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. 2001 Jun;13(6):1293-304.
doi: 10.1105/tpc.13.6.1293.

ELF3 Encodes a Circadian Clock-Regulated Nuclear Protein That Functions in an Arabidopsis PHYB Signal Transduction Pathway

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Free PMC article

ELF3 Encodes a Circadian Clock-Regulated Nuclear Protein That Functions in an Arabidopsis PHYB Signal Transduction Pathway

X L Liu et al. Plant Cell. .
Free PMC article

Abstract

Many aspects of plant development are regulated by photoreceptor function and the circadian clock. Loss-of-function mutations in the Arabidopsis EARLY FLOWERING 3 (ELF3) and PHYTOCHROME B (PHYB) genes cause early flowering and influence the activity of circadian clock-regulated processes. We demonstrate here that the relative abundance of the ELF3 protein, which is a novel nucleus-localized protein, displays circadian regulation that follows the pattern of circadian accumulation of ELF3 transcript. Furthermore, the ELF3 protein interacts with PHYB in the yeast two-hybrid assay and in vitro. Genetic analyses show that ELF3 requires PHYB function in early morphogenesis but not for the regulation of flowering time. This suggests that ELF3 is a component of a PHYB signaling complex that controls early events in plant development but that ELF3 and PHYB control flowering via independent signal transduction pathways.

Figures

Figure 1.
Figure 1.
Sequence Comparison of ELF3 Homologs and Localization of the ELF3 Protein to the Nucleus. (A) Multiple sequence alignment shows four highly conserved regions within ELF3 and putative ELF3 homologs from Arabidopsis (AtEEC) and other plant species (Cardamine oligosperma [cELF3], tomato [tELF3], rice [rELF3], and maize [mELF3]). Protein designations are given at left. Amino acid residues are numbered at both left and right. Residues shaded in black indicate identity of at least three ELF3/ELF3–related sequences in the alignment; light-shaded residues indicate similarity to consensus. Nucleotide sequences from C. oligosperma were obtained by sequencing polymerase chain reaction products using degenerate oligonucleotides to the Arabidopsis ELF3 gene and genomic DNA or cDNA prepared from C. oligosperma seedlings. Sequences were aligned and analyzed using CLUSTAL W (Thompson et al., 1994) and PrettyBox (Genetics Computer Group, Madison, WI). GenBank accession numbers for ELF3 and putative ELF3 homologs are as follows: AtELF3 (AC004747), AtEEC (AB023045), tELF3 (AW093790, AI894513, AI488927, AI486934, and AI894398), rELF3 (AP000399), and mELF3 (AI637184). (B) Total protein and protein from isolated nuclei were prepared from either wild-type (WT) or elf3 mutant lines of Arabidopsis seedlings, electrophoresed, and transferred to a nitrocellulose membrane. Both total protein and nuclear protein immunoblots were probed with ELF3 polyclonal antibody and β-ATPase antibody.
Figure 2.
Figure 2.
Accumulation of the ELF3 Protein Is Regulated by Light and the Circadian Clock. Wild-type seedlings were germinated and grown for 14 days in a 12-hr-light/12-hr-dark photoperiod (12/12). Tissue was then collected every 4 hr (A). Seedlings were also transferred to LL or DD for 48 hr for the collection of LL and DD tissues ([B] and [D], respectively). ELF3 protein was detected with ELF3 antibody, and 0.5% Ponceau S (Pon; Sigma) was used to stain the transferred membrane before probing with ELF3 antibody. (A) Accumulation of the ELF3 protein oscillates in 12/12. (B) Accumulation of the ELF3 protein increases in LL and keeps oscillating in the nucleus. (C) Repeat experiments of ELF3 protein accumulation in LL. Wild-type seedlings were germinated and grown for 14 days in 12/12 and transferred to LL for 48 hr before the collection of tissue every 4 hr during the subsequent 48 hr. The level of ELF3 protein was determined by measuring anti-ELF3 signal relative to the Ponceau staining intensity of the transferred membrane before probing with ELF3 antibody. The results of two independent experiments are shown, with ELF3 protein levels normalized to the highest level detected in each experiment. (D) Accumulation of the ELF3 protein decreases rapidly when plants are transferred to DD.
Figure 3.
Figure 3.
ELF3 and PHYB Proteins Interact in the Yeast Two-Hybrid System and in Vitro. (A) Diagram of the GAL4 DNA binding domain (GBD):ELF3, ELF3 N-terminal domain (N-ELF3), ELF3 intermediate domain (M-ELF3), and ELF3 C-terminal domain (C-ELF3) fusions used as bait to test for protein–protein interactions in the yeast two-hybrid system. (B) Yeast two-hybrid results with various ELF3 bait constructs and the C-terminal PHYB (C-PHYB) prey plasmid isolated in the initial yeast two-hybrid screen performed with ELF3. Different combinations of plasmids are represented by numbers 1 to 7. Yeast colony growth is shown under different selection media, and the quantitation of ELF3–PHYB interactions is shown by the production of β-galactosidase. GAD, Gal 4 activation domain; GBD, Gal 4 DNA binding domain. Error bars indicate ±se. (C) In vitro interaction of ELF3 and full-length PHYB in the far-red light–absorbing form (PfrB) and in the red light–absorbing form (PrB). PHYB was expressed as a glutathione S-transferase (GST) fusion protein (PHYB-GST). Autoradiography of the 35S-labeled ELF3 protein and Coomassie blue staining of total protein are shown on the same gel.
Figure 4.
Figure 4.
Genetic Interaction of elf3-1 with phyB and phyA Mutations. (A) Hypocotyl length of seedlings grown under dark, white light, red light, and far-red light conditions. (B) Flowering time for wild-type (Columbia ecotype [Col]) and mutant plants grown under long-day (18-hr-light/6-hr-dark; 200 μmol m−2 sec−1 white fluorescent light) or short-day (9-hr-light/15-hr-dark; 150 μmol m−2 sec−1 cool-white fluorescent light) growth conditions. Flowering time was determined by the number of rosette leaves produced by a plant with a 1-cm inflorescence height. Error bars indicate ±se.
Figure 5.
Figure 5.
Genetic Interactions of the ELF3 Overexpression (ELF3-OX) Line with the phyB Mutation. (A) Protein gel blot of the total protein of young seedlings with ELF3 antibody, showing that both ELF3-OX and phyB ELF3-OX plants overexpress the ELF3 protein. Pon, Ponceau S; WT, wild type. (B) Hypocotyl length of wild-type, ELF3-OX, elf3-1 mutant, phyB mutant, and phyB ELF3-OX double mutant seedlings (shown left to right) grown under constant red light. Bar = 1 cm. (C) Flowering time for wild-type, ELF3-OX, elf3-1 mutant, phyB mutant, and phyB ELF3-OX double mutant plants (shown left to right) grown in long-day conditions. Bar = 1 cm.
Figure 5.
Figure 5.
Genetic Interactions of the ELF3 Overexpression (ELF3-OX) Line with the phyB Mutation. (A) Protein gel blot of the total protein of young seedlings with ELF3 antibody, showing that both ELF3-OX and phyB ELF3-OX plants overexpress the ELF3 protein. Pon, Ponceau S; WT, wild type. (B) Hypocotyl length of wild-type, ELF3-OX, elf3-1 mutant, phyB mutant, and phyB ELF3-OX double mutant seedlings (shown left to right) grown under constant red light. Bar = 1 cm. (C) Flowering time for wild-type, ELF3-OX, elf3-1 mutant, phyB mutant, and phyB ELF3-OX double mutant plants (shown left to right) grown in long-day conditions. Bar = 1 cm.

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