This research was conducted via 4 work packages (WPs). WP1 comprised the analyses of wild-type, ath1, pentuple della, and sextuple (ath1 della) mutants under different hormonal and light conditions. To assess cell proliferation in their rib meristem (RM), DNA labelling and confocal imaging were used. The combined experiments demonstrated that the ATH1 and DELLA genes act on the control of RM activity. The results showed how ATH1 causes localised changes in the plant’s response to environmental signals, and were published in PNAS. WP2 sought to analyse the interaction between ATH1 and brassinosteroid (BR) in the RM. Early results did not support the hypothesis that BR mediated the role of ATH1 in internode elongation. WP3 involved investigating the biological significance of EAR motif for ATH1 function in stem development. Site-directed mutagenesis and complementation assay were performed revealing no direct involvement of the EAR motif in the ATH1 function in controlling stem growth. In WP4, a CRISPR/Cas9-mediated gene editing system was used to target the promoter of ATH1 gene to identify and disrupt regulatory sequences that mediate the repression of ATH1 during stem growth. The Cas9/gRNAs constructs were prepared, and the transgenic lines generated. Editing of cis-regulatory elements were detected by PCR/restriction enzyme assay followed by sequencing. None of the edited lines were found to be visually different from Arabidopsis wild-type at the flowering stage, suggesting that the cis-elements targeted are either not relevant for ATH1 regulation in the RZ, or that there could be redundancy between different elements in the promoter. Although the results of this screen were negative, they have informed ongoing work on the potential role of the ATH1 promoter in differences in plant architecture in Brassicas.
