induziert wird als nach der ersten. Diese Arbeit beschreibt die funktionelle Charakterisierung eines neuartigen positiven Transkriptionsregulators des Typ-I-Transkriptionsgedächtnisses, des Hitzeschock-Transkriptionsfaktors HSFA3, und vergleicht ihn mit HSFA2, einem bekannten positiven Regulator des Typ-I- und Typ-II-Transkriptionsgedächtnisses. Die Arbeit untersucht das Typ-I-Transkriptionsgedächtnis und seine Abhängigkeit von HSFA2 und HSFA3 zum ersten Mal auf genomweiter Ebene und gibt Einblick in die Bildung heteromerer HSF-Komplexe als Reaktion auf HS. Diese Arbeit bestätigt den engen Zusammenhang zwischen transkriptionellem Gedächtnis und H3K4-Hypermethylierung, über den hier in einer Fallstudie berichtet wird, die darauf abzielt, die H3K4-Hypermethylierung des Typ-II-Transkriptionsgedächtnisgens APX2 durch CRISPR/dCas9-vermitteltes Epigenom-Editing zu reduzieren. Schließlich gibt diese Arbeit einen Einblick in die Anforderungen, die ein Hitzeschock-Transkriptionsfaktor erfüllen muss, damit er als positiver Regulator des Transkriptionsgedächtnisses fungieren kann, und zwar sowohl in Bezug auf sein Expressionsprofil und seine Proteinabundanz nach HS als auch in Bezug auf den Beitrag seiner einzelnen funktionellen Domänen. Zusammenfassend trägt diese Arbeit zu einem genaueren Verständnis der molekularen Prozesse bei, die dem Transkriptionsgedächtnis und damit dem HS-Gedächtnis in Arabidopsis thaliana zugrunde liegen. KW - Arabidopsis thaliana KW - abiotic stress KW - heat stress memory KW - transcription factors KW - HSF KW - epigenome editing KW - histone methylation KW - H3K4me KW - Arabidopsis thaliana KW - H3K4me KW - Hitzeschock-Transkriptionsfaktor KW - abiotischer Stress KW - Epigenom Editierung KW - Hitzestress-Gedächtnis KW - Histon Methylierung KW - Transkriptionsfaktoren Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-569544 ER - TY - THES A1 - Nikolovski, Nino T1 - Pectin: New insights from an old polymer through pectinase-based genetic screens T1 - Pektin: Neue Einblicke in ein altes Polymer durch Pektinase-basierte genetische Screens N2 - Pectic polysaccharides, a class of plant cell wall polymers, form one of the most complex networks known in nature. Despite their complex structure and their importance in plant biology, little is known about the molecular mechanism of their biosynthesis, modification, and turnover, particularly their structure-function relationship. One way to gain insight into pectin metabolism is the identification of mutants with an altered pectin structure. Those were obtained by a recently developed pectinase-based genetic screen. Arabidopsis thaliana seedlings grown in liquid medium containing pectinase solutions exhibited particular phenotypes: they were dwarfed and slightly chlorotic. However, when genetically different A. thaliana seed populations (random T-DNA insertional populations as well as EMS-mutagenized populations and natural variations) were subjected to this treatment, individuals were identified that exhibit a different visible phenotype compared to wild type or other ecotypes and may thus contain a different pectin structure (pec-mutants). After confirming that the altered phenotype occurs only when the pectinase is present, the EMS mutants were subjected to a detailed cell wall analysis with particular emphasis on pectins. This suite of mutants identified in this study is a valuable resource for further analysis on how the pectin network is regulated, synthesized and modified. Flanking sequences of some of the T-DNA lines have pointed toward several interesting genes, one of which is PEC100. This gene encodes a putative sugar transporter gene, which, based on our data, is implicated in rhamnogalacturonan-I synthesis. The subcellular localization of PEC100 was studied by GFP fusion and this protein was found to be localized to the Golgi apparatus, the organelle where pectin biosynthesis occurs. Arabidopsis ecotype C24 was identified as a susceptible one when grown with pectinases in liquid culture and had a different oligogalacturonide mass profile when compared to ecotype Col-0. Pectic oligosaccharides have been postulated to be signal molecules involved in plant pathogen defense mechanisms. Indeed, C24 showed elevated accumulation of reactive oxygen species upon pectinase elicitation and had altered response to the pathogen Alternaria brassicicola in comparison to Col-0. Using a recombinant inbred line population three major QTLs were identified to be responsible for the susceptibility of C24 to pectinases. In a reverse genetic approach members of the qua2 (putative pectin methyltransferase) family were tested for potential target genes that affect pectin methyl-esterification. The list of these genes was determined by in silico study of the pattern of expression and co-expression of all 34 members of this family resulting in 6 candidate genes. For only for one of the 6 analyzed genes a difference in the oligogalacturonide mass profile was observed in the corresponding knock-out lines, confirming the hypothesis that the methyl-esterification pattern of pectin is fine tuned by members of this gene family. This study of pectic polysaccharides through forward and reverse genetic screens gave new insight into how pectin structure is regulated and modified, and how these modifications could influence pectin mediated signalling and pathogenicity. N2 - Pektin Polysaccharide, eine Klasse pflanzlicher Zellwand Polymere, formen eine der komplexesten natürlichen Strukturen. Trotz seiner immensen Bedeutung in der Biologie der Pflanzen sind die Kenntisse über die molekularen Mechanismen der Pektin Biosynthese, dessen Modifikation und Abbau überraschend gering. Eine Möglichkeit neue Einblicke in den pflanzlichen Pektin Metabolismus zu erhalten, ist die Identifizierung von Mutanten mit veränderter Pektinstruktur. Solche Mutanten konnten durch ein neuatiges Selektionsverfahren gefunden werden. Zieht man Keimlinge der Ackerschmalwand (Arabidopsis thaliana) in Flüssigmedium mit Pektinase an, so lässt sich ein typischer Phänotyp beobachten: Die Pflanzen sind kleinwüchsig und leicht chlorotisch. Diesem Verfahren wurden Populationen verschiedener Genotypen (Insertions Linien, EMS Mutanten, natürlich vorkommende Varianten) ausgesetzt. Auf diese Weise wurden Individuen identifiziert, die gegenüber der Pektinase Behandlung eine verminderte oder erhöhte Resistenz aufweisen, was auf eine veränderte Pektinstruktur hindeutet. Die EMS Mutanten wurden einer detaillierten Zellwand Analyse unterzogen. die so in dieser Arbeit identifizierte Kollektion von Mutanten stellt eine wertvolle Ressource für weitere Forschungsansätze zur Regulation, Biosynthese und Modifikation des Pektins dar. Die Lokalisation der Insertionen in den T-DNA Linien führte zur Identifikation interessanter Gene, zu denen der putative Zuckertransporter PEC100 gehört. Dieses Gen steht vermutlich in Verbindung mit der Synthese von Rhamnogalakturonan-I, einem Bestandteil des Pektins. In dieser Arbeit konnte PEC100 im Golgi Apparat, dem Ort der Pektin Biosynthese, lokalisiert werden. Die natürlich vorkommende Variante C24 ist besonders empfindlich gegenüber der Pektinase. Diese Empfindlichkeit konnte anhand rekombinanter Inzucht Linien auf drei bedeutende quantitative Merkmalsloci (QTL) eingegrenzt werden. C24 zeigte zudem ein gegenüber der Referenz verändertes Massenprofil der Oligogalakturonide. Diese werden derzeit als Signalmoleküle in der pflanzlichen Pathogenabwehr diskutiert, was mit der in dieser Arbeit geseigten Resistenz von C24 gegenüber Schwarzfleckigkeit verursachende Pilz (Alternaria brassicicola) korreliert. In einem revers-genetischen Ansatz wurden zudem Mitglieder der Pektin Methyltransferase Familie als potentielle Enzyme getestet, die die Pektin Methylesterifikation beeinflussen könnten. Diese Mutation in einer dieser Methyltransferasen führte zu Veränderungen des Oligogalakturonid Massenprofils. Dies bestätigt die Hypothese, dass Mitglieder dieser Genfamilie an der Regulation der Methylesterifikation von Pektin beteiligt sind. Die vorliegende Studie, in der ein genetishen Selektionverfahren und Methoden der reversen Genetik kombiniert wurden, hat neue Einblicke in die Regulation und Modifikation von Pektin geliefert. KW - Pektin KW - Pektinase KW - genetischer Screen KW - Arabidopsis thaliana KW - Zellwand KW - pectin KW - pectinase KW - genetic screen KW - Arabidopsis thaliana KW - cell wall Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-35255 ER - TY - THES A1 - Nietzsche, Madlen T1 - Identifizierung und Charakterisierung neuer Komponenten der SnRK1-Signaltransduktion in Arabidopsis thaliana T1 - Identification and characterization of novel components of SnRK1-Signalling in Arabidopsis thaliana N2 - Für alle Organismen ist die Aufrechterhaltung ihres energetischen Gleichgewichts unter fluktuierenden Umweltbedingungen lebensnotwendig. In Eukaryoten steuern evolutionär konservierte Proteinkinasen, die in Pflanzen als SNF1-RELATED PROTEIN KINASE1 (SnRK1) bezeichnet werden, die Adaption an Stresssignale aus der Umwelt und an die Limitierung von Nährstoffen und zellulärer Energie. Die Aktivierung von SnRK1 bedingt eine umfangreiche transkriptionelle Umprogrammierung, die allgemein zu einer Repression energiekonsumierender Prozesse wie beispielsweise Zellteilung und Proteinbiosynthese und zu einer Induktion energieerzeugender, katabolischer Stoffwechselwege führt. Wie unterschiedliche Signale zu einer generellen sowie teilweise gewebe- und stressspezifischen SnRK1-vermittelten Antwort führen ist bisher noch nicht ausreichend geklärt, auch weil bislang nur wenige Komponenten der SnRK1-Signaltransduktion identifiziert wurden. In dieser Arbeit konnte ein Protein-Protein-Interaktionsnetzwerk um die SnRK1αUntereinheiten aus Arabidopsis AKIN10/AKIN11 etabliert werden. Dadurch wurden zunächst Mitglieder der pflanzenspezifischen DUF581-Proteinfamilie als Interaktionspartner der SnRK1α-Untereinheiten identifiziert. Diese Proteine sind über ihre konservierte DUF581Domäne, in der ein Zinkfinger-Motiv lokalisiert ist, fähig mit AKIN10/AKIN11 zu interagieren. In planta Ko-Expressionsanalysen zeigten, dass die DUF581-Proteine eine Verschiebung der nucleo-cytoplasmatischen Lokalisierung von AKIN10 hin zu einer nahezu ausschließlichen zellkernspezifischen Lokalisierung begünstigen sowie die Ko-Lokalisierung von AKIN10 und DUF581-Proteinen im Nucleus. In Bimolekularen Fluoreszenzkomplementations-Analysen konnte die zellkernspezifische Interaktion von DUF581-Proteinen mit SnRK1α-Untereinheiten in planta bestätigt werden. Außerhalb der DUF581-Domäne weisen die Proteine einander keine große Sequenzähnlichkeit auf. Aufgrund ihrer Fähigkeit mit SnRK1 zu interagieren, dem Fehlen von SnRK1Phosphorylierungsmotiven sowie ihrer untereinander sehr variabler gewebs-, entwicklungs- und stimulusspezifischer Expression wurde für DUF581-Proteine eine Funktion als Adaptoren postuliert, die unter bestimmten physiologischen Bedingungen spezifische Substratproteine in den SnRK1-Komplex rekrutieren. Auf diese Weise könnten DUF581Proteine die Interaktion von SnRK1 mit deren Zielproteinen modifizieren und eine Feinjustierung der SnRK1-Signalweiterleitung ermöglichen. Durch weiterführende Interaktionsstudien konnten DUF581-interagierende Proteine darunter Transkriptionsfaktoren, Proteinkinasen sowie regulatorische Proteine gefunden werden, die teilweise ebenfalls Wechselwirkungen mit SnRK1α-Untereinheiten aufzeigten. Im Rahmen dieser Arbeit wurde eines dieser Proteine für das eine Beteiligung an der SnRK1Signalweiterleitung als Transkriptionsregulator vermutet wurde näher charakterisiert. STKR1 (STOREKEEPER RELATED 1), ein spezifischer Interaktionspartner von DUF581-18, gehört zu einer pflanzenspezifischen Leucin-Zipper-Transkriptionsfaktorfamilie und interagiert in Hefe sowie in planta mit SnRK1. Die zellkernspezifische Interaktion von STKR1 und AKIN10 in Pflanzen unterstützt die Vermutung der kooperativen Regulation von Zielgenen. Weiterhin stabilisierte die Anwesenheit von AKIN10 die Proteingehalte von STKR1, das wahrscheinlich über das 26S Proteasom abgebaut wird. Da es sich bei STKR1 um ein Phosphoprotein mit SnRK1-Phosphorylierungsmotiv handelt, stellt es sehr wahrscheinlich ein SnRK1-Substrat dar. Allerdings konnte eine SnRK1-vermittelte Phosphorylierung von STKR1 in dieser Arbeit nicht gezeigt werden. Der Verlust von einer Phosphorylierungsstelle beeinflusste die Homo- und Heterodimerisierungsfähigkeit von STKR1 in Hefeinteraktionsstudien, wodurch eine erhöhte Spezifität der Zielgenregulation ermöglicht werden könnte. Außerdem wurden Arabidopsis-Pflanzen mit einer veränderten STKR1-Expression phänotypisch, physiologisch und molekularbiologisch charakterisiert. Während der Verlust der STKR1-Expression zu Pflanzen führte, die sich kaum von Wildtyp-Pflanzen unterschieden, bedingte die konstitutive Überexpression von STKR1 ein stark vermindertes Pflanzenwachstum sowie Entwicklungsverzögerungen hinsichtlich der Blühinduktion und Seneszenz ähnlich wie sie auch bei SnRK1α-Überexpression beschrieben wurden. Pflanzen dieser Linien waren nicht in der Lage Anthocyane zu akkumulieren und enthielten geringere Gehalte an Chlorophyll und Carotinoiden. Neben einem erhöhten nächtlichen Stärkeumsatz waren die Pflanzen durch geringere Saccharosegehalte im Vergleich zum Wildtyp gekennzeichnet. Eine Transkriptomanalyse ergab, dass in den STKR1-überexprimierenden Pflanzen unter Energiemangelbedingungen, hervorgerufen durch eine verlängerte Dunkelphase, eine größere Anzahl an Genen im Vergleich zum Wildtyp differentiell reguliert war als während der Lichtphase. Dies spricht für eine Beteiligung von STKR1 an Prozessen, die während der verlängerten Dunkelphase aktiv sind. Ein solcher ist beispielsweise die SnRK1-Signaltransduktion, die unter energetischem Stress aktiviert wird. Die STKR1Überexpression führte zudem zu einer verstärkten transkriptionellen Induktion von Abwehrassoziierten Genen sowie NAC- und WRKY-Transkriptionsfaktoren nach verlängerter Dunkelphase. Die Transkriptomdaten deuteten auf eine stimulusunabhängige Induktion von Abwehrprozessen hin und konnten eine Erklärung für die phänotypischen und physiologischen Auffälligkeiten der STKR1-Überexprimierer liefern. N2 - For all living organism maintenance of energy homeostasis under changing environmental conditions is indispensable. In eukaryotes, evolutionary conserved protein kinases, such as the SNF1-RELATED PROTEIN KINASE1 (SnRK1) in plants, integrate environmental stress signals, nutrient availability and energy depletion during adaptational responses. Activation of SnRK1 triggers a broad transcriptional reprogramming, which in general represses energy consuming processes such as proliferation and protein biosynthesis and induces energy producing catabolic pathways. Although SnRK1 acts as a convergent point for many different environmental and metabolic signals to control growth and development, it is currently unknown how these many different signals could be translated into a cell-type or stimulusspecific response. This is also due to the fact that only a few proteins participating in SnRK1 signal transduction have yet been identified. In this work, a protein-protein interaction network of the Arabidopsis SnRK1α-subunits AKIN10/AKIN11 was established. Thereby, members of the plant specific DUF581 protein family were identified as SnRK1α interacting proteins. The highly conserved DUF581 domain possesses a zinc finger motif and mediates the interaction with AKIN10/AKIN11. In planta co-expression of AKIN10 with DUF581 proteins leads to a shift of subcellular localization from a nucleo-cytoplasmic distribution of both proteins to a nearly exclusive nuclear localization and show that AKIN10 and DUF581 proteins co-localize in nuclei of plant cells. Bimolecular fluorescence complementation analysis revealed that SnRK1α-subunits interact with DUF581 proteins in plants. Apart from their DUF581 domain there is no strong sequence similarity between DUF581 proteins. Because of their ability to interact with SnRK1, the absence of SnRK1-target motifs and their highly variable transcriptional regulation in a tissue-, development- or stimuli-specific manner, it is possible that DUF581 proteins act as adaptor proteins recruiting substrate proteins into the SnRK1 complex under defined physiological conditions. That said, DUF581 could modify the interaction of SnRK1 with its target proteins and facilitate fine-tuning of SnRK1 signal transduction. Additional interaction studies revealed further DUF581 interacting proteins such as transcription factors, protein kinases and regulatory proteins that in part were also able to interact with SnRK1α. One of these proteins which is supposed to be involved in SnRK1 signaling as a transcriptional regulator was characterized in more detail: Arabidopsis STKR1 (STOREKEPPER RELATED 1) a DUF581-18 interaction partner belongs to a plant specific leucine zipper transcription factor family and is able to interact with SnRK1 in yeast and in planta. Co-operative regulation of target genes by STKR1 and AKIN10 is supported by the specific interaction of these proteins inside the plant nucleus. Furthermore, AKIN10 seems to stabilize protein levels of STKR1 in that it attenuates its proteasomal turnover. Due to the fact that STKR1 is a phosphoprotein with putative SnRK1 target motives it is likely a SnRK1 substrate. However, SnRK1 mediated phosphorylation of STKR1 could not be shown in this work. Though, interaction studies in yeast revealed that a loss of putative phosphorylation sites influences the ability of homo- and hetero-dimerization of STKR1, possibly allowing a higher specificity during target gene regulation. Another part of this work was the phenotypic, physiological and molecular characterization of Arabidopsis plants with altered expression of STKR1. Whereas the absence of STKR1 expression results in plants without strong phenotypic abnormality compared to wildtype the overexpression leads to a strong decrease in plant growth as well as developmental retardations regarding to the induction of flowering and senescence reminiscent of SnRK1overexpressing plants. Plants of these lines were not able to accumulate anthocyanins and also contain reduced levels of chlorophyll and carotenoids. Besides a higher starch turnover in dark, these plants displayed lower sucrose contents. Microarray analysis revealed that under energy deficit stress, induced by extended darkness, a higher number of genes were differentially regulated in plants overexpressing STKR1 compared to wildtype than during the light period. This observation argues for a participation of STKR1 in processes, which are active under extended darkness, being the case for SnRK1 signaling which is strongly activated under energy deficient stress. Overexpression of STKR1 also leads to transcriptional induction of genes associated with defense like NAC and WRKY transcription factors after an extended dark. Results of transcriptome data analysis indicate a stimulus independent induction of defense associated processes and are suitable to explain phenotypical and physiological abnormality of the STKR1 overexpressing lines. KW - SnRK1 KW - Proteinkinase KW - Phosphorylierung KW - Arabidopsis thaliana KW - Energiemangel KW - phosphorylation KW - energy starvation KW - protein kinase Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-98678 ER - TY - JOUR A1 - Nguyen, Hung M. A1 - Schippers, Jos H. M. A1 - Goni-Ramos, Oscar A1 - Christoph, Mathias P. A1 - Dortay, Hakan A1 - van der Hoorn, Renier A. L. A1 - Müller-Röber, Bernd T1 - An upstream regulator of the 26S proteasome modulates organ size in Arabidopsis thaliana JF - The plant journal N2 - In both animal and plant kingdoms, body size is a fundamental but still poorly understood attribute of biological systems. Here we report that the Arabidopsis NAC transcription factor Regulator of Proteasomal Gene Expression' (RPX) controls leaf size by positively modulating proteasome activity. We further show that the cis-element recognized by RPX is evolutionarily conserved between higher plant species. Upon over-expression of RPX, plants exhibit reduced growth, which may be reversed by a low concentration of the pharmacological proteasome inhibitor MG132. These data suggest that the rate of protein turnover during growth is a critical parameter for determining final organ size. KW - Arabidopsis thaliana KW - organ size KW - evolution KW - leaf development KW - proteasome KW - gene regulatory network Y1 - 2013 U6 - https://doi.org/10.1111/tpj.12097 SN - 0960-7412 VL - 74 IS - 1 SP - 25 EP - 36 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Naseri, Gita A1 - Balazadeh, Salma A1 - Machens, Fabian A1 - Kamranfar, Iman A1 - Messerschmidt, Katrin A1 - Müller-Röber, Bernd T1 - Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae JF - ACS synthetic biology N2 - Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast. KW - Arabidopsis thaliana KW - artificial transcription factor KW - NAC transcription factor KW - synthetic biology KW - plant Y1 - 2017 U6 - https://doi.org/10.1021/acssynbio.7b00094 SN - 2161-5063 VL - 6 SP - 1742 EP - 1756 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Muntaha, Sidratul Nur A1 - Li, Xiaoping A1 - Compart, Julia A1 - Apriyanto, Ardha A1 - Fettke, Jörg T1 - Carbon pathways during transitory starch degradation in Arabidopsis differentially affect the starch granule number and morphology in the dpe2/phs1 mutant background JF - Plant physiology and biochemistry : an official journal of the Federation of European Societies of Plant Physiology N2 - The Arabidopsis knockout mutant lacking both the cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) had a dwarf-growth phenotype, a reduced and uneven distribution of starch within the plant rosettes, and a lower starch granule number per chloroplast under standard growth conditions. In contrast, a triple mutant impaired in starch degradation by its additional lack of the glucan, water dikinase (GWD) showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to the wild type. We concluded that ongoing starch degradation is mainly responsible for the observed phenotype of dpe2/phs1. Next, we generated two further triple mutants lacking either the phosphoglucan, water dikinase (PWD), or the disproportionating enzyme 1 (DPE1) in the background of the double mutant. Analysis of the starch metabolism revealed that even minor ongoing starch degradation observed in dpe2/phs1/pwd maintained the double mutant phenotype. In contrast, an additional blockage in the glucose pathway of starch breakdown, as in dpe2/phs1/ dpe1, resulted in a nearly starch-free phenotype and massive chloroplast degradation. The characterized mutants were discussed in the context of starch granule formation. KW - Starch granules KW - Starch metabolism KW - Starch granule number per KW - chloroplast KW - Starch morphology KW - LCSM KW - Arabidopsis thaliana Y1 - 2022 U6 - https://doi.org/10.1016/j.plaphy.2022.03.033 SN - 0981-9428 SN - 1873-2690 VL - 180 SP - 35 EP - 41 PB - Elsevier CY - Paris ER - TY - THES A1 - Moreno Curtidor, Catalina T1 - Elucidating the molecular basis of enhanced growth in the Arabidopsis thaliana accession Bur-0 N2 - The life cycle of flowering plants is a dynamic process that involves successful passing through several developmental phases and tremendous progress has been made to reveal cellular and molecular regulatory mechanisms underlying these phases, morphogenesis, and growth. Although several key regulators of plant growth or developmental phase transitions have been identified in Arabidopsis, little is known about factors that become active during embryogenesis, seed development and also during further postembryonic growth. Much less is known about accession-specific factors that determine plant architecture and organ size. Bur-0 has been reported as a natural Arabidopsis thaliana accession with exceptionally big seeds and a large rosette; its phenotype makes it an interesting candidate to study growth and developmental aspects in plants, however, the molecular basis underlying this big phenotype remains to be elucidated. Thus, the general aim of this PhD project was to investigate and unravel the molecular mechanisms underlying the big phenotype in Bur-0. Several natural Arabidopsis accessions and late flowering mutant lines were analysed in this study, including Bur-0. Phenotypes were characterized by determining rosette size, seed size, flowering time, SAM size and growth in different photoperiods, during embryonic and postembryonic development. Our results demonstrate that Bur-0 stands out as an interesting accession with simultaneously larger rosettes, larger SAM, later flowering phenotype and larger seeds, but also larger embryos. Interestingly, inter-accession crosses (F1) resulted in bigger seeds than the parental self-crossed accessions, particularly when Bur-0 was used as the female parental genotype, suggesting parental effects on seed size that might be maternally controlled. Furthermore, developmental stage-based comparisons revealed that the large embryo size of Bur-0 is achieved during late embryogenesis and the large rosette size is achieved during late postembryonic growth. Interestingly, developmental phase progression analyses revealed that from germination onwards, the length of developmental phases during postembryonic growth is delayed in Bur-0, suggesting that in general, the mechanisms that regulate developmental phase progression are shared across developmental phases. On the other hand, a detailed physiological characterization in different tissues at different developmental stages revealed accession-specific physiological and metabolic traits that underlie accession-specific phenotypes and in particular, more carbon resources during embryonic and postembryonic development were found in Bur-0, suggesting an important role of carbohydrates in determination of the bigger Bur-0 phenotype. Additionally, differences in the cellular organization, nuclei DNA content, as well as ploidy level were analyzed in different tissues/cell types and we found that the large organ size in Bur-0 can be mainly attributed to its larger cells and also to higher cell proliferation in the SAM, but not to a different ploidy level. Furthermore, RNA-seq analysis of embryos at torpedo and mature stage, as well as SAMs at vegetative and floral transition stage from Bur-0 and Col-0 was conducted to identify accession-specific genetic determinants of plant phenotypes, shared across tissues and developmental stages during embryonic and postembryonic growth. Potential candidate genes were identified and further validation of transcriptome data by expression analyses of candidate genes as well as known key regulators of organ size and growth during embryonic and postembryonic development confirmed that the high confidence transcriptome datasets generated in this study are reliable for elucidation of molecular mechanisms regulating plant growth and accession-specific phenotypes in Arabidopsis. Taken together, this PhD project contributes to the plant development research field providing a detailed analysis of mechanisms underlying plant growth and development at different levels of biological organization, focusing on Arabidopsis accessions with remarkable phenotypical differences. For this, the natural accession Bur-0 was an ideal outlier candidate and different mechanisms at organ and tissue level, cell level, metabolism, transcript and gene expression level were identified, providing a better understanding of different factors involved in plant growth regulation and mechanisms underlying different growth patterns in nature. N2 - Der Lebenszyklus blühender Pflanzen ist ein dynamischer Prozess, der das erfolgreiche Durchlaufen mehrerer Entwicklungsphasen impliziert. Es wurden enorme Fortschritte gemacht, um zelluläre und molekulare Regulationsmechanismen zu entschlüsseln, die diesen Phasen, der Morphogenese und dem Wachstum zu Grunde liegen. Obwohl mehrere Schlüsselregulatoren des Pflanzenwachstums oder der Entwicklungsphasenübergänge in Arabidopsis identifiziert wurden, ist nur wenig über Faktoren bekannt, die sowohl während der Embryogenese als auch während der Samenentwicklung und dem weiteren Wachstum aktiv werden. Noch viel weniger ist über akzessionspezifische Faktoren bekannt, die die Pflanzenarchitektur und Organgröße bestimmen. Bur-0 wurde als eine natürliche Arabidopsis-Akzession mit außergewöhnlich großen Samen und großer Blattrosette beschrieben. Ihr Phänotyp macht sie zu einem interessanten Kandidaten für die Untersuchung von Wachstums- und Entwicklungsaspekten in Pflanzen, jedoch muss die molekulare Basis, die diesem großen Phänotyp unterliegt, noch entschlüsselt werden. Daher war das allgemeine Ziel dieser Doktorarbeit, die molekularen Mechanismen, die dem großen Phänotyp in Bur-0 zu Grunde liegen, zu entschlüsseln und zu verstehen. Mehrere natürliche Arabidopsis-Akzessionen und spät blühende Mutantenlinien wurden in dieser Studie analysiert, so auch Bur-0. Die Phänotypen wurden durch eine detaillierte Analyse der Rosettengröße, der Samengröße, der Blütezeit, der Sprossapikalmeristemgröße und des Wachstums in verschiedenen Photoperioden, während der embryonalen und postembryonalen Entwicklung charakterisiert. Unsere Ergebnisse zeigen, dass Bur-0 als interessanter Akzession mit gleichzeitig größeren Blattrosetten, größerem Sprossapikalmeristem (SAM), späterem Blühphänotyp und größeren Samen, aber auch größeren Embryonen auffällt. Interessanterweise führten Kreuzungen zwischen den Akzessionen (F1) zu größeren Samen als die elterlichen selbstgekreuzten Akzessionen, insbesondere wenn Bur-0 als weiblicher elterlicher Genotyp verwendet wurde, was auf elterliche Effekte auf die Samengröße hindeutet, die möglicherweise mütterlicherseits kontrolliert werden. Darüber hinaus ergaben Vergleiche auf Basis von Entwicklungsstadien, dass die große Embryogröße von Bur-0 während der späten Embryogenese erreicht wird und die große Blattrosette während des späten postembryonalen Wachstums. Interessanterweise ergaben Analysen der Entwicklungsphasenprogression, dass ab der Keimung die Länge der Entwicklungsphasen während des postembryonalen Wachstums bei Bur-0 verzögert ist, was darauf hindeutet, dass im Allgemeinen die Mechanismen, die die Entwicklungsphasenprogression regulieren, über die Entwicklungsphasen hinweg geteilt werden. Andererseits ergab eine detaillierte physiologische Charakterisierung in verschiedenen Geweben in unterschiedlichen Entwicklungsstadien akzession-spezifische physiologische und metabolische Merkmale, die den akzession-spezifischen Phänotypen zu Grunde liegen. Insbesondere wurden mehr Kohlenstoff-Ressourcen, während der embryonalen und postembryonalen Entwicklung in Bur-0 gefunden, was auf eine wichtige Rolle von Kohlenhydraten bei der Bestimmung des größeren Bur-0-Phänotyps hindeutet. Zusätzlich wurden Unterschiede in der zellulären Organisation, dem DNA-Gehalt der Nuklei sowie dem Ploidiegrad in verschiedenen Geweben/Zelltypen analysiert und wir fanden heraus, dass die größere Organgröße in Bur-0 hauptsächlich auf die größeren Zellen und auch auf eine höhere Zellproliferation im SAM zurückzuführen ist, aber nicht auf einen anderen Ploidiegrad. Darüber hinaus wurden RNA-seq-Analysen von Embryonen im Torpedo- und Reifestadium sowie SAMs im vegetativen und Florenübergangsstadium von Bur-0 und Col-0 durchgeführt, um akzession-spezifische genetische Faktoren für Pflanzenphänotypen zu identifizieren, die in allen Geweben und Entwicklungsstadien während des embryonalen und postembryonalen Wachstums auftreten. Potenzielle Kandidatengene wurden identifiziert und eine weitere Validierung der Transkriptomdaten durch Expressionsanalysen neuartiger Kandidatengene sowie bekannter Schlüsselregulatoren für Organgröße und -wachstum während der embryonalen und postembryonalen Entwicklung bestätigte, dass die in dieser Studie generierten Transkriptomdatensätze mit hoher Zuverlässigkeit für die Aufklärung molekularer Mechanismen zur Regulierung des Pflanzenwachstums und akzessionspezifischer Phänotypen in Arabidopsis geeignet sind. Insgesamt trägt diese Doktorarbeit zur Forschung im Bereich der Pflanzenentwicklung bei, indem sie eine detaillierte Analyse der Mechanismen liefert, die dem Wachstum und der Entwicklung auf verschiedenen Ebenen der biologischen Organisation zu Grunde liegen, wobei der Schwerpunkt auf Arabidopsis-Akzessionen mit bemerkenswerten phänotypischen Unterschieden liegt. Dafür war die natürliche Akzession Bur-0 ein idealer Ausreißerkandidat und es wurden verschiedene Mechanismen auf Organ- und Gewebeebene, Zellebene, Stoffwechsel, Transkript- und Genexpressionsniveau identifiziert, was ein besseres Verständnis der verschiedenen Faktoren, die an der Regulierung des Pflanzenwachstums beteiligt sind, und der Mechanismen, die den verschiedenen Wachstumsmustern in der Natur zu Grunde liegen, ermöglicht. KW - Plant development KW - Plant growth KW - Arabidopsis thaliana KW - Phenotype KW - Transcriptome KW - Pflanzenentwicklung KW - Pflanzenwachstum KW - Arabidopsis thaliana KW - Phänotyp KW - Transkriptom Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-526814 ER - TY - JOUR A1 - Meyer, Rhonda C. A1 - Witucka-Wall, Hanna A1 - Becher, Martina A1 - Blacha, Anna Maria A1 - Boudichevskaia, Anastassia A1 - Dörmann, Peter A1 - Fiehn, Oliver A1 - Friedel, Svetlana A1 - von Korff, Maria A1 - Lisec, Jan A1 - Melzer, Michael A1 - Repsilber, Dirk A1 - Schmidt, Renate A1 - Scholz, Matthias A1 - Selbig, Joachim A1 - Willmitzer, Lothar A1 - Altmann, Thomas T1 - Heterosis manifestation during early Arabidopsis seedling development is characterized by intermediate gene expression and enhanced metabolic activity in the hybrids JF - The plant journal N2 - Heterosis-associated cellular and molecular processes were analyzed in seeds and seedlings of Arabidopsis thaliana accessions Col-0 and C24 and their heterotic hybrids. Microscopic examination revealed no advantages in terms of hybrid mature embryo organ sizes or cell numbers. Increased cotyledon sizes were detectable 4 days after sowing. Growth heterosis results from elevated cell sizes and numbers, and is well established at 10 days after sowing. The relative growth rates of hybrid seedlings were most enhanced between 3 and 4 days after sowing. Global metabolite profiling and targeted fatty acid analysis revealed maternal inheritance patterns for a large proportion of metabolites in the very early stages. During developmental progression, the distribution shifts to dominant, intermediate and heterotic patterns, with most changes occurring between 4 and 6 days after sowing. The highest incidence of heterotic patterns coincides with establishment of size differences at 4 days after sowing. In contrast, overall transcript patterns at 4, 6 and 10 days after sowing are characterized by intermediate to dominant patterns, with parental transcript levels showing the largest differences. Overall, the results suggest that, during early developmental stages, intermediate gene expression and higher metabolic activity in the hybrids compared to the parents lead to better resource efficiency, and therefore enhanced performance in the hybrids. KW - heterosis KW - seedlings KW - metabolite profiling KW - transcript profiling KW - morphological analysis KW - Arabidopsis thaliana KW - biomass Y1 - 2012 U6 - https://doi.org/10.1111/j.1365-313X.2012.05021.x SN - 0960-7412 VL - 71 IS - 4 SP - 669 EP - 683 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Merida, Angel A1 - Fettke, Jörg T1 - Starch granule initiation in Arabidopsis thaliana chloroplasts JF - The plant journal N2 - The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs. KW - starch granules KW - starch metabolism KW - starch granule initiation KW - starch KW - granule number per chloroplast KW - starch morphology KW - Arabidopsis thaliana Y1 - 2021 U6 - https://doi.org/10.1111/tpj.15359 SN - 0960-7412 SN - 1365-313X VL - 107 IS - 3 SP - 688 EP - 697 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Matallana-Ramirez, Lilian P. A1 - Rauf, Mamoona A1 - Farage-Barhom, Sarit A1 - Dortay, Hakan A1 - Xue, Gang-Ping A1 - Droege-Laser, Wolfgang A1 - Lers, Amnon A1 - Balazadeh, Salma A1 - Müller-Röber, Bernd T1 - NAC Transcription Factor ORE1 and Senescence-Induced BIFUNCTIONAL NUCLEASE1 (BFN1) Constitute a Regulatory Cascade in Arabidopsis JF - Molecular plant N2 - The NAC transcription factor ORE1 is a key regulator of senescence in Arabidopsis thaliana. Here, we demonstrate that senescence-induced and cell death-associated BIFUNCTIONAL NUCLEASE1 (BFN1) is a direct downstream target of ORE1, revealing a previously unknown regulatory cascade.Senescence is a highly regulated process that involves the action of a large number of transcription factors. The NAC transcription factor ORE1 (ANAC092) has recently been shown to play a critical role in positively controlling senescence in Arabidopsis thaliana; however, no direct target gene through which it exerts its molecular function has been identified previously. Here, we report that BIFUNCTIONAL NUCLEASE1 (BFN1), a well-known senescence-enhanced gene, is directly regulated by ORE1. We detected elevated expression of BFN1 already 2 h after induction of ORE1 in estradiol-inducible ORE1 overexpression lines and 6 h after transfection of Arabidopsis mesophyll cell protoplasts with a 35S:ORE1 construct. ORE1 and BFN1 expression patterns largely overlap, as shown by promoterreporter gene (GUS) fusions, while BFN1 expression in senescent leaves and the abscission zones of maturing flower organs was virtually absent in ore1 mutant background. In vitro binding site assays revealed a bipartite ORE1 binding site, similar to that of ORS1, a paralog of ORE1. A bipartite ORE1 binding site was identified in the BFN1 promoter; mutating the cis-element within the context of the full-length BFN1 promoter drastically reduced ORE1-mediated transactivation capacity in transiently transfected Arabidopsis mesophyll cell protoplasts. Furthermore, chromatin immunoprecipitation (ChIP) demonstrates in vivo binding of ORE1 to the BFN1 promoter. We also demonstrate binding of ORE1 in vivo to the promoters of two other senescence-associated genes, namely SAG29/SWEET15 and SINA1, supporting the central role of ORE1 during senescence. KW - Arabidopsis thaliana KW - senescence KW - transcription factor KW - ORE1 KW - BFN1 KW - promoter Y1 - 2013 U6 - https://doi.org/10.1093/mp/sst012 SN - 1674-2052 VL - 6 IS - 5 SP - 1438 EP - 1452 PB - Oxford Univ. Press CY - Oxford ER - TY - THES A1 - Martinez-Seidel, Federico T1 - Ribosome Heterogeneity and Specialization during Temperature Acclimation in Plants N2 - Ribosomes decode mRNA to synthesize proteins. Ribosomes, once considered static, executing machines, are now viewed as dynamic modulators of translation. Increasingly detailed analyses of structural ribosome heterogeneity led to a paradigm shift toward ribosome specialization for selective translation. As sessile organisms, plants cannot escape harmful environments and evolved strategies to withstand. Plant cytosolic ribosomes are in some respects more diverse than those of other metazoans. This diversity may contribute to plant stress acclimation. The goal of this thesis was to determine whether plants use ribosome heterogeneity to regulate protein synthesis through specialized translation. I focused on temperature acclimation, specifically on shifts to low temperatures. During cold acclimation, Arabidopsis ceases growth for seven days while establishing the responses required to resume growth. Earlier results indicate that ribosome biogenesis is essential for cold acclimation. REIL mutants (reil-dkos) lacking a 60S maturation factor do not acclimate successfully and do not resume growth. Using these genotypes, I ascribed cold-induced defects of ribosome biogenesis to the assembly of the polypeptide exit tunnel (PET) by performing spatial statistics of rProtein changes mapped onto the plant 80S structure. I discovered that growth cessation and PET remodeling also occurs in barley, suggesting a general cold response in plants. Cold triggered PET remodeling is consistent with the function of Rei-1, a REIL homolog of yeast, which performs PET quality control. Using seminal data of ribosome specialization, I show that yeast remodels the tRNA entry site of ribosomes upon change of carbon sources and demonstrate that spatially constrained remodeling of ribosomes in metazoans may modulate protein synthesis. I argue that regional remodeling may be a form of ribosome specialization and show that heterogeneous cytosolic polysomes accumulate after cold acclimation, leading to shifts in the translational output that differs between wild-type and reil-dkos. I found that heterogeneous complexes consist of newly synthesized and reused proteins. I propose that tailored ribosome complexes enable free 60S subunits to select specific 48S initiation complexes for translation. Cold acclimated ribosomes through ribosome remodeling synthesize a novel proteome consistent with known mechanisms of cold acclimation. The main hypothesis arising from my thesis is that heterogeneous/ specialized ribosomes alter translation preferences, adjust the proteome and thereby activate plant programs for successful cold acclimation. N2 - Ribosomen dekodieren mRNA, um Proteine zu synthetisieren. Ribosomen, früher als statische, ausführende Maschinen betrachtet, werden heute als dynamische Modulatoren der Translation angesehen. Zunehmend detailliertere Analysen der Strukturheterogenität von Ribosomen führte zu einem Paradigmenwechsel hin zu einer Spezialisierung von Ribosomen für eine selektive Translation. Als sessile Organismen können Pflanzen schädlichen Umwelteinflüssen nicht ausweichen und haben Strategien entwickelt, um diesen zu widerstehen. Zytosolische Ribosomen von Pflanzen sind in mancher Hinsicht vielfältiger, als die von anderen Metazoen. Diese Vielfalt könnte zur Stressakklimatisierung der Pflanzen beitragen. Ziel dieser Arbeit war es, festzustellen, ob Pflanzen die Heterogenität der Ribosomen nutzen, um die Proteinsynthese durch spezialisierte Translation zu regulieren. Ich habe mich auf die Temperaturakklimatisierung konzentriert, insbesondere auf den Wechsel zu niedrigen Temperaturen. Im Verlauf der Kälteakklimatisierung stellt Arabidopsis das Wachstum für sieben Tage ein. Währenddessen etabliert sie die für die Wiederaufnahme des Wachstums erforderlichen Anpassungen. Vorherige Ergebnisse deuten darauf hin, dass Ribosomenbiogenese für die Kälteakklimatisierung essentiell ist. REIL-Mutanten (reil-dkos), denen ein 60S-Reifungsfaktor fehlt, akklimatisieren sich nicht erfolgreich und nehmen das Wachstum nicht wieder auf. Anhand dieser Genotypen habe ich kältebedingte Defekte der Ribosomenbiogenese auf den Aufbau des Polypeptidaustritts-Tunnels (PET) zurückgeführt, indem ich räumliche statistische Analysen von rProtein-Veränderungen auf die pflanzliche 80S-Struktur abgebildet habe. Ich habe entdeckt, dass Wachstumsstillstand und PET-Umbau auch in Gerste auftreten, was auf eine allgemeine Kältereaktion in Pflanzen hindeutet. Der durch Kälte ausgelöste PET-Umbau stimmt über ein mit der Funktion von Rei-1, einem REIL-homologen Protein aus Hefe, in der Rei-1 die PET-Qualitätskontrolle durchführt. Anhand bahnbrechender Daten zur Ribosomenspezialisierung zeige ich, dass Hefe die tRNA-Eintrittsstelle von Ribosomen bei einem Wechsel von Kohlenstoffquellen umbaut, und demonstriere, dass ein räumlich begrenzter Umbau von Ribosomen in Metazoen die Proteinsynthese modulieren kann. Ich argumentiere, dass die regionale Umgestaltung eine Form der Ribosomenspezialisierung sein kann, und zeige, dass nach einer Kälteakklimatisierung heterogene zytosolische Polysomen akkumulieren, was zu Verschiebungen im Translationsoutput führt, der sich zwischen Wildtyp und reil-dkos unterscheidet. Ich habe festgestellt, dass die heterogenen Komplexe aus neu synthetisierten und wiederverwendeten Proteinen bestehen. Ich schlage vor, dass maßgeschneiderte Ribosomenkomplexe freie 60S-Untereinheiten in die Lage versetzen, spezifische 48S-Initiationskomplexe für die Translation auszuwählen. Kälte-akklimatisierte Ribosomen synthetisieren durch Ribosomenumbau ein neues Proteom, das mit bekannten Mechanismen der Kälteakklimatisierung übereinstimmt. Die Haupthypothese, die sich aus meiner Arbeit ergibt, ist, dass heterogene/spezialisierte Ribosomen ihre Translationspräferenzen verändern, das Proteom anpassen und dadurch Pflanzenprogramme für eine erfolgreiche Kälteakklimatisierung aktivieren. T2 - Ribosomenheterogenität und -spezialisierung während der Temperaturakklimatisierung in Pflanzen KW - Ribosome specialization KW - Ribosomal protein heterogeneity KW - Ribosomal protein substoichiometry KW - Protein synthesis KW - Translational regulation KW - Plant cytosolic translation KW - Cold acclimation KW - Ribosome biogenesis KW - 60S maturation KW - Hordeum vulgare KW - Arabidopsis thaliana KW - 60S-Reifung KW - Kälteakklimatisierung KW - Cytosolische Translation in Pflanzen KW - Proteinsynthese KW - Ribosomale Proteinheterogenität KW - Ribosomale Protein Substöchiometrie KW - Ribosomen-Biogenese KW - Ribosomen-Spezialisierung KW - Translationsregulation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-580724 ER - TY - JOUR A1 - Malinova, Irina A1 - Mahto, Harendra A1 - Brandt, Felix A1 - AL-Rawi, Shadha A1 - Qasim, Hadeel A1 - Brust, Henrike A1 - Hejazi, Mahdi A1 - Fettke, Jörg T1 - EARLY STARVATION1 specifically affects the phosphorylation action of starch-related dikinases JF - The plant journal N2 - Starch phosphorylation by starch-related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50-kDa starch-binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various invitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, -glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface. KW - Arabidopsis thaliana KW - EARLY STARVATION1 KW - glucan KW - phosphoglucan KW - starch granule surface KW - starch phosphorylation KW - water dikinase Y1 - 2018 U6 - https://doi.org/10.1111/tpj.13937 SN - 0960-7412 SN - 1365-313X VL - 95 IS - 1 SP - 126 EP - 137 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Malinova, Irina A1 - Kössler, Stella A1 - Orawetz, Tom A1 - Matthes, Ulrike A1 - Orzechowski, Slawomir A1 - Koch, Anke A1 - Fettke, Jörg T1 - Identification of two Arabidopsis thaliana plasma membrane transporters able to transport glucose 1-phosphate JF - Plant & cell physiology N2 - Primary carbohydrate metabolism in plants includes several sugar and sugar-derivative transport processes. Over recent years, evidences have shown that in starch-related transport processes, in addition to glucose 6-phosphate, maltose, glucose and triose-phosphates, glucose 1-phosphate also plays a role and thereby increases the possible fluxes of sugar metabolites in planta. In this study, we report the characterization of two highly similar transporters, At1g34020 and At4g09810, in Arabidopsis thaliana, which allow the import of glucose 1-phosphate through the plasma membrane. Both transporters were expressed in yeast and were biochemically analyzed to reveal an antiport of glucose 1-phosphate/phosphate. Furthermore, we showed that the apoplast of Arabidopsis leaves contained glucose 1-phosphate and that the corresponding mutant of these transporters had higher glucose 1-phosphate amounts in the apoplast and alterations in starch and starch-related metabolism. KW - apoplast KW - Arabidopsis thaliana KW - glucose 1-phosphate transport KW - starch metabolism KW - sugar transport Y1 - 2020 U6 - https://doi.org/10.1093/pcp/pcz206 SN - 0032-0781 SN - 1471-9053 VL - 61 IS - 2 SP - 381 EP - 392 PB - Oxford University Press CY - Oxford ER - TY - THES A1 - Mahto, Harendra T1 - In vitro analysis of Early Starvation 1 (ESV1) and Like Early Starvation 1 (LESV) on starch degradation with focus on glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) N2 - Starch is an insoluble polyglucan, comprises of two polymers, namely, the branched α-1,4: α-1,6-D-glucan amylopectin and the almost unbranched α-1,4-D-glucan amylose. The growth of all plants is directly dependent on the accumulation of transitory starch during the daytime when photosynthesis takes place and subsequently starch degradation during the night. Starch phosphorylation takes place by starch-related dikinases called α-glucan, water dikinase (GWD), and phosphoglucan, water dikinase (PWD), and is a very important step in starch degradation. The biochemical mechanisms of phosphorylation of starch are not properly understood. Recent studies have found that there are two starch binding proteins namely, Early Starvation1 (ESV1) and Like Early Starvation1 (LESV), which play an important role in starch metabolism. It has been shown that ESV1 and LESV proteins affect the starch phosphorylation activity of GWD and PWD enzymes, which control the rate of degradation of starch granules. In this thesis, various in vitro assays were performed to identify and understand the mechanism of recombinant proteins; ESV1 and LESV on the starch degradation. The starch degradation was performed by phosphorylation enzymes, GWD and PWD separately. In various enzymatic assays, the influence of the ESV1 and LESV on the actions of GWD and PWD on the surfaces of different native starch granules were analysed. Furthermore, ESV1 and LESV have specifically shown influences on the phosphorylation activities of GWD and PWD on the starch granule surfaces in an antagonistic pattern in such a way that, the GWD mediated phosphorylation were significantly reduced while PWD mediated phosphorylation were significantly increased respectively. In another set of experiments, ISA and BAM hydrolyzing enzymes were used to alter the structure of starch, and then determine the effect of both dikinases mediated phosphorylation in the presence of ESV1 and LESV on the altered starch granules surfaces. In these results, significant decreases in both GWD and PWD mediated phosphorylation were observed in all the treatments containing either ESV1 or LESV proteins only or both ESV1 and LESV. It was also found that LESV preferentially binds to both amylose and amylopectin, while ESV1 binds to highly ordered glucans such as maltodextrins and amylopectin, which are crystalline in structure. Both ESV1 or LESV proteins either individually or in combination have shown influence on the activity of GWD and PWD phosphate incorporation into the starch granules via reduction even though at different percentages depending on the sources of starch, therefore it is difficult to distinguish the specific function between them. The biochemical studies have shown that protein-glucan interaction specifically between ESV1 or LESV or in combination with different species of starch granules has very strong surface binding, or it might be possible that both the proteins not only bind to the surface of the starch granules but also have entered deep inside the glucan structure of the starch granules. However, the results also revealed that ESV1 and LESV did not alter the autophosphorylation of the dikinases. Also, the chain length distribution pattern of the released glucan chains after treatment of starch with ISA enzyme was evaluated with respect to the degree of polymerization (DP) of the different starch granules. Capillary electrophoresis was employed to study the effect of LESV and ESV1 on the chain length distribution. In summary, this study confirms that ESV1 and LESV play an important role in organizing and regulating the starch metabolism process. In the later half, studies were performed to monitor whether the metabolism of carbohydrates and partitioning, contribute to the higher salt tolerance of the facultative halophyte Hordeum marinum when compared to glycophyte Hordeum vulgare. Seedlings with the same size from both species were hydroponically grown at 0, 150, and 300 mM of NaCl for 3 weeks. H. marinum maintained a high relative growth rate, which was found concomitant in higher aptitude plants to maintain efficient shoot tissue hydration and integrity of membrane under salt conditions when compared to H. vulgare. Hence, our data suggested that the change in the starch storage, distribution of soluble sugar concentrations between source and sink organs, and also changes in the level of enzymes involved in the starch metabolism was significant to give insights into the importance of carbohydrate metabolism in barley species with regards to the salt tolerance. Although these results are still in their nascent state, it could be vital for other researchers to formulate future studies. The preliminary results which were studies about the carbohydrate metabolism and partitioning in salt responses in the halophyte H. marinum and the glycophyte H. vulgare revealed that salt tolerance in barley species is not due to osmotic adjustments, but due to other reasons that were not explored in the past studies. However, the activity of DPE2 in H. vulgare was not hampered by the presence of NaCl as observed. While Pho1 and Pho2, activities were highly increased in cultivated barley. These findings could be suggestive of a possible role of these enzymes in the responses of carbohydrate metabolism to salinity. When sea and cultivated barley species were compared, it was discovered that the former had more versatility in carbohydrate metabolism and distribution. N2 - Stärke ist ein unlösliches Polyglucan, das aus zwei Polymeren besteht, nämlich dem verzweigten α-1,4: α-1,6-D-Glucan Amylopektin und dem fast unverzweigten α-1,4-D-Glucan Amylose. Das Wachstum aller Pflanzen hängt direkt von der Akkumulation transitorischer Stärke während des Tages, wenn die Photosynthese stattfindet, und dem anschließenden Stärkeabbau während der Nacht ab. Die Phosphorylierung von Stärke erfolgt durch stärkeverwandte Dikinasen, die α-Glucan-Wasser-Dikinase (GWD) und Phosphoglucan-Wasser-Dikinase (PWD), und ist ein entscheidender Schritt beim Stärkeabbau. Die biochemischen Mechanismen der Phosphorylierung von Stärke sind nicht genau bekannt. Jüngste Studien haben ergeben, dass es zwei stärkebindende Proteine gibt, nämlich Early Starvation1 (ESV1) und Like Early Starvation1 (LESV), die eine wichtige Rolle im Stärkestoffwechsel spielen. Es hat sich gezeigt, dass ESV1- und LESV-Proteine die Stärkephosphorylierungsaktivität der GWD- und PWD-Enzyme beeinflussen, die die Geschwindigkeit des Abbaus von Stärkekörnern steuern. In dieser Arbeit wurden verschiedene In-vitro-Tests durchgeführt, um den Mechanismus der rekombinanten Proteine ESV1 und LESV auf den Stärkeabbau zu identifizieren und zu verstehen.Der Stärkeabbau wurde von den Phosphorylierungsenzymen GWD und PWD getrennt durchgeführt. In verschiedenen enzymatischen Assays wurde der Einfluss von ESV1 und LESV auf die Wirkung von GWD und PWD auf die Oberflächen verschiedener nativer Stärkekörner analysiert. Darüber hinaus haben ESV1 und LESV spezifisch Einflüsse auf die Phosphorylierungsaktivitäten von GWD und PWD auf den Oberflächen der Stärkekörner in einem antagonistischen Muster gezeigt, so dass die GWD-vermittelte Phosphorylierung signifikant reduziert wurde, während die PWD-vermittelte Phosphorylierung signifikant erhöht wurde. In einer anderen Versuchsreihe wurden ISA- und BAM verwendet, um die Struktur der Stärke zu verändern und dann die Auswirkungen der durch beide Dikinasen vermittelten Phosphorylierung in Gegenwart von ESV1 und LESV auf die veränderten Oberflächen der Stärkekörner zu bestimmen. In diesen Ergebnissen wurde ein signifikanter Rückgang der GWD- und PWD-vermittelten Phosphorylierung in allen Behandlungen beobachtet, die entweder nur ESV1- oder LESV-Proteine oder sowohl ESV1 als auch LESV enthielten. Es wurde auch festgestellt, dass LESV vorzugsweise an Amylose und Amylopektin bindet, während ESV1 an hochgeordnete Glucane wie Maltodextrine und Amylopektin bindet, die eine kristalline Struktur aufweisen. Sowohl ESV1- als auch LESV-Proteine haben entweder einzeln oder in Kombination einen Einfluss auf die Aktivität des GWD- und PWD-Phosphateinbaus in die Stärkekörner durch Reduktion gezeigt, jedoch zu unterschiedlichen Prozentsätzen, je nach Stärkequelle, so dass es schwierig ist, ihre spezifische Funktion zu unterscheiden. Die biochemischen Untersuchungen zeigen, dass die Protein-Glucan-Interaktion speziell zwischen ESV1 oder LESV oder in Kombination mit verschiedenen Arten von Stärkekörnern eine sehr starke Oberflächenbindung aufweist, oder es ist möglich, dass beide Proteine nicht nur an die Oberfläche der Stärkekörner binden, sondern auch tief in die Glucanstruktur der Stärkekörner eingedrungen sind. Die Ergebnisse zeigten jedoch auch, dass ESV1 und LESV die Autophosphorylierung der Dikinasen nicht veränderten. Außerdem wurde die Kettenlängenverteilung der freigesetzten Glucanketten nach Behandlung der Stärke mit dem ISA-Enzym im Hinblick auf den Polymerisationsgrad (DP) der verschiedenen Stärkekörner bewertet. Mit Hilfe der Kapillarelektrophorese wurde die Wirkung von LESV und ESV1 auf die Kettenlängenverteilung untersucht. Zusammenfassend bestätigt diese Studie, dass ESV1 und LESV eine wichtige Rolle bei der Organisation und Regulierung des Stärkestoffwechsels spielen. In der zweiten Hälfte wurden Untersuchungen durchgeführt, um zu prüfen, ob der Stoffwechsel von Kohlenhydraten und deren Verteilung zu der höheren Salztoleranz des fakultativen Halophyten Hordeum marinum im Vergleich zum Glykophyten Hordeum vulgare beitragen. Die gleich großen Sämlinge beider Arten wurden 3 Wochen lang bei 0, 150 und 300 mM NaCl hydroponisch gezogen. H. marinum wies eine hohe relative Wachstumsrate auf, die mit einer höheren Fähigkeit der Pflanzen einherging, unter Salzbedingungen eine effiziente Hydratation des Sprossgewebes und die Integrität der Membran aufrechtzuerhalten, als dies bei H. vulgare der Fall war. Unsere Daten deuten also darauf hin, dass die Veränderungen in der Stärkespeicherung, die Verteilung der Konzentrationen löslicher Zucker zwischen Source- und Sinkorganen und auch die Veränderungen in der Menge der am Stärkestoffwechsel beteiligten Enzyme von Bedeutung sind und Einblicke in die Bedeutung des Kohlenhydratstoffwechsels bei Gerstenarten im Hinblick auf die Salztoleranz geben. Obwohl sich diese Ergebnisse noch im Anfangsstadium befinden, könnten sie für andere Forscher bei der Formulierung künftiger Studien von entscheidender Bedeutung sein. Die vorläufigen Ergebnisse der Studien über den Kohlenhydratstoffwechsel und die Verteilung der Kohlenhydrate bei Salzreaktionen im Halophyten H. marinum und im Glykophyten H. vulgare haben gezeigt, dass die Salztoleranz bei Gerstenarten nicht auf osmotische Anpassungen zurückzuführen ist, sondern auf andere Gründe, die in den bisherigen Studien nicht untersucht wurden. Die Aktivität von DPE2 in H. vulgare wurde jedoch nicht wie beobachtet durch die Anwesenheit von NaCl beeinträchtigt. Dagegen waren die Aktivitäten von Pho1 und Pho2 in kultivierter Gerste stark erhöht. Diese Ergebnisse könnten auf eine mögliche Rolle dieser Enzyme bei der Reaktion des Kohlenhydratstoffwechsels auf den Salzgehalt hinweisen. Beim Vergleich von Meeres- und Kulturgerstenarten wurde festgestellt, dass erstere eine größere Vielseitigkeit im Kohlenhydratstoffwechsel und in der Kohlenhydratverteilung aufweisen. KW - Arabidopsis thaliana KW - starch phosphorylation KW - phosphoglucan KW - starch granule surface KW - Early Starvation 1 Y1 - 2022 ER - TY - JOUR A1 - Mahlow, Sebastian A1 - Hejazi, Mahdi A1 - Kuhnert, Franziska A1 - Garz, Andreas A1 - Brust, Henrike A1 - Baumann, Otto A1 - Fettke, Jörg T1 - Phosphorylation of transitory starch by -glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules JF - New phytologist : international journal of plant science N2 - Glucan, water dikinase (GWD) is a key enzyme of starch metabolism but the physico-chemical properties of starches isolated from GWD-deficient plants and their implications for starch metabolism have so far not been described. Transgenic Arabidopsis thaliana plants with reduced or no GWD activity were used to investigate the properties of starch granules. In addition, using various in vitro assays, the action of recombinant GWD, -amylase, isoamylase and starch synthase 1 on the surface of native starch granules was analysed. The internal structure of granules isolated from GWD mutant plants is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules were similar to wild-type. However, short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD was also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface. A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes. KW - Arabidopsis thaliana KW - glucan KW - water dikinase (GWD) KW - sex1-8 KW - starch granule surface KW - starch phosphorylation Y1 - 2014 U6 - https://doi.org/10.1111/nph.12801 SN - 0028-646X SN - 1469-8137 VL - 203 IS - 2 SP - 495 EP - 507 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Lukoszek, Radoslaw A1 - Müller-Röber, Bernd A1 - Ignatova, Zoya T1 - Interplay between polymerase II- and polymerase III-assisted expression of overlapping genes JF - FEBS letters : the journal for rapid publication of short reports in molecular biosciences N2 - Up to 15% of the genes in different genomes overlap. This architecture, although beneficial for the genome size, represents an obstacle for simultaneous transcription of both genes. Here we analyze the interference between RNA-polymerase II (Pol II) and RNA-polymerase III (Pol III) when transcribing their target genes encoded on opposing strands within the same DNA fragment in Arabidopsis thaliana. The expression of a Pol II-dependent protein-coding gene negatively correlated with the transcription of a Pol III-dependent, tRNA-coding gene set. We suggest that the architecture of the overlapping genes introduces an additional layer of control of gene expression. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. KW - Gene expression KW - Transcription KW - tRNA KW - Nested and overlapping genes KW - Arabidopsis thaliana Y1 - 2013 U6 - https://doi.org/10.1016/j.febslet.2013.09.033 SN - 0014-5793 SN - 1873-3468 VL - 587 IS - 22 SP - 3692 EP - 3695 PB - Elsevier CY - Amsterdam ER - TY - GEN A1 - Liu, Qingting A1 - Zhou, Yuan A1 - Fettke, Jörg T1 - Starch granule size and morphology of Arabidopsis thaliana starch-related mutants analyzed during diurnal rhythm and development T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Transitory starch plays a central role in the life cycle of plants. Many aspects of this important metabolism remain unknown; however, starch granules provide insight into this persistent metabolic process. Therefore, monitoring alterations in starch granules with high temporal resolution provides one significant avenue to improve understanding. Here, a previously established method that combines LCSM and safranin-O staining for in vivo imaging of transitory starch granules in leaves of Arabidopsis thaliana was employed to demonstrate, for the first time, the alterations in starch granule size and morphology that occur both throughout the day and during leaf aging. Several starch-related mutants were included, which revealed differences among the generated granules. In ptst2 and sex1-8, the starch granules in old leaves were much larger than those in young leaves; however, the typical flattened discoid morphology was maintained. In ss4 and dpe2/phs1/ss4, the morphology of starch granules in young leaves was altered, with a more rounded shape observed. With leaf development, the starch granules became spherical exclusively in dpe2/phs1/ss4. Thus, the presented data provide new insights to contribute to the understanding of starch granule morphogenesis. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1245 KW - starch metabolism KW - starch granule KW - starch granule size KW - starch granule morphology KW - LCSM KW - Arabidopsis thaliana Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-550291 SN - 1866-8372 VL - 26 SP - 1 EP - 9 PB - Universitätsverlag Potsdam CY - Potsdam ET - 19 ER - TY - JOUR A1 - Liu, Qingting A1 - Zhou, Yuan A1 - Fettke, Jörg T1 - Starch granule size and morphology of Arabidopsis thaliana starch-related mutants analyzed during diurnal rhythm and development JF - Molecules : a journal of synthetic chemistry and natural product chemistry / Molecular Diversity Preservation International N2 - Transitory starch plays a central role in the life cycle of plants. Many aspects of this important metabolism remain unknown; however, starch granules provide insight into this persistent metabolic process. Therefore, monitoring alterations in starch granules with high temporal resolution provides one significant avenue to improve understanding. Here, a previously established method that combines LCSM and safranin-O staining for in vivo imaging of transitory starch granules in leaves of Arabidopsis thaliana was employed to demonstrate, for the first time, the alterations in starch granule size and morphology that occur both throughout the day and during leaf aging. Several starch-related mutants were included, which revealed differences among the generated granules. In ptst2 and sex1-8, the starch granules in old leaves were much larger than those in young leaves; however, the typical flattened discoid morphology was maintained. In ss4 and dpe2/phs1/ss4, the morphology of starch granules in young leaves was altered, with a more rounded shape observed. With leaf development, the starch granules became spherical exclusively in dpe2/phs1/ss4. Thus, the presented data provide new insights to contribute to the understanding of starch granule morphogenesis. KW - starch metabolism KW - starch granule KW - starch granule size KW - starch granule morphology KW - LCSM KW - Arabidopsis thaliana Y1 - 2021 U6 - https://doi.org/10.3390/molecules26195859 SN - 1420-3049 VL - 26 SP - 1 EP - 9 PB - MDPI CY - Basel, Schweiz ET - 19 ER - TY - JOUR A1 - Liu, Qingting A1 - Li, Xiaoping A1 - Fettke, Jörg T1 - Starch granules in Arabidopsis thaliana mesophyll and guard cells show similar morphology but differences in size and number JF - International journal of molecular sciences N2 - Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells. KW - starch granules KW - starch granule number per chloroplast KW - starch morphology KW - mesophyll cell KW - guard cell KW - LCSM KW - Arabidopsis thaliana KW - starch granule initiation KW - starch metabolism Y1 - 2021 U6 - https://doi.org/10.3390/ijms22115666 SN - 1422-0067 SN - 1661-6596 VL - 22 IS - 11 PB - Molecular Diversity Preservation International CY - Basel ER - TY - GEN A1 - Liu, Qingting A1 - Li, Xiaoping A1 - Fettke, Jörg T1 - Starch granules in Arabidopsis thaliana mesophyll and guard cells show similar morphology but differences in size and number T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1143 KW - starch granules KW - starch metabolism KW - starch granule initiation KW - starch granule number per chloroplast KW - starch morphology KW - mesophyll cell KW - guard cell KW - LCSM KW - Arabidopsis thaliana Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-511067 SN - 1866-8372 IS - 1143 ER - TY - JOUR A1 - Liu, Hsiang-chin A1 - Lämke, Jörn A1 - Lin, Siou-ying A1 - Hung, Meng-Ju A1 - Liu, Kuan-Ming A1 - Charng, Yee-yung A1 - Bäurle, Isabel T1 - Distinct heat shock factors and chromatin modifications mediate the organ-autonomous transcriptional memory of heat stress JF - The plant journal N2 - Plants can be primed by a stress cue to mount a faster or stronger activation of defense mechanisms upon subsequent stress. A crucial component of such stress priming is the modified reactivation of genes upon recurring stress; however, the underlying mechanisms of this are poorly understood. Here, we report that dozens of Arabidopsis thaliana genes display transcriptional memory, i.e. stronger upregulation after a recurring heat stress, that lasts for at least 3 days. We define a set of transcription factors involved in this memory response and show that the transcriptional memory results in enhanced transcriptional activation within minutes of the onset of a heat stress cue. Further, we show that the transcriptional memory is active in all tissues. It may last for up to a week, and is associated during this time with histone H3 lysine 4 hypermethylation. This transcriptional memory is cis-encoded, as we identify a promoter fragment that confers memory onto a heterologous gene. In summary, heat-induced transcriptional memory is a widespread and sustained response, and our study provides a framework for future mechanistic studies of somatic stress memory in higher plants. KW - epigenetics KW - priming KW - heat stress KW - H3K4 methylation KW - transcriptional memory KW - Arabidopsis thaliana KW - HSF Y1 - 2018 U6 - https://doi.org/10.1111/tpj.13958 SN - 0960-7412 SN - 1365-313X VL - 95 IS - 3 SP - 401 EP - 413 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Lisso, Janina A1 - Altmann, Thomas A1 - Müssig, Carsten T1 - The AtNFXL1 gene encodes a NF-X1 type zinc finger protein required for growth under salt stress JF - FEBS letters : the journal for rapid publication of short reports in molecular biosciences N2 - The human NF-X1 protein and homologous proteins in eukaryotes represent a class of transcription factors which are characterised. by NF-X1 type zinc finger motifs. The Arabidopsis genome encodes two NF-X1 homologs, which we termed AtNFXL1 and AtNFXL2. Growth and survival was impaired in atnfxl1 knock-out mutants and AtNFXL1-antisense plants under salt stress in comparison to wild-type plants. In contrast, 35S: :AtNFXL1 plants showed higher survival rates. The AtNFXL2 protein potentially plays an antagonistic role. The Arabidopsis NF-X1 type zinc finger proteins likely are part of regulatory mechanisms, which protect major processes such as photosynthesis. KW - Arabidopsis thaliana KW - NF-X1 KW - salt stress Y1 - 2006 U6 - https://doi.org/10.1016/j.febslet.2006.07.079 SN - 0014-5793 VL - 580 IS - 22 SP - 4851 EP - 4856 PB - Elsevier CY - Amsterdam ER - TY - THES A1 - Lisec, Jan T1 - Identification and characterization of metabolic Quantitative Trait Loci (QTL) in Arabidopsis thaliana T1 - Identifizierung und Charakterisierung von metabolischen Loci quantitativer Merkmale (QTL) in Arabidopsis thaliana N2 - Plants are the primary producers of biomass and thereby the basis of all life. Many varieties are cultivated, mainly to produce food, but to an increasing amount as a source of renewable energy. Because of the limited acreage available, further improvements of cultivated species both with respect to yield and composition are inevitable. One approach to further progress in developing improved plant cultivars is a systems biology oriented approach. This work aimed to investigate the primary metabolism of the model plant A.thaliana and its relation to plant growth using quantitative genetics methods. A special focus was set on the characterization of heterosis, the deviation of hybrids from their parental means for certain traits, on a metabolic level. More than 2000 samples of recombinant inbred lines (RILs) and introgression lines (ILs) developed from the two accessions Col-0 and C24 were analyzed for 181 metabolic traces using gas-chromatography/ mass-spectrometry (GC-MS). The observed variance allowed the detection of 157 metabolic quantitative trait loci (mQTL), genetic regions carrying genes, which are relevant for metabolite abundance. By analyzing several hundred test crosses of RILs and ILs it was further possible to identify 385 heterotic metabolic QTL (hmQTL). Within the scope of this work a robust method for large scale GC-MS analyses was developed. A highly significant canonical correlation between biomass and metabolic profiles (r = 0.73) was found. A comparable analysis of the results of the two independent experiments using RILs and ILs showed a large agreement. The confirmation rate for RIL QTL in ILs was 56 % and 23 % for mQTL and hmQTL respectively. Candidate genes from available databases could be identified for 67 % of the mQTL. To validate some of these candidates, eight genes were re-sequenced and in total 23 polymorphisms could be found. In the hybrids, heterosis is small for most metabolites (< 20%). Heterotic QTL gave rise to less candidate genes and a lower overlap between both populations than was determined for mQTL. This hints that regulatory loci and epistatic effects contribute to metabolite heterosis. The data described in this thesis present a rich source for further investigation and annotation of relevant genes and may pave the way towards a better understanding of plant biology on a system level. N2 - Pflanzen sind die Primärproduzenten von Biomasse und damit Grundlage allen Lebens. Sie werden nicht nur zur Gewinnung von Nahrungsmitteln, sondern zunehmend auch als Quelle erneuerbarer Energien kultiviert. Aufgrund der Begrenztheit der weltweit zu Verfügung stehenden Anbaufläche ist eine zielgerichtete Selektion und Verbesserung der verwendeten Sorten unabdingbar. Um solch eine kontinuierliche Verbesserung zu gewährleisten, ist ein grundlegendes Verständnis des biologischen Systems Pflanze nötig. Diese Arbeit hatte zum Ziel, den Primärmetabolismus der Modellpflanze A. thaliana mit Methoden der quantitativen Genetik zu untersuchen und in Beziehung zu Wachstum und Biomasse zu stellen. Insbesondere sollte Heterosis, die Abweichung von Hybriden in ihren Merkmalen vom Mittelwert der Eltern, auf Stoffwechselebene charakterisiert werden. Mit Hilfe der Gas Chromatographie/ Massen Spektrometrie (GC-MS) wurden über 2000 Proben von rekombinanten Inzucht Linien (RIL) und Introgressions Linien (IL) der Akzessionen Col 0 und C24 bezüglich des Vorkommens von 181 Metaboliten untersucht. Die beobachtete Varianz erlaubte die Bestimmung von 157 metabolischen QTL (mQTL), genetischen Regionen, die für die Metabolitkonzentrationen relevante Gene enthalten. Durch die Untersuchung von Testkreuzungen der RILs und ILs konnten weiterhin 385 heterotische metabolische QTL (hmQTL) identifiziert werden. Im Rahmen dieser Arbeit wurde eine robuste Methode zur Auswertung von GC-MS Analysen entwickelt. Es wurde eine hoch signifikante kanonische Korrelation (r=0.73) zwischen Biomasse und Metabolitprofilen gefunden. Die unterschiedlichen Ansätze zur QTL Analyse, RILs und ILs, wurden verglichen. Dabei konnte gezeigt werden, daß die Methoden komplementär sind, da mit RILs gefundene mQTL zu 56% und hmQTL zu 23% in ILs bestätigt wurden. Durch den Vergleich mit Datenbanken wurden für 67% der mQTL Kandidatengene identifiziert. Um diese zu überprüfen wurden acht dieser Gene resequenziert und insgesamt 23 Polymorphismen darin bestimmt. Die Heterosis in den Hybriden ist für die meisten Metabolite gering (<20%). Für hmQTL konnten weniger Kandidatengene als für mQTL bestimmt werden und sie zeigten eine geringere Übereinstimmung in den beiden Populationen. Dies deutet darauf hin, daß regulatorische Loci und epistatische Effekte einen wichtigen Beitrag zur Heterosis besteuern. Die gewonnenen Daten stellen eine reiche Quelle für die weitergehende Untersuchung und Annotation relevanter Gene dar und ebnen den Weg für ein besseres Verständnis des Systems Pflanze. KW - Arabidopsis thaliana KW - Metabolomics KW - QTL Analyse KW - Arabidopsis thaliana KW - Metabolomics KW - QTL mapping Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-25903 ER - TY - JOUR A1 - Küken, Anika A1 - Gennermann, Kristin A1 - Nikoloski, Zoran T1 - Characterization of maximal enzyme catalytic rates in central metabolism of Arabidopsis thaliana JF - The plant journal N2 - Availability of plant-specific enzyme kinetic data is scarce, limiting the predictive power of metabolic models and precluding identification of genetic factors of enzyme properties. Enzyme kinetic data are measuredin vitro, often under non-physiological conditions, and conclusions elicited from modeling warrant caution. Here we estimate maximalin vivocatalytic rates for 168 plant enzymes, including photosystems I and II, cytochrome-b6f complex, ATP-citrate synthase, sucrose-phosphate synthase as well as enzymes from amino acid synthesis with previously undocumented enzyme kinetic data in BRENDA. The estimations are obtained by integrating condition-specific quantitative proteomics data, maximal rates of selected enzymes, growth measurements fromArabidopsis thalianarosette with and fluxes through canonical pathways in a constraint-based model of leaf metabolism. In comparison to findings inEscherichia coli, we demonstrate weaker concordance between the plant-specificin vitroandin vivoenzyme catalytic rates due to a low degree of enzyme saturation. This is supported by the finding that concentrations of nicotinamide adenine dinucleotide (phosphate), adenosine triphosphate and uridine triphosphate, calculated based on our maximalin vivocatalytic rates, and available quantitative metabolomics data are below reportedKMvalues and, therefore, indicate undersaturation of respective enzymes. Our findings show that genome-wide profiling of enzyme kinetic properties is feasible in plants, paving the way for understanding resource allocation. KW - Arabidopsis thaliana KW - constraint-based modeling KW - enzyme catalytic rates KW - kinetic parameter KW - metabolic network KW - turnover number Y1 - 2020 U6 - https://doi.org/10.1111/tpj.14890 SN - 0960-7412 SN - 1365-313X VL - 103 IS - 6 SP - 2168 EP - 2177 PB - Wiley CY - Oxford ER - TY - THES A1 - Kryvych, Sergiy T1 - Gene expression profiling in different stages of development of Arabidopsis thaliana leaftrichomes at the single cell level T1 - Genexpressionsanalyse basierend auf Einzelzelltechniken in ausgewählten Stadien der Trichomentwicklung von Arabidopsis thaliana N2 - Each organ of a multicellular organism is unique at the level of its tissues and cells. Furthermore, responses to environmental stimuli or developmental signals occur differentially at the single cell or tissue level. This underlines the necessity of precise investigation of the “building block of life” -the individual cell. Although recently large amount of data concerning different aspects of single cell performance was accumulated, our knowledge about development and differentiation of individual cell within specialized tissue are still far from being complete. To get more insight into processes that occur in certain individual cell during its development and differentiation changes in gene expression during life cycle of A. thaliana leaf hair cell (trichome) were explored in this work. After onset of trichome development this cell changes its cell cycle: it starts endoreduplication (a modified cell cycle in which DNA replication continues in the absence of mitosis and cytokinesis). This makes trichomes a suitable model for studying cell cycle regulation, regulation of cell development and differentiation. Cells of interest were sampled by puncturing them with glass microcapillaries. Each sample contained as few as ten single cells. At first time trichomes in initial stage of trichome development were investigated. To allow their sampling they were specifically labelled by green fluorescent protein (GFP). In total three cell types were explored: pavement cells, trichome initials and mature trichomes. Comparison of gene expression profiles of these cells allowed identification of the genes differentially expressed in subsequent stages of trichome development. Bioinformatic analysis of genes preferentially expressed in trichome initials showed their involvement in hormonal, metal, sulphur response and cell-cycle regulation. Expression pattern of three selected candidate genes, involved in hormonal response and early developmental processes was confirmed by independent method. Effects of mutations in these genes on both trichome and plant development as well as on plant metabolism were analysed. As an outcome of this work novel components in the sophisticated machinery of trichome development and cell cycle progression were identified. These factors could integrate hormone stimuli and network interactions between characterized and as yet unknown members of this machinery. I expect findings presented in this work to enhance and complement our current knowledge about cell cycle progression and trichome development, as well as about performance of the individual cell in general. N2 - Jedes Organ eines vielzelligen Organismus weißt einzigartige Merkmale auf seiner Gewebe und Zellebene auf. Darüber hinaus, werden entwicklungsabhängige sowie aus der Umwelt empfangene Signale zelltypspezifisch interpretiert. Aus dieser Spezialisierung einzelner Zellen ergibt sich somit unmittelbar die Notwendigkeit einzelne Zellen, als Bausteine komplexer Organe, individuell zu untersuchen. Obwohl in den letzten Jahrzehnten große Datenmengen über verschiedene Aspekte einzelner Zellen akkumuliert wurden, ist das Gesamtbild der Differenzierung und Entwicklung individueller Zellen in einem vielzelligen Organismus weitgehend unbekannt. Um der Frage nachzugehen, welche Prozesse sich in einer einzelnen Zelle während ihrer Differenzierung und Entwicklung abspielen, wurden Genexpressionsprofile einzelner Blatthaarzellen der Pflanze Arabidopsis thaliana in verschiedene Entwicklungsstadien erstellt. Nach dem Beginn der Entwicklung einer Protodermalzelle in ein Blatthaar (Trichom) kommt es zu einem Umschalten des Zellzyklus; Endoreduplikation setzt ein. Dies bedeutet, dass DNA repliziert wird, aber keine Zellteilung mehr stattfindet. Aus diesem Grunde eignen sich heranwachsende Trichome besonders gut Mechanismen zu erforschen, die in Verbindung mit der Zellzyklusregulation und Zellentwicklung stehen. Die Inhalte ausgewählter Einzelzellen wurden mit Glasmikrokapillaren extrahiert. Jeweils zehn derartige Einzelzellextrakte wurden daraufhin vereint. Als besonders hervorzuheben gilt, dass es uns in dieser Studie zum ersten mal überhaupt gelang die Inhalte einzelner Trichomzellen in ganz frühen Entwicklungsstadien zu extrahieren und anschließend zu analysieren. Um die Extraktion der Inhalte dieser frühen Zellstadien überhaupt zu ermöglichen, war es erforderlich diese mit dem grün fluoreszierenden Protein (GFP) zu markieren. Neben den Trichominitialzellen wurden ausgewachsene Trichomzellen sowie Epidermiszellen (Pavementzellen) mittels der Einzelzelltechnik untersucht. Ein Vergleich der erstellten Genexpressionsprofile dieser drei Zelltypen ermöglichte es Gene zu identifizieren, die in den ausgewählten Entwicklungsstadien der Trichombildung differentiell induziert wurden. Mittels bioinformatischer Analysemethoden gelang es, Gruppen von Genen zu identifieren, die exklusiv in Trichominitialzellen exprimiert sind und den Kategorien, Hormonregulation, Metallhomeostase, Schwefelstoffwechesol sowie Zellzyklusregulation zuzuordnen sind. Weiterhin wurde das Expressionsmuster dreier ausgewählter Kandidatengene mit alternativen Techniken verifiziert. Die ausgewählten Kandidatengene gehörten den Katergorien, Hormonrespons sowie frühe Entwicklungsprozesse, an. Darüber hinaus wurden Mutanten in allen drei Gene erzeugt und der Einfluss dieser Mutationen auf die Trichomentwicklung analysiert. Ein weiterer Aspekt der Mutantenanalyse lag in der Erstellung von Metabolitenprofilen ausgewählter Mutanten. Als ein wesentliches Ziel dieser Arbeit gelang es mir bisher unbekannte Komponenten in der Trichomentwicklung und damit der Zellzyklusregulation zu identifizieren. Diese neu identifizierten Komponenten führen zu einer Integration der hormonellen Kontrolle der Zellteilung und Entwicklung mit bisher unbekannten Faktoren. Ich erwarte, dass die von mir erbrachten Ergebnisse zu einem tieferen Verständnis der Prozesse, die an der Trichomentwicklung sowie an der Zellzyklusregulation beteiligt sind, beitragen. Insbesondere, zu einem erweiterten Verständnis des Verhaltens individueller Zellen in einem vielzelligen Organismus. KW - Arabidopsis thaliana KW - Single cell level KW - Gene expression profiling KW - Cell cycle KW - Plant hormones KW - Leaf trichomes KW - Trichome initial cells Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-17474 ER - TY - THES A1 - Krebs, Jonas T1 - Molecular and physiological characterisation of selected DOF transcription factors in the model plant Arabidopsis thaliana T1 - Molekulare und physiologische Charakterisierung ausgewählter DOF Transkriptionsfaktoren in der Modellpflanze Arabidopsis thaliana N2 - About 2,000 of the more than 27,000 genes of the genetic model plant Arabidopsis thaliana encode for transcription factors (TFs), proteins that bind DNA in the promoter region of their target genes and thus act as transcriptional activators and repressors. Since TFs play essential roles in nearly all biological processes, they are of great scientific and biotechnological interest. This thesis concentrated on the functional characterisation of four selected members of the Arabidopsis DOF-family, namely DOF1.2, DOF3.1, DOF3.5 and DOF5.2, which were selected because of their specific expression pattern in the root tip, a region that comprises the stem cell niche and cells for the perception of environmental stimuli. DOF1.2, DOF3.1 and DOF3.5 are previously uncharacterized members of the Arabidopsis DOF-family, while DOF5.2 has been shown to be involved in the phototrophic flowering response. However, its role in root development has not been described so far. To identify biological processes regulated by the four DOF proteins in detail, molecular and physiological characterization of transgenic plants with modified levels of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 expression (constitutive and inducible over-expression, artificial microRNA) was performed. Additionally expression patterns of the TFs and their target genes were analyzed using promoter-GUS lines and publicly available microarray data. Finally putative protein-protein interaction partners and upstream regulating TFs were identified using the yeast two-hybrid and one-hybrid system. This combinatorial approach revealed distinct biological functions of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 in the context of root development. DOF1.2 and DOF3.5 are specifically and exclusively expressed in the root cap, including the central root cap (columella) and the lateral root cap, organs which are essential to direct oriented root growth. It could be demonstrated that both genes work in the plant hormone auxin signaling pathway and have an impact on distal cell differentiation. Altered levels of gene expression lead to changes in auxin distribution, abnormal cell division patterns and altered root growth orientation. DOF3.1 and DOF5.2 share a specific expression pattern in the organizing centre of the root stem cell niche, called the quiescent centre. Both genes redundantly control cell differentiation in the root´s proximal meristem and unravel a novel transcriptional regulation pathway for genes enriched in the QC cells. Furthermore this work revealed a novel bipartite nuclear localisation signal being present in the protein sequence of the DOF TF family from all sequenced plant species. Summing up, this work provides an important input into our knowledge about the role of DOF TFs during root development. Future work will concentrate on revealing the exact regulatory networks of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 and their possible biotechnological applications. N2 - Mehr noch als Tiere, die ihren Lebensraum unter widrigen Umständen verlassen können, sind Pflanzen mit einem festen Standort auf ihre Anpassungsfähigkeit angewiesen. Einen entscheidenden Beitrag dazu leistet die Genregulation, d.h. das gezielte An- und Ausschalten von Erbanlagen, den Genen. Vermittelt wird dieser Regulationsprozess unter anderem durch Transkriptionsfaktoren: Proteine, die die Fähigkeit besitzen, an bestimmte Regionen der Gene zu binden und damit deren Aktivität zu beeinflussen. In der Ackerschmalwand (Arabidopsis thaliana), die als Modellpflanze in der Genetik verwendet wird, existieren etwa 2000 solcher Transkriptionsfaktoren, eingeteilt in Familien, von denen einige auch in tierischen Organismen auftreten, andere pflanzenspezifisch sind. Auf Grund ihrer Funktion als wichtige Kontrollelemente sind sie von großem wissenschaftlichem und biotechnologischem Interesse. Im Rahmen dieser Doktorarbeit sollte die Funktion von vier pflanzenspezifischen Transkriptionsfaktoren, genannt DOF1.2, DOF3.1, DOF3.5 und DOF5.2, untersucht werden, welche durch ihre spezifische Aktivität in der Wurzelspitze der Ackerschmalwand identifiziert wurden. Um die Funktion dieser vier Regulatoren aufzuklären, wurden an der Modellpflanze gentechnische Veränderungen durchgeführt und die so veränderten, auch als transgen bezeichneten Pflanzen mit molekularbiologischen und physiologischen Methoden untersucht. Es konnte gezeigt werden, dass DOF1.2 und DOF3.5 eine wesentliche Funktion beim gerichteten Wurzelwachstum spielen und ein seitliches Wachsen der Wurzel aufgrund veränderter Umwelteinflüsse verhindern, bzw. hervorrufen können. Die beiden anderen Proteine DOF3.1 und DOF5.2 erfüllen ihre Funktion in der Stammzellnische der Wurzel. Vergleichbar mit tierischen Stammzellen sind auch pflanzliche Stammzellen nicht zu einem bestimmten Zelltyp herangereift, sondern verbleiben in einem sogenannten undifferenzierten Zustand. Es konnte gezeigt werden, dass DOF3.1 und DOF5.2 zum Erhalt dieses Zustands benötigt werden, da nach Inaktivierung beider Proteine Zellspezialisierungen auftreten, die bei gentechnisch unveränderten Pflanzen nicht auftreten. Desweiteren konnte in dieser Arbeit geklärt werden, welcher Proteinabschnitt der DOF-Proteine für ihren Transport in den Zellkern notwendig ist. Denn da die pflanzlichen Erbanlagen im Zellkern vorliegen, muss für eine Einflussnahme auf deren Aktivität zunächst ein Transport der Regulationsproteine in den Zellkern stattfinden. Zusammengenommen konnte mit dieser Doktorarbeit das Wissen über Transkriptionsfaktoren und Entwicklungsprozesse der Wurzel erheblich erweitert werden. Zudem ist die Grundlage für interessante zukünftige Arbeiten gelegt worden. Dabei wird es von zentraler Bedeutung sein, komplexe Regulationsnetzwerke verstehen zu lernen und durch gezielte Manipulationen biotechnologisch nutzen zu können. KW - DOF Transkriptionsfaktoren KW - Arabidopsis thaliana KW - Wurzel KW - Ruhezentrum KW - Columella KW - DOF transcription factors KW - Arabidopsis thaliana KW - root KW - quiescent center KW - columella Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-41831 ER - TY - JOUR A1 - Kappel, Christian A1 - Friedrich, Thomas A1 - Oberkofler, Vicky A1 - Jiang, Li A1 - Crawford, Tim A1 - Lenhard, Michael A1 - Bäurle, Isabel T1 - Genomic and epigenomic determinants of heat stress-induced transcriptional memory in Arabidopsis JF - Genome biology : biology for the post-genomic era N2 - Background Transcriptional regulation is a key aspect of environmental stress responses. Heat stress induces transcriptional memory, i.e., sustained induction or enhanced re-induction of transcription, that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crop plants is an important breeding goal. However, not all heat stress-inducible genes show transcriptional memory, and it is unclear what distinguishes memory from non-memory genes. To address this issue and understand the genome and epigenome architecture of transcriptional memory after heat stress, we identify the global target genes of two key memory heat shock transcription factors, HSFA2 and HSFA3, using time course ChIP-seq. Results HSFA2 and HSFA3 show near identical binding patterns. In vitro and in vivo binding strength is highly correlated, indicating the importance of DNA sequence elements. In particular, genes with transcriptional memory are strongly enriched for a tripartite heat shock element, and are hallmarked by several features: low expression levels in the absence of heat stress, accessible chromatin environment, and heat stress-induced enrichment of H3K4 trimethylation. These results are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes. Conclusions Our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants, enabling the prediction and engineering of genes with transcriptional memory behavior. KW - Transcriptional memory KW - Priming KW - Heat stress KW - HSFA2 KW - HSFA3 KW - Arabidopsis thaliana KW - Histone H3K4 trimethylation KW - ChIP-seq Y1 - 2023 U6 - https://doi.org/10.1186/s13059-023-02970-5 SN - 1474-760X VL - 24 IS - 1 PB - BioMed Central CY - London ER - TY - JOUR A1 - Hansen, Bjoern Oest A1 - Meyer, Etienne H. A1 - Ferrari, Camilla A1 - Vaid, Neha A1 - Movahedi, Sara A1 - Vandepoele, Klaas A1 - Nikoloski, Zoran A1 - Mutwil, Marek T1 - Ensemble gene function prediction database reveals genes important for complex I formation in Arabidopsis thaliana JF - New phytologist : international journal of plant science N2 - Recent advances in gene function prediction rely on ensemble approaches that integrate results from multiple inference methods to produce superior predictions. Yet, these developments remain largely unexplored in plants. We have explored and compared two methods to integrate 10 gene co-function networks for Arabidopsis thaliana and demonstrate how the integration of these networks produces more accurate gene function predictions for a larger fraction of genes with unknown function. These predictions were used to identify genes involved in mitochondrial complex I formation, and for five of them, we confirmed the predictions experimentally. The ensemble predictions are provided as a user-friendly online database, EnsembleNet. The methods presented here demonstrate that ensemble gene function prediction is a powerful method to boost prediction performance, whereas the EnsembleNet database provides a cutting-edge community tool to guide experimentalists. KW - Arabidopsis thaliana KW - co-function network KW - complex I KW - ensemble prediction KW - gene function prediction Y1 - 2017 U6 - https://doi.org/10.1111/nph.14921 SN - 0028-646X SN - 1469-8137 VL - 217 IS - 4 SP - 1521 EP - 1534 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Gonzalez, Wendy A1 - Riedelsberger, Janin A1 - Morales-Navarro, Samuel E. A1 - Caballero, Julio A1 - Alzate-Morales, Jans H. A1 - Gonzalez-Nilo, Fernando D. A1 - Dreyer, Ingo T1 - The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids JF - The biochemical journal N2 - The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2-2.5 pH units) is perceived by voltage-gated inward potassium channels (K-in) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K-in channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K-in channel KAT1, mutations in the unique histidine exposed to the solvent (His(267)) do not affect the pH dependency. We demonstrate in the present study that His(267) of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe(266) shifts its pK(a) to undetectable values through a cation-pi interaction. Instead, we show that Glu(240) placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu(240), several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel. KW - Arabidopsis thaliana KW - channel protein structure KW - channel protein-proton interaction KW - KAT1 KW - pH regulation KW - potassium chanel Y1 - 2012 U6 - https://doi.org/10.1042/BJ20111498 SN - 0264-6021 VL - 442 IS - 7 SP - 57 EP - 63 PB - Portland Press CY - London ER -