Guard-cell phytosterol homeostasis is critical for proper stomatal development

Stomata regulate gas exchange and control water loss in response to the environmental stimuli and their distribution in the leaf epidermis is tightly regulated during development to ensure proper patterns. Although many studies have focused on the function of early stomatal lineage cells, little is known about the role of mature guard cells (GCs) in maintaining stomatal distribution. Here, we identified a previously uncharacterized enzyme, GDSL-type sterol esterase (GSEase), that is specifically expressed in mature guard cells and catalyzes lipid droplet-stored phytosterol ester degradation. Loss of GSEase decreased the level of free campesterol, a biosynthetic precursor of brassinosteroids (BRs), reduced BR level, and increased stomatal density in leaves, which could be further rescued by increasing the BR signaling. Furthermore, selectively reducing the BR response in GCs by utilizing the GSEase promoter-driven det2-1, a mutation causing BR biosynthesis deficiency, resulted in an elevated stomatal count, as demonstrated in gsease plants. These results indicate that GSEase plays a critical role in maintaining phytosterol homeostasis in GCs and the released phytosterols suppress the initiation of stomatal development in adjacent cells though the BR pathway.