Biomedical subjects

Jörg Bohlmann

Publications and source records attributed to Jörg Bohlmann.

34 records · Page 2Linked to original sources

PubMed2004

Characterization of a root-specific Arabidopsis terpene synthase responsible for the formation of the volatile monoterpene 1,8-cineole.

Arabidopsis is emerging as a model system to study the biochemistry, biological functions, and evolution of plant terpene secondary metabolism. It was previously shown that the Arabidopsis genome contains over 30 genes potentially encoding terpene synthases (TPSs). Here we report the characterization of a monoterpene synthase encoded by two identical, closely linked genes, At3g25820 and At3g25830. Transcripts of these genes were detected almost exclusively in roots. An At3g25820/At3g25830 cDNA was expressed in Escherichia coli, and the protein thus produced was shown to catalyze the formation of 10 volatile monoterpenes from geranyl diphosphate, with 1,8-cineole predominating. This protein was therefore designated AtTPS-Cin. The purified recombinant AtTPS-Cin displayed similar biochemical properties to other known monoterpene synthases, except for a relatively low K(m) value for geranyl diphosphate of 0.2 microm. At3g25820/At3g25830 promoter activity, measured with a beta-glucuronidase (GUS) reporter gene, was primarily found in the epidermis, cortex, and stele of mature primary and lateral roots, but not in the root meristem or the elongation zone. Although the products of AtTPS-Cin were not detected by direct extraction of plant tissue, the recent report of 1,8-cineole as an Arabidopsis root volatile (Steeghs M, Bais HP, de Gouw J, Goldan P, Kuster W, Northway M, Fall R, Vivanco JM [2004] Plant Physiol 135: 47-58) suggests that the enzyme products may be released into the rhizosphere rather than accumulated. Among Arabidopsis TPSs, AtTPS-Cin is most similar to the TPS encoded by At3g25810, a closely linked gene previously shown to be exclusively expressed in flowers. At3g25810 TPS catalyzes the formation of a set of monoterpenes that is very similar to those produced by AtTPS-Cin, but its major products are myrcene and (E)-beta-ocimene, and it does not form 1,8-cineole. These data demonstrate that divergence of organ expression pattern and product specificity are ongoing processes within the Arabidopsis TPS family.

Alkyl and Aryl Transferases↗

PubMed2004

Mono and diterpene production in Escherichia coli.

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

Alkyl and Aryl Transferases↗

PubMed2004

Robust simple sequence repeat markers for spruce (Picea spp.) from expressed sequence tags.

Traditionally, simple sequence repeat (SSR)markers have been developed from libraries of genomic DNA. However, the large, repetitive nature of conifer genomes makes development of robust, single-copy SSR markers from genomic DNA difficult. Expressed sequence tags (ESTs), or sequences of messenger RNA, offer the opportunity to exploit single, low-copy, conserved sequence motifs for SSR development. From a 20,275-unigene spruce EST set, we identified 44 candidate EST-SSR markers. Of these, 25 amplified and were polymorphic in white, Sitka, and black spruce; 20 amplified in all 3 spruce species tested; the remaining five amplified in all except one species. In addition, 101 previously described spruce SSRs (mostly developed from genomic DNA), were tested. Of these, 17 amplified across white,Sitka, and black spruce. The 25 EST-SSRs had approximately 9% less heterozygosity than the 17 genomic-derived SSRs (mean H=0.65 vs 0.72), but appeared to have less null alleles, as evidenced by much lower apparent inbreeding (mean F=0.046 vs 0.126). These robust SSRs are of particular use in comparative studies,and as the EST-SSRs are within the expressed portion of the genome, they are more likely to be associated with a particular gene of interest, improving their utility for quantitative trait loci mapping and allowing detection of selective sweeps at specific genes.

Base Sequence↗

PubMed2004

Genomic hardwiring and phenotypic plasticity of terpenoid-based defenses in conifers.

Over evolutionary history, conifers have faced a myriad of threats from phloem- and xylem-feeding insects, defoliating insects, and fungal pathogens. Among the trees' defenses, terpenoids appear to play a major role by harming, disabling, deterring, repelling, or otherwise reducing the fitness of potential invaders. Each of the three classes of terpenoids in conifers, monoterpenes, sesquiterpenes, and diterpenes, are composed of a large number of representative compounds. In most cases, the presence of a particular terpenoid compound in the oleoresin or volatile emissions from a specific conifer can be accounted for by the expression of one of many committed terpene synthase (TPS) genes. However, while each TPS may produce one or a few major products, many produce a variety of minor products with relatively constant component ratios in the product blends. TPS genes exist in conifers in large and functionally diverse, yet monophyletic, gene families. Within these gene families, new biochemical functions of TPS appear to have evolved by gene duplication and changes in the amino acid sequence of the enzyme's active site. In addition, TPS genes may be differentially expressed prior to, during, and following attack by insects or pathogens. Thus, while the production of any particular terpenoid is hardwired into a conifer's genome, these trees have the capacity to change the mixture of terpenoids in oleoresin secretions and volatile emissions. Anatomical changes may also accompany induced terpenoid production, supplementing the plasticity of the molecular and biochemical events.

Alkyl and Aryl Transferases↗

PubMed2004

Identification of Vitis vinifera (-)-alpha-terpineol synthase by in silico screening of full-length cDNA ESTs and functional characterization of recombinant terpene synthase.

The flavour and aroma of certain Vitis vinifera grape varieties is dominated by volatile terpenes and small volatile aldehydes. Monoterpenes contribute to the final grape and wine aroma and flavour in form of free volatiles and as glycoside conjugates of monoterpene alcohols. Typical monoterpenol components of the cultivar Gewürztraminer and other aroma-rich grape varieties are linalool, geraniol, nerol, citronellol, and alpha-terpineol. In a functional genomics effort to identify genes for the formation of monoterpene alcohols in V. vinifera, a database of full-length cDNA sequences was screened in silico and yielded two clones for putative monoterpene synthases. The gene products were functionally characterized by expression in Escherichia coli, in vitro enzyme assay and gas chromatography-mass spectrometry (GC-MS) product identification as multi-product (-)-alpha-terpineol synthases.

Alkyl and Aryl Transferases↗

PubMed2004

Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries.

Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (-)-7-epi-alpha-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (-)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (-)-alpha-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds.

Alkyl and Aryl Transferases↗

PubMed2004

Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid volatiles in hybrid poplar (Populus trichocarpa x deltoides): cDNA cloning, functional characterization, and patterns of gene expression of (-)-germacrene D synthase, PtdTPS1.

Feeding forest tent caterpillars (FTCs) induced local and systemic diurnal emissions of (-)-germacrene D, along with (E)-beta-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), benzene cyanide, and (E,E)-alpha-farnesene, from leaves of hybrid poplar. FTC feeding induced substantially higher levels of volatiles in local and systemic leaves than did mechanical wounding. A full-length poplar sesquiterpene synthase cDNA (PtdTPS1) was isolated and functionally identified as (-)-germacrene D synthase. Expression of PtdTPS1, expression of genes of early, intermediate and late steps in terpenoid biosynthesis, and expression of a lipoxygenase gene (PtdLOX1) were analyzed in local FTC-infested and systemic leaves. Transcript levels of PtdTPS1 and PtdLOX1 were strongly increased in response to herbivory. PtdTPS1 was also induced by mechanical wounding or by methyl jasmonate (MeJA) treatment. FTC feeding did not affect transcript levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), and isoprene synthase (IPS). Two other TPS genes, PtdTPS2 and PtTPS3, and farnesyl diphosphate synthase were only very transiently induced. These results illustrate differential expression of terpenoid pathway genes in response to insect feeding and a key function of (-)-germacrene D synthase PtdTPS1 for herbivore-induced local and systemic volatile emissions in hybrid poplar. FTC-induced transcripts of PtdTPS1 followed diurnal rhythm. Spatial patterns of FTC-induced PtdTPS1 transcript accumulation revealed acropetal but not basipetal direction of the systemic response. Implications for tritrophic poplar-FTC-predator/parasitoid interactions are discussed.

Acetates↗

PubMed2004

Isolation of high-quality RNA from gymnosperm and angiosperm trees.

An improved protocol was developed for efficient and reliable extraction of high-quality total RNA and mRNA from various tissues of spruce (Picea spp.) and poplar (Populus spp.) trees, as well as other plant species. This method was specifically optimized for tissues with high content of polysaccharides, oleoresin terpenoids, and phenolic secondary metabolites, which often co-precipitate with RNA and inhibit subsequent reverse transcription. The improved protocol yielded up to 600 micrograms of total RNA per gram of tissue suitable for standard expressed sequence tags (ESTs), full-length cDNA library construction, and for microarray applications.

Chemical Fractionation↗

PubMed2003

A novel sex-specific and inducible monoterpene synthase activity associated with a pine bark beetle, the pine engraver, Ips pini.

Ecological interactions of conifers and coniferophagous bark beetles are determined in part by terpenoids (isoprenoids), which are major defense metabolites of conifer oleoresin. Curiously, similar compounds are important aggregation pheromones of conifer-attacking bark beetles. Terpene synthases are responsible for generating the enormous variety of terpenoid carbon skeletons found in nature. These catalysts convert short-chain prenyl diphosphates to a diverse assortment of hemiterpene, monoterpene, sesquiterpene, and diterpene natural products. While terpene synthases have frequently been characterized from plant and microbial sources, they have not yet been described in animals. Here we report the discovery of a monoterpene synthase activity in an insect, the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae). Cell-free assays of I. pini revealed that geranyl diphosphate (GDP) is converted to the acyclic monoterpene myrcene in whole-body extracts from males, but not females. Furthermore, the monoterpene synthase activity in males can be induced by prior treatment with juvenile hormone III (JH III) or by feeding on phloem from the host trees, Jeffrey pine (Pinus jeffreyi Grev. & Balf.) or red pine (Pinus resinosa Ait.). The sex-specificity and endocrine induction of this activity argue for its involvement in the biosynthesis of monoterpenoid pheromones mediated by enzymes from insect tissue. This discovery is the first example of a monoterpene synthase in the Metazoa and evokes exciting new questions about the origin, evolution, and occurrence of terpene synthases.

Animals↗

PubMed2003

Traumatic resin defense in Norway spruce (Picea abies): methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase.

Picea abies (L.) Karst. (Norway spruce) employs constitutive and induced resin terpenoids as major chemical and physical defense-shields against insects and pathogens. In recent work, we showed that a suite of terpenoids, monoterpenoids and diterpenoids was induced in stems of Norway spruce after treatment of trees with methyl jasmonate (MeJA) (Martin et al., 2002). Increase of enzyme activities of terpenoid biosynthesis and accumulation of terpenoids was associated with MeJA-induced de novo differentiation of xylem resin ducts. The formation of defense-related traumatic resin ducts was also found in Norway spruce after attack by stem boring insects or after infestation with fungal pathogens. In the present study, we analyzed the traumatic resin response in Norway spruce further at the molecular genetic level. Treatment of trees with MeJA induced transient transcript accumulation of monoterpenoid synthases and diterpenoid synthases in stem tissues of Norway spruce. In screening for defense-related terpenoid synthase (TPS) genes from Norway spruce, a full-length monoterpenoid synthase cDNA, PaJF67, was isolated and the recombinant enzyme expressed in E. coli and functionally characterized in vitro. The cloned PaJF67 cDNA represents a new monoterpenoid synthase gene and the gene product was identified as 3-carene synthase. The enzyme encoded by PaJF67 forms stereospecifically (+)-3-carene (78% of total product) together with minor acyclic and cyclic monoterpenes, including the mechanistically closely related terpinolene (11% of total product). (+)-3-Carene is a characteristic monoterpene of constitutive and induced oleoresin defense of Norway spruce and other members of the Pinaceae.

Acetates↗

PubMed2003

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)-beta-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.

Acetates↗

PubMed2003

Insect attack and wounding induce traumatic resin duct development and gene expression of (-)-pinene synthase in Sitka spruce.

Conifers possess inducible terpenoid defense systems. These systems are associated with the formation of traumatic resin ducts (TRD) and are underpinned by enhanced gene expression and activity of terpene synthases (TPS), enzymes responsible for oleoresin formation. We first determined that Sitka spruce (Picea sitchensis [Bong.] Carriere) had the capacity for TRD formation by mechanically wounding representative trees. We then proceeded to investigate whether the white pine weevil (Pissodes strobi Peck.), a stem-boring insect, can influence the expression of genes encoding monoterpene synthases (mono-tps) in Sitka spruce. We went on to compare this response with the effects of a simulated insect attack by drill wounding. A significant increase in mono-tps transcript level was observed in the leaders of lateral branches of weevil-attacked and mechanically wounded trees. In this study, weevils induced a more rapid enhancement of mono-tps gene expression. A full-length Sitka spruce mono-tps cDNA (PsTPS2) was isolated, expressed in Escherichia coli, and functionally identified as (-)-pinene synthase. The recombinant (-)-pinene synthase catalyzes the formation of (-)-alpha-pinene and (-)-beta-pinene, both of which are known constituents of stem oleoresin in Sitka spruce and increase in abundance after weevil attack. These data suggest that increased (-)-pinene synthase gene expression is an important element of the direct defense system deployed in Sitka spruce after insect attack.

Amino Acid Sequence↗

PubMed2003

(E)-beta-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)-beta-ocimene, derived from geranyl diphosphate, in addition 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)-beta-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)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-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 RRx(8)W 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.

Acyclic Monoterpenes↗

PubMed2002

Functional identification of AtTPS03 as (E)-beta-ocimene synthase: a monoterpene synthase catalyzing jasmonate- and wound-induced volatile formation in Arabidopsis thaliana.

(E)-beta-Ocimene is one of the most commonly found monoterpenes of the volatile blends that are emitted from leaves in response to damage by herbivores or mechanical wounding. (E)-beta-Ocimene is also a component of many floral scents. Airborne (E)-beta-ocimene emitted from plants can serve as a chemical cue for the attraction of parasitoids or predators of plant herbivores and also as an attractant for pollinating insects. Furthermore, exposure of plants to (E)-beta-ocimene can activate defense gene expression. In this paper, we describe cDNA cloning and functional characterization of a gene encoding a highly specialized (E)-beta-ocimene synthase, AtTPS03, from Arabidopsis thaliana (L.) Heynh. AtTPS03 was identified as a member of the large AtTPS gene family of putative terpene synthases. A cDNA for AtTPS03 was expressed in Escherichia coli and the enzyme function identified in vitro. The A. thaliana (E)-beta-ocimene synthase produces almost exclusively (E)-beta-ocimene (94%) with minor amounts of the related acyclic monoterpenes (Z)-beta-ocimene (4%) and myrcene (2%). Transcripts for AtTPS03 were up-regulated in leaves of Arabidopsis in response to mechanical wounding and treatment with jasmonic acid, concurrent with induced emission of (E)-beta-ocimene. AtTPS03 provides an important gene for probing plant-insect and possibly plant-plant interactions mediated by terpenoid volatiles.

Acyclic Monoterpenes↗

PubMed2002

Gene expression of 5-epi-aristolochene synthase and formation of capsidiol in roots of Nicotiana attenuata and N. sylvestris.

Three new copies of a sesquiterpenoid synthase gene encoding 5-epi-aristolochene synthase (EAS) were cloned as cDNAs from Nicotiana attenuata and functionally characterized after expression of the recombinant enzymes in E. coli. Differential patterns of EAS gene expression and formation of the antimicrobial sesquiterpenoid capsidiol were found in roots and shoots of two species of Nicotiana. EAS is expressed constitutively in roots of N. attenuata and N. sylvestris corresponding with constitutive capsidiol formation in roots. Constitutive expression of EAS and capsidiol accumulation were not detectable in shoots of rosette plants of N. attenuata, but accumulation of terpene synthase transcripts could be induced in shoots by feeding of the tobacco hornworm, Manduca sexta. Constitutive expression of EAS has not been previously reported from N. tabacum, where capsidiol is known as an elicitor- and pathogen-inducible phytoalexin. It is conceivable that capsidiol contributes not only to an inducible defense against pathogens, but also to a constitutive defense in an organ-specific manner in some species of Nicotiana.

Alkyl and Aryl Transferases↗

PubMed2002

Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of Norway spruce stems.

Norway spruce (Picea abies L. Karst) produces an oleoresin characterized by a diverse array of terpenoids, monoterpenoids, sesquiterpenoids, and diterpene resin acids that can protect conifers against potential herbivores and pathogens. Oleoresin accumulates constitutively in resin ducts in the cortex and phloem (bark) of Norway spruce stems. De novo formation of traumatic resin ducts (TDs) is observed in the developing secondary xylem (wood) after insect attack, fungal elicitation, and mechanical wounding. Here, we characterize the methyl jasmonate-induced formation of TDs in Norway spruce by microscopy, chemical analyses of resin composition, and assays of terpenoid biosynthetic enzymes. The response involves tissue-specific differentiation of TDs, terpenoid accumulation, and induction of enzyme activities of both prenyltransferases and terpene synthases in the developing xylem, a tissue that constitutively lacks axial resin ducts in spruce. The induction of a complex defense response in Norway spruce by methyl jasmonate application provides new avenues to evaluate the role of resin defenses for protection of conifers against destructive pests such as white pine weevils (Pissodes strobi), bark beetles (Coleoptera, Scolytidae), and insect-associated tree pathogens.

Acetates↗