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The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.
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Proteínas de Arabidopsis , Arabidopsis , Sementes , Ácido alfa-Linolênico , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sementes/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Ácido alfa-Linolênico/metabolismo , Regulação da Expressão Gênica de Plantas , Relógios Circadianos/genética , Ácidos Graxos/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Metabolismo dos LipídeosRESUMO
Fusarium oxysporum is the one of the most common and impactful pathogens of flax. Cultivars of flax that show resistance to this pathogen have previously been identified. To better understand the mechanisms that are responsible for this resistance, we conducted time-lapse analysis of one susceptible and one resistant cultivar over a two-week period following infection. We also monitored changes in some metabolites. The susceptible cultivar showed a strong onset of symptoms from 6 to 8 days after inoculation, which at this time point, was associated with changes in metabolites in both cultivars. The resistant cultivar maintained its height and normal photosynthetic capacity but showed a reduced growth of its secondary stems. This resistance was correlated with the containment of the pathogen at the root level, and an increase in some metabolites related to the phenylpropanoid pathway.
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Current agricultural practices rely heavily on synthetic fertilizers, which not only consume a lot of energy but also disrupt the ecological balance. The overuse of synthetic fertilizers has led to soil degradation. In a more sustainable approach, alternative methods based on biological interactions, such as plant growth-promoting bacteria (PGPRs), are being explored. PGPRs, which include both symbiotic and free-living bacteria, form mutualistic relationships with plants by enhancing nutrient availability, producing growth regulators, and regulating stress responses. This study investigated the potential of Sphingomonas sediminicola Dae20, an α-Proteobacteria species commonly found in the rhizosphere, as a beneficial PGPR. We observed that S. sediminicola Dae20 stimulated the root system and growth of three different plant species in the Brassicaceae family, including Arabidopsis thaliana, mustard, and rapeseed. The bacterium produced auxin, nitric oxide, siderophores and showed ACC deaminase activity. In addition to activating an auxin response in the plant, S. sediminicola Dae20 exhibited the ability to modulate other plant hormones, such as abscisic acid, jasmonic acid and salicylic acid, which are critical for plant development and defense responses. This study highlights the multifunctional properties of S. sediminicola Dae20 as a promising PGPR and underscores the importance of identifying effective and versatile beneficial bacteria to improve plant nutrition and promote sustainable agricultural practices.
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Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with a low overall survival rate of less than 10% and limited therapeutic options. Fluctuations in tumor microenvironment pH are a hallmark of PDAC development and progression. Many ion channels are bona fide cellular sensors of changes in pH. Yet, the interplay between the acidic tumor microenvironment and ion channel regulation in PDAC is poorly understood. In this study, we show that acid adaption increases PANC-1 cell migration but attenuates proliferation and spheroid growth, which are restored upon recovery. Moreover, acid adaptation and recovery conditions favor the plasma membrane localization of the pH-sensitive calcium (Ca2+) channel transient receptor potential C1 (TRPC1), TRPC1-mediated Ca2+ influx, channel interaction with the PI3K p85α subunit and calmodulin (CaM), and AKT and ERK1/2 activation. Knockdown (KD) of TRPC1 suppresses cell migration, proliferation, and spheroid growth, notably in acid-recovered cells. KD of TRPC1 causes the accumulation of cells in G0/G1 and G2/M phases, along with reduced expression of CDK6, -2, and -1, and cyclin A, and increased expression of p21CIP1. TRPC1 silencing decreases the basal Ca2+ influx in acid-adapted and -recovered cells, but not in normal pH conditions, and Ca2+ chelation reduces cell migration and proliferation solely in acid adaptation and recovery conditions. In conclusion, acid adaptation and recovery reinforce the involvement of TRPC1 in migration, proliferation, and cell cycle progression by permitting Ca2+ entry and forming a complex with the PI3K p85α subunit and CaM.
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Pancreatic diseases, such as pancreatitis or pancreatic ductal adenocarcinoma, are characterized by the presence of activated pancreatic stellate cells (PSCs). These cells represent key actors in the tumor stroma, as they actively participate in disease development and progression: reprograming these PSCs into a quiescent phenotype has even been proposed as a promising strategy for restoring the hallmarks of a healthy pancreas. Since TRPM7 channels have been shown to regulate hepatic stellate cells proliferation and survival, we aimed to study the role of these magnesium channels in PSC activation and proliferation. PS-1 cells (isolated from a healthy pancreas) were used as a model of healthy PSCs: quiescence or activation were induced using all-trans retinoic acid or conditioned media of pancreatic cancer cells, respectively. The role of TRPM7 was studied by RNA silencing or by pharmacological inhibition. TRPM7 expression was found to be correlated with the activation status of PS-1 cells. TRPM7 expression was able to regulate proliferation through modulation of cell cycle regulators and most importantly p53, via the PI3K/Akt pathway, in a magnesium-dependent manner. Finally, the analysis of TCGA database showed the overexpression of TRPM7 in cancer-associated fibroblasts. Taken together, we provide strong evidences that TRPM7 can be considered as a marker of activated PSCs.
Assuntos
Neoplasias Pancreáticas , Canais de Cátion TRPM , Humanos , Magnésio/metabolismo , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Neoplasias PancreáticasRESUMO
Dysregulation of the transient receptor canonical ion channel (TRPC1) has been found in several cancer types, yet the underlying molecular mechanisms through which TRPC1 impacts pancreatic ductal adenocarcinoma (PDAC) cell proliferation are incompletely understood. Here, we found that TRPC1 is upregulated in human PDAC tissue compared to adjacent pancreatic tissue and this higher expression correlates with low overall survival. TRPC1 is, as well, upregulated in the aggressive PDAC cell line PANC-1, compared to a duct-like cell line, and its knockdown (KD) reduced cell proliferation along with PANC-1 3D spheroid growth by arresting cells in the G1/S phase whilst decreasing cyclin A, CDK2, CDK6, and increasing p21CIP1 expression. In addition, the KD of TRPC1 neither affected Ca2+ influx nor store-operated Ca2+ entry (SOCE) and reduced cell proliferation independently of extracellular calcium. Interestingly, TRPC1 interacted with the PI3K-p85α subunit and calmodulin (CaM); both the CaM protein level and AKT phosphorylation were reduced upon TRPC1 KD. In conclusion, our results show that TRPC1 regulates PDAC cell proliferation and cell cycle progression by interacting with PI3K-p85α and CaM through a Ca2+-independent pathway.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Cálcio/metabolismo , Calmodulina/metabolismo , Carcinoma Ductal Pancreático/genética , Proliferação de Células , Humanos , Neoplasias Pancreáticas/genética , Fosfatidilinositol 3-Quinases/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Neoplasias PancreáticasRESUMO
Activated pancreatic stellate cells (aPSCs), the crucial mediator of pancreatic desmoplasia, are characterized, among others, by high proliferative potential and abundant transforming growth factor ß1 (TGFß1) secretion. Over the past years, the involvement of Ca2+ channels in PSC pathophysiology has attracted great interest in pancreatic cancer research. We, thus, aimed to investigate the role of the Orai1 Ca2+ channel in these two PSC activation processes. Using the siRNA approach, we invalided Orai1 expression and assessed the channel functionality by Ca2+ imaging, the effect on aPSC proliferation, and TGFß1 secretion. We demonstrated the functional expression of the Orai1 channel in human aPSCs and its implication in the store-operated Ca2+ entry (SOCE). Orai1 silencing led to a decrease in aPSC proliferation, TGFß1 secretion, and AKT activation. Interestingly, TGFß1 induced a higher SOCE response by increasing Orai1 mRNAs and proteins and promoted both AKT phosphorylation and cell proliferation, abolished by Orai1 silencing. Together, our results highlight the role of Orai1-mediated Ca2+ entry in human aPSC pathophysiology by controlling cell proliferation and TGFß1 secretion through the AKT signaling pathway. Moreover, we showed a TGFß1-induced autocrine positive feedback loop by promoting the Orai1/AKT-dependent proliferation via the stimulation of Orai1 expression and function.
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Lignans, phenolic plant secondary metabolites, are derived from the phenylpropanoid biosynthetic pathway. Although, being investigated for their health benefits in terms of antioxidant, antitumor, anti-inflammatory and antiviral properties, the role of these molecules in plants remains incompletely elucidated; a potential role in stress response mechanisms has been, however, proposed. In this study, a non-targeted metabolomic analysis of the roots, stems, and leaves of wild-type and PLR1-RNAi transgenic flax, devoid of (+) secoisolariciresinol diglucoside ((+) SDG)-the main flaxseed lignan, was performed using 1H-NMR and LC-MS, in order to obtain further insight into the involvement of lignan in the response of plant to osmotic stress. Results showed that wild-type and lignan-deficient flax plants have different metabolic responses after being exposed to osmotic stress conditions, but they both showed the capacity to induce an adaptive response to osmotic stress. These findings suggest the indirect involvement of lignans in osmotic stress response.
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Cromatografia Líquida , Linho/metabolismo , Lignanas/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metabolômica , Pressão Osmótica , Linho/química , FenótipoRESUMO
The rapeseed crop (Brassica napus) has to cope with fungal diseases that significantly impacts yields. In particular, the fungal pathogen Leptosphaeria maculans, the causal agent of blackleg disease (also named Phoma stem canker), is a worldwide issue to this crop. Considering environmental concerns, it is essential to propose alternative natural compounds for rapeseed crop protection to reduce chemical fungicide use. Here we report data showing the efficacy of semipurified rhamnolipid (RL) mixes from bacterial origin to protect rapeseed against L. maculans at early stages of infection in controlled conditions. In addition, we show that RL solutions have excellent adhesion properties when sprayed onto rapeseed leaves, without adding any adjuvant. We demonstrate that RL mixes display direct antimycelial properties against the pathogen and stimulate plant defense responses in rapeseed. Our results validate, a preventive action of low RL concentrations to protect rapeseed against L. maculans and a curative effect in specific conditions when applied after the inoculation of the pathogen spores. Semipurified RL mixes therefore appear to be real cost-effective compounds that could be used in fields as biocontrol products to fight L. maculans early infections of rapeseed.
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Ascomicetos , Brassica napus , Infecções , Glicolipídeos , Humanos , Doenças das PlantasRESUMO
Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists-mainly for technical reasons-on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea Indeed, in contrast to previous reports, most OGs were acetyl- and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors.
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Arabidopsis/metabolismo , Botrytis/patogenicidade , Ácidos Hexurônicos/metabolismo , Proteínas de Arabidopsis/metabolismo , Botrytis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Pectinas/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Poligalacturonase/metabolismo , Transdução de SinaisRESUMO
The external seed coat cell layer of certain species is specialized in the production and extrusion of a polysaccharide matrix called mucilage. Variations in the content of the released mucilage have been mainly associated with genetically regulated physiological modifications. Understanding the mucilage extrusion process in crop species is of importance to gain deeper insight into the complex cell wall biosynthesis and dynamics. In this study, we took advantage of the varying polysaccharide composition and the size of the flax mucilage secretory cells (MSCs) to study mucilage composition and extrusion in this species of agricultural interest. We demonstrate herein that flax MSCs are structured in four superimposed layers and that rhamnogalacturonans I (RG I) are firstly synthesized, in the upper face, preceding arabinoxylan and glucan synthesis in MSC lower layers. Our results also reveal that the flax mucilage release originates from inside MSC, between the upper and deeper layers, the latter collaborating to trigger polysaccharide expansion, radial cell wall breaking and mucilage extrusion in a peeling fashion. Here, we provide evidence that the layer organization and polysaccharide composition of the MSCs regulate the mucilage release efficiency like a peeling mechanism. Finally, we propose that flax MSCs may represent an excellent model for further investigations of mucilage biosynthesis and its release.
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In the context of the growing demand for α-linolenic acid due to its high nutritional value as a polyunsaturated fatty acid, we have investigated the contribution of 2-lysophosphatidic acid acyltransferase (LPAAT) enzymes from flax (Linum usitatissimum) in the accumulation of α-linolenic acid into the oil fraction of flax seed. We have isolated the cDNAs encoding three class A microsomal LPAAT2 isoforms from developing flax seeds. The three isoforms, denominated LPAAT2A, LPAAT2A2 and LPAAT2B, are able to complement the LPAAT deficient JC201 E. coli mutant, confirming their functionality. We have performed enzymatic assays showing that the specific activity of the LPAAT2A isoform is significantly higher than that of the LPAAT2A2 and LPAAT2B toward the unsaturated oleic, linoleic and linolenic acids. Moreover, LPAAT2A presents in vitro a high specificity and selectivity for linoleic and linolenic acids as compared to saturated fatty acids. The three isoforms are expressed during all the stages of seed development and in stem and leaf tissues, as shown by an analysis of the transcription level of the corresponding genes. The heterologous expression of LPAAT2A in Arabidopsis seeds leads to an increase in the accumulation of linoleic and linolenic acids in the oil fraction of the seeds from two transgenic lines.
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Aciltransferases/metabolismo , Linho/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Sementes/metabolismo , Ácido alfa-Linolênico/metabolismo , Aciltransferases/genética , Linho/genética , Regulação da Expressão Gênica de Plantas/genética , Sementes/genéticaRESUMO
Modulation of the membrane permeability through a decrease in porin-mediated antibiotic entry and/or an increase in antibiotic efflux is one of the resistance mechanisms to antibiotics evolved by Gram-negative bacteria. To assess whether the outer membrane porin OprD and Resistance-Nodulation-Division (RND) efflux pumps were similarly expressed in 33 ciprofloxacin-resistant clinical strains of Pseudomonas aeruginosa and in 30 non-clinical strains originating from the hospital environment (mainly waterborne Pseudomonas aeruginosa), the expression of oprD, mexB, mexF, and mexY genes was investigated. Overall, the expression of oprD was not detected by RT-qPCR in 14 (22%) strains and underexpressed in 35 (56%) more. No significant difference in oprD expression was detected between clinical and non-clinical strains. As for efflux pumps, 23 (70%) of the clinical strains overexpressed at least one of the tested RND genes. Overexpression of mexB, mexF and mexY was detected in 27, 12, and 45% of the clinical strains, respectively. In the 30 non-clinical strains, no overexpression could be found for mexB, mexF, or mexY. On the contrary, a global underexpression of the tested efflux pump genes was recorded. In both clinical and environmental strains, a positive correlation was found between the expressions of oprD and mexB. Similarly, the expressions of oprD and mexF were positively correlated. This result contrasts with the inverse correlation between both MexAB-OprM/MexEF-OprN and OprD previously described in carbapenem-resistant P. aeruginosa strains. The only positive correlation between phenotypic ciprofloxacin minimum inhibitory concentrations (MICs) and the expression of efflux pump gene was witnessed with mexY (analysis on pooled results for clinical and environmental strains). However, in clinical strains, no statistically significant link could be found between the degree of reduction in ciprofloxacin MICs witnessed with Phenylalanine-Arginine ß-naphthylamide (PAßN) and the expression of any of the 3 RND genes tested.
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AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3-1 loss-of-function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5-kDa protein in the different organs of 4-day-old Atpme3-1 seedlings grown in the dark, as well as in 6-week-old mutant plants. Microarray analysis showed significant down-regulation of the genes encoding several pectin-degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4-day-old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up-regulated. Additional analysis by RT-qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3-1, in which precursors of the α- and ß-subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles.
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Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/fisiologia , Proteínas de Armazenamento de Sementes/metabolismo , Plântula/crescimento & desenvolvimento , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Parede Celular/enzimologia , Genes de Plantas/fisiologia , Germinação , Análise de Sequência com Séries de Oligonucleotídeos , Plântula/metabolismoRESUMO
Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme48-/- pollen grains. In contrast, the PME activity was lower in pme48-/-, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48-/- with the optimum germination medium supplemented with 2.5 mm calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca(2+) necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/metabolismo , Germinação , Pólen/enzimologia , Pólen/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cálcio/farmacologia , Hidrolases de Éster Carboxílico/genética , Meios de Cultura/farmacologia , Esterificação/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Homozigoto , Mutação/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Pectinas/metabolismo , Fenótipo , Pólen/genética , Tubo Polínico/efeitos dos fármacos , Tubo Polínico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
⢠Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. ⢠A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. ⢠We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. ⢠Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Hidrolases de Éster Carboxílico/química , Parede Celular/enzimologia , Ativação Enzimática , Esterificação , Isoenzimas/química , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação/genética , Pectinas/metabolismo , Fenótipo , Feixe Vascular de Plantas/enzimologia , Regiões Promotoras Genéticas/genética , Transporte ProteicoRESUMO
Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative 'housekeeping' genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.
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Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Northern Blotting , Análise de Sequência com Séries de Oligonucleotídeos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.
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Perfilação da Expressão Gênica , Genes de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Arabidopsis/genética , Populus/genética , Reprodutibilidade dos TestesRESUMO
In experimental animals, prenatal diazepam exposure has clearly been associated with behavioral disturbances. Its impact on newborn breathing has not been documented despite potential deleterious consequences for later brain development. We addressed this issue in neonatal rats (0-2 d) born from dams, which consumed 2 mg/kg/d diazepam via drinking fluid throughout gestation. In vivo, prenatal diazepam exposure significantly altered the normoxic-breathing pattern, lowering breathing frequency (105 vs. 125 breaths/min) and increasing tidal volume (16.2 vs. 12.7 mL/kg), and the ventilatory response to hypoxia, inducing an immediate and marked decrease in tidal volume (-30%) absent in controls. In vitro, prenatal diazepam exposure significantly increased the respiratory-like frequency produced by pontomedullary and medullary preparations (+38% and +19%, respectively) and altered the respiratory-like response to application of nonoxygenated superfusate. Both in vivo and in vitro, the recovery from oxygen deprivation challenges was delayed by prenatal diazepam exposure. Finally, real-time PCR showed that prenatal diazepam exposure affected mRNA levels of alpha1 and alpha2 GABAA receptor subunits and of A1 and A2A adenosine receptors in the brainstem. These mRNA changes, which are region-specific, suggest that prenatal diazepam exposure interferes with developmental events whose impact on the respiratory system maturation deserves further studies.
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Diazepam/toxicidade , Moduladores GABAérgicos/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Receptores de GABA-A/efeitos dos fármacos , Receptores Purinérgicos P1/efeitos dos fármacos , Respiração/efeitos dos fármacos , Centro Respiratório/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Feminino , Hipóxia/genética , Hipóxia/fisiopatologia , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor A1 de Adenosina/efeitos dos fármacos , Receptor A2A de Adenosina/efeitos dos fármacos , Receptores de GABA-A/genética , Receptores Purinérgicos P1/genética , Centro Respiratório/crescimento & desenvolvimento , Centro Respiratório/metabolismo , Mecânica Respiratória/efeitos dos fármacos , Volume de Ventilação Pulmonar/efeitos dos fármacosRESUMO
Diazepam (DZP) enhances GABA action at GABA(A) receptor. Chronic prenatal administration of DZP delays the appearance of neonatal reflexes. We examined whether maternal intake of DZP might affect respiratory control system in newborn rats (0-3 day-old). This study was conducted on unrestrained animals and medulla-spinal cord preparations. In addition, the level of expression of the genes encoding for the alpha1 and alpha2 subunits of the GABA(A) receptor was assessed by quantitative real-time RT-PCR. In rats exposed to DZP, the respiratory frequency was significantly lower and the tidal volume higher than in controls with no significant alteration of the minute ventilation. The recovery from moderate hypoxia was delayed compared to controls. The respiratory-like frequency of medullary spinal cord preparation from DZP-exposed neonates was higher than in the control group. Acute applications of DZP (1 microM) to these preparations increased respiratory-like frequency in both groups, but this facilitation was attenuated following prenatal DZP exposure. The present data indicate that prenatal exposure to DZP alters both eupneic breathing and the respiratory response to hypoxia. These effects might partly be ascribed to the down-regulation of the expression of genes encoding GABA(A) receptor subunits. On the other hand, the effects of DZP exposure on reduced preparations suggested changes in the GABA(A) receptor efficiency and/or disruption of the normal development of the medullary respiratory network.