Botanical authentication of globe artichoke-containing foods: Differentiation of Cynara scolymus by a novel HRM approach.

Cynara scolymus L., known as globe artichoke, is a medicinal plant widely used in plant food supplements (PFS) and herbal infusions due to its beneficial health properties. The high demand for artichoke-containing products can lead to adulteration practices. In this work, a real-time polymerase chain reaction (PCR) system coupled to high-resolution melting (HRM) analysis was proposed to differentiate C. scolymus from other Cynara species. Hence, a Cynara-specific real-time PCR assay was successfully developed with high analytical performance, achieving a sensitivity of 0.4 pg of globe artichoke DNA. HRM analysis enabled the discrimination of C. scolymus, with a high level of confidence (>98%), corroborating sequencing data. Application results to artichoke-containing PFS and mixed herbal infusions allowed confirming the presence of C. scolymus in 38% of the samples, suggesting the substitution/mislabelling of globe artichoke in 2 samples and the need for further efforts to increase DNA amplifiability of PFS.

Towards a Cardoon (Cynara cardunculus var. altilis)-Based Biorefinery: A Case Study of Improved Cell Cultures via Genetic Modulation of the Phenylpropanoid Pathway.

Cultivated cardoon (Cynara cardunculus var. altilis L.) is a promising candidate species for the development of plant cell cultures suitable for large-scale biomass production and recovery of nutraceuticals. We set up a protocol for Agrobacterium tumefaciens-mediated transformation, which can be used for the improvement of cardoon cell cultures in a frame of biorefinery. As high lignin content determines lower saccharification yields for the biomass, we opted for a biotechnological approach, with the purpose of reducing lignin content; we generated transgenic lines overexpressing the Arabidopsis thaliana MYB4 transcription factor, a known repressor of lignin/flavonoid biosynthesis. Here, we report a comprehensive characterization, including metabolic and transcriptomic analyses of AtMYB4 overexpression cardoon lines, in comparison to wild type, underlining favorable traits for their use in biorefinery. Among these, the improved accessibility of the lignocellulosic biomass to degrading enzymes due to depletion of lignin content, the unexpected increased growth rates, and the valuable nutraceutical profiles, in particular for hydroxycinnamic/caffeoylquinic and fatty acids profiles.

Genetic diversity and population structure of Cynara cardunculus L. in southern Portugal.

Cynara cardunculus L. is a cardoon species native to the Mediterranean region, which is composed of three botanical taxa, each having distinct biological characteristics. The aim of this study was to examine wild populations of C. cardunculus established in Portugal, in order to determine their genetic diversity, geographic distribution, and population structure. Based on SSR markers, 121 individuals of C. cardunculus from 17 wild populations of the Portuguese Alentejo region were identified and analysed. Ten SSRs were found to be efficient markers in the genetic diversity analysis. The total number of alleles ranged from 9 to 17 per locus. The expected and observed means in heterozygosity, by population analysed, were 0.591 and 0.577, respectively. The wild population exhibited a high level of genetic diversity at the species level. The highest proportion of genetic variation was identified within a geographic group, while variation was lower among groups. Geographic areas having highest genetic diversity were identified in Alvito, Herdade da Abóboda, Herdade da Revilheira and Herdade de São Romão populations. Moreover, significant genetic differentiation existed between wild populations from North-Alentejo geographic locations (Arraiolos, Évora, Monte da Chaminé) and Centro Hortofrutícola, compared with other populations. This study reports genetic diversity among a representative number of wild populations and genotypes of C. cardunculus from Portugal. These results will provide valuable information towards future management of C. cardunculus germplasm.

Hybrid transcriptome sequencing approach improved assembly and gene annotation in Cynara cardunculus (L.).

BACKGROUND: The investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection. In contrast, long-reads sequencing techniques revealed their potential to generate complete transcript assemblies even when a reference genome is lacking. Cynara cardunculus var. altilis (DC) (cultivated cardoon) is a perennial hardy crop adapted to dry environments with many industrial and nutraceutical applications due to the richness of secondary metabolites mostly produced in flower heads. The investigation of this species benefited from the recent release of a draft genome, but the transcriptome profile during the capitula formation still remains unexplored. In the present study we show a transcriptome analysis of vegetative and inflorescence organs of cultivated cardoon through a novel hybrid RNA-seq assembly approach utilizing both long and short RNA-seq reads. RESULTS: The inclusion of a single Nanopore flow-cell output in a hybrid sequencing approach determined an increase of 15% complete assembled genes and 18% transcript isoforms respect to short reads alone. Among 25,463 assembled unigenes, we identified 578 new genes and updated 13,039 gene models, 11,169 of which were alternatively spliced isoforms. During capitulum development, 3424 genes were differentially expressed and approximately two-thirds were identified as transcription factors including bHLH, MYB, NAC, C2H2 and MADS-box which were highly expressed especially after capitulum opening. We also show the expression dynamics of key genes involved in the production of valuable secondary metabolites of which capitulum is rich such as phenylpropanoids, flavonoids and sesquiterpene lactones. Most of their biosynthetic genes were strongly transcribed in the flower heads with alternative isoforms exhibiting differentially expression levels across the tissues. CONCLUSIONS: This novel hybrid sequencing approach allowed to improve the transcriptome assembly, to update more than half of annotated genes and to identify many novel genes and different alternatively spliced isoforms. This study provides new insights on the flowering cycle in an Asteraceae plant, a valuable resource for plant biology and breeding in Cynara and an effective method for improving gene annotation.

Influence of Genotype and Harvest Time on the Cynara cardunculus L. Sesquiterpene Lactone Profile.

The excessive and inappropriate application of herbicides has caused environmental pollution. The use of allelochemicals as bioherbicides could provide a solution to this problem. The allelopathic activity of Cynara cardunculus L. has been studied previously, and sesquiterpene lactones (STLs) were identified as the most relevant allelochemicals. The goal of the study reported here was to investigate the effect of six genotypes and three harvest times on the qualitative and quantitative composition of STLs in C. cardunculus leaves through a new ultra-high-performance liquid chromatography-tandem mass spectrometry analysis method and, thus, the effect on phytotoxicity. Overall, wild cardoon contained the highest levels of STLs of the three botanical varieties studied. Nevertheless, climatic conditions had a marked influence on the presence of STLs among the six genotypes, which was higher in the April harvest. Cynaropicrin was the most abundant STL detected. A close relationship was found between the STL profiles and the allelopathic activity, expressed as inhibition of wheat coleoptile elongation. The data provide a new and important contribution to our understanding of C. cardunculus allelopathy.

Bioactivities, chemical composition and nutritional value of Cynara cardunculus L. seeds.

In the present study, the nutritional value, bioactive properties, and chemical composition of various cardoon (Cynara cardunculus L.) genotypes cultivated in central Greece were investigated. The results demonstrated that Cynara seeds are a good source of fat and protein, while they also contain considerable amounts of K, Mg, and Fe and low amount of Na. Sucrose, oxalic acid, and α-tocopherol were the only free sugar, organic acid, and tocopherol isoform respectively, found among the studied genotypes. The most abundant fatty acids were linoleic, oleic and palmitic acid, while PUFA was the most abundant fatty acid class. All the tested seeds contained only two phenolic compounds, namely 5-O-caffeoylquinic acid and 3,5-O-caffeoylquinic acid, while significant antioxidant activities and cytotoxicity against tumor cell lines and antimicrobial effects were also observed. In conclusion, cardoon seed extracts could be exploited in the food and pharmaceutical industries as alternative sources of natural compounds with bioactive properties.

Reinterpretation of an endangered taxon based on integrative taxonomy: The case of Cynara baetica (Compositae).

The Strait of Gibraltar, the gateway between the Atlantic Ocean and the Mediterranean Sea, has a convulsive geological history, with recurring closing and opening events since the late Miocene. As a consequence, this region has played a major role in the evolutionary history of many species. Cynara baetica (Compositae) is a diploid perennial herb distributed in both sides of this strait. It is currently subdivided into two subspecies: C. baetica subsp. baetica for the Spanish populations, and C. baetica subsp. maroccana for the Moroccan ones. Following three different approximations of species delimitation, including phylogenetic and population genetic analyses (based on three AFLP primer combinations and two intergenic spacers of cpDNA), ecological niche modeling (ENM) and morphological studies, this taxon is investigated and reinterpreted. The results obtained showed a clear genetic, morphological and ecological differentiation between the two taxa and the important role played by the Strait of Gibraltar as a geographical barrier. Based on this evidence, the current taxonomic treatment is modified (both taxa should recover their specific rank) and specific conservation guidelines are proposed for the newly delimited taxa.

Genotyping-by-sequencing highlights patterns of genetic structure and domestication in artichoke and cardoon.

Exploiting the biodiversity of crops and their wild relatives is fundamental for maintaining and increasing food security. The species Cynara cardunculus includes three taxa: the globe artichoke, one of the most important Mediterranean vegetables, the leafy cardoon, and the wild cardoon. In this study, genotyping by sequencing (GBS) was successfully applied to reveal thousands of polymorphisms in a C. cardunculus germplasm collection, including 65 globe artichoke, 9 leafy cardoon, and 21 wild cardoon samples. The collection showed a strong population structure at K = 2, separating the globe artichoke from the leafy and wild cardoon. At higher K values, further substructures were observed, in which the wild cardoon was separated from the leafy cardoon, and the latter included the Spanish wild cardoons, while the wild sample from Portugal was admixed. Moreover, subpopulations within the globe artichoke set were highlighted. Structure analysis restricted to the globe artichoke dataset pointed out genetic differentiation between the ˝Catanesi˝ typology and all the other samples (K = 2). At higher values of K, the separation of the ˝Catanesi˝ group still held true, and green headed landraces from Apulia region, Italy (˝Green Apulian˝) formed a distinct subpopulation. ˝Romaneschi˝ artichoke types fell in a variable group with admixed samples, indicating that they should not be considered as a genetically uniform typology. The results of principal component analysis and Neighbor-Joining hierarchical clustering were consistent with structure results, and in addition provided a measure of genetic relationships among individual genotypes. Both analyses attributed the wild material from Spain and Portugal to the cultivated cardoon group, supporting the idea that this might be indeed a feral form of the leafy cardoon. Different reproductive habit and possibly selective pressure led to a slower LD decay in artichoke compared to cardoon. Genotyping by sequencing has proven a reliable methodology to obtain valuable SNPs and assess population genetics in C. cardunculus.

Phenolic profile and bioactivity of cardoon (Cynara cardunculus L.) inflorescence parts: Selecting the best genotype for food applications.

This study was designed to characterize the phenolic profile and bioactivity of hydroalcoholic extracts from different cardoon (Cynara cardunculus L.) genotypes. The analytical work focussed on the inflorescence stigmas, owing to their application in cheese production. Nevertheless, other parts were concomitantly analysed aiming to define their possible use in related applications. Phenolic profiles obtained by LC-DAD-ESI/MSn showed significant differences among different cardoon genotypes, but apigenin and caffeoylquinic acid derivatives were generally the major molecules in all samples. Genotype influence has also been observed in relation to the antioxidant and antibacterial activities. Besides their strong antioxidant activity, the cardoon inflorescences showed satisfactory antibacterial activity, namely against Gram-positive strains, with particularly low MIC in Listeria monocytogenes. Overall, it was possible to identify the cardoon genotype (within the selected ones) providing the best standardized ingredient (stigma) with considerable added-value to be included in the process of cheese making.

Selected Cardoon (Cynara cardunculus L.) Genotypes Suitable for PDO Cheeses in Mediterranean Regions.

Cardoon flower extract is a traditional and exclusive rennet used for some PDO cheeses in several Mediterranean regions, due to its extremely high concentration in cardosins. In this preliminary study, six individual cardoon genotypes (1M - 6M) were selected because they revealed a wide and consistent diversity of total and specific cardosin concentrations in flowers. During three growing seasons, the stability of 12 biochemical characteristics of flower extracts and 26 plant morphological descriptors was confirmed. Surprisingly, the cardosin profiles of each genotype, based on four main groups A0, A1, A and B, were stable during the annual flower harvesting period and over all three years using ion-exchange chromatography and native-PAGE electrophoresis. This knowledge will allow an improvement in the quality and standardization of cardosin profiles from cardoon flowers used for cheese production and other innovative applications. The results obtained are promising for the development of a plant breeding program based on biochemical and morphological characteristics in order to obtain the most adapted plant architecture for combined purposes related to specific cardosins composition, flower and plant biomass production, and ease of harvesting.

Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase (hqt1) gene from Cynara cardunculus var. scolymus in Nicotiana tabacum cell cultures.

Chlorogenic acids (CGAs) are phenolic compounds biosynthesized in the phenylpropanoid pathway, with hydroxycinnamoyl quinate hydroxycinnamoyltransferase (HQT) as the key enzyme. Variation of CGAs has been noted in different plants, with globe artichoke (Cynara cardunculus var. scolymus L.) producing high amounts and a diverse spectrum of CGAs in its leaves. In the current study, the effect of overexpression of the hqt1 transgene from globe artichoke in tobacco was evaluated at the metabolome level. Here, metabolomic approaches based on ultra-high performance liquid chromatography coupled to mass spectrometry, together with chemometric models such as principal component analysis and orthogonal partial least square discriminant analysis, were employed to evaluate altered metabolic changes due to hqt1 overexpression. CGA profiles (caffeoylquinic acids: 3-CQA, 4-CQA and 5-CQA; p-coumaroylquinic acids: 4-pCoQA and 5-pCoQA; and 4,5-di-caffeoylquinic acid) of transgenic tobacco cell cultures were detected at lower concentrations than in the wild type. Interestingly, the cells were found to rather accumulate, as an unintended effect, abscisic acid - and benzoic acid derivatives. The results suggest that insertion of hqt1 in tobacco, and overexpression in undifferentiated cells, led to rechannelling of the phenylpropanoid pathway to accumulate benzoic acids. These findings proved to be contrary to the results shown elsewhere in leaf tissues, thus indicating differential metabolic control and regulation in the undifferentiated cell culture system.

Haplotype analysis of the germacrene A synthase gene and association with cynaropicrin content and biological activities in Cynara cardunculus.

Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.

Genome reconstruction in Cynara cardunculus taxa gains access to chromosome-scale DNA variation.

The genome sequence of globe artichoke (Cynara cardunculus L. var. scolymus, 2n = 2x = 34) is now available for use. A survey of C. cardunculus genetic resources is essential for understanding the evolution of the species, carrying out genetic studies and for application of breeding strategies. We report on the resequencing analyses (~35×) of four globe artichoke genotypes, representative of the core varietal types, as well as a genotype of the related taxa cultivated cardoon. The genomes were reconstructed at a chromosomal scale and structurally/functionally annotated. Gene prediction indicated a similar number of genes, while distinctive variations in miRNAs and resistance gene analogues (RGAs) were detected. Overall, 23,5 M SNP/indel were discovered (range 6,34 M -14,50 M). The impact of some missense SNPs on the biological functions of genes involved in the biosynthesis of phenylpropanoid and sesquiterpene lactone secondary metabolites was predicted. The identified variants contribute to infer on globe artichoke domestication of the different varietal types, and represent key tools for dissecting the path from sequence variation to phenotype. The new genomic sequences are fully searchable through independent Jbrowse interfaces (www.artichokegenome.unito.it), which allow the analysis of collinearity and the discovery of genomic variants, thus representing a one-stop resource for C. cardunculus genomics.

Genetic diversity and accession structure in European Cynara cardunculus collections.

Understanding the distribution of genetic variations and accession structures is an important factor for managing genetic resources, but also for using proper germplasm in association map analyses and breeding programs. The globe artichoke is the fourth most important horticultural crop in Europe. Here, we report the results of a molecular analysis of a collection including globe artichoke and leafy cardoon germplasm present in the Italian, French and Spanish gene banks. The aims of this study were to: (i) assess the diversity present in European collections, (ii) determine the population structure, (iii) measure the genetic distance between accessions; (iv) cluster the accessions; (v) properly distinguish accessions present in the different national collections carrying the same name; and (vi) understand the diversity distribution in relation to the gene bank and the geographic origin of the germplasm. A total of 556 individuals grouped into 174 accessions of distinct typologies were analyzed by different types of molecular markers, i.e. dominant (ISSR and AFLP) and co-dominant (SSR). The data of the two crops (globe artichoke and leafy cardoon) were analyzed jointly and separately to compute, among other aims, the gene diversity, heterozygosity (He, Ho), fixation indexes, AMOVA, genetic distance and structure. The findings underline the huge diversity present in the analyzed material, and the existence of alleles that are able to discriminate among accessions. The accessions were clustered not only on the basis of their typology, but also on the basis of the gene bank they come from. Probably, the environmental conditions of the different field gene banks affected germplasm conservation. These outcomes will be useful in plant breeding to select accessions and to fingerprint varieties. Moreover, the results highlight the particular attention that should be paid to the method used to conserve the Cynara cardunculus germplasm and suggest to the preference of using accessions from different gene banks to run an association map.

Impact of novel SNPs identified in Cynara cardunculus genes on functionality of proteins regulating phenylpropanoid pathway and their association with biological activities.

BACKGROUND: Cynara cardunculus L. offers a natural source of phenolic compounds with the predominant molecule being chlorogenic acid. Chlorogenic acid is gaining interest due to its involvement in various biological properties such as, antibacterial, antifungal, antioxidant, hepatoprotective, and anticarcinogenic activities. RESULTS: In this work we screened a Cynara cardunculus collection for new allelic variants in key genes involved in the chlorogenic acid biosynthesis pathway. The target genes encode p-coumaroyl ester 3'-hydroxylase (C3'H) and hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (HQT), both participating in the synthesis of chlorogenic acid. Using high-resolution melting, the C3'H gene proved to be highly conserved with only 4 haplotypes while, for HQT, 17 haplotypes were identified de novo. The putative influence of the identified polymorphisms in C3'H and HQT proteins was further evaluated using bioinformatics tools. We could identify some polymorphisms that may lead to protein conformational changes. Chlorogenic acid content, antioxidant and antithrombin activities were also evaluated in Cc leaf extracts and an association analysis was performed to assess a putative correlation between these traits and the identified polymorphisms. CONCLUSION: In this work we identified allelic variants with putative impact on C3'H and HQT proteins which are significantly associated with chlorogenic acid content and antioxidant activity. Further study of these alleles should be explored to assess putative relevance as genetic markers correlating with Cynara cardunculus biological properties with further confirmation by functional analysis.

Spatial genetic structure in wild cardoon, the ancestor of cultivated globe artichoke: Limited gene flow, fragmentation and population history.

Nuclear and chloroplast markers and phenotypic characters were integrated to analyse the population genetic structure of wild cardoon, Cynara cardunculus var. sylvestris, the ancestor of cultivated globe artichoke, Cynara cardunculus var. scolymus on the island of Sardinia, Italy. The spatial scale ranged from a few metres to ∼200km. Wild cardoon appears to be genetically fragmented, with significant genetic divergence at various scales, indicating that gene flow is insufficient to counterbalance the effects of genetic drift or founder effects. Divergence between populations was higher for chloroplast (40%) than for nuclear markers (15%), suggesting that gene flow via seed was lower than via pollen. Two main genetic groups were detected; these correlated with differences in flowering time, capitula size, glossiness, and anthocyanin pigmentation. A complex population structure of wild cardoon emerged over small spatial scales, likely resulting from the interplay between gene dispersal, colonisation history and selective forces. Indeed, Sardinia appears to be a 'hybrid zone' of different gene pools. The island has unique diverse germplasm that has originated from hybridisation among different gene pools. The sampling of seeds from a few plants but from many sites is suggested as the best strategy to harvest the genetic diversity of wild cardoon.

Development of chloroplast genomic resources for Cynara.

In this study, new chloroplast (cp) resources were developed for the genus Cynara, using whole cp genomes from 20 genotypes, by means of high-throughput sequencing technologies. Our target species included seven globe artichokes, two cultivated cardoons, eight wild artichokes, and three other wild Cynara species (C. baetica, C. cornigera and C. syriaca). One complete cp genome was isolated using short reads from a whole-genome sequencing project, while the others were obtained by means of long-range PCR, for which primer pairs are provided here. A de novo assembly strategy combined with a reference-based assembly allowed us to reconstruct each cp genome. Comparative analyses among the newly sequenced genotypes and two additional Cynara cp genomes ('Brindisino' artichoke and C. humilis) retrieved from public databases revealed 126 parsimony informative characters and 258 singletons in Cynara, for a total of 384 variable characters. Thirty-nine SSR loci and 34 other INDEL events were detected. After data analysis, 37 primer pairs for SSR amplification were designed, and these molecular markers were subsequently validated in our Cynara genotypes. Phylogenetic analysis based on all cp variable characters provided the best resolution when compared to what was observed using only parsimony informative characters, or only short 'variable' cp regions. The evaluation of the molecular resources obtained from this study led us to support the 'super-barcode' theory and consider the total cp sequence of Cynara as a reliable and valuable molecular marker for exploring species diversity and examining variation below the species level.

The complete chloroplast genome of Cynara humilis.

The complete chloroplast genome of the wild thistle Cynara humilis L. (Asteraceae) is presented in this study. The genome is 152,585 bp in length and has a quadripartite structure composed by a large single-copy (LSC) of 83,622 bp, a small single-copy (SSC) of 18,651 bp and two inverted repeats (IRb/a) of 25,156 bp each. The GC content corresponds to 37.7%. The amount of unique genes is 114, in which 17 are duplicated in the IRs, for a total of 131 genes. A maximum parsimony phylogenetic analysis revealed that C. humilis chloroplast genome is closely related to that of the globe artichoke within the Carduoideae subfamily.

Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture.

Different sheep and goat cheeses with world-renowned excellence are produced using aqueous extracts of Cynara cardunculus flowers as coagulants. However, the use of this vegetable rennet is mostly limited to artisanal scale production, and no effective solutions to large-scale industrial applications have been reported so far. In this sense, the development of a synthetic rennet based on the most abundant cardoon milk-clotting enzymes (cardosins) would emerge as a solution for scalability of production and for application of these proteases as alternative rennets in dairy industry. In this work, we report the development of a new cardosin B-derived rennet produced in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. Using a stepwise optimization strategy-consisting of culture media screening, complemented with a protein engineering approach with removal of the plant-specific domain, and a codon optimization step-we successfully improved cardosin B production yield (35×) in K. lactis. We demonstrated that the secreted enzyme displays similar proteolytic properties, such as casein digestion profiles as well as optimum pH (pH 4.5) and temperature (40 °C), with those of native cardosin B. From this optimization process resulted the rennet preparation Vegetable Rennet (VRen), requiring no downstream protein purification steps. The effectiveness of VRen in cheese production was demonstrated by manufacturing sheep, goat, and cow cheeses. Interestingly, the use of VRen resulted in a higher cheese yield for all three types of cheese when compared with synthetic chymosin. Altogether, these results clearly position VRen as an alternative/innovative coagulant for the cheese-making industry.

Cytochrome P450s from Cynara cardunculus L. CYP71AV9 and CYP71BL5, catalyze distinct hydroxylations in the sesquiterpene lactone biosynthetic pathway.

Cynara cardunculus (Asteraceae) is a cross pollinated perennial crop which includes the two cultivated taxa globe artichoke and cultivated cardoon. The leaves of these plants contain high concentrations of sesquiterpene lactones (STLs) among which cynaropicrin is the most represented, and has recently attracted attention because of its therapeutic potential as anti-tumor and anti-photoaging agent. Costunolide is considered the common precursor of the STLs and three enzymes are involved in its biosynthetic pathway: i.e. the germacrene A synthase (GAS), the germacrene A oxidase (GAO) and the costunolide synthase (COS). Here we report on the isolation of two P450 genes, (i.e. CYP71AV9 and CYP71BL5), in a set of ∼19,000 C. cardunculus unigenes, and their functional characterization in yeast and in planta. The metabolite analyses revealed that the co-expression of CYP71AV9 together with GAS resulted in the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid in yeast. The co-expression of CYP71BL5 and CYP71AV9 with GAS led to biosynthesis of the free costunolide in yeast and costunolide conjugates in Nicotiana benthamiana, demonstrating their involvement in STL biosynthesis as GAO and COS enzymes. The substrate specificity of CYP71AV9 was investigated by testing its ability to convert amorpha-4,11-diene, (+)-germacrene D and cascarilladiene to their oxidized products when co-expressed in yeast with the corresponding terpene synthases.