Project description:Field-selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola) populations occurring at metal-contaminated mining sites. This tolerance is correlated with heritable increase of metal excretion efficiency; less pronounce cadmium induced growth reduction and, over-expression of the metallothionein gene. We applied transcriptomics to determine differential gene expression caused by this abiotic stress in reference and cadmium tolerant populations. Many cDNAs responded to cadmium exposure in a reference population. Significantly fewer clones were cadmium responsive in tolerant animals. Analysis of variance revealed transcripts that interact between cadmium exposure and population. Hierarchical clustering of these clones revealed two major groups. The first one contained cDNAs that were up regulated by cadmium in the reference culture, but non-responsive or down regulated in tolerant animals. This cluster was also characterized by elevated constitutive expression in the tolerant population. Gene ontology analysis revealed that these cDNAs were involved in structural integrity of the cuticle, anti-microbial defense, calcium-channel blocking, neurotransmitter transport, chromatin remodeling and, endoplasmatic vesicle activity. The second group consisted of cDNAs down regulated in reference animals but not responding or slightly up regulated in tolerant animals. Their functions involved carbohydrate metabolic processes, Ca2+ dependent stress signaling, proteolysis and digestion. The reference population showed a strong signature of stress-induced genome-wide perturbation of gene expression, whereas the tolerant animals maintained normal gene expression upon cadmium exposure. We confirmed the micro-evolutionary processes occurring in soil arthropod populations and suggest a major contribution of gene regulation to the evolution of a stress-adapted phenotype. Orchesella cincta (Collembola) from the laboratory population (LC) at the department of Animal Ecology, Vrije Universiteit Amsterdam were taken as the reference group. This population originated from a pine forest from the reference area (Roggebotzand, the Netherlands, latitude N = 52 º 34’17’’, longitude E = 5 º 47’56’’) that contains on average < 0.5 μg cadmium per gram litter and humus. Tolerant animals were collected from randomly selected litter samples at two areas of the abandoned, but still heavily polluted lead/zinc mining site of Plombières (Belgium, latitude N = 50˚44’03’’, longitude E = 5˚58’02’’): the locations contain an average cadmium concentration between 10 and 52 μg.g-1 soil (Lock et al. 2003; Sterenborg 2003; Van Straalen et al. 1987). To diminish putative environmental effects from the field the animals were reared in a climate room (20°C, 75% humidity, LD 12 h:12 h) in PVC jars on a moist plaster of Paris layer feeding on algae present on twigs for at least three generations before experimental treatment. Animals were exposed to cadmium (nominal concentration of 112,4 μg cadmium.g-1 dry algae) for 3 days, directly after molting, via algal food. Control groups received the same treatment except that water was spiked into the algal food instead of cadmium solution. The cadmium exposure concentration is two times the no observed effect concentration (NOEC) for reproduction in chronically exposed animals. Total RNA was extracted from times 12 pooled animals per treatment using SV Total RNA Isolation system (Promega) and quantified on a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies). Total RNA was visualized on a 1.5% agarose gel to verify its integrity. Each experimental group (pool of 12 animals) was replicated 8 times. Dyes were swapped between biological replicates: 4 Cy-3 labeled replicates and 4 Cy-5 lebeled replicates per experimental group Microarray experiment was designed in a closed loop Lab culture clean vs Cd, Lab culture Cd vs Tolerant culture Cd, Tolerant culture Cd vs Tolerant culture clean, Tolerant culture clean vs lab culture clean

Project description:Marine biomonitoring programs in the U.S. and Europe have historically relied on monitoring tissue concentrations of bivalves to monitor contaminant levels and ecosystem health. By integrating M-bM-^@M-^Xomic methods with these tissue residue approaches we can uncover mechanistic insight to help link tissue concentrations to toxic effects. In an effort to identify novel biomarkers and better understand the molecular toxicology of metal bioaccumulation in bivalves, we exposed the blue mussel, Mytilus edulis L., to sub-lethal concentrations (0.54 M-oM-^AM--M) of cadmium, lead, and a Cd+Pb mixture. Metal concentrations were measured in gill tissues at 1, 2, and 4 weeks, and increased linearly over the 4 week duration. In addition, there was evidence that Pb interfered with Cd uptake in the mixture treatment. Using a 3025 sequence microarray for M. edulis, we performed transcriptomic analysis, identifying 57 differentially expressed sequences. Hierarchical clustering of these sequences successfully distinguished the different treatment groups demonstrating that the expression profiles were reproducible among the treatments. Enrichment analysis of gene ontology terms identified several biological processes that were perturbed by the treatments, including nucleoside phosphate biosynthetic processes, mRNA metabolic processes, and response to stress. To identify transcripts whose expression level correlated with metal bioaccumulation, we performed Pearson correlation analysis. Several transcripts correlated with gill metal concentrations including mt10, mt20, and contig 48, an unknown transcript containing a wsc domain. In addition, three transcripts directly involved in the Unfolded protein response (UPR) were induced in the metal treatments at 2 weeks and were further up-regulated at 4 weeks. Overall, correlation of tissue concentrations and gene expression responses indicates that as mussels accumulate higher concentrations of metals, initial stress responses are mobilized to protect tissues. However, given the role of UPR in apoptosis, it serves as an early indicator of stress, which once overwhelmed will result in adverse physiological effects. A total of 52 samples including initial samples and 3 time-points following exposure to cadmium, lead, or a mixture. There were four replicate samples for each treatment and time-point. An additional three replicate samples were used for RT-qPCR.

Project description:Phytoremediation soil polluted by heavy metal has been drawn on a worldwide attention from human society. However, how to improve the efficiency of plant remediation of soil contaminated by cadmium remains unknown. Previous studies showed that nitrogen (N) significantly enhanced cadmium uptake in poplar plants. In order to further explore the key role of N in the detoxification against cadmium stress in plants, this study try to investigate the poplar proteome and phosphoproteome difference between Cd stress and Cd+N treatment. In total, 5838 of the 6573 identified were quantified. With a fold-change threshold >1.3 and p-value<0.05, 375 and 108 proteins were up- and down-regulated by Cd stress when compared to the control, 42 and 89 proteins were up- and down-regulated, respectively, in Cd+N / Cd group, 522 and 127 proteins were up- and down-regulated, respectively, in Cd+N / CK group. In addition, the phosphoproteome data was obtained after the proteomic difference was normalized, and 1471phosphosites in 721 proteins were quantified. Based on a fold-change threshold >1.2, P-value <0.05, the Cd stress up-regulated 8 phosphosites in 8 proteins and down-regulated 69 phosphosites in 58 proteins, whereas N+Cd treatment up-regulated 95 phosphosites in 86 proteins and down-regulated 17 phosphosites in 17 proteins when compared to sole Cd stress. In addition, N+Cd treatment up-regulated 74 phosphosites in 60 proteins and down-regulated 42 phosphosites in 37 proteins when compared to the control.Several putative responses to stress proteins, transcriptional and translational regulation factors were up-regulated by addition ofexogenous nitrogen followed Cd stress at the proteome and phosphoproteome levels. Especially, heat shock protein 70 (HSP70), peroxidase (POD), zinc finger protein (ZFP), ABC transporter protein (ABC), eukaryotic translation initiation factor (elF) and splicing factor 3B subunit 1-like (SF3BI) were up-regulated by Cd+N treatment whether at the proteome or at the phosphoproteome levels, which was need to further study. In a word, taken together of proteome and phosphoproteome data, nitrogen serves a protective role in plants treated with Cd by multiple ways.

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