113 research outputs found
Novel Regulators in Photosynthetic Redox Control of Plant Metabolism and Gene Expression
No full textDietz K-J, Pfannschmidt T. Novel Regulators in Photosynthetic Redox Control of Plant Metabolism and Gene Expression. Plant Physiology. 2011;155(4):1477-1485
Arabidopsis STN7 Kinase Provides a Link between Short- and Long-Term Photosynthetic Acclimation
No full textFlowering plants control energy allocation to their photosystems in response to light quality changes. This includes the phosphorylation and migration of light-harvesting complex II (LHCII) proteins (state transitions or short-term response) as well as long-term alterations in thylakoid composition (long-term response or LTR). Both responses require the thylakoid protein kinase STN7. Here, we show that the signaling pathways triggering state transitions and LTR diverge at, or immediately downstream from, STN7. Both responses require STN7 activity that can be regulated according to the plastoquinone pool redox state. However, LTR signaling does not involve LHCII phosphorylation or any other state transition step. State transitions appear to play a prominent role in flowering plants, and the ability to perform state transitions becomes critical for photosynthesis in Arabidopsis thaliana mutants that are impaired in thylakoid electron transport but retain a functional LTR. Our data imply that STN7-dependent phosphorylation of an as yet unknown thylakoid protein triggers LTR signaling events, whereby an involvement of the TSP9 protein in the signaling pathway could be excluded. The LTR signaling events then ultimately regulate in chloroplasts the expression of photosynthesis-related genes on the transcript level, whereas expression of nuclear-encoded proteins is regulated at multiple levels, as indicated by transcript and protein profiling in LTR mutants
Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases
No full textIllumination changes elicit modifications of thylakoid proteins
and reorganization of the photosynthetic machinery. This involves, in the short term, phosphorylation of photosystem II
(PSII) and light-harvesting (LHCII) proteins. PSII phosphorylation
is thought to be relevant for PSII turnover1,2, whereas LHCII
phosphorylation is associated with the relocation of LHCII and
the redistribution of excitation energy (state transitions) between
photosystems3,4. In the long term, imbalances in energy distribution
between photosystems are counteracted by adjusting
photosystem stoichiometry5,6. In the green alga Chlamydomonas
and the plant Arabidopsis, state transitions require the orthologous
protein kinases STT7 and STN7, respectively7,8. Here we
show that in Arabidopsis a second protein kinase, STN8, is
required for the quantitative phosphorylation of PSII core proteins.
However, PSII activity under high-intensity light is affected
only slightly in stn8 mutants, and D1 turnover is indistinguishable
from the wild type, implying that reversible protein phosphorylation
is not essential for PSII repair. Acclimation to changes in
light quality is defective in stn7 but not in stn8 mutants, indicating
that short-term and long-term photosynthetic adaptations are
coupled. Therefore the phosphorylation of LHCII, or of an
unknown substrate of STN7, is also crucial for the control of
photosynthetic gene expressio
The plastid-encoded RNA polymerase of plant chloroplasts
No full textPlant chloroplasts possess a dedicated genome (plastome) and a prokaryotic-type plastid-encoded RNA polymerase (PEP) that mediates its expression. PEP is composed of five bacteria-like core proteins and 16 nucleus-encoded PEP-associated proteins (PAPs). These are essential for PEP-driven transcription and chloroplast biogenesis, but their functions and structural arrangement in the PEP complex remained largely enigmatic. Recently, four independently determined cryogenic-electron microscopy (cryo-EM) structures of purified plant PEP complexes reported features of the prokaryotic core and the arrangement of PAPs around it, identified potential functional domains and cofactors, and described the interactions of PEP with DNA. We explore these data and critically discuss the proposed regulatory impact of PAPs on the transcription process. We further address the evolutionary implications and describe fields for future investigation
RNA-protein Interaction Mediating Post-transcriptional Regulation in the Circadian System
No full textSchöning JC, Staiger D. RNA-protein Interaction Mediating Post-transcriptional Regulation in the Circadian System. In: Pfannschmidt T, ed. Plant Signal Transduction. Methods in Molecular Biology. Vol 479. Totowa, NJ: Humana Press; 2009: 337-351.Post-transcriptional control makes an important contribution to shaping transcript profiles of circadianly regulated genes. In Arabidopsis thaliana , the clock-regulated glycine-rich RNA-binding protein AT GRP7 oscillates with a 24-h rhythm and transmits the rhythmicity generated by the central oscillator within the cell. AT GRP7 negatively auto-regulates its own expression at the post-transcriptional level. In response to an elevated protein level, a shift to a cryptic 5′ splice site within the intron occurs, leading to an unproductively spliced transcript that rapidly vanishes due to its short half-life. This feedback regulation relies on direct binding of the RNA-binding protein to its own RNA. Here we describe the analysis of RNA-protein interaction in vitro employing recombinant RNA-binding protein and 32 P-labelled in vitro transcripts or synthetic RNA oligoribonucleotides comprising the binding site under study
Discrete Redox Signaling Pathways Regulate Photosynthetic Light-Harvesting and Chloroplast Gene Transcription
No full textThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Molekulare und funktionelle Charakterisierung der Casein Kinase 2 : vergleichende Phosphoproteom- und Metabolom-Analysen
No full textDas Ziel der Arbei war es die Casein Kinase 2 (CK2), eine lösliche Kinase im Stroma der Chloroplasten, umfassend molekularbiologisch und funktionell zu untersuchen. Dazu wurden mit einer T-DNA Insertionsmutante GK615F11 umfassende Experimente mit dem Ansatz der reversen Genetik durchgeführt. Alle Experimente erfolgten im Vergleich der Mutante zum Wildtyp und während der Arbeit erzeugten komplementierten Linie. Das Wachstum der Rosette und der Wurzel wurde genau dokumentiert, Genexpressionsanalysen mit quantitativer RT-PCR durchgeführt. Es konnte gezeigt werden, dass es sich bei der Mutante um einen funktionalen knock-out, mit einer geringen Restgenexpression handelt.The aim of the thesis was molecularbiological and functional studies of Casein Kinase 2, a soluble kinase in the stroma of the chloroplasts. There for a T-DNA insertion mutant GK615F11 was used for reverse genetics experimenting. All experiments were done in comparison between the mutant to the wild type and to the, during the thesis, generated complementedline. The growth of the rosette and the root were documented, gene expression analysis with quantitative RT-PCR done. It was shown, that the mutant hasa functional knock-out, with little remaining gene expression
Role of orbital fibroblasts in tissue remodeling of Graves' orbitopathy - involvement of hypoxia dependent and independent pathways
No full textGraves’ disease (GD) is an autoimmune disease caused by autoantibodies directed mainly against the thyrotropin stimulating hormone receptor (TSHR) leading to hyperthyroidism. Graves’ orbitopathy (GO) is the common extra-thyroidal manifestation of Graves’ disease is characterized by inflammation and expansion of the retroocular connective/fat tissue and of the extraocular muscles (EOM). The expansion of the orbital tissue within the limited space of the bony orbit leads to proptosis, extraocular muscle dysfunction, and, in severe cases, to an optic nerve compression. Inflammation and enlargement of the orbital tissue can cause hypoxia and consequently induce hypoxia-inducible-factor 1 (HIF-1) pathways. The work presented in this thesis addressed the role of orbital fibroblast (OF) and/or mesenchymal stem cells (MSC) for pathogenesis of GO in patients and mouse model. We aimed to characterize human and mouse OF and/or MSC and involvement of hypoxia depending and independent pathways in the pathophysiological process leading to GO.
The central cell type in this pathophysiologic process are the orbital fibroblasts (OF) which we derived either from fat biopsies of GO patients/control persons or from orbital tissue of GO/control mouse model. Additionally, we identified MSC in the fat tissue of GO patients and showed that OF shared all biological characteristics of MSC: Both the OF and the MSC expressed a typical MSC surface marker set, suppressed T cell proliferation and secreted cytokines. Furthermore, the cells produced hyaluronan and were able to differentiate along adipogenic, osteogenic, chondrogenic, myogenic, and neuronal pathways. It appears that OF population comprises undifferentiated progenitor cells responsible for adipogensis and/or myogenesis in GO.
To analyze whether hypoxia dependent pathways are involved in tissue remodeling of GO we investigated the role of HIF-1 action for angiogenesis and adipogensis. Most likely as a consequence of hypoxia we found increased vascularization and HIF-1α positive cells in the connective/fat tissue of GO patients. OF derived thereof expressed higher HIF-1α levels and secreted elevated levels of VEGF in an HIF-1 dependent manner. Furthermore, hypoxia induced HIF-1 dependent adipogenic differentiation of OF which could be further increased by stimulation of TSHR using TSH indicating an unfavorable marriage between hypoxia and effects of autoimmunity. Smoking is the strongest risk factor for GO and develops it full effect most likely through smoke ingredients and hypoxia. Interestingly, we found that OF derived from GO smoker more strongly induced HIF-1α levels in response to cigarette smoke extract and hypoxia compared to GO non-smoker. Increased HIF-1α expression can contribute to the strong tissue expansion observed GO smoker.
To investigate pathological changes in OF during the course of GO we established a preclinical mouse model of GO induced by genetic immunization of human TSHR A- subunit plasmid. The pathological features of GO in the experimental mouse model were similar in orbital pathology to those present in patients with GO; GO mice developed TSHR antibodies with different levels of stimulating and blocking activity as well as TSHR antigen specific response of splenic T cells. Furthermore GO mice just like GO patients developed thyroid dysfunction and orbital disease with mice showing either more enlargement of orbital adipose tissue or myopathy of EOM. We derived OF from tissue comprise of EOM and adipose of the immunized mice. Mouse OF (mOF) expressed MSC marker, elevated levels of TSHR and insulin-like growth factor 1 receptor (IGF-1R) similar to OF derived from GO patients. Likewise, mOF showed increased adipogenesis and enhanced hyaluronan secretion by activation of TSHR and IGF-1R. Moreover, in response to hypoxia OF derived from GO mice enhanced HIF-1α levels, suggesting that hypoxia dependent signaling is involved in orbital pathogenesis. Thus, OF derived from GO mice display a pathogenic cell type similar to OF derived from GO patients.
In conclusion, OF and MSC are central for GO pathogenesis in patients and mouse model. HIF-1 dependent pathways are involved in tissue remodeling and expansion during pathogenesis of GO. The understanding of the contribution of different orbital cell populations for tissue remodeling in response to hypoxia during the course of the eye disease will be further studied in the GO mouse model. In addition the role of hypoxia for inflammation in context of autoimmunity remains to be discovered. This will lead to a better insight of disease pathogenesis and is fundamental to develop potential novel therapeutic targets.Morbus Basedow (Graves‘ Disease) ist eine Schilddrüsenerkrankung, die durch Autoantikörper verursacht wird, die hauptsächlich gegen den Thyreotropin-stimulierenden Hormonrezeptor (TSHR) gerichtet sind und eine Hyperthyreose zur Folge haben. Endokrine Orbitopathie (Graves' Orbitopathie, GO) ist eine extrathyroidale Manifestation, die üblicherweise im Zusammenhang mit Morbus Basedow auftritt und durch Entzündung und Ausdehnung des retrookularen Bindegewebes und der extraokulären Muskeln (EOM) gekennzeichnet ist. Die Ausdehnung des orbitalen Gewebes in die räumlich begrenzte, knöcherne Orbita führt zu Proptosis, Muskeldysfunktion und in schweren Fällen zu einer Kompression des Sehnervs. Die Entzündung und Ausdehnung des orbitalen Gewebes kann eine Sauerstoffunterversorgung (Hypoxie) des Gewebes verursachen und somit zu einer Induktion von Hypoxie-induzierbarer Faktor 1 (HIF-1) abhängigen Signalwege führen. Die vorliegende Arbeit beschäftigt sich mit der Rolle orbitaler Fibroblasten (OF) und/oder mesenchymaler Stammzellen (MSC) in der Pathogenese von GO Patienten und im GO Mausmodell. Ziel war die Charakterisierung humaner und muriner OF und/oder MSC im Hinblick auf Hypoxie-abhängige und unabhängige pathophysiologische Prozesse der GO.
Orbitale Fibroblasten sind der zentrale Zelltyp im pathophysiologischen Prozess, und wurden entweder aus Fettbiopsien von GO Patienten/Kontrollpersonen oder aus orbitalem Gewebe eines GO Mausmodells/Kontrollmäuse gewonnen. Darüber hinaus identifizierten wir MSC im orbitalen Fettgewebe von GO Patienten und zeigten, dass OF alle biologischen Merkmale der MSC teilten: Sowohl OF als auch MSC exprimierten typische MSC Oberflächen-Marker, unterdrückten die T-Zellproliferation und sezernierten Zytokine. Des Weiteren produzierten sie Hyaluronsäure und waren in der Lage sowohl adipogen, osteogen und chondrogen als auch myogen und neuronal zu differenzieren. Dies zeigte, dass die OF Population undifferenzierte Vorläuferzellen beinhaltet, die für die gesteigerte Adipogenese und/oder Myogenese im Gewebe von GO Patienten verantwortlich sein könnten.
Um zu untersuchen, ob Hypoxie-abhängige Signalwege in den Gewebeumbau bei GO involviert sind, analysierten wir die Rolle von HIF-1 für die Angiogenese und Adipogenese unter hypoxischen Bedingungen. Als eine mögliche Folge der Hypoxiewirkung fanden wir eine erhöhte Vaskularisierung und HIF-1α-positive Zellen im Binde-/Fettgewebe von GO Patienten. Die aus dem Fettgewebe der Patienten gezüchteten OF zeigten eine höhere HIF-1 Expression und sezernierten HIF-1 abhängig mehr VEGF. Des Weiteren wurde unter Hypoxie die adipogene Differenzierung der OF HIF-1 abhängig induziert. Die durch Hypoxie induzierte Adipogenese konnte durch Stimulation des TSHR unter Verwendung von TSH noch weiter gesteigert werden, was das ungünstige Zusammenwirken von Hypoxie und Effekten der Autoimmunität widerspiegelt. Rauchen ist der stärkste Risikofaktor für den Schweregrad einer GO und entfaltet seine Wirkung wahrscheinlich durch die Kombination von Rauchbestandteilen und Hypoxie. Interessanterweise konnten wir feststellen, dass OF von GO Rauchern im Vergleich zu GO Nichtrauchern höhere HIF-1α Mengen in Reaktion auf Zigarettenrauchextrakt und Hypoxie aufwiesen. Die erhöhte HIF-1α Expression bei GO Rauchern könnte die starke Gewebsexpansion, welche bei GO Rauchern beobachtet wird, begünstigen.
Um die pathologischen Veränderungen der OF im Verlauf einer GO untersuchen zu können, etablierten wir ein präklinisches GO Mausmodell, das durch genetische Immunisierung mit einem Plasmid, welches für die humane TSHR A-Untereinheit kodierte, induziert werden konnte. Die TSHR immunisierten Mäuse entwickelten alle Anzeichen von Morbus Basedow mit GO: Die Mäuse entwickelten TSHR-Antikörper mit unterschiedlichen Mengen an stimulierenden- und/oder blockierenden Antikörpern, eine TSHR-Antigen-spezifische Antwort von T-Zellen der Milz, Schilddrüsenfunktionsstörungen und Orbitopathie. Dabei entwickelten die Mäuse ähnlich wie GO Patienten im unterschiedlichen Maße eine Vergrößerung des orbitalen Fettgewebes und/oder eine Myopathie der EOM. Aus dem orbitalen Gewebe der Mäuse, welches überwiegend aus EOM und Fett bestand, isolierten und charakterisierten wir Fibroblasten. Ähnlich wie die OF von Patienten, exprimierten die Maus OF (mOF) typische MSC Marker, TSHR und Insulin Wachstumsfaktor-1-Rezeptor (IGF-1R). Ebenso zeigten mOF eine gesteigerte Adipogenese und vermehrte Hyaluronsäure Sekretion in Antwort auf TSHR und IGF-1R Aktivierung. Darüber hinaus, zeigten OF von GO Mäusen erhöhte HIF-1α Mengen unter hypoxischen Bedingungen, was darauf hindeutete, dass Hypoxie-abhängige Signalwege an der Pathogenese einer GO bei Mäusen beteiligt sein könnten. Somit zeigten die OF von GO Mäusen einen pathogenen Zelltyp, der den OF von GO Patienten ähnlich ist.
Zusammengefasst konnte gezeigt werden, dass OF und/oder MSC eine zentrale Zellpopulation in der Pathogenese von GO Patienten und GO Mausmodell darstellen. HIF-1 abhängige Signalwege sind am Gewebeumbau und -expansion beteiligt. Das Verständnis, wie verschiedene orbitale Zellpopulationen am Gewebeumbau als Reaktion auf Hypoxie mitwirken, wird während des Verlaufs der Augenerkrankung im GO Mausmodell noch weiter untersucht. Darüber hinaus ist es von Interesse die Rolle von Hypoxie-abhängigen Signalwege im Entzündungsprozess im Kontext der Autoimmunität aufzuklären. Dies kann zu einem besseren Einblick in die Pathogenese der GO führen und ist von grundlegender Bedeutung für die Entwicklung neuer therapeutischer Ansätze
Structure of the multi-subunit chloroplast RNA polymerase
No full textChloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression
- …
