Supplemental Fig. S1. Orange- and yellow-flowered cultivars of calendula used in this study.

Alice Orange Orange Star Pompom Orange Orange Gem

Alice Yellow Gold Star Pompom Yellow Golden Gem

Supplemental Fig. S2. Breeding process used to obtain calendula F2 progenies from crosses

between orange- and yellow-flowered lines.

‘Alice Orange’ X ‘Orange Star’(Orange-flowered) (Orange-flowered)

‘Alice Yellow’ X ‘Golden Gem’ (Yellow-flowered) (Yellow-flowered)

Orange-flowered progeny Yellow-flowered progenyX

F1 Progenies (6 individuals, yellow-flowered)

F2 Progenies (146 individuals)

Supplemental Fig. S3. ClustalW tree analysis of calendula CRTISO homologs in various plant

species. Numbers at branch points indicate bootstrap values (1000 replicates). AtCRTISO

(Arabidopsis thaliana, AT1G06820), CmCRTISO (Chrysanthemum morifolium, AB205043),

DcCRTISO (Daucus carota sp. sativus, DQ192188), IpomoeaCRTISO (Ipomoea sp., AB499053),

SlCRTISO (Solanum lycopersicum, AF416727), OncidiumCRTISO (Oncidium Gower Ramsey,

AY973633), ZmCRTISO1 (Zea mays, FJ603466), ZmCRTISO2 (Zea mays, FJ465413), and

SynechocystisCRTISO (Synechocystis sp. PCC 6803, gene sll0033).

SlCRTISO

ZmCRTISO2

AtCRTISO

CmCRTISO

IpomoeaCRTISO

SynechocystisCRTISO

CoCRTISO4

CoCRTISO3

CoCRTISO2

CoCRTISO1-ORb

CoCRTISO1-ORa

CoCRTISO1-Y

0.1

DcCRTISO

OncidiumCRTISO

758

1000977

995

944

996

361 932

359

572

677

TRICHOTOMY

1000ZmCRTISO1

A B

1 2 3 4 5

CoCRTISO1-Y CoCRTISO1-ORa CoCRTISO1-Y CoCRTISO1-ORa

202

116

98

47(kDa)

202

116

98

47(kDa)

1 2 3 4 51 2 3 4 51 2 3 4 5

Supplemental Fig. S4. Expression of CoCRTISO-MBP fusion proteins in E. coli carrying pMAL-

CoCRTISO. The arrowhead shows putative size (100 kDa) of the fusion protein. A, Proteins were

separated by using SDS-PAGE and were stained with Coomassie Brilliant Blue. B, Western blotting

with MBP antibodies. Lane 1, insoluble fraction (pellet) of E. coli lysate under noninducing

conditions; lane 2, insoluble fraction under inducing conditions; lane 3, soluble fraction (supernatant)

under noninducing conditions; lane 4, soluble fraction under inducing conditions; lane 5, affinity-

purified CoCRTISO polypeptide (approximately 1 μg in A and approximately 0.2 μg in B).

CoCRTISO1-Y

CoCRTISO1-ORa

CoCRTISO1-ORb

CoCRTISO2

CoCRTISO3

CoCRTISO4

Control

Abs

orba

nce

at 4

75 n

m

Abs

orba

nce

at 4

50 n

m

0 20 40 60 80 100 [min]

Retention time0 20 40 60 80 100 [min]

Retention time

CoCRTISO1-Y

CoCRTISO1-ORa

CoCRTISO1-ORb

CoCRTISO2

CoCRTISO3

CoCRTISO4

Control

A substrate: (5Z,9Z)- and (5Z,9Z,5’Z)-lycopene B substrate: (5’Z)-γ-carotene

12

Supplemental Fig. S5. HPLC analysis of the in vitro assay products of CoCRTISO1, -2, -3, and -4. Percentages of total peak area are shown in Supplemental Tables S11, S12, and S13. A, (5Z,9Z)- and (5Z,9Z,5Z)-lycopene were used as substrates. B, (5Z)-γ-carotene was used as a substrate. C, (5Z)-rubixanthin was used as a substrate. Peak 1, (5Z,9Z,5Z)-lycopene; peak 2, (5Z,9Z)-lycopene; peak 3, (all-E)-lycopene; peak 4, (all-E)-γ-carotene; peak 5, (5Z)-γ-carotene; peak 6, (all-E)-rubixanthin; and peak 7, (5Z)-rubixanthin.

CoCRTISO1-Y

CoCRTISO1-ORa

CoCRTISO1-ORb

CoCRTISO2

CoCRTISO3

CoCRTISO4

Control

Abs

orba

nce

at 4

50 n

m

0 20 40 60 80 100 [min]

Retention time

C substrate: (5’Z)-rubixanthin

3

12

3

12

3

12

3

12

3

12

3

12

3

45

5

5

5

5

5

5

4

6

7

7

7

7

7

7

7