Spatial and developmental expression of key genes of terpene biosynthesis in Tanacetum parthenium

Feverfew (Tanacetum parthenium) is a medicinal plant belonging to the Asteraceae family. To improve understanding terpene metabolism in feverfew, the relative gene expression of four key genes coding 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR) and germacrene A synthase (GAS) from the mevalonic acid pathway (MVA), as well as 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) from the methyl erythritol phosphate pathway (MEP), were examined. Target organs and tissues included young leaves (not fully expanded), mature leaves (fully expanded), flowers, stems, roots, and glandular trichomes. HMGR, DXR, and HDR were isolated and sequenced for the first time in feverfew. Real-time quantitative PCR analysis revealed differential expression of these genes in feverfew tissues and developmental stages.     

The response of terpenoids to exogenous gibberellic acid in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. at vegetative stage

In this study the influence of gibberellic acid (GA₃) on plastidic and cytosolic terpenoids and on two key enzymes, 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), for terpenoid biosynthesis was compared in vegetative cannabis plants. Treatment with GA₃ resulted in a decrease of DXS activity in comparison with the control plants. The amount of chlorophylls a, b and total carotenoids declined when plants treated by GA₃ in a concentration dependent manner. The α-tocopherol content of cannabis plants decreased in 50 μM GA₃ treatment and increased in 100 μM GA₃ treatment. Exogenous GA₃ caused an increase in HMGR activity. Concomitant with this result, the amount of squalene and phytosterols increased with GA₃ treatment. The amount of THC and CBD did not change at 50 μM GA₃ treatment, but applying of 100 μM GA₃ increased THC and CBD content in leaf plant in comparison with control plants. GA₃ treatment declined number and percentage of monoterpenes in treated plants. Also the number of sesquiterpenes decreased in response to GA₃ treatment but among the remainder of them, the amount of some sesquiterpenes decreased and some sesquiterpenes increased with GA₃ treatment. Our results showed that GA₃ treatment had opposite effect on primary terpenoid biosynthesis by the plastidic 2C-methyl-d-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways. But secondary terpenoids showed different response to GA₃ treatment probably due to interference of two biosynthetic pathways in their formation.     

Evolution of TPS20-related terpene synthases influences chemical diversity in the glandular trichomes of the wild tomato relative Solanum habrochaites

A systematic screen of volatile terpene production in the glandular trichomes of 79 accessions of Solanum habrochaites was conducted and revealed the presence of 21 mono‐ and sesquiterpenes that exhibit a range of qualitative and quantitative variation. Hierarchical clustering identified distinct terpene phenotypic modules with shared patterns of terpene accumulation across accessions. Several terpene modules could be assigned to previously identified terpene synthase (TPS) activities that included members of the TPS‐e/f subfamily that utilize the unusual cis‐prenyl diphosphate substrates neryl diphosphate and 2z,6z‐farnesyl diphosphate. DNA sequencing and in vitro enzyme activity analysis of TPS‐e/f members from S. habrochaites identified three previously unassigned enzyme activities that utilize these cisoid substrates. These produce either the monoterpenes α‐pinene and limonene, or the sesquiterpene 7‐epizingiberene, with the in vitro analyses that recapitulated the trichome chemistry found in planta. Comparison of the distribution of S. habrochaites accessions with terpene content revealed a strong preference for the presence of particular TPS20 alleles at distinct geographic locations. This study reveals that the unusually high intra‐specific variation of volatile terpene synthesis in glandular trichomes of S. habrochaites is due at least in part to evolution at the TPS20 locus.     

Amplification of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase level increases coenzyme Q10 production in recombinant Escherichia coli

For the enhancement of coenzyme Q₁₀ (CoQ₁₀) production, 1-deoxy-d-xylulose 5-phosphate (DXP) synthase of Pseudomonas aeruginosa was constitutively coexpressed in a recombinant Escherichia coli strain, which harbors the ddsA gene from Gluconobacter suboxydans encoding decaprenyl diphosphate synthase. It was found that the expression of the ddsA gene caused depletion of the isopentenyl diphosphate (IPP) pool in E. coli. Amplification of DXP synthase level by installing P. aeruginosa DXP synthase restored the diminished IPP pool and concomitantly resulted in approximately a twofold increase in relative content and productivity of CoQ₁₀. Maximum CoQ₁₀ concentration of 46.1 mg l⁻¹ was achieved from glucose-limited fed-batch cultivation of the recombinant E. coli strain simultaneously harboring the ddsA and dxs genes.     

A Novel Pathway for Sesquiterpene Biosynthesis from Z,Z-Farnesyl Pyrophosphate in the Wild Tomato Solanum habrochaites

In the wild tomato Solanum habrochaites, the Sst2 locus on chromosome 8 is responsible for the biosynthesis of several class II sesquiterpene olefins by glandular trichomes. Analysis of a trichome-specific EST collection from S. habrochaites revealed two candidate genes for the synthesis of Sst2-associated sesquiterpenes. zFPS encodes a protein with homology to Z-isoprenyl pyrophosphate synthases and SBS (for Santalene and Bergamotene Synthase) encodes a terpene synthase with homology to kaurene synthases. Both genes were found to cosegregate with the Sst2 locus. Recombinant zFPS protein catalyzed the synthesis of Z,Z-FPP from isopentenylpyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP), while coincubation of zFPS and SBS with the same substrates yielded a mixture of olefins identical to the Sst2-associated sesquiterpenes, including (+)-α-santalene, (+)-endo-β-bergamotene, and (−)-endo-α-bergamotene. In addition, headspace analysis of tobacco (Nicotiana sylvestris) plants expressing zFPS and SBS in glandular trichomes afforded the same mix of sesquiterpenes. Each of these proteins contains a putative plastid targeting sequence that mediates transport of a fused green fluorescent protein to the chloroplasts, suggesting that the biosynthesis of these sesquiterpenes uses IPP and DMAPP from the plastidic DXP pathway. These results provide novel insights into sesquiterpene biosynthesis and have general implications concerning sesquiterpene engineering in plants.     

Effects of ABA on primary terpenoids and Δ<Superscript>9</Superscript>-tetrahydrocannabinol in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. at flowering stage

This work examined the effects of exogenously applied abscisic acid (ABA) on the content of chlorophyll, carotenoids, α-tocopherol, squalene, phytosterols, Δ⁹-tetrahydrocannabinol (THC) concentration, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS) activity in Cannabis sativa L. at flowering stage. Treatment with 1 and 10 mg l⁻¹ ABA significantly decreased the contents of chlorophyll, carotenoids, squalene, stigmasterol, sitosterol, and HMGR activity in female cannabis plants. ABA caused an increase in α-tocopherol content and DXS activity in leaves and THC concentration in leaves and flowers of female plants. Chlorophyll content decreased with 10 mg l⁻¹ ABA in male plants. Treatment with 1 and 10 mg l⁻¹ ABA showed a decrease in HMGR activity, squalene, stigmasterol, and sitosterol contents in leaves but an increase in THC content of leaves and flowers in male plants. The results suggest that ABA can induce biosynthesis of 2-methyl-d-erythritol-4-phosphate (MEP) pathway secondary metabolites accumulation (α-tocopherol and THC) and down regulated biosynthesis of terpenoid primary metabolites from MEP and mevalonate (MVA) pathways (chlorophyll, carotenoids, and phytosterols) in Cannabis sativa.     

Trichome morphology provides phylogenetically informative characters for Tremandra, Platytheca and Tetratheca (former Tremandraceae)

Summary Trichomes of Tremandra R.Br. ex DC., Platytheca Steetz and Tetratheca Sm. (Elaeocarpaceae, former Tremandraceae), together with two outgroup species of Elaeocarpus L., are illustrated using scanning electron microscopy, and their distribution on various plant organs is documented. Various trichomes types were identified that relate taxa: simple hairs, stellate hairs, short glandular trichomes, long glandular trichomes, and three forms of tubercules. Both outgroup and ingroup taxa have simple hairs. Stellate hairs are confirmed as unique to Tremandra. Prominent and sculptured multicelled tubercules, some bearing a stout hair, are characteristic of Platytheca. Smaller multicelled tubercules occur in both Platytheca and Tetratheca, except for the Western Australian taxon Te. filiformis Benth. (possibly plesiomorphic). Unicellular tubercules (papilla) characterise two species of Tetratheca. Short glandular trichomes, usually found on the ovary, also occur in both of these genera but not in all species (possibly secondary losses), while long glandular trichomes, usually on stems and leaves, occur only in some groups of Tetratheca. Within Tetratheca, Western Australian taxa that have five-merous flowers fall into three ‘groups’: seven species (together with one from South Australia) that have short glandular trichomes but no long glandular trichomes; six species that have long glandular trichomes but no short glandular trichomes; and four species that have both trichome types. All other species of Tetratheca have four-merous flowers and form two ‘groups’: 12 eastern species (including one from South Australia) that have both short glandular trichomes and long glandular trichomes; 4 western species and six eastern species that lack short glandular trichomes. On the basis of these characters, a phylogenetic hypothesis for the three genera is presented.     

Biosynthesis of isoprene in <Emphasis Type="Italic">Escherichia coli</Emphasis> via methylerythritol phosphate (MEP) pathway

Isoprene is an aviation fuel of high quality and an important polymer building block in the synthetic chemistry industry. In light of high oil prices, sustained availability, and environmental concerns, isoprene from renewable materials is contemplated as a substitute for petroleum-based product. Escherichia coli with advantages over other wild microorganisms, is considered as a powerful host for biofuels and chemicals. Here, we constructed a synthetic pathway of isoprene in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra, which catalyzes the conversion of dimethylallyl diphosphate (DMAPP) to isoprene. To improve the isoprene production, we overexpressed the native 1-deoxy-d-xylulose-5-phosphate (DXP) synthase gene (dxs) and DXP reductoisomerase gene (dxr) in E. coli, which catalyzed the first step and the second step of MEP pathway, respectively. The fed-batch fermentation results showed that overexpression of DXS is helpful for the improvement of isoprene production. Surprisingly, heterologous expression of dxs and dxr from Bacillus subtilis in the E. coli expressing ispS resulted in a 2.3-fold enhancement of isoprene production (from 94 to 314 mg/L). The promising results showed that dxs and dxr from B. subtilis functioned more efficiently on the enhancement of isoprene production than native ones. This could be caused by the consequence of great difference in protein structures of the two original DXSs. It could be practical to produce isoprene in E. coli via MEP pathway through metabolic engineering. This work provides an alternative way for production of isoprene by engineered E. coli via MEP pathway through metabolic engineering.     

The 1-deoxy-d-xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine

Muscat flavor is a relevant trait both in winemaking and in fresh grape consumption. From a chemical point of view, it is strongly related to the accumulation of monoterpenes in berries. However, knowledge of the genetic mechanisms underlying its regulation is still limited. The objective of this study was to dissect the genetic determinism of aroma in grapevine by applying the analysis of quantitative trait loci (QTL) and the candidate gene (CG) approach. Two F₁ segregating progenies were evaluated through high-resolution gas chromatography–mass spectrometry (HRGC–MS) for the amounts of individual monoterpenes over 3 and 2 years. In the Italia × Big Perlon cross 34 CGs, chosen according to gene ontology (GO) terms, were placed on a complete map and tested for linkage with QTLs for linalool, nerol and geraniol levels. Two CGs mapped within a QTL for linalool content on LG 10. A third one co-localized with a major QTL for the level of the three monoterpenes on LG 5; this gene encodes 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which is the first enzyme in the plastidial pathway of terpene biosynthesis. Depending on these findings, we report the first in silico analysis of grapevine DXS genes based on the whole genome sequence. Further research on the functional significance of these associations might help to understand the genetic control of Muscat flavor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Battilana and L. Costantini equally contributed to the work.     

Biosynthesis of Resin Terpenes in Leaves and Glandular Hairs of Newcastelia viscida

Label from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid was incorporatedinto resin components of Newcastelia viscida. About 25% of thelabel from [2-¹⁴C]MVA was incorporated into resin componentsafter 4 h. Approximately 80% of the label in the resin was ina TLC band coinciding with the major terpenoid components, with60% associated with the major tricyclic diterpene, a pimaradienewhich contributes 15% to the leaf resin dry weight. Experimentswith detached trichomes showed about 0?01% incorporation oflabel from [2-¹⁴C]MVA into pimaradiene, with most of the [abelbeing associated with ß-sitosterol. It is proposedthat the glandular hairs are the sites of terpene resin synthesis,as well as secretion, in this species.     

Characterization of development and artemisinin biosynthesis in self-pollinated <Emphasis Type="Italic">Artemisia annua</Emphasis> plants

Artemisia annua L. is the only natural resource that produces artemisinin (Qinghaosu), an endoperoxide sesquiterpene lactone used in the artemisinin-combination therapy of malaria. The cross-hybridization properties of A. annua do not favor studying artemisinin biosynthesis. To overcome this problem, in this study, we report on selection of self-pollinated A. annua plants and characterize their development and artemisinin biosynthesis. Self-pollinated F2 plants selected were grown under optimized growth conditions, consisting of long day (16 h of light) and short day (9 h of light) exposures in a phytotron. The life cycles of these plants were approximately 3 months long, and final heights of 30–35 cm were achieved. The leaves on the main stems exhibited obvious morphological changes, from indented single leaves to odd, pinnately compound leaves. Leaves and flowers formed glandular and T-shaped trichomes on their surfaces. The glandular trichome densities increased from the bottom to the top leaves. High performance liquid chromatography–mass spectrometry-based metabolic profiling analyses showed that leaves, flowers, and young seedlings of F2 plants produced artemisinin. In leaves, the levels of artemisinin increased from the bottom to the top of the plants, showing a positive correlation to the density increase of glandular trichomes. RT-PCR analysis showed that progeny of self-pollinated plants expressed the amorpha-4, 11-diene synthase (ADS) and cytochrome P450 monooxygenase 71 AV1 (CYP71AV1) genes, which are involved in artemisinin biosynthesis in leaves and flowers. The use of self-pollinated A. annua plants will be a valuable approach to the study of artemisinin biosynthesis.     

Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development

Despite the commercial importance of Lavandula angustifolia Mill. and L. x intermedia Emeric ex Loisel floral essential oils (EOs), no information is currently available on potential changes in individual volatile organic compound (VOC) content during inflorescence development. Calyces were found to be the main sites of VOC accumulation. The 20 most abundant VOCs could be separated into three sub‐groups according to their patterns of change in concentration The three groups of VOCs sequentially dominated the global scent bouquet of inflorescences, the transition between the first and second groups occurring around the opening of the first flower of the inflorescence and the one between the second and third groups at the start of seed set. Changes in calyx VOC accumulation were linked to the developmental stage of individual flowers. Leaves accumulated a smaller number of VOCs which were a subset of those seen in preflowering inflorescences. Their nature and content remained constant during the growing season. Quantitative real time polymerase chain reaction assessments of the expression of two terpene synthase (TPS) genes, LaLIMS and LaLINS, revealed similar trends between their patterns of expression and those of their VOC products. Molecular and chemical analyses suggest that changes in TPS expression occur during lavender inflorescence development and lead to changes in EO composition. Both molecular data and terpene analysis support the findings that changes in biosynthesis of terpene occurred during inflorescence development.