Timing and the Plant Circadian System
Introduction
Circadian clocks are prevalent biological timing mechanisms used by organisms to predicatively regulate physiology to the anticipated environmental changes present that occur as a consequence of the ever-present day-night cycle. In plants, this clock regulates a suite of developmental and metabolic processes. In fact, around 10% of all transcripts have been found to be clock regulated. In this way, the circadian timer drives molecular outputs in the establishment of fitness in physiological processes and developmental timing. Molecular-genetic analysis has lead to an understanding of the core elements that make up the clock. Mutants that are clock defective have been used to identify loci critical for normal rhythmicity. Intriguingly, many of these clock genes are reciprocally regulated within the clock. Using such molecular-expression analyses in various clock mutants, the first model that partially explained mutant behavior was described. In this model, TIMING OF CAB2 EXPRESSION 1 (TOC1) serves as a positive factor that regulates expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), related genes that both encode Myb-domain containing DNA-binding proteins. CCA1 and LHY in turn function as partially redundant negative factors in TOC1 regulation. As it was noted that this model was oversimplified, mathematical approaches lead to two-loop models, and now a three/four loop model.
The clock is an inter-connective feedback loop that coordinates with parallel-signaling systems. Various inputs to the core include entrainment cues from the light environment and from hormone signaling that fine-tunes oscillator activity. With regard to entrainment regulation, various clock-input factors are required for normal perception of dawn and dusk. As for example, the morning-acting factor TIME FOR COFFEE (TIC) regulates dawn inputs, whereas the factors EARLY FLOWERING 4 (ELF4) and EARLY FLOWERING 3 (ELF3) are key for dusk inputs to the clock. Collectively these input factors regulate clock resetting in response to changing light environments in a process termed re-entrainment. In contrast to this large effect process, hormonal inputs regulate more minor responses to the clock. In particular, and very excitingly, various hormonal-signaling pathways generate specific parameter modulation. It is believed that here the clock is adjusted so that the plant can hone daily anticipatory behaviors in a maximal way to the local abiotic and biotic environment. Collectively, input mechanisms are thus key for normal circadian function in a normal cycling environment, such as that of the rotating earth.
Our group has spent the last years working on three facets of the biological timing in plants. These are on the core-clock mechanism, light input to the clock, and hormonal regulation of biological timing. This has come to particular fruition in the last two years. These themes continue in ongoing research, and in the future, we will include new technical approaches that will be inclusive of our core-oscillator and light input studies as we redefine our efforts in the understanding of biological time keeping.
New findings
Entrainment and the core oscillator:
CCA1 and LHY have been proposed, together with TOC1, to make up the central oscillator of the circadian clock (Davis 2001b; Davis 2002; Davis 2005; Hanano 2005; Kevei 2006). We tested the various clock models genetically. Collectively, we provided direct experimental support for previous modeling efforts where CCA1/LHY along with TOC1 drive the circadian oscillator, and have shown that this clock is essential for correct output regulation (Ding 2007a).
TIC is required for morning activity of the oscillator (Hall 2003). To further investigate how TIC functions within the circadian system, we introduced various circadian markers into the tic mutant and measured evident rhythms. Morning-acting-clock genes were found as transcriptional targets. Unexpectedly we found TIC has a closer relationship with LHY than with CCA1. Epistasis analysis was used to confirm this relationship. To better understand the molecular mechanism of TIC function, we isolated this gene. We found that TIC encodes a pioneer protein continuously present over the diurnal cycle, and that it is strictly nuclear localized. We suggested that TIC encodes a nuclear-acting clock regulator working close to the central oscillator (Ding 2007b).
We previously characterized ELF4 as being important for robust rhythms (Doyle 2002). New efforts were reported in which we showed that ELF4 is necessary for two core-clock functions: entrainment to an environmental cycle, and rhythm sustainability. The elf4 mutant demonstrates clock-input defects in light responsiveness, and thus regulates entrainment. Further, ELF4 was found to be intimately associated with the CCA1/LHY-TOC1 feedback loop, and therefore ELF4 can be considered a component of the clock (McWatters 2007). We extended this further where we proposed how ELF4 functions as a bimodal regulator of the oscillator (Kolmos 2007).
Input/output regulation of hormonal and floral connectivity in biological timing:
We investigated phytohormone effects on plant-circadian rhythms, and found that many phytohormones 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 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. 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 (Hanano 2006). Genomic tests for transcriptional regulators in said processes lead to targeted definitions of controlling factors (Hanano 2007; Hanano, submitted)
A main developmental switch in the life cycle of a flowering plant is the transition from vegetative to reproductive growth. Distinct genetic pathways regulate the timing of this transition. We reported that brassinosteroid (BR) signaling establishes an unexpected and previously unidentified genetic pathway in the developmental-timing transition. The collective conclusion was our proposal that BR signaling acts to repress FLOWERING LOCUS C (FLC) through chromatin remodeling, particularly in genetic situations where FLC is activated (Domagalska 2007a, 2007b). Further studies lead us to the non-establishment of hormone interactions in the developmental transition (Domagalska, submitted).
Previous studies of natural-genetic variation implicated FLC as a circadian-clock regulator. As FLC is best known as a developmental-timing regulator, whose expression is modulated by genes in the autonomous and vernalization pathways, we tested whether these same pathways affect the circadian system. Each pathway was found to regulate circadian period. The genetic mechanisms involved were found to be similar, but not identical, to the control of flowering time. We also revealed an unexpected vernalization-dependent alteration of the clock. This study was used to show that the network affecting circadian timing is partially overlapping with the floral-regulatory network (Salathia 2006).
