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- BBSRC strategic theme: Bioscience for sustainable agriculture and food Wheat is crucial to UK agriculture (https://www.gov.uk/government/statistics/agricultural-land-use-in-the-united-kingdom/agricultural-land-use-in-united-kingdom-at-1-june-2023). The circadian oscillator regulates several pathways underlying yield-related traits, including heading date and temperature response (Asseng et al., 2015; Wittern et al., 2023). However, differences between wheat and the model plant Arabidopsis thaliana obstruct the application of our understanding of these pathways to crops. While CONSTANS regulates flowering time in A. thaliana, wheat heading date is determined by PHOTOPERIOD-1 (Ppd-1) and EARLY FLOWERING 3 (ELF3) (Alvarez et al., 2023; Shaw et al., 2020; Suárez-López et al., 2001). The mechanism of circadian oscillator regulation by temperature is also unclear in wheat; in A. thaliana, ELF3 has been proposed to respond to temperature through a predicted prion domain (PrD) that is not present in wheat ELF3 (Jung et al., 2020; Ronald et al., 2021; Zhu et al., 2023). A better understanding of the wheat circadian clock is thus crucial to breeding strategies targeting clock genes to improve the resilience of wheat to climate change (Steed et al., 2021). In this project, we propose to use molecular and biochemical methods to better understand the structure and function of the wheat circadian clock. To facilitate wheat chronobiology research, we plan to develop a bioluminescent clock gene reporter line to measure circadian rhythms at the genetic level. A reporter can then be crossed into clock gene mutant lines. In addition to this broader aim, we will focus on determining the role of ELF3 within the circadian oscillator. Firstly, we will assess whether an Evening Complex (EC) with LUX ARRHYTHMO (LUX) and EARLY FLOWERING 4 (ELF4) orthologs forms in wheat using in silico and in vivo assays (Herrero et al., 2012; Nusinow et al., 2011). Secondly, we will test the interaction of ELF3 with orthologs of partners from A. thaliana, such as TIMING OF CAB 1, CONSTITUTIVE PHOTOMORPHOGENIC 1, and GIGANTEA (Huang and Nusinow, 2016). To complement this work, we plan to build on ongoing work analysing the wheat circadian transcriptome by investigating the binding of ELF3 to target gene promoters using chromatin immunoprecipitation sequencing (ChIP-seq). We will also investigate the molecular mechanisms of yield-related circadian oscillator output pathways, including flowering time and thermomorphogenesis. In wheat, the regulation of flowering time involves Ppd-1, VERNALIZATION 1 (VRN1), VRN2, and VRN3 and responds to photoperiod and vernalization (Distelfeld et al., 2009). ELF3 integrates this pathway with the clock; we aim to determine whether this regulation occurs through an EC (Alvarez et al., 2023; Wittern et al., 2023). Additionally, thermo-responsive growth can be mediated independent of flowering time (Wang et al., 2024). We will therefore test the involvement of ELF3 through in vivo assays, potentially expanding this to test the broader ELF3 interactome using methods such as affinity purification-mass spectrometry (AP-MS) (Box et al., 2015; Huang and Nusinow, 2016). This work thus aims to elucidate the molecular mechanisms underlying the wheat circadian oscillator and its yield-related output pathways. This can enable the application of this research to agriculture, for example, in the form of breeding targets.