RESUMO
A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5'-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.
Assuntos
Arabidopsis , Proteínas de Transporte , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Ácidos Graxos/metabolismo , Fumaratos/metabolismo , Expressão Gênica , Genes Fúngicos , Genes de Plantas , Cinética , Lipossomos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Nitrogênio/metabolismo , Saccharomyces cerevisiae/genética , Plântula/crescimento & desenvolvimento , Succinatos/metabolismo , Ácidos Tricarboxílicos/metabolismoRESUMO
Despite the fundamental importance of nicotinamide adenine dinucleotide (NAD+) for metabolism, the physiological roles of NAD+ carriers in plants remain unclear. We previously characterized the Arabidopsis thaliana gene (At1g25380), named AtNDT2, encoding a protein located in the mitochondrial inner membrane, which imports NAD+ from the cytosol using ADP and AMP as counter-exchange substrates for NAD+. Here, we further investigated the physiological roles of NDT2, by isolating a T-DNA insertion line, generating an antisense line and characterizing these genotypes in detail. Reduced NDT2 expression affected reproductive phase by reducing total seed yield. In addition, reduced seed germination and retardation in seedling establishment were observed in the mutant lines. Moreover, remarkable changes in primary metabolism were observed in dry and germinated seeds and an increase in fatty acid levels was verified during seedling establishment. Furthermore, flowers and seedlings of NDT2 mutants displayed upregulation of de novo and salvage pathway genes encoding NAD+ biosynthesis enzymes, demonstrating the transcriptional control mediated by NDT2 activity over these genes. Taken together, our results suggest that NDT2 expression is fundamental for maintaining NAD+ balance amongst organelles that modulate metabolism, physiology and developmental processes of heterotrophic tissues.
Assuntos
Proteínas de Arabidopsis/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Germinação/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , NAD/metabolismo , Proteínas de Transporte de Nucleotídeos/genética , Sementes/crescimento & desenvolvimento , Sementes/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Flores/fisiologia , Genótipo , Processos Heterotróficos , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Nucleotídeos/metabolismo , Piridinas/metabolismo , Reprodução/fisiologiaRESUMO
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/metabolismoRESUMO
Lysine (Lys) connects the mitochondrial electron transport chain to amino acid catabolism and the tricarboxylic acid cycle. However, our understanding of how a deficiency in Lys biosynthesis impacts plant metabolism and growth remains limited. Here, we used a previously characterized Arabidopsis mutant (dapat) with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase to investigate the physiological and metabolic impacts of impaired Lys biosynthesis. Despite displaying similar stomatal conductance and internal CO2 concentration, we observed reduced photosynthesis and growth in the dapat mutant. Surprisingly, whilst we did not find differences in dark respiration between genotypes, a lower storage and consumption of starch and sugars was observed in dapat plants. We found higher protein turnover but no differences in total amino acids during a diurnal cycle in dapat plants. Transcriptional and two-dimensional (isoelectric focalization/SDS-PAGE) proteome analyses revealed alterations in the abundance of several transcripts and proteins associated with photosynthesis and photorespiration coupled with a high glycine/serine ratio and increased levels of stress-responsive amino acids. Taken together, our findings demonstrate that biochemical alterations rather than stomatal limitations are responsible for the decreased photosynthesis and growth of the dapat mutant, which we hypothesize mimics stress conditions associated with impairments in the Lys biosynthesis pathway.
Assuntos
Arabidopsis/genética , Lisina/biossíntese , Metaboloma , Proteoma/análise , Transaminases/genética , Transcriptoma , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Mutação , Transaminases/metabolismoRESUMO
INTRODUCTION: The native potatoes (Solanum tuberosum ssp. tuberosum L.) cultivated on Chiloé Island in southern Chile have great variability in terms of tuber shape, size, color and flavor. These traits have been preserved throughout generations due to the geographical position of Chiloé, as well as the different uses given by local farmers. OBJECTIVES: The present study aimed to investigate the diversity of metabolites in skin and pulp tissues of eleven native accessions of potatoes from Chile, and evaluate the metabolite associations between tuber tissues. METHODS: For a deeper characterization of these accessions, we performed a comprehensive metabolic study in skin and pulp tissues of tubers, 3 months after harvesting. Specific targeted quantification of metabolites using 96 well microplates, and high-performance liquid chromatography combined with non-targeted metabolite profiling by gas chromatography time-of-flight mass spectrometry were used in this study. RESULTS: We observed differential levels of antioxidant activity and phenolic compounds between skin and pulp compared to a common commercial cultivar (Desireé). In addition, we uncovered considerable metabolite variability between different tuber tissues and between native potatoes. Network correlation analysis revealed different metabolite associations among tuber tissues that indicate distinct associations between primary metabolite and anthocyanin levels, and antioxidant activity in skin and pulp tissues. Moreover, multivariate analysis lead to the grouping of native and commercial cultivars based on metabolites from both skin and pulp tissues. CONCLUSIONS: As well as providing important information to potato producers and breeding programs on the levels of health relevant phytochemicals and other abundant metabolites such as starch, proteins and amino acids, this study highlights the associations of different metabolites in tuber skins and pulp, indicating the need for distinct strategies for metabolic engineering in these tissues. Furthermore, this study shows that native Chilean potato accessions have great potential as a natural source of phytochemicals.
Assuntos
Tubérculos/metabolismo , Solanum tuberosum/classificação , Solanum tuberosum/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Chile , Fenóis/química , Fenóis/metabolismo , Tubérculos/química , Solanum tuberosum/químicaRESUMO
Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , beta-Frutofuranosidase/fisiologia , Alelos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Conformação Proteica , Locos de Características Quantitativas/genética , Locos de Características Quantitativas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Plântula/crescimento & desenvolvimento , Análise de Sequência de DNA , Vacúolos/enzimologia , Vacúolos/fisiologia , beta-Frutofuranosidase/genéticaRESUMO
Plant metabolic pathways and the molecular and atomic fluxes through them can be deduced using stable isotopically labeled substrates. To this end one prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we summarize the application of gas chromatography-time of flight mass spectrometry (GC-TOF-MS) for metabolic studies using differently labeled elemental isotopes applied to both intact organelles and whole plant tissue. This method allows quantitative evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate) chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabeled substrates. We focus herein on the determination of isotope labeling in organic and amino acids.
Assuntos
Aminoácidos/química , Análise do Fluxo Metabólico , Aminoácidos/metabolismo , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Marcação por Isótopo , Cinética , Plantas/metabolismoRESUMO
Recent years have seen considerable advances in our understanding of the particular physiological roles of the constituent enzymes of the tricarboxylic acid (TCA) cycle. Despite acquiring a fairly comprehensive overview of the functional importance of these proteins relatively little is known concerning how this important pathway is regulated. In this review we concentrate on the mitochondrial reactions since this organelle is the only one in which a full cycle can, at least theoretically, operate. We summarize what is known about the regulation of the enzymes of the pathway both from historical kinetic studies as well as discussing more recent transcriptional and proteomic studies and our enhanced understanding of subcellular compartmentation within the context of metabolic regulation.