RESUMEN
Plants possess a large set of transcription factors both involved in the control of plant development or in plant stress responses coordination. We previously identified PRR2, a Pseudo-Response Regulator, as a plant-specific CML-interacting partner. We reported that PRR2 acts as a positive actor of plant defense by regulating the production of antimicrobial compounds. Here, we report new data on the interaction between PRR2 and transcription factors belonging to the Teosinte branched Cycloidea and PCF (TCP) family. TCPs have been described to be involved in plant development and immunity. We evaluated the ability of PRR2 to interact with seven TCPs representative of the different subclades of the family. PRR2 is able to interact with TCP13, TCP15, TCP19 and TCP20 in yeast two-hybrid system and in planta interactions were validated for TCP19 and TCP20. Transient expression in tobacco highlighted that PRR2 protein is more easily detected when co-expressed with TCP19 or TC20. This stabilization is associated with a specific sub-nuclear localization of the complex in Cajal bodies or in nuclear speckles according to the interaction of PRR2 with TCP19 or TCP20 respectively. The interaction between PRR2 and TCP19 or TCP20 would contribute to the biological function in specific nuclear compartments.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The localization and distribution of non-specific lipid transfer proteins (nsLTP) allergens in the skin and pulp of Rosaceae fruits (apple, peach, apricot, plum) has been investigated. nsLTP essentially concentrate in the pericarp of the fruits whereas the pulp contains lower amounts of allergens. Immunolocalization showed they are primarily located in the cytosol but are subsequently excreted and finally accumulate at the plasmalemma-cell wall interface and in the cell wall. However, high discrepancies were observed in the content of allergens among, e.g. different cultivars of apple. As a consequence, the consumption of peeled-off fruits is recommended to reduce the risk of severe allergic reactions (anaphylactic shock) in individuals sensitized to Rosaceae fruits.
Asunto(s)
Alérgenos/inmunología , Antígenos de Plantas/metabolismo , Proteínas Portadoras/metabolismo , Frutas/citología , Frutas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Plantas/metabolismo , Rosaceae/citología , Rosaceae/metabolismo , Antígenos de Plantas/química , Antígenos de Plantas/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Frutas/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Conformación Proteica , Transporte de Proteínas , Rosaceae/inmunologíaRESUMEN
"Candidatus Glomeribacter gigasporarum" is an endocellular beta-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita. We established a protocol to isolate "Ca. Glomeribacter gigasporarum" from its host which allowed us to carry out morphological, physiological, and genomic investigations on purified bacteria. They are rod shaped, with a cell wall typical of gram-negative bacteria and a cytoplasm rich in ribosomes, and they present no flagella or pili. Isolated bacteria could not be grown in any of the 19 culture media tested, but they could be kept alive for up to 4 weeks. PCR-based investigations of purified DNA from isolated bacteria did not confirm the presence of all genes previously assigned to "Ca. Glomeribacter gigasporarum." In particular, the presence of nif genes could not be detected. Pulsed-field gel electrophoresis analyses allowed us to estimate the genome size of "Ca. Glomeribacter gigasporarum" to approximately 1.4 Mb with a ca. 750-kb chromosome and a 600- to 650-kb plasmid. This is the smallest genome known for a beta-proteobacterium. Such small genome sizes are typically found in endocellular bacteria living permanently in their host. Altogether, our data suggest that "Ca. Glomeribacter gigasporarum" is an ancient obligate endocellular bacterium of the AM fungus G. margarita.
Asunto(s)
Betaproteobacteria , Hongos/crecimiento & desarrollo , Genoma Bacteriano , Micorrizas/crecimiento & desarrollo , Simbiosis , Betaproteobacteria/genética , Betaproteobacteria/crecimiento & desarrollo , Betaproteobacteria/aislamiento & purificación , Betaproteobacteria/ultraestructura , Medios de Cultivo , Electroforesis en Gel de Campo Pulsado , Sorghum/microbiología , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement system activation. C3a and C5a exert several biological activities through binding to their specific receptors, named C3aR and C5aR, respectively. We have previously shown that C3aR and C5aR are constitutively expressed by astrocytes, a cell type that actively participates in inflammatory events in the central nervous system. In this article, we focus on the transduction signal pathways activated by these two receptors on astrocytes. We show that the stimulation of C3aR or C5aR results in the activation of the mitogen activated protein kinase pathway by phosphorylation of the p44 and p42 kinases. On the contrary, the binding of C3a or C5a to their receptors on astrocytes decreases the production of cAMP, revealing an inhibition of the adenylyl cyclase pathway. Stimulation of C3aR and C5aR induces an increase in intracellular calcium concentration, arising from the opening of intracellular calcium channels. The observed calcium wave results from the activation of the phospholipase C pathway. Taken together, our results suggest that the binding of C3a or C5a to their receptors on astrocytes would be of functional importance since it induces the activation of two important transduction pathways leading to several cellular events such as neurotrophin and cytokine production.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Complemento C3a/metabolismo , Complemento C5a/metabolismo , AMP Cíclico/biosíntesis , Sistema de Señalización de MAP Quinasas/fisiología , Transducción de Señal/fisiología , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Astrocitos/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Complemento C3a/análogos & derivados , Complemento C3a/farmacología , Complemento C5a/farmacología , Encefalitis/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (beta- 0-4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.
Asunto(s)
Lignina/metabolismo , Nicotiana/enzimología , Aldehído Oxidorreductasas/metabolismo , Pared Celular/química , Regulación hacia Abajo , Inmunohistoquímica , Lignina/biosíntesis , Lignina/química , Microscopía Fluorescente , Plantas Modificadas GenéticamenteRESUMEN
Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.
Asunto(s)
Arabidopsis/química , Pared Celular/química , Celulasa/química , Celulasa/deficiencia , Pectinas/análisis , Anticuerpos Monoclonales/farmacología , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/metabolismo , Carboximetilcelulosa de Sodio/farmacología , Pared Celular/ultraestructura , Celulasa/farmacología , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa , Epítopos/análisis , Oro/farmacología , Hipocótilo/química , Hipocótilo/ultraestructura , Inmunohistoquímica , Técnicas In Vitro , Pectinas/metabolismo , Epidermis de la Planta/química , Epidermis de la Planta/ultraestructura , Polilisina/farmacología , Polisacáridos/metabolismoRESUMEN
Arbuscular mycorrhizal (AM) symbiosis is an association between obligate biotrophic fungi and more than 80% of land plants. During the pre-symbiotic phase, the host plant releases critical metabolites necessary to trigger fungal growth and root colonization. We describe the isolation of a semipurified fraction from exudates of carrot hairy roots, highly active on germinating spores of Gigaspora gigantea, G. rosea, and G. margarita. This fraction, isolated on the basis of its activity on hyphal branching, contains a root factor (one or several molecules) that stimulates, directly or indirectly, G. gigantea nuclear division. We demonstrate the presence of this active factor in root exudates of all mycotrophic plant species tested (eight species) but not in those of nonhost plant species (four species). We negatively tested the hypothesis that it was a flavonoid or a compound synthesized via the flavonoid pathway. We propose that this root factor, yet to be chemically characterized, is a key plant signal for the development of AM fungi.
Asunto(s)
Daucus carota/microbiología , Hongos/crecimiento & desarrollo , Proteínas de Plantas/aislamiento & purificación , Simbiosis , Daucus carota/fisiología , Flavonoides/química , Flavonoides/metabolismo , Hongos/fisiología , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Especificidad de la Especie , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/fisiologíaRESUMEN
Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.
Asunto(s)
Arabidopsis/enzimología , Membrana Celular/enzimología , Pared Celular/metabolismo , Celulasa/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Arabidopsis/ultraestructura , División Celular , Pared Celular/ultraestructura , Clonación Molecular , Secuencia Conservada , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Hipocótilo , Datos de Secuencia Molecular , Mutación , Polisacáridos/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
The proton spin-spin relaxation time, T2, measured from solid-state NMR, indicates a greater rigidity for cellulose than for the adhesive matrix between the microfibrils of flax ultimate fibres. Cytochemical and biochemical analyses allow the identification of: (1) EDTA-soluble RG I-polymers in the primary walls and cell junctions of fibres; (2) long 1 --> 4-beta-D-galactan chains between primary and secondary wall layers; and (3) arabinogalactan-proteins throughout the secondary walls. These polymers in the adhesive matrix between microfibrils and/or cellulose layers ensure that cracks propagate along the matrix rather than across the fibres and play an important role in allowing flax fibres to approach the tensile strength of advanced synthetic fibres like carbon and Kevlar.
Asunto(s)
Biopolímeros/química , Celulosa/química , Galactanos/química , Plantas/química , Técnica del Anticuerpo Fluorescente Indirecta , Espectroscopía de Resonancia Magnética , Semillas/química , Semillas/inmunología , Solubilidad , Resistencia a la Tracción , AguaRESUMEN
7-Dehydrobrefeldin A (7-oxo-BFA) is a brefeldin A (BFA) analog that, like BFA, is a potent phytotoxin of Alternaria carthami, a fungal pathogen of safflower (Carthamus tinctorius L.) plants. Both BFA and 7-oxo-BFA have been shown to be causal agents of the leaf spot disease of these plants. We have investigated the effects of 7-oxo-BFA on the secretion and the structure of the Golgi stacks of sycamore maple (Acer pseudoplatanus) suspension-cultured cells to determine whether 7-oxo-BFA affects these cells in the same manner as BFA. When applied at 10 micrograms/mL for 1 h, 7-oxo-BFA inhibits secretion of proteins by approximately 80%, the same value obtained for BFA. However, electron micrographs of high-pressure frozen/freeze-substituted cells demonstrated that 7-oxo-BFA is a more potent disrupter of the Golgi stacks of sycamore maple cells than BFA. In cells treated for 1 h with 10 micrograms/mL 7-oxo-BFA, very few Golgi stacks can be discerned. Most of those that are left consist of fewer than three cisternae, all of which stain like trans-Golgi cisternae. They are surrounded by clusters of large (150-300 nm in diameter), darkly staining vesicles that are embedded in a fine-filamentous, ribosome-excluding matrix. Similarly sized and stained vesicles are seen budding from the rims of the residual trans-Golgi cisternae. Both the large vesicles and the residual Golgi stack buds stain with anti-xyloglucan polysaccharide antibodies. Recovery of Golgi stacks after removal of 7-oxo-BFA from 1-h-treated cells takes 2 to 6 h, compared with 1 to 2 h for cells treated with BFA. In contrast to 7-oxo-BFA, the BFA breakdown product BFA acid had no effect either on secretion or on the secretory apparatus. This is the first report, to our knowledge of a BFA analog inhibiting secretion in a eukaryotic cell system.
Asunto(s)
Aparato de Golgi/efectos de los fármacos , Lactonas/farmacología , Micotoxinas/farmacología , Proteínas de Plantas/metabolismo , Árboles/efectos de los fármacos , Brefeldino A , Células Cultivadas , Ciclopentanos/farmacología , Células Eucariotas/efectos de los fármacos , Aparato de Golgi/ultraestructura , Árboles/ultraestructuraRESUMEN
The characteristic features of the pectins present in the walls of immature fibre cells of the hypocotyl of flax seedlings have been studied by a combination of three subtractive methods (treatment with boiling water, calcium chelator, and free endopolygalacturonase), three staining reactions (periodic acid-thiocarbohydrazide-silver, Ruthenium Red, and ferric hydroxylamine) and labelling with an endopolygalacturonase-gold probe. The primary wall and the periphery of the tricellular junctions were shown to contain pectic molecules made of blocks either with free acidic functions or methyl-esterified, these molecules being removed from the wall by splitting alpha (1-4) linkages. On the contrary, the pectic molecules in the core of the tricellular junctions were mainly with free acidic groups, but with an appreciable acetylesterification of their hydroxyl groups; and they were linked with one another chiefly by calcium bonds. This unexpected constitution of the core of the tricellular junctions may be considered to be an early marker of the cells destined to give rise to the fibre bundles of the mature plant.
Asunto(s)
Pared Celular/química , Uniones Intercelulares/química , Plantas/ultraestructura , Polisacáridos/análisis , Pared Celular/ultraestructura , Colorantes Fluorescentes , Histocitoquímica , Uniones Intercelulares/ultraestructura , Microscopía Electrónica , Plantas/química , Coloración y EtiquetadoRESUMEN
We applied the simultaneous use of a subtractive method and two imaging techniques (secondary ion mass spectrometry and electron microscopy after PATAg staining) to correlate the distribution of Ca2+ to pectic substances in cell walls of young flax plants. The calcium images were compared with the structural electron microscopy images. This suggests that the linkage of the pectic substances within the wall is mainly by calcium bridges in the intercellular junctions of most types of cells under study (epidermis, subepidermis, fiber layer, and endodermis) and in the outer part (close to the cuticle) of the wall of the epidermal cells. In the primary walls of the various types of cells under study and in the inner part (close to the cytoplasm) of the wall of the epidermal cells, the linkage of the pectic substances would be mainly by covalent bonds. In the middle lamellae of the various cells, and in the intercellular junctions within the cortical parenchyma, both types of linkages apparently coexist. The mechanism of "ionic condensation" may provide an interpretation for the chemical status of the Ca2+ ions which are associated with the pectic components solubilized in boiling water, and which do not seem to contribute to the linkage of these components within the wall.
Asunto(s)
Calcio/análisis , Pared Celular/química , Pectinas/análisis , Plantas/química , Pared Celular/ultraestructura , Uniones Intercelulares/química , Uniones Intercelulares/ultraestructura , Espectrometría de Masas , Microscopía Electrónica , Plantas/ultraestructura , Coloración y EtiquetadoRESUMEN
After a screening performed on pectinolytic micro-organisms, a strain of B. subtilis was isolated. This strain has a pectic enzyme capable of beta-elimination on highly methylated pectin (degree of esterification = 85%), without the action of a pectinesterase. This activity corresponds to that of a pectin-lyase.