RESUMO
Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.
Assuntos
Alumínio , Monoterpenos Bicíclicos , Citrus , Limoneno , Fotossíntese , Folhas de Planta , Terpenos , Alumínio/toxicidade , Terpenos/metabolismo , Citrus/metabolismo , Citrus/efeitos dos fármacos , Limoneno/metabolismo , Fotossíntese/efeitos dos fármacos , Monoterpenos Bicíclicos/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Monoterpenos/metabolismo , Hemiterpenos/metabolismo , Cicloexenos/metabolismo , Fosfatos Açúcares/metabolismo , Butadienos/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Ácido Mevalônico/metabolismo , Monoterpenos Cicloexânicos , Citrus sinensis/metabolismo , Citrus sinensis/efeitos dos fármacos , Citrus sinensis/genética , Clorofila/metabolismo , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genética , VolatilizaçãoRESUMO
Bacterial cryptic prophage (defective prophage) genes are known to drastically influence host physiology, such as causing cell growth arrest or lysis, upon expression. Many phages encode lytic proteins to destroy the cell envelope. As natural antibiotics, only a few lysis target proteins were identified. ydfD is a lytic gene from the Qin cryptic prophage that encodes a 63-amino-acid protein, the ectopic expression of which in Escherichia coli can cause nearly complete cell lysis rapidly. The bacterial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is responsible for synthesizing the isoprenoids uniquely required for sustaining bacterial growth. In this study, we provide evidence that YdfD can interact with IspG, a key enzyme involved in the MEP pathway, both in vivo and in vitro. We show that intact YdfD is required for the interaction with IspG to perform its lysis function and that the mRNA levels of ydfD increase significantly under certain stress conditions. Crucially, the cell lysis induced by YdfD can be abolished by the overexpression of ispG or the complementation of the IspG enzyme catalysis product methylerythritol 2,4-cyclodiphosphate. We propose that YdfD from the Qin cryptic prophage inhibits IspG to block the MEP pathway, leading to a compromised cell membrane and cell wall biosynthesis and eventual cell lysis.
Assuntos
Biocatálise , Eritritol/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Prófagos/metabolismo , Fosfatos Açúcares/metabolismo , Proteínas Virais/metabolismo , Sequência Conservada , Cisteína/química , Eritritol/metabolismo , Escherichia coli/ultraestrutura , Modelos Biológicos , Ligação Proteica , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Soluções , Estresse Fisiológico , Proteínas Virais/químicaRESUMO
Low-calorie sweeteners are substitutes for sugar and frequently used by patients with cardiometabolic diseases. Erythritol, a natural low-calorie sugar alcohol, was linked to cardiometabolic diseases in several recent metabolomics studies. However, the characterization of its role in disease development is lacking. Macrophage polarization orchestrates the immune response in various inflammatory conditions, most notably cardiometabolic disease. Therefore, the physiological effects of Erythritol on THP-1 macrophages were investigated. We observed an increased cellular abundance of proinflammatory M1 macrophages, characterized by CD11c, TNF-α, CD64, CD38, and HLA-DR markers and decreased anti-inflammatory M2 macrophages, characterized by mannose receptor CD206. The, Erythritol increased ROS generation, and the activation of the AKT pathway, cytosolic calcium overload, and cell cycle arrest at the G1 phase. Concomitantly, an increased population of necroptotic macrophages was observed. In conclusion, we provide evidence that Erythritol induced the proinflammatory phenotype in THP-1 macrophages and this was associated with an increased population of necroptotic macrophages. PRACTICAL APPLICATIONS: This assessment provides evidence of the effects of Erythritol on macrophages, particularly THP-1-derived macrophages. Our results support the role of Erythritol in driving the inflammation that is associated with cardiometabolic diseases and provide insights in the role of Erythritol as an inducer of necroptosis in THP-1 derived macrophages that could be associated the disease.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Fator de Necrose Tumoral alfa , Eritritol/metabolismo , Eritritol/farmacologia , Humanos , Ativação de Macrófagos , Macrófagos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is a kind of serious fat disorder that has become a critical problem to human society. Therefore, finding drugs that are safe and effective has become more and more important. Erythritol (Ery) is a polyol sweetener with a variety of biological functions. However, whether Ery has a relieving effect on NAFLD has not been reported yet. Therefore, we induced HepG2 cells with oleic acid and palmitic acid as our in vitro model. Moreover, we choose wild-type mice with tyloxapol and high-fat diet and nuclear factor E2-related factor 2 (Nrf2) knockout mice with high-fat diet as our in vivo model. We found that Ery could reverse the lipid accumulation, oxidative stress, and endoplasmic reticulum stress caused by the NAFLD model. The mechanism studies showed that Ery promoted the translocation of Nrf2 from cytoplasm to nucleus, and the molecular simulation docking results of Ery and Nrf2 showed that there was a hydrogen bond between them. Moreover, Ery could promote the production of HO-1 and NQO1 antioxidant proteins and inhibit the expression of endoplasmic reticulum stress proteins GPR78, p-PERK, and CHOP. On the contrast, when Nrf2 was knocked out in mice, Ery lost its protective effect on NAFLD. In conclusion, we found that the potential mechanism of Ery's protective effect is that it plays an antioxidant role by activating the Nrf2 signaling pathway, thereby inhibiting endoplasmic reticulum stress and lipid accumulation in NAFLD.
Assuntos
Fator 2 Relacionado a NF-E2 , Hepatopatia Gordurosa não Alcoólica , Animais , Antioxidantes/metabolismo , Dieta Hiperlipídica/efeitos adversos , Chaperona BiP do Retículo Endoplasmático , Eritritol/metabolismo , Fígado/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse OxidativoRESUMO
Terpenes and their derivatives are important biomarkers of grape quality as they contribute to the flavor and aroma of grapes. However, the molecular basis of terpene biosynthesis throughout the grapevine phenological developmental cycle remains elusive. Our current study investigates the free and bound terpene biosynthesis of berries at different phenological stages from preveraison to harvest. Detailed gene expression (transcriptomics) analysis, terpenoid volatile production by gas chromatography-mass spectrometry (GC-MS), and in planta transient expression were employed. Our results show that concentrations of most individual terpenes at different stages are distinctive and increase from preveraison to the veraison stage followed by a decrease from veraison to maturity. The combined transcriptomic analysis and terpene profiling revealed that 22 genes belonging to the MEP pathway and multiple classes of transcription factor family members including bHLH and several hormone biosynthesis- or signaling-related genes likely participate in the regulation of terpenoid biosynthesis according to their specific expression patterns in berries. Quantitative real-time polymerase chain expression analysis of 8 key differentially expressed genes in MEP pathways and further 12 randomly selected genes was performed during 8 sampling stages and validated the RNA-seq-derived expression profiles. To further confirm the function of a subset of the differentially expressed genes, we investigated the effects of combined overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (VvDXS1-LOC100249323), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (VvDXR-LOC100248516), and terpene synthase (VvTPS56-LOC100266449) on the production of terpenes by transient overexpression in Nicotiana benthamiana leaves. The overall developmental patterns of total terpenes and gene expression profiles will help guide the functional analyses of further candidate genes important for terpene biosynthesis of grape as well as identifying the master transcriptional and hormonal regulators of this pathway in the future.
Assuntos
Alquil e Aril Transferases/metabolismo , Eritritol/análogos & derivados , Aromatizantes/metabolismo , Frutas/química , Frutas/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Fosfatos Açúcares/metabolismo , Terpenos/metabolismo , Vitis/genética , Alquil e Aril Transferases/genética , Eritritol/metabolismo , Aromatizantes/química , Frutas/genética , Frutas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Terpenos/química , Vitis/química , Vitis/crescimento & desenvolvimento , Vitis/metabolismoRESUMO
Macroalgae produce a wide range of monoterpenes as secondary metabolites of mevalonate (MVA) and/or methylerythritol phosphate (MEP) pathway (often including haloperoxidase action). Great biodiversity of macroalgal monoterpenes was reported including acyclic, monocyclic, and bicyclic structures. Halogenated monoterpenes exhibited significant biological activity (e.g., anticancer, antiplasmodial, and insecticidal) that is influenced by the number of present halogens (higher halogen content is preferable, especially bromine) and their position within the monoterpene skeleton. In distinction from the existing reviews, the present review provides novelty with respect to: (a) exclusively monoterpenes from red macroalgae are targeted; (b) biosynthesis, isolation, and analysis, as well as bioactivity of monoterpenes are represented; (c) the methods of their isolation, analysis, and structure elucidation are summarized; (d) the bioactivity of macroalgal monoterpenes is systematically presented with emphasis on anticancer activity; (e) the literature references were updated.
Assuntos
Antineoplásicos/farmacologia , Monoterpenos/farmacologia , Rodófitas/química , Alga Marinha/química , Animais , Antineoplásicos/análise , Antineoplásicos/química , Linhagem Celular Tumoral , Eritritol/análogos & derivados , Eritritol/metabolismo , Humanos , Ácido Mevalônico/metabolismo , Estrutura Molecular , Monoterpenos/análise , Monoterpenos/metabolismo , Rodófitas/metabolismo , Alga Marinha/metabolismo , Fosfatos Açúcares/metabolismoRESUMO
The rhizobacterium Serratia plymuthica 4Rx13 releases a unique polymethylated hydrocarbon (C16H26) with a bicyclo[3.2.1]octadiene skeleton called sodorifen. Sodorifen production depends on a gene cluster carrying a C-methyltransferase and a terpene cyclase along with two enzymes of the 2- C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. Comparative analysis of wild-type and mutant volatile organic compound profiles revealed a C-methyltransferase-dependent C16 alcohol called pre-sodorifen, the production of which is upregulated in the terpene cyclase mutant. The monocyclic structure of this putative intermediate in sodorifen biosynthesis was identified by NMR spectroscopy. In vitro assays with the heterologously expressed S. plymuthica C-methyltransferase and terpene cyclase demonstrated that these enzymes act sequentially to convert farnesyl pyrophosphate (FPP) into sodorifen via a pre-sodorifen pyrophosphate intermediate, indicating that the S-adenosyl methionine (SAM)-dependent C-methyltransferase from S. plymuthica exhibits unprecedented cyclase activity. In vivo incorporation experiments with 13C-labeled succinate, l-alanine, and l-methionine confirmed a MEP pathway to FPP via the canonical glyceraldehyde-3-phosphate and pyruvate, as well as its SAM-dependent methylation in pre-sodorifen and sodorifen biosynthesis. 13C{1H} NMR spectroscopy facilitated the localization of 13C labels and provided detailed insights into the biosynthetic pathway from FPP via pre-sodorifen pyrophosphate to sodorifen.
Assuntos
Compostos Bicíclicos com Pontes/metabolismo , Eritritol/análogos & derivados , Metiltransferases/metabolismo , Octanos/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , S-Adenosilmetionina/metabolismo , Serratia/metabolismo , Sesquiterpenos/metabolismo , Fosfatos Açúcares/metabolismo , Compostos Bicíclicos com Pontes/química , Ciclização , Eritritol/química , Eritritol/metabolismo , Metilação , Estrutura Molecular , Octanos/química , Fosfatos de Poli-Isoprenil/química , S-Adenosilmetionina/química , Serratia/enzimologia , Sesquiterpenos/química , Fosfatos Açúcares/químicaRESUMO
Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.
Assuntos
Vias Biossintéticas/genética , Inativação Gênica , Genes de Plantas , Vírus de Plantas/fisiologia , Plantas Medicinais/genética , Withania/genética , Vitanolídeos/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Regulação para Baixo/genética , Eritritol/análogos & derivados , Eritritol/metabolismo , Regulação da Expressão Gênica de Plantas , Ácido Mevalônico/metabolismo , Fenótipo , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Plantas Geneticamente Modificadas , Plantas Medicinais/anatomia & histologia , Plantas Medicinais/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fosfatos Açúcares/metabolismo , Withania/anatomia & histologia , Withania/crescimento & desenvolvimentoRESUMO
Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-d-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/bioproduct production in cyanobacteria.
Assuntos
Cicloexenos/metabolismo , Synechococcus/metabolismo , Terpenos/metabolismo , Trifosfato de Adenosina/metabolismo , Biocombustíveis , Eritritol/análogos & derivados , Eritritol/metabolismo , Microbiologia Industrial , Cinética , Limoneno , Engenharia Metabólica , Redes e Vias Metabólicas , Modelos Biológicos , NADP/metabolismo , Fotossíntese , Proteômica , Fosfatos Açúcares/metabolismoRESUMO
1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway and is also a validated antimicrobial target. Theaflavins, which are polyphenolic compounds isolated from fermented tea, possess a wide range of pharmacological activities, especially an antibacterial effect, but little has been reported on their modes of antimicrobial action. To uncover the antibacterial mechanism of theaflavins and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of theaflavins were investigated in this study. The results show that all four theaflavin compounds could specifically suppress the activity of DXR, with theaflavin displaying the lowest effect against DXR (IC50 162.1 µM) and theaflavin-3,3'-digallate exhibiting the highest (IC50 14.9 µM). Moreover, determination of inhibition kinetics of the theaflavins demonstrates that they are non-competitive inhibitors of DXR against 1-deoxy-D-xylulose 5-phosphate (DXP) and un-competitive inhibitors with respect to NADPH. The possible interactions between DXR and the theaflavins were simulated via docking experiments.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/metabolismo , Antibacterianos/farmacologia , Biflavonoides/farmacologia , Catequina/farmacologia , Escherichia coli/efeitos dos fármacos , Fosfatase Alcalina/antagonistas & inibidores , Biflavonoides/química , Catequina/química , Eritritol/análogos & derivados , Eritritol/metabolismo , Simulação de Acoplamento Molecular , Tamanho da Partícula , Terpenos/metabolismoRESUMO
Little is known about how plant cells regulate the exchange of prenyl diphosphates between the two compartmentalized isoprenoid biosynthesis pathways. Prenylation of proteins is a suitable model to study such interactions between the plastidial methylerythritol phosphate (MEP) and the cytosolic mevalonate (MVA) pathways because prenyl moieties used to modify proteins rely on both origins. Tobacco cells expressing a prenylatable GFP were treated with specific MEP and/or MVA pathways inhibitors to block the formation of prenyl diphosphates and therefore the possibility to modify the proteins. Chemical complementation assays using prenyl alcohol precursors restore the prenylation. Indeed, geranylgeraniol (C20 prenyl alcohol) and to a lesser but significant level C15-farnesol restored the prenylation of a protein bearing a geranylgeranylation CaaX motif, which under standard conditions is modified by a MEP-derived prenyl group. However, the restoration takes place in different ways. While geranylgeraniol operates directly as a metabolic precursor, the C15-prenyl alcohol functions indirectly as a signal that leads to shift the metabolic origin of prenyl groups in modified proteins, here from the plastidial MEP pathway in favor of the cytosolic MVA pathway. Furthermore, farnesol interferes negatively with the MEP pathway in an engineered Escherichia coli strain synthesizing isoprenoids either starting from MVA or from MEP. Following the cellular uptake of a fluorescent analog of farnesol, we showed its close interaction with tobacco plastids and modification of plastid homeostasis. As a consequence, in tobacco farnesol supposedly inhibits the plastidial MEP pathway and activates the cytosolic MVA pathway, leading to the shift in the metabolic origin and thereby acts as a potential regulator of crosstalk between the two pathways. Together, those results suggest a new role for farnesol (or a metabolite thereof) as a central molecule for the regulation of isoprenoid biosynthesis in plants.
Assuntos
Farneseno Álcool/metabolismo , Proteínas de Plantas/metabolismo , Prenilação de Proteína , Linhagem Celular , Eritritol/análogos & derivados , Eritritol/metabolismo , Proteínas de Plantas/química , Plastídeos/metabolismo , Fosfatos Açúcares/metabolismoRESUMO
Terpenoids constitute the main class of secondary metabolites produced in plants with industrial, pharmacological, and agricultural interests. Nicotiana sylvestris has been widely adopted as a diploid model system in plant biology for studies of terpenoid biosynthesis. In this paper, we report the isolation and analysis of the 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (CMS) gene of the MEP (methylerythritol 4-phosphate) pathway from N. sylvestris. We used homologous-based cloning with a RACE method to obtain the full-length coding sequence of the NsCMS. Then, the physical and chemical properties, function, and three-dimensional structure of the NsyCMS protein were predicted. Fluorogenic quantitative PCR was used to conduct an expression analysis at different developmental stages of various tissues of the NsyCMS. The sequence of the NsyCMS consists of a 954-bp open reading frame and encodes a predicted protein of 317 amino acids, with a molecular weight of approximately 49.6 kDa and pi of 6.92. The in vivo localization of the encoded protein was cytoplasmic with no signal peptide, whereas 2 transmembrane regions were found in NsyCMS. The conserved domains of typical 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, aminotransferase, and pyridoxal phosphate-dependent transferase were found in NsyCMS. Differential expression patterns of the NsyCMS were observed throughout the different developmental stages and tissues. NsyCMS messenger RNA was expressed in all tissues, with the highest level of expression in the seedling leaves. NsyMK was expressed at a higher level in the resettling roots. The results from our study set the foundation for exploring the terpenoid biosynthetic pathways in N. sylvestris.
Assuntos
Nicotiana/enzimologia , Fósforo-Oxigênio Liases/genética , Proteínas de Plantas/genética , Terpenos/metabolismo , Clonagem Molecular , Eritritol/análogos & derivados , Eritritol/biossíntese , Eritritol/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas , Modelos Moleculares , Fósforo-Oxigênio Liases/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Fosfatos Açúcares/metabolismo , Nicotiana/genéticaRESUMO
Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding ß-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic ß-amylase encoding genes in pgi1 leaves, which was accompanied by increased ß-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.
Assuntos
Arabidopsis/metabolismo , Citocininas/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Células do Mesofilo/metabolismo , Fotossíntese , Amido/metabolismo , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Loci Gênicos , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/genética , Redes e Vias Metabólicas , Mutação , Fenótipo , Folhas de Planta/metabolismo , Fosfatos Açúcares/metabolismoRESUMO
Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-D-erythritol 4-phosphate pathway enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-D-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits.
Assuntos
Actinidia/enzimologia , Alquil e Aril Transferases/metabolismo , Regulação da Expressão Gênica de Plantas , Monoterpenos/metabolismo , Proteínas de Plantas/metabolismo , Actinidia/genética , Actinidia/crescimento & desenvolvimento , Alquil e Aril Transferases/genética , Sequência de Bases , Eritritol/análogos & derivados , Eritritol/metabolismo , Etilenos/metabolismo , Frutas/enzimologia , Frutas/genética , Frutas/crescimento & desenvolvimento , Expressão Gênica , Dados de Sequência Molecular , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Fosfatos Açúcares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Lavandula/enzimologia , Óleos Voláteis/metabolismo , Óleos de Plantas/metabolismo , Transferases/metabolismo , Aldose-Cetose Isomerases/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Flores/química , Flores/enzimologia , Flores/genética , Expressão Gênica , Lavandula/química , Lavandula/genética , Monoterpenos/metabolismo , Fenótipo , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fosfatos Açúcares/metabolismo , Transferases/genéticaRESUMO
Withanolides biosynthesis in the plant Withania somnifera (L.) Dunal is hypothesized to be diverged from sterol pathway at the level of 24-methylene cholesterol. The conversion and translocation of intermediates for sterols and withanolides are yet to be characterized in this plant. To understand the influence of mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways on sterols and withanolides biosynthesis in planta, we overexpressed the WsHMGR2 and WsDXR2 in tobacco, analyzed the effect of transient suppression through RNAi, inhibited MVA and MEP pathways and fed the leaf tissue with different sterols. Overexpression of WsHMGR2 increased cycloartenol, sitosterol, stigmasterol and campesterol compared to WsDXR2 transgene lines. Increase in cholesterol was, however, marginally higher in WsDXR2 transgenic lines. This was further validated through transient suppression analysis, and pathway inhibition where cholesterol reduction was found higher due to WsDXR2 suppression and all other sterols were affected predominantly by WsHMGR2 suppression in leaf. The transcript abundance and enzyme analysis data also correlate with sterol accumulation. Cholesterol feeding did not increase the withanolide content compared to cycloartenol, sitosterol, stigmasterol and campesterol. Hence, a preferential translocation of carbon from MVA and MEP pathways was found differentiating the sterols types. Overall results suggested that MVA pathway was predominant in contributing intermediates for withanolides synthesis mainly through the campesterol/stigmasterol route in planta.
Assuntos
Colesterol/análogos & derivados , Eritritol/análogos & derivados , Ácido Mevalônico/metabolismo , Nicotiana/enzimologia , Fitosteróis/metabolismo , Fosfatos Açúcares/metabolismo , Withania/enzimologia , Vitanolídeos/metabolismo , Sequência de Bases , Vias Biossintéticas , Carbono/metabolismo , Colesterol/química , Colesterol/metabolismo , Eritritol/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ácido Mevalônico/química , Dados de Sequência Molecular , Filogenia , Fitosteróis/química , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Sitosteroides/química , Sitosteroides/metabolismo , Esteróis/química , Esteróis/metabolismo , Estigmasterol/química , Estigmasterol/metabolismo , Nicotiana/genética , Triterpenos/química , Triterpenos/metabolismo , Withania/química , Withania/genética , Vitanolídeos/químicaRESUMO
S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway-dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism.
Assuntos
Celulase/metabolismo , Monoterpenos/farmacologia , Nicotiana/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Sesquiterpenos/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Biomassa , Morte Celular/efeitos dos fármacos , Monoterpenos Cicloexânicos , Dimetilaliltranstransferase/antagonistas & inibidores , Dimetilaliltranstransferase/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Eritritol/análogos & derivados , Eritritol/metabolismo , Fosfomicina/análogos & derivados , Fosfomicina/farmacologia , Ácido Mevalônico/farmacologia , Modelos Biológicos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Metabolismo Secundário/efeitos dos fármacos , Sesquiterpenos/química , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Fosfatos Açúcares/metabolismo , Nicotiana/citologia , Nicotiana/efeitos dos fármacos , Nicotiana/enzimologiaRESUMO
We applied a parallel pore permeation model based on the Renkin molecular sieving function by using two different-sized pathways to analyze the permeation-enhancing effects of poly-L-arginine (PLA) or a mixed system of spermine (SPM) and sodium taurocholate (STC). Four paracellular markers were simultaneously applied to Caco-2 cell monolayers, and a set of apparent permeability coefficient (P) values was used to obtain membrane parameters. For PLA treatment, the pore occupancy/length ratio (ε/L) of the large pathways increased while the pore radius (R) did not, suggesting that the number of large pathways for the relatively large hydrophilic molecules in the monolayers could be increased by the addition of PLA. In contrast, application of the mixed system comprising SPM and STC significantly increased not only the R of the large pathways but also ε/L of the small pathways. Such changes in membrane parameters could be related to the enhancing mechanism of these compounds. The simulation curves for molecular weight (MW)-P calculated from the membrane parameters could be used to predict the P of drugs with different MWs.
Assuntos
Peptídeos/farmacologia , Espermina/farmacologia , Ácido Taurocólico/farmacologia , Absorção/efeitos dos fármacos , Células CACO-2 , Eritritol/metabolismo , Humanos , Inulina/metabolismo , Isomaltose/metabolismo , Permeabilidade/efeitos dos fármacos , Porosidade , Xilitol/metabolismoRESUMO
Erythritol is a four-carbon sugar preferentially utilized by Brucella spp. The presence of erythritol in the placentas of goats, cows, and pigs has been used to explain the localization of Brucella to these sites and the subsequent accumulation of large amounts of bacteria, eventually leading to abortion. Here we show that Brucella melitensis will also localize to an artificial site of erythritol within a mouse, providing a potential model system to study the pathogenesis of Brucella abortion. Immunohistological staining of the sites of erythritol within infected mice indicated a higher than expected proportion of extracellular bacteria. Ensuing experiments suggested intracellular B. melitensis was unable to replicate within macrophages in the presence of erythritol and that erythritol was able to reach the site of intracellular bacteria. The intracellular inhibition of growth was found to encourage the bacteria to replicate extracellularly rather than intracellularly, a particularly interesting development in Brucella pathogenesis. To determine the effect of erythritol on expression of B. melitensis genes, bacteria grown either with or without erythritol were analyzed by microarray. Two major virulence pathways were up-regulated in response to exposure to erythritol (the type IV secretion system VirB and flagellar proteins), suggesting a role for erythritol in virulence.
Assuntos
Brucella melitensis/metabolismo , Brucella melitensis/patogenicidade , Brucelose/microbiologia , Brucelose/patologia , Eritritol/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fatores de Virulência/biossíntese , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Macrófagos/microbiologia , Camundongos , Análise em MicrossériesRESUMO
Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-D-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-D-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon-intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.