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

第 1 轮：验证参与叶绿体发育的推定 MYB 转录因子

• 批准号: 2887717
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2887717
• 关键词: Rotation; Validation; putative; MYB; transcription

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.

BBSRC战略主题：可持续协议和饮食的生物科学对于英国协议（https://www.gov.uk/government/statistics/agricultural-land-land-land-land-land-the-united-kingdom/agricultural-land-land-use-in---------- use-in-united-united-united-kingdom-at-1-1--2023）至关重要。昼夜节律振荡器调节了与产量相关的几种途径，包括标题日期和温度响应（Asseng等，2015； Wittern等，2023）。但是，小麦和模型植物拟南芥之间的差异阻碍了我们对这些途径在农作物上的理解的应用。当Constans在A. thaliana中调节开花时间，而小麦的标题日期由Photoperiod-1（PPD-1）和早期开花3（Elf3）确定（Alvarez等，2023; Shaw等，2020;Suárez-López等人，2001年）。小麦在小麦中还不清楚昼夜节律振荡器调节的机制。在A. thaliana中，已提出ELF3通过在小麦ELF3中不存在的预测的prion域（PRD）来响应温度（Jung等，2020; Ronald等，2021; Zhu等，2023）。因此，更好地了解小麦昼夜节律时钟对于针对时钟基因的育种策略至关重要，以提高小麦对气候变化的韧性（Steed等，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理解麦小时的结构和功能。为了促进小麦年代生物学研究，我们计划开发生物发光的时钟基因记者线，以测量遗传水平的昼夜节律。然后可以将记者交叉成时钟基因突变线。除了更广泛的目标外，我们还将重点介绍ELF3在昼夜节律振荡器中的作用。首先，我们将评估使用Lux心律失常（lux）和早期开花4（ELF4）直系同源物在小麦中形成的夜间复合物（EC）是否在硅和体内分析中形成（Herrero等，2012; Nusinow et al。，2011）。其次，我们将测试ELF3与来自A. thaliana的伴侣的直系同源物的相互作用，例如CAB 1的时间安排，本构的光性发光1和Gigantea（Huang and Nusinov，2016年）。为了补充这项工作，我们计划通过使用染色质免疫沉淀测序（CHIP-SEQ）研究ELF3与靶基因启动子的结合来建立正在进行的工作，分析小麦昼夜节律转录组。我们还将研究与产量相关的昼夜节律输出途径的分子机制，包括开花时间和热形成。小麦，开花时间的调节涉及PPD-1，春季1（VRN1），VRN2和VRN3，并响应于光周期和白话（Distelfeld等，2009）。 ELF3将此途径与时钟集成在一起；我们旨在确定该调节是否通过EC进行（Alvarez等，2023； Wittern等，2023）。另外，热响应生长可以独立于开花时间介导（Wang等，2024）。因此，我们将通过体内测定测试ELF3的参与，可能会使用诸如亲和力纯化质谱法（AP-MS）等方法来扩展这一点，以测试更广泛的ELF3相互作用组（Box等，2015； Huang和Nusinow，2016）。因此，这项工作旨在阐明小麦昼夜节律振荡器及其与产量相关的输出途径的分子机制。这可以使这项研究的应用以繁殖目标的形式达成共识。