Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1. Genetic analysis of the NSt allele. The NSt allele arose in a niv^rec::Tam3 line with a high somatic reversion frequency. A flower of the progenitor line (HAM5) is presented in Supplemental Figure 1A, showing many somatic reversion sites due to high-frequency excision of Tam3 from the nivealocus. The flower from the plant in which the NSt allele was first detected is shown in Supplemental Figure 1B. Although heterozygous for NSt, very few sites or sectors were observed on flowers of this line compared to a sibling that did not carry the NSt allele (Supplemental Figure 1C). The NSt allele was made homozygous by selfing the line shown in Supplemental Figure 1B to produce line HAM3. This line (NSt/NS) was crossed to the progenitor line HAM5 (nst/nst) and high:medium:low spotting individuals segregated 1:2:1 in the F2 generation (Supplemental Figure 1D) showing that the NSt allele was semi-dominant to thenstallele. In NSt/NStindividuals grown at low temperature, sectors in which a much higher reversion frequency occurred, were occasionally observed (arrowed in Supplemental Figure 1E). This suggested that the NSt allele was unstable, and possibly caused by a transposable element insertion. To test for allelism withSt, anNSt/NSt homozygous line, HAM3, was crossed to St/St (line JI:558, (Carpenter et al., 1987); Supplemental Figure 1F). Individuals in the F1 generation were all identical phenotypically, having a low spotting frequency (less than 1 site per flower; Supplemental Figure 1F). In the F2 generation, high:medium:low spotting plants segregated in the ratio of 21:50:130, suggesting that NSt and St are different, unlinked loci (Supplemental Figure 1F).
• Supplemental Figure 2 - Supplemental Figure 2. Examination of the purity of the extracts of nuclear proteins. A: Examination using fluorescence microscopy. Nuclear and total cellular extracts were stained with DAPI. We detected DAPI-stained blue nuclei and auto-fluorescent red chlorophyll. Both the extracts contained nuclei, while chlorophyll was detected only in the total cellular extracts and not in the nuclear extracts. This result implied that the nuclear extracts were highly purified. Fluorescence images were captured using a fluorescence microscope (Olympus, Tokyo, Japan). Pseudocoloring of the images was performed using the software program IPLab (Scanalytics, Fairfax, VA). B: Dot immuno binding assay (DIBA). DIBA was conducted using an anti-histone H3 antibody (anti-dimethyl-histone H3 Lys4 rabbit antiserum: lot # 26335, Upstate, NY) to test the purity of the extracts of nuclear proteins based on comparison to the extracts of total cellular proteins. The protein concentration in the extracts was measured with a protein assay kit (BioRad, Hercules, CA). The dots within the same columns on the blot contain identical amounts of protein. An initial protein concentration of 100 ng/μl was used for a series of dilutions as indicated in the figure, and each dilution was blotted onto the membrane. The dots interacted with the anti-histone H3 antibody showing that the amount of histone H3 in the 200-fold dilution of the nuclear extracts corresponds to the amount in the 10-fold dilution of the total cellular protein extract. This result showed that the nuclear extracts were 20-fold enriched in histone H3 protein compared to the total protein sample. Taken together with our microscopic observations, we judged that a significant enrichment of nuclear proteins had been obtained for the nuclear extracts from the plants grown at both low and high temperatures. Nu: nuclear proteins, Total: total proteins. H: sample from high-temperature grown plants, L: sample from low-temperature grown plants.