IRANI, N. G., DI RUBBO, S., MYLLE, E., VAN DEN BEGIN, J., SCHNEIDER-PIZON, J., HNILIČKOVÁ, Jaroslava, ŠÍŠA, Miroslav, BUYST, D., VILARRASA-BLASI, J., SZATMÁRI, A. M., VAN DAMME, D., MISHEV, K., CODREANU, M. C., KOHOUT, Ladislav, STRNAD, Miroslav, CANO-DELGADO, A. I., FRIML, J., MADDER, A., RUSSINOVA, E. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nature Chemical Biology. 2012, 8(6), 583-589. ISSN 1552-4450. E-ISSN 1552-4469

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.