Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine

Abstract

In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites.

Keywords: Flavan-3-ol; flavonoid; gallic acid; galloylation; grapevine; shikimate dehydrogenase..

Fig. 1.

Gallic and shikimic acid biosynthesis in plants. Reaction 1: Dehydroquinic acid (DHQ) dehydration catalysed by the dehydroquinate dehydratase (DQD) domain of the dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) producing 3-dehydroshikimic acid (3-DHS). Reaction 2: NADPH-dependent reduction of 3-DHS catalysed by the shikimate dehydrogenase (SDH) domain of DQD/SDH producing shikimic acid (SA). Reaction 3: NADP⁺-dependent oxidation of SA catalysed by the SDH domain of DQD/SDH producing 3-DHS. Reaction 4: NADP⁺-dependent oxidation of 3-DHS catalysed by the SDH domain of DQD/SDH producing gallic acid (GA). Reaction 5: UDP-Glucose-dependent glucosylation of gallic acid catalysed by grapevine glucosyltransferases (Khater et al., 2012). Reaction 6?: Putative galloylation of epicatechin from β-glucogallin. Dotted lines indicate pathways of several steps.

Fig. 2.

Neighbor–Joining tree of selected dehydroquinate dehydratase/shikimate dehydrogenases from dicots. This phylogenetic tree was constructed from the four VvSDHs sequenced in this study and the 28 sequences available on public databases (NCBI and Phytozome) using MEGA6 software. Bootstrap values (percentage of 1 000 replicates) are shown for key branches. The scale bar represents 0.05 substitutions per site. Abbreviations: At (Arabidopsis thaliana), Cs (Citrus sinensis), Cas (Camellia sinensis), Dk (Diospyros kaki), Eg (Eucalyptus grandis), Fv (Fragaria vesca), Jr (Juglans regia), Nt (Nicotiana tabacum), Poptr (Populus trichocarpa), Sl (Solanum lycopersicum), Vv (Vitis vinifera). Accession numbers: Group I: AtSDH (AAF08579), FvSDH3 (XP_004289250), FvSDH4 (XP_004288087), VvSDH1 (KU163040), EgSDH5 (Eucgr.J00263.6), NtSDH1 (AAS90325), Poptr1 (Potri.010G019000), SlSDH1 (AAC17991), CsSDH3 (orange1.1g007151m), JrSHD (AAW65140); Group II: CasSDH1 (AIZ93902), CsSDH1 (orange1.1g010050m), NtSDH2 (AAS90324), VvSDH2 (KU163041), EgSDH4 (Eucgr.B01770.2), Poptr2 (Potri.013G029900), Poptr3 (Potri.005G043400), SlSDH2 (XP_010327280); Group III: EgSDH3 (Eucgr.H04427.1), FvSDH1 (XP_004302480), VvSDH3 (KU163042), CasSDH2 (AJA40947); Group IV: EgSDH2 (Eucgr.H04428.1), VvSDH4 (KU163043), Poptr5 (Potri.013G029800), FvSDH2 (XP_004302479), DkSDH (BAI40147), CasSDH3 (AJA40948); Group V: Poptr4 (Potri.014G135500), EgSDH1 (Eucgr.H01214.1), CsSDH2 (orange1.1g010101m), SlSDH3 (XP_004242317). Accession number corresponds to the NCBI or Phytozome 10.1.2 database. Purple dots show VvSDHs. The presence of a putative chloroplastic target peptide according to ChloroP is shown by a green dot.

Fig. 3.

Comparison of key amino acids in the SDH domain of DQD/SDHs from dicots. Protein alignment has been performed by Clustal Omega. Amino acids numerotation is based on the DQD/SDH from Arabidopsis thaliana (AtSDH; Singh and Christendat, 2006). The key amino acids are shaded in green when they are identical to AtSDH, and in red when they are different from AtSDH. Groups I–V have been determined from the Neighbor–Joining tree. This sequence alignment includes DQD/SDHs from Arabidopsis thaliana (AtSDH), Nicotiana tabacum (NtSDH1 and 2), Solanum lycopersicum (SlSDH1–3), Vitis vinifera (VvSDH1–4), Eucalyptus grandis (EgSDH1–5), Citrus sinensis (CsSDH1–3), Populus trichocarpa (Poptr1–5), Fragaria vesca (FvSDH1–4), Juglans regia (JrSDH), Camellia sinensis (CasSDH1–3), Diospyros kaki (DkSDH).

Fig. 4.

VvSDH expression pattern in grape berry. (A–D) qRT-PCR analysis of the expression level of VvSDH1 (A), VvSDH2 (B), VvSDH3 (C), and VvSDH4 (D) in the pericarp of grape berries harvested at different days after flowering indicated on the x axis. Véraison is marked by the arrow. Data represent the mean of three replicates ±SD. (E–H) qRT-PCR analysis of the expression level of VvSDH1 (E), VvSDH2 (F), VvSDH3 (G), and VvSDH4 (H) in the main tissues of grape berries harvested at different days after flowering indicated on the x axis. Green stage, véraison (marked by the brace), and maturity corresponds to 18, 52, and 99 d after flowering, respectively. Grey, white, and black bars correspond to skin, pulp, and seeds, respectively. Data represent the mean of three replicates ±SD.

Fig. 5.

Gallic acid metabolism in grape berry tissues along development. (A–C) Gallic acid and β-glucogallin in grape berry tissues (A), skin (B), pulp, and (C) seeds. White and grey bars correspond to GA and β-glucogallin, respectively. Data are expressed g^–1 of fresh weight and represent the mean of three replicates ±SD. Samples corresponding to the green stage, véraison, and maturity were collected at 18, 52, and 99 d after flowering, respectively. (D) Galloylated flavan-3-ols in grape berry tissues. Galloylated flavan-3-ols have been quantified after depolymerization by phloroglucinolysis. Grey, white, and black bars correspond to skin, pulp, and seeds, respectively. Data are expressed g^–1 of frozen powder and represent the mean of three replicates ±SD. (E) Galloylation rate (%G) of flavan-3-ols in grape berry tissues. Flavan-3-ols have been quantified after depolymerization by phloroglucinolysis. Grey, white, and black bars correspond to skin, pulp, and seeds, respectively. Data represent the mean of three replicates ±SD.

Fig. 6.

pH effect on VvSDH specific activity. (A) VvSDH activity from shikimic acid and NADP⁺. VvSDH activity was measured by spectrophotometry and recorded NADPH variation 2min after adding the enzyme. Bis-Tris Propane HCl buffer allowed measurements at pH 6.5, 7, 7.5, 8, 8.5, and 9. Data represent the mean of three replicates ±SD. Data are presented independently for VvSDH2 activity due to its lower activity. (B) VvSDH activity from 3-dehydroshikimate and NADPH. VvSDH activity was measured by spectrophotometry and recorded NADPH variation 2min after adding the enzyme. Bis-Tris Propane HCl buffer allowed measurements at pH 6.5, 7, 7.5, 8, 8.5, and 9. Data represent the mean of three replicates ±SD.

Fig. 7.

In vitro production of 3-dehydroshikimate and gallic acid by recombinant VvSDH. (A) Quantification of 3-DHS (white bars) and GA (grey bars) in conditions lacking the enzyme and in the presence of each VvSDH from shikimic acid and NADP⁺, at pH 9. Data represent the mean of three replicates ±SD. The reaction products in the enzymatic assays were quantified by HPLC using UV-DAD (λ=280nm). (B) GA quantification in conditions lacking the enzyme and in the presence of each VvSDH from 3-DHS and NADP⁺, at pH 9. Data represent the mean of three replicates ±SD. The significance of the results was statistically assessed with a Student’s t test using a two-tailed alternative. *: P <0.01. The reaction products in the enzymatic assays were quantified by HPLC using UV-DAD (λ=280nm).

Fig. 8.

Metabolic profiling of grapevine hairy-roots from VvSDH3-over-expression lines. Control: hairy-root devoid of transgene. Lines 3A, 6A, and 9A are three independent lines transformed with VvSDH3. Metabolites are denoted as nmol g⁻¹ hairy-root fresh weight. The dotted lines indicate several enzymatic steps. Each data set represents the mean value of three assays ±SD. The significance of the results was statistically assessed with a Student’s t test using a two-tailed alternative. *: 0.01<P <0.05, **: P <0.01. Abbreviations: 3-DHS, 3-dehydroshikimate; SA, shikimic acid; AAA, aromatic amino acids; HCAs, hydroxycinnamic acids; HCTs, hydroxycinnamoyl tartrates; GA, gallic acid; β-G, β-glucogallin; ECG, epicatechin gallate; nd, not determined.