RESEARCH ARTICLE

Stress granule formation is induced by a threshold temperaturerather than a temperature difference in ArabidopsisTakahiro Hamada1,*, Mako Yako1, Marina Minegishi1, Mayuko Sato2, Yasuhiro Kamei3, Yuki Yanagawa4,Kiminori Toyooka2, Yuichiro Watanabe1 and Ikuko Hara-Nishimura5

ABSTRACTStress granules, a type of cytoplasmic RNA granule in eukaryoticcells, are induced in response to various environmental stresses,including high temperature. However, how high temperatures inducethe formation of these stress granules in plant cells is largelyunknown. Here, we characterized the process of stress granuleformation in Arabidopsis thaliana by combining live imaging andelectron microscopy analysis. In seedlings grown at 22°C, stressgranule formation was induced at temperatures above a criticalthreshold level of 34°C in the absence of transpiration. The thresholdtemperature was the same, regardless of whether the seedlings weregrown at 22°C or 4°C. High-resolution live imaging microscopyrevealed that stress granule formation is not correlated with the sizesof pre-existing RNA processing bodies (P-bodies) but that thetwo structures often associated rapidly. Immunoelectron microscopyrevealed a previously unidentified characteristic of the fine structures ofArabidopsis stress granules and P-bodies: the lack of ribosomes andthe presence of characteristic electron-dense globular and filamentousstructures. These results provide new insights into the universal natureof stress granules in eukaryotic cells.

KEYWORDS: Arabidopsis, Stress granules, P-bodies, Temperature,High-resolution live imagingmicroscopy, Immunoelectronmicroscopy

INTRODUCTIONPlants are sessile organisms that must adapt to environmental changesin order to survive. Temperature is a rapidly changing parameter in theenvironment. For example, the irradiation of leaveswith direct sunlighthas the potential to cause dramatic increases in temperature. Plantsusually release heat by transpiration (Campbell, 1977). However,under some conditions, especially in dry and high-temperatureenvironments, plants are unable to control their body temperature.To survive under severely dry and high-temperature conditions, plantspossess various heat resistance mechanisms, such as the formation ofheat shock proteins (Mittler et al., 2012; Scharf et al., 2012).A wide range of translational repression responses occur in

plants exposed to high temperatures (Nover et al., 1983, 1989). As acause or result of this translational repression, components of the

translational machinery, including mRNAs, eukaryotic translationfactors, poly-A binding proteins (PABP) and 40S ribosomalproteins, begin to form stress granules. Stress granules, a type ofRNA granule, are cytoplasmic aggregates composed of variousRNA molecules and proteins. Stress granules are found in variouseukaryotic cells under a variety of stress conditions. In human cells,stress granule formation is induced by heat stress, arsenite exposure,viral infection and UV irradiation (Kedersha et al., 2013).Stress granules were recently found to be related to human diseases,such as Alzheimer’s disease, amyotrophic lateral sclerosis andfrontotemporal lobar degeneration (Wolozin, 2012; Vanderweydeet al., 2013). In plants, stress granules appear in response tohypoxia, high-salt conditions, and exposure to methyl jasmonate,arsenite, potassium cyanide and myxothiazol (Nover et al., 1983,1989; Weber et al., 2008; Pomeranz et al., 2010; Sorenson andBailey-Serres, 2014; Yan et al., 2014; Gutierrez-Beltran et al.,2015). In addition, several plant-specific stress granule componentshave been identified, revealing some of the molecular mechanismsunderlying stress granule formation (Chantarachot and Bailey-Serres,2018). However, at what time point plants begin to form stressgranules is still largely unknown. For example, how do boththe intensity and duration of stress conditions affect stressgranule formation? Plants usually survive exposure to externaltemperature changes. Therefore, do different plants have differenttemperature thresholds for stress granule formation depending onthe temperature of their habitats?

Here, we examined the process of early stress granule formationin Arabidopsis seedlings under finely set conditions in termsof temperature and duration of treatment. Our experimentsrevealed a threshold of stress granule formation in response to atemperature shift (representing stress intensity) but not duration.The threshold did not vary under different growth conditions.However, the threshold did vary depending on whether transpirationwas allowed to occur, indicating that the cooling effect oftranspiration is closely related to stress granule formation. Time-lapse analyses revealed that stress granules can be generated in aprocessing body (P-body)-independent manner but that they areoften closely associated with P-bodies. Ultrastructural analysis ofArabidopsis stress granules and P-bodies revealed irregularlyshaped, ribosome-free structures with characteristic electron-dense particulates and filamentous structures. These resultsprovide a framework for future studies of plant stress granulesand offer new insights into the roles of stress granules in theresponses of plants to severe environmental conditions.

RESULTSThe threshold temperature of stress granule formation is34°C in seedlings mounted for microscopyTo study stress granule formation in plants, we constructed a plasmidcontaining the eIF4A2 gene fused with GFP at the C-terminusReceived 25 January 2018; Accepted 12 July 2018

1Department of Life Sciences, Graduate School of Arts and Sciences, The Universityof Tokyo, Tokyo 153-8902, Japan. 2RIKEN Center for Sustainable Resource Science,Yokohama 230-0045, Japan. 3National Institute for Basic Biology, Aichi 444-8585,Japan. 4Institute of Agrobiological Sciences, NARO, Tsukuba 305-8602, Japan.5Faculty of Science and Engineering, Konan University, Hyogo 658-8501, Japan.

*Author for correspondence (hama.micro@gmail.com)

T.H., 0000-0002-8829-4001; M.M., 0000-0001-7296-2266; M.S., 0000-0002-9876-5612; Y.K., 0000-0001-6382-1365; K.T., 0000-0002-6414-5191; Y.W., 0000-0002-7139-4903; I.H., 0000-0001-8814-1593

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(eIF4A2-GFP) and obtained stably transformed Arabidopsis linesin which stress granules could be visualized under a fluorescentmicroscope. The transgenic plants emitted strong fluorescencebut did not show noticeable morphological or developmentaldefects (Fig. S1).A rise in temperature is likely to be one of the most critical factors

for stress granule formation in eukaryote cells. We, therefore,immersed eIF4A2-GFP seedlings in water between glass slides(hereafter referred to as the mounting method) and kept them inan incubator at 38°C for 10 or 30 min. The pattern of fluorescencefrom eIF4A2-GFP was diffuse in the cytoplasm at 22°C, and stressgranules formed after heat treatment (Fig. 1A). In the presence ofcycloheximide, an inhibitor of stress granule formation (Kedershaet al., 1999; Sorenson and Bailey-Serres, 2014), stress granuleformation was not induced by 30-min long heat treatment at 38°C(Fig. 1B). These results indicate that eIF4A2-GFP transgenic plantsare good stress granule marker lines. Next, we attempted to determinethe lowest temperature (i.e. threshold temperature) required forseedlings to form stress granules in an incubator, increasing thetemperature from 30°C by 1°C steps to 40°C. In plants subjectedto the mounting method, heat in the cytoplasm could not belost through transpiration. We plotted the diffuse localization offluorescence and granule formation in response to treatment duration

versus temperature. We detected a clear boundary between diffusefluorescence and stress granule formation (Fig. 1C). For example,when treatment was for the duration of 10 min, stress granulesappeared at temperatures above 34°C. We confirmed these resultsusing another stress granule marker line in which GFP was fusedto the N-terminus of Arabidopsis poly(A)-binding protein 6(GFP-PAB6) (Fig. S1C). The GFP-PAB6 and eIF4A2-TagRFPsignals overlapped (Fig. S1G).

Different heat treatments and high-salt stress affect thethreshold temperature required for stress granule formationDifferent heat treatment protocols had been followed in previousphysiological and imaging experiments using plants. We investigatedwhether the application of different heat treatment protocols resultedin any changes in the critical temperature at which stress granulesformed. We examined stress granule formation using the microtubemethod, a typical method used in drug treatments in which seedlingsare immersed in water within a tube. Stress granules formed attemperatures >34°C and treatment duration of 10 min (Fig. 1D).This threshold temperature is identical to that identified by usingthe mounting method, probably because heat in the cytoplasmcannot be lost by transpiration when using the microtube method,which is also true for the mounting method.

Fig. 1. Threshold temperatures of stress granule formation in hypocotyl cells of Arabidopsis seedlings. (A) Induction of stress granule formation byheat treatment. Confocal images showing stress granule formation in the hypocotyls of 5-day-old Arabidopsis seedlings expressing the stress granulemarker eIF4A2-GFP. The seedlings were grown at 22°C and mounted in water between coverslips equipped with a 0.15 mm spacer. Images were taken atthe same time point before and after heat treatment at 38°C for 10 min or 30 min. Scale bar: 10 µm. (B) Effects of cycloheximide treatment on stressgranule formation. eIF4A2-GFP seedlings were pre-treated at 22°C for 30 min before heat treatment at 38°C for 30 min. Scale bar: 10 µm. (C–G) Thresholdtemperatures of stress granule formation under various conditions. Stress granule formation at different temperatures was examined in seedlings. Seedlings weremounted (C) as described for panel A, immersed in water in a tube (D), placed on an agar plate (E) or planted in soil (F). Seedlings exposed to 4°C for2 days (G) were also examined. Stress granules were observed in all cells (○), observed in some cells (Δ) or never observed (×) under each condition.

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Agar plating is another useful treatment for physiologicalexperiments. Here, we heat-treated plants grown on agar plates.Stress granules formed at temperatures >38°C and treatmentduration of 10 min when using the agar plate method (Fig. 1E).When using this method, longer duration and higher temperatureduring heat treatment was required for stress granule formationcompared with the mounting and microtube methods. The fourthmethod, planting in soil, is the closest to natural conditions. Thetemperature required for stress granule formation was >42°Cduring a 10 min treatment period (Fig. 1F). Compared with themounting and microtube methods, seedlings grown on agarplates and in soil can transpire, leading to a reduction intemperature in the cytoplasm. Under conditions that do not allowfor transpiration, 34°C was identified as a constant thresholdtemperature regardless of the duration of the treatment (Fig. 1C,D).Thus, 34°C appears to be the threshold temperature for stressgranule formation in the cytoplasm of Arabidopsis seedlings.Stress granule formation is also induced by other stresses, such as

hypoxia, high-salt conditions and exposure to methyl jasmonate.We investigated whether high-salt conditions affect the thresholdtemperature for stress granule formation by using the microtubemethod. On the basis of previously reported experimental conditions(Yan et al., 2014), we confirmed that stress granule formation wasinduced by treatment with mannitol buffer containing 200 mMNaClbut not by treatment with buffer containing 150 mM NaCl(Fig. S2A). In the presence of 150 mM NaCl, stress granulesformed at temperatures <34°C during a 10 min treatment period(Fig. S2B). These results indicate that a combination of stresses affectthe threshold temperature for the formation of stress granules.

Arabidopsis stress granules do not form in response torelative temperature differencesPlants are exposed to ambient temperatures that fluctuate betweenhigh and low temperatures within a single day.We examined whetherlow-temperature stress also induces stress granule formation. Stressgranules failed to form at low temperatures, such as 10°C and 4°C(Fig. S3). Next, we investigated whether the temperature at whichstress granules form changed when plants had previously been grownat lower temperatures (Fig. 1G). eIF4A2-GFP trasngenic seedlingswere grown at 22°C for 5 days, followed by 4°C for 2 days.Subsequently, the seedlings were transferred from 4 to 22°C.We did not observe stress granule formation, even though theseedlings experienced a temperature change of 18°C, which is aslarge as the change from 22 to 40°C. Stress granules started toform at 34°C but not at 25, 30 or 33°C (Fig. 1G), indicating thatthe threshold temperature was 34°C in seedlings grown at 4°C.This result is almost identical to that of seedlings grown at 22°C(Fig. 1C). We also confirmed these results in GFP-PAB6 transgenicplants (Fig. S1). Our findings indicate that Arabidopsis seedlingsform stress granules by sensing the absolute temperature, not adifference in temperature.

Different sizes and numbers of stress granules develop inresponse to different temperatures and incubation timesDuring our observation of stress granule formation, we noticed thatdifferent numbers and sizes of stress granules developed in responseto different temperatures and incubation times. To quantitativelyinvestigate the effects of different heat treatments on the averagenumber and size of stress granules, we performed heat treatments at34, 38 or 40°C for 10 or 30 min, or 1 or 2 h using the microtubemethod (Fig. 2). After 10 min of heat treatment, the average numberof stress granules increased with increasing temperature (Fig. 2A,B).

Numerous small stress granules were observed at 40°C comparedwith those at 36 and 38°C. Moreover, at 38°C and 40°C, the averagenumber and size of stress granules increased in proportion to theincubation time. However, at 34°C, the average number of stressgranules decreased and average stress granule size significantlyincreased depending on incubation time, indicating that, over time,the stress granules fused with each other at 34°C. Huge stress granulesthat formed at 34°C after 2 h of treatment were observed as brightfoci, confirming the fusion of stress granules.

Cytoplasmic streaming by actin filaments contributes to thefusion of stress granulesTo investigate the mechanism behind the formation of these hugestress granules at 34°C and extended incubation time, we examinedthe behavior of stress granules at 34°C.We observed rapid and long-distance movement of stress granules in the cytoplasm at or nearthe threshold temperature of 34°C (Movie 1). At 38°C, the stressgranules exhibited active movement at 10 min and moved slowly at30 min but not beyond this time point. No movement was observedin samples exposed to higher temperatures such as 40°C for 10 min.However, the size and number of stress granules increased at 40°Cfrom 10 to 30 min in the absence of movement (Fig. S4), indicatingthat movement is not essential for stress granule formation. Therapid, long-distance movement of stress granules suggests thatthey were transported by cytoplasmic streaming, a process driven byinteractions between actin and myosin. In plant cells, both organellesand P-bodies are transported long distances through cytoplasmicstreaming (Boevink et al., 1998; Nebenführ et al., 1999; Mathur et al.,2002; VanGestel et al., 2002; Ueda et al., 2010; Hamada et al., 2012).

On the basis of the results shown in Fig. 1, we chose to treatplants at 38°C for 10 min to observe the formation of stressgranules by using the microtube method. These conditions led tothe formation of well-defined stout stress granules that exhibitedrapid long-distance movements (Movie 1). To test the effects ofcytoskeleton disturbance, we pre-treated eIF4A2-GFP seedlings intubes with the cytoskeleton depolymerization drugs Oryzalin(which depolymerizes microtubules) or latrunculin B (Lat B;which depolymerizes actin filaments) at 22°C for 30 min,followed by incubation at 38°C for 10 min to induce stressgranule formation, and observed them under a microscope at 22°C.Stress granules completely disappeared during the first 10 min ofobservation (Movie 1). In the control samples, some stressgranules were transported long distances at a maximum velocityof ∼0.5 µm/s and an average velocity of ∼0.08 µm/s (Fig. 3A,Movie 1). Treatment with Oryzalin did not affect the motility ofstress granules; the maximum and average velocities were also∼0.5 µm/s and 0.08 µm/s, respectively (Fig. 3B, Movie 2).However, treatment with Lat B reduced stress granule motility(Fig. 3C, Movie 3). The maximum and average velocities in thepresence of Lat B were ∼0.1 µm/s and 0.03 µm/s, respectively.A similar result was obtained when the seedlings were treated with amixture of Lat B andOryzalin (Fig. 3D,Movie 4). These results clearlyindicate that actin-dependent cytoplasmic streaming contributes tothe rapid, long-distance transport of stress granules in plant cells.

Next, we investigated the effect disturbance of the cytoskeleton hason the formation of large stress granules (Fig. 3E–G). After pre-treating eIF4A2-GFP seedlings with Oryzalin or LatB, a combinationof both, or with DMSO (control), they were incubated at 34°C for 2 hto induce the formation of large stress granules. In the presence of0.1% DMSO (control treatment), the median size of stress granuleswas 1.08 µm2 and the average number was 0.004 (n/µm2). Treatmentwith Oryzalin increased the average number to 0.026 and decreased

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the size to 0.11 µm2. These results are consistent with previousstudies reporting that microtubule depolymerization affects stressgranule formation in animals and plants (Ivanov et al., 2003;Gutierrez-Beltran et al., 2015). Treatment with Lat B increased the

average number of granules to 0.064 and decreased their size to0.05 µm2. In the presence of Lat B, the cytoplasm often stagnated andaccumulated at some local areas. Many small stress granules werealso observed in these cytoplasm-accumulated areas. In addition,

Fig. 2. Effects of different temperatures and incubation times on stress granule size and number. (A) Confocal images showing stress granule formationin hypocotyls of 5-day-old eIF4A2-GFP seedlings at different temperatures and incubation times. Seedlings were immersed in water in a tube and incubatedunder each condition. Scale bar: 10 µm. (B,C) Quantification of number and size of stress granules (SGs) at each temperature and incubation time fromexperiments shown in A. At least ten independent images were analyzed. Bar graphs at linear scale (B) and logarithmic scale (C). A total of 19, 23, 16, 34, 7,7, 7, 8, 8, 7, 7 and 10 images were analyzed of seedlings incubated at 34°C for 10 min, 34°C for 30 min, 34°C for 1 h, 34°C for 2 h, 38°C for 10 min,38°C for 30 min, 38°C for 1 h, 38°C for 2 h, 40°C for 10 min, 40°C for 30 min, 40°C for 1 h and 40°C for 2 h, respectively. A total of 4467, 3823, 261, 492, 3245,8660, 8138, 11008, 10421, 7773, 12359 and 9388 stress granules were analyzed of seedlings incubated at 34°C for 10 min, 34°C for 30 min, 34°C for 1 h, 34°Cfor 2 h, 38°C for 10 min, 38°C for 30 min, 38°C for 1 h, 38°C for 2 h, 40°C for 10 min, 40°C for 30 min, 40°C for 1 h and 40°C for 2 h, respectively. *P<0.01,**P<0.05 and ***P=not significant according to Welch’s t-test for unequal variances. Error bars indicate ±s.d.

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treatment with bothOryzalin and Lat B increased the number of stressgranules to 0.053 and decreased their size to 0.08 µm2. These resultsindicate that cytoplasmic streaming induced by actin filaments affectsstress granule size and number in Arabidopsis.

Stress granule formation is not correlated to the sizeof P-bodiesThe identification of the critical threshold temperatures for stressgranule formation allowed us to perform fine-scale analysisof stress granule formation. Stress granules appear adjacent toP-bodies in eukaryotes (Kedersha et al., 2005; Buchan et al., 2008;

Weber et al., 2008). Therefore, to clarify the relationship betweenstress granule formation and the presence of P-bodies in Arabidopsis,we performed time-lapse observations through high-resolution,high-sensitivity confocal microscopy. We increased the temperatureof the samples to 35°C within 30 s by heating the microscope stageusing a heat incubator. In eIF4A2-GFP plants, stress granules beganto form at∼70 s of treatment (Movie 5). Some stress granules formedat the neighboring sites of P-bodies (Fig. 4A, white arrowheads) –identified in Arabidopsis expressing the P-body markermRNA-decapping enzyme-like protein (DCP1) tagged with RFP(DCP1-TagRFP) – and some formed independently of P-bodies

Fig. 3. Effects of cytoskeleton depolymerization drugs on stress granule movement, number and size. (A–D) eIF4A2-GFP seedlings were pre-treatedat 22°C for 30 min with the following cytoskeleton depolymerization drugs: 50 µM Oryzalin (B), 2 µM Lat B (C), a mixture of 50 µM Oryzalin and 2 µM Lat B (D),as well as 0.1% DMSO (control; A). Subsequently, the seedlings were heat-treated at 38°C for 10 min to induce stress granule formation. The averagevelocity (Vave) of stress granules was determined in three independent cells by measuring the distance (in μm/s) the stress granules moved during 5 s. n=225 (A),209 (B), 270 (C) and 210 (D). (E) Confocal images showing stress granules that formed in the presence of each cytoskeleton depolymerization drug.Heat treatment was applied for 2 h. Scale bar: 10 µm. (F,G) Number and size of stress granules (SGs) that formed in the presence of each cytoskeletondepolymerization drug. Heat treatment was applied for 2 h. At least ten independent images were analyzed. Bar graphs at a linear scale (F) and logarithmicscale (G). A total of 14 (DMSO), 15 (Oryzalin), 11 (Lat B) and 8 (Lat B+Oryzalin) images were analyzed. A total of 63 (DMSO), 1049 (Oryzalin), 2219 (Lat B)and 712 (Lat B+Oryzalin) stress granules were analyzed. *P<0.01 according to Welch’s t-test for unequal variances. Error bars indicate ±s.d.

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(Fig. 4A, yellow arrowheads). Half of the P-bodies were notassociated with stress granules at 90 s (Fig. 4B), and someP-bodies were not associated with stress granules, even at 250 s(Fig. 4A, arrows at 250 s). By contrast, there were more stressgranules than P-bodies after 150 s of treatment (Fig. 4B).

During the observation period, stress granules often fused witheach other (Fig. S5B). Fusions also occurred between P-bodies,and between P-bodies and stress granules (Fig. S5B,C). High-resolution images (Fig. S5D) showing the detailed relationshipbetween stress granules and P-bodies indicate that stress granules did

Fig. 4. High-resolution microscopy showing the relationship between stress granule formation and the presence of pre-existing P-bodies.(A) Time-lapse images, corresponding to Movie 5, showing stress granule formation and their association with the P-body marker DCP1-TagRFP.Times were recorded immediately after beginning incubation at 35°C on a microscope stage heater. In the merged images (right), green andmagenta indicate eIF4A2-GFP and DCP1-TagRFP, respectively. White arrowheads indicate stress granule formation at the sites of P-bodies.Yellow arrowheads indicate stress granule formation in the absence of P-bodies. Arrows indicate the moment that stress granules appeared with P-bodies.Scale bar: 4 µm. (B) Number of stress granules (white bars), P-bodies (black bars), and P-body-associated stress granules (striped bars) per μm2 atdifferent time points. Number of stress granules (white bar) including P-body-associated stress granules (striped bar). More than half of the number ofP-bodies did not show eIF4A2-GFP fluorescence (i.e. stress granules) at 90 s. Five independent images were analyzed. Error bars indicate s.d.(C) Correlation analysis of P-body-associated stress granules at 90, 190 and 450 s. The sizes of P-bodies and stress granules in P-body-associatedstress granules were plotted; r indicates the correlation value. n=30 (90 s), 23 (190 s) and 12 (450 s). (D) Confocal images showing the formationof stress granules and their association with the P-body marker DCP1-TagRFP after incubation using the microtube method at 35°C for 60 and 120 min.P-bodies did not associate with huge stress granules (arrowheads). In the merged column, green and magenta indicate eIF4A2-GFP and DCP1-TagRFP, respectively. Scale bar: 10 µm. (E) Number of stress granules (white bars), P-bodies (black bars) and P-body-associated stress granules(striped bars) after incubation using the microtube method at 35°C for 10, 30, 60 and 120 min. Eight independent images were analyzed. Error barsindicate ±s.d.

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not perfectly overlap with P-bodies. Once stress granules associatedwith P-bodies, they did not dissociate from these structures (Fig. S5D,Movie 5). At the sites of P-body and stress granule association, stressgranule sizewas not correlated with P-body size (Fig. 4C).We furtherinvestigated the relationship between stress granules and P-bodiesupon long incubation (60 and 120 min) using the microtubemethod at 35°C (Fig. 4D,E). Fusion of stress granules progressedand the number of stress granules decreased over time. Weexpected all P-bodies to have fused into huge stress granules after2 h. However, contrary to this expectation, most P-bodies were notincorporated into huge stress granules (Fig. 4D), suggesting thatthe interaction between stress granules is stronger than thatbetween stress granules and P-bodies. These observations indicatethat stress granule formation occurs in two steps: stress granulegeneration and fusion. Although it is difficult to separate thesesteps, it appears that stress granules are generated regardless of thepresence of P-bodies. During the stress granule fusion process,stress granules tend to associate quickly and tightly with otherstress granules or P-bodies.

Ultrastructures of stress granules in ArabidopsisUltrastructural analysis of RNA granules by electron microscopyhas not previously been reported in plants. We, therefore, observedArabidopsis stress granules by electron microscopy in eIF4A2-GFPseedlings that had been incubated at 38°C for 15 or 60 min(Fig. 5A). Before and after heating, we fixed the seedlings by high-pressure freezing and subjected them to immunoelectron microscopywith an anti-GFP antibody. Gold nanoparticles representing thepositions of eIF4A2-GFP were scattered throughout the cytoplasm insections produced from samples before heat treatment. In samplesafter heat treatment, gold nanoparticles accumulated at irregularlyshaped, ribosome-free structures (Fig. 5A). The stress granules werenot surrounded by a membrane boundary in the cytoplasm, aspreviously reported for stress granules in animal cells (Souquere et al.,2009).We calculated the diameter of the stress granules bymeasuringthe longest distance between gold particles within each stressgranule (Fig. 5B). The average diameter of the stress granules was191±74.7 nm after heat treatment at 38°C for 15 min. The averagediameter of the stress granules increased to 311±145.7 nm inresponse to treatment at 38°C for 60 min, which is consistent withthe fluorescence microscopy results showing an increase in stressgranule size with increasing heating duration.We then examined the electronmicrographs in detail. No ribosome

particles were observed inside the stress granule regions, which wereidentified by the presence of gold nanoparticles (Fig. 5A). The stressgranules that had formed in response to heat treatment at 38°C for15 and 60 min contained electron-dense particulates and filamentousstructures (Fig. 5A, arrowheads). When seedlings were subjected toheat priming through intermittent heat treatment (38°C for 30 min,22°C for 30 min and 38°C for 30 min), electron-dense particulates inthe stress granules became more prominent and abundant (Fig. 5C,right panel) compared to samples subjected to a simple heat treatmentat 38°C for 60 min (shown in Fig. 5A). As observed by confocalmicroscopy, heat priming did not dramatically alter the size ornumber of stress granules compared to other conditions (Fig. S6). Inaddition, using a combination of heat-priming treatment and osmiumtetroxide (OsO4) fixation, we readily observed similar electron-denseparticulates and filamentous structures, even in wild-type seedlingswithout immunogold labeling (Fig. 5D). Overall, these resultssuggest that stress granules are irregularly shaped, ribosome-freestructures that include characteristic electron-dense particulates andfilamentous structures.

Ribosome-exclusion regions are located adjacent tostress granulesWe noticed that ribosome particles were excluded from regionscontaining stress granules. Interestingly, these ribosome-exclusionregions often extended out of the stress granule areas, which weredefined by immunogold nanoparticles that labeled eIF4A2-GFP(Fig. 5E, left panel). Fortunately, we obtained two images ofsequential ultra-thin sections (Fig. 5E, right panel). In both images,the gold label-free ribosome-exclusion regions also exhibited awispy cloud-like filamentous pattern with electron-dense particulates,thus resembling stress granules. The stress granules and gold label-free areas had distinct shapes in the two sequential sections. Theseresults indicate that, in addition to assuming a typical spherical shape,stress granules can sometimes be amorphous, with a rugged surfacefacing the cytoplasm.

Ultrastructures of P-bodies in ArabidopsisWe reasoned that these gold label-free ribosome-exclusion regionsmight represent (1) stress granules lacking eIF4A2 in a certain area;(2) other RNA granules, such as P-bodies; or (3) unknowncytoplasmic structures. Among these, we investigated the possibilitythat these gold label-free ribosome-exclusion regions representedP-bodies. To examine the ultrastructures of P-bodies in Arabidopsis,we subjected DCP1-GFP-expressing seedlings (Motomura et al.,2015) to immunoelectron microscopy. In samples observed at22°C, immunogold nanoparticles (indicating the localization ofDCP1-GFP) appeared in distinct, non-membranous, circular siteswith electron-dense particulates and filamentous structures (Fig. 6A).We determined that these structures were P-bodies. We found thatribosomes were also excluded from the P-bodies. In samplesobserved at 22°C, electron-dense particulates were not clearlyobserved in the P-bodies. However, after heat treatment, someP-bodies showed prominent electron-dense particulates withelectron-dense filamentous structures (Fig. 6B,C). Thus, theultrastructures of P-bodies after heat treatment resembled thoseof stress granules. As with stress granules, the P-bodies increasedin size after heat treatment, as revealed by confocal microscopy(Motomura et al., 2015). Thus, we measured the sizes of P-bodiesby electron microscopy (Fig. 6D), finding that they increasedfrom 134±77.6 nm (22°C) to 169±59.0 nm (38°C for 15 min) or180±76.5 nm (38°C for 60 min) in size after heat treatment.In addition, gold label-free ribosome-exclusion regions wereoccasionally observed around the P-bodies, especially in heat-treated samples. Some gold label-free ribosome-exclusion regionsexhibited a filamentous pattern (Fig. 6B), and some containedelectron-dense particulates (Fig. 6C). In conclusion, it is difficultto distinguish between stress granules and P-bodies based ontheir ultrastructures alone. To conclusively identify the stressgranule-associated gold label-free ribosome-exclusion regions,comprehensive studies must be performed involving the use ofmulti-label immunoelectron microscopy.

DISCUSSIONOur analyses revealed the relationship between the effects ofthe level and duration of heat stress on stress granule formationin Arabidopsis seedlings. In the absence of transpiration,Arabidopsis seedlings formed stress granules at 34°C,regardless of treatment duration (Fig. 1C,D). Stress intensity(defined by temperature) was responsible for determining thethreshold for stress granule formation. Arabidopsis seedlings thatcould transpire formed stress granules at temperatures >34°C(Fig. 1E,F). For example, in seedlings grown in agar, the duration

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of stress treatment also appeared to be important for determining thestress granule formation threshold (Fig. 1E). However, consideringthat transpiration cools the cytoplasm, it is reasonable to assume thatthe effect of stress duration under this condition was due to the limited

amount of water in the agar plates. Consistent with this speculation,the effect of stress duration was not obvious in seedlings grownin soil, which contained sufficient water for proper transpiration(Fig. 1F). Overall, our results suggest that stress intensity

Fig. 5. Ultrastructural features of stress granules – a single stress granule surrounded by a ribosome-exclusion region. (A) Immunoelectronmicrographs probed with GFP antibodies showing stress granules in hypocotyl cells of 5-day-old eIF4A2-GFP seedlings that had been heat-treated at22°C (left) and 38°C for 15 min (center) and 60 min (right). Stress granules contain electron-dense granular and filamentous structures (arrowheads).Scale bar: 300 nm. (B) Effect of heat treatment on stress granule size. Fifty stress granules were analyzed under each condition. *P<0.01 according toWelch’st-test. Error bars indicate ±s.d. (C) Immunoelectron micrographs of eIF4A2-GFP seedlings treated by heat priming. Seedlings were immersed in water ina tube at 22°C for 90 min (left) and at 38°C for 30 min twice with an in-between break at 22°C for 30 min (right). Scale bar: 300 nm. Enlarged images areshown in the inset. Scale bar: 100 nm. Arrows and arrowheads indicate electron-dense granular and filamentous structures, respectively. (D) OsO4-stainedelectron micrograph of a wild-type seedling treated by heat priming. Seedlings were immersed in water in a tube at 22°C for 90 min (left) and at 38°C for 30 mintwice with an in-between break at 22°C for 30 min (right). Scale bar: 500 nm. Enlarged images are shown in the inset. Scale bar: 100 nm. Arrows andarrowheads indicate electron-dense granular and filamentous structures, respectively. (E) Immunoelectron micrographs of two sequential ∼70 nm-thicksections from an eIF4A2-GFP seedling that had been immersed in water in a tube at 38°C for 60 min. Notice the stress granule (blue line) surrounded bya ribosome-exclusion region (red line). Scale bar: 500 nm.

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(temperature) is the key factor determining the threshold of stressgranule formation.We also found that the threshold temperature for stress granule

formation did not change in seedlings exposed to 4°C for 2 daysbefore the experiment (Fig. 1G). This result indicates thatArabidopsis seedlings have an absolute temperature thresholdfor stress granule formation, and it suggests that stress granulesfunction in stress responses when seedlings planted in soil facesevere environmental conditions, such as temperatures >41°C.Stress granule formation is also induced by hypoxia, drought andhigh-salt conditions in plants (Pomeranz et al., 2010; Sorenson andBailey-Serres, 2014; Yan et al., 2014; Gutierrez-Beltran et al.,2015). Under natural growing conditions (outdoors in soil), plantsare simultaneously exposed to various stresses and may form stressgranules in response to environmental changes. We showed thathigh-salt stress reduced the temperature threshold for stress granuleformation (Fig. S2). Although we do not currently understand how

plants sense and integrate the intensity of stress signals, and exactlywhen stress granules form, the combination of different stresses mayreduce the temperature threshold needed for stress granule formation.

In this study, we determined the minimum heat treatment requiredto trigger stress granule formation (Fig. 1C–F), which enabled us toreduce the amount of unnecessary damage inflicted on the plants.After subjecting the seedlings to the minimum heat treatment, wedetermined that the long-distance transport of stress granules isdriven by actin filaments (Fig. 3), a mechanism similar to that usedto transport other plant organelles and RNA granules (Boevinket al., 1998; Nebenführ et al., 1999; Mathur et al., 2002; Van Gestelet al., 2002; Ueda et al., 2010; Hamada et al., 2012). In the presenceof this long-distance transport, the sizes of stress granules greatlyincreased over time (Figs 2 and 3). The loss of actin filamentsinhibited the formation of huge stress granules (Fig. 3E–G).In addition, the loss of microtubules also inhibited the formation ofhuge stress granules. The result is consistent with previous findings

Fig. 6. Ultrastructural features of P-bodies in Arabidopsis seedlings. (A–C) Immunoelectron micrographs probed with GFP antibodies againstDCP1-GFP showing P-bodies. Five-day-old DCP1-GFP seedlings were immersed in water in a tube at 22°C for 60 min (A) and at 38°C for 15 min (B) andfor 60 min (C). Notice the P-body (surrounded by dashed red line) associated with a ribosome-exclusion region (surrounded by dashed blue line).Scale bar: 500 nm. (D) Effect of heat treatment on P-body size. A total of 25 (22°C), 41 (38°C for 15 min) and 33 (38°C for 15 min) P-bodies were analyzed.*P<0.01 according to Welch’s t-test. Error bars indicate ±s.d.

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regarding the involvement of microtubules in stress granuleformation and transport, in both animal and plant cells (Ivanovet al., 2003; Gutierrez-Beltran et al., 2015).Plant-specific features might explain why plants require not only

microtubules but also actin filament-driven cytoplasmic streamingfor stress granule formation. Plant cells are usually relatively large;for instance, the long axis of Arabidopsis epidermal cells can be>1 mm. In addition, plant cells do not contain a radial microtubulenetwork radiating from a centrosome, as do animal cells. Actinfilaments in plant cells induce rapid cytoplasmic streaming andappear to maintain cellular homeostasis via mixing. The effects ofthe loss of actin filaments and microtubules differ depending ontheir size and number (Fig. 3E–G). We suspect that actin filamentsand microtubules contribute to stress granule formation in differentways. It appears that stress granule formation in plants proceeds intwo steps: stress granule generation and fusion. Actin filaments andmicrotubules might contribute differently to these two processes.Minimum heat treatment also led to the rapid disappearance of

stress granules in plant cells (Movie 1). Stress granules disintegratedcompletely in plant cells within 10 min after the temperature wasreduced to 22°C. The disappearance of hypoxia-induced stressgranules was also observed after 20 min of reoxygenation (Sorensonand Bailey-Serres, 2014). Our results are consistent with the viewthat plant stress granules are flexible and dynamic, rather than rigid,to quickly respond to the need for mRNA release for translation(Sorenson and Bailey-Serres, 2014).Both stress granules and P-bodies in Arabidopsis cells are

irregularly shaped, ribosome-free structures with characteristicelectron-dense particulates and filaments (Figs 5 and 6). Stressgranules and P-bodies in animal cells also have a similar appearance(Yang et al., 2004; Souquere et al., 2009; Cougot et al., 2012),indicating that such ultrastructures are common and shared amongeukaryotes. What the filamentous structures represent and theidentity of the electron-dense particulates are still unclear.Our results may provide clues to help answer these questions. For

example, the electron-dense particulates of plant stress granuleswere observedmore clearly and increased in number with prolongedtreatment time (Fig. 5A). Especially after heat-priming treatment,the stress granules contained higher numbers of electron-denseparticulates and exhibited well-arranged structures (Fig. 5C,D).These results suggest that the presence of electron-dense particulatesin stress granules is a sign of their maturity. Heat priming (or heat pre-treatment) also increases the expression levels of heat-responsivegenes, such as heat shock protein genes (Mittler et al., 2012).Interestingly, heat stress granules (HSGs) in plants also containelectron-dense particulates and filamentous structures (Nover et al.,1983, 1989; Scharf et al., 1998). HSGs are an aggregated form of heatshock proteins or heat shock factors that form in response to heatstress, like stress granules. Plant stress granules and HSGs are distinctstructures at the confocal microscopic level (Weber et al., 2008).These reports indicate that our observed electron-dense particulatesmay be a component of HSGs, not a component of RNA granules.Like the tight connection between stress granules and P-bodiesobserved in the current study (Fig. 4), these contradictory observationsmight be explained if we assume that HSGs and stress granules fusewith each other, and have different compositions over time. In heat-treated animal cells, stress granules and HSGs appeared to overlap toform identical granules at the confocal microscopic level (Kedershaet al., 1999; Piotrowska et al., 2010).Moreover,Weber et al. (2008) hasshown that some stress granules and HSGs are closely attached, andpartially overlap. Additional precise experiments are needed toconclusively address this issue.

The assembly of RNA-binding proteins into RNA granules hasrecently been explained by the liquid–liquid phase separationmechanism (Banani et al., 2017). However, it was also proposedthat the maturation of stress granule cores leads to the enlargement ofstress granules (Wheeler et al., 2016; Protter and Parker, 2016; Panaset al., 2016). Linking ultrastructures to functions or componentsmight clarify the mechanisms underlying stress granule formation.

MATERIALS AND METHODSPlant materials and growth conditionsAll plants of the wild-type and established lines were of the A. thalianaL. (Heynh.) Columbia background. DCP1-GFP and DCP1-TagRFPtransgenic lines (Motomura et al., 2012, 2015) were used as P-bodymarkers. Seeds were sterilized in sterilizing solution (5% NaClO and 1%Triton X-100) or 100% ethanol for 5 min. After sterilization, the seedswere plated on agar containing 1% sucrose, 1% agar, 1% MES-KOH pH5.7, and half-strength A. thaliana nutrient solution as described by Haughnand Somerville (1986). The plates were set in a near-vertical position at22°C under constant light. Five-day-old plants were used for analysis. Forthe soil experiments, 3-day-old plants were transferred to soil in pots andincubated for 2 days under constant light conditions before observation.

Plasmid construction and transformationTo visualize stress granules in seedlings, eIF4A2 (AT1G54270) and PAB6(AT3G16380) cDNAwas amplified by PCR and inserted into the pGWB405,pGWB460 and pGWB406 Gateway vectors (Nakagawa et al., 2007).PCR primers were designed for eIF4A2 (5′-AACCAATTCAGTCGACAT-GGCAGGATCCGCACCGGA-3′ as the left-border primer and 5′-AAGCT-GGGTCTAGATATCCTCACAGCAAATCAGCCACGT-3′ as the right-border primer) and PAB6 (5′-AACCAATTCAGTCGACATGGCGCTCG-TGAAAACGGA-3′ as the left-border primer and 5′-AAGCTGGGTCTA-GATATCCTTGCAATTGTTTTCCTGATTCC-3′ as the right-border primer).Transgenic plants constitutively expressing eIF4A2-GFP and GFP-PAB6were obtained by transformation through the floral dip method (Clough andBent, 1998) using the Agrobacterium tumefaciens strain GV3101.

Heat, drug and high-salt treatments, and confocal microscopyHeat treatments for plants subjected to the mounting, microtube, agarplating and soil methods were applied by using a 93 l heated incubator(MIR-H163, PHC Corporation, Tokyo, Japan). For the microtube method,1 ml water in a 1.5 ml microtube was pre-heated in the incubator before use.For time-lapse analysis, each seedling was immersed in water in a20×10×0.15 mm rectangular chamber between coverslips (60×32 mmNo. 1, 24×18 mm No. 1S, Matsunami Glass, Osaka, Japan) and incubatedin a stage-top incubator system composed of a customized doubleThermoPlate chamber (11.5×7.5×0.3 cm inside size) and a TP-LH lensheater (Tokai Hit, Shizuoka, Japan). The stage-top incubator system canincrease the temperature of a sample up to 35°C within 30 s. For drugtreatments, plants were placed in 1.5 ml tubes containing 1 ml of drugsolution, including 0.1% DMSO (control), 2 µM Lat B, 50 µMOryzalin or30 µM cycloheximide (Sigma, St Louis), and incubated for 30 min at 22°C.Mannitol buffer (400 mM mannitol, 15 mM MgCl2, and 4 mM MES-KOHpH 5.7; Yan et al., 2014) containing 150 or 200 mMNaCl was used for high-salt treatment. Confocal microscopy was performed using two differentconfocal microscope systems; a spinning disc confocal microscope system(Nikon Eclipse Ti microscope equipped with Yokogawa CSU-W1 confocalspinning disc unit, 2× internal magnifying lens, Andor Zyla 4.2 sCMOScamera, Andor IQ3 software) and an Olympus FV1000 confocal microscopesystem. Images were converted to binary images and measured using aParticle Analyzer with Image J software (National Institutes of Health;http://rsbweb.nih.gov/ij/).

Electron microscopyFive-day-old seedlings before and after heat treatment were subjected tohigh-pressure freezing using an EM PACT (Leica Microsystems, Wetzlar,Germany). Each frozen sample was immediately transferred to a sampletube containing freeze-substitution solution (0.25% glutaraldehyde, 0.1%

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uranyl acetate in acetone) and stored at −80°C for 72 h. The tube wasgradually warmed up to room temperature over 2 days. Fixed seedlings werewashed with acetone, dehydrated in methanol and embedded in LR Whiteresin. The embedded seedlings were sliced into ∼70 nm-thin sections withan EM UC7 ultramicrotome (Leica Microsystems). The ultra-thin sectionswere collected on formvar-coated Ni grids and subjected to immunoelectronmicroscopy using anti-GFP antibody (Invitrogen A11122, Thermo FisherScientific, MA) and 12 or 18 nm colloidal gold AffiniPure goat anti-rabbitIgG (#111-205-144 or #111-215-144, Jackson Immunoresearch Inc., PA).The samples were stained with 4% aqueous uranyl acetate and observedunder a transmission electron microscope (JEM-1400, JEOL, Tokyo,Japan). For the ultrastructure observations shown in Fig. 4F, 2% OsO4 inacetone was used as the freeze-substitution solution; the samples were thenembedded in Epon812 resin. The ultra-thin sections were stained with 4%aqueous uranyl acetate and lead citrate.

AcknowledgementsWe thank Mayumi Wakazaki (Riken, Yokohama, Japan) for assistance withelectron microscopy, Kentaro Tamura (Kyoto University, Japan) and Haruko Ueda(Konan University, Kobe, Japan) for helpful discussions, and Misako Taniguchi-Saida [National Institute for Basic Biology (NIBB), Aichi, Japan] for technicalassistance. We thank Dr Tobias I. Baskin at the University of Massachusetts,Amherst, for the critical reading of the manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: T.H., Y.W., I.H.-N.; Methodology: T.H., M.Y., M.M., M.S.,Y.K., K.T.; Validation: T.H., M.Y., M.M., M.S., Y.K., K.T.; Formal analysis: T.H.,M.Y., M.M., M.S., K.T.; Investigation: T.H., M.Y., M.M., M.S., Y.K., K.T.; Resources:T.H., Y.Y.; Data curation: T.H., M.Y., M.M., M.S., K.T.; Writing - original draft: T.H.,Y.W., I.H.-N.; Writing - review & editing: T.H., M.S., Y.K., K.T., Y.W., I.H.-N.;Visualization: T.H.; Supervision: T.H., Y.W., I.H.-N.; Project administration: T.H.;Funding acquisition: T.H., Y.K., Y.W., I.H.-N.

FundingThis study was supported by the Japan Society for the Promotion of Science (JSPS)KAKENHI [Grant Numbers: 16H01229, 26650090, 15H05598 to T.H., 17H06258to Y.K., 15H04628 to Y.W. and 15H05776 to I.H.-N.] and the National Institute forBasic Biology (NIBB) Cooperative Research Programs [Grant Numbers: 15-333 toY.W. and 16-517 and 17-508 to T.H.].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.216051.supplemental

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