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To identify metal adapted bacteria equipped with traits positively influencing the growth of two hyperaccumulator plant species Arabidopsis arenosa and Arabidopsis halleri, we isolated bacteria inhabiting rhizosphere and vegetative tissues (roots, basal and stem leaves) of plants growing on two old Zn-Pb-Cd waste heaps in Bolesław and Bukowno (S. Poland), and characterized their potential plant growth promoting (PGP) traits as well as determined metal concentrations in rhizosphere and plant tissues. To determine taxonomic position of 144 bacterial isolates, 16S rDNA Sanger sequencing was used. A metabolic characterization of isolated strains was performed in vitro using PGP tests. A. arenosa and A. halleri accumulate high amounts of Zn in their tissues, especially in stem leaves. Among in total 22 identified bacterial taxa, the highest level of the taxonomical diversity (H' = 2.01) was revealed in A. halleri basal leaf endophytes originating from Bukowno waste heap area. The 96, 98, 99, and 98% of investigated strains showed tolerant to Cd, Zn, Pb and Cu, respectively. Generally, higher percentages of bacteria could synthesize auxins, siderophores, and acetoin as well as could solubilize phosphate. Nine of waste heap origin bacterial strains were tolerant to toxic metals, showed in vitro PGP traits and are potential candidates for bioremediation., (© 2023. Springer Nature Limited.)

The commonly used Arabidopsis thaliana natural accessions Columbia (Col-0) and Wassilewskija (Ws) are known to differ in their metal sensitivity, with Col-0 being more sensitive to copper (Cu) and cadmium (Cd) than Ws. As both Cu and Cd are known to affect Cu homeostasis, it was investigated whether this process is part of an accession-specific mechanism underlying their difference in metal sensitivity. As roots are the first contact point during metal exposure, responses were compared between roots of both accessions of hydroponically grown plants exposed to excess Cu or Cd for 24 and 72 h. Root Cu levels increased in both accessions under Cu and Cd exposure. However, under Cu exposure, the downregulation of Cu transporter (COPT) genes in combination with a more pronounced upregulation of metallothionein gene MT2b indicated that Ws plants coped better with the elevated Cu concentrations. The Cd-induced disturbance in Cu homeostasis was more efficiently counteracted in roots of Ws plants than in Col-0 plants. This was indicated by a higher upregulation of the SPL7-mediated pathway, crucial in the regulation of the Cu homeostasis response. In conclusion, maintaining the Cu homeostasis response in roots is key to accession-specific differences in Cu and Cd sensitivity., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Glutathione (GSH) is an important player in plant responses to cadmium (Cd) through its dual function as an antioxidant and precursor for metal-chelating phytochelatins (PCs). In addition, it was shown to be involved in cell cycle regulation in Arabidopsis thaliana roots, but its involvement in this process in leaves is largely unknown and has never been evaluated in Cd-exposed plants. This study aimed to elucidate the role of GSH in leaf growth and development, metal chelation, nutrient homeostasis and cell cycle regulation in A. thaliana plants upon prolonged Cd exposure. Responses were compared between wild-type (WT) plants and three GSH-deficient mutants. Our results indicate that PC production remains important in plants exposed to Cd for an extended duration. Furthermore, an important role for GSH in regulating nutrient homeostasis in Cd-exposed plants was revealed. Cell cycle analysis demonstrated that negative effects of Cd exposure on cell division and endoreplication were more pronounced in leaves of the GSH-deficient cadmium-sensitive 2-1 (cad2-1) mutant in comparison to the WT, indicating the involvement of GSH in cell cycle regulation. Finally, a crucial role for GSH in transcriptional activation of the Cd-induced DNA damage response (DDR) was revealed, as the Cd-induced upregulation of DDR-related genes was either less pronounced or completely abolished in leaves of the GSH-deficient mutants., (Copyright © 2020. Published by Elsevier Masson SAS.)

The toxic metal cadmium (Cd) is a major soil pollutant. Knowledge on the acute Cd-induced stress response is required to better understand the triggers and sequence of events that precede plant acclimation. Therefore, we aimed to identify the pressure points of Cd stress using a short-term exposure set-up ranging from 0 h to 24 h. Acute responses related to glutathione (GSH), hydrogen peroxide (H 2 O 2 ), 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene and the oxidative challenge were studied at metabolite and/or transcript level in roots and leaves of Arabidopsis thaliana either exposed or not to 5 µM Cd. Cadmium rapidly induced root GSH depletion, which might serve as an alert response and modulator of H 2 O 2 signalling. Concomitantly, a stimulation of root ACC levels was observed. Leaf responses were delayed and did not involve GSH depletion. After 24 h, a defined oxidative challenge became apparent, which was most pronounced in the leaves and concerted with a strong induction of leaf ACC synthesis. We suggest that root GSH depletion is required for a proper alert response rather than being a merely adverse effect. Furthermore, we propose that roots serve as command centre via a.o. root-derived ACC/ethylene to engage the leaves in a proper stress response.

The natural accession Columbia (Col-0) is considered as the reference genome of the model plant Arabidopsis thaliana. Nonetheless, Col-0 plants are more sensitive to excess copper (Cu) and cadmium (Cd) than other widely used accessions such as Wassilewskija (Ws) plants. In the current study, this accession-specific metal sensitivity is further explored by comparing the responses in leaves of Col-0 and Ws plants exposed to excess Cu and Cd. Our results suggest that different life strategies favored by both accessions under physiological conditions affect their response to metal exposure. While Col-0 plants mainly invest in metal detoxification, Ws plants center on nutrient homeostasis. In particular, the higher expression of genes related to Cu homeostasis genes in non-exposed conditions indicates that Ws plants possess a constitutively efficient metal homeostasis. On the other hand, oxidative stress-related MAPK signaling appears to be boosted in leaves of Col-0 plants exposed to excess Cu. Furthermore, the upregulation of the glutathione (GSH) biosynthesis GSH2 gene and the increased GSH concentration after Cd exposure suggest the activation of detoxification mechanisms, such as phytochelatin production, to counteract the more severe Cd-induced oxidative stress in leaves of Col-0 plants. Exposure to Cd also led to a more pronounced ethylene signaling response in leaves of Col-0 as compared to Ws plants, which could be related to Cd-induced GSH metabolism. In conclusion, accession-specific life strategies clearly affect the way in which leaves of A. thaliana plants cope with excess Cu and Cd., (Copyright © 2018. Published by Elsevier GmbH.)

Cadmium (Cd) exposure can disturb the homeostasis of essential elements. In Arabidopsis thaliana, Cd induces a squamosa promoter binding protein-like 7 (SPL7)-dependent Cu deficiency response. We investigated how Cd induces a Cu deficiency response. The Cu deficiency response consists of the active SPL7 transcription factor binding to GTAC motifs in promoters of among others several Cu transporters, a Cu chaperone, and cupro-miRNAs to regulate Cu homeostasis. We demonstrated that the addition of supplemental Cu to Cd-exposed A. thaliana plants diminished the Cu deficiency response in roots, while it even disappeared in leaves. Exposure of plants to Cd in combination with extra Cu reduced Cd levels in both roots and leaves resulting in an improved cellular oxidative state. Furthermore, we demonstrated a role for phytochelatins (PCs) in the Cd-induced Cu deficiency response, because it was reduced in roots of cad1-3 mutant plants exposed to Cd. In conclusion, a working mechanism is provided in which it is suggested that Cd increases PC levels that can complex both Cd and Cu. This results in cellular Cu deficiency and subsequently the activation of SPL7 and hence the induction of the Cu deficiency response., (© 2016 John Wiley & Sons Ltd.)

When terrestrial environments get contaminated with long-lived gamma emitting radionuclides, plants that grow in these contaminated areas are exposed to gamma radiation during consecutive generations. Therefore it is important to evaluate the gamma induced stress response in plants in and between generations. The objective of this research is to reveal differences at the level of the antioxidative stress response between generations with a different radiation history. An experiment was conducted in which 7-days old Arabidopsis thaliana plants were exposed for 14 days to four different gamma dose rates: 22 mGy/h, 38 mGy/h, 86 mGy/h and 457 mGy/h. Two different plant groups were used: plants that were not exposed to gamma radiation before (P0) and plants that received the aforementioned gamma treatment during their previous generation (S1). Growth, the concentration of the antioxidants ascorbate and glutathione, a number of antioxidative enzyme activities and their gene transcript levels were analysed. A dose-rate dependent induction was seen for catalase (CAT) and guaiacol peroxidase (GPX) in the roots and for syringaldazine peroxidase (SPX) in the shoots. Differences between the two generations were observed for CAT and GPX in the roots, where a significantly higher activity of these reactive oxygen species (ROS) detoxifying enzymes was observed in the S1 generation. For SPX in the shoots, a dose dependent upregulation was observed in the P0 generation. However, high SPX activities were present for all doses in the S1 generation. These differences in enzyme activity between generations for SPX and GPX and the involvement of these enzymes in cell wall biosynthesis, suggest an important role for cell wall strengthening in the response to gamma irradiation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Anthropogenic activities have led to a widespread uranium (U) contamination in many countries. The toxic effects of U at the cellular level have mainly been investigated at a pH around 5.5, the optimal pH for hydroponically grown plants. However, since the speciation of U, and hence its toxicity, is strongly dependent on environmental factors such as the pH, it is important to investigate the effects of U at different environmentally relevant pH levels. Although U is poorly translocated from the roots to the shoots, resulting in a low U concentration in the leaves, it has been demonstrated that toxic effects in the leaves were already visible after 1 day exposure at pH 5.5, although only when exposed to relatively high U concentrations (100 μM). Therefore, the present study aimed to analyse the effects of different U concentrations (ranging from 0 to 100 μM) at pH 4.5 in leaves of Arabidopsis thaliana plants. Results indicate that U induces early senescence in A. thaliana leaves as was suggested by a decreased expression of CAT2 accompanied by an induction of CAT3 expression, a decreased CAT capacity and an increased lipid peroxidation. In addition, miRNA398b/c is involved in the regulation of the SOD response in the leaves. As such, an increased MIR398b/c expression was observed leading to a decreased transcript level of CSD1/2. Finally, the biosynthesis of ascorbate was induced after U exposure. This can point towards an important role for this metabolite in the scavenging of reactive oxygen species under U stress., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Human activity has led to an increasing amount of radionuclides in the environment and subsequently to an increased risk of exposure of the biosphere to ionising radiation. Due to their high linear energy transfer, α-emitters form a threat to biota when absorbed or integrated in living tissue. Among these, (241)Am is of major concern due to high affinity for organic matter and high specific activity. This study examines the dose-dependent biological effects of α-radiation delivered by (241)Am at the morphological, physiological and molecular level in 14-day old seedlings of Arabidopsis thaliana after hydroponic exposure for 4 or 7 days. Our results show that (241)Am has high transfer to the roots but low translocation to the shoots. In the roots, we observed a transcriptional response of reactive oxygen species scavenging and DNA repair pathways. At the physiological and morphological level this resulted in a response which evolved from redox balance control and stable biomass at low dose rates to growth reduction, reduced transfer and redox balance decline at higher dose rates. This situation was also reflected in the shoots where, despite the absence of a transcriptional response, the control of photosynthesis performance and redox balance declined with increasing dose rate. The data further suggest that the effects in both organs were initiated in the roots, where the highest dose rates occurred, ultimately affecting photosynthesis performance and carbon assimilation. Though further detailed study of nutrient balance and (241)Am localisation is necessary, it is clear that radionuclide uptake and distribution is a major parameter in the global exposure effects on plant performance and health., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cadmium (Cd) induces the generation of reactive oxygen species (ROS) and stimulates ethylene biosynthesis. The phytohormone ethylene is a regulator of many developmental and physiological plant processes as well as stress responses. Previous research indicated various links between ethylene signalling and oxidative stress. Our results support a correlation between the Cd-induced oxidative challenge and ethylene signalling in Arabidopsis thaliana leaves. The effects of 24 or 72 h exposure to 5 μM Cd on plant growth and several oxidative stress-related parameters were compared between wild-type (WT) and ethylene insensitive mutants (etr1-1, ein2-1, ein3-1). Cadmium-induced responses observed in WT plants were mainly affected in etr1-1 and ein2-1 mutants, of which the growth was less inhibited by Cd exposure as compared to WT and ein3-1 mutants. Both etr1-1 and ein2-1 showed a delayed response in the glutathione (GSH) metabolism, including GSH levels and transcript levels of GSH synthesising and recycling enzymes. Furthermore, the expression of different oxidative stress marker genes was significantly lower in Cd-exposed ein2-1 mutants, evidencing that ethylene signalling is involved in early responses to Cd stress. A model for the cross-talk between ethylene signalling and oxidative stress is proposed., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

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., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.)

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.

To evaluate the environmental impact of uranium (U) contamination, it is important to investigate the effects of U at ecologically relevant conditions. Since U speciation, and hence its toxicity, strongly depends on environmental pH, the present study aimed to investigate dose-dependent effects of U at pH 7.5. Arabidopsis thaliana plants (Mouse-ear Cress) were exposed for three days to different U concentrations at pH 7.5. In the roots, the increased capacities of ascorbate peroxidase and glutathione reductase indicate an important role for the ascorbate-glutathione cycle during U-induced stress. However, a significant decrease in the ascorbate redox state was observed after exposure to 75 and 100 µM U, indicating that those roots are severely stressed. In accordance with the roots, the ascorbate-glutathione cycle plays an important role in the antioxidative defence systems in A. thaliana leaves exposed to U at pH 7.5 as the ascorbate and glutathione biosynthesis were upregulated. In addition, small inductions of enzymes of the antioxidative defence system were observed at lower U concentrations to counteract the U-induced stress. However, at higher U concentrations it seems that the antioxidative defence system of the leaves collapses as reductions in enzyme activities and gene expression levels were observed.

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., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This study aims to uncover the spatiotemporal involvement of glutathione (GSH) in two major mechanisms of cadmium (Cd)-induced detoxification (i.e. chelation and antioxidative defence). A kinetic study was conducted on hydroponically grown Arabidopsis thaliana (L. Heyhn) to gain insight into the early events after exposure to Cd. Cadmium detoxification was investigated at different levels, including gene transcripts, enzyme activities and metabolite content. Data indicate a time-dependent response both within roots and between plant organs. Early on in roots, GSH was preferentially allocated to phytochelatin (PC) synthesis destined for Cd chelation. This led to decreased GSH levels, without alternative pathways activated to complement GSH's antioxidative functions. After one day however, multiple antioxidative pathways increased including superoxide dismutase (SOD), ascorbate (AsA) and catalase (CAT) to ensure efficient neutralization of Cd-induced reactive oxygen species (ROS). As a consequence of Cd retention and detoxification in roots, a delayed response occurred in leaves. Together with high leaf thiol contents and possibly signalling responses from the roots, the leaves were protected, allowing them sufficient time to activate their defence mechanisms., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

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., (© 2014 American Society of Plant Biologists. All rights reserved.)

To study the impact of environmental uranium (U) contamination, effects should be analysed at different environmentally relevant pH levels as the speciation of U, and hence its toxicity, is strongly dependent on the pH. As photosynthesis is a major energy producing process in plants intimately connected to plant growth and known to be susceptible to metal stress, the effects of different U concentrations on photosynthesis in 18-day-old Arabidopsis thaliana (Columbia ecotype) are investigated at two contrasting pH levels, pH 4.5 and pH 7.5. At pH 4.5, U is highly taken up by the roots but is poorly translocated to the shoots, while at pH 7.5, less U is taken up but the translocation is higher. The lower U concentrations in the shoots at pH 4.5 are accompanied by a more reduced leaf growth as compared to pH 7.5. In addition, U does not influence the photosynthetic machinery at pH 7.5, while an optimization of the photosynthesis takes place after U exposure at pH 4.5. As such, more of the absorbed quanta are effectively used for photosynthesis accompanied by a decreased non-photochemical quenching and an increased electron transport rate. Since the enhanced photosynthesis at pH 4.5 is accompanied by a decreased growth, we suggest that the energy produced during photosynthesis is used for defence reactions against U-induced oxidative stress rather than for growth. As such, a high discrepancy was observed between the two pH levels, with an optimized photosynthetic apparatus at pH 4.5 and almost no effects at pH 7.5., (Copyright © 2014. Published by Elsevier Masson SAS.)

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.

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., (© 2013 Scandinavian Plant Physiology Society.)

Uranium (U) causes oxidative stress in Arabidopsis thaliana plants grown at pH 5.5. However, U speciation and its toxicity strongly depend on environmental parameters, for example pH. It is unknown how different U species determine U uptake and translocation within plants and how they might affect the oxidative defense mechanisms of these plants. The present study analyzed U uptake and oxidative stress-related responses in A. thaliana (Columbia ecotype) under contrasted U chemical speciation conditions. The 18-d-old seedlings were exposed for 3 d to 25 µM U in a nutrient solution of which the pH was adjusted to 4.5, 5.5, 6.5, or 7.5. Results indicate that there is a different rate of U uptake and translocation at the different pHs, with high uptake and low translocation at low pH and lower uptake but higher translocation at high pH. After U exposure, an increased glutathione reductase activity and total glutathione concentration were observed in U-exposed roots, pointing toward an important role for glutathione in the root defense system against U either by chelation or by antioxidative defense mechanisms. In leaves, antioxidative defense mechanisms were activated on U exposure, indicated by increased superoxide dismutase and catalase activity. As it seems that U toxicity is influenced by pH, it is important to consider site-specific characteristics when making U risk assessments., (Copyright © 2013 SETAC.)

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., (© 2012 Blackwell Publishing Ltd.)

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.

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 μM) 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 H(2)O(2) 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., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

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.

Plants exposed to cadmium (Cd) show morphological and physiological disorders. To increase our knowledge regarding Cd-induced signalling, most often the effects of acute exposure are investigated. However, this does not allow in-depth analysis of morphological effects. Therefore, we chronically exposed Arabidopsis thaliana plants to environmentally realistic Cd concentrations (5 or 10 μM) and, using a described phenotypic framework methodology, we determined the impact of Cd on the plant's ability to complete its life cycle and produce germinative seeds. Visible Cd-induced morphological changes were observed within a short exposure period, with chlorotic and anthocyanous leaf colouring occurring dose-dependently. Although rosette growth was severely reduced in Cd-exposed plants, all plants were able to emerge inflorescences and produce siliques containing germinative seeds, thus confirming the non-lethality of the used Cd concentrations. Although the growth inhibition of Cd-exposed plants was dependent on the dose, both concentrations had similar effects on inflorescence height and silique counts. In conclusion, vegetative growth of plants chronically exposed to Cd is inhibited in a concentration-dependent manner. However, the effect on plant regeneration is clearly stress-determined but independent on the Cd concentration applied. In Arabidopsis thaliana, vegetative and reproductive growth are differentially influenced by Cd., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The cellular redox balance seems an important modulator under heavy metal stress. While for other heavy metals these processes are well studied, oxidative stress related responses are also known to be triggered under uranium stress but information remains limited. This study aimed to further unravel the mechanisms by which plants respond to uranium stress. Seventeen-day-old Arabidopsis thaliana seedlings, grown on a modified Hoagland solution under controlled conditions, were exposed to 0, 0.1, 1, 10 and 100 μM uranium for 1, 3 and 7 days. While in Part I of this study oxidative stress related responses in the roots were discussed, this second Part II discusses oxidative stress related responses in the leaves and general conclusions drawn from the results of the roots and the leaves will be presented. As several responses were already visible following 1 day exposure, when uranium concentrations in the leaves were negligible, a root-to-shoot signaling system was suggested in which plastids could be important sensing sites. While lipid peroxidation, based on the amount of thiobarbituric acid reactive compounds, was observed after exposure to 100 μM uranium, affecting membrane structure and function, a transient concentration dependent response pattern was visible for lipoxygenase initiated lipid peroxidation. This transient character of uranium stress responses in leaves was emphasized by results of lipoxygenase (LOX2) and antioxidative enzyme transcript levels, enzyme capacities and glutathione concentrations both in time as with concentration. The ascorbate redox balance seemed an important modulator of uranium stress responses in the leaves as in addition to the previous transient responses, the total ascorbate concentration and ascorbate/dehydroascorbate redox balance increased in a concentration and time dependent manner. This could represent either a slow transient response or a stable increase with regard to plant acclimation to uranium stress., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

When aiming to evaluate the environmental impact of uranium contamination, it is important to unravel the mechanisms by which plants respond to uranium stress. As oxidative stress seems an important modulator under other heavy metal stress, this study aimed to investigate oxidative stress related responses in Arabidopsis thaliana exposed to uranium concentrations ranging from 0.1 to 100 μM for 1, 3 and 7 days. Besides analyzing relevant reactive oxygen species-producing and -scavenging enzymes at protein and transcriptional level, the importance of the ascorbate-glutathione cycle under uranium stress was investigated. These results are reported separately for roots and leaves in two papers: Part I dealing with responses in the roots and Part II unraveling responses in the leaves and presenting general conclusions. Results of Part I indicate that oxidative stress related responses in the roots were only triggered following exposure to the highest uranium concentration of 100 μM. A fast oxidative burst was suggested based on the observed enhancement of lipoxygenase (LOX1) and respiratory burst oxydase homolog (RBOHD) transcript levels already after 1 day. The first line of defense was attributed to superoxide dismutase (SOD), also triggered from the first day. The enhanced SOD-capacity observed at protein level corresponded with an enhanced expression of iron SOD (FSD1) located in the plastids. For the detoxification of H(2)O(2), an early increase in catalase (CAT1) transcript levels was observed while peroxidase capacities were enhanced at the later stage of 3 days. Although the ascorbate peroxidase capacity and gene expression (APX1) increased, the ascorbate/dehydroascorbate redox balance was completely disrupted and shifted toward the oxidized form. This disrupted balance could not be inverted by the glutathione part of the cycle although the glutathione redox balance could be maintained., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The cellular redox state is an important determinant of metal phytotoxicity. In this study we investigated the influence of cadmium (Cd) and copper (Cu) stress on the cellular redox balance in relation to oxidative signalling and damage in Arabidopsis thaliana. Both metals were easily taken up by the roots, but the translocation to the aboveground parts was restricted to Cd stress. In the roots, Cu directly induced an oxidative burst, whereas enzymatic ROS (reactive oxygen species) production via NADPH oxidases seems important in oxidative stress caused by Cd. Furthermore, in the roots, the glutathione metabolism plays a crucial role in controlling the gene regulation of the antioxidative defence mechanism under Cd stress. Metal-specific alterations were also noticed with regard to the microRNA regulation of CuZnSOD gene expression in both roots and leaves. The appearance of lipid peroxidation is dual: it can be an indication of oxidative damage as well as an indication of oxidative signalling as lipoxygenases are induced after metal exposure and are initial enzymes in oxylipin biosynthesis. In conclusion, the metal-induced cellular redox imbalance is strongly dependent on the chemical properties of the metal and the plant organ considered. The stress intensity determines its involvement in downstream responses in relation to oxidative damage or signalling., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Uranium never occurs as a single pollutant in the environment, but always in combination with other stressors such as ionizing radiation. As effects induced by multiple contaminants can differ markedly from the effects induced by the individual stressors, this multiple pollution context should not be neglected. In this study, effects on growth, nutrient uptake and oxidative stress induced by the single stressors uranium and gamma radiation are compared with the effects induced by the combination of both stressors. By doing this, we aim to better understand the effects induced by the combined stressors but also to get more insight in stressor-specific response mechanisms. Eighteen-day-old Arabidopsis thaliana seedlings were exposed for 3 days to 10 muM uranium and 3.5 Gy gamma radiation. Gamma radiation interfered with uranium uptake, resulting in decreased uranium concentrations in the roots, but with higher transport to the leaves. This resulted in a better root growth but increased leaf lipid peroxidation. For the other endpoints studied, effects under combined exposure were mostly determined by uranium presence and only limited influenced by gamma presence. Furthermore, an important role is suggested for CAT1/2/3 gene expression under uranium and mixed stressor conditions in the leaves.

In this study, toxicity effects in plants of uranium in a binary pollution condition were investigated by studying biological responses and unraveling oxidative stress related mechanisms in Arabidopsis thaliana seedlings, grown on hydroponics and exposed for 3 days to 10 μM uranium in combination with 5 μM cadmium. While uranium mostly accumulated in the roots with very low root-to-shoot transport, cadmium was taken up less by the roots but showed higher translocation to the shoots. Under mixed exposure, cadmium influenced uranium uptake highly but not the other way round resulting in a doubled uranium concentration in the roots. Under our mixed exposure conditions, it is clear that micronutrient concentrations in the roots are strongly influenced by addition of cadmium as a second stressor, while leaf macronutrient concentrations are mostly influenced by uranium. Oxidative stress related responses are highly affected by cadmium while uranium influence is more limited. Hereby, an important role was attributed to the ascorbate redox balance together with glutathione as both metabolites, but more explicitly for ascorbate, increased their reduced form, indicating an important defense and regulatory function. While for roots, based on an increase in FSD1 gene expression, oxidative stress was suggested to be superoxide induced, in leaves on the other hand, hydrogen peroxide related genes were mostly altered., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

The leaf proteome of 3-week-old Arabidopsis thaliana seedlings exposed for 1 week to low, environmentally realistic Cd concentrations was investigated. The data indicated that at 1muMCd, A. thaliana plants adapted their metabolism to cope with the Cd exposure. As a result, only moderate protein changes were observed. However, at 10muMCd, severe stress was indicated by growth reduction and chlorosis of rosette leaves at the macroscopic level and by lipid peroxidation and enhanced peroxidase activity at the cellular level. Of the 730 reproducible proteins among all gels, 21 were statistically upregulated in response to Cd. These proteins can be functionally grouped into 5 classes: proteins involved in (1) oxidative stress response, (2) photosynthesis and energy production, (3) protein metabolism, (4) gene expression and finally, (5) proteins with various or unknown function. In order to provide greater insight into the mechanisms induced on Cd exposure, a working model is proposed., (Copyright 2009 Elsevier GmbH. All rights reserved.)

The physiological effects of Cd and Cu have been highlighted in several studies over the last years. At the cellular level, oxidative stress has been reported as a common mechanism in both stress situations. Nevertheless, because of differences in their redox-related properties, the origin of the stress and regulation of these effects can be very different. Our results show a specific Cd-related induction of NADPH oxidases, whereas both metals induced lipid peroxidation via the activation of lipoxygenases. With respect to the antioxidative defense system, metal-specific patterns of superoxide dismutases (SODs) were detected, whereas gene expression levels of the H2O2-quenching enzymes were equally induced by both metals. Because monometallic exposure is very unusual in real-world situations, the metal-specific effects were compared with the mechanisms induced when the plants are exposed to both metals simultaneously. Combined exposure to Cd and Cu enhanced some of the effects that were induced when only one metal was applied to the medium. Other specific monometallically induced effects, such as a copper zinc superoxide dismutase (CSD2) downregulation due to Cd, were also sustained in a multipollution context, irrespective of the other monometallic effects. Furthermore, specific multipollution effects were unravelled, as iron superoxide dismutase 1 (FSD1) upregulation in the leaves was significant only when both Cu and Cd were applied. Additional relationships between these treatments and the common and specific stress induction mechanisms are discussed.

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.

Accurate quantification by real-time RT-PCR relies on normalisation of the measured gene expression data. Normalisation with multiple reference genes is becoming the standard, but the best reference genes for gene expression studies within one organism may depend on the applied treatments or the organs and tissues studied. Ideally, reference genes should be evaluated in all experimental systems. A number of candidate reference genes for Arabidopsis have been proposed, which can be used as a starting point to evaluate their expression stability in individual experimental systems by available computer algorithms like geNorm and NormFinder. Using this approach, we identified the best three reference genes from a set of ten candidates, which included three traditional "housekeeping" genes, for normalisation of gene expression when roots and leaves of Arabidopsis thaliana are exposed to cadmium (Cd) and copper (Cu). The expression stabilities of AT5G15710 (F-box protein), AT2G28390 (SAND family protein) and AT5G08290 (mitosis protein YLS8) were the highest when considering the effect to the roots and shoots of Cd and Cu treatments. Even though the effect of Cd and excess Cu on the plants is very different, the same best reference genes were identified when considering Cd or Cu treatments separately. This suggests that these three genes may also be suitable when studying the gene expression after exposure of Arabidopsis thaliana to increased concentrations of other metals.

Arabidopsis thaliana is one of the most widely used model organisms in plant sciences. Because of the increasing knowledge in the understanding of its molecular pathways, a reproducible and stable growth set-up for obtaining uniform plants becomes more important. In order to be able to easily harvest and study both roots and shoots, and to allow simple exposure to water-soluble toxic substances, a hydroponic system is the desired cultivation method for controlled plant growth. Based on earlier developed hydroponic cultivation protocols, a hydroponic set-up was optimized and statistically validated using linear mixed-effects models. In order to determine important components that influence the level of variability in a hydroponic set-up, stress-related indicators were examined at the biochemical as well as at the molecular level. It is highly recommended that statistical as well as biological assumptions are carried out before post-analyses are performed. Therefore, we suggest a model where factors that influence variability such as the usage of different pots and harvesting on different times are taken into account in the analyses. Furthermore, in contrast to what has been reported in earlier studies, our findings indicate that continuous aeration of the hydroponic solution is highly important.

We examined the subcellular cadmium (Cd) localization in roots and leaves of wild-type Arabidopsis thaliana (ecotype Columbia) exposed to environmentally relevant Cd concentrations. Energy-dispersive X-ray microanalysis (EDXMA) was performed on high-pressure frozen and freeze-substituted tissues. In the root cortex, Cd was associated with phosphorus (Cd/P) in the apoplast and sulfur (Cd/S) in the symplast, suggesting phosphate and phytochelatin sequestration, respectively. In the endodermis, sequestration of Cd/S was present as fine granular deposits in the vacuole and as large granular deposits in the cytoplasm. In the central cylinder, symplastic accumulation followed a distinct pattern illustrating the importance of passage cells for the uptake of Cd. In the apoplast, a shift of Cd/S granular deposits from the middle lamella towards the plasmalemma was observed. Large amounts of precipitated Cd in the phloem suggest retranslocation from the shoot. In leaves, Cd was detected in tracheids but not in the mesophyll tissue. Extensive symplastic and apoplastic sequestration in the root parenchyma combined with retranslocation via the phloem confirms the excluder strategy of Arabidopsis thaliana.

The balance between antioxidants, such as ascorbate (ASC) and glutathione, and oxidative reactive oxygen species (ROS) is known to play a pivotal role in the response of plant cells to abiotic stress. Here cell cultures of Arabidopsis thaliana were investigated with regard to their response to elevated levels of cadmium. At concentrations <100 microM, Cd induces a rapid and concentration-dependent H(2)O(2) accumulation. This response could be inhibited by diphenylene iodonium (DPI, 20 microM). Reverse transcription-PCR analysis of three RBOH (respiratory burst oxidase homologues) genes showed an increased transcription of RBOHF after 15 min. No change in ASC concentration was observed during the first 3 h after Cd addition. In contrast, glutathione levels completely diminished within 1 h. This drop could be attributed to an increase in phytochelatin 4. At the plasma membrane, Cd further induced a significant decrease in dehydroascorbate (DHA) uptake activity (up to 90% inhibition after 4 h). This decrease is not present when cells are treated with LaCl(3) before exposure to CdCl(2). LaCl(3) is a typical inhibitor of Ca channels and prevents Cd uptake in these cells as well as the Cd-induced ROS production. Therefore, these results appear to indicate that Cd uptake is a prerequisite for the change in DHA transport activity. However, DPI did not prevent the drop in DHA uptake activity present in Cd-treated Arabidopsis cells, indicating that this response seems to be independent of the Cd-induced H(2)O(2) production.

• We examined the subcellular cadmium (Cd) localization in roots and leaves of wild-type Arabidopsis thaliana (ecotype Columbia) exposed to environmentally relevant Cd concentrations. • Energy-dispersive X-ray microanalysis (EDXMA) was performed on high-pressure frozen and freeze-substituted tissues. • In the root cortex, Cd was associated with phosphorus (Cd/P) in the apoplast and sulfur (Cd/S) in the symplast, suggesting phosphate and phytochelatin sequestration, respectively. In the endodermis, sequestration of Cd/S was present as fine granular deposits in the vacuole and as large granular deposits in the cytoplasm. In the central cylinder, symplastic accumulation followed a distinct pattern illustrating the importance of passage cells for the uptake of Cd. In the apoplast, a shift of Cd/S granular deposits from the middle lamella towards the plasmalemma was observed. Large amounts of precipitated Cd in the phloem suggest retranslocation from the shoot. In leaves, Cd was detected in tracheids but not in the mesophyll tissue. • Extensive symplastic and apoplastic sequestration in the root parenchyma combined with retranslocation via the phloem confirms the excluder strategy of Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Abstract: The early antioxidative defence mechanisms were studied in Arabidopsis thaliana by applying a range of realistic Cd concentrations. Our data suggest that a 24h exposure to 20μM 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 10μM. 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. [Copyright &y& Elsevier]

Summary: The effect of excess copper on the expression of soluble proteins in 10-day old Phaseolus vulgaris seedlings was studied with two-dimensional electrophoresis and mass spectrometry, to find sensitive biochemical markers of exposure. Despite major differences in root Cu contents, both 15 and 50μM Cu treatments resulted in equal enhancements of Cu in the primary leaves. Three proteins, apparently reacting in a dose-dependent manner to Cu exposure, were identified from roots. The levels of an intracellular pathogenesis-related protein and a newly identified protein homologous to PvPR1, PvPR2, were increased with increasing Cu concentration. The level of a newly identified PR-10 protein decreased in a dose-dependent manner. No significant difference was observed in the leaf protein pattern between controls and 15μM Cu-treated plants. However, at 50μM Cu exposure, the appearance of PvPR1 and a homologue of Arabidopsis thaliana thylakoid lumenal 17.4kDa protein was observed. Another protein slightly enhanced by Cu treatment had sequence homology to a mitochondrial precursor of glycine cleavage system H protein of Flaveria pringlei. [Copyright &y& Elsevier]