Article Figures & Data
Figures
- Figure 1.
PIL5 Is a New Member of the Nuclear Localized Phytochrome-Interacting bHLH Protein Family.
(A) In vitro binding assay between phytochromes (PhyA and PhyB) and PIL5. PhyA and PhyB were preirradiated with red (20 μmol·m−2·s−1) or far-red (3.2 μmol·m−2·s−1) light for 15 min before incubation with PIL5 at 4°C in the dark. After incubation, glutathione sepharose bead-bound proteins were pelleted and analyzed by protein gel blotting using anti-PhyA and PhyB antibodies. Coomassie blue–stained PIL5 was used as the loading control.
(B) Positive and negative controls of the in vitro binding assay. GST-tagged PIF3 (positive control) interacted with both PhyA and PhyB, whereas GST alone (negative control) did not.
(C) PIL5 is localized in the nucleus. A GUS-fused PIL5 gene (PIL5-GUS) was introduced into onion epidermal cells by particle bombardment. The cells were incubated for 16 h either in the dark (D) or under white light (L) and localization of PIL5-GUS was visualized by GUS assay. Nuclei of onion cells were visualized by DAPI staining.
- Figure 2.
PIL5 Is a Negative Component of Phy-Mediated Inhibition of Hypocotyl Elongation.
(A) Genomic structure of PIL5 and a T-DNA–insertion site (pil5-1, salk_072677). No expression of PIL5 was detected in the pil5-1 mutant by RT-PCR. Expression of UBQ was used as a control.
(B) Cosegregation analysis. An ability of pil5-1 mutant seed to germinate after far-red treatment was used for the analysis. Among 300 F2 seeds from a cross between pil5-1 and Col-0, 76 seeds were germinated after far-red light treatment (0.95 mmol·m−2). Genomic DNAs from these germinated seedlings (pool), Col-0, and pil5-1 were used to amplify a PIL5 fragment (PIL5) and PIL5-T-DNA hybrid fragment (PIL5-T-DNA). All germinated seedlings from this F2 population were homozygous pil5 mutants.
(C) Hypocotyl lengths of pil5-1 mutants under 12-h red (20 μmol·m−2·s−1)/12-h dark cycle and 12-h far-red light (3.2 μmol·m−2·s−1)/12-h dark cycle.
(D) Hypocotyl length of pil5-1 phyA-211 and pil5-1 phyB-9 double mutants under red and far-red cycle.
(E) Hypocotyl lengths of pil5-1 pif3-1 double mutants under red and far-red light cycle.
(F) Hypocotyl lengths of PIL5OX1 and PIL5OX2 under red and far-red light cycle. An inset shows the overexpression of PIL5 in two PIL5OX lines. RNAs from 4-d-old light-grown seedlings were used for the RT-PCR analysis. Expression of UBQ was used as a control. White bar = 5 mm. Error bars = sd.
- Figure 3.
PIL5 Is a Negative Component in PhyB-Mediated Promotion of Seed Germination.
(A) Germination patterns of the pil5-1 mutant, the pil3-1 mutant, and the pil5-1 pif3-1 double mutant. No treatment, no extra light illumination after 1-h imbibition; R 5min, red light (6 mmol·m−2) illumination for 5 min after 1-h imbibition; FR 5min, far-red light (0.96 mmol·m−2) illumination for 5 min after 1-h imbibition. WL, continuous white light.
(B) Germination patterns of the PIL5OX transgenic lines.
(C) Quantification of the germination rates of the various mutants under different light conditions. Error bars = sd.
- Figure 4.
Effect of Fluence on Seed Germination.
(A) Increased red light fluence overcame the inhibition of seed germination observed in the PIL5OX lines. After 1-h imbibition, various fluences of red light (0 to 1200 mmol·m−2) were applied to far-red light pretreated seeds, and the samples were then incubated in the dark for 6 d. D indicates 0 mmol·m−2.
(B) Increased far-red light fluence did not inhibit seed germination of pil5 mutants and pil5 pif3 double mutants. After 1-h imbibition, various fluences of far-red light (0 to 57.6 mmol·m−2) were applied to the samples, which were then incubated for 6 d in the dark. D indicates 0 mmol·m−2.
- Figure 5.
Germination Patterns of pil5 phyA and pil5 phyB.
(A) Germination patterns of phyA and pil5 phyA. No treatment, no extra-light illumination after 1-h imbibition; R 5 min, red light (6 mmol·m−2) illumination for 5 min after 1-h imbibition; FR 5 min, far-red light (0.96 mmol·m−2) illumination for 5 min after 1-h imbibition; WL, continuous white light.
(B) Germination patterns of phyB and pil5 phyB.
(C) Quantification of the germination rates of the various mutants under different light conditions. Error bars = sd.
- Figure 6.
PIL5 Regulates PhyA-Mediated Promotion of Seed Germination.
(A) Germination rates of wild-type, pil5, pif3, pil5 pif3, phyA, pil5 phyA, phyB, and pil5 phyB mutants. Seeds were preilluminated with far-red light (0.96 mmol·m−2) after 1-h imbibition and incubated for 56 h in the dark. Then, seeds were illuminated with far-red light (0.96 or 96 mmol·m−2) and incubated further for 5 d in the dark.
(B) Far-red fluence rate response of wild-type and PIL5OX lines. The PILOX lines are hyposensitive to far-red light for germination. D indicates 0 mmol·m−2. Error bars = sd.
- Figure 7.
PIL5 Regulates PhyA-Mediated Inhibition of Hypocotyl Negative Gravitropism.
(A) Hypocotyl negative gravitropic growth patterns of wild-type, pil5, pif3, and pil5 pif3 mutants under continuous white light (8 to 9 μmol·m−2·s−1).
(B) Hypocotyl negative gravitropic growth patterns of wild-type, pil5, pif3, and pil5 pif3 mutants in the dark.
(C) Hypocotyl negative gravitropism of wild-type, pil5, pif3, pil5 pif3, phyA, pil5 phyA, phyB, and pil5 phyB mutants in the dark, under red (20 μmol·m−2·s−1), or far-red light (0.32 μmol·m−2·s−1).
(D) Hypocotyl negative gravitropism of PIL5OX lines either under red (2 μmol·m−2·s−1) or far-red light (0.32 μmol·m−2·s−1).
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