Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L

Abstract

BackgroundCynara 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.ResultsA 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.ConclusionThe 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.

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

“…Two distinct pathways have been proposed for the synthesis of chlorogenic acid: (1) pathway from p-coumaroyl-CoA, involving first a transesterification of this compound and quinic acid via hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) activity and then hydroxylation of p-coumaroylquinate to 5-caffeoylquinic acid, catalyzed by C3 0 H; (2) pathway involving p-coumaroyl-CoA trans-esterification with shikimic acid by means of hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT), then p-coumaroylshikimate hydroxylation to caffeoylshikimate. This compound is then converted by HCT (Hoffmann et al 2003;Comino et al 2007) to caffeoyl-CoA which is conjugated to quinic acid by HQT (Niggeweg et al 2004) activity, to give 5-caffeoylquinic acid.…”

“…The gene encoding HCT in globe artichoke has recently been isolated (Comino et al 2007 LG 10 Some CYP98 genes are expressed constitutively, while others (particularly those involved in the stress response) are inducible. A. thaliana CYP98A3 is constitutively expressed and is upregulated by wounding (Schoch et al 2001).…”

“…Three sequences with high similarity to PAL (phenylalanine ammonia-lyase), the first enzyme involved in the phenylpropanoid pathway, have been isolated (De Paolis et al 2008). Moreover, gene sequences encoding hydroxycinnamoyltransferase (HCT and HQT), involved in the synthesis of chlorogenic acid, have been recently identified (Comino et al 2007(Comino et al , 2009. New insights are thus required for identifying other genes involved in caffeoylquinic acid synthesis and elucidating the causal relationships between the various enzymes.…”

New genetic maps for globe artichoke and wild cardoon and their alignment with an SSR-based consensus map

“…The PCR conditions applied followed the recommendations of the markers developer and the amplicons were separated in the same way as the AFLPs. SNP markers were directed at seven genes involved in the synthesis of caffeoylquinic acids, namely HCT, HQT, C3'H, C4H, 4CL, Acyltransf_1 and _2 (Comino et al 2007;Moglia et al 2009;Menin et al 2010). The assay was based on a tetra-primer ARMS-PCR (amplification refractory mutation system) protocol (Ye et al 2001), with the amplicons being separated by electrophoresis through 2% w/v agarose gels and visualized by ethidium bromide staining.…”

Novel Hydroxycinnamoyl-Coenzyme A Quinate Transferase Genes from Artichoke Are Involved in the Synthesis of Chlorogenic Acid

“…In artichoke, a cDNA coding for HCT has been isolated (Comino et al, 2007). Biochemical characterization suggested that HCT could act either before or after the 3#-hydroxylation step and may contribute to CGA biosynthesis.…”