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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 TRICHOTOMY 1000 ZmCRTISO1
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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