David R. Greenwood

An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor

Apple flavor is characterized by combinations of ester compounds, which increase markedly during fruit ripening. The final step in ester biosynthesis is catalyzed by alcohol acyl transferases (AATs) that use coenzyme A (CoA) donors together with alcohol acceptors as substrates. The gene MpAAT1, which produces a predicted protein containing features of other plant acyl transferases, was isolated from Malus pumila (cv. Royal Gala). The MpAAT1 gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme can utilize a range of alcohol substrates from short to medium straight chain (C3–C10), branched chain, aromatic and terpene alcohols. The enzyme can also utilize a range of short to medium chain CoAs. The binding of the alcohol substrate is rate limiting compared with the binding of the CoA substrate. Among different alcohol substrates there is more variation in turnover compared with Km values. MpAAT1 is capable of producing many esters found in Royal Gala fruit, including hexyl esters, butyl acetate and 2‐methylbutyl acetate. Of these, MpAAT1 prefers to produce the hexyl esters of C3, C6 and C8 CoAs. For the acetate esters, however, MpAAT1 preference depends upon substrate concentration. At low concentrations of alcohol substrate the enzyme prefers utilizing the 2‐methylbutanol over hexanol and butanol, while at high concentrations of substrate hexanol can be used at a greater rate than 2‐methylbutanol and butanol. Such kinetic characteristics of AATs may therefore be another important factor in understanding how the distinct flavor profiles of different fruit are produced during ripening.

Odorant Receptors from the Light brown Apple Moth (Epiphyas postvittana) Recognize Important Volatile Compounds Produced by Plants

Moths recognize a wide range of volatile compounds, which they use to locate mates, food sources, and oviposition sites. These compounds are recognized by odorant receptors (OR) located within the dendritic membrane of sensory neurons that extend into the lymph of sensilla, covering the surface of insect antennae. We have identified 3 genes encoding ORs from the tortricid moth, Epiphyas postvittana, a pest of horticulture. Like Drosophila melanogaster ORs, they contain 7 transmembrane helices with an intracellular N-terminus, an orientation in the plasma membrane opposite to that of classical GPCRs. EpOR2 is orthologous to the coreceptor Or83b from D. melanogaster. EpOR1 and EpOR3 both recognize a range of terpenoids and benzoates produced by plants. Of the compounds tested, EpOR1 shows the best sensitivity to methyl salicylate [EC(50) = 1.8 x 10(-12) M], a common constituent of floral scents and an important signaling compound produced by plants when under attack from insects and pathogens. EpOR3 best recognizes the monoterpene citral to low concentrations [EC(50) = 1.1 x 10(-13) M]. Citral produces the largest amplitude electrophysiological responses in E. postvittana antennae and elicits repellent activity against ovipositing female moths. Orthologues of EpOR3 were found across 6 families within the Lepidoptera, suggesting that the ability to recognize citral may underpin an important behavior.

Chemical communication: Chirality in elephant pheromones

Musth in male elephants is an annual period of heightened sexual activity and aggression that is linked to physical, sexual and social maturation. It is mediated by the release of chemical signals such as the pheromone frontalin, which exists in two chiral forms (molecular mirror images, or enantiomers). Here we show that enantiomers of frontalin are released by Asian elephants in a specific ratio that depends on the animals age and stage of musth, and that different responses are elicited in male and female conspecifics when the ratio alters. This precise control of communication by molecular chirality offers insight into societal interactions in elephants, and may be useful in implementing new conservation protocols.

Metabolic Engineering of Cofactor F420 Production in Mycobacterium smegmatis

Cofactor F420 is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F420 has a direct and important role in archaeal energy metabolism whereas the role of F420 in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F420 has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F420, M. smegmatis has previously been used to provide this cofactor for studies of the F420-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F420 biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F420 metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F420 production. The results indicate that co–expression of the F420 biosynthetic proteins can give rise to a much higher F420 production level. This was achieved by designing and preparing a new T7 promoter–based co-expression shuttle vector. A combination of co–expression of the F420 biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F420 production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F420 produced in this study provide a suitable source of this cofactor for studies of F420-dependent proteins from other microorganisms and for possible biotechnological applications.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

SUMMARY The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for “hot topic” research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.

An unusual plant triterpene synthase with predominant α‐amyrin‐producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica

The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti‐inflammatory, antitumour and antimicrobial properties. α‐Amyrin and β‐amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full‐length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus ×domestica‘Royal Gala’) that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC‐MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α‐amyrin to β‐amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α‐amyrin produced is > 80% of the total product and is, therefore, primarily an α‐amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC‐MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either α‐amyrin and/or β‐amyrin synthase. MdOSC1 and MdOSC3 clustered with the multifunctional triterpene synthases, whereas MdOSC2 was most similar to the β‐amyrin synthases.

Sex Pheromone Evolution Is Associated with Differential Regulation of the Same Desaturase Gene in Two Genera of Leafroller Moths

Chemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation.

Characterisation of a protein from Venturia inaequalis that induces necrosis in Malus carrying the Vm resistance gene

Apple scab (black spot) is caused by the fungus, Venturia inaequalis. Race 1 isolates of this fungus are avirulent on Malus hosts carrying the resistance gene Vm: Detached leaves from a Vm host (resistant, differential host 5) and ‘Royal Gala’ (susceptible, host 1) were inoculated with a conidial suspension of V. inaequalis. In the resistant reaction, a hypersensitive response (HR), characterised by necrosis and the accumulation of autofluorescent materials in epidermal and mesophyll cells, was observed at the site of fungal penetration. No HR was observed in the susceptible host. V. inaequalis grown in vitro produced an elicitor that induced necrosis, similar to the HR, when infiltrated into leaves of the resistant Vm host. No response, however, was observed in the susceptible host. The elicitor was proteinaceous and a fraction with elicitor activity was isolated using ultra-filtration, acetone precipitation and ion-exchange chromatography. The elicitor activity was resistant to boiling but it was abolished by digestion with proteinase K. The protein fraction contained three major proteins all with low isoelectric points (pI 3·0– 4·5). The fraction also elicited necrosis in the differential host 4, but not in any of the other resistant hosts tested, including differential hosts 2, 3, and 6. Therefore, the fraction may contain elicitors with more than one host specificity. q 2003 Elsevier Ltd. All rights reserved.

Crystal Structure of Epiphyas postvittana Takeout 1 with Bound Ubiquinone Supports a Role as Ligand Carriers for Takeout Proteins in Insects

Takeout (To) proteins are found exclusively in insects and have been proposed to have important roles in various aspects of their physiology and behavior. Limited sequence similarity with juvenile hormone-binding proteins (JHBPs), which specifically bind and transport juvenile hormones in Lepidoptera, suggested a role for To proteins in binding hydrophobic ligands. We present the first crystal structure of a To protein, EpTo1 from the light brown apple moth Epiphyas postvittana, solved in-house by the single-wavelength anomalous diffraction technique using sulfur anomalous dispersion, and refined to 1.3 Å resolution. EpTo1 adopts the unusual α/β-wrap fold, seen only for JHBP and several mammalian lipid carrier proteins, a scaffold tailored for the binding and/or transport of hydrophobic ligands. EpTo1 has a 45Å long, purely hydrophobic, internal tunnel that extends for the full length of the protein and accommodates a bound ligand. The latter was shown by mass spectrometry to be ubiquinone-8 and is probably derived from Escherichia coli. The structure provides the first direct experimental evidence that To proteins are ligand carriers; gives insights into the nature of endogenous ligand(s) of EpTo1; shows, by comparison with JHBP, a basis for different ligand specificities; and suggests a mechanism for the binding/release of ligands.

Pernin: a novel, self-aggregating haemolymph protein from the New Zealand green-lipped mussel, Perna canaliculus (Bivalvia: Mytilidae).

A protein, designated pernin, found in the New Zealand green-lipped mussel, comprises almost all of the protein in cell-free haemolymph. It occurs as large, aggregate structures of several hundred units resembling small virus-like particles. Pernin is a non-pigmented, glycosylated protein, composed of 497 amino acids, which has an estimated molecular mass of 60 kDa. It is exceptionally rich in histidine (13.7%) and aspartic acid (12.3%), amino acids both known to participate in the binding of divalent metal cations. In addition, pernin has serine protease inhibitor activity, likely due to a sequence of eight N-terminal amino acid residues, separated from the remainder of the protein via a histidine-aspartate spacer. The pernin monomer comprises three regions of obvious sequence duplication. These make up approximately 95% of the pernin molecule and have sequences clearly homologous to the active-site domain of Cu-Zn SODs (superoxide dismutases). Despite several of the metal ion co-ordinating histidine residues being retained, pernin contains no Cu or Zn. It is, however, associated with Fe with an apparent stoichiometry of 1 atom of Fe to 6 molecules of pernin. Since pernin has no demonstrable SOD activity, these SOD-derived sequences presumably have been modified for another function.