RESUMEN
Chloroplasts and bacterial cells divide by binary fission. The key protein in this constriction division is FtsZ, a self-assembling GTPase similar to eukaryotic tubulin. In prokaryotes, FtsZ is almost always encoded by a single gene, whereas plants harbor several nuclear-encoded FtsZ homologs. In seed plants, these proteins group in two families and all are exclusively imported into plastids. In contrast, the basal land plant Physcomitrella patens, a moss, encodes a third FtsZ family with one member. This protein is dually targeted to the plastids and to the cytosol. Here, we report on the targeted gene disruption of all ftsZ genes in P. patens. Subsequent analysis of single and double knockout mutants revealed a complex interaction of the different FtsZ isoforms not only in plastid division, but also in chloroplast shaping, cell patterning, plant development, and gravity sensing. These results support the concept of a plastoskeleton and its functional integration into the cytoskeleton, at least in the moss P. patens.
Asunto(s)
Bryopsida/genética , Técnicas de Inactivación de Genes/métodos , Secuencia de Bases , Bryopsida/enzimología , Bryopsida/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Cartilla de ADN , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Fenotipo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa , Transcripción GenéticaRESUMEN
An understanding of gene function requires a complementation of gene and gene expression analysis by the systematic analysis of proteins. Progress in plant proteomics has been lagging behind animal and microbial proteomics due to the lack of plant genome data and the problems involved in successful protein extraction from plant material. With the sequencing of more and more plant genomes, this slow progress will soon be overcome. The moss Physcomitrella patens is a model organism in the field of plant functional genomics. P. patens is the first seedless plant for which the complete genome was sequenced. Genome annotation is currently in progress. While identification of proteins requires knowledge of all coding genes of the organism under study, gene annotation and functional characterization benefit greatly from the findings of proteome analysis. The proteome of P. patens is accessible and approaches are under way to increase the spectrum of proteomic methods applied to this plant. Here we provide a protocol for the extraction of proteins from P. patens and describe the basic and still most important method of proteome analysis, two-dimensional polyacrylamide electrophoresis of proteins. As this technique (not entirely unjustifiably) has the reputation of being unpredictably complicated, we provide a detailed protocol intended to reduce the reluctance that many scientists may have in using this technique.
Asunto(s)
Proteínas de Plantas/análisis , Proteómica/métodos , Animales , Bryopsida/química , Bryopsida/genética , Electroforesis en Gel de Poliacrilamida/métodos , Focalización Isoeléctrica/métodos , Proteoma/análisis , Proteómica/instrumentaciónRESUMEN
Plant filamentous temperature-sensitive Z (FtsZ) proteins have been reported to be involved in biological processes related to plastids. However, the precise functions of distinct isoforms are still elusive. Here, the intracellular localization of the FtsZ1-1 isoform in a moss, Physcomitrella patens, was examined. Furthermore, the in vivo interaction behaviour of four distinct FtsZ isoforms was investigated. Localization studies of green fluorescent protein (GFP)-tagged FtsZ1-1 and fluorescence resonance energy transfer (FRET) analyses employing all dual combinations of four FtsZ isoforms were performed in transient protoplast transformation assays. FtsZ1-1 is localized to network structures inside the chloroplasts and exerts influence on plastid division. Interactions between FtsZ isoforms occur in distinct ordered structures in the chloroplasts as well as in the cytosol. The results expand the view of the involvement of Physcomitrella FtsZ proteins in chloroplast and cell division. It is concluded that duplication and diversification of ftsZ genes during plant evolution were the main prerequisites for the successful remodelling and integration of the prokaryotic FtsZ-dependent division mechanism into the cellular machineries of distinct complex processes in plants.
Asunto(s)
Bryopsida/metabolismo , Cloroplastos/metabolismo , Citosol/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis , Proteínas Bacterianas/metabolismo , Bryopsida/genética , Bryopsida/ultraestructura , División Celular , Cloroplastos/ultraestructura , Proteínas del Citoesqueleto/metabolismo , Citosol/ultraestructura , Escherichia coli/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/análisis , Proteínas de Plantas/análisis , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/análisisRESUMEN
Cytokinin hormones are crucial regulators of a large number of processes in plant development. Recently, significant progress has been made toward the elucidation of the molecular details of cytokinin that has led to a model for signal transduction involving a phosphorylation cascade. However, the current knowledge of cytokinin action remains largely unknown and does not explain the different roles of this hormone. To gain further insights into this aspect of cytokinin action and the inducible phosphorelay, we have produced the first large-scale map of a phosphoproteome in the moss Physcomitrella patens. Using a protocol that we recently published (Heintz, D.; et al. Electrophoresis 2004, 25, 1149-1159) that combines IMAC, MALDI-TOF-MS, and LC-MS/MS, a total of 172 phosphopeptide sequences were obtained by a peptide de novo sequencing strategy. Specific P. patens EST and raw genomic databases were interrogated, and protein homology searches resulted in the identification of 112 proteins that were then classified into functional categories. In addition, the temporal dynamics of the phosphoproteome in response to cytokinin stimulation was studied at 2, 4, 6, and 15 min after hormone addition. We identified 13 proteins that were not previously known targets of cytokinin action. Among the responsive proteins, some were involved in metabolism, and several proteins of unknown function were also identified. We have mapped the time course of their activation in response to cytokinin and discussed their hypothetical biological significance. Deciphering these early induced phosphorylation events has shown that the cytokinin effect can be rapid (few minutes), and the duration of this effect can be variable. Also phosphorylation events can be differentially regulated. Taken together our proteomic study provides an enriched look of the multistep phosphorelay system mediating cytokinin response and suggests the existence of a multidirectional interaction between cytokinin and numerous other pathways.
Asunto(s)
Bryopsida/metabolismo , Citocininas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Fosfoproteínas/metabolismo , Proteómica/métodos , Bryopsida/genética , Cromatografía Liquida , Biología Computacional/métodos , Citocininas/farmacología , Fosfoproteínas/genética , Fosfoproteínas/aislamiento & purificación , Fosforilación , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The degradation of aromatic compounds follows different biochemical principles in aerobic and anaerobic microorganisms. While aerobes dearomatize and cleave the aromatic ring by oxygenases, facultative anaerobes utilize an ATP-dependent ring reductase for the dearomatization of the activated key intermediate benzoyl-coenzyme A (CoA). In this work, the aromatic metabolism was studied in the obligately anaerobic model organism Geobacter metallireducens. The gene coding for a putative carboxylic acid-CoA ligase was heterologously overexpressed and the gene product was characterized as a highly specific benzoate-CoA ligase catalysing the initial step of benzoate metabolism. However, no evidence for the presence of an ATP-dependent benzoyl-CoA reductase as observed in facultative anaerobes was obtained. In a proteomic approach benzoate-induced proteins were identified; the corresponding genes are organized in two clusters comprising 44 genes. Induction of representative genes during growth on benzoate was confirmed by reverse transcription polymerase chain reaction. The results obtained suggest that benzoate is activated to benzoyl-CoA, which is then reductively dearomatized to cyclohexa-1,5-diene-1-carbonyl-CoA, followed by beta-oxidation reactions to acetyl-CoA units, as in facultatively anaerobic bacteria. However, in G. metallireducens the process of reductive benzene ring dearomatization appears to be catalysed by a set of completely different protein components comprising putative molybdenum and selenocysteine containing enzymes.
Asunto(s)
Benzoatos/metabolismo , Geobacter/enzimología , Geobacter/crecimiento & desarrollo , Familia de Multigenes , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Anaerobiosis , Biodegradación Ambiental , Coenzima A Ligasas/química , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Geobacter/genética , Datos de Secuencia Molecular , Molibdeno/metabolismo , Selenocisteína/metabolismoRESUMEN
FtsZ is a filament-forming protein that assembles into a ring at the division site of prokaryotic cells. As FtsZ and tubulin share several biochemical and structural similarities, FtsZ is regarded as the ancestor of tubulin. Chloroplasts--the descendants of endosymbiotic bacteria within plant cells--also harbour FtsZ. In contrast to eubacteria, plants have several different FtsZ isoforms. So far, these isoforms have only been implicated with filamentous structures, rings and networks, inside chloroplasts. Here, we demonstrate that a novel FtsZ isoform in the moss Physcomitrella patens is located not only in chloroplasts but also in the cytoplasm, assembling into rings in both cell compartments. These findings comprise the first report on cytosolic localization of a eukaryotic FtsZ isoform, and indicate that this protein might connect cell and organelle division at least in moss.
Asunto(s)
Bryopsida/metabolismo , Cloroplastos/metabolismo , Citoplasma/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis , Genes Reporteros , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.
Asunto(s)
Bryopsida/metabolismo , Mapeo Peptídico/métodos , Proteínas de Plantas/análisis , Proteoma/análisis , Secuencia de Aminoácidos , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismoRESUMEN
We describe a reproducible protocol to explore for the first time the phosphoproteome of a seedless plant, the moss Physcomitrella patens. Following tryptic digestion of a total protein extract, phosphorylated peptides were isolated using the combination of C18 reverse-phase chromatography (RP-C18), immobilized Fe(3+) metal affinity chromatography (IMAC), capillary zone electrophoresis (CZE), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. The total protein extracts were first prepared as usually made for plant two-dimensional gel electrophoresis, the tryptic digest was desalted and concentrated by reverse phase chromatography, and from this mixture the phosphorylated peptides were captured by IMAC. Subsequently, the complex phosphopeptide mixture was separated into ten fractions by RP-C18-HPLC and each analyzed by CZE. This permitted the detection of 253 distinct phosphopeptides. These were identified by nano-LC-MS/MS and MALDI-TOF-MS analysis in conjunction with alkaline phosphatase treatment to remove covalently bound phosphate to specifically identify the phosphopeptides. Among others, several kinases and a transcription factor were identified. This protocol will be taken as a basis to unravel early events in plant signal transduction known to occur via rapid phosphorylation/dephosphorylation of proteins.
Asunto(s)
Bryopsida/metabolismo , Electroforesis Capilar/métodos , Proteínas de Plantas/metabolismo , Proteínas/química , Transducción de Señal , Fosforilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TripsinaRESUMEN
Previously, we showed that an apparent cell wall-plasma membrane interaction in xylem ray parenchyma differed between cold acclimated and non-acclimated red-osier dogwood (Cornus sericea L.) (Ristic and Ashworth 1994). For the present study, a calcium chloride extraction method was used to identify cell-wall-associated xylem proteins that accumulated during periods of cold acclimation. A 24-kDa protein represented the predominant protein in both total protein and CaCl2 extracts during cold acclimation of field-grown plants. Two-dimensional gel electrophoresis separated the 24-kDa protein into four basic isoforms. The most abundant and basic isoform had a high glycine content. In-gel digestion of this basic 24-kDa isoform generated three partial peptide fragments, of which one exhibited homology to the dehydrin protein family. An anti-dehydrin polyclonal antibody cross-reacted with the 24-kDa protein, providing further evidence that this protein is related to dehydrins. The 24-kDa protein began to accumulate in late August, reached a maximum in midwinter, declined during the spring months and was absent in early summer.