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

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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.