Carbon dioxide and water transport through plant aquaporins.

Aquaporins are channel proteins that function to increase the permeability of biological membranes. In plants, aquaporins are encoded by multigene families that have undergone substantial diversification in land plants. The plasma membrane intrinsic proteins (PIPs) subfamily of aquaporins is of particular interest given their potential to improve plant water relations and photosynthesis. Flowering plants have between 7 and 28 PIP genes. Their expression varies with tissue and cell type, through development and in response to a variety of factors, contributing to the dynamic and tissue specific control of permeability. There are a growing number of PIPs shown to act as water channels, but those altering membrane permeability to CO2 are more limited. The structural basis for selective substrate specificities has not yet been resolved, although a few key amino acid positions have been identified. Several regions important for dimerization, gating and trafficking are also known. PIP aquaporins assemble as tetramers and their properties depend on the monomeric composition. PIPs control water flux into and out of veins and stomatal guard cells and also increase membrane permeability to CO2 in mesophyll and stomatal guard cells. The latter increases the effectiveness of Rubisco and can potentially influence transpiration efficiency.

Effects of reduced carbonic anhydrase activity on CO2 assimilation rates in Setaria viridis: a transgenic analysis.

In C4 species, the major ß-carbonic anhydrase (ß-CA) localized in the mesophyll cytosol catalyses the hydration of CO2 to HCO3-, which phosphoenolpyruvate carboxylase uses in the first step of C4 photosynthesis. To address the role of CA in C4 photosynthesis, we generated transgenic Setaria viridis depleted in ß-CA. Independent lines were identified with as little as 13% of wild-type CA. No photosynthetic defect was observed in the transformed lines at ambient CO2 partial pressure (pCO2). At low pCO2, a strong correlation between CO2 assimilation rates and CA hydration rates was observed. C18O16O isotope discrimination was used to estimate the mesophyll conductance to CO2 diffusion from the intercellular air space to the mesophyll cytosol (gm) in control plants, which allowed us to calculate CA activities in the mesophyll cytosol (Cm). This revealed a strong relationship between the initial slope of the response of the CO2 assimilation rate to cytosolic pCO2 (ACm) and cytosolic CA activity. However, the relationship between the initial slope of the response of CO2 assimilation to intercellular pCO2 (ACi) and cytosolic CA activity was curvilinear. This indicated that in S. viridis, mesophyll conductance may be a contributing limiting factor alongside CA activity to CO2 assimilation rates at low pCO2.

Dissolved and particulate copper exposure induces differing gene expression profiles and mechanisms of toxicity in the deposit feeding amphipod Melita plumulosa.

Uptake of metals via ingestion is an important route of exposure for many invertebrates, and it has been suggested that the toxic response to metals accumulated via food differs from that of metals accumulated via the dissolved phase. To test this hypothesis, the deposit-feeding epibenthic amphipod Melita plumulosa was exposed to nontoxic or reproductively toxic concentrations of copper via the overlying water, via ingestion of sediment, or via a combination of the two. Rates of copper uptake from the two exposure routes were predicted using a biokinetic model. Gene expression profiles were measured via microarray analysis and confirmed via quantitative polymerase chain reaction. Differences in expression profiles were related to the exposure route more than to individual or combined rates of copper uptake. Chitinase and digestive protease transcript expression levels correlated to the copper uptake rate from sediment, rather than from the dissolved phase or combined total uptake rate. Overall, this study supports the hypothesis that metals accumulated via ingestion have a different mode of toxic action than metals taken up from water. Consequently, guidelines that only consider dissolved metal exposure, including equilibrium-partitioning-based guidelines, may underestimate the potential effects from deposited or resuspended metal-contaminated sediments.

Roles of Aquaporins in Setaria viridis Stem Development and Sugar Storage.

Setaria viridis is a C4 grass used as a model for bioenergy feedstocks. The elongating internodes in developing S. viridis stems grow from an intercalary meristem at the base, and progress acropetally toward fully expanded cells that store sugar. During stem development and maturation, water flow is a driver of cell expansion and sugar delivery. As aquaporin proteins are implicated in regulating water flow, we analyzed elongating and mature internode transcriptomes to identify putative aquaporin encoding genes that had particularly high transcript levels during the distinct stages of internode cell expansion and maturation. We observed that SvPIP2;1 was highly expressed in internode regions undergoing cell expansion, and SvNIP2;2 was highly expressed in mature sugar accumulating regions. Gene co-expression analysis revealed SvNIP2;2 expression was highly correlated with the expression of five putative sugar transporters expressed in the S. viridis internode. To explore the function of the proteins encoded by SvPIP2;1 and SvNIP2;2, we expressed them in Xenopus laevis oocytes and tested their permeability to water. SvPIP2;1 and SvNIP2;2 functioned as water channels in X. laevis oocytes and their permeability was gated by pH. Our results indicate that SvPIP2;1 may function as a water channel in developing stems undergoing cell expansion and SvNIP2;2 is a candidate for retrieving water and possibly a yet to be determined solute from mature internodes. Future research will investigate whether changing the function of these proteins influences stem growth and sugar yield in S. viridis.

Challenges for using quantitative PCR test batteries as a TIE-type approach to identify metal exposure in benthic invertebrates.

The epibenthic amphipod Melita plumulosa shows unique gene expression profiles when exposed to different contaminants. We hypothesized that specific changes in transcript abundance could be used in a battery of quantitative polymerase chain reaction (qPCR) assays as a toxicity identification evaluation (TIE)-like approach to identify the most relevant stressor in field-contaminated sediments. To test this hypothesis, seven candidate transcriptomic markers were selected, and their specificity following metal exposure was confirmed. The performance of these markers across different levels of added metals was verified. The ability of these transcripts to act as markers was tested by exposing amphipods to metal-contaminated field-collected sediments and measuring changes in transcript abundance via qPCR. For two of the three sediments tested, at least some of the transcriptomic patterns matched our predictions, suggesting that they would be effective in helping to identify metal exposure in field sediments. However, following exposure to the third sediment, transcriptomic patterns were unlike our predictions. These results suggest that the seven transcripts may be insufficient to discern individual contaminants from complex mixtures and that microarray or RNA-Seq global gene expression profiles may be more effective for TIE. Changes in transcriptomics based on laboratory exposures to single compounds should be carefully validated before the results are used to analyze mixtures.

Assessing mechanisms of toxicant response in the amphipod Melita plumulosa through transcriptomic profiling.

This study describes the function of transcripts with altered abundance in the epibenthic amphipod, Melita plumulosa, following whole-sediment exposure to a series of common environmental contaminants. M. plumulosa were exposed for 48 h to sediments spiked and equilibrated with the following contaminants at concentrations predicted to cause sublethal effects to reproduction: porewater ammonia 30 mg L(-1); bifenthrin at 100 µg kg(-1); fipronil at 50 µg kg(-1); 0.6% diesel; 0.3% crude oil; 250 mg Cu kg(-1); 400 mg Ni kg(-1); and 400 mg Zn kg(-1). RNA was extracted and hybridized against a custom Agilent microarray developed for this species. Although the microarray represented a partial transcriptome and not all features on the array could be annotated, unique transcriptomic profiles were generated for each of the contaminant exposures. Hierarchical clustering grouped the expression profiles together by contaminant class, with copper and zinc, the petroleum products and nickel, and the pesticides each forming a distinct cluster. Many of the transcriptional changes observed were consistent with patterns previously described in other crustaceans. The changes in the transcriptome demonstrated that contaminant exposure caused changes in digestive function, growth and moulting, and the cytoskeleton following metal exposure, whereas exposure to petroleum products caused changes in carbohydrate metabolism, xenobiotic metabolism and hormone cycling. Functional analysis of these gene expression profiles can provide a better understanding of modes of toxic action and permits the prediction of mixture effects within contaminated ecosystems.

Effects of atrazine on endocrinology and physiology in juvenile barramundi, Lates calcarifer (Bloch).

Exposure to certain environmental contaminants such as agricultural pesticides can alter normal endocrine and reproductive parameters in wild fish populations. Recent studies have found widespread pesticide contamination across the rivers that discharge into the Great Barrier Reef lagoon. Potential impacts on native fish species exposed to known endocrine disrupting chemicals such as atrazine, simazine, and diuron have not been assessed. In the present study, the authors examined the endocrine and physiological effects of short-term, acute exposure of environmentally relevant concentrations of analytical grade atrazine in juvenile barramundi (Lates calcarifer) in a controlled laboratory experiment. Expression of hepatic vitellogenin was not affected, supporting results of previous studies that showed that atrazine does not have a direct estrogenic effect via mediation of estrogen receptors. The lack of effect on brain cytochrome P19B (CYP19B) expression levels, combined with increases in testosterone (T) and 17ß estradiol and a stable T:17ß estradiol ratio, does not support the hypothesis that atrazine has an indirect estrogenic effect via modulation of aromatase expression. Gill ventilation rate, a measure of oxidative stress, did not change in contrast to other studies finding enhanced osmoregulatory disturbance and gill histopathology after atrazine exposure. To more closely reflect field conditions, the authors recommend that laboratory studies should focus more on examining the effects of commercial pesticide formulations that contain additional ingredients that have been found to be disruptive to endocrine function.

RNA-Seq analysis of the toxicant-induced transcriptome of the marine diatom, Ceratoneis closterium.

Diatoms are of enormous ecological importance as they account for as much as 20% of global primary production, yet they are still understudied from a genomic perspective. The benthic diatom Ceratoneis closterium is well-characterized from an ecotoxicological perspective including its use in ecotoxicological risk assessments and investigating the mode-of-action of metal toxicity. However, this organism has little sequence information available. In this study, 454 pyrosequencing of the stressor-responsive transcriptome was undertaken. These transcripts could be used to characterize general physiological processes such as photosynthesis and respiration, as well as to enable a description of the ecotoxicogenomic responses of this organism. After a 96 h exposure to the concentration of toxicant that inhibited growth rate by 10% (IC10) for the following common coastal contaminants: ammonia, copper, crude oil and simazine (a photosystem II inhibiting herbicide), diatom cells were harvested for RNA extraction and their transcriptomes characterized via 454 pyrosequencing. This resulted in 1.25 million reads, which were assembled into 4768 contigs, when contigs encoding rRNA were removed. More than 80% of the remaining contigs had an ortholog in the BLASTx protein databases. These contigs represented 1660 unique transcripts. The role of these transcripts in stress response, as well as photosynthesis and respiration is discussed. Overall, this study greatly enhances the genomic information available for this important taxonomic group.

Using transcriptomic profiles in the diatom Phaeodactylum tricornutum to identify and prioritize stressors.

The transcriptomic profile of the marine diatom, Phaeodactylum tricornutum, exposed to several ecologically relevant stressors, was used to develop toxicity identification evaluation (TIE)-like gene expression assays. Algal growth inhibition was measured by flow cytometry to determine exposure concentrations that elicited a sublethal toxic response. P. tricornutum was exposed to concentrations of copper (2 µg L⁻¹), cadmium (5 µg L⁻¹), silver (20 µg L⁻¹), simazine (75 µg L⁻¹), the water accommodated fraction (WAF) of weathered crude oil (5 mg L⁻¹), 50 µg L⁻¹ ammonia, a decreased salinity treatment (15‰), and a mixture exposure of ammonia, decreased salinity and cadmium (10 µg L⁻¹). Analysis of the gene expression via microarray indicated that unique transcriptomic signals were generated for each of the individual treatments. Transcriptomic profiles of ammonia and the mixture treatment overlapped substantially. Photosynthesis related transcripts were altered in the simazine (herbicide) treatment. A transcript involved in degrading hydrocarbons, dioxygenase, had increased abundance after crude oil exposure. Overall, transcriptomic responses in the different treatments were associated with stress responses, membrane transport, transcription and translation and could be linked to contaminant mode of action. The transcriptomic profiles were used to design real-time (quantitative) polymerase chain reaction (qPCR) assays that would link changes in transcript abundance to a particular stressor in a TIE-based approach. At least one transcript for each contaminant tested (copper, cadmium, silver, salinity and ammonia) responded exclusively to that contaminant. With further development of additional transcriptomic markers for each contaminant, this new approach has potential to enhance traditional toxicology bioassays by providing additional lines of evidence to identify biologically relevant stressors within a contaminated ecosystem based on changes in the transcriptomic profile.

Comparison of toxicity and transcriptomic profiles in a diatom exposed to oil, dispersants, dispersed oil.

Dispersants are commonly used to mitigate the impact of oil spills, however, the ecological cost associated with their use is uncertain. The toxicity of weathered oil, dispersed weathered oil, and the hydrocarbon-based dispersant Slickgone NS(®), to the diatom Phaeodactylum tricornutum has been examined using standardized toxicity tests. The assumption that most toxicity occurs via narcosis was tested by measuring membrane damage in diatoms after exposure to one of the petroleum products. The mode of toxic action was determined using microarray-based gene expression profiling in diatoms after exposure to one of the petroleum products. The diatoms were found to be much more sensitive to dispersants than to the water accommodated fraction (WAF), and more sensitive to the chemically enhanced WAF (CEWAF) than to either the WAF itself or the dispersants. Exposure to dispersants and CEWAF caused membrane damage, while exposure to WAF did not. The gene expression profiles resulting from exposure to all three petroleum mixtures were highly similar, suggesting a similar mode of action for these compounds. The observed toxicity bore no relationship to PAH concentrations in the water column or to total petroleum hydrocarbon (TPH), suggesting that an undescribed component of the oil was causing toxicity. Taken together, these results suggest that the use of dispersants to clean up oil spills will dramatically increase the oil toxicity to diatoms, and may have implications for ecological processes such as the timing of blooms necessary for recruitment.