Masaharu C. Kato 1, 3, Kouki Hikosaka 1, 4, Naoki Hirotsu 2, Amane Makino 2 and Tadaki Hirose 1. Introduction

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1 Plant Cell Physiol. 44(3): (2003) JSPP 2003 The Excess Light Energy that is neither Utilized in Photosynthesis nor Dissipated by Photoprotective Mechanisms Determines the Rate of Photoinactivation in Photosystem II Masaharu C. Kato 1, 3, Kouki Hikosaka 1, 4, Naoki Hirotsu 2, Amane Makino 2 and Tadaki Hirose 1 1 Graduate School of Life Sciences, Tohoku University, Sendai, Japan 2 Graduate School of Agricultural Sciences, Tohoku University, Sendai, Japan Photoinactivation of PSII is thought to be caused by the excessive light energy that is neither used for photosynthetic electron transport nor dissipated as heat. However, the relationship between the photoinactivation rate and excess energy has not been quantitatively evaluated. Chenopodium album L. plants grown under high-light and high-nitrogen (HL-HN) conditions show higher tolerance to photoinactivation and have higher photosynthetic capacity than the high-light and low-nitrogen (HL-LN)- and lowlight and high-nitrogen (LL-HN)-grown plants. The rate of photoinactivation in the LL-HN plants was faster than that in the HL-LN, which was similar to that in the HL-HN plants, while the LL-HN and HL-LN plants had similar photosynthetic capacities [Kato et al. (2002b) Funct. Plant Biol. 29: 787]. We quantified partitioning of light energy between the electron transport and heat dissipation at the light intensities ranging from 300 to 1,800 mol m 2 s 1. The maximum electron transport rate was highest in the HL- HN plants, heat dissipation was greatest in the HL-LN plants, and the excess energy, which was neither consumed for electron transport nor dissipated as heat, was greatest in the LL-HN plants. The first-order rate constant of the PSII photoinactivation was proportional to the magnitude of excess energy, with a single proportional constant for all the plants, irrespective of their growth conditions. Thus the excess energy primarily determines the rate of PSII photoinactivation. A large photosynthetic capacity in the HL- HN plants and a large heat dissipation capacity in the HL- LN plants both contribute to the protection of PSII against photoinactivation. Keywords: Acclimation Chenopodium album Heat dissipation Photoinhibition Photoprotection Xanthophyll cycle. Abbreviations: A, antheraxanthin; Ci, intercellular CO 2 concentration; D, fraction of light energy that is dissipated in the light; E, fraction of excess energy; F m, maximal fluorescence level in the dark; F o minimal fluorescence level in the dark; F s, actual fluorescence level; F v, variable fluorescence level in the dark; F m, maximal fluorescence in the light; F o, minimal fluorescence in the light; F v /F m, quantum yield of electron transport in open PSII; F/F m, quantum yield of PSII ; electron transport; HL-HN, high-light and high-nitrogen; HL-LN, high-light and low-nitrogen; k pi, rate constant of photoinactivation; L, fraction of light energy that is dissipated in the dark; LL-HN, low-light and high-nitrogen; P, fraction of light energy that is consumed by electron transport; PPFD, photosynthetically active photon flux density; Q A, primary quinone acceptor in PSII; qp, photochemical quenching; V, violaxanthin; Z, zeaxanthin. Introduction Although light is the energy source for plant growth, excessive light can lead to depression in photosynthetic efficiency (photoinhibition), mainly due to oxidative damage to the photosystem II (PSII) (Powles 1984). Low-light-grown plants have been frequently reported to be more susceptible to photoinhibition than high-light grown plants (Chow 1994, Demmig-Adams and Adams 1992, Long et al. 1994, Osmond 1994). Since photosynthetic CO 2 assimilation is a major sink for absorbed light energy, the difference in photosynthetic capacity, which varies depending on growth irradiance, may lead to different susceptibilities to photoinhibition (Powles 1984). Recent studies have suggested that several mechanisms are involved in the protection against photoinhibition of PSII. Photorespiration (Osmond 1981, Kozaki and Takeba 1996, Park et al. 1996), heat dissipation via the xanthophyll cycle (Demmig-Adams and Adams 1992, Demmig-Adams and Adams 1996, Horton et al. 1996, Gilmore 1997), and consumption of reducing power via the water water cycle (Park et al. 1996, Asada 1999) are believed to contribute to reduction and dissipation of excess energy. Although PSII may be damaged in spite of these mechanisms, fast recovery of damaged PSII helps the plant reduce the susceptibility to photoinhibition (Prasil et al. 1992, Aro et al. 1993a, Aro et al. 1993b). Despite these studies, relative contributions of these mechanisms are still not clear, due partly to all these photoprotective mechanisms changing simultaneously with growth irradiance. Kato et al. (2002b) have investigated an involvement of photosynthetic capacity and recovery of damaged PSII via D1 protein turnover in the susceptibility of PSII to photoinhibition in leaves of Chenopodium album L. plants, which were grown 3 Present address: Fuji Oil Co., Ltd., Hannan R&D Center, Izumisano, Osaka, Japan. 4 Corresponding author: , hikosaka@mail.cc.tohoku.ac.jp; Fax,

2 Excess energy determines PSII inactivation rate 319 Table 1 Photosynthetic characteristics in C. album leaves grown at low-light and high-nitrogen (LL-HN), high-light and low-nitrogen (HL-LN), and high-light and high-nitrogen (HL-HN) Growth conditions Chl (mmol m 2 ) Absorptance Initial F v /F m Photosynthetic rate ( mol m 2 s 1 ) Electron transport rate ( mol m 2 s 1 ) LL-HN a a a a 94 5 a HL-LN b b b a b HL-HN c c c b c The photosynthetic and electron transport rate were determined at 1,050 mol quanta m 2 s 1. Different letters within the same column indicate significant differences (P<0.05). Mean SE; n 6. at three different combinations of irradiance and nitrogen availability: high-light and high-nitrogen (HL-HN), high-light and low-nitrogen (HL-LN), and low-light and high-nitrogen (LL- HN). To test the importance of photosynthetic capacity in the susceptibility to photoinhibition, the growth condition was regulated such that photosynthetic capacity was highest in HL-HN plants with LL-HN and HL-LN plants having similar rates to each other. The rate of PSII photoinhibition, which was assessed as a decrease in the quantum yield of photochemistry, was much higher in plants grown at low-light (LL-HN) than in those grown at high-light (HL-HN and HL-LN), suggesting that the susceptibility to photoinhibition is not attributable solely to the photosynthetic capacity. High-light grown plants had a higher rate of concurrent recovery, which was not influenced by the nitrogen availability (Kato et al. 2002b). These results indicate that a higher turnover of D1 protein plays a crucial role in photoprotection in high-light grown plants, irrespective of nitrogen availability. However, when the recovery was inhibited, the LL-HN plants still had higher rates of photoinactivation than high-light grown plants. These results lead us to hypothesize that different abilities of photosynthesis and of photoprotective mechanisms may determine the susceptibility of these differently grown plants to PSII photoinactivation (Kato et al. 2002b). The aim of the present study was to elucidate the factors that determine the rate of photoinactivation. We determined Chl fluorescence parameters and applied the model proposed by Demmig-Adams et al. (1996) to estimate the light energy partitioning among various pathways. In their model, sink of light energy comprises three parts: photosynthetic electron transport, heat dissipation, and excess energy. Electron transport and heat dissipation thus work as photoprotective mechanisms. Energy for electron transport is further utilized either by photosynthesis, photorespiration or the water water cycle. Energy that is neither consumed for electron transport nor dissipated as heat is regarded as excess energy. In the present study, we tested the hypothesis that different abilities of these photoprotective mechanisms bring about the different amount of excess energy and determine the rate of photoinactivation. Results Fig. 1 Comparison of PSII characteristics in leaves of plants grown at low-light and high-nitrogen (LL-HN, closed triangle), high-light and low-nitrogen (HL-LN, open circle), and high-light and high-nitrogen (HL-HN, open triangle). (a) The quantum yield of PSII electron transport ( F/F m ), (b) the fraction of open centers (qp), and (c) the quantum yield of open PSII (F v /F m ), measured after 25 min illumination at each PPFD given on the abscissa. Mean SE; n 3. C. album plants were grown under HL-HN, HL-LN, and LL-HN conditions as described in Kato et al. (2002b). Growth conditions were regulated such that the light-saturated rate of photosynthesis was highest in HL-HN plants with LL-HN and HL-LN plants having similar rates to each other (Table 1, see Kato et al. 2002b for light response curves of photosynthesis).

3 320 Excess energy determines PSII inactivation rate Fig. 2 PPFD response of the fractions of absorbed light in PSII that was lost in the dark (L), that was dissipated thermally (D), and that was utilized in the electron transport (P) in leaves grown at LL-HN (a), HL-LN (b), and HL-HN (c). The fraction of absorbed light neither going into P, D, nor L is defined as excess (E). Mean SE; n 3. Initial F v /F m in dark-adapted leaves was higher than 0.8 in all plants, and there were significant differences; HL-HN plants had the highest value while HL-LN plants had the lowest (Table 1). We determined Chl fluorescence from leaves under various photosynthetically active photon flux densities (PPFD). Fig. 1 shows the quantum yield of electron transport in total PSII (Fig. 1a), the fraction of open PSII center (Fig. 1b), and the quantum yield of electron transport in open PSII (Fig. 1c). The quantum yield of total PSII ( F/F m ) is the product of the fraction of open PSII (qp) and the quantum yield of open PSII (F v /F m ) (Genty et al. 1989). The quantum yield of PSII electron transport decreased with increasing PPFD (Fig. 1a). Such changes were observed in the fraction of open centers (qp) (Fig. 1b) as well as the quantum yield of open PSII (Fig. 1c). The HL-HN plants had the highest F/F m, the HL-LN plants the second highest, and the LL-HN plants the lowest (Fig. 1a). This result indicates that energy consumption for electron transport was highest in HL-HN plants and lowest in LL-HN plants. The HL-HN plants had the highest qp and the HL-LN and LL-HN plants had an intermediate and lowest qp, respectively (Fig. 1b). The HL-HN plants had the highest F v /F m among these plants (Fig. 1c). At low PPFDs the HL-LN plants had higher F v /F m than the LL-HN, whereas at high PPFDs the HL-LN plants had the lowest F v /F m. Partitioning of absorbed light energy was estimated with the model of Demmig-Adams et al. (1996) (Fig. 2). If PSII completely utilizes absorbed light, the maximum quantum yield in dark-adapted leaves (F v /F m ) will be 1.0. Heat dissipation reduces the quantum yield of open PSII (F v /F m ) (Demmig-Adams et al. 1995, Demmig-Adams and Adams 1996, Gilmore and Björkman 1995) and is defined as 1 F v /F m. Heat dissipation is further divided into the fraction of the light energy dissipated in the dark (1 F v /F m, L) and that dissipated in the light (F v /F m F v /F m, D). The fraction of electron transport (P) is defined by F/F m (Genty et al. 1989). The fraction of absorbed light neither going into electron transport nor into heat dissipation is defined as the excess energy (E), i.e. E = 1 L D P (Demmig-Adams et al. 1996). L was slightly higher in the HL-LN plants than in the LL-HN and HL-HN plants. In all the plants, both D and E increased with PPFDs, while P decreased. The magnitudes of P and D differed considerably depending on growth conditions. Compared to the HL-HN plants, the LL-HN plants had a considerably lower P with a higher E, and a higher D (Fig. 2a, c). The HL-LN plants had a lower P but a higher D than the HL-HN plants leading to a similar E (Fig. 2b, c). In order to verify the hypothesis that the PSII photoinacti- Fig. 3 Relationship between the rate constant of photoinhibition (k pi ) and absorbed PPFD. k pi was determined as the first-order rate constant, in which the quantum yield of PSII decreased exponentially upon illumination in the presence of lincomycin, an inhibitor of recovery of the inactivated PSII. Absorbed PPFD was calculated as incident PPFD absorptance. Symbols are the same as those in Fig. 1. Redrawn from Kato et al. (2002b).

4 Excess energy determines PSII inactivation rate 321 Fig. 4 Relationship between the rate constant of photoinactivation (k pi ) and the rate of excess light energy estimated from [E absorption PPFD 0.5] (a), and 1 qp (b). The rate constant of photoinactivation was derived from Kato et al. (2002b). Symbols are the same as those in Fig. 1. vation rate is determined by the excess energy, we determined the quantitative relationship between the inactivation rate and E, both obtained at various PPFDs as above. In the presence of lincomycin, which inhibits the recovery of the inactivated PSII, the quantum yield decreased exponentially upon illumination, with the first-order rate constant k pi (Kato et al. 2002b). Fig. 3 represents the relationship between k pi and the absorbed PPFD determined for the three differently grown plants (data re-cited from Kato et al. 2002b). When the k pi values were plotted against the absolute value of the excess energy that is calculated as E leaf absorptance PPFD 0.5, a simple proportional relationship was obtained (Fig. 4a). All the data points for the plants of the three different growth conditions fell into the same line. Thus, the PSII photoinactivation is linearly correlated with the magnitude of excess energy, and their correlation is not affected by the capacities of photosynthetic electron transport and of heat dissipation of a leaf. To assess the allocation of reducing power produced by electron transport between photosynthesis and other pathways, simultaneous measurements were made of CO 2 gas exchange (photosynthesis), transpiration, and Chl fluorescence under various PPFDs (Fig. 5). The electron transport rate was estimated as F/F m leaf absorptance PPFD 0.5 (Schreiber et al. 1994). The photosynthetic rate increased linearly with the electron transport rate (initial phase), but deviated from the initial slope above a certain level of the electron transport rate (later phase) (Fig. 5a). In the initial phase, there was no difference among growth conditions in the relationship between the rate of photosynthesis and of electron transport. This result suggests that the fraction of reducing power consumed for photosynthesis remains constant against that for the alternative flows (photorespiration and the water water cycle). Therefore, both photorespiration and the water water cycle did not influence susceptibilities to photoinactivation when photosynthetic rates were not saturated. In the later phase, the rate of photosynthesis seemed to reach a steady-state level, and the rate of electron transport increased. This suggests an enhancement of the alternative flows of electron transport under strong light. Although the intercellular CO 2 concentration (Ci) slightly decreased with increasing electron transport rates in all the plants, no difference was found in the relationship among growth conditions (Fig. 5b), suggesting that the ratio of photosynthesis to photorespiration was nearly stable (Farquhar et al. 1980). The electron transport rate at 1,050 mol quanta m 2 s 1 in the Table 2 Carotenoid contents in C. album leaves grown at low-light and high-nitrogen (LL-HN), high-light and low-nitrogen (HL-LN), and high-light and high-nitrogen (HL-HN) Growth conditions V+ A + Z Neoxanthin -carotene Lutein Initial A + Z (mmol mol 1 Chl) LL-HN a a a a a HL-LN c a b b b HL-HN b b b b a Violaxanthin (V), antheraxanthin (A), zeaxanthin (Z), neoxanthin, -carotene, and lutein. Different letters within the same column indicate significant differences (P<0.05). Mean SE; n 10.

5 322 Excess energy determines PSII inactivation rate Fig. 5 Relationship between the electron transport rate and photosynthetic rate (a) and intercellular CO 2 concentration (Ci) (b). Each data point represents one leaf. Symbols are the same as those in Fig. 1. HL-LN plants was slightly higher than that in the LL-HN plants though there was no significant difference in photosynthetic capacity (Table 1). This suggests that a higher rate of electron transport made small contribution to reduce excess energy. Many studies have shown that the ability of heat dissipation is strongly related with the conversion state of the xanthophyll cycle pigments (e.g. Niyogi et al. 1998). We determined the content of carotenoids including the xanthophyll cycle pigments (Table 2). The total size of the xanthophyll cycle pigments was larger in high-light-grown plants than in low-lightgrown plants (violaxanthin [V] + antheraxanthin [A] + zeaxanthin [Z], Table 2). In high-light grown plants, the total size of the xanthophyll cycle pigments was larger in the HL-LN than in the HL-HN plants. The -carotene and lutein levels were higher in the high-light grown than in the low-light grown plants, while these levels were not influenced by nitrogen availabilities. The conversion state of the xanthophyll cycle pigments, (A+Z)/(V+A+Z), changed with PPFD (Fig. 6a). The Fig. 6 The conversion state of the xanthophyll cycle pigments ([A + Z]/[V + A + Z]) as a function of PPFD for plants grown at LL- HN, HL-LN, and HL-HN (a). Leaf discs were collected from individual leaves 25 min after illumination at each PPFD. Relationship between F v /F m and the conversion state of the xanthophyll cycle pigments (A + Z)/(V + A + Z) (b). Symbols are the same as those in Fig. 1. Mean SE; n 3. HL-LN plants showed the highest (A+Z)/(V+A+Z) value at any given PPFDs. Even in darkness 23% of the xanthophyll cycle pigments were retained as (A+Z) in the HL-LN plants. 1 F v /F m (L + D) indicates the fraction of heat-dissipated energy (Demmig-Adams et al. 1996). To examine if the heat dissipation is related to (A + Z)/(V + A + Z) in leaves of C. album grown under different growth conditions, F v /F m was plotted against (A + Z)/(V + A + Z) (Fig. 6b). F v /F m decreased with increasing (A + Z)/(V + A + Z) and the relationship was independent of growth conditions, suggesting that heat dissipation is strongly controlled by the changes in the conversion state of the xanthophyll cycle pigments.

6 Excess energy determines PSII inactivation rate 323 Discussion Relationship between excess light energy and rate of photoinactivation Photoinactivation of PSII has been considered to result from the excess energy that is neither utilized nor dissipated (Powles 1984, Demmig-Adams and Adams 1992, Osmond 1994, Long et al. 1994, Asada 1994, Niyogi 1999), but there seems to be no study that gave quantitative evidence for it. In this study we showed that the rate constant of the photoinactivation was proportional to the magnitude of the excess energy with a single proportion constant for the plants that have varying capacities of photosynthesis and heat dissipation. This is the first study that explained the susceptibility to photoinactivation of PSII by the amount of excess energy. The excess energy can also be expressed as (1 qp) F v / F m (Demmig-Adams et al. 1996). This is the excitation energy absorbed by closed PSII, which was not dissipated as heat. Accumulation of excitation energy in closed PSII may generate long-lived excited states of Chl ( 3 Chl*) and singlet excited oxygen ( 1 O 2 ). 1 O 2 is generated in the PSII reaction center by the interaction of the ground state oxygen ( 3 O 2 ) with the 3 Chl* produced in the recombination reaction of reduced pheophytin with P680 + (Macpherson et al. 1993). 1 O 2 generated within the protein matrix of the reaction center brings about specific damage (Aro et al. 1993a, Andersson and Barber 1996). Thus the increased amount of excess energy may lead to proportional increase in the production of 1 O 2. Two hypotheses have been presented on the determination of photoinactivation of PSII. Park et al. (1995) postulated a photon counter, implying that photoinactivation occurs when a fixed number of photons were absorbed by PSII, irrespective of photoprotective mechanisms. This hypothesis, however, did not explain the different rates of photoinactivation of PSII in plants grown under different conditions (Fig. 3). Another hypothesis is that the susceptibility to photoinhibition depends on the redox state of PSII, measured as 1 qp (excitation pressure) (Ögren 1991, Ögren and Rosenqvist 1992, Öquist et al. 1992a, Öquist et al. 1992b, Öquist et al. 1993, Gray et al. 1996). To test this hypothesis, we plotted k pi against 1 qp (Fig. 4b). Although k pi increased with 1 qp, the relationship was different in different growth conditions. This difference is attributable to the change in heat dissipation. In contrast to our study, Gray et al. (1996) have reported that there was no major change in heat dissipation via the xanthophyll cycle pigments. This suggests that the F v /F m level did not change and therefore the level of excess energy depended only on changes in the 1 qp in their experimental conditions. When heat dissipation changed as in our present study, however, the susceptibility to photoinactivation may not be explained solely by 1 qp. Energy consumption by electron transports The relationship between photosynthesis and electron transport (Fig. 5a) suggested that, under non-saturating light, the fraction of reducing power consumed for photosynthesis remained constant against that for the alternative flows (photorespiration and the water water cycle) irrespective of growth conditions. Under saturating light, on the other hand, the alternative flows of electron transport may become important for protecting PSII. Although Ci tended to decrease with increasing electron transport rates (Fig. 5b), the reduction was too small to increase the fraction of consumption by photorespiration at strong light. Contribution of the water water cycle might have increased with increasing rates of electron transport. This is consistent with the observation by Miyake and Yokota (2000) who showed that the fraction of reducing power consumed by the water water cycle increased when the rates of photosynthesis and photorespiration were saturated under strong light. In the present study, we assumed that half of the total photon flux absorbed by the leaf was absorbed by PSII. Thus, the electron transport rate was calculated as F/F m leaf absorptance PPFD 0.5 (Schreiber et al. 1994). The fraction has been suggested to change with the state transition of LHCII (reviewed in Allen 1992) and with growth conditions (Anderson 1986). However, though in vitro experiments suggested that photoprotection involved the state transition of LHCII (Allen 1992), there is no convincing evidence for its role in photoprotection in vivo (Rintamäki et al. 1997, Haldrup et al. 2001). Hikosaka and Terashima (1996) indicated that the ratio of PSII Chl to total leaf Chl in C. album was independent of growth irradiances that were similar to those used in the present study. These facts suggest that the fraction of light absorbed by PSII Chl was constant irrespective of experiments and of growth conditions. Heat dissipation of absorbed light energy via xanthophyll cycle There was a strong correlation between the quantum yield of open PSII (F v /F m ) and the conversion state of the xanthophyll cycle pigments (Fig. 6b). This suggests that antheraxanthin and zeaxanthin dissipate energy as heat, leading to the reduction in the quantum yield. The largest fraction of heat dissipation was found in the HL-LN plants (Fig. 2) whose (A + Z)/(V + A + Z) was consistently higher than that in the HL-HN and LL-HN plants (Fig. 6b). Several studies have also observed higher (A + Z)/(V + A + Z) in low-nitrogen-grown plants (Demmig-Adams et al. 1995, Verhoeven et al. 1997). What caused the high (A + Z)/(V + A + Z) in the HL-LN plants then? One explanation is that the HL-LN plants had a lower activity of epoxidase that catalyzes the conversion from Z to A, and then V. Another is related with the retention of high levels of (A + Z) in HL-LN plants in the dark (Fig. 6a). Although maximum values of (A + Z)/(V + A + Z) differed among growth conditions, the difference between the maximum and minimum (A + Z)/(V + A + Z) was close to 0.5, irrespective of growth conditions (Fig. 6a). This implies that there is an inherent limitation in the variability of (A + Z)/(V + A + Z) in C. album leaves, i.e. the higher minimum (A + Z)/(V + A + Z) is needed to have higher capacity of heat dissipation. On the other

7 324 Excess energy determines PSII inactivation rate hand, higher heat dissipation due to higher minimum values of (A + Z)/(V + A + Z) may result in lower quantum yields at low light. Actually, HL-LN plants showed lower F v /F m than HL-HN and LL-HN plants (Table 1), as previously reported (Demmig- Adams et al. 1995, Verhoeven et al. 1997). HL-LN plants may have a high capacity of heat dissipation in high light at the expense of the quantum yield in low light. Conclusion In C. album grown under different irradiances and nitrogen availabilities, the susceptibility to photoinhibition was explained by a combination of several factors: the contribution of electron transports, heat dissipation via the xanthophyll cycle, and D1 protein turnover. The excess light energy, which was neither consumed nor dissipated by these mechanisms, determined the rate of photoinactivation irrespective of growth conditions (Fig. 4). To tolerate to strong light, plants need to reduce excess energy. HL-HN plants had the highest tolerance to photoinhibition due to the highest photosynthetic capacity (Fig. 2) with fast recovery of photoinactivated PSII (Kato et al. 2002b). The low photosynthetic capacity of HL-LN plants was compensated for by their high ability of heat dissipation at the expense of the quantum yield at low light (Fig. 2, 6). LL-HN plants had the lowest tolerance to photoinhibition due to their low photosynthetic capacity (Table 1), low heat dissipation (Fig. 2), and slow recovery of inactivated PSII (Kato et al. 2002b). Materials and Methods C. album plants were grown under three different conditions: high-light and high-nitrogen (HL-HN), high-light and low-nitrogen (HL-LN), and low-light and high-nitrogen (LL-HN). Low-light and low-nitrogen treatment was not applied because effects of nitrogen treatment on photosynthetic traits were small at low light conditions (Terashima and Evans 1988). The growth irradiance of high- and lowlight were 650 and 50 mol m 2 s 1, respectively. To obtain the lower light intensity, the plants were placed in a box covered with shade cloth (neutral shading). Standard hydroponic solution contained 12 mm nitrate. Detailed composition is described in Hikosaka et al. (1994). For the treatment of low nitrogen, nitrate concentration was reduced to 0.5 mm and chloride was supplied instead of nitrate to maintain the ionic balance. The nitrate conditions were chosen such that LL-HN and HL-LN plants had the same photosynthetic capacity. The solution was aerated continuously and renewed every week (see Kato et al. 2002b for detailed explanation of growth conditions). Three to four fully expanded young leaves of 8-week-old plants were collected from one plant. Leaves were cut with a sharp razor in water at a petiole length of cm (Kato et al. 2002a). For each treatment, more than five leaves were used. Chl was extracted with dimethylformamide and determined according to Hikosaka and Terashima (1996). Absorptance, transmittance, and reflectance were obtained with an integral sphere (Kato et al. 2002b). For the measurement of xanthophylls, leaf discs (1.77 cm 2 ) were collected from intact leaves and stored at 85 C until processing. It took less than 3 s to remove discs from the leaves and submerge them in liquid nitrogen. Extraction of pigments and analysis of the extracts by HPLC were done as described in Ushio et al. (2003). Chl fluorescence was measured with a PAM-2000 Chl fluorometer and analyzed using the saturating-pulse mode and Data Acquisition Software DA-2000 (Walz, Effetrich, Germany). Leaves were placed in the dark for 45 min prior to estimation of dark level maximal (F m ) and minimal (F o ) fluorescence. The actual fluorescence level, F s, was monitored during illumination. To obtain F m, the leaf was exposed to a saturating flash during illumination. To determine the minimal level of fluorescence during illumination (F o ), far-red light was turned on and a black cloth was quickly placed around the leaf. The leaf was illuminated with far-red light continuously to rapidly reoxidize the PSII centers. All measurements were conducted at 25 C. Simultaneous measurements of photosynthesis and Chl fluorescence were made with an open gas exchange system (Hikosaka et al. 1998). The maximum steady-state photochemical efficiency of PSII was indicated by F v /F m, where F v = F m F o (Krause and Weis 1991). The quantum yield of open PSII was determined by F v /F m, where F v = F m F o (Genty et al. 1989). The photochemical quenching coefficient (qp) was calculated as (F m F s )/(F m F o ) (Schreiber et al. 1994). The quantum yield of PSII electron transport ( F/F m ) was calculated as (F m F s )/F m (Genty et al. 1989). The rate of electron transport was estimated from F/F m 0.5 leaf absorptance PPFD (Schreiber et al. 1994), where the rate of PSI photochemistry was assumed to match that of PSII (see Genty et al. 1990). The rate of excess energy production was estimated from (1 qp) (F v /F m ) 0.5 leaf absorptance PPFD, an analogous expression to the electron transport rate. To determine the rate constant of photoinactivation of PSII in the absence of the recovery of D1 protein (k pi ), petioles of cut leaves were soaked in lincomycin solution (0.7 mm) and the leaf laminae were exposed to a PPFD of 20 mol m 2 s 1 for 2 h at 25 C. Then the leaves were placed in a temperature controlled chamber (25 C) with saturating humidity and illuminated through a glass window. Light was provided by a metalhalide lamp. Different PPFDs, ranging from 300 to 1,800 mol photons m 2 s 1, were obtained by changing the distance between the lamp and the leaves. The k pi was obtained as A = exp( k pi t) where A is F v /F m relative to the initial value and t is illumination time (min) (see Kato et al. 2002b for detail). Kaleidagraph (Abelbeck Software, Japan) was used for curve fitting. Comparisons among growth conditions were done with analysis of variance (ANOVA) using the software StatView (SAS Institute, U.S.A.). Acknowledgments We thank Prof. R. Kanai for valuable comments, T. Yamano and K. Sato for help in the plant growth, S. Yokota and K. Yamabe for measurements of xanthophylls, and O. Muller and an anonymous reviewer for helpful comments on the manuscript. This study was supported in part by grants-in-aid from the Minister of Education, Science and Culture of Japan. References Allen, J.F. (1992) Protein phosphorylation in regulation of photosynthesis. Biochim. Biophys. Acta 1098: Anderson, J.M. (1986) Photoregulation of the composition, function and structure of thylakoid membranes. Annu. Rev. 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