Clathrin-mediated endocytosis of ERECTA family receptors is essential for proper stomatal development in Arabidopsis

Summary Stomata, specialized structures of plant epidermis, are crucial for gas (e.g. CO 2 , O 2 and H 2 O) exchange between plants and the environment. Stomatal density and pattern are governed by ERECTA Family (ERf) receptor-like kinases (RLKs)-controlled signaling. Clathrin-mediated endocytosis (CME) is a new mechanism for the internalization of receptor to activate the downstream signaling in animals. Here, we found that mutation of CME components resulted in formation of stomatal clusters and thus increased stomatal index. The adaptor protein 2 σ (AP2σ) subunit of CME interacted with ERf. ERf receptor internalization required CME. Furthermore, the ERf receptor motifs were identified to be recognized by AP2σ. Genetic analysis showed that CME components CHC2, CLC2, and CLC3 acted downstream of EPIDERMAL PATTERNING FACTOR 1/2 (EPF1/2) while upstream of YODA, and worked together with ERf to regulate stomatal development. Consistently, EPF2-induced MAPK activation was significantly reduced in chc2-2 clc2 clc3 and ap2μ clc2 clc3 mutants or when the ERf endocytic motifs were mutated, leading to stabilized SPEECHLESS protein and misregulated stomatal lineage progression. Overall, our findings demonstrate that activation of the ERf receptors via internalization is essential for stomatal development, establishing a novel mechanism of CME-mediated receptor internalization activation in plants. Significance Statement Stomatal development in Arabidopsis is well characterized, but how EPF-ERf signals activate the MAPK cascade remains unclear. While animals employ clathrin-mediated endocytosis (CME) for receptor internalization and signaling, this mechanism was unknown in plants. We identified CME as a novel regulator of stomatal development. The ERf receptors represent the first identified cargo proteins for AP2σ in plants, undergoing internalization through CME. Clathrin acts downstream of EPF but upstream of YODA, and blocking ERf internalization impairs MAPK activation. Thus, ERf internalization is essential for downstream signaling, revealing a novel CME-dependent activation mechanism in plants.