TY - JOUR
AU - Takahashi, Shunichi
AB - Abstract Dinoflagellates from the genus Symbiodinium form symbiotic relationships with many marine invertebrates, including reef-building corals. Symbiodinium is genetically diverse, and acquiring suitable Symbiodinium phylotypes is crucial for the host to survive in habitat environments, such as high-light conditions. The sensitivity of Symbiodinium to high light differs among Symbiodinium phylotypes, but the mechanism that controls light sensitivity has not yet been fully resolved. In the present study using high-light-tolerant and -sensitive Symbiodinium phylotypes, we examined what determines sensitivity to high light. In growth experiments under different light intensities, Symbiodinium CS-164 (clade B1) and CCMP2459 (clade B2) were identified as high-light-tolerant and -sensitive phylotypes, respectively. Measurements of the maximum quantum yield of photosystem II (PSII) and the maximum photosynthetic oxygen production rate after high-light exposure demonstrated that CCMP2459 is more sensitive to photoinhibition of PSII than CS-164, and tends to lose maximum photosynthetic activity faster. Measurement of photodamage to PSII under light of different wavelength ranges demonstrated that PSII in both Symbiodinium phylotypes was significantly more sensitive to photodamage under shorter wavelength regions of light spectra (<470 nm). Importantly, PSII in CCMP2459, but not CS-164, was also sensitive to photodamage under the regions of light spectra around 470–550 and 630–710 nm, where photosynthetic antenna proteins of Symbiodinium have light absorption peaks. This finding indicates that the high-light-sensitive CCMP2459 has an extra component of photodamage to PSII, resulting in higher sensitivity to high light. Our results demonstrate that sensitivity of PSII to photodamage differs among Symbiodinium phylotypes and this determines their sensitivity to high light. Introduction Reef-building corals and other marine invertebrates harbor endosymbiotic dinoflagellate algae from the genus Symbiodinium. In these symbiotic relationships, the algae provide fixed carbon in the form of photosynthate to their host to support metabolic energy requirements (Muscatine et al. 1984). In return, the symbiotic algae are given protection and receive inorganic compounds such as CO2 and nitrogen compounds from the host (Titlyanov and Titlyanova 2002, Yellowlees et al. 2008). Since the symbiotic algae within corals are the major primary producer in coral reef ecosystems, functional photosynthesis of the symbiont is important for corals and coral reef ecosystems (Hoegh-Guldberg et al. 2007). Therefore, environmental stress, such as strong light (UV and visible light) and increased seawater temperature, which impairs photosynthesis in the symbiotic algae within corals, can cause the destruction of the healthy algae–coral symbiotic relationship and result in coral bleaching (Lesser 2011). Ultimately, when conditions that cause coral bleaching persist, widespread mortality of corals and destruction of coral reef ecosystems occurs (Hoegh-Guldberg 1999). Photosynthetic organisms, including Symbiodinium, tend to lose their photosynthetic activity under conditions where the amount of absorbed light energy exceeds the capacity for light utilization by photosynthesis (Murata et al. 2007, Takahashi and Murata 2008), e.g. under strong light especially with other environmental stressors (such as heat or cold) that limits the photosynthetic CO2 fixation in the Calvin–Benson cycle. This phenomenon is referred to as photoinhibition of photosynthesis (Aro et al. 1993). Photoinhibition is attributed to the photodamage of photosystem II (PSII) and its accumulation. Therefore, a decrease in PSII activity or PSII efficiency owing to photodamage to PSII is also referred to as photoinhibition (here we call it photoinhibition of PSII). To avoid photoinhibition of PSII, the photodamaged PSII is efficiently and quickly repaired through the PSII repair cycle (Aro et al. 1993). Therefore, net photoinhibition of PSII occurs in conditions where the rate of photodamage to PSII exceeds the rate of its repair. In Symbiodinium within corals, strong light leads to severe photoinhibition of PSII, especially at increased seawater temperature (Warner et al. 1996, Warner et al. 1999). Since photoinhibition of PSII happens under excessive light conditions, light energy excessively absorbed by photosynthetic antenna pigments was assumed to cause photodamage to PSII (Melis 1999). However, recent studies using model photosynthetic organisms, such as higher plants, green algae and cyanobacteria, have demonstrated that photodamage to PSII is directly proportional to the intensity of light and is more associated with the light absorbed by manganese in the oxygen-evolving complex in PSII, rather than photosynthetic pigments (Hakala et al. 2005, Ohnishi et al. 2005, Takahashi et al. 2010). Furthermore, excessive absorbed light energy has been demonstrated to accelerate photoinhibition of PSII through inhibition of PSII repair but not acceleration of photodamage to PSII (Takahashi and Murata 2005, Takahashi and Murata 2006). Moreover, reactive oxygen species produced under excessive light conditions have been demonstrated to cause photoinhibition through inhibition of PSII repair but not acceleration of photodamage to PSII (Nishiyama et al. 2001, Nishiyama et al. 2004, Nishiyama et al. 2005, Nishiyama et al. 2006). However, it is still controversial whether this new model is common in all photosynthetic organisms; for example, some studies have shown that photodamage to PSII is attributed to light energy absorbed by photosynthetic pigments and its excess in plants (Oguchi et al. 2011) and Symbiodinium within corals (Hill et al. 2011). Symbiodinium phylotypes are diverse and currently grouped into nine clades from A to I (each clade includes multiple phylotypes) (Pochon and Gates 2010). Sensitivity of PSII to photoinhibition differs among Symbiodinium phylotypes in culture and in hospite within corals under stressful conditions such as strong light and heat stress (Berkelmans and Oppen 2006, Fisher et al. 2012, Krämer et al. 2012). Thus, harboring suitable Symbiodinium phylotypes is important for growth and survival of host corals (LaJeunesse et al. 2009, Kemp et al. 2014). The sensitivity of PSII to photoinhibition under thermal stress has been intensively studied and clarified in Symbiodinium due to its association with coral bleaching. Thermal stress-associated photoinhibition of PSII is primarily due to the inhibition of the PSII repair cycle, and the sensitivity of a Symbiodinium phylotype to PSII photoinhibition under heat stress is determined by the thermal sensitivity of the PSII repair process (Takahashi et al. 2004, Takahashi et al. 2009b). However, the mechanism associated with sensitivity to high light in Symbiodinium phylotypes is not yet fully understood. Sensitivity of Symbiodinium to photoinhibition of PSII under high light differs among phylotypes. Since the extent of photoinhibition of PSII is determined by the balance between the photodamage rate and its repair rate, the different photoinhibition sensitivities among Symbiodinium phylotypes can be due to the difference(s) in the photodamage rate and/or the PSII repair rate under high light. In this study, we focused on the process of photodamage to PSII and examined the effect of high light using high-light-sensitive and -tolerant Symbiodinium phylotypes identified by their growth capability under high light. Our results demonstrate that sensitivity of PSII to photodamage differs among Symbiodinium phylotypes and it determines their photoinhibition sensitivity and growth capability under high light. Furthermore, our results demonstrate that the high-light-sensitive Symbiodinium phylotype has an extra component of photodamage to PSII, resulting in higher sensitivity to high light. These findings provide a new insight into what determines high-light sensitivity in Symbiodinium. Results Light-dependent growth of Symbiodinium phylotypes To examine the effect of high light on growth, two different cultured Symbiodinium phylotypes, CCMP2459 (clade B2) and CS-164 (clade B1), were grown at 80, 300 and 600 µmol photons m−2 s−1 with a light/dark cycle of 16 h/8 h at 23°C. The growth was monitored by measuring the cell concentration with an automatic cell counter every 3 d for 15 d. After 15 d, all CS-164 cultures showed the same brownish coloration, and no difference was found in the culture coloration between the light conditions (Fig. 1A). However, in CCMP2459, the culture coloration differed greatly among light treatments, becoming paler under higher light intensities (Fig. 1A). In CS-164, the cell concentration increased exponentially and there was no difference in the growth rate among the growth light intensities tested (Fig. 1B). In contrast, in CCMP2459, the increase in cell concentration was light dependent, with the growth rate significantly suppressed under higher light intensities (Fig. 1B). Our results demonstrate that growth of Symbiodinium under strong light differs among Symbiodinium phylotypes, with CCMP2459 showing a greater high-light-induced reduction in growth compared with CS-164. Fig. 1 View largeDownload slide Effect of light intensity on the growth of Symbiodinium CS-164 and CCMP2459. Symbiodinium cells (initial cell concentration of 5 × 105 cells ml−1, 5 ml) were grown at 80, 300 and 600 µmol photons m−2 s−1 with a light/dark cycle of 16 h/8 h at 23°C for 15 d. The growth medium was changed once after 6 d. (A) Photographs of cell cultures grown under different light intensities on Day 15. (B) Cell density under different light intensities in CS-164 (upper panel) and CCMP2459 (lower panel). The values are the means ± SD (bars) of results from three independent experiments. The doubling time at 80, 300 and 600 µmol photons m−2 s−1 was 2.6 ± 0.1, 2.8 ± 0.2 and 2.7 ± 0.2 d, respectively, in Symbiodinium C-164, and 2.7 ± 0.2, 3.2 ± 0.1 and 4.6 ± 0.8 d, respectively, in Symbiodinium CCMP2459. Fig. 1 View largeDownload slide Effect of light intensity on the growth of Symbiodinium CS-164 and CCMP2459. Symbiodinium cells (initial cell concentration of 5 × 105 cells ml−1, 5 ml) were grown at 80, 300 and 600 µmol photons m−2 s−1 with a light/dark cycle of 16 h/8 h at 23°C for 15 d. The growth medium was changed once after 6 d. (A) Photographs of cell cultures grown under different light intensities on Day 15. (B) Cell density under different light intensities in CS-164 (upper panel) and CCMP2459 (lower panel). The values are the means ± SD (bars) of results from three independent experiments. The doubling time at 80, 300 and 600 µmol photons m−2 s−1 was 2.6 ± 0.1, 2.8 ± 0.2 and 2.7 ± 0.2 d, respectively, in Symbiodinium C-164, and 2.7 ± 0.2, 3.2 ± 0.1 and 4.6 ± 0.8 d, respectively, in Symbiodinium CCMP2459. Sensitivity of the photosynthetic machinery to high light differs between Symbiodinium phylotypes Measurements of the photosynthetic O2 production rate under different light intensities in seven different Symbiodinium cultures demonstrated that photosynthetic activity and its light response differ among Symbiodinium phylotypes (Supplementary Fig. S1). However, between CCMP2459 and CS-164, there was no significant difference in the light response curve of the photosynthetic O2 production rate; the photosynthetic activity saturated at 200 µmol photons m−2 s−1 and the maximum photosynthetic rate was around 0.23 pmol O2 cell−1 h−1 in both phylotypes measured with short-term illumination (Fig. 2). Furthermore, there was no difference in the components of light-harvesting antenna proteins; both phylotypes had large peridinin–Chl proteins (Supplementary Fig. S2). Thus, CCMP2459 and CS-164 have an equivalent ability for photosynthesis at 23°C at each light intensity in the range 0–800 µmol photons m−2 s−1. Fig. 2 View largeDownload slide Light response curves of photosynthetic O2 production rate in Symbiodinium CS-164 and CCMP2459. Symbiodinium cells (5 × 105 cells ml−1, 1 ml) were placed in a closed cuvette and the photosynthetic O2 production rate was measured in the presence of 500 µM NaHCO3 at 23°C under different light intensities. Fresh samples were used for each measurement under different light intensities. The values are the mean ± SD (bars) from four replicates. Fig. 2 View largeDownload slide Light response curves of photosynthetic O2 production rate in Symbiodinium CS-164 and CCMP2459. Symbiodinium cells (5 × 105 cells ml−1, 1 ml) were placed in a closed cuvette and the photosynthetic O2 production rate was measured in the presence of 500 µM NaHCO3 at 23°C under different light intensities. Fresh samples were used for each measurement under different light intensities. The values are the mean ± SD (bars) from four replicates. To examine the sensitivity of the photosynthetic machinery to strong light, the maximum quantum yield of PSII (Fv/Fm) and the photosynthetic O2 production rate were measured after incubation at different light intensities ranging from 200 to 800 µmol photons m−2 s−1 or darkness (control) for 30 min. In both CS-164 and CCMP2459, Fv/Fm declined after light exposure and the extent of decline was correlated with light intensity, becoming more severe at higher light intensities (Fig. 3A). Importantly, the decline of Fv/Fm was more apparent in CCMP2459 than in CS-164 at any light intensity tested, e.g. Fv/Fm dropped to 90% and 40% of the control after the light exposure at 200 µmol photons m−2 s−1 in CS-164 and CCMP2459, respectively. The maximum photosynthetic O2 production rate also declined in both Symbiodinium phylotypes after the strong light exposure, but more severely in CCMP2459 than in CS-164 (Fig. 3B). However, the decline of the maximum photosynthetic O2 production rate was milder than the decline of Fv/Fm in both CCMP2459 and CS-164 (Fig. 3C). It seems likely that the decline in the maximum photosynthetic O2 production rate follows a significant decline of Fv/Fm (Fig. 3C). Our results demonstrate that CCMP2459 is more sensitive to photoinhibition of PSII than CS-164 under high-light conditions and that photoinhibition of PSII causes a decline of the maximum photosynthetic O2 production rate, namely photoinhibition of photosynthesis, if the photoinhibition of PSII was severe. Fig. 3 View largeDownload slide Photoinhibition of PSII and decline of the maximum photosynthetic O2 production rate after strong light exposure in CS-164 and CCMP2459. Symbiodinium cells (5 × 105 cells ml−1, 1 ml) were exposed to different intensities of light ranging from 200 to 1,000 µmol photons m−2 s−1 or darkness (control) for 30 min. (A) The maximum quantum yield of PSII (Fv/Fm) was measured after incubation in darkness for 10 min. Initial Fv/Fm was 0.59 ± 0.01 in CS-164 and 0.58 ± 0.01 in CCMP2459. (B) The maximum photosynthetic O2 production rate was measured in the light at 600 µmol photons m−2 s−1. The initial rate was 2.9 ± 0.16 pmol O2 cell−1 h−1 in CS-164 and 2.5 pmol O2 cell−1 h−1 in CCMP2459. (C) The correlation between the Fv/Fm and the maximum photosynthetic O2 production rate after exposure to light or darkness. The values are the mean ± SD (bars) from four replicates. Fig. 3 View largeDownload slide Photoinhibition of PSII and decline of the maximum photosynthetic O2 production rate after strong light exposure in CS-164 and CCMP2459. Symbiodinium cells (5 × 105 cells ml−1, 1 ml) were exposed to different intensities of light ranging from 200 to 1,000 µmol photons m−2 s−1 or darkness (control) for 30 min. (A) The maximum quantum yield of PSII (Fv/Fm) was measured after incubation in darkness for 10 min. Initial Fv/Fm was 0.59 ± 0.01 in CS-164 and 0.58 ± 0.01 in CCMP2459. (B) The maximum photosynthetic O2 production rate was measured in the light at 600 µmol photons m−2 s−1. The initial rate was 2.9 ± 0.16 pmol O2 cell−1 h−1 in CS-164 and 2.5 pmol O2 cell−1 h−1 in CCMP2459. (C) The correlation between the Fv/Fm and the maximum photosynthetic O2 production rate after exposure to light or darkness. The values are the mean ± SD (bars) from four replicates. Sensitivity of PSII to photodamage differs among Symbiodinium phylotypes under high light The net extent of photoinhibition of PSII is the result of a balance between photodamage to PSII and its repair. To monitor the process of photodamage, the repair needs to be inhibited. To compare the sensitivity of PSII to the photodamage per se between CS-164 and CCMP2459, we monitored the decline of Fv/Fm in the presence of chloramphenicol (an inhibitor of PSII repair) under 200 µmol photons m−2 s−1 for 3 h. In the absence of chloramphenicol, the decline of Fv/Fm was much faster in CCMP2459 than in CS-164, and Fv/Fm in CS-164 and CCMP2459 declined to 76% and 21%, respectively, of the initial value after 3 h (Fig. 4A). In the presence of chloramphenicol, compared with the absence of the inhibitor, the decline in Fv/Fm was enhanced in both Symbiodinium phylotypes, especially in CS-164. However, even in the presence of chloramphenicol, the decline in Fv/Fm was clearly faster in CCMP2459 than in CS-164, and the Fv/Fm declined to 30% and 0% of the initial value after 3 h in CS-164 and CCMP2459, respectively. These results demonstrate that the sensitivity of PSII to photodamage is higher in CCMP2459 than in CS-164 under the high-light condition tested. Fig. 4 View largeDownload slide Photodamage to PSII and its repair in Symbiodinium CS-164 and CCMP2459. (A) Symbiodinium cells were exposed to light at 200 µmol photons m−2 s−1 in the presence of 1 mM chloramphenicol or in its absence for 3 h. (B) The recovery of Fv/Fm was monitored under 20 µmol photons m−2 s−1 for 3 h after the photoinhibition treatment of 1,000 µmol photons m−2 s−1 for 60 min in CS-164 and at 500 µmol photons m−2 s−1 for 15 min in CCMP2459. The initial Fv/Fm was 0.53 ± 0.010 and 0.51 ± 0.001 in CS-164 and CCMP2459, respectively. The values are the mean ± SD (bars) from four replicates. Fig. 4 View largeDownload slide Photodamage to PSII and its repair in Symbiodinium CS-164 and CCMP2459. (A) Symbiodinium cells were exposed to light at 200 µmol photons m−2 s−1 in the presence of 1 mM chloramphenicol or in its absence for 3 h. (B) The recovery of Fv/Fm was monitored under 20 µmol photons m−2 s−1 for 3 h after the photoinhibition treatment of 1,000 µmol photons m−2 s−1 for 60 min in CS-164 and at 500 µmol photons m−2 s−1 for 15 min in CCMP2459. The initial Fv/Fm was 0.53 ± 0.010 and 0.51 ± 0.001 in CS-164 and CCMP2459, respectively. The values are the mean ± SD (bars) from four replicates. To examine the efficiency of PSII repair, the recovery of Fv/Fm after the photoinhibition treatment was monitored in the absence of chloramphenicol under low light at 20 µmol photons m−2 s−1 in both CS-164 and CCMP2459. The photoinhibition treatment was carried out under 1,000 µmol photons m−2 s−1 (for 1 h) and 500 µmol photons m−2 s−1 (for 15 min) in CS-164 and CCMP2459, respectively (Fig. 4B; Supplementary Fig. S3). After the photoinhibition treatments, Fv/Fm declined to around 20% of the initial level in both phylotypes (Fig. 4B). Under the low-intensity light during recovery, Fv/Fm quickly recovered to around 80% of initial levels in 30 min and reached 90% of its initial level after 3 h in both CS-164 and CCMP2459, with slightly faster recovery in CCMP2459 than in CS-164. Our results demonstrate that the efficiency of the PSII repair process is similar between the two phylotypes. Sensitivity of PSII to photodamage differs among Symbiodinium phylotypes especially in specific regions of the light spectrum The sensitivity of PSII to photodamage was higher in CCMP2459 than in CS-164 (Fig. 4A). To examine which regions of the light spectrum damage PSII more severely in CCMP2459 compared with CS-164, we monitored the decline of Fv/Fm in the presence of chloramphenicol under eight different spectra of light at 150 µmol photons m−2 s−1 (Fig. 5A). We used a xenon lamp with combinations of long-pass and short-pass filters to produce the different spectra (colors) of light: UV/purple, blue, cyan, green, yellow, orange, orange-red and red (see spectra in Supplementary Fig. S4). In CS-164, Fv/Fm declined most quickly under the UV/purple light region (the shortest wavelength region of light used; Fig. 5A). The decline of Fv/Fm was slower under longer wavelength light, and little or no significant difference was found in the decline of Fv/Fm among orange, orange-red and red. The decline of Fv/Fm was faster in CCMP2459 than in CS-164 under light of any selected wavelength range, but the difference between phylotypes was most apparent under cyan, green, orange-red and red. Interestingly, the decline of Fv/Fm was significantly faster under orange-red and red than under orange in CCMP2459 (Fig. 5A). Our results demonstrate that specific wavelength regions of light (cyan, green orange-red and red) damage PSII more effectively in CCMP2459 than in CS-164. The spectra of light absorbance in intact cells demonstrated that both Symbiodinium phylotypes have light absorption peaks at blue (at around 450 nm) and orange-red (at around 675 nm) regions and their spectra were comparable (Fig. 5B). This result indicates that sensitivity of PSII to photodamage is not completely dependent on the light absorbance spectrum from 350 to 800 nm. Fig. 5 View largeDownload slide Photodamage to PSII and light absorbance under different spectra of light in Symbiodinium CS-164 and CCMP2459. (A) In the presence of 1 mM chloramphenicol, Symbiodinium cells were exposed to different spectra of light. The different wavelength regions of light were produced by combinations of long-pass (LP) and short-pass (SP) filters: UV/purple (LP385 and SP425), blue (LP422 and SP470), cyan (LP470 and SP510), green (LP510 and SP550), yellow (LP550 and SP590), orange (LP590 and SP630), orange-red (LP630 and SP670) and red (LP670 and SP710), at 150 µmol photons m−2 s−1. The values are the mean ± SD (bars) from four replicates. (B) Light absorption spectra of intact cells. The spectra were normalized by the peak at 675 nm. Results are an average of three separate measurements. Fig. 5 View largeDownload slide Photodamage to PSII and light absorbance under different spectra of light in Symbiodinium CS-164 and CCMP2459. (A) In the presence of 1 mM chloramphenicol, Symbiodinium cells were exposed to different spectra of light. The different wavelength regions of light were produced by combinations of long-pass (LP) and short-pass (SP) filters: UV/purple (LP385 and SP425), blue (LP422 and SP470), cyan (LP470 and SP510), green (LP510 and SP550), yellow (LP550 and SP590), orange (LP590 and SP630), orange-red (LP630 and SP670) and red (LP670 and SP710), at 150 µmol photons m−2 s−1. The values are the mean ± SD (bars) from four replicates. (B) Light absorption spectra of intact cells. The spectra were normalized by the peak at 675 nm. Results are an average of three separate measurements. Imbalance of light energy absorption and consumption in high light To monitor the state of photosynthetic electron flow at PSII, we measured the effective quantum yield of PSII (ΦPSII), non-photochemical quenching (NPQ) and the redox state of QA (1 – qL) under different light intensities (Fig. 6). ΦPSII represents PSII photochemical efficiency during light exposure and was found to be slightly higher in CS-164 than in CCMP2459 under higher light intensities, although the difference was not significant. NPQ, which is generally enhanced under excessive light conditions for photosynthesis, was found to be higher in CCMP2459 than in CS-164 under high light intensities. The parameter that represents the reduction state of the QA electron acceptor of PSII (1 – qL) showed that QA was more reduced in CCMP2459 than in CS-164 under high light intensities. These results demonstrate that light energy absorbed by antenna pigments was more excessive in CCMP2459 than in CS-164 under high-light conditions. Fig. 6 View largeDownload slide Effect of strong light on ΦPSII, NPQ and 1 – qL in Symbiodinium CS-164 and CCMP2459. Symbiodinium cells (1 × 106 cells ml−1, 0.3 ml) were held at 23°C and incubated in darkness for 10 min and then exposed to increasing light intensities. Each irradiance was applied for 5 min. Parameters for ΦPSII, NPQ and 1 – qL were taken at the end of light exposure for each light intensity. The values are the mean ± SD (bars) from four replicates. Fig. 6 View largeDownload slide Effect of strong light on ΦPSII, NPQ and 1 – qL in Symbiodinium CS-164 and CCMP2459. Symbiodinium cells (1 × 106 cells ml−1, 0.3 ml) were held at 23°C and incubated in darkness for 10 min and then exposed to increasing light intensities. Each irradiance was applied for 5 min. Parameters for ΦPSII, NPQ and 1 – qL were taken at the end of light exposure for each light intensity. The values are the mean ± SD (bars) from four replicates. Discussion The sensitivity of PSII to high light differs among Symbiodinium phylotypes and limits growth under high light conditions Our results demonstrated that high light severely suppresses the growth of CCMP2459 but not CS-164 (Fig. 1). This result indicates that strong light sensitivity differs among the two clade B Symbiodinium phylotypes and limits the growth under high-light conditions. CCMP2459 was found to be more sensitive to photoinhibition of PSII compared with CS-164 under high-light conditions, and experienced a decline in the maximum photosynthetic activity, due to photoinhibition of photosynthesis (Fig. 3). In model organisms using high-light-sensitive mutants, severe photoinhibition of PSII has been demonstrated to cause a decrease in growth under stressful conditions (Niyogi et al. 1997, Sakata et al. 2013). It is therefore conceivable that the suppression of the growth under strong light conditions in CCMP2459 was due to its higher sensitivity of PSII to photoinhibition. In natural conditions, the intensity of light that reaches symbiotic Symbiodinium within corals is dependent on the structural framework of the host coral (Enríquez et al. 2005) and differs across the coral surface, depending on the time of day, tides, weather and season (Lesser et al. 2000, Ulstrup and Van Oppen 2003). For corals growing under conditions of high-light exposure, harboring a high-light-tolerant Symbiodinium phylotype might be important for their growth and survival (Cantin et al. 2009, Cooper et al. 2011, Krämer et al. 2012). Monitoring of the decline of Fv/Fm in the presence of chloramphenicol demonstrated that the rate of photodamage to PSII under high light was faster in CCMP2459 than in CS-164 (Fig. 4A). This result indicates that higher sensitivity of PSII to photoinhibition in CCMP2459 is at least partially due to the higher sensitivity of PSII to photodamage per se. Since the capacity for repair of photodamaged PSII under low light was similar or rather higher in CCMP2459 than in CS-164 (Fig. 4B), the higher sensitivity of CCMP2459 to photoinhibition under high light, in the absence of chloramphenicol, seems not to be due to the difference in their potential PSII repair ability (Hill et al. 2011). However, we need to note that it does not reflect the ability for PSII repair under strong light conditions. Under high-light conditions, the effect of chloramphenicol that inhibits the PSII repair was larger in CS-164 than in CCMP2459 (Fig. 4A), indicating that the PSII repair was less efficient in CCMP2459 than in CS-164 under high light. Therefore, the higher sensitivity of CCMP2459 to photoinhibition under high light might also be at least partially associated with a less efficient PSII repair process. Consistent with this result, in two different clade A Symbiodinium phylotypes, the sensitivity of PSII to photoinhibition has been shown to differ under high light due to the different ability for PSII repair (Ragni et al. 2010). It is conceivable, therefore, that excessive absorbed light energy suppresses PSII repair in high-light-sensitive Symbiodinium phylotypes, as happens in model photosynthetic organisms (Takahashi and Murata 2005, Takahashi and Murata 2006, Takahashi et al. 2007, Takahashi and Murata 2008). This hypothesis is consistent with our results showing that light energy absorbed by photosynthetic antenna pigments is more excessive (i.e. more reduced QA) in high-light-sensitive CCMP2459 than in CS-164 under high-light conditions (Fig. 6) The higher sensitivity of PSII to photodamage in CCMP2459 than in CS-164 was apparent under specific regions of light spectra, such as cyan (470–510 nm), green (510–550 nm), orange-red (630–670 nm) and red (670–710 nm) lights (Fig. 5). Since light-harvesting antenna complexes in Symbiodinium have light absorption peaks in the cyan–green and red light regions (Hennige et al. 2009), the higher sensitivity of PSII to photodamage in CCMP2459 than in CS-164 might be related to the light absorbed by photosynthetic pigments in antenna complexes. It is important to note that light absorption peaks of light-harvesting antenna complexes (Hennige et al. 2009) are slightly different from those of intact cells (Fig. 5B). Recent studies using model photosynthetic organisms have shown that photodamage to PSII is more related to light absorbed by manganese in the oxygen-evolving complex of PSII but not photosynthetic pigments in antenna complexes (Hakala et al. 2005, Ohnishi et al. 2005, Takahashi et al. 2010). This model is referred to as the manganese (or two-step) model and is supported by results showing that model manganese compounds have a strong absorption in the UV and short wavelength region of visible light (e.g. purple and blue) and it fits with the action spectrum of photodamage to PSII (Hakala et al. 2005, Ohnishi et al. 2005). In CS-164, the extent of photodamage to PSII was higher at shorter wavelength regions of visible light (Fig. 5) and it was consistent with the manganese photodamage model. In contrast, in CCMP2459, the extent of photodamage was significant in shorter wavelength regions (UV/purple, blue, cyan and green) and also in longer wavelength regions (orange-red and red) of visible light. It is therefore likely that photodamage to PSII in CCMP2459 includes two components; one is photodamage to PSII caused by light energy absorbed by manganese, as also happens in CS-164, and the other is photodamage by light energy absorbed by photosynthetic pigments in antenna complexes. Under high light conditions, NPQ and 1 – qL were higher in CCMP2459 than in CS-164, indicating that light energy absorbed by photosynthetic antenna pigments in CCMP2459 tended to be excessive under high-light conditions (Fig. 6). The higher photodamage to PSII in CCMP2459 under wavelength regions where photosynthetic pigments have absorption peaks (Fig. 5) might therefore be related to excess light absorbed by antenna pigments. Previously, it has been assumed that excessive light energy causes photodamage to PSII and that photoprotection mechanisms associated with dissipating it, such as thermal energy dissipation, reactive oxygen-scavenging mechanisms and the photorespiratory pathway, alleviate photoinhibition through suppressing photodamage to PSII (Melis 1999). However, recent studies using model photosynthetic organisms have demonstrated that excessive light energy causes photoinhibition of PSII though inhibition of PSII repair but not acceleration of photodamage to PSII (Takahashi and Murata 2005, Takahashi and Murata 2006, Takahashi and Murata 2008). Furthermore, photoprotection mechanisms associated with dissipating the excess energy have been demonstrated to alleviate photoinhibition through suppressing inhibition of PSII repair, but not through suppressing the photodamage to PSII, under excessive light conditions (Nishiyama et al. 2001, Nishiyama et al. 2004, Nishiyama et al. 2005, Nishiyama et al.2006, Takahashi et al. 2007, Takahashi et al. 2009a, Takahashi and Badger 2011). Therefore, even in model photosynthetic organisms, mechanisms associated with photodamage to PSII caused by excess light absorbed by photosynthetic antenna pigments and its protection remain uncertain. Further research is required to understand the mechanism that changes the sensitivity of PSII to photodamage in Symbiodinium phylotypes. Only severe photoinhibition of PSII decreases the maximum photosynthetic activity in Symbiodinium Photodamage to PSII can cause photoinhibition of PSII and subsequent decline of the maximum photosynthetic activity in any photosynthetic organism, including Symbiodinium cells. In the present study, we examined the relationship between the extent of photoinhibition of PSII and the decline of the maximum photosynthetic activity in Symbiodinium (Fig. 3). Our results demonstrated that the decline of the maximum photosynthetic activity happens only after severe photoinhibition of PSII. It is therefore likely that PSII cannot be a rate-limiting step of photosynthesis in Symbiodinium under light-saturating conditions until severe photoinhibition of PSII happens. Indeed, in higher plants, the limiting step is located downstream of PSII under saturated light conditions, e.g. at the Cyt bf complex (Heber et al. 1988) and the Calvin cycle (Miyagawa et al. 2001). Similarly, in capsicum leaves, up to 40% of PSII can be inactivated before the photosynthetic electron transport begins to be limited by PSII (Lee et al. 1999). In Symbiodinium within corals, environmental stress such as moderate heat stress and/or strong light (UV and visible light) has been demonstrated to cause photoinhibition of PSII (Lesser 2011). Our results suggest that photoinhibition of PSII does not always cause a decline in the maximum photosynthetic activity under such conditions. However, we need to note that photoinhibition of PSII can lower the quantum efficiency of photosynthesis through light absorption by inactive PSII under light-limiting conditions. Photoinhibition and coral bleaching Increased seawater temperature is the major factor that causes mass coral bleaching events worldwide (Hoegh-Guldberg et al. 2007). Coral bleaching is associated with loss of symbiotic algae through expulsion from host cells or degradation (or digestion) of Symbiodinium cells within host cells, and also with loss of algal pigments in in hospite Symbiodinium within corals (Gates et al. 1992, Brown 1997, Fitt et al. 2001). Since coral bleaching often follows severe photoinhibition of PSII, photoinhibition of PSII has been assumed to cause coral bleaching. Recent studies have demonstrated that photoinhibition of PSII is associated with coral bleaching caused by loss of algal pigments (photobleaching of algal pigments), but not by the expulsion of algae (Takahashi et al. 2004, Takahashi et al. 2008, Takahashi et al. 2013). We need to note that the expulsion of algae (Belda-Baillie et al. 2002, Tolleter et al. 2013) and the loss of algal pigments (Takahashi et al. 2004, Takahashi et al. 2008, Takahashi et al. 2013) are light independent and dependent, respectively. Since impairment of photosynthesis causes mortality of algae, it is conceivable that severe photoinhibition of PSII is also related to the loss of symbiotic algae through the degradation of Symbiodinium within host corals. Indeed, an inhibitor of the photosynthetic electron transfer at PSII, 3 -(3,4-dichlorophenyl)-1,1-dimethylurea (Diuron), causes degradation of Symbiodinium within corals (Jones 2005). Previously, sensitivity of PSII to photoinhibition in Symbiodinium within corals has been intensively studied under heat stress conditions and it was assumed that the sensitivity of PSII to photoinhibition was determined by the sensitivity of Symbiodinium to heat stress (Lesser 2011). However, our present study has demonstrated that the light sensitivity of PSII differs among Symbiodinium phylotypes, suggesting that the symbiont genotype influences the sensitivity of PSII to photoinhibition. Thus, under conditions that cause coral bleaching, sensitivity of Symbiodinium to photoinhibition is determined by the sensitivity of PSII to heat and high light. Corals that are tolerant to coral bleaching caused by loss of algal pigments might therefore harbor a Symbiodinium phylotype that is tolerant to heat and light (LaJeunesse et al. 2009, Kemp et al. 2014). To understand the sensitivity of corals to bleaching, we need to focus further on the sensitivity of in hospite Symbiodinium within corals to high light as well as heat stress. Materials and Methods Cultures and growth conditions Cultures of Symbiodinium spp., CCMP2457, CCMP2459, CCMP2464, CCMP2467, CCMP2469 and CCMP830, were obtained from the National Center for Marine Algae and Microbiota (USA). CS-164 was obtained from the CSIRO Australian National Algae Culture Collection (Australia). Mf1.05b was a gift from Dr. Mary Alice Coffroth (University of Buffalo, NY, USA). To ensure that cultures were monoclonal, all Symbiodinium phylotypes were subcultured from a single cell isolated by a cell sorter (BD FACS Aria II, BD Biosciences). Symbiodinium cells were grown in artificial seawater (sea salt; Sigma-Aldrich) containing Daigo’s IMK medium for marine microalgae (Wako) after the seawater was filtered with a 0.22 µm pore filter (Steritop-GP Filter Unit, Merck Millipore). The pH of the fresh seawater for the culture was 7.8. Symbiodinium cells were grown at 23°C with photosynthetically active radiation (PAR) at 80 µmol photons m−2 s−1 with 16 h light (white color, fluorescence tubes) each day. Symbiodinium cells that had reached the mid-logarithmic phase of growth were harvested by centrifugation at 2,000 × g for 3 min and resuspended in fresh growth seawater. Measurement of photosynthetic O2 production rate The photosynthetic O2 production rate was measured in the presence of 500 µM NaHCO3 (CO2 supply) in a closed cuvette equipped with the oxygen sensor probe Pst3 using the fiber optic oxygen transmitter (Oxy-4 mini system; PreSens). The cuvette was maintained at 23°C using a cuvette holder that was connected to a temperature controller (Thermomix 1442D/Frigomix 1495; B.Braun), where temperature was measured by a thermometer sensor probe (HI 93530/HI762BL; Hanna). LED white light (see spectrum in Supplementary Fig. S4A) was used to illuminate samples to measure the photosynthetic oxygen production rate. Light intensity was measured with ULM-500 with a mini quantum sensor LS-C (Walz). Before light exposure, 1 ml of Symbiodinium cells (106 cells ml−1) was incubated in darkness for 5 min to stabilize the signal. Cells were then exposed to light for 5 min followed by incubation in darkness for 5 min. The photosynthetic oxygen production rate was calculated as the sum of the oxygen production rate in the light and the oxygen consumption rate in the dark after light exposure. Light treatments Symbiodinium cells (1 ml) were placed in a 24-well microplate in the presence or absence of 1 mM chloramphenicol and exposed to the light from a xenon lamp (MAX-303; Asahi Spectra) with a visible light mirror module (385–740 nm) and a directly attachable collimator lens (MDRLQ1B, Asahi Spectra) (see spectrum in Supplementary Fig. S4A). Chloramphenicol was added to samples 30 min before light exposure. The temperature was maintained at 23°C by placing the micro plate on an aluminum block which was temperature controlled (Thermomix 1442D/Frigomix 1495; B.Braun), and samples were illuminated in the temperature-controlled incubator. The different wavelength ranges of light were selected by combinations of long-pass and short-pass filters (Asahi Spectra) (see spectra in Supplementary Fig. S4B). The intensity of white light was measured using the ULM-500 with the mini quantum sensor LS-C (Walz). The intensity of different wavelength regions of light was measured with a spectroradiometer (PS-200; Apogee). Chl fluorescence measurements Chl fluorescence parameters, such as Fv/Fm, ΦPSII, NPQ and 1 – qL, were all measured with a pulse-amplitude-modulated chlorophyll fluorometer (PAM-2500; Walz). The maximum quantum yield of PSII, Fv/Fm = (Fm – Fo)/Fm, was measured after incubation in darkness for 10 min. To measure ΦPSII, NPQ and 1 – qL, Symbiodinium cells were placed in suspension in a cuvette (KS-2500; Walz) with a stirrer (MKS-2500; Walz) and held at 23°C in a circulating water bath during experiments. These parameters were measured after exposure to different intensities of actinic light for 5 min. A PAM-2500 with a red LED (maximum emission at 630 nm) was used for measuring light, actinic light and saturation pulses. The switch for auto low (200 Hz) and high (20,000 Hz) measuring fluorescent frequency was on for the measurement of parameters. The light intensity of the saturation pulse reached up to 5,000 µmol photons m−2 s−1. ΦPSII was defined as (F′m – Fs)/F′m. NPQ and 1 – qL were calculated as (Fm – F′m)/F′m and 1– (F′o/Fs)(F′m – Fs)/(F′m – F′o), respectively. Fm is the maximum fluorescence in the dark-adapted state; F′m is the maximum fluorescence in any light-adapted state; Fs is the steady-state fluorescence in the light; Fo is the minimal fluorescence in the dark-adapted state; and F′o is the minimal fluorescence in any light-adapted state. Measurement of cell concentration during growth experiments Symbiodinium cells (5 × 105 cells ml−1, 5 ml) were placed in a 12 ml culture tube and grown under light at 80, 300 and 600 µmol photons m−2 s−1 at 23°C. Cell number was measured with an automatic cell counter TC-20 (Bio-Rad). After 6 d, 1 ml of each sample was diluted with 4 ml of fresh growth medium. The cell number counted after 6 d was therefore multiplied by five. Measurement of light absorption spectrum in intact Symbiodinium cells Symbiodinium cells (5 × 105 cells ml−1, 5 ml) were placed in a 1 ml quartz cuvette and the light absorption spectrum was measured using a spectrophotometer (V-650; Jasco) with an integrating sphere (ISV-722; Jasco). The spectra were taken in the range 350–800 nm. The fresh seawater for the culture was used for the baseline. In collected spectra, the baseline was set to zero at 800 nm. Then, the spectra were normalized at 675 nm. SDS–gel analysis of total cellular proteins Symbiodinium cells (10 µg of Chl in 1 ml) were collected by centrifugation (16,000 × g) for 3 min. Cells in a 2 ml tube were dipped in liquid nitrogen and incubated for 5 min. Then, cells were disrupted using a 5 mm stainless steel bead with a TissueLyser II (Qiagen). The operation was carried out for 2 min at 15 Hz with a −80°C cold TissueLyser Adapter (Qiagen). The disrupted cells were suspended in 1 ml of ice-cold 100% methanol and incubated at −80°C for 1 h. The pellet (protein sample) was then collected by centrifugation (16,000 × g) at 3°C for 15 min. The cellular protein was solubilized by LDS sample buffer (Invitrogen) containing NuPAGE-reducing agent (Invitrogen) and incubated at 70°C for 5 min. Cell debris was removed by centrifugation (16,000 × g) for 5 min, and the supernatant (10 µl) was separated by SDS–PAGE (NuPAGE Novex 4–12% Bis–Tris gel; Invitrogen) in MES-SDS running buffer (Invitrogen). Marker 12 (Invitrogen) was used as a protein standard. The gel was fixed by incubation in a 50% methanol and 7% acetic acid solution for 15 min and washed with distilled water. Proteins on the gel were visualized by Coomassie blue staining (GelCode blue stain reagent; Pierce). 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TI - Novel Characteristics of Photodamage to PSII in a High-Light-Sensitive Symbiodinium Phylotype
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcv040
DA - 2015-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/novel-characteristics-of-photodamage-to-psii-in-a-high-light-sensitive-fINUAM7B7E
SP - 1162
EP - 1171
VL - 56
IS - 6
DP - DeepDyve
ER -