Genome-Wide Identification of BAHD Acyltransferases and In vivo Characterization of HQT-like Enzymes Involved in Caffeoylquinic Acid Synthesis in Globe Artichoke

Abstract

Globe artichoke (Cynara cardunculus L. var. scolymus) is a rich source of compounds promoting human health (phytonutrients), among them caffeoylquinic acids (CQAs), mainly represented by chlorogenic acid (CGA), and dicaffeoylquinic acids (diCQAs). The enzymes involved in their biosynthesis belong to the large family of BAHD acyltransferases. Following a survey of the globe artichoke genome, we identified 69 BAHD proteins carrying the catalytic site (HXXXD). Their phylogenetic analysis together with another 43 proteins, from 21 species, representative of the BAHD family, highlighted their grouping in seven major clades. Nine globe artichoke acyltransferases clustered in a sub-group of Clade V, with 3 belonging to hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) and 2 to hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) like proteins. We focused our attention on the former, HQT1, HQT2, and HQT3, as they are known to play a key role in CGA biosynthesis. The expression of genes coding for the three HQTs and correlation of expression with the CQA content is reported for different globe artichoke tissues. For the first time in the globe artichoke, we developed and applied the virus-induced gene silencing approach with the goal of assessing in vivo the effect of HQT1 silencing, which resulted in a marked reduction of both CGA and diCQAs. On the other hand, when the role of the three HQTs was assessed in leaves of Nicotiana benthamiana through their transient overexpression, significant increases in mono- and diCQAs content were observed. Using transient GFP fusion proteins expressed in N. benthamiana leaves we also established the sub-cellular localization of these three enzymes.

Keywords: BAHD acyltransferases; Cynara cardunculus; VIGS; caffeoylquinic acids; functional characterization.

FIGURE 1

Biosynthetic pathways of chlorogenic acid and its related derivatives. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl-CoA ligase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; HQT, hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase; C3’H, p-coumaroyl ester 3′-hydroxylase.

FIGURE 2

UPGMA based phylogenetic tree was constructed of 69 globe artichoke BAHD members sequences and 43 fully/partially characterized sequence from other species. The colors for highlighting clades are: violet (Clade I), pink (Clade II), light red (Clade III), red (Clade IV), orange (Clade V), yellow (Clade VI), and pale yellow (Clade VII). Sequences used for building the tree are reported in the Data Sheet 1.

FIGURE 3

Phylogenetic and bioinformatic analyses on BAHD. (A) UPGMA based phylogenetic tree of five globe artichoke BAHD involved in biosynthesis of lignin and chlorogenic acid and belonging to HQT/HCT branch of the Clade V. (B) Muscle alignment of the CQAs related proteins (in yellow the two functional domains). (C) CQAs related ohnologous genes distribution over the globe artichoke pseudomolecules.

FIGURE 4

qPCR and LC-MS analyses. (A) Relative gene expression of HQT1, HQT2, and HQT3 in globe artichoke leaf (6-weeks, 1 year), stem and bract tissues. Globe artichoke actin was used as reference gene. Error bars represent SD (n = 3). Different letters associated with the set of means indicate significance based on Tukey’s test (P ≤ 0.05). (B) Relative concentration of chlorogenic acid (CGA) and 1,3-dicaffeoylquinic acid (1,3-diCQA) in globe artichoke (F1 hybrid ‘Concerto’) tissues (leaf from 6 weeks-old plants and from 1 year-old plants, stem, bract). Concentrations were compared by measuring mass signals of the molecular ion ([M+H]) in different tissues. Different letters associated with the set of means indicate significance based on Tukey’s’s test (P < 0.05).

FIGURE 5

VIGS of globe artichoke genes using pTRV vectors. Globe artichoke seedlings were infected with Agrobacterium transformed with pTRV2-PDS vectors. Photographs of the leaves were taken 5 weeks after infiltration (A). Relative transcript levels of PDS, HQT1, HQT2, HQT3 genes were evaluated through qPCR analysis (B) in pTRV2, pTRV2-PDS, and pTRV2-PDS-HQT1 infected leaves. ^∗P ≤ 0.05 vs pTRV2 infected (control).

FIGURE 6

LC-PDA profiles of leaf extracts of globe artichoke agro-infiltrated with Agrobacterium transformed with different TRV vectors. Pink/black (pTRV2-PDS biological replicates), Blue/Green (pTRV2-PDS-HQT1 biological replicates) (A). The major peaks correspond to chlorogenic acid (B) and 3,5-dicaffeoylquinic acid (C).

FIGURE 7

LC-MS ESI⁺ MRM profiles. (A) monoCQAs (NeoCGA, CryptoCGA, CGA) and (B) diCQAs (1,3-diCQA, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA) in Nicotiana benthamiana control (CTR) and transformed tissues extracts (HQT1, HQT2, and HQT3). Concentration of (C) monoCQAs (NeoCGA, CryptoCGA, CGA) and (D) diCQAs (1,3-diCQA, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA) in tissue extracts of N. benthamiana control (CTR) and transiently transformed leaves (HQT1, HQT2, and HQT3). Error bars represent SD (n = 3). Asterisk indicates significance based on Tukey’s test (P ≤ 0.05).

FIGURE 8

Subcellular imaging of GFP-tagged constructs expressed in N. benthamiana agroinfiltrated leaves. Confocal microscopy observations of leaf epidermal cells showed that the fluorescence pattern for GFP:HQT1 (A), GFP:HQT2 (B) and GFP:HQT3 (C) was compatible with their localization in the cytosol (arrowheads). For reference, the localization of free GFP in both the cytoplasm and nucleus, and the lace-like fluorescence pattern of ER-localized GFP-KDEL are presented in (D,E), respectively. (n) = nucleus; bars = 50 μm.