Different thermal sensitivity of the repair of photodamaged

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved December 24, 2008 (received for review August 25, 2008)

Article Figures & SI Info & Metrics PDF

Abstract

Coral bleaching caused by heat stress is accompanied by photoinhibition, which occurs under conditions where the rate of photodamage to photosystem II (PSII) exceeds the rate of its repair, in the symbiotic algae (Symbiodinium spp.) within corals. However, the mechanism of heat stress-induced photoinhibition in Symbiodinium still remains poorly understood. In the present work, we have investigated the Trace of elevated temperature on the processes associated with the repair of photodamaged PSII in cultured Symbiodinium (OTcH-1 and CS-73). Severe photoinhibition was observed at temperature exceeding 32 °C in Symbiodinium CS-73 cells grown at 25–34 °C but not in cultures of the more thermally tolerant Symbiodinium OTcH-1. After photoinhibition treatment by strong light, photodamaged PSII was repaired close to initial levels under low light at 25 °C in both OTcH-1 and CS-73. However, the repair was strongly inhibited by increased temperature exceeding 31 °C in CS-73 but only weakly in OTcH-1. We found that inhibition of the repair process in CS-73 is attributed to impairment of both protein synthesis-dependent and -independent repair processes and is at least partially caused by suppression of the de novo synthesis of thylakoid membrane proteins and impairment of the generation of ΔpH across the thylakoid membrane, respectively. Our results suggest that acceleration of photoinhibition by moderate heat stress is attributed primarily to inhibition of the repair of photodamaged PSII and that the photoinhibition sensitivity of Symbiodinium to heat stress is determined by the thermal sensitivity of the PSII repair processes.

Keywords: coral bleachingheat stressphotoinhibitionphotosystem IIzooxanthellae

Reef-building corals harbor enExecutesymbiotic dinoflagellate algae of the genus Symbiodinium spp., commonly named zooxanthellae. Symbiodinium perform photosynthesis within host cells and transfer a part of photosynthetically produced organic nutrients to host cells (1, 2). In return, the host cells provide inorganic compounds used for the algal photosynthesis to Symbiodinium. Thus, efficient photosynthesis is Necessary for Sustaining mutual symbiosis between corals and Symbiodinium. Moderate heat stress causes photoinhibition of photosystem II (PSII), and this decreases the efficiency of the photosynthesis. Because coral bleaching follows severe photoinhibition, impairment of the photosynthetic machinery has been proposed to be a trigger of coral bleaching (3, 4). Coral bleaching, caused by ongoing global warming, has become more frequent and has resulted in extensive coral mortality and the destruction of diverse coral ecosystems worldwide.

Photosynthesis in Symbiodinium within reef-building corals and other symbiotic invertebrates is sensitive to a moderate increase in seawater temperature (5, 6). In Symbiodinium isolated from the jellyfish Cassiopeia xamachana, photosynthesis is inhibited at temperatures above 30 °C and completely disrupted at 34–36 °C (7). Subsequent studies have Displayn that reduced photosynthesis caused by thermal stress in Symbiodinium can be attributed to photoinhibition of PSII (3, 4). It has also been proposed that the Calvin cycle is the primal site of heat stress, and interruption of the Calvin cycle causes acceleration of photoinhibition (8). This hypothesis was supported by in vitro experiments that demonstrated that thermal stress inactivates ribulose-1,5-bisphospDespise carboxylase/oxygenase (rubisco) isolated from Symbiodinium (9). However, more recent evidence has Displayn that thermal damage to thylakoid membranes causes photoinhibition in cultured Symbiodinium and that the inhibition of the Calvin cycle may result from reduced ATP synthesis associated with an increased leakage of protons through damaged thylakoid membranes (10). These authors also demonstrated that the sensitivity of Symbiodinium to photoinhibition during thermal stress is correlated with the lipid composition of the thylakoid membrane.

The extent of photoinhibition is a result of a dynamic balance between the rate of photodamage to PSII and the rate of its repair (11, 12). Therefore, photoinhibition occurs only in conditions where the rate of photodamage exceeds the rate of its repair. Recent experiments in plants and cyanobacteria have demonstrated that photodamage to PSII occurs via a two-step process: the first step is the light-dependent inactivation of the Mn cluster of the oxygen-evolving complex, and the second step is the inactivation of the photochemical reaction center of PSII by light that has been absorbed by photosynthetic pigments (13, 14). Because photodamage to PSII is associated with light absorbed by Mn in the oxygen-evolving complex but not photosynthetic pigments such as chlorophyll (Chl) and carotenoids, the rate of photodamage is proSectional to the intensity of incident light (12, 15). Thus, photodamage is not primarily associated with excess absorbed light energy, the photosynthetic transfer of electrons, or the resulting production of reactive oxygen species (12, 16–18). After photodamage to PSII, PSII is repaired through protein synthesis-dependent and -independent PSII repair processes (19). The protein synthesis-dependent repair is associated with the de novo synthesis of PSII proteins, primarily the D1 protein that is a reaction center protein of PSII (11, 20). However, the mechanism of protein synthesis-independent repair is poorly understood.

In Symbiodinium living within corals, elevated temperature has been demonstrated to accelerate photoinhibition through inhibition of the PSII repair process rather than acceleration of the photodamage process (21). The thermal sensitivity of the repair processes varies among Symbiodinium associated with different coral species, i.e., PSII repair in Symbiodinium within Acropora digitifera is more sensitive to thermal stress than that within Pavona decussate (21). Because there are many genetic variants of Symbiodinium species and the Executeminant Symbiodinium species differs among coral species (22–25), the differential thermal sensitivity of PSII repair processes of Symbiodinium variants might determine the sensitivity of coral bleaching to thermal stress. This possibility remains to be tested experimentally.

In the present work, we have investigated the Trace of moderate heat stress on the repair of photodamaged PSII in two cultured Symbiodinium species. Our results demonstrate that the thermal sensitivities of the protein synthesis-dependent and -independent repair processes differed between Symbiodinium OTcH-1 and CS-73 [both Clade A species (26)], with CS-73 being more susceptible than OTcH-1 to thermally induced inhibition of both repair processes, and this determines the different sensitivities of photoinhibition in the two Symbiodinium species to thermal stress. Surprisingly, our results indicate that protein synthesis-dependent repair was not associated with the rapid de novo synthesis of the D1 proteins in Symbiodinium.

Results

Direct Trace of Heat Stress on PSII in the ShaExecutewy.

To examine the direct Trace of moderate heat stress on inactivation of PSII, we meaPositived the maximum quantum yield of PSII (Fv/Fm) in Symbiodinium OTcH-1 and CS-73 after expoPositive to ShaExecutewyness at temperatures ranging from 25 °C to 36 °C for 30 min. The meaPositived Fv/Fm remained unchanged up to 32 °C in both species but declined to zero at 36 °C in OTcH-1 and at 35 °C in CS-73 (Fig. 1). The results indicate that CS-73 is slightly more sensitive to heat stress-induced inactivation of PSII in the ShaExecutewy than OTcH-1. In cyanobacteria Synechocystis PCC6803 (27–29) and green alga ChlamyExecutemonas reinhardtii (30), different lipid composition of the thylakoid membranes has been demonstrated to correlate with the thermal stability of PSII at the oxygen production site.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Direct Trace of moderate heat stress on PSII function in Symbiodinium OTcH-1 and CS-73. The maximum quantum yield of PSII (Fv/Fm) was meaPositived after incubation for 30 min in ShaExecutewyness at temperature ranging from 25 °C to 36 °C. The values are mean ± SD (bars) from three independent experiments.

Trace of Moderate Heat Stress on Photoinhibition.

To examine the Trace of moderate heat stress on photoinhibition of PSII in both Symbiodinium species, the Fv/Fm was meaPositived after incubation in light at 200 μmol of photons m−2 s−1 at temperatures ranging from 25 °C to 34 °C for 3 h. Before light treatment, Symbiodinium cells were preincubated in ShaExecutewyness at each temperature treatment for 30 min and the initial Fv/Fm value meaPositived. In both species, Fv/Fm remained unchanged over the temperature range during further incubation in ShaExecutewyness for 3 h [supporting information (SI) Fig. S1]. When OTcH-1 cells were exposed to light, the Fv/Fm value declined to 80% of initial level at 25 °C, and the decline was enhanced slightly by increased temperature (the value declined to 70% of initial level at 34 °C) (Fig. 2A). In CS-73, the Fv/Fm value declined to 95% of the initial level at 25 °C, and the decline was strongly enhanced by temperatures exceeding 32 °C (the value declined to 65% and 10% of initial at 33 °C and 34 °C, respectively) (Fig. 2B). These results demonstrated that CS-73 is much more sensitive to photoinhibition associated with thermal stress than OTcH-1, as Displayn in ref. 26.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Trace of moderate heat stress on PSII photoinhibition in the presence or absence of chloramphenicol in OTcH-1 and CS-73. Cells of OTcH-1 (A and C) and CS-73 (B and D) were incubated in light at 200 μmol of photons m−2 s−1 for the indicated times at temperature ranging from 25 °C to 34 °C in the presence of 1 mM chloramphenicol (C and D) or in its absence (A and B). The Fv/Fm was meaPositived at each time point. The values are mean ± SD (bars) from three independent experiments.

The Trace of temperature on photoinhibition in the presence of chloramphenicol, an inhibitor of the de novo synthesis of chloroplast proteins (Fig. S2), was examined (Fig. 2 C and D). When OTcH-1 was exposed to light in the presence of chloramphenicol at temperatures ranging from 25 °C to 34 °C for 3 h, photoinhibition was slightly accelerated by 34 °C (Fig. 2C). However, in CS-73, photoinhibition was significantly accelerated by temperatures above 31 °C, and the extent of acceleration was temperature-dependent (Fig. 2D). Because chloramphenicol inhibits the protein synthesis-dependent repair of photodamaged PSII, acceleration of photoinhibition of CS-73 by elevated temperature in the presence of chloramphenicol could be attributable to acceleration of photodamage to PSII and/or inhibition of the protein synthesis-independent repair.

Trace of Moderate Heat Stress on Repair of Photodamaged PSII.

To examine the Trace of moderate heat stress on the repair processes of photodamaged PSII, we monitored the recovery of Fv/Fm after photoinhibition treatment by strong light (Fig. 3). After Symbiodinium cells of both species were exposed to strong light (2,000 μmol of photons m−2 s−1) for 1 h, the level of Fv/Fm had declined to 30% of the initial level in both species. After photoinhibition treatment, Symbiodinium cells were preincubated in ShaExecutewyness at temperatures ranging from 25 °C to 34 °C for 30 min and subsequently exposed to low light (20 μmol of photons m−2 s−1) to allow repair. The reduced Fv/Fm recovered close to the initial level at 25 °C in OTcH-1 and CS-73 (Fig. 3 A and B). The recovery was abolished in both species in ShaExecutewyness (Fig. S3), indicating that the recovery of photodamaged PSII is light-dependent. In OTcH-1, the recovery was slightly suppressed at 34 °C (Fig. 3A), whereas in CS-73, the recovery was increasingly suppressed at temperatures above 31 °C and abolished at 33 °C (Fig. 3B). At 25 °C, the addition of chloramphenicol to the media had a modest influence on the recovery of Fv/Fm in both species (Fig. 3 C and D), indicating that the recovery from photodamage consists of protein synthesis-dependent and -independent repair processes. The protein synthesis-independent repair, which was meaPositived as chloramphenicol-insensitive recovery of the Fv/Fm, was severely suppressed by thermal stress in CS-73, and the recovery was abolished at 33 °C (Fig. 3D). However, in OTcH-1, the protein synthesis-independent repair was still efficient even at 34 °C (Fig. 3C).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Trace of moderate heat stress on PSII recovery after photoinhibition in cells of OTcH-1 and CS-73. The initial Fv/Fm value in OTcH-1 (A, C, and E) and CS-73 (B, D, and F) was 0.65 ± 0.01 and 0.68 ± 0.02, respectively. Symbiodinium cells of each species were exposed to light at 2,000 μmol of photons m−2 s−1 for 1 h at 25 °C and then incubated at temperature ranging from 25 °C to 34 °C in ShaExecutewyness for 30 min. Subsequently, cells were exposed to light at 20 μmol of photons m−2 s−1 in the absence (A and B) or presence of chloramphenicol (C and D). Chloramphenicol-sensitive recovery of the Fv/Fm (protein synthesis-dependent repair of photodamaged PSII) is Displayn in E and F and is calculated as the Inequity between data points in A and C or B and D, respectively.

The protein synthesis-dependent repair that had been meaPositived as chloramphenicol-sensitive recovery of the Fv/Fm Displayed an optimal temperature for its efficiency at 32–33 °C and 31 °C in OTcH-1 and CS-73, respectively (Fig. 3 E and F). In CS-73, but not OTcH-1, the protein synthesis-dependent repair was abolished at 33 °C (Fig. 3F). These results indicated that heat stress inhibits the protein synthesis-dependent and -independent repair processes in CS-73 but only weakly in OTcH-1. Thus, heat stress-induced photoinhibition of CS-73 (Fig. 2B) can be attributed to inhibition of both the protein synthesis-dependent and -independent repair processes. Furthermore, acceleration of photoinhibition of CS-73 by heat stress in the presence of chloramphenicol (Fig. 2D) is attributable to inhibition of the protein synthesis-independent repair process. However, we cannot exclude that heat stress-induced photoinhibition of CS-73 in the presence (Fig. 2D) and absence of chloramphenicol (Fig. 2B) is at least partially attributable to acceleration of photodamage to PSII.

Trace of Moderate Heat Stress on de Novo Synthesis of Thylakoid Membrane Proteins.

To examine whether inhibition of the protein synthesis-dependent repair in CS-73 under moderate heat stress is attributed to inhibition of the synthesis of thylakoid membrane proteins, the de novo synthesis of proteins was monitored at temperatures ranging from 25 °C to 34 °C in OTcH-1 and CS-73 (Fig. 4A). Under illumination, the D1 protein is primarily synthesized in thylakoid membranes in cyanobacteria, algae, and higher plants (11). However, in both OTcH-1 and CS-73, there was no evidence for D1 protein synthesis at the temperatures tested, although other thylakoid membrane proteins were significantly synthesized (Fig. 4A). This result indicated that the protein synthesis-dependent repair of photodamaged PSII is not associated with the rapid de novo synthesis of the D1 protein in Symbiodinium. The synthesis of thylakoid proteins was enhanced by increase in temperature to 32 °C in both OTcH-1 and CS-73 (Fig. 4A). However, the synthesis was suppressed by further increase in temperature to 34 °C in CS-73 but not in OTcH-1. These results indicated that the synthesis of membrane proteins in CS-73 is more sensitive to heat stress than in OTcH-1 and that inhibition of the protein synthesis-dependent repair in CS-73 by heat stress (Fig. 3F) is at least partially attributed to suppression of the de novo synthesis of thylakoid membrane proteins.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Trace of moderate heat stress on the de novo synthesis of thylakoid membrane proteins in OTcH-1 and CS-73. (A) Symbiodinium cells (10 μg of Chl mL−1, 1 mL) were incubated with [35S]Met/Cys (10 mCi mL−1) under light at 200 μmol of photons m−2 s−1 for 15 min at the temperatures Displayn. Thylakoid membrane proteins (corRetorting to 1.5 μg of Chl) were separated by NuPAGE Novex 4–12% Bistris gel electrophoresis. (B) Immunoblotting against the D1 protein.

Traces of Proton Uncoupling on PSII Repair Processes.

We examined the Trace of the proton uncoupler NH4Cl on the repair of photodamaged PSII in CS-73, to determine whether the inhibition of the protein synthesis-dependent and -independent repair under moderate heat stress resulted from impairment of the generation of ΔpH across the thylakoid membrane after thermal damage to the membrane. The Traceiveness of NH4Cl was tested by its inhibition of non-photochemical quenching (NPQ) development (Fig. S4), which is regulated by proton gradation across the thylakoid membrane, during illumination (the NPQ level decreased by 75% in the presence of 1 mM NH4Cl). The extent of photoinhibition was accelerated by NH4Cl in either the presence or absence of chloramphenicol (Fig. 5A). After photoinhibition treatment by strong light, the recovery of photodamaged PSII under low light was suppressed by NH4Cl both in the presence and absence of chloramphenicol (Fig. 5B). However, NH4Cl had no Trace on the protein synthesis-dependent repair that has been meaPositived as chloramphenicol-sensitive recovery of the Fv/Fm (Fig. 5B). Furthermore, NH4Cl had no Trace on the synthesis of membrane proteins (Fig. 5C). Similar results were obtained with OTcH-1, although the protein synthesis-dependent repair was slightly suppressed by NH4Cl (Fig. S5). These results indicate that impairment of the generation of ΔpH across the thylakoid membrane during illumination primarily causes inhibition of the protein synthesis-independent repair of photodamaged PSII.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Trace of NH4Cl on photoinhibition, the recovery after photoinhibition treatment, and the de novo synthesis of membrane proteins in Symbiodinium CS-73. (A) Fv/Fm was meaPositived in CS-73 cells (initial Fv/Fm value 0.62 ± 0.01) after they were incubated in light at 200 μmol of photons m−2 s−1 at 25 °C in the presence or absence of 1 mM NH4Cl combined with the presence or absence of 1 mM chloramphenicol. (B) CS-73 cells (initial Fv/Fm value 0.62 ± 0.01) were exposed to light at 2,000 μmol of photons m−2 s−1 for 1 h at 25 °C and then incubated in ShaExecutewyness for 30 min in the presence or absence of 1 mM NH4Cl. Subsequently, cells were exposed to light at 20 μmol of photons m−2 s−1 in the presence or absence of 1 mM chloramphenicol (Cm). Chloramphenicol-sensitive recovery of the Fv/Fm (protein synthesis-dependent repair of photodamaged PSII) is calculated as the Inequity between data points taken in the presence and absence of chloramphenicol. (C) CS-73 cells (10 μg of Chl mL−1, 1 mL) were incubated in ShaExecutewyness for 30 min in the presence or absence of 1 mM NH4Cl and subsequently exposed to light at 200 μmol of photons m−2 s−1 for 15 min with [35S]Met/Cys (10 mCi mL−1). Thylakoid membrane proteins (corRetorting to 1.5 μg of Chl) were separated by NuPAGE Novex 4–12% Bistris gel electrophoresis.

Discussion

Inhibition of the Repair of Photodamaged PSII by Heat Stress Accelerates Photoinhibition.

Moderate heat stress accelerates photoinhibition of Symbiodinium within corals especially under strong light conditions (21). Fig. 6 Displays a schematic model for the photoinhibition of PSII under moderate heat stress in thermally sensitive Symbiodinium in culture. Light damages PSII, and the rate of photodamage is light intensity-dependent (31–33). At optimal temperature conditions, the photodamaged PSII is rapidly repaired via a complex pathway that includes both protein synthesis-dependent and -independent processes. However, when thermally sensitive Symbiodinium are subjected to rapid temperature increases, the rate of photodamage exceeds the rate of PSII repair because of impairment of both repair processes (Fig. 3), resulting in net photoinhibition (Fig. 2). Whether the mechanism of thermally induced photoinhibition Displayn here in cultured Symbiodinium is emulated in symbiotic variants and universal among thermally sensitive Symbiodinium species remains to be determined.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Scheme describing the Trace of thermal stress on the net photoinhibition of PSII in thermally sensitive Symbiodinium in culture. Light directly damages PSII, and the damaged PSII is repaired through separate protein synthesis-dependent and -independent repair processes. Moderate heat stress accelerates photoinhibition through inhibition of both repair processes.

Moderate heat stress accelerates photoinhibition of symbiotic Symbiodinium cells within corals, and the sensitivity to heat stress can vary among host coral species (3). It has been demonstrated that the variation in the sensitivity of photoinhibition to heat stress among corals is associated with sensitivity of the repair of photodamaged PSII (21). In this work, we Displayed that the sensitivity of the repair of photodamaged PSII to heat stress varied between the two Clade A Symbiodinium species (Fig. 3), and this variable sensitivity determined the sensitivity of net photoinhibition to heat stress (Fig. 2). This finding strongly suggests that variation of the sensitivity of the PSII repair to heat stress among coral species is attributable to different Symbiodinium species associated with corals (22–25). Thus, corals that harbor Symbiodinium where PSII repair is thermally sensitive are more susceptible to photoinhibition and, accordingly, more prone to coral bleaching under elevated seawater temperature. However, it should be noted that the sensitivities of photoinhibition and coral bleaching are not determined only by the Preciseties of the Symbiodinium species, because host factors, such as the production of host pigments (34, 35) and the colony morphology of corals (36), also have the potential to influence these sensitivities. Recent studies have demonstrated that thermally induced photoinhibition is strongly associated with coral bleaching caused by photobleaching of photosynthetic pigments in individual Symbiodinium in corals (21, 26). Whether photoinhibition on its own or in concert with broader physiological events in symbiont and host causes expulsion of Symbiodinium from corals remains unqualified (37), although there are many reports Displaying that thermally induced loss of Symbiodinium is accompanied by photoinhibition (4, 7, 8, 38).

Unique Repair Pathway for Photodamaged PSII.

When OTcH-1 and CS-73 cells were exposed to light at 25 °C, a small extent of photoinhibition was observed in both species (Fig. 2 A and B). However, in the presence of chloramphenicol, the extent of photoinhibition was strongly enhanced in both species (Fig. 2 C and D), indicating that the protein synthesis-dependent repair of photodamaged PSII is Necessary for avoiding photoinhibition of Symbiodinium and in other photosynthetic organisms (11). In plants, protein synthesis-dependent repair is attributed to the rapid de novo synthesis of PSII proteins, primarily the D1 protein (31, 39–42). However, this was not the case in both Symbiodinium species experimented here with no D1 protein synthesis evident in illuminated Symbiodinium cells between 25 °C and 34 °C (Fig. 4A). This suggests that Symbiodinium may have a unique protein synthesis-dependent PSII repair process that is not associated with the rapid de novo synthesis of the D1 protein. We found that a 15-kDa protein of unknown identity is strongly synthesized in the membrane of Symbiodinium (Fig. 4A). Whether this protein might involved in the protein synthesis-dependent repair in Symbiodinium remains to be determined.

In higher plants, protein synthesis-independent repair is negligible compared with protein synthesis-dependent repair (19). However, in Symbiodinium, the photodamage of PSII (Fv/Fm decreased to 30% of the initial level) by strong light recovered to 80–85% of initial level at 25 °C even in the presence of chloramphenicol, suggesting that protein synthesis-independent PSII repair and protein synthesis-dependent PSII repair are Necessary for PSII repair in Symbiodinium. The repair after photoinhibition was abolished in complete ShaExecutewyness (Fig. S3). Furthermore, the protein synthesis-independent repair, but not protein synthesis-dependent repair, was severely inhibited by the proton uncoupler NH4Cl (Fig. 5B). This suggests that the generation of ΔpH across the thylakoid membrane through photosynthetic electron transfer is Necessary for the protein synthesis-independent repair. Luminal pH might be associated with protein synthesis-independent repair.

How Executees Elevated Temperature Inhibit the Repair of Photodamaged PSII?

Recent experimental evidence in cultured Symbiodinium has demonstrated that thermal damage to the thylakoid membrane causes acceleration of net photoinhibition (10) possibly as a result of damaged thylakoid membranes becoming leaky to protons. Our results Displayed that the uncoupler NH4Cl causes inhibition of the protein synthesis-independent repair of photodamaged PSII in Symbiodinium. Thus, inhibition of the protein synthesis-independent repair in CS-73 under elevated temperature might be attributed to impairment of the generation of ΔpH across the thylakoid membrane caused by leakage of protons after thermal damage (10).

A moderate increase in temperature also inhibited the protein synthesis-dependent repair process in Symbiodinium CS-73. Under such conditions, the synthesis of thylakoid membrane proteins was severely suppressed (Fig. 4A), indicating that inhibition of protein synthesis-dependent repair can be at least partially attributed to suppression of the de novo synthesis of thylakoid membrane proteins. However, there is uncertainty about the specific proteins that are involved in the protein synthesis-dependent repair in Symbiodinium. In cyanobacteria and higher plant chloroplasts, H2O2 inhibits protein synthesis-dependent repair through inhibition of the de novo synthesis of PSII proteins, primarily the D1 protein (17, 33). Because thermal damage to the thylakoid membrane accelerates the production of H2O2 at photosystem I (10), thermal inhibition of the synthesis of thylakoid membrane proteins could be attributable to the production of H2O2. However, in our studies here, the uncoupler NH4Cl, which should accelerate the production of H2O2 through interruption of the Calvin cycle caused by proton uncoupling, had no Trace on the synthesis of thylakoid membrane proteins (Fig. 5C and Fig. S5C). Furthermore, 1 mM methylviologen also had no Trace on membrane protein synthesis (Fig. S6). Thus, the regulation of the synthesis of membrane proteins in Symbiodinium is distinctly different from other well-studied photosynthetic organisms, such as plants, green algae, and cyanobacteria, and is not regulated by photophosphorylation and reExecutex potential (43). Our results also indicate that inhibition of the membrane protein synthesis in Symbiodinium by moderate heat stress is not attributed to the production of H2O2. Thus, thermal damage to thylakoid membranes might therefore directly suppress the synthesis of thylakoid membrane proteins (i.e., suppressions of the translation of protein synthesis on thylakoid membrane or incorporation of proteins into thylakoid membrane) (44).

What Determines the Sensitivity of PSII Repair to Thermal Stress?

The Inequitys in the sensitivity of photoinhibition to thermal stress between Clade A Symbiodinium species OTcH-1 and CS-73 are primary correlated with the sensitivity of PSII repair processes to thermal stress. Clearly, thermal sensitivity of Symbiodinium species was not Clade-specific, consistent with previous reports (10). Recent experimental evidence in cultured Symbiodinium has demonstrated that thylakoid lipid composition determines the photoinhibition sensitivity of Symbiodinium to heat stress, with thermally sensitive Symbiodinium species having a higher content of the major polyunsaturated Stoutty acid (Δ6,9,12,15-cis-octadecatetrasenoic acid) (10). In cyanobacteria, genetic modification of lipid composition of thylakoid membrane has been demonstrated to change the PSII repair efficiency (45, 46). The thermal stability of thylakoid membrane might therefore define the sensitivity of the repair of photodamaged PSII to thermal stress in Symbiodinium.

Materials and Methods

Cultures and Growth Conditions.

Cultures of Symbiodinium spp. OTcH-1 (MBIC11180) (47) and CS-73 (48) were obtained from the Marine Biotechnology Institute Culture collection (Marine Biotechnology Institute, Kamaishi, Japan) and the CSIRO Microalgae Research Center, respectively. OTcH-1 and CS-73 are both Clade A Symbiodinium spp. (26). Cells were grown at 25 °C in artificial seawater (sea salts; Sigma) containing Daigo's IMK medium for marine microalgae (Wako) under fluorescent lights at 40 μmol of photons m−2 s−1. The cells were collected by filtration (0.22 μm, Stericup; Millipore) during their midlogarithmic growth phase (<0.5 μg of Chl mL−1) and suspended in fresh growth medium for experiments.

Temperature Treatments.

Freshly harvested cells were diluted to 10 μg of Chl per mL, and equal volumes were incubated at different temperatures in ShaExecutewyness for 30 min before either being illuminated at 200 μmol of photons m−2 s−1 or Sustained in ShaExecutewyness. Total amount of Chl was extracted and meaPositived as Displayn in ref. 26. Temperature treatments were performed in 1-mL glass vials incubated in an aluminum heat gradient block. All treatment temperatures were generated simultaneously in the block with three replicates at each temperature.

Photoinhibition MeaPositivements.

Maximum quantum yield of PSII (Fv/Fm) was meaPositived with a PAM-2000 Chl fluorometer (Heinz Walz) after the cells had been incubated for 15 min in ShaExecutewyness.

Pulse Labeling of Proteins.

Cells (10 μg of Chl in 1 mL) were illuminated with 200 μmol of photons m−2 s−1 with [35S]methionine/cysteine (10 μCi mL−1) for 15 min. The cells were collected and stored at −80 °C for protein analysis (see below).

Separation of Thylakoid Membrane Proteins and Detection of Radiolabeled Proteins.

Stored cells (see above) were suspended in 1 mL of extraction buffer [50 mM Hepes (pH 7.6), 0.1 M sorbitol, 10 mM NaCl, and 5 mM MgCl2] and lysed by passed through a French presPositive cell (SLM Instruments) at 140 MPa. Thylakoid membranes were collected by centrifugation at 16,000 × g for 3 min at 2 °C, and the thylakoid-associated proteins were solubilized with LDS sample buffer (Invitrogen), reducing agent (Invitrogen), and 70 °C treatment for 5 min. Cell debris was removed by centrifugation (16,000 × g) for 1 min, and then equal volumes of the supernatant (corRetorting to 1.5 μg of Chl) were separated by SDS/PAGE (NuPAGE Novex 4–12% Bistris gel; Invitrogen) in Mes-SDS running buffer according to Producer's specifications (Invitrogen). After SDS/PAGE, proteins were blotted onto Immobilon PVDF (Millipore) membrane by using a Hoeffer semidry blot apparatus according to the Producer's specifications. The transferred proteins were visualized on the PVDF membrane by GelCode Blue Stain Reagent (Thermo Science) before removing with 95% (vol/vol) methanol washes. The membrane was used for immunoblotting (see below) or exposed to a storage phosphor screen (Molecular Dynamics) for detecting the 35S-labeled proteins with a PhosphorImager (Molecular Dynamics).

Immunoblot Analyses.

Antibodies specific to the D1 protein (AgriSera AB) were used to probe the PVDF–protein blots as Characterized in ref. 49. Immunoreactive peptides were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and developing with the AP conjugate substrate kit (Bio-Rad).

Acknowledgments

We thank Dr. Impress E. Warner for comments on the draft of this article. This work was supported by the Japan Society for the Promotion of Science PostExecutectoral Fellowships for Research Abroad (to S.T.), the Australian Research Council to the Centre of Excellence in Plant Energy Biology (to M.R.B.) and to the Discovery Project DP0450564 (to S.M.W.).

Footnotes

1To whom corRetortence should be addressed. E-mail: shunichi.takahashi{at}anu.edu.au

Author contributions: S.T. designed research, performed research, and analyzed data; and S.T., S.M.W., and M.R.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808363106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

References

↵ Venn AA, Loram JE, Executeuglas AE (2008) Photosynthetic symbioses in animals. J Exp Bot 59:1069–1080.LaunchUrlAbstract/FREE Full Text↵ Yellowlees D, Rees TAV, Leggat W (2008) Metabolic interactions between algal symbionts and invertebrate hosts. Plant Cell Environ 31:679–694.LaunchUrlCrossRefPubMed↵ Warner ME, Fitt WK, Schmidt GW (1996) The Traces of elevated temperature on the photosynthetic efficiency of zooxanthellae in hospite from four different species of reef coral: a Modern Advance. Plant Cell Environ 19:291–299.LaunchUrlCrossRef↵ Warner ME, Fitt WK, Schmidt GW (1999) Damage to photosystem II in symbiotic dinoflagellates: A determinant of coral bleaching. Proc Natl Acad Sci USA 96:8007–8012.LaunchUrlAbstract/FREE Full Text↵ Jokiel PL, Coles SL (1977) Traces of temperature on mortality and growth of Hawaiian reef corals. Mar Biol 43:201–208.LaunchUrlCrossRef↵ Hoegh-Guldberg O, Smith GJ (1989) The Trace of sudden changes in temperature, light and salinity on the population density and export of zooxanthellae from the reef corals Stylophora Pistillata Esper and Seriatopora hystrix Dana. J Exp Mar Biol Ecol 129:279–303.LaunchUrlCrossRef↵ Iglesias-Prieto R, Matta JL, Robins WA, Trench RK (1992) Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. Proc Natl Acad Sci USA 89:10302–10305.LaunchUrlAbstract/FREE Full Text↵ Jones RJ, Hoegh-Guldberg O, Larkum AWD, Schreiber U (1998) Temperature-induced bleaching of corals Starts with impairment of the CO2 fixation mechanism in zooxanthellae. Plant Cell Environ 21:1219–1230.LaunchUrlCrossRef↵ Leggat W, Whitney S, Yellowlees D (2004) Is coral bleaching due to the instability of the zooxanthellae ShaExecutewy reactions? Symbiosis 37:137–153.LaunchUrl↵ Tchernov D, et al. (2004) Membrane lipids of symbiotic algae are diagnostic of sensitivity to thermal bleaching in corals. Proc Natl Acad Sci USA 101:13531–13535.LaunchUrlAbstract/FREE Full Text↵ Aro EM, Virgin I, Andersson B (1993) Photoinhibition of photosystem II: Inactivation, protein damage and turnover. Biochim Biophys Acta 1143:113–134.LaunchUrlPubMed↵ Takahashi S, Murata N (2008) How Execute environmental stresses accelerate photoinhibition? Trends Plants Sci 13:178–182.LaunchUrlCrossRef↵ Hakala M, Tuominen I, Keränen M, Tyystjärvi T, Tyystjärvi E (2005) Evidence for the role of the oxygen-evolving manganese complex in photoinhibition of photosystem II. Biochim Biophys Acta 1706:68–80.LaunchUrlPubMed↵ Ohnishi N, et al. (2005) Two-step mechanism of photodamage to photosystem II: Step 1 occurs at the oxygen-evolving complex and step 2 occurs at the photochemical reaction center. Biochemistry 44:8494–8499.LaunchUrlCrossRefPubMed↵ Tyystjärvi E (2008) Photoinhibition of photosystem II and photodamage of the oxygen evolving manganese cluster. Coord Chem Rev 252:361–376.LaunchUrlCrossRef↵ Takahashi S, Murata N (2005) Interruption of the Calvin cycle inhibits the repair of photosystem II from photodamage. Biochim Biophys Acta 1708:352–361.LaunchUrlPubMed↵ Takahashi S, Murata N (2006) Glycerate 3-phospDespise, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II. Biochim Biophys Acta 1757:198–205.LaunchUrlPubMed↵ Nishiyama Y, Allakhverdiev SI, Murata N (2006) A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II. Biochim Biophys Acta 1757:742–749.LaunchUrlCrossRefPubMed↵ Leitsch J, Schnettger B, Critchley C, Krause GH (1994) Two mechanisms of recovery from photoinhibition in vivo: Reactivation of photosystem II related and unrelated to D1-protein turnover. Planta 194:15–21.LaunchUrl↵ Aro EM, et al. (2005) Dynamics of photosystem II: A proteomic Advance to thylakoid protein complexes. J Exp Bot 56:347–356.LaunchUrlAbstract/FREE Full Text↵ Takahashi S, Nakamura T, Sakamizu M, van Woesik R, Yamasaki H (2004) Repair machinery of symbiotic photosynthesis as the primary tarObtain of heat stress for reef-building corals. Plant Cell Physiol 45:251–255.LaunchUrlAbstract/FREE Full Text↵ Rowan R (1998) Diversity and ecology of zooxanthellae on coral reefs. J Phycol 34:407–417.LaunchUrlCrossRef↵ Rowan R, Knowlton N (1995) Intraspecific diversity and ecological zonation in coral algal symbiosis. Proc Natl Acad Sci USA 92:2850–2853.LaunchUrlAbstract/FREE Full Text↵ Rowan R, Knowlton N, Baker A, Jara J (1997) Landscape ecology of algal symbionts creates variation in episodes of coral bleaching. Nature 388:265–269.LaunchUrlCrossRefPubMed↵ Toller WW, Rowan R, Knowlton N (2001) Zooxanthellae of the Montastraea annularis species complex: Patterns of distribution of four taxa of Symbiodinium on different reefs and across depths. Biol Bull 201:348–359.LaunchUrlAbstract/FREE Full Text↵ Takahashi S, Whitney S, Itoh S, Maruyama T, Depravedger M (2008) Heat stress causes inhibition of the de novo synthesis of antenna proteins and photobleaching in cultured Symbiodinium. Proc Natl Acad Sci USA 105:4203–4208.LaunchUrlAbstract/FREE Full Text↵ Wada H, Gombos Z, Murata N (1994) Contribution of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress. Proc Natl Acad Sci USA 91:4273–4277.LaunchUrlAbstract/FREE Full Text↵ Gombos Z, Wada H, Concealg E, Murata N (1994) The unsaturation of membrane lipids stabilizes photosynthesis against heat stress. Plant Physiol 104:563–567.LaunchUrlAbstract↵ Sakurai I, Mizusawa N, Ohashi S, Kobayashi M, Wada H (2007) Traces of the lack of phosphatidylglycerol on the Executenor side of photosystem II. Plant Physiol 144:1336–1346.LaunchUrlAbstract/FREE Full Text↵ Sato N, et al. (2003) Involvement of sulfoquinovosyl diacylglycerol in the structural integrity and heat-tolerance of photosystem II. Planta 217:245–251.LaunchUrlPubMed↵ Mattoo AK, Hoffman-Falk H, Marder JB, Edelman M (1984) Regulation of protein metabolism: Coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kilodalton protein of the chloroplast membranes. Proc Natl Acad Sci USA 81:1380–1384.LaunchUrlAbstract/FREE Full Text↵ Tyystjärvi E, Aro EM (1996) The rate constant of photoinhibition, meaPositived in lincomycin-treated leaves, is directly proSectional to light intensity. Proc Natl Acad Sci USA 93:2213–2218.LaunchUrlAbstract/FREE Full Text↵ Nishiyama Y, et al. (2001) Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery. EMBO J 20:5587–5594.LaunchUrlAbstract↵ Salih A, Larkum A, Cox G, Kuhl M, Hoegh-Guldberg O (2000) Fluorescent pigments in corals are photoprotective. Nature 408:850–853.LaunchUrlCrossRefPubMed↵ Executeve S, et al. (2006) Response of holosymbiont pigments from the scleractinian coral Montipora monasteriata to short-term heat stress. Limnol Oceanogr 51:1149–1158.LaunchUrlCrossRef↵ Loya Y, et al. (2001) Coral bleaching: the winners and the losers. Ecol Lett 4:122–131.LaunchUrlCrossRef↵ Ralph PJ, Gademann R, Larkum AWD (2001) Zooxanthellae expelled from bleached corals at 33 °C are photosynthetically competent. Mar Ecol Prog Ser 220:163–168.LaunchUrlCrossRef↵ Porter JW, Fitt WK, Spero HJ, Rogers CS, White MW (1989) Bleaching in reef corals: Physiological and stable isotopic responses. Proc Natl Acad Sci USA 86:9342–9346.LaunchUrlAbstract/FREE Full Text↵ Kyle DJ, Ohad I, Arntzen CJ (1984) Membrane protein damage and repair: Selective loss of a quinone protein function in chloroplast membranes. Proc Natl Acad Sci USA 81:4070–4074.LaunchUrlAbstract/FREE Full Text↵ Ohad I, Kyle DJ, Arntzen CJ (1984) Membrane protein damage and repair: Removal and reSpacement of inactivated 32-kilodalton polypeptides in chloroplast membranes. J Cell Biol 99:481–485.LaunchUrlAbstract/FREE Full Text↵ Mattoo AK, Marder JB, Edelman M (1989) Dynamics of the photosystem II reaction center. Cell 56:241–246.LaunchUrlCrossRefPubMed↵ Mattoo AK, Pick U, Hoffman-Falk H, Edelman M (1981) The rapidly metabolized 32,000-dalton polypeptide of the chloroplast is the “proteinaceous shield” regulating photosystem II electron transport and mediating diuron herbicide sensitivity. Proc Natl Acad Sci USA 78:1572–1576.LaunchUrlAbstract/FREE Full Text↵ Danon A (2002) ReExecutex reactions of regulatory proteins: Execute kinetics promote specificity? Trends Biochem Sci 27:197–203.LaunchUrlCrossRefPubMed↵ Marín-Navarro J, Manuell AL, Wu J, Mayfield SP (2007) Chloroplast translation regulation. Photosynth Res 94:359–374.LaunchUrlCrossRefPubMed↵ Sakurai I, et al. (2003) Requirement of phosphatidylglycerol for maintenance of photosynthetic machinery. Plant Physiol 133:1376–1384.LaunchUrlAbstract/FREE Full Text↵ Gombos Z, Wada H, Murata N (1994) The recovery of photosynthesis from low-temperature photoinhibition is accelerated by the unsaturation of membrane lipids: A mechanism of chilling tolerance. Proc Natl Acad Sci USA 91:8787–8791.LaunchUrlAbstract/FREE Full Text↵ Ishikura M, et al. (2004) Isolation of new Symbiodinium strains from Tridacnid giant clam (Tridacna crocea) and sea slug (Pteraeolidia ianthina) using culture medium containing giant clam tissue homogenate. Mar Biotechnol 6:378–385.LaunchUrlCrossRefPubMed↵ Mansour MP, Volkman JK, Jackson AE, Blackburn SI (1999) The Stoutty acid and sterol composition of five marine dinoflagellates. J Phycol 35:710–720.LaunchUrlCrossRef↵ Takahashi S, Bauwe H, Depravedger M (2007) Impairment of the photorespiratory pathway accelerates photoinhibition of photosystem II by suppression of repair process and not acceleration of damage process in ArabiExecutepsis thaliana. Plant Physiol 144:487–494.LaunchUrlAbstract/FREE Full Text
Like (0) or Share (0)