Phase Separation as a Molecular Thermosensor

How organisms sense temperature is a long-standing question. However, the identification of molecular thermosensors has been limited. Now, in a recent issue of Nature, Jung et al. demonstrate that phase separation of ELF3, a component of the circadian clock, acts as a thermosensor. How organisms sense temperature is a long-standing question. However, the identification of molecular thermosensors has been limited. Now, in a recent issue of Nature, Jung et al. demonstrate that phase separation of ELF3, a component of the circadian clock, acts as a thermosensor. Temperature is a major environmental factor that affects every aspect of plant life. Molecular strategies have thus evolved to enable plants to register seasonal fluctuations in temperature and survive stressful temperature extremes. With respect to seasonal temperature, plants monitor daily peaks and troughs of temperature as well as longer-term averages (Zhao et al., 2020Zhao Y. Antoniou-Kourounioti R.L. Calder G. Dean C. Howard M. Temperature-dependent growth contributes to long-term cold sensing.Nature. 2020; 583: 825-829Crossref PubMed Scopus (17) Google Scholar; Hepworth et al., 2018Hepworth J. Antoniou-Kourounioti R.L. Bloomer R.H. Selga C. Berggren K. Cox D. Collier Harris B.R. Irwin J.A. Holm S. Säll T. et al.Absence of warmth permits epigenetic memory of winter in Arabidopsis.Nat. Commun. 2018; 9: 639Crossref PubMed Scopus (41) Google Scholar; Antoniou-Kourounioti et al., 2018Antoniou-Kourounioti R.L. Hepworth J. Heckmann A. Duncan S. Questa J. Rosa S. Sall T. Holm S. Dean C. Howard M. Temperature sensing is distributed throughout the regulatory network that controls FLC epigenetic silencing in vernalization.Cell Syst. 2018; 7: 643-655Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). However, the molecular mechanisms that underpin temperature sensing are still largely unknown. The circadian clock provides a mechanism for plants to align internal physiology with external environmental cues. At the hub of the circadian clock, an assembly of proteins called the evening complex (EC) directly coordinates gene expression with temperature information (Ezer et al., 2017Ezer D. Jung J.H. Lan H. Biswas S. Gregoire L. Box M.S. Charoensawan V. Cortijo S. Lai X. Stöckle D. et al.The evening complex coordinates environmental and endogenous signals in Arabidopsis.Nat. Plants. 2017; 3: 17087Crossref PubMed Scopus (107) Google Scholar). The evening complex is comprised of EARLY FLOWERING3 and EARLY FLOWERING4 (ELF3 and ELF4) and LUX ARRYTHMO (LUX). In a recent study in Nature, Jung et al., 2020Jung J.H. Barbosa A.D. Hutin S. Kumita J.R. Gao M. Derwort D. Silva C.S. Lai X. Pierre E. Geng F. et al.A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis.Nature. 2020; 585: 256-260https://doi.org/10.1038/s41586-020-2644-7Crossref PubMed Scopus (60) Google Scholar now address the molecular mechanism by which the EC mediates thermal responsiveness. When temperature increases, ELF3 phase separates to form molecular condensates in which ELF3 is inactive. This process can be reversed by decreasing temperature (Figure 1). These findings are exciting not only from the thermo-sensing perspective but also because they add to the growing realization that phase separation is a mechanism functionally relevant to many biological processes. ELF3 contains a polyQ repeat, the length of which varies from 7 to 29 residues in natural Arabidopsis populations. Jung and colleagues first tested the association between polyQ length and ELF3 activity in response to higher temperature and found that this association was weak. In Arabidopsis ELF3, the polyQ repeat is embedded in a prion domain (PrD). This prompted the authors to hypothesize that regions surrounding the polyQ repeat may confer temperature responsiveness for ELF3. Indeed, they found that the PrD varies in size in plants that are adapted to different climates. In Brachypodium distachyon, a grass species adapted to warmer climates, ELF3 (BdELF3) does not contain a PrD, and in the potato Solanum tuberosum, which prefers moderate climates, ELF3 (StELF3) had a much smaller PrD than in Arabidopsis. The authors next sought to test the role of ELF3-PrD in temperature responsiveness. Overexpressing BdELF3 and StELF3 rescued the early-flowering phenotype of Arabidopsis elf3-1 at normal (22°C), but not higher (27°C), temperatures. The chimeric ELF3-BdPrD, in which the PrD of Arabidopsis ELF3 was replaced with the corresponding sequence of BdELF3, was unable to fully support temperature-responsive flowering. These results unambiguously showed that the PrD is responsible for temperature responsiveness of ELF3. Jung and colleagues next tested the function of ELF3-PrD at the molecular level. Previously, the Wigge group had shown that ELF3 is a transcriptional repressor that occupies and regulates its target genes in a temperature-dependent manner (Ezer et al., 2017Ezer D. Jung J.H. Lan H. Biswas S. Gregoire L. Box M.S. Charoensawan V. Cortijo S. Lai X. Stöckle D. et al.The evening complex coordinates environmental and endogenous signals in Arabidopsis.Nat. Plants. 2017; 3: 17087Crossref PubMed Scopus (107) Google Scholar). In the current work, the authors found that both BdELF3 and the chimeric ELF3-BdPrD lost the temperature responsiveness of binding and regulation of target genes. These results then led the authors to investigate whether temperature controls the activity of ELF3 directly. Surprisingly, they observed formation of nuclear speckles at higher temperature when expressing ELF3 fused with green fluorescent protein (ELF3-GFP) in planta. This behavior was dependent on PrD, as BdELF3 and chimeric ELF3-BdPrD were evenly distributed even at higher temperature. The ability to form thermo-responsive speckles is intrinsic to ELF3, because the authors observed the same phenomenon upon expressing ELF3 in Saccharomyces cerevisiae, a heterologous system lacking ELF3-related genes. PrDs are known to mediate phase separation of proteins (Franzmann et al., 2018Franzmann T.M. Jahnel M. Pozniakovsky A. Mahamid J. Holehouse A.S. Nüske E. Richter D. Baumeister W. Grill S.W. Pappu R.V. et al.Phase separation of a yeast prion protein promotes cellular fitness.Science. 2018; 359: 39-47Crossref Scopus (250) Google Scholar), prompting the authors to analyze the behavior of ELF3 in vitro. The results showed that purified Arabidopsis ELF3 PrD, but not BdELF3 PrD, rapidly and reversibly formed liquid droplets in a temperature-dependent manner. These observations suggest that in response to high temperature ELF3 phase separates into molecular condensates in order to inactivate itself. Within the EC, the small protein ELF4 is known to regulate the activity of ELF3 via an as-of-yet-unclear mechanism. The authors observed that overexpression of ELF4 largely abolished the thermal responsiveness of flowering and ELF3 binding to and regulation of its target genes, indicating that ELF4 is able to maintain ELF3 in an active state at high temperature. Collectively, the elegant study of Jung et al., 2020Jung J.H. Barbosa A.D. Hutin S. Kumita J.R. Gao M. Derwort D. Silva C.S. Lai X. Pierre E. Geng F. et al.A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis.Nature. 2020; 585: 256-260https://doi.org/10.1038/s41586-020-2644-7Crossref PubMed Scopus (60) Google Scholar showed that Arabidopsis ELF3 can sense warm temperature through its PrD and adopt two conformations: an active diffused form and an inactive form in phase-separated condensates. These findings shed light on how temperature can be perceived and demonstrate that phase separation is functionally important. Yet, this work also raises many interesting questions that need further investigation. For example, as mentioned earlier, it remains an open question as to how the evening complex component ELF4 modulates the temperature responsiveness of ELF3. ELF4 binds a region in ELF3 neighboring the PrD. It is possible that the binding of ELF4 tunes ELF3 structure as shown in vitro (Silva et al., 2020Silva C.S. Nayak A. Lai X. Hutin S. Hugouvieux V. Jung J.H. López-Vidriero I. Franco-Zorrilla J.M. Panigrahi K.C.S. Nanao M.H. et al.Molecular mechanisms of Evening Complex activity in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2020; 117: 6901-6909Crossref PubMed Scopus (32) Google Scholar) or masks the PrD of ELF3, precluding ELF3 association with itself and the formation of condensates (Figure 1). However, this must be reconciled with a previous study showing that ELF4 caused ELF3 to form speckles in the nucleus (Herrero et al., 2012Herrero E. Kolmos E. Bujdoso N. Yuan Y. Wang M. Berns M.C. Uhlworm H. Coupland G. Saini R. Jaskolski M. et al.EARLY FLOWERING4 recruitment of EARLY FLOWERING3 in the nucleus sustains the Arabidopsis circadian clock.Plant Cell. 2012; 24: 428-443Crossref PubMed Scopus (190) Google Scholar). Also, it is intriguing to know whether the speckles formed by ELF3 in vivo exhibit liquid-like behavior, and related to this, whether these speckles are reversible when the temperature drops. If reversible, how quickly do these speckles dissolve or disassemble, given that daily temperature fluctuates extensively (Hepworth et al., 2018Hepworth J. Antoniou-Kourounioti R.L. Bloomer R.H. Selga C. Berggren K. Cox D. Collier Harris B.R. Irwin J.A. Holm S. Säll T. et al.Absence of warmth permits epigenetic memory of winter in Arabidopsis.Nat. Commun. 2018; 9: 639Crossref PubMed Scopus (41) Google Scholar)? As phase separation has been shown to be a mechanism for buffering noise in gene expression (Klosin et al., 2020Klosin A. Oltsch F. Harmon T. Honigmann A. Jülicher F. Hyman A.A. Zechner C. Phase separation provides a mechanism to reduce noise in cells.Science. 2020; 367: 464-468Crossref PubMed Scopus (67) Google Scholar), it would be valuable to explore the role of phase separation of ELF3 in buffering the consequences of daily temperature fluctuations. Above all, the study from Jung and colleagues demonstrated that phase separation of a PrD-containing protein acts as a molecular thermosensor. About 500 Arabidopsis proteins harbor PrDs based on prediction (Chakrabortee et al., 2016Chakrabortee S. Kayatekin C. Newby G.A. Mendillo M.L. Lancaster A. Lindquist S. Luminidependens (LD) is an Arabidopsis protein with prion behavior.Proc. Natl. Acad. Sci. USA. 2016; 113: 6065-6070Crossref PubMed Scopus (81) Google Scholar). Therefore, the findings of this study might extend to other PrD-containing proteins, with other PrDs serving as sensors of different external stimuli.