The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway
© Comino et al; licensee BioMed Central Ltd. 2009
Received: 25 September 2008
Accepted: 18 March 2009
Published: 18 March 2009
The leaves of globe artichoke and cultivated cardoon (Cynara cardunculus L.) have significant pharmaceutical properties, which mainly result from their high content of polyphenolic compounds such as monocaffeoylquinic and dicaffeoylquinic acid (DCQ), and a range of flavonoid compounds.
Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase (HQT) encoding genes have been isolated from both globe artichoke and cultivated cardoon (GenBank accessions DQ915589 and DQ915590, respectively) using CODEHOP and PCR-RACE. A phylogenetic analysis revealed that their sequences belong to one of the major acyltransferase groups (anthranilate N-hydroxycinnamoyl/benzoyltransferase). The heterologous expression of globe artichoke HQT in E. coli showed that this enzyme can catalyze the esterification of quinic acid with caffeoyl-CoA or p-coumaroyl-CoA to generate, respectively, chlorogenic acid (CGA) and p-coumaroyl quinate. Real time PCR experiments demonstrated an increase in the expression level of HQT in UV-C treated leaves, and established a correlation between the synthesis of phenolic acids and protection against damage due to abiotic stress. The HQT gene, together with a gene encoding hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT) previously isolated from globe artichoke, have been incorporated within the developing globe artichoke linkage maps.
A novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated, characterized and mapped. This is a good basis for our effort to understand the genetic basis of phenylpropanoid (PP) biosynthesis in C. cardunculus.
Cynara cardunculus L. (2n = 2x = 34) is an allogamous species native to the Mediterranean basin, belonging to the family Asteraceae, order Asterales. The species includes three subspecies: the globe artichoke (var. scolymus L.), which is grown for its edible immature inflorescence; the cultivated cardoon (var. altilis DC.), which produces fleshy stalks; and their common ancestor, the wild cardoon (var. sylvestris (Lamk) Fiori) [1–3]. Leaf extracts contain molecules of some pharmaceutical interest, including antibacterial [4–7] antioxidative [8, 9] anti-HIV [10–12], hepatoprotective, choleretic , cholesterol biosynthesis inhibitory [14, 15] and anticancer  activities. Many of these properties rely on specific phenylpropanoids (PPs), particularly 5-caffeoylquinic acid (chlorogenic acid, CGA) and di-caffeoylquinic (DCQ) acids, along with various flavonoid compounds [17, 18]. The level and composition of the PP pool can vary considerably between organisms, tissues, developmental stages and in response to environmental conditions [19, 20]. PP metabolism is induced by biotic and abiotic stresses such as wounding, UV-irradiation and pathogen attack [21, 22]. Recently, Moglia et al.  have established that UV-C radiation enhances the level of caffeoylquinic acid in the globe artichoke.
Linkage maps, created for genes in biosynthetic pathways in several species, can be used to locate known genes of a pathway within a specific genomic region. [33, 34]. The presence of allelic variation at the sequence level in genes of known biochemical functional is useful for candidate gene approaches . Genetic maps of globe artichoke  have been based on observed segregation behaviour in an F1 population formed by the intercrossing of the two contrasting varieties 'Romanesco C3' (a late-maturing, non-spiny type) and 'Spinoso di Palermo' (an early-maturing spiny type).
Here, we report the isolation of the cDNA of a novel acyltransferase involved in C. cardunculus PP biosynthesis, and assess its leaf expression level as induced by UV-C irradiation. We also derive the map location of this gene, along with that of the HCT gene described by Comino et al. .
Isolation and cloning of a full length HQT cDNA of globe artichoke and cardoon
Heterologous expression of globe artichoke HQT in E. coliand enzyme assays
Each reaction product was identified by comparing its retention time and absorbance spectrum with authentic samples or isolated compounds previously characterized. The ability of the isolated acyltransferases to catalyse the reverse reaction (i.e. the production of caffeoyl-CoA from CGA) was also successfully achieved, as has been described in other systems [24, 28, 42]. Caffeoyl-CoA was detected when CGA was incubated with Coenzyme A in the presence of the recombinant protein (Fig. 4), whereas no metabolic product was detected from cultures carrying an empty plasmid.
In order to assess the involvement in the response to UV-C irradiation, the expression levels of HQT and HCT were analysed by real-time PCR. Based on normalized levels (using actin as an internal standard), it was clear that UV-C treatment induced a significant increase in transcription (12.3 ± 1.8 fold for HCT and 4.4 ± 0.7 fold for HQT). Comparison between the standard curves for each enzyme revealed a correlation coefficient of > 0.98 and an efficiency (slope of the curve) > 0.90 (data not shown).
Model, expected and observed segregation ratios of SNPs developed from HQT and HCT genes in the F1 progeny.
(Female × Male)
Expected ratios and F1 plant genotypes
ab × ab
1: 2: 1
(aa: ab: bb)
ab × aa
Only the female parent was heterozygous at HCTsnp97, delivering a segregation ratio of 1:1 with no significant distortion (Table 1, Fig. 6). As a result, the HCT gene could only be located on the maternal map, where it maps to LG9, separated by 3 cM from the AFLP locus p12/m61-04 and by 8 cM from the SSAP locus cyre5/m47-02 (Fig. 7b). A further six markers are present on this 58.4 cM LG, including one SSR (CELMS-10), two M-AFLPs (polyGT and polyGA) and three AFLPs. The marker density is 7.3 cM (range 1.6–7.7), with two gaps of > 10 cM.
Plants synthesize a variety of secondary metabolites, which function as UV protectants, phytoalexins, flower pigments, signalling molecules and building blocks for lignin. Some have significance in the area of human health, both as 'phytomedicines', which target specific health problems, and/or as 'nutraceuticals', which provide long term nutritional benefit . Particular plant PPs have been associated with anti-oxidant, estrogen-like and vasodilatory activity, while others have proven anti-inflammatory and anti-cancer chemopreventive action [29, 44–48]
CGA is the most widespread plant PP. Progress is being made in relation to the definition of its biosynthetic pathway, with the characterisation of two acyltransferases (HCT,  and HQT, ) able to synthesize p-coumaroylshikimate and p-coumaroyl quinate esters and a cytochrome P450 p-coumaroyl ester 3'-hydroxylase (C3'H) from a p-coumaroyl ester substrate [49, 50].
The major phenolic compounds present in the leaves of the globe artichoke are the DCQs, and their precursor CGA. Although there is no firm proof as yet that DCQ originates from CGA, the structural similarity of the two molecules makes this rather likely. A globe artichoke acyltransferase involved in PP synthesis responded to both p-coumaroyl-CoA and caffeoyl-CoA esters as acyl donors .
In the present study, we have described C. cardunculus sequences carrying peptide motifs characteristic of the plant acyltransferase family. These sequences cluster within the N-hydroxycinnamoyl/benzoyltransferase group  and are closely related to their tobacco and tomato orthologues. The hydroxycinnamoyltransferase activity of the enzyme and its involvement in PP biosynthesis have been confirmed by heterologous expression assays, which showed that it can use either p-coumaroyl-CoA or caffeoyl-CoA esters as an acyl donor, and can use quinic acid as an acceptor. As the HQT gene product failed to utilize shikimic acid, we believe that it is involved in the transesterification of caffeoyl-CoA and quinic acid, a reaction which occurs in the first route of CGA biosynthesis, but also at the end of the third pathway, following the action of HCT and C3'H resulting in the formation caffeoyl-CoA.
PP metabolism can be induced by the application of abiotic stresses [21, 52] and it has been shown that PP leaf content of globe artichoke mostly responds to UV-C irradiation, as compared to other treatments such as methyljasmonate and salicylate that are inactive . Here, we have investigated the effect of UV-C irradiation on the transcription level of the HCT and HQT genes involved in the caffeoylquinic acid pathway. The transcription of both genes was induced by UV-C, suggesting their involvement in the higher production of PPs observed as the response to this stress. Previous work on globe artichoke demonstrated that UV-C application led to large increases of leaf DCQs whereas no significant effect was observed on CGA . On the basis of our data this might be a consequence of the rapid conversion of CGA to DCQs as by means of an unknown downstream enzymatic step. Indeed the involvement of the HQT gene in the profile of phenolic acids accumulated can influence the kind of response to the UV stress as reported in a previous study on tomato by Clè et al. .
The genetic mapping of biosynthetic pathway genes of known biochemical function can help unravel the complexity of plant secondary metabolism. The precision of both marker order and inter-marker distances on LG5 and LG9 have been improved with the integration of the HQT and HCT genes. The former increased the number of bridge markers on LG5, and reduced some large gaps (of 10 cM and 8 cM) affecting the female and the male LGs (Fig. 7a). Its incorporation has caused some readjustment in the marker orders and inter-marker distances determined previously . Thus in the female LG, the order of CELMS24 and e38/m47-01 was inverted, as were those of CELMS-24, e35/m62-16 and pGA/e33-02, p13/m60-05 on the male LG. The placement of the HCT gene on female LG9 did not increase the number of bridge markers, nor did it affect marker order. However, it did succeed in filling a large (13 cM) gap, and in reducing the mean inter-marker distance. Increasing marker density by the addition of genes to a map can be accomplished via the exploitation of mapping populations which segregate for traits and markers in common across the populations [53, 54]. We are currently constructing further genetic maps based on combinations between 'Romanesco C3' and either cultivated or wild cardoon accessions, primarily as a means of initiating comparative QTL mapping. Within gene markers, such as the ones described here for the HCT and HQT genes, are particularly suitable for general mapping, and should prove useful as anchor points among diverse populations.
A novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated and characterized. Its activity and involvement in CGA biosynthesis have been confirmed by heterologous expression assays, demonstrating that it can use either p-coumaroyl-CoA or caffeoyl-CoA as an acyl donor, and quinic acid as an acceptor. We previously observed that the PP metabolism can be induced by UV-C irradiation, whose effect on the transcription level of the HCT and HQT genes has been investigated. The HQT as well as HCT genes have been located in our previously developed globe artichoke genetic maps; the linkage analyses of genes having known biochemical function can help elucidate the complexity of plant secondary metabolism.
This work is a further contribution in the understanding of the genetic basis of phenylpropanoid (PP) biosynthesis in C. cardunculus; our future research activity will be focused on the analysis of the expression in vivo of both HQT and HCT, as well as on isolating further acyltransferases involved in the phenylpropanoid pathway of the species.
Plant material and RNA extraction
Leaves of globe artichoke, and cultivated cardoon were collected from experimental fields in Scalenghe, Torino (Italy).
Total RNA was extracted from approximately 100 mg fresh tissue using the "Trizol" reagent (Invitrogen, USA), following the manufacturer's instructions. Final RNA concentration was determined by spectrophotometry, and its integrity was assessed by electrophoresis in 1% (w/v) formaldehyde-agarose gel .
Isolation and cloning of full length cDNA of globe artichoke and cardoon
Oligonucleotide sequences used to study HQT gene in C. cardunculus.
Heterologous expression of globe artichoke HQT in E. coliand enzymatic assays
The globe artichoke HQT open reading frame (ORF) was amplified using HQT-For and HQT-Rev primers (Table 2), which contain additional restriction sites, respectively, NdeI (5'-end) and BamHI (3'-end). In a first step the amplified fragment was digested with NdeI and partially with BamHI (15 min at 37°C with 1 unit of BamHI). This partial second digestion being necessary because of the presence of an internal BamHI restriction site. The restricted PCR fragment was finally ligated into the cloning site of Nde I – Bam HI digested pET3a plasmid (Novagen, USA). The resulting recombinant pET3a-HQT plasmid was transferred into E. coli strain BL21(DE)pLysE, and grown on a selective medium (LB in presence of 50 mg/l ampicillin and 34 mg/l chloramphenicol). Individual colonies were transferred to 4 ml LB medium and incubated for 12 h at 37°C. Two ml of this bacterial preculture were transferred in 50 ml LB medium and grown for 3 h at 28°C prior to an isopropyl-β-D-thiogalactopyranoside (IPTG) induction (final concentration of 1 mM) during 8 h at 28°C. After centrifugation for 10 min at 5000 g, the pellet was resuspended in 1 ml of phosphate-buffered saline pH 7.5 and lysed by three cycles of freezing (in liquid nitrogen) and thawing (at 37°C), followed by three bursts of 30 s sonication on ice. Sonicated cells were centrifuged at 4°C and 14,000 g for 5 min, and the supernatant was assayed for HQT activity, and profiled by SDS-PAGE (10% resolving gel, 5% stacking gel) using Coomassie brilliant blue staining . Negative controls used comparable preparations harbouring an empty vector.
The recombinant proteins were used for enzyme assays. CGA was purchased from Sigma-Aldrich (Germany), and quinic acid from Fluka (Switzerland). CoA esters (substrates) were synthesised using the procedure proposed by Beuerle and Pichersky . 4CL enzyme was kindly provided by Dr. Douglas (University of British Columbia, Vancouver).
The 20 μl reaction mixture contained 100 mM phosphate buffer (pH 7.5), 1 mM dithiothreitol, between 50 ng and 1 μg of protein, and the various substrates (p-coumaroyl-CoA, caffeoyl-CoA, quinic acid and shikimic acid) at concentrations ranging from 0.1 mM to 5 mM. The reverse reaction, i.e. conversion of chlorogenic acid and CoA-SH (Sigma) into caffeoyl-CoA, was tested as follow: 50 ng to 1 μg protein was incubated in presence of 1 mM dithiothreitol, 100 μM of chlorogenic acid and 100 μM CoA. Reactions were incubated at 30°C for 30 min, stopped by the addition of 20 μl of acetonitrile/HCl (99:1) and products were analysed by reverse-phase HPLC on a C18 column (LiChroCART 125-4, Merck). The two solvents used are 90% H2O, 9.9% CH3CN, 0.1% HCOOH and 80% CH3CN, 19.9% H2O, 0.1% CH3COOH. The percentage of the latter reached the 60% over a 15 min run time, and 100% after 28 min.
Real-time PCR experiments
For real-time PCR assays, UV-C stress experiments are performed as described in Moglia et al., . Total RNA was extracted as described above. The first-strand cDNA was synthesised using iScript cDNA Synthesis Kit (Biorad), following manufacturer's instructions.
Primers (HCT-ForRT, HCT-RevRT, HQT-ForRT, HQT-RevRT, Table 2) were designed on HCT (DQ104740), and HQT (DQ915589) sequences using the Primer 3 software http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi. As a housekeeping gene, actin was chosen for its stability and level of expression, which is comparable to the genes of interest and whose expression remained stable after the UV-C stress. The primers (ACT-ForRT, ACT-RevRT, Table 2) for its amplification were designed on the artichoke actin (ACT, AM744951). All primers were purchased from Metabion (Germany).
Standard curves were prepared for both the housekeeping ACT and target genes. The cDNAs were performed in triplicate for each sample in 20 μl. Reaction mixes contained 2× iQ SYBR Green Supermix (Bio-Rad Laboratories, USA), specific primers at 300 nM, and 3 μl of cDNA. PCR reactions were carried out in 48-well optical plates using the iCycler Real-time PCR Detection System (Bio-Rad Laboratories, USA). Cycling parameters were as follows: one cycle at 95°C for 5 min for DNA polymerase activation, followed by 35 cycles of 5 sec at 95°C (denaturation) and 20 sec at 60°C (annealing and extension). In all experiments, appropriate negative controls containing no template were subjected to the same procedure to exclude or detect any possible contamination. Melting curve analysis was performed at the end of amplification. Standard curves were analyzed with the iCycler iQ software. This quantification system was designed to automate analysis options, including quantitative and melting curve analysis. The results of amplification were analyzed by the comparative threshold cycle method, also known as the 2-ΔΔCt method . This method compares, for each time-point considered, the Ct values of the samples of interest (CtI) with the appropriate calibrator (CtM). The Ct values of both the calibrator and the samples of interest are normalized to the housekeeping gene.
SNP detection and linkage analysis
The allelic forms of globe artichoke HCT (isolated in the previous work)  and HQT (this work) were analysed in the two globe artichoke genotypes ('Romanesco C3' and 'Spinoso di Palermo'), previously used for map development . The full length HCT and HQT sequences were amplified on parental genome with 2 sets of primers (one for each isolated gene, HCT-For, HCT-Rev, HQT-For and HQT-Rev reported in Table 2) and PCR products were sequenced for SNP identification. SNPs genotyping were carried out with the tetra-primers ARMS-PCR method [61, 62] by using two sets of outer and inner primers (Table 2), designed using the software made available on line http://cedar.genetics.soton.ac.uk/public_html/primer1.html. PCR products were separated by 2% agarose gel electrophoresis.
Segregation data of HCT- and HQT-SNP markers were monitored and analyzed together with those of AFLP, S-SAP, M-AFLP and SSR markers previously applied for globe artichoke maps construction . The goodness-of fit between observed and expected segregation data was assessed using the chi-square (χ2) test. Independent linkage maps were constructed for each parent using the two way-pseudo testcross mapping strategy  by using JoinMap 2.0 software . For both maps, linkage groups were accepted at a LOD threshold of 4.0. To determine marker order within a linkage group, the following JoinMap parameter settings were used: Rec = 0.40, LOD = 1.0, Jump = 5. Map distances were converted to centiMorgans using the Kosambi mapping function . Linkage groups were drawn using MapChart 2.1 software .
We are particularly grateful to Martine Callier for technical assistance. We are grateful to Dr. C.J. Douglas (University of British Columbia, Vancouver) for providing 4CL enzyme.
This work was financially supported by Italian Ministry of Education, University and Research and by French Ministry of Research.
- Rottenberg A, Zohary D: The wild ancestry of the cultivated artichoke. Genetic Resources and Crop Evolution. 1996, 43 (1): 53-58.View ArticleGoogle Scholar
- Lanteri S, Saba E, Cadinu M, Mallica G, Baghino L, Portis E: Amplified fragment length polymorphism for genetic diversity assessment in globe artichoke. Theor Appl Genet. 2004, 108 (8): 1534-1544.PubMedView ArticleGoogle Scholar
- Acquadro A, Portis E, Lee D, Donini P, Lanteri S: Development and characterization of microsatellite markers in Cynara cardunculus L. Genome. 2005, 48 (2): 217-225.PubMedView ArticleGoogle Scholar
- Martino V, Caffini N, Phillipson J, Lappa A, Tchernitchin A, Ferraro G, Debenedelli S, Schilcher H, Acevedo C: Identification and characterization of antimicrobial components in leaf extracts of globe artichoke (Cynara scolymus L.). Acta Horticulturae. 1999, 501: 111-114.Google Scholar
- Zhu X, Zhang H, Lo R: Phenolic compounds from the leaf extract of artichoke (Cynara scolymus L.) and their antimicrobial activities. J Agric Food Chem. 2004, 52 (24): 7272-7278.PubMedView ArticleGoogle Scholar
- Zhu X, Zhang H, Lo R: Antifungal activity of Cynara scolymus L. extracts. Fitoterapia. 2005, 76 (1): 108-111.PubMedView ArticleGoogle Scholar
- Kukic J, Popovic V, Petrovic S, Mucaji P, Ciric A, Stoikovic D, Sokovic M: Antioxidant and antimicrobial activity of Cynara cardunculus extracts. Food Chemistry. 2008, 107 (2): 861-868.View ArticleGoogle Scholar
- Wang M, Simon J, Aviles I, He K, Zheng Q, Tadmor Y: Analysis of antioxidative phenolic compounds in artichoke (Cynara scolymus L.). J Agric Food Chem. 2003, 51 (3): 601-608.PubMedView ArticleGoogle Scholar
- Zang L, Cosma G, Gardner H, Castranova V, Vallyathan V: Effect of chlorogenic acid on hydroxyl radical. Mol Cell Biochem. 2003, 247 (1–2): 205-210.PubMedView ArticleGoogle Scholar
- McDougall B, King P, Wu B, Hostomsky Z, Reinecke M, Robinson W: Dicaffeoylquinic and dicaffeoyltartaric acids are selective inhibitors of human immunodeficiency virus type 1 integrase. Antimicrob Agents Chemother. 1998, 42 (1): 140-146.PubMedPubMed CentralGoogle Scholar
- Slanina J, Taborska E, Bochorakova H, Slaninova I, Humpa O, Robinson W, Schram K: New and facile method of preparation of the anti-HIV-1 agent, 1,3-dicaffeoylquinic acid. Tetrahedron Letters. 2001, 42 (19): 3383-3385.View ArticleGoogle Scholar
- Gu R, Dou G, Wang J, Dong J, Meng Z: Simultaneous determination of 1,5-dicaffeoylquinic acid and its active metabolites in human plasma by liquid chromatography-tandem mass spectrometry for pharmacokinetic studies. J Chromatogr B Analyt Technol Biomed Life Sci. 2007, 852 (1–2): 85-91.PubMedView ArticleGoogle Scholar
- Wagenbreth D: Evaluation of artichoke cultivars for growing and pharmaceutical use. Beitr Zuchtungsforsch. 1996, 2: 400-403.Google Scholar
- Pittlern M, Ernst E: Artichoke leaf extract for serum cholesterol reduction. Perfusion. 1998, 11: 338-340.Google Scholar
- Azzini E, Bugianesi R, Romano F, Di Venere D, Miccadei S, Durazzo A, Foddai M, Catasta G, Linsalata V, Maiani G: Absorption and metabolism of bioactive molecules after oral consumption of cooked edible heads of Cynara scolymus L. (cultivar Violetto di Provenza) in human subjects: a pilot study. British Journal of Nutrition. 2007, 97 (5): 963-969.PubMedView ArticleGoogle Scholar
- Kurata R, Adachi M, Yamakawa O, Yoshimoto M: Growth suppression of human cancer cells by polyphenolics from sweetpotato (Ipomoea batatas L.) leaves. J Agric Food Chem. 2007, 55 (1): 185-190.PubMedView ArticleGoogle Scholar
- Lattanzio V, Cardinali A, Di Venere D, Linsalata V, Palmieri S: Browning phenomena in stored artichoke (Cynara scolymus L.) heads: enzymic or chemical reactions?. Food Chemistry. 1994, 50: 1-7.View ArticleGoogle Scholar
- Schutz K, Kammerer D, Carle R, Schieber A: Identification and quantification of caffeoylquinic acids and flavonolds from artichoke (Cynara scolymus L.) heads, juice, and pomace by HPLC-DAD-ESI/MSn. Journal of Agricultural and Food Chemistry. 2004, 52 (13): 4090-4096.PubMedView ArticleGoogle Scholar
- Winkel-Shirley B: Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Physiologia Plantarum. 1999, 107 (1): 142-149.View ArticleGoogle Scholar
- Cle C, Hill L, Niggeweg R, Martin C, Guisez Y, Prinsen E, Jansen M: Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum; consequences for phenolic accumulation and UV-tolerance. Phytochemistry. 2008, 69 (11): 2149-2156.PubMedView ArticleGoogle Scholar
- Dixon R, Paiva N: Stress-induced phenylpropanoid metabolism. Plant Cell. 1995, 7 (7): 1085-1097.PubMedPubMed CentralView ArticleGoogle Scholar
- Treutter D: Biosynthesis of phenolic compounds and its regulation in apple. Plant Growth Regulation. 2001, 34 (1): 71-89.View ArticleGoogle Scholar
- Moglia A, Lanteri S, Comino C, Acquadro A, R dV, Beekwilder J: Stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke. J Agric Food Chem. 2008, 56 (18): 8641-8649.PubMedView ArticleGoogle Scholar
- Hoffmann L, Maury S, Martz F, Geoffroy P, Legrand M: Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism. Journal of Biological Chemistry. 2003, 278 (1): 95-103.PubMedView ArticleGoogle Scholar
- Niggeweg R, Michael A, Martin C: Engineering plants with increased levels of the antioxidant chlorogenic acid. Nature Biotechnology. 2004, 22 (6): 746-754.PubMedView ArticleGoogle Scholar
- Villegas R, Kojima M: Purification and characterization of Hydroxycinnamoyl D-Glucose: quinate hydroxycinnamoyl transferase in the root of sweet potato, Ipomoea batatas lam. The journal of biological chemistry. 1986, 261: 8729-8733.PubMedGoogle Scholar
- Stöckigt J, Zenk M: Enzymatic synthesis of chlorogenic acid from caffeoyl coenzyme A and quinic acid. FEBS Lett. 1974, 42 (2): 131-134.PubMedView ArticleGoogle Scholar
- Ulbrich B, Zenk M: Partial purification and properties of p-hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase from higher plants. Phytochemistry. 1979, 18: 929-933.View ArticleGoogle Scholar
- Hoffmann L, Besseau S, Geoffroy P, Ritzenthaler C, Meyer D, Lapierre C, Pollet B, Legrand M: Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis. Plant Cell. 2004, 16 (6): 1446-1465.PubMedPubMed CentralView ArticleGoogle Scholar
- Wagner A, Ralph J, Akiyama T, Flint H, Phillips L, Torr K, Nanayakkara B, Kiri L: Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase in Pinus radiata. Proceedings of the National Academy of Sciences of The United States of America. 2007, 104 (28): 11856-11861.PubMedPubMed CentralView ArticleGoogle Scholar
- Shadle G, Chen F, Reddy M, Jackson L, Nakashima J, Dixon R: Down-regulation of hydroxycinnamoyl CoA: Shikimate hydroxycinnamoyl transferase in transgenic alfalfa affects lignification, development and forage quality. Phytochemistry. 2007, 68 (11): 1521-1529.PubMedView ArticleGoogle Scholar
- Comino C, Lanteri S, Portis E, Acquadro A, Romani A, Hehn A, Larbat R, Bourgaud F: Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L. BMC Plant Biology. 2007, 7: 14PubMedPubMed CentralView ArticleGoogle Scholar
- Thorup T, Tanyolac B, Livingstone K, Popovsky S, Paran I, Jahn M: Candidate gene analysis of organ pigmentation loci in the Solanaceae. Proceedings of The National Academy of Sciences of The United States of America. 2000, 97 (21): 11192-11197.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen X, Salamini F, Gebhardt C: A potato molecular-function map for carbohydrate metabolism and transport. Theoretical and Applied Genetics. 2001, 102 (2–3): 284-295.View ArticleGoogle Scholar
- Just B, Santos C, Fonseca M, Boiteux L, Oloizia B, Simon P: Carotenoid biosynthesis structural genes in carrot (Daucus carota): isolation, sequence-characterization, single nucleotide polymorphism (SNP) markers and genome mapping. Theor Appl Genet. 2007, 114 (4): 693-704.PubMedView ArticleGoogle Scholar
- Lanteri S, Acquadro A, Comino C, Mauro R, Mauromicale G, Portis E: A first linkage map of globe artichoke (Cynara cardunculus var. scolymus L.) based on AFLP, S-SAP, M-AFLP and microsatellite markers. Theor Appl Genet. 2006, 112 (8): 1532-1542.PubMedView ArticleGoogle Scholar
- Rose T, Schultz E, Henikoff J, Pietrokovski S, McCallum C, Henikoff S: Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 1998, 26: 1628-1635.PubMedPubMed CentralView ArticleGoogle Scholar
- St Pierre B, Laflamme P, Alarco A, De Luca V: The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for Coenzyme A dependent acyl transfer. Plant Journal. 1998, 14: 703-713.PubMedView ArticleGoogle Scholar
- Suzuki H, Nakayama T, Nishino T: Proposed mechanism and functional amino acid residues of Malonyl-CoA: Anthocyanin 5-O-Glucoside-6"'-O-Malonyltransferase from flowers of Salvia splendens, a member of the Versatile Plant Acyltransferase Family. Biochemistry. 2003, 42: 1764-1771.PubMedView ArticleGoogle Scholar
- D'Auria J: Acyltransferases in plants: a good time to be BAHD. Current Opinion in Plant Biology. 2006, 9 (3): 331-340.PubMedView ArticleGoogle Scholar
- Lepelley M, Cheminade G, Tremillon N, Simkin A, Caillet V, McCarthy J: Chlorogenic acid synthesis in coffee: An analysis of CGA content and real-time RT-PCR expression of HCT, HQT, C3H1, and CCoAOMT1 genes during grain development in C. canephora. Plant Science. 2007, 172 (5): 978-996.View ArticleGoogle Scholar
- Rhodes M, Wooltorton L: The enzymic conversion of hydroxycinnamic acids to p- coumarylquinic and chlorogenic acids in tomato fruits. Phytochemistry. 1976, 15: 947-951.View ArticleGoogle Scholar
- Dixon R: Engineering of plant natural product pathways. Current Opinion in Plant Biology. 2005, 8 (3): 329-336.PubMedView ArticleGoogle Scholar
- Jang M, Cai E, Udeani G, Slowing K, Thomas C, Beecher C, Fong H, Farnsworth N, Kinghorn A, Mehta R, et al: Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science. 1997, 275 (5297): 218-220.PubMedView ArticleGoogle Scholar
- Kahkonen M, Hopia A, Vuorela H, Rauha J, Pihlaja K, Kujala T, Heinonen M: Antioxidant activity of plant extracts containing phenolic compounds. J Agric Food Chem. 1999, 47 (10): 3954-3962.PubMedView ArticleGoogle Scholar
- Burns J, Gardner P, O'Neil J, Crawford S, Morecroft I, McPhail D, Lister C, Matthews D, MacLean M, Lean M, et al: Relationship among antioxidant activity, vasodilation capacity, and phenolic content of red wines. J Agric Food Chem. 2000, 48 (2): 220-230.PubMedView ArticleGoogle Scholar
- Bandoniene D, Murkovic M: On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.). J Agric Food Chem. 2002, 50 (9): 2482-2487.PubMedView ArticleGoogle Scholar
- Dixon R, Ferreira D: Genistein. Phytochemistry. 2002, 60 (3): 205-211.PubMedView ArticleGoogle Scholar
- Schoch G, Goepfert S, Morant M, Hehn A, Meyer D, Ullmann P, Werck-Reichhart D: CYP98A3 from Arabidopsis thaliana is a 3 '-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. Journal of Biological Chemistry. 2001, 276 (39): 36566-36574.PubMedView ArticleGoogle Scholar
- Franke R, Humphreys J, Hemm M, Denault J, Ruegger M, Cusumano J, Chapple C: The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant Journal. 2002, 30 (1): 33-45.PubMedView ArticleGoogle Scholar
- Burhenne K, Kristensen B, Rasmussen S: A new class of N-hydroxycinnamoyltransferases – Purification, cloning, and expression of a barley agmatine coumaroyltransferase (Ec 22.214.171.124). Journal of Biological Chemistry. 2003, 278 (16): 13919-13927.PubMedView ArticleGoogle Scholar
- Treutter D: Significance of flavonoids in plant resistance and enhancement of their biosynthesis. Plant Biology. 2005, 7 (6): 581-591.PubMedView ArticleGoogle Scholar
- Hayes P, Schmitt K, Jones H, Gyapay G, Weissenbach J, Goodfellow P: Regional assignment of human ESTs by whole-genome radiation hybrid mapping. Mammalian Genome. 1996, 7 (6): 446-450.PubMedView ArticleGoogle Scholar
- Weeden N, Ellis T, Timmerman-Vaughan G, Simon C, Torres A, Wolko B: How similar are the genomes of the cool season food legumes?. Linking research and marketing opportunities for pulses in the 21st Century. Proceedings of the Third International Food Legumes Research Conference, Adelaide, Australia, 22–26 September 1997. Kluwer Academic Publishers; Dordrecht, The Netherlands; 2000:397-410.Google Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
- Morant M, Hehn A, Werck-Reichhart D: Conservation and diversity of gene families explored using the CODEHOP strategy in higher plants. BMC Plant Biol. 2002, 2: 7PubMedPubMed CentralView ArticleGoogle Scholar
- Kumar S, Tamura K, Nei M: MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics. 2004, 5 (2): 150-163.PubMedView ArticleGoogle Scholar
- Beuerle T, Pichersky E: Enzymatic Synthesis and Purification of Aromatic Coenzyme A Esters. Analytical Biochemistry. 2002, 302: 305-312.PubMedView ArticleGoogle Scholar
- Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols. New Jersey: Humana Press; 1999:365-386.View ArticleGoogle Scholar
- Livak KJ, Schmittgen T: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408.PubMedView ArticleGoogle Scholar
- Ye S, Dhillon S, Ke X, Collins A, Day I: An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Research. 2001, 29 (17): art no. -e88Google Scholar
- Chiapparino E, Lee D, Donini P: Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS-PCR. Genome. 2004, 47 (2): 414-420.PubMedView ArticleGoogle Scholar
- Weeden N: Approaches to mapping in horticultural crops. Plant genome analysis. Edited by: Greshoff PM. Boca Raton: CRC Press; 1994:57-68.Google Scholar
- Stam P, Van Ooijen J: JoinMap version 2.0: software for the calculation of genetic linkage maps. CPRO-DLO (ed.), Wageningen, The Netherlands, 60; 1995.Google Scholar
- Kosambi D: The estimation of map distances from recombination values. Ann Eugen. 1944, 12: 172-175.View ArticleGoogle Scholar
- Voorrips R: MapChart: Software for the graphical presentation of linkage maps and QTLs. Journal of Heredity. 2002, 93 (1): 77-78.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.