Supplemental Data

Files in this Data Supplement:

• Supplemental Table 1
• Supplemental Table 2
• Supplemental Methods
• Supplemental Figure 1 - Lesions and Transcript Accumulation in Mutant Lines. For each allele used in this study, RT-PCR was used to detect the presence of transcripts. The locations of the mutations and primers used for RT-PCR (connected by bidirectional arrows) are indicated for each gene. Arrows in T-DNA insertion rectangles indicate the likely locations of P_35Spromoter sequences. With the exception of clv3-2, clv3-2 cna-1, and clv3-2 cna-2 inflorescence apices, RNA was extracted from the aerial portions of 2-week-old seedlings and reverse transcribed using a poly-dT primer as previously described (Prigge and Wagner, 2001). After amplification, samples were electrophoresed along with 1 kb plus ladder (Invitrogen, Carlsbad, CA) on agarose gels and photographed. Primers for the β-ATPase gene were assayed for each RNA sample to control for quality and quantity of cDNA (Prigge and Wagner, 2001). (A) REV transcript was detected for the rev-6 allele, but levels may be reduced relative to REV+. Because the rev-6 allele contains a premature stop codon and displays a phenotype similar to that of several other alleles, it is a null or strongly hypomorphic allele (Otsuga et al., 2001). (B) The phb-11 allele contains a Ds insertion in the homeodomain-encoding region of the gene and is thus likely to be a null allele despite transcripts being detected downstream from the insertion site. Although the phb-13 allele appeared to express the 5? portion of the PHB gene, it is also likely a strong loss-of-function allele because the phenotypes of rev phb and of rev phb phv plants are similar using either the phb-11 and phb-13 alleles (Table 1). No transcripts could be detected spanning or downstream from the phb-13 T-DNA insertion. The phb-12 allele accumulates a reduced amount of full-length transcript and is a weak allele based upon genetic interactions in post-embryonic tissues. The residual expression of the phb-12 allele may be due to a Cauliflower Mosaic Virus 35S promoter sequences on the T-DNA directed towards the gene. This would require that RNA polymerase transcribe through Agrobacterium nos gene terminator sequences. (C) PHV transcripts were detected for the phv-11 allele upstream and downstream from the insertion site, but no transcripts were detected across the middle of the gene, spanning the insertion site. While we cannot rule out that this allele retains some PHV function, it is unlikely a weak allele because strong rev alleles contain lesions downstream of the site corresponding to the phv-11 insertion location (Otsuga et al., 2001). Also, we found no evidence to suggest phv-11 is dominant-negative. (D) The cna-2 allele contains a T-DNA insertion at the 3? end of the region encoding the leucine-zipper domain and thus is likely to be a complete loss-of-function allele. CNA transcripts were detected upstream and downstream from the insertion site in RNA extracted from cna-2 seedlings, but no 3? transcripts were detected from clv3-2 cna-2 inflorescence apices. No transcripts spanning the insertion site were detected. The cna-1 allele contains a point mutation that confers a dominant-negative phenotype (K. A. Green, M. J. Prigge, R. B. Katzman, and S. E. Clark, unpublished data). (E) athb8-11 deletes the entire gene, and no transcripts were detected. The athb8-12 allele is expressed downstream from the insertion site, but the transcript is less abundant than the wild-type ATHB8+ gene. As with phb-12, the transcripts from the athb8-12 allele may result from T-DNA-derived promoter sequences. Because the athb8-12 allele conferred similar phenotypes as athb8-11 in phb phv cna backgrounds (data not shown), it may also be a strong loss-of-function allele.
• Supplemental Figure 2 - Comparison of HD-Zip III Gene Expression Patterns. The expression patterns of each HD-Zip III gene in wild-type embryos and inflorescences were determined by in situ hybridization. The gene hybridized is indicated at the top of each column of panels. Gene expression is indicated by the indigo staining while the dark orange-brown tissue is the seed coat. SM, shoot meristem; C, cotyledon; IM, inflorescence meristem. (A) While strong signal was observed in early globular-stage embryos after hybridization with the REV and CNA probes, little or no staining was observed using probes to the other genes. (B) In late globular-stage embryos, REV expression continued to be detectable throughout all except the basal-most cells of the embryo proper while CNA expression was reduced. The PHB and PHV genes were upregulated in cells of the apical portion of late globular embryos. (C) Upon cotyledon initiation in heart-stage embryos, the REV, PHB, and PHV genes were expressed in the adaxial portion of the emerging cotyledons and in the presumptive shoot meristem. The REV and PHB genes were also expressed in provascular tissues. The CNA gene was expressed in a diffuse pattern, enriched in the apical end and in provascular tissues. The first detectible expression of ATHB8 was restricted to a small patch of provascular cells in heart-stage embryos. (D) and (E) The expression domains seen in torpedo-stage and walking-stick-stage embryos were similar to those of heart-stage embryos except that the PHV gene was no longer expressed in the shoot meristem and that the CNA gene expression pattern resolved to one similar to those of REV and PHB. (F) The REV and PHB genes were expressed in floral meristems and at presumptive floral meristem initiation sites in inflorescence meristems while PHV appeared at later stages in floral meristem development. The CNA gene was highly expressed throughout the inflorescence, particularly in floral meristems and developing vasculature. ATHB8 gene expression was restricted to vascular tissues. (G) During flower development, REV, PHB, and PHV were each detected in adaxial tissues of floral organs, but only REV and PHB expression persisted in the floral meristem. (H) Expression of each of the HD-Zip III genes was detected in vascular tissue in the inflorescence stem, although PHV is only weakly expressed in pith cells.