Seth J. Davis

The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana

Many plants use day length as an environmental cue to ensure proper timing of the switch from vegetative to reproductive growth. Day-length sensing involves an interaction between the relative length of day and night, and endogenous rhythms that are controlled by the plant circadian clock. Thus, plants with defects in circadian regulation cannot properly regulate the timing of the floral transition. Here we describe the gene EARLY FLOWERING 4 (ELF4), which is involved in photoperiod perception and circadian regulation. ELF4 promotes clock accuracy and is required for sustained rhythms in the absence of daily light/dark cycles. elf4 mutants show attenuated expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a gene that is thought to function as a central oscillator component. In addition, elf4 plants transiently show output rhythms with highly variable period lengths before becoming arrhythmic. Mutations in elf4 result in early flowering in non-inductive photoperiods, which is probably caused by elevated amounts of CONSTANS (CO), a gene that promotes floral induction.

The Molecular Basis of Temperature Compensation in the Arabidopsis Circadian Clock

Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In Arabidopsis thaliana, ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 27°C), whereas they decrease for LHY. However, as temperatures decrease (from 17 to 12°C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 27°C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in lhy and gi mutant plants. However, at 12°C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than lhy at the lower temperature. At 17°C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperature-specific phenotypes of gi mutants.

The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidopsis circadian clocks

Plants synchronize developmental and metabolic processes with the earths 24-h rotation through the integration of circadian rhythms and responses to light. We characterize the time for coffee (tic) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b-binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 (ELF3) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify TIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. Even the robust circadian clock of plants cannot function with defects at two different phases.

EARLY FLOWERING4 Recruitment of EARLY FLOWERING3 in the Nucleus Sustains the Arabidopsis Circadian Clock

ELF3 and ELF4 play pivotal roles in the circadian clock mechanism and in the integration of light signals to the clock, but the molecular basis of ELF3 and ELF4 action is poorly understood. This work uses multidisciplinary approaches to identify and characterize these clock factors as members of a dusk complex that works as a repressor to sustain rhythms of the circadian oscillator. The plant circadian clock is proposed to be a network of several interconnected feedback loops, and loss of any component leads to changes in oscillator speed. We previously reported that Arabidopsis thaliana EARLY FLOWERING4 (ELF4) is required to sustain this oscillator and that the elf4 mutant is arrhythmic. This phenotype is shared with both elf3 and lux. Here, we show that overexpression of either ELF3 or LUX ARRHYTHMO (LUX) complements the elf4 mutant phenotype. Furthermore, ELF4 causes ELF3 to form foci in the nucleus. We used expression data to direct a mathematical position of ELF3 in the clock network. This revealed direct effects on the morning clock gene PRR9, and we determined association of ELF3 to a conserved region of the PRR9 promoter. A cis-element in this region was suggestive of ELF3 recruitment by the transcription factor LUX, consistent with both ELF3 and LUX acting genetically downstream of ELF4. Taken together, using integrated approaches, we identified ELF4/ELF3 together with LUX to be pivotal for sustenance of plant circadian rhythms.

Multiple phytohormones influence distinct parameters of the plant circadian clock

Circadian systems coordinate endogenous events with external signals. In mammals, hormone‐clock feedbacks are a well‐known integration system. Here, we investigated phytohormone effects on plant‐circadian rhythms via the promoter:luciferase system. We report that many hormones control specific features of the plant‐circadian system, and do so in distinct ways. In particular, cytokinins delay circadian phase, auxins regulate circadian amplitude and clock precision, and brassinosteroid and abscisic acid modulate circadian periodicity. We confirmed the pharmacology in hormone synthesis and perception mutants, as rhythmic expression is predictably altered in an array of hormone‐related mutants. We genetically dissected one mechanism that integrates hormone signals into the clock, and showed that the hormone‐activated ARABIDOPSIS RESPONSE REGULATOR 4 and the photoreceptor phytochrome B are elements in the input of the cytokinin signal to circadian phase. Furthermore, molecular‐expression targets of this signal were found. Collectively, we found that plants have multiple input/output feedbacks, implying that many hormones can function on the circadian system to adjust the clock to external signals to properly maintain the clock system.

ELF4 Is Required for Oscillatory Properties of the Circadian Clock

Circadian clocks are required to coordinate metabolism and physiology with daily changes in the environment. Such clocks have several distinctive features, including a free-running rhythm of approximately 24 h and the ability to entrain to both light or temperature cycles (zeitgebers). We have previously characterized the EARLY FLOWERING4 (ELF4) locus of Arabidopsis (Arabidopsis thaliana) as being important for robust rhythms. Here, it is shown that ELF4 is necessary for at least two core clock functions: entrainment to an environmental cycle and rhythm sustainability under constant conditions. We show that elf4 demonstrates clock input defects in light responsiveness and in circadian gating. Rhythmicity in elf4 could be driven by an environmental cycle, but an increased sensitivity to light means the circadian system of elf4 plants does not entrain normally. Expression of putative core clock genes and outputs were characterized in various ELF4 backgrounds to establish the molecular network of action. ELF4 was found to be intimately associated with the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LONG ELONGATED HYPOCOTYL (LHY)-TIMING OF CAB EXPRESSION1 (TOC1) feedback loop because, under free run, ELF4 is required to regulate the expression of CCA1 and TOC1 and, further, elf4 is locked in the evening phase of this feedback loop. ELF4, therefore, can be considered a component of the central CCA1/LHY-TOC1 feedback loop in the plant circadian clock.

Ambient Thermometers in Plants: From Physiological Outputs towards Mechanisms of Thermal Sensing

Plants respond to ambient temperature changes over a series of timescales. Genetic and physiological studies over the last decades have revealed myriad thermally sensitive pathways in plants. A recent study provides a genetic and biochemical mechanistic description of how thermal changes can be transduced to influence gene expression. What remains to be revealed in this, and other thermally controlled responses, is a description of the primary temperature-sensing event. Cooling and warming alter membrane fluidity and elicit intracellular free-calcium elevations, a process that has been considered the primary event controlling plant responses to temperature. Such direct thermal sensors appear to process temperature information. Future efforts will be required to identify the effector proteins linking perception to response. This review considers the evidence for plant thermometers to date, provides a description of several notable physiological and developmental processes under ambient temperature control, and outlines major questions that remain to be addressed in the understanding of thermometers in plants.

Mutation at the circadian clock gene EARLY MATURITY 8 adapts domesticated barley (Hordeum vulgare) to short growing seasons

The circadian clock is an autonomous oscillator that produces endogenous biological rhythms with a period of about 24 h. This clock allows organisms to coordinate their metabolism and development with predicted daily and seasonal changes of the environment. In plants, circadian rhythms contribute to both evolutionary fitness and agricultural productivity. Nevertheless, we show that commercial barley varieties bred for short growing seasons by use of early maturity 8 (eam8) mutations, also termed mat-a, are severely compromised in clock gene expression and clock outputs. We identified EAM8 as a barley ortholog of the Arabidopsis thaliana circadian clock regulator EARLY FLOWERING3 (ELF3) and demonstrate that eam8 accelerates the transition from vegetative to reproductive growth and inflorescence development. We propose that eam8 was selected as barley cultivation moved to high-latitude short-season environments in Europe because it allowed rapid flowering in genetic backgrounds that contained a previously selected late-flowering mutation of the photoperiod response gene Ppd-H1. We show that eam8 mutants have increased expression of the floral activator HvFT1, which is independent of allelic variation at Ppd-H1. The selection of independent eam8 mutations shows that this strategy facilitates short growth-season adaptation and expansion of the geographic range of barley, despite the pronounced clock defect.

Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering

A main developmental switch in the life cycle of a flowering plant is the transition from vegetative to reproductive growth. In Arabidopsis thaliana, distinct genetic pathways regulate the timing of this transition. We report here that brassinosteroid (BR) signaling establishes an unexpected and previously unidentified genetic pathway in the floral-regulating network. We isolated two alleles of brassinosteroid-insensitive 1 (bri1) as enhancers of the late-flowering autonomous-pathway mutant luminidependens (ld). bri1 was found to predominantly function as a flowering-time enhancer. Further analyses of double mutants between bri1 and known flowering-time mutants revealed that bri1 also enhances the phenotype of the autonomous mutant fca and of the dominant FRI line. Moreover, all of these double mutants exhibited elevated expression of the potent floral repressor FLOWERING LOCUS C (FLC). This molecular response could be efficiently suppressed by vernalization, leading to accelerated flowering. Additionally, specific reduction of the expression of FLC via RNA interference accelerated flowering in bri1 ld double mutants. Importantly, combining the BR-deficient mutant cpd with ld also resulted in delayed flowering and led to elevated FLC expression. Finally, we found increased histone H3 acetylation at FLC chromatin in bri1 ld mutants, as compared with ld single mutants. In conclusion, we propose that BR signaling acts to repress FLC expression, particularly in genetic situations, with, for example, dominant FRI alleles or autonomous-pathway mutants, in which FLC is activated.

TIME FOR COFFEE Represses Accumulation of the MYC2 Transcription Factor to Provide Time-of-Day Regulation of Jasmonate Signaling in Arabidopsis

The morning-acting clock factor TIME FOR COFFEE (TIC) was found to work in the jasmonate (JA) hormone pathway through a protein-depletion role at the positive JA regulator MYC2. The Arabidopsis thaliana circadian clock thus allows JA hormone responses to be phased in the morning. Plants are confronted with predictable daily biotic and abiotic stresses that result from the day–night cycle. The circadian clock provides an anticipation mechanism to respond to these daily stress signals to increase fitness. Jasmonate (JA) is a phytohormone that mediates various growth and stress responses. Here, we found that the circadian-clock component TIME FOR COFFEE (TIC) acts as a negative factor in the JA-signaling pathway. We showed that the tic mutant is hypersensitive to growth-repressive effects of JA and displays altered JA-regulated gene expression. TIC was found to interact with MYC2, a key transcription factor of JA signaling. From this, we discovered that the circadian clock rhythmically regulates JA signaling. TIC is a key determinant in this circadian-gated process, and as a result, the tic mutant is defective in rhythmic JA responses to pathogen infection. TIC acts here by inhibiting MYC2 protein accumulation and by controlling the transcriptional repression of CORONATINE INSENSITIVE1 in an evening-phase–specific manner. Taken together, we propose that TIC acts as an output component of the circadian oscillator to influence JA signaling directly.