Fig 1.
Biosynthesis of hydrolyzable tannins.
(A) An ester bond is formed between gallic acid and glucose by a UDP-glucosyltransferase (UGT) activity to produce β-glucogallin. Multiple acyltransferases convert β-glucogallin to pentagalloylglucose, which is further transformed into gallotannins or ellagitannins. Dotted arrows denote multiple enzymatic steps. (B) Chemical structures of punicalagin (α and β isomers), ellagic acid and hexahydroxydiphenic acid (HHDP) are illustrated.
Fig 2.
Pomegranate UGT84A23 and UGT84A24 cluster with glucose ester forming UGTs.
A neighbor-joining (NJ) tree was constructed using PgUGTs with selected plant family 1 UGTs that represent fifteen phylogenetic groups [30] and glucose ester forming UGTs that recognize phenolic acids as substrates. Bootstrap values (1,000 iterations) greater than 60% are shown next to the branches. UGT84A23, UGT84A24, UGT73AL1 and UGT85K15 are highlighted in bold text.
Fig 3.
Pomegranate UGT84A23 and UGT84A24 convert UDP-glucose (UDPG) and gallic acid to β-glucogallin.
(A) HPLC elution profiles of the enzyme assay products (indicated with asterisks) and the authentic standards at 280 nm are shown at a 1% offset retention time and 50% offset absorbance. (B) Chemical structures of galloyl glucose conjugates. The ester (β-glucogallin) or ether (GA-3-O-Glc and GA-4-O-Glc) linkages between gallic acid and glucose are indicated by arrows. (C) Pomegranate UGT84A23 and UGT84A24 enzyme assay products were identified via HPLC, MS and MS/MS analyses.
Fig 4.
Pomegranate UGT84A23 and UGT84A24 are active towards a range of phenolic substrates.
Specific activities of recombinant UGT84A23 (A) and UGT84A24 (B) at pH 5 (phenolic acids) and pH 8 (flavonoids) are shown. C. HPLC retention times, maximum absorbance (λmax), MS and MS/MS fragmentation patterns of the phenolic substrates and their glucose (Glc) conjugates formed by UGT84A23 and UGT84A24. 4-HBA, 4-hydroxybenzoic acid; 3,4-DHBA, 3,4-dihydroxybenzoic acid.
Table 1.
Kinetic parameters of pomegranate UGT84A23 and UGT84A24 towards selected phenolic acid substrates.
Fig 5.
Gene expression and punicalagin accumulation in overexpression (A) and RNAi knockdown (B) hairy root lines of UGT84A23 or UGT84A24. Changes in gene expression are represented on the primary y-axis as fold change relative to the vector transformed control, pK7WG2D-1 for overexpression lines and pHG8-1 for RNAi knockdown lines. Gene expression data presented are mean ± SD of three technical replicates for each line. Punicalagin accumulation (α and β isomers combined) is represented on the secondary y-axis as peak areas at 378 nm.
Fig 6.
Reduced punicalagin accumulation and increased galloyl glucoside production in UGT84A23 and UGT84A24 double RNAi knockdown hairy root lines.
(A) Gene expression and punicalagin accumulation in vector control and UGT84A23 and UGT84A24 double RNAi knockdown lines. Changes in gene expression are represented on the primary y-axis as fold change relative to the vector control pHG8-1. Gene expression data presented are mean ± SD of three technical replicates for each line. Punicalagin accumulation (α and β isomers combined) is represented on the secondary y-axis as peak areas at 378 nm. The RNAi constructs were derived from a chimera of the coding sequences (CDS) or the 3’ untranslated region (3’ UTR) of UGT84A23 and UGT84A24. (B) Overlay of HPLC chromatograms from a representative vector transformed control (pHG8-1) and a representative double knockdown line of UGT84A23 and UGT84A24 (3’ UTR-2). Peaks that are only present in the double knockdown lines and those that show varied accumulation in vector control and double knockdown lines are indicated. (C) MS and MS/MS analyses of peaks indicated in (B). HPLC retention times and λmax of standards and the respective peaks in the 3’ UTR-2 hairy root line are also shown.
Fig 7.
Subcellular localization of pomegranate UGT84A23 and UGT84A24 in Nicotiana benthamiana leaves analyzed by confocal microscopy.
Green fluorescent protein (GFP) was fused to the N- (denoted GFP-UGT84A23 or GFP-UGT84A24) or C-terminus (denoted UGT84A23-GFP or UGT84A24-GFP) of the UGT proteins. (A) The free GFP expression alone or merged with spRFP-AFVY or ER-mCherry. (B) The expression of the GFP fused UGT84A23 and UGT84A24 proteins alone or merged with spRFP-AFVY. (C) The expression of the GFP fused UGT84A23 and UGT84A24 proteins alone or merged with chlorophyll autofluorescence. (D) The expression of the GFP fused UGT84A23 and UGT84A24 proteins alone or merged with ER-mCherry.
Fig 8.
Subcellular localization of pomegranate UGT84A23 and UGT84A24 in root cells of pomegranate seedlings analyzed by immunogold electron microscopy.
The grids were incubated with 1.5% goat serum only (A, D, G), anti-UGT84A24 saturated with UGT84A23 and UGT84A24 proteins (B, E, H), or anti-UGT84A24 (C, F, I). Representative 9 μm2 image areas encompassing various organelles are shown. Arrows indicate immunogold labels. C, cytosol; E, ER; V, vacuole; M, mitochondria; W, cell wall; P, plastid; N, nucleus. Scale bar, 1 nm.
Table 2.
Distribution of immunogold particles in root cells of pomegranate seedlings.
Fig 9.
UGT84A24 expression in pomegranate peel, stem, root, leaf and aril.
Relative expression of UGT84A24 and a pomegranate Actin gene [18] was determined by semi-quantitative RT-PCR.
Fig 10.
Phenolic metabolites from different pomegranate tissues.
(A) β-glucogallin, gallic acid-3-O-β-D-glucopyranoside (GA-3-O-Glc) and gallic acid-4-O-β-D-glucopyranoside (GA-4-O-Glc) standards. HPLC elution profiles of phenolic metabolites from fruit peel (B), stem (C), root (D), leaf (E), and aril (F), as well as absorption spectra of β-glucogallin (G), GA-3-O-Glc (H), and GA-4-O-Glc (I) are shown. Metabolites eluted from 1 min to 8 min are shown as an inset in the corresponding panel. The peaks eluted at 3.26 min (the same retention time as that of GA-3-O-Glc, but with a different absorption spectrum) and 6.38 min (a similar retention time as that of GA-4-O-Glc, but with a different absorption spectrum) in peel, stem and root tissues are indicated with arrows and their absorption spectra are shown in (J) and (K), respectively. These two peaks were not detected in extracts obtained from leaf and aril tissues. α, punicalagin isomer α; β, punicalagin isomer β.