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1.
Plant J ; 100(3): 487-504, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31278825

RESUMO

Nicotinamide adenine dinucleotide (NAD+ ) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD+ biosynthesis is exclusively cytosolic. Hence, NAD+ must be imported into organelles to support their metabolic functions. Three NAD+ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T-DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD+ homeostasis for both metabolic and developmental processes in plants.


Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , NAD/metabolismo , Antiporters/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloroplastos/metabolismo , Citosol/metabolismo , Proteínas de Fluorescência Verde , Homeostase , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutagênese Insercional , Proteínas de Transporte de Nucleotídeos , Peroxissomos/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/fisiologia , Amido/metabolismo
2.
J Exp Bot ; 69(22): 5355-5371, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30169823

RESUMO

In eudicotyledons, accumulation of trihydroxycinnamoyl spermidine that is restricted to the pollen wall constitutes an evolutionary conserved trait. However, the role of this compound, which is synthetized by the BAHD enzyme spermidine hydroxycinnamoyl transferase (SHT), is still a matter of debate. Here, we show that this particular phenolamide is replaced by tetrahydroxycinnamoyl spermine in the pollen coat of the Asteraceae. Phylogenetic analyses combined with quantitative RT-PCR experiments allowed the identification of two homologous genes from Cichorium intybus (chicory) putatively involved in its metabolism. In vitro biochemical characterization of the two enzymes, named CiSHT1 and CiSHT2, confirmed the capability of recombinant proteins to synthesize spermine as well as spermidine derivatives. The wild-type metabolic phenotype was partially restored in an Arabidopsis sht mutant expressing CiSHT2. Strikingly, the transgenic plants also accumulated spermine derivatives that were absent in the wild-type. Overexpression of CiSHT2 in chicory hairy roots led to the accumulation of spermine derivatives, confirming its in vivo function. Complementary sequence analyses revealed the presence of an amino acid motif typical of the SHTs among the BAHD enzyme family. Our results highlight a recent neofunctionalization among the SHTs that has promoted the emergence of new phenolamides in the Asteraceae, which could potentially have contributed to the evolutionary success of this family.


Assuntos
Arabidopsis/genética , Cichorium intybus/genética , Proteínas de Plantas/genética , Pólen/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Cichorium intybus/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Espermina/metabolismo
3.
Methods Mol Biol ; 1595: 291-304, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28409472

RESUMO

Peroxisomal ß-oxidation in plants is essential for mobilization of storage oil in seed-oil storing plants, such as Arabidopsis thaliana. In plants, degradation of fatty acids occurs exclusively in peroxisomes via ß-oxidation, driving seedling growth and development upon germination. Thus, the determination of storage oil breakdown rates is a useful approach to investigate defects in peroxisomal ß-oxidation. Here we describe an acid catalyzed derivatization process of fatty acids representing a fast and efficient procedure to generate high yields of fatty acid methyl esters (FAMEs). The subsequent analysis by gas chromatography coupled to mass spectrometry (GC-MS) allows the quantification of total fatty acid content. The results provide detailed information of the complete storage oil breakdown process via peroxisomal ß-oxidation during seedling growth.


Assuntos
Arabidopsis/metabolismo , Oxirredução , Peroxissomos/metabolismo , Óleos de Plantas/metabolismo , Plântula/metabolismo , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Germinação , Extratos Vegetais , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
4.
Plant J ; 69(1): 1-13, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21895810

RESUMO

The existence of a transport protein that imports cytosolic NAD(+) into peroxisomes has been controversially discussed for decades. Nevertheless, the biosynthesis of NAD(+) in the cytosol necessitates the import of NAD(+) into peroxisomes for numerous reduction/oxidation (redox) reactions. However, a gene encoding such a transport system has not yet been identified in any eukaryotic organism. Here, we describe the peroxisomal NAD(+) carrier in Arabidopsis. Our candidate gene At2g39970 encodes for a member of the mitochondrial carrier family. We confirmed its peroxisomal localization using fluorescence microscopy. For a long time At2g39970 was assumed to represent the peroxisomal ATP transporter. In this study, we could show that the recombinant protein mediated the transport of NAD(+) . Hence, At2g39970 was named PXN for peroxisomal NAD(+) carrier. The loss of PXN in Arabidopsis causes defects in NAD(+) -dependent ß-oxidation during seedling establishment. The breakdown of fatty acid released from storage oil was delayed, which led to the retention of oil bodies in pxn mutant seedlings. Based on our results, we propose that PXN delivers NAD(+) for optimal fatty acid degradation during storage oil mobilization.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , NAD/metabolismo , Peroxissomos/metabolismo , Óleos de Plantas/metabolismo , Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Ácido 2,4-Diclorofenoxiacético/farmacologia , Nucleotídeos de Adenina/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Proteínas de Transporte da Membrana Mitocondrial/genética , Mutação , Oxirredução , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Sacarose/metabolismo
5.
Plant Mol Biol ; 55(1): 17-32, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15604662

RESUMO

When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first. However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0-4 and temperatures of up to 56 degrees C. This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols. This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology. The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G. sulphuraria a valuable model for physical studies on the photosynthetic apparatus. In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes. Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation. To begin to explore the unique biology of G. sulphuraria , 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated. Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism. For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases. Sequence data and additional information are available at http://genomics.msu.edu/galdieria.


Assuntos
Carbono/metabolismo , Etiquetas de Sequências Expressas , Lipídeo A/biossíntese , Plastídeos/metabolismo , Rodófitas/genética , Proteínas de Algas/genética , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , DNA Complementar/química , DNA Complementar/genética , Metabolismo Energético/genética , Ácidos Graxos/metabolismo , Biblioteca Gênica , Hexoquinase/genética , Concentração de Íons de Hidrogênio , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Consumo de Oxigênio , Proteínas de Transporte de Fosfato/genética , Fotossíntese/genética , Filogenia , Rodófitas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Temperatura
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