Ligand-induced ubiquitination regulates endocytosis and homeostasis of the ERECTA receptor kinase for stomatal development

Stomata, valves on the plant epidermis, control gas and water vapor exchange. The patterning and spacing of stomata are primarily regulated by the ERECTA leucine-rich repeat receptor kinase (LRR-RK). Ubiquitination is a crucial mechanism to regulate the homeostasis of RKs by impacting their protein stability and localization. It has been shown that plant U-box ubiquitin E3 ligases, PUB30 and PUB31, act as key attenuators of the ERECTA signaling pathway. However, the molecular link between ubiquitination and function of ERECTA remains unclear. Here, we reveal that perception of the peptide ligand EPIDERMAL PATTERNING FACTOR2 (EPF2) by ERECTA induces the K63-linked ubiquitination of ERECTA mediated by PUB30 and PUB31. We further identify the specific ubiquitination sites within the juxtamembrane and kinase domains of ERECTA. Importantly, the site-directed mutagenesis of these K63-linked ubiquitination sites overly inhibited stomatal development, indicating that the ubiquitination-deficient ERECTA is hyperactive. The ubiquitination-deficient ERECTA stably accumulates at the plasma membrane and fails to internalize upon EPF2 application. Our findings thus uncover a mechanism by which ligand-induced ubiquitination orchestrates receptor homeostasis and endocytosis for proper stomatal patterning and differentiation.