EARLY FLOWERING 4 functions in phytochrome B-regulated seedling de-etiolation

Abstract

To define the functions of genes previously identified by expression profiling as being rapidly light induced under phytochrome (phy) control, we are investigating the seedling de-etiolation phenotypes of mutants carrying T-DNA insertional disruptions at these loci. Mutants at one such locus displayed reduced responsiveness to continuous red, but not continuous far-red light, suggesting a role in phyB signaling but not phyA signaling. Consistent with such a role, expression of this gene is induced by continuous red light in wild-type seedlings, but the level of induction is strongly reduced in phyB-null mutants. The locus encodes a novel protein that we show localizes to the nucleus, thus suggesting a function in light-regulated gene expression. Recently, this locus was identified as EARLY FLOWERING 4, a gene implicated in floral induction and regulating the expression of the gene CIRCADIAN CLOCK-ASSOCIATED 1. Together with these previous data, our findings suggest that EARLY FLOWERING 4 functions as a signaling intermediate in phy-regulated gene expression involved in promotion of seedling de-etiolation, circadian clock function, and photoperiod perception.

Figure 1.

Comparison of predicted amino acid sequences of the ELF4 family. A, Amino acid sequence alignments of ELF4 protein family members, including ELF4 (At2g40080), ELF4-L1 (At2g29950), ELF4-L2 (At1g72630), ELF4-L3 (At2g06255), ELF4-L4 (At1g17455) from Arabidopsis, ELF4Os (AAD27669) from rice (Oryza sativa) and ELF4Sb (AAD27564) from sorghum (Sorghum bicolor). Reverse font, Identical residues; gray boxes, similar residues. Numbers at the right indicate amino acid residues. All sequences shown are full length, except for ELF4Sb (1–141 of the 438 amino acids shown). The alignments were performed using MultiAlign (Corpet, 1988). The putative nuclear localization signal (NLS) in ELF4 is underlined. B, Phylogenetic neighbor-joining tree of the aligned sequences. The unrooted tree was constructed using PAUP 4.0 software, showing the putative evolutionary relationships of the ELF4 family members. The branch lengths are proportional to the indicated distance values (changes) between sequences.

Figure 2.

elf4 mutant seedlings have reduced responsiveness to Rc. A, Structure of the ELF4 gene, showing the positions of the T-DNA insertion sites in the elf4-101 and the elf4-102 mutants, three EEs in the promoter, and the putative NLS (black rectangle). The numbers indicate nucleotide positions relative to the first nucleotide of the ATG start codon. B, Four-day-old wild-type sibling (Col-sib), elf4-101, and elf4-102 mutant seedlings grown in the dark, Rc (9.5 μmol m^-2 s^-1), or FRc (2.7 μmol m^-2 s^-1).

Figure 3.

elf4 mutants have reduced sensitivity to Rc but not to FRc. Fluence rate response curves for Col-sib, elf4-101, and elf4-102 mutants under Rc (A) and FRc (B). The phyB-9 mutant in Rc and phyA-211 mutant in Rc and FRc, are included for hypocotyl length comparisons. C, elf4 mutant seedlings show reduced cotyledon expansion. Four-day-old seedlings grown in Rc (9.5 μmol m^-2 s^-1).

Figure 4.

Rc-induced expression of the ELF4 gene is phyB dependent. Representative northern blots probed with ELF4 riboprobes. A, ELF4 transcript levels in Col-sib, elf4-101, and elf4-102 mutant seedlings grown in FRc for 12 h. B, ELF4 expression in wild-type, Col-sib, and phyB-9 mutant seedlings grown in Rc (7 μmol m^-2 s^-1) for 6, 12, or 24 h. RNA from dark controls (0D and 24D) are also included. C, Quantitation of the ELF4 transcript levels from four independent replicates. The values were normalized to the 18S rRNA signal, and the mean values for each time point were plotted with ses.

Figure 5.

ELF4 localizes to the nucleus. Transient transfection assays in onion epidermal cells using EGFP-ELF4 constructs. A and B, Cells expressing the EGFP-ELF4 fusion protein; C and D, cells expressing the EGFP-protein control. GFP florescence (A) and propidium iodide (PI)-stained (B) nuclei show that all of the detectable EGFP-ELF4 fusion protein is nuclear localized. GFP fluorescence (C) and PI-stained (D) nuclei show that the EGFP protein control is distributed throughout the cell, including the cytoplasm and nucleus.

Figure 6.

Simplified model of ELF4 function in phy-regulated seedling de-etiolation, circadian rhythms, and flowering time. ELF4 expression is regulated by phy in response to light signals, as is the expression of CCA1 and LHY. ELF4 functions as a positive regulator of phyB-mediated seedling de-etiolation. ELF4 function is closely linked to the central oscillator, thereby functioning in clock maintenance and regulating circadian rhythmicity. Like TOC1, ELF4 has EE in its promoter, and it induces the expression of CCA1. We propose that the ELF4 expression is regulated in a negative manner, possibly by CCA1/LHY binding to the EE in its promoter, similar to the regulation of TOC1. ELF4 represses CO expression exerting control on flowering time.