Determining the roles and regulation of MIRNA156 genes in reproductive development of annual and perennial Brassicaceae species

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MicroRNAs (miRNAs) are short non-coding RNAs that provide robustness to regulatory circuits and stabilize developmental decisions by annealing to and repressing expression of target messenger RNAs (mRNAs). Mature miRNAs are generated by processing longer pri-miRNAs that are encoded by MIRNA (MIR) genes. Understanding the regulation and patterns of expression of mature miRNAs is complicated because each miRNA is encoded by many MIRs, which often precludes precisely positioning the miRNA in regulatory pathways. In plants, miR156/7 negatively regulates expression of SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors, which have important roles in diverse plant species in controlling different aspects of development, including initiation of flowering and flower development. Mature mir156/7 is encoded by 12 genes in the model plant species Arabidopsis thaliana, but the function or expression patterns of individual MIR156/7 genes in the shoot apex where flowering occurs is almost unknown. We and our Chinese co-operation partner (Professor Jiawei Wang, Chinese Academy of Sciences, Shanghai), with whom we have co-operated in analysing the role of mature miR156 in controlling flowering time, propose to collaborate to systematically analyse the functions of the MIR156/7 genes in A. thaliana. By sharing the work involved, we aim to define which of these MIR genes are important in regulating flowering time and floral meristem identity. CRISPR-cas9 genome editing is being used in each laboratory to efficiently generate mutations in each of the MIR156/7 genes, and at least two loss of function alleles will be isolated in each gene. In parallel the temporal and spatial patterns of each MIR gene in the shoot meristem and developing flower will be assessed using gene fusions to fluorescent proteins. Based on their patterns of expression, combinations of mir156/7 mutations will be generated and their effects on flowering assessed phenotypically. Similarly, the gene fusions will be used to follow transcriptional regulation of each MIR156/7 gene at the shoot meristem by transcription factors, environmental cues, hormones and metabolites implicated in the regulation of mature miR156/7 levels. Finally, the flowering response of perennial relatives of A. thaliana to cold winter temperatures has been shown by the applicant and co-operating partner to be dependent on miRNA156/7 levels and based on the results obtained in A. thaliana, we will assess which of the MIR156/7 genes are responsible for this effect. Overall these systematic analyses will define which MIR156/7 genes are involved in regulating floral induction and floral meristem identity, and form an essential basis for positioning these genes within the regulatory circuits that control the early stages of plant reproductive development.