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

1. 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

2. 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 X Yellow-flowered progeny F1 Progenies (6 individuals, yellow-flowered) F2 Progenies (146 individuals)

3. 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). CoCRTISO1-Y CoCRTISO1-ORa 758 CoCRTISO1-ORb TRICHOTOMY CoCRTISO2 CoCRTISO3 977 1000 CoCRTISO4 995 CmCRTISO 944 DcCRTISO AtCRTISO 996 OncidiumCRTISO 361 932 SynechocystisCRTISO 572 ZmCRTISO1 1000 359 ZmCRTISO2 IpomoeaCRTISO 677 SlCRTISO 0.1

4. 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). A B CoCRTISO1-Y CoCRTISO1-ORa CoCRTISO1-Y CoCRTISO1-ORa 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 202 116 98 47 (kDa) 202 116 98 47 (kDa)

5. 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,5Z)-lycopene were used as substrates. B, (5Z)-γ-carotene was used as a substrate. C, (5Z)-rubixanthin was used as a substrate. Peak 1, (5Z,9Z,5Z)-lycopene; peak 2, (5Z,9Z)-lycopene; peak 3, (all-E)-lycopene; peak 4, (all-E)-γ-carotene; peak 5, (5Z)-γ-carotene; peak 6, (all-E)-rubixanthin; and peak 7, (5Z)-rubixanthin. A substrate: (5Z,9Z)- and (5Z,9Z,5’Z)-lycopene B substrate: (5’Z)-γ-carotene 5 CoCRTISO1-Y CoCRTISO1-ORa CoCRTISO1-ORb CoCRTISO2 CoCRTISO3 CoCRTISO4 Control 2 CoCRTISO1-Y CoCRTISO1-ORa CoCRTISO1-ORb CoCRTISO2 CoCRTISO3 CoCRTISO4 Control 4 1 3 5 2 1 3 5 2 1 3 5 2 4 1 Absorbance at 475 nm Absorbance at 450 nm 3 5 2 1 3 5 2 1 3 5 2 1 3 0 20 40 60 80 100 [min] 0 20 40 60 80 100 [min] Retention time Retention time C substrate: (5’Z)-rubixanthin 7 CoCRTISO1-Y CoCRTISO1-ORa CoCRTISO1-ORb CoCRTISO2 CoCRTISO3 CoCRTISO4 Control 6 7 7 7 Absorbance at 450 nm 7 7 7 0 20 40 60 80 100 [min] Retention time