Comprehensive Flavonol Profiling and TranscriptomeCoexpression Analysis Leading to Decoding Gene–MetaboliteCorrelations in Arabidopsis W OA

Keiko Yonekura-Sakakibara,a,1 Takayuki Tohge,a,1,2 Fumio Matsuda,a Ryo Nakabayashi,b Hiromitsu Takayama,b

Rie Niida,a Akiko Watanabe-Takahashi,a Eri Inoue,a and Kazuki Saitoa,b,3

a RIKEN Plant Science Center, Tsurumi-ku, Yokohama 230-0045, JapanbGraduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan

To complete the metabolic map for an entire class of compounds, it is essential to identify gene–metabolite correlations of a

metabolic pathway. We used liquid chromatography–mass spectrometry (LC-MS) to identify the flavonoids produced by

Arabidopsis thaliana wild-type and flavonoid biosynthetic mutant lines. The structures of 15 newly identified and eight

known flavonols were deduced by LC-MS profiling of these mutants. Candidate genes presumably involved in the flavonoid

pathway were delimited by transcriptome coexpression network analysis using public databases, leading to the detailed

analysis of two flavonoid pathway genes, UGT78D3 (At5g17030) and RHM1 (At1g78570). The levels of flavonol 3-O-

arabinosides were reduced in ugt78d3 knockdown mutants, suggesting that UGT78D3 is a flavonol arabinosyltransferase.

Recombinant UGT78D3 protein could convert quercetin to quercetin 3-O-arabinoside. The strict substrate specificity of

UGT78D3 for flavonol aglycones and UDP-arabinose indicate that UGT78D3 is a flavonol arabinosyltransferase. A

comparison of flavonol profile in RHM knockout mutants indicated that RHM1 plays a major role in supplying UDP-

rhamnose for flavonol modification. The rate of flavonol 3-O-glycosylation is more affected than those of 7-O-glycosylation

by the supply of UDP-rhamnose. The precise identification of flavonoids in conjunction with transcriptomics thus led to the

identification of a gene function and a more complete understanding of a plant metabolic network.

INTRODUCTION

Plants employ many diverse metabolic pathways to produce

>200,000 compounds (Dixon and Strack, 2003). Completion of

the Arabidopsis thaliana and rice (Oryza sativa) genome se-

quencing projects has allowed (1) the annotation of genes

involved in metabolic pathways based on homology and (2)

genome-wide identification of whole metabolic pathways. As-

signing functions to each of the genes that correlate with me-

tabolites in a pathway is a prerequisite for developing a

comprehensive understanding of complicated metabolic path-

ways. Because of its small genome and the development of

extensive genetic, molecular, and physiological tools, Arabidop-

sis may be the only plant in which the complete network of

secondary metabolism can currently be characterized within a

single plant species.

The flavonoids comprise one of the major secondary metab-

olite groups, with >7000 known compounds (Harborne et al.,

1999; Andersen and Markham, 2006). Formation of the basic

flavonoid skeleton has been well studied in terms of molecular

biology and natural products chemistry. Flavonol and anthocya-

nidin regulatory and biosynthetic pathways have been charac-

terized, and the corresponding genes have been isolated from

various plants (Figure 1; Andersen and Markham, 2006; Tanaka

and Filippa, 2006). However, the pathways for sequential mod-

ification, such as glycosylation, acylation, and methylation, are

still relatively unexplored even though modification produces a

huge chemical diversity and is essential for the stable accumu-

lation of flavonoids. A wide variety of flavonoids have been

isolated and identified from specific organs, such as flowers or

fruits (Andersen and Markham, 2006), but there has been no

comprehensive description of flavonoid metabolism in the or-

gans of a single plant species. Although the flavonoids are

important as flower pigments, UV-B protectants, phytoalexins,

signaling molecules, and regulators of auxin transport (Dooner

et al., 1991; Dixon and Paiva, 1995), the precise relationship

between structure and function is also largely unclear.

In Arabidopsis, at least 13 structures of flavonoids have been

identified by nuclear magnetic resonance (NMR) and/or liquid

chromatography–mass spectrometry (LC-MS ) with standards,

and more flavonoid compounds were reported to be present

(Graham, 1998; Veit and Pauli, 1999; Bloor and Abrahams, 2002;

Kerhoas et al., 2006). Thirty-five flavonoid biosynthetic genes,

including genes encoding 12 transcription factors, 10 structural

enzymes, and 10 modification enzymes, have been identified

(Tohge et al., 2005, 2007; Lepiniec et al., 2006; Fraser et al., 2007;

1 These authors contributed equally to this work.2 Current address: Max Planck Institute of Molecular Plant Physiology,Am Muhlenberg 1, 14476 Potsdam Golm, Germany.3 Address correspondence to ksaito@faculty.chiba-u.jp.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Kazuki Saito(ksaito@faculty.chiba-u.jp).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.108.058040

The Plant Cell, Vol. 20: 2160–2176, August 2008, www.plantcell.org ã 2008 American Society of Plant Biologists

Luo et al., 2007; Stracke et al., 2007; Yonekura-Sakakibara et al.,

2007), but even in this highly studied species, the full details of

the flavonoid pathway, including modification, are still unknown.

The major flavonoid structures in Arabidopsis suggest that there

are as yet unidentifiedmodification enzyme genes and pathways

in flavonoid metabolism. Generally, modification enzymes are

encoded bymultigene families. For example, there are 107 UDP-

dependent glycosyltransferase (UGT) genes in Arabidopsis

(D’Auria and Gershenzon, 2005). The presence of multiple genes

hampers efforts to determine precise gene functions by sequence

homology and to draw a complete pathway, even in Arabidopsis.

The presence of unidentified minor flavonoids also suggests

additional missing genes and pathways (Tohge et al., 2007).

Because of the rich research resources available for Arabi-

dopsis, it is the best candidate for cataloging the entire flavonoid

pathway. The integration of metabolome analysis with tran-

scriptome analysis has been successfully used in Arabidopsis to

decipher gene functions in biological processes (Hirai et al.,

2005, 2007; Tohge et al., 2005). Transcriptome coexpression

analysis is also a powerful tool for the efficient targeting of genes

within a multigene family (Yonekura-Sakakibara et al., 2007;

Saito et al., 2008). Use of these novel tools in combination with

reverse genetics and a biochemical approach using recombinant

proteins can provide robust evidence from which a whole path-

way can be constructed.

In this study, we attempted to complete the identification of

flavonols in each of the organs and flavonol annotation using

flavonoid mutant lines. Taken together with previous reports, a

total of 32 flavonol end or intermediate products were listed.

Thirty of 32 flavonols were detected, and the structures of 19

compounds were annotated. Transcriptome coexpression anal-

ysis using known flavonoid genes as queries suggested 139

genes that may be involved in flavonoid metabolism. Finally,

integration of comprehensive flavonol identification/annotation

with transcriptome coexpression analysis identified a gene

encoding flavonol 3-O-arabinosyltransferase and revealed the

physiological role of a gene coding for UDP-rhamnose synthase

in flavonoid biosynthesis.

RESULTS

Comprehensive Flavonol Profiling in Wild-Type and

tt4Mutants

To catalog all of the flavonol derivatives in Arabidopsis, flavonol

profiling in the flowers, leaves, stems, and roots of the wild type

and tt4 mutants were performed using ultraperformance liquid

chromatography (UPLC)–photodiode array (PDA)–electrospray

ionization (ESI)/quadrupole time-of-flight (Q-TOF)/MS (Figure

2A). The metabolites detected only in the wild type should be

flavonoid derivatives because TT4 encodes chalcone synthase,

the first committed enzyme in flavonoid biosynthesis, and no

flavonoid derivatives were detected in the tt4 mutant (Shirley

et al., 1995; von Roepenack-Lahaye et al., 2004).

The flavonols were differentially distributed in Arabidopsis

organs (Figure 3). Kaempferol derivatives (f1 to f3 in Figure 3)

were the major flavonols in leaves, stems, and flowers. In roots,

there were high levels of quercetin 3-O-glucoside-7-O-rhamno-

side (f6) in addition to kaempferol glycosides (f2 and f3), but there

was very little kaempferol 3-O-rhamnoside-7-O-rhamnoside (f1).

As for other phenolic compounds, a sinapoyl derivative s2 was

detected predominantly in the leaves and stems of both the wild

type and tt4.

The MS-detectable peaks in the wild type and tt4 were

compared to extract all flavonoid-related peaks (see

Figure 1. The Arabidopsis Flavonoid Biosynthetic Pathway.

4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone

isomerase; F3H, flavanone 3-hydroxylase; F39H, flavonoid 39-hydroxy-

lase; FLS, flavonol synthase; OMT1, O-methyltransferase; UGT, UDP-

dependent glycosyltransferase; DFR, dihydroflavonol 4-reductase;

LDOX/ANS, leucoanthocyanidin dioxygenase/anthocyanidin synthase;

AAT, anthocyanin acyltransferase.

Flavonol Metabolism in Arabidopsis 2161

Supplemental Figure 1 and Supplemental Data Set 1 online).

Principal component analysis was conducted with 1804 MS

peaks in leaves, 1864 in flowers, 1576 in roots, and 2138 in

stems, indicating the presence of clear clusters according to the

wild-type and tt4 genotypes (Figure 2B). The wild-type–specific

peaks (53 peaks in leaves, 322 in flowers, 152 in roots, and 75 in

stems) were picked up using a high PC1 loading value, and most

of the peaks are due to the ions of flavonols (see Supplemental

Figure 1 online). For example, 42 of 53 peaks are three major

kaempferol glycosides in leaves (f1 to f3). A total of 30 flavonol

derivatives were detected in Arabidopsis (four in leaves, 25 in

flowers, 14 in roots, and four in stems). Flowers, in which most

flavonol derivatives were detected, were used for further anal-

ysis.

Figure 2. UPLC-PDA-MS Analyses of the Extracts from Leaves, Flowers, Roots, and Stems of Wild-Type (Col-0) and tt4 Plants.

(A) UPLC-PDA and mass chromatograms of aqueous methanol extracts of the Arabidopsis wild type and the tt4 knockout mutant. Absorbance at 320

nm was used for the detection of flavonols. Labels correspond to compounds shown in Figure 3.

(B) Principal component analyses of the data obtained by LC-MS. Proportions of the first and second components are in parentheses. Wild-type results

are in red, and those from the tt4 mutant are in blue.

2162 The Plant Cell

Peak Annotation by Comparison with Flavonoid

Biosynthetic Gene Knockout Mutants

Twelve flavonols (f1 to f8, f17, f18, f20, and f24) in Arabidopsis

have been identified by UV spectroscopy, MS, 1H-NMR, and13C-NMR spectroscopy or annotated using UPLC-PDA-ESI/

Q-TOF/MS (Veit and Pauli, 1999; Jones et al., 2003; Tohge et al.,

2005, 2007; Kerhoas et al., 2006). We identified two additional

flavonols (f16 and f28) by comparing retention times andmass-to-

charge ratio (m/z) values with the standard compounds (Table 1;

seeSupplementalDataSet 2 online). Toannotate the remaining18

uncharacterized flavonols, mutants lacking CHS, F39H, flavonol39-O-methyltransferase (OMT1), flavonoid 3-O-glucosyltransfer-

ase (UGT78D2), flavonol 3-O-rhamnosyltransferase (UGT78D1),

flavonol 7-O-rhamnosyltransferase (UGT89C1), or anthocyanin

5-O-glucosyltransferase (UGT75C1) were used for flavonol anal-

ysis. Of the 14 identified (f1 to f8, f16 to f18, f20, f24, and f28),

flavonol derivativesmissing fromeachmutantwerewell correlated

with the loss of gene function (Figure 4, Table 1). No quercetin and

isorhamnetin conjugates (f5, f6, f8, f16, f24, and f28) accumulated

in tt7, which lacks F39H. No isorhamnetin conjugates (f28) were

detected in omt1mutants. Themutants ugt78d1 and ugt89c1 had

Figure 3. Flavonol Glycosides in Arabidopsis.

Asterisks indicate that the compounds were identified based on a comparison of retention times and UV/mass spectra of the standards used in this

study. R1=H, kaempferol; R1=OH, quercetin; R1=OMe, isorhamnetin. The presence of a compound in the indicated tissue is denoted by a gray box.

Flavonol Metabolism in Arabidopsis 2163

no conjugates of flavonol 3-O-rhamnoside (f1, f5, f17, f20, and f24)

and flavonol 7-O-rhamnoside (f1 to f3, f5, f6, and f8), respec-

tively. Interestingly, in the ugt78d2 mutant, accumulation of

the kaempferol/quercetin 3-O-glucosides (f2, f3, f6, and f8) was

drastically reduced but detectable. Other kaempferol/quercetin

3-O-glucosides (f16 and f18) also decreased. This result is in

agreement with the finding that cyanidin derivatives were detec-

ted at low levels in the ugt78d2mutant (Tohge et al., 2005).On the

other hand, accumulation of the isorhamnetin 3-O-glucosides

(f28) was similar or slightly increased. These data indicate that

another enzyme is involved in 3-O-glucosylation of isorhamnetin.

Flavonol profiles in the flowers of the wild type and ugt75c1were

essentially identical, demonstrating thatUGT75C1 is not involved

in flavonol glycosylation at any detectable level.

The basic flavonol structures and glycosylation patterns were

revealed by superimposing the flavonol profiles of mutants over

thewild type (Table 1). The structures of additional nine flavonols,

including pentosides (f14, f15, f19, f23, f25, f27, f29, f30, and f32),

were identified in Arabidopsis. A total of 23 flavonol structures and

their distribution in the mutants show that at least four flavonol

glycosyltransferases, anadditional flavonol 3-O-glucosyltransferase,

a flavonol 3-O-pentosyltransferase, a flavonol 3-O-glycoside:299-O-rhamnosyltransferase, and a flavonol 3-O-glycoside:699-O-

glucosyltransferase remain to be identified.

New Flavonoid Biosynthetic Genes Deduced by

Transcriptome Coexpression Analysis

Transcriptome coexpression analysis (Saito et al., 2008) was

used to identify all of the flavonoid-related genes and was

executed with the 24 genes encoding flavonoid biosynthetic

enzymes or transcription factors listed in Table 2 as query. The

lowest correlation coefficient (r) between query genes, except

for the flavonol 7-O-glucosyltransferase gene (UGT73C6) and

OMT1 is 0.527. UGT73C6 and OMT1 were excluded because

UGT73C6 may cross-hybridize with UGT73C5, a brassino-

steroid-related UGT, and the OMT1 coexpression profile sug-

gests that it is under the regulation of lignin biosynthesis but

not of flavonoid biosynthesis. The coexpression relationships of

all Arabidopsis genes exhibiting correlation coefficients > 0.525

(r > 0.525, all data sets version 3) with the query genes are shown

in Supplemental Figure 2 online and Table 3. The cutoff valuewas

set according to Aoki et al. (2007). The clusters of coexpressed

flavonoid genes were classified into two major groups. One

cluster contains the general flavonoid/flavonol subgroup; an-

other cluster represents the anthocyanin subgroup. UGT73C6

and OMT1 did not belong to either of these clusters. The 139

genes were highly correlated with the general flavonoid-related

or anthocyanin-related genes (see Supplemental Figure 2 online).

Table 1. The Flavonol Profiles in Acidic Methanol-Water Flower Extracts of Wild-Type Plants and Flavonoid Mutant Lines

Peak No.

Rt ESI-MS Fragment

Col-0 tt4 tt7 omt1

ugt78d2 ugt78d1 ugt89c1 ugt75c1(min) (m/z) (m/z)a (Fd3GlcT) (F3RhaT) (F7RhaT) (A5GlcT)

f1b 5.38 579 433, 287 5.14 ND 6.05 5.71 5.61 ND ND 4.63

f2b 4.88 595 433, 287 3.33 ND 4.87 3.96 0.19 4.25 ND 2.94

f3b 4.14 741 595, 449, 287 6.47 ND 7.75 7.49 0.29 7.33 ND 5.93

f5b 4.94 595 449, 303 2.09 ND ND 4.14 2.04 ND ND 1.86

f6b 4.47 611 449, 303 2.16 ND ND 3.51 0.12 2.54 ND 1.97

f8b 3.88 757 611, 449, 303 1.11 ND ND 2.37 0.04 1.58 ND 1.09

f14 4.99 625 463, 317 2.71 ND ND ND 2.08 3.47 ND 2.51

f15 4.49 625 479, 317 0.05 ND ND ND 0.41 ND 1.12 0.05

f16b 5.33 465 303 0.07 ND ND 0.12 ND 0.10 2.36 0.08

f17b 6.52 433 287 0.03 ND 0.13 0.04 0.02 ND 3.54 0.02

f18b 5.84 449 287 0.08 ND 0.19 0.06 ND 0.10 3.18 0.05

f19 4.95 565 433, 287 0.07 ND 0.28 0.11 0.66 0.13 ND 0.08

f20 4.34 595 449, 287 0.10 ND 0.32 0.18 0.14 ND 1.96 0.09

f21 5.02 611 449, 287 2.37 ND 3.27 3.10 ND 2.07 3.00 1.97

f22 4.83 711 565, 433, 287 0.02 ND 0.03 0.03 0.18 0.02 ND 0.02

f23 5.72 435 303 0.01 ND ND 0.02 0.02 0.01 0.50 0.01

f24b 5.92 449 303 0.05 ND ND 0.14 0.07 ND 2.09 0.06

f25 4.66 581 449, 303 0.36 ND ND 0.72 1.16 0.40 ND 0.33

f26 4.60 627 465, 303 1.66 ND ND 1.86 0.06 1.74 1.77 1.35

f27 6.64 463 317 0.05 ND ND ND 0.07 ND 1.89 0.04

f28b 5.97 479 317 0.14 ND ND ND 0.15 0.16 2.99 0.15

f29 5.17 595 463, 317 0.25 ND ND ND 2.46 0.34 ND 0.24

f30 5.50 609 463, 317 2.09 ND ND ND 4.38 ND ND 1.83

f31 5.07 641 479, 317 Trace ND ND Trace 0.11 0.01 0.01 0.01

f32 4.27 771 625, 463, 317 0.03 ND ND ND 0.03 0.05 ND 0.05

s1b 4.00 387 207 0.06 0.12 0.07 0.02 0.07 0.05 0.07 0.05

s2b 5.46 341 207 0.08 0.21 0.08 0.03 0.10 0.10 0.12 0.07

Flavonols were quantified by measuring mass peak area using a response value of the peak area of an internal standard (naringenin 7-O-glucoside).

ND, not detected; trace, <0.01; s1 and s2, sinapoyl derivatives.a Detected in mass and/or tandem mass data.b Identified based on a comparison of retention times and UV/mass spectra of the standards.

2164 The Plant Cell

Twenty-four genes hadmore than two correlationswith flavonoid-

related genes. One-third of the genes listed in Table 3 were

coregulated with flavonoid-related genes by flavonoid-specific

MYBs (PAP1, MYB11, MYB12, and MYB111; Tohge et al.,

2005, Stracke et al., 2007). The genes encoding embryo-specific

protein (At5g62210), 3-keto-acyl-CoA thiolase (At5g48880), cryp-

tochrome 3 (At5g24850), UGT84A1 (At4g15480), cinnamoyl-CoA

reductase-like protein (At2g23910), early light-induced protein

(At3g22840), TT8 (At4g09820), and glutathione S-transferase

(GST; At1g10370) were regulated in a manner similar to the

flavonoid biosynthetic genes by high irradiance and blue light in a

CRYPTOCHROME1 (CRY1) photoreceptor and/or LONG HYPO-

COTYL5 (HY5) transcription factor-dependent manner (Kleine

et al., 2007). Other genes were also regulated by HY5 under

UV-B radiation, much like the flavonoid biosynthetic genes (Table

3; Brown et al., 2005). For further analysis, we focused on

UGT78D3 as a putative flavonol glycosyltransferase and RHM1,

which was selected from the genes highly correlated with the

flavonol biosynthetic genes.

UGT78D3 Encodes Flavonol 3-O-Arabinosyltransferase

UGT78D3 is correlated with MYB111 (r = 0.572) but not with the

other query genes. However, UGT78D3 belongs to the UGT78D

Figure 4. UPLC-PDA-MS Analyses of the Extracts from Flowers of Wild-Type (Col-0) and Flavonoid-Defective Mutants.

Absorbance at 320 nm was used for detection. A5GlcT, anthocyanin 5-O-glucosyltransferase; F3RhaT, flavonol 3-O-rhamnosyltransferase; F7RhaT,

flavonol 7-O-rhamnosyltransferase; Fd3GlcT, flavonoid 3-O-glucosyltransferase. Labels correspond to compounds shown in Figure 3. The labels in

parentheses indicate compounds at <5% in the peak.

Flavonol Metabolism in Arabidopsis 2165

subfamily, which also contains flavonoid 3-O-glucosyltrans-

ferase (UGT78D2) and flavonol 3-O-rhamnosyltransferase

(UGT78D1), suggesting that UGT78D3 encodes a flavonoid

3-O-glycosyltransferase. UGT78D3 has high sequence identity

with UGT78D2 (75.7% at the amino acid level) and UGT78D1

(68.3%). UGT78D2 has an amino acid sequence identity with

UGT78D1 of 71.7%.

To test our hypothesis, T-DNA insertionmutant SALK_114099,

designated as ugt78d3, was used for reverse genetics analysis.

T-DNAwas inserted between2122 and2100 bp upstream from

the start codon of UGT78D3 (Figure 5A). UGT78D3 transcripts

were abundant in floral buds but present at low levels in siliques

and stems (Figure 5B). Real-time PCR showed that the accu-

mulation level of UGT78D3 transcripts in flowers of the homozy-

gous ugt78d3mutant decreased to 30% of those in the wild type

(Figure 5C). Flavonol target analysis revealed that three flavonol

3-O-pentoside conjugates (f19, f25, and f29) were under the

detection limit in flowers of the ugt78d3mutant (Figure 5D). This

flavonol 3-O-pentoside conjugate-deficient phenotype was

complemented by the constitutive expression of UGT78D3

cDNA under the control of the cauliflower mosaic virus 35S

promoter (Figure 5D), indicating that UGT78D3 encodes a fla-

vonol 3-O-pentosyltransferase.

Flavonoid analysis of ugt78d3 mutants provided no additional

information on the structure of flavonol 3-pentoside conjugates

(f19, f25, and f29). Therefore, a biochemical approach was used

to identify the exact structure of the pentose at the C-3 position.

UGT78D3 recombinant protein was produced in Escherichia coli

as a GST fusion and purified. The sugar donor specificity of

UGT78D3 was examined with UDP-arabinose and UDP-xylose

as UDP-pentoses. UDP-galactose, UDP-glucose, and UDP-

rhamnose were also tested. The GST-UGT78D3 fusion protein

catalyzed the conversion of quercetin to a quercetin 3-O-arabi-

noside, as confirmed by UPLC retention time, UV absorbance,

and MS (Figure 6). No UGT activity was observed with UDP-

xylose, UDP-galactose, UDP-glucose, or UDP-rhamnose as

substrates, indicating that UGT78D3 is specific for UDP-arabi-

nose as a sugar donor (Table 4). The sugar acceptor specificity of

UGT78D3 was examined with kaempferol, quercetin, myricetin,

isorhamnetin, cyanidin, and their glycosides as substrates.

UGT78D3 activity was specific for flavonol aglycones (kaemp-

ferol, quercetin, myricetin, and isorhamnetin) and kaempferol

7-O-rhamnoside but had no activity on cyanidin or the flavonoid

3-O-glycosides. These results indicate that UGT78D3 arabino-

sylates flavonols at the C-3 position.

To confirm UGT78D3 function in planta, the structures of

f19 and f25 were confirmed by direct comparison with the

enzymatically synthesized standard compounds. Kaempferol

3-O-arabinoside-7-O-rhamnoside was prepared by the reaction

catalyzed by UGT89C1 with kaempferol 3-O-a-L-arabinoside

and UDP-b-L-rhamnose as substrates. An enzymatic product

(1.8 mg) was isolated by multistep chromatography, and its

structure was determined by 1H-NMR and rotating frame over-

hauser enhancement (ROE). The ROE correlation of protons at

C-6 (d 6.43) and C-8 (d 6.73) by the irradiation of anomeric proton

of rhamnose (rhamnose H-1, d 5.52) indicated that rhamnose

was attached at the C-7 position of kaempferol (Mulinacci

et al., 1995). The chromatographic behavior and MS data of

Table 2. Query Genes Used for Transcriptome Coexpression Analysis

Name AGI No. Gene Annotation

4CL3 At1g65060 4-Coumarate:CoA ligase

CHS, TT4 At5g13930 Chalcone synthase

CHI, TT5 At3g55120 Chalcone isomerase

F3H, TT6 At3g51240 Flavanone 3-hydroxylase

F39H, TT7 At5g07990 Flavonoid 39-hydroxylase

FLS1 At5g08640 Flavonol synthase

DFR, TT3 At5g42800 Dihydroflavonol reductase

ANS, TT18 At4g22880 Anthocyanidin synthase

GST, TT19 At5g17220 GST

UGT73C6 At2g36790 Flavonol 7-O-glucosyltransferase

UGT75C1 At4g14090 Anthocyanin 5-O-glucosyltransferase

UGT78D1 At1g30530 Flavonol 3-O-rhamnosyltransferase

UGT78D2 At5g17050 Flavonoid 3-O-glucosyltransferase

UGT89C1 At1g06000 Flavonol 7-O-rhamnosyltransferase

A5G6999MaT At3g29590 Anthocyanin 5-O-glucoside:6999-O-malonyltransferase

A3G699p-CouT At1g03940 Anthocyanin 3-O-glucoside:699-O-p-coumaroyltransferase

A3G699p-CouT At1g03495 Anthocyanin 3-O-glucoside:699-O-p-coumaroyltransferase

SCPL10 At2g23000 Sinapoylglucose:anthocyanin acyltransferase

OMT1 At5g54160 Caffeic acid/5-hydroxyferulic acid O-methyltransferase, flavonol

39-O-methyltransferase

MYB11 At3g62610 R2R3 MYB protein

MYB12 At2g47460 R2R3 MYB protein

MYB111 At5g49330 R2R3 MYB protein

PAP1, MYB75 At1g56650 R2R3 MYB protein

PAP2, MYB90 At1g66390 R2R3 MYB protein

2166 The Plant Cell

kaempferol 3-O-L-arabinoside-7-O-L-rhamnoside were identical

with those of f19 and enzymatically synthesized products by

UGT78D3 with kaempferol 7-O-a-L-rhamnoside and UDP-b-L-

arabinose (Figure 7). These results indicated that f19 is kaemp-

ferol 3-O-L-arabinoside-7-O-L-rhamnoside. The component of

f25 was also identified to be quercetin 3-O-L-arabinoside-7-O-L-

rhamnoside by the similar procedure.

The UDP-Rhamnose Synthase 1Gene Plays a Role in

Rhamnosylation of Flavonoids

In Arabidopsis, there are three UDP-rhamnose synthase genes

(RHM1, RHM2, and RHM3) (Usadel et al., 2004; Western et al.,

2004; Oka et al., 2007). Expression of the UDP-Rhamnose

Synthase 1 (RHM1) gene was highly correlated with the general

flavonoid pathway genes, for example, 4CL3 (r= 0.736), FLS1 (r =

0.696), UGT89C1 (r = 0.669), CHI (r = 0.647), F3H (r = 0.611), and

CHS (r = 0.603), based on data from all experiments. RHM2/

MUM4 and RHM3 expression profiles differ from the flavonoid

biosynthetic genes (r < 0.4). The accumulation of RHM1-RHM3

transcripts in the target organs was analyzed by real-time

PCR (Figure 8A). Major flavonol glycosides were predominantly

accumulated in floral buds and flowers (Figure 2; Yonekura-

Sakakibara et al., 2007), which is consistent with accumulation

pattern of RHM1 transcripts. RHM1 transcripts also made up

the bulk of RHM transcript accumulation in all of the tested

organs except for siliques. In siliques, the transcripts of RHM2/

MUM4, which are involved in seed mucilage pectin synthesis

(Usadel et al., 2004; Western et al., 2004), were predominantly

accumulated.

To verify the physiological relationship between RHM1 and

the flavonoid biosynthetic genes, the flavonol profiles of the

knockout mutants of RHM1 (rol1-1 and rol1-2) and RHM2

(rhm2-1 and rhm2-3) were analyzed (Figure 8). Flavonol profiles

in leaves and flowers of the rhm2mutants showed no significant

Table 3. The Genes Coexpressed with Those in Flavonoid Metabolism

MYB11Correlated MYB12 CRY1

Gene No. AGI No. PAP1 MYB111 HY5

Flavonoid/phenylpropanoid biosynthesis

9 At5g05270 Chalcone isomerase family + + +

7 At5g54060 UDP-glucosyltransferase, UGT79B1 +

5 At4g15480 UDP-glucosyltransferase, UGT84A1 + +

3 At2g23910 Cinnamoyl-CoA reductase-like, CCRL9 + +

Transporter

6 At5g17010 Sugar transporter family

2 At1g10370 Glutathione S-transferase, GST30 +

2 At5g02270 ABC transporter, AtNAP9 +

2 At5g44110 ABC transporter, AtNAP2 +

Protein kinase

2 At1g10850 Ser/Thr kinase family

2 At3g56370 Leu-rich repeat transmembrane protein kinase

Transcription factor

2 At2g37260 WRKY transcription factor, TTG2 +

2 At4g09820 Basic helix-loop-helix family, TT8 + +

2 At5g60140 Transcriptional factor B3 family

Others

8 At5g62210 Embryo-specific protein + +

7 At1g65560 Allyl alcohol dehydrogenase

7 At5g48880 3-Keto-acyl-CoA thiolase, KAT5 + +

6 At1g78570 Rhamnose synthase, RHM1

5 At5g24850 Cryptochrome 3, CRY3 +

4 At1g06550 Enoyl-CoA hydratase/isomerase family

3 At5g20070 Nudix hydrolase homolog, ATNUDT19

2 At1g36160 Acetyl-CoA carboxylase

2 At1g72970 HOTHEAD

2 At3g22840 Early light-induced protein1, ELIP1 +

2 At5g39220 Hydrolase family

Only the genes that correlated with at least two genes are shown. The plus sign means the similar regulatory behavior with flavonoid biosynthetic

genes in knockout mutants (CRY1/HY5, HY5 or MYB11/MYB12/MYB111) or an overexpressed plant (PAP1).

Flavonol Metabolism in Arabidopsis 2167

alteration. However, in leaves of rol-1 and rol1-2, accumulation

levels of kaempferol 3-O-rhamnoside-7-O-rhmanoside (f1)

were considerably reduced and those of kaempferol 3-O-gluco-

side-7-O-rhmanoside (f2) were elevated. In flowers of the rol1

mutants, levels of kaempferol/quercetin 3-O-rhamnoside-7-O-

rhamnosides (f1 and f5) and kaempferol 3-O-rhamnosyl(1/2)-

glucoside-7-O-rhamnoside (f3) were reduced and those of

flavonol 3-O-glucosides (f16, f18, and f28) were elevated con-

siderably. These results imply that UDP-rhamnose produced

by RHM1 is used for flavonol rhamnosylation and RHM1 plays

a major role in supplying UDP-rhamnose for modification of

flavonols.

Figure 5. A T-DNA Insertion Mutant of UGT78D3.

(A) Schematic representation ofUGT78D3with a T-DNA insertion mutant

used in this work. The thick black line indicates coding sequence.

Numbers indicate the position of the T-DNA insertion. The gray box

adjacent to T-DNA indicates the region containing the UGT78D3 pro-

moter from �77 bp to +4 bp. LB, left border; RB, right border.

(B) Real-time PCR analysis of UGT78D3 transcripts in organs of the

Arabidopsis wild type (Col-0). Error bars represent SD of three technical

replicates per sample.

(C) Real-time PCR analysis of UGT78D3 transcripts in wild-type (Col-0)

and ugt78d3 mutant flowers. Error bars represent SD of three technical

replicates per sample.

(D) Extracted fragment mass chromatograms of aqueous methanol

extracts from flowers of the wild type (Col-0), the ugt78d3 mutant, and

ugt78d3 complemented with p35S:UGT78D3. The extracted fragment

mass ionsm/z 565,m/z 581, andm/z 595 correspond to compounds f19,

f25, and f29 in Figure 3, respectively.

Figure 6. UPLC Analyses of the Reaction Products of UGT78D3 Re-

combinant Protein.

Elution profiles of reaction products of GST protein (control) or GST-

fused UGT78D3 protein (UGT78D3) and the standards (quercetin

3-O-arabinoside) are shown.

2168 The Plant Cell

DISCUSSION

The Power of Detailed Targeted Flavonol Analysis and

Transcriptome Coexpression Analysis for

Functional Genomics

Here, we used a novel strategy for functional genomics in

Arabidopsis using flavonol as a case study (Figure 9). Coupling

the detailed analysis of secondary metabolites with coexpression

can be used as a more general method for identifying the biosyn-

thetic pathways of plant compounds associated with specific

organs and developmental stages as well as their genetic com-

ponents. The useof T-DNAmutations ingenes encodingflavonoid

biosynthetic enzymes to provide comprehensive annotation of the

flavonols in Arabidopsis is effective and can provide an efficient

method for elucidating flavonol structures. The integration of

metabolic profiling and transcriptome coexpression could effi-

ciently narrow the pool of candidate genes. Using these methods,

a flavonol 3-O-arabinosyltransferase gene was found, and the

relationship between flavonols and UDP-rhamnose synthesis has

emerged. After listing the candidates potentially involved in the

reactions or regulation of a metabolic pathway, the specific

identification of a gene function could be accomplished using a

traditional reverse genetics approachwith knockoutmutants and/

or by biochemical characterization with the recombinant proteins,

as has been exemplified in this study. The above strategy will be

much improved as large metabolome data sets follow transcrip-

tome data sets into to the public domain.

This combined strategy can also be useful for the detailed

characterization of relatively minor flavonoid pathways that are

evident only in mutants. The detailed flavonoid analyses of

ugt78d2 mutants suggest the existence of another flavonoid

3-O-glucosyltransferase because a small amount of flavonoid

3-O-glucoside is present in the knockout mutant of UGT78D2.

The ugt89c1mutants accumulate flavonol 3-O-glucosides but not

3-O-rhamnosyl(1/2)glucosides, suggesting that 7-O-rhamnosy-

lation likely occurs prior to 299-O-rhamnosylation. Accumulation of

f19, f25, and f29 may also suggest that 7-O-rhamnosylation of

kaempferol 3-O-arabinoside likely occurs prior to further glyco-

sylation with deoxyhexose. The presence of f21, f26, and f31

suggests that a novel flavonol 3-O-glucoside:hexosyltransferase

using glucose, galactose, or mannose may be involved in flavonol

modification because those sugars have been so far identified as

a sugar moiety of flavonol glycosides and have the molecular

weight corresponding to unidentified hexose of f21, f26, and f30

(Andersen and Markham, 2006).

Transcriptome Coexpression Analysis Delimits Genes

Related to Flavonoid Metabolism

Transcriptome coexpression analyses suggest other candidate

genes that might be related to flavonoid metabolism. Eight of

24 candidate genes were regulated in PAP1-overexpressing

or in MYB11/MYB12/MYB111 triple knockout plants. CHIput(At5g05270) may play a supplementary role to the primary CHI

(At3g55120). The coordinated expression of CHIput with CHI

tends to support this hypothesis. UGT79B1 belongs to a cluster

containing the anthocyanin biosynthetic group. Phylogenetic

trees of flavonoid UGTs suggest that UGT79B1 encodes antho-

cyanin 3-O-glucoside 299-xylosyltransferase (Tohge et al., 2005).

These data suggest thatCHIput andUGT79B1 are involved in the

flavonoid pathway. UGT84A1 (At4g15480) has 1-O-glucosyl-

transferase activity for phenylpropanoids such as p-coumaric

acid and sinapic acid (Lim et al., 2001). Sinapoylmalate con-

verted from 1-O-sinapoylglucose by malate sinapoyltransferase

(SNG1), is a UV-B protective compound much as the flavonoids

are (Ruegger and Chapple, 2001). Under UV stress conditions,

accumulation of sinapoylmalate was higher in tt4mutants than in

the wild type (Kusano et al., 2007), suggesting that sinapoylma-

late biosynthesis acts as a backup system for flavonoid metab-

olism under stress conditions. The positive correlation between

MYB12 and UGT84A1 (r = 0.781) and the negative correlation of

MYB12/UGT84A1 and SNG1 (r = 20.357/20.323) tends to

support this hypothesis. 1-O-Sinapoylglucose is also a substrate

for anthocyanin sinapoyltransferase (Fraser et al., 2007), and

anthocyanins accumulate with UV exposure and high sucrose

stress. Arabidopsis may thus coregulate 1-O-sinapoylglucose

with the flavonoids for prompt responses to stress. Cinnamoyl-

CoA reductase–related protein (At2g23910) alsomay be involved

in phenylpropanoid biosynthesis. Although coexpression does

not necessarily indicate a functional relationship (Stuart et al.,

2003), we believe that the significance of transcriptome coex-

pression analysis for functional gene identification has been

established, at least for secondary metabolism.

A Flavonol Glycosyltransferase:Flavonol

3-O-Arabinosyltransferase

We demonstrated that UGT78D3 encodes a flavonol 3-O-

arabinosyltransferase. The UGT78D3 function in planta was

Table 4. Substrate Specificity of Arabidopsis UGT78D3

Relative Activity (%)

Sugar donora

UDP-arabinose 100.0 6 10.0

UDP-glucose ND

UDP-rhamnose ND

UDP-xylose ND

UDP-galactose ND

Sugar acceptorb

Kaempferol (Kae) 100.0 6 8.5

Kae 3-O-glucoside ND

Kae 7-O-rhamnoside 140.4 6 13.9

Quercetin (Que) 282.2 6 9.6

Que 3-O-glucoside ND

Que 3-O-rhamnoside ND

Myricetin 148.2 6 2.0

Isorhamnetin 164.9 6 34.4

Cyanidin (Cya) ND

Cya 3-O-glucoside ND

Cya 3-O-rhamnoside ND

Cya 3,5-O-diglucoside ND

ND, not detected.aThe reactions were performed with kaempferol as the sugar acceptor.bThe reactions were performed with UDP-arabinose as the sugar donor.

Flavonol Metabolism in Arabidopsis 2169

established by the identification of f19 and f25 as kaempferol/

quercetin 3-O-arabinoside-7-O-rhamnoside, respectively. The

conformation of substrates (kaempferol/quercetin 3-O-a-L-arab-

inosides and UDP-b-L-rhamnose) and previous data are consis-

tent with the hypothesis that f19 and f25 are kaempferol/

quercetin 3-O-a-L-arabinoside-7-O-a-L-rhamnosides, respec-

tively (Mulinacci et al., 1995; Sakar et al., 2005). Compared

with UGT78D1 and UGT78D2, UGT78D3 is weakly correlated

with genes in the flavonoid metabolic pathway, which is consis-

tent with the anatomical distribution of flavonol glycosides. The

flavonol 3-O-arabinosides make up only a minor fraction of

flavonols or are under the detection limit in organs except for

flowers, whereas the flavonol 3-O-glucosides and 3-O-rhamno-

sides are distributed in all tested organs as major flavonols.

Among the 6850 flavonoids registered in the Flavonoid Viewer

database (http://www.metabolome.jp/software/FlavonoidViewer/

viewer), 2.9% are arabinosylated flavonoids (M. Arita, personal

communication). To find genes involved in such minor metabolite

biosynthesis, an in-depth analysis of gene-to-metabolite correla-

tion is essential.

Functional identification of three Arabidopsis flavonol 3-O-

glycosyltransferases (3GlyTs) in the UGT78D subfamily with

Figure 7. UPLC-MS Analyses of the Reaction Products of UGT78D3 or UGT89C1 Recombinant Protein.

Extracted fragment mass chromatograms of aqueous methanol extracts from flowers of wild type (Col-0) ([A] and [F]), the reaction products of

UGT89C1 with kaempferol 3-O-arabinoside (B), (A) coeluted with (B) (C), the reaction products of UGT78D3 with kaempferol 7-O-rhamnoside (D), (A)

coeluted with (D) (E), the reaction products of UGT89C1 with quercetin 3-O-arabinoside (G), and (F) coeluted with (G) (H). The extracted fragment mass

ionsm/z 565 ([A] to [E]) andm/z 581 ([F] to [H]) correspond to compounds f19 and f25 in Figure 3, respectively. Kae 3-Ara, kaempferol 3-O-arabinoside;

Kae 7-Rha, kaempferol 7-O-rhamnoside; Que 3-Ara, quercetin 3-O-arabinoside.

2170 The Plant Cell

Figure 8. T-DNA Insertion Mutants of RHM1 and RHM2, and UPLC-MS Analyses of the rhm Mutant Lines.

(A) Real-time PCR analysis of RHMs transcripts in Arabidopsis organs. Error bars represent SD of three technical replicates per sample.

(B) Schematic representation of RHM1 and RHM2 with the mutations used in this work. rol1-1, a nonsense mutation at position 318; rol1-2, a missense

Flavonol Metabolism in Arabidopsis 2171

distinct UDP-sugar specificity allowed us to deduce the amino

acid residues involved in substrate recognition. The three-dimen-

sional structure of grape (Vitis vinifera) flavonoid 3-O-glucosyl-

transferase (3GlcT) was determined, and a structural analysis

suggests the presence of several key residues that interact with

UDP-sugar and the flavonoid backbone (Offen et al., 2006). The

amino acids Gln-375, Asp-374, and Thr-141 have been pro-

posed to interact with hydroxyl groups at the C-2 and C-3, C-3,

and C-4, and C-6 positions of the glucose moiety of UDP-

glucose, respectively, but Gln-375 and Thr-141 are not con-

served in the Arabidopsis 3GlyTs (see Supplemental Figure 3

online). His residues corresponding to Gln-375 are conserved

among the galactosyltransferases (Kubo et al., 2004). A His

residue was also found as the corresponding residue in

UGT78D3. The configurations of hydroxyl groups at C-2, C-3,

and C-4 positions are the same in arabinose and galactose.

However, UGT78D3 could not use UDP-galactose. Asp-374 in

grape flavonoid 3GlcT is conserved in the three Arabidopsis

3GlyTs, although the configurations of hydroxyl groups at the

C-3 and C-4 positions are different (see Supplemental Figure 3

online). Given this disparity, the residues involved in sugar donor

specificity cannot be ascribed to a single amino acid residue.

RHM1 Is Involved in Flavonol Biosynthesis

Rhamnosylation is a major glycosylation of flavonols in Arabi-

dopsis. Twenty-one of 32 detected flavonol derivatives in wild-

type Arabidopsis are rhamnosides (Figure 3). A comparison of

flavonol profile indicates that RHM1 plays a major role in sup-

plying UDP-rhamnose for flavonol biosynthesis. RHM1 tran-

scripts are accumulated as themajorRHM species inmost of the

tested Arabidopsis organs. The expression pattern ofRHM1was

well coordinated with the known flavonoid biosynthetic genes.

The RHM1 correlation coefficients for flavonol rhamnosyltrans-

ferase (UGT89C1, r = 0.67; UGT78D1, r = 0.51) are higher than

those for flavonol glucosyltransferase (UGT78D2, r = 0.45;

UGT78D3, r = 0.32). In leaves, glycosylation at the C-3 position

was strongly affected by the restriction of UDP-rhamnose supply

because subsequent glycosylations at the C-7 or C-299 positionsfor major flavonols are rhamnosylation only. In flowers of rol1s,

additional flavonol 3-O-glucosides were detected, suggesting

that more UDP-rhamnose may be necessary in flowers and

glycosylation at the C-7 position is also affected in the severe

limitation of UDP-rhamnose. In fact, major six flavonol contents

in flowers are ;10- to 70-fold higher than in leaves (Yonekura-

Sakakibara et al., 2007). The role of RHM1 for UDP-rhamnose

supply to flavonols was also supported by a recent report

with young shoots of the rol1 mutants (Ringli et al., 2008) and

our data with RHM1 knockdown mutants (SALK_027926 and

SALK_143589; see Supplemental Figure 4 online).

The extent of the RMH3 contributions is unclear, and it may

be committed to other pathways. RHM2/MUM4 is involved in

the synthesis of pectinaceous rhamnogalacturonan I in seed

mucilage (Usadel et al., 2004; Western et al., 2004). Interestingly,

RHM3 has a low correlation coefficient with RHM1 (r = 0.46, all

data sets version 3). The molecular evolution of RHMs may

explain this correlation. The Arabidopsis genome contains a

number of large duplicated chromosomal segments. Blanc et al.

(2003) proposed that the Arabidopsis lineage underwent at least

two distinct episodes (old and recent) of duplication. According to

Blanc et al. on their website (http://wolfe.gen.tcd.ie/athal/dup) for

exploring paralogous blocks identified within the Arabidopsis

genome, RHM1 is the ancestral gene form. RHM3 was duplica-

ted and diverged from RHM1 during the old duplication, and

RHM2/MUM4 emerged from RHM3 by some recent duplication

(see Supplemental Figure 5 online). Comprehensive analysis of

rhamnose-containing metabolites using RHM knockout mutants

will be helpful to clarify the additional functions of RHM1, RHM2/

MUM4, and RHM3.

METHODS

Plant Materials

Arabidopsis thaliana (accession Columbia-0; Lehle Seeds) was used as

the wild type in this study. The mutants ugt75c1, ugt78d1, ugt78d2,

ugt89c1, rol1-1, rol1-2, and rhm2-1 were described previously (Jones

et al., 2003; Usadel et al., 2004; Tohge et al., 2005; Diet et al., 2006;

Yonekura-Sakakibara et al., 2007). The T-DNA–inserted knockout mutants

of omt1 (CS25167) and tt7 (CS6509) were obtained from the ABRC. The

tt4 (C1) mutant was obtained from tt mutant lines induced by ion beam

irradiation of Arabidopsis (Shikazono et al., 2003). The T-DNA–inserted

mutantofArabidopsis, linesSALK_114099 forUGT78D3andSALK_085051

(rhm2-3, a homozygous knockout line) for RHM2, were obtained from the

Salk Institute. T-DNA insertion lines were screened by PCR using specific

primers for UGT78D3, RHM2 ,and T-DNA: UGT78D3f, UGT78D3r, RHM2f,

RHM2r, LBa1, and RBa1 (see Supplemental Table 1 online). PCR products

were sequenced to determine the exact insertion points.

Plants were cultured on Murashige and Skoog–agar medium contain-

ing 1% sucrose (Valvekens et al., 1988) in a growth chamber at 228Cwith

16 h/ 8 h light and dark cycles for 18 d or in a greenhouse at 228Cwith 16 h/

8 h light/dark for 4 weeks. Plants were harvested, immediately frozenwith

liquid nitrogen, and stored at –308C until use. Five biological replicates

were used for flavonoid profiling.

Figure 8. (continued).

mutation changing an Arg at position 283 to a Lys; rhm2-1 and rhm2-3, T-DNA insertion mutants. LB/RB, left/right borders. Numbers indicate the

position of T-DNA insertion.

(C) UPLC-PDA-MS analyses of the extracts from leaves and flowers in the wild type (Col-0) and the rhm mutants. Absorbance at 320 nm was used for

detection. Labels correspond to compounds shown in Figure 3. The labels in parentheses indicate compounds at <5% in the peak.

(D) The ratio of flavonol derivatives to total flavonol in leaves of the wild type (Col-0) and the mutants. Error bars represent SD for five independent

experiments.

(E) The ratio of flavonol derivatives to total flavonol in flowers of the wild type (Col-0) and the mutants. Error bars represent SD for five independent

experiments. N.D., not determined.

2172 The Plant Cell

Chemicals

Flavonoid standards, including quercetin 3-O-arabinoside, were purchased

from Extrasynthese and Analyticon. Kaempferol 3-O-a-L-rhamnoside-7-O-

a-L-rhamnoside, kaempferol 3-O-a-L-rhamnosyl(1/2)-b-D-glucoside-7-

O-a-L-rhamnoside, quercetin 3-O-a-L-rhamnoside-7-O-a-L-rhamnoside,

and quercetin 3-O-a-L-rhamnosyl(1/2)-b-D-glucoside-7-O-a-L-rhamno-

side were from our laboratory collection. UDP-b-L-arabinose and UDP-a-

D-xylosewere purchased fromCarboSource Services (supported in part by

National Science Foundation–Plant Cell Wall Biosynthesis Research Net-

work Grant 0090281).

Flavonol Profiling by UPLC-PDA-ESI/Q-TOF/MS

Flavonol analyses were performed in quintuplicate. Frozen leaves were

homogenized in 5 mL of extraction solvent (methanol: CH3COOH:H2O =

9:1:10, 0.02 mM naringenin-7-O-glucoside) per milligram of fresh weight

of tissue in a mixer mill (MM300; Retsch) for 5 min at 30 Hz. After

centrifugation at 12,000g, the supernatants were immediately used for

flavonoid analysis.

For flavonol profiling, a Waters Acquity UPLC system (Waters) fitted

with a Q-TOF Premier mass spectrometer (Micromass MS Technologies)

was used. UPLCwas performed on aUPLCphenyl C18 column (F2.1mm

Figure 9. Proposed Comprehensive Flavonol Pathway in Arabidopsis.

Each number in blue corresponds to the compounds shown in Figure 3. The compounds detected in Arabidopsis are shown in a blue background.

Dotted lines indicate proposed but unidentified pathways. F3AraT, flavonol 3-O-arabinosyltransferase; Fd3GlcT, flavonoid 3-O-glucosyltransferase;

F3RhaT, flavonol 3-O-rhamnosyltransferase; F7GlcT, flavonol 7-O-glucosyltransferase; F7RhaT, flavonol 7-O-rhamnosyltransferase; Ara, arabinose;

DeoxyHex, deoxyhexose; Glc, glucose; Hex, hexose; Rha, rhamnose.

Flavonol Metabolism in Arabidopsis 2173

3 100mm;Waters) at a flow rate of 0.5mL/min at 358C.Compoundswere

separatedwith a linear elution gradient with solvent A (0.1% formic acid in

water) and solvent B (0.1% formic acid in acetonitrile) from 0min, 100%A

to 10 min, 40% B. PDA was used for the detection of UV-visible

absorption in the range of 210 to 500 nm. The TOF mass analyzer was

used for the detection of flavonol glycosides [M + H]+ and the peak of

fragment ions in a positive ion scanning mode with the following setting:

desolvation temperature, 4008C with a nitrogen gas flow of 600 L/h;

capillary spray, 3.0 kV; source temperature, 1508C; and cone voltage of

10 V for flavonoid glycosides [M + H]+ or 30 V for fragment ions. For

comprehensive flavonol profiling, nonprocessedMSdatawere converted

to NetCDF format by MassLynx software (Micromass MS Technologies).

Data analyses including principal component analysis were performed

by the Phenomenome Profiler (Phenomenome Discoveries). Flavonol

glycoside standards were used for the identification of the peaks in the

plant extracts based on retention times, UV-visible absorption spectra,

and mass fragmentation by tandem MS analysis. Other flavonol peaks

were annotated by comparing their UV-visible absorption spectra, elution

times, m/z values, and MS2 fragmentation patterns with 85 reference

flavonoid compounds and the reported data (Tohge et al., 2005, 2007;

Routaboul et al., 2006; Yonekura-Sakakibara et al., 2007). The mass

spectrum data of standard compounds (see Supplemental Data Set

2 online) were recorded in the MASSBANK Database (http://www.

massbank.jp/index-e.html).

Transcriptome Coexpression Analysis

Coexpression analyses were performed using a Coexpression Gene

Search algorithm on the RIKEN PRIMe website (http://prime.psc.riken.

jp/; Akiyama et al., 2008) as described previously (Yonekura-Sakakibara

et al., 2007; Saito et al., 2008). Twenty-four genes (listed in Table 2) were

used as core query genes. The genes exhibiting positive correlations (r >

0.525) against the flavonoid biosynthetic genes were extracted using

ATTED-II (Obayashi et al., 2007) based on all data sets version 3 in

AtGenExpress. The coexpression graph was depicted by the Pajek

program.

Evaluation of T-DNA Insertion Mutants

For complementation tests, full-length UGT78D3 cDNA was amplified by

PCR using Arabidopsis flower cDNA as a template with primers

UGT78D3-GWf and UGT78D3-GWr (see Supplemental Table 1 online).

The Gateway system was used for construction of the binary vectors for

Arabidopsis transformation. cDNA was cloned into the pCR8/GW/TOPO

vector (Invitrogen) as an entry vector and sequenced to confirm the

absence of PCR errors. pGWB2 was used as a destination vector.

Transformation into Agrobacterium tumefaciens and Arabidopsis and the

selection of transformants were performed as described previously

(Yonekura-Sakakibara et al., 2007).

Arabidopsis plants were grown at 228C under 16 h/8 h light and dark

cycles in a plant growth room for ;5 weeks. The leaves and flowers of

plants were harvested, immediately frozen with liquid nitrogen, and

stored at 2308C until use for metabolite profiles.

Quantitative Real-Time PCR

RNA Extraction and cDNA synthesis were performed as described

previously (Yonekura-Sakakibara et al., 2004). The developmental stage

of each organ used for analysis was as described previously (Yonekura-

Sakakibara et al., 2007). Accumulation levels ofUGT78D3,RHM1,RHM2,

and RHM3 transcripts were analyzed by real-time PCR with an ABI

PRISM 7500 real-time PCR system (Applied Biosystems), monitoring

amplification with SYBR-Green I dye (Applied Biosystems). The primers

UGT78D3-RTf, UGT78D3-RTr, RHM1-RTf, RHM1-RTr, RHM2-RTf,

RHM2-RTr, RHM3-RTf, andRHM3-RTr (see Supplemental Table 1 online)

were designed using Primer Express software (Applied Biosystems) and

checked for specific product formation by a dissociation program. In

each case, plasmid DNA containing the corresponding gene was used as

a template to generate a calibration curve. Real-time PCRwas performed

in triplicate on a single biological sample for each genotype.

Production of Recombinant UGT78D3 Protein and Assay

of Glycosyltransferase

Full-length UGT78D3 cDNA was amplified and cloned into pCR8/GW/

TOPO (Invitrogen) as described above. To construct the protein expres-

sion vector, the resultant plasmid was amplified by PCR with the primers

UGT78D3-pET41bf and UGT78D3-pET41br (see Supplemental Table

1 online), the PCR product was digested with StuI and XhoI, and ligated

with StuI-XhoI-digested pET-41b(+) vector (Novagen). The sequence of

the resultant plasmid, pKYS342, was confirmed. Escherichia coli strain

BL21star(DE3) was used as a host for expression. Transformed cells were

cultivated at 378C until A600 reached 0.5. After the addition of isopropyl-

b-D-thiogalactopyranoside to a final concentration of 1 mM, cells were

cultured at 258C for 4 h. The cells were collected, and the proteinwaspurified

as a GST fusion according to the manufacturer’s instructions (Qiagen).

The standard enzyme assay reaction mixture (final volume of 50 mL)

consisted of 50 mM HEPES-KOH, pH 7.5, 150 mM flavonoid substrate,

and 500 mMUDP-sugar. The mixture was preincubated at 308C for 2 min,

and the reaction was started by the addition of enzyme. Reactions were

stopped after 0, 4, 8, 12, or 30min of incubation at 308C by the addition of

50 mL ice-cold 0.5% (v/v) trifluoroacetic acid/methanol for flavonols or 50

mL ice-cold 1.0% (v/v) HCl/methanol for anthocyanidins and anthocyanins,

and the supernatant was recovered by centrifugation at 12,000g for 3 min.

Flavonoids in the resultant solution were analyzed as described above.

Preparation and NMR Analysis of Kaempferol 3-O-Arabinoside-

7-O-RhamnosideandQuercetin 3-O-Arabinoside-7-O-Rhamnoside

A reactionmixture of UGT89C1with kaempferol 3-O-a-L-arabinoside and

UDP-b-L-rhamnose as substrates (total 300 mL) was concentrated

and chromatographed on COSMOSIL 75C18-OPN (Nacalai Tesque) and

stepwise elutedwithwater, 10%aqueousCH3CN, 30%aqueousCH3CN,

50% aqueous CH3CN, 70% aqueous CH3CN, and 100% methanol.

Kaempferol glycosides in the fractions of 30% aqueous CH3CN, 50%

aqueous CH3CN, 70% aqueous CH3CN, and 100% methanol were

further separated by preparative HPLC using a C18 reversed-phase

column (10 mm 3150 mm I.D., particle size 5 mm, Inertsil ODS-EP; GL

Science) using 30% aqueous CH3CN with flow rates of 2.0 mL/min at

308C. Kaempferol 3-O-L-arabinoside-7-O-L-rhamnoside (1.8 mg) was

obtained and the structure was supported by 1H-NMR (600 MHz, meth-

anol-d4): d 5.16 (1H, d, J = 6.6 Hz, arabinose H-1), d 5.52 (1H, br. s,

rhamnose H-1), d 6.43 (1H, d, J = 1.8 Hz, H-6), d 6.73 (1H, d, J = 1.2 Hz,

H-8), d 6.86 (2H, d, J= 9.0Hz, H-39andH-59), d 8.06 (2H,d, J= 8.4Hz, H-29

and H-69), ROE correlation between the anomeric proton of rhamnose

(rhamnose H-1, d 5.52), and the protons at C-6 (H-6, d 6.43) and C-8

position (H-8, d 6.73), ESI-TOF-MS (positive ion mode): m/z 565.1554

[M+H]+, calculated for C26H29O14, 565.1557, ESI-TOF-MS/MS (positive

ion mode, collision energy 30 eV):m/z (relative intensity) 433.1145 [M+H-

arabinose]+ (39), 287.0605 [M+H-arabinose-rhamnose]+ (100), and UV

lmax nm: 345 (on flow).

A large-scale enzymatic product of UGT89C1 with quercetin 3-O-a-L-

arabinoside and UDP-b-L-rhamnose was isolated by a similar procedure

except for the mobile phase (20% aqueous CH3CN) of preparative HPLC

step. Quercetin 3-O-L-arabinoside-7-O-L-rhamnoside (2.1 mg) was ob-

tained and the structure was supported by 1H-NMR (600MHz, methanol-

d4): d 5.22 (1H, d, J = 6.0 Hz, arabinose H-1), d 5.56 (1H, br. s, rhamnose

H-1), d 6.46 (1H, br. s, H-6), d 6.75 (1H, br. s, H-8), d 6.88 (1H, d, J = 7.8 Hz,

H-59), d 7.61 (1H, br. d, J = 8.4 Hz, H-69), d 7.76 (1H, br. s, H-29). ROE

2174 The Plant Cell

(irradiation d 5.56 [rhamnose H-1]): H-6 (d 6.46) and H-8 (d 6.75), ROE

correlation between the anomeric proton of rhamnose (rhamnose H-1, d

5.52) and the protons at C-6 (H-6, d 6.43) and C-8 position (H-8, d 6.73),

ESI-TOF-MS (positive ion mode): m/z 581.1496 [M+H]+, calculated for

C26H29O15, 581.1506. ESI-TOF-MS/MS (positive ion mode, collision

energy 30 eV): m/z (relative intensity) 449.1086 [M+H-arabinose]+ (41),

303.0554 [M+H-arabinose-rhamnose]+ (100), and UV lmax nm:340 (on

flow). Monitoring was accomplished by PDA. 1H-NMR spectra were

recorded on a JNM ECP-600 spectrometer (JEOL).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative database under accession numbers TT4 (At5g13930), TT7

(At5g07990), UGT75C1 (At4g14090), UGT78D1 (At1g30530), UGT78D2

(At5g17050), UGT78D3 (At5g17030), UGT89C1 (At1g06000), OMT1

(At5g54160),RHM1 (At1g78570),RHM2 (At1g53500), andRHM3 (At3g14790).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Venn Diagrams of MS Peaks Detected in

Various Organs of Col-0 and tt4.

Supplemental Figure 2. Coexpression Relationships of Genes In-

volved in the Flavonoid Pathway in Arabidopsis.

Supplemental Figure 3. Multiple Alignment of the Deduced Amino

Acid Sequences of Flavonoid 3-O-Glucosyltransferases from Arabi-

dopsis and Grape (Vitis vinifera).

Supplemental Figure 4. T-DNA Insertion Mutants of RHM1 and

UPLC/MS Analyses of the rhm1 Mutant Lines.

Supplemental Figure 5. Schematic Representation of Duplicated

Regions Containing RHMs in the Arabidopsis Genome.

Supplemental Table 1. Primers Used in This Study.

Supplemental Data Set 1. MS-Detectable Peaks in the Wild Type

and tt4.

Supplemental Data Set 2. Characteristics of Standard Compounds.

ACKNOWLEDGMENTS

We thank C. Ringli (University of Zurich) for the rol1-1 and rol1-2

mutants, M. Pauly (Michigan State University) for the rhm2-1 mutant, S.

Kitamura (Japan Atomic Energy Research Institute) for the tt4 mutant, T.

Nakagawa (Shimane University) for the pGWB2 vector, Y. Sekiyama

(RIKEN) for helpful comments on NMR analysis, T. Obayashi (University

of Tokyo), H. Suzuki, and N. Sakurai (Kazusa DNA Research Institute) for

advice on coexpression analysis, and K. Akiyama (RIKEN) for excellent

technical support for using the RIKEN-PRIMe website. This study was

supported in part by grants-in-aid from the Ministry of Education,

Culture, Sports, Science, and Technology of Japan and the Uehara

Memorial Foundation, Japan.

Received January 15, 2008; revised July 8, 2008; accepted August 10,

2008; published August 29, 2008.

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2176 The Plant Cell

DOI 10.1105/tpc.108.058040; originally published online August 29, 2008; 2008;20;2160-2176Plant Cell

Rie Niida, Akiko Watanabe-Takahashi, Eri Inoue and Kazuki SaitoKeiko Yonekura-Sakakibara, Takayuki Tohge, Fumio Matsuda, Ryo Nakabayashi, Hiromitsu Takayama,

ArabidopsisMetabolite Correlations in −GeneComprehensive Flavonol Profiling and Transcriptome Coexpression Analysis Leading to Decoding

 This information is current as of December 8, 2020

 

Supplemental Data /content/suppl/2008/08/19/tpc.108.058040.DC1.html

References /content/20/8/2160.full.html#ref-list-1

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