Unravelling the role of an autonomous pathway component in FTi control in Arabidopsis and barley

揭示自主途径成分在拟南芥和大麦 FTi 控制中的作用

• 批准号: 196931130
• 负责人: Professorin Dr. Dorothee Staiger
• 金额: --
• 依托单位: Department of Plant Sciences & Plant Pathology
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/196931130?language=en
• 关键词: Unravelling; role; autonomous; pathway; component

The circadian clock regulated glycine-rich RNA-binding protein AtGRP7 (Arabidopsis thaliana gylcine-rich RNA binding protein 7) promotes floral transition in the long day plant Arabidopsis thaliana. We will compare the role of this glycine-rich RNA-binding protein in flowering time (FTi) control in Arabidopsis thaliana and the role of its homologue HvGR-RBP1 in Hordeum vulgare. AtGRP7 is associated with several of the known flowering pathways in Arabidopsis. Global profiling of small RNAs by RNA-seq identified a suite of miRNAs including miRNAs associated with FTi control that were differentially expressed in AtGRP7-ox plants vs. wt plants. While the level of these miRNAs was reduced in AtGRP7-ox plants, the levels of the corresponding precursors were elevated, indicating that AtGRP7 affects the processing of microRNA precursor transcripts. In line with this, we could show that AtGRP7 interacts with pri-miRNAs in vivo. We will investigate in detail the impact of AtGRP7 on these FTi-related miRNAs during floral transition. Furthermore, we will investigate how alternative splicing of pri-miRNAs by AtGRP7 affects accumulation of FTi-related miRNA under noninductive and inductive conditions. The relevance of the miRNAs and their corresponding targets for the effect of AtGRP7 on FTi will be investigated. Furthermore, we will monitor differences in expression of other types of small RNAs including siRNAs in plants with altered AtGRP7 level during floral transition.A major goal of the project is to establish the CRISPR/Cas 9 system to disentangle the function of redundant FTi regulators. In barley, we have obtained TILLING lines with mutations in HvGR-RBP1. We will characterize the flowering behaviour of these lines both in defined light-dark cycles and in the field. Furthermore, we will take a candidate gene approach to identify potential downstream targets of HvGR-RBP1 including FTi related miRNAs and provide insights into the signalling pathways through which HvGR-RBP1 influences FTi. To correlate the physiological phenotypes with a molecular function we will test recombinant proteins with the mutations in the RNA binding domain and the glycine rich stretch corresponding to the HvGR-RBP1 variants for their RNA binding behaviour and subcellular localization. Finally, we will address whether the role of AtGRP7 at the intersection between clock control, FTi and pathogen defense in Arabidopsis is conserved for HvGR-RBP1 by testing the pathogen response of the TILLING lines. Overall, the project contributes to the development of a functional cross-species network of FTi regulators, the major strategic aim of SPP1530.

生物钟调控的富含甘氨酸的RNA结合蛋白AtGRP7(拟南芥富含甘氨酸的RNA结合蛋白7)促进长日照植物拟南芥的花转换。我们将比较这种富含甘氨酸的RNA结合蛋白在拟南芥开花时间(FTI)控制中的作用，以及它的同源物HvGR-RBP1在大麦中的作用。AtGRP7与拟南芥中已知的几条开花途径相关。通过RNA-seq对小RNA的全球图谱分析发现了一组miRNAs，包括与FTI控制相关的miRNAs，这些miRNAs在AtGRP7-ox植物和wt植物中差异表达。在AtGRP7-OX植物中，这些miRNAs的水平降低，而相应的前体水平上升，表明AtGRP7影响microRNA前体转录本的加工。与此相一致，我们可以证明AtGRP7在体内与pri-miRNAs相互作用。我们将详细研究AtGRP7在花转换过程中对这些FTI相关miRNAs的影响。此外，我们将研究在非诱导和诱导条件下，AtGRP7对pri-miRNAs的选择性剪接如何影响FTI相关miRNA的积累。将调查miRNAs及其相应目标与AtGRP7对FTI影响的相关性。此外，我们将监测其他类型的小RNA的表达差异，包括在花转换过程中AtGRP7水平改变的植物中的siRNAs。该项目的一个主要目标是建立CRISPR/Cas 9系统，以解开多余的FTI调节因子的功能。在大麦中，我们获得了HvGR-RBP1突变的分蘖系。我们将描述这些品系在明确的明暗周期和田间开花行为。此外，我们将采用候选基因的方法来确定HvGR-RBP1的潜在下游靶点，包括与FTI相关的miRNAs，并深入了解HvGR-RBP1影响FTI的信号通路。为了将生理表型与分子功能联系起来，我们将测试与HvGR-RBP1变体对应的RNA结合域和富含甘氨酸的部分突变的重组蛋白的RNA结合行为和亚细胞定位。最后，我们将通过测试分耕系的病原菌响应来研究AtGRP7在拟南芥中时钟控制、FTI和病原菌防御之间的交叉点上的作用是否在HvGR-RBP1中保守。总体而言，该项目有助于发展跨物种的功能性快车道监管机构网络，这是SPP1530的主要战略目标。