2025/06/01 by Gwendolyn K. Kirschner · 1 voice
Biochemistry, Genetics and Molecular Biology · Energy · #Endoplasmic Reticulum Stress and Disease #Photovoltaic System Optimization Techniques #Plant Gene Expression Analysis
paper · pdf · doi:10.1111/tpj.70276
openalex publication_date 2025/06/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/06/15
Liquid–liquid phase separation (LLPS) describes a process in which biomolecules spontaneously separate from a uniform solution into two liquid layers—a dense condensate phase and a dilute phase. These so-called biomolecular condensates are composed of concentrated proteins and nucleic acids and can be found in the nucleus, the cytoplasm and membranes. They play roles in many biological processes, including transcriptional regulation, RNA splicing, protein degradation and stress granule formation (Liu et al., 2024). In plants, stress granule formation is a mechanism for temperature sensing: high temperature induces a condensation of thermosensors by LLPS into nuclear subdomains, where they interact with heat stress-responsive genes. One example is the temperature-sensing transcriptional co-regulator THERMO-WITH ABA-RESPONSIVE 1 (TWA1), which interacts with the co-repressor TOPLESS (TPL) in nuclear stress granules and thereby regulates the expression of heat stress-responsive genes, such as genes encoding HEAT SHOCK PROTEINs (HSPs) and HEAT SHOCK FACTORs (Hsfs) (Bohn et al., 2024). Condensate formation by LLPS is also a way to integrate light signalling with a response to high temperature (Wang & Zhu, 2022). During their research on thermomorphogenesis, Qi Wang, Zhen Gong and Ziqiang Zhu, authors of the highlighted publication, read a publication by Shimada and colleagues, who had generated a collection of Arabidopsis lines with inducible transcription factors and screened them for light-related phenotypes after induction (Shimada et al., 2022). Among these transcription factors, the DEHYDRATION-RESPONSIVE ELEMENT BINDING (DREB) transcription factor, DREB AND EAR MOTIF PROTEIN 4 (DEAR4), caught the attention of Wang and colleagues. Overexpression of DEAR4 caused hypocotyl elongation under both far-red/red and blue light (Shimada et al., 2022). Since some DEARs are also responsive to temperature stress, Wang and colleagues decided to analyse the response of DEAR4 overexpression to high temperature (Wang et al., 2025). Dear4 single mutants resembled the wild-type phenotype, but DEAR4 overexpressors had a higher survival rate under elevated heat stress temperature (43°C) (Figure 1a), suggesting that there is a high redundancy between family members. Interestingly, while DEAR4 transcription decreased under heat stress, the protein accumulation increased. After a temperature shift from 22°C to 38°C, the proteins formed speckle-like condensations in the nuclei of Arabidopsis roots (Figure 1b), which were reversible upon a return to normal temperature. To analyse the nature of the condensates, the authors used fluorescence recovery after photobleaching (FRAP). Unlike solid aggregates, the condensates formed by LLPS exhibit liquid-like behaviours such as fluidity, fusion and dripping. In FRAP experiments, fluorescently labelled molecules within a localized region of the condensate are photobleached. The subsequent recovery of the fluorescence signal is monitored as unbleached molecules diffuse back into the bleached area. Rapid fluorescence recovery, which the authors observed for DEAR4-GFP, indicates liquid-like properties, as it demonstrates fast molecular redistribution and dynamic exchange within the condensate. DEAR4 confers thermotolerance in Arabidopsis. (a) β-estradiol-induced overexpression of DREA4 increases the seedling survival rate under heat stress (HS). (b) High temperature induces the formation of DEAR4-GFP nuclear condensates. Modified from Wang et al. (2025). The DREB transcription factor family is named after the ability of family members to specifically recognize and bind to the dehydration-responsive cis element (A/GCCGA). The promoter of RD29A contains three copies of this cis element and is therefore a common choice for studying the transcriptional activity of DREB transcription factors (Tsutsui et al., 2009). A luciferase assay with the RD29A promoter and DEAR4 showed that the EAR motif is necessary for DEAR4 transcriptional repressor activity. Overexpressing a truncated form of DEAR4 lacking the ERF-associated amphiphilic repression (EAR) domain did not confer heat stress resistance, suggesting that the role of the EAR motif in transcriptional repression was essential for DEAR4-mediated thermotolerance. To understand the functional mechanism of the DEAR4-mediated thermotolerance, the authors compared the transcriptomes of wild-type and DEAR4-overexpressing plants under normal temperature and heat stress. Because DEAR4 is a transcriptional repressor, they focused on genes with decreased transcript levels in the DEAR4 overexpressors and identified GA-STIMULATED ARABIDOPSIS 5 (GASA5). GASA5 was shown to be a negative regulator of some HSPs under heat stress (Zhang & Wang, 2011). ChIP-qPCR assay and electrophoretic mobility shift assay (EMSA) showed that DEAR4 could bind the GASA5 promoter and repress GASA5 expression. By immunoprecipitation and mass spectrometry (IP-MS), the authors screened for DREA4 interaction partners and identified TPL and TOPLESS-RELATED PROTEIN 1 (TPR1). As expected, the interaction was facilitated by the EAR motif, and the DEAR4-TPL complex formed speckle-like structures, with more speckles forming under higher temperatures. The C-terminus of DEAR4 contains a long intrinsically disordered region (IDR), which is often associated with LLPS (Liu et al., 2024). The EAR motif within the IDR region had the highest Predictor of Natural Disordered Regions score; therefore, the authors tested the relevance of this motif for the condensation. DEAR4-GFP without the EAR motif in the IDR region did not form condensates under high temperature, suggesting that this motif is important for LLPS-mediated condensation. TPL/TPR corepressors interact with histone deacetylases to regulate chromatin deacetylation (Kagale & Rozwadowski, 2011). The H3K9ac histone acetylation level of GASA5 was reduced under heat stress in both wild-type and DEAR4 overexpressors, and the heat stress-triggered repression of GASA5 expression was attenuated in tpl mutants, suggesting that the inhibition of GASA5 expression could be caused by histone deacetylation. In this study, the authors showed that DEAR4 forms reversible, LLPS-dependent speckles under high temperature. In these speckles, DEAR4 interacts with TPL corepressors to repress the negative thermotolerance regulator GASA5 through chromatin modification. This demonstrates another way how LLPS-mediated speckle formation could be used to integrate heat stress into transcriptional pathways.