Thermal acclimation of the symbiotic alga Symbiodinium spp. alleviates photobleaching under heat stress.

A moderate increase in seawater temperature causes coral bleaching, at least partially through photobleaching of the symbiotic algae Symbiodinium spp. Photobleaching of Symbiodinium spp. is primarily associated with the loss of light-harvesting proteins of photosystem II (PSII) and follows the inactivation of PSII under heat stress. Here, we examined the effect of increased growth temperature on the change in sensitivity of Symbiodinium spp. PSII inactivation and photobleaching under heat stress. When Symbiodinium spp. cells were grown at 25°C and 30°C, the thermal tolerance of PSII, measured by the thermal stability of the maximum quantum yield of PSII in darkness, was commonly enhanced in all six Symbiodinium spp. tested. In Symbiodinium sp. CCMP827, it took 6 h to acquire the maximum PSII thermal tolerance after transfer from 25°C to 30°C. The effect of increased growth temperature on the thermal tolerance of PSII was completely abolished by chloramphenicol, indicating that the acclimation mechanism of PSII is associated with the de novo synthesis of proteins. When CCMP827 cells were exposed to light at temperature ranging from 25°C to 35°C, the sensitivity of cells to both high temperature-induced photoinhibition and photobleaching was ameliorated by increased growth temperatures. These results demonstrate that thermal acclimation of Symbiodinium spp. helps to improve the thermal tolerance of PSII, resulting in reduced inactivation of PSII and algal photobleaching. These results suggest that whole-organism coral bleaching associated with algal photobleaching can be at least partially suppressed by the thermal acclimation of Symbiodinium spp. at higher growth temperatures.

Reef-building corals harbor symbiotic dinoflagellate algae of the genus Symbiodinium. Corals generally show a brownish coloration due to algal photosynthetic pigments, such as peridinin and chlorophylls a and c 2 present in in situ Symbiodinium spp. However, under increased seawater temperatures, corals become pale through the loss of Symbiodinium spp. cells and/or the loss of photosynthetic pigments of in situ Symbiodinium spp. (Glynn, 1993(Glynn, , 1996Hoeghguldberg, 1999;Fitt et al., 2001;Coles and Brown, 2003). This phenomenon is so-called coral bleaching. Since a healthy algae-coral symbiotic relationship is important for coral survival (Yellowlees et al., 2008), severe coral bleaching leads to the mortality of corals and even the destruction of entire coral reef ecosystems. The frequency and intensity of coral bleaching have been increasing since the early 1980s, and it is predicted to become more severe in the future due to ongoing global climate change and warming (Hughes et al., 2003). Coral reef ecosystems are in serious decline, with an estimated 30% already severely damaged, and it is predicted that globally as much as 60% of the world's coral reef ecosystems may be lost by 2030 (Hughes et al., 2003).
Coral bleaching caused by heat stress is at least partially attributed to the photobleaching of photosynthetic pigments in Symbiodinium spp. within corals (Kleppel et al., 1989;Porter et al., 1989;Fitt et al., 2001;Takahashi et al., 2004;Venn et al., 2006). The photobleaching commonly occurs in photosynthetic organisms under conditions where the absorbed light energy for photosynthesis is in excess of the capacity to use it, particularly under environmental stress conditions in high light (Niyogi, 1999). In cultured Symbiodinium spp. cells, heat stress-associated algal photobleaching is attributed to the loss of major light-harvesting proteins, such as the peridinin-chlorophyll a-binding proteins and the chlorophyll a-chlorophyll c 2 -peridin protein complexes . A recent study has also demonstrated that the heat stressassociated loss of light-harvesting proteins in Symbiodinium spp. is attributed to suppression of the de novo synthesis of light-harvesting proteins but not acceleration of the photodamage and subsequent degradation of light-harvesting proteins . High-temperature sensitivity of Symbiodinium spp. cells to photobleaching differs among Symbiodinium spp., and this is at least partially attributed to the thermal sensitivity of the de novo synthesis of light-harvesting proteins .
Heat stress-associated photobleaching in Symbiodinium spp. follows severe photoinhibition of PSII . The extent of photoinhibition is a result of the dynamic balance between the rate of photodamage to PSII and the rate of its repair. In plants and green algae, the PSII repair process is primarily composed of the degradation and the de novo synthesis of the D1 proteins in photodamaged PSII protein complexes (Aro et al., 1993;Takahashi and Murata, 2008;Takahashi and Badger, 2011). However, this differs in Symbiodinium spp., in that the photodamaged PSII can be repaired without the de novo synthesis of D1 proteins (Takahashi et al., 2009b). Furthermore, a part of photodamaged PSII is repaired without protein synthesis (Takahashi et al., 2009b), indicating that Symbiodinium spp. have a unique PSII repair mechanism. In Symbiodinium spp. found within corals and also in culture, heat stress accelerates photoinhibition at least partially through the suppression of PSII repair (Warner et al., 1999;Takahashi et al., 2004Takahashi et al., , 2009b. However, the sensitivity of PSII repair to heat stress differs among Symbiodinium spp. and is strongly related to the sensitivity of PSII to photoinhibition under heat stress (Takahashi et al., 2009b).
The high-temperature sensitivity of corals to bleaching is changed by their growth temperature, and this is suggested to be due to changing in situ Symbiodinium spp. populations from heat-sensitive to heat-resistant ecotypes (Baker, 2001(Baker, , 2003Baker et al., 2004;Berkelmans and van Oppen, 2006;Jones et al., 2008;Jones and Berkelmans, 2010). However, thermal tolerance of the population might also be enhanced by thermal acclimation mechanism(s) associated with both the corals and Symbiodinium spp., although experimental data that directly support this hypothesis are lacking. In this study, we examine the effect of increased growth temperature (thermal acclimation treatment) on the extent of heat stress-associated algal photobleaching using cultured Symbiodinium spp. Our results demonstrate that Symbiodinium spp. commonly have thermal acclimation mechanisms that enhance the high-temperature tolerance of PSII and alleviate heat stress-associated photobleaching. Our results strongly suggest that thermal acclimation of Symbiodinium spp. plays a role in alleviating algal photobleaching-associated coral bleaching under heat stress.

Effect of Growth Temperature on Thermal Tolerance of PSII
To determine if the growth of Symbiodinium spp. at moderately elevated temperatures results in an acclimatory shift in the thermal tolerance of PSII, the maximum quantum yield of PSII (F v /F m ) was measured in six different Symbiodinium spp. grown at 25°C or 30°C after exposure treatments at temperatures ranging from 25°C to 38°C for 1 h in darkness (Fig. 1). In Symbiodinium spp. cells grown at 25°C, the measured F v /F m remained unchanged up to 33°C in all species but declined to zero at 34°C in CCMP827, at 35°C in CCMP831, CCMP830, and CCMP421, and at 36°C in Mf1.05b and OAH-1. When Symbiodinium spp. cells were grown at 30°C for 8 d, the thermal tolerance of PSII (the minimum temperature that leads to a reduction of F v /F m to zero) increased 1°C in CCMP827, CCMP831, and Mf1.05b and 2°C in OAH-1, CCMP830, and CCMP421. These results demonstrate that all Symbiodinium spp. tested in our study commonly had a thermal acclimation mechanism that was able to improve the high-temperature tolerance of PSII. Furthermore, our results indicate that the thermal acclimation ability shows slight differences among Symbiodinium spp.
To further understand the thermal acclimation mechanism in Symbiodinium spp., we examined the effect of different growth temperatures ranging from 25°C to 32°C on the thermal tolerance of PSII using Symbiodinium sp. CCMP827 ( Fig. 2A). When growth temperature increased from 25°C to 28°C, the thermal tolerance of PSII apparently increased 1°C. There was no difference in the thermal tolerance of PSII between cells grown at 28°C and 30°C. In cells grown at 32°C, the thermal tolerance of PSII was improved as much as in cells grown at 28°C and 30°C, but the F v /F m value after incubation at each growth temperature was much lower in cells grown at 32°C, indicating that PSII was severely impaired in cells grown at 32°C. These results demonstrated that a small increase (less than 3°C) in growth temperature is enough to fully activate the thermal acclimation mechanism in CCMP827. To test whether the Figure 1. Effect of growth temperature on the thermal tolerance of PSII in six different Symbiodinium spp. Cells were grown at 25˚C or 30˚C for 8 d. F v /F m was measured after incubation of cells for 1 h in darkness at temperatures ranging from 25˚C to 38˚C. Values are means 6 SD from three independent experiments. minimum temperature that fully activates the thermal acclimation mechanism is common in Symbiodinium spp., we performed the same experiments in OAH-1 and CCMP830. In OAH-1, the thermal tolerance showed improvement in cells grown at 30°C compared with those at 28°C (Supplemental Fig. S1A). Furthermore, in CCMP830, the thermal tolerance was increased further in cells grown at 32°C (Supplemental Fig. S1B). These results demonstrate that the minimum temperature that fully activates the thermal acclimation mechanism shows differences among Symbiodinium spp.
We examined how long it takes to acquire the maximum thermal tolerance of PSII in CCMP827 (Fig.   2B). Cells grown at 25°C were transferred to 30°C and incubated for 3 h, 6 h, 1 d, or 8 d. The thermal tolerance of PSII was slightly enhanced after 3 h of incubation at 30°C and was significantly enhanced after 6 h. Further incubation had no effect on the thermal tolerance of PSII up to 8 d. These results demonstrate that the process of thermal acclimation was completed after 6 h in CCMP827.
We examined how long it takes to lose the acquired thermal tolerance of PSII in CCMP827 (Fig. 2C). Cells grown at 30°C for 3 d were transferred to 25°C and incubated for 2, 4, or 6 d. The thermal tolerance of PSII was slightly reduced after 2 d and was significantly reduced after 4 d. After 6 d of incubation at 25°C, there was no significant difference in the thermal tolerance of PSII with cells continuously grown at 25°C. These results demonstrate that the acquired thermal tolerance of PSII is completely lost in 6 d in CCMP827.
Thermal acclimation may be associated with the de novo synthesis of proteins. To examine this, CCMP827 cells were transferred from 25°C to 30°C and incubated in the presence or absence of chloramphenicol in darkness for 12 h. In general, chloramphenicol inhibits the de novo synthesis of chloroplast-encoded proteins. However, in Symbiodinium spp., chloramphenicol has been demonstrated to inhibit the de novo synthesis of both chloroplast-and nucleus-encoded proteins (Takahashi et al., 2009b). Since chloramphenicol inhibits protein synthesis-dependent repair of photodamaged PSII and also causes photoinhibition in the light, experiments were carried out under darkness. In the absence of chloramphenicol, the thermal tolerance of PSII was enhanced by moderately increased temperature in darkness ( Fig. 3A) to an extent similar to that in the light (Figs. 1A and 2B), indicating that the thermal acclimation of PSII in Symbiodinium spp. is not light dependent. The effect of moderately increased growth temperature on the thermal tolerance of PSII was completely abolished by chloramphenicol (Fig.  3B). These results demonstrate that the thermal acclimation mechanism in CCMP827 is associated with the de novo synthesis of proteins. Figure 2. Thermal acclimation of PSII stability in Symbiodinium sp. CCMP827. A, Symbiodinium sp. CCMP827 cells were grown at different temperatures ranging from 25˚C to 32˚C for 8 d. B, Symbiodinium sp. CCMP827 cells grown at 25˚C were transferred to 30˚C and incubated for different periods (3 h, 6 h, 1 d, or 8 d). C, Symbiodinium sp. CCMP827 cells grown at 30˚C for 3 d were transferred to 25˚C and incubated for different periods (2, 4, or 6 d). Control (Cont.) results are from experiments with cells continuously grown at 25˚C. F v /F m was measured after incubation for 1 h in darkness at temperatures ranging from 25˚C to 35˚C. Values are means 6 SD from three independent experiments.

Thermal Acclimation Helps Maintain Higher Photosynthetic Performance under Heat Stress in Light
An increase in seawater temperature causes acceleration of the photoinhibition of PSII in the light in Symbiodinium spp. (Warner et al., 1999;Takahashi et al., 2004Takahashi et al., , 2009b. To examine the effects of thermal acclimation on the sensitivity of PSII to photoinhibition under heat stress, the extent of photoinhibition was monitored in CCMP827 cells grown at 25°C or 30°C at temperatures ranging from 25°C to 34°C under exposure to light (Fig. 4A). In cells grown at 25°C, the extent of photoinhibition (decrease in the value of F v /F m during light exposure) was drastically enhanced at temperatures above 31°C. However, in cells grown at 30°C, the temperature that caused a rapid onset of photoinhibition was 1°C higher than that in cells grown at 25°C. These results demonstrate that thermal acclimation not only improves the stability of PSII in the dark but also alleviates photoinhibition of PSII in the light under heat stress in CCMP827. Thus, thermal acclimation improves photosynthetic performance under heat stress conditions through enhancing the thermal tolerance of PSII (Figs. 1A and 2A) and suppressing the thermal sensitivity of PSII to photoinhibition in the light in CCMP827 (Fig. 4A). However, this was different in other Symbiodinium spp. (Supplemental Fig. S2). In OAH-1 and CCMP830 cells, an increase in growth temperature did not help alleviate photoinhibition under heat stress (Supplemental Fig. S2), although it enhanced the thermal tolerance of PSII (Fig. 1, D and E). These results demonstrate that thermal acclimation improves photosynthetic performance in OAH-1 and CCMP830 primarily through enhancing the thermal tolerance of PSII but not through suppressing the thermal sensitivity of PSII to photoinhibition in the light.
To further understand how thermal acclimation alleviates photoinhibition under heat stress, the thermal sensitivity of photoinhibition in the presence of chloramphenicol was examined in CCMP827 cells grown at 25°C or 30°C (Fig. 4B). When cells grown at 25°C and 30°C were exposed to light at 30°C, there was no difference in the extent of photoinhibition in the presence of chloramphenicol between them. However, when cells were exposed to light at 33°C, the extent of photoinhibition was much higher in cells grown at 25°C than 30°C. These results show that thermal acclimation reduces photoinhibition in the presence of chloramphenicol under heat stress in CCMP827. As chloramphenicol inhibits the protein synthesis-dependent repair of photodamaged PSII, it is possible that thermal acclimation may suppress either or both acceleration of photodamage and/or inhibition of the protein synthesisindependent repair under heat stress.
To examine the effect of growth temperatures (25°C and 30°C) on the sensitivity of the PSII repair process to heat stress, we monitored the recovery of F v /F m after photoinhibition treatment by strong light in Symbiodinium sp. CCMP827 (Fig. 4C). Cells grown at 25°C and 30°C were exposed to strong light (2,000 mmol m 22 s 21 ) at each growth temperature for 1 h. Cells were then preincubated in darkness at 30°C or 33°C for 1 h and subsequently exposed to low light (20 mmol m 22 s 21 ) to allow repair. The value of F v /F m before the low-light exposure (after photoinhibition and preincubation treatments) was 40% of the initial level in all conditions except at 33°C in cells grown at 25°C (in this case, F v /F m declined to 20% of initial values). This is presumably because PSII is thermally inactivated under 33°C in cells grown at 25°C, as shown in Figure 1A. In cells grown at both 25°C and 30°C, the recovery of F v /F m was Figure 4. Effect of moderately increased growth temperature on the thermal sensitivity of PSII to photoinhibition and subsequent repair in Symbiodinium sp. CCMP827. CCMP827 cells grown at 25˚C or 30˚C for more than 3 d were used for experiments. A, Cells were incubated at temperatures ranging from 25˚C to 34˚C in darkness for 1 h and then exposed to light at 200 mmol m 22 s 21 for 12 h. B, Cells were incubated at 30˚C or 33˚C in darkness for 1 h and then exposed to light at 200 mmol m 22 s 21 for 3 h in the presence of 1 mM chloramphenicol. C, Cells were preexposed to light at 2,000 mmol m 22 s 21 at their respective growth temperatures for 1 h. Cells were then incubated at 30˚C or 33˚C in darkness for 1 h before monitoring the recovery of F v /F m for 3 h at 20 mmol m 22 s 21 . In all experiments, F v /F m was measured after 10 min of incubation in darkness. Values are means 6 SD from three independent experiments. g.t., Growth temperature; t.t., treated temperature. significantly lower at 33°C than 30°C, indicating that heat stress reduces the repair of photodamaged PSII in CCMP827 cells (Fig. 4C). Importantly, there was no effect of growth temperature on the recovery of F v /F m at either 30°C or 33°C (Fig. 4C). This demonstrates that thermal acclimation had no influence on the sensitivity of PSII repair to heat stress. Thus, in the presence of chloramphenicol, suppression of heat stress-associated photoinhibition by increased growth temperature (Fig.  4B) could be attributed to reducing photodamage to PSII caused by heat stress. This demonstrates that heat stress accelerates the photoinhibition process through both increasing photodamage to PSII and reducing the PSII repair process and that, as a consequence, thermal acclimation alleviates photoinhibition through avoiding acceleration of photodamage to PSII under heat stress.

Thermal Acclimation Alleviates Photobleaching under Heat Stress
A moderate increase in seawater temperature causes acceleration of photobleaching in Symbiodinium spp. (Takahashi et al., 2004. To examine the effect of thermal acclimation processes on the extent of photobleaching under heat stress, total chlorophyll (chlorophylls a and c 2 ) content was measured before and after light exposure for 12 h at temperatures ranging from 25°C to 35°C in Symbiodinium sp. CCMP827 cells grown at 25°C or 30°C (Fig. 5). In cells grown at 25°C, loss of chlorophyll content was drastically enhanced at temperatures above 33°C, and 50% of initial chlorophyll content was lost at 34°C. However, in cells grown at 30°C, the temperature that initiated the drastic loss of chlorophyll was 1°C higher than that in cells grown at 25°C, and only 20% of initial chlorophyll content was lost at 34°C. These results demonstrated that thermal acclimation increased the thermal threshold for initiating photobleaching in Symbiodinium sp. CCMP827.
Similar results were obtained in Symbiodinium spp. OAH-1 and CCMP830 (Supplemental Fig. S3), where the thermal threshold for initiating drastic photobleaching increased 1°C after growing cells at moderately increased temperatures.
Photobleaching of Symbiodinium spp. Is Not Associated with Inactivation of PSII Photobleaching of Symbiodinium spp. commonly follows severe inactivation of PSII under heat stress. To determine whether the heat stress-associated photobleaching is due to inactivation of PSII, the effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the extent of photobleaching was examined. When Symbiodinium sp. CCMP827 cells were incubated with 5 mM DCMU, the photosynthetic oxygen production rate decreased to 6% of that in the absence of DCMU (Fig.  6A). After incubation of light at 200 mmol m 22 s 21 for 12 h, DCMU slightly enhanced the extent of photobleaching (Fig. 6B). However, the effect of DCMU on photobleaching was much lower than the effect of heat stress on photobleaching in Figure 5. These results demonstrate that heat stress-associated photobleaching in Symbiodinium sp. CCMP827 is not due to inactivation of PSII.

Thermal Acclimation Alleviates Photobleaching in Symbiodinium spp. under Heat Stress
Heat stress causes coral bleaching at least partially through the acceleration of photobleaching in Symbiodinium spp. living within corals (Kleppel et al., 1989;Porter et al., 1989;Fitt et al., 2001;Takahashi et al., 2004;Venn et al., 2006). In this study, we demonstrate that the thermal tolerance threshold of Symbiodinium spp. for initiating photobleaching increases after growing at moderately increased temperature (Fig. 5). Extrapolation of our results strongly suggests that photobleachingassociated coral bleaching can be alleviated by thermal acclimation of Symbiodinium spp. growing within corals.
Consistent with previous studies , the sensitivity of Symbiodinium spp. to bleaching under heat stress differs among species: CCMP827 (Fig.  5) showed more severe photobleaching under heat stress conditions than OAH-1 (Supplemental Fig. S3A) and CCMP830 (Supplemental Fig. S3B) in cells grown at 25°C. In all three Symbiodinium spp. tested in this study, thermal acclimation commonly showed a 1°C increase in the thermal threshold for initiating photobleaching ( Fig. 5; Supplemental Fig. S3). However, the results demonstrate that different thermal sensitivity among Symbiodinium spp. is not due to different thermal acclimation ability.
The thermal sensitivity of Symbiodinium spp. to photobleaching corresponds with the sensitivity of PSII to inactivation under heat stress (Chen et al., 2004;. Since the process of photobleaching Figure 5. Effect of moderately increased growth temperature on the thermal sensitivity of Symbiodinium spp. to photobleaching. Symbiodinium sp. CCMP827 cells grown at 25˚C or 30˚C were incubated at different temperatures ranging from 25˚C to 35˚C for 1 h in darkness. Subsequently, cells (5 mg chlorophyll mL 21 ) were exposed to light at 200 mmol m 22 s 21 for 12 h at the same temperature. Total chlorophyll content (chlorophylls a and c 2 ) was measured before and after light exposure, and the loss of chlorophyll content (percentage of initial) was calculated. Values are means 6 SD from three independent experiments.
occurs after severe inactivation of PSII under heat stress, inactivation of PSII is expected to cause photobleaching in Symbiodinium spp. However, in this study, inhibition of photosynthetic oxygen production at PSII by DCMU had little effect on the extent of photobleaching under our experimental conditions (Fig. 6B). In a previous study using Symbiodinium spp., heat stress-associated photobleaching was demonstrated to be primarily due to suppression of the de novo synthesis of light-harvesting proteins in thylakoid membranes . However, there was no effect of DCMU on the de novo synthesis of any membrane proteins (Supplemental Fig.  S4). Therefore, it is likely that heat stress-associated photobleaching of Symbiodinium spp. is not due to a lack of PSII activity. Thus, the alleviation of photobleaching in thermally acclimated Symbiodinium spp. cells (Fig. 5) is not due to remaining PSII activity. Our results suggest that heat stress causes both inactivation of PSII and photobleaching with a time delay. This might be because photobleaching is a slow process through proteolytic degradation of light-harvesting proteins, while inactivation of PSII is a quick process though nonenzymatic photochemical reactions.
In OAH-1 and CCMP830, the thermal threshold for initiating apparent photobleaching increased by 1°C after thermal acclimation (Supplemental Fig. S3), although the thermal tolerance of PSII in the dark increased 2°C to 3°C (Supplemental Fig. S1). These results demonstrate that the thermal tolerance of PSII that was examined in the dark does not always correspond with photobleaching sensitivity under heat stress. This lack of correlation might be because the thermal sensitivity of PSII to inactivation is determined not only by the thermal tolerance of PSII but also the thermal sensitivity of PSII to photoinhibition. In OAH-1 and CCMP830, PSII performance was much higher in cells grown at 30°C or 32°C than in cells grown at 25°C under heat stress (more than 34°C) before light exposure (Supplemental Fig. S2). However, the difference was gradually minimized during light exposure due to the acceleration of photoinhibition in cells grown at higher temperature ( Supplemental Fig, S2). Thus, the thermal sensitivity of PSII in Symbiodinium spp. differs before and after light exposure treatments. The thermal sensitivity of Symbiodinium spp. to photobleaching seems to correspond more closely with the thermal tolerance of PSII in the light rather than its stability in the dark.
How Does Thermal Acclimation Enhance the Thermal Tolerance of PSII in Symbiodinium spp.?
Moderately increased growth temperature has been demonstrated to enhance the thermal tolerance of PSII in photosynthetic organisms (Armond et al., 1978;Nishiyama et al., 1999;Tanaka et al., 2000;Kimura et al., 2002;Nanjo et al., 2010), including Symbiodinium spp. (Díaz-Almeyda et al., 2011). In this study, small increases in temperature (less than 3°C) were found to be sufficient to activate the thermal acclimation mechanism of PSII in Symbiodinium spp. (Fig. 2A). Furthermore, we found that it takes only 6 h to acquire the maximum thermal tolerance of PSII in Symbiodinium sp. CCMP827 (Fig. 2B). The thermal acclimation mechanism of Symbiodinium spp. was associated with the de novo synthesis of proteins (Fig. 3), as has been shown in other photosynthetic organisms (Tanaka et al., 2000;Nanjo et al., 2010). Furthermore, the thermal tolerance of PSII was enhanced in darkness (Fig.  3A) to a similar extent as in the light (Figs. 1A and 2). Thus, the synthesis of proteins that are responsible for the thermal acclimation of PSII in Symbiodinium spp. is regulated solely by temperature, as has been shown in the green alga Chlamydomonas reinhardtii (Tanaka et al., 2000). Our results suggest that Symbiodinium spp. cells acquire the thermal tolerance of PSII at any time of the day under increased seawater temperature.
A variety of factors have been shown to enhance the thermal tolerance of PSII in studies using plants, algae, and cyanobacteria, such as psbU protein in PSII (Nishiyama et al., 1997;Kimura et al., 2002), lipid compounds (Sato et al., 2003;Sakurai et al., 2007aSakurai et al., , 2007bMizusawa et al., 2009), xanthophyll zeaxanthin (Havaux et al., 1996;Havaux, 1998), heat shock proteins (Stapel et al., 1993;Eriksson and Clarke, 1996;Heckathorn et al., 1998;Tsvetkova et al., 2002), s factors (Singh et al., 2006;Tuominen et al., 2006), and isoprene (Sharkey and Singsaas, 1995). In Symbiodinium spp., membrane lipid composition has been suggested to determine the thermal tolerance of PSII (Tchernov et al., 2004). However, it is still controversial whether changes in lipid composition are responsible for the thermal acclimation of PSII in Symbiodinium spp. (Díaz-Almeyda et al., 2011). In cyanobacteria, the synthesis of fatty acids is involved, but stability increases of PSII are not associated with either the total amount of fatty acids or the levels of unsaturated Figure 6. Effect of DCMU on the sensitivity of Symbiodinium spp. to photobleaching. Symbiodinium sp. CCMP827 cells grown at 25˚C were incubated for 1 h in darkness with (+) or without (2) 5 mM DCMU and used for experiments. All experiments were carried out at 25˚C. A, Effect of DCMU on photosynthesis. Photosynthetic oxygen production rate was measured under the light at 1,000 mmol photons m 22 s 21 . The photosynthetic oxygen production rate was 115 6 7.6 mmol oxygen mg 21 chlorophyll h 21 in the absence of DCMU (control). B, Effect of DCMU on photobleaching. Cells (5 mg chlorophyll mL 21 ) were exposed to light at 200 mmol m 22 s 21 for 12 h. Total chlorophyll content (chlorophylls a and c 2 ) was measured before and after light exposure, and the loss of chlorophyll content (percentage of initial) was calculated. Values are means 6 SD from three independent experiments. fatty acids in thylakoid membrane (Nanjo et al., 2010). Models suggest that the synthesis of fatty acids is expected to be associated with binding the PSIIstabilizing proteins, such as lipoprotein. The inhibition of thermal acclimation of PSII by an inhibitor of protein synthesis, therefore, might be due to inhibition of the synthesis of proteins associated with the synthesis of fatty acids or stabilizing PSII under heat stress (Nishiyama et al., 1999;Kimura et al., 2002;Nanjo et al., 2010).

Thermal Acclimation Decreases the Sensitivity of PSII to Photoinhibition under Heat Stress
Heat stress causes the acceleration of photoinhibition of PSII in cultured Symbiodinium spp. and in situ Symbiodinium spp. within corals (Warner et al., 1999;Takahashi et al., 2004Takahashi et al., , 2009b. Heat stress-associated photoinhibition in Symbiodinium sp. CCMP827 cells grown at 25°C was due to the acceleration of photodamage to PSII (Fig. 4B) and also through the inhibition of PSII repair (Fig. 4C). We found that increased growth temperature alleviates heat stress-associated photoinhibition (Fig. 4A) through suppressing photodamage to PSII (Fig. 4B) but not suppressing the inhibition of PSII repair (Fig. 4C). In Symbiodinium spp., heat stress has been demonstrated to damage thylakoid membranes (Tchernov et al., 2004), which impairs the generation of the proton gradient across the thylakoid membrane. Since impairment of the proton gradation causes the acceleration of photodamage to PSII (Takahashi et al., 2009a(Takahashi et al., , 2009b, alleviation of thermal stress-associated photodamage to PSII in Symbiodinium spp. after growing at moderately increased temperature might be due to suppressing damage to thylakoid membranes under heat stress. When corals are grown under moderately increased temperature, heat stress-associated coral bleaching is suppressed (Coles and Jokiel, 1978;Coles and Brown, 2003). Since the sensitivity of corals to bleaching under heat stress is changed by the thermal sensitivity of Symbiodinium spp. living within corals and this differs among Symbiodinium spp., the sensitivity of corals to bleaching under heat stress can be decreased by changing the dominant in situ Symbiodinium spp. population from heat sensitive to heat tolerant (Baker, 2001(Baker, , 2003Baker et al., 2004;Berkelmans and van Oppen, 2006;Jones et al., 2008;Jones and Berkelmans, 2010). In this study, we found that the sensitivity of Symbiodinium spp. to photobleaching under heat stress is suppressed by acclimation to moderately increased growth temperatures. In three cultured Symbiodinium spp. tested in this study, the thermal threshold for initiating apparent photobleaching commonly increased by 1°C after thermal acclimation ( Fig. 5; Supplemental Fig. S3). By extrapolation, these findings suggest that the threshold for initiating photobleaching-associated coral bleaching might increase by at least 1°C through a thermal acclimation process of Symbiodinium spp. within corals without involving a change in the overall Symbiodinium spp. population. It is still uncertain whether there are Symbiodinium spp. that show a greater acclimation ability and that increase their thermal threshold for initiating photobleaching by more than 1°C after thermal acclimation.
In this study, thermal acclimation was demonstrated to alleviate the inactivation of PSII through both enhancing the thermal tolerance of PSII (Fig. 1) and suppressing the photoinhibition of PSII (Fig. 4A) under heat stress. Since inactivation of PSII has been hypothesized to be a trigger of expulsion of Symbiodinium spp. from host cells (Brown, 1997;Hoeghguldberg, 1999;Warner et al., 1999), thermal acclimation of Symbiodinium spp. might also reduce coral bleaching that is associated with the loss of Symbiodinium spp. under heat stress. However, further study is needed to elucidate this hypothesis.
The thermal acclimation of Symbiodinium spp. occurs in a period of hours (Fig. 2B), while dominant Symbiodinium spp. within corals are changed in the order of days (Baker, 2003). Therefore, it is conceivable that thermal acclimation of Symbiodinium spp. and changes of dominant Symbiodinium spp. are associated with shortand long-term thermal acclimation, respectively, and that both help to alleviate coral bleaching under heat stress.

Cultures and Growth Conditions
Cultures of Symbiodinium sp. CCMP827 (clade A), CCMP831 (clade A), CCMP830 (clade B), and CCMP421 (clade E) were obtained from the National Center for Marine Algae and Microbiota (Tchernov et al., 2004). OAH-1 (clade B; Ishikura et al., 2004) and Mf1.05b (clade B; Voolstra et al., 2009) were gifts from Dr. Tadashi Maruyama and Dr. Mary Alice Coffroth, respectively. Clades of these Symbiodinium spp. cultures are according to previous studies (Ishikura et al., 2004;Tchernov et al., 2004;Voolstra et al., 2009). Symbiodinium spp. cells (200-400 mL) were grown in artificial seawater (sea salts; Sigma) containing Daigo's IMK medium for marine microalgae (Wako) in 2-L shaker flasks with a filter cap under fluorescent light at 80 mmol photons m 22 s 21 with a light/dark cycle of 12 h/12 h. The flask was mixed once per day for aeration. Growth temperatures were controlled with an electronic aquarium heater (A-761; Hagen) in a 40-L aquarium tank. The cells were collected by filtration (0.22-mm Stericup; Millipore) during their midlogarithmic growth phase (less than 0.5 mg chlorophyll mL 21 ) and suspended in fresh growth medium for experiments.

Temperature Treatments
Freshly harvested cells were diluted to 5 mg chlorophyll mL 21 , and equal volumes were incubated at different temperatures in darkness for 1 h before either being illuminated at 200 mmol photons m 22 s 21 with halogen lamps or maintained in darkness. All temperature treatments represented in each experiment were performed simultaneously using an aluminum gradient heat bar with glass vials containing cells at appropriate temperatures along the bar in wells and illuminated with halogen lamps from the top where necessary. Light intensity (400-700 nm) was measured with a LI-250 light meter (LI-COR).

Photoinhibition, Chlorophyll, and Photosynthetic Oxygen Production Rate Measurements
F v /F m was measured with a PAM-2000 chlorophyll fluorometer (Heinz Walz) after the cells had been incubated for 10 min in darkness. The concentrations of chlorophylls a and c 2 were measured by treating cells collected by centrifugation (16,000g, 1 min) with 80% (v/v) methanol at 70°C for 10 min . Cell debris was removed by centrifugation (16,000g, 1 min), the absorption spectrum of the supernatant was measured using a diode-array spectrophotometer (Cary 50 Bio; Varian), and the total chlorophyll a and c 2 concentrations were calculated according to Jeffrey and Humphrey (1975). To measure the photosynthetic oxygen production rate, light-dependent oxygen production was measured in Symbiodinium spp. cells (10 mg of total chlorophyll a and c 2 in 1 mL) with a Clark-type oxygen electrode (Hansatech Instruments) in a closed cuvette in the light at 1,000 mmol photons m 22 s 21 at 25°C.

Pulse Labeling of Proteins and Separation of Membrane Proteins
Symbiodinium sp. CCMP827 cells (5 mg chlorophyll mL 21 ) were incubated with or without 5 mM DCMU in the dark at 25°C for 1 h. Then, cells were exposed to light at 200 mmol photons m 22 s 21 in the presence of [ 35 S]Met/Cys (10 mCi mL 21 ) at 25°C for 15 min. Cells were collected by centrifugation, and membrane proteins (corresponding to 1.5 mg of chlorophyll) were separated by NuPAGE Novex 4% to 12% Bis-Tris gel electrophoresis (Invitrogen; . The gel was stained with Coomassie Brilliant Blue G-250 (GelCode Blue Stain Reagent; Thermo Scientific) and dried on paper with the gel dryer (model 583; Bio-Rad) at 80°C for 1 h. The dried gel was exposed to an imaging Screen-K (Kodak) and visualized with the Molecular Imager PharosFX Plus System (Bio-Rad).

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S2. Effect of moderately increased growth temperature on the thermal sensitivity of PSII to photoinhibition in Symbiodinium sp. OAH-1 and CCMP830.
Supplemental Figure S3. Effect of moderately increased growth temperature on the thermal sensitivity of Symbiodinium spp. to photobleaching.
Supplemental Figure S4. Effect of DCMU on the de novo synthesis of membrane proteins in Symbiodinium sp. CCMP827.