Abstract:
Phototropism directs plant shoot growth toward a light source by establishing a lateral auxin gradient. This process relies on the modification of the polar localization of PIN-FORMED (PIN) auxin efflux carriers, such as PIN3, at the plasma membrane (PM). Blue light (BL) perception by phototropin1 (phot1) initiates this response; however, the mechanisms linking phot1 to auxin transport remain unknown. The plant-specific protein NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) acts immediately downstream of phot1 activation and is essential for phototropic bending. In darkness, NPH3 localizes predominantly to the PM, whereas BL induces phot1-dependent phosphorylation at Ser744, enabling the binding of 14-3-3 proteins and triggering partial dissociation from the PM. Following release, NPH3 assembles into biomolecular condensates in the cytosol. Remarkably, cytosolic NPH3 can re-associate with the PM upon return to darkness or prolonged BL exposure.
Yet, how this dynamic NPH3 cycling contributes to phot1 signalling and what biochemical function NPH3 has remain unclear.
To address this, we mapped the protein-interaction networks of NPH3 in its two distinct subcellular localizations: at the PM and within cytosolic condensates. Using complementary proteomic approaches, including TurboID-based proximity labelling, GFP-based immunoprecipitation, and Fluorescence-Activated Particle Sorting in etiolated transgenic Arabidopsis seedlings, we identified reproducible but distinct sets of putative NPH3 interaction partners in each compartment. These results suggest that NPH3 activity is closely linked to its subcellular localization.
Proteomic analysis of NPH3 condensates indicates that they consist predominantly of NPH3 itself and are also enriched in general regulators of condensate dynamics, namely molecular chaperones of the HSP70-family. Validation through partial co-localization and co-immunoprecipitation in a transient plant expression system supported that NPH3 can associate with HSP70-1 in condensates. These observations, together with structural predictions, suggest that NPH3 likely forms a single-component condensate that serves to transiently sequester (and thus inactivate) NPH3 in the cytosol, thereby delaying its re-association with the PM.
In contrast, PM-associated NPH3 appears to perform a distinct biochemical function. Here, our proteomic analyses indicate that NPH3 is in close proximity to the endocytic machinery, in line with a potential role in organizing or stabilizing molecular environments that promote clathrin-mediated endocytosis (CME) at the PM. We focused on two putative NPH3 interaction partners, the subunits TPLATE and AtEH1 of a CME-adaptor known as the TPLATE complex. Interactions between NPH3 variants and TPLATE or AtEH1 were investigated by transient co-expression and co-localization studies, Förster Resonance Energy Transfer using Fluorescence Lifetime Imaging Microscopy (FRET-FLIM), and targeted co-immunoprecipitation. Our results indicate that NPH3 associates with TPLATE and AtEH1 at the PM. In addition, cell biological experiments were conducted to elucidate the physiological significance and functional consequences of these interactions. In Arabidopsis nph3-7 loss-of-function mutants, phototropic bending is abolished, bulk endocytosis is reduced, and PIN3 fails to polarize under unilateral BL, supporting a potential role for NPH3 in coordinating membrane trafficking. NPH3-organized molecular PM environments likely facilitate the BL-induced polarization of PIN3 in hypocotyl endodermis cells, required for the establishment of the lateral auxin gradient.
The results presented in this thesis support a model in which NPH3 functions as a dynamic PM-domain organizer, contributing to PIN3 trafficking through spatially regulated endocytic processes at the PM. The formation of NPH3 cytosolic condensates as a result of phot1-dependent phosphorylation likely serves as an inactivation mechanism that supports signal attenuation and proper PIN3 dynamics at the PM, which are necessary for phototropic growth.
NPH3