Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering

Abstract

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

Figure 1.

Structures of the flavan-3-ol monomers.

Figure 2.

Unrooted phylogram comparison of LAR, ANR, DFR, and related proteins from the reductase-epimerase-dehydrogenase superfamily. The tree was constructed from the ClustalW alignment using the neighbor-joining method and MrBayes software. The proteins are labeled according to the species followed by the name of the protein (e.g. CsDFR is DFR from tea; AAT66505). The protein names not listed in Supplemental Figures S3 or S4 are as follows: ANR from Ginkgo biloba (AAU95082); DFR from Arabidopsis (P51102); DFR1 and DFR2 from M. truncatula (AAR27014 and AAR27015); DFR from Cymbidium hybrid (AAC17843); IFR from Pisum sativum (AAB31368); IFR from alfalfa (P52575); pinoresinol:lariciresinol reductase (PLR) from Forsythia × intermedia (AAC49608); PLR from Thuja plicata (AAF63507); phenylcoumaran benzylic ether reductase (PCBER) from P. trichocarpa (CAA06707); and PCBER from Pinus taeda (AAF64173). The scale bar indicates the estimated number of amino acid substitutions per site.

Figure 3.

HPLC chromatograms from assays of recombinant tea LAR and ANR proteins. A, Chromatograms of products from assay of recombinant CsLAR protein (bottom panel) and boiled protein as a control (top panel). B, Profile of radioactivity in the peaks shown in A, bottom panel. The main product peaks in A and B elute at the same retention time as an authentic standard of (+)-catechin (for comparison of assays with M. truncatula LAR, see Pang et al., 2007). C to E, Chromatograms of products from assay of recombinant CsANR1 (top panels) and boiled protein as a control (bottom panels) incubated with the substrates cyanidin (C), delphinidin (D), and pelargonidin (E). F to H, Chromatograms of products from assay of recombinant CsANR2 protein (top panels) and boiled protein as a control (bottom panels) incubated with the substrates cyanidin (F), delphinidin (G), and pelargonidin (H). I and J, Chromatograms of products from assay of recombinant CsANR1 (I) and CsANR2 (J) on a chiral column. Solid lines represent enzyme products, and dashed lines represent authentic standards. An authentic standard of (+)-E was not available; this peak is surmised based on its retention time, UV spectrum, and the fact that the other three isomers had been identified. AFZ, Afzelechin; C, catechin; EC, epicatechin; EFZ, epiafzelechin (Fig. 1).

Figure 4.

Ectopic expression of CsLAR and CsANRs in M. truncatula hairy roots. A, Anthocyanin pigmentation phenotype of CsANR2-expressing M. truncatula hairy roots under light microscopy for regular observation (top left), and GFP signal observed by fluorescence microscopy for screening of positive events (bottom left). The remaining photographs were taken under white light for the vector control line (top middle), roots expressing CsANR2 (bottom middle), vector control roots stained with DMACA (top right), and a CsANR2-expressing line stained with DMACA (bottom right). B to D, Levels of anthocyanins (B), soluble PAs (C), and insoluble PAs (D) in transgenic M. truncatula hairy roots and a vector control (CK). Values show means and sd of triplicate analytical replicates from independent transgenic lines and controls that were pooled from several clonally propagated replicas of the same transgenic line. Asterisks indicate values that are significantly different from that of the vector control by Student’s t test (P < 0.05). FW, Fresh weight.

Figure 5.

PA profiles of transgenic M. truncatula hairy roots expressing tea ANR and LAR. Normal-phase HPLC chromatograms show DMACA-reactive PA peaks (by postcolumn derivatization) in extracts from hairy roots expressing CsLAR1 (line 5; A), CsANR1 (line 82; B), CsANR2 (line 9; C), CsANR2(GFP) (line 3; D), and wild-type control (CK; E) detected at 640 nm. Arrowheads indicate peaks that coelute with authentic standards of catechin and epicatechin. mAU, Milliabsorbance units.

Figure 6.

Expression of CsLAR and CsANRs in PAP1-expressing tobacco. A, Anthocyanin pigmentation phenotype of transgenic tobacco expressing CsLAR, CsANR1, or CsANR2 in the PAP1 background, together with PAP1, PAP1 × MtANR, and wild-type (CK) plants. B to D, Levels of anthocyanins (B), soluble PAs (C), and insoluble PAs (D) in transgenic tobacco expressing CsLAR, CsANR1, or CsANR2 in the PAP1 background, along with PAP1 × MtANR, PAP1 tobacco, and wild-type control (CK). Values show means and sd of triplicate analytical replicates from independent transgenic lines and controls that were pooled from several clonally propagated plants of the same transgenic line. Asterisks indicate values that are significantly different from that of the PAP1 line by Student’s t test (P < 0.05). FW, Fresh weight.

Figure 7.

PA profiles from transgenic tobacco expressing the PAP1 transcription factor and tea ANR and LAR enzymes. Normal-phase HPLC chromatograms show DMACA-reactive PA peaks (by postcolumn derivatization) in extracts from CsLAR (line 15; A), CsANR1 (line 45; B), CsANR2 (line 69; C), PAP1 × MtANR (D), PAP1 (E), and wild-type tobacco (CK; F) detected at 640 nm. The arrow indicates the peak that coelutes with an authentic catechin standard.