The circadian clock is an endogenous mechanism that coordinates biological processes with daily and seasonal changes in the environment. Heterodimerization of central clock components is an important way of controlling clock function in several different circadian systems. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are Myb-related proteins that function in or close to the central oscillator in Arabidopsis (Arabidopsis thaliana). Single mutants of cca1 and lhy have a phenotype of short-period rhythms. cca1 lhy double mutants show an even shorter period phenotype than the cca1 single mutant, suggesting that CCA1 and LHY are only partially functionally redundant. To determine whether CCA1 and LHY act in parallel or synergistically in the circadian clock, we examined their expression in both light-grown and etiolated seedlings. We have shown that LHY and CCA1 bind to the same region of the promoter of a Light-harvesting chlorophyll a/b protein (Lhcb, also known as CAB). CCA1 and LHY can form homodimers, and they also colocalize in the nucleus and heterodimerize in vitro and in vivo. In Arabidopsis, CCA1 and LHY physically interact in a manner independent of photoperiod. Moreover, results from gel filtration chromatography indicate that CCA1 and LHY are present in the same large complex in plants. Taken together, these results imply that CCA1 and LHY function synergistically in regulating circadian rhythms of Arabidopsis.

The circadian clock is an endogenous oscillator with a period of approximately 24 h that allows organisms to anticipate, and respond to, changes in the environment. In Arabidopsis (Arabidopsis thaliana), the circadian clock regulates a wide variety of physiological processes, including hypocotyl elongation and flowering time. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a central clock component, and CCA1 overexpression causes circadian dysfunction, elongated hypocotyls, and late flowering. EARLY FLOWERING3 (ELF3) modulates light input to the clock and is also postulated to be part of the clock mechanism. elf3 mutations cause light-dependent arrhythmicity, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is not clear. Here, we show that CCA1 represses ELF3 by associating with its promoter, completing a CCA1-ELF3 negative feedback loop that places ELF3 within the oscillator. We also show that ELF3 acts downstream of CCA1, mediating the repression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5 in the control of hypocotyl elongation. In the regulation of flowering, our findings show that ELF3 and CCA1 either cooperate or act in parallel through the CONSTANS/ FLOWERING LOCUS T pathway. In addition, we show that CCA1 represses GIGANTEA and SUPPRESSOR OF CONSTANS1 by direct interaction with their promoters, revealing additional connections between the circadian clock and the flowering pathways.

Histone methylation plays an essential role in regulating chromatin structure and gene expression. Jumonji C (JmjC) domaincontaining proteins are generally known as histone demethylases. Circadian clocks regulate a large number of biological processes, and recent studies suggest that chromatin remodeling has evolved as an important mechanism for regulating both plant and mammalian circadian systems. Here, we analyzed a subgroup of JmjC domain-containing proteins and identified Arabidopsis (Arabidopsis thaliana) JMJ30 as a novel clock component involved in controlling the circadian period. Analysis of loss-and gain-of-function mutants of JMJ30 indicates that this evening-expressed gene is a genetic regulator of period length in the Arabidopsis circadian clock. Furthermore, two key components of the central oscillator of plants, transcription factors CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL, bind directly to the JMJ30 promoter to repress its expression, suggesting that JMJ30 regulates the pace of the circadian clock in close association with the central oscillator. JMJ30 represents, to our knowledge, the first JmjC domain-containing protein involved in circadian function, and we envision that this provides a possible molecular connection between chromatin remodeling and the circadian clock.

The circadian clock regulates the expression of an array of Arabidopsis genes such as those encoding the LIGHT-HARVESTING CHLOROPHYLL A/B (Lhcb) proteins. We have previously studied the promoters of two of these Arabidopsis genes--Lhcb1*1 and Lhcb1*3--and identified a sequence that binds the clock protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). This sequence, designated CCA1-binding site (CBS), is necessary for phytochrome and circadian responsiveness of these genes. In close proximity to this sequence, there exists a G-box core element that has been shown to bind the bZIP transcription factor HY5 in other light-regulated plant promoters. In the present study, we examined the importance of the interaction of transcription factors binding the CBS and the G-box core element in the control of normal circadian rhythmic expression of Lhcb genes. Our results show that HY5 is able to specifically bind the G-box element in the Lhcb promoters and that CCA1 can alter the binding activity of HY5. We further show that CCA1 and HY5 can physically interact and that they can act synergistically on transcription in a yeast reporter gene assay. An absence of HY5 leads to a shorter period of Lhcb1*1 circadian expression but does not affect the circadian expression of CATALASE3 (CAT3), whose promoter lacks a G-box element. Our results suggest that interaction of the HY5 and CCA1 proteins on Lhcb promoters is necessary for normal circadian expression of the Lhcb genes.

Circadian rhythms are generated by endogenous central oscillators that respond to input from the environment and regulate rhythmic outputs. In Arabidopsis, more than a dozen components that affect rhythms have been identified and used to propose models of the central oscillator. However, none has been shown to fulfill one of the expected characteristics of an oscillator component: that a pulse of its expression shifts the phase of circadian rhythms. Here we show that a pulse of the proposed oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) causes dramatic phase shifts in rhythms of expression of the circadian reporter CAB2∷LUC, as well as of the clock-associated genes TIMING OF CAB EXPRESSION 1 (TOC1) and GIGANTEA (GI). These results demonstrate that pulses of either CCA1 or LHY are capable of resetting the circadian clock. In contrast, a pulse of TOC1 expression did not elicit phase shifts. Control of TOC1 protein level is in part posttranscriptional; thus a pulse of TOC1 protein could be induced only at times when it is already high. Our work also shows that the ethanol-inducible system can be useful for achieving relatively short (<8 h) pulses of gene expression in seedlings.