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Fig. 7. RNA gel blot of RNA extracts of pif3-3 and poc1 mutant seedlings and their respective wild types (Col-0 and Ws) grown for 4 days in continuous red light (Rc) (Upper) or far-red light (FRc) (Lower) probed with a full-length PIF3 cDNA probe.

Fig. 8. pif3 mutant deetiolation in prolonged continuous red (Rc) and far red (FRc) light. (A) Rc fluence-rate response curves for hypocotyl length in WT, WT-s, pif3-1, pif3-2, and pif3-3 grown for 4 days in Rc. (B) Cotyledon area of wild-type and pif3 seedlings grown under Rc (5 and 8 mmol·m^-2·s^-1 for WT-s and pif3-3, and 8 mmol·m^-2·s^-1 for WT and pif3-2) for 4 days. (C) pif3 mutants respond normally to FRc. FRc fluence rate-response curves for hypocotyl length in WT, WT-s, pif3-1, pif3-2, and pif3-3 grown for 4 days in FRc from germination onwards.

Fig. 9. Phytochrome B (phyB) levels in pif3 mutants. (A) Immunoblot of protein extracts of wild-type, phyB-9, and pif3 mutant seedlings grown in the dark for 4 days probed with monoclonal antibodies B1 and B7 specific for phyB (Upper). The Coomassie blue-stained membrane after hybridization is shown (Lower). (B) Immunoblot of protein extracts of wild-type, phyB-9, and pif3 mutant seedlings grown in continuous red light (Rc) (10 mmol·m^-2·s^-1) for 4 days probed with monoclonal antibodies B1 and B7 specific for phyB (Upper). The Coomassie blue-stained membrane after hybridization is shown (Lower). (C) Immunoblot of a dilution series of protein extracts of wild-type Col-0 and pif3-3 mutant seedlings grown in Rc (10 mmol·m^-2·s^-1) for 4 days probed with monoclonal antibodies B1 and B7 specific for phyB (Upper). The Coomassie blue-stained membrane after hybridization is shown (Lower). (D) RNA gel blot of RNA extracts from Col-0, Ws, and pif3-3 and poc1 mutant seedlings grown in the dark or Rc (10 mmol·m^-2·s^-1) for 4 days probed with a specific PHYB cDNA probe. As a control, the blot was reprobed to detect 18S RNA. (E) Immunoblot of protein extracts of wild-type Col-0 and pif3-3 mutant seedlings grown in the dark for 4 days, transferred to Rc (10 mmol·m^-2·s^-1) for 0, 0.5, 1, 3, and 6 h, and probed with monoclonal antibodies B1 and B7 specific for phyB (Upper). The Coomassie blue-stained membrane after hybridization is shown (Lower). Protein extraction and Western blot analysis for detection of phyB levels in pif3 mutants was done following the procedure described by Monte et al. (1).

1. Monte, E., Alonso, J. M., Ecker, J. R., Zhang, Y., Li, X., Young, J., Austin-Phillips, S. & Quail, P. H. (2003) Plant Cell 15, 1962-1980.

Fig. 10. Delayed chlorophyll accumulation in pif3 mutants is not allele specific. Both pif3-2 and pif3-3 alleles display delayed chlorophyll accumulation upon transfer to continuous red light (Rc). Wild-type and mutant seedlings were grown for 4 days in darkness and then transferred to Rc (10 m mol·m^-2·s^-1) for 0, 6, 12, and 24 h.

Fig. 11. The difference in chlorophyll accumulation rate per seedling between wild-type and pif3 in Fig. 2B is not due to differences in cotyledon expansion rate. Cotyledon area of wild-type and pif3-3 mutant seedlings grown in the dark for 4 days and transferred to Rc (10 m mol·m^-2·s^-1) for 0, 6, 12, and 24 h.

Fig. 12. PIF3 functions in early continuous red light (Rc)-repressed gene expression. (A) Repressed genes arrayed in rank order of relative responsiveness to 1 h of Rc in wild type (WT) compared to pif3-3 mutant seedlings. The curves depict the distribution of the 204 genes defined statistically as significantly repressed by 1 h of Rc in WT, and as significantly dependent on PIF3 for that Rc responsiveness, arrayed in order of descending mean fold repression ratio (MFRR) for WT/pif3 [MFRR(WT/pif3)] (blue), and the reciprocal for pif3/WT [MFRR(pif3/WT)] (red). Vertical dashed lines divide the array into bins (A-F) according to MFRR value. (Inset) Histogram showing total number of genes in each bin. (B) Genes in each bin arrayed by fold-repression (D1/R1) of expression imposed by 1 h Rc (R1) relative to the level in unirradiated, dark-control seedlings (D1), in WT seedlings for bins A-D, and in pif3 seedlings for bins E and F. Curves depict distribution of fold-repression values for WT (blue) and pif3 (red) seedlings. Note that scale varies between panels. Horizontal dashed line indicates 2-fold repression in each bin. Value above bin indicates range of MFRR(WT/pif3) values for Bins A-D, and MFRR(pif3/WT) values for Bins E and F. Numbers in parentheses are the numbers of genes in each bin. (C) Distribution of genes repressed 2-fold or more by 1-h Rc among functional categories. Both the number and percent of annotated genes in each category within each bin are tabulated. TX, transcription; P/C, photosynthesis/chloroplast; CM, cellular metabolism; S, signaling; TR, transport; G/D, growth/development; H, hormones; S/D, stress/defense.

Fig. 13. PIF3 is not necessary for free-running circadian oscillations in LHY expression in continuous light. Wild type (WT) and pif3-3 seedlings were entrained under 12 h WL/12 dark diurnal conditions for 5 days and then transferred to continuous WLc. RNA was extracted at the times shown and LHY expression level was determined by Northern blot analysis as described by Kaczorowski and Quail (1). Average of three independent replicates is shown, scaled relative to the WT 21 h average value. Error bars represent standard error. Top bar shows subjective night in gray shading. One of the 3 replicate RNA blots is shown.

1. Kaczorowski, K. A. & Quail, P. H. (2003) Plant Cell 15, 2654-2665.

Fig. 14. Rapid light-induced disappearance of histochemically detectable, nuclear-localized GUS:PIF3 protein. Transgenic Arabidopsis seedlings expressing GUS:PIF3 fusion protein driven by the 35S cauliflower mosaic virus promoter were grown in the dark for 4 days and then either exposed to continuous red light (Rc) for 1 h (1 h Rc) or retained in darkness (Dark). Seedlings were then stained for GUS and DNA (4',6-diamidino-2-phenylindole) and examined microscopically. Seedlings were vacuum infiltrated for 10 min under green safe light in GUS staining solution [100 mM sodium phosphate, pH 7.0/5 mM ferricyanide/5 mM ferrocyanide/1% triton X-100/1 mM EDTA/2 mg/ml 5-bromo-4chloro-3indolyl b-D-glucuronide (Gold BioTechnology, St. Louis)], followed by a 48-72 h incubation at 37°C in darkness. Seedlings were then moved to WL, de-stained at room temperature in an ascending concentration gradient of ethanol (30%–50%-70%, 20min each), and kept in 70% ethanol. DAPI staining and imaging was performed as previously described (1).The presence of GUS:PIF3 is apparent in the nuclei of dark-control seedlings, but has dropped below detectable levels within 1 h of Rc.

1. Ni, M., Tepperman, J. M. & Quail, P. H. (1998) Cell 95, 657-667.

Fig. 15. GUS protein is intrinsically stable in continuous red light (Rc). GUS activity was measured in 4-day, dark-grown Arabidopsis seedlings transferred to Rc for the indicated periods. A transgenic line expressing GUS protein alone driven by the constitutive 35S cauliflower mosaic virus promoter (35S::GUS) is compared with three independent lines expressing a GUS:PIF3 fusion protein (GP15.1; GP15.3; GP15.5) also driven by the 35S promoter (35S::GUS:PIF3).