Dissecting brassinosteroid signaling mediated regulation of root growth angle in wheat

不同根型（初生根、种子根和冠状根）的RGA通常是不同的，以限制竞争，并被称为向重力设定点角（GSA）。初生根的垂直GSA由正向重力机制维持，而其他根型的非垂直GSA由竞争向重力和反向重力抵消（AGO）机制决定。虽然过去在模式植物拟南芥中的研究揭示了向重力性机制，但对AGO机制的理解仍然有限，因为拟南芥中不存在关键的根类型（种子和冠）。此外，由于遗传资源有限，这些机制在作物中基本上尚未探索。小麦是英国和世界的重要作物，代表了研究RGA的理想系统，因为所有的根类型都很容易分离，并且具有可用的遗传和基因组资源。最近，我们筛选了各种小麦植物激素信号突变体（生长素，乙烯，油菜素类固醇和bibberellin）RGA和观察BR信号突变体（突出tabri 1）显示显着陡峭的种子和冠根相比，wildtype.Aim：在这个项目中，我们将确定油菜素类固醇控制RGA在小麦的分子机制。（Year 1）tabri 1（A-同源物）显示比野生型更陡的>30%的精液和冠RGA。我们将表型剩余的单个tabri 1突变体（B和D同源物）和它们的组合双重和三重突变体，并评估所观察到的tabri 1表型是否特异于A同源物或通过其他TaBRI 1同源物中的堆叠突变而增强。此外，我们将使用垂直重力仪和90重力仪筛选其他可用的油菜素类固醇信号传导、生物合成和受体突变体的RGA表型。TaBR 1依赖性RGA调节发生在哪里？（1 -2年级）我们将研究TaBRI 1在小麦根中的何处产生，并通过用绿色荧光蛋白（GFP）标记BRI 1蛋白来控制RGA，然后将该转基因导入tabri 1植物中，以测试它是否可以拯救突变体并揭示其表达位置。探索Watkins collection（Year 3）中BR信号传导和RGA的遗传多样性我们目前正在筛选>来自小麦群体的800个品系的RGA表型。使用生物信息学管道，我们将研究TaBR 1内的多态性解释了RGA的多少自然变异。这一分析将有助于将这些品系的等位基因、生境和环境条件联系起来，并产生进一步的可检验的假设。TaBR 1调节哪些下游成分来控制RGA？（3 -4年级）从WT和在垂直和重力刺激条件下生长的单一或更高阶Tabri 1突变体的根样品中提取RNA，并进行RNAseq分析，以确定BRI 1介导的RGA控制期间涉及的关键差异表达基因及其富集过程。TaBR 1介导的RGA的相关性是什么？（4年级）已知养分、水和热应力会影响RGA，以优化土壤资源的捕获。我们将在控制和胁迫条件下对突变体和野生型进行生长室、温室和田间试验，并评估RGA、根和地上部生物量以及谷物产量。输出量：对控制RGA的分子机制的进一步理解将指导可以通过DFW育种工具包利用的等位基因的选择。

RGA of different root-types (primary, seminal and crown) are often distinct to limit competition and are referred to as gravitropic setpoint angle (GSA). The vertical GSA of primary roots is maintained by the positive gravitropic mechanism, while non-vertical GSA in other root-types is determined by competing gravitropic and anti-gravitropic offset (AGO) mechanisms. Although past research in the model plant Arabidopsis unraveled gravitropic mechanisms, understanding of AGO mechanisms remains limited as crucial root-types (seminal and crown) do not exist in Arabidopsis. Moreover, these mechanisms remain largely unexplored in crops due to limited genetic resources. Wheat, an important crop for the UK and world, represents an ideal system for studying RGA as all root-types are easily distinguiishable and have available genetic and genomic resources. Recently, we screened various wheat phytohormone signaling mutants (auxin, ethylene, brassinosteroid and bibberellin) for RGA and observed that BR signaling mutants (prominently for tabri1) show significantly steeper seminal and crown roots compared to wildtype.Aim:In this project, we will determine the molecular mechanisms by which brassinosteroids control RGA in wheat.Work Plan:Do other Brassinosteroid pathway genes regulate RGA? (Year1) tabri1 (A-homeologue) showed >30% steeper seminal and crown RGA than the wildtype. We will phenotype remaining single tabri1 mutants (of B- and D-homeologues) and their combinatorial double and triple mutants and assess if the observed tabri1 pheotype is specific to A-homeolgoue or enhanced by stacking mutations in other TaBRI1 homeologues. Additionally, we will screen other available brassinosteroid signaling, biosynthetic and receptor mutants for RGA phenotype using vertical and 90 gravistmulus.Where does TaBR1-dependant RGA regulation takes place? (Year1-2)We will investigate where TaBRI1 is made in the wheat root and functions to control RGA by tagging the BRI1 protein with green fluorescent protein (GFP), then putting this transgene into tabri1 plants to test if it can rescue the mutant and reveal its location of expression.Explore the genetic diversity of BR signaling and RGA in Watkins collection (Year3)We are currently screening >800 lines from a wheat population for RGA phenotype. Using bioinformatics pipeline, we will investigate how much natural variation of RGA is explained by the polymorphism within the TaBR1. This analyss will help link the ancestory, habitat and environmental conditions from which these lines were collected and generate further testable hypothesis.What downstream components TaBR1 regulate to control RGA? (Year3-4)Extract RNA from root samples of WT and single or higher order Tabri1 mutants growing under vertical and gravistimulus conditions and perform RNAseq analysis to determine key differentially expressed genes and their enriched processes involved during BRI1 mediated RGA control. What is the relevane of TaBR1 mediated RGA? (Year4)Nutrient, water and heat stresses are known to influence RGA to optimise capture of soil resources. We will perform growth-room, glasshouse and field experiments on mutants and WT under control and stress coditions and assess the RGA, root and shoot biomass and grain yield. Output: Improved understanding of molecular mechanisms controlling RGA will guide selection of alleles that can be exploited through DFW breeders toolkit.