Get closer and make hotspots: liquid-liquid phase separation in plants

Abstract

Liquid-liquid phase separation (LLPS) facilitates the formation of membraneless compartments in a cell and allows the spatiotemporal organization of biochemical reactions by concentrating macromolecules locally. In plants, LLPS defines cellular reaction hotspots, and stimulus-responsive LLPS is tightly linked to a variety of cellular and biological functions triggered by exposure to various internal and external stimuli, such as stress responses, hormone signaling, and temperature sensing. Here, we provide an overview of the current understanding of physicochemical forces and molecular factors that drive LLPS in plant cells. We illustrate how the biochemical features of cellular condensates contribute to their biological functions. Additionally, we highlight major challenges for the comprehensive understanding of biological LLPS, especially in view of the dynamic and robust organization of biochemical reactions underlying plastic responses to environmental fluctuations in plants.

Keywords: condensate; intrinsically disordered protein; liquid-liquid phase separation; multivalent interaction; prion-like domain.

Figure 1. Molecular mechanism of liquid–liquid phase separation (LLPS)

The presence of intrinsically disordered regions (IDRs) attracts multivalent interactions and induces LLPS. Post‐translational modifications (PTMs), such as phosphorylation, acetylation, methylation, and deamidation, can influence LLPS by inhibiting electrostatic and other types of interactions, thus serving as on/off switches. Multiple environmental factors including ionic strength, pH, and temperature can also affect LLPS by inducing changes in various types of interactions among macromolecules.

Figure 2. Functional diagrams of cytosolic condensates in plants

(A) Heat‐responsive stress granules (SGs). UBP1b inhibits the degradation of DnaJ and SAP3 mRNAs, activating gene expression. Heat shock recovery releases ribosomal protein (rProtein)‐encoding transcripts from the SGs, possibly through HSP101, and promotes translation. (B–D) Processing bodies (PBs). Dark‐induced PBs containing DCP5 attenuate the translation of mRNAs involved in photomorphogenesis (B). Upon ethylene perception, EIN2 associates with the 3'‐UTRs of EBF1 and EBF2 transcripts and sequesters them within PBs, derepressing ethylene responses (C). The PB component TZF9 is phosphorylated by MAPKs upon PAMP recognition, releasing defense‐related mRNAs from PBs to trigger the immune response (D). (E) NPR1 condensate. Salicylic acid (SA)‐induced NPR1 monomer forms cytosolic condensates, together with CUL3–E3 ligase complexes, and ubiquitinates effector‐triggered immunity (ETI) proteins, such as NB‐LRRs, to inhibit the hypersensitive response (HR).

Figure 3. Nuclear condensates in plants

(A) FCA body. The phase‐separated FCA body enhances polyadenylation by compartmentalizing 3'‐end processing factors. The FCA body is further regulated by FLL2 and other autonomous proteins. (B) Photobody. Left: Upon blue light exposure, the active forms of CRYs enter the nucleus and form a photobody, together with core light signaling proteins, such as ELF3, COP1, SPA1, and HY5. Right: The phyB‐associated photobody induces the phosphorylation and consequently the degradation of the PHYTOCHROME‐INTERACTING FACTOR 3 (PIF3) protein. (C) ELF3 body. At low temperature, ELF3 forms the Evening Complex, along with ELF4 and LUX, and acts as a transcriptional repressor. At high temperature, the PrD‐containing ELF3 is assembled into nuclear bodies. The temperature‐responsive LLPS of ELF3 prevents its binding to EC‐target genes, thus allowing gene expression. (D) PcG body. PRC2 interacts with PWO1, EMB1579, CUL4, DDB1, and MSI4 to form hotspots for establishing H3K27me3. The IDR‐containing LHP1 protein also binds to PcG‐deposited H3K27me3 and contributes to the formation of PcG body.

Figure 4. Liquid–liquid phase separation in chloroplasts

(A) Pyrenoid of C. reinhardtii. The pyrenoid matrix exhibits liquid‐like properties through multivalent interactions between Rubisco and EPYC1. EPYC1 has five Rubisco‐binding regions, and Rubisco has eight binding sites for EPYC1. The multivalent interactions allow regular spacing of Rubiscos in pyrenoid matrix to maximize CO₂ fixation and photosynthetic efficiency. (B) STT body. STT1 and STT2 form heterodimers through C‐terminal ankyrin domain interaction. N‐terminal intrinsically disordered regions of STT proteins enable LLPS along with cpTat pathway substrates, such as OE23. After the STT body moves to thylakoid membrane, Hcf106 allows reverse phase separation and the release of OE23 to translocate across thylakoid membrane.