carbocyanines and castasterone

Synthetic derivatization of hormonally active brassinosteroids (BRs) can provide useful small molecule tools to probe BR signaling pathways, such as fluorescent analogs. However, most biologically active BRs are not suitable for direct chemical conjugation techniques because their derivatization typically requires extensive synthetic work and chemistry expertise. Here, we describe an operationally simple, two-step procedure to prepare and purify an Alexa Fluor 647-castasterone (AFCS) from commercially available materials. The reported strategy is also amenable to the introduction of various other amine-based labeling groups.

Topics: Brassinosteroids; Carbocyanines; Chemistry Techniques, Synthetic; Cholestanols; Chromatography, Thin Layer; Fluorescent Dyes; Oximes; Plant Growth Regulators; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staining and Labeling

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.

Topics: Arabidopsis; Arabidopsis Proteins; Brassinosteroids; Carbocyanines; Cell Membrane; Cholestanols; Dose-Response Relationship, Drug; Endocytosis; Endosomes; Fluorescent Dyes; Green Fluorescent Proteins; Kinetics; Meristem; Microscopy, Confocal; Molecular Structure; Plant Growth Regulators; Protein Kinases; Protein Transport; Seedlings; Signal Transduction; Vacuoles