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

生物钟调节的富含甘氨酸的RNA结合蛋白AtGRP 7（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变。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）控制中的作用及其同源物HvGR-RBP 1在大麦中的作用。AtGRP 7与拟南芥中几种已知的开花途径相关。通过RNA-seq对小RNA的全局分析鉴定了一组miRNA，包括与FTi对照相关的miRNA，其在AtGRP 7-ox植物与wt植物中差异表达。虽然AtGRP 7-ox植物中这些miRNA的水平降低，但相应前体的水平升高，表明AtGRP 7影响microRNA前体转录物的加工。 与此一致，我们可以证明AtGRP 7与pri-miRNAs在体内相互作用。我们将详细研究AtGRP 7在花转变过程中对这些FTi相关miRNAs的影响。此外，我们将研究AtGRP 7对pri-miRNA的选择性剪接如何影响非诱导和诱导条件下FTi相关miRNA的积累。将研究miRNAs及其相应靶标对AtGRP 7对FTi的作用的相关性。此外，我们还将研究AtGRP 7水平改变的植物在花转变过程中其他类型的小RNA（包括siRNA）的表达差异。该项目的主要目标之一是建立CRISPR/Cas 9系统，以解开多余FTi调节子的功能。在大麦中，我们已经获得了在HvGR-RBP 1中具有突变的TILLING系。我们将描述这些线在定义的光暗周期和在该领域的开花行为。此外，我们将采用候选基因的方法来识别HvGR-RBP 1的潜在下游靶点，包括FTi相关的miRNA，并提供对HvGR-RBP 1影响FTi的信号通路的见解。为了将生理表型与分子功能相关联，我们将测试具有对应于HvGR-RBP 1变体的RNA结合结构域和富含甘氨酸的片段中的突变的重组蛋白的RNA结合行为和亚细胞定位。最后，我们将通过测试TILLING系的病原体响应来解决AtGRP 7在拟南芥中的时钟控制、FTi和病原体防御之间的交叉点处的作用是否对于HvGR-RBP 1是保守的。总的来说，该项目有助于开发FTi监管机构的功能性跨物种网络，这是SPP 1530的主要战略目标。

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.