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

Stomata, specialized structures of plant epidermis, are crucial for gas (e.g. CO2, O2 and H2O) 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 2σ (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.