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

Summary Stomatal patterning on the plant epidermis is regulated by the ERECTA family of leucine‐rich repeat receptor kinases (LRR‐RKs). While ubiquitination has emerged as a key regulatory mechanism for the endocytosis of receptor kinases, the precise molecular connections among ERECTA ubiquitination, endocytosis, and its biological functions remain unclear. Here, we uncover a critical role of ligand‐induced ubiquitination in regulating the endocytosis and homeostasis of the ERECTA receptor kinase for stomatal development and elongation of inflorescence pedicels in Arabidopsis thaliana . Previous ubiquitome profiling of plant membrane proteins identified one ubiquitination site in ERECTA, and our sequence alignment analysis identified two additional ubiquitination sites within the juxtamembrane domain. We subsequently performed site‐directed mutagenesis of these lysine residues (K617, K625, and K668) to arginine (ERECTA 3KR ). Our phenotypic analyses demonstrate that the mutant version of ERECTA confers over‐inhibition of stomatal development and excessive pedicel growth, indicating that the absence of ubiquitination confers hyperactivity of ERECTA. Our biochemical analyses further demonstrate that the perception of EPIDERMAL PATTERNING FACTOR2 (EPF2) peptide ligand induces K63‐linked ubiquitination of ERECTA mediated by PUB30 and PUB31. By contrast, the ligand‐induced K63‐linked polyubiquitination is impaired in ERECTA 3KR . K63‐ubiquitin chains are considered to be a general signal for endocytosis for plasma membrane proteins. Notably, impaired K63‐linked ubiquitination of ERECTA compromises its ligand‐induced endocytosis, as well as subsequent trafficking to multivesicular bodies and the vacuole for degradation. Moreover, ERECTA 3KR exhibits reduced co‐localization with endocytic trafficking markers (ARA6 and SYP43). Consistently, ERECTA 3KR accumulates to a higher protein level than wild‐type ERECTA and exhibits reduced sensitivity to ligand application, as well as pharmacological inhibition of clathrin‐mediated endocytosis or vacuolar degradation. Together, our findings reveal that K63‐linked ubiquitination of ERECTA is essential for its ligand‐induced endocytosis and vacuolar degradation, thereby ensuring proper signal attenuation. The absence of this post‐translational modification leads to sustained plasma membrane accumulation of active receptors, resulting in excessive inhibition of stomatal development. Therefore, our study highlights that ERECTA signals at the plasma membrane and that ubiquitination serves as a key mechanism for fine‐tuning receptor kinase activity and developmental outcomes in plants.