Supporting Legends
Figure S1. Flower phenotypes of the efp mutants
Ipomoea efp mutants and germinal revertants (a), and efp knockdown transformants of
Petunia (b) and Torenia (c). The TT48 and TT49 series of Torenia transformants harbor
the Torenia ThEFP-A and ThEFP-B RNAi constructs, respectively (Figure S5).
Figure S2. Comparison of the EFP protein with its relatives
(a) A phylogenetic tree for the CHI superfamily. The amino acid sequences were
aligned with the ClustalW program of the MEGA 5 software (Tamura et al., 2011). The
tree was constructed using the neighbor-joining method of the MEGA 5 with the
Poisson correction model. Bootstrap values of 1000 replicates are shown next to the
branches, and the scale bar indicates 0.1 amino acid substitutions per site. Accession
numbers of the sequences are shown in parentheses, and the amino acid sequences of
InCHI and InFAP were deduced from EST sequences. (b) Multiple alignments of the
deduced amino acid sequences of Arabidopsis AtCHIL and EFP proteins. (c)
Comparison of the catalytic residues of CHI with AtCHIL and EFP proteins. Sequences
were aligned with the ClustalW program v2.1. Identical amino acids, highly similar
residues, less similar residues, and gaps are indicated by asterisks (*), colons (:), periods
(.), and hyphens (-), respectively. The conserved catalytic residues among CHI proteins
are indicated by red characters (Jez et al., 2000; Ngaki et al., 2012). Abbreviations
shown in front of each protein indicate the plant species: At, Arabidopsis thaliana; Gm,
Glycine max; In, Ipomoea nil; Ms, Medicago sativa; Og, Oncidium cv. Gower Ramsey;
Ph, Petunia hybrida; and Pp, Physcomitrella patens; and Th, Torenia hybrida. OgEFP,
PpEFPa, PpEFPb, PpFAPb were originally called OgCHI, CHILa, CHILb, and FAPb,
respectively (Chiou and Yeh, 2008; Ngaki et al., 2012).
Chiou, C.Y. and Yeh, K.W. (2008) Differential expression of MYB gene (OgMYB1)
determines color patterning in floral tissue of Oncidium Gower Ramsey. Plant Mol.
Biol., 66, 379-388.
Jez, J.M., Bowman, M.E., Dixon, R.A. and Noel, J.P. (2000) Structure and
mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nat.
Struct. Biol., 7, 786-791.
Ngaki, M.N., Louie, G.V., Philippe, R.N., Manning, G., Pojer, F., Bowman, M.E., Li,
L., Larsen, E., Wurtele, E.S. and Noel, J.P. (2012) Evolution of the
chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis. Nature,
485, 530-533.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011)
MEGA5: molecular evolutionary genetics analysis using maximum likelihood,
evolutionary distance, and maximum parsimony methods. Molecular biology and
evolution, 28, 2731-2739.
Figure S3. HPLC analysis of anthocyanins in Ipomoea
Anthocyanin structures (a) and HPLC analysis of anthocyanins in EFP (b) and efp-1(c)
flowers.
WBA
(Wedding
Bells
Anthocyanin),
pelargonidin
3-O-[2-O-(6-O-(trans-3-O-(
β
-D-glucopyranosyl)caffeoyl)β
-D-glucopyranosyl)-6-O-(trans-4-O-(6-O-(trans-3-O-(β-D-glucopyranosyl)caffeoyl-β
-D-glucopyranosyl)caffeoyl)-D-glucopyranoside]-5-O-[ β -D-glucopyranoside]; A1,
pelargonidin
3-O-[2-O-(6-O-(trans-3-O-( β -D-glucopyranosyl)caffeoyl)- β
-D-glucopyranosyl)- β -D-glucopyranoside]-5-O-[ β -D-glucopyranoside]; A2,
pelargonidin
3-O-[2-O-(6-O-(trans-caffeoyl)β
-D-glucopyranosyl)β
-D-glucopyranoside]-5-O-[
β
-D-glucopyranoside];
A3,
pelargonidin
3-O-[2-O-(6-O-(trans-3-O-(
β
-D-glucopyranosyl)caffeoyl)β
-D-glucopyranosyl)-6-O-(trans-caffeoyl)β
-D-glucopyranoside]-5-O-[
β
-D-glucopyranoside]. Absorbance at 530 nm was used for the detection of anthocyanin
pigments.
Figure S4. HPLC analysis of colorless flavonoids in Ipomoea
HPLC analysis of colorless flavonoids in Ipomoea EFP (a) and efp-1 (b) flowers.
Absorbance at 360 nm was used to detect colorless flavonoids. A1–3 are the
anthocyanins presented in Figure S3a, and F1 is the flavonol kaempferol glycoside.
C1–4 are caffeic acid derivatives: C1, chlorogenic acid; C2, 1-O-caffeoyl glucoside.
Note that chalcone 2´-O-glucoside (retention time: 25.9 min), which is a major yellow
flavonoid pigment that accumulates in CHI null mutants (Saito et al., 2011), was
virtually absent in the efp-1 mutant.
Saito, N., Tatsuzawa, F., Hoshino, A., Abe, Y., Ichimura, M., Yokoi, M., Toki, K.,
Morita, Y., Iida, S. and Honda, T. (2011) The anthocyanin pigmentation
controlled by the speckled and c-1 mutations of the Japanese morning glory. J.
Japan. Soc. Hort. Sci., 80, 452-460.
Figure S5. RNAi vectors for Petunia and Torenia EFP knockdowns
Restriction sites used for vector construction are indicated, and the sites within the
parentheses are from cloning vectors. The restriction fragments were cloned into the
pBINPLUS binary vector (van Engelen et al., 1995). MAC-1P and MAST are the Ti
plasmid mannopine synthase promoter (Comai et al., 1990) and terminator, respectively.
El235SP and NOST denote an enhanced cauliflower mosaic virus 35S promoter
(Mitsuhara et al., 1996) and Agrobacterium tumefaciens nopaline synthase terminator,
respectively.
Comai, L., Moran, P. and Maslyar, D. (1990) Novel and useful properties of a
chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol.
Biol., 15, 373-381.
Mitsuhara, I., Ugaki, M., Hirochika, H., Ohshima, M., Murakami, T., Gotoh, Y.,
Katayose, Y., Nakamura, S., Honkura, R., Nishimiya, S., Ueno, K., Mochizuki,
A., Tanimoto, H., Tsugawa, H., Otsuki, Y. and Ohashi, Y. (1996) Efficient
promoter cassettes for enhanced expression of foreign genes in dicotyledonous and
monocotyledonous plants. Plant Cell Physiol., 37, 49-59.
van Engelen, F.A., Molthoff, J.W., Conner, A.J., Nap, J.P., Pereira, A. and
Stiekema, W.J. (1995) pBINPLUS: an improved plant transformation vector based
on pBIN19. Transgenic Res., 4, 288-290.