Photoacclimation Strategy in Photosystem II of Prymnesiophyceae Isochrysis galbana

Photoacclimation Strategy in Photosystem II of Prymnesiophyceae Isochrysis galbana プリムネシウム藻綱 Isochrysis galbana の光化学系 II における光適応戦略 6D551 小幡光子 指導教員山本修一 SYNOPSIS 海洋に生息する藻類は 海水の鉛直混合や昼夜により 弱光から強光までの様々な光強度 (Photon flux density; PFD) にさらされる 藻類にとって 弱光は光合成の制限要因となり 強光は光合成の阻害要因となるため 藻類は 光合成とともに光合成阻害の回避 ( 光保護 ) を行う 藻類は さらされる PFD に対して光合成と光保護のバランスが最適となるように適応すると考えられていることから 藻類の光適応戦略は 供給される光エネルギーと藻類が要求する光エネルギーのバランスに関係があることが予想される 光エネルギーの供給と要求の関係から PFD は光エネルギー供給が要求を下回る 光制限下 と光エネルギー供給が要求を上回る 光飽和下 に分けられる また 光合成や光保護の調節は 主に 光吸収を行う集光アンテナと電子伝達を行う反応中心からなる光化学系 II(Photosystem II; PSII) で行われる そこで本研究では 最も簡単な PSII システムを持つプリムネシウム藻綱 Isochrysis galbana をモデル種として 光制限下と光飽和下に対する光合成と光保護のバランスの適応を調べることにより 藻類の光適応戦略を明らかにすることとした 光合成と光保護の指標として 1 つには 集光アンテナの集光能力と熱放散能力を用い もう 1 つには 反応中心の電子伝達とクロロフィル蛍光の放出を用いた まず 比成長速度と適応させた PFD の関係から I. galbana では 365μmol photons m -2 s -1 以下が光制限下 それ以上が光飽和下であることが推定された 集光アンテナの集光能力と熱放散能力のバランスは 365μmol photons m -2 s -1 付近以下で集光能力が熱放散能力に比べて大きくなったのに対して それ以上では熱放散能力が集光能力に比べて大きくなった 一方 反応中心の電子伝達とクロロフィル蛍光の放出のバランスは 365μmol photons m -2 s -1 付近以下で電子伝達が優占し それ以上ではクロロフィル蛍光の放出が優占した 本研究は 光制限下では光エネルギーの要求を満たすために集光能力及び電子伝達に用いられる光エネルギーの割合を増加させて光合成能力を高め 光飽和下では過剰な光エネルギーを排出するために熱放散とクロロフィル蛍光の放出を増加させて光保護能力を高めるという PSII における I. galbana の光適応戦略を明らかにした また PSII における光適応戦略が光エネルギーの供給と要求のバランスに依存することを明らかにした さらに 本研究は アイスアルジ群集の光適応戦略との比較から 本研究が明らかにした種レベルの光適応戦略を群集レベルに応用できる可能性を示した Keywords: electron transport, fluorescence emission, light harvesting, light limited, light saturated, photoprotection, photosynthesis, thermal dissipation Introduction Algae live in diverse and highly variable light environments and are often subjected to changes in the photon flux density (PFD) imposed by the natural physics of the ocean. The PFD received by algal cells varies from to 2 μmol photons m -2 s -1 (Cullen & Lewis 1988). Light is often a limiting factor for algal growth in the ocean. However, light can be also harmful at supraoptimal PFD leading to a damage of photosystem II (PSII) and a reduction in the photosynthetic rate. Algae have evolved a number of acclimation responses to accommodate the change in PFD of photosynthetically active radiation (PAR) to achieve a balance between maximizing rates of photosynthesis and avoiding the damaging effects of excess PFD on photosynthesis (La Roche et al. 1991). Algae need to enhance photosynthetic capability for maximizing rates of photosynthesis under low PFDs while enhance photoprotective capability to maintain maximum photosynthetic rate and to avoid the damage from excess light energy under high PFDs. Photoacclimation strategy of algae must be associated with adjustments of the balance between photosynthetic and photoprotective capability of algae. Photoacclimation responses depend in part on whether the PFD is light-limiting or light-saturating for growth (Raven & Geider 23). This implies that strategy of algae may be associated with the balance of light energy supply and demand of algae. When growth rate depends on growth PFD (light limited [] condition), the light energy supply is insufficient to algal light energy demand, while growth rate becomes saturated (light saturated [] condition), light energy supply is enough or excess for the light energy demand (Fig.1). I hypothesize that algae enhance photosynthetic capability under condition, while algae enhance the photoprotective capability under condition. Photoacclimation strategy of algae may be explained by adjustments of the balance between photosynthetic and photoprotective capability to light conditions. μ rel ( ) E s /E d ( ) μ rel K G 15 E s /E d ratio Figure 1. Relationship between relative growth rate, μ rel or ratio of light

energy supply to demand, E s /E d and growth PFD., and K G indicate light limited, light saturated conditions and growth saturation PFD, respectively. Since both processes of photosynthesis and photoprotection occur within PSII, photoacclimation strategy of algae is closely associated with the photoacclimation of PSII. The PS II is one of the elements of the photosynthetic electron transport system and is composed of many different proteins and pigment molecules. A couple of functions of PSII can be categorized for light-harvesting antenna and reaction center. To study the balance between photosynthetic and photoprotective capability in light-harvesting antenna of Isochrysis galbana, diadinoxanthin (DD) and diatoxanthin (DT) could be considered as a representative pigments. In fact, DD can transfer excitation energy to and play a role in the light energy acquisition as photosynthetic pigments, while DT can absorb excitation energy from and play a role in the thermal dissipation as photoprotective pigments (Frank et al. 1994). DD and DT have a reversible relation mutually. This reversible conversion can be considered as a reversible transition of light-harvesting into thermal dissipation. Thus, DD and DT are critical to examine the balance between light-harvesting and thermal dissipative capability of light-harvesting antenna in PSII of I. galbana. Information on status and functions of reaction center of PSII can be derived from the measurement of chlorophyll a fluorescence induced by light, because fluorescence yield depends on status and functions of the reaction center (Krause & Weis 1991). The variable chlorophyll a fluorescence provides chlorophyll a fluorescence parameters. The chlorophyll a fluorescence parameters can be categorized by quantum yields and quenching parameters in the reaction center. The quantum yields and the quenching parameters in the reaction center are measured by pulse amplitude modulation fluorometer (PAM). The quantum yield of PSII (F v /F m ) is considered as effective efficiency of electron transport in a reaction center under a light exposure. The maximum quantum yield of PSII (F v /F m ) can be measured under dark condition and is considered as potential efficiency of electron transport in a reaction center. On the other hand, the quenching parameters are consisted of operating efficiency (qp) and excitation pressure (1 qp). These parameters are considered as the proportions of oxidative and reductive reaction centers, respectively. Most reaction centers are oxidative at high operating efficiency, while most reaction centers are reductive at high excitation pressure. Allocation of the light energy which is quantifying the fate of light energy absorbed by the algal cell has become an important aspect of algal photosynthetic research Recently, Kato et al. (23) suggests that the model of the allocation of light energy absorbed by the algal cell can be estimated from both quantum yields and quenching parameters. The determination of the quantum yields provides a measurement of constitutive energy loss at reaction center (Φ C ). The determination of both quantum yields and quenching parameters allow estimating electron transport (Φ E ) and fluorescence emission (Φ F ). Summation of those three energy allocations, however, does not equal to the absorbed light energy. The difference is considered as a non-photochemical quenching (NPQ). This quenching is derived from a thermal dissipation of the absorbed light energy through DD-cycle in light-harvesting antenna. This allocation is estimated as a regulated thermal dissipation (Φ R ). Variability in the allocation of the absorbed light energy should be associated with photoacclimation strategy of PSII. In the present study, in order to study on the photoacclimation in reaction center of PSII, the allocation of the absorbed light energy is investigated in I. galbana grown under different PFDs. The present study focused on the adjustment of the balance between photosynthetic and photoprotective capability to meet the balance between light energy supply and demand in the both light-harvesting antenna and reaction center of PSII in Prymnesiophyceae Isochrysis galbana. I. galbana which belong to algal group has one main light-harvesting system and energy regulation mechanism. The objectives in this study are (1) to examine the relationship between growth rate and growth PFD to delimit the two light conditions in relative to light energy supply and demand, (2) to determine the photoacclimation of DD and DT in the light-harvesting antenna in relative to the light conditions, and (3) to determine similarly the photoacclimation of variable chlorophyll a fluorescence in the reaction center. Finally, a model of the photoacclimation strategy in PSII of I. galbana and requirements for future research are presented. Materials and Methods Study 1. The relationship between light energy supply and demand of I. galbana I. galbana (NEPECC633) was obtained from the North East Pacific Culture Collection at the University of British Columbia. I. galbana was grown in 25 ml batch cultures in enriched f/2 seawater medium at 25 ºC and PFD of 3, 6, 125, 25, 5 and 996 μmol photons m -2 s -1 provided by cool-white fluorescent lamps with a 12h light and 12h dark cycle. Cells of I. galbana were counted on an inverted microscope (Olympus). The growth rate and growth PFD curve was estimated by fitting the following equations; μ rel = 1 exp (-E/K E ) (1) where μ rel is relative growth rate, E is the growth PFD, K E is light saturation parameter. Growth saturation PFD (K G ) was defined as the growth PFD value when μ rel was assumed to be equal to.99. Study 2. Photoacclimation of light-harvesting antenna of I. galbana I. galbana was preconditioned in 3 or 5 L continuous culture which was maintained in enriched f/2 seawater medium at 25 ºC and 35PSU salinity. of 45, 25, 425 and 137 μmol photons m -2 s -1 provided by cool-white fluorescent lamps with a 12h light and 12h dark cycle. The steady state of growth rates was established at.3 day -1 by controlling the dilution rate with a peristaltic pump. The PFD was determined by a scalar quantum sensor. Subsamples were collected every 3 hours for the analysis of, DD and DT concentrations by HPLC. Light-harvesting capability (LHC) was estimated from a slope of least square regression analysis between specific DD (DD ) and specific DD+DT ([DD+DT] ). On

the other hand, thermal dissipative capability (TDC) was estimated from a slope of least square regression analysis between specific DT (DT ) and (DD+DT). Study 3. Photacclimation of reaction center of I. galbana I. galbana was preconditioned in continuous culture and was acclimated under the same experimental conditions described in study 2. Subsamples for the measurements of quantum yields of PSII and quenching parameters were collected every 3 hours. The quantum yields of PSII under dark and light conditions (F v /F m and F v '/F m '), and the quenching parameters (qp and 1 qp) were measured by PAM (Obata et al. 29). The F v '/F m ', qp and 1 qp were determined at step-wise increasing actinic light up to 2 μmol photons m -2 s -1 with 3 sec illumination periods at each. Allocation of light energy absorbed by algal cell to constitutive energy loss (Φ C ), electron transport (Φ E ), fluorescence emission (Φ F ), and regulated thermal dissipation by DT (Φ R ) were estimated by qp F v '/F m ', 1 F v /F m, (1 qp) F v '/F m ', and F v /F m F v '/F m ', respectively (Demming-Adams et al. 1996). Results and Discussions Study 1. The relationship between light energy supply and demand of I. galbana The intrinsic growth rate and PFD curve of I. galbana was obtained by normalizing growth rate to the maximum growth rate (μ rel ) (Fig.2). The growth saturation PFD, K G of I. galbana was 365 μmol photons m -2 s -1. This suggests that a range of light limited () and light saturated () conditions for I. galbana correspond to lower and higher than 365 μmol photons m -2 s -1, respectively. μ rel K G 1.4 1.2 n=18 r 2 =.94 p<1 365 15 Figure 2. Relationship between the relative growth rate, μ rel and growth PFD in I. galbana., and K G indicate light limited, light saturated conditions and growth saturation PFD, respectively. Data sources are as follows: Falkowski et al. (1985) (square), Tzovenis et al. (1997) (triangle), Jokiel and York (1984) (diamond) and the present study (circle). Study 2. Photoacclimation of light-harvesting antenna of I. galbana Light-harvesting capability (LHC) and thermal dissipative capability (TDC) of I. galbana showed sigmoid response to growth PFD (Fig.3). This sigmoid relationship was crossed at 349 μmol photons m -2 s -1. The maximum LHC and minimum TDC observed under condition suggest that I. galbana might have to enhance the light-harvesting capability to gain much light energy. In contrast, the minimum LHC and the maximum TDC obtained under condition suggest that I. galbana do not require much light energy and might have to enhance the performance of dissipation of excitation energy rather than absorption of photons. LHC and TDC 5 1 15 Figure 3. Light dependence of light-harvesting capability, LHC (circles) and thermal dissipative capability, TDC (squares) in I. galbana. Study 3. Photacclimation of reaction center of I. galbana Decrease of the operating efficiency (qp) and increase of the excitation pressure (1 qp) with actinic PFD were observed regardless of growth PFD (Fig.4). In contrast, the maximum quantum yield of PSII (F v /F m ) and quantum yield (F v '/F m ') were relatively constant regardless of growth PFD. These results suggest that the photoacclimation of reaction center can be characterized for the redox state of reaction centers rather than the efficiency of electron transport of a reaction center. The qp and the 1 qp crossed under all growth PFD (Fig.4). Average of the PFD at the cross point was 294±57 μmol photons m -2 s -1. These results suggests that the state of reaction centers of PSII in Isochrysis galbana change from oxidative to reductive state upon a shift and condition. qp and 1 qp (A) (C) 5 1 15 2 (B) (D) Actinic PFD 5 1 15 2 Figure 4. Response of operating efficiency, qp (circles) and excitation pressure, 1-qP (squares) to actinic PFD in I. galbana acclimated at 45 (A), 25 (B), 425 (C) and 137 (D) μmol photons m -2 s -1. Allocation of light energy absorbed by the algal cell varied with growth PFD in I. galbana (Fig.5). The Φ C ranged from.3 to. In contrast, the Φ E decreased from to.1 and the Φ F increased from.1 to.5 with growth PFD. The Φ E was high under condition while the Φ F was high under condition.

This result suggests that I. galbana enhance the electron transport under condition and the fluorescence emission under condition. At the highest growth PFD, the Φ F reached to.5 and Φ R was appeared. These results suggested that energy loss depend on Φ F in I. galbana. Fluorescence emission through the adjustment of the redox state of reaction centers may be a critical photoprotective mechanism for I. galbana. Energy allocation (dimension less) 45 25 425 137 Φ R Φ F Φ E Φ C Figure 5. Allocation of absorbed light energy for I. galbana. Bars of white, right striped line, left striped line and black indicate constitutive energy loss, Φ C, electron transport, Φ E, fluorescence emission, Φ F, and regulated thermal dissipation by DT, Φ R, respectively. Sum of these four fractions become unity. Conclusions Photoacclimation strategy of algae can be explained by adjustments of the balance between photosynthetic and photoprotective capability of PSII to meet the balance of light energy supply and demand of algae. The growth PFD was delimited at growth saturation PFD (K G ) for two light conditions; light limited () and light saturated () conditions. Under the condition, the algal light energy demand is overwhelmed the light energy supply while the light energy supply is enough or excess for the light energy demand under the condition. In both the light-harvesting antenna and reaction center, photosynthetic and photoprotective capability of Isochrysis galbana depend on the light conditions. The acclimation of the light-harvesting antenna and the reaction centers leads enhancement of allocation of absorbed light energy to electron transport under condition and to fluorescence emission under condition. Thermal dissipation by DT is also occurred under condition. The similarity in all estimates suggests that the photoacclimation strategy in PSII of I. galbana shift from photosynthesis to photoprotection around K G (Table 1). Table 1. The growth saturation PFD, K G, the PFD of the cross point of LHC and TDC and that of qp and 1-qP of I. galbana. PFD μmol photons m -2 s -1 K G 365 Cross point of LHC and TDC 349 Cross point of qp and 1-qP 294±57 Source Study 1 Study 2 Study 3 Under condition, I. galbana enhance photosynthetic capability of PSII by increase of light-harvesting capability and electron transport for maximizing photosynthetic rate while enhance photoprotective capability of PSII by increase of thermal dissipative capability in addition to fluorescence emission for avoiding the damage from excess light energy under condition (Fig.6). In I. galbana, main photoprotective mechanism may be the fluorescence emission through the adjustment of the redox state of reaction centers rather than thermal dissipation by DT. A better knowledge of the photoacclimation strategy is required to take account of the balance between light energy supply and demand. Fluorescence emission Fluorescence emission Electron transport P68 Light DD Light Electron transport P68 DD DT DT Thermal dissipation Figure 6. Simplified schema of PSII related to photoacclimation strategy in PSII of I. galbana under light limited () and light saturated () condition. Dotted and shaded areas indicate light-harvesting antenna while stripe area indicates reaction center. Expansive application The findings in the present study can support the importance of the scaling of growth PFD for understanding of photoacclimation strategy. The growth PFD normalized to K G can serve effectively as the scaling of photoacclimiation for comparative studies. By scaling of growth PFD in this manner, photoacclimation strategy can be incorporated into the photoadaptation strategy of algal community. I tested the applicability of the photoacclimation strategy at the levels of species to photoadaptation strategy at the level of community by investigation of photoadaptation in PSII of ice algal community which inhabits under condition defined in study 1. I conducted in situ incubation experiment at Saroma-ko Lagoon, Hokkaido, Japan (Obata & Taguchi 29). Ice algal community in Saroma-Ko Lagoon, Hokkaido enhanced the light-harvesting capability and electron transport in PSII and allocation of absorbed light energy to electron transport. The validity in applicability of the photoacclimation strategy to the photoadaptation strategy was demonstrated by the experimental observations of the ice algal community as well as I. galbana under condition. This can be supported by the photophysiological studies of ice algal community in Saroma-Ko Lagoon, Hokkaido (Obata & Taguchi 29). References Cullen, J. J. & M. R. Lewis, 1988. Journal of Plankton Research 1: 139-163. Demming-Adams, B. W., W. W. 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