title: Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry.
abstract: Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways.
authors.affiliation: From the ‡Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, Missouri, 63132;
authors.forename: He
authors.identifier: http://orcid.org/0000-0002-4564-7604
authors.initials: H
authors.lastname: Huang
authors.affiliation: From the ‡Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, Missouri, 63132;
authors.forename: Sophie
authors.identifier: http://orcid.org/0000-0001-8550-2832
authors.initials: S
authors.lastname: Alvarez
authors.affiliation: From the ‡Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, Missouri, 63132;
authors.forename: Rebecca
authors.identifier: http://orcid.org/0000-0001-7994-2073
authors.initials: R
authors.lastname: Bindbeutel
authors.affiliation: §University of California San Diego, Division of Biological Sciences, Cell and Developmental Biology Section, 9500 Gilman Drive, La Jolla, California 92093-0116;
authors.forename: Zhouxin
authors.initials: Z
authors.lastname: Shen
authors.affiliation: From the ‡Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, Missouri, 63132;
authors.forename: Michael J
authors.identifier: http://orcid.org/0000-0002-6899-5652
authors.initials: MJ
authors.lastname: Naldrett
authors.affiliation: From the ‡Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, Missouri, 63132;
authors.forename: Bradley S
authors.identifier: http://orcid.org/0000-0002-1207-9006
authors.initials: BS
authors.lastname: Evans
authors.affiliation: §University of California San Diego, Division of Biological Sciences, Cell and Developmental Biology Section, 9500 Gilman Drive, La Jolla, California 92093-0116;
authors.forename: Steven P
authors.identifier: http://orcid.org/0000-0002-7226-8618
authors.initials: SP
authors.lastname: Briggs
authors.affiliation: ¶The University of North Carolina at Chapel Hill, Department of Chemistry, Chapel Hill, North Carolina 27599;
authors.forename: Leslie M
authors.identifier: http://orcid.org/0000-0002-8008-3998
authors.initials: LM
authors.lastname: Hicks
authors.affiliation: ‖University of Southern California, Molecular and Computational Biology Section, Los Angeles, California 90089.
authors.forename: Steve A
authors.initials: SA
authors.lastname: Kay
authors.affiliation: From the ‡Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, Missouri, 63132; meter@danforthcenter.org.
authors.forename: Dmitri A
authors.identifier: http://orcid.org/0000-0002-0497-1723
authors.initials: DA
authors.lastname: Nusinow
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D002940:Circadian Rhythm; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D055537:Light Signal Transduction / Q000235:genetics; D018613:Microscopy, Confocal; D009154:Mutation; D030821:Plants, Genetically Modified; D011485:Protein Binding; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D049452:Green Fluorescent Proteins; D020543:Proteome; D014157:Transcription Factors; C432430:At2g25930 protein, Arabidopsis; C465973:ELF4 protein, Arabidopsis; C504848:Lux Arrhythmo protein, Arabidopsis