Jörg Bohlmann

Sesquiterpene synthases from grand fir (Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of delta-selinene synthase and gamma-humulene synthase.

Grand fir (Abies grandis) has been developed as a model system for the study of oleoresin production in response to stem wounding and insect attack. The turpentine fraction of the oleoresin was shown to contain at least 38 sesquiterpenes that represent 12.5% of the turpentine, with the monoterpenes comprising the remainder. Assays of cell-free extracts from grand fir stem with farnesyl diphosphate as substrate indicated that the constitutive sesquiterpene synthases produced the same sesquiterpenes found in the oleoresin and that, in response to wounding, only two new products were synthesized, δ-cadinene and (E)-α-bisabolene. A similarity based cloning strategy yielded two new cDNA species from a stem cDNA library that, when expressed in Escherichia coli and the gene products subsequently assayed, yielded a remarkable number of sesquiterpene products. The encoded enzymes have been named δ-selinene synthase and γ-humulene synthase based on the principal products formed; however, each enzyme synthesizes three major products and produces 34 and 52 total sesquiterpenes, respectively, thereby accounting for many of the sesquiterpenes of the oleoresin. The deduced amino acid sequence of the δ-selinene synthase cDNA open reading frame encodes a protein of 581 residues (at 67.6 kDa), whereas that of the γ-humulene synthase cDNA encodes a protein of 593 residues (at 67.9 kDa). The two amino acid sequences are 83% similar and 65% identical to each other and range in similarity from 65 to 67% and in identity from 43 to 46% when compared with the known sequences of monoterpene and diterpene synthases from grand fir. Although the two sesquiterpene synthases from this gymnosperm do not very closely resemble terpene synthases from angiosperm species (52–56% similarity and 26–30% identity), there are clustered regions of significant apparent homology between the enzymes of these two plant classes. The multi-step, multi-product reactions catalyzed by the sesquiterpene synthases from grand fir are among the most complex of any terpenoid cyclase thus far described.

Induction of Volatile Terpene Biosynthesis and Diurnal Emission by Methyl Jasmonate in Foliage of Norway Spruce

Terpenoids are characteristic constitutive and inducible defense chemicals of conifers. The biochemical regulation of terpene formation, accumulation, and release from conifer needles was studied in Norway spruce [Picea abies L. (Karst)] saplings using methyl jasmonate (MeJA) to induce defensive responses without inflicting physical damage to terpene storage structures. MeJA treatment caused a 2-fold increase in monoterpene and sesquiterpene accumulation in needles without changes in terpene composition, much less than the 10- and 40-fold increases in monoterpenes and diterpenes, respectively, observed in wood tissue after MeJA treatment (D. Martin, D. Tholl, J. Gershenzon, J. Bohlmann [2002] Plant Physiol 129: 1003–1018). At the same time, MeJA triggered a 5-fold increase in total terpene emission from foliage, with a shift in composition to a blend dominated by oxygenated monoterpenes (e.g. linalool) and sesquiterpenes [e.g. (E)-β-farnesene] that also included methyl salicylate. The rate of linalool emission increased more than 100-fold and that of sesquiterpenes increased more than 30-fold. Emission of these compounds followed a pronounced diurnal rhythm with the maximum amount released during the light period. The major MeJA-induced volatile terpenes appear to be synthesized de novo after treatment, rather than being released from stored terpene pools, because they are almost completely absent from needle oleoresin and are the major products of terpene synthase activity measured after MeJA treatment. Based on precedents in other species, the induced emission of terpenes from Norway spruce foliage may have ecological and physiological significance.

Functional Characterization of Nine Norway Spruce TPS Genes and Evolution of Gymnosperm Terpene Synthases of the TPS-d Subfamily

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (−)-limonene synthase, (−)-α/β-pinene synthase, and (−)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-α-farnesene synthase, and E-α-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.

(E)-β-Ocimene and Myrcene Synthase Genes of Floral Scent Biosynthesis in Snapdragon: Function and Expression of Three Terpene Synthase Genes of a New Terpene Synthase Subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-β-ocimene, derived from geranyl diphosphate, in ad-dition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-β-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-β-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-β-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-β-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx8W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.

Genomic analysis of the terpenoid synthase (AtTPS) gene family of Arabidopsis thaliana

Abstract. A family of 40 terpenoid synthase genes (AtTPS) was discovered by genome sequence analysis in Arabidopsis thaliana. This is the largest and most diverse group of TPS genes currently known for any species. AtTPS genes cluster into five phylogenetic subfamilies of the plant TPS superfamily. Surprisingly, thirty AtTPS closely resemble, in all aspects of gene architecture, sequence relatedness and phylogenetic placement, the genes for plant monoterpene synthases, sesquiterpene synthases or diterpene synthases of secondary metabolism. Rapid evolution of these AtTPS resulted from repeated gene duplication and sequence divergence with minor changes in gene architecture. In contrast, only two AtTPS genes have known functions in basic (primary) metabolism, namely gibberellin biosynthesis. This striking difference in rates of gene diversification in primary and secondary metabolism is relevant for an understanding of the evolution of terpenoid natural product diversity. Eight AtTPS genes are interrupted and are likely to be inactive pseudogenes. The localization of AtTPS genes on all five chromosomes reflects the dynamics of the Arabidopsis genome; however, several AtTPS genes are clustered and organized in tandem repeats. Furthermore, some AtTPS genes are localized with prenyltransferase genes (AtGGPPS, geranylgeranyl diphosphate synthase) in contiguous genomic clusters encoding consecutive steps in terpenoid biosynthesis. The clustered organization may have implications for TPS gene evolution and the evolution of pathway segments for the synthesis of terpenoid natural products. Phylogenetic analyses highlight events in the divergence of the TPS paralogs and suggest orthologous genes and a model for the evolution of the TPS gene family.

Insect-induced conifer defense. White pine weevil and methyl jasmonate induce traumatic resinosis, de novo formed volatile emissions, and accumulation of terpenoid synthase and putative octadecanoid pathway transcripts in Sitka spruce.

Stem-boring insects and methyl jasmonate (MeJA) are thought to induce similar complex chemical and anatomical defenses in conifers. To compare insect- and MeJA-induced terpenoid responses, we analyzed traumatic oleoresin mixtures, emissions of terpenoid volatiles, and expression of terpenoid synthase (TPS) genes in Sitka spruce (Picea sitchensis) following attack by white pine weevils (Pissodes strobi) or application of MeJA. Both insects and MeJA caused traumatic resin accumulation in stems, with more accumulation induced by the weevils. Weevil-induced terpenoid emission profiles were also more complex than emissions induced by MeJA. Weevil feeding caused a rapid release of a blend of monoterpene olefins, presumably by passive evaporation of resin compounds from stem feeding sites. These compounds were not found in MeJA-induced emissions. Both weevils and MeJA caused delayed, diurnal emissions of (−)-linalool, indicating induced de novo biosynthesis of this compound. TPS transcripts strongly increased in stems upon insect attack or MeJA treatment. Time courses and intensity of induced TPS transcripts were different for monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Increased levels of weevil- and MeJA-induced TPS transcripts accompanied major changes in terpenoid accumulation in stems. Induced TPS expression profiles in needles were less complex than those in stems and matched induced de novo emissions of (−)-linalool. Overall, weevils and MeJA induced similar, but not identical, terpenoid defense responses in Sitka spruce. Findings of insect- and MeJA-induced accumulation of allene oxide synthase-like and allene oxide cyclase-like transcripts are discussed in the context of traumatic resinosis and induced volatile emissions in this gymnosperm system.

Monoterpene Synthases from Grand Fir (Abies grandis) cDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF MYRCENE SYNTHASE, (−)-(4S)-LIMONENE SYNTHASE, AND (−)-(1S,5S)-PINENE SYNTHASE

Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (−)-limonene and (−)-α- and (−)-β-pinene are prominent components; (−)-limonene and (−)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression inEscherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (−)-limonene synthase, a myrcene synthase, and a (−)-pinene synthase that produces both α-pinene and β-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA blot hybridization using probes derived from the three monoterpene synthase cDNAs. The availability of cDNA species encoding these monoterpene synthases will allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects. These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic olefin products.

Assembling the 20 Gb white spruce (Picea glauca) genome from whole-genome shotgun sequencing data

White spruce (Picea glauca) is a dominant conifer of the boreal forests of North America, and providing genomics resources for this commercially valuable tree will help improve forest management and conservation efforts. Sequencing and assembling the large and highly repetitive spruce genome though pushes the boundaries of the current technology. Here, we describe a whole-genome shotgun sequencing strategy using two Illumina sequencing platforms and an assembly approach using the ABySS software. We report a 20.8 giga base pairs draft genome in 4.9 million scaffolds, with a scaffold N50 of 20 356 bp. We demonstrate how recent improvements in the sequencing technology, especially increasing read lengths and paired end reads from longer fragments have a major impact on the assembly contiguity. We also note that scalable bioinformatics tools are instrumental in providing rapid draft assemblies. Availability: The Picea glauca genome sequencing and assembly data are available through NCBI (Accession#: ALWZ0100000000 PID: PRJNA83435). http://www.ncbi.nlm.nih.gov/bioproject/83435. Contact: ibirol@bcgsc.ca Supplementary information: Supplementary data are available at Bioinformatics online.

Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera) Terpene Synthase Gene Family Based on Genome Assembly, FLcDNA Cloning, and Enzyme Assays

BackgroundTerpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.ResultsWe present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation.ConclusionsThe highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently known VvTPS gene family.

Genomics of hybrid poplar (Populus trichocarpa× deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in poplar

As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar (Populus spp.) ESTs by focusing on herbivore‐ and elicitor‐treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normalized or full‐length cDNA libraries, we generated 139 007 3′‐ or 5′‐end sequenced ESTs, representing more than one‐third of the c. 385 000 publicly available Populus ESTs. Clustering and assembly of 107 519 3′‐end ESTs resulted in 14 451 contigs and 20 560 singletons, altogether representing 35 011 putative unique transcripts, or potentially more than three‐quarters of the predicted c. 45 000 genes in the poplar genome. Using this EST resource, we developed a cDNA microarray containing 15 496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (Malacosoma disstria). After 24 h of feeding, 1191 genes were classified as up‐regulated, compared to only 537 down‐regulated. Functional classification of this induced gene set revealed genes with roles in plant defence (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signalling (e.g. lipoxygenase, allene oxide synthase, 1‐aminocyclopropane‐1‐carboxylate oxidase), transport (e.g. ABC proteins, calreticulin), secondary metabolism [e.g. polyphenol oxidase, isoflavone reductase, (–)‐germacrene D synthase] and transcriptional regulation [e.g. leucine‐rich repeat transmembrane kinase, several transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome‐scale approach to characterize insect‐induced defences in a woody perennial providing a solid platform for functional investigation of plant–insect interactions in poplar.