RESUMO
Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a13C-HCO3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.
Assuntos
Dióxido de Carbono , Malatos , Malatos/metabolismo , Dióxido de Carbono/metabolismo , Células do Mesofilo/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Citratos/metabolismoRESUMO
BACKGROUND: The development of biomass crops aims to meet industrial yield demands, in order to optimize profitability and sustainability. Achieving these goals in an energy crop like sugarcane relies on breeding for sucrose accumulation, fiber content and stalk number. To expand the understanding of the biological pathways related to these traits, we evaluated gene expression of two groups of genotypes contrasting in biomass composition. RESULTS: First visible dewlap leaves were collected from 12 genotypes, six per group, to perform RNA-Seq. We found a high number of differentially expressed genes, showing how hybridization in a complex polyploid system caused extensive modifications in genome functioning. We found evidence that differences in transposition and defense related genes may arise due to the complex nature of the polyploid Saccharum genomes. Genotypes within both biomass groups showed substantial variability in genes involved in photosynthesis. However, most genes coding for photosystem components or those coding for phosphoenolpyruvate carboxylases (PEPCs) were upregulated in the high biomass group. Sucrose synthase (SuSy) coding genes were upregulated in the low biomass group, showing that this enzyme class can be involved with sucrose synthesis in leaves, similarly to sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP). Genes in pathways related to biosynthesis of cell wall components and expansins coding genes showed low average expression levels and were mostly upregulated in the high biomass group. CONCLUSIONS: Together, these results show differences in carbohydrate synthesis and carbon partitioning in the source tissue of distinct phenotypic groups. Our data from sugarcane leaves revealed how hybridization in a complex polyploid system resulted in noticeably different transcriptomic profiles between contrasting genotypes.
Assuntos
Biomassa , Carbono/metabolismo , Genótipo , Saccharum/genética , Sacarose/metabolismo , Transcriptoma , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poliploidia , Saccharum/crescimento & desenvolvimento , Saccharum/metabolismo , Regulação para CimaRESUMO
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Assuntos
Glucose-6-Fosfato/química , Fosfoenolpiruvato Carboxilase/química , Fosfoenolpiruvato/química , Proteínas de Plantas/química , Zea mays/enzimologia , Regulação Alostérica , Domínio Catalítico , Glucose-6-Fosfato/metabolismo , Cinética , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genéticaRESUMO
Water deficit is a major environmental constraint on crop productivity and performance and nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. This study aims to test the hypothesis that leaf spraying of S-nitrosoglutathione (GSNO), an NO donor, improves the antioxidant defense in both roots and leaves of sugarcane plants under water deficit, with positive consequences for photosynthesis. In addition, the roles of key photosynthetic enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) in maintaining CO2 assimilation of GSNO-sprayed plants under water deficit were evaluated. Sugarcane plants were sprayed with water or GSNO 100 µM and subjected to water deficit, by adding polyethylene glycol (PEG-8000) to the nutrient solution. Sugarcane plants supplied with GSNO presented increases in the activity of antioxidant enzymes such as superoxide dismutase in leaves and catalase in roots, indicating higher antioxidant capacity under water deficit. Such adjustments induced by GSNO were sufficient to prevent oxidative damage in both organs and were associated with better leaf water status. As a consequence, GSNO spraying alleviated the negative impact of water deficit on stomatal conductance and photosynthetic rates, with plants also showing increases in Rubisco activity under water deficit.
Assuntos
Doadores de Óxido Nítrico/farmacologia , Fosfoenolpiruvato Carboxilase/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/efeitos dos fármacos , S-Nitrosoglutationa/farmacologia , Saccharum/efeitos dos fármacos , Antioxidantes/metabolismo , Catalase/metabolismo , Desidratação , Oxirredução , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/fisiologia , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/enzimologia , Estômatos de Plantas/fisiologia , Transpiração Vegetal/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/metabolismo , Saccharum/enzimologia , Saccharum/fisiologia , Superóxido Dismutase/metabolismo , Água/fisiologiaRESUMO
The morphology and photosynthetic enzyme activity were studied in maize phosphoenolpyruvate carboxylase transgenic rice and non-transgenic rice. The results showed that compared with non-transgenic rice, phosphoenolpyruvate carboxylase transgenic rice was taller and had a stronger stalk, wider leaves, and more exuberant root system, with increased photosynthetic enzyme activity and improved yield components. Therefore, given the superiority of this plant type and heterosis, this is a novel breeding strategy for rice for the introduction of C4 photosynthesis genes into high-yielding rice.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oryza/fisiologia , Fenótipo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente ModificadasRESUMO
The objective of this study was to investigate the mRNA expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) after gastric bypass surgery (GBS) in rats with type 2 diabetic mellitus (T2DM). Thirty-six male Goto-Kakizaki rats, aged 12 weeks, were randomly divided into the GBS, sham operation with diet restriction (SO), and sham operation alone (control) groups (N = 12 per group). Liver specimens from all rats were obtained during the operation and 8 weeks after operation. Blood lipid levels were measured before and 8 weeks after operation. Fasting blood glucose (FBG), food intake, and body weight were recorded at weekly time points after operation. The blood glucose area under the curve (AUC) was calculated, and insulin sensitivity indices (ISI) were assessed. The expression PEPCK mRNA and protein were measured by real-time polymerase chain reaction and western blot. Compared with those of the SO and control groups, the blood lipid levels and the FBG in the GBS group was significantly decreased (P < 0.05), as was the AUC (P < 0.05), whereas the ISI was significantly increased (P < 0.05). PEPCK mRNA and protein levels in the GBS group were lower than those in the control group, whereas those in the SO group were significantly higher than those in controls (P < 0.05). In conclusion, GBS can reduce blood glucose in T2DM rats while improving glucose tolerance and hyperglycemia, and the mechanism appears to be associated with a decrease of hepatic PEPCK mRNA and protein expression.
Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Derivação Gástrica , Expressão Gênica , Fígado/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Animais , Área Sob a Curva , Biomarcadores , Glicemia , Peso Corporal , Diabetes Mellitus Experimental , Modelos Animais de Doenças , Derivação Gástrica/métodos , Insulina/metabolismo , Resistência à Insulina , Lipídeos/sangue , Masculino , Fosfoenolpiruvato Carboxilase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
Transcriptomic and proteomic studies have improved our knowledge of guard cell function; however, metabolic changes in guard cells remain relatively poorly understood. Here we analysed metabolic changes in guard cell-enriched epidermal fragments from tobacco during light-induced stomatal opening. Increases in sucrose, glucose and fructose were observed during light-induced stomatal opening in the presence of sucrose in the medium while no changes in starch were observed, suggesting that the elevated fructose and glucose levels were a consequence of sucrose rather than starch breakdown. Conversely, reduction in sucrose was observed during light- plus potassium-induced stomatal opening. Concomitant with the decrease in sucrose, we observed an increase in the level as well as in the (13) C enrichment in metabolites of, or associated with, the tricarboxylic acid cycle following incubation of the guard cell-enriched preparations in (13) C-labelled bicarbonate. Collectively, the results obtained support the hypothesis that sucrose is catabolized within guard cells in order to provide carbon skeletons for organic acid production. Furthermore, they provide a qualitative demonstration that CO2 fixation occurs both via ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPcase). The combined data are discussed with respect to current models of guard cell metabolism and function.
Assuntos
Dióxido de Carbono/metabolismo , Nicotiana/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Estômatos de Plantas/fisiologia , Ribulose-Bifosfato Carboxilase/metabolismo , Sacarose/metabolismo , Cinética , Células Vegetais/metabolismo , Células Vegetais/fisiologia , Estômatos de Plantas/efeitos da radiação , Nicotiana/citologiaRESUMO
Photosynthetic modulation by sugars has been known for many years, but the biochemical and molecular comprehension of this process is lacking. We studied how the exogenous sucrose supplied to leaves could affect sugar metabolism in leaf, sheath and stalk and inhibit photosynthesis in four-month old sugarcane plants. Exogenous sucrose 50mM sprayed on attached leaves strongly impaired the net CO2 assimilation (PN) and decreased the instantaneous carboxylation efficiency (PN/Ci), suggesting that the impairment in photosynthesis was caused by biochemical restrictions. The photosystem II activity was also affected by excess sucrose as indicated by the reduction in the apparent electron transport rate, effective quantum yield and increase in non-photochemical quenching. In leaf segments, sucrose accumulation was related to increases in the activities of soluble acid and neutral invertases, sucrose synthase and sucrose phosphate synthase, whereas the contents of fructose increased and glucose slightly decreased. Changes in the activities of sucrose hydrolyzing and synthesizing enzymes in leaf, sheath and stalk and sugar profile in intact plants were not enough to identify which sugar(s) or enzyme(s) were directly involved in photosynthesis modulation. However, exogenous sucrose was able to trigger down-regulation in the Rubisco abundance, activation state and enzymatic activity. Despite the fact that PN/Ci had been notably decreased by sucrose, in vitro activity and abundance of PEPCase did not change, suggesting an in vivo modulation of this enzyme. The data reveal that sucrose and/or other derivative sugars in leaves inhibited sugarcane photosynthesis by down-regulation of Rubisco synthesis and activity. Our data also suggest that sugar modulation was not exerted by a feedback mechanism induced by the accumulation of sugars in immature sugarcane stalk.
Assuntos
Metabolismo dos Carboidratos/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/metabolismo , Saccharum/fisiologia , Sacarose/farmacologia , Western Blotting , Dióxido de Carbono/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/enzimologia , Estômatos de Plantas/fisiologia , Saccharum/efeitos dos fármacos , Sacarose/metabolismo , Fatores de TempoRESUMO
Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans.
Assuntos
Gluconeogênese , Glicerol Quinase/metabolismo , Leishmania mexicana/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Protozoários/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Southern Blotting , Western Blotting , DNA de Protozoário/genética , Glucose/metabolismo , Glucose/farmacologia , Glicerol Quinase/genética , Humanos , Leishmania mexicana/genética , Leishmania mexicana/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Mutação , Fosfoenolpiruvato Carboxilase/genética , Proteínas de Protozoários/genética , Piruvato Ortofosfato Diquinase/genéticaRESUMO
Phosphoenolpyruvate carboxykinase 1 (PCK1), also named PEPCK-C, is a multiple-function gene that is involved in gluconeogenesis, glyceroneogenesis, reproduction, female fertility, and development of obesity and diabetes. How its many functions are regulated was largely unknown. Therefore, we investigated mRNA expression and possible splice variants of PCK1 by screening cDNA in nine tissues from Holstein bulls and cows. PCK1 mRNA was highly expressed in the liver, kidney, ovary and testis; expression levels were low in the heart, spleen, and lung tissues. Expression of this gene was not detected in skeletal muscle. This led to the discovery of five novel bovine splice variants, named PCK1-AS1-PCK1-AS5. In PCK1-AS1, 51 nucleotides in the interior of exon 2 were spliced out. In PCK1-AS2, exons 2 and 3 were altered by the alternative 3' and 5' splice sites, respectively. PCK1-AS3 was truncated from the 3' end of exon 2 to the 5' end of exon 4. In PCK1-AS4, exon 5 was completely spliced out. In PCK1-AS5, exons 5 and 6 and the 5' end of exon 7 were spliced out. These splice variants (PCK1-AS1-PCK1-AS5) potentially encoded shorter proteins (605, 546, 373, 246 and 274 amino acids, respectively), when compared to the complete protein (622 amino acids). Considering the functional domains of the PCK1 protein, it is likely that these splice variants considerably affect the function of this protein; alternative splicing could be one of the mechanisms by which the diverse functions of PCK1 are regulated.
Assuntos
Bovinos/genética , Fosfoenolpiruvato Carboxilase/genética , Sequência de Aminoácidos , Animais , China , Clonagem Molecular , DNA Complementar , Éxons , Feminino , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Conformação Proteica , RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Crassulacean acid metabolism (CAM) is a physiological adaptation of plants that live in stress environment conditions. A good model of CAM modulation is the epiphytic bromeliad, Guzmania monostachia, which switches between two photosynthetic pathways (C3-CAM) in response to different environmental conditions, such as light stress and water availability. Along the leaf length a gradient of acidity can be observed when G. monostachia plants are kept under water deficiency. Previous studies showed that the apical portions of the leaves present higher expression of CAM, while the basal regions exhibit lower expression of this photosynthetic pathway. The present study has demonstrated that it is possible to induce the CAM pathway in detached leaves of G. monostachia kept under water deficit for 7 d. Also, it was evaluated whether CAM expression can be modulated in detached leaves of Guzmania and whether some spatial separation between NO3(-) reduction and CO2 fixation occurs in basal and apical portions of the leaf. In addition, we analyzed the involvement of endogenous cytokinins (free and ribosylated forms) as possible signal modulating both NO3(-) reduction and CO2 fixation along the leaf blade of this bromeliad. Besides demonstrating a clear spatial and functional separation of carbon and nitrogen metabolism along G. monostachia leaves, the results obtained also indicated a probable negative correlation between endogenous free cytokinins - zeatin (Z) and isopentenyladenine (iP) - concentration and PEPC activity in the apical portions of G. monostachia leaves kept under water deficit. On the other hand, a possible positive correlation between endogenous Z and iP levels and NR activity in basal portions of drought-exposed and control leaves was verified. Together with the observations presented above, results obtained with exogenous cytokinins treatments, strongly suggest that free cytokinins might act as a stimulatory signal involved in NR activity regulation and as a negative regulator of PEPC activity in CAM-induced leaves of G. monostachia during a diel cycle.
Assuntos
Bromeliaceae/enzimologia , Bromeliaceae/metabolismo , Citocininas/metabolismo , Nitrato Redutase/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Bromeliaceae/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Citocininas/farmacologia , Folhas de Planta/efeitos dos fármacos , Zeatina/metabolismoRESUMO
BACKGROUND AND AIMS: A positive correlation between tissue thickness and crassulacean acid metabolism (CAM) expression has been frequently suggested. Therefore, this study addressed the question of whether water availability modulates photosynthetic plasticity in different organs of two epiphytic orchids with distinct leaf thickness. METHODS: Tissue morphology and photosynthetic mode (C3 and/or CAM) were examined in leaves, pseudobulbs and roots of a thick-leaved (Cattleya walkeriana) and a thin-leaved (Oncidium 'Aloha') epiphytic orchid. Morphological features were studied comparing the drought-induced physiological responses observed in each organ after 30 d of either drought or well-watered treatments. KEY RESULTS: Cattleya walkeriana, which is considered a constitutive CAM orchid, displayed a clear drought-induced up-regulation of CAM in its thick leaves but not in its non-leaf organs (pseudobulbs and roots). The set of morphological traits of Cattleya leaves suggested the drought-inducible CAM up-regulation as a possible mechanism of increasing water-use efficiency and carbon economy. Conversely, although belonging to an orchid genus classically considered as performing C3 photosynthesis, Oncidium 'Aloha' under drought seemed to express facultative CAM in its roots and pseudobulbs but not in its leaves, indicating that such photosynthetic responses might compensate for the lack of capacity to perform CAM in its thin leaves. Morphological features of Oncidium leaves also indicated lower efficiency in preventing water and CO2 losses, while aerenchyma ducts connecting pseudobulbs and leaves suggested a compartmentalized mechanism of nighttime carboxylation via phosphoenolpyruvate carboxylase (PEPC) (pseudobulbs) and daytime carboxylation via Rubisco (leaves) in drought-exposed Oncidium plants. CONCLUSIONS: Water availability modulated CAM expression in an organ-compartmented manner in both orchids studied. As distinct regions of the same orchid could perform different photosynthetic pathways and variable degrees of CAM expression depending on the water availability, more attention should be addressed to this in future studies concerning the abundance of CAM plants.
Assuntos
Orchidaceae/anatomia & histologia , Orchidaceae/metabolismo , Fotossíntese/fisiologia , Folhas de Planta/anatomia & histologia , Carbono/metabolismo , Secas , Malato Desidrogenase/metabolismo , Orchidaceae/fisiologia , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Água/metabolismoRESUMO
Guzmania monostachia is an epiphyte tank bromeliad capable of up-regulating crassulacean acid metabolism (CAM) in response to several environmental stimuli, including drought and light stress. In other plant species, abscisic acid (ABA) and nitric oxide (NO) seem to be involved in CAM induction. Because the leaves of tank bromeliads perform different functions along their length, this study attempted to investigate whether ABA and NO are involved in regulation of CAM expression in this species by quantifying these compounds in apical and basal portions of the leaf, and whether there would be differences in this event for each leaf portion. Detached leaves exposed to a 30% polyethylene glycol solution showed a significant upregulation of CAM on the seventh day of treatment only in the apical portion, as indicated by nocturnal acid accumulation and phosphoenolpyruvate carboxylase (PEPC) activity. On the three days prior to CAM induction, ABA, NO and H2O2 were quantified. The amounts of ABA were higher in PEG-exposed leaves, along their entire length. NO, however, was higher only in the apical portion, precisely where CAM was up-regulated. H2O2 was higher only in the basal portion of PEG-exposed leaves. Our results suggest that ABA might be a systemic signal to drought, occurring in the entire leaf. NO and H2O2, however, may be signals restricted only to the apical or basal portions, respectively.
Assuntos
Ácido Abscísico/metabolismo , Bromeliaceae/metabolismo , Secas , Óxido Nítrico/metabolismo , Folhas de Planta/metabolismo , Bromeliaceae/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: In Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS-) and pyruvate kinase isozymes PykA and PykF (PTS-pykA- and PTS-pykF-). To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined. RESULTS: When compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc) and PEP carboxykinase (Pck) was detected in all strains. In strains W3110, VH33 (PTS-) and VH35 (PTS-, pykF-), the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation. CONCLUSIONS: Metabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high degree of flexibility to perturbations of the central metabolic network in E. coli. The observed responses to reduced glucose uptake and PEP to pyruvate rate of conversion caused by PTS, PykA and PykF inactivation included flux rerouting in several central metabolism nodes towards anabolic biosynthetic reactions, thus compensating for carbon limitation in these mutant strains. The detected cycle involving Ppc and Pck was found to be required for maintaining the specific growth and glucose consumption rates in all studied strains. Strains VH33 (PTS-), VH34 (PTS-pykA-) and VH35 (PTS-pykF-) have useful properties for biotechnological processes, such as increased PEP availability and high biomass yields from glucose, making them useful for the production of aromatic compounds or recombinant proteins.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Piruvato Quinase/metabolismo , Aminoácidos Aromáticos/metabolismo , Biomassa , Ciclo do Carbono , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Isoenzimas/metabolismo , Mutação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismoRESUMO
In bacteria, anaplerotic carbon fixation necessary for growth on carbon sources that are metabolized to three-carbon intermediates is provided by the activity of pyruvate carboxylase (PYC) and/or phosphoenolpyruvate carboxylase (PPC). In contrast to other rhizobia, which encode only one of these enzymes in their genomes, Bradyrhizobium japonicum USDA110 encodes both. Streptavidin-HRP western blot analysis of B. japonicum extracts demonstrated the presence of a biotin-containing protein whose molecular mass was indistinguishable from those of PYCs produced by Sinorhizobium meliloti and Rhizobium etli. Sequence analysis of the possible B. japonicum PYC revealed the lack of a pyruvate binding site as well as other characteristics indicating that the enzyme is non-functional, and PPC activity, but not PYC activity, was detectible in extracts prepared from strain USDA110. A B. japonicum cosmid genomic library was used to clone the ppc by functional complementation of S. meliloti pyc mutant RmF991. S. meliloti RmF991-carrying plasmids containing the B. japonicum ppc regained the ability to grow with glucose as a carbon source and produced PPC activity. The cloned ppc gene was inactivated by insertion mutagenesis and recombined into the USDA110 genome. The resulting ppc mutant was essentially devoid of PPC activity and grew poorly with glucose as carbon source in comparison to the wild-type strain. These data indicate that B. japonicum utilizes PPC, and not PYC, as an anaplerotic enzyme for growth on carbon sources metabolized to three-carbon intermediates.
Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/enzimologia , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Carbono/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismoRESUMO
This study investigated whether Euphorbia subgenus Chamaesyce subsection Acutae contains C(3)-C(4) intermediate species utilizing C(2) photosynthesis, the process where photorespired CO(2) is concentrated into bundle sheath cells. Euphorbia species in subgenus Chamaesyce are generally C(4), but three species in subsection Acutae (E. acuta, E. angusta, and E. johnstonii) have C(3) isotopic ratios. Phylogenetically, subsection Acutae branches between basal C(3) clades within Euphorbia and the C(4) clade in subgenus Chamaesyce. Euphorbia angusta is C(3), as indicated by a photosynthetic CO(2) compensation point (Ð) of 69 µmol mol(-1) at 30 °C, a lack of Kranz anatomy, and the occurrence of glycine decarboxylase in mesophyll tissues. Euphorbia acuta utilizes C(2) photosynthesis, as indicated by a Ð of 33 µmol mol(-1) at 30 °C, Kranz-like anatomy with mitochondria restricted to the centripetal (inner) wall of the bundle sheath cells, and localization of glycine decarboxlyase to bundle sheath mitochondria. Low activities of PEP carboxylase, NADP malic enzyme, and NAD malic enzyme demonstrated no C(4) cycle activity occurs in E. acuta thereby classifying it as a Type I C(3)-C(4) intermediate. Kranz-like anatomy in E. johnstonii indicates it also utilizes C(2) photosynthesis. Given the phylogenetically intermediate position of E. acuta and E. johnstonii, these results support the hypothesis that C(2) photosynthesis is an evolutionary intermediate condition between C(3) and C(4) photosynthesis.
Assuntos
Euphorbia/fisiologia , Fotossíntese/fisiologia , Evolução Biológica , Dióxido de Carbono/análise , Dióxido de Carbono/metabolismo , Isótopos de Carbono/análise , Região do Caribe , Respiração Celular/fisiologia , Cloroplastos/ultraestrutura , Euphorbia/enzimologia , Euphorbia/ultraestrutura , Malato Desidrogenase/metabolismo , México , Mitocôndrias/ultraestrutura , Fosfoenolpiruvato Carboxilase/metabolismo , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Folhas de Planta/ultraestrutura , Transpiração Vegetal/fisiologia , Ribulose-Bifosfato Carboxilase/metabolismo , Temperatura , TexasAssuntos
Evolução Molecular , Fotossíntese , Plantas/metabolismo , Dióxido de Carbono/metabolismo , Cloroplastos/metabolismo , Descarboxilação , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Malatos/metabolismo , Mutação , Fenótipo , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/genética , Folhas de Planta/metabolismo , Estômatos de Plantas/fisiologia , Plantas/genética , Elementos Reguladores de Transcrição , Ribulose-Bifosfato Carboxilase/metabolismo , Estresse FisiológicoRESUMO
We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.
Assuntos
Ozônio/farmacologia , Fosfoenolpiruvato Carboxilase/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Zea mays/efeitos dos fármacos , Zea mays/enzimologia , Fosfoenolpiruvato Carboxilase/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA de Plantas/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribulose-Bifosfato Carboxilase/efeitos dos fármacos , Zea mays/genéticaRESUMO
We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.
Assuntos
Ozônio/farmacologia , Fosfoenolpiruvato Carboxilase/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Zea mays/efeitos dos fármacos , Zea mays/enzimologia , Fosfoenolpiruvato Carboxilase/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/efeitos dos fármacos , RNA de Plantas/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/efeitos dos fármacos , Zea mays/genéticaRESUMO
Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C(4) photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C(4) photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C(4) photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO(2) via PEPC from C(4) acid decarboxylation and CO(2) donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C(4) species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C(3) (Suaeda linifolia) chenopods. It was found that PEPC from both types of C(4) chenopods displays higher specific activity than that of the C(3) species and shows kinetic and regulatory characteristics similar to those of C(4) species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C(4) species possesses a Kranz-type C(4) isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C(4) species are in a clade with the C(3) and Kranz C(4) Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C(4)-type PEPC similar to that in Kranz C(4) plants, which likely is required for effective function of C(4) photosynthesis.