Independent roles for EARLY FLOWERING 3 and ZEITLUPE in the control of circadian timing, hypocotyl length, and flowering time

Abstract

The circadian clock regulates many aspects of plant development, including hypocotyl elongation and photoperiodic induction of flowering. ZEITLUPE (ZTL) is a clock-related F-box protein, and altered ZTL expression causes fluence rate-dependent circadian period effects, and altered hypocotyl elongation and flowering time. EARLY FLOWERING 3 (ELF3) is a novel protein of unknown biochemical function. elf3 mutations cause light-dependent circadian dysfunction, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is unclear. Here we show that the effects of ZTL and ELF3 on circadian clock function and early photomorphogenesis are additive. The long period of ztl mutations and ELF3 overexpressors are more severe than either alone. Dark-release experiments showing additivity in phase advances suggest that the arrthymicity caused by ZTL overexpression and that of the elf3-1 mutation arise through independent pathways. A similar additive effect on hypocotyl elongation in red and blue light is also observed. In contrast, ELF3 and ZTL overexpressors act similarly to control flowering time in long days through the CONSTANS/FLOWERING LOCUS T (CO/FT) pathway. ZTL overexpression does not delay flowering through changes in GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F-BOX levels, but through a ZTL-mediated reduction in CO expression. In contrast, ELF3 negatively regulates CO, FT, and GIGANTEA transcript levels, as the expression of all three genes is increased in elf3-1. The elf3-1 co-1 double mutant flowers much earlier in long days than co-1, although FT message levels remain very low. These results show that elf3-1 can derepress late flowering through a CO-independent mechanism. ELF3 may act at more than one juncture, possibly posttranscriptionally.

Figure 1.

Effects of ZTL and ELF3 expression levels on free-running period. A and B, Rhythmicity of CAB:LUC expression in elf3-1, ZTL OX (C24 ecotype), elf3-1 ztl-3, and elf3-1 ZTL OX plants (A), and in ELF3 OX, ztl-1 (Col), and ELF3 OX ztl-1 double mutant plants (B). Plants were entrained in 12-h-white light /12-h-dark cycles and transferred to constant red light (RR; 20 μmol m⁻² s⁻¹), and bioluminescence was monitored every 2 h for 4 to 5 d. Traces represent averages from at least 20 seedlings from two independent experiments.

Figure 2.

Effects of ZTL and ELF3 expression levels on clock-controlled expression in extended dark. A to C, Timing of peak CAB:LUC expression in wild type (Col WT), elf3-1, ztl-3, and elf3-1 ztl-3 plants (A); elf3-1, ZTL OX (C24), and elf3-1 ZTL OX plants (B); and ELF3 OX, ztl-1 (Col), and ELF3 OX ztl-1 plants (C). D, Peak CAB:LUC expression in Col and C24 wild type and an F₂ population of a cross between Col and C24 are shown as controls. Seedlings were entrained in 12-h-light (white)/12-h-dark cycles, transferred to DD, and measured for luminescence expression every hour. Mean values (±SEM [A, C, and D]) and mean values normalized to mean expression level of 1 for each genotype (B) are shown.

Figure 3.

ZTL and ELF3 control of hypocotyl growth during early photomorphogenesis. The wild-type (Col), elf3-1, ztl-3, ELF3 OX, ZTL OX (Col), elf3-1 ztl-3, and elf3-1 ZTL OX plants were grown for 7 to 10 d under constant red light (RL; A and B) or constant blue light (BL; C and D) at the fluence rates indicated and measured for hypocotyl length. Unconnected data points show lengths for dark-grown seedlings. Values (mean hypocotyl length ± SEM) are representative of two trials; n = 16 to 25.

Figure 4.

Control of flowering time by ZTL and ELF3 expression levels. A, Total number of leaves (rosette + cauline) produced at flowering under long days (16 h light/ 8 h dark) in wild-type (Col), elf3-1, ztl-3, ELF3 OX, ZTL OX (Col), elf3-1 ztl-3, and elf3-1 ZTL OX plants. Values (±SEM) are representative of two independent trials; n = 13 to 16. B and C, Expression levels of CO (B) and FT (C) transcripts under long days were determined by semiquantitative PCR for the lines described in A. Values are expressed relative to ACTIN2 (ACT2) control. The same appropriate wild-type data are plotted in both sections of B and C to facilitate comparisons. White and black boxes represent light and dark periods, respectively. Data are representative of three independent trials.

Figure 5.

Effects of ZTL and ELF3 expression levels on GI and FKF1 expression. Expression levels of GI (A) and FKF1 (B) transcripts under long days were determined by semiquantitative PCR for the same lines tested for flowering-time effects in Figure 4. Values are expressed relative to ACTIN2 (ACT2) control. White and black boxes represent light and dark periods, respectively. Data are representative of three independent trials.

Figure 6.

Effect of the elf3-1 co-1 double mutant on flowering and FT expression. A, Total number of leaves (rosette + cauline; ±SEM) produced at flowering under long days (16 h light/ 8 h dark) in wild-type (Ler), elf3-1 (2× introgressed into Ler), elf3-1 co-1, co-1 (Ler), and ELF3 co-1 segregants isolated from the elf3-1 × co-1 F₂ population (co-1 [seg]). n = 8 to 20. B, Expression levels of FT transcripts under long days were determined by quantitative PCR for the lines described in A. Values are expressed relative to ACTIN2 (ACT2) control. White and black boxes represent light and dark periods, respectively. Data are representative of two independent trials.

Figure 7.

ELF3 may regulate flowering through multiple flowering time-related genes in the CO/FT pathway. ELF3 negatively regulates GI, CO, and FT expression; modest derepression of FKF1 expression by elf3-1 is not shown. Epistasis tests show elf3-1 early flowering requires GI and FT, but not CO, indicating ELF3 may regulate CO/FT expression via GI (Fig. 7A). The dotted line leading from GI to X (Mizoguchi et al., 2005) is supported by our data, but our results additionally suggest a posttranslational control of FT by GI (Fig. 7A), based on low FT levels in the elf3-1 co-1 double mutant. Alternatively, this result plus high FT expression in the elf3-1 ZTL OX background, when CO expression is wild type or lower, indicates ELF3 may regulate FT expression posttranscriptionally (Fig. 7B). Other models are also possible; see text for more details.