removed from this tube as a standard for comparison with subsequent time samples. Cells were diluted 1:100 into various media and incubated at the appropriate temperature until OD 600 was 0.5, whereupon they were diluted 1:100 (about once or twice a day). Samples were removed at various time points and lysed by incubation with Zymolyase and boiling. The lysates were subjected to PCR. The PCR product was sequenced in accordance with the standard protocol provided by Applied Biosystems and analysed on an ABI Prism 3700 DNA Analyzer with DNA Sequencing Analysis Software Version 3.6.1 (Applied Biosystems, Foster City, California). Mutant frequencies within the culture were estimated with the sequencing-based protocol developed by Kwok and Duan 29 . Data were fitted to exponential equations that accounted for changes in growth of the mutant of interest and changes in growth of competitors. Received 7 August; accepted 23 October 2003; doi:10.1038/nature02178. 1. Pawson, T. & Nash, P. 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Yeh, and members of the Lim laboratory for assistance and discussion. This work was supported by grants from the NIH and the Packard Foundation. S.-H.P. is a Jane Coffin Child Fellow. A.Z. is supported by the UCSF Medical Scientist Training Program. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to W.A.L. (wlim@itsa.ucsf.edu). .............................................................. Gating of the rapid shade-avoidance response by the circadian clock in plants Michael G. Salter*, Keara A. Franklin* & Garry C. Whitelam Department of Biology, University of Leicester, Leicester LE1 7RH, UK * These authors contributed equally to this work ............................................................................................................................................................................. The phytochromes are a family of plant photoreceptor proteins that control several adaptive developmental strategies 1,2 . For example, the phytochromes perceive far-red light (wavelengths between 700 and 800 nm) reflected or scattered from the leaves of nearby vegetation. This provides an early warning of potential shading, and triggers a series of ‘shade-avoidance’ responses, such as a rapid increase in elongation 3 , by which the plant attempts to overgrow its neighbours 3 . Other, less immediate, responses include accelerated flowering and early production of seeds. However, little is known about the molecular events that connect light perception with increased growth in shade avoid- ance. Here we show that the circadian clock gates this rapid shade-avoidance response. It is most apparent around dusk and is accompanied by altered expression of several genes. One of these rapidly responsive genes encodes a basic helix–loop–helix protein, PIL1, previously shown to interact with the clock protein TOC1 (ref. 4). Furthermore PIL1 and TOC1 are both required for the accelerated growth associated with the shade-avoidance response. Selective absorption of blue and of red (600–700 nm) wave- lengths by the chlorophylls means that the radiation reflected/ scattered by green leaves is relatively enriched in the far-red (700– 800 nm). This far-red-rich light signal (that is, a decrease in the ratio of red to far-red (R/FR)) is detected by nearby plants as a change in the equilibrium between the P r and P fr forms of phytochromes B, D and E (ref. 5), providing an unambiguous signal that potential competitors are nearby. In response to a low R/FR many plants evoke a suite of adaptive reactions, shade avoidance, including rapidly increased elongation of internodes 6 and/or petioles, reduced leaf growth and increased apical dom- inance in an attempt to avoid being shaded. Prolonged exposure to the low-R:FR signal evokes a survival reaction: the acceleration of flowering 3,7 . Shade avoidance is displayed by most angiosperms, including crop species, conferring high relative fitness in dense stands 3 and is one of the best-studied examples of adaptive phenotypic plasticity in plants. To gain insight into the molecular events involved in rapid shade- avoidance responses, we carried out Affymetrix Arabidopsis oligoar- ray analysis on plants exposed to low R/FR (see Supplementary Information). Among those genes displaying the most marked changes in expression in response to 1 h of low R/FR is the ATHB-2 gene, encoding a homeodomain ZIP transcription factor; this gene is known to be rapidly and reversibly regulated by changes in R/FR (ref. 8). However, the greatest increase in transcript level in response to low R/FR was observed for a gene annotated as encoding an unknown protein. The transcript of this gene increases in abundance by ,35-fold at 1 h. After correcting for errors in the annotation of this gene, we identified it as PIL1 (for PIF3-like 1) encoding a basic helix–loop–helix protein, previously identified as a protein that interacts with the circadian clock protein TOC1 (ref. 4). The increase in PIL1 transcript level in response to low R/FR starting 1 h after dawn is extremely rapid. Quantitative reverse transcriptase polymerase chain reaction (RT–PCR) shows that letters to nature NATURE | VOL 426 | 11 DECEMBER 2003 | www.nature.com/nature 680 © 2003 Nature Publishing Group