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1.
Proc Natl Acad Sci U S A ; 120(16): e2218334120, 2023 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-37036995

RESUMEN

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.


Asunto(s)
Toxinas Bacterianas , Avispas , Animales , Domesticación , Toxinas Bacterianas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Transferencia de Gen Horizontal , Avispas/metabolismo , Inmunidad Innata/genética
2.
Environ Microbiol ; 16(12): 3714-29, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25156344

RESUMEN

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti carries a large number of toxin-antitoxin (TA) modules both on the chromosome and megaplasmids. One of them, the vapBC-5 module that belongs to the type II systems was characterized here. It encodes an active toxin vapC-5, and was shown to be controlled negatively by the complex of its own proteins. Different mutants of the vapBC-5 genes exhibited diverse effects on symbiotic efficiency during interaction with the host plant Medicago sativa. The absence of the entire vapBC-5 region had no influence on nodule formation and nitrogen fixation properties. The strain carrying an insertion in the antitoxin gene showed a reduced nitrogen fixation capacity resulting in a lower plant yield. In contrast, when the toxin gene was mutated, the strain developed more efficient symbiosis with the host plant. The nitrogen fixing root nodules had a delayed senescent phenotype and contained elevated level of plant-derived molecules characteristic of later steps of nodule development. The longer bacteroid viability and abundance of active nitrogen fixing zone resulted in increased production of plant material. These data indicate that modification of the toxin/antitoxin production may influence bacteroid metabolism and may have an impact on the adaptation to changing environmental conditions.


Asunto(s)
Antitoxinas/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Medicago sativa/microbiología , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/fisiología , Simbiosis , Antitoxinas/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Medicago sativa/crecimiento & desarrollo , Medicago sativa/fisiología , Mutación , Fijación del Nitrógeno/genética , Operón , Fenotipo , Nódulos de las Raíces de las Plantas/crecimiento & desarrollo , Nódulos de las Raíces de las Plantas/microbiología
3.
Cells ; 13(7)2024 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-38607032

RESUMEN

Coevolution of hosts and their parasites has shaped heterogeneity of effector hemocyte types, providing immune defense reactions with variable effectiveness. In this work, we characterize hemocytes of Drosophila willistoni, a species that has evolved a cellular immune system with extensive variation and a high degree of plasticity. Monoclonal antibodies were raised and used in indirect immunofluorescence experiments to characterize hemocyte subpopulations, follow their functional features and differentiation. Pagocytosis and parasitization assays were used to determine the functional characteristics of hemocyte types. Samples were visualized using confocal and epifluorescence microscopy. We identified a new multinucleated giant hemocyte (MGH) type, which differentiates in the course of the cellular immune response to parasitoids. These cells differentiate in the circulation through nuclear division and cell fusion, and can also be derived from the central hematopoietic organ, the lymph gland. They have a binary function as they take up bacteria by phagocytosis and are involved in the encapsulation and elimination of the parasitoid. Here, we show that, in response to large foreign particles, such as parasitoids, MGHs differentiate, have a binary function and contribute to a highly effective cellular immune response, similar to the foreign body giant cells of vertebrates.


Asunto(s)
Drosophila , Parásitos , Animales , Diferenciación Celular , Fagocitosis , Inmunidad Celular
4.
Front Immunol ; 14: 1322381, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38187383

RESUMEN

Background: Insects have specialized cell types that participate in the elimination of parasites, for instance, the lamellocytes of the broadly studied species Drosophila melanogaster. Other drosophilids, such as Drosophila ananassae and the invasive Zaprionus indianus, have multinucleated giant hemocytes, a syncytium of blood cells that participate in the encapsulation of the eggs or larvae of parasitoid wasps. These cells can be formed by the fusion of hemocytes in circulation or originate from the lymph gland. Their ultrastructure highly resembles that of the mammalian megakaryocytes. Methods: Morphological, protein expressional, and functional features of blood cells were revealed using epifluorescence and confocal microscopy. The respective hemocyte subpopulations were identified using monoclonal antibodies in indirect immunofluorescence assays. Fluorescein isothiocyanate (FITC)-labeled Escherichia coli bacteria were used in phagocytosis tests. Gene expression analysis was performed following mRNA sequencing of blood cells. Results: D. ananassae and Z. indianus encapsulate foreign particles with the involvement of multinucleated giant hemocytes and mount a highly efficient immune response against parasitoid wasps. Morphological, protein expressional, and functional assays of Z. indianus blood cells suggested that these cells could be derived from large plasmatocytes, a unique cell type developing specifically after parasitoid wasp infection. Transcriptomic analysis of blood cells, isolated from naïve and wasp-infected Z. indianus larvae, revealed several differentially expressed genes involved in signal transduction, cell movements, encapsulation of foreign targets, energy production, and melanization, suggesting their role in the anti-parasitoid response. A large number of genes that encode proteins associated with coagulation and wound healing, such as phenoloxidase activity factor-like proteins, fibrinogen-related proteins, lectins, and proteins involved in the differentiation and function of platelets, were constitutively expressed. The remarkable ultrastructural similarities between giant hemocytes and mammalian megakaryocytes, and presence of platelets, and giant cell-derived anucleated fragments at wound sites hint at the involvement of this cell subpopulation in wound healing processes, in addition to participation in the encapsulation reaction. Conclusion: Our observations provide insights into the broad repertoire of blood cell functions required for efficient defense reactions to maintain the homeostasis of the organism. The analysis of the differentiation and function of multinucleated giant hemocytes gives an insight into the diversification of the immune mechanisms.


Asunto(s)
Hemocitos , Avispas , Animales , Drosophila melanogaster , Diferenciación Celular , Drosophila , Plaquetas , Mamíferos
5.
J Innate Immun ; 14(4): 335-354, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34864742

RESUMEN

Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, we showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. Here, we used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in Drosophila ananassae after parasitoid wasp infection. We found dynamic structural changes, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, we used RNA sequencing to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. Our results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response.


Asunto(s)
Hemocitos , Avispas , Animales , Drosophila , Interacciones Huésped-Parásitos , Inmunidad Innata , Transcriptoma , Avispas/genética
6.
Mol Plant Microbe Interact ; 23(5): 638-50, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20367472

RESUMEN

A toxin-antitoxin (TA)-like system (designated as bat/bto genes) was identified in Bradyrhizobium japonicum, based on sequence homology and similarities in organization and size to known TA systems. Deletion of the bat/bto module resulted in pleiotropic alterations in cell morphology and metabolism. The generation time of the mutant was considerably decreased in rich media. Atomic force microscopy revealed the modified shape (shorter and wider) and softness of mutant cells. The synthesis of phosphatidylcholine was completely blocked in the mutant bacteria, and vaccenic acid, the predominant fatty acid of membranes of the wild-type cell, was replaced by palmitic acid in the mutant membranes. The mutant bacteria synthesized incomplete lipopolysaccharide molecules. Remarkable changes in the membrane lipid composition may explain the observed morphological alterations and growth properties of the mutant bacteria. The overlapping promoter region of bat/bto and glpD (coding for the aerobic sn-glycerol-3-phosphate dehydrogenase) genes suggests a complex regulation and the involvement of bat/bto in the control of main metabolic pathways and an important role in the maintenance of a normal physiological state of B. japonicum. These data reveal new aspects of the role of TA systems in bacteria.


Asunto(s)
Antitoxinas/genética , Toxinas Bacterianas/genética , Bradyrhizobium/genética , Regulación Bacteriana de la Expresión Génica , Metabolismo de los Lípidos/genética , Transcripción Genética , Secuencia de Aminoácidos , Antitoxinas/química , Antitoxinas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Secuencia de Bases , Fenómenos Biomecánicos/efectos de los fármacos , Bradyrhizobium/citología , Bradyrhizobium/enzimología , Bradyrhizobium/crecimiento & desarrollo , Carbono/farmacología , División Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Escherichia coli/citología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Sitios Genéticos/genética , Genoma Bacteriano/genética , Metabolismo de los Lípidos/efectos de los fármacos , Lipopolisacáridos/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Datos de Secuencia Molecular , Mutación/genética , Nitrógeno/farmacología , Operón/genética , Fenotipo , Fosfolípidos/metabolismo , Regiones Promotoras Genéticas/genética , Simbiosis/genética , Transcripción Genética/efectos de los fármacos
7.
Dev Comp Immunol ; 109: 103701, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32320738

RESUMEN

Cell mediated immunity of the honey bee (Apis mellifera) involves the activity of several hemocyte populations, currently defined by morphological features and lectin binding characteristics. The objective of the present study was to identify molecular markers capable of characterizing subsets of honey bee hemocytes. We developed and employed monoclonal antibodies with restricted reactions to functionally distinct hemocyte subpopulations. Melanizing cells, known as oenocytoids, were defined by an antibody to prophenoloxidase, aggregating cells were identified by the expression of Hemolectin, and phagocytic cells were identified by a marker expressed on granulocytes. We anticipate that this combination of antibodies not only allows for the detection of functionally distinct hemocyte subtypes, but will help to further the exploration of hematopoietic compartments, as well as reveal details of the honey bee cellular immune defense against parasites and microbes.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Abejas/inmunología , Hemocitos/inmunología , Hemolinfa/inmunología , Animales , Anticuerpos Monoclonales/análisis , Abejas/citología , Abejas/microbiología , Biomarcadores/análisis , Escherichia coli/inmunología , Hemocitos/citología , Hemocitos/microbiología , Hemolinfa/citología , Hemolinfa/microbiología , Larva/citología , Larva/inmunología , Larva/microbiología , Microscopía Fluorescente , Fagocitosis/inmunología
8.
J Innate Immun ; 12(3): 257-272, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31553970

RESUMEN

Previously, a novel cell type, the multinucleated giant hemocyte (MGH) was identified in the ananassae subgroup of Drosophilidae. These cells share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation. We were able to show that MGHs also differentiate in Zaprionus indianus, an invasive species belonging to the vittiger subgroup of the family, highly resistant to a large number of parasitoid wasp species. We have classified the MGHs of Z. indianusas giant hemocytes belonging to a class of cells which also include elongated blood cells carrying a single nucleus and anuclear structures. They are involved in encapsulating parasites, originate from the lymph gland, can develop by cell fusion, and generally carry many nuclei, while possessing an elaborated system of canals and sinuses, resulting in a spongiform appearance. Their nuclei are all transcriptionally active and show accretion of genetic material. Multinucleation and accumulation of the genetic material in the giant hemocytes represents a two-stage amplification of the genome, while their spongy ultrastructure substantially increases the contact surface with the extracellular space. These features may furnish the giant hemocytes with a considerable metabolic advantage, hence contributing to the mechanism of the effective immune response.


Asunto(s)
Drosophilidae/inmunología , Genoma de los Insectos , Células Gigantes/inmunología , Hemocitos/inmunología , Inmunidad Celular , Animales , Drosophilidae/genética
9.
Genes (Basel) ; 10(3)2019 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-30841641

RESUMEN

Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, we investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. We found that naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. We demonstrate that hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, our findings contribute to the better understanding of HSC maintenance.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Hemolinfa/citología , Transducción de Señal , Animales , Diferenciación Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hemolinfa/metabolismo , Modelos Animales
10.
Dev Comp Immunol ; 76: 403-411, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28713010

RESUMEN

The identification of molecular markers considerably facilitated the classification and functional analysis of blood cell types. Apis mellifera hemocytes have been classified by morphological criteria and lectin binding properties; however, the use of molecular markers has been minimal. Here we describe a monoclonal antibody to a non-phagocytic subpopulation of A. mellifera hemocytes and to a constituent of the hemolymph clot. We demonstrate that the antibody identifies the A. mellifera hemolectin, a protein carrying human von Willebrand factor homology domains, characteristic of proteins involved in blood coagulation and platelet aggregation in mammals. Hemolectin expressing A. mellifera hemocytes contain the protein as cytoplasmic granules and contribute to the formation of a protein matrix, building up around foreign particles. Consequently, hemolectin as a marker molecule reveals a clear functional heterogeneity of hemocytes, allowing for the analytical separation of hemocyte classes, and could promote the molecular identification of hemocyte lineages in A. mellifera.


Asunto(s)
Abejas/inmunología , Hemocitos/fisiología , Hemolinfa/metabolismo , Lectinas/metabolismo , Trombosis/metabolismo , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Biodiversidad , Separación Celular , Lectinas/genética , Lectinas/inmunología , Mamíferos , Fagocitosis , Agregación Plaquetaria/genética , Homología de Secuencia de Aminoácido , Transcriptoma , Factor de von Willebrand/genética
11.
Insect Biochem Mol Biol ; 87: 45-54, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28633893

RESUMEN

The Nimrod gene cluster, located on the second chromosome of Drosophila melanogaster, is the largest synthenic unit of the Drosophila genome. Nimrod genes show blood cell specific expression and code for phagocytosis receptors that play a major role in fruit fly innate immune functions. We previously identified three homologous genes (vajk-1, vajk-2 and vajk-3) located within the Nimrod cluster, which are unrelated to the Nimrod genes, but are homologous to a fourth gene (vajk-4) located outside the cluster. Here we show that, unlike the Nimrod candidates, the Vajk proteins are expressed in cuticular structures of the late embryo and the late pupa, indicating that they contribute to cuticular barrier functions.


Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insecto , Familia de Multigenes , Animales , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero , Pupa/genética , Pupa/crecimiento & desarrollo
12.
PLoS One ; 11(3): e0150910, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26942456

RESUMEN

Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. Here, we show that the Drosophila inosine 5'-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid.


Asunto(s)
Proteínas de Drosophila/inmunología , Drosophila melanogaster/inmunología , Drosophila melanogaster/parasitología , IMP Deshidrogenasa/inmunología , Avispas , Alelos , Animales , Diferenciación Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Guanina/química , Hemocitos/citología , Interacciones Huésped-Parásitos/inmunología , IMP Deshidrogenasa/genética , Inmunidad Celular , Larva/inmunología , Mutación , Interferencia de ARN
13.
J Innate Immun ; 7(4): 340-53, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25659341

RESUMEN

We identified and characterized a so far unrecognized cell type, dubbed the multinucleated giant hemocyte (MGH), in the ananassae subgroup of Drosophilidae. Here, we describe the functional and ultrastructural characteristics of this novel blood cell type as well as its characterization with a set of discriminative immunological markers. MGHs are encapsulating cells that isolate and kill the parasite without melanization. They share some properties with but differ considerably from lamellocytes, the encapsulating cells of Drosophila melanogaster, the broadly used model organism in studies of innate immunity. MGHs are nonproliferative effector cells that are derived from phagocytic cells of the sessile tissue and the circulation, but do not exhibit phagocytic activity. In contrast to lamellocytes, MGHs are gigantic cells with filamentous projections and contain many nuclei, which are the result of the fusion of several cells. Although the structure of lamellocytes and MGHs differ remarkably, their function in the elimination of parasites is similar, which is potentially the result of the convergent evolution of interactions between hosts and parasites in different geographic regions. MGHs are highly motile and share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation.


Asunto(s)
Movimiento Celular/inmunología , Células Gigantes/inmunología , Inmunidad Celular , Fagocitosis , Animales , Drosophila , Células Gigantes/citología , Hemocitos
14.
Fly (Austin) ; 7(4): 263-6, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23899817

RESUMEN

The NimC1 molecule has been described as a phagocytosis receptor, and is being used as a marker for professional phagocytes, the plasmatocytes, in Drosophila melanogaster. In studies including tumor-biology, developmental biology, and cell mediated immunity, monoclonal antibodies (P1a and P1b) to the NimC1 antigen are used. As we observed that these antibodies did not react with plasmatocytes of several strains and genetic combinations, a molecular analysis was performed on the structure of the nimC1 gene. In these strains we found 2 deletions and an insertion within the nimC1 gene, which may result in the production of a truncated NimC1 protein. The NimC1 positivity was regained by recombining the mutation with a wild-type allele or by using nimC1 mutant lines under heterozygous conditions. By means of these procedures or using the recombined stock, NimC1 can be used as a marker for phagocytic cells in the majority of the possible genetic backgrounds.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fagocitos/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Expresión Génica , Variación Genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología
15.
Plant Mol Biol ; 69(5): 593-604, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19067180

RESUMEN

Isoprene synthase (ISPS) catalyzes the formation of isoprene, an important volatile terpenoid with strong effects on global atmospheric chemistry and protective physiological functions in plant leaves. Many terpene synthase genes including isoprene synthase, a member of the TPS-b cluster of this numerous gene family, were already functionally analysed but much less is known about regulation of their promoters. To study regulation of the PcISPS gene in detail we developed transgenic Grey poplar (Populus x canescens) and Arabidopsis thaliana plants in which the PcISPS promoter is fused to enhanced green fluorescent protein (E-GFP) and beta-glucuronidase (GUS) reporter genes. We analysed these reporters during plant development, for organ specificity and in plants subjected to different light and temperature regimes. We observed low promoter activity in non-isoprene emitting tissue like roots where ISPS gene is transcribed but no active enzyme is detectable. In leaves we demonstrate that light and temperature directly modulate ISPS promoter activity. Moreover, with confocal laser scanning microscopy we show a cell specific gradient of ISPS promoter activity within the leaf parenchyma depending on light direction. Our results indicate that ISPS promoter activity, which correlates with basal isoprene emission capacity, is not uniformly distributed within leaf tissue and that it can adapt rapidly towards internal as well as external environmental stimuli.


Asunto(s)
Transferasas Alquil y Aril/genética , Ambiente , Regulación de la Expresión Génica de las Plantas , Populus/enzimología , Populus/genética , Regiones Promotoras Genéticas/genética , Arabidopsis/citología , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Butadienos/metabolismo , Clorofila/metabolismo , Fluorescencia , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Glucuronidasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Hemiterpenos/metabolismo , Luz , Especificidad de Órganos/efectos de la radiación , Pentanos/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/efectos de la radiación , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/efectos de la radiación , Populus/citología , Populus/efectos de la radiación , Temperatura
16.
Plant Physiol ; 143(1): 540-51, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17122071

RESUMEN

Isoprene (2-methyl-1,3-butadiene) emission varies diurnally in different species. In poplar (Populus spp.), it has recently been shown that the gene encoding the synthesizing enzyme for isoprene, isoprene synthase (ISPS), displays diurnal variation in expression. Working on shoot cultures of Grey poplar (Populus x canescens) placed under a different light regime in phytochambers, we showed that these variations in PcISPS gene expression, measured by quantitative real-time polymerase chain reaction, are not only due to day-night changes, but also are linked to an internal circadian clock. Measurement of additional selected isoprenoid genes revealed that phytoene synthase (carotenoid pathway) displays similar fluctuations, whereas 1-deoxy-d-xylulose 5-phosphate reductoisomerase, possibly the first committed enzyme of the 1-deoxy-d-xylulose 5-phosphate pathway, only shows light regulation. On the protein level, it appeared that PcISPS activity and protein content became reduced under constant darkness, whereas under constant light, activity and protein content of this enzyme were kept high. In contrast, isoprene emission rates under continuous irradiation displayed circadian changes as is the case for gene expression of PcISPS. Furthermore, binding assays with Arabidopsis (Arabidopsis thaliana) late elongated hypocotyl, a transcription factor of Arabidopsis involved in circadian regulation, clearly revealed the presence of circadian-determining regulatory elements in the promoter region of PcISPS.


Asunto(s)
Ritmo Circadiano , Hemiterpenos/biosíntesis , Populus/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Secuencia de Bases , Butadienos , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Pentanos , Fotoperiodo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Proteínas de Plantas/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Brotes de la Planta/efectos de la radiación , Populus/genética , Populus/efectos de la radiación , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo
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