Spatial and single-cell transcriptomics uncover brassinosteroid-mediated coordination of sepal elongation in Arabidopsis

BACKGROUND: Arabidopsis sepals must grow in a coordinated and robust manner to achieve consistent size and shape, ensuring proper closure and protecting the developing flower bud. To understand how this robust coordination occurs, we analyze the loss-of-robustness mutant development related myb-like1 (drmy1), which exhibits variable sepal initiation and growth, causing failure of bud closure. Specifically, drmy1 displays disproportionate sepal growth, with overgrown outer sepals and undergrown inner sepals, resulting in marked size variability within individual flower buds. RESULTS: Using single cell and spatial RNA-seq, we identify changes in expression of key genes related to brassinosteroid (BR) signaling in drmy1, particularly in cell types important to early flower bud development such as epidermal cells, boundary cells, and meristematic cells. Confocal imaging of a BRI1-EMS-SUPPRESSOR1 (BES1) ratiometric reporter confirms that BR signaling is upregulated and more variable in young drmy1 sepals. Genetic and chemical perturbations of BR signaling further demonstrate its role in growth coordination. Crossing drmy1 with BR mutants or applying brassinolide (a potent brassinosteroid) or brassinazole (a brassinosteroid biosynthesis inhibitor) rescues the sepal elongation defect by differentially altering inner and outer sepal growth. Specifically, in drmy1 mutants, enhanced BR signaling preferentially promotes inner sepal growth, while reduced BR signaling preferentially decreases outer sepal elongation, thereby restoring more balanced sepal growth. CONCLUSIONS: These results suggest that DRMY1 is required for proper brassinosteroid-mediated coordination of sepal elongation, ensuring balanced growth between inner and outer sepals during early development.