Biomedical subjects

Dorothea Tholl

Publications and source records attributed to Dorothea Tholl.

8 recordsLinked to original sources

PubMed2006

Terpene synthases and the regulation, diversity and biological roles of terpene metabolism.

Terpene synthases are the primary enzymes in the formation of low-molecular-weight terpene metabolites. Rapid progress in the biochemical and molecular analysis of terpene synthases has allowed significant investigations of their evolution, structural and mechanistic properties, and regulation. The organization of terpene synthases in large gene families, their characteristic ability to form multiple products, and their spatial and temporal regulation during development and in response to biotic and abiotic factors contribute to the time-variable formation of a diverse group of terpene metabolites. The structural diversity and complexity of terpenes generates an enormous potential for mediating plant-environment interactions. Engineering the activities of terpene synthases provides opportunities for detailed functional evaluations of terpene metabolites in planta.

Alkyl and Aryl Transferases↗

PubMed2006

Practical approaches to plant volatile analysis.

Plants emit volatile organic compounds (VOCs) that play important roles in their interaction with the environment and have a major impact on atmospheric chemistry. The development of static and dynamic techniques for headspace collection of volatiles in combination with gas chromatography-mass spectrometry analysis has significantly improved our understanding of the biosynthesis and ecology of plant VOCs. Advances in automated analysis of VOCs have allowed the monitoring of fast changes in VOC emissions and facilitated in vivo studies of VOC biosynthesis. This review presents an overview of methods for the analysis of plant VOCs, including their advantages and disadvantages, with a focus on the latest technical developments. It provides guidance on how to select appropriate instrumentation and protocols for biochemical, physiological and ecologically relevant applications. These include headspace analyses of plant VOCs emitted by the whole organism, organs or enzymes as well as advanced on-line analysis methods for simultaneous measurements of VOC emissions with other physiological parameters.

Gas Chromatography-Mass Spectrometry↗

PubMed2005

Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers.

Despite the fact that Arabidopsis is largely self-pollinating, its flowers emit a complex mixture of terpene volatiles consisting predominantly of a large group of over 20 sesquiterpenes. Here we report that only two terpene synthases, encoded by the florally expressed genes At5g23960 and At5g44630, are responsible for the formation of virtually all sesquiterpenes found in the Arabidopsis floral volatile blend. Two independent mutant lines with T-DNA insertions in the previously identified At5g23960 gene lacked the emission of three sesquiterpenes, including the main sesquiterpene volatile (E)-beta-caryophyllene, confirming the previous in vitro functional assignment for this gene. Flowers of a mutant line carrying a T-DNA insertion in gene At5g44630 emitted these three sesquiterpenes, but did not emit any of the remaining sesquiterpene volatiles. An At5g44630 cDNA was expressed in Escherichia coli and the produced protein catalyzed the conversion of farnesyl diphosphate into over 15 sesquiterpenes in similar proportions to those found in the floral volatile blend. At5g23960 and At5g44630 promoter-beta-glucuronidase (GUS) fusion experiments demonstrated that both genes are expressed in several parts of the Arabidopsis flower, with strong At5g23960 promoter-GUS activity in the stigma and strong expression of At5g44630 in intrafloral nectaries. Given the previously reported antimicrobial activity of terpenes, their production in stigmas and nectaries may serve to inhibit microbial infection at these vulnerable sites. A survey of 37 Arabidopsis thaliana ecotypes revealed quantitative, but almost no qualitative, variations of floral monoterpene and sesquiterpene emissions suggesting that floral terpene volatiles must play some significant role in the life of the Arabidopsis plant.

Alkyl and Aryl Transferases↗

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

Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases.

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.

Alkyl and Aryl Transferases↗

PubMed2003

An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense.

Emission of methylsalicylate (MeSA), and occasionally of methylbenzoate (MeBA), from Arabidopsis thaliana leaves was detected following the application of some forms of both biotic and abiotic stresses to the plant. Maximal emission of MeSA was observed following alamethicin treatment of leaves. A gene (AtBSMT1) encoding a protein with both benzoic acid (BA) and salicylic acid (SA) carboxyl methyltransferase activities was identified using a biochemical genomics approach. Its ortholog (AlBSMT1) in A. lyrata, a close relative of A. thaliana, was also isolated. The AtBSMT1 protein utilizes SA more efficiently than BA, whereas AlBSMT1 catalyzes the methylation of SA less effectively than that of BA. The AtBSMT1 and AlBSMT1 genes showed expression in leaves under normal growth conditions and were more highly expressed in the flowers. In A. thaliana leaves, the expression of AtBSMT1 was induced by alamethicin, Plutella xylostella herbivory, uprooting, physical wounding, and methyl jasmonate. SA was not an effective inducer. Using a beta-glucuronidase (GUS) reporter approach, the promoter activity of AtBSMT1 was localized to the sepals of flowers, and also to leaf trichomes and hydathodes. Upon thrip damage to leaves, AtBSMT1 promoter activity was induced specifically around the lesions.

Amino Acid Sequence↗

PubMed2003

Biosynthesis and emission of terpenoid volatiles from Arabidopsis flowers.

Arabidopsis is believed to be mostly self-pollinated, although several lines of genetic and morphological evidence indicate that insect-mediated outcrossing occurs with at least a low frequency in wild populations. Here, we show that Arabidopsis flowers emit both monoterpenes and sesquiterpenes, potential olfactory cues for pollinating insects. Of the 32 terpene synthase genes in the Arabidopsis genome, 20 were found to be expressed in flowers, 6 of these exclusively or almost exclusively so. Two terpene synthase genes expressed exclusively in the flowers and one terpene synthase gene expressed almost exclusively in the flowers were characterized and found to encode proteins that catalyze the formation of major floral volatiles. A beta-glucuronidase fusion construct with a promoter of one of these genes demonstrated that gene expression was restricted to the sepals, stigmas, anther filaments, and receptacles, reaching a peak when the stigma was receptive to cross pollen. The observation that Arabidopsis flowers synthesize and emit volatiles raises intriguing questions about the reproductive behavior of Arabidopsis in the wild and allows detailed investigations of floral volatile biosynthesis and its regulation to be performed with this model plant system.

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↗