1 Natural variation reveals that intracellular distribution of ELF3 protein is 2 associated with function in the circadian clock 3 4 Muhammad Usman Anwer1,3, Eleni Boikoglou1,2,a, Eva Herrero1,b, Marc Hallstein1, 5 Amanda Melaragno Davis1,3, Geo Velikkakam James1, Ferenc Nagy2 and Seth Jon 6 Davis1,3,c 7 1 Max Planck Institute for Plant Breeding Research, 50829, Cologne, Germany 8 2 Biological Research Centre of the Hungarian Academy of Sciences, H–6726 9 Szeged, Hungary 10 3 Department of Biology, University of York, York YO10 5DD, UK 11 12 a Present address: Cold Spring Harbor Laboratory, Laurel Hollow, NY 11791, USA 13 b Present address: MRC National Institute for Medical Research, NW7 1AA, London, 14 United Kingdom 15 16 c Corresponding author: E-mail: [email protected], [email protected] 17 18 Running title: 19 Circadian repression through ELF3 localization 20 1 21 Abstract: 22 Natural selection of variants within the Arabidopsis thaliana circadian clock can be 23 attributed to adaptation to varying environments. To define a basis for such variation, 24 we examined clock speed in a reporter-modified Bay-0 x Shakdara recombinant 25 inbred line and localized heritable variation. Extensive variation led us to identify 26 EARLY FLOWERING3 (ELF3) as a major quantitative trait locus (QTL). The causal 27 nucleotide polymorphism caused a short-period phenotype under light and severely 28 dampened rhythm generation in darkness, and entrainment alterations resulted. We 29 found that ELF3-Sha protein failed to properly localize to the nucleus, and its ability 30 to accumulate in darkness was compromised. Evidence was provided that the ELF3- 31 Sha allele originated in Central Asia. Collectively we showed that ELF3 protein plays 32 a vital role in defining its light-repressor action in the circadian clock and that its 33 functional abilities are largely dependent on its cellular localization. 34 2 35 Introduction: 36 The rotation of the earth leads to environmental changes in ambient light and 37 temperature that define a 24-hour cycle. During these diurnal cycles, light is correlated with 38 warmth, while darkness is correlated with coolness. Thus, to ensure fitness in response to 39 such regular environmental oscillations, many organisms have evolved an internal timing 40 mechanism to predict these cyclic environmental cues, and this is termed the circadian clock. 41 This internal clock synchronizes major developmental and physiological processes. For 42 example, a considerable proportion of metabolism is under clock control [1-4], and seasonal 43 developmental programs require a circadian oscillator [5]. The coordination of molecular and 44 physiological processes with the external environment thus provides an adaptive advantage 45 under diverse climatic conditions [6]. 46 Circadian rhythms are produced by molecular oscillators, which are comprised of 47 interlocking feedback loops having several components [7]. In Arabidopsis, the central loop 48 consists of two closely related single-myb transcription factors, CIRCADIAN CLOCK 49 ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), as well as the 50 pseudo response regulator (PRR) TIMING OF CAB EXPRESSION 1 (TOC1). In the morning, 51 CCA1 and LHY repress TOC1 by directly binding to its promoter, resulting in the evening 52 accumulation of TOC1, which, in turn, represses CCA1 and LHY expression [8,9]. The core 53 oscillator is further tuned by an evening- and morning-phased loop. The evening loop is 54 proposed to include GIGANTEA (GI) and TOC1, where GI activates the expression of TOC1, 55 and TOC1 represses GI [9-11]. Two members of the pseudo response regulator gene family, 56 PRR7 and PRR9, repress CCA1 and LHY during the day, and this establishes the morning 57 loop [10,11]. Recently, an evening complex comprising EARLY FLOWERING 3 (ELF3), 58 EARLY FLOWERING 4 (ELF4), and LUX ARRYTHMO (LUX) has been found to repress the 59 morning loop by specifically binding to the PRR9 promoter to mediate transcription 60 repression [12-19]. 61 Ambient light and temperature are two important environmental factors, termed 62 zeitgebers, which reset the clock in a process referred to as entrainment [20,21]. PRR7 and 63 PRR9 have been reported to play a role in temperature compensation, which is the 64 resistance of period change under differing mean ambient temperatures [22,23]. However, 65 diurnal temperature regulation of the circadian clock is still poorly understood [21]. In 66 contrast, light has been established as a key factor for the entrainment of the clock where 67 continuous irradiation shortens free-running period length in a fluence-rate-dependent 68 manner as a process termed parametric entrainment [24-26]. Most genetic components of 69 the clock were originally discovered from a light-entrained oscillator. Several studies have 3 70 shown that many observable rhythms in plants dampen in constant darkness (DD), where 71 dampening is defined as a reduction in circadian amplitude. For example, many genes have 72 been associated with circadian rhythms of mRNA abundance that are expressed robustly 73 under constant light (LL), but dampen in DD [27,28]. Furthermore, the cyclic protein 74 accumulation of several clock-regulated components, such as GI and ELF3 [29,30], was 75 found to be reduced when shifted to darkness. Thus, the effects of various light-input signals 76 on the Arabidopsis core oscillator remain to be elucidated. 77 Molecular and genetic studies support the involvement of ELF3 in light gating to the 78 circadian clock, and ELF3 is also a required component of the core oscillator [16,25,31,32]. 79 The elf3 mutant was originally isolated in a screen for lines displaying photoperiod- 80 independent early flowering [33,34]. Further characterization of elf3 revealed other severe 81 phenotypes, including defects in clock-regulated leaf movement rhythms, rhythmic hypocotyl 82 elongation, and arrhythmic expression of gene expression under the free running condition of 83 LL and in DD [16,31,32,35,36]. Natural variation at ELF3 has been detected that could be 84 associated to an alteration in shade-avoidance responses, which included increased 85 elongation of the hypocotyl, delay of cotyledon opening in seedlings, increased elongation of 86 stems and petioles, and reduced developmental timing in adults, and to changes in circadian 87 function [47,48,49,62]. ELF3 was cloned and reported to encode a nuclear protein of 88 unknown function, and it was proposed to work as a transcription factor [30,37]. Based on 89 the accumulation of ELF3 protein upon shifting plants to LL, and its decrease when plants 90 were shifted to DD, it was concluded that its abundance is dependent on ambient light [30]. 91 To extend this hypothesis based on the reported light-dependent phenotypes of elf3, 92 McWatters et al. proposed that ELF3 fulfills the so-called zeitnehmer concept in that this 93 factor acts to bridge light perception to the clock [38]. This idea was further strengthened 94 when ELF3 was found to physically interact with the phytochromeB photoreceptor [30]. The 95 previously noted role of ELF3 in light input to the circadian clock [31] is not altered by new 96 findings that ELF3 is core to the oscillator. 97 Many elements in the clock were isolated in screens for rhythm mutants using 98 bioluminescence readout of the molecular oscillator. Worldwide, the species distribution of 99 Arabidopsis ranges from the equator to extreme northern latitudes near the Arctic Circle [39]. 100 Therefore, as an alternative to induced mutants, another source of genetic variation can be 101 found among naturally occurring populations of Arabidopsis. More specifically, environmental 102 variations across and within local populations of Arabidopsis act as a discriminatory force on 103 the gene pool from which only a few genetic variants of adaptive phenotypes will be selected 104 and pass through reproduction. Arabidopsis populations based on Recombinant Inbred Lines 105 (RILs) have been derived from parental accessions. Such populations are advantageous in 4 106 mapping novel gene interactions. For example, quantitative trait locus (QTL) mapping was 107 used to explain circadian parameters associated with natural variation in the circadian 108 rhythmicity of leaf movement [40-43]. More recently, the rhythm of bioluminescence from 109 modified firefly LUCIFERASE (LUC) genes coupled to the clock-controlled promoter was 110 successfully used to map QTLs for circadian parameters [44,45]. Thus, the luciferase-based 111 system can be employed to accurately measure variation in circadian-rhythm parameters 112 within RIL populations to detect phenotypically expressed genetic variation in circadian clock 113 genes [40]. 114 In this study, we characterized a natural allele of ELF3 to determine its effect on 115 rhythmicity in the Arabidopsis circadian clock. Classical QTL mapping in a modified Bay-0 x 116 Shakdara mapping population, followed by positional cloning, revealed an allele of ELF3 117 (ELF3-Sha) that accelerates circadian oscillations in a light-dependent manner and a 118 dampened oscillator in darkness, We determined that the periodicity phenotype of ELF3-Sha 119 results from a single encoded amino-acid change A362V, which is an allelic variant that is 120 largely confined to a latitudinal stripe in Central Asia. Furthermore, we identified that the 121 circadian abnormalities in ELF3-Sha are associated to cellular localization defects of ELF3 122 protein that results in an inability to properly function under light and in darkness. Thus, by 123 characterizing ELF3-Sha, we clarified the molecular mode-of-action of ELF3 in the circadian 124 clock. 125 5 126 Results: 127 Analysis of circadian periodicity detected QTLs in Bay-0 X Sha RILS 128 A total of 71 RILs derived from the genetic cross of Bayreuth-0 and Shakdara (BxS) 129 were modified to harbor the CCR2 promoter, also termed GRP7 [46], fused to luciferase 130 (CCR2::LUC).