Single-Particle Tracking of Genetically Encoded Multimeric Nanoparticles Reveals Regional Heterogeneity and Osmotic Stress-Induced Convergence of Cytoplasmic Crowding in Plant Root Cells

Abstract Macromolecular crowding is a fundamental physical property of the cytoplasm that governs intracellular diffusion and biochemical reactions. However, in situ quantitative characterization of intracellular dynamics and associated biophysical states in intact plant tissues remains challenging. Using 40-nm genetically encoded multimeric nanoparticles (GEMs) and single-particle tracking in Arabidopsis roots, we quantitatively map the regional heterogeneity of cytoplasmic diffusion dynamics and crowding along the root developmental axis: elongation zone cells exhibit a dense, low-mobility baseline, whereas maturation zone and root hair cells display higher mobility. These regions exhibit different sensitivities to osmotic stress. Notably, under severe ionic stress, both the diffusion coefficients and non-Gaussian parameters of the maturation zone and root hair cells converge toward the levels of the elongation zone cells, suggesting an intrinsic physical baseline for cytoplasmic crowding. This kinetic convergence in these cells is accompanied by vacuolar retraction and an increase in cytoplasmic thickness. Together, our study establishes a GEMs-based platform for in situ biophysical analysis in plant cells and uncovers a spatially-resolved physical landscape of cytoplasmic crowding and its dynamic reorganization under osmotic stress.