Development 142: doi:10.1242/dev.121905: Supplementary Material
Supplementary Material
SUPPLEMENTARY FIGURES AND TABLES
Petunia
A
lp
b sm
Tomato
lp
se
se
se
b
lp
sam
b sm
fm
b
ilp
FA
AN
C
Arabidopsis
fm
se
ilp
ALF DOT
B
lp
lp
se
sam
2
0 sam
LFY UFO
D
Antirrhinum
lp
sam
lp
?
lp
FLO
?
se
2
0 sam
se
1
sam
ilp
3
3
se
1
FIM
Fig. S1. Schematic representation of the expression pattern of ALF and DOT and their homologs
in tomato Arabidopsis and Antirrhinum. (A-D) Expression in the vegetative SAM (left), in the
flowering meristems and young FMs (middle) and in a stage 3 flower (right). Expression of the
homologs ALF, FA, LFY and FLO is indicated in yellow, and of the homologs DOT, AN, UFO and
FIM in blue. Regions co-expressing both genes are marked in green. Abbreviations: br, bract; fm,
floral meristem; ilp, incipient leaf primordium; lp, leaf primordium; sam, shoot apical meristem; se,
sepal; sm, sympodial meristem. Redrawn after Moyroud et al (Moyroud et al., 2010).
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
+1 ATG
GUS
pDOT :GUS
1
# 5636
# 2720
# 2662
pDOT3.1(3.1Kb)
1
# 5767
1
# 5907
GUS
t35S
GUS
t35S
GUS
t35S
2
# 2691
1
# 2695
# 2720
# 2694
GUS
pLFY (2.3 Kb)
pLFY:GUS
pUFO (3.1 Kb)
pUFO:GUS
3
# 2720
+1 ATG
ALF cDNA
DOT cDNA
# 3890
# 2595
pLFY (2.3 Kb)
pLFY:LFY
# 2696
# 2720
# 2694
LFY cDNA
1
pUFO (3.1 Kb)
# 5551
UFO cDNA
# 3571
2
# 2721
tNOS
# 2720
pUFO:UFO
# 3888
tDOT
3
# 3759
1
# 3896
3
# 5566
DOT cDNA
# 3896
t35S
2
# 3895
pDOT4.6(4.6Kb)
pDOT4.6:DOT:tDOT
3
# 3887
# 3890
1
# 5636
# 2721
t35S
2
pDOT4.6(4.6Kb)
pDOT4.6:DOT
# 2721
# 3962
# 3895
1
# 0530
2
DOT cDNA
pDOT3.1(3.1Kb)
# 5636
# 2664
3
tNOS
1
# 3988
pDOT3.1:DOT
# 2721
tNOS
# 3757
pALF(2.8Kb)
pALF:ALF
GUS
# 2691
1
# 3756
# 2720
# 2697
# 2664
3
tNOS
2
# 2691
1
# 2696
# 2721
# 5927
pFIM (3.6Kb)
pFIM:GUS
tNOS
# 5768
pAN (5 Kb)
pAN:GUS
# 2664
3
GUS
# 5936
pDOT4.6(4.6Kb)
pDOT :GUS
4.6
tNOS
2
# 2691
1
# 2661
3.1
# 2722
# 2614
pALF (2.8Kb)
pALF:GUS
2
# 2615
1
# 2610
# 3572
2
# 2721
tNOS
Fig. S2. Schematic representation of the transgenes used in this study. Constructs are in the
pRD400 (Datla et al., 1992) and Gateway™ vectors (see Materials and Methods). The primers used
for construction are indicated with arrows, sequences are listed in Table S1 and numbers of the
amplicons indicate the order in which digested PCR products were cloned into the vector. Black bars,
5’ non-coding regions; blue bars, GUS coding sequence; red bars, NOS or t35S terminator; green bars,
ALF, DOT, LFY or UFO coding sequence, magenta bar tDOT; open circle, ATG start codon; closed
circle, stop codon.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Fig. S3. Complementation of alf by pALF:ALF. (A) alf mutant in hybrid W138/W115 background.
(B) alf fully complemented by the pALF:ALF transgene; structure of the inflorescence and flowers is
indistinguishable from wild type. Note that the outgrowth of axillary shoots (ax) is not repressed like
in (A). (C) alf partially complemented by pALF:ALF. The cymose inflorescence structure is restored,
but the second and third whorl of the flower consist of sepals instead of petals and stamens. Each
panel depicts the inflorescence phenotype on the left and a diagrammatic representation of
inflorescence structure on the right. Consecutive flowers are numbered from old to young (f1, f2); b,
bract.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Figure S4 Sequence comparison of pLFY and homologs from other species. (A) Promoter
comparison performed by Phytozome software (Goodstein et al., 2012). Sequence similarity in coding
regions is indicated with blue peaks, and in non-coding regions with grey peaks, if similarity is
between 50 and 70%, and with beige peaks if similarity is above 70%. (B) Comparison of pDOT with
pAN and pUFO from tomato and Arabidopsis respectively, using mVISTA (Frazer et al., 2004). Light
gray indicate conserved blocks between petunia and tomato. Note that pLFY has little or no sequence
similarity with pAN and pDOT. Arabidopsis thaliana, A. lyrata, Brassica rapa, Capsella rubella,
Capsella grandiflora and Eutrema salsugineum are Rosids belonging to the Brassicaceae, whereas
Medicago truncatula, Phaseolus vulgaris and Vitis vinifera are distantly related Rosids. Mimulus
gutatis and Solanum lycospersicum (tomato) belong to the Asterids.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Fig. S5. The promoter:cDNA transgenes have no effect on Arabidopsis development. (A) The
inflorescences of Arabidopsis containing the promoter:cDNA transgenes appeared wild type, similar to
those of the empty vector control (left). (B) Wild type flowers were observed in Arabidopsis
inflorescences transformed with the promoter:cDNA constructs. (C) Bar plot showing the mean
number of rosette plus cauline leaves of primary transformants: empty vector control. The difference
between the means is not significant in a two-tailed test with α = 0.05, P ≥ 0,783 for each of the
comparisons in One-Way ANOVA.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Fig. S6. Complementation of alf by pLFY:LFY. (A) alf inflorescence in W115/W138 hybrid
background with schematic representation of the flower-to-shoot phenotype. Outgrowth of axillaries is
suppressed. (B) Inflorescence of wild type W115/W138 hybrid. (C) alf partially complemented by
pLFY:LFY; note that the cymose inflorescence structure was restored, but that flowers consist of
whorls of sepals (green dots in the diagram). See also (F). (D) Transformants showing almost full
complementation of alf, only small floral organ defects remained. See (G-H). (E) Wild type
W115/W138 hybrid flower. (F) Partially complemented “green flower” as seen on inflorescences such
as in (C). (G-H) Nearly completely complemented flower of pLFY:LFY alf. In (H) the flower was
opened to show presence of all floral organs. Some stamens contained ‘flags’ of petal tissue (arrow),
or appeared filamentous (arrowhead). Consecutive flowers are numbered from old to young (f1, f2); b,
bract; ax, axillary; c, carpel, diagrams as in Fig. 1.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Fig. S7. Strong pDOT4.6:GUS Arabidopsis expressors. (A) In flowering pDOT4.6:GUS Arabidopsis
(At), both the SAM and young flower primordia strongly expressed GUS. In older flowers blue
staining was confined to the base of the flower. (B) Longitudinal section showing GUS mRNA
expression in Arabidopsis inflorescence, detected by in situ hybridization. Expression is absent in
apical IM but limited to sepal and petal primordial. *, apical meristem.
Fig. S8. pUFO:GUS is expressed in mature petunia embryos. (A) Wild type W115 and (B)
pDOT3.1:GUS embryos stained for GUS. No blue signal was observed. (C) pUFO:GUS is expressed in
the SAM (arrow) and in the root of mature petunia embryos. c, cotyledons.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Figure S9. Developmental effects of pUFO:UFO in petunia. (A-B) pUFO:UFO flowers have
supernumerary organs. Wild type petunia flowers (left) have five petals (A) and five stamens (B),
whereas pUFO:UFO flowers have six or more. Carpels (c) appeared wild type. (C) Repetitively
branching inflorescence of dot in W115/W138 background. (D) pUFO:UFO converted the dot
inflorescence to a single, aberrant flower, which terminated in a wild type carpel (inset).
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Fig. S10 Expression of pAN:GUS in petunia and Arabidopsis inflorescences, and pFIM:GUS in
petunia. (A) In petunia (Ph) transformants with moderate pAN:GUS expression GUS activity is first
seen as a stripe (arrow) within the apical FM at the adaxial side of the first emerging sepal. (B). In
petunia transformants with high pAN:GUS expression, GUS activity stains the entire FM dome, but no
staining is seen in the emerging SIM. (C) In high Arabidopsis (At) expressors, pAN:GUS is active in
both IM and FM. (D) In low petunia (Ph) expressors pFIM:GUS is first seen as a stripe (arrow) at the
sepal/petal boundary. (E) In high petunia expressors, pFIM:GUS was observed in whole FM dome. *,
(sympodial) inflorescence meristem.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Relative expression level
A
UFO
LFY
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
B
Vegetative Phase
(seedlings)
Reproductive Phase
(infloresence tips)
FIM
Relative expression level
FLO
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Vegetative Phase
(seedlings)
Reproductive Phase
(infloresence tips)
Fig. S11: LFY, UFO and FIM mRNA are expressed in the vegetative and reproductive phase,
while FLO is only expressed in the reproductive phase. (A) Relative expression level of LFY and
UFO mRNA in vegetative (2-week old seedlings) and reproductive phase of Arabidopsis. (B) Relative
expression level of FLO and FIM in vegetative and reproductive phase of Antirrhinum.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Block 1 P h
Block 2
Block 3
Block 4
Sl
Am
At
1
1
1
1
A TA CA T TT CC T CT AC A CA C A G T A G A A A G GA G A C C A A GA A C T C C ACT TG GT T AT GT G TG TA
A TA CA - -- -C T CT AC T CA C - - T A - A A A G CA A A C C T A GA A C T C C A-- TA GT T AA TC A AT CA
- TA C- - -- -- - -T AC T TG - - - T G A C T A G GA A A T - A A AA G T T T C AA- TA TT T TC TT G -- CA
- -- -- - -- -- - -- -- - -- - - - - - - - - - - -- - - - - - - -- - C T C C A-- -A AT T -- -- - -- -A
Ph
Sl
Am
At
61
52
45
11
T CA TA T C- AC T CT GC T GA T T G T A C T T C C T- - - - C G G CC C T T G A CAA GA AA A CA CT G AA AT
T AA TA A C- TT G TC TC T TT A T T T G T G T G T GT G T - - - - -- - - T G T CTG TT GA A CT AC - -A GT
T GT TG G CA TT A AC TC T G- A T C T C T G T C T GA A A - - - - -- - - - - - CT- TT TA C TT GC C AG GT
- -- -- - -A TA A TT TC - -- - - - - - - G - A T G- - - - - - - -- - - - - - --- -- -- - -- -- - -- --
Ph
Sl
Am
At
11 6
10 1
92
23
G AT AG A TG TT G TC TC T TT A T T T G T G T G T GT G T G T G T GT G T T G T CTG TT GA A CT GC - -A GT
- -- CT G TA GA T TG TC C TT C C T T C C T C G G CC - T T G A C AA G A A A A CAC TG AA A TG AT A CA TG
- -- CT G AA CC A CG TC C TT C C T T C C T C G G CA - T T G A C AA G A A A A CAC TG AA A CG AT A GA TG
- -- -- - -- -- - -- -- - -- - C T - - - - - G G GC A T T G A C AA G A T A A CTC TG AA G -- -- - -- --
Ph
Sl
Am
At
17 4
15 7
14 8
50
T AC CC A TC CA A TT CA A GA C T T A G C T T C C AG T A C T T T AT CC A TC -A C TC CA - -- C C A A C C A A A A AA T A T A - T AT GT G TC -A A T- -A A AA A A T G A C T T T C AG T G C T C A
- -- -C T CT CA A CT T- - -- - - - - - - - - - - -- - - - - - -
Ph
Sl
1 A AT GTA AA GT T TG CA T AG G G A TA T A A A A T GA A A C C C T CA A - - - AA C AA TT T AT TAC TG CT
1 A AT GAA AA CT T GA CA T TG G G A TG T G A A A T AA A A C C C T AG A T C G AA C AA TT T GT TAC TG AT
Ph
Sl
5 8 A CG ACT A- -- - -- -- - -C C A C TT A T T T G T AC T T C T A T T- - - - - -- T TA AA A AT --G AT TT
6 1 A CG ACT AG CT A TA CA T AC C A C TT G T T A G A AC T A T A A T TA A T A G GA T TA AA A AT ATG CT TT
Ph
Sl
9 9 A TT ATC TC TC T GT TG T TT T G T TT A A T G T G AT A T T T A A AG A A G T AT G AA -G A CC AGG TG AC
12 1 A TT ATT TG TG T GA TG T T- - A A TT A T T G T G AT G T T T A A AC A A G T AG A AA AG A CC AGG TG AC
Ph
Sl
15 8 A CT TGG AG AT T TA GA C AA A T C GG G T A A A A -T G T T A A - TA A G A A TA C AA AA G AA TAG TT GC
17 9 A CT TGA AA TT T TA GA C AA A T C GG G T A A A A AT G T T A A G TA A C A A CA C AA AA A AA TAG TT GC
Ph
Sl
21 6 A -- -AA CT TG C AA TG T TG A C T AT A G - C T T CT G C A C T G AT G A G T TG T TA TA T CC TTC CT TC
23 9 T GC CAA CT TT C AA TC T TG A C T AT A G T C T T TT G C A C T C A- - - - - -G C CT TT T CT TTT CT TC
Ph
Sl
27 2 T AT TTT GA CG A TG TT G A
29 3 T AT TTT GA CG A TG TT G A
Ph
Sl
Am
1 G TA CA A CT TG G AA GC A TA G GT T T C T T G A AG G A A T G C AC A A T T T CCA AA TC T GG GG A CT AA
1 G TA GA A GT TG G AA AC A TA G GT T T C T T G A TG G A A T G C AC A A T T T CCA AA TG T GG TG A GA AA
1 - -- -A A CT TG G TT TG A TA G CT T C C T T G A AG A A A T G C AT A A T T T CCA AG TT T GA TG T GT AC
Ph
Sl
Am
6 1 A CC TT T TA -C C TT AA A A- G GA G A T T T C T TG A A T G G G AA A C G C A ATT TC CA A AT CT G AT CA
6 1 A GC TT T AT -C C TT AA A AA G GA G G T T T C T TC A A T G G G AA - C A C A ATT TC CA A AT GT G AT GA
5 7 T CC TT T TT GC C AT TT A A- G GA G G T T T C T TG A A - G C A AA A C A C G ATT TC CA T AT CT G AT CA
Ph
Sl
Am
11 9 C CA CA A CT TT T TC CA T TT T TG G G T - C T T AA C C A C T G GC T T G G G TAG GG GT C TA GG T GT GG
11 9 G TA CT A CT TT T TC CA T TT T TG G G T T C T T GA T A A - - A AC T - - - - --- GT GT C TT G- T AG GG
11 5 C TC AA A CT TT T TC TA T TT T TA G A - - - - - -- - - - - - - -- - - - - - --- -- -- - -- -- - AG GG
Ph
Sl
Am
17 8 C AA TC A TA AT A AA TT G AT T GA C A T A A A C AA G A C A A T
16 9 T AA TC A TA AT A AG TA G AT T GA T A G A A A C AA G A C A - 14 2 C AA TC A TG TT A AA GA G AT T GA C A G A A A C AA G A C A - -
Ph
Sl
1 T AA GA T TG AT T GC -C A GT TG C G A A A C TC A A G T A A - -- - - A C A A AC A CC TT T AA AA A CC TT
1 T AA GA T TG AT T GG GC A GT TG C - - A A C TG A A G T A A T TT A A G C A A A- A CC TT A TA AA A CC CT
Ph
Sl
5 5 A GT AA C TC CT A TG AG T AT CT A A G A G T AA A C A C G A G GG A T T C C A CT A TT TT A AC CA A GA GA
5 8 A GT GA C TC CC A TG A- - AT CT A A G A G T AA A C A T G A G GG A T T C C A TT A TT TT A AC CA A GA GA
Ph
Sl
11 5 T TA TT G AA GG T AG AA T TT TA A A A G T C AA A A C T T T G AA A C A G T A GC G CT AT C CA -T C AA TC
11 6 C AA TT G GA GG C A- AA T TT AA A A A G T C AA A A C T T T G AA A C A G T A GC T TT TT T CA AT C AA TC
Ph
Sl
17 4 T AG CA C TA AA A TC AG A TA GC A A G T C A AA C T T C T C T GA T G T A A C CG G CC AA
17 5 T AG CA C TA AA - TC AG G AA CA A G G T C A AA C T T C - - T GA T G T A A C TA G CC AA
Figure S12. Sequence aligments of blocks 1-4 from pDOT blocks aligned with regions in .
Sequence alignment of the four blocks found in pDOT (Ph), pAN (Sl), pUFO (At) and pFIM (Am) (cf.
Fig 6). Putative binding sites for SBP and MADS-box protein were identified using the JASPAR
database (http://jaspar.genereg.net) and are indicated with green and orange boxes respectively.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
A
Solyc02g081660.2.1
AN
Solyc02g081680.2.1
S. lycopersicum
1 Kb
S. tuberosum
A. thaliana
G. max
V. vinifera
E. grandis
M. guttatus
P. trichocarpa
B
UFO
AT1G30930
A. thaliana
1 Kb
A. lyrata
B. rapa
C. rubella
C. grandiflora
E. salsugineum
Figure S13. Promoter comparison of DOT-like genes. (A) Similarity of AN and flanking sequences
with homologs from other species. Note that some regions upstream AN are conserved in the Rosid
Mimulus guttatis and the distantly related Asterids Glycine max, Eucalyptus grandis, Populus
trichocarpa and Vitis vinifera, but not in Arabidopsis thaliana. (B) Conservation of pUFO sequences
in other Brassicaceae. Both diagrams were obtained using the Phytozome portal (Goodstein et al.,
2012). Sequence similarity in coding regions is indicated with blue peaks, and in non-coding regions
with grey peaks, if similarity is between 50 and 70% with beige peaks if similarity is above 70%.
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Table S1. Primers used for construction of transgenes
Name
flo9R
flo22F
flo48F
flo52R
flo53R
flo59F
flo60R
flo61F
flo65R
GUS F4
GUS F6
GUS R5
lfy4R
lfy5F
lfy10R
lfy11F
NOS F1
NOS F2
NOS F3
NOS R1
NOS R2
NOS R4
NOS R5
puf3R
puf5R
puf14F
puf46F
puf47R
puf56R
puf57F
ufo4F
ufo5R
ufo8R
PhBOX4attB1
PhpDOTE/SattB2
DOTstopattB2
DOTattB2-5510Rev
SlANpromattB1
SlANpromattB2
AmFIMp-attB1
AmFIMp-attB2
pDOT-attB1
pDOT-attB4
DOT-attB4r
DOT-attB3r
GUS-attB4r
GUS-attB3r
t35S-attB3
t35S-attB2
tDOT-attB3
tDOT-attB2
Number
0530
1287
2610
2614
3107
3756
3757
3758
4229
2615
2691
2664
2694
2695
3571
3759
2720
3576
3577
2721
2722
3578
3579
0827
0836
1511
2661
2662
3962
3988
2696
2697
3572
5636
5936
4192
5551
5767
5768
5907
5927
5636
3890
3895
3896
5024
3914
3887
3888
5566
5551
Description
ALF STOP R BamHI
ALF dTPH1 F
ALF -2800F BamHI
ALF ATG R HinDIII
ALF intron R
ALF -2800F KpnI
ALF ATG R BamHI
ALF ATG F BamHI
ALF dTPH1 R
GUS ATG F HinDIII
GUS ATG F BamHI
GUS STOP R BamHI
LFY ATG R BamHI
LFY -2300F EcoRI
LFY STOP R BamHI
LFY ATG F BamHI
tNOS F SalI
tNOS F BamHI
tNOS F XbaI
tNOS R SalI
tNOS R HinDIII
tNOS R BamHI
tNOS R XbaI
DOT dTPH1 R
DOT dTPH1 R
DOT dTPH1 F
DOT -3100F EcoRI
DOT ATG R BamHI
DOT STOP R SalI
DOT -3100 TOPO
UFO -3800F EcoRI
UFO ATG R BamHI
UFO STOP R XbaI
pDOT4.6 attB1
pDOT4.6 attB2
DOT-attB2
tDOT-attB2
pAN-attB1
pAN-attB2
pFIM-attB1
pFIM-attB2
pDOT-attB1
pDOT-attB4
DOT-attB4r
DOT-attB3r
GUS-attB4r
GUS-attB3r
t35S-attB3
t35S-attB2
tDOT-attB3
tDOT-attB2
Sequence
CGGGATCCTTAGAATGACAACCTAA CAGATGGGAACTGCTTGTTGGAG CGCGGATCCGTGACTTGGAAGTTGGAACAAACG CCCAAGCTTACTTGCTGAGAAAGCCTCTGGGTCC TGGCCTTCCAAAAGTTATGCATGTC GGGGTACCGTGACTTGGAAGTTGGAACAAACG CTTGCTGAGAAAGCCTCTGGATCCATGTTG CAACATGGATCCAGAGGCTTTCTCAGCAAG CTCATTCTGCCACCGCCTGGC CCCAAGCTTGTCCGTCCTGTAGAAACCCCAACC CGCGGATCCGGTCCGTCCTGTAGAAACCCCAAC CGCGGATCCTCATTGTTTGCCTCCCTGCTGC CACCGGAATAAGCCACTCGTGAAACCTTCAGGATCC GGAATTCGGCCTATACGACGTCGTTTGAAAGAGATCC CGGGATCCCTAGAAACGCAAGTCGTCG CGGGATCCTGAAGGTTTCACGAGTGGC ACGCGTCGACCCCGATCGTTCAAACATTTGGCAATA CGGGATCCCCCGATCGTTCAAACATTTGGCAATA GCTCTAGACCCGATCGTTCAAACATTTGGCAATA ACGCGTCGACCCGATCTAGTAACATAGATGACACCG CCCAAGCTTCCGATCTAGTAACATAGATGACACCG CGGGATCCCCGATCTAGTAACATAGATGACACCG GCTCTAGACCGATCTAGTAACATAGATGACACCG TGGACAAGGAGGAATCCAAAC CCGCATGGGCGCTTGAATTTTAG CTATTGACTTAGCTGTGGCTGG CGGAATTCGATTTCATTGCGGTTGGTATTTACGCC CGGGATCCCTAATAGGTGCATGATGAAAAGCTTCC ACGCGTCGACGTATCAGTTGAAAGATTGAAAGGGTAATGTCAAC CACCGATTTCATTGCGGTTGGTATTTACGCC CCGGAATTCTCTGTTTTAATTGCCCCACTTC CGCGGATCCTTATTGATGAACACAGTTGAATCC GCTCTAGACTAACAGACTCCAGGAAATGG GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAACATTAAGATAAACTCA GGGGACCACTTTGTACAAGAAAGCTGGGTGTCTAGGATAATTGAAGGTAT GGGGACCACTTTGTACAAGAAAGCTGGGTAGTTGAAAGATTGAAAGGGTAATGTC GGGGACCACTTTGTACAAGAAAGCTGGGT AATGAAAACGTAATGCAATAC GGGGACAAGTTTGTACAAAAAAGCAGGCTCAAAGGTGGTAAGATTGATTG GGGGACCACTTTGTACAAGAAAGCTGGGTTTGAGTTTGAAGCTAGGAGAG GGGGACAAGTTTGTACAAAAAAGCAGGCTCACCAATCTCATGATTCCACT GGGGACCACTTTGTACAAGAAAGCTGGGTAGTTTTGGATTTGCTAAAGAA GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAACATTAAGATAAACTCA GGGGACAACTTTGTATAGAAAAGTTGGGTCTTGTCTAGGATAATTGAAGGTATAC GGGGACAACTTTTCTATACAAAGTTGATGGAAGCTTTTCATCATGCA GGGGACAACTTTATTATACAAAGTTGTCAGTTGAAAGATTGAAAGGGTAA GGGGACAACTTTTCTATACAAAGTTGCCATGGTCCGTCCTGTAGAAACC GGGGACAACTTTATTATACAAAGTTGGGTAGCAATTCCCGAGGCT GGGGACAACTTTGTATAATAAAGTTGCGGCCATGCTAGAGTCCGC GGGGACCACTTTGTACAAGAAAGCTGGGTAGGTCACTGGATTTTGGTTTTAG GGGGACAACTTTGTATAATAAAGTTGTACTTTTGAACTGTTTCAAGTGG GGGGACCACTTTGTACAAGAAAGCTGGGTAATGAAAACGTAATGCAATAC Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
Table S2. Primers used for Real-Time PCR
Primer
Primer ID
Sequence
AtACTIN-FW
6231
AGTGGTCGTACAACCGGTATTGT
AtACTIN-RV
6232
GATGGCATGAGGAAGAGAGAAAC
AmACTIN-FW
6951
GCCAAGACAAGCTCCTCTGT
AmACTIN-RV
6952
ATTCCGATCATTGATGGCTGGA
LFY-FW
5606
CCTCGTCTCTCTATTTGGTATG
LFY-RV
6955
CATCCACCACGTCCAGACGT
UFO-FW
6956
CTACACACAGTTTGCAGCAGAAG
UFO-RV
6957
CTCACTATATCAAACAGCAACTGC
FLO-FW
6947
AACCAAAAATGCGCCACTAC
FLO-RV
6948
TGTAGCATGCCTGACGCCAT
FIM-FW
5780
GATGGACTGCAGAATCTGGAGC
FIM-RV
6945
GTAGCCTTCGTAGTTTGTCGGTC
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
SUPPLEMENTARY MATERIALS AND METHODS
Preparation of gene constructs
The pALF, pLFY, pUFO and the pDOT3.1:GUS constructs were made by digestion (restriction sites
listed in supplementary material, table S1 and Fig. S2) and ligation into pRD400tNOS a version of
pRD400 (Datla et al., 1992) in which we had ligated tNOS as a SalI fragment.
pALF:ALF:tNOS was made by digesting of pALF with KpnI/BamHI and ALF with BamHI and
ligation into pRD400tNOS digested with KpnI/BamHI. pLFY:LFY:tNOS and pUFO:UFO:tNOS were
made by ligating the corresponding genomic region of LFY or UFO as an EcoRI/BamHI or
EcoRI/XbaI fragment respectively into pRD400tNOS.
With the same procedure we made the promoter:GUS constructs. For the pALF:GUS, we digested
pALF with BamHI/HinDIII and GUS with HinDIII at both sites. For pDOT3.1:GUS, pLFY:GUS and
pUFO:GUS constructs we used EcoRI/BamHI for all the promoter fragments and BamHI at both sites
for GUS. After ligation, the promoter:GUS fragments were introduced into pRD400tNOS digested with
BamHI/HinDIII and EcoRI/BamHI respectively. pDOT3.1:DOT:tNOS was made by introducing the
genomic pDOT:DOT fragment into the Gateway™ TOPO Entry vector according to the instructions of
the manufacturer and subsequent recombination into the binary vector pK2GW7.
The pFIM and pAN PCR fragments were introduced into entry vector via BP reaction with attB1B2 site. The entry clones were subcloned according to Gateway Technology with Clonase II
(Invitrogen) into the destination vectors (Karimi et al., 2002): we used the pKGW,0 for
promoter:cDNA constructs and pKGWFS7.0 with GUS for promoter analysis (supplementary
material, Fig. S2).
pDOT4.6:DOT:t35S, pDOT4.6:DOT:tDOT, pDOT4.6:GUS:t35S and pDOT4.6:GUS:tDOT were
constructed with Gateway Multisite Recombination. pDOT4.6 was introduced in pDONR221 P1-P4 by
a BP reaction using primer with attB1-B4 sites; GUS cDNA was inserted into pDONR221 P4r-P3r by
a BP reaction using primer with attB4r-B3r; DOT cDNA sequence was also cloned into pDONR221
P4r-P3r clone by a BP reaction using primer with attB4r-B3r; tDOT was inserted by a BP reaction into
pDONR221 P3-P2 using primer with attB3-B2; t35S was also inserted by a BP reaction into
pDONR221 P3-P2 using primer with attB3-B2. By LR Clonase reaction the fragments were subcloned into pKGW,0 destination vectors in the different combination to obtain the desired construct.
All the primers used for cloning are listed in Table S1.
All transgenes were (re)sequenced before introduction into the transformable petunia line W115 or
homozygous alf and dot mutants using Agrobacterium tumefaciens (strain AGL0) mediated leaf disc
transformation (Horsch et al., 1985). Arabidopsis thaliana Columbia was transformed with
Agrobacterium tumefaciens strain C58C1 (MP90) using the floral dip method (Clough and Bent,
Development | Supplementary Material
Development 142: doi:10.1242/dev.121905: Supplementary Material
1998), and transformants were selected on Murashige and Skoog medium (Duchefa) containing 50
mg/l kanamycin mono-sulfate.
SUPPLEMENTARY REFERENCES
Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana. Plant J. 16(6): 735-43.
Datla, R. S., Hammerlindl, J. K., Panchuk, B., Pelcher, L. E. and Keller, W. (1992) Modified
binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 122(2): 383-4.
Frazer, K. A., Pachter, L., Poliakov, A., Rubin, E. M. and Dubchak, I. (2004) VISTA:
computational tools for comparative genomics. Nucleic Acids Res 32(Web Server issue): W2739.
Goodstein, D. M., Shu, S., Howson, R., Neupane, R., Hayes, R. D., Fazo, J., Mitros, T., Dirks, W.,
Hellsten, U., Putnam, N. et al. (2012) Phytozome: a comparative platform for green plant
genomics. Nucleic Acids Res 40(Database issue): D1178-86.
Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G. and Fraley, R. T. (1985)
A simple and general method for transferring genes into plants. Science 227(4691): 1229-1231.
Karimi, M., Inze, D. and Depicker, A. (2002) GATEWAY vectors for Agrobacterium-mediated
plant transformation. Trends in Plant Science 7(5): 193-5.
Moyroud, E., Kusters, E., Monniaux, M., Koes, R. and Parcy, F. (2010) LEAFY blossoms. Trends
in Plant Science 15(6): 346-52.
Development | Supplementary Material