RESUMO
Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of many Gram-negative bacteria, providing a barrier against the entry of toxic molecules. In Escherichia coli, LPS is exported to the cell surface by seven essential proteins (LptA-G) that form a transenvelope complex. At the inner membrane, the ATP-binding cassette (ABC) transporter LptB2FG associates with LptC to power LPS extraction from the membrane and transfer to the periplasmic LptA protein, which is in complex with the OM translocon LptDE. LptC interacts both with LptB2FG and LptADE to mediate the formation of the transenvelope bridge and regulates the ATPase activity of LptB2FG. A genetic screen has previously identified suppressor mutants at a residue (R212) of LptF that are viable in the absence of LptC. Here, we present in vivo evidence that the LptF R212G mutant assembles a six-protein transenvelope complex in which LptA mediates interactions with LptF and LptD in the absence of LptC. Furthermore, we present in vitro evidence that the mutant LptB2FG complexes restore the regulation of ATP hydrolysis as it occurs in the LptB2FGC complex to achieve wild-type efficient coupling of ATP hydrolysis and LPS movement. We also show the suppressor mutations restore the wild-type levels of LPS transport both in vivo and in vitro, but remarkably, without restoring the affinity of the inner membrane complex for LptA. Based on the sensitivity of lptF suppressor mutants to selected stress conditions relative to wild-type cells, we show that there are additional regulatory functions of LptF and LptC that had not been identified. IMPORTANCE The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli, by seven essential proteins. LptC is the unconventional regulatory subunit of the LptB2FGC ABC transporter, involved in coordinating energy production and LPS transport. Surprisingly, despite being essential for bacterial growth, LptC can be deleted, provided that a specific residue in the periplasmic domain of LptF is mutated and LptA is overexpressed. Here, we apply biochemical techniques to investigate the suppression mechanism. The data produced in this work disclose an unknown regulatory function of LptF in the transporter that not only expands the knowledge about the Lpt complex but can also be targeted by novel LPS biogenesis inhibitors.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Supressão Genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Transporte Biológico/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Improvement of drought tolerance of crops is a great challenge in conditions of increasing climate change. This report describes that the silencing of the synaptotagmin-5 (OsSYT-5) gene encoding the rice Ca2+ sensing protein with a C2 domain led to a significant improvement of rice tolerance to water deficit stress. Transgenic lines with suppressed expression of the OsSYT-5 gene exhibited an enhanced photosynthetic rate but reduced stomatal conductance and transpiration during water deficit stress. The abscisic acid (ABA) content under both normal and drought conditions was elevated in the leaves of the transgenic rice as compared to the wild type. The silencing of the OsSYT-5 gene affected the expression of several genes associated with ABA-related stress signaling in the transgenic rice plants. In the water deficit experiment, the transgenic lines with a silenced OsSYT-5 gene exhibited symptoms of drought stress seven days later than the wild type. Transgenic lines with suppressed OsSYT-5 gene expression exhibited higher pollen viability and produced more grains compared to the wild type at both normal and drought stress conditions.
Assuntos
Adaptação Fisiológica/genética , Secas , Inativação Gênica , Genes de Plantas , Oryza/genética , Oryza/fisiologia , Ácido Abscísico/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Oryza/anatomia & histologia , Fenótipo , Fotossíntese/genética , Folhas de Planta/genética , Folhas de Planta/fisiologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/fisiologia , Estresse Fisiológico/genética , Supressão Genética , Regulação para Cima/genéticaRESUMO
Several rare neurodegenerative diseases, including chorea acanthocytosis, are caused by mutations in the VPS13A-D genes. Only symptomatic treatments for these diseases are available. Saccharomyces cerevisiae contains a unique VPS13 gene and the yeast vps13Δ mutant has been proven as a suitable model for drug tests. A library of drugs and an in-house library of natural compounds and their derivatives were screened for molecules preventing the growth defect of vps13Δ cells on medium with sodium dodecyl sulfate (SDS). Seven polyphenols, including the iron-binding flavone luteolin, were identified. The structure-activity relationship and molecular mechanisms underlying the action of luteolin were characterized. The FET4 gene, which encodes an iron transporter, was found to be a multicopy suppressor of vps13Δ, pointing out the importance of iron in response to SDS stress. The growth defect of vps13Δ in SDS-supplemented medium was also alleviated by the addition of iron salts. Suppression did not involve cell antioxidant responses, as chemical antioxidants were not active. Our findings support that luteolin and iron may target the same cellular process, possibly the synthesis of sphingolipids. Unveiling the mechanisms of action of chemical and genetic suppressors of vps13Δ may help to better understand VPS13A-D-dependent pathogenesis and to develop novel therapeutic strategies.
Assuntos
Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Luteolina/farmacologia , Fármacos Neuroprotetores/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Produtos Biológicos/química , Proliferação de Células/efeitos dos fármacos , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Luteolina/química , Fármacos Neuroprotetores/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Supressão GenéticaRESUMO
Mutant screens remain among the most powerful unbiased methods for identifying key genes in a pathway or process of interest. However, mutants impacting pollen function pose special challenges due to their genetic behavior. Here we describe an approach for isolating pollen mutants based on screening for suppressors of a low pollen fertility starting genotype. By identifying suppressor mutants with improved pollen fertility, we are able to identify new genes which are functionally relevant to pathway(s) causing low seed set in the original background. With this method, the low fertility of the genetic background may be due to one or more mutations or transgenes disrupting any aspect of pollen development or function. Furthermore, screening for improved pollen fertility biases toward recovery of the desired mutants due to their enhanced male transmission. The causative mutation is cloned using next-generation sequencing. The procedure uses both genetic and bioinformatics approaches to ultimately yield a very small list of candidate causative mutations speeding the transition from mutant phenotype to underlying gene.
Assuntos
Clonagem Molecular/métodos , Infertilidade das Plantas/genética , Pólen/genética , Supressão Genética , Arabidopsis , Cruzamentos Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pólen/fisiologia , Análise de Sequência de DNA/métodosRESUMO
Dwarfism and drought tolerance are 2 valuable traits in breeding of many crops. In this study, we report the novel physiological roles of cholesterol in regulation of plant growth and drought tolerance. Compared with the wild type, sterol-C24-methyltransferase 1 (SMT1) gene transcript was greatly reduced in a bermudagrass mutant with dwarfism and enhanced drought tolerance, accompanied with cholesterol accumulation, elevated transcript levels of a small group of genes including SAMDC, and increased concentrations of putrescine (Put), spermidine (Spd), and spermine (Spm). Knock-down of OsSMT1 expression by RNA interference resulted in similar phenotypic changes in transgenic rice. Moreover, exogenously applied cholesterol also led to elevated transcripts of a similar set of genes, higher levels of Put, Spd, and Spm, improved drought tolerance, and reduced plant height in both bermudagrass and rice. We revealed that it is Spm, but not Spd, that is responsible for the height reduction in bermudagrass and rice. In conclusion, we suggest that cholesterol induces expression of SAMDC and leads to dwarfism and elevated drought tolerance in plants as a result of the promoted Spd and Spm synthesis.
Assuntos
Adaptação Fisiológica , Colesterol/metabolismo , Cynodon/anatomia & histologia , Secas , Oryza/anatomia & histologia , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Supressão Genética , Adaptação Fisiológica/genética , Cynodon/genética , Cynodon/fisiologia , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Mutação/genética , Oryza/genética , Plantas Geneticamente Modificadas , Poliaminas/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Potato cyst nematode Globodera rostochiensis is an obligate parasite of solanaceous plants, triggering metabolic and morphological changes in roots which may result in substantial crop yield losses. Previously, we used the cDNA-AFLP to study the transcriptional dynamics in nematode infected tomato roots. Now, we present the rescreening of already published, upregulated transcript-derived fragment dataset using the most current tomato transcriptome sequences. Our reanalysis allowed to add 54 novel genes to 135, already found as upregulated in tomato roots upon G. rostochiensis infection (in total - 189). We also created completely new catalogue of downregulated sequences leading to the discovery of 76 novel genes. Functional classification of candidates showed that the 'wound, stress and defence response' category was enriched in the downregulated genes. We confirmed the transcriptional dynamics of six genes by qRT-PCR. To place our results in a broader context, we compared the tomato data with Arabidopsis thaliana, revealing similar proportions of upregulated and downregulated genes as well as similar enrichment of defence related transcripts in the downregulated group. Since transcript suppression is quite common in plant-nematode interactions, we assessed the possibility of miRNA-mediated inverse correlation on several tomato sequences belonging to NB-LRR and receptor-like kinase families. The qRT-PCR of miRNAs and putative target transcripts showed an opposite expression pattern in 9 cases. These results together with in silico analyses of potential miRNA targeting to the full repertoire of tomato R-genes show that miRNA mediated gene suppression may be a key regulatory mechanism during nematode parasitism.
Assuntos
Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Solanum lycopersicum/genética , Solanum lycopersicum/parasitologia , Tylenchoidea/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Animais , Arabidopsis/genética , Sequência de Bases , Resistência à Doença , Perfilação da Expressão Gênica , Genes de Plantas , Raízes de Plantas/genética , Proteínas Quinases/genética , Solanum tuberosum/genética , Supressão Genética , Transcriptoma/genéticaRESUMO
Cell-to-cell adhesion in plants is mediated by the cell wall and the presence of a pectin-rich middle lamella. However, we know very little about how the plant actually controls and maintains cell adhesion during growth and development and how it deals with the dynamic cell wall remodeling that takes place. Here we investigate the molecular mechanisms that control cell adhesion in plants. We carried out a genetic suppressor screen and a genetic analysis of cell adhesion-defective Arabidopsis thaliana mutants. We identified a genetic suppressor of a cell adhesion defect affecting a putative O-fucosyltransferase. Furthermore, we show that the state of cell adhesion is not directly linked with pectin content in the cell wall but instead is associated with altered pectin-related signaling. Our results suggest that cell adhesion is under the control of a feedback signal from the state of the pectin in the cell wall. Such a mechanism could be necessary for the control and maintenance of cell adhesion during growth and development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/enzimologia , Fucosiltransferases/metabolismo , Arabidopsis/genética , Adesão Celular , Parede Celular/metabolismo , Genes de Plantas , Testes Genéticos , Complexo de Golgi/metabolismo , Modelos Biológicos , Mutação/genética , Pectinas/metabolismo , Transdução de Sinais , Especificidade por Substrato , Supressão GenéticaRESUMO
Lipid-derived reactive carbonyl species (RCS) possess electrophilic moieties and cause oxidative stress by reacting with cellular components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the detoxification of lipid-derived RCS, especially α,ß-unsaturated carbonyls. In this study, we aimed to evaluate the physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA), and RNA interference knockdown, aor (RNAi), lines. We found that both aor mutants showed smaller plant sizes than wild-type plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wild-type and aor (RNAi) plants showed similar CO2 assimilation rates; however, the respiration rate was lower in aor (RNAi) than in wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation during the night was suppressed in aor (RNAi). In contrast, the phenotype of aor (RNAi) was rescued when aor (RNAi) plants were grown under constant light conditions. These results indicate that the smaller plant sizes observed in aor mutants grown under day/night cycle conditions were attributable to the decrease in carbon utilization during the night. Here, we propose that the detoxification of lipid-derived RCS by AtAOR in chloroplasts contributes to the protection of dark respiration and supports plant growth during the night.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Carbono/metabolismo , Cloroplastos/enzimologia , Escuridão , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/metabolismo , Oxirredutases/metabolismo , Folhas de Planta/enzimologia , Supressão Genética , Acroleína/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Respiração Celular/efeitos da radiação , Clorofila/metabolismo , Cloroplastos/efeitos da radiação , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Mutação/genética , Nitrogênio/metabolismo , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/genética , Fenótipo , Fotossíntese , Extratos Vegetais/metabolismo , Folhas de Planta/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Amido/metabolismoRESUMO
Amber suppression is a useful method of genetically incorporating a non-natural amino acid (NAA) into a protein during translation by utilizing an NAA-charged amber suppressor tRNA (sup-tRNA). A wheat germ extract (WGE) is suitable for this method by virtue of its high productivity and versatility in addition to its advantages as a cell-free translation system. However, in spite of this high potential, a genetic NAA incorporation system in WGE has not been sufficiently optimized in terms of sup-tRNAs, in contrast to that in E. coli and its cell extracts. We herein rationally optimized amber sup-tRNAs to efficiently incorporate a model NAA, p-acetyl-phenylalanine (AcPhe), into a protein in WGE, via flexizyme-based aminoacylation. The optimized sup-tRNA (named tLys-opt) that was pre-charged with AcPhe exclusively yielded up to 220 µg mL(-1) of AcPhe-incorporated protein (yellow fluorescent protein, YPet) under the optimal conditions. This high productivity is comparable to the best reported yield of a similar NAA-incorporated protein synthesized with an engineered aminoacyl-tRNA synthetase/sup-tRNA pair in WGE, despite the fact that tLys-opt that has released AcPhe was not reused at all in this study. The results clearly show both the necessity of optimizing sup-tRNAs for efficient NAA incorporation and the validity of our strategy for their optimization. Because the optimization strategy described here is expected to be applicable not only to amber sup-tRNAs for other NAAs but also to ones used in other acylation methods, it would facilitate the synthesis of large amounts of various types of NAA-incorporated proteins in WGE.
Assuntos
Fenilalanina/análogos & derivados , Extratos Vegetais/química , Biossíntese de Proteínas , Proteínas/química , RNA de Transferência/genética , Supressão Genética , Triticum/química , Estrutura Molecular , Fenilalanina/genética , Fenilalanina/metabolismo , Proteínas/metabolismoRESUMO
We have developed a novel type of biofunction-assisted aptasensor that harnesses ligand-dependent 3' processing of a premature amber suppressor tRNA and the subsequent amber suppression of a reporter gene in a wheat germ extract.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Extratos Vegetais/genética , RNA de Transferência/química , Teofilina/análise , Triticum/genética , Vasodilatadores/análise , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Genes Reporter , Extratos Vegetais/química , RNA de Transferência/genética , Supressão Genética , Triticum/químicaRESUMO
PURPOSE: Curcumin, a natural phytochemical, exhibits potent anticancer activities. Here, we sought to determine the molecular mechanisms underlying the cytotoxic effects of curcumin against human non-small cell lung cancer (NSCLC) cells. METHODS: MTT assay and annexin-V/PI staining were used to analyze the effects of curcumin on the proliferation and apoptosis of A549 cells. The expression of microRNA-21 in curcumin-treated A549 cells was measured by quantitative real-time polymerase chain reaction assay. The protein level of phosphatase and tensin homolog (PTEN), a putative target of microRNA-21, was determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA was performed to modulate the expression of microRNA-21 and PTEN under the treatment of curcumin. RESULTS: Curcumin at 20-40 μM inhibited cell proliferation and induced apoptosis in A549 cells. Curcumin treatment produced a dose-dependent and significant (P < 0.05) suppression of microRNA-21 expression, compared to untreated A549 cells. Moreover, the protein level of PTEN, a putative target of microRNA-21, was significantly elevated in curcumin-treated A549 cells, as determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA significantly (P < 0.05) reversed the growth suppression and apoptosis induction by curcumin, compared to corresponding controls. CONCLUSIONS: Our data suggest a novel molecular mechanism in which inhibition of microRNA-21 and upregulation of PTEN mediate the anticancer activities of curcumin in NSCLC cells. Suppression of microRNA-21 may thus have therapeutic benefits against this malignancy (AU)
No disponible
Assuntos
Humanos , Masculino , Feminino , Curcumina/metabolismo , Curcumina/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Apoptose , Supressão Genética/efeitos dos fármacos , Genes Supressores de Tumor , Proteína Supressora de Tumor p53/efeitos dos fármacos , Fitoterapia/métodos , RNA , RNA Neoplásico , Proliferação de Células/efeitos dos fármacos , Supressão GenéticaRESUMO
Magnesium is the most abundant divalent metal in cells and is required for many structural and enzymatic functions. For bacteria, at least three families of proteins function as magnesium transporters. In recent years, it has been shown that a subset of these transport proteins is regulated by magnesium-responsive genetic control elements. In this study, we investigated the cellular requirements for magnesium homeostasis in the model microorganism Bacillus subtilis. Putative magnesium transporter genes were mutationally disrupted, singly and in combination, in order to assess their general importance. Mutation of only one of these genes resulted in strong dependency on supplemental extracellular magnesium. Notably, this transporter gene, mgtE, is known to be under magnesium-responsive genetic regulatory control. This suggests that the identification of magnesium-responsive genetic mechanisms may generally denote primary transport proteins for bacteria. To investigate whether B. subtilis encodes yet additional classes of transport mechanisms, suppressor strains that permitted the growth of a transporter-defective mutant were identified. Several of these strains were sequenced to determine the genetic basis of the suppressor phenotypes. None of these mutations occurred in transport protein homologues; instead, they affected housekeeping functions, such as signal recognition particle components and ATP synthase machinery. From these aggregate data, we speculate that the mgtE protein provides the primary route of magnesium import in B. subtilis and that the other putative transport proteins are likely to be utilized for more-specialized growth conditions.
Assuntos
Antiporters/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Magnésio/metabolismo , Antiporters/genética , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Técnicas de Inativação de Genes , Supressão GenéticaRESUMO
Polyamines play very important role in various cellular metabolic functions, including floral induction, floral differentiation and fertility regulation. In the present study, S-adenosylmethionine decarboxylase (SAMDC), a key gene involved in polyamine biosynthesis, has been targeted in tapetal tissue of tomato using RNAi to examine its effect on tapetum development and pollen viability. The target SAMDC gene fragments of three homologues were cloned in a hairpin RNA construct under the control of tapetal-specific A9 promoter, which was used to generate several RNAi tomato plants. These RNAi lines expressed the intended small interfering RNAs in the anther and showed the aborted and sterile pollen exhibiting shrunken and distorted morphology. These RNAi tomato plants having sterile pollen, failed to set fruits but female fertility of the plants remained unaffected as cross pollination resulted in fruit setting. Expression profiling of SAMDC genes showed considerable decrease in transcripts of SAMDC1 (5-8 fold) and SAMDC2 and SAMDC3 (2-3 fold) in the anthers of RNAi plants. The other polyamine biosynthesis genes, ADC and SPDSYN exhibited ~1.5 fold decrease in their transcript levels. Presence of siRNA molecules specific to SAMDC homologues in anther and tapetal-specific activity of A9 promoter as shown with GUS reporter system of RNAi plants suggested down-regulation of the target genes in tapetum by RNAi. These observations indicate the importance of SAMDC, in turn polyamines in pollen development, and thus tapetum-specific down-regulation of SAMDC genes using RNAi can be used for developing male sterile plants.
Assuntos
Adenosilmetionina Descarboxilase/genética , Genes de Plantas/genética , Infertilidade das Plantas/genética , Pólen/enzimologia , Interferência de RNA , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Arabidopsis/genética , Sequência de Bases , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/ultraestrutura , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/ultraestrutura , Poliaminas/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos , Supressão GenéticaRESUMO
Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton-pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H(+) -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531 and 595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.
Assuntos
Candida albicans/enzimologia , Expressão Gênica , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Candida albicans/genética , Meios de Cultura/química , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/isolamento & purificação , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , ATPases Translocadoras de Prótons/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Supressão GenéticaRESUMO
BACKGROUND: The Arabidopsis microRNA156 (miR156) regulates 11 members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) family by base pairing to complementary target mRNAs. Each SPL gene further regulates a set of other genes; thus, miR156 controls numerous genes through a complex gene regulation network. Increased axillary branching occurs in transgenic Arabidopsis overexpressing miR156b, similar to that observed in loss-of-function max3 and max4 mutants with lesions in carotenoid cleavage dioxygenases. Arabidopsis miR156b was found to enhance carotenoid levels and reproductive shoot branching when expressed in Brassica napus, suggesting a link between miR156b expression and carotenoid metabolism. However, details of the miR156 regulatory network of SPL genes related to carotenoid metabolism are not known. RESULTS: In this study, an Arabidopsis T-DNA enhancer mutant, sk156, was identified due to its altered branching and trichome morphology and increased seed carotenoid levels compared to wild type (WT) ecovar Columbia. Enhanced miR156b expression due to the 35S enhancers present on the T-DNA insert was responsible for these phenotypes. Constitutive and leaf primodium-specific expression of a miR156-insensitive (mutated) SPL15 (SPL15m) largely restored WT seed carotenoid levels and plant morphology when expressed in sk156. The Arabidopsis native miR156-sensitive SPL15 (SPL15n) and SPL15m driven by a native SPL15 promoter did not restore the WT phenotype in sk156. Our findings suggest that SPL15 function is somewhat redundant with other SPL family members, which collectively affect plant phenotypes. Moreover, substantially decreased miR156b transcript levels in sk156 expressing SPL15m, together with the presence of multiple repeats of SPL-binding GTAC core sequence close to the miR156b transcription start site, suggested feedback regulation of miR156b expression by SPL15. This was supported by the demonstration of specific in vitro interaction between DNA-binding SBP domain of SPL15 and the proximal promoter sequence of miR156b. CONCLUSIONS: Enhanced miR156b expression in sk156 leads to the mutant phenotype including carotenoid levels in the seed through suppression of SPL15 and other SPL target genes. Moreover, SPL15 has a regulatory role not only for downstream components, but also for its own upstream regulator miR156b.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes/genética , MicroRNAs/metabolismo , Mutação/genética , Fatores de Transcrição/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Carotenoides/metabolismo , DNA Bacteriano/genética , Regulação para Baixo/genética , Genes de Plantas/genética , Dados de Sequência Molecular , Mutagênese Insercional/genética , Motivos de Nucleotídeos/genética , Fenótipo , Caules de Planta/anatomia & histologia , Caules de Planta/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Supressão Genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , TransgenesRESUMO
BACKGROUND: While there is now a significant body of research correlating apple (Malus x domestica) fruit softening with the cell wall hydrolase ENDO-POLYGALACTURONASE1 (PG1), there is currently little knowledge of its physiological effects in planta. This study examined the effect of down regulation of PG1 expression in 'Royal Gala' apples, a cultivar that typically has high levels of PG1, and softens during fruit ripening. RESULTS: PG1-suppressed 'Royal Gala' apples harvested from multiple seasons were firmer than controls after ripening, and intercellular adhesion was higher. Cell wall analyses indicated changes in yield and composition of pectin, and a higher molecular weight distribution of CDTA-soluble pectin. Structural analyses revealed more ruptured cells and free juice in pulled apart sections, suggesting improved integrity of intercellular connections and consequent cell rupture due to failure of the primary cell walls under stress. PG1-suppressed lines also had reduced expansion of cells in the hypodermis of ripe apples, resulting in more densely packed cells in this layer. This change in morphology appears to be linked with reduced transpirational water loss in the fruit. CONCLUSIONS: These findings confirm PG1's role in apple fruit softening and suggests that this is achieved in part by reducing cellular adhesion. This is consistent with previous studies carried out in strawberry but not with those performed in tomato. In apple PG1 also appears to influence other fruit texture characters such as juiciness and water loss.
Assuntos
Regulação para Baixo/genética , Frutas/enzimologia , Frutas/fisiologia , Malus/enzimologia , Transpiração Vegetal , Resistência à Tração , Água/metabolismo , Adesão Celular , Parede Celular/metabolismo , Cruzamentos Genéticos , Frutas/genética , Frutas/ultraestrutura , Regulação da Expressão Gênica de Plantas , Malus/genética , Malus/fisiologia , Malus/ultraestrutura , Pectinas/metabolismo , Transpiração Vegetal/genética , Plantas Geneticamente Modificadas , Poligalacturonase/genética , Poligalacturonase/metabolismo , Polimerização , Estações do Ano , Supressão Genética , Ácidos Urônicos/metabolismoRESUMO
Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N(2) fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N(2) fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on ß-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro (33)P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance.
Assuntos
Glycine max/fisiologia , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Nodulação/fisiologia , Nódulos Radiculares de Plantas/metabolismo , Bioensaio , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucuronidase/metabolismo , Hidroponia , Dados de Sequência Molecular , Nitrogênio/metabolismo , Proteínas de Transporte de Fosfato/genética , Fósforo/metabolismo , Proteínas de Plantas/genética , Nodulação/genética , Plantas Geneticamente Modificadas , Interferência de RNA , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/genética , Saccharomyces cerevisiae/metabolismo , Solubilidade , Glycine max/genética , Glycine max/crescimento & desenvolvimento , Glycine max/microbiologia , Supressão GenéticaRESUMO
The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , DNA Helicases/metabolismo , Anemia de Fanconi/metabolismo , Recombinação Homóloga/genética , Meiose/genética , Homologia de Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência de Bases , Troca Genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/química , DNA Helicases/genética , Reparo do DNA/genética , Epistasia Genética , Humanos , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Infertilidade das Plantas/genética , Pólen/citologia , Pólen/genética , Supressão GenéticaRESUMO
The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing the activity of PTOX in the desaturation steps of carotenogenesis. Similar results were obtained when AOX1a was reengineered to target the plastid. Chloroplast-localized AOX2 formed monomers and dimers, reminiscent of AOX regulation in mitochondria. Both AOX2 and AOX1a were present in higher molecular weight complexes in plastid membranes. The presence of these proteins did not generally affect steady state photosynthesis, aside from causing enhanced nonphotochemical quenching in both lines. Because AOX2 was imported into chloroplasts using its own transpeptide, we propose that AOX2 is able to function in chloroplasts to supplement PTOX activity during early events in chloroplast biogenesis. We conclude that the ability of AOX1a and AOX2 to substitute for PTOX in the correct physiological and developmental contexts is a striking example of the capacity of a mitochondrial protein to replace the function of a chloroplast protein and illustrates the plasticity of the photosynthetic apparatus.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Plastídeos/enzimologia , Arabidopsis/genética , Carotenoides/biossíntese , Clorofila/metabolismo , Clorofila A , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Ensaios Enzimáticos , Éxons/genética , Fluorescência , Genes Supressores , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Oxirredutases/química , Oxirredutases/genética , Fotossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plasmídeos/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Frações Subcelulares/enzimologia , Supressão GenéticaRESUMO
In plants and animals, the SCF-type ubiquitin protein ligases play an important role in many different physiological processes by regulating protein stability such as S-RNase-based self-compatibility, flower development, hormone responses and meiosis. This study identified an SlFbf gene in tomato that encodes 381 amino acid residues containing a typical F-box motif and an FBA_1 motif associated proteasome pathway; the transcripts of SlFbf was detected in all the tissues (root, stem, leaf, sepal, petal, stamen, pistil, green fruit, breaker fruit and red fruit), with the highest in stamen specifically during flowering stage; SlFbf responded to gibberellins, abscisic acid and light. Suppressed SlFbf leads to bigger pollen and less seeds showing that SlFbf might have an effect on fertilization through regulating stamen development. These findings provide more information about the functions of Fbf gene family.