Abstract
Control of protein turnover is a key post-translational control point in the oscillatory network of the circadian clock. Some elements, such as TOC1 and PRR5 are engaged by a well-described F-box protein, ZEITLUPE, dedicated to their proteolytic turnover to shape their expression profile to a specific time of night. For most other clock components the regulation of their protein abundance is unknown, though turnover is often rapid and often lags the cycling of the respective mRNA. Here we report the design and results of an unbiased genetic screen in Arabidopsis to uncover proteolytic regulatory factors of PSEUDO-RESPONSE REGULATOR 7 (PRR7), a transcriptional repressor that peaks in the late afternoon. We performed EMS mutagenesis on a transgenic line expressing a PRR7::PRR7-luciferase (PRR7-LUC) translational fusion that accurately recapitulates the diurnal and circadian oscillations of the endogenous PRR7 protein. Using continuous luciferase imaging under constant light, we recovered mutants that alter the PRR7-LUC waveform and some that change period. We have identified novel alleles of ELF3 and ELF4, core components of the ELF3-ELF4-LUX Evening Complex (EC), that dampen the oscillation of PRR7-LUC. We report the characterization of two new hypomorphic alleles of ELF3 that help to understand the relationship between molecular potency and phenotype.
Highlights
The circadian clock system helps to coordinate daily oscillations in gene expression, metabolism and physiology to help optimize growth and reproduction under daily light/dark cycles
We reasoned that mutants exhibiting luminescence oscillations with reduced amplitude and/or higher trough levels would be candidates for a loss-of-function in factors involved in PSEUDO-RESPONSE REGULATOR 7 (PRR7)-LUC turnover. 41,433 ethyl methanesulfonate (EMS)-mutagenized seedlings from 44 pools (50–150 plants/pool) were screened and 31 candidates were identified
Higher levels of PRR7LUC during normal trough times could indicate a more stable protein, suggesting a loss-of-function in PRR7 proteolytic factors. This was observed in ztl mutants where strongly flattened rhythms of SCFZTL complex targets, TIMING OF CAB EXPRESION 1 (TOC1) and PRR5, were seen under LD (Mas et al, 2003; Kiba et al, 2007; Fujiwara et al, 2008)
Summary
The circadian clock system helps to coordinate daily oscillations in gene expression, metabolism and physiology to help optimize growth and reproduction under daily light/dark cycles. It is primarily comprised of interlocked autoregulatory feedback loops of gene transcription and translation, but relies strongly on numerous post-transcriptional and post-translational processes (Seo and Mas, 2014; Mateos et al, 2018). Numerous studies have highlighted the dual role that most of these PRRs play as transcriptional repressors (Farre and Liu, 2013) At one level they act to repress transcription of certain core clock genes, helping to maintain the correct period and robustness of the central oscillator. The precise phase-specific expression of each of the PRRs contributes to an orchestration of concomitant specific phasing of output gene expression (Nakamichi et al, 2012; Farre and Liu, 2013; Liu et al, 2013, 2016)
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