title: Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways.
abstract: The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (early flowering4; ELF4 and pseudoresponse regulator3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4-211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with early flowering3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4-211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range.
authors.affiliation: From the ‡Division of Integrative Biosciences and Biotechnology, POSTECH, Hyojadong, Pohang, Kyungbuk, 790-784, Republic of Korea.
authors.forename: Mani Kant
authors.initials: MK
authors.lastname: Choudhary
authors.affiliation: ¶Plant Proteomics Research Unit, RIKEN Center for Sustainable Resource Science (CSRS), Yokohama, Kanagawa, 230-0045, Japan;
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nomura
authors.affiliation: From the ‡Division of Integrative Biosciences and Biotechnology, POSTECH, Hyojadong, Pohang, Kyungbuk, 790-784, Republic of Korea §Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210; ‖Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: ¶Plant Proteomics Research Unit, RIKEN Center for Sustainable Resource Science (CSRS), Yokohama, Kanagawa, 230-0045, Japan;
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nakagami
authors.affiliation: From the ‡Division of Integrative Biosciences and Biotechnology, POSTECH, Hyojadong, Pohang, Kyungbuk, 790-784, Republic of Korea §Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210; somers.24@osu.edu.
authors.forename: David E
authors.initials: DE
authors.lastname: Somers
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020346:Aquaporins / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology* / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057906:Circadian Clocks / Q000528:radiation effects; D002940:Circadian Rhythm / Q000528:radiation effects; D016000:Cluster Analysis; D030562:Databases, Protein; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects; D063990:Gene Ontology; D008027:Light; D053858:Metabolic Networks and Pathways / Q000528:radiation effects; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D014176:Protein Biosynthesis / Q000528:radiation effects; D060066:Protein Interaction Maps / Q000528:radiation effects; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000528:radiation effects
chemical_list: D020346:Aquaporins; D029681:Arabidopsis Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D012333:RNA, Messenger