Materials and Methods for Supporting Analyses
Phylogenetic analyses
Sequences of SEP and DEF/AP3 from other plant species used in the PHYLOGENETIC
analysis
(Figs.
S7
and
S8)
were
obtained
from
NCBI
(http://www.ncbi.nlm.nih.gov/nuccore; see Supplementary Tables S3 and S6 for
accession numbers). The full lengths of each amino acid sequence for these SEP and
DEF/AP3, including the torenia-homologous genes, were used for the phylogenetic
analysis. Protein sequences were aligned using GENETYX ver. 8.0.0 (Genetyx
Corporation, Tokyo, Japan) and refined by hand with minor adjustment, taking into
account the amino acid sequences around the MADS domain (Davies et al. 1996). After
these sequences had been aligned, they were used for phylogenetic analysis using the
neighbor-joining method in GENETYX ver. 8.0.0. Statistical significance was tested by
bootstrap analysis for 10,000 replicates.
Expression analysis by qRT-PCR
For qRT-PCR, total RNAs were prepared from petals, stamens, and carpels using TRIzol
(Invitrogen). Three independent wild-type torenias and transgenic lines were used (Fig.
S3). Because torenia stamens are small, it is difficult to prepare a sample of sufficient
volume for a number of the qRT-PCR materials. Thus, 12–16 stamens were combined as
a single sample (Figs. S6 and S10). Sampling of petals for this analysis was done
similarly, with 3 or 4 petals being combined as one sample. Next, cDNAs were
synthesized from total RNA using a ReverTra Ace® qPCR RT Kit (Toyobo). The
qRT-PCR reaction was performed using SYBR Premix Ex Taq II (TIi RNaseH Plus;
TaKaRa), and the signals were detected on a Thermal Cycler Dice® Real Time System
1
TP800 (TaKaRa), according to the manufacturer’s instructions. The sequences of the
specific primers are shown in Supplementary Tables S2 and S4.
Southern gel blot analysis
Genomic DNA was prepared from torenia leaves using ISOPLANT II (Nippon Gene,
Tokyo, Japan). Twenty micrograms of torenia genomic DNA was digested with EcoRI,
HindIII, and PstI. The genomic DNAs were separated on 0.8% agarose gel and
transferred
to
a
nylon
membrane
(Hybond
N +;
GE
Healthcare,
http://www.gelifesciences.co.jp/index.html). As probes for the analysis, we used the
3′-UTR region, including a partial coding region of TfDEF and TfGLO without MADS
domains. The DNA probe was labeled using a DIG DNA labeling kit (Roche Applied
Science, Mannheim, Germany) by PCR. The sequences of specific primers used here are
shown in Supplementary Table S5. Hybridization signals were detected by
chemiluminescence with CSPD-Star (Roche Applied Science, Mannheim, Germany) as
the substrate and visualized with a light-capture system (AE-6962; ATTO, Tokyo, Japan).
2
Fig. S1
3
Fig. S2
4
Fig. S3
5
Fig. S4
6
Fig. S5
7
Fig. S6
8
Fig. S7
9
Fig. S8
10
Fig. S9
11
Fig. S10
12
Fig. S11
13
Supplementary Figure Legends
Fig. S1. Confirmation of expression of introduced class B genes by RT-PCR. (A) TfDEF
and TfGLO expression. (B) TfDEF-SRDX and TfGLO-SRDX expression. TfACT3 was
used as an internal control. PCR cycles are indicated to the right.
Fig. S2. Floral phenotypes of TfDEF/TfGLO-ox plants. (A) Partial petaloid phenotype in
carpels of TfDEF/TfGLO-ox plants. (B) TfDEF/TfGLO-ox7 and -ox11 plants exhibited
floral phenotypes similar to those of TfDEF/TfGLO-ox5 plants. These TfDEF/TfGLO-ox
plants showed petal-like sepal phenotypes (left panels) and semi-petaloid carpels (right
panels). Red arrowheads indicate the extremely short peduncles. (C) Interior of carpels of
wild-type and TfDEF/TfGLO-ox torenias, observed after cutting longitudinally. Blue
and red arrowheads indicate the ovule and petaloid funicle, respectively. Scale bar = 5
mm.
Fig. S3. Comparison of TfFAR and TfPLE expressions in carpels of TfDEF/TfGLO-ox
and TfDEF/TfGLO-RD plants. Quantitative RT-PCR was performed to examine
endogenous TfFAR and TfPLE expressions using petals and carpels of wild-type and
carpels of TfDEF/TfGLO-ox and TfDEF/TfGLO-RD torenias. TfACT3 was used as an
internal control. The vertical axis represents the mean of three independent transgenic
lines.
Fig. S4. Cell shapes of carpels examined by SEM. Top panels indicate sites of
observation of styles (red arrowhead) and ovaries (blue arrowhead) in wild-type torenias
14
(left) and TfDEF/TfGLO-ox plants (right); the arrowhead colors at the sites of observation
correspond to those of the outlines of each SEM image. The black arrowhead indicates
apiculate cells not seen in carpels of wild-type torenias. Scale bar = 20 µm.
Fig. S5. SEM analysis of floral organs of wild-type and transgenic torenias. (A) Cell
shapes of petals and sepals in wild-type plants. Scale bar = 20 µm (petals and all sepals
with yellow outline) and 50 µm (all other petals). (B) Cell shapes of sepals in TfDEF-ox
plants. Scale bar = 20 µm. (C) Cell shapes of sepaloid petals in TfDEF/TfGLO-RD plants.
Scale bar = 20 µm. Colors of arrowheads at the sites of observation correspond to the
colors of the image outlines.
Fig. S6. Comparison of expressions of class B and C genes in stamens and carpels of
TfDEF/TfGLO-RD plants. (A) RT-PCR analysis of expressions of endogenous TfDEF,
TfGLO, TfFAR, and TfPLE using stamens of wild-type and TfDEF/TfGLO-RD plants.
(B) RT-PCR analysis of expressions of endogenous TfDEF and TfGLO in carpels of
wild-type and TfDEF/TfGLO-RD plants. TfACT3 was used as an internal control. PCR
cycles are indicated to the right of each column.
Fig. S7. Phylogenetic tree of SEP-related proteins. (A) Amino acid sequence alignment
of the MADS domains of SEP proteins from Antirrhinum, Arabidopsis, and torenia. (B)
Phylogenetic relationship among SEP proteins. The neighbor-joining tree was generated
based on amino acid sequences with coding regions of SEP genes. 10,000 bootstrap
samples were generated to assess support for the relationships. Origins and accession
numbers of each protein used are described in Supplemental Table S3. Local bootstrap
15
values (%) are indicated near the branch points (values below 50% are omitted).
Fig. S8. Phylogenetic tree of DEF/AP3-related proteins. The neighbor-joining tree is based
on amino acid sequences with coding regions of DEF/AP3 genes. Bootstrap samples
(10,000) were generated to assess support for the relationships. Origins and accession
numbers of each protein used are described in Supplementary Table S6. Local bootstrap
values (%) are indicated near the branch points (values below 50% are omitted). Gray
boxes; DEF/AP3 proteins, which are similar to TfDEF (green box), have counterpart
TM6 proteins.
Fig. S9. Southern gel blot analysis to identify genes homologous with torenia class B
genes. Torenia genomic DNA was digested with EcoRI, HindIII, and PstI as indicated,
and hybridized with TfDEF (left) and TfGLO (right) probes.
Fig. S10. Comparison of transgene expressions in petals and stamens using
TfDEF/TfGLO-ox plants. Quantitative RT-PCR analysis was performed to examine
endogenous TfGLO (pink) and transgene TfGLO-SRDX (green) expression using petals
and stamens of wild-type and TfDEF/TfGLO-ox torenias. TfACT3 was used as an internal
control. The vertical axis represents the mean of three experiments using the same
samples (see Supplementary Materials and Methods for Supporting Analyses) with
standard deviation.
Fig. S11. Putative biosynthetic pathway of anthocyanins and flavones in torenia. This is a
modified version of an earlier biosynthetic pathway (Sasaki et al. 2010). ANS,
16
anthocyanin synthase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR,
dihydroflavonol 4-reductase; FNS, flavone synthase; F3H, flavanone hydroxylase; F3H,
flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; 3GT, glucose: flavonoid
3-O-glucosyltransferase; MT, methyl transferase.
17