The circadian clock is essential for coordinating the proper phasing of many important cellular processes. Robust cycling of key clock elements is required to maintain strong circadian oscillations of these clock-controlled outputs. Rhythmic expression of the Arabidopsis thaliana F-box protein ZEITLUPE (ZTL) is necessary to sustain a normal circadian period by controlling the proteasome-dependent degradation of a central clock protein, TIMING OF CAB EXPRESSION 1 (TOC1)1,2. ZTL messenger RNA is constitutively expressed, but ZTL protein levels oscillate with a threefold change in amplitude through an unknown mechanism3. Here we show that GIGANTEA (GI) is essential to establish and sustain oscillations of ZTL by a direct protein–protein interaction. GI, a large plant-specific protein with a previously undefined molecular role, stabilizes ZTL in vivo. Furthermore, the ZTL–GI interaction is strongly and specifically enhanced by blue light, through the amino-terminal flavin-binding LIGHT, OXYGEN OR VOLTAGE (LOV) domain of ZTL. Mutations within this domain greatly diminish ZTL–GI interactions, leading to strongly reduced ZTL levels. Notably, a C82A mutation in the LOV domain, implicated in the flavin-dependent photochemistry, eliminates blue-light-enhanced binding of GI to ZTL. These data establish ZTL as a blue-light photoreceptor, which facilitates its own stability through a blue-light-enhanced GI interaction. Because the regulation of GI transcription is clock-controlled, consequent GI protein cycling confers a post-translational rhythm on ZTL protein. This mechanism of establishing and sustaining robust oscillations of ZTL results in the high-amplitude TOC1 rhythms necessary for proper clock function.
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We thank S. L. Harmer for providing ztl-103 in advance of publication, S. Kay for TOC1-TOC1-YFP and LKP2-OX seed, D. Bisaro for the adenosine kinase antibody and J. Na for excellent technical assistance. T. Mizoguchi provided gi-3, gi-4, gi-5, gi-6, and GI-OX (Ler). W.-Y. K. was supported in part by a grant from the MOST/KOSEF to the Environmental Biotechnology National Core Research Center (BK21 program grant), Korea. S.F. was funded in part by Support for Long-term Visit from the Yamada Science Foundation. Support is acknowledged from a Korea Science and Engineering Foundation (KOSEF) grant funded by MOST and the National Core Research Center for Systems Biodynamics (to H.G.N.), from the NZ Marsden grant (to J.P.), and from the NSF (IBN and MCB) to D.E.S.
Author Contributions W.-Y.K., S.F., S.-S.S., L.H., J.K. and Y.K. performed experiments. W.-Y.K. and H.-G.N. co-directed and designed experiments. J.P., K.D. and H.G.N. contributed novel reagents. J.P., S.F. and H.G.N. commented on and edited the manuscript. D.E.S. directed research, designed experiments and wrote the manuscript.
This file contains Supplementary Figures S1-S6 with Legends.
About this article
Frontiers in Plant Science (2019)