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1.
Plant J ; 96(3): 635-650, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30079488

RESUMO

Characterizing the natural diversity of gene expression across environments is an important step in understanding how genotype-by-environment interactions shape phenotypes. Here, we analyzed the impact of water deficit onto gene expression levels in tomato at the genome-wide scale. We sequenced the transcriptome of growing leaves and fruit pericarps at cell expansion stage in a cherry and a large fruited accession and their F1 hybrid grown under two watering regimes. Gene expression levels were steadily affected by the genotype and the watering regime. Whereas phenotypes showed mostly additive inheritance, ~80% of the genes displayed non-additive inheritance. By comparing allele-specific expression (ASE) in the F1 hybrid to the allelic expression in both parental lines, respectively, 3005 genes in leaf and 2857 genes in fruit deviated from 1:1 ratio independently of the watering regime. Among these genes, ~55% were controlled by cis factors, ~25% by trans factors and ~20% by a combination of both types of factors. A total of 328 genes in leaf and 113 in fruit exhibited significant ASE-by-watering regime interaction, among which ~80% presented trans-by-watering regime interaction, suggesting a response to water deficit mediated through a majority of trans-acting loci in tomato. We cross-validated the expression levels of 274 transcripts in fruit and leaves of 124 recombinant inbred lines (RILs) and identified 163 expression quantitative trait loci (eQTLs) mostly confirming the divergences identified by ASE. Combining phenotypic and expression data, we observed a complex network of variation between genes encoding enzymes involved in the sugar metabolism.


Assuntos
Locos de Características Quantitativas/genética , Solanum lycopersicum/genética , Transcriptoma , Água/fisiologia , Alelos , Desidratação , Frutas/genética , Frutas/fisiologia , Genótipo , Solanum lycopersicum/fisiologia , Fenótipo
2.
Front Plant Sci ; 9: 137, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29491875

RESUMO

Changing the balance between ascorbate, monodehydroascorbate, and dehydroascorbate in plant cells by manipulating the activity of enzymes involved in ascorbate synthesis or recycling of oxidized and reduced forms leads to multiple phenotypes. A systems biology approach including network analysis of the transcriptome, proteome and metabolites of RNAi lines for ascorbate oxidase, monodehydroascorbate reductase and galactonolactone dehydrogenase has been carried out in orange fruit pericarp of tomato (Solanum lycopersicum). The transcriptome of the RNAi ascorbate oxidase lines is inversed compared to the monodehydroascorbate reductase and galactonolactone dehydrogenase lines. Differentially expressed genes are involved in ribosome biogenesis and translation. This transcriptome inversion is also seen in response to different stresses in Arabidopsis. The transcriptome response is not well correlated with the proteome which, with the metabolites, are correlated to the activity of the ascorbate redox enzymes-ascorbate oxidase and monodehydroascorbate reductase. Differentially accumulated proteins include metacaspase, protein disulphide isomerase, chaperone DnaK and carbonic anhydrase and the metabolites chlorogenic acid, dehydroascorbate and alanine. The hub genes identified from the network analysis are involved in signaling, the heat-shock response and ribosome biogenesis. The results from this study therefore reveal one or several putative signals from the ascorbate pool which modify the transcriptional response and elements downstream.

3.
Plant Sci ; 242: 120-130, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26566830

RESUMO

Quantitative trait loci (QTL) have been identified using traditional linkage mapping and positional cloning identified several QTLs. However linkage mapping is limited to the analysis of traits differing between two lines and the impact of the genetic background on QTL effect has been underlined. Genome-wide association studies (GWAs) were proposed to circumvent these limitations. In tomato, we have shown that GWAs is possible, using the admixed nature of cherry tomato genomes that reduces the impact of population structure. Nevertheless, GWAs success might be limited due to the low decay of linkage disequilibrium, which varies along the genome in this species. Multi-parent advanced generation intercross (MAGIC) populations offer an alternative to traditional linkage and GWAs by increasing the precision of QTL mapping. We have developed a MAGIC population by crossing eight tomato lines whose genomes were resequenced. We showed the potential of the MAGIC population when coupled with whole genome sequencing to detect candidate single nucleotide polymorphisms (SNPs) underlying the QTLs. QTLs for fruit quality traits were mapped and related to the variations detected at the genome sequence and expression levels. The advantages and limitations of the three types of population, in the context of the available genome sequence and resequencing facilities, are discussed.


Assuntos
Variação Genética , Genoma de Planta/genética , Locos de Características Quantitativas/genética , Análise de Sequência de DNA/métodos , Solanum lycopersicum/genética , Mapeamento Cromossômico/métodos , Cromossomos de Plantas/genética , Cruzamentos Genéticos , Efeito Fundador , Frutas/genética , Genética Populacional/métodos , Genótipo , Endogamia , Polimorfismo de Nucleotídeo Único
4.
Plant Biotechnol J ; 13(4): 565-77, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25382275

RESUMO

Identification of the polymorphisms controlling quantitative traits remains a challenge for plant geneticists. Multiparent advanced generation intercross (MAGIC) populations offer an alternative to traditional linkage or association mapping populations by increasing the precision of quantitative trait loci (QTL) mapping. Here, we present the first tomato MAGIC population and highlight its potential for the valorization of intraspecific variation, QTL mapping and causal polymorphism identification. The population was developed by crossing eight founder lines, selected to include a wide range of genetic diversity, whose genomes have been previously resequenced. We selected 1536 SNPs among the 4 million available to enhance haplotype prediction and recombination detection in the population. The linkage map obtained showed an 87% increase in recombination frequencies compared to biparental populations. The prediction of the haplotype origin was possible for 89% of the MAGIC line genomes, allowing QTL detection at the haplotype level. We grew the population in two greenhouse trials and detected QTLs for fruit weight. We mapped three stable QTLs and six specific of a location. Finally, we showed the potential of the MAGIC population when coupled with whole genome sequencing of founder lines to detect candidate SNPs underlying the QTLs. For a previously cloned QTL on chromosome 3, we used the predicted allelic effect of each founder and their genome sequences to select putative causal polymorphisms in the supporting interval. The number of candidate polymorphisms was reduced from 12 284 (in 800 genes) to 96 (in 54 genes), including the actual causal polymorphism. This population represents a new permanent resource for the tomato genetics community.


Assuntos
Locos de Características Quantitativas , Solanum lycopersicum/genética , Genes de Plantas , Polimorfismo de Nucleotídeo Único
5.
BMC Genomics ; 14: 791, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24228636

RESUMO

BACKGROUND: One of the goals of genomics is to identify the genetic loci responsible for variation in phenotypic traits. The completion of the tomato genome sequence and recent advances in DNA sequencing technology allow for in-depth characterization of genetic variation present in the tomato genome. Like many self-pollinated crops, cultivated tomato accessions show a low molecular but high phenotypic diversity. Here we describe the whole-genome resequencing of eight accessions (four cherry-type and four large fruited lines) chosen to represent a large range of intra-specific variability and the identification and annotation of novel polymorphisms. RESULTS: The eight genomes were sequenced using the GAII Illumina platform. Comparison of the sequences with the reference genome yielded more than 4 million single nucleotide polymorphisms (SNPs). This number varied from 80,000 to 1.5 million according to the accessions. Almost 128,000 InDels were detected. The distribution of SNPs and InDels across and within chromosomes was highly heterogeneous revealing introgressions from wild species and the mosaic structure of the genomes of the cherry tomato accessions. In-depth annotation of the polymorphisms identified more than 16,000 unique non-synonymous SNPs. In addition 1,686 putative copy-number variations (CNVs) were identified. CONCLUSIONS: This study represents the first whole genome resequencing experiment in cultivated tomato. Substantial genetic differences exist between the sequenced tomato accessions and the reference sequence. The heterogeneous distribution of the polymorphisms may be related to introgressions that occurred during domestication or breeding. The annotated SNPs, InDels and CNVs identified in this resequencing study will serve as useful genetic tools, and as candidate polymorphisms in the search for phenotype-altering DNA variations.


Assuntos
Genoma de Planta , Solanum lycopersicum/genética , Cruzamento , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Variações do Número de Cópias de DNA , Evolução Molecular , Heterozigoto , Mutação INDEL , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
6.
J Exp Bot ; 64(18): 5737-52, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24151307

RESUMO

Integrative systems biology proposes new approaches to decipher the variation of phenotypic traits. In an effort to link the genetic variation and the physiological and molecular bases of fruit composition, the proteome (424 protein spots), metabolome (26 compounds), enzymatic profile (26 enzymes), and phenotypes of eight tomato accessions, covering the genetic diversity of the species, and four of their F1 hybrids, were characterized at two fruit developmental stages (cell expansion and orange-red). The contents of metabolites varied among the genetic backgrounds, while enzyme profiles were less variable, particularly at the cell expansion stage. Frequent genotype by stage interactions suggested that the trends observed for one accession at a physiological level may change in another accession. In agreement with this, the inheritance modes varied between crosses and stages. Although additivity was predominant, 40% of the traits were non-additively inherited. Relationships among traits revealed associations between different levels of expression and provided information on several key proteins. Notably, the role of frucktokinase, invertase, and cysteine synthase in the variation of metabolites was highlighted. Several stress-related proteins also appeared related to fruit weight differences. These key proteins might be targets for improving metabolite contents of the fruit. This systems biology approach provides better understanding of networks controlling the genetic variation of tomato fruit composition. In addition, the wide data sets generated provide an ideal framework to develop innovative integrated hypothesis and will be highly valuable for the research community.


Assuntos
Frutas/química , Frutas/fisiologia , Variação Genética , Proteínas de Plantas/metabolismo , Característica Quantitativa Herdável , Solanum lycopersicum/fisiologia , Biologia de Sistemas/métodos , Enzimas/genética , Enzimas/metabolismo , Genótipo , Análise dos Mínimos Quadrados , Solanum lycopersicum/genética , Redes e Vias Metabólicas , Tamanho do Órgão , Proteínas de Plantas/genética , Proteoma
7.
Proteomics ; 13(20): 3059-63, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23929585

RESUMO

Tomato (Solanum lycopersicum) is the model species for studying fleshy fruit development. An extensive proteome map of the fruit pericarp is described in light of the high-quality genome sequence. The proteomes of fruit pericarp from 12 tomato genotypes at two developmental stages (cell expansion and orange-red) were analyzed. The 2DE reference map included 506 spots identified by nano-LC/MS and the International Tomato Annotation Group Database searching. A total of 425 spots corresponded to unique proteins. Thirty-four spots resulted from the transcription of genes belonging to multigene families involving two to six genes. A total of 47 spots corresponded to a mixture of different proteins. The whole protein set was classified according to Gene Ontology annotation. The quantitative protein variation was analyzed in relation to genotype and developmental stage. This tomato fruit proteome dataset is currently the largest available and constitutes a valuable tool for comparative genetic studies of tomato genome expression at the protein level. All MS data have been deposited in the ProteomeXchange with identifier PXD000105.


Assuntos
Frutas/anatomia & histologia , Frutas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Solanum lycopersicum/anatomia & histologia , Solanum lycopersicum/metabolismo , Ontologia Genética , Espectrometria de Massas , Proteínas de Plantas/metabolismo , Análise de Componente Principal
8.
OMICS ; 17(6): 338-52, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23692365

RESUMO

Salinity is a major abiotic stress that adversely affects plant growth and productivity. The physiology of the tomato in salty and nonsalty conditions has been extensively studied, providing an invaluable base to understand the responses of the plants to cultural practices. However few data are yet available at the proteomic level looking for the physiological basis of fruit development, under salt stress. Here, we report the effects of salinity and calcium on fruit proteome variations of two tomato genotypes (Cervil and Levovil). Tomato plants were irrigated with a control solution (3 dSm(-1)) or with saline solutions (Na or Ca+Na at 7.6 dSm(-1)). Tomato fruits were harvested at two ripening stages: green (14 days post-anthesis) and red ripe. Total proteins were extracted from pericarp tissue and separated by two-dimensional gel electrophoresis. Among the 600 protein spots reproducibly detected, 53 spots exhibited significant abundance variations between samples and were submitted to mass spectrometry for identification. Most of the identified proteins were involved in carbon and energy metabolism, salt stress, oxidative stress, and proteins associated with ripening process. Overall, there was a large variation on proteins abundance between the two genotypes that can be correlated to salt treatment or/and fruit ripening stage. The results showed a protective effect of calcium that limited the impact of salinization on metabolism, ripening process, and induced plant salt tolerance. Collectively, this work has improved our knowledge about salt and calcium effect on tomato fruit proteome.


Assuntos
Cálcio/metabolismo , Frutas/metabolismo , Proteoma , Salinidade , Solanum lycopersicum/metabolismo , Metabolismo Energético , Frutas/efeitos dos fármacos , Frutas/crescimento & desenvolvimento , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/crescimento & desenvolvimento , Biossíntese de Proteínas , Proteômica , Cloreto de Sódio/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico
9.
Theor Appl Genet ; 126(3): 567-81, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23124430

RESUMO

Association mapping has been proposed as an efficient approach to assist in the identification of the molecular basis of agronomical traits in plants. For this purpose, we analyzed the phenotypic and genetic diversity of a large collection of tomato accessions including 44 heirloom and vintage cultivars (Solanum lycopersicum), 127 S. lycopersicum var. cerasiforme (cherry tomato) and 17 Solanum pimpinellifolium accessions. The accessions were genotyped using a SNPlex™ assay of 192 SNPs, among which 121 were informative for subsequent analysis. Linkage disequilibrium (LD) of pairwise loci and population structure were analyzed, and the association analysis between SNP genotypes and ten fruit quality traits was performed using a mixed linear model. High level of LD was found in the collection at the whole genome level. It was lower when considering only the 127 S. lycopersicum var. cerasiforme accessions. Genetic structure analysis showed that the population was structured into two main groups, corresponding to cultivated and wild types and many intermediates. The number of associations detected per trait varied, according to the way the structure was taken into account, with 0-41 associations detected per trait in the whole collection and a maximum of four associations in the S. lycopersicum var. cerasiforme accessions. A total of 40 associations (30 %) were co-localized with previously identified quantitative trait loci. This study thus showed the potential and limits of using association mapping in tomato populations.


Assuntos
Mapeamento Cromossômico , Frutas/genética , Genoma de Planta , Fenótipo , Polimorfismo de Nucleotídeo Único , Solanum lycopersicum/genética , DNA de Plantas/genética , Genótipo , Desequilíbrio de Ligação , Locos de Características Quantitativas
10.
G3 (Bethesda) ; 2(8): 853-64, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22908034

RESUMO

Genome-wide association mapping is an efficient way to identify quantitative trait loci controlling the variation of phenotypes, but the approach suffers severe limitations when one is studying inbred crops like cultivated tomato (Solanum lycopersicum). Such crops exhibit low rates of molecular polymorphism and high linkage disequilibrium, which reduces mapping resolution. The cherry type tomato (S. lycopersicum var. cerasiforme) genome has been described as an admixture between the cultivated tomato and its wild ancestor, S. pimpinellifolium. We have thus taken advantage of the properties of this admixture to improve the resolution of association mapping in tomato. As a proof of concept, we sequenced 81 DNA fragments distributed on chromosome 2 at different distances in a core collection of 90 tomato accessions, including mostly cherry type tomato accessions. The 81 Sequence Tag Sites revealed 352 SNPs and indels. Molecular diversity was greatest for S. pimpinellifolium accessions, intermediate for S. l. cerasiforme accessions, and lowest for the cultivated group. We assessed the structure of molecular polymorphism and the extent of linkage disequilibrium over genetic and physical distances. Linkage disequilibrium decreased under r(2) = 0.3 within 1 cM, and minimal estimated value (r(2) = 0.13) was reached within 20 kb over the physical regions studied. Associations between polymorphisms and fruit weight, locule number, and soluble solid content were detected. Several candidate genes and quantitative trait loci previously identified were validated and new associations detected. This study shows the advantages of using a collection of S. l. cerasiforme accessions to overcome the low resolution of association mapping in tomato.


Assuntos
Genoma de Planta , Solanum lycopersicum/genética , Cromossomos de Plantas , Frutas/genética , Variação Genética , Estudo de Associação Genômica Ampla , Desequilíbrio de Ligação , Fases de Leitura Aberta , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Análise de Sequência de DNA
11.
J Exp Bot ; 62(8): 2797-813, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21330356

RESUMO

To evaluate the genotypic variation of salt stress response in tomato, physiological analyses and a proteomic approach have been conducted in parallel on four contrasting tomato genotypes. After a 14 d period of salt stress in hydroponic conditions, the genotypes exhibited different responses in terms of plant growth, particularly root growth, foliar accumulation of Na(+), and foliar K/Na ratio. As a whole, Levovil appeared to be the most tolerant genotype while Cervil was the most sensitive one. Roma and Supermarmande exhibited intermediary behaviours. Among the 1300 protein spots reproducibly detected by two-dimensional electrophoresis, 90 exhibited significant abundance variations between samples and were submitted to mass spectrometry for identification. A common set of proteins (nine spots), up- or down-regulated by salt-stress whatever the genotype, was detected. But the impact of the tomato genotype on the proteome variations was much higher than the salt effect: 33 spots that were not variable with salt stress varied with the genotype. The remaining number of variable spots (48) exhibited combined effects of the genotype and the salt factors, putatively linked to the degrees of genotype tolerance. The carbon metabolism and energy-related proteins were mainly up-regulated by salt stress and exhibited most-tolerant versus most-sensitive abundance variations. Unexpectedly, some antioxidant and defence proteins were also down-regulated, while some proteins putatively involved in osmoprotectant synthesis and cell wall reinforcement were up-regulated by salt stress mainly in tolerant genotypes. The results showed the effect of 14 d stress on the tomato root proteome and underlined significant genotype differences, suggesting the importance of making use of genetic variability.


Assuntos
Raízes de Plantas/metabolismo , Proteoma/metabolismo , Cloreto de Sódio/farmacologia , Solanum lycopersicum/genética , Solanum lycopersicum/fisiologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Antioxidantes/metabolismo , Carbono/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Cloretos/metabolismo , Cromatografia Líquida , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Eletroforese em Gel Bidimensional , Metabolismo Energético/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Variação Genética/efeitos dos fármacos , Genótipo , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/crescimento & desenvolvimento , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Osmose/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Salinidade , Tolerância ao Sal/efeitos dos fármacos , Tolerância ao Sal/genética , Sódio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
12.
J Exp Bot ; 62(3): 907-19, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21036926

RESUMO

A mechanistic model predicting the accumulation of tomato fruit sugars was developed in order (i) to dissect the relative influence of three underlying processes: assimilate supply (S), metabolic transformation of sugars into other compounds (M), and dilution by water uptake (D); and (ii) to estimate the genetic variability of S, M, and D. The latter was estimated in a population of 20 introgression lines derived from the introgression of a wild tomato species (Solanum chmielewskii) into S. lycopersicum, grown under two contrasted fruit load conditions. Low load systematically decreased D in the whole population, while S and M were targets of genotype × fruit load interactions. The sugar concentration positively correlated to S and D when the variation was due to genetic introgressions, while it positively correlated to S and M when the variation was due to changes in fruit load. Co-localizations between quantitative trait loci (QTLs) for sugar concentration and QTLs for S, M, and D allowed hypotheses to be proposed on the processes putatively involved at the QTLs. Among the five QTLs for sugar concentration, four co-localized with QTLs for S, M, and D with similar allele effects. Moreover, the processes underlying QTLs for sugar accumulation changed according to the fruit load condition. Finally, for some genotypes, the processes underlying sugar concentration compensated in such a way that they did not modify the sugar concentration. By uncoupling genetic from physiological relationships between processes, these results provide new insights into further understanding of tomato fruit sugar accumulation.


Assuntos
Metabolismo dos Carboidratos , Frutas/metabolismo , Locos de Características Quantitativas , Solanum lycopersicum/genética , Frutas/genética , Genótipo , Solanum lycopersicum/metabolismo , Modelos Genéticos
13.
C R Biol ; 332(11): 1007-21, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19909923

RESUMO

Very few reports have studied the interactions between ascorbate and fruit metabolism. In order to get insights into the complex relationships between ascorbate biosynthesis/recycling and other metabolic pathways in the fruit, we undertook a fruit systems biology approach. To this end, we have produced tomato transgenic lines altered in ascorbate content and redox ratio by RNAi-targeting several key enzymes involved in ascorbate biosynthesis (2 enzymes) and recycling (2 enzymes). In the VTC (ViTamin C) Fruit project, we then generated phenotypic and genomic (transcriptome, proteome, metabolome) data from wild type and mutant tomato fruit at two stages of fruit development, and developed or implemented statistical and bioinformatic tools as a web application (named VTC Tool box) necessary to store, analyse and integrate experimental data in tomato. By using Kohonen's self-organizing maps (SOMs) to cluster the biological data, pair-wise Pearson correlation analyses and simultaneous visualization of transcript/protein and metabolites (MapMan), this approach allowed us to uncover major relationships between ascorbate and other metabolic pathways.


Assuntos
Ácido Ascórbico/metabolismo , Frutas/crescimento & desenvolvimento , Genômica/métodos , Solanum lycopersicum/crescimento & desenvolvimento , Análise de Variância , Ascorbato Oxidase/genética , Ascorbato Oxidase/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Solanum lycopersicum/genética , Solanum lycopersicum/efeitos da radiação , Redes e Vias Metabólicas , Metaboloma , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteoma , Integração de Sistemas
14.
J Pineal Res ; 45(4): 524-31, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18826489

RESUMO

Quinone reductase 2 (QR2, E.C. 1.10.99.2) is implicated in cell reactive oxygen species production. The catalytic activity of this enzyme is inhibited by 1 microM of melatonin. QR2 was identified as the third melatonin binding site (MT3). It is of major importance to understand the exact roles of melatonin and QR2 in oxidative stress. A fascinating possibility that melatonin could serve as a co-substrate or substrate of QR2 was hypothesized recently. In the current investigation, nuclear magnetic resonance studies of the QR2 catalytic reaction were performed, the results led us to conclude that, whatever the conditions, melatonin is not cleaved off to form N1-acetyl-N2-formyl-5-methoxykynurenine by a catalytically active QR2, very strongly indicating that melatonin is neither a substrate nor a co-substrate of this enzyme. Further studies are needed in order to better understand the relationship between MT3/QR2, melatonin and redox status of the cells, in order to better explain the anti-oxidant activities of melatonin at pharmacological concentrations (>1 microM).


Assuntos
Cinuramina/análogos & derivados , Melatonina/metabolismo , Quinona Redutases/metabolismo , Sítios de Ligação , Humanos , Cinuramina/química , Cinuramina/metabolismo , Melatonina/química , Ressonância Magnética Nuclear Biomolecular , Estresse Oxidativo , Quinona Redutases/química , Quinona Redutases/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Melatonina/química , Receptores de Melatonina/metabolismo
15.
Genome ; 50(4): 422-7, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17546100

RESUMO

Using a complementary (c)DNA-amplified fragment length polymorphism (AFLP) approach, we investigated differential gene expression linked to resistance mechanisms during the incompatible potato - Globodera pallida interaction. Expression was compared between a resistant and a susceptible potato clone, inoculated or not inoculated with G. pallida. These clones were issued from a cross between the resistant Solanum sparsipilum spl329.18 accession and the susceptible dihaploid S. tuberosum Caspar H3, and carried, respectively, resistant and susceptible alleles at the resistance quantitative trait loci (QTLs). Analysis was done on root fragments picked up at 4 time points, during a period of 6 days after infection, from penetration of the nematode in the root to degradation of the feeding site in resistant plants. A total of 2560 transcript-derived fragments (TDFs) were analyzed, resulting in the detection of 46 TDFs that were up- or downregulated. The number of TDFs that were up- or downregulated increased with time after inoculation. The majority of TDFs were upregulated at only 1 or 2 time points in response to infection. After isolation and sequencing of the TDFs of interest, a subset of 36 sequences were identified, among which 22 matched plant sequences and 2 matched nematode sequences. Some of the TDFs that matched plant genes showed clear homologies to genes involved in cell-cycle regulation, transcription regulation, resistance downstream signalling pathways, and defense mechanisms. Other sequences with homologies to plant genes of unknown function or without any significant similarity to known proteins were also found. Although not exhaustive, these results represent the most extensive list of genes with altered RNA levels after the incompatible G. pallida-potato interaction that has been published to date. The function of these genes could provide insight into resistance or plant defense mechanisms during incompatible potato-cyst nematode interactions.


Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Imunidade Inata/genética , Solanum/genética , Solanum/parasitologia , Tylenchoidea/fisiologia , Animais , Interações Hospedeiro-Parasita/genética
16.
Plant Physiol ; 144(1): 18-31, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17351058

RESUMO

The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3beta-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome.


Assuntos
Lactuca/genética , Nicotiana/genética , Retroelementos , Genoma de Planta , Glucuronidase/análise , Lactuca/anatomia & histologia , Lactuca/enzimologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional/métodos , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/anatomia & histologia , Plantas Geneticamente Modificadas/metabolismo , Transformação Genética
17.
Biochem J ; 388(Pt 1): 205-15, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15636586

RESUMO

The catabolism of melatonin, whether naturally occurring or ingested, takes place via two pathways: approximately 70% can be accounted for by conjugation (sulpho- and glucurono-conjugation), and approximately 30% by oxidation. It is commonly thought that the interferon-induced enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42), which oxidizes tryptophan, is also responsible for the oxidation of 5-hydroxytryptamine (serotonin) and its derivative, melatonin. Using the recombinant enzyme expressed in Escherichia coli, we show in the present work that indoleamine 2,3-dioxygenase indeed cleaves tryptophan; however, under the same conditions, it is incapable of cleaving the two other indoleamines. By contrast, myeloperoxidase (EC 1.11.1.7) is capable of cleaving the indole moiety of melatonin. However, when using the peroxidase conditions of assay -- with H2O2 as co-substrate -- indoleamine 2,3-dioxygenase is able to cleave melatonin into its main metabolite, a kynurenine derivative. The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H2O2, to the activities of both myeloperoxidase and indoleamine 2,3-dioxygenase (with lower potency), since both enzymes have Km values for melatonin in the micromolar range. Under these conditions, several indolic compounds can be cleaved by both enzymes, such as tryptamine and 5-hydroxytryptamine. Furthermore, melatonin metabolism results in a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.


Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Melatonina/metabolismo , Peroxidase/metabolismo , Triptofano/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/química , Melatonina/química , Modelos Químicos , Estrutura Molecular , Oxirredução , Peroxidase/química , Especificidade por Substrato , Triptofano/química
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