Rotation 1: Validation of a putative MYB transcription factor involved in chloroplast development

BBSRC战略主题：可持续农业和食品生物科学小麦对英国农业至关重要（https：//www.gov.uk/government/statistics/agricultural-land-use-in-the-united-kingdom/agricultural-land-use-in-united-kingdom-at-1-june-2023）。昼夜节律振荡器调节产量相关性状的几种潜在途径，包括抽穗期和温度反应（Asseng et al.，2015; Wittern等人，2023年）。然而，小麦和模式植物拟南芥之间的差异阻碍了我们对这些途径的理解应用于作物。而CONSTANS则调控A. thaliana，小麦抽穗期通过光周期-1（Ppd-1）和早花期3（ELF 3）确定（Alvarez等，2023; Shaw等人，2020; Suárez-López等人，2001年）。在小麦中，昼夜节律振荡器受温度调节的机制也不清楚;已经提出ELF 3通过小麦ELF 3中不存在的预测朊病毒结构域（PrD）来响应温度（Jung等人，2020;罗纳德等人，2021年; Zhu等人，2023年）。因此，更好地了解小麦生物钟对于育种策略至关重要，这些策略靶向时钟基因以提高小麦对气候变化的适应性（Steed et al.，2021）。在这个项目中，我们建议使用分子和生物化学方法来更好地了解小麦昼夜节律钟的结构和功能。为了促进小麦时间生物学的研究，我们计划开发一个生物发光时钟基因报告线，以测量在遗传水平上的昼夜节律。然后可以将报告基因杂交到时钟基因突变株系中。除了这个更广泛的目标，我们将集中精力确定ELF 3在昼夜节律振荡器中的作用。首先，我们将使用计算机模拟和体内测定来评估是否在小麦中形成具有LUX ARRHYTHMO（LUX）和EARLY FLOWARTH 4（ELF 4）直系同源物的晚间复合物（EC）（Herrero等人，2012; Nusinow等人，2011年）。其次，我们将测试ELF 3与来自A. thaliana，如CAB 1的时间，组成光形态发生1，和巨人茶（Huang和Nusinow，2016）。为了补充这项工作，我们计划在正在进行的工作的基础上，通过使用染色质免疫沉淀测序（ChIP-seq）研究ELF 3与靶基因启动子的结合来分析小麦昼夜节律转录组。我们还将研究产量相关的昼夜节律振荡器输出途径的分子机制，包括开花时间和热形态建成。在小麦中，开花时间的调节涉及Ppd-1、VERNALIZATION 1（VRN 1）、VRN 2和VRN 3，并且响应于光周期和春化作用（Distelfeld et al.，2009年）。ELF 3将该途径与时钟整合;我们的目标是确定该调节是否通过EC发生（Alvarez et al.，2023; Wittern等人，2023年）。另外，热响应性生长可以独立于开花时间来介导（Wang et al.，2024年）。因此，我们将通过体内测定来测试ELF 3的参与，潜在地将其扩展以使用诸如亲和纯化-质谱（AP-MS）的方法来测试更广泛的ELF 3相互作用组（Box等人，2015; Huang和Nusinow，2016）。因此，本研究旨在阐明小麦昼夜节律振荡器及其产量相关输出途径的分子机制。这可以使这项研究应用于农业，例如，以育种目标的形式。

BBSRC strategic theme: Bioscience for sustainable agriculture and foodWheat 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.