Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Changes in the expression of At3g21150 and At3g21330 genes in response to 1h Rc light treatment. Representative Northern blots (A and B) showing early (within 1h) light-regulated expression of genes identified in a more recent study (Tepperman et al., 2006), as induced, At3g21150 (A and C) and repressed, At3g21330 (B and D) by 1 h Rc. Wild type (Col), 4-day old seedlings were either grown in darkness (0D) or were treated with 1h Rc (R1) (7 μmol m^-2 s^-1). Average values of three biological replicates are plotted (C and D). Values were normalized for loading using 18S rRNA. Transcript levels were calculated relative to values of 0D set at 1. Error bars represent ranges.
• Supplemental Figure 10 - pil1 mutation does not alter light-induced expression pattern of central circadian oscillator genes CCA1, LHY, or TOC1. Analysis of (A) CCA1, (B) LHY, (C) TOC1 transcript levels in the pil1 mutant and wild type (WT) seedlings grown in response to Rc. Seedlings were grown for 96 h in darkness and then transferred to Rc (∼8 μmol m^-2 s^-1) for 24h. Tissue was collected at the indicated times, including a dark control at 24 h (24D). Average values of two biological replicates are plotted (normalized for loading using 18S rRNA), and error bars represent ranges. Representative northern blots are shown.
• Supplemental Figure 2 - Assessment of the effect of T-DNA insertion on gene expression for the homozygous mutant lines analyzed in this study. Total RNA isolated from Col (WT) and the indicated T-DNA mutant-lines was subjected to RT-PCR (A and B) or Northern blot (C) analysis. In order to detect possible partial transcripts, gene-specific primers targeting specific nucleotide sequences closely downstream of the start codons were used for the analysis. For each gene, RT-PCR was performed using seedlings grown under inductive conditions, 1h Rc (R1) (7 μmol m^-2 s^-1) for genes induced in light (A) and dark grown seedlings for genes repressed in light (B and C). Transcripts detected in 3 mutant-lines were either greatly reduced in levels or were altered in size (*). Actin 2 (AT3G18780) and 18S rRNA were used as controls for RT-PCR and Northern blot analysis, respectively. Genes are listed in numerical order in each panel with gene numbers corresponding to Table 1 indicated in parenthesis. Representative (of triplicate) ethidium bromide-stained gels and northern blots are shown.
• Supplemental Figure 3 - Statistical significance of the differences in (A) hypocotyl length and (B) cotyledon area, between 4-day old wild type and mutant seedlings grown in Rc. The wild type hypocotyl length and cotyledon area were used (set at unity) to determine the relative differences displayed by the mutant seedlings. Corrected probability (p) values indicate the statistical significance of the differences in mean hypocotyl lengths and cotyledon areas between the wild type and 32 mutant lines, as determined by t-test analysis of data from 30 seedlings for each genotype. Values below p=0.05 (horizontal dashed lines) were considered as being statistically significant for differences in hypocotyl length and cotyledon area between wild type and mutant seedlings. Values in the color-shaded areas represent statistically insignificant differences from the wild type. The numbers correspond to the genes listed in Table 1. Data from phyB-9 mutant seedlings are included to provide a frame of reference for the seedling phenotypes observed in Rc.
• Supplemental Figure 4 - Statistical significance of the differences in hypocotyl length between 4-day old wild type and mutant seedlings grown (A) in FRc light, or (B) in darkness. The wild type hypocotyl length was used (set at unity) to determine the relative differences displayed by the mutant seedlings. Corrected probability (p) values indicate the statistical significance of the differences in mean hypocotyl lengths between the wild type and 32 mutant lines, as determined by t-test analysis of data from 30 seedlings for each genotype. Values below p=0.05 were considered as being statistically significant for differences in hypocotyl length between wild type and mutant seedlings. The values in the color-shaded area represent statistically insignificant differences from the wild type. The numbers correspond to the genes listed in Table 1. Data from phyA-211 mutant seedlings are included in (A),to provide a frame of reference for the seedling phenotypes observed in FRc.
• Supplemental Figure 5 - Northern blot analysis of pil1-mutant allele expression. (A) Schematic representation of the PIL1 gene. T-DNA insertion sites for two independent lines, pil1-1 (Salter, 2003) and pil1-2 (new allele isolated from SIGnAL collection) are indicated (arrows). The numbers indicate nucleotide position of the insertion within the PIL1 gene. In addition, a fast neutron allele that deletes the whole PIL1 gene, pil1-3 (not indicated) was isolated. Homozygous lines were isolated and wild-type (WT) siblings were used as controls. (B) Northern blot analysis of pil1-mutant alleles. For each PIL1 allele, RNA was isolated from 4d old etiolated seedlings (mutant and corresponding wild-type siblings) and a PIL1 specific probe was used to determine mRNA levels. For each allele, wild-type siblings were used as controls. No mRNA was detected in any of the pil1 alleles, suggesting that they are likely null alleles.
• Supplemental Figure 6 - pil1-mutant seedlings are hyposensitive to Rc. (A) Visible phenotypes of pil1 mutant seedlings (pil1-3 used as representative allele) grown in Rc (two different fluence rates are shown) or in darkness for 4 days. The pictures show the effect of pil1 mutation on the hypocotyl length (left panel) and cotyledon area (right panel). (B) Rc and FRc fluence rate response curves for pil1-3. The other two alleles display similar response to these treatments (data not shown). (C) Quantification of cotyledon area for pil1-2 and pil1-3 mutant seedlings. For the quantification graph, the area of 80 cotyledons was measured at different fluence rates of Rc with NIH image software and the corresponding wild-type siblings were used for comparison. Standard errors were determined.
• Supplemental Figure 7 - Effect of pil1 mutation on petiole elongation and flowering time. (A) Elongated petiole phenotype of pil1-2 mutant plants compared with Wt (Col-0) plants when grown under dim white light (2.5 μmol m^-2 s^-1) for 3 or 4 weeks. Quantification of the response is shown below the pictures. Measurements were performed using NIH Image for 30 plants, normalized to Col values, and standard errors were determined. (B) Flowering time was estimated for 40 plants of each genotype grown in three different photoperiods (Long days, LD; Short Days, SD; and 12h light / 12h dark cycles) and recorded as number of leaves at the time of bolting. The graph shows WT in blue and pil1-2 in red. The data are shown as (+/- standard error).
• Supplemental Figure 8 - PHYB protein levels are not affected in pil1 mutants. Immunoblot of total protein extracted from seedlings grown for 3 days in Rc and probed with phyB monoclonal antibodies.
• Supplemental Figure 9 - PIL1 is nuclear localized. (A) Bright field image showing PIL1:GUS fusion protein localized to the nucleus in leek epidermal cells. (B) DAPI staining of nuclei of cells shown in A. (C) Bright field image showing GUS control protein distributed throughout the cell. (D) DAPI staining of nuclei of cells shown in C.