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At least 37 records · Page 2Linked to original sources

PubMed2006

Disruption of essential plastid gene expression caused by T7 RNA polymerase-mediated transcription of plastid transgenes during early seedling development.

Transcription of plastid transgenes by plastid-targeted T7 RNA polymerase (ptT7RNAP) during early seedling development in tobacco was associated with a pale-green leaf phenotype, depletion of plastid rRNAs and arrest of shoot development. Extensive analysis of mutant seedlings at the transcript level using DNA microarrays and RNA gel blotting revealed severe disruption of plastid rRNA accumulation at 4-days post-germination and reduced transcript accumulation for the essential gene clpP. Several nuclear genes encoding plastid proteins were differentially regulated in mutant seedlings over time. Ef-Tu was upregulated at 4-days post-germination and then subsequently downregulated, while RbcS was already downregulated at this early time point. The downregulation of nuclear genes encoding plastid proteins suggests disruption of plastid-to-nucleus signalling. In contrast, transcripts of three plastid genes showed increased accumulation in mutant seedlings. Transcripts of ndhC and ndhK accumulated at high levels possibly due to T7RNAP-mediated enhancement of transcription, while ptT7RNAP-mediated transcription through the phage T7 Tphi terminator into the adjacent plastome increased the level of accD transcripts. The leakiness of the Tphi terminator has implications for the use of T7RNAP-based expression systems in plastid biotechnology.

Bacteriophage T7↗

PubMed2004

Phylogeny of nuclear-encoded plastid-targeted GAPDH gene supports separate origins for the peridinin- and the fucoxanthin derivative-containing plastids of dinoflagellates.

Although most photosynthetic dinoflagellates have plastids with peridinin, the three dinoflagellate genera Karenia, Karlodinium, and Takayama possess anomalously pigmented plastids that contain fucoxanthin and its derivatives (19'-hexanoyloxy-fucoxanthin and 19'-butanoyloxy-fucoxanthin) instead of the peridinin. This pigment composition is similar to that of haptophytes. All peridinin-containing dinoflagellates investigated so far have at least two types of glyceraldehyde-3-phosphate dehydrogenase (GAPDH): cytosolic and plastid-targeted forms. In the present study, we cloned and sequenced genes encoding cytosolic and plastid-targeted GAPDH proteins from three species of the fucoxanthin derivative-containing dinoflagellates. Based on the molecular phylogeny, the plastid-targeted GAPDH genes of the fucoxanthin derivative-containing dinoflagellates were closely related to those of haptophyte algae rather than to the peridinin-containing dinoflagellates, while one of several cytosolic versions from the peridinin- and the fucoxanthin derivative-containing dinoflagellates are closely related to each other. Considering a previously reported theory that the plastid-targeted GAPDH from the peridinin-containing dinoflagellates originated by a gene duplication of the cytosolic form before the splitting of the dinoflagellate lineage, it is highly likely that the plastid-targeted GAPDH gene of the peridinin-containing dinoflagellates is original in this algal group and that in the fucoxanthin-containing dinoflagellates, the original plastid-targeted GAPDH was replaced by that of a haptophyte endosymbiont during a tertiary endosymbiosis. The present results strongly support the hypothesis that the plastids of the peridinin- and the fucoxanthin derivative-containing dinoflagellates are of separate origin.

Amino Acid Sequence↗

PubMed2004

The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation.

The rice virescent-2 mutant (v(2)) is temperature conditional and develops chlorotic, chloroplast-deficient leaves at the restrictive temperature. In the v(2) mutant, plastid-encoded proteins involved in photosynthesis and plastid transcriptional regulation were not detectable at any time during chloroplast differentiation. However, the plastid transcripts for these two classes of proteins behaved differently in the mutant, with those for the plastid transcription/translation apparatus accumulating to wild-type levels and those for photosynthetic apparatus being suppressed. Polysome analysis showed that translation of the plastid transcripts encoding the plastid transcription/translation apparatus was blocked at an early stage of chloroplast differentiation. Accumulation of transcripts of nuclear-encoded photosynthetic genes, such as cab and rbcS, was strongly suppressed in the mutant at later stages of chloroplast differentiation, whereas transcripts of genes for the plastid transcription apparatus, such as OsRpoTp and OsSIG2A, accumulated to abnormally high levels at these stages. These results suggest that activation of the plastid translation machinery at an early stage of chloroplast differentiation is important for triggering the transmission of information about plastid developmental state to the nucleus, which in turn is required for the induction of nuclear-encoded chloroplast proteins at later stages of chloroplast differentiation.

Cell Differentiation↗

PubMed1998

RNA polymerase subunits encoded by the plastid rpo genes are not shared with the nucleus-encoded plastid enzyme.

Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases. The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme. The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus. Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion of rpoB (encoding the beta-subunit) from the plastid genome. To determine if NEP and PEP share any essential subunits, the rpoA, rpoC1, and rpoC2 genes encoding the PEP alpha-, beta'-, and beta"-subunits were removed by targeted gene deletion from the plastid genome. We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters. Therefore, rpoA, rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery. Furthermore, our data indicate that no functional copy of rpoA, rpoB, rpoC1, or rpoC2 that could complement the deleted plastid rpo genes exists outside the plastids.

Base Sequence↗

PubMed2005

Development of molecular probes for dinophysis (dinophyceae) plastid: a tool to predict blooming and explore plastid origin.

Dinophysis are species of dinoflagellates that cause diarrhetic shellfish poisoning. We have previously reported that they probably acquire plastids from cryptophytes in the environment, after which they bloom. Thus monitoring the intracellular plastid density in Dinophysis and the source cryptophytes occurring in the field should allow prediction of Dinophysis blooming. In this study the nucleotide sequences of the plastid-encoded small subunit ribosomal RNA gene and rbcL (encoding the large subunit of RuBisCO) from Dinophysis spp. were compared with those of cryptophytes, and genetic probes specific for the Dinophysis plastid were designed. Fluorescent in situ hybridization (FISH) showed that the probes bound specifically to Dinophysis plastids. Also, FISH on collected nanoplankton showed the presence of probe-hybridized eukaryotes, possibly cryptophytes with plastids identical to those of Dinophysis. These probes are useful not only as markers for plastid density and activity of Dinophysis, but also as tools for monitoring cryptophytes that may be sources of Dinophysis plastids.

Animals↗

PubMed1991

Plastid genome structure and plastid-related transcript levels in albino barley plants derived from another culture.

Southern analysis of DNA from four albino barley plants regenerated from microspores by direct embryogenesis revealed the presence of plastid genomes which had undergone deletion or alteration of specific restriction fragments (delta ptDNAs). In contrast, a fifth plant appeared to contain an intact plastid genome. All the albino plants studied contained reduced amounts of ptDNA, the most abundant restriction fragments being present at levels between 6% and 20% of those found in the leaves of green seedlings. Steady-state levels of transcripts from plastid and nuclear genes encoding plastid components were estimated by Northern analysis of RNA from albino plants. Transcripts from the plastid genes rbcL, psbD-psbC and the 16S and 23S rRNAs were undetectable or were present at greatly reduced levels in albino plants compared to those found in green leaves. Transcripts from the nuclear genes rbcS and cab, which encode chloroplast localised proteins, were also present at reduced levels in albino pollen plants. Levels of the nuclear encoded 25S rRNA, which is not a plastid component, were found to be identical in albino plants and green leaves suggesting that only the expression of plastid-related genes may be affected in albino plants. The general reduction of plastid-related transcripts was independent of the different patterns of ptDNA alteration seen in albino pollen plants.

Blotting, Southern↗

PubMed2004

Comparative analysis of the complete plastid genome sequence of the red alga Gracilaria tenuistipitata var. liui provides insights into the evolution of rhodoplasts and their relationship to other plastids.

We sequenced to completion the circular plastid genome of the red alga Gracilaria tenuistipitata var. liui. This is the first plastid genome sequence from the subclass Florideophycidae (Rhodophyta). The genome is composed of 183,883 bp and contains 238 predicted genes, including a single copy of the ribosomal RNA operon. Comparisons with the plastid genome of Porphyra pupurea reveal strong conservation of gene content and order, but we found major genomic rearrangements and the presence of coding regions that are specific to Gracilaria. Phylogenetic analysis of a data set of 41 concatenated proteins from 23 plastid and two cyanobacterial genomes support red algal plastid monophyly and a specific evolutionary relationship between the Florideophycidae and the Bangiales. Gracilaria maintains a surprisingly ancient gene content in its plastid genome and, together with other Rhodophyta, contains the most complete repertoire of plastid genes known in photosynthetic eukaryotes.

Consensus Sequence↗

PubMed2002

Protein transport into secondary plastids and the evolution of primary and secondary plastids.

Chloroplasts are key organelles in algae and plants due to their photosynthetic abilities. They are thought to have evolved from prokaryotic cyanobacteria taken up by a eukaryotic host cell in a process termed primary endocytobiosis. In addition, a variety of organisms have evolved by subsequent secondary endocytobioses, in which a heterotrophic host cell engulfed a eukaryotic alga. Both processes dramatically enhanced the complexity of the resulting cells. Since the first version of the endosymbiotic theory was proposed more than 100 years ago, morphological, physiological, biochemical, and molecular data have been collected substantiating the emerging picture about the origin and the relationship of individual organisms with different primary or secondary chloroplast types. Depending on their origin, plastids in different lineages may have two, three, or four envelope membranes. The evolutionary success of endocytobioses depends, among other factors, on the specific exchange of molecules between the host and endosymbiont. This raises questions concerning how targeting of nucleus-encoded proteins into the different plastid types occurs and how these processes may have developed. Most studies of protein translocation into plastids have been performed on primary plastids, but in recent years more complex protein-translocation systems of secondary plastids have been investigated. Analyses of transport systems in different algal lineages with secondary plastids reveal that during evolution existing translocation machineries were recycled or recombined rather than being developed de novo. This review deals with current knowledge about the evolution and function of primary and secondary plastids and the respective protein-targeting systems.

Endocytosis↗

PubMed2005

The complete plastid genome sequence of the haptophyte Emiliania huxleyi: a comparison to other plastid genomes.

The complete nucleotide sequence of the plastid genome of the haptophyte Emiliania huxleyi has been determined. E. huxleyi is the most abundant coccolithophorid and has a key role in the carbon cycle. It is also implicated in the production of dimethylsulphide (DMS), which is involved in cloud nucleation and may affect the global climate. Here, we report the plastid genome sequence of this ecologically and economically important species and compare its gene content and arrangement to other known plastid genomes. The genome is circular and consists of 105,309 bp with an inverted repeat of 4,841 bp. In terms of both genome size and gene content E. huxleyi cpDNA is substantially smaller than any other from the red plastid lineage. The genetic information is densely packed, with 86.8% of the genome specifying 110 identified protein-coding genes, 9 open reading frames, 28 different tRNAs, and 3 rRNAs. A detailed comparison to other plastid genomes, based on gene content, gene function, and gene cluster analysis is discussed. These analyses suggest a close relationship of the E. huxleyi cpDNA to the chlorophyll c-containing plastids from heterokonts and cryptophytes, and they support the origin of the chromophyte plastids from the red algal lineage.

Evolution, Molecular↗

PubMed2004

GFP-labelled Rubisco and aspartate aminotransferase are present in plastid stromules and traffic between plastids.

Plastid stromules are membrane-bound protrusions of the plastid envelope that contain soluble stroma. Stromules are often found connecting plastids within a cell and fluorescence recovery after photobleaching (FRAP) experiments have demonstrated that green fluorescent protein (GFP) can move between plastids via these connections. In this report, the ability of endogenous plastid proteins to travel through stromules was investigated. The motility of GFP-labelled plastid aspartate aminotransferase and the Rubisco small subunit was studied in stromules by FRAP. Both fusion proteins assemble into protein complexes that appear to behave similarly to their endogenous counterparts. In addition, both enzymes are capable of trafficking between plastids via stromules.

Arabidopsis↗

PubMed2004

The nuclear genes Lhcb and HEMA1 are differentially sensitive to plastid signals and suggest distinct roles for the GUN1 and GUN5 plastid-signalling pathways during de-etiolation.

Feedback mechanisms are critical to the regulation of chloroplast development and signals from functional plastids are required to maintain nuclear gene expression of chloroplast proteins. To understand the role of these signals in de-etiolating Arabidopsis thaliana L. seedlings, we followed the expression of three nuclear genes, Lhcb, HEMA1 and GSA, under a variety of treatments (Norflurazon, lincomycin and a far-red light pre-treatment) leading to plastid damage in white light and in a range of genetic backgrounds known to modulate plastid signalling: the genomes uncoupled mutants, gun1, gun4, gun5 and the gun1,5 double mutant, and in a transgenic line over-expressing NADPH:protochlorophyllide oxidoreductase. The three nuclear genes were differentially sensitive to changes in plastid signalling, with Lhcb the most strongly repressed and GSA insensitive to all but the most severe treatments. Analysis of plastid morphology in seedlings grown under identical conditions demonstrated that these responses corresponded closely to the degree of plastid damage. Furthermore, although Lhcb and HEMA1 were responsive to both GUN1 and GUN5 signals, the relative inputs from these pathways differed for each transcript with GUN1 being dominant for HEMA1 regulation. Further analysis of HEMA1 expression in gun1 seedlings under non-photobleaching conditions indicates that GUN1 is an important suppressor of HEMA1 expression in the dark and under saturating white light. These results are consistent with plastid signals functioning in a feedback regulatory mechanism during chloroplast biogenesis, and suggest a key role for GUN1 during the early stages of chloroplast development.

Arabidopsis↗

PubMed2004

Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for selection and tracking of plastid-transformed cells in tobacco.

A plastid transformation vector was constructed for dicistronic expression of the aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp) genes under the control of the plastid rrn promoter. Gold particles coated with the vector DNA were bombarded onto tobacco leaf explants using a particle delivery system. Leaf explants produced adventitious shoots when cultured on shoot-inducing medium containing 500 mg l(-1) spectinomycin. Shoots that exhibited green fluorescence under UV light were selected. Southern blot analysis detected the presence of the aadA and gfp genes between trnA and trnI in the plastid genome. Northern blot analysis revealed that the aadA and gfp genes were both properly transcribed into a dicistronic transcriptional unit. The expression of the gfp gene in the plastid enabled separation of transformed chloroplasts from wild-type chloroplasts in the protoplast under a fluorescent microscope. The overall results indicate that dicistronic expression of the aadA and gfp genes in the plastid simplifies gene manipulation, facilitating selection and tracking of plastid-transformed cells.

Chloroplasts↗

PubMed2002

Plastid genes transcribed by the nucleus-encoded plastid RNA polymerase show increased transcript accumulation in transgenic plants expressing a chloroplast-localized phage T7 RNA polymerase.

A gene fusion encoding a plastid-targeted bacteriophage T7 RNA polymerase (T7RNAP) under the transcriptional control of the light-regulated promoter and the plastid-targeting signals of a ribulose-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene was introduced into the nuclear genome of Nicotiana tabacum (tobacco). Immunoblot analysis, in vitro transcription assays and protease treatment of isolated chloroplasts revealed that T7RNAP activity was localized within chloroplasts. RNA gel blot analyses showed a substantial increase in transcript abundance for several plastid genes that are normally transcribed by the nucleus-encoded plastid RNA polymerase (NEP) including rpoC1, rpl33, rps18, rps12, and clpP. By contrast, no significant changes were observed in the levels of psbD, 16SrDNA, and ndhA transcripts. These results suggest a possible direct or indirect T7RNAP-mediated enhancement of transcription of a subset of plastid genes that contain NEP promoters. Despite these alterations in plastid transcript levels, the plants showed no visible abberant phenotype.

Bacteriophage T7↗

PubMed2002

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants.

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.

Aldose-Ketose Isomerases↗

PubMed2003

Expression of the plastid-located glutamine synthetase of Medicago truncatula. Accumulation of the precursor in root nodules reveals an in vivo control at the level of protein import into plastids.

In this paper, we report the cloning and characterization of the plastid-located glutamine synthetase (GS) of Medicago truncatula Gaertn (MtGS2). A cDNA was isolated encoding a GS2 precursor polypeptide of 428 amino acids composing an N-terminal transit peptide of 49 amino acids. Expression analysis, by Westerns and by northern hybridization, revealed that MtGS2 is expressed in both photosynthetic and non-photosynthetic organs. Both transcripts and proteins of MtGS2 were detected in substantial amounts in root nodules, suggesting that the enzyme might be performing some important role in this organ. Surprisingly, about 40% of the plastid GS in nodules occurred in the non-processed precursor form (preGS2). This precursor was not detected in any other organ studied and moreover was not observed in non-fixing nodules. Cellular fractionation of nodule extracts revealed that preGS2 is associated with the plastids and that it is catalytically inactive. Immunogold electron microscopy revealed a frequent coincidence of GS with the plastid envelope. Taken together, these results suggest a nodule-specific accumulation of the GS2 precursor at the surface of the plastids in nitrogen-fixing nodules. These results may reflect a regulation of GS2 activity in relation to nitrogen fixation at the level of protein import into nodule plastids.

Amino Acid Sequence↗

PubMed2005

pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression.

Transcription in plastids is mediated by a plastid-encoded multimeric (PEP) and a nuclear-encoded single-subunit RNA polymerase (NEP) and a still unknown number of nuclear-encoded factors. By combining gel filtration and affinity chromatography purification steps, we isolated transcriptionally active chromosomes from Arabidopsis thaliana and mustard (Sinapis alba) chloroplasts and identified 35 components by electrospray ionization ion trap tandem mass spectrometry. Eighteen components, called plastid transcriptionally active chromosome proteins (pTACs), have not yet been described. T-DNA insertions in three corresponding genes, ptac2, -6, and -12, are lethal without exogenous carbon sources. Expression patterns of the plastid-encoded genes in the corresponding knockout lines resemble those of Deltarpo mutants. For instance, expression of plastid genes with PEP promoters is downregulated, while expression of genes with NEP promoters is either not affected or upregulated in the mutants. All three components might also be involved in posttranscriptional processes, such as RNA processing and/or mRNA stability. Thus, pTAC2, -6, and -12 are clearly involved in plastid gene expression.

Arabidopsis↗

PubMed1992

Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth.

Albino rice plants derived from pollen contain plastid genomes that have suffered large-scale deletions. From the roots of albino plants, we obtained several calli containing homogeneous plastid DNA differing in the size and position of the deletion. DNA differing in the size and position of the deletion. Southern blotting and pulsed field gel electrophoresis experiments revealed that the DNAs were linear molecules having a hairpin structure at both termini, existing as monomers (19 kb) or dimers, trimers and tetramers linked to form head-to-head and tail-to-tail multimers. This characteristic form is similar to that of the vaccinia virus, in which the replication origin is thought to lie at or near the hairpin termini. Furthermore, polymerase chain reaction experiments revealed complete loss of the ribosomal RNA genes of the plastid DNA. The results suggest that plant cells can grow without translation occurring in plastids. All of the deleted plastid DNAs commonly retained the region containing the tRNA(Glu) gene (trnE), which is essential for biosynthesis of porphyrin. As porphyrin is the precursor of heme for mitochondria and other organelles, it is considered that trnE on the remnant plastid genome may be transcribed by an RNA polymerase encoded on nuclear DNA.

Base Sequence↗

PubMed1998

Site-selective inhibition of plastid RNA editing by heat shock and antibiotics: a role for plastid translation in RNA editing.

RNA editing in higher plant plastids changes single cytidine residues to uridine through an unknown mechanism. In order to investigate the relation of editing to physiological processes and to other steps in plastid gene expression, we have tested the sensitivity of chloroplast RNA editing to heat shock and antibiotics. We show that heat shock conditions as well as treatment of plants with prokaryotic translational inhibitors can inhibit plastid RNA editing. Surprisingly, this inhibitory effect is confined to a limited number of plastid editing sites suggesting that some site-specific factor(s) but none of the general components of the plastid RNA editing machinery are compromised. Contrary to previous expectations, our results provide evidence for a role of plastid translation in RNA editing.

Anti-Bacterial Agents↗