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Solving the tangled ontogenesis of the stem for sustainable crops

Periodic Reporting for period 1 - SOS-CROPS (Solving the tangled ontogenesis of the stem for sustainable crops)

Reporting period: 2019-05-20 to 2021-05-19

The SOS-CROPS looks at a region of the shoot apical meristem called the rib zone (RZ), where the stem initiates and develops, and it is one of the least understood aspects of plant development, despite its importance in crop productivity. The research questions how the ARABIDOPSIS THALIANA HOMEODOMAIN GENE 1 (ATH1) functions as a central node in the regulatory network that controls RZ activity and stem growth through the regulation of organ boundary genes and integration of hormone signalling. Such topic is important because there is a pressing need for redesigning the crop production systems to achieve sustainability, i.e. lesser impact on the environment, while maximizing yield to ensure adequate food production for the society.

Objectives of this Action have been to (a) test whether ATH1 restricts GA level and signalling in the RZ; (b) test whether ATH1 and BR have antagonistic roles in the RZ; (c) assess whether the transcriptional corepressor TOPLESS protein mediates repressive interactions in the ATH1 regulatory network; (d) use genome editing to select regulatory mutations in ATH1 that can be used to inhibit stem growth with fewer side effects than current semi-dwarf mutations.
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.
There are some broad implications of our work for our understanding of stem development and plant growth. Firstly, this project shed light on a poorly understood but strategically important aspect of plant development. The obtained data indicate that ATH1 is part of a regulatory hub that integrates gibberellin signalling for the control of the RZ function, and consequently stem growth. Secondly, the gene network controlling rib meristem activity and stem growth is an attractive target for engineering or selecting regulatory mutations to select or engineer dwarfing alleles in the RZ.
ATH1 and DELLA genes act on the control of rib zone acitivity