Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry

Authors

He Huang, Donald Danforth Plant Science Center
Sophie Alvarez, University of Nebraska-LincolnFollow
Rebecca Bindbeutel, Donald Danforth Plant Science Center
Zhouxin Shen, University of California - San Diego
Michael J. Naldrett, University of Nebraska-LincolnFollow
Bradley S. Evans, Donald Danforth Plant Science CenterFollow
Steven P. Briggs, University of California - San Diego
Leslie M. Hicks, University of North Carolina at Chapel Hill
Steve A. Kay, University of Southern California
Dmitri A. Nusinow, Donald Danforth Plant Science CenterFollow

Document Type

Article

Date of this Version

2016

Citation

Molecular & Cellular Proteomics 15.1, 201–217

Comments

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI: 10.1074/mcp.M115.054064

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.