Minisymposium 7: Protein turnover

Abs # 17005: The Arabidopsis F-Box Proteins EBF1 and 2 Form SCF E3s That Repress Ethylene Action and Promote Growth By Directing EIN3 Degradation.

Presenter:	Gagne, Jennifer M., jmgagne@wisc.edu
Authors	Gagne, Jennifer M. (A)   Binder, Brad M. (B)   Smalle, Jan  (A)   Gingerich, Derek J. (A)   Walker, Joseph M. (A)   Bleecker, Anthony B. (B)   Vierstra, Richard D. (A)
Affiliations:	(A): Department of Genetics, University of Wisconsin-Madison (B): Department of Botany, University of Wisconsin-Madison

Ubiquitination of intracellular proteins by ubiquitin protein ligases (or E3s) plays an essential role in eukaryotic cell regulation, primarily through its ability to selectively target proteins for degradation by the 26S proteasome. SCF complexes are one influential E3 class that use F-Box proteins to deliver targets to a core ligase activity provided by the Skp1, Cullin and Rbx1 subunits. Almost 700 F-Box proteins have been identified in Arabidopsis, indicating that SCF E3s likely play a pervasive role in plant physiology and development. Here, we describe the reverse genetic analysis of two F-Box proteins EIN3-binding F-box (EBF)-1 and -2 that work coordinately in SCF complexes to repress ethylene action. Phenotypic and molecular analyses show that ebf1 and ebf2 mutants are hypersensitive to exogenous ethylene and its precursor ACC. EBF1 and 2 interact directly with ETHYLENE INSENSITIVE-3 (EIN3), a transcriptional regulator important for ethylene signaling suggesting that EIN3 is a target of these E3s. In support of this, EIN3 levels are increased in mutants affecting either EBF1 or 2. Double ebf1 ebf2 mutants display a substantial arrest of seedling growth including swollen hypocotyls, an inhibition of cotyledon expansion, ectopic emergence of root hairs, a strong anthocyanin accumulation and accelerated senescence of the cotyledons. The mutants also have elevated EIN3 levels in the absence of ethylene treatment, indicating that control of EIN3 levels by SCF^EBF1/2 is important for regulating plant growth. Phenotypic and kinetic analyses suggest that EBF1 and EBF2 work together to degrade EIN3 but have different temporal roles. Rapid kinetic and immunoblot analyses have been used to further elucidate these roles. Preliminary data suggest that EBF1 constitutively represses EIN3 levels to prevent signaling in the absence of the hormone whereas EBF2 is necessary following the engagement of the ethylene signaling pathway to dampen further signaling. Collectively these results show that SCF^EBF1/2-dependent ubiquitination and subsequent removal of EIN3 is critical not only for proper ethylene signaling but also for plant growth and development.