Claudia Voelckel

An analysis of plant–aphid interactions by different microarray hybridization strategies

Aphids have long been considered ‘stealthy’ herbivores that subvert a plants induced defenses and manipulate its source‐sink signaling, but these hypotheses are largely untested at a transcriptional level. We analysed gene expression in native tobacco plants (Nicotiana attenuata) infested with Myzus nicotianae aphids, without resorting to the use of clip‐cages, with a cDNA microarray containing 240 defense‐related N. attenuata genes. Using a hybridization scheme (‘ratio analysis’ and ‘state analysis’) broadly applicable in two‐factor analyses, we examined how the aphids influenced source–sink relationships and determined if their feeding preference, apart from benefiting from the sink strength of young leaves, was associated with the expression of known plant defense genes. In contrast to the responses elicited by attack from tissue‐feeding lepidopteran larvae and mesophyll‐sucking insects, attack from phloem‐feeding aphids elicited only weak responses. Similar to other herbivores, M. nicotianae feeding increased the expression of trypsin protease inhibitors (TPI), lipoxygenase, and xyloglucan‐endotransglycosylase genes, and decreased small RUBISCO subunit and ubiquitin carrier protein transcripts. Aphid‐specific changes included the up‐regulation of glutamate synthase and the down‐regulation of a germin‐like protein. Aphids preferentially settled on younger leaves, which expressed more hydroperoxide lyase and TPI than did older leaves, suggesting that these genes, which mediate the synthesis of compounds reported to be toxic for aphids in other plant systems, are either not under transcriptional control or not important in this system. By identifying aphid‐responsive genes, we have made a first step in identifying the ‘genes that matter’ in plant–aphid interactions.

Convergence, constraint and the role of gene expression during adaptive radiation: floral anthocyanins in Aquilegia

Convergent phenotypes are testament to the role of natural selection in evolution. However, little is known about whether convergence in phenotype extends to convergence at the molecular level. We use the independent losses of floral anthocyanins in columbines (Aquilegia) to determine the degree of molecular convergence in gene expression across the anthocyanin biosynthetic pathway (ABP). Using a phylogeny of the North American Aquilegia clade, we inferred six independent losses of floral anthocyanins. Via semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR), we monitored developmental and tissue‐specific variation in expression of the six major structural ABP loci in three Aquilegia species, two that produce anthocyanins (A+) and one that does not (A−). We then compared ABP expression in petals of old‐bud and pre‐anthesis flowers of 13 Aquilegia species, eight wild species and two horticultural lines representing seven independent A− lineages as well as three wild A+ species. We only found evidence of down‐regulation of ABP loci in A− lineages and losses of expression were significantly more prevalent for genes late in the pathway. Independent contrast analysis indicates that changes in expression of dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) are strongly phylogenetically correlated consistent with the multilocus targets of trans‐regulatory elements in the ABP of other systems. Our findings strongly suggest that pleiotropy constrains the evolution of loss of floral anthocyanins to mutations affecting genes late in the ABP mostly through convergent changes in regulatory genes. These patterns support the hypothesis that rapid evolutionary change occurs largely through regulatory rather than structural mutations.

Specificity in Ecological Interactions. Attack from the Same Lepidopteran Herbivore Results in Species-Specific Transcriptional Responses in Two Solanaceous Host Plants

Model systems have proven enormously useful in elucidating the biochemical function of plant genes. However their ecological function, having been sculpted by evolutionary forces specific to a species, may be less conserved across taxa. Responses to wounding and herbivore attack differ among plant families and are known to be mediated by oxylipin, ethylene, and systemin-signaling networks. We analyzed transcriptional responses of two native Solanaceous species to the attack of an herbivore whose elicitors are known not to be influenced by diet. With The Institute for Genomic Research 10k-cDNA potato (Solanum tuberosum) microarray, we compared the transcriptional responses of Nicotiana attenuata with those of black nightshade (Solanum nigrum) when both were attacked by the Solanaceous generalist herbivore, Manduca sexta. Based on an NADH dehydrogenase subunit F phylogeny, S. nigrum is more closely related to potato than N. attenuata but responded significantly less to M. sexta attack. Apart from transcriptional differences anticipated from their differences in secondary metabolism, both species showed distinct transcriptional patterns (with only 10% overlap in significantly regulated genes), which point to fundamental differences in the signaling cascades and downstream genes mediating herbivore resistance. The lackluster transcriptional response of S. nigrum could not be attributed to its inability to respond to elicitation, because methyl jasmonate elicitation of S. nigrum resulted in a strong transcriptional response. Given that attack from the same herbivore elicits profoundly different responses in two Solanaceaous taxa, we conclude that blueprints for commonly regulated responses to plant-herbivore interactions appear unlikely.

Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta

Summary. Several lines of evidence support the defensive function of nicotine production in the Nicotiana genus against a range of herbivores, but the evidence is largely correlative. To suppress nicotine production in planta and to test its defensive function, we expressed DNA of putrescine N-methyl transferase in an anti-sense orientation (AS-PMT) in N. sylvestris and fed leaf material from two lines of transformed and wild type plants to Manduca sexta larvae. Larvae consumed more leaf area and gained more mass on the foliage of plants with low PMT expression and low nicotine levels as compared to plants with high PMT expression and high nicotine levels and wild type plants. Overall, larval consumption and performance were negatively correlated with constitutive nicotine levels. We conclude that nicotine decreases the palatability of N. sylvestris leaves to the nicotine-resistant M. sexta larvae.

AN APPROACH TO TRANSCRIPTOME ANALYSIS OF NON-MODEL ORGANISMS USING SHORT-READ SEQUENCES

Transcriptome analysis using high-throughput short-read sequencing technology is straightforward when the sequenced genome is the same species or extremely similar to the reference genome. We present an analysis approach for when the sequenced organism does not have an already sequenced genome that can be used for a reference, as will be the case of many non-model organisms. As proof of concept, data from Solexa sequencing of the polyploid plant Pachycladon enysii was analysed using our approach with its nearest model reference genome being the diploid plant Arabidopsis thaliana. By using a combination of mapping and de novo assembly tools we could determine duplicate genes belonging to one or other of the genome copies. Our approach demonstrates that transcriptome analysis using high-throughput short-read sequencing need not be restricted to the genomes of model organisms.

Cutoffs and k-mers: implications from a transcriptome study in allopolyploid plants

BackgroundTranscriptome analysis is increasingly being used to study the evolutionary origins and ecology of non-model plants. One issue for both transcriptome assembly and differential gene expression analyses is the common occurrence in plants of hybridisation and whole genome duplication (WGD) and hybridization resulting in allopolyploidy. The divergence of duplicated genes following WGD creates near identical homeologues that can be problematic for de novo assembly and also reference based assembly protocols that use short reads (35 - 100 bp).ResultsHere we report a successful strategy for the assembly of two transcriptomes made using 75 bp Illumina reads from Pachycladon fastigiatum and Pachycladon cheesemanii. Both are allopolyploid plant species (2n = 20) that originated in the New Zealand Alps about 0.8 million years ago. In a systematic analysis of 19 different coverage cutoffs and 20 different k-mer sizes we showed that i) none of the genes could be assembled across all of the parameter space ii) assembly of each gene required an optimal set of parameter values and iii) these parameter values could be explained in part by different gene expression levels and different degrees of similarity between genes.ConclusionsTo obtain optimal transcriptome assemblies for allopolyploid plants, k-mer size and k-mer coverage need to be considered simultaneously across a broad parameter space. This is important for assembling a maximum number of full length ESTs and for avoiding chimeric assemblies of homeologous and paralogous gene copies.

Hybridization may facilitate in situ survival of endemic species through periods of climate change

Predicting species’ chances of survival under climate change requires an understanding of their adaptive potential. Now research into hybridization—one mechanism that could facilitate adaptation—shows that species of the plant genus Pachycladon that survived the Last Glacial Maximum benefited from the transfer of genetic information through hybridization. Predicting survival and extinction scenarios for climate change requires an understanding of the present day ecological characteristics of species and future available habitats, but also the adaptive potential of species to cope with environmental change. Hybridization is one mechanism that could facilitate this. Here we report statistical evidence that the transfer of genetic information through hybridization is a feature of species from the plant genus Pachycladon that survived the Last Glacial Maximum in geographically separated alpine refugia in New Zealand’s South Island. We show that transferred glucosinolate hydrolysis genes also exhibit evidence of intra-locus recombination. Such gene exchange and recombination has the potential to alter the chemical defence in the offspring of hybridizing species. We use a mathematical model to show that when hybridization increases the adaptive potential of species, future biodiversity will be best protected by preserving closely related species that hybridize rather than by conserving distantly related species that are genetically isolated.

De novo sequence assembly and characterisation of a partial transcriptome for an evolutionarily distinct reptile, the tuatara (Sphenodon punctatus)

BackgroundThe tuatara (Sphenodon punctatus) is a species of extraordinary zoological interest, being the only surviving member of an entire order of reptiles which diverged early in amniote evolution. In addition to their unique phylogenetic placement, many aspects of tuatara biology, including temperature-dependent sex determination, cold adaptation and extreme longevity have the potential to inform studies of genome evolution and development. Despite increasing interest in the tuatara genome, genomic resources for the species are still very limited. We aimed to address this by assembling a transcriptome for tuatara from an early-stage embryo, which will provide a resource for genome annotation, molecular marker development and studies of development and adaptation in tuatara.ResultsWe obtained 30 million paired-end 50 bp reads from an Illumina Genome Analyzer and assembled them with Velvet and Oases using a range of kmers. After removing redundancy and filtering out low quality transcripts, our transcriptome dataset contained 32911 transcripts, with an N50 of 675 and a mean length of 451 bp. Almost 50% (15965) of these transcripts could be annotated by comparison with the NCBI non-redundant (NR) protein database or the chicken, green anole and zebrafish UniGene sets. A scan of candidate genes and repetitive elements revealed genes involved in immune function, sex differentiation and temperature-sensitivity, as well as over 200 microsatellite markers.ConclusionsThis dataset represents a major increase in genomic resources for the tuatara, increasing the number of annotated gene sequences from just 60 to almost 16,000. This will facilitate future research in sex determination, genome evolution, local adaptation and population genetics of tuatara, as well as inform studies on amniote evolution.

Transcriptional and biochemical signatures of divergence in natural populations of two species of New Zealand alpine Pachycladon

New Zealand is diverse in alpine and subalpine environments, a consequence of Late Tertiary tectonic and climatic change. However, few studies have sought to evaluate the importance of these environments as abiotic drivers in the diversification of plant species. Of particular interest is the Late Tertiary radiation of Pachycladon, an endemic New Zealand genus of alpine cress. Here we report observations on genome‐wide levels of differential expression measured in the habitats of two closely related species of Pachycladon with distinct altitudinal preferences. Using Arabidopsis microarrays, we have identified 310 predominantly hormone‐ and stress‐response genes up‐regulated in Pachycladon fastigiata and 324 genes up‐regulated in Pachycladon enysii. Expression patterns for glucosinolate biosynthesis and hydrolysis genes (MAM1, MAM‐I, MAM‐D, AOP2, ESP, ESM1) as well as flavonoid biosynthesis genes (F3’H, FLS, FAH1) were found to be species specific. Predicted differences in flavonoid contents were partly confirmed by high performance liquid chromatography analysis. Differences in glucosinolate profiles and glucosinolate hydrolysis products obtained by high performance liquid chromatography and gas chromatography–mass spectrometry analysis, respectively, also supported inferences from expression analyses. Five glucosinolate chemotypes were matched to known Arabidopsis ecotypes, and the potential adaptive significance of these chemotypes has been discussed. Our findings, in contrast to expectations for evolution of the New Zealand flora, suggest that biotic drivers, such as plant–herbivore interactions, are likely to be as important as abiotic drivers in the diversification of Pachycladon.

Transcript and protein profiling identify candidate gene sets of potential adaptive significance in New Zealand Pachycladon

BackgroundTranscript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three Pachycladon species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species.ResultsEvidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins) and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase), the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases), water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase) and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis). Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not.ConclusionsWe have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three Pachycladon species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. Our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence.