Identification of Molecular Determinants Contributing to the Function of NPH3 in Phototropic Hypocotyl Bending

As sessile organisms, plants have to adapt to environmental changes such as varying light conditions. Through the phototropic response plants can adapt their growth according to the direction of light. Perception of the light stimulus in Arabidopsis thaliana occurs via the blue light (BL) photoreceptor phototropin 1 (phot1), finally resulting in a reallocation of the PIN auxin efflux carriers at the plasma membrane (PM). This causes a lateral auxin gradient associated with a higher auxin concentration on the shaded side of the shoot, resulting in a bending of the hypocotyl towards the light stimulus. NON PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a plant specific protein that is essential for the phototropic response and acts downstream of phot1. In darkness, the hydrophilic NPH3 localizes to the PM and exists in a phosphorylated state, but relocalizes to cytosolic particles upon BL irradiation correlating with a dephosphorylation. Former members of our group identified NPH3 as an BL-dependent interaction partner of 14-3-3 proteins. Via an in-depth characterization of numerous NPH3 variants combined with confocal laser scanning microscopy (CLSM) we could show that an amphipathic helix located in the C-terminal NPH3 region is required for electrostatic interaction-based PM association in vivo. Furthermore, analysis of BL-triggered 14-3-3 association with NPH3 turned out to be required for PM dissociation of NPH3. CLSM videos illustrated that PM-detachment of NPH3 and formation of particle-like structures in the cytosol are separate and consecutive processes. Furthermore, our findings suggest that NPH3 forms membraneless, biomolecular condensates upon BL treatment. Serial deletions revealed that condensate formation requires two regions, the N-terminal BTB domain and a region upstream of the C-terminal coiled coil domain. Moreover, the phosphorylation pattern of NPH3 and its light-induced changes are highly complex. BL-irradiation initially induces phosphorylation of the 14-3-3 binding site, followed by an association of 14-3-3 proteins which - in addition to the PM dissociation - are also required for the dephosphorylation of various NPH3 residues. Formation of cytosolic condensates, however, is neither a consequence of the BL-induced 14-3-3 interaction nor dephosphorylation and might thus be due to intrinsic properties of NPH3. Remarkably, the BL-induced modification of both the phosphorylation pattern and the subcellular localization of NPH3 are reversible upon retransfer to darkness. Based on our data, we propose, that light-dependent cycling of NPH3 between the PM and cytosolic condensates is essential for NPH3 function during the phototropic response