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In accordance with realistic application approaches, a short-term 1-factorial experiment was set up to investigate the phytotoxic impact of pre-emergent application of the chloroacetamide herbicide metazachlor on Brassica napus. In addition to morphological parameters, the underlying processes that ultimately determine the extent of herbicide-induced phytotoxicity (i.e., herbicide metabolization and cellular antioxidant defense) were examined. The present study demonstrated that metazachlor provoked fasciation of the leaves closely after emergence, which might be linked to its mode of action whereby cell division is impaired through the inhibition of very long chain fatty acid synthesis. The increased activities of antioxidative enzymes and metabolites in leaf tissue indicated the presence of reactive oxygen species under the influence of metazachlor. This resulted in oxidative damage in the form of membrane lipid peroxidation. Simultaneously, the increased activity of glutathione S-transferase and the shift in glutathione redox state suggested activation of the detoxification metabolism. This occurred, however, at the expense of growth, with a temporary reduction in plant height and weight after application. The results indicated that metazachlor disappeared within 3 mo to 4 mo after application, which resulted in the recovery of the crop. In conclusion, metazachlor induces phytotoxicity in the short term, either directly through its mode of action or indirectly through the induction of oxidative stress, which resulted in a temporary reduction in growth. Environ Toxicol Chem 2017;36:59-70. (c) 2016 SETAC We thank A. Wijgaerts and C. Put for their technical assistance in the microcosm experiment. The present study was supported by a PhD grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen, to H. Vercampt).

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. Summary statement We demonstrated that addition of supplemental Cu to Cd-exposed Arabidopsis thaliana plants diminished the SPL7-dependent Cu deficiency response in roots, while it even disappeared in leaves. Moreover, in roots of phytochelatin (PC-) deficient cad1-3 mutants, this Cu deficiency response was reduced. Therefore, 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. This work was supported by Hasselt University by a PhD grant for Heidi Gielen and through the Methusalem project [08M03VGRJ].

Air pollution, containing fossil fuel-related volatile organic compounds (VOCs), constitutes a major problem in most urban areas. According to recent data, more people die from exposure to air pollution than from malaria and HIV infection together. Many years of research show that plant–microbe interactions can be exploited to significantly enhance phytoremediation of contaminated environments. Microorganisms living on plant leaves constitute promising candidates to detoxify harmful VOCs and promote plant growth. Hedera helix, an evergreen plant known for its hardiness and climbing ability, proves to be an excellent host species in the context of phytoremediation of air pollution. We employed 16S rRNA gene amplicon-based metagenomics and real-time PCR to characterize the taxonomic structure of phyllospheric microbial communities associated with H. helix, and compared the communities present at polluted high-traffic locations with non-polluted sites. Further, we investigated bacterial isolates using culture-dependent approaches and sequenced the genomes of promising bacteria with regard to phytoremediation applications. Our results indicate that Actinobacteria, Firmicutes and Betaproteobacteria are more abundantly present on H. helix leaves in a polluted environment. Moreover, the relative composition of the H. helix phyllobiome appears to be similar to that of the model plant Arabidopsis thaliana. We further show that air pollution increases the prevalence of bacterial functional traits affiliated to plant growth and detoxification of fossil fuel-related VOCs. The presence of relevant pathways could be confirmed by genome-level analyses of bacterial isolates showing a high in vitro capacity to metabolize selected VOCs and promote plant growth. In conclusion, an integrated description of the H. helix phyllobiome is presented, including the identification of interesting bacteria with significant potential as inoculants in phytoremediation approaches. This work is supported by the Hasselt University Methusalem project 08M03VGRJ

The INTENSE project: Intensify production, transform biomass to energy and novel goods and protect soils in Europe. International Conference on the Biogeochemistry of Trace Elements ICOBTE

Phytoextraction makes use of trace element-accumulating plants that concentrate the pollutants in their tissues. Pollutants can be then removed by harvesting plants. The success of phytoextraction depends on trace element availability to the roots and the ability of the plant to intercept, take up, and accumulate trace elements in shoots. Current phytoextraction practises either employ hyperaccumulators or fast-growing high biomass plants; the phytoextraction process may be enhanced by soil amendments that increase trace element availability in the soil. This review will focus on the role of plant-associated bacteria to enhance trace element availability in the rhizosphere. We report on the kind of bacteria typically found in association with trace element – tolerating or – accumulating plants and discuss how they can contribute to improve trace element uptake by plants and thus the efficiency and rate of phytoextraction. This enhanced trace element uptake can be attributed to a microbial modification of the absorptive properties of the roots such as increasing the root length and surface area and numbers of root hairs, or by increasing the plant availability of trace elements in the rhizosphere and the subsequent translocation to shoots via beneficial effects on plant growth, trace element complexation and alleviation of phytotoxicity. An analysis of data from literature shows that effects of bacterial inoculation on phytoextraction efficiency are currently inconsistent. Some key processes in plant–bacteria interactions and colonization by inoculated strains still need to be unravelled more in detail to allow full-scale application of bacteria assisted phytoremediation of trace element contaminated soils.

Phytoextraction has been reported as an economically and ecologically sound alternative for the remediation of metal-contaminated soils. Willow is a metal phytoextractor of interest because it allows to combine a gradual contaminant removal with production of biomass that can be valorized in different ways. In this work two willow clones growing on a metal-contaminated site were selected: ‘Belgisch Rood’ (BR) with a moderate metal extraction capacity and ‘Tora’ (TO) with a twice as high metal accumulation. All cultivable bacteria associated with both willow clones were isolated and identified using 16SrDNA ARDRA analysis followed by 16SrDNA sequencing. Further all isolated bacteria were investigated for characteristics that might promote plant growth (production of siderophores, organic acids and indol acetic acid) and for their metal resistance. The genotypic and phenotypic characterization of the isolated bacteria showed that the TO endophytic bacterial population is more diverse and contains a higher percentage of metal-resistant plant growth promoting bacteria than the endophytic population associated with BR. We hypothesize that the difference in the metal accumulation capacity between BR and TO clones might be at least partly related to differences in characteristics of their associated bacterial population. Funding Information This research was funded by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), PhD grant for J.J., and by the Fund for Scientific Research Flanders (FWO-Vlaanderen), PhD grant for S.C. and post doc grant for N. We. and N. Wi. This work has been financially supported by the UHasselt Methusalem project 08M03VGRJ and by the European Commission under the Seventh Framwork Programme for Research (FP7-KBBE-266124, GREEN-LAND).

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

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

Gentle remediation options (GROs) are risk management strategies or technologies involving plant (phyto-), fungi (myco-), and/or bacteria-based methods that result in a net gain (or at least no gross reduction) in soil function as well as effective risk management. GRO strategies can be customised along contaminant linkages, and can generate a range of wider economic, environmental and societal benefits in contaminated land management (and in brownfields management more widely). The application of GROs as practical on-site remedial solutions is still limited however, particularly in Europe and at trace element (typically metal and metalloid) contaminated sites. This paper discusses challenges to the practical adoption of GROs in contaminated land management, and outlines the decision support tools and best practice guidance developed in the European Commission FP7-funded GREENLAND project aimed at overcoming these challenges. The GREENLAND guidance promotes a refocus from phytoremediation to wider GROs- or phyto-management based approaches which place realisation of wider benefits at the core of site design, and where gentle remediation technologies can be applied as part of integrated, mixed, site risk management solutions or as part of “holding strategies” for vacant sites. The combination of GROs with renewables, both in terms of biomass generation but also with green technologies such as wind and solar power, can provide a range of economic and other benefits and can potentially support the return of low-level contaminated sites to productive usage, while combining GROs with urban design and landscape architecture, and integrating GRO strategies with sustainable urban drainage systems and community gardens/parkland (particularly for health and leisure benefits), has large potential for triggering GRO application and in realising wider benefits in urban and suburban systems. Quantifying these wider benefits and value (above standard economic returns) will be important in leveraging funding for GRO application and soft site end-use more widely at vacant or underutilized sites. Further details of the GREENLAND project, and downloadable versions of the GREENLAND decision support tool and GRO best practice guidance, can be found at http://www.greenland-project.eu. The authors are grateful for financial support from the European Commission under the Seventh Framework Programme for Research (FP7-KBBE-266124), and thank the wider GREENLAND consortium and project advisory board for their support and input during the development of the DST and GRO best practice guidance.

International audience; BACKGROUND The disposal of metal-enriched plant biomass is an important and still unresolved issue associated with phytoextraction. Therefore, the aim of this study was to valorise these contaminated biomasses into valuable products, i.e. bio-chars and activated carbons (ACs), through slow pyrolysis followed by steam activation. The effects of pyrolysis/steam activation on metal availability in the chars/ACs were also evaluated. In addition, Cr(VI) adsorption by these ACs in aqueous solutions was studied. RESULTS Relevant chars/ACs yields were obtained due to applied thermochemical treatments. Leachability of heavy metals from those products was limited indicating that they may be safely applied as adsorption media. Prepared ACs demonstrated good Cr(VI) adsorption capacities occurring through a ‘coupled adsorption–reduction’ mechanism. It was found that Cr(VI) removal strongly depended on pH of Cr(VI) solutions and was higher for ACs activated at 800 °C. Among applied isotherm adsorption models, i.e. Langmuir and Freundlich, a better fit was obtained with the Langmuir model corresponding to monolayer coverage sorption of Cr(VI) on ACs surface. CONCLUSIONS Heavy metals enriched biomass can be successfully valorised through slow pyrolysis/steam activation into value added products, i.e. ACs that can be used as efficient and safe adsorbents for Cr(VI) removal.

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

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

Additional file 1. Soil characteristics analyzed in 2012 and 2013. a organic matter, b electrical conductivity of a saturated soil paste extract (ECe), c Total soil N by Kjeldahl method.

Phytoremediation is a promising technology to clean-up contaminated soils based on the synergistic actions of plants and microorganisms. However, to become a widely accepted, and predictable remediation alternative, a deeper understanding of the plant-microbe interactions is needed. A number of studies link the success of phytoremediation to the plant-associated microbiome functioning, though whether the microbiome can exist in alternative, functional states for soil remediation, is incompletely understood. Moreover, current approaches that target the plant host, and environment separately to improve phytoremediation, potentially overlook microbial functions and properties that are part of the multiscale complexity of the plant-environment wherein biodegradation takes place. In contrast, in situ studies of phytoremediation research at the metaorganism level (host and microbiome together) are lacking. Here, we discuss a competition-driven model, based on recent evidence from the metagenomics level, and hypotheses generated by microbial community ecology, to explain the establishment of a catabolic rhizosphere microbiome in a contaminated soil. There is evidence to ground that if the host provides the right level and mix of resources (exudates) over which the microbes can compete, then a competitive catabolic and plant-growth promoting (PGP) microbiome can be selected for as long as it provides a competitive superiority in the niche. The competition-driven model indicates four strategies to interfere with the microbiome. Specifically, the rhizosphere microbiome community can be shifted using treatments that alter the host, resources, environment, and that take advantage of prioritization in inoculation. Our model and suggestions, considering the metaorganism in its natural context, would allow to gain further knowledge on the plant-microbial functions, and facilitate translation to more effective, and predictable phytotechnologies. This work was supported by a PhD grant to ST and a post doc grant for NW from the Fund for Scientific Research Flanders, Belgium (FWO-Vlaanderen) and the Methusalem project 08M03VGRJ.

Plants exposed to excess metals are challenged by an increased generation of reactive oxygen species (ROS) such as superoxide (O-2(center dot-)), hydrogen peroxide (H2O2) and the hydroxyl radical ((OH)-O-center dot). The mechanisms underlying this oxidative challenge are often dependent on metal-specific properties and might play a role in stress perception, signaling and acclimation. Although ROS were initially considered as toxic compounds causing damage to various cellular structures, their role as signaling molecules became a topic of intense research over the last decade. Hydrogen peroxide in particular is important in signaling because of its relatively low toxicity, long lifespan and its ability to cross cellular membranes. The delicate balance between its production and scavenging by a plethora of enzymatic and metabolic antioxidants is crucial in the onset of diverse signaling cascades that finally lead to plant acclimation to metal stress. In this review, our current knowledge on the dual role of ROS in metal-exposed plants is presented. Evidence for a relationship between H2O2 and plant metal tolerance is provided. Furthermore, emphasis is put on recent advances in understanding cellular damage and downstream signaling responses as a result of metal-induced H2O2 production. Finally, special attention is paid to the interaction between H2O2 and other signaling components such as transcription factors, mitogen-activated protein kinases, phytohormones and regulating systems (e.g. microRNAs). These responses potentially underlie metal-induced senescence in plants. Elucidating the signaling network activated during metal stress is a pivotal step to make progress in applied technologies like phytoremediation of polluted soils. This work was supported by the Research Foundation Flanders (FWO) by a postdoctoral grant for EK and projects [G0D3414] and [G0D11141]. Additional funding came from Hasselt University (BOF12NI28, BOF14DOC04) and PhD grants from the Agency for Innovation by Science and Technology (IWT-Flanders).

Widespread pollution of terrestrial ecosystems with petroleum hydrocarbons (PHGs) has generated a need for remediation and, given that many PHCs are biodegradable, bio- and phyto-remediation are often viable approaches for active and passive remediation. This review focuses on phytoremediation with particular interest on the interactions between and use of plant-associated bacteria to restore PHC polluted sites. Plant-associated bacteria include endophytic, phyllospheric, and rhizospheric bacteria, and cooperation between these bacteria and their host plants allows for greater plant survivability and treatment outcomes in contaminated sites. Bacterially driven PHC bioremediation is attributed to the presence of diverse suites of metabolic genes for aliphatic and aromatic hydrocarbons, along with a broader suite of physiological properties including biosurfactant production, biofilm formation, chemotaxis to hydrocarbons, and flexibility in cell-surface hydrophobicity. In soils impacted by PHC contamination, microbial bioremediation generally relies on the addition of high-energy electron acceptors (e.g., oxygen) and fertilization to supply limiting nutrients (e.g., nitrogen, phosphorous, potassium) in the face of excess PHC carbon. As an alternative, the addition of plants can greatly improve bioremediation rates and outcomes as plants provide microbial habitats, improve soil porosity (thereby increasing mass transfer of substrates and electron acceptors), and exchange limiting nutrients with their microbial counterparts. In return, plant-associated microorganisms improve plant growth by reducing soil toxicity through contaminant removal, producing plant growth promoting metabolites, liberating sequestered plant nutrients from soil, fixing nitrogen, and more generally establishing the foundations of soil nutrient cycling. In a practical and applied sense, the collective action of plants and their associated microorganisms is advantageous for remediation of PHC contaminated soil in terms of overall cost and success rates for in situ implementation in a diversity of environments. Mechanistically, there remain biological unknowns that present challenges for applying bio- and phyto-remediation technologies without having a deep prior understanding of individual target sites. In this review, evidence from traditional and modern omics technologies is discussed to provide a framework for plant microbe interactions during PHC remediation. The potential for integrating multiple molecular and computational techniques to evaluate linkages between microbial communities, plant communities and ecosystem processes is explored with an eye on improving phytoremediation of PHC contaminated sites. This work was supported by the Hasselt University BOF project 06G02 and the Methusalem project 08M03VGRJ.

The phytohormone ethylene is known to mediate a diverse array of signaling processes during abiotic stress in plants. Whereas many reports have demonstrated enhanced ethylene production in metal-exposed plants, the underlying molecular mechanisms are only recently investigated. Increasing evidence supports a role for ethylene in the regulation of plant metal stress responses. Moreover, crosstalk appears to exist between ethylene and the cellular redox balance, nutrients and other phytohormones. This review highlights our current understanding of the key role ethylene plays during responses to metal exposure. Moreover, particular attention is paid to the integration of ethylene within the broad network of plant responses to metal stress. This work was supported by the Research Foundation Flanders (FWO) by a grant for EK and project [G0D3414]. Additional funding came from Hasselt University through a PhD grant for KS and the Methusalem project [08M03VGRJ].

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