hy5 seeds are less sensitive to the inhibition of ABA, salt, and sugar on germination and seedling growth. (A and B) hy5 is less sensitive to ABA inhibition of seed germination and seedling growth. Morphology of seedlings in the absence (A) or presence (B) of 1.0 μM ABA. The pictures were taken 12 days after the seeds were incubated at 22°C. (C) Germination rates of wild-type and hy5 seeds on 150 mM NaCl. (D) Germination rates of wild-type and hy5 seeds on 333 mM glucose. After sowing on the respective plates, seeds were incubated at 4°C for 3 days before being placed at 22°C under constant white light for germination. Seeds with emerged radicals were counted as germinated. Numbers are averages of two independent experiments with ≈100 seeds for each genotype.

Reduced ABI5::GUS activity in hy5 mutants. (A–C) Two-day-old seedlings of light-grown Col-0 (A), light-grown hy5 (B), and dark-grown Col-0 (C). (D and E) Floral tissues of Col-0 (D) and hy5 (E). (F) Siliques of Col-0 (Left) and hy5 (Right) 14 days after pollination. (G–L) Four-day-old seedlings of Col-0 and hy5 without (G and J) or with 100 mM NaCl (H and K) or 10 μM ABA (I and L) treatment. (M) GUS activity (arbitrary units) of light- or dark-grown Col-0 and hy5 seedlings. (N) GUS activity (arbitrary units) of Col-0 and hy5 without or with 10 μM ABA or 100 mM NaCl treatment. Data in M and N are means and standard errors of three independent experiments.

hy5 seedlings are less sensitive to ABA inhibition of lateral root growth and are more susceptible to stress. (A) Morphology of wild-type and hy5 seedlings on plates without or with 0.5 or 1.0 μM ABA. Five-day-old seedlings grown on a half MS medium were transferred to half MS media supplemented with or without ABA. The pictures were taken 5 days after the transfer. (B) Relative root length of wild-type and hy5 seedlings at 5 days after being transferred to plates with the indicated concentrations of ABA. (C) Length of total lateral roots of two-week-old wild-type and hy5 seedlings as shown in A. (D) Morphology of wild-type and hy5 seedlings on plates without or with 50 or 100 mM mannitol. The pictures were taken 3 weeks after seedlings were transferred to the shown plates. (E) Relative root length of wild-type and hy5 seedlings at 8 days after being transferred to plates with the indicated concentrations of NaCl. (F) Length of lateral roots of the wild-type and hy5 seedlings on media supplemented with the indicated concentrations of NaCl. Data in B, C, E, and F represent means and SE (n = 6).

HY5 regulates the expression of a subset of ABA-inducible genes in young seedlings or dry seeds and binds to ABI5 regulatory sequences with high affinity. (A) RD29B transcript levels in response to ABA treatment. Two-week-old seedlings were sprayed with 100 μM ABA and incubated under light for 0.5 or 2 h before RNA extraction. Ten μg of the total RNA was loaded per lane. A β-tubulin gene was used as a loading control. (B) Transcript levels of the indicated genes in dry seeds of Col-0, hy5, abi5 (Ws background), and Ws. The 18s rRNA gene was used as a loading control. (C–F) Binding of HY5 to ABI5 regulatory sequences. (C) Schematic of the ABI5 promoter. The black filled box represents the promoter region, gray filled box stands for intron, box with lines represents the 5′ UTR, and empty boxes stand for G-boxes. (D and E) Gel-retardation assays of ABI5 promoter P1 and P2 (D), P3 and P4 (E) with 150 μg GST (lanes 2 and 7) or increased amounts (50, 100, 150 μg) of GST-HY5 protein (lanes 3–5 and 8–10). Lanes 1 and 6 are labeled probes alone to show the position of the free probes. The DNA–protein complexes are indicated by arrowheads. (F) Competition gel-retardation assay of P1 with cold P1 or P4 in the presence of 100 μg of GST-HY5. Lane 1 is labeled P1 alone, and lane 2 is the labeled P1 with 100 μg of GST-HY5. Increased amounts (30×, 300×) of unlabeled P1 or P4 were added to lanes 3 and 4 and lanes 5 and 6, respectively. The DNA–protein complexes are indicated by arrowheads. (G) HY5 binds to the P1 motif of ABI5 in vivo and ABA specifically enhances the binding. Shown are anti-HA ChIP assays with Col-0 and HA-HY5 transgenic plants with or without 0.5 μM ABA treatment. P1–P4 are fragments of the ABI5 promoter. RbcSA and CHS are light-regulated HY5 target genes. Input control: genomic DNA not subject to immunoprecipitation. Mock control: ChIP without HA antibody.

Overexpression of ABI5 restores ABA sensitivity in hy5 and enhances light response in the wild type. (A) ABI5 transcript levels in the ABI5 overexpression lines of Col-0 and hy5. (B and C) Morphology of 7-day-old seedlings overexpressing ABI5 in the wild-type or hy5 background without (B) or with (C) 1.0 μM ABA. (D) Germination rates of ABI5 overexpression lines in wild-type and hy5 backgrounds. Data are means from three independent experiments with ≈100 seeds for each genotype. (E) Primary root length of ABI5 overexpression lines in the wild-type or hy5 background under the indicated concentrations of ABA (0, 0.5, 1.0, and 2.5 μM). Data are means and standard errors (n = 10). (F–H) Hypocotyl lengths of seedlings of Col-0 and Col-0 transformed with 35S::ABI5 under the indicated fluence rates of blue (F), far-red (G), or red light (H). Data are means and standard errors (n = 30).