A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation - PubMed (original) (raw)

. 2021 Aug 5;184(16):4284-4298.e27.

doi: 10.1016/j.cell.2021.06.009. Epub 2021 Jul 6.

Steven Boeynaems 2, Eduardo Flores 3, Benjamin Jin 4, Shannon Hateley 4, Flavia Bossi 4, Elena Lazarus 4, Janice G Pennington 5, Emiel Michiels 6, Mathias De Decker 7, Katlijn Vints 8, Pieter Baatsen 8, George W Bassel 9, Marisa S Otegui 10, Alex S Holehouse 11, Moises Exposito-Alonso 1, Shahar Sukenik 3, Aaron D Gitler 12, Seung Y Rhee 13

Affiliations

A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation

Yanniv Dorone et al. Cell. 2021.

Abstract

Many organisms evolved strategies to survive desiccation. Plant seeds protect dehydrated embryos from various stressors and can lay dormant for millennia. Hydration is the key trigger to initiate germination, but the mechanism by which seeds sense water remains unresolved. We identified an uncharacterized Arabidopsis thaliana prion-like protein we named FLOE1, which phase separates upon hydration and allows the embryo to sense water stress. We demonstrate that biophysical states of FLOE1 condensates modulate its biological function in vivo in suppressing seed germination under unfavorable environments. We find intragenic, intraspecific, and interspecific natural variation in FLOE1 expression and phase separation and show that intragenic variation is associated with adaptive germination strategies in natural populations. This combination of molecular, organismal, and ecological studies uncovers FLOE1 as a tunable environmental sensor with direct implications for the design of drought-resistant crops, in the face of climate change.

Keywords: adaptation; bet hedging; biomolecular condensate; intrinsically disordered proteins; phase separation; prion-like; salt stress; seed germination; water sensing; water stress.

Copyright © 2021 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of interests Y.D., S.B., A.D.G., and S.Y.R. are inventors on a provisional patent application filed with the U.S. Patent and Trademark Office on August 7(th) 2020 by Leland Stanford University & Carnegie Institution for Science (application number 63063009). Aspect of the manuscript covered in the patent application: regulation of seed germination via FLOE1 modulation.

Figures

Figure 1:

Figure 1:. FLOE1 is an uncharacterized seed protein that undergoes biomolecular condensation in a hydration-dependent manner.

(A) Identification of genes enriched in dry Arabidopsis seeds. (B–C) The seed proteome is enriched for specific amino acids (B) and intrinsic disorder (C). Mann-Whitney test. (D) The seed proteome is enriched for prion-like proteins. Binomial test. AT4G28300 is an uncharacterized prion-like protein, which we named FLOE1. (E) FLOE1p:FLOE1-GFP is expressed during embryonic development and forms condensates. (F) FLOE1-GFP forms condensates in embryos dissected from dry seed in a hydration-dependent and reversible manner. Embryonic cotyledons are shown. PSV denotes autofluorescent protein storage vacuoles that are more prominent in the dry state than in the hydrated state (see Fig. S1E). (G) Cell-to-cell variation in subcellular FLOE1-GFP heterogeneity in response to salt. Radicles are shown. * denotes nuclear localization. (H) Quantification of cellular FLOE1 heterogeneity as a function of salt concentration. Black line denotes the 95th percentile of the 2M NaCl heterogeneity distribution. (I) Quantification of the percentage of cells per radicle that show FLOE1 condensation as a function of salt concentration. Mean ± SEM. Four-parameter dose-response fit. (J) Quantification of the percentage of cells per radicle that show FLOE1 nuclear localization as a function of salt concentration. Mean ± SEM. Gaussian fit. (K) FLOE1-GFP condensation exhibits reversibility between high and no salt treatment. Radicles are shown. (L) Scheme highlighting different FLOE1 behaviors upon imbibition. Fluorescence microscopy images are maximum projections. GFP signal is displayed as an inverted gray scale.

Figure 2:

Figure 2:. FLOE1 attenuates germination under water stress.

(A) floe1-1 seeds show higher germination levels under salt stress. Two-way ANOVA, four-parameter dose-response fit, *** p-value < 0.001. Representative of three independent experiments. ΔIC50 is 18.5mM. (B) Seeds retain full germination potential under standard conditions after a 15-day 230mM salt stress treatment. Representative of three independent experiments. (C) Condensates are largely absent in ungerminated seeds after 15 days of incubation under salt stress. FLOE1 condensates appear within two hours after transfer to standard conditions (MS medium). Maximum projection images of radicle cells. GFP signal is displayed as an inverted gray scale. (D) Scheme highlighting the potential function of FLOE1 in attenuating germination when water potential is low. Droplets indicate water availability. (E) floe1-1 seeds show high numbers of differentially expressed genes (DEGs) after imbibition under salt stress, as opposed to unimbibed (dry) and normally imbibed (water) seeds. Imbibition was performed on MS medium by first stratifying for 5 days followed by 4h incubation in a growth cabinet. (F) floe1-1 seeds upregulate stress response genes and genes implicated in metabolism compared to wildtype seeds, and have relatively lower expression of genes involved in ribosomal biogenesis. The only KEGG pathway enriched for the WT was “ribosome” (p-value = 3.88E-17, not shown). See also Table S2. Font size correlates to −log10 (p-value). P-values at bottom-right for scale.

Figure 3:

Figure 3:. Molecular dissection of FLOE1 phase separation.

(A) Recombinant MBP-FLOE1 phase separates in the test tube upon MBP cleavage with TEV protease. Irregular small aggregates can be seen pre-cleavage highlighting FLOE1 aggregation-propensity. DIC imaging. (B) FLOE1 domain structure. CC = predicted coiled coil, DUF = DUF1421. Balloon plots show amino acid composition of the disordered domains. (C–D) Expression of FLOE1 domain deletion mutants in tobacco epidermal pavement cells (C) and human U2OS cells (D). V = vacuole, C = cytoplasm, N = nucleus. (E) Summary of FLOE1 behavior in tobacco and human cells. Fluorescence microscopy images are single optical sections. GFP signal is displayed as a false-colored intensity scale.

Figure 4:

Figure 4:. Molecular dissection of FLOE1 phase separation.

(A) Chimeric proteins containing both the FLOE1 nucleation domain and the PrLDs from FLOE1 (QPS) or the human FUS protein form cytoplasmic condensates. Percentages display number of cells lacking or containing condensates. Average of three experiments. Arrowheads point at cytoplasmic condensates. (B) The number of QPS tyrosine residues alters FLOE1 phase separation in human and tobacco cells. (C) FLOE1 phase diagram as a function of concentration and number of QPS tyrosines. (D) Number of QPS tyrosines affects intracondensate FLOE1 dynamics. Mobile fraction as assayed by FRAP is shown. One-way ANOVA. (E–F) QPS tyrosine-phenylalanine and tyrosine-tryptophan substitutions alter condensate morphology (E) and intracondensate dynamics compared to WT (F). One-way ANOVA. (G) DS deletion or DS tyrosine/phenylalanine-serine substitutions alter condensate morphology. (H) TEM shows that mutant DS FLOE1 condensates have filamentous substructure that is absent in the WT. U2OS cells. (I) DS tyrosine/phenylalanine-serine substitutions alter intracondensate dynamics. Student’s t-test. (J) DS tyrosine/phenylalanine-serine substitutions alter condensate morphology. Mann-Whitney. (K) Scheme summarizing synergistic and opposing roles of FLOE1 domains on the material property spectrum. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001. Purple band denotes WT mean ± SD (D, F, I, J). Fluorescence microscopy images are single optical sections. GFP signal is displayed as a false-colored intensity scale (scale in panel B).

Figure 5:

Figure 5:. FLOE1 condensate material properties regulate its role in seed germination under salt stress.

(A) Scheme highlighting position of tested FLOE61 mutants on the material properties spectrum. (B) Representative images of floe1-1 mutants complemented with ΔQPS, ΔDUF and ΔDS forms of FLOE1 upon dissection in water. Maximum projection images from embryo radicles. (C) Close-up images of WT and mutant FLOE1 condensates. Single optical sections from embryo radicles. (D) Quantification of FLOE1 condensate size. One-way ANOVA. (E) ΔDS FLOE1 condensates are not dependent on hydration. Maximum projection images from embryo radicles. (F) Germination levels of WT, floe1-1 and complemented lines. One-way ANOVA. Representative of three independent experiments. (G) Scheme highlighting FLOE1’s role in regulating germination and the effect of mutants with altered material properties. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001. GFP signal is displayed as an inverted gray scale.

Figure 6:

Figure 6:. The DS-rich domain drives variation in condensate properties of FLOE1 isoforms and paralogs.

(A–B) A. thaliana has two FLOE1 isoforms. FLOE1.2 is missing most of the DS-rich region (A) and forms larger condensates than FLOE1.1 in tobacco leaves (B). (C) The large FLOE1.2 condensates recruit FLOE1.1. (D) FLOE1 has two A. thaliana paralogs that form larger condensates in tobacco leaves. (E) Disorder plots show strong length- and disorder variation between FLOE1 and FLOE2/3 in their DS-rich domains, but not in their QPS-rich domains. (F) FLOE1 condensates do not mix with FLOE2 and FLOE3 condensates. Deletion of the FLOE1 DS-rich domain partially disrupts mixing with wildtype FLOE1, but drives uniform mixing with FLOE2/3 condensates. (G) Scheme highlighting the switch-like role of the FLOE1 DS-domain in condensate mixing and the corresponding phenotypes. All images are single optical sections of tobacco epidermal pavement cells. (B, D) GFP signal is displayed as false-colored intensity scale (scale in panel D). (C, F) GFP and RFP signal are false-colored green and magenta.

Figure 7:

Figure 7:. Natural sequence variation tunes FLOE phase separation.

(A) A species tree of the plant kingdom with example species and their number of FLOE homologs. (B) Gene tree of FLOE homologs. Numbers correspond to FLOE1, FLOE2, and FLOE3. (C) Distribution of DS and QPS length differences between the FLOE1-like (FLOE1L) and FLOE2-like (FLOE2L) clades among monocots and eudicots. Mann-Whitney. (D) Examples of FLOE homologs from across the plant kingdom. N denotes nuclear localization. Single optical sections of tobacco epidermal pavement cells. GFP signal is displayed as false-colored intensity scale. Full species names for (B,D) in Fig. S6J. Each panel indicates the specific clade of the homolog (FLOE1L or FLOE2L).

Comment in

References

    1. 1001 Genomes Consortium, 2016. 1,135 Genomes Reveal the Global Pattern of Polymorphism in Arabidopsis thaliana. Cell 166, 481–491. - PMC - PubMed
    1. Abatzoglou JT, Dobrowski SZ, Parks SA, and Hegewisch KC (2018). TerraClimate, a high-resolution global dataset of monthly climate and climatic water balance from 1958–2015. Sci Data 5, 170191. - PMC - PubMed
    1. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Cech M, Chilton J, Clements D, Coraor N, Gruning BA, et al. (2018). The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46, W537–W544. - PMC - PubMed
    1. Alberti S, Halfmann R, King O, Kapila A, and Lindquist S (2009). A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell 137, 146–158. - PMC - PubMed
    1. Atwell S, Huang YS, Vilhjalmsson BJ, Willems G, Horton M, Li Y, Meng D, Platt A, Tarone AM, Hu TT, et al. (2010). Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature 465, 627–631. - PMC - PubMed

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