Biomolecular condensates formed through phase separation have emerged as a central principle of cellular organization, enabling the dynamic regulation of gene expression, signaling, metabolism, and stress responses. While early conceptual advances in condensate biology have largely originated from animal and in vitro systems, plant cells present a unique set of biological and technical challenges, including rigid cell walls, turgor pressure, plastid autofluorescence, complex endomembrane organization, and acute environmental responsiveness. These distinctive features impede the direct transfer of existing methodologies and drive the development of heterogeneous experimental practices. In this community comment, we present a comprehensive methodological framework for studying biomolecular condensates in plants, spanning in silico prediction, in vitro reconstitution, molecular dynamics simulations, live-cell and super-resolution imaging, material property measurements, membrane-associated condensates, and synthetic condensate engineering. We highlight best practices, common pitfalls, and plant-specific considerations, emphasizing the need for orthogonal validation, quantitative interpretation, and physiological relevance. By consolidating current methodologies and articulating shared principles, this review aims to establish a foundation for rigorous, reproducible, and conceptually coherent research in condensate biology of plants and beyond, with emerging implications for crop genetic improvement and synthetic biology applications.
Jiaxuan Peng, J. Agudo-Canalejo, Monika Chodasiewicz et al.· Science China Life Sciences· 0 citations
Intracellular biomolecular condensation forms multicomponent signaling hubs that regulate development, stress responses, and environmental adaptation. While the molecular grammar encoded within scaffold proteins defines the basal associative features driving condensation, heterotypic condensates are intrinsically dynamic, multicomponent, and far-from-equilibrium systems. Consequently, how condensates organize component composition, stoichiometry, and functional specificity in space and time under physiological conditions remains poorly understood. Addressing this challenge requires integrative frameworks that combine predictive biophysical features with experimental information on protein abundance, interaction networks, subcellular localization, and evolutionary conservation. Here, we first analyzed phase separation (PS) proteins across the Tree of Life in 1,106 species, revealing a stark contrast in computationally predicted phase-separation propensity between eukaryotes and prokaryotes, with genome size as a key determinant. Through a broad analysis of amino acid homorepeat-containing proteins (HRPs) across all species, we uncovered how phase separation evolves via a balance between functional condensation and avoidance of harmful, aggregation-prone sequences. We further identified potential signaling hubs and components across kingdoms by integrating PS-positive proteins with experimentally derived abundance and interactome data from four model eukaryotic species. Using Arabidopsis as a model, we dissect the relationships among PS propensity, condensation hub prediction, HRPs, subcellular localization, and structural conservation. Consequently, we developed PhaseHub (https://phasehub.sbs.ntu.edu.sg/), a user-friendly interface for exploring scaffold-client dynamics, PS components, sequence signatures within each PS protein, and hubs. Our work provides an evolutionary framework for understanding multicomponent PS hubs by integrating molecular grammar with physiological context, thereby facilitating hypothesis generation and rational design.
