a, In vitro phosphorylation of BASL by BIN2. Alanine substitutions in five BIN2 phosphorylated residues (BASL(5A)) reduced the BASL phosphorylation. The S72A substitution in the truncated BASL (BASL(1–84/S72A)) abolished phosphorylation. b, BASL protein. The blue dots, red asterisks, and green dot mark the identified in vitro BIN2 phosphorylation sites (this study), previously described MAPK phosphorylation sites, and the predicted S72 phosphorylation site, respectively. The nuclear localization signal (NLS) is marked in blue. The putative GSK3 phosphorylation motifs are marked in yellow. c, Confocal images of abaxial epidermis of cotyledons at 2.5 d.p.g. of BASL:GFP-BASL;basl-2 or SPCH:SPCH-GFP;spch-3 plants grown in the presence of 50 μM bikinin (BIK) or mock (DMSO). Scale bars, 20 μm. d, Wild-type (Col-0) seedlings at 2.5 d.p.g. grown in the presence of 50 μm BIK or mock (DMSO). Western blot analysis of MPK3 and MPK6 activation upon treatment with BIK. Active MPK3 and MPK6 were detected by a phospho-p44/42 MAPK antibody. For blot source data, see Supplementary Fig. 1. e, Gene expression of SPCH, POLAR and BASL as in d, measured by qRT–PCR. n = 3 biologically independent experiments. Transcript levels were normalized to the UBIQUITIN10 gene. f, Model for the function of BIN2 and POLAR during ACDs. In the MMC or young meristemoid (M), POLAR is highly expressed, nonpolar, whereas the BIN2 expression is absent or undetectable. In mature MMCs or meristemoids, BIN2 is expressed, is upregulated and initiates BASL polarization redundantly with the MAPK module. The BIN2–POLAR–BASL complex migrates to the cell cortex and polarizes before the ACD, thereby relieving the BIN2 inhibition on SPCH in the nucleus and attenuating the MAPK signalling in the polar region. After the ACD, the smaller daughter cell (meristemoid) accumulates SPCH to sustain POLAR and BASL transcription, allowing further amplifying ACDs. When the POLAR expression remains in the larger daughter cell (SLGC), the BIN2–POLAR–BASL polarity module is reoriented at the opposite side, allowing the occurrence of spacing divisions. When the POLAR expression in the SLGC is low, BIN2 is more abundant in the nucleus, relieving its inhibitory function on the MAPK signalling, leading to SPCH degradation and pavement cell differentiation. Meristemoids destined for a guard mother cell fate lose the POLAR expression, while maintaining the BIN2 expression. For a–e, experiments were repeated independently three times. Box plots show the first and third quartiles, split by the median (line) and mean (cross).