RESUMO
This study determines the functional role of the plant ultraviolet-B radiation (UV-B) photoreceptor, UV RESISTANCE LOCUS 8 (UVR8) under natural conditions using a large-scale 'synchronized-genetic-perturbation-field-experiment'. Laboratory experiments have demonstrated a role for UVR8 in UV-B responses but do not reflect the complexity of outdoor conditions where 'genotype × environment' interactions can mask laboratory-observed responses. Arabidopsis thaliana knockout mutant, uvr8-7, and the corresponding Wassilewskija wild type, were sown outdoors on the same date at 21 locations across Europe, ranging from 39°N to 67°N latitude. Growth and climatic data were monitored until bolting. At the onset of bolting, rosette size, dry weight, and phenolics and glucosinolates were quantified. The uvr8-7 mutant developed a larger rosette and contained less kaempferol glycosides, quercetin glycosides and hydroxycinnamic acid derivatives than the wild type across all locations, demonstrating a role for UVR8 under field conditions. UV effects on rosette size and kaempferol glycoside content were UVR8 dependent, but independent of latitude. In contrast, differences between wild type and uvr8-7 in total quercetin glycosides, and the quercetin-to-kaempferol ratio decreased with increasing latitude, that is, a more variable UV response. Thus, the large-scale synchronized approach applied demonstrates a location-dependent functional role of UVR8 under natural conditions.
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Asymmetrical flow field-flow fractionation (AF4) efficiently separates various macromolecules and nano-components of natural waters according to their hydrodynamic sizes. The online coupling of AF4 with fluorescence (Fluo) and UV absorbance (UV) detectors (FluoD and UVD, respectively) and inductively coupled plasma-mass spectrometry (ICP-MS) provides multidimensional information. This makes it a powerful tool to characterize and quantify the size distributions of organic and inorganic nano-sized components and their interaction with trace metals. In this study, we developed a method combining thiol labeling by monobromo(trimethylammonio)bimane bromide (qBBr) with AF4-FluoD to determine the size distribution and the quantities of thiols in the macromolecular dissolved organic matter (DOM) present in highly colored DOM-rich water sampled from Shuya River and Lake Onego, Russia. We found that the qBBr-labeled components of DOM (qB-DOM) were of humic type, characterized by a low hydrodynamic size (d h < 2 nm), and have concentrations <0.3 µM. After enrichment with mercury, the complexes formed between the nano-sized components and Hg were analyzed using AF4-ICP-MS. The elution profile of Hg followed the distribution of the UV-absorbing components of DOM, characterized by slightly higher sizes than qB-DOM. Only a small proportion of Hg was associated with the larger-sized components containing Fe and Mn, probably inorganic oxides that were identified in most of the samples from river to lake. The size distribution of the Hg-DOM complexes was enlarged when the concentration of added Hg increased (from 10 to 100 nM). This was explained by the presence of small iron oxides, overlapping the size distribution of Hg-DOM, on which Hg bound to a small proportion. In addition, to provide information on the dispersion of macromolecular thiols in colored DOM-rich natural water, our study also illustrated the potential of AF4-FluoD-UVD-ICP-MS to trace or quantify dynamic changes while Hg binds to the natural nano-colloidal components of surface water.
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Metabolomics characterizes low-molecular-weight molecules involved in different biochemical reactions and provides an integrated assessment of the physiological state of an organism. By using liquid chromatography-mass spectrometry targeted metabolomics, we examined the response of green alga Chlamydomonas reinhardtii to sublethal concentrations of inorganic mercury (IHg) and monomethylmercury (MeHg). We quantified the changes in the levels of 93 metabolites preselected based on the disturbed metabolic pathways obtained in a previous transcriptomics study. Metabolites are downstream products of the gene transcription; hence, metabolite quantification provided information about the biochemical status of the algal cells exposed to Hg compounds. The results showed that the alga adjusts its metabolism during 2 h exposure to 5 × 10-9 and 5 × 10-8 mol L-1 IHg and MeHg by increasing the level of various metabolites involved in amino acid and nucleotide metabolism, photorespiration, and tricarboxylic acid (TCA) cycle, as well as the metabolism of fatty acids, carbohydrates, and antioxidants. Most of the metabolic perturbations in the alga were common for IHg and MeHg treatments. However, the exposure to IHg resulted in more pronounced perturbations in the fatty acid and TCA metabolism as compared with the exposure to MeHg. The observed metabolic perturbations were generally consistent with our previously published transcriptomics results for C. reinhardtii exposed to the comparable level of IHg and MeHg. The results highlight the potential of metabolomics for toxicity evaluation, especially to detect effects at an early stage of exposure prior to their physiological appearance.
Assuntos
Chlamydomonas reinhardtii , Mercúrio , Compostos de Metilmercúrio , Antioxidantes , Chlamydomonas reinhardtii/genética , Mercúrio/toxicidade , Metabolômica , Compostos de Metilmercúrio/toxicidadeRESUMO
As plants and associated insects are at the bottom of some terrestrial food webs, they are the primary contributors to mercury (Hg) fluxes in ecosystems. In addition to the trophic position of these organisms, factors related to their life traits have been hypothesized to influence their exposure to Hg. This study investigates the transfer of Hg in a soil-nettle-insect system and the insect-related factors affecting their Hg concentrations in a revegetated chlor-alkali landfill. Twenty-three insect species were identified and classified according to their life traits, their relationship with nettle, and their morphological characteristics. We observed low total mercury (THg) concentrations in nettles, with only 1% methylmercury (MeHg) being detected, while concentrations ranged from 5 to 3700 µg/kg dry wt. in insects with a MeHg percentage of up to 75%. The nettle-related insects were primarily exposed to Hg through the food web with significant biomagnification, particularly at the level of secondary predators. Within the nettle-unrelated group, the insect habitat was the most explanatory factor, with the highest enrichment being for the insects that spent part of their cycle in direct contact with Hg sources. Therefore, these insects require special attention because they are an essential vector of Hg transfer for terrestrial top predators.
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Mercúrio , Compostos de Metilmercúrio , Poluentes Químicos da Água , Animais , Ecossistema , Monitoramento Ambiental , Peixes , Cadeia Alimentar , InsetosRESUMO
The hypothesis that increased UV radiation result in co-tolerance to Hg toxicity in aquatic plants was studied at the physiological and transcriptomic level in Elodea nuttallii. At the transcriptomic level, combined exposure to UV+Hg enhanced the stress response in comparison with single treatments, affecting the expression level of transcripts involved in energy metabolism, lipid metabolism, nutrition, and redox homeostasis. Single and combined UV and Hg treatments dysregulated different genes but with similar functions, suggesting a fine regulation of the plant to stresses triggered by Hg, UV and their combination but lack of co-tolerance. At the physiological level, UV+Hg treatment reduced chlorophyll content and depleted antioxidative compounds such as anthocyanin and GSH/GSSG in E. nuttallii. Nonetheless, combined exposure to UV+Hg resulted in about 30% reduction of Hg accumulation into shoots vs exposure to Hg alone, which was congruent with the level of expression of several transporter genes, as well as the UV effect on Hg bioavailability in water. The findings of the present work underlined the importance of performing experimentation under environmentally realistic conditions and to consider the interplay between contaminants and environmental variables such as light that might have confounding effects to better understand and anticipate the effects of multiple stressors in aquatic environment.
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Hydrocharitaceae/efeitos dos fármacos , Hydrocharitaceae/efeitos da radiação , Mercúrio/toxicidade , Raios Ultravioleta/efeitos adversos , Poluentes Químicos da Água/toxicidade , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Clorofila/metabolismo , Hydrocharitaceae/metabolismo , Inativação Metabólica , Mercúrio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Testes de Toxicidade , Transcriptoma/efeitos dos fármacos , Transcriptoma/efeitos da radiação , Poluentes Químicos da Água/metabolismoRESUMO
In this study, the uptake and sub-toxic effects of CuO nanoparticles (CuO-NPs), dissolved Cu(II) alone or in combination with UV radiation on the aquatic macrophyte Elodea nuttallii were studied. Emphasis was on Cu accumulation, growth, photosynthesis and the oxidative stress related enzymes peroxidase (POD) and superoxide dismutase (SOD). The results showed stronger Cu accumulation in plants exposed to 10 mg L(-1) CuO-NPs, corresponding to 1.4-2 mg L(-1) dissolved Cu(II), than to 256 µg L(-1) Cu(II). However, the ratio between the accumulated Cu and dissolved Cu in CuO treatments was lower than in Cu(II) treatments. Additional UV exposure increased accumulation in both treatments, with the effect being stronger for Cu accumulation from CuO-NPs than for dissolved Cu(II). Photosynthetic capacity was strongly reduced by UV treatment, whereas remained unaffected by Cu(II) or CuO-NP treatments. Similarly, the increase of SOD activity was more pronounced in the UV treatments. On the other hand, POD activity enhancement was strongest in the plants exposed to CuO-NPs for 24 h. Expression of the copper transporter COPT1 as revealed by RT-qPCR was inhibited by Cu(II) and CuO-NP treatment, limiting the uptake of excess Cu into the cells. Overall, the combined exposure of E. nuttallii to UV radiation with CuO-NPs or Cu(II) has a higher impact than exposure to CuO-NPs or Cu(II) alone. The results imply that heavy pollution of natural water with CuO-NPs or dissolved Cu might have stronger effects in combination with natural UV irradiation on organisms in situ.
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Hydrocharitaceae/efeitos dos fármacos , Hydrocharitaceae/efeitos da radiação , Raios Ultravioleta , Poluentes Químicos da Água/toxicidade , Cobre/toxicidade , Hydrocharitaceae/metabolismo , Nanopartículas Metálicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Fotossíntese/efeitos dos fármacos , Fotossíntese/efeitos da radiaçãoRESUMO
Vegetated and shallow areas such as wetlands and salt marshes, as well as freshwater lakes and rivers, have been identified as hotspots for Hg methylation. The presence of aquatic macrophytes, the predominant primary producers in shallow waters, plays an important but still poorly understood role in the fate of Hg in these environments. The present review focuses on the influences of macrophytes on Hg speciation and distribution in sediments, the rhizosphere, and the water column; on Hg transformation; and on Hg release to the environment, including transfer to the trophic web. Future research will require an improved understanding of the mechanisms and the factors controlling these aspects as well as a broader general view. Thus, the main gaps in knowledge are also discussed.
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Organismos Aquáticos/metabolismo , Mercúrio/metabolismo , Plantas/metabolismo , Biotransformação , Cadeia AlimentarRESUMO
The present article reviews current knowledge and recent progress on the bioavailability and toxicity of mercury to aquatic primary producers. Mercury is a ubiquitous toxic trace element of global concern. At the base of the food web, primary producers are central for mercury incorporation into the food web. Here, the emphasis is on key, but still poorly understood, processes governing the interactions between mercury species and phytoplankton, and macrophytes, two representatives of primary producers. Mass transfer to biota surface, adsorption to cell wall, internalization and release from cells, as well as underlying toxicity mechanisms of both inorganic mercury and methylmercury are discussed critically. In addition, the intracellular distribution and transformation processes, their importance for mercury toxicity, species-sensitivity differences and trophic transfer are presented. The mini-review is illustrated with examples of our own research.
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Mercúrio , Compostos de Metilmercúrio , Poluentes Químicos da Água/análise , Cadeia AlimentarRESUMO
Macrophytes bioaccumulate metals, the suggestion being made that they be considered for phytoremediation. However, a thorough understanding of the mechanisms of metal tolerance in these plants is necessary to allow full optimization of this approach. The present study was undertaken to gain insight into Hg and Cd accumulation and their effects in a representative macrophyte, Elodea nuttallii. Exposure to methyl-Hg (23 ng dm(-3)) had no significant effect while inorganic Hg (70 ng dm(-3)) and Cd (281 µg dm(-3)) affected root growth but did not affect shoots growth, photosynthesis, or antioxidant enzymes. Phytochelatins were confirmed as having a role in Cd tolerance in this plant while Hg tolerance seems to rely on different mechanisms. Histology and subcellular distribution revealed a localized increase in lignification, and an increased proportion of metal accumulation in cell wall over time. Proteomics further suggested that E. nuttallii was able to efficiently adapt its energy sources and the structure of its cells during Hg and Cd exposure. Storage in cell walls to protect cellular machinery is certainly predominant at environmental concentrations of metals in this plant resulting in a high tolerance highlighted by the absence of toxicity symptoms in shoots despite the significant accumulation of metals.
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Parede Celular/efeitos dos fármacos , Hydrocharitaceae/efeitos dos fármacos , Metais/química , Proteômica/métodos , Antioxidantes/química , Biodegradação Ambiental , Butionina Sulfoximina/química , Cádmio/química , Cloreto de Cádmio/química , Eletroforese em Gel Bidimensional , Géis/química , Cinética , Lignina/química , Cloreto de Mercúrio/química , Mercúrio/química , Peroxidase/química , Fotossíntese/efeitos dos fármacos , Fitoquelatinas/química , Proteínas de Plantas/química , Raízes de Plantas/efeitos dos fármacos , Brotos de Planta/efeitos dos fármacos , Poluentes do Solo/análise , Superóxido Dismutase/químicaRESUMO
Toxic metals polluting aquatic ecosystems are taken up by inhabitants and accumulate in the food web, affecting species at all trophic levels. It is therefore important to have good tools to assess the level of risk represented by toxic metals in the environment. Macrophytes are potential organisms for the identification of metal-responsive biomarkers but are still underrepresented in ecotoxicology. In the present study, we used next-generation sequencing to investigate the transcriptomic response of Elodea nuttallii exposed to enhanced concentrations of Hg and Cd. We de novo assembled more than 60 000 contigs, of which we found 170 to be regulated dose-dependently by Hg and 212 by Cd. Functional analysis showed that these genes were notably related to energy and metal homeostasis. Expression analysis using nCounter of a subset of genes showed that the gene expression pattern was able to assess toxic metal exposure in complex environmental samples and was more sensitive than other end points (e.g., bioaccumulation, photosynthesis, etc.). In conclusion, we demonstrate the feasibility of using gene expression signatures for the assessment of environmental contamination, using an organism without previous genetic information. This is of interest to ecotoxicology in a wider sense given the possibility to develop specific and sensitive bioassays.
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Cádmio/toxicidade , Hydrocharitaceae/genética , Mercúrio/toxicidade , Transcriptoma , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores , Expressão Gênica , RNA de Plantas/genéticaRESUMO
Previous studies suggest that macrophytes might participate in bioaccumulation and biomagnification of toxic mercury (Hg) in aquatic environment. Hg bioaccumulation and uptake mechanisms in macrophytes need therefore to be studied. Amongst several macrophytes collected in an Hg contaminated reservoir in Romania, Elodea nuttallii showed a high organic and inorganic Hg accumulation and was then further studied in the laboratory. Tolerance and accumulation of Hg of this plant was also high in the microcosm. Basipetal transport of inorganic Hg was predominant, whereas acropetal transport of methyl-Hg was observed with apparently negligible methylation or demethylation in planta. Hg concentrations were higher in roots>leaves>stems and in top>middle>bottom of shoots. In shoots, more than 60% Hg was found intracellularly where it is believed to be highly available to predators. Accumulation in shoots was highly reduced by cold, death and by competition with Cu(+). Hg in E. nuttallii shoots seems to mainly originate from the water column, but methyl-Hg could also be remobilized from the sediments and might drive in part its entry in the food web. At the cellular level, uptake of Hg into the cell sap of shoots seems linked to the metabolism and to copper transporters. The present work highlights an important breakthrough in our understanding of Hg accumulation and biomagnifications: the remobilization of methyl-Hg from sediments to aquatic plants and differences in uptake mechanisms of inorganic and methyl-Hg in a macrophyte.
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Hydrocharitaceae/metabolismo , Mercúrio/metabolismo , Brotos de Planta/metabolismo , Poluentes Químicos da Água/metabolismo , Biodegradação Ambiental , Cobre/metabolismo , Ecossistema , Cadeia Alimentar , Mercúrio/análise , Romênia , Poluentes Químicos da Água/análiseRESUMO
The objective of this study was to assess the effect of a rooted macrophyte Elodea nuttallii on rhizosphere bacterial communities in Hg contaminated sediments. Specimens of E. nuttallii were exposed to sediments from the Hg contaminated Babeni reservoir (Olt River, Romania) in our microcosm. Plants were allowed to grow for two months until they occupied the entirety of the sediments. Total Hg and MMHg were analysed in sediments where an increased MMHg percentage of the total Hg in pore water of rhizosphere sediments was found. E. nuttallii roots also significantly changed the bacterial community structure in rhizosphere sediments compared to bulk sediments. Deltaproteobacteria dominated the rhizosphere bacterial community where members of Geobacteraceae within the Desulfuromonadales and Desulfobacteraceae were identified. Two bacterial operational taxonomic units (OTUs) which were phylogenetically related to sulfate-reducing bacteria (SRB) became abundant in the rhizosphere. We suggest that these phylotypes could be potentially methylating bacteria and might be responsible for the higher MMHg percentage of the total Hg in rhizosphere sediments. However, SRB were not significantly favoured in rhizosphere sediments as shown by qPCR. Our findings support the hypothesis that rooted macrophytes created a microenvironment favorable for Hg methylation. The presence of E. nuttallii in Hg contaminated sediments should therefore not be overlooked.
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Bactérias , Sedimentos Geológicos/química , Sedimentos Geológicos/microbiologia , Hydrocharitaceae/fisiologia , Compostos de Metilmercúrio/química , Raízes de Plantas/fisiologia , Bactérias/classificação , Bactérias/genética , Poluição Ambiental , Mercúrio/química , Filogenia , RNA Ribossômico 16S , Rios/química , Rios/microbiologia , Poluição Química da ÁguaRESUMO
This study characterized the fecal indicator bacteria (FIB), including Escherichia coli (E. coli) and Enteroccocus (ENT), disseminated over time in the Bay of Vidy, which is the most contaminated area of Lake Geneva. Sediments were collected from a site located at â¼500 m from the present waste water treatment plant (WWTP) outlet pipe, in front of the former WWTP outlet pipe, which was located at only 300 m from the coastal recreational area (before 2001). E. coli and ENT were enumerated in sediment suspension using the membrane filter method. The FIB characterization was performed for human Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and human specific bacteroides by PCR using specific primers and a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bacterial cultures revealed that maximum values of 35.2 × 10(8) and 6.6 × 10(6)CFU g(-1) dry sediment for E. coli and ENT, respectively, were found in the sediments deposited following eutrophication of Lake Geneva in the 1970s, whereas the WWTP started operating in 1964. The same tendency was observed for the presence of human fecal pollution: the percentage of PCR amplification with primers ESP-1/ESP-2 for E. faecalis and E. faecium indicated that more than 90% of these bacteria were from human origin. Interestingly, the PCR assays for specific-human bacteroides HF183/HF134 were positive for DNA extracted from all isolated strains of sediment surrounding WWPT outlet pipe discharge. The MALDI-TOF MS confirmed the presence of general E. coli and predominance E. faecium in isolated strains. Our results demonstrated that human fecal bacteria highly increased in the sediments contaminated with WWTP effluent following the eutrophication of Lake Geneva. Additionally, other FIB cultivable strains from animals or adapted environmental strains were detected in the sediment of the bay. The approaches used in this research are valuable to assess the temporal distribution and the source of the human fecal pollution in aquatic environments.
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Bactérias/crescimento & desenvolvimento , Fezes/microbiologia , Sedimentos Geológicos/microbiologia , Lagos/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bacteroides/classificação , Bacteroides/genética , Bacteroides/crescimento & desenvolvimento , Bacteroides/isolamento & purificação , Monitoramento Ambiental , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Eutrofização , Humanos , SuíçaRESUMO
BACKGROUND: RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably influenced by the chosen method. RESULTS: We quantified gene expression of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the roots of two black poplar clones, 58-861 and Poli, which were subjected to drought stress. After proving the chosen reference genes actin (ACT), elongation factor 1 (EF1) and ubiquitin (UBQ) to be constantly expressed in the different watering regimes, we applied different approaches for relative quantification to the same raw fluorescence data. The results obtained using the comparative Cq method, LinRegPCR, qBase software and the Pfaffl model showed a good correlation, whereas calculation according to the Liu and Saint method produced highly variable results. However, it has been shown that the most reliable approach for calculation of the amplification efficiency is using the mean increase in fluorescence during PCR in each individual reaction. Accordingly, we could improve the quality of our results by applying the mean amplification efficiencies for each amplicon to the Liu and Saint method. CONCLUSIONS: As we could show that gene expression results can vary depending on the approach used for quantification, we recommend to carefully evaluate different quantification approaches before using them in studies analysing gene expression.
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Expressão Gênica , Populus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ascorbato Peroxidases , Secas , Peroxidases/genética , Peroxidases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Populus/enzimologia , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , ÁguaRESUMO
Trees need to store reserves to allow their survival during winter and for bud flush and leaf growth in the following spring. In many tree species, these reserve functions are mainly covered by starch, which is degraded to soluble carbohydrates during the dormant season for maintenance respiration and in spring during bud flush. We conducted girdling experiments on poplar (Populus deltoides x nigra cv. Dorskamp) in order to elucidate how interrupted transport of carbohydrates to the roots during autumn affects plant survival during winter and bud flush in spring. We measured the content of starch, sucrose, glucose, fructose, raffinose and stachyose in stems (above and below the girdle), coarse roots and fine roots over 1 year. We found that, in response to girdling, carbohydrates accumulated in stems above the girdle. As a result of interrupted reserve allocation, girdled plants depleted their root starch reserves nearly to zero, whereas in stems below the girdle, reserves were maintained close to control values, presumably in order to facilitate dormancy release and re-sprouting from buds below the girdle. Furthermore, we showed that stachyose accumulated during winter also in the roots, even in girdled plants, consistent with its importance as freezing protectant. The lower stachyose content of roots compared with shoots was likely due to protection of the roots from cold by the soil.
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Carboidratos/fisiologia , Raízes de Plantas/fisiologia , Caules de Planta/fisiologia , Populus/fisiologia , Amido/fisiologia , Transporte Biológico , Carboidratos/química , Nitrogênio/metabolismo , Populus/crescimento & desenvolvimento , Estações do Ano , Amido/química , Fatores de TempoRESUMO
Drought is expected to become an increasingly important factor limiting tree growth caused by climate change. Two divergent clones of Populus nigra (58-861 and Poli) originating from contrasting environments were subjected to water limitation (WL) to elucidate whether they differ in tolerance to drought, which mechanisms to avoid stress they exhibit and whether drought has an impact on the interactions between roots and shoots. Limiting water availability caused photosynthetic rate and total non-structural carbohydrate (TNC) levels to decrease in 58-861. However, starch-degrading enzyme activity and gene expression were induced in roots, and soluble sugar levels were higher than in well-watered (WW) plants. These data suggest that assimilation and partitioning of carbon to the roots are decreased, resulting in mobilization of stored starch. In contrast, the photosynthetic rate of Poli was reduced only late in the treatment, and carbohydrate levels in WL plants were higher than in WW plants. Superoxide dismutase (SOD) activity and gene expression were higher in Poli than in 58-861, even in WW plants, leading to a higher capacity to defend against oxidative stress.