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

Abstract

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.

Conclusion: 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.

Figure 1

Sequence alignment of HCT from artichoke with representative members of the plant hydroxycinnamoyl transferase family. CAD47830 from N. tabacum; NP_199704 from A. thaliana; CAB06427 from D. caryophyllus; NP_200592 from A. thaliana; NP_179497 from A. thaliana; DQ104740 (in bold) from C. cardunculus. Black frames indicate regions chosen to design CODEHOP; gray boxes indicate structural motifs conserved in the acyltransferase family.

Figure 2

Phylogenetic analysis of acyltransferases. The tree was constructed by the neighbour-joining method. The length of the lines indicates the relative distances between nodes. Protein sequences used for the alignment are: DcHCBT, anthranilate N-hydroxycinnamoyl/benzoyltransferase of D. caryophyllus (Z84383); IbHCBT, N-hydroxycinnamoyl/benzoyltransferase from I. batatas (AB035183); AtHCT, shikimate/quinate hydroxycinnamoyltransferase of A. thaliana (At5g48930); NtHCT, shikimate/quinate hydroxycinnamoyltransferase of N. tabacum (AJ507825); NtHQT, hydroxycinnamoyl CoA quinate transferase of N. tabacum (CAE46932); LeHQT, hydroxycinnamoyl CoA quinate transferase of L. esculentum (CAE46933); At2G19070 and At5G57840, A. thaliana genes encoding putative acyltransferases; CcHCT, hydroxycinnamoyl CoA quinate transferase from C. cardunculus (DQ104740, this work).

Figure 3

Expression of recombinant HCT in E. coli. Protein content of non-induced (T = 0 h) and induced (T = 8 h) non-transformed cells (1) were compared with those of non-induced (T = 0 h) and induced (T = 8 h) transformed cells (2). After induction at 37°C for 8 h, HCT protein cannot be detected in the soluble fraction (2 S at T = 8 h), but is present in the soluble fraction after induction at 28°C. Arrows indicate the ~50 kDa HCT protein. Molecular marker masses indicated on the left. (1 = empty vector; 2 = vector -HCT; S = soluble fraction; P = pellet; M = molecular weight marker).

Figure 4

HPLC analysis of the HCT reaction products. An aliquot of the incubation reaction without (black line) or with (gray line) recombinant HCT was analysed. (a) HCT reaction with p-coumaroyl-CoA and quinate; standard of p-coumaroyl-quinate (dotted line) is used as reaction control; (b) HCT reverse reaction with chlorogenic acid and CoA.

Figure 5

Example of comparison between absorption spectrum of the reaction product and authentic standard. Absorption spectra of p-coumaroyl-quinate: standard (dotted line) and product by reaction with HCT (black line).

Figure 6

HPLC/DAD profiles at 330 nm of the compounds identified in C. cardunculus. (a) globe artichoke, (b) cultivated cardoon, and (c) wild cardoon. Peaks: 1. caffeoylquinic acid; 2. chlorogenic acid (5-CQA); 3. p-coumaroylquinic acid; 4. feruloylquinic acid; 5. and 6. di-caffeoylquinic acids; 7. luteolin 7-O-rutinoside; 8. luteolin 7-O-glucoside; 9. luteolin 7-O-glucuronide; 10. luteolin malonylglucoside; 11. apigenin 7-O-glucuronide; 12. luteolin; 13. apigenin. In bold are indicated the caffeoylquinic acids and in brackets others compounds detected in C.cardunculus.

Figure 7

Northern blot analyses for HCT expression. First and second panels are, respectively, total RNA ethidium bromide-stained prior to membrane transfer and 18S expression to control RNA quality and sample loading. Third panel is the HCT expression with probe1 in 'Violet Margot' (1), Romanesco C3' (2), and with probe 2 in wild cardoon (3) and cultivated cardoon (4).

Figure 8

The three routes in phenylpropanoid metabolism (labelled 1, 2 and 3) proposed for the chlorogenic acid synthesis in plants. Enzymes involved in the pathway are: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase; C3H, p-coumarate 3'-hydroxylase; HQT, hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase; UGCT, UDP glucose: cinnamate glucosyl transferase; HCGQT, hydroxycinnamoyl D-glucose: quinate hydroxycinnamoyl transferase (adapted from [25]).