Novel hydroxycinnamoyl-coenzyme A quinate transferase genes from artichoke are involved in the synthesis of chlorogenic acid

Abstract

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.

Figure 1.

Protein sequence alignment of artichoke HQT1 and HQT2 with representative members of the HQT family, HCBT, artichoke HCT, and 2E1T. Accession numbers are as follows: artichoke HQT1, AM690438; artichoke HQT2, EU839580; artichoke HQT, ABK79689; cardoon HQT, ABK79690; tobacco HQT, Q70G33; tomato HQT, Q70G32; sweet potato HCBT, Q9SST8; coffee HQT, A4ZKM4; potato HQT, Q3HRZ5; artichoke HCT, AAZ80046. 2E1T refers to DmAT A4PHY4. Black bars indicate the conserved motifs for the acyl transferase family: HXXXDG and DFGWG. Gray bars identify conserved amino acid residues for HQT.

Figure 2.

Neighbor-joining phylogenetic tree of acyltransferase proteins from different plant species. Line lengths indicate the relative distances between nodes. Protein accession numbers are indicated. Den. morifolium DmAT and R. serpentina ACT refer to acyltransferase sequences for which the crystal structures are available (2e1t and 2bgh, respectively). ACY, Hydroxyanthranilate hydroxycinnamoyl transferase. Numbers on the nodes refer to bootstrap test values.

Figure 3.

HPLC/LC/MS analysis of HQT1 and HQT2 acyltransferase assays. A and B, HPLC profiles of HQT1 and HQT2 reaction products without (control) or with recombinant protein obtained using caffeoyl-CoA and quinate (A) or p-coumaroyl-CoA and quinate (B). Peak 1, Chlorogenic acid (CGA); peak 2, caffeoyl-CoA; peak 3, p-coumaroylquinate; peak 4, p-coumaroyl-CoA. C, Mass spectrum of HPLC peak 1 obtained from HQT1 reaction; m/z (mass-to-charge ratio) = 355 ([M+H]⁺); M = CGA. D, Mass spectrum of HPLC peak 3 obtained from HQT1 reaction; m/z = 339 ([M+H]⁺); M = p-coumaroylquinate. mAU, Milliabsorbance units.

Figure 4.

Proposed binding sites for HQT1, HQT2, and HCT. Views of the comparative three-dimensional models of HQT1 (A), HQT2 (B), and HCT (C) highlighting the proposed binding sites are shown. α-Helices and β-sheets are shown in gray cartoon representation. The substrates caffeoylquinate (A), p-coumaroylquinate (B), and caffeoylshikimate (C) are shown in stick representation using the PyMOL color code: cyan, carbon atoms; red, oxygen atoms; white, hydrogen atoms; and blue, nitrogen atoms. Possible interactions between the substrates and residues of the proposed binding sites are indicated by black dashed lines. Residues of the binding sites are in pink cartoon and stick representation for HQT1, in yellow cartoon and stick representation for HQT2, and in green cartoon and stick representation for HCT.

Figure 5.

Levels of expression of hqt1 and hqt2 and levels of chlorogenic acid in various artichoke tissues and two genotypes. A and B, Real-time PCR of hqt1 and hqt2 transcripts of external bracts (ExB), intermediate bracts (ImB), internal bracts (InB), receptacle (Rec), and leaves from the productive (PrL) or vegetative (VeL) period in two genotypes, S. Erasmo (white bars) and Mola (black bars). The data represent mean normalized expression (±sd) of biological and experimental replicates. C, Chlorogenic acid content as mg g⁻¹ fresh weight of the same genotypes and tissues analyzed by real-time PCR. Values are means ± sd of three replicates.

Figure 6.

Transient and stable overexpression of artichoke hqt1 in N. benthamiana and N. tabacum, respectively, molecular characterization of stable transgenic lines, and determination of CGA and cynarin contents. A and B, Contents of CGA (A) and cynarin (B) as μg g⁻¹ fresh weight (FW) in N. benthamiana wild-type plants (NbWT) and in transiently transformed leaves. Transformants either carried the empty vector (pKY) or expressed the silencing inhibitor protein p19 (p19), HQT1 (hqt1), or both HQT1 and p19 proteins (hqt1+p19). C, RT-PCR analysis of hqt1 (top) and the tobacco actin gene used as control (bottom) in a tobacco wild-type plant (NtWT), in a tobacco line transformed with the empty vector (pKY), and in hqt1-overexpressing lines (lines 5, 10, 22, 31, 34, and 35). D and E, Contents of CGA (D) and cynarin (E) as μg g⁻¹ fresh weight in N. tabacum wild-type plants (NtWT) and in stably HQT1-transformed leaves (hqt1). The data represent means ± sd of eight transiently transformed leaves and leaves from three independent, stably transformed lines (lines 5, 31, and 35).

Figure 7.

Proposed pathways for the biosynthesis of chlorogenic acid in artichoke (redrawn from Mahesh et al., 2007). PAL, Phe ammonia-lyase.