DEV02836 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 (Adobe Acrobat) -

  Fig. S1. Alignment of subgroup 9 R2R3 MYB proteins. The regions above the black bar were used to construct the phylogenetic tree shown in Fig. 1.

• Supplemental Figure 2 (Adobe Acrobat) -

  Fig. S2. Phylogeny of subgroup 9 R2R3 MYB proteins. (A). Phylogenetic tree of all subgroup 9 R2R3 MYB proteins. (B) Protein sequences were aligned using the ClustalW (version 1.83) program (Thompson et al., 1994). Phylogenetic analysis was performed with Phylip programs (version 3.63) using only the MYB domain and the adjacent MYB subgroup 9 motif (Kranz et al., 1998). A distance matrix method employing the Jones-Taylor-Thornton model was used to compare the sequences and a tree was built using the neighbour-joining clustering method (Saitou and Nei, 1987). 1000 bootstrapped data sets were used to indicate the confidence of each tree clade. Only values greater than 500 are shown. Accessions additional to those used for Fig. 1: Ac_EST682340 (Allium cepa); Aq_TC15922 (Aquilegia); Aq_TC8966; Bn_TC8918 (Brassicca napus); Dspp.MYB1-AF485892 (GenBank); Dendrobium sp. XMW-2002-1; GhMYB25-AF336283 (GenBank) (Gossypium hirsutum); Gh_TC69374; Gh_Ea0029G04f; Gh_TC71908; Hv_SMEc0019F08f (Hordeum vulgare); Le_TC189627 (Lycopersicon esculentum/Solanum lycopersicum); Le_TC187610; Pp_BJ185129 (GenBank) (Physcomitrella patens); Pinus_TC63515 (Pinus); Pop_TC62999 (Poplar); Pop_TC47690; Ptr17g259196−protein ID: 259196 (Popular trichocarpa); Ptr08g421128−protein ID: 421128; Ptr02g799038−protein ID: 799038; Ptr10g226354−protein ID: 226354; Ptr08g720191−protein ID: 720191; Ptr14g731056−protein ID: 731056; Os02g36890 (Oryza sativa); Os08g33660; Os04g38740; Os04g45060; Os02g42870; SdMYBML1 (Solanum dulcamara); SdMYBML2; Ta_BE490032 (GenBank)(Triticum aestivum); Ta_BE497135 (GenBank); Vv_I137F11_604650 (Vitis vinifera); Zm_TC364156 (Zea mays); Zm_TC337113; Zm_TC333047. Accessions labelled TC# were obtained from the TIGR Plant Gene Indices (http://compbio.dfci.harvard.edu/tgi/plant.html); those from GenBank are indicated; rice locus identifiers or new sequences from the Populus trichocarpa genome sequencing project (http://genome.jgi-psf.org/Poptr1_1/). Sequences from Solanum dulcamara are unpublished (C.M., unpublished).

• Supplemental Figure 3 (Adobe Acrobat) -

  Fig. S3. Southern blot analysis of Petunia DNA. Petunia genomic DNA was digested with BamHI and EcoRI, separated by electrophoresis through a 0.8% agarose gel and blotted onto nitrocellulose. The image on the left shows one half of the filter probed with a radioactive fragment of DNA from the 3′ end of the MIXTA cDNA, washed at low stringency. The right-hand image shows the duplicate half of the filter probed with the 3′ end of the PhMYB1 cDNA and washed at low stringency. A single gene fragment homologous to PhMYB1 (arrowed) was detected with the PhMYB1 probe. This band was weakly detected by the MIXTA probe.

• Supplemental Figure 4 (Adobe Acrobat) -

  Fig. S4. SEMs of the adaxial surface of 2x35S::PhMYB1-transformed tobacco. The left-hand image shows the adaxial surface of an inflorescence leaf; the right-hand image is the adaxial surface of a vegetative leaf. Ectopic expression of AmMYBML2 or AtMYB16 gave the same phenotype (not shown). Only a few epidermal cells produce outgrowths; often, these protrusions are observed in cells overlying the veins (left-hand image). Scale bars: 100 μm.

• Supplemental Figure 5 -

  Fig. S5. Comparison of the effects of expression of MIXTA, AmMYBML1, AmMYBML2 and PhMYB1 (under the control of the CaMV 35S promoter) on the shape of carpel epidermal cells of tobacco. SEMs show that MIXTA promotes the formation of multicellular trichomes on carpels and that AmMYBML1 promotes the formation of multicellular trichomes and conical cells, whereas AmMYBML2 and PhMYB1 promote the formation of conical cells only.

• Supplemental Figure 6 -

  Fig. S6. Freeze-fracture SEM through mixta mutant and wild-type revertant sector of epidermal cells from the petal of the unstable mixta mutant line of A. majus. mixta mutant cells are completely flat in contrast to those that lack PhMYB1 activity (which are shallow cones) in the phmyb1 unstable mutant of Petunia (see Fig. 7D).