Microarrays in ecological research: A case study of a cDNA microarray for plant-herbivore interactions

BACKGROUND: Microarray technology allows researchers to simultaneously monitor changes in the expression ratios (ERs) of hundreds of genes and has thereby revolutionized most of biology. Although this technique has the potential of elucidating early stages in an organism's phenotypic response to complex ecological interactions, to date, it has not been fully incorporated into ecological research. This is partially due to a lack of simple procedures of handling and analyzing the expression ratio (ER) data produced from microarrays. RESULTS: We describe an analysis of the sources of variation in ERs from 73 hybridized cDNA microarrays, each with 234 herbivory-elicited genes from the model ecological expression system, Nicotiana attenuata, using procedures that are commonly used in ecologic research. Each gene is represented by two independently labeled PCR products and each product was arrayed in quadruplicate. We present a robust method of normalizing and analyzing ERs based on arbitrary thresholds and statistical criteria, and characterize a "norm of reaction" of ERs for 6 genes (4 of known function, 2 of unknown) with different ERs as determined across all analyzed arrays to provide a biologically-informed alternative to the use of arbitrary expression ratios in determining significance of expression. These gene-specific ERs and their variance (gene CV) were used to calculate array-based variances (array CV), which, in turn, were used to study the effects of array age, probe cDNA quantity and quality, and quality of spotted PCR products as estimates of technical variation. Cluster analysis and a Principal Component Analysis (PCA) were used to reveal associations among the transcriptional "imprints" of arrays hybridized with cDNA probes derived from mRNA from N. attenuata plants variously elicited and attacked by different herbivore species and from three congeners: N. quadrivalis, N. longiflora and N. clevelandii. Additionally, the PCA revealed the contribution of individual gene ERs to the associations among arrays. CONCLUSIONS: While the costs of 'boutique' array fabrication are rapidly declining, familiar methods for the analysis of the data they create are still missing. The case history illustrated here demonstrates the ease with which this powerful technology can be adapted to ecological research

Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR

BACKGROUND: After transformation, plants that are homozygous and contain one copy of the transgene are typically selected for further study. If real-time PCR is to be used to determine copy number and zygosity, it must be able to distinguish hemizygous from homozygous and one-copy from two-copy plants. That is, it must be able to detect two-fold differences. RESULTS: When transgenic Nicotiana attenuata plants which had been previously determined by Southern analysis to contain one or two copies of the transgene, were analyzed by real-time PCR (2(-ΔΔCt )method), the method failed to confirm the results from the Southern analysis. In a second data set we analyzed offspring of a hemizygous one-copy plant, which were expected to segregate into three groups of offspring in a 1:2:1 ratio: no transgene, hemizygous, homozygous. Because it was not possible to distinguish homozygous from hemizygous plants with real-time PCR, we could not verify this segregation ratio. CONCLUSIONS: Detection of two-fold differences by real-time PCR is essential if this procedure is to be used for the characterization of transgenic plants. However, given the high variability between replicates, a detection of two-fold differences is in many cases not possible; in such cases Southern analysis is the more reliable procedure

An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining,we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(−)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions.We used these lines to test ovipositingmoths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but themagnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity

A persistent major mutation in canonical jasmonate signaling is embedded in an herbivory-elicited gene network

When insect herbivores attack plants, elicitors from oral secretions and regurgitants (OS) enter wounds during feeding, eliciting defense responses. These generally require plant jasmonate (JA) signaling, specifically, a jasmonoyl-L-isoleucine (JA-Ile) burst, for their activation and are well studied in the native tobacco Nicotiana attenuata. We used intraspecific diversity captured in a 26-parent MAGIC population planted in nature and an updated genome assembly to impute natural variation in the OS-elicited JA-Ile burst linked to a mutation in the JA-Ile biosynthetic gene NaJAR4. Experiments revealed that NaJAR4 variants were associated with higher fitness in the absence of herbivores but compromised foliar defenses, with two NaJAR homologues (4 and 6) complementing each other spatially and temporally. From decade-long seed collections of natural populations, we uncovered enzymatically inactive variants occurring at variable frequencies, consistent with a balancing selection regime maintaining variants. Integrative analyses of OS-induced transcriptomes and metabolomes of natural accessions revealed that NaJAR4 is embedded in a nonlinear complex gene coexpression network orchestrating responses to OS, which we tested by silencing four hub genes in two connected coexpressed networks and examining their OS-elicited metabolic responses. Lines silenced in two hub genes (NaGLR and NaFB67) co-occurring in the NaJAR4/6 module showed responses proportional to JA-Ile accumulations; two from an adjacent module (NaERF and NaFB61) had constitutively expressed defenses with high resistance. We infer that mutations with large fitness consequences can persist in natural populations due to compensatory responses from gene networks, which allow for diversification in conserved signaling pathways and are generally consistent with predictions of an omnigene model

Surface Enhanced Second Harmonic Generation from Macrocycle, Catenane, and Rotaxane Thin Films: Experiments and Theory

Surface enhanced second harmonic generation (SE SHG) experiments on molecular structures, macrocycles, catenanes, and rotaxanes, deposited as monolayers and multilayers by vacuum sublimation on silver, are reported. The measurements show that the molecules form ordered thin films, where the highest degree of order is observed in the case of macrocycle monolayers and the lowest in the case of rotaxane multilayers. The second harmonic generation activity is interpreted in terms of electric field induced second harmonic (EFISH) generation where the electric field is created by the substrate silver atoms. The measured second order nonlinear optical susceptibility for a rotaxane thin film is compared with that obtained by considering only EFISH contribution to SHG intensity. The electric field on the surface of a silver layer is calculated by using the Delphi4 program for structures obtained with TINKER molecular mechanics/dynamics simulations. An excellent agreement is observed between the calculated and the measured SHG susceptibilities.

Molecular evolution and diversification of the Argonaute family of proteins in plants

BACKGROUND: Argonaute (AGO) proteins form the core of the RNA-induced silencing complex, a central component of the smRNA machinery. Although reported from several plant species, little is known about their evolution. Moreover, these genes have not yet been cloned from the ecological model plant, Nicotiana attenuata, in which the smRNA machinery is known to mediate important ecological traits. RESULTS: Here, we not only identify 11 AGOs in N. attenuata, we further annotate 133 genes in 17 plant species, previously not annotated in the Phytozome database, to increase the number of plant AGOs to 263 genes from 37 plant species. We report the phylogenetic classification, expansion, and diversification of AGOs in the plant kingdom, which resulted in the following hypothesis about their evolutionary history: an ancestral AGO underwent duplication events after the divergence of unicellular green algae, giving rise to four major classes with subsequent gains/losses during the radiation of higher plants, resulting in the large number of extant AGOs. Class-specific signatures in the RNA-binding and catalytic domains, which may contribute to the functional diversity of plant AGOs, as well as context-dependent changes in sequence and domain architecture that may have consequences for gene function were found. CONCLUSIONS: Together, the results demonstrate that the evolution of AGOs has been a dynamic process producing the signatures of functional diversification in the smRNA pathways of higher plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-014-0364-6) contains supplementary material, which is available to authorized users

Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference

Background: RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant’s lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata’s dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. Methodology/Principal Findings: Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for tw

What makes health impact assessments successful? Factors contributing to effectiveness in Australia and New Zealand

Background: While many guidelines explain how to conduct Health Impact Assessments (HIAs), less is known about the factors that determine the extent to which HIAs affect health considerations in the decision making process. We investigated which factors are associated with increased or reduced effectiveness of HIAs in changing decisions and in the implementation of policies, programs or projects. This study builds on and tests the Harris and Harris-Roxas' conceptual framework for evaluating HIA effectiveness, which emphasises context, process and output as key domains. Methods: We reviewed 55 HIA reports in Australia and New Zealand from 2005 to 2009 and conducted surveys and interviews for 48 of these HIAs. Eleven detailed case studies were undertaken using document review and stakeholder interviews. Case study participants were selected through purposeful and snowball sampling. The data were analysed by thematic content analysis. Findings were synthesised and mapped against the conceptual framework. A stakeholder forum was utilised to test face validity and practical adequacy of the findings. Results: We found that some features of HIA are essential, such as the stepwise but flexible process, and evidence based approach. Non-essential features that can enhance the impact of HIAs include capacity and experience; 'right person right level'; involvement of decision-makers and communities; and relationships and partnerships. There are contextual factors outside of HIA such as fit with planning and decision making context, broader global context and unanticipated events, and shared values and goals that may influence a HIA. Crosscutting factors include proactive positioning, and time and timeliness. These all operate within complex open systems, involving multiple decision-makers, levels of decision-making, and points of influence. The Harris and Harris-Roxas framework was generally supported. Conclusion: We have confirmed previously identified factors influencing effectiveness of HIA and identified new factors such as proactive positioning. Our findings challenge some presumptions about 'right' timing for HIA and the rationality and linearity of decision-making processes. The influence of right timing on decision making needs to be seen within the context of other factors such as proactive positioning. This research can help HIA practitioners and researchers understand and identify what can be enhanced within the HIA process. Practitioners can adapt the flexible HIA process to accommodate the external contextual factors identified in this report