RESUMO
Plant polyphenolics exhibit a broad spectrum of health-promoting effects when consumed as part of the diet, and there is considerable interest in enhancing the levels of these bioactive molecules in plants used as foods. AtMYB12 was originally identified as a flavonol-specific transcriptional activator in Arabidopsis thaliana, and this has been confirmed by ectopic expression in tobacco. AtMYB12 is able to induce the expression of additional target genes in tobacco, leading to the accumulation of very high levels of flavonols. When expressed in a tissue-specific manner in tomato, AtMYB12 activates the caffeoyl quinic acid biosynthetic pathway, in addition to the flavonol biosynthetic pathway, an activity which probably mirrors that of the orthologous MYB12-like protein in tomato. As a result of its broad specificity for transcriptional activation in tomato, AtMYB12 can be used to produce fruit with extremely high levels of multiple polyphenolic anti-oxidants. Our data indicate that transcription factors may have different specificities for target genes in different plants, which is of significance when designing strategies to improve metabolite accumulation and the anti-oxidant capacity of foods.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Flavonóis/biossíntese , Ácido Quínico/análogos & derivados , Solanum lycopersicum/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Antioxidantes/metabolismo , Proteínas de Arabidopsis/genética , Carotenoides/biossíntese , Clonagem Molecular , Flavonoides/biossíntese , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Dados de Sequência Molecular , Fenóis , Filogenia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmídeos , Polifenóis , Ácido Quínico/metabolismo , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Fatores de Transcrição/genética , Transformação GenéticaRESUMO
Chlorogenic acid (CGA) is one of the most abundant phenolic compounds in tomato (Solanum lycopersicum). Hydroxycinnamoyl CoA quinate transferase (HQT) is the key enzyme catalysing CGA biosynthesis in tomato. We have studied the relationship between phenolic accumulation and UV-susceptibility in transgenic tomato plants with altered HQT expression. Overall, increased CGA accumulation was associated with increased UV-protection. However, the genetic manipulation of HQT expression also resulted in more complex alterations in the profiles of phenolics. Levels of rutin were relatively high in both HQT gene-silenced and HQT-overexpressing plants raised in plant growth tunnels. This suggests plasticity in the flux along different branches of phenylpropanoid metabolism and the existence of regulatory mechanisms that direct the flow of phenolic precursors in response to both metabolic parameters and environmental conditions. These changes in composition of the phenolic pool affected the relative levels of UV-tolerance. We conclude that the capability of the phenolic compounds to protect against potentially harmful UV radiation is determined both by the total levels of phenolics that accumulate in leaves as well as by the specific composition of the phenolic profile.
Assuntos
Ácido Clorogênico/metabolismo , Fenóis/metabolismo , Solanum/metabolismo , Solanum/efeitos da radiação , Ácido Clorogênico/química , Clorofila/química , Clorofila/metabolismo , Solanum lycopersicum/química , Solanum lycopersicum/metabolismo , Espectrometria de Massas , Extratos Vegetais/química , Solubilidade , Análise EspectralRESUMO
BACKGROUND: The herbicidal mode of action of flamprop-M-methyl [methyl N-benzoyl-N-(3-chloro-4-fluorophenyl)-D-alaninate] was investigated. RESULTS: For initial characterization, a series of bioassays was used, which indicated a mode of action similar to that of mitotic disrupter herbicides. Cytochemical fluorescence studies, which included monoclonal antibodies against polymerized tubulin, were applied to elucidate effects on mitosis and microtubule assembly in maize roots. When seedlings were root treated with 50 microM of flamprop-M-methyl, cell division activity in meristematic root tip cells ceased within 4 h. The compound severely disturbed the orientation of spindle and phragmoblast microtubules, leading to defective spindle and phragmoblast structures. Cortical microtubules were only slightly affected. In late anaphase and early telophase cells, phragmoblast microtubules were disorganized in multiple arrays that hampered regular cell plate deposition in cytokinesis. Microtubules of the spindle apparatus were found attached to chromosomal kinetochores, but did not show regular organization associated with a zone of microtubule-organizing centres at the opposite ends of the cell. On account of this loss of spindle organization, chromosomes remained in a condensed state of prometaphase or metaphase. Unlike known microtubule disrupter herbicides, flamprop-M-methyl and its biologically active metabolite flamprop did not inhibit soybean tubulin polymerization to microtubules in vitro at 50 microM. In contrast, soybean plants responded sensitively to the compounds. CONCLUSION: The results indicate that flamprop-M-methyl is a mitotic disrupter herbicide with a new antimicrotubule mechanism of action that affects orientation of spindle and phragmoblast microtubules, possibly by minus-end microtubule disassembly.
Assuntos
Alanina/análogos & derivados , Herbicidas/farmacologia , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Zea mays/efeitos dos fármacos , Alanina/farmacologia , Imuno-HistoquímicaRESUMO
Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Animais , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Dimerização , Drosophila melanogaster/metabolismo , Humanos , Carioferinas/metabolismo , Chaperonas Moleculares , Interferência de RNA , Proteína Exportina 1RESUMO
The trend to view many foods not only as sustenance but also as medicine, so-called functional foods, is increasing. Phenolics are the most widespread dietary antioxidants, and among these, chlorogenic acid (CGA) accumulates to high levels in some crop plants. CGA acts as an antioxidant in plants and protects against degenerative, age-related diseases in animals when supplied in their diet. cDNA clones encoding the enzyme that synthesizes CGA, hydroxycinnamoyl-CoA quinate: hydroxycinnamoyl transferase (HQT), were characterized from tomato and tobacco. Gene silencing proved HQT to be the principal route for accumulation of CGA in solanaceous species. Overexpression of HQT in tomato caused plants to accumulate higher levels of CGA, with no side-effects on the levels of other soluble phenolics, and to show improved antioxidant capacity and resistance to infection by a bacterial pathogen. Tomatoes with elevated CGA levels could be used in foods with specific benefits for human health.
Assuntos
Aciltransferases/genética , Ácido Clorogênico/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Acil Coenzima A/metabolismo , Aciltransferases/isolamento & purificação , Aciltransferases/metabolismo , Agrobacterium tumefaciens/genética , Sequência de Aminoácidos , Northern Blotting , Western Blotting , Ácido Clorogênico/análise , Coenzima A-Transferases/genética , DNA Complementar/química , DNA Complementar/genética , Eletrólitos/metabolismo , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Cinética , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , Estresse Oxidativo/fisiologia , Paraquat/farmacologia , Fotossíntese/efeitos dos fármacos , Fotossíntese/fisiologia , Filogenia , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Pseudomonas syringae/crescimento & desenvolvimento , Ácido Quínico/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/metabolismo , TransfecçãoRESUMO
In higher plants, activation sequence-1 (as-1) of the cauliflower mosaic virus 35S promoter mediates both salicylic acid- and auxin-inducible transcriptional activation. Originally found in viral and T-DNA promoters, as-1-like elements are also functional elements of plant promoters activated in the course of a defence response upon pathogen attack. as-1-like elements are characterised by two imperfect palindromes with the palindromic centres being spaced by 12 bp. They are recognised by plant nuclear as-1-binding factor ASF-1, the major component of which is basic/leucine zipper (bZIP) protein TGA2.2 (approximately 80%) in Nicotiana tabacum. In electrophoretic mobility shift assays, ASF-1 as well as bZIP proteins TGA2.2, TGA2.1 and TGA1a showed a 3-10-fold reduced binding affinity to mutant as-1 elements encoding insertions of 2, 4, 6, 8 or 10 bp between the palindromes, respectively. This correlated with a 5-10-fold reduction in transcriptional activation from these elements in transient expression assays. Although ASF-1 and TGA factors bound efficiently to a mutant element carrying a 2 bp deletion between the palindromes [as-1/(-2)], the latter was strongly compromised with respect to mediating gene expression in vivo. A fusion protein consisting of TGA2.2 and a constitutive activation domain mediated transactivation from as-1/(-2) demonstrating binding of TGA factors in vivo. We therefore conclude that both DNA binding and transactivation require optimal positioning of TGA factors on the as-1 element.
Assuntos
DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nicotiana/metabolismo , Proteínas Nucleares , Vírus de Plantas/genética , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação , DNA Viral/química , Proteínas de Ligação a DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Viral da Expressão Gênica , Genes Reporter , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Deleção de Sequência/genética , Especificidade por Substrato , Termodinâmica , Nicotiana/genética , Nicotiana/virologia , Fatores de Transcrição/químicaAssuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Arabidopsis/enzimologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Pirimidinas/síntese química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The first five steps of the non-mevalonate pathway have been tested in high-throughput screening (HTS) campaigns, using enzymes of plant origin. Hit rates were in general relatively low, which could be attributed to the high polarity and charged nature of substrates and active sites of these enzymes. Still, for all the enzymes, apart from IspF (2-methylerythritol 2,4-cyclodiphosphate synthase), inhibitors could be identified with activities below 100 µM, and these were followed up to identify structure-activity relationships (SARs). For the enzyme IspD (2C-methyl-D-erythritol 4-phosphate cytidyltransferase), inhibitors with IC50 down to 35 nM were identified that also showed herbicidal activity.
Assuntos
Herbicidas/química , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/fisiologia , Plantas/enzimologia , Fenômenos Fisiológicos Vegetais/efeitos dos fármacos , Terpenos/metabolismoRESUMO
MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode--implemented on a quadrupole-TOF instrument--that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/instrumentação , Acetilação , Bases de Dados de Proteínas , Proteínas de Escherichia coli/química , Glicosilação , Espectrometria de Massas/métodos , Nanotecnologia , Ribonucleases/química , Sensibilidade e EspecificidadeRESUMO
Legionella pneumophila, the causal agent of Legionnaires' disease, is an intracellular parasite and invades and proliferates within different eukaryotic cells, including human alveolar macrophages. After several 100-fold multiplication within host cells, the pathogens are released for new invasion by induction of apoptosis or necrosis. Here we report that L. pneumophila produces a glucosyltransferase, which selectively modifies an approximately 50-kDa mammalian protein by using UDP-glucose as a cosubstrate. MS analysis identified the protein substrate as the mammalian elongation factor (EF)1A. Legionella glucosyltransferase modifies its eukaryotic protein substrate at serine-53, which is located in the GTPase domain of the EF. Glucosylation of EF1A results in inhibition of eukaryotic protein synthesis and death of target cells. Our findings show a mode of inhibition of protein synthesis by microbial pathogens and offer a perspective for understanding of the host-pathogen interaction of L. pneumophila.
Assuntos
Glucosiltransferases/metabolismo , Legionella pneumophila/enzimologia , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células CACO-2 , Bovinos , Linhagem Celular , Glucosiltransferases/genética , Humanos , Técnicas In Vitro , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
MSL-2 (male-specific lethal 2) is the limiting component of the Drosophila dosage compensation complex (DCC) that specifically increases transcription from the male X chromosome. Ectopic expression of MSL-2 protein in females causes DCC assembly on both X chromosomes and lethality. Inhibition of MSL-2 synthesis requires the female-specific protein sex-lethal (SXL), which binds to the msl-2 mRNA 5' and 3' untranslated regions (UTRs) and blocks translation through distinct UTR-specific mechanisms. Here, we purify translationally silenced msl-2 mRNPs and identify UNR (upstream of N-ras) as a protein recruited to the 3' UTR by SXL. We demonstrate that SXL requires UNR as a corepressor for 3'-UTR-mediated regulation, imparting a female-specific function to the ubiquitously expressed UNR protein. Our results reveal a novel functional role for UNR as a translational repressor and indicate that UNR is a key component of a "fail-safe" dosage compensation regulatory system that prevents toxic MSL-2 synthesis in female cells.