Arabidopsis thaliana mTERF proteins: evolution and functional classification

Organellar gene expression (OGE) is crucial for plant development, photosynthesis, and respiration, but our understanding of the mechanisms that control it is still relatively poor. Thus, OGE requires various nucleus-encoded proteins that promote transcription, splicing, trimming, and editing of organellar RNAs, and regulate translation. In metazoans, proteins of the mitochondrial Transcription tERmination Factor (mTERF) family interact with the mitochondrial chromosome and regulate transcriptional initiation and termination. Sequencing of the Arabidopsis thaliana genome led to the identification of a diversified MTERF gene family but, in contrast to mammalian mTERFs, knowledge about the function of these proteins in photosynthetic organisms is scarce. In this hypothesis article, I show that tandem duplications and one block duplication contributed to the large number of MTERF genes in A. thaliana, and propose that the expansion of the family is related to the evolution of land plants. The MTERF genes especially the duplicated genes display a number of distinct mRNA accumulation patterns, suggesting functional diversification of mTERF proteins to increase adaptability to environmental changes. Indeed, hypothetical functions for the different mTERF proteins can be predicted using co-expression analysis and gene ontology (GO) annotations. On this basis, mTERF proteins can be sorted into five groups. Members of the ``chloroplast'' and ``chloroplast-associated'' clusters are principally involved in chloroplast gene expression, embryogenesis, and protein catabolism, while representatives of the ``mitochondrial'' cluster seem to participate in DNA and RNA metabolism in that organelle. Moreover, members of the ``mitochondrion-associated'' cluster and the ``low expression'' group may act in the nucleus and/or the cytosol. As proteins involved in OGE and presumably nuclear gene expression (NGE), mTERFs are ideal candidates for the coordination of the expression of organelle and nuclear genomes

Photolyase/Cryptochrom-Homologe aus Synechocystis sp. PCC 6803 und Arabidopsis thaliana: Funktion, Lokalisation und biochemische Eigenschaften

In dieser Arbeit wurden Photolyase/Cryptochrom-homologe Proteine (cry und phr) aus dem Cyanobakterium Synechocystis sp. PCC 6803 und ein neues Cryptochrom (At-cry3) aus der Hoeheren Pflanze Arabidopsis thaliana charakterisiert. Synechocystis CRY liegt mit den offenen Leserastern sll1628 und sll1630 in einem Operon. Die durch sll1628 kodierte Aminosaeuresequenz besitzt eine schwache Homologie zu einem TPR (tetratricopeptide repeat)-Domaenen Protein; die sll1630 Aminosaeuresequenz ist zu keinem bekannten Protein homolog. Für das in E. coli heterolog exprimierte Synechocystis cry konnte nachgewiesen werden, dass es nicht kovalent FAD bindet. Die phr und cry Mutanten zeigten hinsichtlich Wachstum und Pigmentzusammensetzung weder unter photoautotrophen und heterotrophen Wachstumsbedingungen noch im Dauer-Blau- oder Rotlicht einen vom Wildtyp abweichenden Phaenotyp. Im UV-B ist in der cry Mutante im Gegensatz zur phr Mutante und dem Wildtyp die de novo Synthese fuer das D1 Protein des Photosystem II nicht induziert. In der cry Mutante ist ferner die Phototaxis zum Rot- und Dunkelrotlicht reduziert. Mittels quantitativer RT-PCR-Analysen wurde die lichtabhaengige Induktion der Transkription des psbA3 Gens, das fuer das Photosystem II D1 Protein kodiert, ueber einen großen Spektralbereich und beim Wechsel von niedriger zu hoher Fluenzrate untersucht. Unter den gewaehlten Lichtbedingungen ist mit Ausnahme von Dunkelrot in der cry Mutante die psbA3 Induktion abgeschwaecht, besonders drastisch im UV-A-Bereich. Somit wurde in dieser Arbeit gezeigt, dass sll1629 fuer ein Cryptochrom kodiert. Im zweiten Teil der Arbeit wurde das Protein At5g24850 (At-cry3) aus Arabidopsis thaliana untersucht. At-cry3 ist 569 Aminosaeuren gross und besitzt ueber einen Bereich von 400 Aminosaeuren ca. 50 Prozent Identitaet zu Synechocystis cry. Durch in vitro Import und GFP-Lokalisationsstudien wurde nachgewiesen, dass die N-terminalen 63 Aminosaeuren von At-cry3 notwendig und hinreichend fuer den Import dieses Proteins sowohl in Mitochondrien als auch in Chloroplasten sind. At-cry3 wurde als His-tag-Fusion heterolog in E. coli ueberexprimiert. Spektroskopische Analysen zeigten, dass At-cry3 nicht kovalent FAD bindet. Weder spektroskopisch noch mit Duennschichtchromatographie konnte ein zweiter Kofaktor nachgewiesen werden. Durch Expression von At-cry3 in einem Photolyase-defizienten E.-coli-Stamm und in vitro und in vivo Untersuchungen konnte gezeigt werden, dass At-cry3 keine Photolyaseaktivitaet besitzt. Ferner zeigten in vitro DNA-Bindungsstudien, dass At-cry3 sequenzunspezifisch an einzel- und doppelstraengige DNA bindet. Die Frage, ob At-cry3 eine Mutation in Synechocystis cry komplementieren kann, kann mit den vorliegenden Daten noch nicht abschliessend beantwortet werden. In Zusammenarbeit mit unserer AG hat Peter Lockhart, Massey University, New Zealand, einen Stammbaum mit ueber 70 Mitgliedern der Photolyase/Cryptochrom-Familie erstellt. Dieser deutet an, dass Pflanzen ihre Cryptochrome durch einen dualen horizontalen Gentransfer erhalten haben koennten: das CRY3 Gen von Cyanobakterien, den Vorlaeufern der Plastiden, Gene der CRY1/CRY2-Familie von Aplpha-Proteobakterien, den Vorlaeufern heutiger Mitochondrien

Intracompartmental and Intercompartmental Transcriptional Networks Coordinate the Expression of Genes for Organellar Functions

Genes for mitochondrial and chloroplast proteins are distributed between the nuclear and organellar genomes. Organelle biogenesis and metabolism, therefore, require appropriate coordination of gene expression in the different compartments to ensure efficient synthesis of essential multiprotein complexes of mixed genetic origin. Whereas organelle-to-nucleus signaling influences nuclear gene expression at the transcriptional level, organellar gene expression (OGE) is thought to be primarily regulated posttranscriptionally. Here, we show that intracompartmental and intercompartmental transcriptional networks coordinate the expression of genes for organellar functions. Nearly 1,300 ATH1 microarray-based transcriptional profiles of nuclear and organellar genes for mitochondrial and chloroplast proteins in the model plant Arabidopsis (Arabidopsis thaliana) were analyzed. The activity of genes involved in organellar energy production (OEP) or OGE in each of the organelles and in the nucleus is highly coordinated. Intracompartmental networks that link the OEP and OGE gene sets serve to synchronize the expression of nucleus- and organelle-encoded proteins. At a higher regulatory level, coexpression of organellar and nuclear OEP/OGE genes typically modulates chloroplast functions but affects mitochondria only when chloroplast functions are perturbed. Under conditions that induce energy shortage, the intercompartmental coregulation of photosynthesis genes can even override intracompartmental networks. We conclude that dynamic intracompartmental and intercompartmental transcriptional networks for OEP and OGE genes adjust the activity of organelles in response to the cellular energy state and environmental stresses, and we identify candidate cis-elements involved in the transcriptional coregulation of nuclear genes. Regarding the transcriptional regulation of chloroplast genes, novel tentative target genes of σ factors are identified

Chloroplast development and genomes uncoupled signaling are independent of the RNA-directed DNA methylation pathway

The Arabidopsis genome is methylated in CG and non-CG (CHG, and CHH in which H stands for A, T, or C) sequence contexts. DNA methylation has been suggested to be critical for seed development, and CHH methylation patterns change during stratification and germination. In plants, CHH methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. To test for an involvement of the RdDM pathway in chloroplast development, we analyzed seedling greening and the maximum quantum yield of photosystem II (F-v/F-m) in Arabidopsis thaliana seedlings perturbed in components of that pathway. Neither seedling greening nor F-v/F-m in seedlings and adult plants were affected in this comprehensive set of mutants, indicating that alterations in the RdDM pathway do not affect chloroplast development. Application of inhibitors like lincomycin or norflurazon inhibits greening of seedlings and represses the expression of photosynthesis-related genes including LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN1.2 (LHCB1.2) in the nucleus. Our results indicate that the LHCB1.2 promoter is poorly methylated under both control conditions and after inhibitor treatment. Therefore no correlation between LHCB1.2 mRNA transcription and methylation changes of the LHCB1.2 promoter could be established. Moreover, we conclude that perturbations in the RdDM pathway do not interfere with gun signaling

Detecting Functional Groups of Arabidopsis Mutants by Metabolic Profiling and Evaluation of Pleiotropic Responses

Metabolic profiles and fingerprints of Arabidopsis thaliana plants with various defects in plastidic sugar metabolism or photosynthesis were analyzed to elucidate if the genetic mutations can be traced by comparing their metabolic status. Using a platform of chromatographic and spectrometric tools data from untargeted full MS scans as well as from selected metabolites including major carbohydrates, phosphorylated intermediates, carboxylates, free amino acids, major antioxidants, and plastidic pigments were evaluated. Our key observations are that by multivariate statistical analysis each mutant can be separated by a unique metabolic signature. Closely related mutants come close. Thus metabolic profiles of sugar mutants are different but more similar than those of photosynthesis mutants. All mutants show pleiotropic responses mirrored in their metabolic status. These pleiotropic responses are typical and can be used for separating and grouping of the mutants. Our findings show that metabolite fingerprints can be taken to classify mutants and hence may be used to sort genes into functional groups

Arabidopsis thaliana mTERF10 and mTERF11, but Not mTERF12, Are Involved in the Response to Salt Stress

Plastid gene expression (PGE) is crucial for plant development and acclimation to various environmental stress conditions. Members of the "mitochondrial transcription termination factor" (mTERF) family, which are present in both metazoans and plants, are involved in organellar gene expression. Arabidopsis thaliana contains 35 mTERF proteins, of which mTERF10, mTERF11, and mTERF12 were previously assigned to the "chloroplast-associated" group. Here, we show that all three are localized to chloroplast nucleoids, which are associated with PGE. Knock-down of MTERF10, MTERF11, or MTERF12 has no overt phenotypic effect under normal growth conditions. However, in silico analysis of MTERF10, -11, and -12 expression levels points to a possible involvement of mTERF10 and mTERF11 in responses to abiotic stress. Exposing mutant lines for 7 days to moderate heat (30 degrees C) or light stress (400 mu mol photons m(-2) s(-1)) fails to induce a phenotype in mterf mutant lines. However, growth on MS medium supplemented with NaCl reveals that overexpression of MTERF11 results in higher salt tolerance. Conversely, mterf10 mutants are hypersensitive to salt stress, while plants that modestly overexpress MTERF10 are markedly less susceptible. Furthermore, MTERF10 overexpression leads to enhanced germination and growth on MS medium supplemented with ABA. These findings point to an involvement of mTERF10 in salt tolerance, possibly through an ABA-mediated mechanism. Thus, characterization of an increasing number of plant mTERF proteins reveals their roles in the response, tolerance and acclimation to different abiotic stresses

Organellar Gene Expression and Acclimation of Plants to Environmental Stress

Organelles produce ATP and a variety of vital metabolites, and are indispensable for plant development. While most of their original gene complements have been transferred to the nucleus in the course of evolution, they retain their own genomes and gene-expression machineries. Hence, organellar function requires tight coordination between organellar gene expression (OGE) and nuclear gene expression (NGE). OGE requires various nucleus-encoded proteins that regulate transcription, splicing, trimming, editing, and translation of organellar RNAs, which necessitates nucleus-to-organelle (anterograde) communication. Conversely, changes in OGE trigger retrograde signaling that modulates NGE in accordance with the current status of the organelle. Changes in OGE occur naturally in response to developmental and environmental changes, and can be artificially induced by inhibitors such as lincomycin or mutations that perturb OGE. Focusing on the model plant Arabidopsis thaliana and its plastids, we review here recent findings which suggest that perturbations of OGE homeostasis regularly result in the activation of acclimation and tolerance responses, presumably via retrograde signaling

Cellulose defects in the Arabidopsis secondary cell wall promote early chloroplast development

Lincomycin (LIN)‐mediated inhibition of protein synthesis in chloroplasts prevents the greening of seedlings, represses the activity of photosynthesis‐related genes in the nucleus, including LHCB1.2, and induces the phenylpropanoid pathway, resulting in the production of anthocyanins. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of LIN or other inhibitors of early chloroplast development. In a screen using concentrations of LIN lower than those employed to isolate gun mutants, we have identified happy on lincomycin (holi) mutants. Several holi mutants show an increased tolerance to LIN, exhibiting de‐repressed LHCB1.2 expression and chlorophyll synthesis in seedlings. The mutations responsible were identified by whole‐genome single‐nucleotide polymorphism (SNP) mapping, and most were found to affect the phenylpropanoid pathway; however, LHCB1.2 expression does not appear to be directly regulated by phenylpropanoids, as indicated by the metabolic profiling of mutants. The most potent holi mutant is defective in a subunit of cellulose synthase encoded by IRREGULAR XYLEM 3, and comparative analysis of this and other cell‐wall mutants establishes a link between secondary cell‐wall integrity and early chloroplast development, possibly involving altered ABA metabolism or sensing

Evolutionary tinkering: birth of a novel chloroplast protein

The term ‘evolutionary tinkering’ refers to evolutionary innovation by recombination of functional units, and includes the creation of novel proteins from pre-existing modules. A novel instance of evolutionary tinkering was recently discovered in the flowering plant genus Nicotiana: the conversion of a nuclear transcription factor into the plastid-resident protein WIN4 (wound-induced clone 4) involved in environmental stress responses. In this issue of the Biochemical Journal, Kodama and Sano now show that two steps are necessary for the establishment of the novel plastid protein: the acquisition of an internal translation initiation site and the use of multiple transcription starts to produce short mRNA variants that encode the plastid-targeted protein form