Content uploaded by Loic Lepiniec
Author content
All content in this area was uploaded by Loic Lepiniec
Content may be subject to copyright.
TRANSPARENT TESTA10 Encodes a Laccase-Like Enzyme
Involved in Oxidative Polymerization of Flavonoids in
Arabidopsis Seed Coat
W
Lucille Pourcel,
a
Jean-Marc Routaboul,
a
Lucien Kerhoas,
b
Michel Caboche,
a
Loı¨c Lepiniec,
a
and Isabelle Debeaujon
a
,
1
a
Laboratoire de Biologie des Semences, Unite
´
Mixte de Recherche 204, Institut National de la Recherche Agronomique/Institut
National Agronomique Paris-Grignon, Institut Jean-Pierre Bourgin, 78026 Versailles, France
b
LaboratoiredePhytopharmacieetMe
´
diateurs Chimiques, Institut National de la Recherche Agronomique,
78026 Versaill es, France
The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the
seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocya-
nidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate
more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10
cannot trigger H
2
O
2
-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV–
visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone
are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of
their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin
rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach.
TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing
testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish
that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.
INTRODUCTION
Flavonoids are plant secondary metabolites derived from the
phenylpropanoid pathway. In seeds, they act in defense against
predators and pathogens (Dixon et al., 2002), increase seed coat
(testa)–imposed dormancy (Debeaujon et al., 2000), and protect
against UV radiation (Winkel-Shirley, 2002a). Arabidopsis thaliana
seeds accumulate two types of flavonoid pigments during their
development. Flavonols (yellow pigments) are localized in the seed
envelopes and in the embryo, mainly as glycosylated quercetin
derivatives (Routaboul et al., 2005). Proanthocyanidins (PAs), also
called condensed tannins, accumulate exclusively in the inner
integument (essentially the endothelium cell layer) and in the
chalaza zone (Debeaujon et al., 2003). In Arabidopsis testa, PAs
consist of oligomers of the flavan-3-ol 2,3-cis-(!)-epicatechin,
also called procyanidins (Abrahams et al., 2003). PAs are synthe-
sized as colorless polymers in vesicles derived from the endo-
plasmic reticulum, which further coalesce into the central vacuole
(Stafford, 1988). In dead cells in which the cytoplasm has disinte-
grated, PAs are oxidized into brown complexes that cross-bond
within the cell. These modifications contribute the typical brown
color of Arabidopsis wild-type testa during seed desiccation.
The flavonoid pathway in Arabidopsis (Figure 1) has been
characterized mainly using mutants called transparent testa (tt)
and tannin-deficient seeds that are affected in seed coat pig-
mentation (Winkel-Shirley, 2002b; Abrahams et al., 2003). At
least 20 loci have been identified. They correspond to enzymes,
regulatory factors, and transporters (Winkel-Shirley, 2002b;
Debeaujon et al., 2003; Baxter et al., 2005).
In plants, browning reactions are usually caused by the
oxidation of phenolic compounds and result in the formation of
quinones, which are unstable molecules prone to form brown
polymers (Guyot et al., 1996). These reactions may be catalyzed
by polyphenol oxidases such as laccase (EC 1.10.3.2), catechol
oxidase (EC 1.10.3.1), and tyrosinase (EC 1.14.18.1) and by
peroxidases (EC 1.11.1.7). Oxidation proceeds in the presence
of molecular oxygen for polyphenol oxidase and with H
2
O
2
for
peroxidase (Dehon et al., 2002; Lopez-Serrano and Barcelo,
2002). In addition, quinones can be obtained by autoxidation or
chemical oxidation (Guyot et al., 1996). Although valued in tea
and wine processing, browning reactions are particularly detri-
mental for fresh fruits and vegetables (Fraignier et al., 1995).
Moreover, the coupling of phenols in the presence of catechol
oxidase is responsible for browning and cloudiness in beverages
(Guyot et al., 1996).
1
To whom correspondence should be addressed. E-mail debeaujo@
versailles.inra.fr; fax 33-0-1-30-83-30-99.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Isabelle Debeaujon
(debeaujo@versailles.inra.fr).
W
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.105.035154.
The Plant Cell, Vol. 17, 2966–2980, November 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
Laccases (o- and p-diphenol:dioxygen oxidoreductases) are
part of a larger group of enzymes called blue copper oxidases
that includes, among others, ascorbate oxidase and ceruloplas-
min. These enzymes are copper-containing glycoproteins that
catalyze the reduction of molecular oxygen to water with
concomitant oxidation of the phenolic substrate, which results
in the formation of quinones (Messerschmidt and Huber, 1990).
Laccases are widely present in eukaryotes (higher plants, fungi,
and insects) and in prokaryotes (Claus, 2004). They generally
have a low specificity with regard to the reducing substrate and
can oxidize o- and p-diphenols, monophenols, and ascorbic acid
(Mayer and Staples, 2002). In some reports, plant laccase
activities have been associated with lignification (Bao et al.,
1993; Chabanet et al., 1994; Ranocha et al., 2002), but a direct
activity of the enzyme on the polymerization of monolignols
within the cell wall matrix has not been demonstrated (Mayer and
Staples, 2002). Plant laccases were also reported to play a role in
iron metabolism (Hoopes and Dean, 2004). The expression of
a fungal laccase gene in transgenic maize was associated with
kernel browning and limited germination (Hood et al., 2003).
In this study, we report the isolation and functional character-
ization of the TT10 gene from Arabidopsis. Browning of the tt10
mutant seed coat is delayed compared with that of the wild type.
On the other hand, tt10 showed wild-type levels of flavonoids in
all its tissues, as established by thin layer chromatography
(Shirley et al., 1995). This phenotype suggested that TT10 may
act at the level of the unknown step(s) leading to PA oxidative
browning (Figure 1). TT10 encodes a predicted protein exhibiting
strong similarity with polyphenol oxidases of the laccase type.
The activity of its promoter colocalized with PA- and flavonol-
accumulating cells of the testa. Moreover, TT10 is involved in the
oxidative polymerization of flavonoids. We propose that a func-
tion of TT10 would be to oxidize epicatechin into the corre-
sponding quinones, initiating subsequent polymerization and the
formation of brown epicatechin oligomers different from procya-
nidin oligomers.
RESULTS
Phenotypic and Genetic Characterization of the tt10 Mutant
Afirsttt10 allele (tt10-1) was characterized previously (Koornneef,
1990). Shirley et al. (1995) genetically mapped the mutation and
showed that it was recessive and that the seed phenotype was
determined by the maternal genotype. We isolated five new
independent alleles, tt10-2 to tt10-6. They display the same
seed phenotype at harvest, which is a pale-brown seed coat
with a dark-brown chalaza zone (Figures 2A and 2B). After 6 to
12 months of storage, the tt10 seed coat goes brown until it re-
sembles the wild type. The presence of flavan-3-ols and flavo-
nols in tt10 seeds was detected histochemically using vanillin and
diphenylboric acid 2-aminoethyl ester staining, respectively. No
difference was observed between mutant and wild-type seeds
with these techniques. Moreover, no modification in seed coat
structure was detected after cytological observations.
Identification of the TT10 Gene by a Candidate
Gene Approach
To explain the delay in browning of the tt10 testa, we proposed
that early seed coat browning could be caused by the enzymatic
oxidation of phenolic compounds by a laccase, a catechol
oxidase, or a peroxidase. Chemical oxidation by air molecular
oxygen (autoxidation) would explain the late browning observed
in tt10 mutant seeds.
The tt10 mutation had been located previously on chromo-
some 5, between the TT3 and LEAFY (LFY) loci, corresponding to
the two molecular markers DFR (for dihydroflavonol reductase)
and LFY located at 16.8 and 24.5 Mb, respectively (Shirley et al.,
1995). Consequently, in silico analyses were performed to
identify candidate genes that mapped around this region. The
Arabidopsis genome was screened for genes annotated as
laccase, catechol oxidase, diphenol oxidase, and peroxidase.
Eleven genes were identified in the LFY to DFR interval, corre-
sponding to two laccases and nine peroxidases.
The expression pattern of the candidate genes was estab-
lished by RT-PCR analyses with flowers and immature siliques of
Figure 1. Scheme of the Flavonoid Pathway in Arabidopsis.
The flavonoid pathway leads to the formation of three major compounds:
PAs that get brown after oxidation, flavonol glycosides, and anthocya-
nins. Enzymes are represented in uppercase boldface letters, the corre-
sponding mutants in lowercase italic letters, and the regulatory mutants
in parentheses. ANR, anthocyanidin reductase; CE, condensing enzyme;
CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol
reductase; F3H, flavanone 3-hydroxylase; F39H, flavanone 39-hydroxy-
lase; FLS, flavonol synthase; GT, glycosyltransferase; LDOX, leucoan-
thocyanidin dioxygenase; POD, peroxidase; PPO, polyphenol oxidase;
tt(g), transparent testa (glabra). (Adapted from Debeaujon et al., 2003.)
Oxidation of Flavonoids by TT10 Laccase 2967
the wild type and then with flowers of the tt10-1 and tt10-2
alleles. The results revealed that only one gene, At5g48100, was
strongly expressed in siliques and flowers of the wild type and
not detected in the tt10-2 mutant. To provide further evidence
that At5g48100 was TT10, we analyzed its expression and DNA
sequence in the six tt10 alleles.
Semiquantitative RT-PCR did not detect the TT10 cDNA in all
of the alleles except tt10-1 and tt10-3 (Figure 4A). DNA gel blot
analyses revealed a restriction length polymorphism between
the alleles and the corresponding wild types, except for tt10-1
(see Supplemental Figure 1 online). However, sequencing of the
cDNA amplified in the tt10-1 allele showed the presence of two
point mutations leading to modifications of two amino acids
(152 D/G and 509 G/D). Finally, by sequencing the
At5g48100 gene in tt10-6, a T-DNA insertion was found in the
39 untranslated region (UTR), whereas no mutation appeared in
the coding region. These data suggest that the 39 UTR is
important for TT10 expression. The localization of the T-DNA
insertion in the tt10-4 and tt10-5 alleles was confirmed by PCR
analysis (Figure 3B).
To confirm that the phenotype conferred by tt10 is caused by
a mutation in the At5g48100 gene, we conducted a functional
complementation of the tt10-2 mutant with an 8-kb genomic
clone containing the At5g48100 gene with a 2.8-kb promoter and
a 1.6-kb 39 flanking region (Figure 3A). The T2 seed progeny
originating from 15 independent T1 hygromycin-resistant trans-
formants exhibited a reversion of the phenotype to that of the
wild type on the basis of seed color (Figure 2C) and flavonoid
composition (see Supplemental Figure 2 online). In addition,
hygromycin resistance cosegregated with wild-type seed color
in the progeny from selfing of T2 plants. Together, these data
demonstrate that the mutation in At5g48100 is responsible for
the phenotype conferred by tt10. Thus, the At5g48100 gene is
called TT10 below.
Structure of TT10
The structure of TT10 was inferred from comparison of a
1698-bp full-length cDNA sequence with the genomic DNA
sequence. It contains six exons and five introns. The UTRs
were determined by rapid amplification of cDNA ends to be 34 bp
for the 59 UTR and 50 bp for the 39 UTR (Figure 3B). Functional
complementation of the tt10-2 mutant with the TT10 cDNA
(cTT10) confirmed its structure. The reversion to the wild-type
phenotype was observed in the T2 progeny of T1 hygromycin-
resistant transformants expressing the Pro
35Sdual
:59UTR-cTT10-
39UTR construct but not in those expressing Pro
35Sdual
:cTT10.
Ectopic expression of TT10 did not have any obvious detrimental
effect on plant development and growth (see Supplemental
Figure 3 online).
Expression of th e TT10 Gene
The expression pattern was investigated in various vegetative
and reproductive tissues (Figure 4B). The TT10 mRNA was
detected predominantly in developing siliques, with an increase
during seed development. We observed an increase of accu-
mulation at
;3 to 4 d after fertilization (DAF). TT10 mRNA was
also detected at low levels in stems, seedlings, and flowers but
not in roots. Our results are consistent with data from in silico
microarrays (https://www.genevestigator.ethz.ch/) and EST
databases (http://www.tigr.org/) showing that TT10 is expressed
mainly in developing siliques and seeds. We reproducibly ob-
served that TT10 was expressed at a lower level in Landsberg
erecta (Ler) ecotype than in Wassilewskija-2 (Ws-2) and Columbia
(Col) ecotypes (Figure 4A).
Analysis of TT10 Promoter A ctiv ity
To investigate further the regulation of TT10 at the transcriptional
level, a 2.0-kb promoter was translationally fused to the uidA
reporter gene encoding a b-glucuronidase (GUS) protein and to
the mGFP5-ER reporter gene. Wild-type plants were trans-
formed with the constructs. In seed coat, at early stages of
embryo morphogenesis (1 to 3 DAF), TT10 promoter activity was
detected in the endothelium and in the pigment strand at the
chalaza zone (Figures 5A, 5C, 5D, and 5G). Later, the activity
increased and spread to the outer integument, mostly in the oil
Figure 2. Seed Phenotypes.
(A) and (B) Phenotype of a tt10 null mutant allele. At harvest, tt10 seeds present a pale-brown seed coat color with a dark-brown chalaza zone
(arrowheads).
(C) Complementation of the tt10 mutant. Genotypes are tt10-2 seeds; T2 progeny of tt10-2 homozygous plants transformed with the wild-type TT10
genomic region; and wild-type seeds. All seeds were obtained under the same conditions and observed at harvest.
Bars ¼ 600 mmin(A),240mmin(B), and 480 mmin(C).
2968 The Plant Cell
penultimate cell layer (Figures 5E, 5F, and 5H to 5K). Therefore,
promoter activity colocalizes first with PA-producing cells and
afterwards with flavonol-producing cells of the testa (Figures 5L
to 5N). The uidA gene was also strongly expressed in early
aborted seeds (Figure 5B) and in the transmitting tissue of the
silique (see Supplemental Figure 4 online). Three regulatory
mutants, tt2, tt8, and ttg1 (Nesi et al., 2000), were also trans-
formed with the Pro
TT10
:uidA construction. Resulting trans-
formants did not show any difference in GUS activity
compared with the wild type, suggesting that these regulators
do not affect TT10 promoter activity. Consistently, no difference
in TT10 mRNA accumulation was observed in regulatory mutant
backgrounds.
The TT10 Protein Belon gs t o the Laccase-Like
Multigene Family
The TT10 gene encodes a putative laccase protein with 565
amino acids (Figure 3C), a predicted molecular mass of 64 kD,
and a pI of 7.09 (http://www.expasy.org/tools/protparam/). The
TT10 protein sequence contains four His-rich copper binding
domains, L1 to L4 (Kumar et al., 2003), corresponding to the pu-
tative catalytic sites (http://www.expasy.ch/tools/scanprosite/).
These domains are characteristic of the multi-copper oxidase
family and are highly conserved in laccases of various organisms
(Figure 6A). The protein harbors many putative glycosylation
sites (Figure 3C). A putative cleavage site was predicted in the
Figure 3. Molecular Analysis of the TT10 Locus, and Structure of the Putative TT10 Protein.
(A) Scheme of the TT10 gene on the MDN11 P1 clone. The arrows represent the orientation of gene transcription. An 8-kb XhoI genomic fragment (thick
line) containing TT10 was used for tt10-2 complementation.
(B) Structure of the TT10 gene and localization of the mutations in the tt10 alleles. Gray rectangles represent T-DNA insertions, with the left border (LB)
indicated inside. The positions of the conserved copper binding residues are indicated by asterisks. Lengths are in base pairs.
(C) TT10 encodes a putative laccase with 565 amino acids. The N-terminal predicted signal peptide is shaded in black. The cleavage site is indicated
with an arrow. The 10 putative N-glycosylation sites are shown in boldface. The amino acids involved in copper binding are boxed.
(D) Analysis of the hydrophobicity profile using the method of Kyte and Doolittle (1982). The hydropathy value was calculated over a window of 11 amino
acids (DNA Strider 1.3. program).
Oxidation of Flavonoids by TT10 Laccase 2969
N-terminal region (http://www.cbs.dtu.dk/services/SignalP/),
defining a hydrophobic signal peptide of 21 amino acids (Figures
3C and 3D). According to PSORT website predictions (http://
psort.ims.u-tokyo.ac.jp/), the TT10 protein would be secreted in
the apoplast.
A search for homologous proteins in the Arabidopsis genome
identified 16 other putative laccases (http://www.arabidopsis.
org/Blast/). The corresponding genes are mostly localized on
chromosome 2 and at the extremities of chromosome 5. We
named them At LAC1 to At LAC17 according to their positions on
the genome (from the top of chromosome 1 to the bottom of
chromosome 5) (Figures 6B and 6C). A multialignment of the 17
full-length Arabidopsis proteins showed that TT10 (At LAC15)
was close to the At5g09360 protein (At LAC14), with 49% identity
at the amino acid level. From in silico microarray data (https://
www.genevestigator.ethz.ch/) and EST databases (http://
www.tigr.org/), we inferred that TT10 is by far the most abundant
laccase in Arabidopsis seeds.
An analysis of the distance relationships between plant lac-
case sequences was performed and is illustrated by the simpli-
fied tree presented in Figure 6D (for the complete tree, see
Supplemental Figure 5 online). It is fully consistent with the recent
phylogenetic analysis of McCaig et al. (2005), proposing the
existence of six groups. TT10/At LAC15 belongs to group 4, with
four dicotyledon laccases: At LAC14, Rhus vernicifera Rv LAC2
(47% identity), Acer pseudoplatanus Ap LAC1 (49% identity), and
Gossypium arboreum Ga LAC1 (43% identity), and one mono-
cotyledon laccase, Oryza sativa Os LAC1 (44% identity). In ad-
dition, the use of ascorbate oxidase sequences as an outgroup
suggests that the TT10 group diverged early from the other
groups of plant laccases.
Flavonoid Com positi on of tt10 Seed s
To investigate further the function of TT10, we extracted flavo-
noids from freshly harvested seeds. Epicatechin monomers and
PA polymers were measured, combining acid-catalyzed hydro-
lysis and liquid chromatography–mass spectrometry (LC-MS)
analysis. Upon cleavage and oxidation under strongly acidic
conditions, PAs yield pink-colored anthocyanidin monomers that
can be easily quantified (Porter et al., 1986). The tt10-2 mutant
contained 4.6-fold more soluble PAs than did the wild-type
control (Figure 7A). These results were confirmed by detecting epi-
catechin and procyanidin polymers by LC-MS analysis of the
soluble fraction (Figure 7B). Additionally, we measured insoluble
PAs by performing acid-catalyzed hydrolysis directly on the
pellet remaining after solvent extraction. The difference between
tt10-2 and wild-type content was smaller in this PA fraction
(Figure 7A).
LC-MS also allowed the analysis of flavonols. Quercetin-3-O-
rhamnoside was the main flavonol present in seeds and was 50%
more abundant in tt10-2 mutants than in wild-type controls
(Figure 7C). On the other hand, dimers from quercetin rhamno-
side were 12-fold less in tt10-2 seeds (Figure 7D). The abundance
of other flavonols was not modified by the mutation of TT10.
In Situ Localization and Analy sis of TT10 Activity in
Immature Seed Coat
A histochemical assay was developed to search for any accel-
eration of seed coat browning after adding different phenolic
substrates exogenously (Figure 8). Immature seeds at 7 to 8 DAF
were used because, at this time, the testa is still colorless and the
TT10 gene is strongly expressed. The genotypes tested were the
wild type Ws-2, the tt10-2 mutant, and a control tt4-8 without
flavonoids (chalcone synthase knockout mutant). After an over-
night incubation in control buffer, the seed coat stayed trans-
parent for all of the genotypes. When epicatechin or catechin
(flavan-3-ol monomers) was added with or without the addition of
catalase (preventing H
2
O
2
accumulation and therefore any
peroxidase activity), wild-type as well as tt4-8 mutant seed coats
went brown (Figures 8A and 8C). On the contrary, tt10-2 stayed
colorless. A tt4-8 seed section after reaction showed that the
brown color was localized in the endothelium and the oi1 cell
layers of the seed coat, where TT10 is expressed (Figure 8B). The
addition of quercetin and quercetin rhamnoside did not modify
seed coat color in any of the tested genotypes (Figure 8C).
Peroxidase activity in the chalaza region of wild-type seeds was
revealed by brown staining in the presence of H
2
O
2
, whereas no
reaction was observed in the absence of H
2
O
2
.
To investigate further the activity of the TT10 enzyme, we
performed LC-MS/MS analysis of flavonoid products extracted
from wild-type, tt10-2, and tt4-8 immature seeds after incubation
overnight with epicatechin. The wild-type and tt4-8 extracts
Figure 4. Expression Pattern of the TT10 Gene in Mutant Alleles and the
Wild Type.
(A) TT10 gene expression in the tt10 alleles was detected in flowers
(tt10-1 to tt10-3 alleles, Ler, and Ws) and siliques at 2 to 3 DAF (tt10-4 to
tt10-6 and Col) by semiquantitative RT-PCR (amplification of cTT10 from
ATG to STOP). Hybridization was performed using the TT10 cDNA as
a probe. EFaA4 gene expression was used as a control.
(B) The TT10 mRNA accumulation pattern was investigated in various
wild-type Ws-2 tissues by semiquantitative RT-PCR: roots (R), stems
(St), leaves (L), seedlings (S), flowers (F), and siliques at different stages
of development (1, 2þ3, 4þ5, 6þ7, and 8þ9 d after fertilization), as
described above. Drawings represent corresponding developmental
embryo stages.
2970 The Plant Cell
exhibited a bright yellow color with absorbance maxima at 280
and 380 nm, whereas the tt10-2 extract was weakly colored and
displayed a single absorbance maximum at 280 nm. The tt4-8
extract yielded no red color when submitted to acid-catalyzed
hydrolysis, showing that it did not contain procyanidins. HPLC
analysis at 380 nm (Figure 8D) revealed eight major peaks
present in the wild type and tt4-8, but not in tt10-2, that were
denoted 1 to 8 in increasing retention time order. These peaks
were absent from extracts of wild-type and mutant seeds in-
cubated in control buffer. Importantly, all of the peaks detected in
tt4-8 represent only oxidation products of epicatechin by TT10,
because this mutant does not have endogenous flavonoids.
Interestingly, these oxidation products were more abundant in
tt4-8 than in the wild type.
Figure 5. Pattern of TT10 Promoter Activity in Wild-Type Seeds.
(A) to (F) Expression of the Pro
TT10
:uidA cassette in developing seeds at 1 DAF (A), 3 DAF ([C] and [D]), 8 DAF ([E] and [F]), and in an aborted seed (B) .
GUS activity was observed with Nomarski optics on whole mounts for (A) to (C) and (F) and on sections for (D) and (E).
(G) to (K) Expression of the Pro
TT10
:mGFP5-ER cassette in developing seeds at 2 DAF (G), 3 to 4 DAF ([H] to [J]), and 6 DAF (K) visualized on confocal
cross sections showing GFP activity. (I) to (J) show overlays.
(L) Scheme of an immature seed transverse section showing the location of flavonoid pigments at the seed coat level (integuments and chalaza).
(M) Acc umulation of PAs and precursors in an immature seed at 3 to 4 DAF (whole mount vanillin staining).
(N) Acc umulation of flavonols in an immature seed at 3 to 4 DAF (diphenylboric acid 2-aminoethyl ester staining on seed section).
c, chalaza; e, endothelium; em, embryo; ii, inner integument; m, micropyle; oi, outer integument. Bars ¼ 24 mmin(F) and (K),70mmin(A) to (D) and (G),
80 mmin(M),90mmin(H), (L),and(N), 110 mmin(I) to (J), and 150 mmin(E).
Oxidation of Flavonoids by TT10 Laccase 2971
Figure 6. Relationships between TT10 and Other Laccase-Like Proteins.
(A) Alignment of the amino acid sequences of TT10 (At LAC15) and other laccases around the four His-rich copper binding domains. Rv LAC2, Rhus
vernicifera laccase; Pt LAC2, Pinus taeda laccase; Tv LAC4, Trametes versicolor laccase; Ag LAC1, Anopheles gambiae laccase; Bh LAC, Bacillus
halodurans alkaline laccase. The amino acids involved in binding copper are numbered in boldface (types 1, 2, and 3) and boxed. Identical amino acids
are indicated with asterisks.
(B) Genetic localization of the putative Arabidopsis laccases (LAC). Chromosome numbers are written below in roman numerals. The number of laccase
genes on each chromosome is shown in parentheses.
(C) References of Arabidopsis laccases in the Arabidopsis Genome Initiative and National Center for Biotechnology Information databases.
(D) Simplified phylogenetic relationships between TT10/At LAC15 and other plant putative laccases. Alignment was performed between TT10 and the
100 most homologous protein sequences using the ClustalX program (version 1.8; Thompson et al., 1997) and optimized manually. The distance matrix
was subjected to clustering using the neighbor-joining method. Bootstrap values with 1000 repetitions were used for statistical analysis and are
indicated at each branch point. The tree is rooted by ascorbate oxidase (AOX) sequences as an outgroup. Circled numbers represent the six laccase
groups established by McCaig et al. (2005). Only the composition of group 4, to which Arabidopsis TT10/At LAC15 laccase belongs, is presented (for
complete tree, see Supplemental Figure 5 online). Protein sequences used in this analysis are as follows. The two letters preceding the protein name
(LAC and AOX) describe the organism from which the sequence is derived: Ap, Acer pseudoplatanus;At,Arabidopsis thaliana; Ga, Gossypium
arboreum;Os,Oryza sativa;Rv,Rhus vernicifera. The bar at bottom indicates relative branch length.
2972 The Plant Cell
The electrospray mass spectra (in both positive and negative
modes) gave molecular masses of 576 (for peaks 2, 4, and 6), 862
(for peaks 3, 5, 7, and 8), and 864 (for peak 1). Thus, compounds
2, 4, and 6 may correspond to epicatechin dimers with one
additional unsaturation (two interflavan linkages and/or one
additional unsaturation), peaks 3, 5, 7, and 8 may correspond
to epicatechin trimers with two additional unsaturations, and
compound 1 may correspond to an epicatechin trimer with one
additional unsaturation. Fragments of the MS/MS spectrum of
compound 6 (Figure 8E) were also found in the MS spectrum of
dehydrodicatechin of the A typ e characterized previously after
catechin oxidation by grape (Vitis vinifera) polyphenol oxidase
in vitro (Guyot et al., 1996). By analogy with these previous
results, we propose that compound 6 is a dehydrodiepicat echin
A (s ee Supplement al Figure 6 online). The similarity between the
MS/MS spectra obtained for the eight oxidati on products
(result not shown for products 2 to 4, 7, and 8), especially at
m/z < 575, suggests that they all derive from ep icatechin
through s imila r oxidative me chanis ms. Notably, i f these oxida-
tion products are isomers of procyanidi n dim ers and trimers,
they differ from them by the nature and position of their
interflavan linkage.
DISCUSSION
The biology of fungal laccases has been a subject of intense
research for some time; however, analyses of the physiological
Figure 7. Flavon o id Composition of tt10-2 and Wild-Type Mature Seeds.
(A) Analysis of soluble and insoluble PAs measured after acid-catalyzed hydrolysis.
(B) Detection of epicatechin and soluble PAs by LC-MS.
(C) and (D) Detection of flavonol derivatives by LC-MS.
Values represent avera ges 6
SE of three independent measurements. EC, epicatechin; G, glucoside; H, hexoside; I, isorhamnetin; K, kaempferol; PC,
procyanidin; Q, quercetin; R, rhamnoside.
Oxidation of Flavonoids by TT10 Laccase 2973
functions of higher plant laccases are scarce (Mayer and Staples,
2002). Experimental approaches based on enzyme purification
are rendered difficult because plant extracts contain many
oxidative enzymes with overlapping substrate specificities
(Ranocha et al., 1999). Genetic strategies based on the use of
mutants, antisense knockouts, and overexpressors represent
powerful alternatives. For instance, Ranocha et al. (2002)
analyzed populations of transgenic poplar (Populus trichocarpa)
showing reduced levels of a laccase and demonstrated alter-
ations in phenolic metabolism and cell wall structure. In this
Figure 8. Analysis of in Situ Enz ymatic Activity.
(A) Detection of browning in immature seed coats (7 to 8 DAF) from the wild type, tt10-2,andtt4-8 after incubation in epicatechin overnight. Incubation
in phosphate buffer was used as a control. Bar ¼ 90 mm.
(B) Vibratome section of a tt4-8 seed after incubation in epicatechin overnight. e, endothelium; oi, outer integument. Bar ¼ 25 mm.
(C) Observation of seed coat color after the addition of various phenolic substrates. !, colorless; þ < þþ < þþþ, browning intensity. The chalaza is
brown only with the peroxidase substrate 3,39-diaminobenzidine (DAB) þ H
2
O
2
. Color observations are for seed coat color or seed coat color/chalaza
color. Q-3-O-R, quercetin 3-O-rhamnoside.
(D) HPLC analysis of flavonoid products extracted from wild-type, tt10-2,andtt4-8 immature seeds after incubation overnight with epicatechin. The
seeds were thoroughly rinsed in control buffer before extraction to eliminate residues of exogenous epicatechin.
(E) MS/MS spectra in the negative mode of products 6 (at 30 eV), 5 (at 40 eV), and 1 (at 35 eV) formed from epicatechin.
2974 The Plant Cell
study, we have characterized several Arabidopsis tt10 mutant
alleles affected in seed coat browning and demonstrated that the
corresponding protein is involved in the oxidative polymerization
of flavonoids. We provide additional evidence that the spectrum
of physiological functions of plant laccases extends beyond their
potential role in the oxidative polymerization of monolignols.
The TT10 protein contains the four conserved copper binding
domains characteristic of the multi-copper oxidase family, which
were thoroughly described for fungal laccases (Bertrand et al.,
2002; Kumar et al., 2003). From both of these motifs and the
overall sequence similarity with laccases of other organisms, par-
ticularly with the prototype laccase enzyme from lacquer tree
(Nitta et al., 2002), we infer that TT10 is a laccase-like protein.
Semiquantitative RT-PCR experiments and analysis of pro-
moter activity with reporter genes showed that TT10 is ex-
pressed mainly in the testa during seed development, although it
is also detected in a few other tissues. Our results contradict
those of McCaig et al. (2005), who detected a strong expression
of At5g48100 in Arabidopsis roots and leaves by RT-PCR. One
possible explanation for this discrepancy could be that expres-
sion of the gene was induced by stressful environmental con-
ditions that were not present in our experiments. These results
suggest the need to examine further the response of TT10 to
various stresses. The detection of a strong GUS activity driven by
the TT10 promoter in early aborted seeds strongly suggests that
TT10 may be transcriptionally induced by senescence. It is well
known that oxidative browning is observed during the aging of
various tissues of plants as a process preventing infections
through the production of antimicrobial components and re-
active oxygen species. Some peroxidases oxidize vacuolar and
apoplastic phenolics to produce o-quinones that react further to
produce brown components (Takahama, 2004). Our data sug-
gest that the TT10 laccase may also be involved in such a pro-
cess, albeit at the apoplast level.
From the analysis of the tt10-6 allele and functional comple-
mentation with the TT10 cDNA, we deduced that the 39 UTR may
be important for TT10 expression. The lack of mRNA in the tt10-6
background suggests that the 39 UTR would exhibit regulatory
sequences increasing the levels of TT10 expression, possibly by
stabilizing mRNA. Such a situation has been described for plant
genes (Gutierrez et al., 1999).
TT10 expression is lower in the Ler ecotype than in Ws-2 and
Col. This reduction fits a reduction in enzyme activity, because
Ler exhibits twice the amount of soluble PAs compared with
Ws-2 and Col (Routaboul et al., 2005).
TT10 promoter activity appears to be tightly regulated in time
and space. The activity appears first in the endothelium and then
in the oi1 cell layer of the outer integument. This pattern probably
requires the action of specific regulatory factors. However, in
contrast with the BAN gene expressed essentially in PA-pro-
ducing cells (Debeaujon et al., 2003), TT10 does not seem to be
regulated by the TT2, TT8, and TTG1 regulatory factors.
Importantly, the cellular pattern of TT10 promoter activity
correlates with PA- and flavonol-producing cells, which is
consistent with a role for TT10 in flavonoid metabolism in the
seed coat. Whether TT10 may also play a role in lignin metab-
olism remains to be investigated. It appears from phloroglucinol-
HCl staining that lignin in the Arabidopsis seed coat is restricted
to the xylem vessels at the chalaza (data not shown), where no
TT10 promoter activity was detected.
The TT10 protein, which is predicted to be glycosylated, is
likely to transit within the secretory pathway and be delivered into
the apoplast after cleavage of the transit peptide. TT10 mRNA
accumulation increases during seed development at the same
time as epicatechin biosynthesis, whereas seed browning oc-
curs concomitantly with seed desiccation, at 10 DAF. The dif-
ference in intracellular compartmentation between substrate and
enzyme may explain this delay.
Kitamura et al. (2004) showed that epicatechin and PAs
accumulate in the vacuole in early developmental stages and
migrate to the cell wall in senescing testa, until they are not visible
in the vacuole any longer. Epicatechin and PA secretion into the
apoplast can be explained by a vacuole burst caused by cell
death during the seed desiccation period. Then, epicatechin and
PAs would interact with TT10 and become oxidized and poly-
merized. Interactions between laccases and phenolic com-
pounds in the cell wall have been observed in the case of
monolignol polymerization (Liu et al., 1994). TT10 would act in the
same way with epicatechin and PAs. Similar to tt10, the tt19
mutant exhibits a delay in seed coat browning compared with
the wild type. The corresponding gene encodes a glutathione
S-transferase putatively involved in the transport of flavan-3-ols
(Kitamura et al., 2004). The delay in enzymatic browning in this
mutant may be explained by a late interaction between flavan-3-
ols and TT10 in the cell wall. A better knowledge of the PA
secretory pathway will be necessary to understand the complete
mechanisms of seed coat browning.
LC-MS analyses of mature seed extracts revealed a role for
TT10 in the oxidative polymerization of flavan-3-ols. During seed
desiccation, the initially colorless PAs form oxidized complexes
with cell wall polysaccharides and other phenolics, a process
that causes the tissue to darken (Marles et al., 2003). Epicatechin
does not bind to cell walls, in contrast with procyanidins, which
form noncovalent interactions with polysaccharides (Renard
et al., 2001). The higher amount of extractable PAs detected in
the tt10 mutant than in the wild type may be attributable to the
absence of such interactions.
As expected, the browning process observed during the in situ
activity assay was triggered by providing flavan-3-ol monomers,
subsequently oxidized by the resident enzyme expressed in the
seed coat. The major products resulting from TT10 activity are
yellow quinone-methide epicatechin dimers and trimers. They
are similar to those reported by Guyot et al. (1996), which are
formed in vitro when a grape polyphenol oxidase acts on
catechin (an epimer of epicatechin) to form the corresponding
quinones. As established in model oxidation experiments, the
o-quinones generated are generally colored and are highly
reactive: for instance, they may polymerize and also oxidize
other compounds. Some of the secondary reaction products are
colored or may be oxidized to colored quinoid compounds.
Therefore, the color of enzymatically oxidized phenols depends
mainly on the nature and relative importance of their subsequent
reactions, although it may be influenced by that of the primary
quinones (Nicolas et al., 1993). The brown color observed in seed
coats during maturation probably results from successive oxi-
dation reactions leading to more complex oxidation levels.
Oxidation of Flavonoids by TT10 Laccase 2975
Dixon et al. (2005) proposed a model for PA formation from
flavan-3-ols. They speculated that epicatechin or catechin may
be converted to the corresponding o-quinones by a polyphenol
oxidase enzyme. The quinones would then be converted to
carbocations through coupled nonenzymatic oxidation. Nucleo-
philic attack on the carbocations by epicatechin or catechin
would produce dimers and then oligomeric PAs linked through
C
4
–C
8
or C
4
–C
6
. In our experimental system, if the yellow
polymers are isomers of the unoxidized, colorless PA polymers,
they differ from them by the interflavan linkages (for structural
details, see Supplemental Figure 6 online). No PAs were detected
in tt4-8 seeds incubated with epicatechin, which suggests that
TT10 is not a condensing enzyme that catalyzes the polymeri-
zation of flavan-3-ols into the typical unoxidized PAs. We show
here that TT10 is a laccase-like flavonoid oxidase that is able to
catalyze the oxidative polymerization of epicatechin into yellow
to brown pigments different from the colorless PAs. Whether
TT10 can also oxidize PAs remains to be investigated (see
Supplemental Figure 6 online). Feeding PAs to immature seeds in
the frame of our in situ activity assay will help answer this
question. Dissecting the activity of the TT10 recombinant protein
toward given substrates in vitro will also be essential before
definitive conclusions on the function of this enzyme can be
drawn.
Dirigent proteins have been demonstrated to ensure the
stereoselective coupling step leading to the formation of (þ)-
pinoresinol, which is a specific coniferyl alcohol dimer and the
only product formed after the oxidation of coniferyl alcohol by
a still unknown one-electron oxidase (Halls and Lewis, 2002).
Whether such proteins are responsible for some linkage spec-
ificity in the polymerization process initiated by TT10 requires
further investigation. Comparing the oxidation products obtained
in situ and in vitro may help answer this question.
The fact that more oxidation products are made in the tt4-8
background than in the wild-type background during the in situ ac-
tivity assay may be attributable to an inhibitory effect of procya-
nidins on TT10 activity in the wild type, as described previously for
apple polyphenol oxidase (Le Bourvellec et al., 2004).
TT10 activity on flavan-3-ols is H
2
O
2
-independent, demon-
strating that TT10 does not act as a peroxidase. We have
detected a peroxidase browning activity in the chalaza zone
using a peroxidase-specific substrate (DAB). This result may
explain the presence of dark-brown chalaza in tt10 mutant seeds
at harvest. A peroxidase activity leading to the formation of
brown products was also reported in the chalaza of developing
barley (Hordeum vulgare) grain (Cochrane et al., 2000). The
authors suggest that the enzyme may be involved in mechanisms
of defense against pathogen invasion, which may also be the
situation in Arabidopsis. During seed development, the presence
of several oxidases may be needed to ensure different functions.
TT10 is also involved in quercetin rhamnoside oxidative
polymerization. The products formed are quercetin rhamnoside
dimers, which are apparently not brown, in contrast with oxidized
epicatechin derivatives. A mutant without flavonols would allow
a better understanding of the role of TT10 in quercetin rhamno-
side dimerization in relation to browning. Protection against UV
light–induced damage is an important function of flavonols in
plants (Winkel-Shirley, 2002a).
The 16 other Arabidopsis laccase-like proteins cannot com-
pensate for the loss of TT10 activity in tt10 seeds. This finding
suggests either that they are not localized in similar tissues or
that they have a different activity or substrate specificity. To date,
none of these laccases has been functionally characterized. We
also infer from the tt10 mutant seed coat phenotype that no other
oxidase enzyme, such as catechol oxidase or peroxidase, car-
ries out epicatechin oxidation in the absence of TT10 during seed
development. The seeds of tt10 plants do finally turn brown,
probably by autoxidation. This takes more time than enzymat-
ically, which explains why tt10 seeds are pale at harvest but
darken with storage time and not during seed maturation.
The mechanism of oxidative polymerization involving a laccase
has been suggested in the case of monolignols (Sterjiades et al.,
1992; Boerjan et al., 2003). TT10 displays strong similarity with
Rhus vernicifera, Acer pseudoplatanus, and Gossypium arbor-
eum laccases (Rv LAC2, Ap LAC1, and Ga LAC1, respectively),
the activities of which have been linked to polyphenol metabo-
lism. Rv LAC2 was found to catalyze the polymerization of urishiol
into lacquer for wound healing in response to pathogen attack
(Nitta et al., 2002). Ap LAC1, purified from cell suspension, was
able to oxidize monolignols to form water-insoluble polymers
in vitro (Sterjiades et al., 1992). The Ga LAC1 secretory laccase
expressed in Arabidopsis was responsible for the conversion of
sinapic acid into a monolactone-type dimer and for the trans-
formation of 2,4,6-trichlorophenol (Wang et al., 2004). Therefore,
phenolic polymerization may be a common function for plant
laccases. The biochemical step catalyzed by these enzymes is
the formation of highly reactive quinones, which afterward
spontaneously polymerize to give yellow to brown products
according to the extent of polymerization (Rouet-Mayer et al.,
1990). Therefore, polymerization is an indirect consequence of
quinone formation.
The plant laccases can be divided into at least six groups
present in both monocots and dicots (McCaig et al., 2005). TT10
belongs to group 4, which probably diverged early from the other
group. Laccases may have acquired specific functions very early
in evolution, such as the induction of resistance to biotic and
abiotic stresses, on the model of tomato catechol oxidase
(Thipyapong et al., 2004). The Arabidopsis genome does not
contain any typical catechol oxidase, as assessed by searching
for Arabidopsis sequences homologous with those of a poplar
catechol oxidase protein (Constabel et al., 2000). Laccases may
fill the absence of catechol oxidase in the sense of a specializa-
tion toward o-diphenol oxidation.
The biological role of TT10 during seed development may be to
form flavonoid derivatives that strengthen the testa to protect the
embryo and endosperm from biotic and abiotic stresses. Werker
et al. (1979) showed that quinones reinforced the barrier to water
permeation of Pisum seed coats. The physiological role of TT10
in the seed coat may be also to create a barrier against
pathogens by triggering the formation of antimicrobial quinones,
thus protecting the seed during storage and germination. Ex-
pression data (https://www.genevestigator.ethz.ch/) indicated
that TT10 is significantly induced by nematode attack and by
heat and osmotic stresses that can be related to drought.
Moreover, putative binding sites for regulatory factors induced
by desiccation, pathogens, and elicitors are present in the TT10
2976 The Plant Cell
promoter (http://www.dna.affrc.go.jp/PLACE/signalscan.html).
An in-depth functional study of the promoter, as well as an
analysis of tt10 seed longevity, germination, and resistance to
pathogens, will have to be performed to determine the precise
roles of TT10 in seed biology.
METHODS
Plant Materials, Growth Conditions, and Genetic Analyses
The tt10-1 allele (CS110) comes from an ethyl methanesulfonate muta-
genesis of the Ler ecotype of Arabidopsis thaliana (Koornneef, 1990). The
tt10-2 and tt10-3 alleles (CPI13 and CQK31 lines, respectively) are T-DNA
insertion lines in the Ws-2 ecotype, generated at the Institut National de la
Recherche Agronomique Versailles (Bechtold et al., 1993). They were
visually screened from T3 seed lots for an altered seed color. These lines
were untagged by T-DNA. The tt10-4, tt10-5,andtt10-6 alleles (N502972,
N128292, and N114753 lines, respectively) are T-DNA insertion lines in
the Col ecotype obtained from the Salk Institute (http://signal.salk.edu/).
They were identified by reverse genetics based on the At5g48100 gene
sequence. The tt4-8 allele, corresponding to the DFW34 line, is a T-DNA
line in the Ws-2 ecotype from the Versailles collection (Debeaujon et al.,
2003). The tt4-8 allele is completely devoid of flavonoids (Routaboul et al.,
2005). In vitro and greenhouse culture condit ions were described pre-
viously by Debeaujon et al. (2001). Crosses were performed between
tt10-1 and the other alleles. In all cases, F2 seeds exhibited a mutant
phenotype, indicating that the mutations were allelic. Reciprocal crosses
with the corresponding wild types were also realized to assess the
maternal determinism of the mutations.
Nucleic Acid Analyses
DNA and RNA experiments were performed according to Nesi et al.
(2000) and Debeaujon et al. (2001). The 59 and 39 ends of the mRNA
transcript were identified with the Ambion kit FirstChoice RLM-rapid
amplification of cDNA ends. Nucleic acids were sequenced using the
Applied Biosystems DNA sequencing kit (Bigdye Terminator version 3.0)
and the ABI Prism 310 genetic analyzer. RT-PCR for amplification of the
1.6-kb TT10 cDNA in wild-type and mutant tissues was realized using
primers TT10-ATG and TT10-STOP defined in the coding region (see
Supplemental Table 1 online).
Constructs for Functional Complementation
PCR amplifications were performed using the proofreading Pfu Ultra DNA
polymerase (Stratagene) with the primer sequences presented in Sup-
plemental Table 1 online. Complementation was performed with a geno-
mic 8-kb XhoI subclone of the MDN11 Mitsui P1 clone, containing the
TT10 gene. The sub clone was ligated into the SalI site of the pBIB-HYG
binary vector (Becker, 1990) to generate the TT10/pBIB-HYG plasmid.
Complementation was also performed with cTT10, either with or without
the 59 and 39 UTRs, and placed under the control of the dual 35S
promoter. A full-length cDNA (clone RAFL15-04-D21) isolated from an
Arabidopsis siliques/flowers library (Seki et al., 2004) was obtained from
RIKEN. The cDNA was amplified from 59 to 3 9 UTR using primers TT10-
59UTR-SalIandTT10-39UTR-SalI and from ATG to STOP using primers
TT10-ATG-SalIandTT10-STOP-SalI. Finally, the cDNA was SalI-digested
and cloned at the SalI sites of the pMagic plasmid (Nesi et al., 2002) to
generate the Pro
35Sdual
:cTT10/pBIB-HYG construction.
Constructs for the Analysis of Promoter Activity
To construct the Pro
TT10
:uidA:t35S cassette, a 2.0-kb TT10 promoter was
amplified by PCR from the TT10/pBIB-HYG plasmid using primers
pTT10-59XhoIandpTT10-39NcoI. The XhoI-NcoI–digested fragment
was cloned into the NcoI and XhoI sites of the pBS-GUS vector described
previously (Debeaujon et al., 2003). The Pro
TT10
:uidA:t35S cassette was
cloned as a SmaI-KpnI fragment into the pBIB-HYG binary vector.
To build the Pro
TT10
:mGFP5-ER cassette, a 2.0-kb TT10 promoter was
amplified with primers pTT10-39Bam HI and pTT10-59SalI, generating
a BamHI-SalI PCR product that was cloned at the BamHI-SalI sites of
the pBI101-mGFP5-ER plasmid. To generate pBI101-mGFP5-ER,the
mGFP5-ER cassette was released from pBIN-mGFP5-ER plasmid with
BamHI-SstI and cloned into a BamHI-SstI–digested pBI101 binary vector
(Clontech).
Stable transformation of Arabidopsis plants was performed as de-
scribed previously (Nesi et al., 2000).
Microscopy
GUS staining, together with sample mounting in a chloral hydrate solution
and inclusion in resin for the realization of sections, were performed
according to Debeaujon et al. (2003). Samples were observed with
a fluorescence Axioplan microscope (Zeiss) equipped with Nomarski
differential interference contrast optics. GFP observations of living seeds
were made as described by Debeaujon et al. (2003). For the histochemical
detection of flavan-3-ols, vanillin staining was handled as described
previously (Debeaujon et al., 2000). Flavonol localization was assayed
with diphenylboric acid 2-aminoethyl ester staining according to Sheahan
and Rechnitz (1993).
In Situ Enzyme Activity
The accelerated browning assay was performed on opened immature
siliques (7 to 8 DAF) in 100 mM phosphate buffer, pH 6.6, with 50 mM (þ)-
catechin hydrate, 50 mM (!)-epicatechin, 10 mM quercetin dihydrate, or
50 mM quercetin rhamnoside hydrate (Sigma-Aldrich for all products).
Assays were run with or without catalase (125 units/mL) to remove any
possible endogenous H
2
O
2
and therefore inhibit peroxidase activity.
Siliques were preincubated in the catalase solution for 1 h before the
addition of substrate. Histochemical staining for peroxidase activity was
performed with 2.5 mM DAB in the presence of 0.02% H
2
O
2
. Control
staining was performed without H
2
O
2
. For all assays, vacuum was applied
for 1 h before incubation at 378C in the dark, overnight. Tissues were
observed directly with a microscope.
Flavonoid Extraction and Analysis
Fifteen milligrams of seeds was ground in a 2-mL homogenizer (Potter)
with 1 mL of acetonitrile:water (75:25, v/v) for 5 min at 48C and were
sonicated for 20 min. Four micrograms of apigenin-7-O-glucoside was
used as an internal standard. After centrifugation, the pellet was re-
extracted with 1 mL of acetonitrile:water (75:25, v/v) overnight at 48C. The
two extracts were pooled and concentrated under nitrogen and the dry
extract was dissolved in 200 mL of acetonitrile:water (75:25, v/v) to give
the final extract used for the analysis of flavonols and soluble PAs. The
pellet was preserved for insoluble PA analysis.
Analyses of soluble and insoluble PAs were performed after acid-
catalyzed hydrolysis (acid butanol assay) according to Porter et al. (1986)
on aliquots of the final extracts and on the entire remaining pellets,
respectively. They are expressed in milligrams per gram of leucocyanidin
equivalent assuming that the effective E
1%,1cm,550nm
of leucocyanidin is
460. Five grams of seeds was ground in a 40-mL conical glass (Potter)
with 30 mL of acetonitrile:water (75:25, v/v) at 48C and sonicated for
20 min. After centrifugation, the pellet was extracted overnight at 48C,
with an additional 30 mL of acetonitrile:water (75:25, v/v). To minimize PA
oxidation, solutions were saturated with nitrogen and kept in the dark
at 48C. The two extracts were pooled, concentrated under reduced
Oxidation of Flavonoids by TT10 Laccase 2977
pressure at 408C, and dissolved in 1 mL of methanol:water (1:1, v/v). The
crude PA extract was purified using Toyopearl TSK HW 40-F (Sigma-
Aldrich) as described previously (Kennedy and Jones, 2001). We checked
that epicatechin monomer was removed after this purification step using
LC-MS. The purified PA extract was dissolved in 1 mL of 0.1 N HCl in
methanol containing 50 mg of phloroglucinol and 10 mg of ascorbic acid
and reacted for 20 min at 508C. The reaction was stopped with 5 mL of
40 mM sodium acetate. Reaction products were analyzed using LC-MS
and quantified at 280 nm using published values for the molar response of
epicatechin and epicatechin-phloroglucinol (Kennedy and Jones, 2001).
Mass analysis was conducted on an aliquot of the final extract using
a Quattro liquid chromatograph with an electrospray ionization Z-spray
interface (MicroMass), MassLynx software, an Alliance 2695 reversed-
phase HPLC system (Waters), and a Waters 2487 UV detector set at
280 nm. An Uptisphere C
18
column (150 3 2 mm, 5 mm; Interchrom) was
used with a mix comprising solvent A acetonitrile:water (95:5, v/v; 0.2%
acetic acid) and solvent B acetonitrile:water (5:95, v/v; 0.2% acetic acid)
with a gradient profile (starting with 10:90 A/B [v/v] for 5 min; linear
gradient up to 70:30 A/B over 35 min; a washing step of 100:0 A/B for
15 min, and a final equilibration at 10:90 A/B for 2 0 min) at a flow rate of
200 mL/min. Quantification was based on the area of major MS sig-
nals ([MþH]
þ
and fragment). Flavonoid content was expressed relative to
quercetin 3-O-rhamnoside, rutin, and epicatechin (Ex trasynthe
`
se) for
monoglycosylated flavonols, diglycosylated flavonols, and procyanidin
derivatives, respectively.
Assignment of epicatechin itself was made by comparison with an
authentic standard (same retention time at 6 min, instead of 3.5 min for
catechin, and same MS/MS fragmentation pattern for ion [MþH]
þ
at m/z
291). Procyanidins (up to 4 units) were characterized during LC-MS
analysis by their [MþH]
þ
mass signals at m/z 579, 867, and 1155 and
characteristic fragments resulting from losses of 289/290 neutrals.
Assignment of quercetin-3-O-rhamnoside was made by comparison
with an authentic standard; it was characterized by its retention time at
23.1 min, ions at m/z 449 [MþH]
þ
and 303 [MþH-rhamnose]
þ
in the
positive mode. Quercetin rhamnoside dimer characteristics were 17.5,
19.7, 20.5, and 21.5 min, m/z 895 [MþH]
þ
, 749 [MþH-rhamnose]
þ
,and
603 [MþH-rhamnose rhamnose]
þ
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: Ag LAC1 (AAN17505),
Ap LAC1 (AAB09228), Bh LAC (AY228142), Ga LAC1 (AAR83118), MDN11
P1 clone (AB017064), Os LAC1 (NP_918753), Pt LAC2 (AAK37824),
RAFL15-04-D21 cDNA clone (BT002919), Rv LAC2 (BAB63411), and
Tv LAC4 (Q12719).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Table 1. Primers Used for PCR and RT-PCR Analysis
of the TT10 Gene, cDNA, and Promoter.
Supplemental Figure 1. Molecular Characterization of the tt10
Mutant Alleles.
Supplemental Figure 2. Flavonoid Composition of Mature Seeds from
Complementing Lines Expressing the Wild-Type Genomic Region.
Supplemental Figure 3. Analysis of TT10 Gene Expression in
Overexpressing Lines.
Supplemental Figure 4. Pattern of TT10 Promoter Activity in the
Transmitting Tissue of the Silique.
Supplemental Figure 5. Phylogenetic Relationships between TT10/
AtLAC15 and Other Plant Putative Laccases.
Supplemental Figure 6. Partia l Arabidopsis Flavonoid Pathway
Showing the Various Levels of TT10 Action: A Working Model.
ACKNOWLEDGMENTS
We thank Ve
´
ronique Cheynier, Lise Jouanin, the coeditor, and the three
reviewers for their comments on the manuscript. We also acknowledge
Olivier Grandjean, Bertrand Dubreucq, and Jocelyne Kronenberger for
technical advice in microscopy. We are grateful to Bertrand Dubreucq
and Sandra Moreau for the pBI101-mGFP5-ER plasm id and to Jim
Haseloff for pBIN-mGFP5-ER. Finally, we also thank the Institut National
de la Recherche Agronomique Versailles and the Salk Institute for giving
access to their collections of T-DNA insertion mutants and RIKEN BRC
for providing the RAFL15-04-D21 cDNA clone. This research was funded
in part by the French Ministry of Research (ACI Development). L.P. was
supported by the Institut National de la Recherche Agronomique and the
Centre Technique Interprofessionnel des Ole
´
agineux Me
´
tropolitains.
Received June 12, 2005; revised August 30, 2005; accepted September
28, 2005; published October 21, 2005.
REFERENCES
Abrahams, S., Lee, E., Walker, A.R., Tanner, G.J., Larkin, P.J., and
Ashton, A.R. (2003). The Arabidopsis TDS4 gene encodes leucoan-
thocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin
synthesis and vacuole development. Plant J. 35, 624–636.
Bao, W., O’Malley, D.M., Whetten, R., and Seder off, R.R. (1993). A
laccase associated with lignification in loblolly-pine xylem. Science
260, 672–674.
Baxter, I.R., Young, J.C., Armstrong, G., Foster, N., Bogenschutz,
N., Cordova, T., Peer, W.A., Hazen, S.P., Murphy, A.S., and Harper,
J.F. (2005). A plasma membrane H
þ
-ATPase is required for the
formation of proanthocyanidins in the seed coat endothelium of
Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 102, 2649–2654.
Bechtold, N., Ellis, J., and Pelletier, G. (1993). In-planta Agrobacte-
rium-mediated gene transfer by infiltration of adult Arabidopsis
thaliana plants. C. R. Acad. Sci. III 316, 1194–1199.
Becker, D. (1990). Binary vectors which allow the exchange of plant
selectable markers and reporter genes. Nucleic Acids Res. 18, 203.
Bertrand, T., Jolivalt, C., Briozzo, P., Caminade, E., Joly, N., Madzak,
C., and Mougin, C. (2002). Crystal structure of a four-copper laccase
complexed with an arylamine: Insights into substrate recognition and
correlation with kinetics. Biochemistry 41, 7325–7333.
Boerjan, W., Ralph, J., and Baucher, M. (2003). Lignin biosynthesis.
Annu. Rev. Plant Biol. 54, 519–546.
Chabanet, A., Goldberg, R., Catesson, A.M., Quinet-Sze
´
ly, M.,
Delaunay, A.M., and Faye, L. (1994). Characterization and local ization
of a phenoloxidase in mung bean hypocotyl cell walls. Plant Physiol .
106, 1095–1102.
Claus, H. (2004). Laccases: Structure, reactions, distribution. Micron
35, 93–96.
Cochrane, M.P., Paterson, L., and Gould, E. (2000). Changes in
chalazal cell walls and in the peroxid ase enzymes of the crease region
during grain development in barley. J. Exp. Bot. 51, 507–520.
Constabel, C.P., Yip, L., Patton, J.J., and Christopher, M.E. (2000).
Polyphenol oxidase from hybrid poplar. Cloning and expression in
response to wounding and herbivory. Plant Physiol. 124, 285–295.
Debeaujon, I., Le
´
on-Kloosterziel, K.M., and Koornneef, M. (2000).
Influence of the testa on seed dormancy, germination, and longe vity in
Arabidopsis. Plant Physiol. 122, 403–414.
2978 The Plant Cell
Debeaujon, I., Nesi, N., Perez, P., Devic, M., Grandjean, O., Caboche,
M., and Lepiniec, L. (2003). Proanthocyanidin-accumulating cells in
Arabidopsis testa: Regulation of differentiation and role in seed
development. Plant Cell 15, 2514–2531.
Debeaujon, I., Peeters, A.J., Le
´
on-Kloosterziel, K.M., and
Koornneef, M. (2001). The TRANSPARENT TESTA12 gene of Arabi-
dopsis encodes a multidrug secondary transporter-like protein re-
quired for flavonoid sequestration in vacuoles of the seed coat
endothelium. Plant Cell 13, 853–871.
Dehon, L., Ma cheix, J.J., and Durand, M. (2002). Involvement of
peroxidases in the formation of the brown coloration of heartwood in
Juglans nigra. J. Exp. Bot. 53, 303–311.
Dixon, R.A., Achnine, L., Kota, P., Liu, C.J., Reddy, M.S.S., and
Wang, L.J. (2002). The phenylpropanoid pathway and plant defence—
A genomics perspective. Mol. Plant Pathol. 3, 371–390.
Dixon, R.A., Xie, D.Y., and Sharma, S.B. (2005). Proanthocyanidins—A
final frontier in flavonoid research? New Phytol. 165, 9–28.
Fraignier, M.P., Marques, L., Fleuriet, A., and Macheix, J.J. (1995).
Biochemical and immunochemical characteristics of polyphenol ox-
idases from diff erent fruits of Prunus. J. Agric. Food Chem. 43, 2375–
2380.
Gutierrez, R.A., MacIntosh, G.C., and Green, P.J. (1999). Current
perspectives on mRNA stability in plants: Multiple levels and mech-
anisms of control. Trends Plant Sci. 4, 429–438.
Guyot, S., Vercauteren, J., and Cheynier, V. (1996). Structural de-
termination of colourless and yellow dimers resulting from (þ)-cate-
chin coupling catalysed by grape polyphenoloxidase. Phytochemistry
42, 1279–1288.
Halls, S.C., and Lewis, N.G. (2002). Secondary and quaternary struc-
tures of the (þ)-pinoresinol-forming dirigent protein. Biochemistry 41,
9455–9461.
Hood, E.E., Bailey, M.R., Beifuss, K., Magallanes-Lundback, M.,
Horn, M.E., Callaway, E., Drees, C., Delaney, D.E., Clough, R.,
and Howard, J.A. (2003). Criteria for high-le vel expression of a fun-
gal laccase gene in transgenic maize. Plant Biotechnol. J. 1, 129–140.
Hoopes, J.T., and Dean, J.F. (2004). Ferroxidase activity in a laccase-
like multicopper oxidase from Liriodendron tulipifera. Plant Physiol.
Biochem. 42, 27–33.
Kennedy, J.A., and Jones, G.P. (2001). Analysis of proanthocyanidin
cleavage products following acid-catalysis in the presence of excess
phloroglucinol. J. Agric. Food Chem. 49, 1740–1746.
Kitamura, S., Shikazono, N., and Tanaka, A. (2004). TRANSPARENT
TESTA 19 is involved in the accumulation of both anthocyanins and
proanthocyanidins in Arabidopsis. Plant J. 37, 104–114.
Koornneef, M. (1990). Mutations affecting the testa colour in Arabi-
dopsis. Arabidopsis Inf. Serv. 27, 1–4.
Kumar, S.V.S., Phale, P.S., Durani, S., and Wangikar, P.P. (2003).
Combined sequence and structure analysis of the fungal laccase
family. Biotechnol. Bioeng. 83, 386–394.
Kyte, J., and Doolittle, R.F. (1982). A simple method for displaying the
hydropathic character of a protein. J. Mol. Biol. 157, 105–132.
Le Bourvellec, C., Le Quere, J.M., Sanoner, P., Drilleau, J.F., and
Guyot, S. (2004). Inhibition of apple polyphenol oxidase activity by
procyanidins and polyphenol oxidation products. J. Agric. Food
Chem. 52, 122–130.
Liu, L., Dean, J.F.D., Friedman, W.E., and Eriksson, K.E.L. (1994). A
laccase-like phenoloxidase is correlated with lignin biosynthesis in
Zinnia elegans stem tissues. Plant J. 6, 213–224.
Lopez-Serrano, M., and Barcelo, A.R. (2002). Comparative study of
the products of the peroxidase-catalyzed and the polyphenoloxidase-
catalyzed (þ)-catechin oxidation. Their possible implications in straw-
berry (Fragaria 3 ananassa) browning reactions. J. Agric. Food Chem.
50, 1218–1224.
Marles, M.A.S., Ray, H., and Gruber, M.Y. (2003). New perspectives
on proanthocyanidin biochemistry and molecular regulation. Phyto-
chemistry 64, 367–383.
Mayer, A.M., and Staples, R.C. (2002). Laccase: New functions for an
old enzyme. Phytochemistry 60, 551–565.
McCaig, B.C., Meagher, R.B., and Dean, J.F. (2005). Gene struc-
ture and molecular analysis of the laccase-like multicopper
oxidase (LMCO) gene family in Arabidopsis thaliana. Planta 221,
619–636.
Messerschmidt, A., and Huber, R. (1990). The blue oxidases, ascor-
bate oxidase, laccase and ceruloplasmin—Modelling and structural
relationships. Eur. J. Biochem. 187, 341–352.
Nesi, N., Debeaujon, I., Jond, C., Pelletier, G., Caboche, M., and
Lepiniec, L. (2000). The TT8 gene encodes a basic helix-loop-helix
domain protein required for expression of DFR and BAN genes in
Arabidopsis siliques. Plant Cell 12, 1863–1878.
Nesi, N., Debeaujon, I., Jond, C., Stewart, A.J., Jenkins, G.I.,
Caboche, M., and Lepiniec, L. (2002). The TRANSPARENT TESTA16
locus encodes the ARABIDOPSIS BSISTER MADS domain protein
and is required for proper development and pigmentation of the seed
coat. Plant Cell 14, 2463–2479.
Nicolas, J., Cheynier, V., Fleuriet, A., and Rouet-Mayer, M.A. (1993).
Polyphenols and enzymatic browning. In Polyphenolic Phenomena, A.
Scalbert, ed (Versailles, France: INRA Editions), pp. 165–175.
Nitta, K., Kataoka, K., and Sakurai, T. (2002). Primary structure of
a Japanese lacquer tree laccase as a prototype enzyme of multi-
copper oxidases. J. Inorg. Biochem. 91, 125–131.
Porter, L.J., Hrstich, L.N., and Chan, B.G. (1986). The conversion of
procyanidins and prodelphinidins to cyanidins and delphinidins.
Phytochemistry 25, 223–230.
Ranocha, P., Chabannes, M., Chamayou, S., Danoun, S., Jauneau,
A., Boudet, A.M., and Goffner, D. (2002). Laccase down-regulation
causes alterations in phenolic metabolism and cell wall structure in
poplar. Plant Physiol. 129, 145–155.
Ranocha, P., McDougall, G., Hawkins, S., Sterjiades, R., Borderies,
G., Stewart, D., Cabanes-Macheteau, M., Boudet, A.M., and
Goffner, D. (1999). Biochemical characterization, molecular cloning
and expression of laccases—a divergent gene family—in poplar. Eur.
J. Biochem. 259, 485–495.
Renard, C.M.G.C., Baron, A., Guyot, S., and Drilleau, J.F. (2001).
Interactions between apple cell walls and native apple polyphenols:
Quantification and some consequences. Int. J. Biol. Macromol. 29,
115–125.
Rouet-Mayer, M.A., Ralambosoa, J., and Philippon, J. (1990). Roles
of o-quinones and their polymers in the enzymic browning of apples.
Phytochemistry 29, 435–440.
Routaboul, J.-M., Kerhoas, L., Debeaujon, I., Pourcel, L., Caboche,
M., Einhorn, J., and Lepiniec, L. (2005). Flavonol and proanthocya-
nidin diversity and biosynthesis in seed of Arabidopsis thaliana.
Planta, in press.
Seki, M., et al. (2004). RIKEN Arabidopsis full-length (RAFL) cDNA and
its applications for expression profiling under abiotic stress condi-
tions. J. Exp. Bot. 55, 213–223.
Sheahan, J.J., and Rechnitz, G.A. (1993). Differential visualisation of
transparent testa mutants in Arabidopsis thaliana. Anal. Chem. 65,
961–963.
Shirley, B.W., Kubasek, W.L., Storz, G., Bruggemann, E., Koornneef,
M., Ausubel, F.M., and Goodman, H.M. (1995). Analysis of
Arabidopsis mutants deficient in flavonoid biosynthesis. Plant J. 8,
659–671.
Stafford, H.A. (1988). Proanthocyanidins and the lignin connection.
Phytochemistry 27, 1–6.
Sterjiades, R., Dean, J.F.D., and Eriksson, K.E.L. (1992). Laccase
Oxidation of Flavonoids by TT10 Laccase 2979
from sycamore maple (Acer pseudoplatanus) polymerizes monoli-
gnols. Plant Physiol. 99, 1162–1168.
Takahama, U. (2004). Oxidation of vacuolar and apoplastic phenolic
substrates by peroxidase: Physiological significance of the oxidation
reactions. Phytochem. Rev. 3, 207–219.
Thipyapong, P., Hunt, M.D., and Steffens, J.C. (2004). Antisense
downregulation of polyphenol oxidase results in enhanced disease
susceptibility. Planta 220, 105–117.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and
Higgins, D.G. (1997). The CLUSTAL X windows interface: Flexible
strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res. 25, 4876–4882.
Wang, G.D., Li, Q.J., Luo, B., and Chen, X.Y. (2004). Ex planta
phytoremediation of trichlorophenol and phenolic allelochemicals
via an engineered secretory laccase. Nat. Biotechnol. 22, 893–897.
Werker, E., Marbach, I., and Mayer, A.M. (1979). Relation between the
anatomy of the testa, water permeability and the presence of
phenolics in the genus Pisum. Ann. Bot. (Lond.) 43, 765–771.
Winkel-Shirley, B. (2002a). Biosynthesis of flavonoids and effects of
stress. Curr. Opin. Plant Biol. 5, 218–223.
Winkel-Shirley, B. (2002b). A mutational approach to dissection
of flavonoid biosynthesis in Arabidopsis. In Phytochemistry in
the Genomics and Post-Genomics Eras, J.T. Romeo and R.A.
Dixon, eds (Oxford, UK: Pergamon-Elsevier Science), pp. 95–110.
2980 The Plant Cell
5’-ATGTCACATTCCTTCTTCAATTTATTC-3’
TT10-ATG
5’-TTATTCATAGCAAGGCGGCAAATC-3’
TT10-STOP
5’-GTAGTCGACCACACTGATTTTGC-3’
pTT10-5’SalI
5’-GTAGGATCCTTTGGAAGAGTTTTA-3’
pTT10-3’BamHI
5’-GAAGGAATGTGCCATGGTGGAAGAG-3’
pTT10-3’NcoI
5’-GTACTCGAGCACACTGATTTTGCTTGGAATG-3’
pTT10-5’XhoI
5’-GTAGTCGACTTATTCATAGCAAGGCGGCA-3’
TT10-STOP-SalI
5’-GTAGTCGACATGTCACATTCCTTCTTCAA-3’
TT10-ATG-SalI
5’-GTAGTCGACTAAAATAATTTATTACATAAATG-3’
TT10-3'UTR-SalI
5’-GTAGTCGACTGAGGGTAATTAATTTACTAAAAC-3’
TT10-5'UTR-SalI
Primer sequencePrimer name
Supplemental Table 1. Primers used for PCR and RT-PCR analysis of the TT10 gene,
cDNA and promoter.
kb
• EcoRV
Col
tt10-4
4
2.3
1.6
• HindIII
12
5
Col
tt10-5 tt10-6
• EcoRI
kb
12.5
L
er tt10-1
kb
tt10-3
5.1
6
9
Ws
tt10-2
kb
Supplemental Figure 1. Molecular characterization of the tt10 mutant
alleles.
Restriction length polymorphism between the tt10 alleles and the
corresponding WT was studied with DNA Blot analyses. Hybridization was
performed using the TT10 cDNA (from ATG to stop) as a probe.
Supplemental Figure 2. Flavonoid composition of mature seeds from
complementing lines expressing the wild-type genomic region.
Analysis was performed by LC-MS in two independent T2 complementing lines
(C1 and C2), in tt10-2 and in corresponding WT.
EC, epicatechin; G, glucoside; H, hexoside; I, isorhamnetin; K, kaempferol; PC,
procyanidins; Q, quercetin; R, rhamnoside.
Q3-O-R
Q-R
dimers
Q-H-R
K-3,7-O-di-R
K-3-O-G-7-O-R
K-R
I-R
I-H-R
0
1
2
3
Flavonols (mg.g-1 seeds)
C1
C2
WT
tt10-2
0
0,04
0,08
0,12
0,16
C1 C2 WT tt10-2
Flavonol derivatives
0
1
EC
PC dimer
PC trimer
PC tetramer
C1 C2 WT tt10-2
Arbitrary units
Soluble PAs
A B
OE1 OE2 WT
L St L St L St
TT10
EF1aA4
Supplemental Figure 3. Analysis of TT10 gene expression in overexpressing
lines.
TT10 mRNA accumulation was analyzed by RT-PCR in leaves and stems of
two independent TT10 overexpressors (OE) and in the Ws-2 wild type. The OE
genotypes express the Pro
35Sdual
:5’UTR-cTT10-3’UTR construct in a tt10-2
background. EF1
a
A4 gene expression was used as control.
Supplemental Figure 4. Pattern of TT10 promoter activity in the transmitting
tissue of the silique.
(A) and (B) Expression of the Pro
TT10
:uidA cassette in the transmitting tissue
of the silique at around 2 daf. GUS activity was observed with Nomarski optics
on whole mounts for (A), and on a magnified style section for (B).
Abbreviations: pg, pollen grain; se, seed coat endothelium; st, stigmata; sy,
style; tt, transmitting tissue.
Bars = 280 mm in (A), and 90 mm in (B)
A B
st
tt
pg
se
sy
606
1000
0.05
Plant LAC
At LAC11
Os LAC4
Nt LAC1
At LAC4
At LAC10
At LAC16
797
Pt LAC8
923
1000
902
987
998
890
At LAC1
2
Lt LAC1
At LAC17
Pb LAC1
At LAC2
801
Os LAC3
747
1000
1
At LAC9
At LAC8
At LAC7
1000
Os LAC5
Lp LAC1
1000
1000
998
5
At LAC6
At LAC5
At LAC12
Os LAC2
1000
At LAC3
At LAC13
964
1000
Pt LAC2
865
1000
3
4
Ap LAC1
Rv LAC2
Ga LAC1
871
923
At LAC15
At LAC14
Os LAC1
Plant AOX
At AOX1
At AOX2
Mt AOX1
1000
6
Supplemental Figure 5. Phylogenetic relationships between TT10 / At LAC15 and other plant putative
laccases.
Alignment was carried out between TT10 protein and the 100 most similar protein sequences using the
Clustal X program (version 1.8 ; Thompson et al., 1997, Nucleic Acids Res. 25, 4876-4882), and
optimized manually. The distance matrix was subjected to clustering using the neighbour-joining
method. Bootstrap values with 1000 repetitions were used for statistical analysis, and indicated at each
branch point. Tree is rooted by the ascorbate oxidase sequences as outgroup. Arabidopsis putative
laccases are on gray background, and TT10 / At LAC15 is written in bold characters. Circled numbers
represent the number of laccase ancestors shared by monocotyledons, dicotyledons and
gymnosperms. Protein sequences used in this analysis are as follows. The two letters preceding the
protein name (LAC, laccase; AOX, ascorbate oxidase) describe the organism from which the sequence
is derived: Ap, Acer pseudoplatanus; At, Arabidopsis thaliana; Ga, Gossypium arboreum; Lp, Lolium
perenne; Lt, Liriodendron tulipifera; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, Oryza sativa;
Pb, Populus balsamifera; Pt, Pinus taeda; Rv, Rhus vernicifera. Ap LAC1 (AAB09228), Ga LAC1
(AAR83118), Lp LAC1 (AAL73970), Lt LAC1 (AAB17194), Nt LAC1 (AAC49536), Os LAC1
(NP_918753), Os LAC2, (NP_915305), Os LAC3 (NP_915445), Os LAC4 (NP_917849), Os LAC5
(XP_463491), Pb LAC1 (CAA74105), Pt LAC2 (AAK37824), Pt LAC8 (AAK37830), Rv LAC2
(BAB63411), At AOX1 (AAU95421), At AOX2 (AAO73900), Mt AOX1 (CAA75577). Bar indicates
relative branch length.
Supplemental Figure 6. Partial Arabidopsis flavonoid pathway showing
the various levels of TT10 action: a working model.
The dashed arrow stands for a hypothetical step. Chemical structures of
the end-products are also presented.
Enzymes are in uppercase boldface letters, and the corresponding mutants
in lowercase italic letters. ANR, anthocyanidin reductase; CE, hypothetical
condensing enzyme; DFR, dihydroflavonol reductase; F3’H, flavonol 3’-
hydroxylase; FLS, flavonol synthase; RT, rhamnosyltransferase; LDOX,
leucocyanidin dioxygenase; POD, peroxidase; PPO, polyphenol oxidase,
tt, transparent testa.
Dihydroquercetin
Leucocyanidin
Cyanidin
DFR
LDOX
RT
ANR
tt3
tt18/ tds4
ban
Quercetin
Epicatechin
CE ?
PPO
tt10
PPO
tt10
FLS
Quercetin rhamnoside
F3’H
tt7
O
O
O
O
OH
HO
OH
OH
OH
OH
OH
A C
B
D F
E
O
4
8
4a
3
6
7
5
2
6’
5’
4’
2’
1’
3’
8a
Oxidized epicatechin derivatives
e.g.
dihydrodiepicatechin A
(brown)
4
8
4a
3
6
7
5
2
6’
5’
4’
2’
1’
3’
8a
(Adapted from Guyot et al., 1996,
Phytochemistry 42, 1279-1288)
(colorless)
OHO
OH
OH
OHO
OH
OH
OH
OH
OH
OH
4
8
4a 3
6
7
5
2
6’
5’
4’
2’
1’
3’
8a
4
8
4a
3
6
7
5
2
6’
5’
4’
2’
1’
3’
8a
A
B
C
D
E
F
PROCYANIDINS
(condensed tannins)
e.g.
procyanidin B2
Oxidized PROCYANIDIN derivatives
PPO
tt10 ?
(brown)
Quercetin-rhamnoside
dimers
OHO
OH O
O-Rhamnose
OH
OH
2
4
8
4a 3
6
7
5
2
6’
5’
4’
2’
1’
3’
8a
A
B
C
