Abstract
The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation of the plant clock.
Highlights
We examined the function of the feedback loop between CLOCK ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION1 (TOC1) in more detail by analysis of CCA1::LUC+ and TOC1::LUC+ reporters in plants free running in constant artificial white light after entrainment to light/dark conditions of 12/12 h (LD 12):12
We have shown that ebi-1 is a mutant of Arabidopsis that displays a short-free-running period (FRP), early-phase phenotype of the circadian clock
Our data indicate that the likely cause of this phenotype is misregulation of the feedback loop between CCA1, LATE ELONGATED HYPOCOTYL (LHY), and TOC1 that lies at the heart of the plant clockwork
Summary
The ebi mutant was isolated from an ethyl methanesulfonate (EMS)-mutagenized population of Wassilewskija (Ws-2) Arabidopsis plants homozygous for the CHLOROPHYLL A/B_BINDING PROTEIN 2 fused to LUCIFERASE (CAB2::LUC+) reporter construct (Kevei et al, 2006). Similar to CAB2::LUC+ expression, the FRP of leaf movement rhythms measured in constant white light (25 mmol m22 s21) was shorter for ebi-1 than for the wild type (Fig. 1, B and C; Ws-2, mean period = 24.2 h, SE = 0.14, n = 11; ebi-1, mean period = 23.1, SE = 0.07, n = 12). Periods of CAB2::LUC+ expression in the wild type, ebi-1, cca1-11;lhy-21, ztl-21, and double and triple mutants (as indicated) were measured under different quality and quantities of light. We transfected protoplasts with Myc-tagged wild-type or mutated EBI protein in the presence or absence of transfected HA-tagged ZTL and measured the expression of CCA1 and TOC1 from their endogenous promoters at zeitgeber time (ZT) ZT 6 (middle of the light period) and ZT 18 (middle of the dark period). At ZT 6, transfection with either wild-type EBI or the mutated form, ebi, reduced CCA1 expression (Fig. 8); transfection with ZTL plus either form of EBI
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