The transcriptional apparatus of Chlamydomonas chloroplasts

The transcriptional apparatus of higher plant chloroplasts is well characterised and consists of a plastid-encoded polymerase (PEP) and a nuclear encoded polymerase (NEP). PEP is dispensable to cell viability. The situation in green algal species, however, is less clear. Chloroplast genes encoding subunits of the PEP have been cloned and sequenced in the green alga Chlamydomonas reinhardtii and preliminary reverse-genetic studies suggest that PEP is essential to cell viability, which is in contrast to the situation in higher plants. To investigate this further a series of gene knockouts were constructed using the chloroplast gene rpoC2, encoding the " subunit of PEP. Results indicate that PEP is essential to C. reinhardtii cell viability. In addition, inhibitors of PEP have been used in an in vivo transcription assay to try to identify a second RNA polymerase activity in C. reinhardtii chloroplasts. In all higher plant and red algal species so far studied the PEP factor is encoded in the nuclear genome. A C. reinhardtii nuclear gene (rpoD) encoding a putative PEP factor has been cloned and partially sequenced. This is the first factor cloned from a green algal species. A transcript of ~2.9 kb was detected for the rpoD gene by northern analysis. Finally, epitope tagging technology was developed for chloroplast and bacterial gene products. The rpoC2 gene of C. reinhardtii was modified to produce a 6x-histidine tagged polypeptide and an attempt was made to purify this polypeptide from C. reinhardtii cells using IMAC. In addition, a 3x haemagglutinin (HA) epitope tag was codon optimised for use in C. reinhardtii chloroplasts and this epitope was used to tag -galactosidase in E. coli. The protein was detected in a western blot using anti-HA monoclonal antibodies. This epitope will prove useful as a tool to tag C. reinhardtii chloroplast proteins

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont

CHARACTERIZATION OF PLANT POLYADENYLATION TRANSACTING FACTORS-FACTORS THAT MODIFY POLY(A) POLYMERSE ACTIVITY

Plant polyadenylation factors have proven difficult to purify and characterize, owing to the presence of excessive nuclease activity in plant nuclear extracts, thereby precluding the identification of polyadenylation signal-dependent processing and polyadenylation in crude extracts. As an alternative approach to identifying such factors, a screen was conducted for activities that inhibit the non-specific activity of plant poly(A) polymerases (PAP). One such factor (termed here as Putative Polyadenylation Factor B, or PPF-B) was identified in a screen of DEAE-Sepharose column fractions using a partially purified preparation of a plant nuclear poly(A) polymerase. This factor was purified to near homogeneity. Surprisingly, in addition to being an effective inhibitor of the nuclear PAP, PPF-B inhibited the activity of a chloroplast PAP. In contrast, this factor stimulated the activity of the yeast PAP. Direct assays of ATPase, proteinase, and nuclease activities indicated that inhibition of PAP activity was not due to depletion of substrates or degradation of products of the PAP reaction. The major polypeptide component of PPF-B proved to be a novel linker histone (RSP), which copurified with inhibitory activity by affinity chromatography on DNA-cellulose. The association of inhibitory activity with a linker histone and the spectrum of inhibitory activity, raise interesting possibilities regarding the role of PPF-B in nuclear RNA metabolism. These include a link between DNA damage and polyadenylation, as well as a role for limiting the polyadenylation of stable RNAs in the nucleus and nucleolus. The Arabidopsis genome possesses genes encoding probable homologs of most of the polyadenylation subunits that have been identified in mammals and yeast. Two of these reside on chromosome III and V and have the potential to encode a protein that is related to the yeast and mammalian Fip1 subunit (AtFip1-III and AtFip1-V). These genes are universally expressed in Arabidopsis tissues. AtFip1-V stimulates the non-specific activity of at least one Arabidopsis nuclear PAP, binds RNA, and interacts with other polyadenylation homologs AtCstF77 and AtCPSF30. These studies suggest that AtFip1- V is an authentic polyadenylation factor that coordinates other subunits and plays a role in regulating the activityof PAP in plants

Identification of the Physiological Role of Carbonic Anhydrase Using the Antisense Technique, And, Investigation of Its Transcriptional Regulation in Arabidopsis Thaliana (L.) Heynh.

Carbonic anhydrase (CA, EC 4.2.1.1), catalyzes the interconversion of CO\sb2 and \rm HCO\sb3\sp- in many organisms. The majority of CA activity in C3 leaves is in the chloroplast and expression of the chloroplast CA is regulated by light. To investigate the physiological role of the chloroplast CA, its expression was suppressed by insertion of a CA transgene in the antisense orientation in Arabidopsis thaliana (L.) Heynhold. Activity assays and immunoblots show that in five independent lines of antisense transgenic plants CA activity was strongly suppressed. All five independent lines died or grew poorly in the absence of sucrose whereas wild type plants grew well in the same media. Antisense plants exhibited a phenotype comparable to wild type plants on sucrose-free media only when grown in an atmosphere with elevated levels of CO\sb2. The results support the notion that CA facilitates CO\sb2 diffusion from the atmosphere to the chloroplast and clearly show that CA plays a pivotal role in carbon assimilation in C3 plants. In order to investigate the regulation of expression of the chloroplastic CA, a CA gene from Arabidopsis thaliana was cloned, sequenced, and characterized. Sequence analysis of the 5\sp\prime end of the gene encoding the chloroplastic CA revealed the presence of several cis-acting elements previously shown to mediate expression of genes encoding chloroplast proteins. Serial deletions from the 5\sp\prime end of the CA promoter were fused with a reporter gene encoding $\beta$-glucuronidase and introduced into Arabidopsis thaliana. Deletion of an AT-rich palindromic sequence, designated AT-1P, resulted in the largest decrease in GUS activity in transgenic plants. However, the presence of only a second AT-rich palindromic sequence, designated TA-1P, upstream of the CAAT box conferred leaf-specific expression. An expression library from Arabidopsis thaliana was screened with a concatemer of AT-1P and yielded a partial clone. A cDNA containing the entire coding sequence was obtained by 5\sp\prime RACE. Conceptual translation of this cDNA reveals a unique protein containing two putative \rm C\sb2/C\sb2 zinc fingers near the N-terminus followed by four putative CCHC zinc fingers

The nucleotide-binding sub-proteome of mustard chloroplasts and its involvement in plastid redox signaling

The actual keen about redox signal development at the plastid photosynthetic apparatus, transmission and the reply to the signal was highlighted by the contribution of three review article to this work. Pfannschmidt et al. 2008 summarizes short and long term acclimation responses (STR and LTR respectively) to redox signals of the Plastoquinone (PQ) pool and the involvement of putative phosphorylation cascades and thioredoxins as well as the influence of the redox state on primary target genes in plastids and nucleus. Further on experimental approaches for the generation of a defined redox state at the photosynthetic electron transport (PET) chain was discussed. Dietzel et al. 2008 reviews the different types of retrograde signals between plastids and nucleus as well as the complexity and interaction of the signaling cascades and networks and in Pfalz et al. 2012 the environmental influences on gene expression and recent findings within plastid redox signaling were discussed. For a detailed investigation of the adaption of plastid gene expression responding to plastid redox signals the gene expression machinery of chloroplasts itself was studied. An experimental approach was used for the generation of a defined redox signal in mustard cotyledons, the following isolation of its chloroplasts and further on the nucleotide binding sub-proteome using heparin-Sepharose (HS) (Steiner et al. 2009; Schröter et al. 2010). The characterization and comparison of mustard cotyledons acclimated to redox signal inducing Light-qualities with Arabidopsis thaliana cotyledons was important for the integration of new findings within Sinapis alba into established models (Steiner et al. 2009). An effect on the transcriptional regulation of the two plastome-encoded genes psaAB and psbA was studied here concerning promoter recognition and specificity (Steiner et al. 2009). The impact of phosphorylation events on gene expression was surveyed and confirmed by determination of the phosphorylation state of the HS fractions, the endogenous kinase activity and the cooperative influence of kinase activity and thiol redox state on Chloroplast transcription (Steiner et al. 2009). HS proteins fractions contain a high degree of DNA and especially psaA and psbA binding proteins which were identified using mass spectrometry and Brassicales databases (Steiner et al. 2009; Schröter et al. 2010; Steiner et al. 2011). Special emphasis was on the analysis of the essential subunits of the plastid-encoded plastid RNA-polymerase (PEP) which was well to prepare by 2 dimensional (2D) blue native (BN) gel electrophoresis (Schröter et al. 2010; Steiner et al. 2011). The degree of proteins involved in gene expression was strongly increased by the use of a second chromatographic step with Phosphocellulose (PC) additional to HS (Schröter et al. 2014). Visualization and identification of this nucleotide binding sub-proteome was the aim of the last publication included into this work giving access to a precise view on the gene expression related proteome of mustard plastids (Schröter et al. 2014).Drei review Artikel beleuchten das aktuelle Wissen über die Redoxsignalentwicklung im plastidären Photosyntheseapparat, die Signalübermittlung und –beantwotung. Pfannschmidt et al. 2008 fasst Kurzzeitund Langzeitantworten auf Redoxsignale des Plastochinonpools zusammen und darüberhinaus die Einbeziehung von Phosphorylierungskaskaden und Thioredoxinen sowie den Einfluss des Redoxstatus auf primäre Zielgene in Plastiden und dem Zellkern. Desweiteren wurden experimentelle Ansätze für die Erzeugung eines definierten Redoxstatus in der photosynthetischen Elektronentransportkette diskutiert. Dietzel et al. 2008 fasst die verschiedenen Typen retrograder Signale zwischen Plastiden und Zellkern zusammen sowie die Komplexität und Interaktion der Signalkaskaden und –netzwerke und in Pfalz et al. 2012 werden die Umwelteinflüsse auf die Genexpression und aktuelle Erkenntnisse über Redoxsignale diskutiert. Für eine detailierte Untersuchung der Genexpressionsadaption als Antwort auf plastidäre Redoxsignale wurde die Genexpressionsmaschinerie der Chloroplasten direkt studiert. Ein definiertes Redoxsignal wurde in Senfkeimlingen generiert, anschließend die Chloroplasten und schließlich die nukleotidbindenden Proteine mittels Chromatographie über Heparinsepharose (HS) isoliert (Steiner et al. 2009; Schröter et al. 2010). Der Vergleich und die Charakterisierung der Senfkeimlinge, die an das Redoxsignal induzierende Licht akklimatisiert waren, mit Arabidopsis thaliana Keimlingen war wichtig für die Integration neuer Erkenntnisse über Sinapis alba in etablierte Modelle (Steiner et al. 2009). Der Effekt auf die transkriptionale Regulierung der zwei plastomkodierten Gene psaAB und psbA wurde hinsichtlich Promotererkennung und –spezifität untersucht (Steiner et al. 2009). Die Auswirkung von Phosphorylierungen auf die Genexpression wurde, durch die Bestimmung des Phosphorylierungsgrades der HS Fraktionen, der endogene Kinaseaktivität und des kooperativen Einflusses der Kinaseaktivität und des Thiolredoxstatus auf die Chloroplastentranskription, untersucht (Steiner et al. 2009). HS Fraktionen besitzen einen hohen Grad an DNA- und speziell psaA- und psbA-bindenden Proteinen, die durch Massenspektrometrie und Analyse mit Brassicales-Datenbanken identifiziert werden können (Steiner et al. 2009; Schröter et al. 2010; Steiner et al. 2011). Der Schwerpunkt lag bei der Analyse der essentiellen Untereinheiten der plastidenkodierten plastidären RNAPolymerase (PEP), die gut durch 2 dimensionale (2D) blue native (BN) Gelelektrophorese präpariert werden konnte (Schröter et al. 2010; Steiner et al. 2011). Der Anteil an Proteinen der Genexpression konnte durch eine zweite Chromatographie über Phosphocellulose (PC) zusätzlich zur HSChromatographie erzielt werden (Schröter et al. 2014). In der letzten Publikation dieser Arbeit geht es vorrangig um die Visualisierung und Identifizierung dieses nukleotidbindenden Teilproteoms, wodurch ein Zugang zu einem detaillierteren Einblick in das genexpressionsrelevante Proteom der Senfplastiden erzielt wurde (Schröter et al. 2014)

Spinach Carbonic Anhydrase: Primary Sequence, Transport Into Chloroplasts and Expression in Escherichia Coli.

Carbonic anhydrase (CA) catalyzes the reversible hydration of CO\sb2. Plant carbonic anhydrases are structurally distinct from those found in animals. In C\sb3 plants, the enzyme is located in chloroplasts. To date, little structural information on the enzyme from plants has been available. In this study the full-length cDNA for the spinach chloroplast CA was sequenced. The cDNA contained 1,156 base pairs. The open reading frame encoded a protein of 34,569 daltons. Comparison of the N-terminal sequence of this protein with that of other chloroplast precursors indicated that CA is synthesized as a precursor. The transit peptide likely contains about 60 amino acids. Indirect immunoprecipitation of translation products synthesized using pea poly A RNA indicated that the pea precursor is approximately 36,000 daltons. Incubation of either the pea or spinach precursor with isolated intact chloroplasts resulted in import of the precursor and cleavage to a polypeptide of about 30,000 daltons. Various spinach CAs containing deletions ranging from 34 to 78 amino acids were expressed in E. coli, under the control of the trc promotor. Each of the CAs assembled to yield the enzymatically active hexameric enzyme. Immunological techniques performed in E. coli expressed spinach CA as well as total plant extracts demonstrated the susceptibility of CA towards proteolysis at the N-terminal end. Furthermore, a cross-reacting protein, which is distinct from the well characterized periplasmic CA, was demonstrated to be present in extracts of Chlamydomonas reinhardti. Western blotting of CA extracts from leaves, chloroplasts or E. coli expressing CA indicated that CA is susceptible to proteolysis. N-terminal sequencing data obtained by others suggests that the degradation is from the N-terminus. Also, extracts from pea stems and leaves contained a polypeptide which cross-reacted with antibodies raised against spinach CA. Root extracts contained no cross-reacting polypeptides. These results suggest that, like other chloroplast proteins, expression of CA is regulated in a tissue specific manner

Membrane proteins in the outer mebrane of plastids and mitochondria

Channels of the plastid and mitochondrial outer membranes facilitate the turnover of molecules and ions via these membranes. Although channels have been studied many questions pertaining to the whole diversity of plastid and mitochondrial channels in Arabidopsis thaliana and Pisum sativum remain unanswered. In this thesis I studied OEP16, OEP37 and VDAC families in two model plants, in Arabidopsis and pea. The Arabidopsis OEP16 family represents four channels of α-helical structure, similar to the pea OEP16 protein. These channels are suggested to transport amino acids and compounds with primary amino groups. Immunoblot analysis, GFP/RFP protein fusion expression, as well as proteomic analysis showed that AtOEP16.1, AtOEP16.2 and AtOEP16.4 are located in the outer envelope membrane of plastids, while AtOEP16.3 is in mitochondria. The gene expression and immunoblot analyses revealed that AtOEP16.1 and AtOEP16.3 proteins are highly abundant and ubiquitous; expression of AtOEP16.1 is regulated by light and cold. AtOEP16.2 is highly expressed in pollen, seeds and seedlings. AtOEP16.4 is a low expressed housekeeping protein. Single knockout mutants of AtOEP16.1, AtOEP16.2 and AtOEP16.4, and double mutants of AtOEP16 gene family did not show any remarkable phenotype. However, macroarray analysis of Atoep16.1-p T-DNA mutant revealed 10 down-regulated and 6 up-regulated genes. In contrast to the α-helical OEP16 proteins, the OEP37 and VDAC proteins are of β-barrel structure. The PsOEP37 and AtOEP37 channel proteins form a selective barrier in the outer envelope of chloroplasts. Electrophysiological studies in lipid bilayer membranes showed that the PsOEP37 channel is permeable for cations. Specific expression profiles showed that AtOEP37 and PsOEP37 are highly expressed in the entire plant. The isolated PsVDAC gene encodes a protein, which is located in mitochondria. In Arabidopsis gene database, five Arabidopsis genes, which code for VDAC-like proteins were announced. One gene was not detected, whereas four of these genes expressed in leaves, roots, flower buds and pollen