Stress Granule Formation Is Induced by a Threshold Temperature Rather

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

ABSTRACT translational machinery, including mRNAs, eukaryotic translation Stress granules, a type of cytoplasmic RNA granule in eukaryotic factors, poly-A binding proteins (PABP) and 40S ribosomal cells, are induced in response to various environmental stresses, proteins, begin to form stress granules. Stress granules, a type of including high temperature. However, how high temperatures induce RNA granule, are cytoplasmic aggregates composed of various the formation of these stress granules in plant cells is largely RNA molecules and proteins. Stress granules are found in various unknown. Here, we characterized the process of stress granule eukaryotic cells under a variety of stress conditions. In human cells, formation in Arabidopsis thaliana by combining live imaging and stress granule formation is induced by heat stress, arsenite exposure, electron microscopy analysis. In seedlings grown at 22°C, stress viral infection and UV irradiation (Kedersha et al., 2013). granule formation was induced at temperatures above a critical Stress granules were recently found to be related to human diseases, ’ threshold level of 34°C in the absence of transpiration. The threshold such as Alzheimer s disease, amyotrophic lateral sclerosis and temperature was the same, regardless of whether the seedlings were frontotemporal lobar degeneration (Wolozin, 2012; Vanderweyde grown at 22°C or 4°C. High-resolution live imaging microscopy et al., 2013). In plants, stress granules appear in response to revealed that stress granule formation is not correlated with the sizes hypoxia, high-salt conditions, and exposure to methyl jasmonate, of pre-existing RNA processing bodies (P-bodies) but that the arsenite, potassium cyanide and myxothiazol (Nover et al., 1983, two structures often associated rapidly. Immunoelectron microscopy 1989; Weber et al., 2008; Pomeranz et al., 2010; Sorenson and revealed a previously unidentified characteristic of the fine structures of Bailey-Serres, 2014; Yan et al., 2014; Gutierrez-Beltran et al., Arabidopsis stress granules and P-bodies: the lack of ribosomes and 2015). In addition, several plant-specific stress granule components the presence of characteristic electron-dense globular and filamentous have been identified, revealing some of the molecular mechanisms structures. These results provide new insights into the universal nature underlying stress granule formation (Chantarachot and Bailey-Serres, of stress granules in eukaryotic cells. 2018). However, at what time point plants begin to form stress granules is still largely unknown. For example, how do both KEY WORDS: Arabidopsis, Stress granules, P-bodies, Temperature, the intensity and duration of stress conditions affect stress High-resolution live imaging microscopy, Immunoelectron microscopy granule formation? Plants usually survive exposure to external temperature changes. Therefore, do different plants have different INTRODUCTION temperature thresholds for stress granule formation depending on Plants are sessile organisms that must adapt to environmental changes the temperature of their habitats? in order to survive. Temperature is a rapidly changing parameter in the Here, we examined the process of early stress granule formation environment. For example, the irradiation of leaves with direct sunlight in Arabidopsis seedlings under finely set conditions in terms has the potential to cause dramatic increases in temperature. Plants of temperature and duration of treatment. Our experiments usually release heat by transpiration (Campbell, 1977). However, revealed a threshold of stress granule formation in response to a under some conditions, especially in dry and high-temperature temperature shift (representing stress intensity) but not duration. environments, plants are unable to control their body temperature. The threshold did not vary under different growth conditions. To survive under severely dry and high-temperature conditions, plants However, the threshold did vary depending on whether transpiration possess various heat resistance mechanisms, such as the formation of was allowed to occur, indicating that the cooling effect of heat shock proteins (Mittler et al., 2012; Scharf et al., 2012). transpiration is closely related to stress granule formation. Time- A wide range of translational repression responses occur in lapse analyses revealed that stress granules can be generated in a plants exposed to high temperatures (Nover et al., 1983, 1989). As a processing body (P-body)-independent manner but that they are cause or result of this translational repression, components of the often closely associated with P-bodies. Ultrastructural analysis of Arabidopsis stress granules and P-bodies revealed irregularly shaped, ribosome-free structures with characteristic electron- 1Department of Life Sciences, Graduate School of Arts and Sciences, The University dense particulates and filamentous structures. These results of Tokyo, Tokyo 153-8902, Japan. 2RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan. 3National Institute for Basic Biology, Aichi 444-8585, provide a framework for future studies of plant stress granules Japan. 4Institute of Agrobiological Sciences, NARO, Tsukuba 305-8602, Japan. and offer new insights into the roles of stress granules in the 5Faculty of Science and Engineering, Konan University, Hyogo 658-8501, Japan. responses of plants to severe environmental conditions. *Author for correspondence ([email protected]) RESULTS T.H., 0000-0002-8829-4001; M.M., 0000-0001-7296-2266; M.S., 0000-0002- The threshold temperature of stress granule formation is 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 34°C in seedlings mounted for microscopy To study stress granule formation in plants, we constructed a plasmid

Received 25 January 2018; Accepted 12 July 2018 containing the eIF4A2 gene fused with GFP at the C-terminus Journal of Cell Science

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(eIF4A2-GFP) and obtained stably transformed Arabidopsis lines versus temperature. We detected a clear boundary between diffuse in which stress granules could be visualized under a fluorescent fluorescence and stress granule formation (Fig. 1C). For example, microscope. The transgenic plants emitted strong fluorescence when treatment was for the duration of 10 min, stress granules but did not show noticeable morphological or developmental appeared at temperatures above 34°C. We confirmed these results defects (Fig. S1). using another stress granule marker line in which GFP was fused A rise in temperature is likely to be one of the most critical factors to the N-terminus of Arabidopsis poly(A)-binding protein 6 for stress granule formation in eukaryote cells. We, therefore, (GFP-PAB6) (Fig. S1C). The GFP-PAB6 and eIF4A2-TagRFP immersed eIF4A2-GFP seedlings in water between glass slides signals overlapped (Fig. S1G). (hereafter referred to as the mounting method) and kept them in an incubator at 38°C for 10 or 30 min. The pattern of fluorescence Different heat treatments and high-salt stress affect the from eIF4A2-GFP was diffuse in the cytoplasm at 22°C, and stress threshold temperature required for stress granule formation granules formed after heat treatment (Fig. 1A). In the presence of Different heat treatment protocols had been followed in previous cycloheximide, an inhibitor of stress granule formation (Kedersha physiological and imaging experiments using plants. We investigated et al., 1999; Sorenson and Bailey-Serres, 2014), stress granule whether the application of different heat treatment protocols resulted formation was not induced by 30-min long heat treatment at 38°C in any changes in the critical temperature at which stress granules (Fig. 1B). These results indicate that eIF4A2-GFP transgenic plants formed. We examined stress granule formation using the microtube are good stress granule marker lines. Next, we attempted to determine method, a typical method used in drug treatments in which seedlings the lowest temperature (i.e. threshold temperature) required for are immersed in water within a tube. Stress granules formed at seedlings to form stress granules in an incubator, increasing the temperatures >34°C and treatment duration of 10 min (Fig. 1D). temperature from 30°C by 1°C steps to 40°C. In plants subjected This threshold temperature is identical to that identified by using to the mounting method, heat in the cytoplasm could not be the mounting method, probably because heat in the cytoplasm lost through transpiration. We plotted the diffuse localization of cannot be lost by transpiration when using the microtube method, fluorescence and granule formation in response to treatment duration 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 by heat treatment. Confocal images showing stress granule formation in the hypocotyls of 5-day-old Arabidopsis seedlings expressing the stress granule marker 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 at the 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 stress granule 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) Threshold temperatures of stress granule formation under various conditions. Stress granule formation at different temperatures was examined in seedlings. Seedlings were mounted (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 for

2 days (G) were also examined. Stress granules were observed in all cells (○), observed in some cells (Δ) or never observed (×) under each condition. Journal of Cell Science

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Agar plating is another useful treatment for physiological Numerous small stress granules were observed at 40°C compared experiments. Here, we heat-treated plants grown on agar plates. with those at 36 and 38°C. Moreover, at 38°C and 40°C, the average Stress granules formed at temperatures >38°C and treatment number and size of stress granules increased in proportion to the duration of 10 min when using the agar plate method (Fig. 1E). incubation time. However, at 34°C, the average number of stress When using this method, longer duration and higher temperature granules decreased and average stress granule size significantly during heat treatment was required for stress granule formation increased depending on incubation time, indicating that, over time, compared with the mounting and microtube methods. The fourth the stress granules fused with each other at 34°C. Huge stress granules method, planting in soil, is the closest to natural conditions. The that formed at 34°C after 2 h of treatment were observed as bright temperature required for stress granule formation was >42°C foci, confirming the fusion of stress granules. during a 10 min treatment period (Fig. 1F). Compared with the mounting and microtube methods, seedlings grown on agar Cytoplasmic streaming by actin filaments contributes to the plates and in soil can transpire, leading to a reduction in fusion of stress granules temperature in the cytoplasm. Under conditions that do not allow To investigate the mechanism behind the formation of these huge for transpiration, 34°C was identified as a constant threshold stress granules at 34°C and extended incubation time, we examined temperature regardless of the duration of the treatment (Fig. 1C,D). the behavior of stress granules at 34°C. We observed rapid and long- Thus, 34°C appears to be the threshold temperature for stress distance movement of stress granules in the cytoplasm at or near granule formation in the cytoplasm of Arabidopsis seedlings. the threshold temperature of 34°C (Movie 1). At 38°C, the stress Stress granule formation is also induced by other stresses, such as granules exhibited active movement at 10 min and moved slowly at hypoxia, high-salt conditions and exposure to methyl jasmonate. 30 min but not beyond this time point. No movement was observed We investigated whether high-salt conditions affect the threshold in samples exposed to higher temperatures such as 40°C for 10 min. temperature for stress granule formation by using the microtube However, the size and number of stress granules increased at 40°C method. On the basis of previously reported experimental conditions from 10 to 30 min in the absence of movement (Fig. S4), indicating (Yan et al., 2014), we confirmed that stress granule formation was that movement is not essential for stress granule formation. The induced by treatment with mannitol buffer containing 200 mM NaCl rapid, long-distance movement of stress granules suggests that but not by treatment with buffer containing 150 mM NaCl they were transported by cytoplasmic streaming, a process driven by (Fig. S2A). In the presence of 150 mM NaCl, stress granules interactions between actin and myosin. In plant cells, both organelles formed at temperatures <34°C during a 10 min treatment period and P-bodies are transported long distances through cytoplasmic (Fig. S2B). These results indicate that a combination of stresses affect streaming (Boevink et al., 1998; Nebenführ et al., 1999; Mathur et al., the threshold temperature for the formation of stress granules. 2002; Van Gestel et al., 2002; Ueda et al., 2010; Hamada et al., 2012). On the basis of the results shown in Fig. 1, we chose to treat Arabidopsis stress granules do not form in response to plants at 38°C for 10 min to observe the formation of stress relative temperature differences granules by using the microtube method. These conditions led to Plants are exposed to ambient temperatures that fluctuate between the formation of well-defined stout stress granules that exhibited high and low temperatures within a single day. We examined whether rapid long-distance movements (Movie 1). To test the effects of low-temperature stress also induces stress granule formation. Stress cytoskeleton disturbance, we pre-treated eIF4A2-GFP seedlings in granules failed to form at low temperatures, such as 10°C and 4°C tubes with the cytoskeleton depolymerization drugs Oryzalin (Fig. S3). Next, we investigated whether the temperature at which (which depolymerizes microtubules) or latrunculin B (Lat B; stress granules form changed when plants had previously been grown which depolymerizes actin filaments) at 22°C for 30 min, at lower temperatures (Fig. 1G). eIF4A2-GFP trasngenic seedlings followedbyincubationat38°Cfor10mintoinducestress were grown at 22°C for 5 days, followed by 4°C for 2 days. granule formation, and observed them under a microscope at 22°C. Subsequently, the seedlings were transferred from 4 to 22°C. Stress granules completely disappeared during the first 10 min of We did not observe stress granule formation, even though the observation (Movie 1). In the control samples, some stress seedlings experienced a temperature change of 18°C, which is as granules were transported long distances at a maximum velocity large as the change from 22 to 40°C. Stress granules started to of ∼0.5 µm/s and an average velocity of ∼0.08 µm/s (Fig. 3A, form at 34°C but not at 25, 30 or 33°C (Fig. 1G), indicating that Movie 1). Treatment with Oryzalin did not affect the motility of the threshold temperature was 34°C in seedlings grown at 4°C. stress granules; the maximum and average velocities were also This result is almost identical to that of seedlings grown at 22°C ∼0.5 µm/s and 0.08 µm/s, respectively (Fig. 3B, Movie 2). (Fig. 1C). We also confirmed these results in GFP-PAB6 transgenic However, treatment with Lat B reduced stress granule motility plants (Fig. S1). Our findings indicate that Arabidopsis seedlings (Fig. 3C, Movie 3). The maximum and average velocities in the form stress granules by sensing the absolute temperature, not a presence of Lat B were ∼0.1 µm/s and 0.03 µm/s, respectively. difference in temperature. A similar result was obtained when the seedlings were treated with a mixture of Lat B and Oryzalin (Fig. 3D, Movie 4). These results clearly Different sizes and numbers of stress granules develop in indicate that actin-dependent cytoplasmic streaming contributes to response to different temperatures and incubation times the rapid, long-distance transport of stress granules in plant cells. During our observation of stress granule formation, we noticed that Next, we investigated the effect disturbance of the cytoskeleton has different numbers and sizes of stress granules developed in response on the formation of large stress granules (Fig. 3E–G). After pre- to different temperatures and incubation times. To quantitatively treating eIF4A2-GFP seedlings with Oryzalin or LatB, a combination investigate the effects of different heat treatments on the average of both, or with DMSO (control), they were incubated at 34°C for 2 h number and size of stress granules, we performed heat treatments at to induce the formation of large stress granules. In the presence of 34, 38 or 40°C for 10 or 30 min, or 1 or 2 h using the microtube 0.1% DMSO (control treatment), the median size of stress granules method (Fig. 2). After 10 min of heat treatment, the average number was 1.08 µm2 and the average number was 0.004 (n/µm2). Treatment of stress granules increased with increasing temperature (Fig. 2A,B). with Oryzalin increased the average number to 0.026 and decreased Journal of Cell Science

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Fig. 2. Effects of different temperatures and incubation times on stress granule size and number. (A) Confocal images showing stress granule formation in hypocotyls of 5-day-old eIF4A2-GFP seedlings at different temperatures and incubation times. Seedlings were immersed in water in a tube and incubated under each condition. Scale bar: 10 µm. (B,C) Quantification of number and size of stress granules (SGs) at each temperature and incubation time from experiments 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°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. *P<0.01, **P<0.05 and ***P=not significant according to Welch’s t-test for unequal variances. Error bars indicate ±s.d. the size to 0.11 µm2. These results are consistent with previous average number of granules to 0.064 and decreased their size to studies reporting that microtubule depolymerization affects stress 0.05 µm2. In the presence of Lat B, the cytoplasm often stagnated and granule formation in animals and plants (Ivanov et al., 2003; accumulated at some local areas. Many small stress granules were

Gutierrez-Beltran et al., 2015). Treatment with Lat B increased the also observed in these cytoplasm-accumulated areas. In addition, Journal of Cell Science

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Fig. 3. Effects of cytoskeleton depolymerization drugs on stress granule movement, number and size. (A–D) eIF4A2-GFP seedlings were pre-treated at 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 average velocity (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 cytoskeleton depolymerization drug. Heat treatment was applied for 2 h. At least ten independent images were analyzed. Bar graphs at a linear scale (F) and logarithmic scale (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. treatment with both Oryzalin and Lat B increased the number of stress Weber et al., 2008). Therefore, to clarify the relationship between granules to 0.053 and decreased their size to 0.08 µm2. These results stress granule formation and the presence of P-bodies in Arabidopsis, indicate that cytoplasmic streaming induced by actin filaments affects we performed time-lapse observations through high-resolution, stress granule size and number in Arabidopsis. high-sensitivity confocal microscopy. We increased the temperature of the samples to 35°C within 30 s by heating the microscope stage Stress granule formation is not correlated to the size using a heat incubator. In eIF4A2-GFP plants, stress granules began of P-bodies to form at ∼70 s of treatment (Movie 5). Some stress granules formed The identification of the critical threshold temperatures for stress at the neighboring sites of P-bodies (Fig. 4A, white arrowheads) – granule formation allowed us to perform fine-scale analysis identified in Arabidopsis expressing the P-body marker of stress granule formation. Stress granules appear adjacent to mRNA-decapping enzyme-like protein (DCP1) tagged with RFP

P-bodies in eukaryotes (Kedersha et al., 2005; Buchan et al., 2008; (DCP1-TagRFP) – and some formed independently of P-bodies Journal of Cell Science

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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 and magenta 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 at different time points. Number of stress granules (white bar) including P-body-associated stress granules (striped bar). More than half of the numberof P-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-associated stress granules were plotted; r indicates the correlation value. n=30 (90 s), 23 (190 s) and 12 (450 s). (D) Confocal images showing the formation of 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 bars indicate ±s.d.

(Fig. 4A, yellow arrowheads). Half of the P-bodies were not During the observation period, stress granules often fused with associated with stress granules at 90 s (Fig. 4B), and some each other (Fig. S5B). Fusions also occurred between P-bodies, P-bodies were not associated with stress granules, even at 250 s and between P-bodies and stress granules (Fig. S5B,C). High- (Fig. 4A, arrows at 250 s). By contrast, there were more stress resolution images (Fig. S5D) showing the detailed relationship granules than P-bodies after 150 s of treatment (Fig. 4B). between stress granules and P-bodies indicate that stress granules did Journal of Cell Science

6 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216051. doi:10.1242/jcs.216051 not perfectly overlap with P-bodies. Once stress granules associated Ribosome-exclusion regions are located adjacent to with P-bodies, they did not dissociate from these structures (Fig. S5D, stress granules Movie 5). At the sites of P-body and stress granule association, stress We noticed that ribosome particles were excluded from regions granule size was not correlated with P-body size (Fig. 4C). We further containing stress granules. Interestingly, these ribosome-exclusion investigated the relationship between stress granules and P-bodies regions often extended out of the stress granule areas, which were upon long incubation (60 and 120 min) using the microtube defined by immunogold nanoparticles that labeled eIF4A2-GFP method at 35°C (Fig. 4D,E). Fusion of stress granules progressed (Fig. 5E, left panel). Fortunately, we obtained two images of and the number of stress granules decreased over time. We sequential ultra-thin sections (Fig. 5E, right panel). In both images, expected all P-bodies to have fused into huge stress granules after the gold label-free ribosome-exclusion regions also exhibited a 2 h. However, contrary to this expectation, most P-bodies were not wispy cloud-like filamentous pattern with electron-dense particulates, incorporated into huge stress granules (Fig. 4D), suggesting that thus resembling stress granules. The stress granules and gold label- the interaction between stress granules is stronger than that free areas had distinct shapes in the two sequential sections. These between stress granules and P-bodies. These observations indicate results indicate that, in addition to assuming a typical spherical shape, that stress granule formation occurs in two steps: stress granule stress granules can sometimes be amorphous, with a rugged surface generation and fusion. Although it is difficult to separate these facing the cytoplasm. steps, it appears that stress granules are generated regardless of the presence of P-bodies. During the stress granule fusion process, Ultrastructures of P-bodies in Arabidopsis stress granules tend to associate quickly and tightly with other We reasoned that these gold label-free ribosome-exclusion regions stress granules or P-bodies. might represent (1) stress granules lacking eIF4A2 in a certain area; (2) other RNA granules, such as P-bodies; or (3) unknown Ultrastructures of stress granules in Arabidopsis cytoplasmic structures. Among these, we investigated the possibility Ultrastructural analysis of RNA granules by electron microscopy that these gold label-free ribosome-exclusion regions represented has not previously been reported in plants. We, therefore, observed P-bodies. To examine the ultrastructures of P-bodies in Arabidopsis, Arabidopsis stress granules by electron microscopy in eIF4A2-GFP we subjected DCP1-GFP-expressing seedlings (Motomura et al., seedlings that had been incubated at 38°C for 15 or 60 min 2015) to immunoelectron microscopy. In samples observed at (Fig. 5A). Before and after heating, we fixed the seedlings by high- 22°C, immunogold nanoparticles (indicating the localization of pressure freezing and subjected them to immunoelectron microscopy DCP1-GFP) appeared in distinct, non-membranous, circular sites with an anti-GFP antibody. Gold nanoparticles representing the with electron-dense particulates and filamentous structures (Fig. 6A). positions of eIF4A2-GFP were scattered throughout the cytoplasm in We determined that these structures were P-bodies. We found that sections produced from samples before heat treatment. In samples ribosomes were also excluded from the P-bodies. In samples after heat treatment, gold nanoparticles accumulated at irregularly observed at 22°C, electron-dense particulates were not clearly shaped, ribosome-free structures (Fig. 5A). The stress granules were observed in the P-bodies. However, after heat treatment, some not surrounded by a membrane boundary in the cytoplasm, as P-bodies showed prominent electron-dense particulates with previously reported for stress granules in animal cells (Souquere et al., electron-dense filamentous structures (Fig. 6B,C). Thus, the 2009). We calculated the diameter of the stress granules by measuring ultrastructures of P-bodies after heat treatment resembled those the longest distance between gold particles within each stress of stress granules. As with stress granules, the P-bodies increased granule (Fig. 5B). The average diameter of the stress granules was in size after heat treatment, as revealed by confocal microscopy 191±74.7 nm after heat treatment at 38°C for 15 min. The average (Motomura et al., 2015). Thus, we measured the sizes of P-bodies diameter of the stress granules increased to 311±145.7 nm in by electron microscopy (Fig. 6D), finding that they increased response to treatment at 38°C for 60 min, which is consistent with from 134±77.6 nm (22°C) to 169±59.0 nm (38°C for 15 min) or the fluorescence microscopy results showing an increase in stress 180±76.5 nm (38°C for 60 min) in size after heat treatment. granule size with increasing heating duration. In addition, gold label-free ribosome-exclusion regions were We then examined the electron micrographs in detail. No ribosome occasionally observed around the P-bodies, especially in heat- particles were observed inside the stress granule regions, which were treated samples. Some gold label-free ribosome-exclusion regions identified by the presence of gold nanoparticles (Fig. 5A). The stress exhibited a filamentous pattern (Fig. 6B), and some contained granules that had formed in response to heat treatment at 38°C for electron-dense particulates (Fig. 6C). In conclusion, it is difficult 15 and 60 min contained electron-dense particulates and filamentous to distinguish between stress granules and P-bodies based on structures (Fig. 5A, arrowheads). When seedlings were subjected to their ultrastructures alone. To conclusively identify the stress heat priming through intermittent heat treatment (38°C for 30 min, granule-associated gold label-free ribosome-exclusion regions, 22°C for 30 min and 38°C for 30 min), electron-dense particulates in comprehensive studies must be performed involving the use of the stress granules became more prominent and abundant (Fig. 5C, multi-label immunoelectron microscopy. right panel) compared to samples subjected to a simple heat treatment at 38°C for 60 min (shown in Fig. 5A). As observed by confocal DISCUSSION microscopy, heat priming did not dramatically alter the size or Our analyses revealed the relationship between the effects of number of stress granules compared to other conditions (Fig. S6). In the level and duration of heat stress on stress granule formation addition, using a combination of heat-priming treatment and osmium in Arabidopsis seedlings. In the absence of transpiration, tetroxide (OsO4) fixation, we readily observed similar electron-dense Arabidopsis seedlings formed stress granules at 34°C, particulates and filamentous structures, even in wild-type seedlings regardless of treatment duration (Fig. 1C,D). Stress intensity without immunogold labeling (Fig. 5D). Overall, these results (defined by temperature) was responsible for determining the suggest that stress granules are irregularly shaped, ribosome-free threshold for stress granule formation. Arabidopsis seedlings that structures that include characteristic electron-dense particulates and could transpire formed stress granules at temperatures >34°C filamentous structures. (Fig. 1E,F). For example, in seedlings grown in agar, the duration Journal of Cell Science

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Fig. 5. Ultrastructural features of stress granules – a single stress granule surrounded by a ribosome-exclusion region. (A) Immunoelectron micrographs probed with GFP antibodies showing stress granules in hypocotyl cells of 5-day-old eIF4A2-GFP seedlings that had been heat-treated at 22°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 to Welch’s t-test. Error bars indicate ±s.d. (C) Immunoelectron micrographs of eIF4A2-GFP seedlings 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 min twice with an in-between break at 22°C for 30 min (right). Scale bar: 300 nm. Enlarged images are shown in the inset. Scale bar: 100 nm. Arrows and arrowheads indicate electron-dense granular and filamentous structures, respectively. (D) OsO4-stained electron 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 30min twice 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 and arrowheads indicate electron-dense granular and filamentous structures, respectively. (E) Immunoelectron micrographs of two sequential ∼70 nm-thick sections 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 by a ribosome-exclusion region (red line). Scale bar: 500 nm. of stress treatment also appeared to be important for determining the amount of water in the agar plates. Consistent with this speculation, stress granule formation threshold (Fig. 1E). However, considering the effect of stress duration was not obvious in seedlings grown that transpiration cools the cytoplasm, it is reasonable to assume that in soil, which contained sufficient water for proper transpiration the effect of stress duration under this condition was due to the limited (Fig. 1F). Overall, our results suggest that stress intensity Journal of Cell Science

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Fig. 6. Ultrastructural features of P-bodies in Arabidopsis seedlings. (A–C) Immunoelectron micrographs probed with GFP antibodies against DCP1-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) and for 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.

(temperature) is the key factor determining the threshold of stress plants sense and integrate the intensity of stress signals, and exactly granule formation. when stress granules form, the combination of different stresses may We also found that the threshold temperature for stress granule reduce the temperature threshold needed for stress granule formation. formation did not change in seedlings exposed to 4°C for 2 days In this study, we determined the minimum heat treatment required before the experiment (Fig. 1G). This result indicates that to trigger stress granule formation (Fig. 1C–F), which enabled us to Arabidopsis seedlings have an absolute temperature threshold reduce the amount of unnecessary damage inflicted on the plants. for stress granule formation, and it suggests that stress granules After subjecting the seedlings to the minimum heat treatment, we function in stress responses when seedlings planted in soil face determined that the long-distance transport of stress granules is severe environmental conditions, such as temperatures >41°C. driven by actin filaments (Fig. 3), a mechanism similar to that used Stress granule formation is also induced by hypoxia, drought and to transport other plant organelles and RNA granules (Boevink high-salt conditions in plants (Pomeranz et al., 2010; Sorenson and et al., 1998; Nebenführ et al., 1999; Mathur et al., 2002; Van Gestel Bailey-Serres, 2014; Yan et al., 2014; Gutierrez-Beltran et al., et al., 2002; Ueda et al., 2010; Hamada et al., 2012). In the presence 2015). Under natural growing conditions (outdoors in soil), plants of this long-distance transport, the sizes of stress granules greatly are simultaneously exposed to various stresses and may form stress increased over time (Figs 2 and 3). The loss of actin filaments granules in response to environmental changes. We showed that inhibited the formation of huge stress granules (Fig. 3E–G). high-salt stress reduced the temperature threshold for stress granule In addition, the loss of microtubules also inhibited the formation of formation (Fig. S2). Although we do not currently understand how huge stress granules. The result is consistent with previous findings Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216051. doi:10.1242/jcs.216051 regarding the involvement of microtubules in stress granule The assembly of RNA-binding proteins into RNA granules has formation and transport, in both animal and plant cells (Ivanov recently been explained by the liquid–liquid phase separation et al., 2003; Gutierrez-Beltran et al., 2015). mechanism (Banani et al., 2017). However, it was also proposed Plant-specific features might explain why plants require not only that the maturation of stress granule cores leads to the enlargement of microtubules but also actin filament-driven cytoplasmic streaming stress granules (Wheeler et al., 2016; Protter and Parker, 2016; Panas for stress granule formation. Plant cells are usually relatively large; et al., 2016). Linking ultrastructures to functions or components for instance, the long axis of Arabidopsis epidermal cells can be might clarify the mechanisms underlying stress granule formation. >1 mm. In addition, plant cells do not contain a radial microtubule network radiating from a centrosome, as do animal cells. Actin MATERIALS AND METHODS filaments in plant cells induce rapid cytoplasmic streaming and Plant materials and growth conditions appear to maintain cellular homeostasis via mixing. The effects of All plants of the wild-type and established lines were of the A. thaliana the loss of actin filaments and microtubules differ depending on L. (Heynh.) Columbia background. DCP1-GFP and DCP1-TagRFP transgenic lines (Motomura et al., 2012, 2015) were used as P-body their size and number (Fig. 3E–G). We suspect that actin filaments markers. Seeds were sterilized in sterilizing solution (5% NaClO and 1% and microtubules contribute to stress granule formation in different Triton X-100) or 100% ethanol for 5 min. After sterilization, the seeds ways. It appears that stress granule formation in plants proceeds in were plated on agar containing 1% sucrose, 1% agar, 1% MES-KOH pH two steps: stress granule generation and fusion. Actin filaments and 5.7, and half-strength A. thaliana nutrient solution as described by Haughn microtubules might contribute differently to these two processes. and Somerville (1986). The plates were set in a near-vertical position at Minimum heat treatment also led to the rapid disappearance of 22°C under constant light. Five-day-old plants were used for analysis. For stress granules in plant cells (Movie 1). Stress granules disintegrated the soil experiments, 3-day-old plants were transferred to soil in pots and completely in plant cells within 10 min after the temperature was incubated for 2 days under constant light conditions before observation. reduced to 22°C. The disappearance of hypoxia-induced stress granules was also observed after 20 min of reoxygenation (Sorenson Plasmid construction and transformation and Bailey-Serres, 2014). Our results are consistent with the view To visualize stress granules in seedlings, eIF4A2 (AT1G54270) and PAB6 that plant stress granules are flexible and dynamic, rather than rigid, (AT3G16380) cDNA was amplified by PCR and inserted into the pGWB405, pGWB460 and pGWB406 Gateway vectors (Nakagawa et al., 2007). to quickly respond to the need for mRNA release for translation PCR primers were designed for eIF4A2 (5′-AACCAATTCAGTCGACAT- (Sorenson and Bailey-Serres, 2014). GGCAGGATCCGCACCGGA-3′ as the left-border primer and 5′-AAGCT- Both stress granules and P-bodies in Arabidopsis cells are GGGTCTAGATATCCTCACAGCAAATCAGCCACGT-3′ as the right- irregularly shaped, ribosome-free structures with characteristic border primer) and PAB6 (5′-AACCAATTCAGTCGACATGGCGCTCG- electron-dense particulates and filaments (Figs 5 and 6). Stress TGAAAACGGA-3′ as the left-border primer and 5′-AAGCTGGGTCTA- granules and P-bodies in animal cells also have a similar appearance GATATCCTTGCAATTGTTTTCCTGATTCC-3′ as the right-border primer). (Yang et al., 2004; Souquere et al., 2009; Cougot et al., 2012), Transgenic plants constitutively expressing eIF4A2-GFP and GFP-PAB6 indicating that such ultrastructures are common and shared among were obtained by transformation through the floral dip method (Clough and eukaryotes. What the filamentous structures represent and the Bent, 1998) using the Agrobacterium tumefaciens strain GV3101. identity of the electron-dense particulates are still unclear. Our results may provide clues to help answer these questions. For Heat, drug and high-salt treatments, and confocal microscopy Heat treatments for plants subjected to the mounting, microtube, agar example, the electron-dense particulates of plant stress granules plating and soil methods were applied by using a 93 l heated incubator were observed more clearly and increased in number with prolonged (MIR-H163, PHC Corporation, Tokyo, Japan). For the microtube method, treatment time (Fig. 5A). Especially after heat-priming treatment, 1 ml water in a 1.5 ml microtube was pre-heated in the incubator before use. the stress granules contained higher numbers of electron-dense For time-lapse analysis, each seedling was immersed in water in a particulates and exhibited well-arranged structures (Fig. 5C,D). 20×10×0.15 mm rectangular chamber between coverslips (60×32 mm These results suggest that the presence of electron-dense particulates No.1,24×18mmNo.1S,MatsunamiGlass,Osaka,Japan)andincubated in stress granules is a sign of their maturity. Heat priming (or heat pre- in a stage-top incubator system composed of a customized double treatment) also increases the expression levels of heat-responsive ThermoPlate chamber (11.5×7.5×0.3 cm inside size) and a TP-LH lens genes, such as heat shock protein genes (Mittler et al., 2012). heater (Tokai Hit, Shizuoka, Japan). The stage-top incubator system can Interestingly, heat stress granules (HSGs) in plants also contain increase the temperature of a sample up to 35°C within 30 s. For drug treatments, plants were placed in 1.5 ml tubes containing 1 ml of drug electron-dense particulates and filamentous structures (Nover et al., solution, including 0.1% DMSO (control), 2 µM Lat B, 50 µM Oryzalin or 1983, 1989; Scharf et al., 1998). HSGs are an aggregated form of heat 30 µM cycloheximide (Sigma, St Louis), and incubated for 30 min at 22°C. shock proteins or heat shock factors that form in response to heat Mannitol buffer (400 mM mannitol, 15 mM MgCl2,and4mMMES-KOH stress, like stress granules. Plant stress granules and HSGs are distinct pH 5.7; Yan et al., 2014) containing 150 or 200 mM NaCl was used for high- structures at the confocal microscopic level (Weber et al., 2008). salt treatment. Confocal microscopy was performed using two different These reports indicate that our observed electron-dense particulates confocal microscope systems; a spinning disc confocal microscope system may be a component of HSGs, not a component of RNA granules. (Nikon Eclipse Ti microscope equipped with Yokogawa CSU-W1 confocal Like the tight connection between stress granules and P-bodies spinning disc unit, 2× internal magnifying lens, Andor Zyla 4.2 sCMOS observed in the current study (Fig. 4), these contradictory observations camera, Andor IQ3 software) and an Olympus FV1000 confocal microscope might be explained if we assume that HSGs and stress granules fuse system. Images were converted to binary images and measured using a Particle Analyzer with Image J software (National Institutes of Health; with each other, and have different compositions over time. In heat- http://rsbweb.nih.gov/ij/). treated animal cells, stress granules and HSGs appeared to overlap to form identical granules at the confocal microscopic level (Kedersha Electron microscopy et al., 1999; Piotrowska et al., 2010). Moreover, Weber et al. (2008) has Five-day-old seedlings before and after heat treatment were subjected to shown that some stress granules and HSGs are closely attached, and high-pressure freezing using an EM PACT (Leica Microsystems, Wetzlar, partially overlap. Additional precise experiments are needed to Germany). Each frozen sample was immediately transferred to a sample conclusively address this issue. tube containing freeze-substitution solution (0.25% glutaraldehyde, 0.1% Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216051. doi:10.1242/jcs.216051 uranyl acetate in acetone) and stored at −80°C for 72 h. The tube was Ivanov, P. A., Chudinova, E. M. and Nadezhdina, E. S. (2003). Disruption of gradually warmed up to room temperature over 2 days. Fixed seedlings were microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation. Exp. washed with acetone, dehydrated in methanol and embedded in LR White Cell Res. 290, 227-233. ∼ Kedersha, N. L., Gupta, M., Li, W., Miller, I. and Anderson, P. (1999). resin. The embedded seedlings were sliced into 70 nm-thin sections with RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2α to the an EM UC7 ultramicrotome (Leica Microsystems). The ultra-thin sections assembly of mammalian stress granules. J. Cell Biol. 147, 1431-1442. were collected on formvar-coated Ni grids and subjected to immunoelectron Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., microscopy using anti-GFP antibody (Invitrogen A11122, Thermo Fisher Fritzler, M. J., Scheuner, D., Kaufman, R. J., Golan, D. E. and Anderson, P. Scientific, MA) and 12 or 18 nm colloidal gold AffiniPure goat anti-rabbit (2005). Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol. 169, 871-884. IgG (#111-205-144 or #111-215-144, Jackson Immunoresearch Inc., PA). Kedersha, N., Ivanov, P. and Anderson, P. (2013). Stress granules and cell The samples were stained with 4% aqueous uranyl acetate and observed signaling: more than just a passing phase? Trends Biochem. Sci. 38, 494-506. under a transmission electron microscope (JEM-1400, JEOL, Tokyo, Mathur, J., Mathur, N. and Hülskamp, M. (2002). Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based Japan). For the ultrastructure observations shown in Fig. 4F, 2% OsO4 in acetone was used as the freeze-substitution solution; the samples were then peroxisome motility in plants. Plant Physiol. 128, 1031-1045. Mittler, R., Finka, A. and Goloubinoff, P. (2012). How do plants feel the heat? embedded in Epon812 resin. The ultra-thin sections were stained with 4% Trends Biochem. Sci. 37, 118-125. aqueous uranyl acetate and lead citrate. Motomura, K., Le, Q. T. N., Kumakura, N., Fukaya, T., Takeda, A. and Watanabe, Y. (2012). The role of decapping proteins in the miRNA Acknowledgements accumulation in Arabidopsis thaliana. RNA Biol. 9, 644-652. We thank Mayumi Wakazaki (Riken, Yokohama, Japan) for assistance with Motomura, K., Le, Q. T. N., Hamada, T., Kutsuna, N., Mano, S., Nishimura, M. and electron microscopy, Kentaro Tamura (Kyoto University, Japan) and Haruko Ueda Watanabe, Y. (2015). Diffuse decapping enzyme DCP2 accumulates in DCP1 foci (Konan University, Kobe, Japan) for helpful discussions, and Misako Taniguchi- under heat stress in Arabidopsis thaliana. Plant Cell Physiol. 56, 107-115. Saida [National Institute for Basic Biology (NIBB), Aichi, Japan] for technical Nakagawa, T., Suzuki, T., Murata, S., Nakamura, S., Hino, T., Maeo, K., Tabata, R., Kawai, T., Tanaka, K., Niwa, Y. et al. (2007). Improved gateway assistance. We thank Dr Tobias I. Baskin at the University of Massachusetts, binary vectors: high-performance vectors for creation of fusion constructs in Amherst, for the critical reading of the manuscript. transgenic analysis of plants. Biosci. Biotechnol. Biochem. 71, 2095-2100. Nebenführ, A., Gallagher, L. A., Dunahay, T. G., Frohlick, J. A., Mazurkiewicz, A. M., Competing interests Meehl, J. B. and Staehelin, L. A. (1999). Stop-and-go movements of plant Golgi The authors declare no competing or financial interests. stacks are mediated by the Acto-myosin system. Plant Physiol. 121, 1127-1141. Nover, L., Scharf, K. D. and Neumann, D. (1983). Formation of cytoplasmic heat Author contributions shock granules in tomato cell cultures and leaves. Mol. Cell. Biol. 3, 1648-1655. Conceptualization: T.H., Y.W., I.H.-N.; Methodology: T.H., M.Y., M.M., M.S., Nover, L., Scharf, K. D. and Neumann, D. (1989). Cytoplasmic heat shock Y.K., K.T.; Validation: T.H., M.Y., M.M., M.S., Y.K., K.T.; Formal analysis: T.H., granules are formed from precursor particles and are associated with a specific M.Y., M.M., M.S., K.T.; Investigation: T.H., M.Y., M.M., M.S., Y.K., K.T.; Resources: set of mRNAs. Mol. Cell. Biol. 9, 1298-1308. T.H., Y.Y.; Data curation: T.H., M.Y., M.M., M.S., K.T.; Writing - original draft: T.H., Panas, M. D., Ivanov, P. and Anderson, P. (2016). Mechanistic insights into Y.W., I.H.-N.; Writing - review & editing: T.H., M.S., Y.K., K.T., Y.W., I.H.-N.; mammalian stress granule dynamics. J. Cell Biol. 215, 313-323. Visualization: T.H.; Supervision: T.H., Y.W., I.H.-N.; Project administration: T.H.; Piotrowska, J., Hansen, S. J., Park, N., Jamka, K., Sarnow, P. and Gustin, K. E. Funding acquisition: T.H., Y.K., Y.W., I.H.-N. (2010). Stable formation of compositionally unique stress granules in virus-infected cells. J. Virol. 84, 3654-3665. Pomeranz, M. C., Hah, C., Lin, P.-C., Kang, S. G., Finer, J. J., Blackshear, P. J. Funding and Jang, J.-C. (2010). The Arabidopsis tandem zinc finger protein AtTZF1 This study was supported by the Japan Society for the Promotion of Science (JSPS) traffics between the nucleus and cytoplasmic foci and binds both DNA and RNA. KAKENHI [Grant Numbers: 16H01229, 26650090, 15H05598 to T.H., 17H06258 Plant Physiol. 152, 151-165. to Y.K., 15H04628 to Y.W. and 15H05776 to I.H.-N.] and the National Institute for Protter, D. S. W. and Parker, R. (2016). Principles and properties of stress granules. Basic Biology (NIBB) Cooperative Research Programs [Grant Numbers: 15-333 to Trends Cell Biol. 26, 668-679. Y.W. and 16-517 and 17-508 to T.H.]. Scharf, K.-D., Berberich, T., Ebersberger, I. and Nover, L. (2012). The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim. Supplementary information Biophys. Acta 1819, 104-119. ̈ Supplementary information available online at Scharf, K. D., Heider, H., Hohfeld, I., Lyck, R., Schmidt, E. and Nover, L. (1998). The tomato Hsf system: HsfA2 needs interaction with Hsfa1 for efficient nuclear http://jcs.biologists.org/lookup/doi/10.1242/jcs.216051.supplemental import and may be localized in cytoplasmic heat stress granules. Mol. 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