PIL5, a phytochrome-interacting basic helix-loop-helix protein, is a key negative regulator of seed germination in Arabidopsis thaliana

Abstract

The first decision made by an angiosperm seed, whether to germinate or not, is based on integration of various environmental signals such as water and light. The phytochromes (Phys) act as red and far-red light (Pfr) photoreceptors to mediate light signaling through yet uncharacterized pathways. We report here that the PIF3-like 5 (PIL5) protein, a basic helix-loop-helix transcription factor, is a key negative regulator of phytochrome-mediated seed germination. PIL5 preferentially interacts with the Pfr forms of Phytochrome A (PhyA) and Phytochrome B (PhyB). Analyses of a pil5 mutant in conjunction with phyA and phyB mutants, a pif3 pil5 double mutant, and PIL5 overexpression lines indicate that PIL5 is a negative factor in Phy-mediated promotion of seed germination, inhibition of hypocotyl negative gravitropism, and inhibition of hypocotyl elongation. Our data identify PIL5 as the first Phy-interacting protein that regulates seed germination.

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).