MATE2 Mediates Vacuolar Sequestration of Flavonoid Glycosides …€¦ · Supplemental Figure 2: Uptake of Cyanidin 3-O-Glucoside by MATE2 (A) Concentration dependent-uptake of cyanidin

Supplemental Data. Zhao et al. (2011). Plant Cell 10.1105/tpc.111.080804

MATE2 Mediates Vacuolar Sequestration of Flavonoid Glycosides and

Glycoside Malonates in Medicago truncatula[W].

Jian Zhao, David Huhman, Gail Shadle, Xian-Zhi He, Lloyd W. Sumner, Yuhong Tang, and

Richard A. Dixon1

Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore,

Oklahoma 73401, USA

SUPPLEMENTAL DATA

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

Supplemental Figure 1. Amino Acid Alignments of MATE2 with MATE Transporters from

Other Plant Species.

Supplemental Figure 2. Uptake of Cyanidin 3-O-Glucoside by MATE2.

Supplemental Figure 3. Microarray and Quantitative RT-PCR Analysis of Tissue Level

Expression of Flavonoid Biosynthetic, Modification, and Transporter Genes in Wild-Type M.

truncatula R108.

Supplemental Figure 4. Heat Map Mosaic Representation of Pearson Correlation Values

between each Gene in Figure 3A.

Supplemental Figure 5. Amino Acid Alignments of Conserved Motifs in MaT4, MaT5, and

MaT6 from M. truncatula with Other Plant Malonyltransferases.

Supplemental Figure 6. HPLC Analysis of MaT4 Malonyltransferase Activity Toward Various

Flavonoid Glucosides.

Supplemental Figure 7. HPLC Analysis of Anthocyanin Malonyltransferase Reactions

Catalyzed by MaT5 or MaT6.

Supplemental Figure 8. ESI-LC-MS Analysis and Identification of Flavonoid Glucoside

Malonates from MaT4 Malonyltransferase Enzyme Assays.

Supplemental Figure 9. Schematic of MaT4-, 5- and 6-Catalyzed Malonylation of Flavonols,

Flavones, Isoflavones and Anthocyanins.


Supplemental Figure 10. Concentration Dependent Uptake of Pelargonidin and Delphinidin

Mono- and Di-Glucosides by MATE2.

Supplemental Figure 11. Concentration Dependent Uptake of Flavones and Flavonol

(Malonyl)Glucosides by MATE2.

Supplemental Figure 12. Topological Analysis of MATE2 and Related MATE Transporters

from Other Plant Species.

Supplemental Figure 13. Localization and Transport Activity of MATE2-GFP Expressed in

Yeast.

Supplemental Figure 14. Localization of MATE2-GFP Expressed in Tobacco and Onion Cells.

Supplemental Figure 154. Protein Gel-Blot Analysis of the Subcellular Localization of MaT4,

MaT5, and MaT6 in Tobacco Plants.

Supplemental Figure 16. M. truncatula Leaf Pigmentation and Leaf Cross-Sections of mate2

Mutant and Wild-Type R108.

Supplemental Figure 17. Transcript Analysis of Flavonoid Pathway Genes in Wild-Type R108

and mate2 Mutants.

Supplemental Figure 18. Flavonoid Profiles of M. truncatula mate2 Mutants and their Null

Segregant Controls.

Supplemental Figure 19. Anthocyanin Levels in Seeds of Mature mate2 Mutant and Null

Segregant Controls.

Supplemental Table 1. Kinetic Parameters of MaT4 and MaT5 Malonyltransferases from M.

truncatula Toward Various Flavonoid Glucosides.

Supplemental Table 2. Primers Used in the Present Study

Supplemental Dataset 1. Alignment corresponding to the Phylogenetic Analysis in Figure 2A.

Supplemental Dataset 2. Alignment corresponding to the Phylogenetic Analysis in Figure 4A.

Supplemental Figure 1. Amino Acid

Alignments of MATE2 with MATE Transporters

From Other Plant Species.

Alignment of protein sequences was done with

ClustalW. Formatting of aligned sequences was

done with box shade program

(http://www.ch.embnet.org/software/BOX_form.

html). Protein sequences and their accession

numbers are: tomato MTP77 (AAQ55183),

grapevine AM2 (FJ264202) and AM3

(FJ264203), Arabidopsis FFT (BAE98568) and

TT12 (NP_191462), and M. truncatula MATE1

(ACX37118) and MATE2 (HM856605). The

putative transmembrane domains (TMDs) are

underlined. Amino acids identical in the two

proteins are highlighted in black and

conservative substitutions are highlighted in

gray.

TMD1 TMD2

TMD3

TMD4

TMD6

TMD5

TMD7

TMD8

TMD9 TMD10

TMD11 TMD12


Supplemental Figure 2: Uptake of Cyanidin 3-O-Glucoside by MATE2

(A) Concentration dependent-uptake of cyanidin 3-O-glucoside (Cy3G) into membrane vesicles

expressing MATE2 or empty vector.

(B) Double reciprocal plot of initial rate data for Cy3G transport by MATE2.

(C) Uptake of flavonoid aglycones (100 μM) into MATE2-expressing membrane vesicles and

vesicles from yeast expressing empty pYES vector. Assays were for 8 min of incubation.

Data are means and standard deviations from triplicate experiments.

A B

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 50 100 150 200 250 300 350

Upta

ke(n

mol/m

g p

rote

in/m

in)

Cy3G (μM)

MATE2

Vector

No-ATP-Mg

0

1

2

3

4

5

-0.02 -0.01 0 0.01 0.02 0.03

1/(

nm

ol/m

g p

rote

in/m

in)

1/[Cy3G] ] (μM-1)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Up

take

(n

mo

l/m

g p

rote

in) pYES-MATE2

pYES

C


Supplemental Figure 3. Microarray and Quantitative RT-PCR Analysis of Tissue Specific Expression of

Flavonoid Biosynthetic, Modification, and Transporter Genes in Wild-Type M. truncatula R108.

(A) Microarray analysis. Tissue level-expression pattern of MATE2 and UGT78G1in M. truncatula. Data

were obtained from the Medicago Gene Atlas database (http://mtgea.noble.org/v2/) using the probesets

Mtr.51063.1.S1_at (MATE2) and Mtr.39747.1.S1_at (UGT78G1) as queries.

(B) and (C) qRT-PCR analysis. Tissues were from M. truncatula leaf (3-4 weeks old), root, stem (5-6

internodes from top),vegetative bud, flower (1-2 days after opening of petals), and pod (6-12 days post-

flowering). Amplified genes are: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone

3-hydroxylase; DFR, dihydroflavonol reductase; IFS, isoflavone synthase; IFR, , isoflavone

reductase; FLS, flavonol synthase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase;

ACTIN was used as control. Data are means and SD of triplicate experiments.


http://mtgea.noble.org/v2/

Supplemental Figure 4. Heat Map Mosaic Representation of Pearson Correlation Values

between each Gene in Figure 3A.

Genes are ordered as given in Figure 3A. The red color shows higher correlation values

between gene(s).


Supplemental Figure 5. Amino Acid Alignments of Conserved Motifs in MaT4, MaT5, and MaT6 from M.

truncatula with Other Plant Malonyltransferases.

Alignment of protein sequences was done with ClustalW (http://www.ebi.ac.uk/clustalw/). Formatting of sequences

was done with box shade program (http://www.ch.embnet.org/software/BOX_form.html). Proteins and their accession

numbers are: Salvia splendens Ss-5MaT1(AAR26386) and Ss-5MaT2 (AAL50566); Dendranthema×morifolium Dm-

3MaT1 (AAQ63615) and Dm-3MaT2 (AAQ63616); Senecio cruentus Sc-3MaT (AAO38058); Dahlia variabilis Dv-

3MaT (AAO12206); Perilla frutescens Pf-5MaT (AAL50565), Medicago truncatula MaT1 (ABY91220), MaT2

(ABY91222), MaT3 (ABY91221), MaT4 (HM856606), MaT5(HM856607) and MaT6 (HM856608). The conserved

motifs are boxed.

-HXXXD- (motif 1)

-NYXGNC- (motif 2)

-DFGWG- (motif 3)


http://www.ebi.ac.uk/clustalw/

Abundance

Retention time (min)

Abundance

Abundance

Abundance

Abundance

Abundance


Abundance

Abundance

Retention time (min) Retention time (min)

A7G A7GM

A7G

K7G

K7GM

K7G

G7G

G7GM

G7G

B7G

B7GM

B7G


Supplemental Figure 6. HPLC Analysis of MaT4 Malonyltransferase Activity Toward Various Flavonoid

Glucosides.

Recombinant His-MaT4 was purified and used in enzyme assays. In each group of HPLC chromatographs,

the top shows substrate, and the bottom shows reaction mixture containing both substrate and malonylated product.

Substrates are: A7G, apigenin 7-O-glucoside; L7G, luteolin 7-O-glucoside; N7G, naringenin 7-O-glucoside;

K7G, kaempferol 7-O-glucoside; D7G, daidzein 7-O glucoside; G7G, genistein 7-O-glucoside; F7G, formononetin

7-O-glucoside; B7G, biochanin A 7-O-glucoside.

F7G

Abundance

Abundance

Ab

un

da

nce

A

bundance

Abundance

Abundance

Abundance

Abundance



D7GD7GM

D7G

L7G

L7GM

L7G

F7G

F7GM

F7G

N7G

N7GM

N7G


Supplemental Figure 7. HPLC Analysis of Anthocyanin Malonyltransferase Reactions Catalyzed by MaT5

or MaT6.

Recombinant His-tagged MaT5 and MaT6 purified from E. coli extracts were used in enzyme reactions.

In each group of HPLC chromatographs, the top shows substrate and the bottom shows reaction mixture

containing both substrate and malonylated products (indicated by arrows). Substrates are: Cy3G,

cyanidin 3-O-glucoside; P3G, pelargonidin 3-O-glucoside; P35G, pelargonidin 3,5-di-O glucoside, D3G,

delphinidin 3-O-glucoside; D35G, delphinidin 3,5-di-O-glucoside, as well as their malonylated products.


λ5

30

nm

Cy3G Cy3GMs

Cy3G

D35G

D35G

D3G

D3G

D3GMs

P3G

P3GMs

P3Gλ5

30

nm

λ5

30

nm

λ5

30

nm

λ5

30

nm

λ5

30

nm

λ5

30

nm

λ5

30

nm


P35G P35G

λ5

30

nm

λ5

30

nm



239.4

271.2

433.4

455.1

477.1

499.1 543.2

+MS, 30.8min (#604)

0

1

2

3

4

4x10

Intens.

100 200 300 400 500 600 700 m/z

239.3

271.2

519.3

541.2

563.2 779.4

+MS, 34.3min (#673)

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Intens.

100 200 300 400 500 600 700 m/z

10 20 30 40 50 60 70Time [min]

0.0

0.2

0.4

0.6

0.8

1.0

1.2

5x10

Intens.

G7G

G7GM

G7G

G7GM


m / z

Ab

un

da

nce

A

bun

da

nce

10 20 30 40 50 60 70Time [min]

0

1

2

3

5x10

Intens.

L7G

L7GM

239.4

287.0

449.0

470.9

492.9

+MS, 30.5min (#597)

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Intens.

100 200 300 400 500 600 700 m/z

287.2

535.3

557.2

+MS, 32.7min (#644)

0.0

0.5

1.0

1.5

2.0

2.5

5x10

Intens.

100 200 300 400 500 600 700 m/z

L7G

L7GM


m / z

269.0

430.9 452.9

+MS, 35.9min (#710)

0

1

2

3

4

5

6

5x10

Intens.

100 200 300 400 500 600 700 m/z

239.4

269.2

495.6

517.3

539.2

561.2

777.4

+MS, 39.0min (#771)

0

2

4

6

4x10

Intens.

100 200 300 400 500 600 700 m/z

F7G

F7GM

10 20 30 40 50 60 Time [min]

0.0

0.5

1.0

1.5

2.0

2.5

5x10

Intens.

153.3239.3

273.0

434.9

456.9

619.3

+MS, 32.3min (#638)

0.0

0.5

1.0

1.5

5x10

Intens.

100 200 300 400 500 600 700 m/z

153.3 239.4

273.2

401.1

521.5

543.2

565.0

+MS, 35.0min (#692)

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Intens.

100 200 300 400 500 600 700 m/z

10 20 30 40 50 60 70 Time [min]

0

2

4

6

5x10

Intens.

F7G

F7GM

N7G

N7GM

N7G

N7GM


Ab

un

da

nce

A

bun

da

nce

m / z m / z


10 20 30 40 50 60 Time [min]

0

1

2

3

5x10

Intens.

10 20 30 40 50 60 Time [min]

0.0

0.2

0.4

0.6

0.8

5x10

Intens.

D7G

D7GMB7G

B7GM


285.0

447.1

469.0

555.1

+MS, 42.7min (#833)

0.0

0.5

1.0

1.5

5x10

Intens.

100 200 300 400 500 600 700 m/z

285.2

533.3

555.2

+MS, 45.0min (#884)

0

1

2

3

5x10

Intens.

100 200 300 400 500 600 700 m/z

239.4

255.2

283.0

417.3

439.0

460.9

+MS, 26.3min (#516)

0

2

4

6

4x10

Intens.

100 200 300 400 500 600 700 m/z

239.3

255.2

283.0 481.5

503.3

525.2

547.1

763.4

785.4

+MS, 30.2min (#593)

0

1

2

3

4x10

Intens.

100 200 300 400 500 600 700 m/z

B7G D7G

D7GMB7GM

m / zm / z


Ab

un

da

nce

Supplemental Figure 8. ESI-LC-MS Analysis and Identification of Flavonoid Glucoside Malonates

from MaT4 Malonyltransferase Enzyme Assays.

Recombinant MaT4-catalyzed malonylation of flavonoid 7-O-glucosides was determined by ESI-LC-MS.

In each group of HPLC and LC-MS chromatographs, the top shows the liquid chromatograph,

the middle shows the mass spectrum of the substrate, and the bottom shows the mass spectrum of the product.

Flavonoid 7-O-glucosides are: L7G, luteolin 7-O-glucoside; N7G, naringenin 7-O-glucoside;

D7G, daidzein 7-O glucoside; G7G, genistein 7-O-glucoside; F7G, formononetin 7-O-glucoside;

B7G, biochanin A 7-O-glucoside, and their corresponding malonylated products, L7GM,

N7GM, D7GM, G7GM, F7GM, and B7GM, respectively.

Ab

un

da

nce


Supplemental Figure 9. Schematic of MaT4-, 5- and 6-catalyzed malonylation of flavones and

flavonols (A), isoflavones (B), and anthocyanins (C). Note that the exact position of malonylation remains

to be determined; the illustrated substitution of the 6’’-position of the glucose units is based on

previous studies of the structures of isoflavone glucoside malonates.


Supplemental Figure 10. Concentration Dependent Uptake of Pelargonidin and Delphinidin Mono-

and Di-Glucosides by MATE2.

The uptake of pelargonidin 3-O-glucoside (P3G), pelargonidin 3,5-O-diglucoside (P35G),

delphinidin 3-O-glucoside, or delphinidin 3,5-O-diglucoside (D35G) into MATE2 or empty vector-

expressing yeast microsomal vesicles was assayed. MATE2-mediated uptake was obtained by

subtracting vector controls from MATE2 values. Data are means and standard deviations from

triplicate experiments. The insets are double reciprocal plots of initial rate data at different

concentrations of the anthocyanins


Supplemental Figure 11. Concentration Dependent Uptake of Flavones and Flavonol

(Malonyl)Glucosides by MATE2.

(A) and (B) Apigenin 7-O-glucoside (A7G) or kaempferol 7-O-glucoside (K7G) and their malonates,

A7GM and K7GM, respectively, were generated and purified from MaT4-catalyzed malonylation

reactions. Their uptake by MATE2-expressing yeast microsomal vesicles was assayed. MATE2-

mediated uptake was obtained by subtracting vector controls from MATE2 values. Data are means and

standard deviations from triplicate experiments.

(C) and (D), Double reciprocal plots of initial rate data at different concentrations of A7G, A7GM,

K7G, and K7GM.


Supplemental Figure 12. Topological Analysis of MATE2 and Related MATE Transporters from Other Plants.

The twelve membrane-spanning domains of MATE2 and their orthologs were determined using the TMHMM2

program in SMART (http://smart.embl-heidelberg.de). Protein sequences and their accesion numbers are:

tomato MTP77 (AAQ55183), AM2 (FJ264202), AM3 (FJ264203), FFT (BAE98568), and MATE2

(HM856605).

FFT

AM3

AM1

MATE2

MTP77


Supplemental Figure 13. Localization and Transport Activity of MATE2-GFP Expressed in Yeast.

Wild-type yeast W303A cells were transformed with pYES-MATE2-GFP and grown in YPD medium

overnight for microscopy. Alternatively, yeast cells expressing pYES-MATE2 (MATE2), pYES-MATE2-

GFP (MATE2-GFP) and pYES (vector control) were induced with galactose for activity assay.

(A) MATE2-GFP expressed in yeast shows similar transport activity as MATE2 toward anthocyanin

glucosides and apigenin 7-O-glucoside (A7G). Asterisks indicate statistically significant difference

(** p< 0.01, * p< 0.05) compared with pYES vector controls (from triplicate experiments).

(B) MATE2-GFP imaging in a yeast cell. i, fluorescence image; ii, DIC image; iii, merged images.

Bars =5 mm.

(C) MATE2-GFP co-fractionates with vacuolar membrane vesicles. Overnight grown yeast cells

expressing pYES-MATE2-GFP were used for fractionation of microsomes in a 15-50 % non-continuous-

sucrose gradient.Fractions were probed with antibodies against GFP, V-H+-ATPase (tonoplast marker),

BiP (endoplasmic reticulum marker), and PM-H+-ATPase (plasma membrane marker).


Supplemental Figure 14. Localization of MATE2-GFP Expressed in Tobacco and Onion Cells

MATE2-GFP was driven by the cauliflower mosaic virus 35S promoter and transiently expressed in

tobacco leaf or onion epidermal cells. Materials were viewed by confocal microscopy.

(A) Fluorescence image of free GFP in a tobacco epidermal cell. Arrow shows nucleus. Bar = 20 μm.

(B) and (C), GFP fluorescence images of MATE2-GFP in tobacco (B) and Arabidopsis (C) leaf

epidermal cells. Bar = 50 μm

(D) to (F) Fluorescence images of onion epidermal cells expressing free GFP or MATE2-GFP. (D)

Fluorescence image of free GFP; (E) and (F) Fluorescence images of MATE2-GFP. Arrows show

nucleus. Bars = 35 μm.


Supplemental Figure 15. Protein Gel-Blot Analysis of the Subcellular Localization of MaT4, MaT5, and

MaT6 in Tobacco Leaves.

Nicotina benthamiana leaves were transformed with Agrobacteria harboring pKGFP-MaTs by infiltration.

Leaves were examined for GFP expression by confocal microscopy after 48h. The leaves expressing free

GFP, pGFP-MaT4, pGFP-MaT5, and pGFP-MaT6 were collected and frozen in liquid nitrogen for

extraction of proteins. Soluble and microsomal fractions were prepared from total protein extracts by

differential centrifugation. Twenty μg of soluble and microsomal proteins were loaded for protein gel

blotting. GFP-MaT4, GFP-MaT5, and GFP-MaT6 were detected with anti-GFP antibody, and ER-derived

microsomes were revealed with anti-BiP antibody (endoplasmic reticulum marker).


Supplemental Figure 16. M. truncatula Leaf Pigmentation and Leaf Cross-Sections of mate2

Mutant and Wild-Type R108.

Leaves of 4 week old mate2-2 and R108 were cut with a microtome into slices of 100 μm thickness

and viewed under the light microscope for detection of anthocyanin pigments.

(A) and (D), Enlarged image of pigments on the bottom surface of a leaf

(B) and (E), cross-sections of leaf vein area

(C) and (F), cross-sections of leaf far from the vein area


Supplemental Figure 17. Transcript Analysis of Flavonoid Pathway Genes in Wild-Type R108

and mate2 Mutants.

Quantitative RT-PCR was performed with flower samples of wild-type R108 and mate2 mutants.

Amplified genes are: DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase;

ANR, anthocyanidin reductase; IFR, isoflavone reductase; and MATE2. ACTIN was used as internal

control.

Data are means and standard deviations from triplicate experiments.


Supplemental Figure 18. Flavonoid Profiles of M. truncatula mate2 Mutants and their Null Segregant Controls.

Leaves from 4-week old seedlings and flower at 2-3 days post flowering were harvested from homozygous mate2

mutants (mate2Ho) and null segregant controls (mate2WT) for metabolite analysis by UPLC-ESI-TOF-MS. Data

are from three biological replicates .

(A) Profiles of flavonoids. Flavonoid glucosides are: L3,7G, luteolin 3,7-di-O-glucoside, Rutin, quercetin 3-O-

rutinoside; L3’,7G, luteolin 3’,7-di-O-glucoside; Vitexin, apigenin-8-C-glucoside; K3R7G, kaempferol 3-O-

rhamnoside 7-O-glucoside; Q3R, quercetin 3-O-rhamnoside; L4G, luteolin 4-O-glucoside; N7G, naringenin 7-O

glucoside; L4’,7G, luteolin 4’,7-O-diglucoside; and B7G, biochanin A 7-O-glucoside.

(B) Profiles of anthocyanins. P3G, pelargonidin 3-O-glucoside; P35G, pelargonidin 3,5-O-diglucoside, D3G,

delphinidin 3-O-glucoside; D35G, delphinidin 3,5-O-diglucoside.

Asterisks indicate where the flavonoid levels in mate2 mutants are statistically different (P < 0.05; two-paired t

test) from those in the corresponding null segregant plants (with a minimum of three biological replicates).


Supplemental Figure 19. Anthocyanin Levels in Seeds of Mature mate2 Mutants and Null

Segregant Controls.

Mature seeds from homozygous mate2 mutants (mate2-1Ho, mate2-2Ho, mate2-3 Ho, and mate2-4

Ho) and null segregant controls (mate2-1WT, mate2-2WT, mate2-3WT, and mate2-4 WT) were

analyzed for anthocyanins. Asterisks indicate that the anthocyanin levels in mate2 mutants are

statistically different (P < 0.05; two-paired t test) from those in the corresponding wild-type plants

(with a minimum of three biological replicates).


A7G B7G D7G G7G K7G N7G L7G F7G Cy3

G

D3G P3G P3,5 G

MaT4 100 93 90 76 97 47 93 78 nd nd nd nd

MaT5 nd nd nd nd nd nd nd nd 100 53 120 49

MaT6 nd nd nd nd nd nd nd nd 100 50 120 48

Substrate Km (μM)

Vmax

(nmol/mg/min)

Kcat

(s-1 )A7G 12.5 353 21.3

B7G 35.3 197 50.4

D7G 49.3 153 34.5

L7G 36.5 152 34.3

N7G 24.5 198 32.7

K7G 14.9 242 30.8Malonyl CoA 48.4 157 84.5

p-Coumaroyl CoA nd nd nd

nd: not detected

(C) Kinetic parameters of MaT5

Substrate Km (μm)

Vmax

(nmol/mg/min)

Kcat

(s-1 )

Cy3G 89 124 12

D3G 100 94 23

P3G 56 242 23

Malonyl CoA 80 143 21

p-Coumaroyl CoA nd nd nd

nd: not detected;

MaT6 shows the same substrate preference as MaT5

(A) Relative activity of MaTs toward flavonoid glucosides

Notes:

nd: not detected; Malonyltransferase activity toward apigenin 7-O-glucoside (A7G) was set at

100 %, activity toward other compounds was compared with A7G. In MaT5 and MaT6 activity

assays, activity toward cyanidin 3-O-glucoside (Cy3G) was set at 100 %.

(B) Kinetic parameters of MaT4

Supplemental Table 1. Kinetic parameters of MaT4, MaT5, and MaT6 malonyltransferases from M.

truncatula toward various flavonoid glucosides.

Flavonoid 7-O-glucosides are: A7G, apigenin 7-O-glucoside; L7G, luteolin 7-O-glucoside; N7G, naringenin

7-O-glucoside; K7G, kaempferol 7-O-glucoside; D7G, daidzein 7-O glucoside; G7G, genistein 7-O-

glucoside; F7G, formononetin 7-O-glucoside; B7G, biochanin A 7-O-glucoside. Anthocyanins are:

Cy3G, cyanidin 3-O-glucoside; P3G, pelargonidin 3-O-glucoside; P35G, pelargonidin 3,5-di-O glucoside;

and D3G, delphinidin 3-O-glucoside.


Primer name Sequence Restriction site and purpose

MATE_F 5’-CAGAGTTTCCAACGAACTCGGACT-3’ In situ hybridization

MATE_R 5’-CCCATTGCTGTCAACTTCTACGGT-3’ In situ hybridization

MATE_T7_F

5’GCGTAATACGACTCACTATAGGGCAGAGTTTCCAACGAACT

CGGACT-3’

In situ hybridization

MATE_T7_R

5’-GCGTAATACGACTCACTATAGGGCCCATTGCTGTCA

ACTTCTACGGT-3’

In situ hybridization

MATE2-5’ Forward 5’-CACCATGGACTCTCACACTCCTCTTCTCAACACC-3’ Cloning MATE2

MATE2-3’ Reverse 5’-GCGAGCTCCTATGGCTTGTCCACCCCATTGCTG-3’ Cloning MATE2 i

MATE2tnt , Forward 5’- GACGGTGGCGACTACTTGG-3’ Tnt1 mutant screening

MATE2tnt, reverse 5’-TATCCCCGAGAGCAGCAAG-3’ Tnt1 mutant screening

MATE2-GFP-5’ Forward 5’-GAATTCACCACTAGTATGGACTCTCACACTCCTCTT-3’

(Spe1)

Making MATE2-GFP fusion

MATE2-GFP-3’Reverse 5’-GAATGCGGCCGCTGGCTTGTCCACCCCATTGCTG-3’ (Not1) Making MATE2-GFP fusion

MATE2 SFI-5’Forward 5’-CACCGGCCAAATCGGCCATGGACTCTCACACTCCTC

TT-3’ (Sfi1A)

Subcloning MATE2-GFP into plant

expression vector

MATE2 SFI-3’Reverse 5’-CGGCCCTTATGGCCTTATTATTACTTGTACAGCTC GTC

CAT-3’ (Sfi1B)

Subcloning MATE2-GFP into plant

expression vector

MaT4, Forward 5’-CACCATGGCATTTAACAAGAACAATATC-3’ For cloning

MaT4, Reverse 5’- TCAATTGGAGCATAGTCCTTCAAG-3’ For cloning

MaT5, Forward 5’-CACCATGTCCACCATTCCTTTCATT-3’ For cloning

MaT5, Reverse 5’-TCATGGTAGATCCTCTAGTCCATTC-3’ For cloning

MaT6, Forward 5’- CACCATGGGAAAACCTATAGGAGCA-3’ For cloning

MaT6, Reverse 5’- CTAATATTTAAAAACTTCAAGTCCTTGTT-3’ For cloning

ACTIN, forward 5’-TCAATGTGCCTGCCATGTATGT -3’ For RT-PCR

ACTIN, reverse 5’-ACTCACACCGTCACCAGAATCC -3’ For RT-PCR

ANR, forward 5’-TTGTGGCAGAGAAAGAATCAACTT-3’ For RT-PCR

ANR, reverse 5’-CTCGGGAACACTGGTATTGTGA -3’ For RT-PCR

ANS, forward 5’-TCCACCTCGCACTTTTGCT-3’ For RT-PCR

ANS, reverse 5’-TCTTCTCCTCCTCATCCTTCCTAA-3’ For RT-PCR

MATE2F, forward (P1) 5’-TTTCCGACCCAAAAGTTCCTT-3’ cDNA 563-583, For RT-PCR

MATE2R, reverse (P2) 5’-CACTAACCCAATCCAAGCAATG-3’ cDNA 628-607, For RT-PCR

FLSF, forward 5’- CCACAATTCATTCGCTTAGCAA-3’ For RT-PCR

FLSR, reverse 5’- CACCCTCCATGGCCTTTG-3’ For RT-PCR

DFRF, forward 5’- TCGTCCACTTGGATGATCTTTG-3’ For RT-PCR

DFRR, reverse 5’- CTCCCTTCTACTTCCATATGCTCAA-3’ For RT-PCR

F3HF, forward 5’- CACCAGCTCAAACTCTCACCTATC-3’ For RT-PCR

F3HR, reverse 5’- TTCGCGAACAAAACTGGATTC-3’ For RT-PCR

CHIF, forward 5’- GATTAAGGGTGCACAGTATGGTGTT-3’ For RT-PCR

CHIR, reverse 5’- TCATCGGCTGCCAAACG-3’ For RT-PCR

CHSF, forward 5’- CCAACCAAAATCAAAGATTACACACTT-3’ For RT-PCR

CHSR, reverse 5’- CAGCGCCAGGCATGTCTA-3’ For RT-PCR

IFSF, forward 5’- CAATCCTCCGAGTCCCAAAC-3’ For RT-PCR

IFSR, reverse 5’- GGGTTATCCAAAAGGTGAAGATGA-3’ For RT-PCR

IFRF, forward 5’- AAAGCGAGTATCCGAAGAGTAATTG-3’ For RT-PCR

IFRR, reverse5’- GCGTGGCAACAAAGGTAAGTG-3’

For RT-PCR


Primer name Sequence Restriction site and purpose

UGT78G1F 5’-GTTTTGGCATTCCCATTTGG-3’ For RT-PCR

UGT78G1R 5’-GAGCCTCTGTAGCAATTTTTTTCAC-3’ For RT-PCR

MaT4F 5’-TCATCTTTTGTTCTCACTTGTGCTT -3’ For RT-PCR

MaT4R 5’-CCATATCTGCTCGGTGAATCG-3’ For RT-PCR

MaT5F 5’-CAGTCTCTTCCCTCCCCCTAA-3’ For RT-PCR

MaT5R 5’-TGGTTGGCTTGGAGAGAAAAG-3’ For RT-PCR

MaT6F 5’-TGCAACAAGAACAGCAACAACTAC-3’ For RT-PCR

MaT6R 5’-GATTGGACCAGCAAAATGCAA-3’ For RT-PCR

Tnt15 forward 5’-ACAGTGCTACCTCCTCTGGATG -3’ Tnt1 mutant screening

Tnt13 reverse 5’- CAGTGAACGAGCAGAACCTGTG -3’ Tnt1 mutant screening

Tnt1-R1 5’-TGTAGCACCGAGATACGGTAATTAACA -3’ Tnt1 mutant screening

Tnt1-R2 5’-AGTTGGCTACCAATCCAACAAGGA-3’ Tnt1 mutant screening

Supplemental Table 2. Primers used in the present study

Documents

MATE2 Mediates Vacuolar Sequestration of Flavonoid Glycosides …€¦ · Supplemental Figure 2: Uptake of Cyanidin 3-O-Glucoside by MATE2 (A) Concentration dependent-uptake of cyanidin