Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - HPLC analysis of methanol-extractable anthocyanins and phenylopropanoids in petal limbs of PH4 and ph4-V2153 flowers in the R27 genetic background. (A) Examples of HPLC profiles of methanolic petal limb extracts from wild type (PH4) and mutant (ph4) flower buds (stage 4) or open flowers (stage 6). The numbers of distinct peaks as they were used for quantification are indicated in red in the top panels. Peak 7 in the 220nm and 280 nm profiles actually consists of two distinct peaks (indicated with the horizontal red line) that were difficult to quantify separately. Purified cyanidin 3-glucoside co-migrated with peak 5 of the 530 nm profile. (B) Quantification of peaks in 220 nm, 280 nm and 530 nm profiles. Peaks in different profiles were numbered as indicated in (A), their abundance was measured from the peak area and expressed as % of total absorption at the same wavelength. The graphs show average % of absorption (bars) ± standard deviation (error bars) (n≥5).
• Supplemental Figure 2 - Alignment of MYB domains of PH4 and other R2R3 MYB proteins. Identical amino acids are shown in black and similar amino acids in grey. A phylogenetic tree based on this alignment is shown in Figure 4A.
• Supplemental Figure 3 - Co-Immunoprecipitation of An1 and PH4 synthesized in vitro by coupled transcription and translation. AN1 protein was synthesized either in a ³⁵S-labeled form (indicated with an asterisk) or unlabelled. AN1 and PH4 proteins, either alone or after mixing (AN1+PH4), were precipitated with anti-AN1 antibodies (x Ab) and the precipitated radio-labeled proteins were analyzed by gel-electrophoresis. After treatment of labeled PH4 with anti AN1 serum a small fraction of the input is pulled down. This may be due to non specific binding of PH4 to the anti-AN1 serum or to the Sepharose beads, or, because it could not be eliminated with different washing conditions, co-immunoprecipitation with an AN1-like protein present in the wheat germ translation extract. However, when unlabelled AN1 was added prior to immunoprecipitation, PH4 was pulled down efficiently (> 90% of input).
• Supplemental Figure 4 - Alignment of the protein encoded by a partial CAC16.5 cDNA of petunia to cysteine/thiol proteases of other species. Cystein protease sequences were from Anthurium andreanum (AaCYSPROT ; Genbank accession number AAP41846.1), Gossypium hirsutum (GhCYSPROT; Accession AAO18731.1), Matricaria chamomilla (McTHIOLPROT; Accession AAD54424.1), Daucus carota (DcCYSPROT, Accession BAD29960.1), Zingiber officinale (ZoCYSPROT, Accession AAW34136.1) and maize (ZmCYSPROT: BAA88898.1).
• Supplemental Figure 5 - Phenotype of 35S:AN1 35S:PH4 double transgenic plants (A) Phenotype of 35S:AN1 35S:PH4 plant #2 showing purple pigmentation in leaves. (B) Mean pH of leaf extracts from 35S:AN1 35S:PH4 double transgenics (plants 2-4) ± standard deviation (n=4). All three plants express 35S:PH4 to similar levels. Plant #2 expresses at 35S:AN1 at high level, whereas 35S:AN1 expression in plants 3 and 4 is reduced (see Figure 7D).
• Supplemental Table 1 - Complementation analysis of ph2, ph3 and ph4 mutants.