This proposal is designed to investigate three different post-translational mechanisms of circadian clock control in Arabidopsis. The first is the role of protein phosphorylation, and the effect on activity, localization and stability of key clock proteins. Robust-phase specific phosphorylation of five key proteins in the Arabidopsis clock raises the question of its significance in their activity, localization and turnover. We will focus on the first validated phosphorylation-dependent protein-protein interaction in the Arabidopsis clock to begin to understand the dynamics of TOC1 and PRR3 post-translationally. Additionally, we propose a novel approach to discovering new kinases and phosphatases that act on clock proteins. We also propose to begin modeling this portion of the circadian system to address circuitry of the clock that is not based on transcription. The second is the effect chaperonins play in the maturation of the F-box protein ZEITLUPE to its functional state, necessary to the maintenance of robust circadian amplitude and period. We have identified two components new to any circadian system which likely contribute to ZTL maturation. Through effects on ZTL, these two components indirectly regulate the circadian system. The role of these two proteins in obtaining fully active ZTL, and their potential interaction is the underlying question addressed. The third mechanism is the role of the nuclear pore in the regulation of nuclear import/export of mRNA and/or protein of clock genes. The nuclear pore acts as a gatekeeper to the nucleus and all transcription factors and other regulators of nuclear function must pass through this highly complex structure. All eukaryotic circadian systems involve nucleocytoplasmic shuttling of mRNA and protein which may contribute substantially to establishing the timing delays necessary to establishing a 24 h molecular periodicity. We have identified a nuclear pore component that slows the circadian clock when absent. Understanding the molecular basis of this delay should lead to a greater understanding of how circadian timing is tied to intracellular transport. In a related way, the TOC1/PRR5 interaction appears to facilitate TOC1 nuclear entry and may also serve to recruit a kinase to the interaction. The molecular basis of this interaction and the effect on period will lead to increased understanding of the role of regulated nuclear entry in the clock. While the early heuristic models of the clock focused on transcription/translation feedback loops, recent findings across all circadian systems have highlighted the inadequacy of this view and how post- translational mechanisms contribute substantially to sustaining circadian oscillation. The studies described below will contribute to a greater understanding of circadian clock in particular, and to oscillatory feedback systems in general.

Public Health Relevance

Circadian rhythms control a wide variety of processes in all eukaryotes, including recent linkages to the cell cycle and cancer. The fundamental organization of the clock as consisting of two or more, linked autoregulatory feedback loops is shared among all model systems, but the individual molecular players often vary among the kingdoms. Here post-translational mechanism of circadian clock control in Arabidopsis is investigated, with potential relevance to other eukaryotic oscillatory systems.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Sesma, Michael A
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Ohio State University
Other Basic Sciences
Schools of Arts and Sciences
United States
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Foo, Mathias; Somers, David E; Kim, Pan-Jun (2016) Kernel Architecture of the Genetic Circuitry of the Arabidopsis Circadian System. PLoS Comput Biol 12:e1004748
Choudhary, Mani Kant; Nomura, Yuko; Wang, Lei et al. (2015) Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways. Mol Cell Proteomics 14:2243-60
Liu, Hongtao; Wang, Qin; Liu, Yawen et al. (2013) Arabidopsis CRY2 and ZTL mediate blue-light regulation of the transcription factor CIB1 by distinct mechanisms. Proc Natl Acad Sci U S A 110:17582-7
Kim, Yumi; Lim, Junhyun; Yeom, Miji et al. (2013) ELF4 regulates GIGANTEA chromatin access through subnuclear sequestration. Cell Rep 3:671-7
Kim, Jeongsik; Geng, Ruishuang; Gallenstein, Richard A et al. (2013) The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA. Development 140:4060-9
Wang, Lei; Kim, Jeongsik; Somers, David E (2013) Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription. Proc Natl Acad Sci U S A 110:761-6
Kim, Yumi; Han, Seungmin; Yeom, Miji et al. (2013) Balanced nucleocytosolic partitioning defines a spatial network to coordinate circadian physiology in plants. Dev Cell 26:73-85
Somers, David E (2012) The Arabidopsis clock: time for an about-face? Genome Biol 13:153
Kim, Yumi; Yeom, Miji; Kim, Hyunmin et al. (2012) GIGANTEA and EARLY FLOWERING 4 in Arabidopsis exhibit differential phase-specific genetic influences over a diurnal cycle. Mol Plant 5:678-87
Kim, Tae-sung; Kim, Woe Yeon; Fujiwara, Sumire et al. (2011) HSP90 functions in the circadian clock through stabilization of the client F-box protein ZEITLUPE. Proc Natl Acad Sci U S A 108:16843-8

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