Phytochemical, Morphological and Genetic Characterisation of Anacyclus pyrethrum var. depressus (Ball.) Maire and Anacyclus pyrethrum var. pyrethrum (L.) Link.

The present study is based on a multidisciplinary approach carried out for the first time on Anacyclus pyrethrum var. pyrethrum and Anacyclus pyrethrum var. depressus, two varieties from the endemic and endangered medicinal species listed in the IUCN red list, Anacyclus pyrethrum (L.) Link. Therefore, morphological, phytochemical, and genetic characterisations were carried out in the present work. Morphological characterisation was established based on 23 qualitative and quantitative characters describing the vegetative and floral parts. The phytochemical compounds were determined by UHPLC. Genetic characterisation of extracted DNA was subjected to PCR using two sets of universal primers, rbcL a-f/rbcL a-R and rpocL1-2/rpocL1-4, followed by sequencing analysis using the Sanger method. The results revealed a significant difference between the two varieties studied. Furthermore, phytochemical analysis of the studied extracts revealed a quantitative and qualitative variation in the chemical profile, as well as the presence of interesting compounds, including new compounds that have never been reported in A. pyrethrum. The phylogenetic analysis of the DNA sequences indicated a similarity percentage of 91%. Based on the morphological characterisation and congruence with the phytochemical characterisation and molecular data, we can confirm that A. pyrethrum var. pyrethrum and A. pyrethrum var. depressus represent two different taxa.

TcbHLH14 a Jasmonate Associated MYC2-like Transcription Factor Positively Regulates Pyrethrin Biosynthesis in Tanacetum cinerariifolium.

Natural pyrethrins have high application value, and are widely used as a green pesticide in crop pest prevention and control. Pyrethrins are mainly extracted from the flower heads of Tanacetum cinerariifolium; however, the natural content is low. Therefore, it is essential to understand the regulatory mechanisms underlying the synthesis of pyrethrins through identification of key transcription factors. We identified a gene encoding a MYC2-like transcription factor named TcbHLH14 from T. cinerariifolium transcriptome, which is induced by methyl jasmonate. In the present study, we evaluated the regulatory effects and mechanisms of TcbHLH14 using expression analysis, a yeast one-hybrid assay, electrophoretic mobility shift assay, and overexpression/virus-induced gene silencing experiments. We found that TcbHLH14 can directly bind to the cis-elements of the pyrethrins synthesis genes TcAOC and TcGLIP to activate their expression. The transient overexpression of TcbHLH14 enhanced expression of the TcAOC and TcGLIP genes. Conversely, transient silencing of TcbHLH14 downregulated the expression of TcAOC and TcGLIP and reduced the content of pyrethrins. In summary, these results indicate that the potential application of TcbHLH14 in improving the germplasm resources and provide a new insight into the regulatory network of pyrethrins biosynthesis of T. cinerariifolium to further inform the development of engineering strategies for increasing pyrethrins contents.

TcMYB8, a R3-MYB Transcription Factor, Positively Regulates Pyrethrin Biosynthesis in Tanacetum cinerariifolium.

Pyrethrins are a mixture of terpenes, with insecticidal properties, that accumulate in the aboveground parts of the pyrethrum (Tanacetum cinerariifolium). Numerous studies have been published on the positive role of MYB transcription factors (TFs) in terpenoid biosynthesis; however, the role of MYB TFs in pyrethrin biosynthesis remains unknown. Here, we report the isolation and characterization of a T. cinerariifolium MYB gene encoding a R3-MYB protein, TcMYB8, containing a large number of hormone-responsive elements in its promoter. The expression of the TcMYB8 gene showed a downward trend during the development stage of flowers and leaves, and was induced by methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA). Transient overexpression of TcMYB8 enhanced the expression of key enzyme-encoding genes, TcCHS and TcGLIP, and increased the content of pyrethrins. By contrast, transient silencing of TcMYB8 decreased pyrethrin contents and downregulated TcCHS and TcGLIP expression. Further analysis indicated that TcMYB8 directly binds to cis-elements in proTcCHS and proTcGLIP to activate their expression, thus regulating pyrethrin biosynthesis. Together, these results highlight the potential application of TcMYB8 for improving the T. cinerariifolium germplasm, and provide insight into the pyrethrin biosynthesis regulation network.

Overexpression of chrysanthemyl diphosphate synthase (CDS) gene in Tagetes erecta leads to the overproduction of pyrethrin.

Pyrethrins are widely accepted as natural insecticides and offers several advantages of synthetic compounds, i.e., rapidity of action, bioactivity against a wide range of insects, comparatively lesser costs and the like. A significant source of pyrethrin is Chrysanthemum cinerariaefolium; cultivated in restricted areas, as a result; natural pyrethrins are not produced in a large amount that would meet the ongoing global market demand. However, increasing its content and harnessing the desired molecule did not attract much attention. To enhance the production of pyrethrins in Tagetes erecta, the Chrysanthemyl diphosphate synthase (CDS) gene was overexpressed under the promoter CaMV35S. Hypocotyls were used as explant for transformation, and direct regeneration was achieved on MS medium with 1.5 mg L-1 BAP and 5.0 mg L-1 GA3. Putative transgenics were screened on 10 mgL-1 hygromycin. After successful regeneration, screening and rooting process, the transgenic plants were raised inside the glass house and PCR amplification of CDS and HYG-II was used to confirm the transformation. Biochemical analysis using HPLC demonstrated the expression levels of the pyrethrin, which was approx. twenty-six fold higher than the non-transformed Tagetes plant.

Draft Genome of Tanacetum Coccineum: Genomic Comparison of Closely Related Tanacetum-Family Plants.

The plant Tanacetum coccineum (painted daisy) is closely related to Tanacetum cinerariifolium (pyrethrum daisy). However, T. cinerariifolium produces large amounts of pyrethrins, a class of natural insecticides, whereas T. coccineum produces much smaller amounts of these compounds. Thus, comparative genomic analysis is expected to contribute a great deal to investigating the differences in biological defense systems, including pyrethrin biosynthesis. Here, we elucidated the 9.4 Gb draft genome of T. coccineum, consisting of 2,836,647 scaffolds and 103,680 genes. Comparative analyses of the draft genome of T. coccineum and that of T. cinerariifolium, generated in our previous study, revealed distinct features of T. coccineum genes. While the T. coccineum genome contains more numerous ribosome-inactivating protein (RIP)-encoding genes, the number of higher-toxicity type-II RIP-encoding genes is larger in T. cinerariifolium. Furthermore, the number of histidine kinases encoded by the T. coccineum genome is smaller than that of T. cinerariifolium, suggesting a biological correlation with pyrethrin biosynthesis. Moreover, the flanking regions of pyrethrin biosynthesis-related genes are also distinct between these two plants. These results provide clues to the elucidation of species-specific biodefense systems, including the regulatory mechanisms underlying pyrethrin production.

Engineering of tomato type VI glandular trichomes for trans-chrysanthemic acid biosynthesis, the acid moiety of natural pyrethrin insecticides.

Glandular trichomes, known as metabolic cell factories, have been proposed as highly suitable for metabolically engineering the production of plant high-value specialized metabolites. Natural pyrethrins, found only in Dalmatian pyrethrum (Tanacetum cinerariifolium), are insecticides with low mammalian toxicity and short environmental persistence. Type I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of the three rethrolone-type alcohols. To test if glandular trichomes can be made to synthesize trans-chrysanthemic acid, we reconstructed its biosynthetic pathway in tomato type VI glandular trichomes, which produce large amounts of terpenoids that share the precursor dimethylallyl diphosphate (DMAPP) with this acid. This was achieved by coexpressing the trans-chrysanthemic acid pathway related genes including TcCDS encoding chrysanthemyl diphosphate synthase and the fusion gene of TcADH2 encoding the alcohol dehydrogenase 2 linked with TcALDH1 encoding the aldehyde dehydrogenase 1 under the control of a newly identified type VI glandular trichome-specific metallocarboxypeptidase inhibitor promoter. Whole tomato leaves harboring type VI glandular trichomes expressing all three aformentioned genes had a concentration of total trans-chrysanthemic acid that was about 1.5-fold higher (by mole number) than the levels of ß-phellandrene, the dominant monoterpene present in non-transgenic leaves, while the levels of ß-phellandrene and the representative sesquiterpene ß-caryophyllene in transgenic leaves were reduced by 96% and 81%, respectively. These results suggest that the tomato type VI glandular trichome is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.

Transcriptional Responses and GCMS Analysis for the Biosynthesis of Pyrethrins and Volatile Terpenes in Tanacetum coccineum.

Natural pyrethrins have been widely used as natural pesticides due to their low mammalian toxicity and environmental friendliness. Previous studies have mainly focused on Tanacetumcinerariifolium, which contains high levels of pyrethrins and volatile terpenes that play significant roles in plant defense and pollination. However, there is little information on T. coccineum due to its lower pyrethrin content and low commercial value. In this study, we measured the transcriptome and metabolites of the leaves (L), flower buds (S1), and fully blossomed flowers (S4) of T. coccineum. The results show that the expression of pyrethrins and precursor terpene backbone genes was low in the leaves, and then rapidly increased in the S1 stage before decreasing again in the S4 stage. The results also show that pyrethrins primarily accumulated at the S4 stage. However, the content of volatile terpenes was consistently low. This perhaps suggests that, despite T. coccineum and T. cinerariifolium having similar gene expression patterns and accumulation of pyrethrins, T. coccineum attracts pollinators via its large and colorful flowers rather than via inefficient and metabolically expensive volatile terpenes, as in T. cinerariifolium. This is the first instance of de novo transcriptome sequencing reported for T. coccineum. The present results could provide insights into pyrethrin biosynthetic pathways and will be helpful for further understanding how plants balance the cost-benefit relationship between plant defense and pollination.

A dual-target molecular mechanism of pyrethrum repellency against mosquitoes.

Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-ß-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.

Genetic improvement of pyrethrum (Tanacetum cinerariifolium Sch. Bip.) through gamma radiation and selection of high yield stable mutants through seven post-radiation generations.

PURPOSE: To increase the size of the flowers for easy plucking, flower yield, pyrethrins content (%), and elite mutant selection in pyrethrum. MATERIALS AND METHODS: To increase pyrethrum production and acclimatize in north Indian plain condition, a genetic improvement program was undertaken to widen the range of variations for size and yield of flowers and pyrethrins content (%) in pyrethrum crop. Pyrethrum seeds of the variety Avadh were irradiated with gamma rays at 20 to 300 Gy doses in Gamma chamber 5000 (cobalt-60 research irradiator). RESULTS: Observations gathered visually in M1 based on vigor, synchronization of flowering, and flower's size. Out of 90 M2 families, 20 mutants were raised in M3 along with the check-in preliminary evaluation trial. The four promising mutants, 1 (20 Gy-3), 7 (40 Gy-5), 10 (40 Gy-8), 14 (60 Gy19-10) was grown for four years in a bench-scale trial (randomized block design, replicated thrice) to test the yield performance and selection of high yielding elite mutant (s). It has been found that pyrethrum is sensitive to gamma rays irradiation and produced a high range of qualitative and quantitative variations. After massive screening over four years, two promising mutants for high dry flower yield and pyrethrins content, namely 7 (40 Gy-5), and 10 (40GY-8) were isolated. CONCLUSIONS: The mutagenesis changed traits mean in positive or negative directions. Pyrethrum plant is highly sensitive to gamma irradiation and produced a high range of variability in the qualitative and quantitative traits. The mutagenesis changed the mean of traits in both positive and negative directions. Due to mutagenic efficacy, two mutants 7 (40 Gy-5), and 10 (40GY-8) were expressed high performance for pyrethrin percent i.e., 87.23 and 59.78% improvement over the check variety 'Avadh', with synchronous flowering. These two mutants are in the pipeline for release as a variety for cultivation in the North Indian plains.

How Plants Synthesize Pyrethrins: Safe and Biodegradable Insecticides.

Natural pyrethrin insecticides produced by Dalmatian pyrethrum (Tanacetum cinerariifolium) have low mammalian toxicity and short environmental persistence, providing an alternative to widely used synthetic agricultural insecticides that pose a threat to human health and the environment. A recent surge of interest in the use of pyrethrins as agricultural insecticides coincides with the discovery of several new genes in the pyrethrin biosynthetic pathway. Elucidation of this pathway facilitates efforts to breed improved pyrethrum varieties and to engineer plants with improved endogenous defenses or hosts for heterologous pyrethrin production. We describe the current state of knowledge related to global pyrethrum production, the pyrethrin biosynthetic pathway and its regulation, and recent efforts to engineer the pyrethrin pathway in diverse plant hosts.

Draft genome of Tanacetum cinerariifolium, the natural source of mosquito coil.

Pyrethrum (Tanacetum cinerariifolium), which is a perennial Asteraceae plant with white daisy-like flowers, is the original source of mosquito coils and is known for the biosynthesis of the pyrethrin class of natural insecticides. However, the molecular basis of the production of pyrethrins by T. cinerariifolium has yet to be fully elucidated. Here, we present the 7.1-Gb draft genome of T. cinerariifolium, consisting of 2,016,451 scaffolds and 60,080 genes predicted with high confidence. Notably, analyses of transposable elements (TEs) indicated that TEs occupy 33.84% of the genome sequence. Furthermore, TEs of the sire and oryco clades were found to be enriched in the T. cinerariifolium-specific evolutionary lineage, occupying a total of 13% of the genome sequence, a proportion approximately 8-fold higher than that in other plants. InterProScan analysis demonstrated that biodefense-related toxic proteins (e.g., ribosome inactivating proteins), signal transduction-related proteins (e.g., histidine kinases), and metabolic enzymes (e.g., lipoxygenases, acyl-CoA dehydrogenases/oxygenases, and P450s) are also highly enriched in the T. cinerariifolium genome. Molecular phylogenetic analysis detected a variety of enzymes with genus-specific multiplication, including both common enzymes and others that appear to be specific to pyrethrin biosynthesis. Together, these data identify possible novel components of the pyrethrin biosynthesis pathway and provide new insights into the unique genomic features of T. cinerariifolium.

Defense of pyrethrum flowers: repelling herbivores and recruiting carnivores by producing aphid alarm pheromone.

(E)-ß-Farnesene (EßF) is the predominant constituent of the alarm pheromone of most aphid pest species. Moreover, natural enemies of aphids use EßF to locate their aphid prey. Some plant species emit EßF, potentially as a defense against aphids, but field demonstrations are lacking. Here, we present field and laboratory studies of flower defense showing that ladybird beetles are predominantly attracted to young stage-2 pyrethrum flowers that emitted the highest and purest levels of EßF. By contrast, aphids were repelled by EßF emitted by S2 pyrethrum flowers. Although peach aphids can adapt to pyrethrum plants in the laboratory, aphids were not recorded in the field. Pyrethrum's (E)-ß-farnesene synthase (EbFS) gene is strongly expressed in inner cortex tissue surrounding the vascular system of the aphid-preferred flower receptacle and peduncle, leading to elongated cells filled with EßF. Aphids that probe these tissues during settlement encounter and ingest plant EßF, as evidenced by the release in honeydew. These EßF concentrations in honeydew induce aphid alarm responses, suggesting an extra layer of this defense. Collectively, our data elucidate a defensive mimicry in pyrethrum flowers: the developmentally regulated and tissue-specific EßF accumulation and emission both prevents attack by aphids and recruits aphid predators as bodyguards.

Jasmone Hydroxylase, a Key Enzyme in the Synthesis of the Alcohol Moiety of Pyrethrin Insecticides.

Pyrethrins are synthesized by the plant pyrethrum (Tanacetum cinerariifolium), a chrysanthemum relative. These compounds possess efficient insecticidal properties and are not toxic to humans and most vertebrates. Pyrethrum flowers, and to a smaller extent leaves, synthesize six main types of pyrethrins, which are all esters of a monoterpenoid acid moiety and an alcohol moiety derived from jasmonic acid. Here, we identified and characterized the enzyme responsible for the conversion of jasmone, a derivative of jasmonic acid, to jasmolone. Feeding pyrethrum flowers with jasmone resulted in a 4-fold increase in the concentration of free jasmolone as well as smaller but significant proportional increases in free pyrethrolone and all three type I pyrethrins. We used floral transcriptomic data to identify cytochrome P450 genes whose expression patterns were most highly correlated with that of a key gene in pyrethrin biosynthesis, T. cinerariifolium chrysanthemyl diphosphate synthase The candidate genes were screened for jasmone hydroxylase activity through transient expression in Nicotiana benthamiana leaves fed with jasmone. The expression of only one of these candidate genes produced jasmolone; therefore, this gene was named T. cinerariifolium jasmolone hydroxylase (TcJMH) and given the CYP designation CYP71AT148. The protein encoded by TcJMH localized to the endoplasmic reticulum, and microsomal preparations from N. benthamiana leaves expressing TcJMH were capable of catalyzing the hydroxylation of jasmone to jasmolone in vitro, with a Km value of 53.9 µm TcJMH was expressed almost exclusively in trichomes of floral ovaries and was induced in leaves by jasmonate.

Production of trans-chrysanthemic acid, the monoterpene acid moiety of natural pyrethrin insecticides, in tomato fruit.

The pyrethrum plant, Tanacetum cinerariifolium (Asteraceae) synthesizes a class of compounds called pyrethrins that have strong insecticidal properties but are safe to humans. Class I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of three jasmonic-acid derived alcohols. We reconstructed the trans-chrysanthemic acid biosynthetic pathway in tomato fruits, which naturally produce high levels of the tetraterpene pigment lycopene, an isoprenoid which shares a common precursor, dimethylallyl diphosphate (DMAPP), with trans-chrysanthemic acid. trans-Chrysanthemic acid biosynthesis in tomato fruit was achieved by expressing the chrysanthemyl diphosphate synthase gene from T. cinerariifolium, encoding the enzyme that uses DMAPP to make trans-chrysanthemol, under the control of the fruit specific promoter PG, as well as an alcohol dehydrogenease (ADH) gene and aldehyde dehydrogenase (ALDH) gene from a wild tomato species, also under the control of the PG promoter. Tomato fruits expressing all three genes had a concentration of trans-chrysanthemic acid that was about 1.7-fold higher (by weight) than the levels of lycopene present in non-transgenic fruit, while the level of lycopene in the transgenic plants was reduced by 68%. Ninety seven percent of the diverted DMAPP was converted to trans-chrysanthemic acid, but 62% of this acid was further glycosylated. We conclude that the tomato fruit is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.

Coexpression Analysis Identifies Two Oxidoreductases Involved in the Biosynthesis of the Monoterpene Acid Moiety of Natural Pyrethrin Insecticides in Tanacetum cinerariifolium.

Flowers of Tanacetum cinerariifolium produce a set of compounds known collectively as pyrethrins, which are commercially important pesticides that are strongly toxic to flying insects but not to most vertebrates. A pyrethrin molecule is an ester consisting of either trans-chrysanthemic acid or its modified form, pyrethric acid, and one of three alcohols, jasmolone, pyrethrolone, and cinerolone, that appear to be derived from jasmonic acid. Chrysanthemyl diphosphate synthase (CDS), the first enzyme involved in the synthesis of trans-chrysanthemic acid, was characterized previously and its gene isolated. TcCDS produces free trans-chrysanthemol in addition to trans-chrysanthemyl diphosphate, but the enzymes responsible for the conversion of trans-chrysanthemol to the corresponding aldehyde and then to the acid have not been reported. We used an RNA sequencing-based approach and coexpression correlation analysis to identify several candidate genes encoding putative trans-chrysanthemol and trans-chrysanthemal dehydrogenases. We functionally characterized the proteins encoded by these genes using a combination of in vitro biochemical assays and heterologous expression in planta to demonstrate that TcADH2 encodes an enzyme that oxidizes trans-chrysanthemol to trans-chrysanthemal, while TcALDH1 encodes an enzyme that oxidizes trans-chrysanthemal into trans-chrysanthemic acid. Transient coexpression of TcADH2 and TcALDH1 together with TcCDS in Nicotiana benthamiana leaves results in the production of trans-chrysanthemic acid as well as several other side products. The majority (58%) of trans-chrysanthemic acid was glycosylated or otherwise modified. Overall, these data identify key steps in the biosynthesis of pyrethrins and demonstrate the feasibility of metabolic engineering to produce components of these defense compounds in a heterologous host.

Comparative transcriptome analysis reveals candidate genes for the biosynthesis of natural insecticide in Tanacetum cinerariifolium.

BACKGROUND: Pyrethrins are monoterpenoids and consist of either a chrysanthemic acid or pyrethric acid with a rethrolone moiety. Natural pyrethrins are safe and eco-friendly while possessing strong insecticidal properties. Despite such advantages of commercial value coming with the eco-friendly tag, most enzymes/genes involved in the pyrethrin biosynthesis pathway remain unidentified and uncharacterized. Since the flowers of Tanacetum cinerariifolium are rich in major pyrethrins, next generation transcriptome sequencing was undertaken to compare the flowers and the leaves of the plant de novo to identify differentially expressed transcripts and ascertain which among them might be involved in and responsible for the differential accumulation of pyrethrins in T. cinerariifolium flowers. RESULTS: In this first tissue specific transcriptome analysis of the non-model plant T. cinerariifolium, a total of 23,200,000 and 28,500,110 high quality Illumina next generation sequence reads, with a length of 101 bp, were generated for the flower and leaf tissue respectively. After functional enrichment analysis and GO based annotation using public protein databases such as UniRef, PFAM, SMART, KEGG and NR, 4443 and 8901 unigenes were identified in the flower and leaf tissue respectively. These could be assigned to 13344 KEGG pathways and the pyrethrin biosynthesis contextualized. The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was involved in the biosynthesis of acid moiety of pyrethrin and this pathway predominated in the flowers as compared to the leaves. However, enzymes related to oxylipin biosynthesis were found predominantly in the leaf tissue, which suggested that major steps of pyrethrin biosynthesis occurred in the flowers. CONCLUSIONS: Transcriptome comparison between the flower and leaf tissue of T. cinerariifolium provided an elaborate list of tissue specific transcripts that was useful in elucidating the differences in the expression of the biosynthetic pathways leading to differential presence of pyrethrin in the flowers. The information generated on genes, pathways and markers related to pyrethrin biosynthesis in this study will be helpful in enhancing the production of these useful compounds for value added breeding programs. Related proteome comparison to overlay our transcriptome comparison can generate more relevant information to better understand flower specific accumulation of secondary metabolites in general and pyrethrin accumulation in particular.

Selective regulation of pyrethrin biosynthesis by the specific blend of wound induced volatiles in Tanacetum cinerariifolium.

Natural pyrethrins are used to control household and agricultural pests, and it is of value to understand biosynthesis in Tanacetum cinerariifolium for enhanced production. We previously found that a blend of four green leaf volatiles (GLVs) and (E)-ß-farnesene emitted by T. cinerariifolium seedlings enhanced gene expressions of certain biosynthetic enzymes in unwounded seedlings; however, the extent to which such a regulation facilitates pyrethrin biosynthesis remains unknown. Here we have investigated the effects of the blend of the volatile organic compounds (VOCs) on gene expressions of seven biosynthetic enzymes. VOC treatment resulted in enhanced chrysanthemyl diphosphate synthase (CDS), chrysanthemic acid synthase (CAS), Tanacetum cinerariifolium GDSL lipase (TcGLIP) and acyl-Coenzyme A oxidase 1 (ACX1) gene expressions that reached a peak at a 12 h VOC treatment, whereas the treatment minimally influenced the expressions of other biosynthetic genes. In undifferentiated Tanacetum tissues, such VOC-induced amplification of CDS, CAS, TcGLIP and ACX1 gene expressions were markedly reduced, suggesting that a high-resolution, VOC-mediated communication is an event selective to differentiated plants.

Genetic diversity and structure of Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir./Sch./Bip., Asteraceae) within the Balkan refugium.

Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir./Sch./Bip.) is an outcrossing, perennial insecticidal plant, restricted to the eastern Adriatic coast (Mediterranean). Amplified fragment-length polymorphisms (AFLP) were used to investigate the genetic diversity and structure within and among 20 natural plant populations. The highest level of gene diversity, the number of private alleles and the frequency down-weighted marker values (DW) were found in northern Adriatic populations and gradually decreased towards the southern boundary of the species range. Genetic impoverishment of these southern populations is most likely the result of human-related activities. An analysis of molecular variance (AMOVA) indicated that most of the genetic diversity was attributed to differences among individuals within populations (85.78%), which are expected due to the outcrossing nature of the species. A Bayesian analysis of the population structure identified two dominant genetic clusters. A spatial analysis of the genetic diversity indicated that 5.6% of the genetic differentiation resulted from isolation by distance (IBD), while 12.3% of the genetic differentiation among populations followed the pattern of isolation by environmental distance (IBED). Knowledge of the genetic diversity patterns of the natural populations and the mechanism behind these patterns is required for the exploitation and possible conservation management of this endemic and economically important species.

A trichome-specific linoleate lipoxygenase expressed during pyrethrin biosynthesis in pyrethrum.

The lipid precursor alcohols of pyrethrins-jasmolone, pyrethrolone and cinerolone-have been proposed as sharing parts of the oxylipin pathway with jasmonic acid. This implies that one of the first committed steps of pyrethrin biosynthesis is catalyzed by a lipoxygenase, catalyzing the hydroperoxidation of linolenic acid at position 13. Previously, we showed that the expression and activity of chrysanthemyl diphosphate synthase (TcCDS), the enzyme catalyzing the first committed step in the biosynthesis of the acid moiety of pyrethrins, is trichome-specific and developmentally regulated in flowers. In the present study we characterized the expression pattern of 25 lipoxygenase EST contigs, and subsequently carried out the molecular cloning of two pyrethrum lipoxygenases, TcLOX1 and TcLOX2, that have a similar pattern to TcCDS. Only recombinant TcLOX1 catalyzed the peroxidation of the linolenic acid substrate. Just as TcCDS, TcLOX1, are exclusively expressed in trichomes. Phylogenetic analysis showed that the enzyme shared the highest homology with chloroplast-localized 13-type-lipoxygenases that are involved in maintaining basal levels of jasmonate.

Biosynthesis of sesquiterpene lactones in pyrethrum (Tanacetum cinerariifolium).

The daisy-like flowers of pyrethrum (Tanacetum cinerariifolium) are used to extract pyrethrins, a botanical insecticide with a long history of safe and effective use. Pyrethrum flowers also contain other potential defense compounds, particularly sesquiterpene lactones (STLs), which represent problematic allergenic residues in the extracts that are removed by the pyrethrum industry. The STLs are stored in glandular trichomes present on the pyrethrum achenes, and have been shown to be active against herbivores, micro-organisms and in the below-ground competition with other plants. Despite these reported bioactivities and industrial significance, the biosynthetic origin of pyrethrum sesquiterpene lactones remains unknown. In the present study, we show that germacratrien-12-oic acid is most likely the central precursor for all sesquiterpene lactones present in pyrethrum. The formation of the lactone ring depends on the regio- (C6 or C8) and stereo-selective (α or ß) hydroxylation of germacratrien-12-oic acid. Candidate genes implicated in three committed steps leading from farnesyl diphosphate to STL and other oxygenated derivatives of germacratrien-12-oic acid were retrieved from a pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. The diversity and distribution of sesquiterpene lactones in different tissues and the correlation with the expression of these genes are shown and discussed.