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Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

Yanniv Dorone, Steven Boeynaems, Benjamin Jin, Flavia Bossi, Eduardo Flores, Elena Lazarus, Emiel Michiels, Mathias De Decker, Pieter Baatsen, Alex S. Holehouse, Shahar Sukenik, Aaron D. Gitler, Seung Y. Rhee

Preprint posted on August 07, 2020 https://www.biorxiv.org/content/10.1101/2020.08.07.242172v1

Like oil in water: The prion-like protein FLOE1 phase-separates upon hydration to enable seed germination

Selected by Martin Balcerowicz

Background: Sensing water – a crucial task in the decision to germinate

Water is essential for plant growth and survival: it acts as a solvent for countless ions and molecules, enables biochemical reactions, transports nutrients and metabolites and keeps a plant erect through hydrostatic pressure. A water deficit thus substantially affects plant physiology and development. While plants have devised multiple mechanisms to cope with limited water supply, one of the most profound strategies is to avoid it altogether: Under adverse conditions, seeds remain dormant and can persist in this state for years, in some cases for centuries. Once germination is induced, however, the process is irreversible, and the emerging seedling must cope with whatever environment it is exposed to. It is therefore vital for the seed to perceive environmental conditions, particularly water supply, prior to germination.

While we know that plants are clearly aware of their water environment (roots for instance can grow towards a water source), our understanding of how water is sensed is still limited. Mechanosensitive ion channels have been on the forefront of putative water sensors; they would be able to register changes in hydraulic pressure that result from a changing water supply (Scharwies and Dinneny, 2019). More recently, intrinsically disordered proteins (IDPs) have taken the spotlight. These proteins are prone to alter conformation with changes in their environment, e.g. the cell’s osmotic potential (Covarrubias et al., 2017). In their preprint, Dorone, Boeynaems et al. identify an IDP that undergoes phase separation upon hydration and thereby can serve as a water sensor in seeds.

Key findings: Phase-separation of FLOE1 upon hydration signals the seed to germinate

Dorone, Boeynaems et al. leveraged published transcriptomics data to get an idea which proteins accumulate in seeds. They found that prion-like proteins (a type of IDPs) were over-represented in seeds and further investigated the function of a previously uncharacterised IDP that they later named FLOE1 (for it acting similar to a sheet of floating ice). A FLOE1-GFP fusion protein was evenly distributed in embryo cells when seeds were dissected in glycerine or high salt solution (i.e. a low water environment), but formed cytoplasmic accumulations – so-called condensates – when transferred to water. This observation suggested that FLOE1 undergoes liquid-liquid phase separation, a process by which one liquid separates from another, as for instance oil droplets do when mixed with water. The same behaviour was also observed when the protein was produced in and purified from bacteria and turned out to be reversible both in vitro and in planta.

Investigating FLOE1 protein structure, the authors found that the protein harbours two predicted disordered domains, one enriched with aspartic acid and serine (DS domain) and one enriched in glutamine, proline and serine (QPS domain). Deletion mutants of FLOE1 were subsequently analysed in human cell culture, tobacco leaves as well as in stable Arabidopsis transformants. Two tendencies were observed in all systems:

  • The QPS domain underpins condensate formation, its deletion reduces the number of condensates formed.
  • The DS domain in contrast seems necessary to maintain liquid droplet state; its deletion causes the condensates to adopt a more solid gel-like state and renders their formation less dependent on hydration.

The relevance of FLOE1 and its deletion variants for seed germination was then investigated in planta: A floe1 loss-of-function mutant displayed normal germination behaviour under well-hydrated conditions, but increased germination rates under high salt conditions. Deletion of the QPS domain resulted in similar effects as a complete loss-of-function, suggesting that FLOE1 and its QPS domain are essential to monitor hydration state in seedlings. Intriguingly, lines carrying a deletion in the DS domain showed even greater germination rates than the floe1 mutant, under both well hydrated and salt stress conditions. Thus, it seems that deletion of the DS domain has a gain-of-function effect.

Taking these results together, the authors hypothesise that FLOE1 acts as a “water stress-dependent resistor” which determines how fast a seed triggers germination upon hydration. While its precise signalling mechanism is yet unclear, liquid-liquid phase separation appears to be at the heart of FLOE1’s functionality. Its phase-separation is triggered by hydration rather than dehydration – the latter being known for several other IDPs (Covarrubias et al., 2017) – and thus possesses some intriguing characteristics that require further investigation.

Figure 1: FLOE1 phase-separates into liquid condensates when seeds are hydrated and thereby allows germination to occur. Loss of its disordered domains strongly affect these condensates: deletion of the QPS domain (ΔQPS) interferes with condensate formation; deletion of the DS domain (ΔDS) renders condensates more gel-like (reproduced from Dorone, Boeynaems et al., Figure 3K, with permission of the authors).

Why I chose this preprint

Phase separation is one of the “hot topics” in current biological research. But while phase-separating proteins have been described in a number of systems, this is one of the instances where the process of phase separation can be directly linked to a developmental response (i.e. germination of the seed). It is also particularly exiting as FLOE1 appears to act as a true hydration sensor in plants.

Open questions/future directions

  1. FLOE1 takes on a key role in the decision to germinate; how does it connect to the known pathways that control germination, e.g. signalling of plant hormones abscisic acid (ABA) and gibberellin (GA)?
  2. The fact that FLOE1 forms condensates upon hydration – rather than upon dehydration – is quite unusual. Is there evidence for a specific (peculiar?) property in its structure that could explain this phenomenon?

References/further reading

Covarrubias, A. A., Cuevas-Velazquez, C. L., Romero-Pérez, P. S., Rendón-Luna, D. F. and Chater, C. C. C. (2017). Structural disorder in plant proteins : where plasticity meets sessility. Cellular and Molecular Life Sciences 74, 3119–3147.

Scharwies, J. D. and Dinneny, J. R. (2019). Water transport, perception, and response in plants. J Plant Res 132, 311–324.

Tags: arabidopsis, germination, intrinsic disorder, phase separation, prion-like protein, water sensing

Posted on: 28th August 2020

doi: https://doi.org/10.1242/prelights.24287

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