Phenotype of 4-d-old wild type, eid1 mutant, and transgenic seedlings under different light conditions. (WS) Wassilewskija wild type; (Col) Columbia wild type. eid1-1 and eid1-2 were isolated from the phyB-5 mutant in the Landsberg erecta (Ler) background. The eid1-3, eid1-4, and eid1-5 alleles were derived from a WS background and the eid1-6 from a Col background. Scale bars, 3 mm. (A) Seedlings grown under the weak red-light field used for screening. (B) Seedlings grown under the weak far-red light field used for screening. (C) Etiolated seedlings grown in darkness. (D) Phenotype of wild-type, eid1-3, and rescued eid1-3 lines under the weak red light used for screening. The phenotype of independent transgenic lines transformed with a 35S-promoter–EID1-ORF–nos-terminator construct (P35S) or a genomic EID1 fragment (gene) is shown.

Nucleotide and amino acid sequences of EID1. (A) Nucleotide and predicted amino acid sequences of wild-type and mutant alleles. The sequences of the Ler and WS gene are given as a standard. Deviations of the published Col sequence (accession no. AL161494) and the eid1 mutant alleles are shown. The start of the longest EST clone (161I3T7) is marked by a number sign and its polyadenylation site is indicated by a slash (/). Stop codons are indicated by asterisks (*). Amino acid sequences with similarities to an F-box are underlined with a bold line and the leucine zipper domain is double underlined. The acidic domain is single underlined and the basic domain is underlined with a wave line. (B) Alignment of F-box domains identified from F-box proteins known to be expressed in Arabidopsis. Unusual flower organs (UFO), accession no. X89224; transport inhibitor response 1 (TIR1), accession no. AF005048; Zeitlupe (ZTL), accession no. AF254413. (1) S or T; (2) large hydrophobic amino acids; (3) basic amino acids. Highly conserved amino acids are capitalized. Amino acids identical to the consensus are boxed in black. Gray shadings with white letters indicate amino acids similar to the consensus and gray shadings with black letters mark similarities between the Arabidopsis F-box proteins.

EID1 interacts with the Skp1-like proteins ASK1 and ASK2. (A) Analysis of the interaction in the yeast two-hybrid system. Growth on control plates lacking tryptophan and leucine (T⁻ L⁻) and on selective plates lacking both amino acids and adenine (T⁻ L⁻ A⁻) is shown. The plates were photographed after 4 d. Galactosidase activity is given as the mean ± SD of five independent yeast lines for each combination of constructs. (AD) GAL4 activation domain; (BD) GAL4 binding domain. (B) ASK1 and ASK2 interaction with EID1 in GST pull-down experiments. The SDS-PAGE of ³⁵S-labeled ASK1, ASK2, and the Luciferase control are shown. An aliquot (0.5 μL) of the labeled proteins was loaded as a reference. The proteins used in the pull-down experiments are indicated on the top. (GST) Glutathione-S-Transferase; (resin) control with empty Glutathione-S-Sepharose.

Cellular distribution of EID1–GFP in transiently transformed parsley protoplasts. (A) Protoplasts were transfected with a plasmid directing expression of EID1–GFP. (B) Protoplasts were transfected with a plasmid directing expression of GFP linked to the Cterminus of EID^mNLS, which contains mutations in the putative NLS. After transformation cells were kept in darkness for 24 h before analysis. The top panels show bright light field images and the lower panels show fluorescence images. The position of the nucleus is indicated by an arrow. Scale bars, 10 μm.

Action spectra for hypocotyl elongation in phyB-5 and phyB-5 eid1-1 seedlings. Fluence rate response curves were measured at different wavelengths and the fluence rate that led to 40% inhibition compared with dark controls was determined. The value obtained for phyB-5 at 716 nm (0.12 μmole/m²sec) was set to 1 and the relative photon effectiveness for phyB-5 and phyB-5 eid1-1 at different wavelengths was calculated. The determined values are symbolized by squares or circles connected with a bold line. Photoconversion cross-sections are plotted as thin lines by using published data for purified oat phytochrome (Mancinelli 1994). (A) Action spectra for phyB-5. (B) Action spectra for phyB-5 eid1-1.