RESUMO
Aluminum (Al) toxicity is a crucial limiting factor for crop growth in acid soils. Recently, Liu et al. demonstrated that the root microbiota of rice modulates the responses to Al toxicity and phosphorus limitation, offering intriguing insights into microbiome function and opening new research opportunities.
Assuntos
Microbiota , Oryza , Solo , Plantas , Fósforo , Alumínio , Concentração de Íons de Hidrogênio , Raízes de PlantasRESUMO
Nitrogen (N) nutrition and meiosis demand large amounts of energy and widely affect crop yield. Recently, Yang and colleagues connected both processes by demonstrating that meiosis initiation depends on the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) system, whereas meiotic defects of the etfß mutant can be rescued using N supplementation.
Assuntos
Aminoácidos , Ubiquinona , Aminoácidos/metabolismo , Meiose/genética , Nitrogênio , Sementes/genética , Sementes/metabolismoRESUMO
Phosphorus (P) deficiency affects agricultural systems by limiting crop quality and yield. Studies have suggested that silicon (Si) improves P uptake in plants grown under P deficiency. However, the effects of Si on photosynthesis and carbohydrate metabolism under P stress remain unclear. We performed a hydroponic study using two wheat cultivars with contrasting sensitivity to P deficiency (Púrpura, sensitive; Fritz, semi-tolerant) that were exposed to P (0, 0.01, or 0.1 mM) and Si (0 or 2 mM), and we evaluated the photosynthetic performance and metabolic alterations. In plants from the sensitive cultivar undergoing P deficiency, Si application increased sucrose levels, starch breakdown and length of shoots, and also improved plant dry weight. In Fritz (the semi-tolerant cultivar), Si exposure reduced P concentration, and increased shoot length and P use efficiency (PUE) under P shortage. Interestingly, Si application altered cell wall composition, which was associated with higher mesophyll conductance and net CO2 assimilation in Fritz plants grown under P stress. Together, our results indicate that under P deficiency, Si nutrition positively affects photosynthesis and carbohydrate levels in a genotype-dependent manner. Furthermore, these results suggest that Si plays an important role in maintaining high photosynthetic rates in wheat plants undergoing P deficiency.
Assuntos
Silício , Triticum , Metabolismo dos Carboidratos , Fósforo , Fotossíntese , Folhas de Planta , Silício/farmacologiaRESUMO
This study aimed to evaluate the simultaneous interferences of Cu and Zn found in swine wastewater (SW) in the development of microalgae considering real conditions of cultivation in high rate algal ponds (HRAPs). Ten HRAPs on a pilot scale were fed with SW with different mixtures of Cu (0.5-3.0 mg/L) and Zn (5.0-25.0 mg/L). The interferences of these metals in removing nutrients (N-NH4+ and soluble phosphorus (Ps)) from the SW were determined. In addition, this study evaluated the effects on biomass growth and biochemical composition. Chlorella sp. was dominant in all HRAPs and the condition that potentiated its growth occurred in medium containing 1.8 mg Cu/L + 15.0 mg Zn/L, while higher concentrations conferred inhibition. Only Cu compromised the removal rates of N-NH4+ while the effects of Zn were not significant. Contrary, Zn interfered with Ps removal rates, but the impact of Cu was not significant. The greatest Cu applications increased the protein levels by biomass (50.5-55.2 %). Carbohydrate accumulation was favored by conditions that inhibited the development of microalgae due to either limitation or excess of metals. Copper and Zn compromised the levels of lipids, and the control treatment had the highest content (24.5 %). The presence of Cu and Zn changed the dynamics of HRAPs regarding nutrient removal, productivity, and biochemical composition of the biomass.
Assuntos
Chlorella , Microalgas , Purificação da Água , Animais , Biomassa , Nitrogênio/análise , Nutrientes , Lagoas , Suínos , Águas Residuárias , ZincoRESUMO
Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Plantas/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Coenzima A/metabolismo , Regulação da Expressão Gênica de Plantas , Ferro/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Desacoplamento Mitocondrial/genética , Proteínas de Desacoplamento Mitocondrial/metabolismo , Modelos Biológicos , NAD/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Plantas/genéticaRESUMO
We evaluated whether phosphorus (P) ameliorates manganese (Mn) excess harmful effects on photosynthetic performance, growth, oxidative stress, and antioxidants in ryegrass. Two perennial ryegrass genotypes, Banquet-II as Mn-resistant and One-50 as Mn-sensitive genotype, were growth under hydroponic conditions subjected to increased P (25, 50, 100, 200 and 400⯵M), excess (750⯵M) and sufficient Mn (2.4⯵M) for 15 days. Growth rate, lipid peroxidation (LP), enzymatic and non-enzymatic antioxidants, photosynthetic parameters, and pigments were determined. Significant reduction of photosynthesis and growth in One-50 was observed under Mn-excess combined with low and adequate P, recovering under greater P-doses. The P concentration of both genotypes was enhanced towards increased P-supply, regardless of Mn treatments. Shoots Mn-concentration remained constant in both genotypes under Mn-excess, independently of P-levels; meanwhile, Banquet-II roots Mn-concentration increased 23% by P-supply. Furthermore, Banquet-II roots showed higher superoxide dismutase (SOD) activity than One-50, which increased towards the highest P dose under sufficient and excess of Mn. A high dose of phosphorus amendment alleviated Mn-toxicity in Mn-sensitive genotype (One-50). Besides, in the Mn-resistant genotype, enhanced plant performance is highlighted, explained by a high Mn-accumulation in roots and increased SOD activity, decreasing Mn translocation to shoots and therefore protecting the photosynthetic apparatus.
Assuntos
Lolium/efeitos dos fármacos , Lolium/metabolismo , Manganês/toxicidade , Fósforo/farmacologia , Regulação da Expressão Gênica de Plantas , Genótipo , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fotossíntese/efeitos dos fármacosRESUMO
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
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
Naturally occurring genetic variation in plants can be very useful to dissect the complex regulation of primary metabolism as well as of physiological traits such as photosynthesis and photorespiration. The physiological and genetic mechanisms underlying natural variation in closely related species or accessions may provide important information that can be used to improve crop yield. In this chapter we describe in detail the use of a population of introgression lines (ILs), with the Solanum pennellii IL population as a study case, as a tool for the identification of genomic regions involved in the control of photosynthetic efficiency.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Fotossíntese/genética , Característica Quantitativa Herdável , Solanum/genética , Dióxido de Carbono/análise , Dióxido de Carbono/metabolismo , Quimera , Clorofila/metabolismo , Clorofila A , Mapeamento Cromossômico , Cromossomos de Plantas/química , Cruzamentos Genéticos , Fluorescência , Marcadores Genéticos , Genótipo , Imagem Óptica/métodos , Oxigênio/análise , Oxigênio/metabolismo , Consumo de Oxigênio/genética , Fenótipo , Melhoramento Vegetal , Folhas de Planta/genética , Folhas de Planta/metabolismo , Locos de Características Quantitativas , Solanum/metabolismoRESUMO
BACKGROUND AND AIMS: Leaf gas exchange is influenced by stomatal size, density, distribution between the leaf adaxial and abaxial sides, as well as by pore dimensions. This study aims to quantify which of these traits mainly underlie genetic differences in operating stomatal conductance (gs) and addresses possible links between anatomical traits and regulation of pore width. METHODS: Stomatal responsiveness to desiccation, gs-related anatomical traits of each leaf side and estimated gs (based on these traits) were determined for 54 introgression lines (ILs) generated by introgressing segments of Solanum pennelli into the S. lycopersicum 'M82'. A quantitative trait locus (QTL) analysis for stomatal traits was also performed. KEY RESULTS: A wide genetic variation in stomatal responsiveness to desiccation was observed, a large part of which was explained by stomatal length. Operating gs ranged over a factor of five between ILs. The pore area per stomatal area varied 8-fold among ILs (2-16 %), and was the main determinant of differences in operating gs between ILs. Operating gs was primarily positioned on the abaxial surface (60-83 %), due to higher abaxial stomatal density and, secondarily, to larger abaxial pore area. An analysis revealed 64 QTLs for stomatal traits in the ILs, most of which were in the direction of S. pennellii. CONCLUSIONS: The data indicate that operating and maximum gs of non-stressed leaves maintained under stable conditions deviate considerably (by 45-91 %), because stomatal size inadequately reflects operating pore area (R(2) = 0·46). Furthermore, it was found that variation between ILs in both stomatal sensitivity to desiccation and operating gs is associated with features of individual stoma. In contrast, genotypic variation in gs partitioning depends on the distribution of stomata between the leaf adaxial and abaxial epidermis.
Assuntos
Folhas de Planta/fisiologia , Estômatos de Plantas/fisiologia , Solanum/fisiologia , Dessecação , Variação Genética , Hibridização Genética , Solanum lycopersicum/anatomia & histologia , Solanum lycopersicum/genética , Solanum lycopersicum/fisiologia , Modelos Biológicos , Fenótipo , Folhas de Planta/anatomia & histologia , Estômatos de Plantas/anatomia & histologia , Solanum/anatomia & histologia , Solanum/genéticaRESUMO
Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering.
Assuntos
Glucose-1-Fosfato Adenililtransferase/metabolismo , Malatos/metabolismo , Mitocôndrias/metabolismo , Tubérculos/enzimologia , Plastídeos/metabolismo , Solanum tuberosum/enzimologia , Amido/biossíntese , Isótopos de Carbono , Respiração Celular , Fumaratos/metabolismo , Metabolômica , Oxirredução , Plantas Geneticamente Modificadas , Interferência de RNA , Solanum tuberosum/genética , Solanum tuberosum/fisiologiaRESUMO
Biochemical, molecular and genetic studies emphasize the role of the potato vacuolar invertase Pain-1 in the accumulation of reducing sugars in potato tubers upon cold storage, and thereby its influence on the quality of potato chips and French fries. Previous studies showed that natural Pain-1 cDNA alleles were associated with better chip quality and higher tuber starch content. In this study, we focused on the functional characterization of these alleles. A genotype-dependent transient increase of total Pain-1 transcript levels in cold-stored tubers of six different genotypes as well as allele-specific expression patterns were detected. 3D modelling revealed putative structural differences between allelic Pain-1 proteins at the molecule's surface and at the substrate binding site. Furthermore, the yeast SUC2 mutant was complemented with Pain-1 cDNA alleles and enzymatic parameters of the heterologous expressed proteins were measured at 30 and 4 °C. Significant differences between the alleles were detected. The observed functional differences between Pain-1 alleles did not permit final conclusions on the mechanism of their association with tuber quality traits. Our results show that natural allelic variation at the functional level is present in potato, and that the heterozygous genetic background influences the manifestation of this variation.
Assuntos
Alelos , Solanum tuberosum/enzimologia , beta-Frutofuranosidase/genética , Sequência de Bases , Primers do DNA , DNA Complementar , Genótipo , Modelos Moleculares , Reação em Cadeia da Polimerase , Conformação Proteica , RNA Mensageiro/genética , beta-Frutofuranosidase/químicaRESUMO
Trehalose-6-phosphate (T6P) is a signaling metabolite that regulates carbon metabolism, developmental processes, and growth in plants. In Arabidopsis (Arabidopsis thaliana), T6P signaling is, at least in part, mediated through inhibition of the SNF1-related protein kinase SnRK1. To investigate the role of T6P signaling in a heterotrophic, starch-accumulating storage organ, transgenic potato (Solanum tuberosum) plants with altered T6P levels specifically in their tubers were generated. Transgenic lines with elevated T6P levels (B33-TPS, expressing Escherichia coli osmoregulatory trehalose synthesis A [OtsA], which encodes a T6P synthase) displayed reduced starch content, decreased ATP contents, and increased respiration rate diagnostic for high metabolic activity. On the other hand, lines with significantly reduced T6P (B33-TPP, expressing E. coli OtsB, which encodes a T6P phosphatase) showed accumulation of soluble carbohydrates, hexose phosphates, and ATP, no change in starch when calculated on a fresh weight basis, and a strongly reduced tuber yield. [¹4C]glucose feeding to transgenic tubers indicated that carbon partitioning between starch and soluble carbohydrates was not altered. Transcriptional profiling of B33-TPP tubers revealed that target genes of SnRK1 were strongly up-regulated and that T6P inhibited potato tuber SnRK1 activity in vitro. Among the SnRK1 target genes in B33-TPP tubers, those involved in the promotion of cell proliferation and growth were down-regulated, while an inhibitor of cell cycle progression was up-regulated. T6P-accumulating tubers were strongly delayed in sprouting, while those with reduced T6P sprouted earlier than the wild type. Early sprouting of B33-TPP tubers correlated with a reduced abscisic acid content. Collectively, our data indicate that T6P plays an important role for potato tuber growth.
Assuntos
Germinação/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Tubérculos/crescimento & desenvolvimento , Tubérculos/metabolismo , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/metabolismo , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Ácido Abscísico/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Radioisótopos de Carbono , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Marcadores Genéticos , Germinação/genética , Glucose/farmacologia , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Fenótipo , Tubérculos/enzimologia , Tubérculos/genética , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Sacarose/farmacologia , Transcrição Gênica/efeitos dos fármacos , Trealose/metabolismoRESUMO
Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (HIPs) are expected to exist. Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Fosforilases/metabolismo , Polissacarídeos/metabolismo , Arabidopsis/citologia , Soluções Tampão , Cromatografia de Afinidade , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica de Plantas , Metabolômica , Monossacarídeos/metabolismo , Mutação/genética , Especificidade de Órgãos , Fenótipo , Extratos Vegetais/metabolismo , Folhas de Planta/metabolismo , Ligação Proteica , Transporte Proteico , Solanum tuberosum/metabolismo , Solubilidade , Especificidade da Espécie , Amido/metabolismoRESUMO
Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (F (v)/F (m)), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism.
Assuntos
Aminoácidos/análise , Isocitrato Desidrogenase/metabolismo , Fotossíntese , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Respiração Celular , Ciclo do Ácido Cítrico/fisiologia , Citosol/enzimologia , Citosol/metabolismo , DNA Complementar , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ácidos Cetoglutáricos/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , NADP/metabolismo , Nitrogênio/metabolismo , Fotossíntese/genética , Pigmentação , Pigmentos Biológicos/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Reação em Cadeia da Polimerase , RNA de Plantas/genéticaRESUMO
Plant metabolism is highly coordinated with development. However, an understanding of the whole picture of metabolism and its interactions with plant development is scarce. In this work, we show that the deficiency in the plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) leads to male sterility in Arabidopsis (Arabidopsis thaliana). Pollen from homozygous gapcp double mutant plants (gapcp1gapcp2) displayed shrunken and collapsed forms and were unable to germinate when cultured in vitro. The pollen alterations observed in gapcp1gapcp2 were attributed to a disorganized tapetum layer. Accordingly, the expression of several of the genes involved in tapetum development was down-regulated in gapcp1gapcp2. The fertility of gapcp1gapcp2 was rescued by transforming this mutant with a construct carrying the GAPCp1 cDNA under the control of its native promoter (pGAPCp1::GAPCp1c). However, the GAPCp1 or GAPCp2 cDNA under the control of the 35S promoter (p35S::GAPCp), which is poorly expressed in the tapetum, did not complement the mutant fertility. Mutant GAPCp isoforms deficient in the catalytic activity of the enzyme were unable to complement the sterile phenotype of gapcp1gapcp2, thus confirming that both the expression and catalytic activity of GAPCp in anthers are necessary for mature pollen development. A metabolomic study in flower buds indicated that the most important difference between the sterile (gapcp1gapcp2, gapcp1gapcp2-p35S::GAPCp) and the fertile (wild-type plants, gapcp1gapcp2-pGAPCp1::GAPCp1c) lines was the increase in the signaling molecule trehalose. This work corroborates the importance of plastidial glycolysis in plant metabolism and provides evidence for the crucial role of GAPCps in pollen development. It additionally brings new insights into the complex interactions between metabolism and development.
Assuntos
Arabidopsis/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Plastídeos/enzimologia , Pólen/metabolismo , Arabidopsis/genéticaRESUMO
Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+) ratio was unaffected, the stem NADPH/NADP(+) ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+) ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.
Assuntos
Proteínas Mitocondriais/metabolismo , NADPH Desidrogenase/metabolismo , Nicotiana/enzimologia , Oxirredução , Proteínas de Plantas/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/genética , NADPH Desidrogenase/genética , Proteínas de Plantas/genética , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Solanum tuberosum/genética , Nicotiana/genética , Nicotiana/crescimento & desenvolvimentoRESUMO
The 2-oxoglutarate dehydrogenase complex constitutes a mitochondrially localized tricarboxylic acid cycle multienzyme system responsible for the conversion of 2-oxoglutarate to succinyl-coenzyme A concomitant with NAD(+) reduction. Although regulatory mechanisms of plant enzyme complexes have been characterized in vitro, little is known concerning their role in plant metabolism in situ. This issue has recently been addressed at the cellular level in nonplant systems via the use of specific phosphonate inhibitors of the enzyme. Here, we describe the application of these inhibitors for the functional analysis of the potato (Solanum tuberosum) tuber 2-oxoglutarate dehydrogenase complex. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-oxoglutarate dehydrogenase complex, displaying greater inhibitory effects than a diethyl ester of SP, a phosphono ethyl ester of SP, or a triethyl ester of SP. Incubation of potato tuber slices with the inhibitors revealed that they were adequately taken up by the tissue and produced the anticipated effects on the in situ enzyme activity. In order to assess the metabolic consequences of the 2-oxoglutarate dehydrogenase complex inhibition, we evaluated the levels of a broad range of primary metabolites using an established gas chromatography-mass spectrometry method. We additionally analyzed the rate of respiration in both tuber discs and isolated mitochondria. Finally, we evaluated the metabolic fate of radiolabeled acetate, 2-oxoglutarate or glucose, and (13)C-labeled pyruvate and glutamate following incubation of tuber discs in the presence or absence of either SP or the carboxy ethyl ester of SP. The data obtained are discussed in the context of the roles of the 2-oxoglutarate dehydrogenase complex in respiration and carbon-nitrogen interactions.
Assuntos
Complexo Cetoglutarato Desidrogenase/fisiologia , Nitrogênio/metabolismo , Proteínas de Plantas/fisiologia , Solanum tuberosum/enzimologia , Dióxido de Carbono/metabolismo , Inibidores Enzimáticos/farmacologia , Ésteres/farmacologia , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Organofosfonatos/farmacologia , Oxigênio/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Tubérculos/efeitos dos fármacos , Tubérculos/enzimologia , Tubérculos/metabolismo , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/metabolismo , Succinatos/farmacologiaRESUMO
The cytosolic pools of glucose-1-phosphate (Glc-1-P) and glucose-6-phosphate are essential intermediates in several biosynthetic paths, including the formation of sucrose and cell wall constituents, and they are also linked to the cytosolic starch-related heteroglycans. In this work, structural features and biochemical properties of starch-related heteroglycans were analyzed as affected by the cytosolic glucose monophosphate metabolism using both source and sink organs from wild-type and various transgenic potato (Solanum tuberosum) plants. In leaves, increased levels of the cytosolic phosphoglucomutase (cPGM) did affect the cytosolic heteroglycans, as both the glucosyl content and the size distribution were diminished. By contrast, underexpression of cPGM resulted in an unchanged size distribution and an unaltered or even increased glucosyl content of the heteroglycans. Heteroglycans prepared from potato tubers were found to be similar to those from leaves but were not significantly affected by the level of cPGM activity. However, external glucose or Glc-1-P exerted entirely different effects on the cytosolic heteroglycans when added to tuber discs. Glucose was directed mainly toward starch and cell wall material, but incorporation into the constituents of the cytosolic heteroglycans was very low and roughly reflected the relative monomeric abundance. By contrast, Glc-1-P was selectively taken up by the tuber discs and resulted in a fast increase in the glucosyl content of the heteroglycans that quantitatively reflected the level of the cytosolic phosphorylase activity. Based on (14)C labeling experiments, we propose that in the cytosol, glucose and Glc-1-P are metabolized by largely separated paths.
Assuntos
Citosol/metabolismo , Glucofosfatos/metabolismo , Polissacarídeos/metabolismo , Solanum tuberosum/metabolismo , Amido/metabolismo , Configuração de Carboidratos , Plantas Geneticamente Modificadas/metabolismoRESUMO
In the present article we evaluate the consequence of tuber-specific expression of yeast invertase, on the pathways of carbohydrate oxidation, in potato (Solanum tuberosum L. cv. Desiree). We analysed the relative rates of glycolysis and the oxidative pentose phosphate pathway that these lines exhibited as well as the relative contributions of the cytochrome and alternative pathways of mitochondrial respiration. Enzymatic and protein abundance analysis revealed concerted upregulation of the glycolytic pathway and of specific enzymes of the tricarboxylic acid cycle and the alternative oxidase but invariant levels of enzymes of the oxidative pentose phosphate pathway and proteins of the cytochrome pathway. When taken together these experiments suggest that the overexpression of a cytosolic invertase (EC 3.2.1.26) results in a general upregulation of carbohydrate oxidation with increased flux through both the glycolytic and oxidative pentose phosphate pathways as well as the cytochrome and alternative pathways of oxidative phosphorylation. Moreover these data suggest that the upregulation of respiration is a consequence of enhanced efficient mitochondrial metabolism.