Phase separation and biomolecular condensate formation drive plant endomembrane and autophagy crosstalk

Abstract

Like other eukaryotes, plants are a rich hub of proteins, lipids, and nucleic acid biomolecules that undergo liquid-liquid phase separation to form liquid-like biomolecular condensates that facilitate diverse cellular functions, especially upon biotic and abiotic stresses. Current plant-related research highlights the emerging role of biomolecular condensates in stress sensing, modulation, and response as an intricate mechanism for rapid and efficient stress adaptation. The cellular functions of condensates and their localization emphasize the importance of endomembrane systems in bridging the understanding of membrane-bound and membrane-less organelles and their compartmentalization. This review provides an overview of the recent updates and findings in plant phase separation and biomolecular condensate formation. With the increasing evidence of research pointing to a link between membrane-less condensates, autophagy, and the endomembrane system, we discuss the crosstalk between the multivesicular body (MVB), autophagosome, and vacuole. We also elaborate on the functional and regulatory roles of biomolecular condensates in plant autophagosome formation at the early and late stages. Finally, we provide insights for future investigations on plant cellular biomolecular condensates to pave the way for new frontiers of studies in improving agricultural plant yield, resilience, and other biotechnological applications.

Keywords: Autophagosome; autophagy; biomolecular condensates; endomembrane system; liquid–liquid phase separation; membrane trafficking; multivesicular body; vacuole.

Fig. 1.

Compartmentalization of membrane-bound and membrane-less organelles in the plant endomembrane system and autophagic pathway. (A) Diverse environmental signals and conditions trigger intercellular modifications that may initiate liquid–liquid phase separation (LLPS) and its transition within and across several cellular compartments. (B) LLPS is a stage in the phase transition process during biomolecular condensate formation. Cells under different conditions undergo phase transition processes. (C) Common cellular condensates C1–C4 formed from aggregation-prone proteins, and subsequent LLPS are localized in the nucleus (nuclear speckle, C1; and Mediator condensate, C2) and cytoplasm (stress granule, C3; and P-body C4). The endomembrane system and autophagy are intricately linked through the coordinated activity of several organelles including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN) or early endosome (EE), multivesicular body (MVB) or pre-vacuolar compartment (PVC) or late endosome (LE), and the vacuole, which characterize the secretory pathway. Clathrin-coated vesicles (CCVs) play a crucial role in the endocytic pathway by internalizing plasma membrane components and extracellular materials into CCVs, facilitating their cellular uptake as cargo to the sorting endosome for recycling back to the plasma membrane via recycling endosomes or targeted degradation channelled to the vacuole. Coat protein complex II (COP II) vesicles transport cargo (primarily proteins) from the ER to the Golgi. In contrast, COP I vesicles mediate the retrograde transport from the Golgi to the ER, ensuring proper organelle function. Some specific COPII components (Sar1 and Sec23 proteins) are involved in the conventional ER–Golgi trafficking and also regulate plant autophagic flux by interacting with the core autophagy machinery. Remarkably, these vesicular trafficking machineries have been reported to provide membrane sources for autophagosome formation. The Golgi, TGN, and MVB contribute to the sorting and delivery of autophagic cargo, with the vacuole serving as the final destination for degradation, highlighting the dynamic interplay between these compartments in maintaining cellular homeostasis. Within plant cells, the MVB, autophagosome, and vacuole are closely related endomembrane organelles. Notably, the autophagy pathways can be bulk or selective depending on the sequestered cargoes. Created in BioRender. Mgbechidinma et al. (2025) https://BioRender.com/ai1pc3r.

Fig. 2.

The eight broad identification and characterization methodologies for studying plant biomolecular condensates. (A) Sample-based methods involve the use of plant seedlings for leaf and root observation, leaf infiltration, and protoplast expression; heterologous expression and purified proteins reveal the biological system with an improved expression level for the target condensate-forming protein, providing an efficient system for fast testing of the full-length proteins and their domains for phase separation. (B) Cellular-based methods involve observing the plant condensate-rich region (shoot or root), FRAP analysis, 1,6-hexandiol treatment, condensate subcellular localization assay via confocal, super-resolution microscopy, and TEM imaging, measuring and detecting condensate formation (via disrupting LLPS), fluidity, viscosity, dynamics, subcellular localization, and structures. (C) Molecular-based methods using total internal reflection fluorescence microscopy (TIRFM), cryo-EM/ET, and immunoblot assay facilitate high-resolution single-molecule analysis and identification/quantification of target macromolecules and condensates. (D) Biophysical-based methods, involving the use of NMR, giant unilamellar vesicles (GUVs), small unilamellar vesicles (SUVs), liposomes, and other synthetically synthesized vesicles and membranes, provide information on the coordination, phase changes, and membrane–condensate interactions. (E) Component/property-based methods, involving biotinylated isoxazole precipitation, proximity biotin labelling, immunoprecipitation, LC-MS, and in vitro reconstitution, reveal the type and abundance of biomolecules present or interacting with the condensates. (F) Technique-based methods include the in silico study for a guided prediction and structural insights into the biomolecules, the in vitro study validating phase separation, and the in vivo studies confirming the physiological relevance of LLPS and the formed condensates. (G) Functional-based methods, involving phenotypic analysis (growth and developmental patterns) and genotypic analysis (transcription, mRNA processing, and translation), provide information about the relevance of the condensate presence or absence, its function, and possible future prospects that can be harnessed for a scale-up crop improvement. (H) Integrated methods reveal the importance of incorporating several methodologies to study plant biomolecular condensates. Created in BioRender. Mgbechidinma et al. (2025) https://BioRender.com/zr3a0nd.

Fig. 3.

Summary of known condensate-forming proteins, functions, and key techniques in plants. The table highlights recent studies on plant organelle-localized condensates and the key technologies. ALIX, ALG2-interacting protein X; ARF3, AUXIN RESPONSE FACTOR 3; ATG, autophagy; BiFC, bimolecular fluorescence complementation assay; CaLB1, calcium-dependent lipid-binding protein 1; CP29A, chloroplast-localized RNA-binding protein 29A; ER, endoplasmic reticulum; FRAP, fluorescence recovery after photobleaching; FREE1, FYVE domain protein required for endosomal sorting 1; GUVs, giant unilamellar vesicles; HEM1, haematopoietic protein-1; IDR, intrinsically disordered region; LCD, low-complexity domain; MVB, multivesicular body; PEG, polyethylene glycol; PM, plasma membrane; PrLDs, prion-like domains; SAP18, Sin3-associated protein 18 kDa; SLCA, split-luciferase complementation assay; SSLB, solid-supported lipid bilayers; SUVs, small unilamellar vesicles; UBP1c, oligouridylate-binding protein 1c. Most of the studies also used confocal microscopy, TEM and immunogold labelling, electron tomography (ET), correlative light and electron microscopy (CLEM), stimulated emission depletion microscopy (STED), and conducted small-angle measurements. The condensate component and functional analysis were also analysed using immunoprecipitation–MS (IP-MS), ShinyGO for Gene Ontology analysis, split-luciferase complementation assay, co-localization assays, and inhibitor treatment assay. Created in BioRender. Mgbechidinma et al. (2025) https://BioRender.com/uf5r0hj.

Fig. 4.

The endosomal–vacuolar trafficking through the ESCRT machinery and condensate-dependent pathway. In the typical MVB to vacuole and autophagosome to vacuole pathways, MVBs and autophagosomes deliver cargoes into the vacuole via direct membrane fusion, regulated by ESCRT machinery and Rab GTPases. As a plant-specific ESCRT component, FREE1 can form condensates with cargo, wetting and creating intraluminal vesicles (ILVs) within MVBs. Additionally, protein storage vacuoles (PSVs) can separate into two distinct phases, α and β, due to the presence of cargo and remodelling of new tonoplasts at the interface. ESCRT, endosomal sorting complex required for transport; FREE1, FYVE domain protein required for endosomal sorting 1; VPS, vacuolar protein sorting; GEFs, guanine nucleotide exchange factors; Rab, Ras-related protein; SH3P2, SH3 domain-containing protein2; and Ub, Ubiquitinated. Created in BioRender. Mgbechidinma et al. (2025) https://BioRender.com/xuyfhvu.

Fig. 5.

Biomolecular condensates as a platform for autophagy initiation and selective autophagy in plants. (A) The evolutionarily conserved autophagy pathway occurs at the basal level but can be induced under stress conditions. Schematic diagram of the early steps of plant autophagy. The ATG1/13 complex, ATG9 complex, and the PI3K complex initiate phagophore formation from the endoplasmic reticulum (ER). ATG9, together with ATG2 and ATG18, mediates phagophore expansion through lipid transfer and scramblases as the only transmembrane protein initially localized at the endosome or trans-Golgi network (TGN). Two ubiquitin-like conjugation systems [ATG7(E1) and ATG10(E2)] catalyse ATG12–ATG5 conjugation, while ATG7(E1), ATG3(E2), and ATG12–ATG5/ATG16 complex(E3) catalyse ATG8 lipidation. Lipidation in most ATG8 isoforms is preceded by the ATG4 cleavage that exposes the C-terminal glycine residue, enabling phosphatidylethanolamine (PE) covalent attachment and the subsequent incorporation of ATG8–PE into the autophagosome membrane. While TORC1, SnRK1, and SINAT are known regulators of autophagy, the plant hormone brassinosteroid (BR) signalling component ‘BAK1’ also plays an important role. (B) Upon heat stress recovery, the disassembly of stress granules and subsequent release of the ATG proteins activate autophagy. (C) ATG3 promotes autophagy by facilitating LLPS of ATG8e in Arabidopsis. (D) Condensate formation by IDR-rich complex ATG1/13 sets the stage for autophagy initiation upon nutrient starvation in yeast; however, whether phase separation occurs during ATG1/13 complex assembly remains to be explored in plants. (E) Ubiquitinated and non-ubiquitinated proteins are selectively degraded via condensate formation through the autophagy pathway in plants and mammals. ATGs, autophagy-related proteins; FIP200, focal adhesion kinase interacting protein of 200 kDa; LLPS, liquid–liquid phase separation; LC3, Microtubule-Associated Protein 1 Light Chain 3 (a functional homologue of plant ATG8); NBR1, Neighbor of BRCA1 gene 1; P, phosphorylation; Ptc2, PP2C phosphatases; SINAT, SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA; SnRK1, Sucrose-nonfermentation1-related protein kinase1; TAX1BP1, Tax1-binding protein 1; TOR, TARGET OF RAPAMYCIN; and TORC1, TOR complex 1. Created in BioRender. Mgbechidinma et al. (2025) https://BioRender.com/pg8wq57.

Fig. 6.

Autophagosome formation, trafficking, and vacuolar fusion require condensates. (A) Upon phagophore conjugation with lipidated ATG8 (ATG8–PE), an autophagosome as an enclosed double-membraned organelle is formed and transported to the vacuolar tonoplast for fusion via the microtubule (MT) cytoskeleton controlled by the ESCRT machinery and the v-SNARE-type mechanism. ESCRT mediates the phagophore closure to form an autophagosome. SNARE proteins, GTPase, and tethers mediate autophagosome–endosome (amphisome) and autophagosome–vacuole fusion. The released autophagic bodies are degraded within the vacuolar lumen, although the fate of the recycled components remains largely unknown. (Bi) ARLA1s regulate VPS41 LLPS-driven condensate formation under normal conditions and their dynamic conversion into VAPVs during autophagy. On losing liquidity, the condensate converts to puncta that decorate the expanding phagophore and, upon multivesicular body (MVB) fusion in a RAB7 GTPase independent manner, forms a VPS41-associated phagic vacuole (VAPVs), which completes the autophagy pathway through vacuolar degradation. (Bii) In a salt stress-specific condition, Arabidopsis Ca²⁺-dependent lipid-binding protein CaLB1 interacts with the ESCRT-associated protein ALIX at the autophagosome to undergo phase separation, facilitating the recruitment and positioning of ESCRT-III components for efficient closure and maturation of autophagosome. Created in BioRender. Mgbechidinma et al. (2025) https://BioRender.com/9ox3tn0.