Complexity in the wiring and regulation of plant circadian networks

Abstract

Endogenous circadian rhythms regulate many aspects of an organism's behavior, physiology and development. These daily oscillations synchronize with the environment to generate robust rhythms, resulting in enhanced fitness and growth vigor in plants. Collective studies over the years have focused on understanding the transcription-based oscillator in Arabidopsis. Recent advances combining mechanistic data with genome-wide approaches have contributed significantly to a more comprehensive understanding of the molecular interactions within the oscillator, and with clock-controlled pathways. This review focuses on the regulatory mechanisms within the oscillator, highlighting key connections between new and existing components, and direct mechanistic links to downstream pathways that control overt rhythms in the whole plant.

Figure 1. Monitoring clock function underlying oscillator and output phenotypes

Endogenous circadian rhythms were first observed by daily leaf movement in plants. Genetic and biochemical approaches were then instrumental in discovering the regulatory units responsible for these rhythms. Subsequently, forward and reverse genetic approaches were used to identify additional components and monitor clock function. A) A 24 hour (24 hr) period of diurnal cycles (12 hr light: 12 hr dark) are often used to entrain the clock, and subsequently released to free running conditions (circadian) of continuous light. Clock gene promoter fusions to the firefly luciferase gene (LUC) are imaged over a period of several days to monitor altered clock phenotypes based on bioluminescence (Luminescence). B) Alteration in clock function is reflected in rhythmic changes of the 24 hr periodicity (short, long or arrhythmic). Loss of function (lower cased) or constitutive expression (upper cased - overexpression) confers these changes in period. Black sinusoidal waves represent normal circadian oscillations. Green dashed and blue dashed waves represent short and long period phenotypes respectively. Circadian oscillations can also be abolished (arrhythmic - red dashed lines). Alterations in clock gene expression are also reflected in changes in phase and amplitude. C) An altered clock confers changes in clock-controlled outputs. The circadian clock regulates hypocotyl elongation; as such loss of function or overexpression of clock genes confers short (S) or long (L) hypocotyls. In Arabidopsis, photoperiod flowering is dependent on day length. Long days (16 hr of light and 8 hr of dark) promote flowering, and short days (8 hr of light and 16 hr of dark) delay flowering. Loss of function or overexpression of clock genes confers either early flowering (EF) or late flowering (LF) relative to wild-type (WT).

Figure 2. A model for transcription based interactions in the Arabidopsis clock network

In vitro and invivo assays were instrumental in validating direct molecular interactions between oscillator components. The core of the oscillator consists of two Myb transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB EXPRESSION 1 (TOC1). Other components expressed throughout the day interconnect with the core oscillator to form multiple feedback loops and a complex clock network. CCA1 and LHY directly repress TOC1, LUX, GI, ELF3, ELF4, CHE, JMJD5 (also known as JMJD30), and NOX (also known as BROTHER OF LUX ARRHYTHMO) by binding to their promoters. In return, TOC1, LUX, GI, ELF3, positively regulates CCA1 and LHY via an unknown mechanism. NOX directly activates CCA1 by binding to its promoter. LUX binds to its promoter and repress its own expression (indicated by black dashed lines). CHE and JMJD5 function as a direct repressor of CCA1. TOC1 inhibits the expression of CCA1, LHY, PRR9, PRR7, PRR5, LUX, ELF4, and GI. Sequential expression of PRR9, PRR7, and PRR5 directly inhibit the expression of CCA1 and LHY. In turn, PRR9 and PRR7 are positively regulated by CCA1 and LHY. PRR9 and PRR5 are also positively regulated by LWD1 and PRR5, respectively. For simplification, other components that affect clock function such as PRR3, TIC, and SRR1 are not illustrated in the figure above. Though not illustrated in the above figure, protein-protein interactions often occur between clock components and are an important mechanism regulating clock function. CCA1 and LHY physically interact. TOC1 interacts with CHE and PRR5, and interacts with JMJD5 genetically. LUX interacts with ELF3 and ELF4 to form the evening complex (EC). Direct mechanistic connections exist between clock components and modulators of physiological processes. The EC regulates hypocotyl growth by directly binding to the promoters of PIF4 and PIF5. Direct interaction between GI, CO, and FT, and GI and FT, modulates photoperiod flowering. Arrows represents transcriptional activation, and horizontal lines represent repression. Dashed lines in grey indicate the protein and gene associations.

Figure 3. Multiple layers of regulation within the Oscillator

Post-transcriptional and posttranslational-based regulation are mechanisms underlying robust clock function. Alternative splicing events have been observed for CCA1, LHY, TOC1, PRR5, PRR7, PRR9, and GI. PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5) a protein involved in methylation of histones, RNA binding and spliceosomal proteins, is required for the alternative splicing of PRR9. Casein Kinase2 (CK2), an evolutionarily conserved serine/threonine protein kinase, phosphorylates both CCA1 and LHY; and SINAT5, a E3 ubiquitin ligase is involved in the ubiquitination of LHY. PRR5 and TOC1 are specifically targeted for proteosome dependent degradation by the E3 ubiquitin ligase Skp/Cullin/F-box (SCF) complex members ZEITLUPE (ZTL), FLAVIN BINDING KELCH F-BOX 1 (FKF1) and LOV KELCH PROTEIN 2 (LKP2). Other oscillator components PRR3, PRR7, PRR9, and GI are subjected to proteosome degradation, though the mechanism is unknown. A role for chromatin remodeling in regulating clock gene expression and function is exist for a few oscillator components. TOC1 expression correlates with histone 3 (H3) acetylation (Ac). However, TOC1, CCA1, LHY and GI expression also correlates with H3 lysine 9 acetylation (K9Ac), and H3 lysine 4 (K4) dimetylation (Me2), via an unknown mechanism. H3Ac, H3K9Ac, and H3K4Me2 are all defined as marks for gene activation. For simplicity, PRR3 is not included in the above illustration though alternative spliced transcripts have been detected for this component.