Misregulation of MYB16 causes stomatal cluster formation by disrupting polarity in asymmetric cell division

Stomata and leaf cuticle regulate water evaporation from the plant body and balance the trade-off between photosynthesis and water loss. We identified MYB16, a key transcription factor controlling cutin biosynthesis, from previous stomatal lineage ground cell (SLGC)-enriched transcriptome study. The preferential localization of MYB16 in SLGCs but not meristemoids suggests a link between cutin synthesis and stomatal development. Here, we showed that downregulation of MYB16 in meristemoids was directly mediated by the stomatal master transcription factor, SPEECHLESS (SPCH). The suppression of MYB16 before asymmetric division was crucial for stomatal patterning because overexpression or ectopic expression of MYB16 in meristemoids increased impermeability and elevated stomatal density and clusters. The aberrant pattern of stomata was due to reduced and disrupted establishment of polarity during asymmetric cell division. Manipulating polarity by growing seedlings on hard agar rescued stomatal clusters and polarity defects in MYB16 ectopic lines. By expressing a cutinase in MYB16 ectopic lines, stomatal clustering was reduced, which suggests that the ectopic accumulation of cuticle affects the polarity in asymmetrically dividing cells and causes clustered stomata. Taken together, inhibiting MYB16 expression by SPCH in early stomatal lineage is required to correctly place the polarity complex for proper stomatal patterning during leaf morphogenesis.