supplemental data. souer et al. (2008). patterning of ... · souer et al. (2008). patterning of...

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Supplemental Data. Souer et al. (2008). Patterning of inflorescences and flowers by the F-box protein

DOUBLE TOP and the LEAFY-homolog ABERRANT LEAF AND FLOWER of petunia.

Supplemental Figure 1. Phenotypes of 35S:ALF and 35S:LFY in petunia and Arabidopsis.

(A) Wild type (WT) and 35S:ALF petunia.

(B) Detail of wild type and 35S:ALF petunia inflorescence.

(C) Wild type and 35S:LFY petunia.

(D, E) Inflorescence of alfw2167/w2167

mutant (D) and a partial complementant containing 35S:LFY (E). Note

that LFY restores the determinacy of floral meristems and the development of floral organs.

(F) RT-PCR analysis of ALF and LFY mRNA expressed in leaves from 35S:ALF and 35S:LFY petunia

transformants and untransformed controls (WT).

(G) Immuno-detection of ALF and LFY protein in leaves from 35S:ALF, and 35S:LFY petunia

transformants and control plants (WT). The arrow indicates ALF and LFY protein; the asterisks indicate

unrelated proteins that react a-specifically with the LFY antibody.

(H) Wild type Arabidopsis (Columbia).

(I) 35S:ALF Arabidopsis. Note that the co-florescences are solitary flowers (arrow).

(J) Detail of wild type and 35S:ALF Arabidopsis showing the transformation of co-florescences into

solitary flowers (arrow).

(K) Aberrant flower terminating the primary 35S:ALF Arabidopsis inflorescence.

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Supplemental Figure 2. cDNA-AFLP analysis of the alf, dot and alf dot inflorescences.

(A) Phenotype of alf, dot and alf dot mutants.

(B) cDNA-AFLP analysis of polyA+ RNA isolated from inflorescence apices (top 3-4 mm) of alf, dot,

evergreen (evg) single, double and triple mutants. evg mutants define a new locus and have a floral

meristem identity phenotype that is very similar to alf and dot (unpublished data from Rebocho, 2007). The

gel images show products amplified from 14 RNA samples using 6 different primer combinations in which

two (constant) adapter primers (Eco and Mse) were extended with different dinucleotides at their 3’ end. In

total we used 224 different primer combinations to display ~ 6000 mRNA fragments. Except for the down-

regulation of DOT mRNA in all genotypes containing evg (Rebocho, 2007), we detected no other

reproducible differences between the analyzed genotypes.

Reference:

Rebocho, A. B. (2007). Genetic control of diverse inflorescence architectures. PhD thesis, Vrije

Universiteit, Amsterdam, The Netherlands.

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Figure 3. Sequence alterations in dot alleles.

(A) Map of the DOT gene. Yellow triangles indicate dTPH1 insertions and the green triangle a dTPH7

insertion. All transposon insertion alleles exhibit identical phenotypes.

(B) Sequence of the DOT cDNA. Because DOT lacks introns, the genomic sequence is identical to the

cDNA sequence shown, except for the polyA tail. The 8-bp target site duplications (TSDs) that flank the

transposon insertions in mutant alleles are indicated in blue. The sequence of the F-box is indicated in red.

(C) Sequence of the weak dotH2082

allele, the progenitor dotA2232

and DOT+. Nucleotides shown in blue

represent the TSD. Blue dots in dotH2082

denote TSD nucleotides that were deleted upon dTPH1 excision

and red lettering TSD nucleotides that were duplicated in an inverted orientation.

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Supplemental Figure 4. Alignment of Petunia DOT and homologous F-box proteins from Solanum

lycopersicum (Sl DOT), Antirrhinum majus (FIM), Arabidopsis thaliana (UFO) and Pisum sativum

(STP).

Black shading indicates sequence identity and grey shading sequence similarity. For a text file of this

alignment see Supplemental Data Set 1 on line.

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Supplemental Figure 5. Analysis of Sl DOT and Sp DOT in an-1 and an-3 mutants.

(A) Alignment of DOT, Sl DOT and Sp DOT cDNA. Start and stop codons are indicated by underlining, as

well as the 39-bp deletion in S. pimpinellifolium an-3 mutants.

(B) DNA gel blot of BclI, HinDIII and HinDIII/BamHI digested DNA of four wild type (wt) and two an-1

S. lycopersicum plants hybridized with Sl DOT cDNA. Asterisks indicate bands that match with predicted

restriction fragments shown in (C).

(C) Map of Sl DOT showing the size (in bp) and position of the predicted genomic restriction fragments.

(D) PCR analysis of an S. pimpinellifolium family showing co-segregation of Sp DOT and AN. Plants with

a wild type or an-3 phenotype are indicated by + and – respectively.

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Supplemental Figure 6. 35S:DOT activates B and E class organ identity genes in green tissues.

(A) Phenotype of wild type (left) 35S:DOT leaves (right) in a genetic background specifying a strong

magenta flower color. Note that the leaf to petal transformation in these leaves is weaker than that seen in

Figure 3H.

(B) RT-PCR analysis of ALF, DOT, and floral organ identity gene mRNAs in wild type leaves and

inflorescences compared to 35S:DOT leaves. The strength of the phenotype of the 35S:DOT leaves that

were used was similar to that shown in (A). ACTIN mRNA was used as a constitutive control.

(C) Phenotype of a 35S:DOT ph def (greenpetals) mutant (left) and 35S:DOT petunia (right). The 35S:DOT

sepals contain patches of petal tissue (arrow), which are absent in 35S:DOT phdef sepals.

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Supplemental Figure 7. Phenotype of dot, 35S:DOT and 35S:DOT dot progeny.

(A) Wild type inflorescence.

(B) dot inflorescence.

(C) Older 35S:DOT plant. Note that the primary shoot terminated with a solitary flower. This breaks the

dormancy of meristems in lower leaf axils, which grow out and again terminate with a solitary flower. The

repetition of this process results in plants with decreased plant height bearing many branches each ending in

a solitary flower.

(D-G) Variable phenotype of 35S:DOT dot plants. All flower precociously and display a leaf phenotype.

Only a small fraction generate (solitary) flowers with petals and stamens (D), but most generate “leafy”

flowers (E-F), which lack petals and stamens and form green sepal-like organs surrounding a carpel (ca)

(G). Such leafy flowers may occur solitary (D-E) or in larger numbers along branches in a cymose (zigzag)

pattern (F).

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Supplemental Figure 8. Phenotype of 35S:UFO in petunia.

(A) Wild type (WT) and two 35S:UFO petunia transformants.

(B) Detail of 35S:UFO transformant showing that the cymose inflorescence is converted into a solitary

flower.

(C) Leaf of wild type (WT) and 35S:UFO petunia.

(D) Top view of 35S:UFO petunia flower. Note the patches of white petal tissue in sepals and bracts.

All plants shown are in a background (an2) specifying a white color of the petal limb.

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Supplemental Figure 9. Analysis of petunia SKP1 homologs (PSK1 to PSK4) isolated by yeast two-

hybrid screening with DOT bait.

(A) Neighbor joining 50% majority rule consensus tree of four petunia SKP1 homologs (PSK1-PSK4) and

SKP1 homologs from Arabidopsis (ASK1-ASK19), tomato (TC172459 and TC 171678), man

(EAW62274) and yeast (SKP1). SKP1 from yeast and its human homolog were used as outgroup. Number

indicate percentage bootstrap support (1000 replicates). For a text file of the alignments that were used to

calculate the tree see Supplemental Data Set 2 online

(B) Alignment of PSK1-4 with SKP1 homologs from Arabidopsis, man and yeast.

(C) Alignment of petunia, Arabidopsis, tomato, yeast and human SKP1 homologs use to build the tree

shown in (A).

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Supplemental Figure 10. Intracellular localization of PSK1, ALF and DOT.

(A) Confocal images of different agro-infected cell types that express GFP-PSK1, ALF-GFP or DOT-GFP.

(B) Confocal images of epidermal petal cells after co-infection with constructs expressing a GFP fusion

protein and a construct expressing an unlabelled partner protein. Scale bars equal 10 !m.

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Supplemental Figure 11. Immunodetection of LFY protein expressed in petunia seedlings.

(A) Immunoblot probed with anti-LFY of proteins extracted from wild type petunia seedlings (WT) or

petunia seedlings containing 35S:LFY and/or 35S:DOT that were treated with the proteasome inhibitor 50

!M of MG132 (+) or mock treatment (-) (16 hours).

(B) Immunoblot probed with anti-LFY of protein extracts from seedlings containing 35S:LFY, 35S:DOT

35S:LFY double transgenics with a growth-arrested phenotype that contained (35S:LFY + 35S:DOT) and a

mixture of siblings that contain either 35S:LFY or 35S:DOT or no transgene (35S:LFY or 35S:DOT).

The asterisk indicates a protein species that is specific for 35S:DOT and 35S:UFO seedlings, which may

represent modified version of LFY and/or endogenous ALF (±55 kD), or an unrelated petal specific protein

that cross-reacts with anti-LFY.

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Supplemental Tables.

Supplemental Table 1. PCR primers used for analysis of Sl FALSIFLORA, Sl DOT, and Sp DOT.

Gene- primer pair1 Primer sequences

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Sl FALSIFLORA-A

F – CCGACGGTGGAACGAATGTCC (#2957)

R – CCTCATCCTCTTCCCCTCTTC (#2958)

Sl DOT- B

F – CTGCCAAACATTCATATCTCTTTCC (#3282)

R – GTTCCAAGAAATTGGTTGTTGGTGTTGGGATAGG (#3395)

Sl DOT- C

F – CTGCCAAACATTCATATCTCTTTCC (#3282)

R – GGATCAAACAAGTAACCTTCAACAGAACAAACTCCTG (#3393)

Sl DOT-D

F – CTGCCAAACATTCATATCTCTTTCC (#3282)

R – CCACTTCAGCAAACTGCACATACAATTGTTGTG (#3400)

Sl DOT-E

F – CCAAATTCATCATCAGTTAATGG (#2951)

R – GGATCAAACAAGTAACCTTCAACAGAACAAACTCCTG (#3393)

Sl DOT- F

F – CCAAATTCATCATCAGTTAATGG (#2951)

R – CCACTTCAGCAAACTGCACATACAATTGTTGTG (#3400)

Sl DOT-G

F – GGAATGATCATCAACACTTGGATGGATAGTAGAATTGGAG (#3391)

R – GGATCAAACAAGTAACCTTCAACAGAACAAACTCCTG (#3393)

Sl DOT-H

F – CTTTGTTGGTATAGGCTTTCTTTTGCTTTAATCCCAC (#3397)

R – CCACTTCAGCAAACTGCACATACAATTGTTGTG (#3400)

Sp DOT-I

F –GCAGCAGTTGAAAAGAGTAAACTG (#4038)

R - CCTCCAACTCCAAATTCTCCAC (#3283)

1Primer pairs are indicated by capital letters (A-I) and correspond to numbering shown in Figure 2.

2For each primer forward or reverse orientation relative to the mRNA is indicated as F or R. Primer numbers are e

indicated in brackets. Sl, Solanum lycopersicum; Sp, Solanum pimpinellifolium.

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Supplemental Table 2. Primers used for RT-PCR analysis.

Gene /RNA Primer sequence1

Sl DOT

F – CTTTGTTGGTATAGGCTTTCTTTTGCTTTAATCCCAC (#3394)

R – CCACTTCAGCAAACTGCACATACAATTGTTGTG (#3394)

Sl FALSIFLORA

F – CCGACGGTGGAACGAATGTCC (#2957)

R – CCTCATCCTCTTCCCCTCTTC (#2958)

ACTIN

F - AGATCTGGCATCATACCTTCTACA (#2602)

R - CCMGCAGCTTCCATRCCAATCA (#2603)

DOT

F – CCGCTCGAGGGTACATGTTGAGGG (#1212)

R – CTACATACTATTTTCGGACCCA (#828)

ALF

F – GCTCTAGATGAACAATGCAGGGATTTC (#264)

R – CGGGATCCTTAGAATGACAACCTAA (#530)

GLO2

F – CCATAGAGAGCTCATGATATTG (#1103)

R – CGATAAGGTACACGAGTCTTG (#1104)

DEF2

F – CGATCTGAACTATGAGCAGTTG (#1880)

R – GCATGTCCAGAAACATTGCATG (#1879)

FBP2

F – GGTTGATGGAAGGAAGCACAC (#1456)

R – GGTTGATGGAAGGAAGCACAC (#1457)

FBP5

F - CAAGGTTCTTGAAAGAAAGCTGG (#1460)

R - CAAGCATTAAAGTGTCCAGAATG (#1461)

FBP9

F – GCTTGAGCACCAATTGGATTC (#1903)

R – GCCACAGCAAAAAAGGAAAATAC (#1904)

FBP23

F – GCTGGAAGAAAGTGTAGCAGG (#1464)

R – GGCAGGACTGTTCAAGTGTCG (#1465)

FBP6

F – CTCAGAAATAGAGCTCATGC (#1890)

R – CTGAAGGACGTTTCTGAAGATG (#1891)

FBP14

F – CTCAGAGATCTGAGGAACCTGG (#1881)

R – CAGTTGATCTTCGTTCCCGAG (#1882)

PFG

F – CAAACTCAATGGGAGCAGCAG (#1105)

R – CTTCTAGGGCCAGCAAATTCTG (#1106)

FZY

F – CCAGATCTGCTATGGGTTAATGGACCTA (#0902)

R – CCAGATCTCCAAGAAGGTACATTGCTTT (#0820

FBP26

F – CCAGATCTAAGCATGAAAGAGCTTCAAAATTTGG (#0504)

R- CGGGATCCGTTGTGTTTGTTTGATCACATGATA (#0505)

FBP13

F – GCCTCAACATGAGATTAAGC (#1896)

R – CATTGCATGAAGTGTCATCAC (#1897)

TER

F – GGAGTTGATCCCTACTCTTC (#1883)

R – CGTAGTACATGGCCAAATTAG (#1884)

At LFY

F – GGGAATTCTCCGGTACTCATCACGCTCTTGATG (#1512)

R – GGCTCGAGCTAGAAACGCAAGTCGTCGCC (#1487)

At UFO

F – ATGGATTCAACTGTGTTCATC (#1262)

R – CTCACTAACCCTCCTGATGATCCTG (#1400) 1For each primer forward or reverse orientation relative to the mRNA is indicated as F or R. Primer numbers are

indicated in brackets. 2

Petunia DEF and GLO were formerly known as and GREENPETALS and FBP1.

At, Arabidopsis thaliana; Sl, Solanum lycopersicum;all other sequences are from Petunia hybrida.