DEV02599 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. A novel FAS1 mutant allele. (A) Seeds of the fas1-4 mutant were obtained from the Nottingham Arabidopsis Stock Centre (seed stock ID N828822). The T-DNA insertion is located within the sixth intron of the FAS1 gene, 2110 bp after the ATG. LB, left border of the T-DNA. (B) The amount of full-length FAS1 transcript is reduced in fas1-4 plants, as shown by RT-PCR. Shown are the results of independent cDNA preparations from wild-type (Col) and fas1-4 flowers, and from 4-week-old wild-type and fas1-4 seedlings. n.t., no template control. (C) 8-day-old representative light-grown seedlings of wild type (Col) and fas1-4. (D) 3-week-old wild-type (Col) and fas1-4 plants. Note the narrower and serrated rosette leaves in fas1-4. fas1-4 mutants exhibit a strong phenotype similar to fas1 and fas2 null alleles, including fasciation, the production of more and thinner secondary shoots, serrated rosette and cauline leaves, and a slightly altered floral morphology. The plants are viable and fertile, and the overall morphology is highly similar to fas2-4 and fas1-1 (accession En).

• Supplemental Figure 2 -

  Fig. S2. A novel FAS2 null allele. (A) Seeds of the fas2-4 mutant were obtained from the Nottingham Arabidopsis Stock Centre (seed stock ID N533228). The T-DNA insertion is located within the fourth intron of the FAS2 gene, 796 bp after the ATG. LB, left border of the T-DNA. (B) No full-length FAS2 transcripts could be detected in fas2-4 plants by RT-PCR. Shown are the results of two independent cDNA preparations, each from 2-week-old wild-type (Col) and fas2-4 seedlings. n.t., no template control. (C) 9-day-old representative light-grown seedlings of wild type (Col) and fas2-4. (D) 3-week-old wild-type (Col) and fas2-4 plants. Note that the rosette leaves are serrated and narrower in fas2-4 than in wild-type plants. fas2-4 plants are fully viable; however, they show fasciation and altered floral morphology similar to fas2-1 mutants (accession Ler). Furthermore, they also have more and thinner secondary branches, as well as serrated rosette and cauline leaves. Thus, the leaf shape of fas2-4 is more similar to that of fas1-1 and fas1-4 than to that of fas2-1.

• Supplemental Figure 3 -

  Fig. S3. Transgenic MSI1 antisense (msi1-as) plants. (A) Protein was extracted from leaves of Col wild-type control plants (WT) and three transgenic lines expressing a fragment of the MSI1 cDNA in antisense orientation. Protein (10 μg per sample) was subjected to immunoblotting with affinity-purified, anti-MSI1-specific antiserum (upper panel). Ponceau Red staining of the blot is shown as a loading control (lower panel). (B) RNA was isolated from leaves of Col wild-type (WT) and msi1-as plants before bolting. After treatment with DNaseI, RNA was subjected to reverse transcription in the presence (+) or absence (−) of reverse transcriptase using oligo(dT) primers. Semi-quantitative PCR with different cDNA-specific primers was performed on aliquots of the produced cDNA. GAPDH was used as a control. (C) Rosettes of 3-week-old wild-type and msi1-as plants. (D) Ectopic expression of floral homeotic genes in leaves of msi1-cs but not of msi1-as plants. RNA was isolated from floral buds and open flowers of wild-type plants, and leaves of wild-type, msi1-cs and msi1-as plants before bolting. After treatment with DNaseI, RNA was subjected to reverse transcription in the presence (+) or absence (−) of reverse transcriptase using oligo(dT) primers. PCR with different cDNA-specific primers was performed on aliquots of the produced cDNA (10 ng total RNA from flowers and 50 ng total RNA from leaves). (E) The average rosette diameter of 3-week-old plants was determined for CAF-1 mutants and their respective wild types. Shown are the means±s.e.m. (n&γτ;7). (F) The growth rate of the primary shoot was determined for CAF-1 mutant plants and their respective wild types during the linear growth phase (usually 5-15 days after bolting). Shown are the means±s.e.m. of the growth rates expressed relative to the value of its corresponding wild-type (n&γτ;10).

• Supplemental Figure 4 -

  Fig. S4. Expression of CAF-1 subunit genes is correlated with cell proliferation activity. (A) Expression of genes encoding Arabidopsis CAF-1 subunits during the cell cycle. Suspension cells were synchronised using aphidicolin. Gray areas denote periods of DNA synthesis activity. (B) Expression of genes encoding Arabidopsis CAF-1 subunits during the subculture regime of the suspension culture. Gray areas denote periods of logarithmic growth of the culture. (C) Expression of genes encoding yeast CAF-1 subunits during the cell cycle. Synchronization of yeast was established using an α-factor-dependent procedure. Gray areas denote periods of DNA synthesis activity. Data were taken from the microarray data sets of (A,B) Menges et al. (Menges et al., 2003) and (C) Spellman et al. (Spellman et al., 1998).

  Menges, M., Hennig, L., Gruissem, W. and Murray, J. A. (2003). Genome-wide gene expression in an Arabidopsis cell suspension. Plant Mol. Biol. 53, 423-442.

  Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., Brown, P. O., Botstein, D. and Futcher, B. (1998). Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell 9, 3273-3297.