Liquid-liquid phase separation (LLPS) is increasingly recognized as a key cellular mechanism for protecting and organizing biomolecules in response to stress. These dynamic assemblies, known as biomolecular condensates, are formed through the self-association of macromolecules such as proteins and nucleic acids. Intrinsically disordered proteins (IDPs) are known to undergo LLPS, yet the molecular determinants governing this process remain poorly understood. Gaining insight into these mechanisms is crucial for understanding how cells respond to environmental stress and how LLPS may be dysregulated in disease. Here, we investigated LLPS behavior in ERD14, a plant dehydrin from Arabidopsis that is upregulated during drought stress. ERD14 is an IDP previously shown to prevent protein denaturation and stabilize membranes. We examined the structural and phase-separation properties of ERD14 using a combination of biophysical techniques, including small-angle X-ray and neutron scattering, dynamic light scattering, analytical ultracentrifugation, and confocal microscopy. We focused on how LLPS is modulated by macromolecular crowding, salt concentration, pH, and RNA. We found that LLPS is highly dependent on ERD14 concentration, with a cooperative transition and an estimated EC50 of 83 μM in the presence of PEG6000. LLPS was favored at mildly acidic pH (∼6.6) and suppressed by increasing ionic strength, with both NaCl and KCl reducing turbidity at concentrations ≥100 mM. PolyU RNA exhibited a biphasic effect—low concentrations disrupted LLPS, while the addition of salt to RNA-containing mixtures restored turbidity, suggesting a complex interplay of electrostatics and multivalent interactions. The role of ERD14 phosphorylation in LLPS regulation is currently under investigation. Together, our findings show that ERD14 LLPS is governed by a combination of protein concentration, crowding, pH, ionic strength, and RNA interactions. These results offer new insights into the biophysical regulation of stress-responsive condensates in plants.
O'Neill et al. (Sun,) studied this question.