Search

Quantitative 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.

Lipid peroxidation in leaves and roots of A. thaliana wild-type and spl7 knockout plants. Nineteen-days-old plants were exposed for 24 h to 2 μM CuSO4, 5 μM CdSO4 or grown under control conditions. Data are mean ± S.E. of 4 biological replicates. Significant treatment differences (P

Forward and reverse primers used to determine gene expression levels via quantitative real-time PCR. E-E-jn, exon-exon junction; E-I-b, exon-intron boundary; YLS, yellow-leaf-specific; UBQ10, ubiquitin; ACT, actin ; EF, elongation factor; APS, ATP sulfurylase; SULTR, sulfate transporter; LAC, laccase; CSD, Cu/Zn superoxide dismutase; FSD, Fe superoxide dismutase. *Pri-miRNA primer concentrations were increased to 900 nM. (DOCX 18 kb)

Verification of the T-DNA insertion in the spl7 mutant. Primers were designed using T-DNA primer design. (DOCX 32 kb)

Quantitative real-time PCR parameters according to the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines derived from Bustin et al. [51]. (DOCX 16 kb)

Plant survival under abiotic stress conditions requires morphological and physiological adaptations. Adverse soil conditions directly affect root development, although the underlying mechanisms remain largely to be discovered. Plant hormones regulate normal root growth and mediate root morphological responses to abiotic stress. Hormone synthesis, signal transduction, perception and cross-talk create a complex network in which metal stress can interfere, resulting in root growth alterations. We focus on Arabidopsis thaliana, for which gene networks in root development have been intensively studied, and supply essential terminology of anatomy and growth of roots. Knowledge of gene networks, mechanisms and interactions related to the role of plant hormones is reviewed. Most knowledge has been generated for auxin, the best-studied hormone with a pronounced primary role in root development. Furthermore, cytokinins, gibberellins, abscisic acid, ethylene, jasmonic acid, strigolactones, brassinosteroids and salicylic acid are discussed. Interactions between hormones that are of potential importance for root growth are described. This creates a framework that can be used for investigating the impact of abiotic stress factors on molecular mechanisms related to plant hormones, with the limited knowledge of the effects of the metals cadmium, copper and zinc on plant hormones and root development included as case example.

A 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 authors apologize to any researcher whose work is not cited here due to limitations of space and scope. This work was supported by the Research Foundation—Flanders (FWO) by a PhD grant for Els Keunen and the project G.0807.09. Support was also obtained from the Methusalem project 08G03VGRJ.

Worldwide, metals have been distributed to excessive levels in the environment due to industrial and agricultural activities. Plants growing on soils contaminated with excess levels of metals experience a disturbance of the cellular redox balance, which leads to an augmentation of reactive oxygen species (ROS). Even though the increased ROS levels can cause cellular damage, controlled levels play an important role in modulating signaling networks that control physiological processes and stress responses. Plants control ROS levels using their antioxidative defense system both under non-stress conditions, as well as under stress conditions such as exposure to excess metals. Ascorbate (AsA) is a well-known and important component of the plant's antioxidative system. As primary antioxidant, it can reduce ROS directly and indirectly via ascorbate peroxidase in the ascorbate-glutathione cycle. Furthermore, AsA fulfills an essential role in physiological processes, some of which are disturbed by excess metals. In this review, known direct effects of excess metals on AsA biosynthesis and functioning will be discussed, as well as the possible interference of metals with the role of AsA in physiological and biochemical processes. The authors apologize to any researcher whose work is not cited here due to limitations of space and scope. This work was supported by Hasselt University through BOF (Bijzonder Onderzoeksfonds) project BOF08G01 and the Methusalem project 08G03VGRJ.

Since the industrial revolution, the production, and consequently the emission of metals, has increased exponentially, overwhelming the natural cycles of metals in many ecosystems. Metals display a diverse array of physico-chemical properties such as essential versus non-essential and redox-active versus non-redox-active. In general, all metals can lead to toxicity and oxidative stress when taken up in excessive amounts, imposing a serious threat to the environment and human health. In order to cope with different kinds of metals, plants possess defense strategies in which glutathione (GSH; gamma-glu-cys-gly) plays a central role as chelating agent, antioxidant and signaling component. Therefore, this review highlights the role of GSH in: (1) metal homeostasis; (2) antioxidative defense; and (3) signal transduction under metal stress. The diverse functions of GSH originate from the sulfhydryl group in cysteine, enabling GSH to chelate metals and participate in redox cycling. The authors apologize to any researcher whose work is not cited here due to limitations of space and scope. This work was supported by the Agency for Innovation by Science and Technology with a PhD grant for Marijke Jozefczak. Additional financial support was obtained from the Research Foundation-Flanders (FWO) project G.0807.09 and from the Methusalem project 08G03VGRJ.

Comprehending the decomposition process is crucial for our understanding of the mechanisms of carbon (C) sequestration in soils. The decomposition of plant biomass has been extensively studied. It revealed that extrinsic biomass properties that restrict its access to decomposers influence decomposition more than intrinsic ones that are only related to its chemical structure. Fungal biomass has been much less investigated, even though it contributes to a large extent to soil organic matter, and is characterized by specific biochemical properties. In this study, we investigated the extent to which decomposition of heathland fungal biomass was affected by its hydrophobicity (extrinsic property) and melanin content (intrinsic property). We hypothesized that, as for plant biomass, hydrophobicity would have a greater impact on decomposition than melanin content. Mineralization was determined as the mineralization of soil organic carbon (SOC) into CO2 by headspace GC/MS after inoculation by a heathland soil microbial community. Results show that decomposition was not affected by hydrophobicity, but was negatively correlated with melanin content. We argue that it may indicate that either melanin content is both an intrinsic and extrinsic property, or that some soil decomposers evolved the ability to use surfactants to access to hydrophobic biomass. In the latter case, biomass hydrophobicity should not be considered as a crucial extrinsic factor. We also explored the ecology of decomposition, melanin content, and hydrophobicity, among heathland soil fungal guilds. Ascomycete black yeasts had the highest melanin content, and hyaline Basidiomycete yeasts the lowest. Hydrophobicity was an all-or-nothing trait, with most isolates being hydrophobic. The authors are thankful to the BOF (Special Research Fund) from Hasselt University for financing their research, as well as METHUZALEM provided to Jaco Vangronsveld under grant number 08M03VGRJ.

The reactive oxygen species (ROS)-signaling pathway is very important in heavy metal toxicity. Induction of the antioxidative defense mechanism, comprising ROS-scavenging enzymes and metabolites, in plants after environmental uranium contamination has been insufficiently studied in the past. This study aimed to analyze oxidative stress related responses in Arabidopsis thaliana after uranium exposure. Seventeen-day-old seedlings were exposed to 0, 0.1, 1, 10 and 100 μM uranium for 3 days. After exposure to 100 μM uranium, a decrease in fresh weight for leaves and roots was observed, leaves colored anthocyanous and roots were stunted and yellow. To reveal the importance of oxidative stress in uranium toxicity, alterations in ROS-scavenging enzymes were studied at protein and transcriptional level. Superoxide dismutase (SOD) capacities increased in leaves and roots after exposure to 100 μM uranium but no differences were observed for catalase (CAT) capacities. Transcript levels of different SODs located at various cellular compartments were affected depending on the place of action. Gene expression of CAT in leaves and roots was also affected after uranium exposure. Results indicate that oxidative stress plays an important role in uranium toxicity but suggest that plant responses differ for leaves and roots.

Over the past decades, large areas have been contaminated with cadmium (Cd) and copper (Cu) due to anthropogenic activities. These toxic metals are able to accumulate in plants where they interfere with essential physiological processes necessary for normal functioning. A common cellular consequence of toxic metal exposure is an increase of reactive oxygen species (ROS), thereby disturbing the cellular redox status and causing oxidative damage to cellular components. However, controlled levels of ROS can also act as signaling molecules in normal cell metabolism as well as in defense responses. Hydrogen peroxide (H2O2) is a well-known example involved in the control of such responses and its production is elevated in plants exposed to Cd or Cu. Therefore, H2O2 could be involved in metal-induced signal transduction events. Ascorbate peroxidases (APX) are important H2O2 scavengers and show a high affinity in the presence of ascorbate (AsA). Therefore APXs are involved in protecting the plant cell against harmful amounts of H2O2 as well as controlling H2O2 levels for signaling involved in defense responses. To understand plant responses to toxic concentrations of metals, it is necessary to gain knowledge about stress signaling caused by this stress factor at the cellular level. Because AsA and APX are involved in controlling H2O2 levels, they play a role in stress signaling pathways hence in many cellular processes. However, until today, a potential role for APX and AsA in biological processes dealing with metal-induced oxidative stress and downstream responses in plants has not been studied. Therefore, the main objective of this work was to unravel the role of APX and AsA in oxidative signaling and in the regulation of oxidative stress responses during Cu and Cd exposure. In de afgelopen decennia werden grote gebieden verontreinigd met cadmium (Cd) en koper (Cu) als gevolg van humane activiteiten. Deze toxische metalen kunnen accumuleren in planten en interfereren met essentiële fysiologische processen die noodzakelijk zijn voor een normale plantontwikkeling. Een algemeen cellulair gevolg van blootstelling aan toxische metalen is een verhoging van reactieve zuurstofvormen (ROS), waardoor de cellulaire redox toestand wordt verstoord en oxidatieve schade aan cellulaire componenten optreedt. Daarnaast hebben gecontroleerde niveaus van ROS ook een belangrijke rol als signaalmolecule zowel in het controleren van het normale celmetabolisme alsook verdedigingsresponsen. Waterstofperoxide (H2O2) is een bekend voorbeeld betrokken bij de controle van deze responsen en de productie ervan wordt aanzienlijk verhoogd in planten blootgesteld aan Cd en Cu. Hierdoor is het mogelijk dat H2O2 betrokken is in metaal-geïnduceerde signalisatie netwerken. Ascorbaatperoxidasen (APX) zijn belangrijke scavengers van H2O2 en vertonen een hoge affiniteit voor H2O2 in aanwezigheid van ascorbaat (ASA). Hierdoor staan APXs in voor een bescherming van de plantencel tegen schadelijke hoeveelheden H2O2 alsook voor de regulatie van de hoeveelheid H2O2 voor signalisatie betrokken in verdedigingsresponsen. Om te kunnen begrijpen hoe planten reageren op toxische metaalconcentraties, is het noodzakelijk om meer inzicht te krijgen in de signaaltransductie pathways die deze stress-factor op cellulair niveau veroorzaakt. Omdat AsA en APX betrokken zijn in de regulatie van de hoeveelheid H2O2, spelen ze een rol in signaaltransductie pathways en dus in vele cellulaire processen. Een mogelijke rol voor APX en AsA in biologische processen die omgaan met metaalgeïnduceerde oxidatieve stress en downstream responsen in planten zijn echter tot op heden nog niet onderzocht. Daarom is de algemene doelstelling van deze doctoraatsstudie om de functie van APX en AsA te ontrafelen in oxidatieve signalisatie en in de regulatie van oxidatieve stress responsen tijdens Cu en Cd blootstelling.

Anthropogenic 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.

Background: Understanding more about root development under stress conditions is essential for future crop improvement, which may be aimed at safe biomass production or clean up of polluted soils. Sublethal stress-exposed roots exhibit stress-induced morphogenic responses (SIMRs). However, for the metals cadmium (Cd), copper (Cu) and Zinc (Zc) in Arabidopsis thaliana seedlings, metalspecific growth responses were found instead of the general SIMR. It is hypothesised that LOX-genes are involved in these metal-specific growth adaptations. Objectives: The aim of this study is to participate in unravelling the molecular mechanims underlying these metal-specific growth responses in Arabidopsis thaliana and to understand more about local and systemic effects on root development in a more relevant context of heterogeneous exposure. Methodology: The phenotypic change under metal-stress conditions of lox-mutants (lox1-1, lox3A, lox5-1) compared to wild-type was investigated in split-root systems. Furthermore, the expression of genes involved in plant hormone synthesis or metabolism was examined in metal-exposed wild-type roots using quantitative reverse transcription PCR. Results: Phenotypic comparison of lox-mutants with wild-type after growth in split-root vertical agar plates revealed that Cd, Cu and Zn in the lower zone stimulate lateral root elongation and number in the upper control zone, which can be due to the elevation of the inhibition on lateral root elongation by LOX3 and LOX5. An incomplete primary root avoidance was observed in the Cd- and Zn-containing lower zone. The incomplete avoidance under Cd-exposure may be due to 9-LOX-signalling. LOX-genes did not seem to be involved in primary root growth under Cu-exposure, which may be due to the fact that Cu can induce reactive oxygen species directly. The systemic Zn-effect in wild-types is not present in lox3. The gene expression data revealed an significant increase in AAO1-expression under Cu- and Zn-exposure. Conclusion: LOX-genes are involved in the metal-specific growth responses and have a preference for the metal-free zone under heterogeneous exposure.