RESUMO
Three plant-type phosphoenolpyruvate carboxylase (PPC1 to PPC3) and two phosphoenolpyruvate carboxylase kinase (PPCKs: PPCK1 and 2) genes are present in the Arabidopsis thaliana genome. In seeds, all PPC genes were found to be expressed. Examination of individual ppc mutants showed little reduction of PEPC protein and global activity, with the notable exception of PPC2 which represent the most abundant PEPC in dry seeds. Ppc mutants exhibited moderately lower seed parameters (weight, area, yield, germination kinetics) than wild type. In contrast, ppck1-had much altered (decreased) yield. At the molecular level, ppc3-was found to be significantly deficient in global seed nitrogen (nitrate, amino-acids, and soluble protein pools). Also, N-deficiency was much more marked in ppck1-, which exhibited a tremendous loss of 95% and 90% in nitrate and proteins, respectively. The line ppck2-had accumulated amino-acids but lower levels of soluble proteins. Regarding carboxylic acid pools, Krebs cycle intermediates were found to be diminished in all mutants; this was accompanied by a consistent decrease in ATP. Lipids were stable in ppc mutants, however ppck1-seeds accumulated more lipids while ppck2-seeds showed high level of polyunsaturated fatty acid oleic and linolenic (omega 3). Altogether, the results indicate that the complete PEPC and PPCK family are needed for normal C/N metabolism ratio, growth, development, yield and quality of the seed.
Assuntos
Arabidopsis , Fosfoenolpiruvato Carboxilase , Trifosfato de Adenosina , Ácidos Carboxílicos , Isoenzimas/genética , Isoenzimas/metabolismo , Lipídeos , Nitratos , Nitrogênio/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas Serina-Treonina Quinases , SementesRESUMO
BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) plays an important role in the primary metabolism of higher plants. Several studies have revealed the critical importance of PEPC in the interaction of carbon and nitrogen metabolism. However, the function mechanism of PEPC in nitrogen metabolism is unclear and needs further investigation. RESULTS: This study indicates that transgenic rice expressing the sugarcane C4-PEPC gene displayed shorter primary roots and fewer crown roots at the seedling stage. However, total nitrogen content was significantly higher in transgenic rice than in wild type (WT) plants. Proteomic analysis revealed that there were more differentially expressed proteins (DEPs) responding to nitrogen changes in transgenic rice. In particular, the most enriched pathway "glutathione (GSH) metabolism", which mainly contains GSH S-transferase (GST), was identified in transgenic rice. The expression of endogenous PEPC, GST and several genes involved in the TCA cycle, glycolysis and nitrogen assimilation changed in transgenic rice. Correspondingly, the activity of enzymes including GST, citrate synthase, 6-phosphofructokinase, pyruvate kinase and ferredoxin-dependent glutamate synthase significantly changed. In addition, the levels of organic acids in the TCA cycle and carbohydrates including sucrose, starch and soluble sugar altered in transgenic rice under different nitrogen source concentrations. GSH that the substrate of GST and its components including glutamic acid, cysteine and glycine accumulated in transgenic rice. Moreover, the levels of phytohormones including indoleacetic acid (IAA), zeatin (ZT) and isopentenyladenosine (2ip) were lower in the roots of transgenic rice under total nutrients. Taken together, the phenotype, physiological and biochemical characteristics of transgenic rice expressing C4-PEPC were different from WT under different nitrogen levels. CONCLUSIONS: Our results revealed the possibility that PEPC affects nitrogen metabolism through regulating GST, which provide a new direction and concepts for the further study of the PEPC functional mechanism in nitrogen metabolism.
Assuntos
Glutationa Transferase/metabolismo , Nitrogênio/metabolismo , Oryza/enzimologia , Fosfoenolpiruvato Carboxilase/metabolismo , Saccharum/enzimologia , Carbono/metabolismo , Oryza/genética , Oryza/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Plantas Geneticamente Modificadas , Proteômica , Saccharum/genética , TranscriptomaRESUMO
It has been challenging to simultaneously improve photosynthesis and stress tolerance in plants. Crassulacean acid metabolism (CAM) is a CO2-concentrating mechanism that facilitates plant adaptation to water-limited environments. We hypothesized that the ectopic expression of a CAM-specific phosphoenolpyruvate carboxylase (PEPC), an enzyme that catalyzes primary CO2 fixation in CAM plants, would enhance both photosynthesis and abiotic stress tolerance. To test this hypothesis, we engineered a CAM-specific PEPC gene (named AaPEPC1) from Agave americana into tobacco. In comparison with wild-type and empty vector controls, transgenic tobacco plants constitutively expressing AaPEPC1 showed a higher photosynthetic rate and biomass production under normal conditions, along with significant carbon metabolism changes in malate accumulation, the carbon isotope ratio δ13C, and the expression of multiple orthologs of CAM-related genes. Furthermore, AaPEPC1 overexpression enhanced proline biosynthesis, and improved salt and drought tolerance in the transgenic plants. Under salt and drought stress conditions, the dry weight of transgenic tobacco plants overexpressing AaPEPC1 was increased by up to 81.8% and 37.2%, respectively, in comparison with wild-type plants. Our findings open a new door to the simultaneous improvement of photosynthesis and stress tolerance in plants.
Assuntos
Adaptação Fisiológica/genética , Agave/genética , Metabolismo Ácido das Crassuláceas/genética , Nicotiana/genética , Fosfoenolpiruvato Carboxilase/genética , Proteínas de Plantas/genética , Agave/metabolismo , Dióxido de Carbono/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Malatos/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Prolina/biossíntese , Salinidade , Estresse Fisiológico , Nicotiana/metabolismo , TransgenesRESUMO
Hepatic gluconeogenesis is the central pathway for glucose generation in the body. The imbalance between glucose synthesis and uptake leads to metabolic diseases such as obesity, diabetes, and cardiovascular diseases. Small leucine zipper protein (sLZIP) is an isoform of LZIP and it mainly functions as a transcription factor. Although sLZIP is known to regulate the transcription of genes involved in various cellular processes, the role of sLZIP in hepatic glucose metabolism is not known. In this study, we investigated the regulatory role of sLZIP in hepatic gluconeogenesis and its involvement in metabolic disorder. We found that sLZIP expression was elevated during glucose starvation, leading to the promotion of phosphoenolpyruvate carboxylase and glucose-6-phosphatase expression in hepatocytes. However, sLZIP knockdown suppressed the expression of the gluconeogenic enzymes under low glucose conditions. sLZIP also enhanced glucose production in the human liver cells and mouse primary hepatic cells. Fasting-induced cyclic adenosine monophosphate impeded sLZIP degradation. Results of glucose and pyruvate tolerance tests showed that sLZIP transgenic mice exhibited abnormal blood glucose metabolism. These findings suggest that sLZIP is a novel regulator of gluconeogenic enzyme expression and plays a role in blood glucose homeostasis during starvation.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Gluconeogênese/genética , Hepatócitos/patologia , Zíper de Leucina/genética , Fígado/patologia , Doenças Metabólicas/genética , Animais , AMP Cíclico/genética , Regulação da Expressão Gênica/genética , Glucose/genética , Glucose-6-Fosfatase/genética , Células Hep G2 , Homeostase/genética , Humanos , Masculino , Doenças Metabólicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfoenolpiruvato Carboxilase/genética , Transcrição Gênica/genéticaRESUMO
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
Cottonseed oil is one of the most important renewable resources for edible oil and biodiesel. To detect QTLs associated with cottonseed oil content (OC) and identify candidate genes that regulate oil biosynthesis, a panel of upland cotton germplasm lines was selected among those previously used to perform GWASs in China. In the present study, 13 QTLs associated with 53 common SNPs on 13 chromosomes were identified in multiple environments based on 15,369 polymorphic SNPs using the Cotton63 KSNP array. Of these, the OC QTL qOC-Dt5-1 delineated by nine SNPs occurred in a confidence interval of 4 SSRs with previously reported OC QTLs. A combined transcriptome and qRT-PCR analysis revealed that a peroxidase gene (GhPRXR1) was predominantly expressed during the middle-late stage (20-35 days post anthesis) of ovule development. The overexpression of GhPRXR1 in yeast significantly increased the OC by 20.01-37.25 %. Suppression of GhPRXR1 gene expression in the virus-induced gene-silenced cotton reduced the OC by 18.11%. Our results contribute to identifying more OC QTLs and verifying a candidate gene that influences cottonseed oil biosynthesis.
Assuntos
Estudo de Associação Genômica Ampla , Gossypium/genética , Fosfoenolpiruvato Carboxilase/genética , Óleos de Plantas/química , Proteínas de Plantas/genética , China , Gossypium/química , Gossypium/enzimologia , Gossypium/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/metabolismo , Locos de Características QuantitativasRESUMO
BACKGROUND: Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements. RESULTS: We found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export. CONCLUSIONS: Since more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Escherichia coli/metabolismo , Malatos/metabolismo , Engenharia Metabólica/métodos , Trifosfato de Adenosina/metabolismo , Ciclo do Ácido Cítrico/genética , Escherichia coli/genética , NAD/metabolismo , NADP/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismoRESUMO
Under fasting conditions, activation of several hepatic genes sets the stage for gluconeogenesis in the liver. cAMP response element-binding protein (CREB), CREB-regulated transcription coactivator 2 (CRTC2), and peroxisome proliferator-activated receptor γ coactivator 1-alpha (PGC-1α) are essential for this transcriptional induction of gluconeogenic genes. PGC-1α induction is mediated by activation of a CREB/CRTC2 signaling complex, and recent findings have revealed that small heterodimer partner-interacting leucine zipper protein (SMILE), a member of the CREB/ATF family of basic region-leucine zipper (bZIP) transcription factors, is an insulin-inducible corepressor that decreases PGC-1α expression and abrogates its stimulatory effect on hepatic gluconeogenesis. However, the molecular mechanism whereby SMILE suppresses PGC-1α expression is unknown. Here, we investigated SMILE's effects on the CREB/CRTC2 signaling pathway and glucose metabolism. We found that SMILE significantly inhibits CREB/CRTC2-induced PGC-1α expression by interacting with and disrupting the CREB/CRTC2 complex. Consequently, SMILE decreased PGC-1α-induced hepatic gluconeogenic gene expression. Furthermore, SMILE inhibited CREB/CRTC2-induced phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) gene expression by directly repressing the expression of these genes and by indirectly inhibiting the expression of PGC-1α via CREB/CRTC2 repression. Indeed, enhanced gluconeogenesis and circulating blood glucose levels in mice injected with an adenovirus construct containing a constitutively active CRTC2 variant (CRTC2-S171A) were significantly reduced by WT SMILE, but not by leucine zipper-mutated SMILE. These results reveal that SMILE represses CREB/CRTC2-induced PGC-1α expression, an insight that may help inform potential therapeutic approaches targeting PGC-1α-mediated regulation of hepatic glucose metabolism.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Gluconeogênese , Glucose-6-Fosfatase/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica , Glucose-6-Fosfatase/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fosfoenolpiruvato Carboxilase/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
KEY MESSAGE: Hexaploid wheat is more responsive than tetraploid to the interactive effects of elevated [CO2] and low P in terms of carboxylate efflux, enzyme activity and gene expression (TaPT1 and TaPAP). Availability of mineral nutrients to plants under changing climate has become a serious challenge to food security and economic development. An understanding of how elevated [CO2] influences phosphorus (P) acquisition processes at the whole-plant level would be critical in selecting cultivars as well as to maintain optimum yield in limited-P conditions. Wheat (Triticum aestivum and T. durum) grown hydroponically with sufficient and low P concentration were exposed to elevated and ambient [CO2]. Improved dry matter partitioning towards root resulted in increased root-to-shoot ratio, root length, volume, surface area, root hair length and density at elevated [CO2] with low P. Interaction of low P and [CO2] induced activity of enzymes (phosphoenolpyruvate carboxylase, malate dehydrogenase and citrate synthase) in root tissue resulting in twofold increase in carboxylates and acid phosphatase exudation. Physiological absorption capacity of roots showed that plants alter their uptake kinetics by increasing affinity (low Km) in response to elevated [CO2] under low P supply. Increased relative expression of genes, purple acid phosphatase (TaPAP) and high-affinity Pi transporter (TaPT1) in roots induced by elevated [CO2] and low P supported our physiological observations. Hexaploid wheat (PBW-396) being more responsive to elevated [CO2] at low P supply as compared to tetraploid (PDW-233) necessitates the ploidy effect to be explored further which might be advantageous under changing climate.
Assuntos
Dióxido de Carbono/metabolismo , Fósforo/metabolismo , Tetraploidia , Triticum/genética , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/genética , Fotossíntese/fisiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Triticum/metabolismoRESUMO
We investigated the effect of the heterologous expression of phosphoenolpyruvate carboxylase (ZmPepcase), aspartate aminotransferase (GmAspAT), and glutamine synthetase (NtGS) on carbon (C) and nitrogen (N) metabolism in Arabidopsis (Arabidopsis thaliana). These transgenes were expressed either separately or in different combinations. The highest gains in shoot dry weight were observed in transgenic lines coexpressing all three genes. Tracer experiments using NaH14CO3 suggested that the coexpression of ZmPepcase, GmAspAT, and NtGS resulted in a higher flux of assimilated CO2 toward sugars and amino acids. Upon feeding the leaf discs with glycine-1-14C, transgenic lines evolved significantly lower 14CO2 levels than the wild type, suggesting that a higher reassimilation of CO2 evolved during photorespiration. Leaves of transgenic plants accumulated significantly lower ammonium without any significant difference in the levels of photorespiratory ammonium relative to the wild type, suggesting a higher reassimilation of photorespired NH3 Transgenic lines also showed improved photosynthetic rates, higher shoot biomass accumulation, and improved seed yield in comparison with wild-type plants under both optimum and limiting N conditions. This work demonstrates that the heterologous coexpression of ZmPepcase, GmAspAT, and NtGS reduced the photorespiratory loss of C and N with concomitant enhancements in shoot biomass and seed yield.
Assuntos
Arabidopsis/fisiologia , Aspartato Aminotransferases/genética , Glutamato-Amônia Ligase/genética , Fosfoenolpiruvato Carboxilase/genética , Aminoácidos/metabolismo , Amônia/metabolismo , Arabidopsis/genética , Aspartato Aminotransferases/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Glutamato-Amônia Ligase/metabolismo , Nitrogênio/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/genética , Brotos de Planta/genética , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/crescimento & desenvolvimento , Glycine max/genética , Nicotiana/genética , Zea mays/genéticaRESUMO
Phosphoenolpyruvate carboxylase (PEPC) is an important regulatory enzyme situated at a key branch point of central plant metabolism. Plant genomes encode several plant-type PEPC (PTPC) isozymes, along with a distantly related bacterial-type PEPC (BTPC). BTPC is expressed at high levels in developing castor oil seeds where it tightly interacts with co-expressed PTPC polypeptides to form unusual hetero-octameric Class-2 PEPC complexes that are desensitized to allosteric inhibition by L-malate. Analysis of RNA-Seq and microarray transcriptome datasets revealed two distinct patterns of tissue-specific BTPC expression in vascular plants. Species such as Arabidopsis thaliana, strawberry, rice, maize, and poplar mainly exhibited pollen- or floral-specific BTPC expression. By contrast, BTPC transcripts were relatively abundant in developing castor, cotton, and soybean seeds, cassava tubers, as well as immature tomato, cucumber, grape, and avocado fruit. Immunoreactive 118 kDa BTPC polypeptides were detected on immunoblots of cucumber and tomato fruit extracts. Co-immunoprecipitation established that as in castor, BTPCs physically interact with endogenous PTPCs to form Class-2 PEPC complexes in tomato and cucumber fruit. We hypothesize that Class-2 PEPCs simultaneously maintain rapid anaplerotic PEP carboxylation and respiratory CO2 refixation in diverse, biosynthetically active sinks that accumulate high malate levels.
Assuntos
Magnoliopsida/genética , Malatos/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Proteínas de Plantas/genética , Transcriptoma/genética , Perfilação da Expressão Gênica , Magnoliopsida/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/metabolismoRESUMO
We compared the drought tolerance of wild-type (WT) and transgenic rice plants (PC) over-expressing the maize C4PEPC gene, which encodes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) gene, and evaluated the roles of saccharide and sugar-related enzymes in the drought response. Pot-grown seedlings were subjected to real drought conditions outdoors, and the yield components were compared between PC and untransformed wild-type (WT) plants. The stable yield from PC plants was associated with higher net photosynthetic rate under the real drought treatment. The physiological characters of WT and PC seedlings under a simulated drought treatment (25% (w/v) polyethylene glycol-6000 for 3 h; PEG 6000 treatment) were analyzed in detail for the early response of drought. The relative water content was higher in PC than in WT, and PEPC activity and the C4-PEPC transcript level in PC were elevated under the simulated drought conditions. The endogenous saccharide responses also differed between PC and WT under simulated drought stress. The higher sugar decomposition rate in PC than in WT under drought analog stress was related to the increased activities of sucrose phosphate synthase, sucrose synthase, acid invertase, and neutral invertase, increased transcript levels of VIN1, CIN1, NIN1, SUT2, SUT4, and SUT5, and increased activities of superoxide dismutase and peroxidase in the leaves. The greater antioxidant defense capacity of PC and its relationship with saccharide metabolism was one of the reasons for the improved drought tolerance. In conclusion, PEPC effectively alleviated oxidative damage and enhanced the drought tolerance in rice plants, which were more related to the increase of the endogenous saccharide decomposition. These findings show that components of C4 photosynthesis can be used to increase the yield of rice under drought conditions.
Assuntos
Secas , Oryza/enzimologia , Oryza/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , Antioxidantes/metabolismo , Metabolismo dos Carboidratos/genética , Metabolismo dos Carboidratos/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/fisiologia , Fosfoenolpiruvato Carboxilase/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Plântula/enzimologia , Plântula/metabolismo , Plântula/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Zea mays/enzimologia , Zea mays/metabolismo , Zea mays/fisiologiaRESUMO
Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP+ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.
Assuntos
Leishmania donovani/enzimologia , Leishmania donovani/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Autofagia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Gluconeogênese/genética , Gluconeogênese/fisiologia , Glucose/metabolismo , Leishmania donovani/crescimento & desenvolvimento , Macrófagos/parasitologia , Estresse Oxidativo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Inanição/metabolismo , Ativação Transcricional , Regulação para Cima , Virulência , Fatores de Virulência/metabolismoRESUMO
Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.
Assuntos
Fosfoenolpiruvato Carboxilase/genética , Proteínas de Plantas/genética , Sorghum/genética , Germinação , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Fosforilação , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sorghum/enzimologia , Sorghum/crescimento & desenvolvimento , Espectrometria de Massas em Tandem , UbiquitinaçãoRESUMO
Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.
Assuntos
Adaptação Fisiológica , Secas , Genes de Plantas , Fosfoenolpiruvato Carboxilase/genética , Proteômica/métodos , Triticum/genética , Triticum/fisiologia , Zea mays/enzimologia , Adaptação Fisiológica/genética , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Reprodutibilidade dos Testes , Estresse Fisiológico/genéticaRESUMO
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
This work investigates proline synthesis in six barley varieties subjected to iron deficiency, salinity or both stresses. The highest growth under Fe sufficiency corresponded to Belgrano and Shakira. A moderate augment of leaf phosphoenolpyruvate carboxylase (PEPC) activity was observed in all six varieties in response to Fe deficiency, consistently in leaves and sporadically in roots. All six varieties accumulated proline under Fe deficiency, to a higher extent in leaves than in roots. The decrease of Fe supply from 100 µM NaFe(III)-EDTA to 0.5 µM NaFe(III)-EDTA reduced growth and photosynthetic pigments similarly in the six barley varieties. On the contrary, differences between varieties could be observed with respect to increased or, conversely, decreased proline content as a function of the amount of NaFe(III)-EDTA supplied. These two opposite types were represented by Belgrano (higher proline under Fe deficiency) and Shakira (higher proline under Fe sufficiency). Time-course experiments suggested that leaf PEPC activity was not directly responsible for supplying C for proline synthesis under Fe deficiency. High proline levels in the leaves of Fe-deficient Belgrano plants in salinity were associated to a better performance of this variety under these combined stresses.
Assuntos
Hordeum/fisiologia , Deficiências de Ferro , Prolina/metabolismo , Salinidade , Cloreto de Sódio/farmacologia , Hordeum/efeitos dos fármacos , Hordeum/enzimologia , Hordeum/genética , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse FisiológicoRESUMO
Overexpression of phosphoenolpyruvate carboxykinase (PCK) was reported to cause the harboring of higher intracellular ATP concentration in Escherichia coli, accompanied with a slower growth rate. For systematic determination of the relationship between the artificial increase of ATP and growth retardation, PCKWT enzyme was directly evolved in vitro and further overexpressed. The evolved PCK67 showed a 60% greater catalytic efficiency than that of PCKWT. Consequently, the PCK67-overexpressing E. coli showed the highest ATP concentration at the log phase of 1.45 µmol/gcell, with the slowest growth rate of 0.66 h(-1), while the PCKWT-overexpressing cells displayed 1.00 µmol/gcell ATP concentration with the growth rate of 0.84 h(-1) and the control had 0.28 µmol/gcell with 1.03 h(-1). To find a plausible reason, PCK-overexpressing cells in a steady state during chemostat growth were applied to monitor intracellular reactive oxygen species (ROS). Higher amount of intracellular ROS were observed as the ATP levels increased. To confirm the hypothesis of slower growth rate without perturbation of the carbon flux by PCK-overexpression, phototrophic Gloeobacter rhodopsin (GR) was expressed. The GR-expressing strain under illumination harbored 81% more ATP concentration along with 82% higher ROS, with a 54% slower maximum growth rate than the control, while both the GR-expressing strain under dark and dicarboxylate transporter (a control membrane protein)-expressing strain showed a lower ATP and increased ROS, and slower growth rate. Regardless of carbon flux changes, the artificial ATP increase was related to the ROS increase and it was reciprocally correlated to the maximum growth rate. To verify that the accumulated intracellular ROS were responsible for the growth retardation, glutathione was added to the medium to reduce the ROS. As a result, the growth retardation was restored by the addition of 0.1 mM glutathione. Anaerobic culture even enabled the artificial ATP-increased E. coli to grow faster than control. Collectively, it was concluded that artificial ATP increases inhibit the growth of E. coli due to the overproduction of ROS.
Assuntos
Trifosfato de Adenosina/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Anaerobiose , Biocatálise/efeitos dos fármacos , Ciclo do Carbono , Cianobactérias/genética , Cianobactérias/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Glutationa/metabolismo , Glutationa/farmacologia , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rodopsina/metabolismoRESUMO
Drought stress is one of the most frequent forms of abiotic stresses, which occurs under condition of limited water availability. In this work, the possible participation of phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC), NADP-malic enzyme (EC 1.1.1.40; NADP-ME), and pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) in response to drought of tobacco plants (Nicotiana tabacum L., cv. W38) was investigated. Enzyme specific activities in tobacco leaves of drought stressed plants were significantly increased after 11 days of stress, PEPC 2.3-fold, NADP-ME 3.9-fold, and PPDK 2.7-fold compared to control plants. The regulation of PEPC and NADP-ME activities were studied on transcriptional level by the quantitative RT PCR and on translational level - immunochemically. The amount of NADP-ME protein and transcription of mRNA for chloroplastic NADP-ME isoform were increased indicating their enhanced synthesis de novo. On the other hand, mRNA for cytosolic isoform of NADP-ME was decreased. The changes in PEPC protein and PEPC mRNA were not substantial. Therefore regulation of PEPC activity by phosphorylation was evaluated and found to be involved in the stress response. During recovery, activities of the tested enzymes returned close to their basal levels.
Assuntos
Secas , Malato Desidrogenase/metabolismo , Nicotiana/enzimologia , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/genética , Piruvato Ortofosfato Diquinase/metabolismo , Estresse Fisiológico/fisiologia , Aclimatação , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Malato Desidrogenase/genética , Fosfoenolpiruvato Carboxilase/genética , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Piruvato Ortofosfato Diquinase/genética , Reação em Cadeia da Polimerase em Tempo Real , Nicotiana/genéticaRESUMO
In the present agricultural scenario, the major thrust is to increase crop productivity so as to ensure sustainability. In an earlier study, foliar application of thiourea (TU; a non physiological thiol based ROS scavenger) has been demonstrated to enhance the stress tolerance and yield of different crops under field condition. Towards this endeavor, present work deals with the effect of TU on photosynthetic efficiency and source-to-sink relationship of Indian mustard (Brassica juncea) for understanding its mode of action. The application of TU increased the efficiency of both PSI and PSII photosystems and vegetative growth of plant. The comparative analysis of sucrose to starch ratio and expression level of sugar transporters confirmed the higher source and sink strength in response to TU treatment. The biochemical evidence in support of this was derived from higher activities of sucrose phosphate synthase and fructose-1,6-bis-phosphatase at source; and sucrose synthase and different classes of invertases at both source and sink. This indicated an overall increase in photoassimilate level at sink. An additional contribution through pod photosynthesis was confirmed through the analysis of phosphoenol pyruvate carboxylase enzyme activity and level of organic acids. The increased photoassimilate level was also co-ordinated with acetyl coA carboxylase mediated oil biosynthesis. All these changes were ultimately reflected in the form of 10 and 20% increase in total yield and oil content, respectively under TU treatment as compared to control. Additionally, no change was observed in oil composition of seeds derived from TU treated plants. The study thus signifies the co-ordinated regulation of key steps of photosynthesis and source-to-sink relationship through the external application of TU resulting in increased crop yield and oil content.