Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L.
BACKGROUND
Cynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant.


RESULTS
A gene sequence encoding a hydroxycinnamoyltransferase (HCT) involved in the synthesis of CGA, was identified. Isolation of the gene sequence was achieved by using a PCR strategy with degenerated primers targeted to conserved regions of orthologous HCT sequences available. We have isolated a 717 bp cDNA which shares 84% aminoacid identity and 92% similarity with a tobacco gene responsible for the biosynthesis of CGA from p-coumaroyl-CoA and quinic acid. In silico studies revealed the globe artichoke HCT sequence clustering with one of the main acyltransferase groups (i.e. anthranilate N-hydroxycinnamoyl/benzoyltransferase). Heterologous expression of the full length HCT (GenBank accession DQ104740) cDNA in E. coli demonstrated that the recombinant enzyme efficiently synthesizes both chlorogenic acid and p-coumaroyl quinate from quinic acid and caffeoyl-CoA or p-coumaroyl-CoA, respectively, confirming its identity as a hydroxycinnamoyl-CoA: quinate HCT. Variable levels of HCT expression were shown among wild and cultivated forms of C. cardunculus subspecies. The level of expression was correlated with CGA content.


CONCLUSIONS
The data support the predicted involvement of the Cynara cardunculus HCT in the biosynthesis of CGA before and/or after the hydroxylation step of hydroxycinnamoyl esters.
Comino
Cinzia
C
Di.Va.P.R.A. Plant Genetics and Breeding, University of Torino 10095, Grugliasco (Turin), Italy. cinzia.comino@unito.it
Lanteri
Sergio
S
Portis
Ezio
E
Acquadro
Alberto
A
Romani
Annalisa
A
Hehn
Alain
A
Larbat
Romain
R
Bourgaud
Frédéric
F
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D015152:Blotting, Northern; D002726:Chlorogenic Acid / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D031942:Cynara / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli; D005419:Flavonoids / Q000032:analysis; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D010636:Phenols / Q000032:analysis; D010802:Phylogeny; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology; D059808:Polyphenols; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D018076:DNA, Complementary; D005419:Flavonoids; D010636:Phenols; D059808:Polyphenols; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002726:Chlorogenic Acid; D000217:Acyltransferases; C021394:hydroxycinnamoyl-CoA-quinate transferase