Characterisation of two alcohol acyltransferases from kiwifruit (Actinidia spp.) reveals distinct substrate preferences.

Volatile esters are key compounds of kiwifruit flavour and are formed by alcohol acyltransferases that belong to the BAHD acyltransferase superfamily. Quantitative RT-PCR was used to screen kiwifruitderived expressed sequence tags with proposed acyltransferase function in order to select ripeningspecific sequences and test their involvement in alcohol acylation. The screening criterion was for at least 10-fold increased transcript accumulation in ripe compared with unripe kiwifruit and in response to ethylene. Recombinant expression in yeast revealed alcohol acyltransferase activity for Actinidia-derived AT1, AT16 and the phylogenetically distinct AT9, using various alcohol and acyl-CoA substrates. Functional characterisation of AT16 and AT9 demonstrated striking differences in their substrate preferences and apparent catalytic efficiencies ðV0 max K�1 m Þ. Thus revealing benzoyl-CoA:alcohol O-acyltransferase activity for AT16 and acetyl-CoA:alcohol O-acyltransferase activity for AT9. Both kiwifruit-derived enzymes displayed higher reaction rates with butanol compared with ethanol, even though ethanol is the main alcohol in ripe fruit. Since ethyl acetate and ethyl benzoate are major esters in ripe kiwifruit, we suggest that fruit characteristic volatile profiles result from a combination of substrate availability and specificity of individual alcohol acyltransferases

Insect olfaction and the evolution of receptor tuning

Insects detect odorants primarily using odorant receptors (OR) housed in the dendritic membrane of olfactory sensory neurons (OSN). Pioneering studies indicated that insects, like mammals, detect odorants in a combinatorial fashion with a specific odor ligand activating several broadly tuned ORs, and each OR being activated by several ligands. Several recent studies, however, challenge this view by providing examples where ecologically relevant odorants are detected by high-specificity ORs activating dedicated neuronal circuits. Here we review these contrasting findings on the ligand selectivity of insect ORs and their neuronal wiring, and outline scenarios describing how adaptive and neutral evolution might shape both narrow and broad receptor tuning. The fact that not all ORs display narrow tuning might partly be due to key ligands having been missed from screens or too high stimuli concentrations being used. However, the birth-and-death model of OR evolution, involving both adaptive and neutral events, could also explain the evolution of broad tuning in certain receptors due to positive selection or relaxed constraint. If the insect olfactory system indeed contains both narrowly and broadly tuned ORs, this suggests that it is a hybrid between dedicated channels and combinatorial coding. The relative extent of the two coding modes is then likely to differ between species, depending on requirements of perceived chemical space and the size of the OR repertoire. We address this by outlining scenarios where certain insect groups may be more likely to have evolved combinatorial coding as their dominant coding strategy. Combinatorial coding may have evolved predominantly in insects that benefit from the ability to discriminate between a larger number of odorants and odor objects, such as polyphagous or social species. Alternatively, combinatorial coding may have evolved simply as a mechanism to increase perceived odor space in species with small OR repertoires

Divergent Gene Expression Following Duplication of Meiotic Genes in the Stick Insect Clitarchus hookeri

evab060Some animal groups, such as stick insects (Phasmatodea), have repeatedly evolved alternative reproductive strategies, including parthenogenesis. Genomic studies have found modification of the genes underlying meiosis exists in some of these animals. Here we examine the evolution of copy number, evolutionary rate, and gene expression in candidate meiotic genes of the New Zealand geographic parthenogenetic stick insect Clitarchus hookeri. We characterized 101 genes from a de novo transcriptome assembly from female and male gonads that have homology with meiotic genes from other arthropods. For each gene we determined copy number, the pattern of gene duplication relative to other arthropod orthologs, and the potential for meiosis-specific expression. There are five genes duplicated in C hookers; including one also duplicated in the stick insect Timema cristinae, that are not or are uncommonly duplicated in other arthropods. These included two sister chromatid cohesion associated genes (SA2 and SCC2), a recombination gene (HOPI), an RNA-silencing gene (AGO2) and a cell-cycle regulation gene (WEE1). Interestingly, WEE1 and SA2 are also duplicated in the cyclical parthenogenetic aphid Acyrthosiphon pisum and Daphnia duplex, respectively, indicating possible roles in the evolution of reproductive mode. Three of these genes (SA2, SCC2, and WEE1) have one copy displaying gonad-specific expression. All genes, with the exception of WEE1, have significantly different nonsynonymous/synonymous ratios between the gene duplicates, indicative of a shift in evolutionary constraints following duplication. These results suggest that stick insects may have evolved genes with novel functions in gamete production by gene duplication.Peer reviewe

Ester biosynthesis in kiwifruit: from genes to enzymes to pathways

The distinctive flavours of different kiwifruit (Actinidia) genotypes are determined by a unique combination of volatile compounds. We are using a genomics approach to identify genes responsible for the production of esters in kiwifruit. From an extensive database of kiwifruit ESTs we have mined acyl transferase genes, including putative alcohol acyl transferases predicted to be involved in the final step of ester biosynthesis. In this paper we report the first functional characterisation of two acyl transferases in kiwifruit, AeAT9 and AdAT17, using either transient expression in planta or by recombinant protein expression in yeast. Evidence is provided that both enzymes are alcohol acyl transferases and could potentially have roles in the biosynthesis of esters in kiwifruit. Our results suggest these two alcohol acyl transferases enzymes contribute to the production of branched, straight chain and sulphur esters

Divergent transcriptional responses to low temperature among populations of alpine and lowland species of New Zealand stick insects (Micrarchus).

In widespread and genetically structured populations, temperature variation may lead to among-population differentiation of thermal biology. The New Zealand stick insect genus Micrarchus contains four species that inhabit different thermal environments, two of which are geographically widespread. RNA-Seq and quantitative PCR were used to investigate the transcriptional responses to cold shock among lowland and alpine species to identify cold-responsive transcripts that differ between the species and to determine whether there is intraspecific geographical variation in gene expression. We also used mitochondrial DNA, nuclear 28S ribosomal DNA and transcriptome-wide SNPs to determine phylogeographic structure and the potential for differences in genetic backgrounds to contribute to variation in gene expression. RNA-Seq identified 2160 unigenes that were differentially expressed as a result of low-temperature exposure across three populations from two species (M. hystriculeus and M. nov. sp. 2), with a majority (68% ± 20%) being population specific. This extensive geographical variation is consistent across years and is likely a result of background genetic differences among populations caused by genetic drift and possibly local adaptation. Responses to cold shock shared among alpine M. nov. sp. 2 populations included the enrichment of cuticular structure-associated transcripts, suggesting that cuticle modification may have accompanied colonization of low-temperature alpine environments and the development of a more cold-hardy phenotype

Identification of cold-responsive genes in a New Zealand alpine stick insect using RNA-Seq.

The endemic New Zealand alpine stick insect Micrarchus nov. sp. 2 regularly experiences sub-zero temperatures in the wild. 454-based RNA-Seq was used to generate a de novo transcriptome and differentiate between treatments to investigate the genetic basis of cold tolerance. Non cold-treated individuals were compared to those exposed to 0°C for 1 h followed by a 1 h recovery period at 20°C. We aligned 607,410 Roche 454 reads, generating a transcriptome of 5235 contigs. Differential expression analysis ranked candidate cold responsive genes for qPCR validation by P-value. The top nine up-regulated candidates, together with eight a priori targets identified from previous studies, had their relative expression quantified using qPCR. Three candidate cold responsive genes from the RNA-Seq data were verified as significantly up-regulated, annotated as: prolyl 4-hydroxylase subunit alpha-1 (P4HA1), staphylococcal nuclease domain-containing protein 1 (snd1) and cuticular protein analogous to peritrophins 3-D2 (Cpap3-d2). All three are novel candidate genes, illustrating the varied response to low temperature across insects

Recent Work on the Design and Construction of Air Inflated Structures

AbstractOver the last few years our practice has been responsible for the structural design of a number of lightweight structures that achieve their structural stability by inflating elements of fabric structure. The paper will explain the design rationale of key examples; describing the structural forms, methods of analysis and design, and the detailing, materials, and methods of fabrication. The largest and most complex of these structures to date is a demountable stage set structure for the Cirque du Soleil “Toruk: First Flight” show, comprising two 15 metre high and 20 metre wide structures. The stage set structures form ‘trees’ on which acrobats climb and perform. Each structure incorporates a high level access gantry with acrobat suspension and counterweight systems, as well as a secondary inflatable skin cladding. The entire primary structure and cladding system is constructed from inflated fabric elements using internal pressures ranging from 0.5 KN/m2 up to 90 KN/m2, and are deflated down to less than 5% of their erected volume for transport between venues. Other examples of our work described will include temporary pavilions with clear spans of up to 25 metres, using a combination of air tight and air permeable elements. Also a range of wide span structures that we describe as “semi-permanent” that are capable of resisting full environmental wind and snow loading, and that are fully demountable but that can also be erected for extended periods of time in a single location

Enhanced Angular Momentum Transport in Accretion Disks

The status of our current understanding of angular momentum transport in accretion disks is reviewed. The last decade has seen a dramatic increase both in the recognition of key physical processes and in our ability to carry through direct numerical simulations of turbulent flow. Magnetic fields have at once powerful and subtle influences on the behavior of (sufficiently) ionized gas, rendering them directly unstable to free energy gradients. Outwardly decreasing angular velocity profiles are unstable. The breakdown of Keplerian rotation into MHD turbulence may be studied in some numerical detail, and key transport coefficients may be evaluated. Chandra observations of the Galactic Center support the existence of low luminosity accretion, which may ultimately prove amenable to global three-dimensional numerical simulation.Comment: 43 pages, 2 figures, to appear v.43 A.R.A.A. October 200