Abstract
As an adaptation to life in a world with predictable daily changes, most eukaryotes and some prokaryotes have endogenous circadian (approximately 24 h) clocks. In plants, the circadian clock regulates a diverse range of cellular and physiological events from gene expression and protein phosphorylation to cellular calcium oscillations, hypocotyl growth, leaf movements, and photoperiod-dependent flowering. In Arabidopsis (Arabidopsis thaliana), as in other model organisms, such as Drosophila (Drosophila melanogaster) and mice, circadian rhythms are generated by molecular oscillators that consist of interlocking feedback loops involving a number of elements. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYLS (LHY) are closely related single myb transcription factors that have been identified as key elements in the Arabidopsis oscillator. Research in other model organisms has shown that posttranslational regulation of oscillator components plays a critical role in the generation of the approximately 24-h cycles. To examine the role of posttranslational regulation of CCA1 and LHY in the Arabidopsis oscillator, we generated transgenic plants with tagged CCA1 and LHY under the control of their own promoters. We have shown that these tagged proteins are functional and can restore normal circadian rhythms to CCA1- and LHY-null plants. Using the tagged proteins, we demonstrate that CCA1 can form both homodimers and heterodimers with LHY. Furthermore, we also show that CCA1 is localized to the nucleus in vivo and that there is no significant delay between the translation of CCA1 and its translocation to the nucleus. We discuss our findings in the context of the functioning of the Arabidopsis oscillator.
Highlights
As an adaptation to life in a world with predictable daily changes, most eukaryotes and some prokaryotes have endogenous circadian clocks
The model of the Arabidopsis oscillator consists of interlocking feedback loops of several components, including CIRCADIAN CLOCK ASSOCIATED1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB EXPRESSION1 (TOC1)
Our results show that the levels of CCA1-HA-yellow fluorescent protein (YFP) protein in both transgenic lines reach a maximum at 1.5 h after lights-on (Fig. 1C)
Summary
As an adaptation to life in a world with predictable daily changes, most eukaryotes and some prokaryotes have endogenous circadian (approximately 24 h) clocks. The model of the Arabidopsis oscillator consists of interlocking feedback loops of several components, including CIRCADIAN CLOCK ASSOCIATED1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB EXPRESSION1 (TOC1). Overexpression of CCA1 or LHY in transgenic Arabidopsis plants abrogates the circadian rhythmicity of clock-controlled processes, including gene expression and leaf movements (Schaffer et al, 1998; Wang and Tobin, 1998; Thain et al, 2004). Posttranslational Regulation of CCA1 ments than wild-type plants, while plants with mutations in both genes appear to be unable to maintain sustained oscillations (Green and Tobin, 1999; Alabadi et al, 2002; Mizoguchi et al, 2002) Despite their homology, CCA1 and LHY may have somewhat different roles in the oscillator mechanism (Gould et al, 2006; Zeilinger et al, 2006). A combination of experimental and mathematical modeling techniques (Locke et al, 2006; Zeilinger et al, 2006) have supported the idea that other morning and evening genes, such as PRR7, PRR9, and GI, are involved in the oscillator, possibly by forming two other additional feedback loops interacting with the CCA1/LHY/TOC1 loop
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