Posttranslational Regulation of CIRCADIAN CLOCK ASSOCIATED1 in the Circadian Oscillator of Arabidopsis    

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.The circadian, approximately 24 h, clock has an enormous influence on the biology of plants and controls a plethora of processes, including hypocotyl growth, shade avoidance, leaf movements, scent production, and stomatal opening (Yakir et al., 2007). Consistent with the role of the circadian clock in the regulation of a wide range of activities, transcription from approximately one-third of the genome (including noncoding genes) of the model plant Arabidopsis (Arabidopsis thaliana) is under circadian control (Michael and McClung, 2003; Covington et al., 2008;Michael et al., 2008;Hazen et al., 2009). In addition, the circadian clock serves as a timekeeper to regulate daylengthdependent processes, such as flowering time and tuberization. The circadian systems responsible for generating circadian rhythms are ubiquitous in eukaryotes and have also been found in some prokaryotes (Dunlap, 1999). Conceptually, a circadian system can be divided into three parts: the oscillator mechanism, input pathways, and output pathways. Interestingly, although their components differ, the basic oscillator mechanism appears to be well conserved in all the eukaryotic model organisms that have been studied (Dunlap, 2006). Oscillators are comprised of interlocking positive/negative feedback loops made from clock proteins that control t...

Circadian clock gene LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia

“…Although the circadian oscillation patterns of PhLHY vary depending on tissues and light conditions, the daily expression patterns of PhLHY resemble those of LHY/CCA1. Next we examined the intracellular localization of PhLHY protein, as its homologs CCA1 and LHY are nuclear-localized proteins (22,23). In comparing the intracellular localization patterns of the PhLHY-GFP protein with that of RFP-tagged histone 2B (H2B), PhLHY-GFP localized in the nuclei of Petunia flower and leaf cells (Fig.…”

LIGHT-REGULATED WD1 and PSEUDO-RESPONSE REGULATOR9 Form a Positive Feedback Regulatory Loop in the Arabidopsis Circadian Clock

“…The clock mutants used in this study were cca1-1 (Yakir et al, 2009), lhy-101 (Khanna et al, 2006), toc1-101 (Kaczorowski, 2004;Kikis et al, 2005), elf4-101 (Khanna et al, 2003), gi-2 (Park et al, 1999), prr5-11, prr7-11 (Yamamoto et al, 2003, and prr9-10 (Ito et al, 2003). The results of the LWD1 and LWD2 promoter activity assay with each mutant are shown in Figure 5.…”

Unraveling the circadian clock inArabidopsis

“…15,54 The homodimerization and heterodimerization of CCA1α and LHY are important for the ability of these proteins to regulate circadian rhythms. 54,81,82 The splice variant CCA1β represses CCA1α and LHY activity by competitively forming nonfunctional CCA1α/CCA1β and CCA1β/LHY heterodimers, creating a self-regulatory circuit for CCA1 and LHY through the alternative splicing of CCA1. 80 Thus, transcription factors can auto-regulate their expression by generating competitive inhibitors through alternative splicing.…”