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Supporting Information Legends Table S1. Localization of PTL protein following transient expression in onion epidermal cells. Table S2. Primers used in the study. Figure S1. Full alignment of PTL orthologs from Angiosperms. Sequences were selected based on tblastn searches using the N trihelix of PETAL LOSS as the query. The sequences are the closest matches to PTL, and in each case there are 23 amino acids between the second and third conserved aromatic amino acid (all other GT clade trihelix proteins have 22). Sequences are from Phytozome v9.1 (phytozome.net) for all species except Amborella trichpoda (amborella.org) and Musa acuminata (bananagenome.cirad.fr). Other species are Vitis vinifera, Populus trichopoda, Manihot esculenta, Ricinus communis, Linum usitasissimum, Glycine max, Phaseolus vulgaris, Medicago truncatula, Prunus persica, Malus domestica, Cucumis sativa, Eucalyptus grandis, Citrus sinensis, Citrus clementina, Gossypium raimondii, Theobroma cacao, Carica papaya, Arabidopsis thaliana, Mimulus guttatus, Solanum lycopersicum, and Solanum tuberosum. All representatives containing the N-terminal trihelix are included. In some cases sequences lacking some or all of the C terminal trihelix are present. The alignment was performed using ClustalW (with manual adjustment) in BioEdit v7.2.5 (mbio.ncsu.edu/BioEdit/page2.html). Amino acids are colour coded based on their chemical properties. Boxes enclose the five conserved domains: C-terminal, N trihelix, Central, C trihelix and C-terminal. The three conserved aromatic residues in each trihelix are shown above the alignment. Figure S2. Full alignment of GT2 clade members from Arabidopsis thaliana. Domains conserved in PTL are boxed, and the conserved tryptophans in the trihelix domains are indicated. Arrowhead indicates the diagnostic extra amino acid present in the second helix of the N-terminal trihelix of PTL and EDA31. Note that EDA31 lacks most of the C terminal trihelix, and that At5g47660 lacks the N-terminal trihelix. Figure S3. Full alignment of PTL and EDA31 orthologs from the Brassicaceae. Domains conserved in PTL are boxed, and the conserved tryptophans in the trihelix domains are indicated. (a). Orthologs of PTL were present in all Brassicaeae examined (Arabidopsis thaliana, A. lyrata, Capsella rubella, Thellungiella halophila, and Brassica rapa.) (b). The most closely related paralog of PTL, EDA31 was present only in Arabidopsis thaliana, A. lyrata, and Capsella rubella. Figure S4. PTL complementation of individual ptl-1 T1 plants following deletion of the C terminal region and addition of the VP16 activation domain. pPTL(1.3i):PTL was transformed into ptl-1 mutant plants. Up to 15 T1 transformants were selected and scored for PTL function by recording the mean number of second whorl organs (petals, petal mosaics and filamentous structures) per flower in the first 10 flowers on the main stem. The data for individual T1 plants are ranked from left to right in order of increasing means. Bars show standard errors of the means. Control data for transformants carrying unmodified PTL sequence and from the empty vector were collected in each transformation experiment. (a). Effect of deletion of C-terminal amino acids of PTL from codon 521 (PTLΔC2). (b). Effect of addition of VP16 activation sequences to PTLΔC2. (c). Effect of addition of VP16 to PTL. Figure S5. PTL complementation of individual ptl-1 T1 plants following site directed mutagenesis of the trihelix DNA binding domains. (a). Substitution of the conserved tryptophan and the following two acidic residues to alanine in the second helix of the N-terminal trihelix (WDE to AAA), or the C-terminal trihelix (WEE to AAA). (b). Substitution of the basic residues to alanine upstream of the conserved phenylalanine in the third helix of the N-terminal trihelix (KKCR to AACA). (See legend of Figure S4 for experimental details.) Figure S6. PTL complementation of individual ptl-1 T1 plants following substitution of the trihelix DNA-binding domains, or the full length PTL, with relatives from the GT2 clade. (a). Substitution of the N-terminal or C-terminal trihelix domains of PTL with EDA31 or GT2. (b). Substitution of full length PTL with EDA31, GT2, DF1, or At5g28300. (See legend of Figure S4 for experimental details.)