Snapdragon flowers emit two monoterpene olefins, myrcene and ( E )-␤ -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 )-␤ -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 )-␤ -Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly ( E )-␤ -ocimene (97% of total monoterpene olefin product) with small amounts of ( Z )-␤ -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.

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 (2)-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 (2) Chemical and physical defense of conifers against potential herbivores and pathogens depends on a large array of structurally diverse monoterpenoids (C10), sesquiterpenoids (C15), and diterpenoids (C20;

For the first time, the complete functional gene for isoprene synthase has been isolated from poplar (Populus alba x Populus tremula). The gene was quite similar to known limonene and other monoterpene synthases, but was found to specifically catalyze the formation of isoprene from the precursor dimethylallyl diphosphate with only a marginal activity for the formation of the monoterpene limonene from geranyl diphosphate as compared with limonene synthases. Omitting the part of the gene that putatively encoded the signal peptide necessary for transport into the chloroplast led to an enhanced rate of isoprene formation by the recombinant protein.

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.

The present work aimed to proof the functionality of the non-mevalonate pathway in cyanobacteria. It was intended to isolate the 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase gene (dxr), as this gene encodes the enzyme which catalyzes a pathway-specific, indicative step of this pathway. For this purpose, a segment of dxr was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with the PCR segment led to the identification of a 26.5 kbp locus on which a dxr homologous gene and two adjacent open reading frames organized in one operon were localized by DNA sequencing. The functionality of the gene was demonstrated expressing the gene in Escherichia coli and using the purified gene product in a photometrical NADPH dependent test based on the substrate DXP generating system. While the content of one of the central intermediates of the isoprenoid biosynthesis (dimethylallyl diphosphate = DMADP) was significantly (P 9 9 0.001) increased in E. coli cells overexpressing the DXP synthase gene (dxs) of S. leopoliensis, overexpression of dxr does not lead to an elevated DMADP level. Since even in strains harboring an expression fusion of dxs the additional overexpression of dxr does not influence the DMADP content, it is concluded that Dxs but not Dxr catalyzes a rate limiting step of the non-mevalonate isoprenoid biosynthesis. ß

Three derivatization agents used in GC analysis of amino acids were compared: N,O‐bis(trimethylsilyl)trifluoroacetamide, (BSTFA), N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide (MTBSTFA), and isobutyl chloroformate (iBuCF). It was shown that the analytical characteristics achieved in the case of silylation with MTBSTFA are comparable to those obtained for esterification/acylation. However, since the former approach requires laborious sample preparation to isolate the compounds in question prior to derivatization, determination of amino acids as N(O,S)‐alkoxycarbonyl alkyl esters seems to be preferable in many cases. Application of the esterification/acylation procedure to analysis of lyophilized E. coli microbial culture was demonstrated.

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.

The mass spectra of tert-butyldimethylsilyl (TBDMS) derivatives of 17 amino acids were obtained using electron ionization (EI) and atmospheric pressure photochemical ionization (APPhCI) mass spectrometry. The APPhCI mass spectra for all of the derivatives except arginine were shown to consist of only molecular [M](+.) and quasimolecular [MH](+) ions whereas, in the case of EI, the compounds in question underwent a drastic fragmentation. The application of APPhCI to gas chromatography-mass spectrometry enables a reliable identification of the TBDMS derivatives of amino acids in a mixture, even if its components are only partially resolved, due to the unique molecular masses for each compound. Comparison of the respective positive-ion chemical ionization (PICI) mass spectra available in the literature with APPhCI spectra has shown that, in the case of PICI, unlike APPhCI, noticeable fragmentation occurs.