Links Between Heathland Fungal Biomass Mineralization, Melanization, and Hydrophobicity.

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.

Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data.

Identification and Quantification of Celery Allergens Using Fiber Optic Surface Plasmon Resonance PCR.

Abstract: Accurate identification and quantification of allergens is key in healthcare, biotechnology and food quality and safety. Celery (Apium graveolens) is one of the most important elicitors of food allergic reactions in Europe. Currently, the golden standards to identify, quantify and discriminate celery in a biological sample are immunoassays and two-step molecular detection assays in which quantitative PCR (qPCR) is followed by a high-resolution melting analysis (HRM). In order to provide a DNA-based, rapid and simple detection method suitable for one-step quantification, a fiber optic PCR melting assay (FO-PCR-MA) was developed to determine different concentrations of celery DNA (1 pM-0.1 fM). The presented method is based on the hybridization and melting of DNA-coated gold nanoparticles to the FO sensor surface in the presence of the target gene (mannitol dehydrogenase, Mtd). The concept was not only able to reveal the presence of celery DNA, but also allowed for the cycle-to-cycle quantification of the target sequence through melting analysis. Furthermore, the developed bioassay was benchmarked against qPCR followed by HRM, showing excellent agreement (R² = 0.96). In conclusion, this innovative and sensitive diagnostic test could further improve food quality control and thus have a large impact on allergen induced healthcare problems.

Toxicity responses of Cu and Cd: the involvement of miRNAs and the transcription factor SPL7.

BACKGROUND: MicroRNAs are important posttranscriptional regulators of gene expression playing a role in developmental processes as well as in stress responses, including metal stress responses. Despite the identification of several metal-responsive miRNAs, the regulation and the role of these miRNAs and their targets remain to be explored. In this study, miRNAs involved in the response to Cd and Cu excess in Arabidopsis thaliana are identified. In addition, the involvement of the transcription factor SPL7, namely the key regulator of Cu homeostasis, in these metal stress responses is demonstrated by the use of an spl7 knockout mutant. Furthermore, more insight is given in the Cd-induced Cu deficiency response through determining the effects of adding supplemental Cu to Cd-exposed plants. RESULTS: Thirteen miRNAs were identified in response to Cu and Cd excess in A. thaliana. Several of these miRNAs (miR397a, miR398b/c and miR857) were oppositely affected under Cu and Cd exposure. The induced expression of these miRNAs after Cd exposure was totally abolished in the spl7 mutant (SQUAMOSA promoter binding protein like7), indicating a major role for SPL7 in the Cd response. Plants exposed to Cd showed a higher Cu content in the roots, whereas the Cu content in the leaves of the spl7 mutant was reduced. Furthermore, the Cd-induced Cu deficiency response disappeared when supplemental Cu was added. CONCLUSIONS: Copper- and Cd-responsive miRNAs were identified and several of them are SPL7-dependently regulated. SPL7 seems to be a shared component between both the Cu toxicity and the Cd toxicity response, yet oppositely regulated, that is inactivated after Cu exposure and activated after Cd exposure. Since SPL7 is the key regulator of Cu homeostasis, and Cd affects the Cu homeostasis, we hypothesize that SPL7 is activated in response to Cd possibly due to a Cd-induced Cu deficiency. Since adding additional Cu to Cd-exposed plants resulted in the disappearance of the Cu deficiency response, Cd possibly provokes Cu deficiency, thereby activating SPL7 and inducing subsequently the Cu deficiency response.

ALTERNATIVE OXIDASE1a modulates the oxidative challenge during moderate Cd exposure in Arabidopsis thaliana leaves.

This study aims to unravel the functional significance of alternative oxidase1a (AOX1a) induction in Arabidopsis thaliana leaves exposed to cadmium (Cd) by comparing wild-type (WT) plants and aox1a knockout mutants. In the absence of AOX1a, differences in stress-responsive transcript and glutathione levels suggest an increased oxidative challenge during moderate (5 µM) and prolonged (72h) Cd exposure. Nevertheless, aox1a knockout leaves showed lower hydrogen peroxide (H2O2) accumulation as compared to the WT due to both acute (24h) and prolonged (72h) exposure to 5 µM Cd, but not to 10 µM Cd. Taken together, we propose a working model where AOX1a acts early in the response to Cd and activates or maintains a mitochondrial signalling pathway impacting on cellular antioxidative defence at the post-transcriptional level. This fine-tuning pathway is suggested to function during moderate (5 µM) Cd exposure while being overwhelmed during more severe (10 µM) Cd stress. Within this framework, ethylene is required - either directly or indirectly via NADPH oxidase isoform C - to fully induce AOX1 expression. In addition, reciprocal crosstalk between these components was demonstrated in leaves of A. thaliana plants exposed to Cd.

Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of arabidopsis to cadmium.

BACKGROUND AND AIMS: Cadmium (Cd) is a non-essential trace element that elicits oxidative stress. Plants respond to Cd toxicity via increasing their Cd-chelating and antioxidative capacities. They predominantly chelate Cd via glutathione (GSH) and phytochelatins (PCs), while antioxidative defence is mainly based on the use and recycling of both GSH and ascorbate (AsA), complemented by superoxide dismutase (SOD) and catalase (CAT). In addition, both metabolites act as a substrate for the regeneration of other essential antioxidants, which neutralize and regulate reactive oxygen species (ROS). Together, these functions influence the concentration and cellular redox state of GSH and AsA. In this study, these two parameters were examined in plants of Arabidopsis thaliana exposed to sub-lethal Cd concentrations. METHODS: Wild-type plants and mutant arabidopsis plants containing 30-45 % of wild-type levels of GSH (cad2-1) or 40-50 % of AsA (vtc1-1), together with the double-mutant (cad2-1 vtc1-1) were cultivated in a hydroponic system and exposed to sub-lethal Cd concentrations. Cadmium detoxification was investigated at different levels including gene expression and metabolite concentrations. KEY RESULTS: In comparison with wild-type plants, elevated basal thiol levels and enhanced PC synthesis upon exposure to Cd efficiently compensated AsA deficiency in vtc1-1 plants and contributed to decreased sensitivity towards Cd. Glutathione-deficient (cad2-1 and cad2-1 vtc1-1) mutants, however, showed a more oxidized GSH redox state, resulting in initial oxidative stress and a higher sensitivity to Cd. In order to cope with the Cd stress to which they were exposed, GSH-deficient mutants activated multiple alternative pathways. CONCLUSIONS: Our observations indicate that GSH and AsA deficiency differentially alter plant GSH homeostasis, resulting in opposite Cd sensitivities relative to wild-type plants. Upon Cd exposure, GSH-deficient mutants were hampered in chelation. They experienced phenotypic disturbances and even more oxidative stress, and therefore activated multiple alternative pathways such as SOD, CAT and ascorbate peroxidase, indicating a higher Cd sensitivity. Ascorbate deficiency, however, was associated with enhanced PC synthesis in comparison with wild-type plants after Cd exposure, which contributed to decreased sensitivity towards Cd.

Gene Networks Involved in Hormonal Control of Root Development in Arabidopsis thaliana: A Framework for Studying Its Disturbance by Metal Stress.

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.

Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression.

BACKGROUND: Anthropogenic activities cause metal pollution worldwide. Plants can absorb and accumulate these metals through their root system, inducing stress as a result of excess metal concentrations inside the plant. Ethylene is a regulator of multiple plant processes, and is affected by many biotic and abiotic stresses. Increased ethylene levels have been observed after exposure to excess metals but it remains unclear how the increased ethylene levels are achieved at the molecular level. In this study, the effects of cadmium (Cd) exposure on the production of ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and on the expression of the ACC Synthase (ACS) and ACC Oxidase (ACO) multigene families were investigated in Arabidopsis thaliana. RESULTS: Increased ethylene release after Cd exposure was directly measurable in a system using rockwool-cultivated plants; enhanced levels of the ethylene precursor ACC together with higher mRNA levels of ethylene responsive genes: ACO2, ETR2 and ERF1 also indicated increased ethylene production in hydroponic culture. Regarding underlying mechanisms, it was found that the transcript levels of ACO2 and ACO4, the most abundantly expressed members of the ACO multigene family, were increased upon Cd exposure. ACC synthesis is the rate-limiting step in ethylene biosynthesis, and transcript levels of both ACS2 and ACS6 showed the highest increase and became the most abundant isoforms after Cd exposure, suggesting their importance in the Cd-induced increase of ethylene production. CONCLUSIONS: Cadmium induced the biosynthesis of ACC and ethylene in Arabidopsis thaliana plants mainly via the increased expression of ACS2 and ACS6. This was confirmed in the acs2-1acs6-1 double knockout mutants, which showed a decreased ethylene production, positively affecting leaf biomass and resulting in a delayed induction of ethylene responsive gene expressions without significant differences in Cd contents between wild-type and mutant plants.

The role of the kinase OXI1 in cadmium- and copper-induced molecular responses in Arabidopsis thaliana.

The hypothesis that mitogen-activated protein kinase (MAPK) signalling is important in plant defences against metal stress has become accepted in recent years. To test the role of oxidative signal-inducible kinase (OXI1) in metal-induced oxidative signalling, the responses of oxi1 knockout lines to environmentally realistic cadmium (Cd) and copper (Cu) concentrations were compared with those of wild-type plants. A relationship between OXI1 and the activation of lipoxygenases and other initiators of oxylipin production was observed under these stress conditions, suggesting that lipoxygenase-1 may be a downstream component of OXI1 signalling. Metal-specific differences in OXI1 action were observed. For example, OXI1 was required for the up-regulation of antioxidative defences such as catalase in leaves and Fe-superoxide dismutase in roots, following exposure to Cu, processes that may involve the MEKK1-MKK2-WRKY25 cascade. Moreover, the induction of Cu/Zn superoxide dismutases in Cu-exposed leaves was regulated by OXI1 in a manner that involves fluctuations in the expression of miRNA398. These observations contrast markedly with the responses to Cd exposure, which also involves OXI1-independent pathways but rather involves changes in components mediating intracellular communication.

Correlation between hormonal homeostasis and morphogenic responses in Arabidopsis thaliana seedlings growing in a Cd/Cu/Zn multi-pollution context.

To date, almost no information is available in roots and shoots of the model plant Arabidopsis thaliana about the hierarchic relationship between metal accumulation, phytohormone levels, and glutathione/phytochelatin content, and how this relation affects root development. For this purpose, specific concentrations of cadmium, copper and zinc, alone or in triple combination, were supplied for 12 days to in vitro growing seedlings. The accumulation of these metals was measured in roots and shoots, and a significant competition in metal uptake was observed. Microscopic analyses revealed that root morphology was affected by metal exposure, and that the levels of trans-zeatin riboside, dihydrozeatin riboside, indole-3-acetic acid and the auxin/cytokinin ratio varied accordingly. By contrast, under metal treatments, minor modifications in gibberellic acid and abscisic acid levels occurred. Real-time polymerase chain reaction analysis of some genes involved in auxin and cytokinin synthesis (e.g. AtNIT in roots and AtIPT in shoots) showed on average a metal up-regulated transcription. The production of thiol-peptides was induced by all the metals, alone or in combination, and the expression of the genes involved in thiol-peptide synthesis (AtGSH1, AtGSH2, AtPCS1 and AtPCS2) was not stimulated by the metals, suggesting a full post-transcriptional control. Results show that the Cd/Cu/Zn-induced changes in root morphology are caused by a hormonal unbalance, mainly governed by the auxin/cytokinin ratio.

The influence of metal stress on the availability and redox state of ascorbate, and possible interference with its cellular functions.

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.

Auxin and cytokinin metabolism and root morphological modifications in Arabidopsis thaliana seedlings infected with Cucumber mosaic virus (CMV) or exposed to cadmium.

Arabidopsis thaliana L. is a model plant but little information is available about morphological root changes as part of a phytohormonal common response against both biotic and abiotic stressors. For this purpose, two-week-old Arabidopsis seedlings were treated with 10 µM CdSO4 or infected with CMV. After 12 days the entire aerial parts and the root system were analyzed, and the presence of CMV or the accumulation of Cd were detected. Microscopic analysis revealed that both CMV and Cd influenced root morphology by a marked development in the length of root hairs and an intense root branching if compared to controls. Among the three treatments, Cd-treated seedlings showed a shorter root axis length and doubled their lateral root diameter, while the lateral roots of CMV-infected seedlings were the longest. The root growth patterns were accompanied by significant changes in the levels of indole-3-acetic acid, trans-zeatin riboside, dihydrozeatin riboside, as a probable consequence of the regulation of some genes involved in their biosynthesis/degradation. The opposite role on root development played by the phythormones studied is discussed in detail. The results obtained could provide insights into novel strategies for plant defense against pathogens and plant protection against pollutants.

Understanding the development of roots exposed to contaminants and the potential of plant-associated bacteria for optimization of growth.

BACKGROUND AND SCOPE: Plant responses to the toxic effects of soil contaminants, such as excess metals or organic substances, have been studied mainly at physiological, biochemical and molecular levels, but the influence on root system architecture has received little attention. Nevertheless, the precise position, morphology and extent of roots can influence contaminant uptake. Here, data are discussed that aim to increase the molecular and ecological understanding of the influence of contaminants on root system architecture. Furthermore, the potential of plant-associated bacteria to influence root growth by their growth-promoting and stress-relieving capacities is explored. METHODS: Root growth parameters of Arabidopsis thaliana seedlings grown in vertical agar plates are quantified. Mutants are used in a reverse genetics approach to identify molecular components underlying quantitative changes in root architecture after exposure to excess cadmium, copper or zinc. Plant-associated bacteria are isolated from contaminated environments, genotypically and phenotypically characterized, and used to test plant root growth improvement in the presence of contaminants. KEY RESULTS: The molecular determinants of primary root growth inhibition and effects on lateral root density by cadmium were identified. A vertical split-root system revealed local effects of cadmium and copper on root development. However, systemic effects of zinc exposure on root growth reduced both the avoidance of contaminated areas and colonization of non-contaminated areas. The potential for growth promotion and contaminant degradation of plant-associated bacteria was demonstrated by improved root growth of inoculated plants exposed to 2,4-di-nitro-toluene (DNT) or cadmium. CONCLUSIONS: Knowledge concerning the specific influence of different contaminants on root system architecture and the molecular mechanisms by which this is achieved can be combined with the exploitation of plant-associated bacteria to influence root development and increase plant stress tolerance, which should lead to more optimal root systems for application in phytoremediation or safer biomass production.

MicroRNAs in metal stress: specific roles or secondary responses?

In plants, microRNAs (miRNAs) control various biological processes by negatively regulating the expression of complementary target genes, either (1) post-transcriptionally by cleavage or translational inhibition of target mRNA, or (2) transcriptionally by methylation of target DNA. Besides their role in developmental processes, miRNAs are main players in stress responses, including metal stress responses. Exposure of plants to excess metal concentrations disturbs the cellular redox balance and enhances ROS accumulation, eventually leading to oxidative damage or signaling. Plants modify their gene expression by the activity of miRNAs in response to metal toxicity to regulate (1) complexation of excess metals, (2) defense against oxidative stress and (3) signal transduction for controlling various biological responses. This review focuses on the biogenesis, working mechanisms and functioning of miRNAs in plants. In a final part, our current knowledge on the regulatory roles of miRNAs in plant metal stress responses is highlighted, and whether stress-regulated miRNAs have specific roles or are secondary consequences is discussed.

Glutathione is a key player in metal-induced oxidative stress defenses.

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; γ-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.

Mitogen-Activated Protein (MAP) kinases in plant metal stress: regulation and responses in comparison to other biotic and abiotic stresses.

Exposure of plants to toxic concentrations of metals leads to disruption of the cellular redox status followed by an accumulation of reactive oxygen species (ROS). ROS, like hydrogen peroxide, can act as signaling molecules in the cell and induce signaling via mitogen-activated protein kinase (MAPK) cascades. MAPK cascades are evolutionary conserved signal transduction modules, able to convert extracellular signals to appropriate cellular responses. In this review, our current understanding about MAPK signaling in plant metal stress is discussed. However, this knowledge is scarce compared to research into the role of MAPK signaling in the case of other abiotic and biotic stresses. ROS production is a common response induced by different stresses and undiscovered analogies may exist with metal stress. Therefore, further attention is given to MAPK signaling in other biotic and abiotic stresses and its interplay with other signaling pathways to create a framework in which the involvement of MAPK signaling in metal stress may be studied.

Metal-induced oxidative stress and plant mitochondria.

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.

Cadmium stress: an oxidative challenge.

At the cellular level, cadmium (Cd) induces both damaging and repair processes in which the cellular redox status plays a crucial role. Being not redox-active, Cd is unable to generate reactive oxygen species (ROS) directly, but Cd-induced oxidative stress is a common phenomenon observed in multiple studies. The current review gives an overview on Cd-induced ROS production and anti-oxidative defense in organisms under different Cd regimes. Moreover, the Cd-induced oxidative challenge is discussed with a focus on damage and signaling as downstream responses. Gathering these data, it was clear that oxidative stress related responses are affected during Cd stress, but the apparent discrepancies observed in between the different studies points towards the necessity to increase our knowledge on the spatial and temporal ROS signature under Cd stress. This information is essential in order to reveal the exact role of Cd-induced oxidative stress in the modulation of downstream responses under a diverse array of conditions.

Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana tabacum.

Selection and use of appropriate reference genes as internal controls in real-time reverse transcription PCR (RT-PCR) assays is highly important for accurate quantification of gene expression levels. Since some photosynthetic genes are encoded in the nuclear genome and others in the chloroplast genome, we evaluated both nuclear- and plastid-encoded candidate reference genes. Six plastid-encoded candidate reference genes were derived from Arabidopsis microarray data and three plastid- and five nuclear-encoded reference genes were derived from literature. Cytokinins influence photosynthetic gene expression, so we evaluated the expression stability of the candidate reference genes in transgenic Nicotiana tabacum plants with elevated or diminished cytokinin content. We found that the most reliable strategy makes use of plastid-encoded genes for normalizing plastid photosynthetic genes and nuclear-encoded reference genes for normalizing nuclear photosynthetic genes. Compared to the use of nuclear reference genes only, this approach assimilates any effects on transcriptional activity of chloroplasts or number of chloroplast. The best expression stabilities in Nicotiana tabacum were observed for the plastid-encoded references genes Nt-RPS3, Nt-NDHI and Nt-IN1 and for the nuclear-encoded genes Nt-ACT9, Nt-alphaTUB and Nt-SSU. These genes may be suitable for normalization of photosynthetic genes under other experimental conditions in Nicotiana tabacum, and orthologues of these genes may be suitable candidates for normalizing photosynthetic gene expression in other species.

Effects of uranium and phosphate concentrations on oxidative stress related responses induced in Arabidopsis thaliana.

The 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 4days to 100microM 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 25microM P in combination with 100microM 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 (25microM P) and high phosphate (100microM P) treatments were observed justifying the use of the low phosphate concentration in combination with uranium.