Tsonko D. Tsonev 1, 2 and Kouki Hikosaka 1, 3. Introduction

Transcription

1 Plant Cell Physiol. 44(8): (2003) JSPP 2003 Contribution of Photosynthetic Electron Transport, Heat Dissipation, and Recovery of Photoinactivated Photosystem II to Photoprotection at Different Temperatures in Chenopodium album Leaves Tsonko D. Tsonev 1, 2 and Kouki Hikosaka 1, 3 1 Graduate School of Life Sciences, Tohoku University, Aoba, Sendai, Japan 2 Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria Temperature dependence of photoinhibition and photoprotective mechanisms (10 35 C) was investigated for Chenopodium album leaves grown at 25 C under 500 mol quanta m 2 s 1. The fraction of active photosystem II (PSII) was determined after photoinhibitory treatment at different temperatures in the presence and absence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis. In the absence of lincomycin, leaves were more tolerant to photoinhibition at high (25 35 C) than at low (11 15 C) temperatures. In the presence of lincomycin, the variation in the tolerance to photoinactivation became relatively small. The rate constant of photoinactivation (k pi ) was stable at C and increased by 50% with temperature decrease from 25 to 11 C. The rate constant of recovery of inactivated PSII (k rec ) was more sensitive to temperature; it was very low at 11 C and increased by an order of magnitude at 35 C. We conclude that the recovery of photoinactivated PSII plays an essential role in photoprotection at C. Partitioning of light energy to various photoprotective mechanisms was further analyzed to reveal the factor responsible for k pi. The fraction of energy utilized in photochemistry was lower at lower temperatures. Although the fraction of heat dissipation increased with decreasing temperatures, the excess energy that is neither utilized by photochemistry nor dissipated by heat dissipation was found to be greater at lower temperatures. The k pi value was strongly correlated with the excess energy, suggesting that the excess energy determines the rate of photoinactivation. Keywords: Chenopodium album D1 protein turnover Excess light energy Photoinhibition Photoprotective mechanisms Temperature dependence. Abbreviations: a, fraction of active PSII; D, fraction of the heat dissipation; E, fraction of the excess energy; F m, F 0, F v, maximum, initial and variable chlorophyll fluorescence; F m maximum chlorophyll fluorescence during illumination; F s, steady state chlorophyll fluorescence; F v variable chlorophyll fluorescence during illumination; k pi, k rec, rate constants for photoinactivation and recovery; L, fraction of the light energy that is lost in the dark; NPQ, non-photochemical quenching; PSII, Photosystem II; q P, photochemical quenching coefficient. Introduction Although light is the energy source for plant growth, strong light can cause photoinhibition in photosynthesis. Photosystem II (PSII), which is the major site of photoinhibition, is believed to be inactivated when absorbed energy exceeds that consumed by photosynthetic processes (Powles 1984, Demmig-Adams and Adams 1992, Osmond 1994, Kato et al. 2003). Plants have evolved several photoprotective mechanisms that reduce excess energy. It has been suggested that consumption of reducing power by photosynthetic CO 2 fixation (Powles 1984), photorespiration (Powles and Osmond 1979), heat dissipation via the xanthophyll cycle (Demmig- Adams and Adams 1992, Gilmore 1997), the water water cycle (Osmond and Grace 1995, Asada 1999, Makino et al. 2002) and by cyclic electron flow around PSII (Miyake and Yokota 2001) contribute to photoprotection of PSII. Furthermore, fast recovery of photoinactivated PSII via degradation and re-synthesis of damaged D1 protein (Aro et al. 1993a, Aro et al. 1993b) also contributes to maintaining the fraction of functional PSII in leaves. The susceptibility to photoinhibition depends on temperature. A number of studies have shown that leaves are more susceptible at low temperatures (Powles 1984, Osmond 1994, Huner et al. 1993, Huner et al. 1998, Allen and Ort 2001). It has been suggested that temperature dependence in recovery of inactivated PSII is related with the higher susceptibility to photoinhibition at lower temperatures (Allen and Ort 2001). The rate of recovery is often comparable to that of photoinactivation in PSII at normal temperatures (Wünschmann and Brand 1992, Aro et al. 1993b, Lee et al. 2001, Kato et al. 2002b), but is very low at low temperatures (Greer et al. 1986, Aro et al. 1990a, Salonen et al. 1998), suggesting that low temperatures accelerate photoinhibition through the suppression of the recovery. However, it has been indicated that the susceptibility to photoinhibition cannot be accounted for solely by a differential capacity for repair of inactivated PSII (Huner et al. 1993). For example, the recovery rate of inactivated PSII is not correlated with the susceptibility to photoinhibition when species with various chilling-sensitivity were compared (Öquist and Huner 1991). Hurry and Huner (1992) also showed that, even when recovery of inactivated PSII is inhibited, cold-hardened ; 3 Corresponding author: , hikosaka@mail.tains.tohoku.ac.jp; Fax,

2 Temperature dependence of photoprotective processes 829 wheat was still more tolerant to photoinhibition than non-hardened wheat. It has been proposed that photoinhibition is related to the redox state of PSII (Öquist et al. 1992, Huner et al. 1993, Huner et al. 1998). Low temperature inhibits consumption of light energy by photosynthesis and photorespiration, and thus absorbed light energy likely becomes excessive, presumably leading to over-reduction in PSII. Susceptibility to photoinhibition has been shown to be correlated with the redox state of PSII, regardless of the environmental constraints on photosynthesis caused by low temperature or light acclimation (Ögren and Rosenqvist 1992, Öquist et al. 1992, Gray et al. 1996). This suggests that reduction in the consumption of light energy is responsible for higher susceptibility to photoinhibition under lower temperatures. Although knowledge is accumulating on photoprotection at low temperature, it is still unclear what photoprotective process is quantitatively responsible for temperature dependence of the susceptibility to photoinhibition. When there exist plural photoprotective processes, a strong correlation between photoinhibitory susceptibility and activity of one process does not necessarily mean that contribution of this process is quantitatively large. We have demonstrated that quantitative contribution of photoprotective mechanisms to photoinhibitory susceptibility varies among leaves grown under different conditions (Kato et al. 2002b, Kato et al. 2003). We raised Chenopodium album plants under high-light and high-nitrogen (HL-HN), high-light and low-nitrogen (HL-LN), and low-light and highnitrogen (LL-HN) conditions. The HL plants were more tolerant to photoinhibition than the LL-HN plants. Although the HL plants had a higher recovery rate of inactivated PSII irrespective of nitrogen availability, there was still a difference in the rate of photoinactivation in the presence of an inhibitor of the PSII recovery between the HL and LL plants (Kato et al. 2002b). We further estimated partitioning of light energy to photochemistry, heat dissipation and excess energy using the model of Demmig-Adams et al. (1996). The HL-HN plants had the highest rate of photochemistry and the HL-LN plants had greater heat dissipation fraction, both contributing to reduction in excess energy. The LL-HN plants had the lower rate of photochemistry and the smaller fraction of heat dissipation, leading to the greatest excess energy (Kato et al. 2003). Although there was no single photoprotective process that solely explained the tolerance to photoinhibition, the rate constant of photoinactivation without recovery was strongly correlated with the excess energy that is neither utilized in photosynthesis nor dissipated as heat, suggesting that the excess energy determines the rate of photoinactivation (Kato et al. 2003). These facts suggest that the tolerance to photoinhibition results from a combination of various photoprotective mechanisms. The aim of the present study was to evaluate quantitative contribution of photoprotective mechanisms to photoinhibitory susceptibility under various temperatures. C. album plants were grown under HL-HN conditions, and their leaves were exposed Fig. 1 Temperature dependence of the maximum quantum yield of PSII photochemistry in the dark (F v /F m ) (a) and photosynthetic rates at 1,000 mol quanta m 2 s 1 under ambient CO 2 partial pressure (b) for C. album leaves. Means and SE of eight replications are shown. to various temperatures under strong light. Using the methods of Kato et al. (Kato et al. 2002b, Kato et al. 2003), temperature dependence of the susceptibility to photoinhibition, photosynthetic rates, the rate constant of photoinactivation and recovery, and of partitioning of absorbed light energy was determined. We discuss how temperature affects the susceptibility to photoinhibition. Results C. album plants were grown at 25/20 C (day/night) under 500 mol quanta m 2 s 1 with sufficient nutrient supply (see Materials and Methods). Fig. 1a shows the maximal quantum yield of photochemistry (F v /F m ) of leaves adapted to the dark at a given temperature. F v /F m was higher than 0.8 at all temperatures, suggesting that the maximal quantum yield is less sensitive to temperatures if leaves are in the dark. Fig. 1b shows the photosynthetic rate at 1,000 mol quanta m 2 s 1. The photosynthetic rate was highest at 25 C. In the present study we defined photoinhibition as the reduction of F v /F m as there was a linear relationship between quantum yield of CO 2 assimilation and F v /F m (data not shown). At all temperatures F v /F m decreased with illumination time in the absence of lincomycin (Fig. 2). This reduction was mitigated with time and F v /F m reached in a near-equilibrium level after about 300 min. Such an equilibration to a steady-state level of active PSII can be expected if a first-order degradation

3 830 Temperature dependence of photoprotective processes Fig. 2 Photoinactivation of PSII (indicated by relative reduction in F v /F m ) measured in the presence (open circles) and absence (closed circles) of the chloroplast-encoded protein synthesis inhibitor, lincomycin, for C. album leaves illuminated with 900 1,000 mol quanta m 2 s 1 at different temperatures. Fitted curves are a = {k rec + k pi exp[ (k pi + k rec )t]}(k pi + k rec ) 1 (continuous line) and a = exp( k pi t) (broken line), where a is the fraction of active PSII and t is the illumination time. See Fig. 3 for values of k pi and k rec. a f represent results obtained at 11, 15, 20, 25, 30, and 35 C, respectively. is counteracted by a first-order recovery reaction during highlight treatment (Tyystjärvi et al. 1992). At higher temperatures (25 30 C) the F v /F m values at 300 min decreased only by 10% from the initial values while larger decreases were found at lower temperatures. Photoinactivation can be revealed only when the rate of de novo synthesis of D1 protein is inhibited. Lincomycin was used to inhibit the D1 protein synthesis. It is known that lincomycin exacerbates photoinhibition without causing any other effects (Tyystjärvi et al. 1992, Aro et al. 1993b). The reduction of F v /F m was thus greater in lincomycin-treated leaves than in untreated ones. Temperature dependence of the reduction of F v /F m in lincomycin-treated leaves was not remarkable, in contrast to that in untreated leaves. We assumed that reduction in the fraction of active PSII with illumination time follows a model of two opposing firstorder reactions with different rate constants for photoinactivation (k pi ) and concurrent recovery (k rec ). Assuming that the rates of photoinactivation and recovery are proportional to the concentration of active and inactivated PSII, respectively, the fraction of active PSII (a) is expressed as: a = {k rec + k pi exp[ (k pi + k rec )t]}(k pi + k rec ) 1, (1) (see Tyystjärvi et al. 1992, Kato et al. 2002b), where t is illumination time (min). If lincomycin is added, k rec becomes zero. Then Eqn. 1 is simplified as: a = exp( k pi t). (2) First, k pi was determined by applying Eqn. 2 to a time-course of F v /F m during illumination in the presence of lincomycin. With the k pi value, then k rec was obtained from a time-course of F v /F m during illumination in the absence of lincomycin by Eqn. 1. Fitted curves of Eqn. 1 and 2 are shown in Fig. 2. Calculated rate constants (k pi and k rec ) were plotted against temperature (Fig. 3a, b). The curve of the rate constant of photoinactivation (k pi ) had weak temperature dependence; it was similar at higher temperatures (25 35 C) and increased by 50% with temperature decline from 25 to 11 C. The rate constant of recovery (k rec ) showed a strong temperature dependence; it increased by an order of magnitude with temperature and reached a maximum at about C. Fig. 3c shows the fraction of active PSII at the equilibrium calculated from Eqn. 1 (t >10,000 min). At 11 C, half of PSII were inactive at the equilibrium because the values of k pi and k rec were similar to each other. At higher temperatures, the fraction of active PSII

4 Temperature dependence of photoprotective processes 831 Fig. 3 Temperature dependence of the rate constants for photoinactivation, k pi (a), and recovery, k rec (b), and the fraction of active PSII at the equilibrium (c) in C. album leaves illuminated with 1,000 mol quanta m 2 s 1 calculated according to Tyystjärvi et al. (1992). See text for the fraction of active PSII at the equilibrium. Fig. 4 Temperature dependence of the quantum yield of PSII photochemistry in the light ( F/F m.) (a), the fraction of open PSII (q P ) (b), the quantum yield of open PSII (F v /F m.) (c), and NPQ (d) in C. album leaves at 1,000 mol quanta m 2 s 1. Means and SE of eight replications are shown. is greater due to higher values of k rec. Fig. 4 shows the quantum yield of photochemistry in the light ( F/F m ) as the product of the fraction of open PSII (q P ) and the quantum yield of open PSII (F v /F m ) (Genty et al. 1989). F/F m (Fig. 4a) and q P (Fig. 4b) increased with increasing temperature at low temperatures and had stable values at C. F v /F m also tended to be higher at higher temperatures but its temperature dependence was smaller than those of F/F m and q P (Fig. 4c). Conversely, non-photochemical quenching (NPQ) decreased and showed a minimum at about 30 C (Fig. 4d). We used these data to estimate partitioning of light energy to various pathways as a function of temperature (Fig. 5). If PSII could completely utilize absorbed photon energy for photochemistry, the maximum quantum yield in the dark (F v /F m ) and in the light (F v /F m ) should be 1.0 (Demmig-Adams et al. 1996). The difference between 1 and F v /F m (L) can result from some form of energy loss in the dark. F v /F m F v /F m defines the fraction of the variable heat dissipation in the light (D) (Demmig-Adams et al. 1996). P is the fraction of photochemical electron transport defined by F/F m (Genty et al. 1989). The fraction of absorbed light neither going into photochemical electron transport nor heat dissipation defines excess energy (E) and is given by 1 L D P (Demmig-Adams et al. 1996, Kato et al. 2003). The fraction of energy consumed by photochemistry decreased by a half as temperature decreases from 35 to 10 C (Fig. 5). The fraction of heat dissipation increased in a counteracting manner, which partly contributed to decrease in the excess energy. However, the decrease in photochemistry was larger than that of heat dissipation, resulting in doubling of excess energy as temperature decreases from 35 C to 10 C. To test the hypothesis that the rate of photoinactivation depends on the excess light energy we plotted the rate constant of photoinactivation (k pi ) against excess light energy (Fig. 6). Excess light

5 832 Temperature dependence of photoprotective processes Fig. 5 Temperature dependence of the fractions of absorbed light in PSII that is lost in the dark (L), that is dissipated as heat (D), that is utilized in photochemistry (P) in C. album leaves at 1,000 mol quanta m 2 s 1. The fraction of absorbed light neither going into P, D, nor L defines excess (E). Means and SE of eight replications are shown. energy gave a strong linear relationship with the k pi value. Discussion As it has been known in various plant species, strong temperature dependence was found in the susceptibility to photoinhibition in C. album leaves (Fig. 2). The fraction of active PSII at the equilibrium was found to be lowest (49%) at 11 C while it was about 90% at C (Fig. 3c). However, when the repair of D1 protein is blocked, the photoinactivation was found at all temperatures in a range of C. Although the rate constant of photoinactivation (k pi ) was found to be temperature dependent, the difference was only 50% (Fig. 3a). On the other hand, the rate constant of recovery (k rec ) was highly sensitive to temperature. The value of k rec was similar to that of k pi at 11 C, leading to 49% of the fraction of active PSII at the equilibrium (Fig. 3c) and increased by an order of magnitude from 11 to 35 C (Fig. 3b). To evaluate their quantative contribution to photoprotection, we conducted a simple sensitivity analysis. Leaves photoinhibited at 25 C had the k pi and k rec values of and min 1, respectively, resulting in the fraction of active PSII of 89% at the equilibrium. When we replace the k pi value by , which was observed at 11 C, the fraction of active PSII is calculated to be 85%. When we similarly replace the k rec value by , which was observed at 11 C, the fraction greatly decreases to 60%. Thus the temperature dependence of recovery of inactivated PSII was most responsible for the susceptibility to photoinhibition. Inactivated PSII is recovered in the following processes: inactivated PSII complex is transferred from the appressed region to the non-appressed region of thylakoid membranes Fig. 6 Relationship between the rate constant of photoinactivation (k pi ) and the excess energy (E). Data from Fig. 3 and 5 are used. and then is repaired after degradation and de novo synthesis of D1 protein (Aro et al. 1993a). It was shown that low temperature markedly reduces the rate of enzymatic PSII repair processes and a decrease in membrane fluidity limits the migration of inactive PSII complexes between stroma and grana thylakoids (Aro et al. 1990b, Baroli and Melis 1998, Salonen et al. 1998). Nishiyama et al. (2001) suggested that oxidative stress inhibits the repair of inactivated PSII. These factors might cause the large decrease in k rec at lower temperatures. Although temperature dependence of k pi was smaller than that of k rec, temperature had a significant effect on the rate of photoinactivation: k pi increased by 50% as temperature declines from 25 to 11 C (Fig. 3a). Decrease in the quantum yield of PSII photochemistry in the light ( F/F m ) was partly related to the increase in the rate of photoinactivation at low temperatures (Fig. 4). The reducing equivalents produced by photochemistry are consumed by photosynthetic CO 2 fixation, photorespiration, the PSII cyclic electron transport, and the water water cycle. Temperature dependence of the photosynthetic rate, which had a maximum at 25 C, was slightly different from that of F/F m. In C 3 plants, inhibition of photosynthetic rates at moderately high temperature is usually attributed to an increase in the ratio of oxygenation/carboxylation activities in ribulose-1,5-bisphosphate carboxylase (RuBPCase). As temperature increases, the ratio of dissolved O 2 /CO 2 and the specificity of RuBPCase for O 2 increases, thus favoring oxygenase activity (Sage and Sharkey 1987, Crafts-Brandner and Salvucci 2002). Therefore, at higher temperatures photorespiration is suggested to have larger contribution to photoprotection. On the other hand, low temperatures suppress the activity of many photosynthetic processes (Berry and Björkman 1980). Many authors have indicated that electron transport in the thylakoid membranes is strongly temperature dependent (Armond et al. 1978, Kirschbaum and Farquhar 1984, Laisk and Oja

6 Temperature dependence of photoprotective processes , Hikosaka et al. 1999). Thus the electron transport may mainly explain temperature dependence of F/F m at lower temperatures. In contrast to F/F m, NPQ and 1 F v /F m were found to be higher at lower temperatures (Fig. 4). This suggests that leaves could dissipate more energy as heat at lower temperatures. However, according to the model of Demmig-Adams et al. (1996), the fraction of energy dissipated as heat increased only by 10% from 25 to 10 C, which was smaller than the decrease in the fraction of energy consumed in photochemistry (30%) (Fig. 5). Thus, although heat dissipation contributed to photoprotection at low temperatures, its contribution was not sufficient to fully compensate for the reduction in the electron transport activity. The excess energy (E) that is neither consumed by photochemistry nor dissipated as heat increased with decreased temperature (Fig. 5). k pi was positively correlated with E, suggesting that the excess energy determines the rate of photoinactivation. This is consistent with our previous finding that k pi is proportional to E irrespective of photoinhibitory irradiance and growth conditions (Kato et al. 2003). E can also be expressed as (1 q P ) F v /F m (Demmig-Adams et al. 1996). This is the excitation energy absorbed by closed PSII, which was not dissipated by 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 centre by 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 centre 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. It should be noted that the k pi -E relationship was a linear function from the origin in the study of Kato et al. (2003), while it had a positive Y-intercept in the present study (Fig. 6), suggesting that the effect of the excess energy on photoinactivation is relatively weak at lower temperatures. This may be explained partly by the re-activation of PSII without turnover of D1 protein. It is known that recovery after low temperature photoinhibition exhibits a rapid initial phase which seems to be independent of protein synthesis (Hurry and Huner 1992, Leitsch et al. 1994). This rapid recovery has been attributed to epoxidation of zeaxanthin via the xanthophyll cycle in leaves of several plant species (Thiele et al. 1996, Thiele et al. 1998, Jahns and Miehe 1996, Xu et al. 2000). Huner et al. (1993) speculated that a population of inactivated PSII can revert rapidly to active PSII at low temperatures through conformational changes. In the present study, recovery of inactivated PSII through processes other than protein synthesis was ignored. If the population of recovered PSII through such processes is large at low temperatures, the rate of photoinactivation might be underestimated. We can summarize the quantitative contribution of photoprotective mechanisms at different temperatures as follows. The energy utilized by photochemistry, which may be eventually consumed either by photosynthesis, photorespiration, the water water cycle, and the PSII cyclic flow, decreased as temperature reduces. The energy dissipated as heat partly increased with decreased temperature in a counteracting manner to the decrease in photochemistry, but the increase was insufficient to compensate for the reduction in photochemistry. As a result, the excess energy increased with decreased temperature, accelerating the rate constant of photoinactivation by 50%. The rate constant of recovery of inactivated PSII had stronger temperature dependence than the rate constant of photoinactivation, indicating that the rate of recovery was the factor most responsible for temperature dependence of the susceptibility to photoinhibition. One may consider that the present result contradicts previous results suggesting that reduction in consumption of light energy, especially by photosynthesis, is responsible for higher susceptibility to photoinhibition (Öquist et al. 1992, Huner et al. 1993, Huner et al. 1998, Gray et al. 1996). However, these previous authors did not necessarily evaluate quantitative contribution of photoprotective mechanisms. Consider a situation where two leaves have similar rate constants of recovery to each other but have different photosynthetic capacities, leading to different susceptibilities to photoinhibition. There will be a correlation between the susceptibility and photosynthetic capacity irrespective of contribution of the recovery. We stress that quantitative evaluation is indispensable to clarify how tolerance to photoinhibition is determined, because it results from a combination of various photoprotective mechanisms. Materials and Methods C. album L. is a broad-leaved summer annual, which colonizes disturbed habitats. Leaves of C. album do not exhibit apparent chloroplast movement (Oguchi et al. 2003). Plants were grown in a phytotron (14/10 h photoperiod, 25/20 C day/night temperature, 80% relative air humidity). Metal halide lamps (DR400/T; Toshiba, Tokyo, Japan) served as a light source. Two-week-old seedlings germinated in sand were transplanted to pots (1.5 liter volume) filled with sand and grown at a photosynthetic photon flux density of 500 mol quanta m 2 s 1. The standard hydroponic solution as described by Hikosaka et al. (1994) (12 mm N) was fed at 1 liter per pot. The solution was continually aerated and was renewed every week. Fully expanded young leaves (4 5) of 6-week-old plants were sampled. Leaves were cut with a sharp razor in water at a petiole length of cm, such treatment having been observed not to affect photosynthetic characteristics (Kato et al. 2002a). Four independent experiments were done. The first experiment was performed to study the role of chloroplast-encoded protein turnover in the susceptibility to photoinhibition at different temperatures (11, 15, 20, 25, 30, and 35 C). The petioles were soaked in water or lincomycin solution (0.7 mm) and the leaf laminae were exposed to 20 mol quanta m 2 s 1 for 2 h at 25 C. The leaves were placed in a temperature-controlled chamber with saturating humidity and illuminated through a glass window. Light (900 1,000 mol quanta m 2 s 1 ) was provided by metalhalide lamps. Air

7 834 Temperature dependence of photoprotective processes was passed through soda lime to remove CO 2 and mixed with 100% (v/v) CO 2 using mass-flow controllers (RK-150; Kojima, Utsunomiya, Japan). Then the air was passed through water whose temperature was regulated to adjust air humidity. The concentration of CO 2 in the entering air was adjusted such that the concentration in the outlet air was at 360 mol mol 1. Leaf temperature was measured with a copper-constantan thermocouple. Ten leaves were placed in the chamber and samples were taken every 40 or 60 min. The samples were kept in the dark for 40 min at the corresponding temperature and later initial fluorescence (F 0 ) and maximum fluorescence after a saturating pulse of light (F m ) were measured using a chlorophyll fluorescence measuring device (PAM-2000, Walz, Effeltrich, Germany). The fraction of active PSII was evaluated as F v /F m = (F m F o )/F m. In the second experiment simultaneous measurements of the rate of leaf net gas exchange and chlorophyll fluorescence were done using a portable photosynthesis measurement system with fluorescence measuring supplement (Li-6400, LiCor, Lincoln, NE, U.S.A.) to assess the different fates for absorbed light energy. Intact leaves were used. After 40 min adaptation of a plant to the corresponding temperature and to dark, the initial fluorescence (F o ) and maximum fluorescence after a saturating pulse of light (F m ) were measured. Later the light (1,000 mol quanta m 2 s 1, 10% blue light) was switched on. About 30 min were necessary for the photosynthetic rate to reach steady state. Then it was registered as well as the actual fluorescence level, F s. 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 to rapidly deoxidize the PSII centres. The quantum yield of open PSII was determined by F v /F m (Genty et al. 1989). The photochemical quenching coefficient (q P ) was calculated as (F m F s )/(F m F o ) (Harbinson et al. 1989, Schreiber et al. 1994). The quantum yield of PSII photochemistry was calculated as F/F m =(F m F s )/F m (Genty et al. 1989). The level of energy dissipation activity was estimated from the non-photochemical quenching of F m as F m /F m 1 (NPQ, see Bilger and Björkman 1990). Acknowledgment We thank R. Oguchi for his technical support to the study. This work was supported in part by fellowship No from Japan Society for Promotion of Science to TDT and by grants from the Ministry of Education, Science, Sports and Culture of Japan to KH. References Allen, D.J. and Ort, D.R. (2001) Impacts of chilling temperatures on photosynthesis in warm-climate plants. Trends Plant Sci. 6: Andersson, B. and Barber, J. (1996) Mechanisms of photodamage and protein degradation during photoinhibition of photosystem II. 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