1,721,027 research outputs found
Gene expression studies in different genotypes of an ectomycorrhizal fungus require a high number of reliable reference genes
No full textQuantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.</jats:p
Metal-Induced Oxidative Stress and Plant Mitochondria
No full textA general status of oxidative stress in plants caused by exposure to elevated metal concentrations in the environment coincides with a constraint on mitochondrial electron transport, which enhances ROS accumulation at the mitochondrial level. As mitochondria are suggested to be involved in redox signaling under environmental stress conditions, mitochondrial ROS can initiate a signaling cascade mediating the overall stress response, i.e., damage versus adaptation. This review highlights our current understanding of metal-induced responses in plants, with focus on the production and detoxification of mitochondrial ROS. In addition, the potential involvement of retrograde signaling in these processes will be discussed
The in planta proteome of wild type strains of the fire blight pathogen, Erwinia amylovora
No full textErwinia amylovora is a Gram-negative plant pathogen that causes fire blight. This disease affects most members of the Rosaceae family including apple and pear. Here, an infection model is introduced to study proteomic changes in a highly virulent E. amylovora strain upon interaction with its host as compared to a lower virulent strain. For this purpose separate shoots of apple rootstocks were wound-infected and when infection became systemic, bacterial cells were isolated and processed for analysis in a proteomics platform combining 2-D fluorescence difference gel electrophoresis and mass spectrometry. Comparing the proteome of the isolates, significant abundance changes were observed in proteins involved in sorbitol metabolism, amylovoran production as well as in protection against plant defense mechanisms. Furthermore several proteins associated with virulence were more abundant in the higher virulent strain. Changes at the proteome level showed good accordance at the transcript level, as was verified by RT-qPCR. In conclusion, this infection model may be a valuable tool to unravel the complexity of plant-pathogen interactions and to gain insight in the molecular mechanisms associated with virulence of E. amylovora, paving the way for the development of plant-protective interventions against this detrimental disease. Significance: During this research a first time investigation was performed on the proteome of E. amylovora, grown inside a susceptible host plant. This bacterium is the causal agent of fire blight, which can affect most members of the Rosaceae family including apple and pear. To do so, an artificial infection model on shoots of apple rootstocks was optimized and employed. When infection was systemic, bacterial cells were extracted from the plant tissue followed by extraction of the proteins from the bacteria. Further processing of the proteins was done by using a 2-D fluorescence difference gel electrophoresis analysis followed by mass spectrometry. By the use of two strains differing in their virulent ability, we were able to draw conclusions concerning virulence and behavior of different strains inside the host. This research provides a model to investigate plant-pathogen interactions and more importantly, we identified possible new targets for the development of novel control methods against this devastating disease. (C) 2016 Elsevier B.V. All rights reserved.Partial funding by project no 101513 of the Agency of Innovation by Science and Technology (IWT-Flanders, Belgium) is acknowledged. Michelle Holtappels was indebted to the IWT for a predoctoral fellowship. We thank Erik Royackers for technical assistance. We acknowledge the financial support from the Hercules Foundation in the framework of the project R-3986 'LC-MS@UHasselt: Linear Trap Quadrupool-Orbitrap mass spectrometer'. We also acknowledge Greet Clerx, Elke Knoops and Annelies Eyskens for their technical support. Dr. Sacha Bohler is acknowledged for his help with the proteomics procedures. The authors have no conflict of interest
A mutant of the Arabidopsis thaliana LIPOXYGENASE1 gene shows altered signalling and oxidative stress related responses after cadmium exposure
No full textAbstract: Lipoxygenases (LOXes, EC 1.13.11.12) are involved in growth, development and responses to stress. Earlier results suggested a role in stress generation, signalling and/or responses when Arabidopsis thaliana is exposed to cadmium (Cd), and expression of the cytosolic LOX1 was highly upregulated in the roots after Cd exposure. To investigate the involvement of LOX1 in early metal stress responses, three-week-old wild-type and lox1-1 mutant A. thaliana plants were acutely (24 h) exposed to realistic Cd concentrations (5 and 10 mu M) and several oxidative stress and signalling related parameters were studied at transcriptional and biochemical levels. Transcription of several genes encoding ROS producing and scavenging enzymes failed to be induced up to wild-type levels after Cd exposure. Expression of 9-LOX enzymes was inhibited in lox1-1 mutant roots due to lack of functional LOX1 and downregulated LOX5 expression, and the lox1-1 mutation also interfered with the expression of genes involved in jasmonate biosynthesis. LOX1 and RBOHD may be involved in stress signalling from roots to shoots, as the induction of APX2 expression, which is dependent on RBOHD activity, was disrupted in lox1-1 while RBOHD failed to be upregulated. A different pattern of H2O2 production and ascorbate and glutathione levels in lox1-1 mutants after Cd exposure may have indirectly influenced gene expression patterns. Although indirect effects of the lox1-1 mutation on gene expression complicate the determination of exact sensing - signalling - response pathways, the results presented here outline a more refined LOX1 functioning in Cd-induced stress responses that could be used in studies determining the exact involvement of LOX1 in these pathways. (c) 2012 Elsevier Masson SAS. All rights reserved
Cadmium-induced transcriptional and enzymatic alterations related to oxidative stress
No full textThe early antioxidative defence mechanisms were studied in Arabidopsis thaliana by applying a range of realistic Cd concentrations. Our data suggest that a 24 h exposure to 20 mu M CdSO4 is already too toxic to study moderate toxicity, whereas a highly coordinated oxidative stress-related defence response could be observed after Cd application of 5 and 10 mu M. Significant differences in transcript abundance of several genes involved in antioxidative defence were observed. The generation of superoxide seems the main cause of oxidative stress in the roots, whereas in the leaves hydrogen peroxide appears to be an important player. Furthermore, an increased transcript level of lipoxygenase, a potential inducer of oxidative stress, suggests a central role of this gene in causing the Cd-related redox imbalance. Our results show that Cd as a non-redox-active metal induces oxidative stress and indicate that the antioxidative defence system is moderated by the activation of different genes in different organs and cellular compartments.This work was supported by a PhD grant for Karen Smeets
from Hasselt University (tUl-impulsfinanciering-toxicology) and two BOF (Bijzonder onderzoeksfonds) projects of Hasselt University, BOF02G01 and BOF04G01
Effects of uranium and phosphate concentrations on oxidative stress related responses induced in Arabidopsis thaliana
No full textThe production of reactive oxygen species (ROS) and the induction of the antioxidative defense mechanism are very important in heavy metal toxicity. In this study, biological effects induced after uranium contamination were investigated for Arabidopsis thaliana. Three-week-old seedlings were exposed for 4 days to 100 mu M U in an adjusted Hoagland solution. Uranium exposure caused a decreased growth of leaves (38%) and roots (70%) and a modified nutrient profile was observed. Investigation of lipid peroxidation products indicated a significant increase of membrane damage. Important ROS-producing and -scavenging enzymes were studied at transcriptional and protein level to investigate the importance of the ROS-signature in uranium toxicity. Elevated gene expression was observed for NADPH-oxidase, a ROS-producing enzyme. Changes in gene expression for different ROS-scavenging enzymes as Cu/ZnSOD. FeSOD and APX were also observed. Analysis of enzyme capacities showed little effects after uranium contamination. Higher ascorbate levels in uranium exposed leaves suggested an increase of antioxidative defense via the ascorbate-glutathione pathway after uranium exposure. Theoretical calculations indicated rapid formation of uranium-phosphate precipitates if normal phosphate concentrations are used. Precipitation tests recommend the use of 25 mu M P in combination with 100 mu M U to inhibit uranium precipitation. Because this combination was used for uranium toxicity investigation, the influence of this low phosphate concentration on plant growth and oxidative stress had to be evaluated. Minor differences between low phosphate (25 mu M P) and high phosphate ( 100 mu M P) treatments were observed justifying the use of the low phosphate concentration in combination with uranium. (C) 2008 Elsevier Masson SAS. All rights reserved.This work was supported by a PhD grant for Nathalie Vanhoudt
from the Belgian Nuclear Research Centre (SCKCEN)
Involved or not involved - the role of the glutamate receptor 2.5 gene in root development under stress
No full textAnthropogenic activities have become a worldwide problem as they have led to environmental contamination, which is not biodegradable and persistent. The contamination causes and effect on plant roots and because plants lack mobility, they possess high plasticity in root development. Research showed that amino acids and metals induce the same morphogenic response: primary root growth inhibition and lateral root growth. But which genes are involved is still not clear. This research investigates a gene for primary root growth inhibition: the AtGLR2.5 gene, belonging to the family of glutamate receptor-like proteins. This gene has four splicing variants: AtGLR2.5a, AtGLR2.5b, AtGLR2.5c and AtGLR2.5d and unpublished data showed that transformation of a glr2.5 mutant with the AtGLR2.5c cDNA can restore L-glutamate sensitivity. The first hypothesis states that AtGLR2.5c could be involved in the impairment of the primary root growth under L-glutamate stress, and a second hypothesis states its involvement in metal-induced primary root inhibition. The results of this research project indicated a primary root growth inhibition under the L-glutamate, copper, cadmium and zinc treatments. The gene expression of the splicing variant AtGLR2.5c was also measured under the different treatments. Analysing the data showed that the splicing variant AtGLR2.5c was not involved in the primary root growth under L-glutamate stress, but it was involved in the primary root growth under metal stress
A forward genetics screen towards identifying mechanisms and genes involved in lateral root inhibition by excess zinc
No full text
Metaalspecifieke regulaties in de promotoractiviteit van de lipoxygenase genen in Arabidopsis thaliana
No full text- …
