Arabidopsis blue light receptor phototropin 1 undergoes blue light-induced activation in membrane microdomains

Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and step-wise photobleaching analysis we demonstrated that in the dark phot1-GFP proteins remain in an inactive state and mostly present as a monomer. The phot1-GFP diffusion rate and its dimerization increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive phot1 -GFP, whereas it did enhance its dimerization, suggesting that phot1 dimerization is independent of its phosphorylation. Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) analysis revealed that the interaction between phot1-GFP and AtRem1.3-mCherry was enhanced along with increased time of BL treatment. However, the BL-dependent interaction was not obvious in plants co-expressing phot1 -GFP and AtRem1.3-mCherry, implicating that BL facilitated the translocation of functional phot1-GFP into AtRem1.3-labeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated phot1-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane microdomains act as an organizing platform essential for proper function of activated phot1 at the PM