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J. Biol. Chem., Vol. 278, Issue 34, 31647-31656, August 22, 2003
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Proanthocyanidin Biosynthesis in Plants
PURIFICATION OF LEGUME LEUCOANTHOCYANIDIN REDUCTASE AND MOLECULAR CLONING OF ITS cDNA*,
From the CSIRO Plant Industry, GPO Box 1600, Canberra, Australian Capital Territory 2601, Australia
Received for publication, March 19, 2003 , and in revised form, June 4, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and thus is the first committed step in PA biosynthesis. The enzyme was purified to near homogeneity from PA-rich leaves of the legume Desmodium uncinatum (Jacq.) DC, partially sequenced and the corresponding cDNA cloned. The identity of the enzyme was confirmed by expressing active recombinant LAR in Escherichia coli and in tobacco and white clover. The enzyme is a monomer of 43 kDa (382 amino acids) and is most active synthesizing catechin (specific activity of
10 µmol
min+1 mg of protein+1) but also
synthesizes afzelechin and gallocatechin. LAR is most closely related to the
isoflavone reductase group of plant enzymes that are part of the
Reductase-Epimerase-Dehydrogenase (RED) family of proteins. Unlike all other
plant isoflavone reductase homologues that are about 320 amino acids long, LAR
has an additional 65-amino acid C-terminal extension whose function is not
known. Curiously, although Arabidopsis makes PA, there is no obvious
LAR orthologue in the Arabidopsis genome. This may be because
Arabidopsis seems to produce only an epicatechin, rather than a dual
catechin/epicatechin-based PA similar to many other plants. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Proanthocyanidins (PA)1 or condensed tannins are polymers of flavonoid molecules that occur widely in plants and interact strongly with proteins (1–3). The aromatic rings and multiplicity of hydroxyl groups on the flavanol subunits can interact with protein amino acid residues by hydrophobic and hydrogen bonds. These interactions are further increased in multidentate PA polymers. PA polymers are heterogeneous in their monomer composition, size, cross-linking bonds, and topology in different species and in different tissues and developmental stages of a single plant. The degree of polymerization ranges up to about 70, while the subunits are usually linked by C4–C8 bonds, less often by C4–C6 bonds and occasionally the polymer may be branched. The constituent subunit flavanols usually consist of combinatorial variants of two epimers of the C-ring 3-hydroxyl and the isomers that differ in the extent of hydroxylation of the B-ring (Fig. 1). Examples of the first group are catechin and epicatechin where the 3-hydroxyl is either trans or cis to the B-ring, respectively. The second group can be exemplified by afzelechin, catechin, and gallocatechin where the B-ring has one, two, or three hydroxyls, respectively. Only the broad outlines of the enzymology and transport processes of PA synthesis are known. The initial steps in the pathway take place in the cytoplasm, but the PA polymer accumulates in the vacuole. The first committed step in PA biosynthesis that diverges from the pathway common with anthocyanins is believed to be the synthesis of an initiating flavan-3-ol such as catechin or epicatechin directly or indirectly from flavan-3,4-diols. Based on chemical models, polymerization is then believed to occur by attack of the C4 atom of the electrophilic flavan-3,4-diol such as leucocyanidin onto the C8 atom of the nucleophilic flavan-3-ol. Polymerization continues by sequential addition of flavan-3,4-diols to the growing chain. This makes the pathway unusual because during the synthesis of a polymer of ten units only 10% of the monomer flux need proceed the full-length of the pathway while the 90% contributing to extension units does not pass through the catechin or epicatechin steps (Fig. 1.) The only biochemical steps that have been demonstrated in vitro are the NADPH-dependent reduction of (+)3,4-cis-leucocyanidin to catechin catalyzed by leucoanthocyanidin reductase (LAR) that removes the C4 hydroxyl group (Fig. 1) and an anthocyanidin reductase that converts anthocyanidin to epicatechin (4). The LAR reaction has been demonstrated in extracts of barley (5, 6), legumes (6–9), and gymnosperms (10) but until now has not been extensively characterized or purified, while the anthocyanidin reductase activity has been demonstrated in recombinant bacterial extracts (4) but not in plant extracts.
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Other steps that are likely to be necessary for PA biosynthesis include the transport of flavan-3-ols and flavan-3,4-diols into the vacuole and the polymerization of the monomers. The anthocyanin pathway from which PA diverges produces flavanol molecules with the C3 hydroxyl trans to the B-ring, whereas most PA extension units have the C3 hydroxyl cis to the B-ring. It is not known how or where in the pathway this apparent epimerization of the extension units occurs and whether separate enzymes and transporters then utilize the different epimers. Compared with the LAR reaction, the anthocyanidin reductase activity of the BANYULS protein requires a double reduction (presumably utilizing two NADPH molecules) to produce epicatechin. It is not clear what the intermediate molecule is or whether it is released from the enzyme active site during catalysis. Conceivably such an intermediate could function as an extension unit in PA biosynthesis.
Molecular genetic studies in Arabidopsis of the PA-containing testa or seed coat have revealed mutants specifically deficient in PA accumulation. Transparent Testa (TT) mutants that have light-colored seed coats have been found to be defective in the anthocyanin/PA pathway and some of these seem to be specifically deficient in PA accumulation (11). Using the PA-specific stain dimethylaminocinnamaldehyde (DMACA) as a screen, we have recently described six novel Tannin-deficient seed (TDS) Arabidopsis mutants (3). More than 12 genes identified by mutation are required specifically for PA biosynthesis but many are not cloned (3). Several of the cloned genes are transcription factors that regulate the expression and tissue specificity of enzymes in the anthocyanin/PA pathway. Two cloned genes that seem to function in the pathway are TT12, a MATE-type membrane transporter that may transport PA precursors into the vacuole (11) and BANYULS (4, 12). The TT12 protein seems to be a transporter but its activity has not been defined biochemically. We do not know whether TT12 transports initiating units, namely epicatechin, which occurs in Arabidopsis, and catechin, which does not seem to occur in Arabidopsis but is found in other PA-containing plants, as well as the unknown extension units for the PA polymer. Double mutant analysis indicates that BANYULS functions after DFR and before TT12 in the PA pathway (11). Mutant banyuls plants no longer produce PA in the seed coat but produce extra anthocyanin. The anthocyanin-accumulating phenotype is consistent with BANYULS being the Arabidopsis gene for LAR (12) but the recent demonstrations that the BANYULS protein converts anthocyanidin to epicatechin (4) and that the tds4 mutation occurs in the Arabidopsis Leucocyanidin DiOXygenase (LDOX) gene that synthesizes the BANYULS substrate anthocyanidin2 suggests that the pathway to PA is more convoluted than previously thought because the two potential initiating units catechin and epicatechin are made from leucocyanidin by one and two enzymatic steps, respectively.
We report here on the purification and cloning of LAR from PA-rich leaves of the legume D. uncinatum (Jacq.) DC. The deduced sequence revealed that LAR is most closely related to the isoflavone reductase (IFR) group of enzymes that are common in plants and is a member of the larger and more widespread Reductase-Epimerase Dehydrogenase (RED) family of proteins (13, 14). Expression of the active, recombinant LAR in Escherichia coli, and plants confirmed the identity of the purified protein.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials—Afzelechin was obtained from Prof. E Malan, (+)dihydromyricetin and (+)dihydrokaempferol from Industrial Research (Lower Hutt, NZ), and (+)dihydroquercetin from Appin Biochemicals (Abingdon, UK). Catechin ((+)catechin or (2R,3S)-catechin) and (–) catechin (2S,3R)-catechin) were obtained from Sigma. Unlabeled and 3H-labeled flavan-3,4-diols were prepared essentially described by Tanner and Kristiansen (6) using the HPLC systems in "Supplementary Materials." Flavan-3,4-diol was dried under N2 immediately before adding other assay components. Dyes were coupled to Sepharose as described previously (15, 16). Standard molecular biology procedures were conducted as described in (17). An Arabidopsis line (Salk 040250) with a T-DNA insertion in the BANYULS (At1g61720) gene produced by The Salk Institute Genomic Analysis Laboratory and obtained from The Arabidopsis Biological Resource Center. Homozygous mutant plants were obtained by growing supplied T3 seed and examining seed of T4 plants for the banyuls phenotype, namely red (anthocyanidin-containing) developing seed, dark grayish mature seed and PA deficient when stained with DMACA. Mutant plants were resistant to kanamycin showing the nptII selectable marker gene was still functioning. D. uncinatum (cv Silverleaf), tobacco (Nicotiana tabacum), and white clover (Trifolium repens) were grown in a glasshouse at 20 °C under ambient daylight and 15 °C nights.
Assay of LAR Activity—LAR activity was determined by
monitoring the conversion of 3,4-cis-[4-3H]leucocyanidin
to [3H]catechin by HPLC
(6). The 100-µl assay
mixture contained 10% (w/v) glycerol, 100 mM phosphate, 1
mM dithiothreitol adjusted to pH 7.0 with NaOH and the substrates,
0.5 mM NADPH and 2.5 µM
3,4-cis-[4-3H]leucocyanidin (1 µCi), or other
3,4-cis-diol substrates. The assay was initiated by the addition of
enzyme and incubated at 30 °C for 30 min. The assay was terminated by
extraction with 0.2 ml of ethyl acetate containing 10 nmol of unlabeled
flavan-3-ol product e.g. catechin, as carrier followed by two
additional extractions with ethyl acetate. The ethyl acetate extracts
containing flavan-3,4-diol and flavan-3-ol were dried under a stream of
nitrogen at room temperature, dissolved in 100 µl of water, and separated
by HPLC while monitoring 3H and A280 for
product synthesis and recovery, respectively. Reverse phase HPLC of LAR
reactions was performed at 35 °C in systems IIa and III that are described
under "Supplementary Materials." Routine assays were separated by
a reverse phase procedure using a Goldpak 3 µm C18 column, 50 x 4.5
mm (Activon), eluted with 2% (v/v) acetic acid at 2
ml·min–1 (System II) that included both
substrate and product in the chromatogram. No additional radioactive peaks
were seen beyond catechin on the isocratic HPLC system II. In this isocratic
system epicatechin would be retained on the column while in a gradient system
that eluted epicatechin after catechin (System III) the radioactive product
comigrated with catechin and no radioactivity was detected after the catechin
peak. The 3H was detected with 10% efficiency so 200,000
cpm were recovered in the HPLC chromatogram. In the presence of excess LAR
more than 95% of the 3,4-cis-[4-3H]leucocyanidin could be
converted to [3H]catechin product. When the LAR assay was conducted
with higher concentrations of unlabeled leucocyanidin and the separation
carried out without addition of carrier catechin an A280
peak of newly synthesized catechin was observed. When other
[3H]flavan-3,4-diol substrates were used the 3H products
of the LAR reaction always comigrated with the appropriate unlabeled carrier
flavan-3-ol product, i.e. afzelechin, catechin, and gallocatechin. A
more stringent separation on a chiral column that separates (+) catechin from
the rare (–) catechin confirmed that the product of the LAR reaction
comigrated with (+) catechin (Fig.
2). In this separation the leucocyanidin substrate remained
adsorbed to the column and was eluted in the postchromatography washing
procedure. Chiral separation of the ethyl acetate-extracted reaction mixture
was achieved on a 250 x 4.6 mm Chiralcel OJ-H (Daicel) column, 5-µm
packing, protected with a similar guard column (50 x 4.6 mm) eluted with
a solvent consisting of hexane: ethanol (70:30 v/v) at 1 ml/min. HPLC followed
by electrospray ionization (negative mode) mass spectrometry of LAR reaction
products was conducted as described under "Supplementary
Materials." Kinetic constants were determined with LAR purified beyond
the dye columns. Transformation of 3,4-cis-leucocyanidin and NADPH by
the purified or recombinant LAR only occurred in the presence of enzyme and
both substrates.
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Protein Determinations—Protein was usually measured using a
dye-binding method (18) using
-globulin as standard. In the final enzyme preparation, the presence of
detergent interfered with this method so protein was measured either by
fluorescence of the o-phthalaldehyde amino acid adducts after acid
hydrolysis of the protein (19)
using -globulin as standard, or by densitometry of the Coomassie
Blue-stained protein band after SDS-PAGE. Both methods yielded comparable
results.
PA Determination—PA was determined using a method modified from Ref. 20 in a final reaction containing 0.18% DMACA, 86% butyl alcohol, 6.3% ethanol, 0.27 M HCl, and 7.5% water by measuring the absorbance at 650 nm.
Purification of LAR—Small unexpanded leaflets ( 15 mm
long) of D. uncinatum were harvested directly into liquid nitrogen
and stored at –80 °C. Frozen leaves were ground to a powder in
liquid N2, and the powder stored for no more than a few weeks at
–80 °C. When required, the frozen leaf powder (100 g) was warmed to
–20 °C (0.5 h), then homogenized in 200 ml of Grinding Buffer (50
mM phosphate, 10% (w/v) glycerol, 1% (w/v) polyethylene glycol-6000
(PEG), 1 mM Na2EDTA, 25 mM sodium ascorbate,
5 mM dithiothreitol, 20 mM 2-mercaptoethanol, 2 µg of
leupeptin·ml–1, 1 µg of
pepstatin·ml–1, 1 µg of
E64·ml–1, 0.1 mM
phenylmethylsulfonyl fluoride, adjusted to pH 8.0 at room temperature with
NaOH). The homogenate was filtered through Miracloth, centrifuged at 20,000
x g for 30 min and the pH of the supernatant readjusted to pH
8.0.
PEG (0.3 g/ml) was dissolved in the supernatant, and the precipitate removed by centrifugation as above. The PEG supernatant was adjusted to pH 5.8 with 1 M acetic acid, centrifuged as above and the acid precipitate resuspended in 20 ml of Buffer 1 (10 mM phosphate, 0.1% (v/v) Tween 20, 20% (w/v) glycerol, 1 mM Na2EDTA, 5 mM dithiothreitol adjusted to pH 7.0 with NaOH) containing 2 µg of leupeptin·ml–1, 1 µg of pepstatin·ml–1, and 1% w/v PEG.
The acid precipitate was applied to a column of Sepharose CL 4B-Procion Yellow H3R (5.1 cm2 x 17 cm) at 2.5 ml·min–1, the second unbound protein peak collected, and applied to a column of Sepharose S200-Bayer 4 (5.1 cm2 x 16.5 cm) at 2 ml·min–1. The column was washed extensively with Buffer 1 and bound LAR eluted with a 400-ml linear salt gradient to 1 M NaCl in Buffer 1. Fractions (10 ml) containing LAR activity were pooled, concentrated over a YM10 membrane (Amicon), desalted into Buffer 2 (10 mM phosphate, 0.01% (w/v) Tween 20, 20% (w/v) glycerol, 5 mM dithiothreitol, 2 µg of leupeptin·ml–1, 1 µg of pepstatin·ml–1 adjusted to pH 7.0 with NaOH), on PD10 columns (Amersham Biosciences), and applied to a column (0.65 cm2 x 8.5 cm) of Sepharose CL4B-Cibacron Orange F-R (Ciba-Geigy), at 1 ml·min–1. The column was washed extensively with Buffer 2 and bound LAR eluted with 5 µM NADPH in Buffer 2 (10 ml). Fractions (1 ml) containing LAR activity were pooled and applied to a 5-ml column of hydroxylapatite (Bio-Rad EconPak CHTII) at 0.5 ml·min–1 and eluted with Buffer 2.
Fractions with LAR activity were combined from two experiments, the buffer exchanged to Buffer 3 (25 mM triethanolamine, 20% (w/v) glycerol, 0.01% (w/v) Tween 20, 1 mM dithiothreitol adjusted to pH 7.0 with HCl) and applied to a Mono Q HR5 x 5 column (Amersham Biosciences) at 1 ml·min–1. The column was washed with 10 ml of Buffer 3, bound LAR eluted with a linear gradient from 0 to 250 mM NaCl in Buffer 3 over 20 min, and 1-ml fractions were collected (Fig. 3).
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The two fractions containing peak LAR activity were pooled, diluted to 10 ml with Buffer 3, re-applied to the Mono Q column, washed as before, and LAR eluted at 1 ml·min–1 with a linear gradient from 0 to 150 mM NaCl in Buffer 3 over 20 min. Fractions of 0.5 ml were collected. The two peak LAR fractions were pooled, concentrated to 0.2 ml with an Ultrafree concentrator (Millipore), and stored at –20 °C before protein sequencing. Aliquots were also diluted with an equal volume of glycerol and stored at –20 °C with no loss of activity for at least 6 months.
Amino Acid Sequencing—Purified LAR protein (1.3 µg) was separated by SDS-PAGE. The 45-kDa protein band (Fig. 2) was excised, dried, and digested with trypsin. The tryptic peptides were analyzed by ESI-TOF MS/MS at the Australian Proteome Analysis Facility (APAF). The sequences obtained were Pep1: T(L/I)VVGGTGF(L/I)GQF(L/I)TK, Pep2: (L/I)GFGYPTF(L/I)(L/I)VR, Pep3: EYE(L/I)DVV(L/I)S(L/I)VG-GAR, Pep4: (L/I)(L/I)D Q(L/I)T(L/I)(L/I)EA(L/I)K, Pep5: F(L/I)PSEF-GHDVDR, Pep6: AYFID, Pep7: (L/I)EVDFGPVEPG(L/I), and Pep8: (L/I)(L/I)SGW.
For N-terminal sequencing 0.6 µg of purified LAR was separated by SDS-PAGE, transferred to Problot membrane (Applied Biosystems), the 45-kDa band excised, and subjected to Edman degradation at the Australian Proteome Analysis Facility. The sequence obtained, (PepN) was TVSGAIPSMTKNRTLVVGGTGFIGQFIT.
Amplification of LAR Gene Fragments—Blast analysis of the initial peptides Pep1, Pep2, and Pep4 suggested that they could belong to an IFR-like protein and would occur in the order: N terminus, 1, 2, 4, and C terminus. Degenerate oligonucleotides coding for these peptides were used in PCR mixes with D. uncinatum leaf cDNA. A 228-bp DNA fragment was produced with primers based on Pep2, (5'-GGITT(C/T)GGITA(C/T)CCIACITT(T/C)) and Pep4, (5'-(T/C)TTIAIIGC(C/T)TCI-AII-AIIGTIAI(T/C)-TG(G/A)TCA). The PCR fragment was cloned and sequenced.
Cloning of LAR cDNA—D. uncinatum mRNA was purified from
total RNA isolated from newly emerged leaves using PolyATract (Promega).
Double-stranded cDNA was synthesized using oligo-dT primers and directionally
cloned into 
Uni-ZAP XR (Stratagene). The library (106
plaques) was screened with the 228-bp LAR PCR product. Ten positive clones
from many hundreds initially identified were plaque-purified. Three clones
were sequenced, and the others were characterized by PCR analysis using
LAR-specific and plasmid-specific primers.
Expression of LAR Activity in E. coli—The coding region of
the LAR cDNA (nucleotides 122–1270) was amplified by PCR and introduced
into the BamHI site of the pET3a expression vector (Novagen) to
express the LAR polypeptide containing the 14-amino acid N-terminal T7 tag.
This plasmid was then introduced into E. coli strain XL1 blue.
Bacteria were grown in LB at 37° for 2 h, 1 mM
isopropyl-1-thio-
-D-galactopyranoside added and the bacteria
grown for another 4 h. The cells were collected, disrupted by sonication in 5
volumes of Grinding buffer, and insoluble material removed by centrifugation.
Portions of 1–20 µl were assayed for LAR activity.
Plant Transformation—The LAR cDNA was excised from pBlue-script SK II using the 5' polylinker SmaI site and the SpeI site in the 3'-UTR of the cDNA and ligated into the SnaBI and AvrII sites of the binary plasmid pPLEX X002 (21). This inserts the cDNA between the subterranean clover stunt virus double S4 promoter and the Flaveria Me1 terminator. The binary plasmid was transferred into Agrobacterium tumefaciens and used to transform T. repens and N. tabacum (22, 23). For detection of LAR activity, plants were extracted in Grinding Buffer and insoluble material removed by centrifugation. Portions were assayed for LAR activity in the standard assay.
DNA and RNA Analysis—Genomic DNA was isolated from leaves of D. uncinatum using a CTAB/PVP (cetyltrimethylammonium bromide/polyvinylpyrrolidone) extraction procedure, digested with restriction enzymes and separated by electrophoresis (10 µg per track) and transferred to a Hybond N+ nylon membrane. The membrane was probed with a 32P-labeled C-terminal fragment obtained by PCR (bases 1007–1599) from the full-length cDNA. The membrane was hybridized at 65° in 2x SSC and washed in 2x SSC.
Total RNA was isolated from D. uncinatum leaves using the Qiagen RNeasy system, separated (10 µg per track) in formaldehyde gels and transferred to Hybond N+ nylon membrane. The membrane was probed with a C-terminal fragment of the LAR cDNA in 2x SSC at 65° and washed with 2x SSC at 60°.
Immunological Procedures—Two peptides comprising LAR peptides C1, His321–Lys338 and C2, Ile349–Lys370 were synthesized and an octavalent multi-antigenic peptide produced (24). Rabbit antisera were raised to each peptide and the IgG purified by chromatography on protein G. Immunoprecipitations with crude antisera utilized killed Staphylococcus aureus cells to precipitate antibody-bound LAR from free LAR. Interaction between purified IgG and LAR was also demonstrated by monitoring the formation of a 180-kDa complex between the antibody and active LAR by Superdex gel filtration chromatography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Enzyme Purification—LAR activity was purified
50,000-fold from young PA-producing leaves of D. uncinatum to a
specific activity of
10µmol·min–1·mg of
protein–1
(Table I). The procedure
utilized initial fractionation with polyethylene glycol to protect and
separate the enzyme from the inhibitory PA present in the leaf extract and to
concentrate the enzyme in preparation for column chromatography. The amount of
PA was also monitored during the purification. More than 95% of the extracted
PA remained in the 30% PEG/pH 5.8 supernatant while about 1% of the PA was
present in the protein precipitated by 30% PEG and a further 1% in the LAR
fraction precipitated at pH 5.8. PA was undetectable in the LAR fractions
after the first chromatography step. The subsequent series of dye-ligand
chromatography steps made use of the strategy outlined in Scopes
(25) to exploit the unique
interaction of the enzyme active site with a range of dyes. About 40 dye
columns were screened to select the best dye column combinations
(16). The initial dye-column
did not bind LAR but presumably bound proteins that might otherwise co-purify
with LAR on subsequent dye columns. The next dye column bound LAR, which could
then be eluted by the substrate NADPH. The elution step also utilized the
extraordinary affinity of LAR for NADPH (0.4 µM) to help
separate LAR from many other enzymes that also bind to the dye column via
their NADPH binding site but with lower affinity for NADPH. LAR could only be
released from the final dye-column by high salt. The purification was
completed by hydroxylapatite and Mono Q anion exchange chromatography. The
final preparation contained a major polypeptide (Mr
45 kDa by SDS-PAGE, Figs.
3 and
4) that always co-purified with
LAR activity in the final purification steps as well as on a Superdex 200 gel
filtration column where it elutes as a 52-kDa protein.
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Enzyme Kinetic Properties—LAR had a broad pH optimum around pH 7.0, maintaining 95% of maximum activity from pH 6.2 to 7.8 in phosphate buffer. LAR showed normal Michaelis-Menten kinetics for all substrates. LAR has a very low Km for NADPH (
0.4
µM) but can utilize NADH (Km 60
µM) at 30% of the rate with NADPH.
3,4-cis-leucocyanidin was the preferred flavan-3,4-diol substrate
(Km 6 µM) while
3,4-cis-leucodelphinidin had a comparable
Km (5 µM) and 20% of the
Vmax and 3,4-cis-leucopelargonidin had a
significantly higher Km (26 µM) and
lower Vmax (4% of leucocyanidin). Although the LAR
purification was followed using leucocyanidin as a substrate the ratio of
activity with these three flavanol-diol substrates in a crude D.
uncinatum leaf extract was similar to that of the purified enzyme. This
suggests that the enzyme we purified can account for the leucopelargonidin and
leucodelphinidin reductase activity present in the crude extract. Both
products of the LAR reaction inhibited catalysis. NADP+ weakly
inhibited (I50 of 500 µM) while the I50
for the flavan-3-ol products, afzelechin, catechin, and gallocatechin were 14,
12, and 280 µM, respectively. The 2,3-trans
flavan-4-ols, epi-catechin (I50 1 mM) and
epi-gallocatechin (I50 1.4 mM), which are also potential
LAR products, were much less inhibitory than the 2,3-cis-flavan-4-ols
above. The non-physiological 3,4-trans-isomers were not detectably
converted by the LAR enzyme, rather they inhibited the reaction with
3,4-cis-leucocyanidin; 3,4-trans-leucocyanidin and
3,4-trans-leucopelargonidin had I50 values of 120 and 460
µM, respectively. No significant inhibition was observed with 1
mM 3,4-trans-leucodelphinidin. Cyanidin and pelargonidin,
products of leucoanthocyanidin dioxygenase (anthocyanidin synthase) and
substrates of anthocyanidin reductase inhibited LAR by 48 and 55% at 6
µM while delphinidin inhibited by 97% at 60 µM.
Among other phenylpropanoid metabolites, LAR inhibition was most marked for
the dihydroxylated B-ring compounds, eriodictyol and dihydroquercetin, which
gave 53 and 67% inhibition at 10 µM, respectively
(Table II). Desalting the
enzyme into buffer without reducing agents led to loss of activity. Addition
of 1 mM dithiothreitol or 10 mM 2-mercaptoethanol to the
inactive enzyme restored 40% of the activity, but prolonged incubation with
dithiothreitol or 2-mercaptoethanol did not increase LAR activity further.
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Amino Acid Sequence and cDNA Cloning—Amino acid sequence from the 45-kDa polypeptide was obtained by N-terminal sequencing as well as mass spectrometric analysis of eight tryptic peptides. BlastP analysis of GenBankTM indicated that many of these peptides showed similarity to plant IFR-like proteins. RT-PCR of Desmodium leaf RNA using redundant oligonucleotides coding for two of these peptides (Pep2 and Pep4), and subsequent cloning gave a PCR product of the expected size (228 bp) for an IFR-like protein. The deduced 59 amino acid sequence of the region internal to the primers revealed 21 amino acids identical to the tryptic peptides Pep2, Pep3, and Pep4 of LAR. This PCR product was then used to screen a D. uncinatum leaf cDNA library. Ten clones were purified and three apparently full-length clones were sequenced. All were essentially identical, differing only in length at the 5'-end and were identical to the internal sequence of the PCR product used to screen the library. The cDNA contained an open reading frame of 382 amino acids that included the N-terminal sequence (which lacked the initiating Met) as well as six of eight tryptic peptides (Pep1-Pep6) found in the purified LAR protein (Fig. 5). The 1657 nucleotide cDNA contained 121 nucleotides upstream of the first in-frame ATG and 387 nucleotides after the stop codon and before the poly(A) tail.
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Immunological Analysis—Antibodies were raised to two peptides present in the C-terminal extension that does not occur in other IFR-like proteins. Both antibodies recognized the 45-kDa denatured LAR after SDS-PAGE and blotting of either the purified protein or crude extracts (Figs. 4 and 6). The antibodies did not inhibit LAR activity but we could show that the antibodies bound to native LAR because immunoprecipitation experiments removed all LAR activity from solution. Furthermore, incubating antibody and LAR resulted in formation of an active 180-kDa complex (presumably 1 LAR:1 IgG) that could be separated by gel filtration from the 52-kDa native LAR. Preimmune serum did not interact with denatured or native LAR. Monitoring the purification with these antibodies showed recovery comparable to that found with LAR activity (Fig. 4).
LAR Gene and Transcript Analysis—Southern blot analysis of D. uncinatum genomic DNA probed with the LAR-specific 3'-probe showed a single band after digestion with either EcoRI, HindIII, SacI, SacII, SmaI, XbaI, or BamHI (Fig. 7).
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Northern blot analysis with the C-terminal probe showed a major band of
1800 nucleotides. The intensity of this mRNA species was highest in young
leaves and declined in older leaves in parallel with LAR
activity.3
Expression of the D. uncinatum LAR in E. coli and Transgenic
Plants—Introduction of the LAR coding region into E. coli
in a pET expression plasmid enabled the bacteria to produce the 45-kDa LAR
polypeptide, detected by blotting and probing with the C2 antibody
(Fig. 4) as well as LAR
activity (Fig. 8). E.
coli extracts without the LAR coding region produced no LAR protein or
activity. The tobacco and white clover plants transformed with the LAR cDNA
also expressed LAR protein and activity. Activities were about 0.5–20
pmol·min–1·mg of
protein–1 or 100-fold less than found in the
PA-rich Desmodium leaf extracts, but significantly higher than the
activity found in the untransformed plants, which produced no detectable
activity(< 0.5 pmol·min–1·mg of
protein–1).
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No Detectable LAR Activity in PA-synthesizing Siliques of
Arabidopsis—Measurement of LAR activity in developing
Arabidopsis siliques (< 1 cm) revealed no detectable activity
either in PA-synthesizing wild-type plants or PA-deficient banyuls
plants. The use of the banyuls extract served as a PA-negative
control to ensure any LAR activity was not inactivated by endogenous PA of
wild-type seeds. We estimate the LAR activity to be <50
pmol·min–1·g fresh
wt–1 compared with 7
nmol·min–1·g fresh
wt–1 in PA-synthesizing Desmodium leaf
extracts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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LAR Catalytic Activity—LAR catalyzed the NADPH-dependent conversion of 2,3-trans-3,4-cis-[3H]leucocyanidin to catechin (2R,3S)-catechin) in a widely used assay (5–9). The validity of the assay was confirmed by cochromatography of the 3H product with authentic catechin in three HPLC systems including a chiral column (Fig. 2) that resolves catechin from (–) catechin (2S,3R)-catechin). Incubation of LAR and NADPH with unlabeled 2,3-trans-3,4-cis-leucocyanidin transformed the leucocyanidin into an A280 absorbing peak that eluted at the same retention value as authentic catechin. LAR also converted the related B-ring isomeric substrates leucopelargonidin and leucodelphinidin to afzelechin and gallocatechin respectively as judged by cochromatography with authentic standards on HPLC. The expected molecular masses for the negative ions of these flavan-3-ol products as determined by HPLC followed by MS were obtained, namely 273.3, 289.3, and 305.3 Da for afzelechin, catechin, and gallocatechin, respectively.
While LAR can synthesize catechin it is possible that it could also synthesize epicatechin from 2,3-cis-3,4-trans-leucocyanidin in which case LAR could function as a redundant step to the anthocyanidin reductase activity of BANYULS. The molecule 2,3-cis-3,4-trans-leucocyanidin is not commercially available nor is it known to occur free in plants so we could not test this potential substrate. Another way to explore the active site is to determine how well potential products inhibit the enzyme reaction. We found that epicatechin was about 100-fold less effective as an inhibitor of LAR than the known product, catechin, suggesting that LAR may not synthesize epicatechin efficiently. While a number of flavanoid-derived substances inhibit LAR it may be physiologically significant that substrates of anthocyanidin reductase, pelargonidin, cyanidin, and delphinidin are potent inhibitors of LAR. Little is known of the cytoplasmic levels of these compounds as well as leucocyanidin so more detailed analysis of these interactions between the enzymes synthesizing catechin and epicatechin must wait.
Protein and Gene—LAR activity is highest in very young
leaves of D. uncinatum that are actively synthesizing PA, so it was
necessary to purify the enzyme from tissue that plant biochemists normally
avoid, namely tannin- or PA-rich tissue. The inclusion of 1% PEG in the
Grinding Buffer was essential to obtain high levels of soluble LAR activity,
while fractionation with PEG and isoelectric precipitation in the presence of
30% PEG removed 99% of the leaf PA. The remaining PA was removed in the first
chromatographic step. The final specific activity of purified LAR (10
µmol·min–1·mg of
protein–1) is significantly greater than that of
other sequence-related enzymes, notably Chickpea IFR, Forsythia
intermedia pinoresinol-lariciresinol reductase (PLR) and Pinus
taeda phenylcoumaran-benzylic ether reductase (PCBER) that have specific
activities of 3, 0.25, and .002
µmol·min·mg–1 protein,
respectively
(26–28).
This suggests that the most of the protein in the final preparation can be
largely accounted for by LAR enzyme and that it is unlikely either that two
45-kDa proteins are present, or that LAR activity is due to a minor
contaminating protein in the final preparation. It also suggests that the LAR
reaction we have documented is unlikely to be a side reaction of an enzyme
that catalyzes another reaction at a greater rate. However given the very low
specific activity of the PCBER enzyme, it is conceivable that a better
substrate may be found for it. The specific activity of the anthocyanidin
reductase activity of BANYULS has not been reported. Seven of nine peptide
sequences could be accounted for by the deduced LAR protein sequence further
indicating that the 45-kDa protein consisted largely of LAR. Although the
crude leaf extract can transform other flavan-3,4-diols to flavan-3-ols, it is
likely that only a single LAR enzyme is expressed in D. uncinatum
leaves because the ratio of activities with leucocyanidin and leucodelphinidin
are the same in the unfractionated extract as for the purified LAR. The
activity with leucopelargonidin in crude extracts was too low to measure. This
is consistent with the properties of the purified enzyme that is least
efficient with leucopelargonidin as substrate. Analysis of genomic DNA also
indicated there is only a single LAR gene in D. uncinatum
The predicted protein was most closely related (40% identical) to
various plant IFR-like proteins including chickpea IFR, Forsythia
intermedia PLR and Pinus taeda PCBER and many uncharacterised,
related homologues. These proteins belong to the RED or
SDR(short-[polypeptide] chain dehydrogenase/reductase) protein superfamily
(13,
14).
The LAR protein contains sequence motifs present in other RED proteins (13, 14, 29, Fig. 5, "Supplementary Material" and Fig. 1). These include the glycine-rich cluster from Gly18 to Gly25 that interacts with the pyrophosphate backbone of NAD(P) and the three residues of the catalytic triad Ser120, Tyr139, and Lys142. The tyrosine residue that seems to be important for high catalytic activity in the more distant RED family members is not absolutely conserved in plant IFR homologues. The catalytically verified IFR and PLR enzymes contain phenylalanine and leucine4 instead of tyrosine while 6 of 8 Arabidopsis IFR homologues contain phenylalanine rather than tyrosine. Based on homology modeling to known RED family structures it seems likely that Arg44 interacts with the 2'-phosphate of NADPH and contributes to the 100-fold greater affinity of LAR for NADPH compared with NADH. This Arg is absolutely conserved in the top 100 matches to plant IFR-like proteins in GenBankTM. In NAD-binding RED proteins an acidic residue is usually found in this position where it would repel the 2'-phosphate of NADP. All other IFR-like proteins (and DFR, BANYULS, and Sqd1, Ref. 30) have the conserved Arg whereas the comparable residue in NAD-utilizing enzymes is Asp/Gly. The crystal structure of Sqd1, an enzyme of sulfo-lipid synthesis, was determined with NAD+ and uridine diphosphate-glucose bound to the active site, but since Sqd1 (At4g33030) contains a chloroplast transit peptide it is likely to be located in the chloroplast and use NADP+ in preference to NAD+ like most chloroplast enzymes.
LAR and the IFR Family—LAR is a 42.7-kDa protein containing
382 amino acids and exists in solution as a monomer as judged by gel
filtration. Chick pea IFR is also a monomer in solution but PLR and PCBER seem
to be dimers
(26–28).
All the most closely related plant homologues of LAR are proteins of about 320
amino acids in length. The predicted LAR protein contains an extra 65 amino
acids at the C terminus that seems beyond the minimal catalytic domain defined
by structures of all other IFR proteins. Interrogation of the non-redundant
data base with this 65 amino acid sequence does not show any significant
matches. The predicted molecular mass and pI of the 382 amino acid protein
(42.7 kDa and 5.9) are comparable to those of purified LAR (45 kDa and 5.8)
suggesting that this C-terminal extension is present in the mature protein.
The size of the mRNA (1.8 kb) determined by electrophoresis and blotting
is also consistent with the cloned cDNA (1.7 kb). Furthermore, antibodies
raised to synthetic peptide sequences present in this C-terminal extension
bind to denatured LAR in protein blots of crude and purified enzyme
(Fig. 6) as well as to native
LAR in immunoprecipitation and gel filtration mobility shift experiments.
These antibodies do not inhibit LAR activity further suggesting that the
C-terminal extension is not essential for catalytic activity. The C-terminal
extension largely survives purification suggesting that it is not very
susceptible to proteolysis and probably folds in a specific conformation, but
with at least both C1 and C2 antigenic sites accessible to antibodies. Some
other eukaryotic enzymes have extra polypeptide beyond what is required for
catalysis. Plant sucrose phosphate phosphatase (SPP), a member of the HAD
(Halo Acid Dehalogenase) family, is a good example of this
(16). Cyanobacterial SPP is a
260 amino acid protein that is similar in size to many other HAD domain
proteins and probably comprises the minimal domain necessary for catalysis.
Plant SPPs are about 420 amino acids in length, consisting of the N-terminal
260 amino acid catalytic domain followed by a 160-amino acid C-terminal
extension. Similarly, compared with homologous malate dehydrogenases from
bacteria and eukaryotes, chloroplast NADP-malate dehydrogenase has both N- and
C-terminal extensions that mediate the light-dependent regulation of enzyme
activity (31). It is not clear
what function the LAR C terminus serves but we can speculate that it may be
involved in regulation, localization, helping to fold the catalytic domain or
interactions with other proteins. Sqd1, a RED family enzyme that is involved
in synthesis of UDP-sulfoquinivose in plants is 394 residues long and also has
a longer C-terminal region than most other RED members
(30). The Sqd1C-terminal
extension forms three helices linked by extended structure, which folds
intimately around residues 230–330 that cover the UDP-glucose site
(equivalent to the leucocyanidin binding site) of the active site groove. The
LAR C-terminal extension may form a similar folding structure and possibly
shield the reactive leucocyanidin molecule from the solvent.
The Arabidopsis genome has no gene closely related to
Desmodium LAR that could be regarded as an LAR orthologue but does
have eight IFR-like genes. These are a three gene tandem repeat on chromosome
1 (At1g75280, At1g75290, and At1g75300) and isolated genes on chromosome 1
(At1g19540 and At1g32100) and three isolated genes on chromosome 4 (At4g13660,
At4g34540, and At4g39230). Moreover, we could not detect LAR activity in
developing Arabidopsis siliques that were actively accumulating PA
intermediates. The Arabidopsis IFR-like proteins are more closely
related to each other and to the catalytically defined IFR, PLR, and PCBER
from other plant species (including a gymnosperm) than they are to LAR, as
illustrated in the phylogenetic tree of
Fig. 9. Unlike LAR, they are
all about 320 amino acids long and do not have a C-terminal extension. Our
re-examination of the annotated genes and their ESTs indicates that the
annotations are correct at the C-terminal end. BANYULS orthologues are readily
identifiable among plant ESTs including grape vine, cotton, and bean as well
as the anthocyanin reductase of Medicago truncatula
(4). These proteins are
60–80% identical to the archetypal Arabidopsis BANYULS whereas
the nearest Arabidopsis IFR homologues have only 40% identity to
Desmodium LAR. The phylogenetic tree of
Fig. 9 shows that these BANYULS
orthologues from other species cluster together with the Arabidopsis
BANYULS and are more closely related to Arabidopsis BANYULS than they
are to Arabidopsis DFR. Arabidopsis makes PA in the seed
coat (3) so it is surprising
that a readily recognizable LAR gene is not present. Many Arabidopsis
mutants deficient in tannin accumulation have been described including some of
the TT mutants as well as more recently described Tannin-deficient
Seed (TDS) mutants
(3). One mutant that was
considered a possible candidate for an LAR gene is BANYULS but the
BANYULS protein is only 25% identical to LAR, although since it is also a
member of the RED family, like LAR, it is plausible that it could catalyze a
similar reaction. The TDS4 mutant does not accumulate epicatechin
(3) and epistasis analysis
places TDS4 before BANYULS in the Arabidopsis PA pathway.2
Recombinant BANYULS expressed in E. coli does not catalyze the
synthesis of catechin in the standard LAR assay.3 BANYULS has
recently been shown to catalyze the conversion of anthocyanidin to epicatechin
(4) and the TDS4 gene
has been identified as the Arabidopsis LDOX (At4g22880)
gene2 indicating that there are two pathways from leucocyanidin to
catechin-like molecules, a single step reaction catalyzed by LAR and a two
step reaction catalyzed by LDOX and BANYULS. Xie et al.
(4) reported that ectopic
expression of Medicago truncatula BANYULS in transgenic tobacco
resulted in white rather than pink petals, consistent with a diversion of
intermediates from anthocyanin to PA or PA precursors and further claimed that
condensed tannin was present in these petals because they were positive to
DMACA and butyl alcohol-HCl reagents. We have obtained similar phenotypes with
tobacco containing ectopically expressed Arabidopsis BANYULS and
while we can detect a range of molecules that react with DMACA and butyl
alcohol-HCl, we can detect no polymers by thin layer
chromatography.3 Our observations suggest that the claim that the
expression of BANYULS alone results in the production of condensed tannin
based on the specificity of DMACA and butyl alcohol-HCl reagents
(4) is premature. This is
consistent with our study of Arabidopsis TDS mutants that accumulate
epicatechin but not polymers
(3) indicating that genes
downstream of epicatechin are essential for PA (condensed tannin)
synthesis.
|
Arabidopsis differs from most other plant species because it seems to contain only monomeric epicatechin without detectable monomeric catechin (3) while Arabidopsis PA contains only epicatechin initiating units and no detectable catechin initiating units.2 It may be that Arabidopsis does not make catechin because it does not have an LAR gene. Since most PA-containing plants have both catechin- and epicatechin-containing polymers it may be that part of the success in finding PA mutants in Arabidopsis is that two partially redundant pathways to PA do not occur in this model plant.
|
FOOTNOTES |
|---|
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) LAR nucleotide AJ550154 [GenBank] and protein CAD79341 [GenBank] , BANYULS or anthocyanidin reductase from Phaseolus coccineus (BN000164 [GenBank] ), Gossypium arboreum (BN000165 [GenBank] ), and Vitis vinifera (BN000166 [GenBank] ).
* This work was supported by Meat and Livestock Australia and Pioneer Hi-bred
International Inc (a DuPont Company). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at
http://www.jbc.org)
contains supplementary data.
To whom correspondence may be addressed: CSIRO Plant Industry, GPO Box 1600,
Canberra, ACT 2601, Australia. Tel.: 61-2-6246-5044; E-mail:
greg.tanner{at}csiro.au.
To whom correspondence may be addressed: CSIRO Plant Industry, GPO Box 1600,
Canberra, ACT 2601, Australia. Tel.: 61-2-6246-5219; E-mail:
tony.ashton{at}csiro.au.
1 The abbreviations used are: PA, proanthocyanidin; BAN, BANYULS or
anthocyanidin reductase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR,
dihydroflavanol reductase; DMACA, dimethylaminocinnamaldehyde; F3'H,
flavonoid 3'-hydroxylase, F3H, flavanone 3-hydroxylase; HPLC, high
performance liquid chromatography; IFR, isoflavone reductase; LAR,
leucoanthocyanidin reductase; LDOX, leucocyanidin dioxygenase; MATE, multidrug
and toxin extrusion; PEG, polyethylene glycol; PLR, pinoresinol-lariciresinol
reductase; PCBER, phenylcoumaran-benzylic ether reductase; RED,
Reductase-Epimerase-Dehydrogenase; SPP, sucrose phosphate phosphatase; TDS,
tannin-deficient seed; TT, transparent testa.
2 Abrahams, S., Lee, E., Walker, A. R., Tanner, G. J., Larkin, P. J., and
Ashton, A. R. (2003) Plant J. 35, in press.
3 G. J. Tanner, S. Abrahams, P. J. Larkin, A. R. Ashton, unpublished
observations.
4 The twenty most closely related proteins in GenBankTM to this
Forsthysia PLR all have Phe at this position.
|
ACKNOWLEDGMENTS |
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We thank Anna Mechanicos for technical assistance; Heidy Hewetson and Kelly Barr for harvesting the tiny leaves used for the enzyme purification; Prof. E. Malan (Bloemfontein) for the kind gift of afzelechin, Dr. Stephen Bloor (BioDiscovery New Zealand Ltd) for mass spectrometry and Klaus Kristiansen for introducing G. J. T. to such a rewarding enzyme. This research has been facilitated by access to the Australian Proteome Analysis Facility established under the Australian government's major National Research Facilities Program.
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