BRASSINOSTEROIDS (BRs) play important roles in regulating plant growth and development. The Arabidopsis GSK3-like kinase BIN2, a major negative regulator of BR signaling, plays diverse roles in plant growth and developmental processes. In a previous study, based on the relationship between BIN2 and its substrate, we reported the possibility that BIN2 protein stability is controlled by BRASSINOSTEROID F-BOX Protein 1 and 2, BRF1 and BRF2, (BRF1/2, renamed BRFP1/2). The aim of the study was to characterize the functions of the F-box proteins BRFP1 and BRFP2, and to confirm their relationship with the BIN2 proteins and the role of the BR signaling for plant growth and development. BRFP1-overexpressing (BRFP1-FLAG) plants displayed better growth compared to the wild-type Col-0 plants in contrast to brfp1/2 mutants. BRFP1 plants were sensitive to BL, the most active BR, as opposed to the brfp1/2 mutants. We confirmed that BRFP1 overexpression suppressed the dwarf BIN2-HA phenotype by reducing BIN2 protein levels through BRFP1–BIN2 interactions in vivo. These results suggest the possibility that the stability of BIN2 is regulated by BRFP1. Altogether, results of our finding propose that the F-box proteins BRFP1/2 are involved in BR signaling for regulating plant growth.

The Glycogen synthase kinase 3 (GSK3)-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2), a major negative regulator in the Brassinosteroids (BRs) signaling, is involved in a variety of plant signaling pathways by interacting with novel substrates and plays a major role in cellular, growth and developmental regulation. Despite BIN2 functional studies including BR signaling related proteasome-mediated BIN2 degradation, the molecular regulating mechanisms and it’s related the protein component for regulating BIN2 degradation has not been completely known. This study aimed to i) identify BIN2 protein and its interacting partner from HA-Immunoprecipitation (IP) of the BL-treated BIN2-HA and bin2-6D-HA lines, using liquid chromatography tandem mass spectrometry (LC–MS/MS) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI–TOF/TOF MS) and ii) characterize relationships between BIN2 and interacting partner proteins. We generated transgenic plants constitutively expressing BIN2-HA, bin2-6D (BIN2E264K)-HA, and BIN2KD (BIN2K69R)-HA construct. IP of the HA-tagged bin2-6D/BIN2 protein followed by mass spectrometry identified F-box protein, BRASSINOSTEROID F-BOX 1 (BRF1) and BRF2 containing the LRR and FBD domain, as interacting proteins with BIN2. Validation in vitro by yeast two-hybrid analyses further confirmed the interacting protein. These results, together with phylogeny and sequence alignments of BRF1 and BRF2 homologs multiple methods, suggest that f-box protein BRF1 and BRF2 play redundant or overlapping roles in regulating BIN2. It is likely that the BIN2 protein stability is controlled by BRF1 and BRF2.