EARLY FLOWERING3 encodes a novel protein that regulates circadian clock function and flowering in Arabidopsis

Abstract

Higher plants use photoperiodic cues to regulate many aspects of development, including the transition from vegetative to floral development. The EARLY FLOWERING3 (ELF3) gene is required for photoperiodic flowering and normal circadian regulation in Arabidopsis. We have cloned ELF3 by positional methods and found that it encodes a novel 695-amino acid protein that may function as a transcriptional regulator. ELF3 transcript level is regulated in a circadian manner, as is expected of a zeitnehmer input pathway component. Overexpression of the LATE ELONGATED HYPOCOTYL gene, which has been proposed to function as a clock component, did not abolish circadian regulation of ELF3 transcription, providing further evidence that ELF3 is a circadian clock input pathway component.

Figure 1.

Map-Based Cloning of ELF3. (A) Physical mapping of ELF3. Molecular markers are shown above the line with relevant recombination distances, and the YAC contig of ∼500 kb of Arabidopsis genomic DNA is shown below the line. YAC 6D6 is chimeric, with the unlinked region represented by a dashed line. cM, centimorgan. (B) YAC end clone 10A10L was used as a starting point for identifying λ and cosmid clones. RFLP analysis localized ELF3 to the 60-kb minimal recombinant interval shown. The closed boxes indicate the RFLPs. BAC clones spanning this region were identified, and cosmid subclones of BAC 4D15 were used for transformation rescue. Cosmid B8 complemented elf3-1 and elf3-3 mutant phenotypes (see Figure 2). (C) The positions and structures of two transcription units within cosmid B8 are shown. ORF, open reading frame.

Figure 2.

Complementation of elf3 Mutants. (A) Ten-day-old seedlings grown in SD conditions. Left to right: wild-type Columbia, elf3-1, transgenic elf3-1 containing cosmid B8, and transgenic elf3-1 containing cosmid E11. (B) Hypocotyl lengths of 10-day-old seedlings grown in SD conditions. Col, wild-type Columbia; Ws, Wassilewskija. Error bars indicate ±sd (n = 20). (C) Twenty-five-day-old plants grown in LD conditions. Left to right: wild-type Columbia, elf3-1, transgenic elf3-1 containing cosmid B8, and transgenic elf3-1 containing cosmid E11. (D) Flowering time expressed as vegetative leaves produced before flowering. Plants were grown in either SD or LD conditions. Error bars indicate ±sd (n ≥ 16).

Figure 3.

DNA Coding and Amino Acid Sequence of ELF3 and Sequence Comparison with Putative Orthologs. (A) The DNA sequence corresponding to ELF3 cDNA clone 8.2 is shown, along with the predicted amino acid sequence of the longest open reading frame. Amino acid numbering is shown at right, and intron positions are marked with inverted triangles. Molecular changes in eight elf3 alleles are shown above the DNA sequence. The closed inverted triangle is a cryptic splice site used in some elf3-7 transcripts (see text). A potential nuclear targeting signal is shown in boldface. Translation stop codon is indicated by an asterisk. (B) Black boxes indicate identical residues, and gray boxes indicate conserved residues between ELF3 and ELF3-like predicted proteins in other plant species. Dashes indicate gaps in the sequences. Tomato, soybean, and maize ELF3-like proteins were predicted from partial sequences from expressed sequence tag clones. The rice ELF3-like protein was predicted from genomic sequence.

Figure 4.

ELF3 Transcript Levels in Wild-Type and Mutant Backgrounds. Ten micrograms of total RNA was hybridized with antisense riboprobes for ELF3 or CCR2 and then subjected to RNase digestion, as described in Methods. β-ATPase served as an internal control, and tRNA was used as a negative control. Time 0 (T0) indicates dawn. Quantitative representation of expression data normalized to β-ATPase is shown in all panels except (A). Open and closed bars along x-axis represent light and dark photoperiods, respectively. (A) Wild-type seedlings were grown in 12-hr-light/12-hr-dark conditions for ∼1 week, and samples were collected every 4 hr. The specific probe was complementary to ELF3 at top and to CCR2 at bottom. (B) Quantification of ELF3 and CCR2 mRNA levels shown in (A) after normalization to β-ATPase mRNA. The experiment was repeated at least three times. (C) ELF3 transcript level in mature wild-type plants grown in 9-hr-light/15-hr-dark SD conditions. Tissue samples were collected at T3, T13, and T21. (D) ELF3 transcript level in elf3 mutant seedlings grown in 12-hr-light/12-hr-dark conditions. Samples were collected every 4 hr. The experiment was repeated five times for elf3-1. (E) ELF3 transcript level in wild-type seedlings grown in either 9-hr-light/15-hr-dark SD conditions (9L:15D) or 18-hr-light/6-hr-dark LD conditions (18L:6D). Samples were collected every 2 hr. The experiment was repeated twice. (F) and (G) ELF3 transcript level in wild-type and mutant seedlings grown in 9-hr-light/15-hr-dark SD conditions (F) or 18-hr-light/6-hr-dark LD conditions (G). Samples were collected at T4 and T20. Col, Columbia; Ler, Landsberg; phy, phytochrome; Ws, Wassilewskija.

Figure 5.

ELF3 Transcript Level Continues to Cycle in Constant Conditions. Wild-type ([A] and [C]) and elf3 mutant (B) seedlings were entrained under 12-hr-light/12-hr-dark conditions and then shifted to either constant light ([A] and [B]) or constant dark (C) at time 0, which corresponds to normal dawn. Samples were taken every 4 hr and analyzed by RNase protection. ELF3 and CCR2 transcript levels were normalized to β-ATPase. Experiments were repeated at least three times. Col, Columbia.

Figure 6.

ELF3 Transcript Level Continues to Cycle in lhy Mutant Seedlings. Wild-type Landsberg (Ler) and lhy mutant seedlings were entrained under 12-hr-light/12-hr-dark conditions and then shifted to constant light at time 0, which corresponds to normal dawn. Samples were taken every 4 hr and analyzed by RNase protection. ELF3 (A) and CCR2 (B) transcript levels were normalized to β-ATPase. Scales for ELF3 (A) differ between wild type and lhy. The experiment was repeated twice.