Cell type-specific attenuation of brassinosteroid signaling precedes stomatal asymmetric cell division

In Arabidopsis thaliana, brassinosteroid (BR) signaling and stomatal development are connected through the SHAGGY/GSK3-like kinase BR INSENSITIVE2 (BIN2). BIN2 is a key negative regulator of BR signaling but it plays a dual role in stomatal development. BIN2 promotes or restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffold protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages after triggering BR signaling with either exogenous BRs or the specific BIN2 inhibitor, bikinin, revealed that the two modes of BR signaling activation generate spatiotemporally distinct transcriptional responses. We established that BIN2 is always sensitive to the inhibitor but, when in a complex with POLAR and its closest homolog POLAR-LIKE1, it becomes protected from BR-mediated inactivation. Subsequently, BR signaling in ACD precursors is attenuated, while it remains active in epidermal cells devoid of scaffolds and undergoing differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants. Single-cell RNA-seq experiments were performed on protoplast cells extracted from the aerial tissue of 5 day old Arabidopsis thaliana seedlings.