Douglas-Fir ( Pseudotsuga menziesii (Mirb.) Franco) Transcriptome Profile Changes Induced by Diesel Emissions Generated with CeO2 Nanoparticle Fuel Borne Catalyst.

It is important to understand molecular effects on plants exposed to compounds released from use of products containing engineered nanomaterials. Here, we present mRNA sequencing data on transcriptome impacts to Douglas-fir following 2 weeks of sublethal exposure to 30:1 diluted airborne emissions released from combustion of diesel fuel containing engineered CeO2 nanoparticle catalysts (DECe). Our hypothesis was that chamber exposure to DECe would induce distinct transcriptome changes in seedling needles compared with responses to conventional diesel exhaust (DE) or filtered DECe Gas Phase. Significantly increased uptake/binding of Ce in needles of DECe treated seedlings was 2.7X above background levels and was associated with altered gene expression patterns. All 225 Blast2GO gene ontologies (GOs) enriched by up-regulated DECe transcripts were nested within GOs for DE, however, 29 of 31 enriched GOs for down-regulated DECe transcripts were unique. MapMan analysis also identified three pathways enriched with DECe down-regulated transcripts. There was prominent representation of genes with attenuated expression in transferase, transporter, RNA regulation and protein degradation GOs and pathways. CeO2 nanoparticle additive decreased and shifted molecular impact of diesel emissions. Wide-spread use of such products and chronic environmental exposure to DECe may adversely affect plant physiology and development.

A comprehensive framework for evaluating the environmental health and safety implications of engineered nanomaterials.

Engineered nanomaterials (ENM) are a growing aspect of the global economy, and their safe and sustainable development, use, and eventual disposal requires the capability to forecast and avoid potential problems. This review provides a framework to evaluate the health and safety implications of ENM releases into the environment, including purposeful releases such as for antimicrobial sprays or nano-enabled pesticides, and inadvertent releases as a consequence of other intended applications. Considerations encompass product life cycles, environmental media, exposed populations, and possible adverse outcomes. This framework is presented as a series of compartmental flow diagrams that serve as a basis to help derive future quantitative predictive models, guide research, and support development of tools for making risk-based decisions. After use, ENM are not expected to remain in their original form due to reactivity and/or propensity for hetero-agglomeration in environmental media. Therefore, emphasis is placed on characterizing ENM as they occur in environmental or biological matrices. In addition, predicting the activity of ENM in the environment is difficult due to the multiple dynamic interactions between the physical/chemical aspects of ENM and similarly complex environmental conditions. Others have proposed the use of simple predictive functional assays as an intermediate step to address the challenge of using physical/chemical properties to predict environmental fate and behavior of ENM. The nodes and interactions of the framework presented here reflect phase transitions that could be targets for development of such assays to estimate kinetic reaction rates and simplify model predictions. Application, refinement, and demonstration of this framework, along with an associated knowledgebase that includes targeted functional assay data, will allow better de novo predictions of potential exposures and adverse outcomes.

Focused Microbiome Shifts in Reconstructed Wetlands Correlated with Elevated Copper Concentrations Originating from Micronized Copper Azole-Treated Wood.

Micronized copper (Cu) azole (MCA) wood preservative formulations include Cu in nano form, and relatively little is known about longer term effects of Cu leached from MCA into wetland ecosystems. We tested the hypothesis that changes in soil microbiomes within reconstructed freshwater wetlands will be associated with exposure to elevated Cu concentrations originating from immersed MCA-treated wood stakes. Eight replicate communities were assembled with Willamette Valley (OR, USA) flood plain soil and clonally propagated wetland plants within mesocosms. Inundated communities were equilibrated for 5 months before installation of MCA or control southern yellow pine stakes (n = 4 communities/experimental group). Soil samples were collected for 16S and internal transcribed spacer amplicon sequencing to quantify responses in prokaryotes and eukaryotes, respectively, at 15 time points, spanning two simulated seasonal dry downs, for up to 678 days. Physiochemical properties of water and soil were monitored at 20 and 12 time points respectively, over the same period. For both taxonomic groups of organisms, phylogenetic diversity increased and was positively correlated with elapsed days. Furthermore, there was significant divergence among eukaryotes during the second year based on experimental group. Although the composition of taxa underwent succession over time, there was significantly reduced relative abundance of sequence variants from Gomphonema diatoms and Scutellinia fungi in communities where MCA wood stakes were present compared with the controls. These focused microbiome shifts were positively correlated with surface water Cu and soil Cu concentrations, which were significantly elevated in treated communities. The reconstructed communities were effective systems for assessing potential impacts to wetland microbiomes after exposure to released copper. The results further inform postcommercialization risk assessments on MCA-treated wood. Environ Toxicol Chem 2021;40:3351-3368. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.

Molecular and physiological responses to titanium dioxide and cerium oxide nanoparticles in Arabidopsis.

Changes in tissue transcriptomes and productivity of Arabidopsis thaliana were investigated during exposure of plants to 2 widely used engineered metal oxide nanoparticles, titanium dioxide (nano-titania) and cerium dioxide (nano-ceria). Microarray analyses confirmed that exposure to either nanoparticle altered the transcriptomes of rosette leaves and roots, with comparatively larger numbers of differentially expressed genes found under nano-titania exposure. Nano-titania induced more differentially expressed genes in rosette leaves, whereas roots had more differentially expressed genes under nano-ceria exposure. MapMan analyses indicated that although nano-titania up-regulated overall metabolism in both tissues, metabolic processes under nano-ceria remained mostly unchanged. Gene enrichment analysis indicated that both nanoparticles mainly enriched ontology groups such as responses to stress (abiotic and biotic), and defense responses (pathogens), and responses to endogenous stimuli (hormones). Nano-titania specifically induced genes associated with photosynthesis, whereas nano-ceria induced expression of genes related to activating transcription factors, most notably those belonging to the ethylene responsive element binding protein family. Interestingly, there were also increased numbers of rosette leaves and plant biomass under nano-ceria exposure, but not under nano-titania. Other transcriptomic responses did not clearly relate to responses observed at the organism level, possibly because of functional and genomic redundancy in Arabidopsis, which may mask expression of morphological changes, despite discernable responses at the transcriptome level. In addition, transcriptomic changes often relate to transgenerational phenotypic development, and hence it may be productive to direct further experimental work to integrate high-throughput genomic results with longer term changes in subsequent generations. Environ Toxicol Chem 2017;36:71-82. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Soil bacterial diversity in a loblolly pine plantation: influence of ectomycorrhizas and fertilization.

We studied the effect of ectomycorrhizas and fertilization on soil microbial communities associated with roots of 10-year-old loblolly pine. Ectomycorrhizas were identified using a combination of community terminal restriction fragment profiling and matching of individual terminal restriction fragments to those produced from ectomycorrhizal clones and sequences recovered from roots and sporocarps. Differences between bacterial communities were initially determined using cluster analysis on community terminal restriction fragment profiles and through subsequent recovery of 16S rDNA clones. Analysis of bacterial clones revealed that terminal restriction fragment length was often shared between taxonomically dissimilar bacterial types. Consequently, we could not reliably infer the identity of peaks in the bacterial community profile with some exceptions, notably chloroplast rDNA that generated an approximate peak size of 80.2 bp. Fertilization increased the frequency of a Piloderma-like ectomycorrhiza. However, we did not detect clear effects of fertilization or the presence of viable ectomycorrhizas on bacterial communities. Bacterial communities seemed to be determined largely by the carbon and nitrogen content of soil. These results suggest that important soil microbial groups respond differently to soil conditions and management practices, with ectomycorrhizal communities reflecting past nutrient conditions and bacterial communities reflecting current environmental conditions of soil microsites.

Germination and early plant development of ten plant species exposed to titanium dioxide and cerium oxide nanoparticles.

Ten agronomic plant species were exposed to different concentrations of nano-titanium dioxide (nTiO2 ) or nano-cerium oxide (nCeO2 ) (0 µg/mL, 250 µg/mL, 500 µg/mL, and 1000 µg/mL) to examine potential effects on germination and early seedling development. The authors modified a standard test protocol developed for soluble chemicals (OPPTS 850.4200) to determine if such an approach might be useful for screening engineered nanomaterials (ENMs) and whether there were differences in response across a range of commercially important plant species to 2 common metal oxide ENMs. Eight of 10 species responded to nTiO2 , and 5 species responded to nCeO2 . Overall, it appeared that early root growth may be a more sensitive indicator of potential effects from ENM exposure than germination. The observed effects did not always relate to the exposure concentration, indicating that mass-based concentration may not fully explain the developmental effects of these 2 ENMs. The results suggest that nTiO2 and nCeO2 have different effects on early plant growth of agronomic species, with unknown effects at later stages of the life cycle. In addition, standard germination tests, which are commonly used for toxicity screening of new materials, may not detect the subtle but potentially more important changes associated with early growth and development in terrestrial plants. Environ Toxicol Chem 2016;35:2223-2229. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.

Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts.

BACKGROUND: The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set. RESULTS: Numerous sequences for PCR primers were tested to develop PCR assays with a wide range of fungal compatibility and high discrimination from plant DNA. A nested set of 4 primers was developed that reflected these criteria and performed well amplifying ITS regions of fungal rDNA. Primers in the 5.8S sequence were also developed that would permit separate amplifications of ITS1 and ITS2. A range of basidiomycete fruiting bodies and ascomycete cultures were analyzed with the nested set of primers and Restriction Fragment Length Polymorphism (RFLP) fingerprinting to demonstrate the specificity of the assay. Single ectomycorrhizal root tips were similarly analyzed. These primers have also been successfully applied to Quantitative PCR (QPCR), Length Heterogeneity PCR (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses of fungi. A set of wide-range plant-specific primers were developed at positions corresponding to one pair of the fungal primers. These were used to verify that the host plant DNA was not being amplified with the fungal primers. CONCLUSION: These plant primers have been successfully applied to PCR-RFLP analyses of forest plant tissues from above- and below-ground samples and work well at distinguishing a selection of plants to the species level. The complete set of primers was developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of fungi where samples also contain high levels of background plant DNA, such as verifying ectomycorrhizal morphotypes or characterizing phylosphere communities.

Phenotypic and genomic responses to titanium dioxide and cerium oxide nanoparticles in Arabidopsis germinants.

The effects of exposure to nanoparticles of titanium dioxide (nano-titanium) and cerium oxide (nano-cerium) on gene expression and growth in Arabidopsis thaliana germinants were studied by using microarrays and quantitative real-time polymerase chain reaction (qPCR), and by evaluating germinant phenotypic plasticity. Exposure to 12 d of either nano-titania or nano-ceria altered the regulation of 204 and 142 genes, respectively. Genes induced by the nanoparticles mainly include ontology groups annotated as stimuli responsive, including both abiotic (oxidative stress, salt stress, water transport) and biotic (respiratory burst as a defense against pathogens) stimuli. Further analysis of the differentially expressed genes indicates that both nanoparticles affected a range of metabolic processes (deoxyribonucleic acid [DNA] metabolism, hormone metabolism, tetrapyrrole synthesis, and photosynthesis). Individual exposures to the nanoparticles increased percentages of seeds with emergent radicles, early development of hypocotyls and cotyledons, and those with fully grown leaves. Although there were distinct differences between the nanoparticles in their affect on molecular mechanisms attributable to enhancing germinant growth, both particles altered similar suites of genes related to various pathways and processes related to enhanced growth.

Allocation of carbon in mycorrhizal Pinus ponderosa seedlings exposed to ozone.

The effect of ozone on tree growth and metabolism has been studied widely. Despite the research emphasis, relatively little is known about how the below-ground component responds when shoots are exposed to ozone, even though evidence suggests that ozone can affect roots more than shoots. Undemanding how ozone affects carbohydrate allocation throughout the plant is essential to understanding the mechanisms of response to ozone. The purpose of this study was to follow the allocation and metabolism of carbon in a Pinus Ponderosa Laws.-Hebeloma crustuliniforme (Bull.: St. Amans) Quel seedling system under ozone stress. The hypothesis that ozone affects carbon transport below ground and overall sink strength of roots. similarly in mycorrhizal and non-mycorrhizal seedlings was tested. To test the hypothesis, a unique culturing system was used to quantify carbon movement to all components of the symbiosis and to construct an overall budget for carbon for both mycorrhizal and non-mycorrhizal seedlings. Fluxes of CO2 and carbon allocation were followed by measuring instantaneous CO2 flux and by 14 C labelling. Two experiments were conducted that differed in their total ozone exposure (39.3 ppm h in expt 1, and 58.1 ppm h in expt 2). Mycorrhizal inoculation significantly increased CO., assimilation rates (A) and A/R (R = shoot respiration) ratios in both experiments compared with non-mycorrhizal seedlings. Ozone exposure in expt 2 significantly decreased the A/R ratio (P < 0.003) in both mycorrhizal treatments. Below-ground respiration was significantly greater in mycorrhizal than in non-mycorrhizal seedlings in both experiments, and was not affected by ozone exposure, Intact, extramatrical hyphal respiration was lower by 33% in seedlings exposed to ozone, but differences were not statistically significant (P ≤ (0.167). Mycorrhizal seedling roots reached maximum respiratory 14 CO2 release rates c. 5 h and < 20 h earlier than non-mycorrhizal seedlings in expts 1 and 2, respectively, suggesting accelerated transport of 14 C below ground in mycorrhizal seedlings. Mycorrhizal seedlings also exhibited greater rates of 14 C release below ground than non-mycorrhizal controls. The maximum rate of respiratory release of 14 CO2 below ground was significantly reduced by exposure to ozone in both mycorrhizal and non-mycorrhizal treatments. Ozone significantly reduced 14 C activity in the fungus of mycorrhizal plants. This constitutes the first report of an ozone-induced reduction in carbon allocation to the fungal symbiont in a mycorrhizal association. The results suggest a substantial impact of ozone on the carbon balance of the mycorrhiza: however, there was no evidence to suggest that mycorrhizal and non-mycorrhizal ponderosa pine seedlings responded differently to ozone stress.

Nitrogen and carbon stable isotope abundances support the myco-heterotrophic nature and host-specificity of certain achlorophyllous plants.

• Over 400 species of achlorophyllous vascular plants are thought to obtain all C from symbiotic fungi. Consequently, they are termed 'myco-heterotrophic.' However, direct evidence of myco-heterotrophy in these plants is limited. • During an investigation of the patterns of N and C stable isotopes of various ecosystem pools in two old-growth conifer forests, we sampled six species of myco-heterotrophic achlorophyllous plants to determine the ability of stable isotope ratios to provide evidence of myco-heterotrophy and host-specificity within these symbioses. • Dual-isotope signatures of the myco-heterotrophic plants differed from those of all other pools. They were most similar to the signatures of ectomycorrhizal fungi, and least like those of green plants. δ15 N values of the myco-heterotrophic plants correlated strongly and positively with those of putative mycobionts. • Used in conjunction with other techniques, N and C stable isotope ratios can be used to demonstrate myco-heterotrophy and host-specificity in these plants when other ecosystem pools are well characterized. They also appear promising for estimating the degree of heterotrophy in photosynthetic, partially myco-heterotrophic plants.

Patterns of nitrogen and carbon stable isotope ratios in macrofungi, plants and soils in two old-growth conifer forests.

• To further assess the usefulness of stable isotope ratios for understanding elemental cycling and fungal ecology, we measured δ15 N and δ13 C in ectomycorrhizal and saprotrophic macrofungi, plants, woody debris and soils from two old-growth conifer forests in Olympic National Park, Washington, USA. • Ecosystem isotope patterns were similar at the two forests, but differences existed that appear to reflect soil nitrogen availability and C allocation within the ectomycorrhizal symbioses. δ15 N and δ13 C of ectomycorrhizal and saprotrophic fungi differed in both forests, and a dual δ15 N/δ13 C plot provided the best means of distinguishing them. Within both groups, δ15 N and δ13 C differed among genera and species, and the difference in species composition was an important determinant of the different overall δ15 N of the ectomycorrhizal fungi at the two forests. • Variation in multiple ecophysiological traits such as organic N use, mycelial morphology and transfer of N to phytobionts appears to underlie the variation in the isotope signatures of ectomycorrhizal fungi. • The varied isotope signatures of ectomycorrhizal fungi suggest considerable functional diversity among them. Life-history strategies could provide a framework for interpreting these patterns.

Preferential interaction of Na+ over K+ with carboxylate-functionalized silver nanoparticles.

Elucidating mechanistic interactions between monovalent cations (Na(+)/K(+)) and engineered nanoparticle surfaces to alter particle stability in polar media have received little attention. We investigated relative preferential interaction of Na(+) and K(+) with carboxylate-functionalized silver nanoparticles (carboxylate-AgNPs) to determine if interaction preference followed the Hofmeister series (Na(+)>K(+)). We hypothesized that Na(+) will show greater affinity than K(+) to pair with carboxylates on AgNP surfaces, thereby destabilizing the colloidal system. Destabilization upon Na(+) or K(+) interacting with carboxylate-AgNPs was evaluated probing changes in multiple physicochemical characteristics: surface plasmon resonance/optical absorbance, electrical conductivity, pH, hydrodynamic diameter, electrophoretic mobility, surface charge, amount of Na(+)/K(+) directly associated with AgNPs, and Ag(+) dissociation kinetics. We show that Na(+) and K(+) react differently, indicating local Na(+) pairing with carboxylates on AgNP surfaces is kinetically faster and remarkably favored over K(+), thus supporting Hofmeister ordering. Our results suggest that AgNPs may transform into micron-size aggregates upon release into aqueous environments and that the fate of such aggregates may need consideration when assessing environmental risk.

Potential for metal contamination by direct sonication of nanoparticle suspensions.

While conducting toxicity tests with nano titanium dioxide, the authors found that test suspensions were being contaminated with aluminum and titanium from tip erosion during direct sonication. The contaminating alloy particles had a measurable size distribution and zeta potential using dynamic light scattering, which changed the measured characteristics of the suspensions. Caution should be used when employing direct sonication for preparing test suspensions due to potential interferences of these particles in toxicological assessments.

Elevated CO(2) and temperature alter net ecosystem C exchange in a young Douglas fir mesocosm experiment.

We investigated the effects of elevated CO(2) (EC) [ambient CO(2) (AC) + 190 ppm] and elevated temperature (ET) [ambient temperature (AT) + 3.6 degrees C] on net ecosystem exchange (NEE) of seedling Douglas fir (Pseudotsuga menziesii) mesocosms. As the study utilized seedlings in reconstructed soil-litter-plant systems, we anticipated greater C losses through ecosystem respiration (R(e)) than gains through gross photosynthesis (GPP), i.e. negative NEE. We hypothesized that: (1) EC would increase GPP more than R(e), resulting in NEE being less negative; and (2) ET would increase R(e) more than GPP, resulting in NEE being more negative. We also evaluated effects of CO(2) and temperature on light inhibition of dark respiration. Consistent with our hypothesis, NEE was a smaller C source in EC, not because EC increased photosynthesis but rather because of decreased respiration resulting in less C loss. Consistent with our hypothesis, NEE was more negative in ET because R(e) increased more than GPP. The light level that inhibited respiration varied seasonally with little difference among CO(2) and temperature treatments. In contrast, the degree of light inhibition of respiration was greater in AC than EC. In our system, respiration was the primary control on NEE, as EC and ET caused greater changes in respiration than photosynthesis.

Carbon use, nitrogen use, and isotopic fractionation of ectomycorrhizal and saprotrophic fungi in natural abundance and 13C-labelled cultures.

Stable isotopes in fruit bodies from field studies have been used to infer ectomycorrhizal or saprotrophic status and to understand carbon and nitrogen use, but few controlled culture studies have correlated source and fungal isotopic patterns. Here, we measured natural abundances of 15N and 13C in ten strains of ectomycorrhizal fungi and seven strains of saprotrophic fungi grown on agar with three different primary carbon sources: glucose, glucose plus malt extract, and potato dextrose agar. Eight fungal strains were also grown using position-specific, 13C-labelled glucose (C-1 through C-6 labelled). Most fungi resembled nitrogen sources in delta 15N, suggesting that growth on agar media minimizes isotopic fractionation on uptake compared to growth on liquid media, and that in general saprotrophic and mycorrhizal fungi process nitrogen similarly. Saprotrophic fungi were more depleted in 13C than ectomycorrhizal fungi on all media, presumably because of assimilation of 13C-depleted, agar-derived carbon. Results on 13C-enriched glucose indicated that saprotrophic fungi obtained up to 45 % of their carbon from the agar substrate. Fungi generally incorporated the individual carbon atoms of glucose in the order, C-4 < C-1 < C-2, C-3, C-5 < C-6, ranging from a mean of 9 % for the C-4 atom to 21 % for the C-6 atom. Based on these incorporation patterns and intramolecular 13C patterns within glucose, differential incorporation of carbon atoms within glucose among fungal taxa contributed less than 1% to isotopic differences among taxa, whereas isotopic fractionation among taxa during metabolism varied up to 4%. Parallel studies of 13C-enriched and natural abundance substrates were crucial to interpreting our results.