Deciphering the role of GRF4 in barley and durum wheat

Advanced knowledge of the molecular mechanisms affecting the expression of yield components in cereals is crucial for breeders to improve capacity. GRFs (Growth Regulating Factors) are plant-specific transcription factors involved in plant development and stress response. GRF mRNAs ar target of miR396 and GRF proteins interact with GIF (GRF Intera Factor) to regulate the expression of target genes. In rice a mutat leading in the miR396 binding site makOisnGgRF4 insensitive to post transcriptional regulation results in an increased seed size and promotes nitrogen assimilation and carbon fixation. In durum wheat, a locus carrying the wheat ortholog ofOsGRF4 from wild emmer has been associated wit larger seeds. Using the reverse genetics approach, our project aims to identify and characterize in barley and durum wheat the genes homologous to OsGRF4 and assess their functions. We identified 18 GRF sequences in H. vulgare cultivar Golden Promise and 31 in T. durum cultivar Svevo, through a phylogenetic analysis we identified the genes orthologous to rice GRF4: HvGRF4A, HvGRF4B in barley and TdGRF4A, TdGRF4B in durum wheat. A gene expression analysis across a range of barley tissues has revealed that HvGRF4A and HvGRF4B are mainly expressed in young reproductive tissues, with a peak in the seeds 0-3 days pollination, these results find validation in literature data. To study the function of GRF4A, we developed a set of barley and du wheat transgenic and edited lines: 1) ectopic expression of HvGRF4A in barley and durum wheat 2) ectopic expression of HvmiR396 in barley 3) ectopic expression of a miR396-insensitive HvGRF4A sequence generate through mutation in the mRNA binding site in barley and durum wheat 4) CRISPR-Cas9 genome editing in barley to produce grf4 mutants 5) CRISPR-Cas9 construct for homologous recombination to abolish the miR396 regulation in barley. We have also FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR) barley lines with specific single nucleotide changes in th miR396 binding site. Molecular and phenotypic analysis of these lines are ongoing. Preliminary data suggests that in barley GRF4A may influence flowering time, anth formation and seed length. Interestingly, these functions ar completely conserved in durum wheat where we observed a further effect on spike architecture.