Supplemental Figure 1. “Aranci, Bergamotti, Cedri, Limoni e Lumie” (1715),

oil on canvas by Bartolomeo Bimbi, Villa Medicea di Poggio a Caiano.

The cut orange at the bottom of the display is labeled ’18’ but is unnamed in

the legend. The flesh of this orange is red.

Supplemental Data. Butelli et al. (2012). Plant Cell 10.1105/tpc.111.095232

1

   

Supplemental Figure 2. The production of anthocyanin pigment as a result

of 35S:Ruby expression in tobacco is particularly strong in older senescent

leaves. Left hand leaf is a tobacco leaf from a plant transformed with the

binary vector pBin19 alone, right hand leaf is older mature leaf from a

35S:Ruby plant.

Supplemental Data. Butelli et al. (2012). Plant Cell 10.1105/tpc.111.095232

2

   

rela

tive

expr

essi

on

0

50000

100000

Rosea1

MYC2Rose

a1

+ MYC2

contro

l

pF3H:GUS pDFR:GUS

Rosea1

MYC2Rose

a1

+ MYC2

contro

l

Supplemental Figure 3. Cs MYC2 regulates anthocyanin biosynthetic gene

expression

MYB transcription factors interact with bHLH and WDR proteins in a complex

known as the MBW complex. The WDR partner is generally constitutively

expressed (Walker et al., 1999) but activity of the bHLH partner can limit

anthocyanin production (Hellens et al., 2010; Bradley et al., 1999; Lauter et

al., 2004).

Transfection assays in tobacco protoplasts using reporter genes driven by the

F3H and DFR promoters from Antirrhinum majus were used to assay the

ability of different transcription factors to activate the expression of

anthocyanin biosynthetic genes. No activation of either promoter was

observed with Cs MYC2 on its own. The MYB gene Rosea1 activated

expression of both promoters slightly on its own. The combination of Cs MYC2

with Rosea1 significantly enhanced expression from both the F3H and DFR

promoters, showing that Cs MYC2 can interact in the MBW complex to

activate anthocyanin biosynthesis. Control lanes show GUS activity from

protoplasts transfected with the reporter construct alone without added

regulatory genes.

Data are presented as means (± SD) of three biological replicates.

Supplemental Data. Butelli et al. (2012). Plant Cell 10.1105/tpc.111.095232

3

   

0

1500

3000

dopp

io m

osca

to II

dopp

io s

angu

igno

sang

uine

llo c

omun

esa

ngui

nello

mar

ches

e ro

meo

sang

uine

llo tu

mm

ulid

dusa

ngui

nello

con

zam

ustu

sang

uine

llo n

ucel

lare

sang

uine

llo S

SA

nuce

llare

sang

uign

o zu

cche

rino

mos

cato

spi

nasa

ngui

nello

luci

ano

brog

nasa

ngui

nello

dop

pio

mos

cato

IIdo

ppio

san

guig

no s

igno

relli

sang

uine

llo s

poto

pul

eosa

ngui

nello

66

SS

Aov

alet

to s

angu

igno

sang

uign

ello

pas

qual

e br

ogna

vani

glia

san

guig

no

taro

cco

tapi

Supplemental Figure 4. Expression analysis of Ruby.

Expression analysis of Ruby in varieties of the ‘Sanguigni,’ and ‘Sanguinelli’

blood orange groups. Red pigmentation in the last sample, Vaniglia

Sanguigno, is due to the carotenoid lycopene, not anthocyanins.

 

Supplemental Data. Butelli et al. (2012). Plant Cell 10.1105/tpc.111.095232

4

   

Supplemental Table 1. Sequences of the primers used in the study.

Name

Sequence

Description

BUT-F3 GGRKTKAGRAARGGTDCATGGAC degenerate primers to BUT-R3 CCARWARTTYTTSACATCRTTWGC isolate Ruby partial cDNA PMC-R1 TTCCCGGAAGCCTGCCCACAATCA Ruby 5’ and 3’ RACE PMC-F1 CATGGACAGGAGAGGAAGATGATCT from Moro flesh cDNA PMC-Z CTTACTTTGCATTGAGAAGATCCCA Ruby 5’ and 3’ RACE PMC-R1 TTCCCGGAAGCCTGCCCACAATCA from Jingxian juice cDNA LeLe-R1 CAACTTCATCTGCTGCAAATTCTCCT PMC-GWF GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCGGATTCCTTAGGAGTT Expression of Ruby in PMC-GWR GGGACCACTTTGTACAAGAAAGCTGGGTCTTACTTTGCATTGAGAAGATC tobacco PMC-47 TCCTCTCCTGTCCATGCACCTTTACGAAC Chromosome walking for PMC-109 GAGGAACTTGATGCCATTTTGCTTCCCCA Isolation of Ruby promoters PMC-CF CATTGAAGCAGGCCAGAGTTGTCCGACTGATGAC iPCR for isolation of Tcs1 PMC-NR1 CTCTCCTGTCCATGCACCTTTACGAACTCCTAAG PMC-G3 GAGAGTATACCGTATGCGTACACA Isolation of full-length Tcs1 PMC-U ACACGTAGCTATTGGACCACCCT PMCi4

GCCGAAAAGTCTCCAGTAGTGACAAAGGTGACAG iPCR for isolation of Ruby PMCiD TCGCTGTTCTTCCCGGAAGCCTGCCCACAATCAG deletion allele and Tcs2 PMC-G2 CCGAAATACAGAATGCTCAAATGGGA Isolation of full-length Tcs2 PMC-S5 AGCACCTACACTTACTCAACTCTC PMC2ES-Fw AGCTGCTGGGCAACAGATGGT Ruby expression PMC2ES-Rev CTTCACATCGTTCGCTGTTC qRT-PCR GAL-POL1-Fw GCCCGTGGACGTAGGCTAA Tcs1 LTR expression GAL-POL1-Rev AAGAACAAGCACAAAAGAAAATACCA qRT-PCR GAL-POL4-Fw TGACAGTCAGAGTGCCTTGCA Tcs1 Gag-Pol expression GAL-POL4- Rev TCCTATGTGCTTTGTCCTGGAA qRT-PCR

 

 

 

Supplemental Data. Butelli et al. (2012). Plant Cell 10.1105/tpc.111.095232

5