Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Abstract

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.

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

“…Fluorescently tagged BRI1 and BAK1(SERK3) receptor proteins expressed in root epidermal cells, a cell file shown to exhibit active BR signaling (Hacham et al, 2011), were used for investigating receptor colocalization and heterooligomerization in an intact plant organ. Our results show that only around 10% of BRI1 receptors form heterooligomers with BAK1(SERK3) during active BR signaling in the PM, the main site of BRI1 signaling activity (Irani et al, 2012), of root epidermal cells. Pretreatment of roots with the BR biosynthesis inhibitors brassinazole (BRZ) or propiconazole (PPC) clearly decreased the amount of BRI1-BAK1(SERK3) heterooligomers.…”

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

“…Treatment with TyrA23 inhibits internalization of the PIN-FORMED auxin transporters and the water channel PLASMA MEMBRANE INTRINSIC PROTEIN2 (Dhonukshe et al, 2007), the iron transporter IRON-REGULATED TRANSPORTER1 (Barberon et al, 2011), the plant-specific endocytic SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN727 (Ebine et al, 2011), and the ligand-activated brassinosteroid receptor BRASSI-NOSTEROID INSENSITIVE1 (Irani et al, 2012). Amino acid substitutions of the YXXF motif eliminate polar localization of the Arabidopsis boron transporter REQUIRES HIGH BORON1 in the plasma membrane of root tip cells (Takano et al, 2010).…”

Clathrin in plant defense signaling and execution

“…1). Interestingly, temporal dynamics for endocytotic internalization of the steroid receptor BRI1 were speedier when compared with pathogen-related pattern recognition ligandbound FLS2 or PEPR1 receptors, but the final destination was the same, indicating a possible existence of the late-endosomal compartment as a general depot for internalized activated RKs in plant cells (6)(7)(8).…”

“…Although flg22-induced/FLS2 RK-dependent transient activation of reactive oxygen species production (via RBOH NADPH oxidase/NOX) and MAPK signaling was not inhibited concomitantly with clathrin endocytosis inhibition (7), in the case of Pep1-induced/PEPR-dependent activation, MAPK signaling output was strongly inhibited by endocytosis down-regulation (8). As in the case of FLS2, activated BRI1 internalization is not necessary for the signaling; in contrast, retention of activated brassinosteroid-bound BRI1 at the PM stimulated signal output (6). Although early MAPK/NOX response to FLS2 activation is not affected by CME inhibition, following callose deposition and stomata closure, defense responses are inhibited (7).…”

“…A new era in plant ligand-receptor interaction studies is indicated by the use of signaling-competent fluorescent ligand analogs [5′-carboxytetramethylrhodamine at the N-terminal (TAMRA)-modified ligands in these two PNAS reports (7,8)] to monitor real-time dynamics of ligand binding to receptors and subsequent internalization by CME, demonstrating the feasibility of this approach in plant cell signaling studies (6)(7)(8). Despite the general functioning of the BAK1 coreceptor in all activated receptor complexes studied in both reports (7,8), specific receptor-ligand interactions result in exclusive activation of a single pathway in response to a particular ligand.…”