ELF3 modulates resetting of the circadian clock in Arabidopsis

Abstract

The Arabidopsis early flowering 3 (elf3) mutation causes arrhythmic circadian output in continuous light, but there is some evidence of clock function in darkness. Here, we show conclusively that normal circadian function occurs with no alteration of period length in elf3 mutants in dark conditions and that the light-dependent arrhythmia observed in elf3 mutants is pleiotropic on multiple outputs normally expressed at different times of day. Plants overexpressing ELF3 have an increased period length in both constant blue and red light; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by using light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and constitutive overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. The phase of ELF3 function correlates with its peak expression levels in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting.

Figure 1.

ELF3 Transcription Is Regulated by the Circadian Clock. Seedlings were grown under 12-hr-light (white fluorescent; 50 to 60 μmol·m⁻²·sec⁻¹)/12-hr-dark cycles for 6 days before transfer to LL (time 0) or DD (time 12). Shaded bars indicate subjective day and night. WT, wild type. (A) elf3::luc expression is rhythmic in continuous white light. Mean bioluminescence traces (±se) from wild-type elf3::luc (closed circles; n = 5) and cab2::luc (gray squares; n = 6) seedlings are plotted. (B) elf3 mutations cause arrhythmic expression of elf3::luc in LL. Mean elf3::luc bioluminescence traces (±se) from wild-type (closed circles; n = 7), elf3-1 (open triangles; n = 12), and elf3-7 (closed gray triangles; n = 9) seedlings are plotted. (C) elf3 mutations lead to arrhythmic ccr2::luc expression in LL. Mean ccr2::luc bioluminescence traces (±se) from wild-type (closed circles; n = 12), elf3-1 (open triangles; n = 12), and elf3-7 (closed gray triangles; n = 12) are plotted. (D) elf3::luc expression is rhythmic in DD. Representative bioluminescence traces from individual wild-type elf3::luc (closed circles) and ccr2::luc (open diamonds) seedlings are plotted. Data from elf3::luc plants are plotted on the left y axis, and ccr2::luc data are plotted on the right y axis. (E) ccr2::luc rhythms are robust in DD, and period length is unaffected by ELF3 levels. Mean ccr2::luc bioluminescence traces from wild-type (closed circles; n = 27), elf3-1 (open triangles; n = 27), and ELF3-OX (open circles; n = 27) are plotted. Data from wild-type and ELF3-OX plants are plotted on the left y axis, and elf3-1 data are plotted on the right y axis.

Figure 2.

The Circadian Clock Remains Intact in Seedlings Overexpressing ELF3. Luminescence in seedlings constitutively overexpressing a genomic copy of ELF3 under the control of the cauliflower mosaic virus 35S promoter (ELF3-OX) was determined as described in Figure 1 and Methods. After entrainment in 12-hr-light/12-hr-dark cycles, plants were transferred to continuous red light (∼200 μmol·m⁻²·sec⁻¹) (time 0). (A) Expression of genes closely associated with circadian function, elf3::luc (closed circles; n = 36) and cca1::luc (open squares; n = 9), is rhythmic in ELF3-OX seedlings. Data from elf3::luc plants are plotted on the left y axis, and cca1::luc data are plotted on the right y axis. (B) Expression of the circadian output genes cab2::luc (closed gray squares; n = 14) and ccr2::luc (open diamonds; n = 10) is rhythmic in ELF3-OX seedlings. Data from cab2::luc plants are plotted on the left y axis, and ccr2::luc data are plotted on the right y axis. Error bars indicate ±se.

Figure 3.

ELF3 Overexpression Lengthens the Period of the Circadian Clock in Red Light and Blue Light. After entrainment, wild-type (WT) and ELF3-OX seedlings were transferred to continuous red light or blue light at specific fluence rates, and luminescence was determined as described in Figure 1 and Methods. Variance-weighted means (± variance-weighted se) after >112 hr in free-running conditions are presented in FRCs. Asterisks, P < 0.01 (Student's two-tailed heteroscedastic t test). (A) Effect of red light fluence rate on free-running period of cab2::luc expression in wild-type (closed circles; n = 18, 18, 18, 18, 17, 16, and 18) and ELF3-OX (open circles; n = 16, 18, 18, 18, 13, 11, and 36) seedlings. (B) Effect of blue light fluence rate on free-running period of cab2::luc expression in wild-type (closed circles; n = 14, 13, 12, 18, 22, 18, 18, and 17) and ELF3-OX (open circles; n = 6, 17, 17, 18, 18, 18, and 18) seedlings.

Figure 4.

ELF3 Attenuates Phytochrome-Mediated Induction of Circadian Outputs. Luminescence from clusters of ∼50 etiolated cab2::luc seedlings was measured before and after a 2-min red light (RL) pulse (∼25 μmol·m⁻²·sec⁻¹ at time 0; red arrow). Preflash levels were normalized to 1 so that induction levels could be evaluated and compared. (A) ELF3 levels affect the magnitude of cab2::luc acute induction. Normalized mean cab2::luc bioluminescence traces (±se) from the wild type (WT; blue circles; n = 3), elf3-1 (open purple triangles; n = 4), and ELF3-OX (open green circles; n = 4) are plotted. (B) Complete phytochrome response is present in the elf3-7 weak allele. Representative individual traces from the wild type (blue circles), elf3-1 (open purple triangles), and elf3-7 (closed orange triangles) are plotted.

Figure 5.

ELF3 Levels Affect Sensitivity of the Clock to Resetting by Light. PRCs for the wild type (WT; blue circles), elf3-1 (open purple triangles), and ELF3-OX (open green circles) constructed by plotting phase shifts of ccr2::luc expression (± pooled se; circadian hour) elicited by hour-long light pulses are plotted against the circadian times at which the light pulses were administered. Phase advances are plotted as positive values, and delays are plotted as negative values. Circadian times at which a light pulse induces arrhythmicity in elf3-1 seedlings are indicated by purple ×'s above the x axis. PRCs have been double plotted to aid in identification of the circadian features. (A) Red light PRC. (B) Blue light PRC.

Figure 6.

Model of the Role of ELF3 in the Plant Circadian System. ELF3 has a unique role within the circadian system of Arabidopsis. Circadian regulation of ELF3 expression enables the oscillator to control the light sensitivity of both clock resetting and the induction of circadian outputs in a phase-specific manner. This is a prime example of a gating mechanism in which a circadian output feeds back to regulate input pathways.