Article Figures & Data
Figures
- Figure 1.
Blue Light–Dependent Phosphorylation of cry1.
(A) Five-day-old etiolated seedlings were cut above the roots and incubated with 300 μCi of 32P-H3PO4 in water for 3 h in the dark. The tissue aliquots were exposed to blue light (30 μmol·m−2·s−1) for the time indicated or kept in darkness. The cry1 protein was immunoprecipitated with anti-CRY1 antibodies, separated on a 10% SDS-PAGE gel, blotted to a membrane, and analyzed by autoradiography (Autoradiograph) and then by probing an immunoblot with anti-cry1 antibodies (Immuno).
(B) Immunoblots of samples prepared from wild-type seedlings exposed to blue light for 15 min (15′) or 30 min (30′) at the fluence rate indicated were probed with the anti-CRY1 antibody. The signals of the slow-migrating bands (indicated by the arrow-bracket) are normalized to the fast-migrating band in the same lane (arrowhead), represented as [cry1(Pi)/cry1 (%)], and plotted against the fluence rates (graph at bottom).
(C) Immunoblot showing dephosphorylation of cry1 in the dark. Etiolated seedlings were exposed to blue light (B; 30 μmol·m−2·s−1) for 1 h and then transferred to dark. Samples were prepared before or after seedlings were transferred to darkness for 15 or 30 min (D15 and D30).
Arrows with brackets indicate phosphorylated cry1; arrowheads represent nonphosphorylated cry1. wt, wild type.
- Figure 2.
Blue Light Specificity and the Effect of Phytochrome Mutations on cry1 Phosphorylation.
(A) Immunoblot showing cry1 phosphorylation in seedlings exposed to blue light (B; 30 μmol·m−2·s−1), far-red light (FR; 50 μmol·m−2·s−1), or red light (R; 50 μmol·m−2·s−1) for the time indicated or kept in the dark (D).
(B) Immunoblot showing cry1 phosphorylation in samples prepared from the wild type or from phyA, phyB, phyA phyB, phyBDE, and phyABD mutants. Seedlings were grown in the dark and then exposed to blue light (20 μmol·m−2·s−1) for the time indicated. wt, wild type.
(C) Immunoblot showing cry1 phosphorylation in samples prepared from wild-type or phyA seedlings grown in the dark and then exposed to blue light (30 μmol·m−2·s−1) for 1 h. Protein extracts from light-treated seedlings were incubated with alkaline phosphatase (Alk. PPase) at 30°C for 30 min (+) or analyzed without phosphatase treatment (−).
Asterisks indicated a band nonspecifically recognized by the antibody.
- Figure 3.
Long-Hypocotyl Phenotype of the Newly Isolated cry1 Mutants.
(A) Seedlings were grown in continuous blue light (25 μmol·m−2·s−1) for 5 days, and hypocotyl lengths were measured. wt, wild type.
(B) Representative seedlings of parental control (phyA), a cry1 reference allele (cry1-304), and nine newly isolated cry1 alleles are shown. cry1-304 and cry1-371 express no CRY1 apoprotein, whereas the other alleles express full-length CRY1 apoprotein.
- Figure 4.
Immunoblots Showing the Lack of cry1 Phosphorylation and Normal cry2 Phosphorylation in Different cry1 Mutant Alleles.
(A) Samples were prepared from etiolated seedlings exposed to 25 μmol·m−2·s−1 blue light for the time indicated or from samples kept in the dark (0 h), and immunoblots were probed with anti-CRY1 antibodies (cry1). For a loading control, the membrane was stained with Ponceau red, and a portion of the stained blot showing unspecified proteins is included (stained).
(B) Samples were prepared from etiolated seedlings exposed to 7 μmol·m−2·s−1 blue light for the time indicated or from samples kept in the dark (0 min). The immunoblot was probed first with anti-CRY2 antibody (cry2), stripped, and reprobed with anti-CRY1 antibody (cry1).
- Figure 5.
Lesions of the cry1 Mutants.
Sequences of the cry1 mutants were aligned with the deduced amino acid sequence of CRY1 of the Columbia accession (WT; At4g08920). Residue numbers of CRY1 are indicated above the sequence. White and gray boxes highlight mutant residues of the different alleles.
- Figure 6.
Blue Light–Dependent Phosphorylation of cry1 in Vitro.
(A) The cry1 protein purified from insect cells (cry1; 1 μg) and the molecular mass marker proteins (M; 1 μg per band) were fractionated on a SDS-PAGE gel and stained with Coomassie blue.
(B) to (D) Autoradiographs showing cry1 phosphorylation in vitro. Arabidopsis cry1 ([B] and [C]) and the control protein ubc9 (B) were incubated with γ-32P-ATP, and the reaction products were kept in the dark or exposed to blue light (33 μmol·m−2·s−1) for the time indicated (15 to 60 min). cry1 phosphorylation reaction products incubated with γ-32P-ATP or γ-32P-GTP under blue light (33 μmol·m−2·s−1) for the time indicated (15 to 60 min) are shown in (D).
(E) The immunoblot of the same membrane used in (C) was probed with the anti-CRY1 antibody.
