Introduction. Keywords: A/Ci CO 2 Cyclic electron flow Non-photochemical quenching (NPQ) Photosynthesis PSI.

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1 Plant Cell Physiol. 46(4): (2005) doi: /pcp/pci067, available online at JSPP 2005 CO 2 Response of Cyclic Electron Flow around PSI (CEF-PSI) in Tobacco Leaves Relative Electron fluxes through PSI and PSII Determine the Magnitude of Non-photochemical Quenching (NPQ) of Chl Fluorescence Chikahiro Miyake 1, Momoko Miyata, Yuki Shinzaki and Ken-ichi Tomizawa Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto, Japan We hypothesized that cyclic electron flow around photosystem I (CEF-PSI) participates in the induction of non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence when the rate of photosynthetic linear electron flow (LEF) is electron-acceptor limited. To test this hypothesis, the relationships among photosynthesis rate, electron fluxes through both PSI and PSII [Je(PSI) and Je(PSII)] and Chl fluorescence parameters were analyzed simultaneously in intact leaves of tobacco plants at several light intensities and partial pressures of ambient CO 2 (Ca). At low light intensities, decreasing Ca lowered the photosynthesis rate, but Je(PSI) and Je(PSII) remained constant. Je(PSI) was larger than Je(PSII), indicating the existence of CEF-PSI. Increasing the light intensity enhanced photosynthesis and both Je(PSI) and Je (PSII). Je(PSI)/Je(PSII) also increased at high light and at high light and low Ca combined, showing a strong, positive relationship with NPQ of Chl fluorescence. These results indicated that CEF-PSI contributed to the dissipation of photon energy in excess of that consumed by photosynthesis by driving NPQ of Chl fluorescence. The main physiological function of CEF-PSI in photosynthesis of higher plants is discussed. Keywords: A/Ci CO 2 Cyclic electron flow Non-photochemical quenching (NPQ) Photosynthesis PSI. Abbreviations: Ca, ambient CO 2 ; CEF-PSI, cyclic electron flow around PSI; Ci, intercellular partial pressure of CO 2 ; Je(PSI)/(PSII), electron fluxes through PSI/PSII; LEF, linear electron flow; LHC, light-harvesting complex; NPQ, non-photochemical quenching; PCO, photorespiratory carbon oxidation; PCR, photosynthetic carbon reduction; Φ(PSI)/(PSII), quantum yield of PSI/PSII; Rubisco, ribulose-1,5- bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate. Introduction Photon energy in excess of that consumed by photosynthetic CO 2 assimilation causes photoinhibition of photosystem II (PSII) in thylakoid membranes (Asada 1999, Kato et al. 2003, Miyake and Okamura 2003). Photon energy absorbed by chloroplasts is transformed in thylakoid membranes to the biochemical energy, NADPH and ATP, required for CO 2 fixation ; by the Calvin cycle. These transformations become less efficient when CO 2 supply to ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) or regeneration of RuBP by the Calvin cycle limits photosynthesis. Under these conditions, the photosynthetic electron transport system becomes filled up with electrons, due to insufficient regeneration of both NADP + and ADP (Melis 1999, Niyogi 2000, Ort and Baker 2002). As a result, charge separation of photoexcited PSII reaction center chlorophyll (Chl), P680, is suppressed, leading to accumulation of excited triplet P680, 3 P680* (Hideg et al. 1994a, Hideg et al. 1994b, Hideg et al. 1998). The 3 P680* reacts rapidly with O 2, producing reactive, singlet O 2 ( 1 O 2 ). The 1 O 2 oxidatively degrades the PSII reaction center protein, D 1 protein, and inactivates PSII. Furthermore, under conditions of limited photosynthetic linear electron flow (LEF), photoexcited Chl, 1 Chl*, in light-harvesting complex II (LHCII) protein cannot transfer excitons to P680, resulting in accumulation of 3 Chl*. As in the PSII reaction center, 1 O 2 then accumulates in thylakoid membranes (Macpherson et al. 1993, Mishra et al. 1994, Miyao 1994). Thylakoid membranes are rich in unsaturated fatty acids, which are very susceptible to oxidative attack by 1 O 2. The production of 1 O 2 in the membranes stimulates peroxidation of the fatty acids and degrades the thylakoid membrane bilayers (Asada 1996). Therefore, excess photoexcitation of P680 needs to be suppressed to protect PSII from photoinhibition. To suppress PSII photoinhibition, plant chloroplasts have a protection mechanism by which excess photon energy is dissipated as heat, as observed in non-photochemical quenching (NPQ) of Chl fluorescence (Demmig-Adams and Adams 1996, Niyogi et al. 1998, Pogson et al. 1998, Niyogi 1999, Yamamoto et al. 1999, Niyogi 2000, Pogson and Rissler 2000, Müller et al. 2001). In NPQ of Chl fluorescence, the xanthophyll cycle enzyme violaxanthin de-epoxidase is activated by acidification of the thylakoid lumen space and catalyzes the de-epoxidation of violaxanthin to zeaxanthin (Eskling et al. 2001). Zeaxanthin accepts excitons directly from LHCII protein 1 Chl* and dissipates the exciton energy as heat (Niyogi 2000). Furthermore, zeaxanthin reacts directly with 1 O 2 and dissipates its energy as heat (Müller et al. 2001). Zeaxanthin also stabilizes membrane bilayer structures and helps maintain the integrity of oxidatively damaged thylakoid membranes (Yamamoto et al. 1999). As described above, the induction of NPQ of Chl fluorescence requires a ph gradient ( ph) across the thylakoid mem- 1 Corresponding author: , cmiyake@rite.or.jp; Fax,

2 630 CO 2 response of cyclic electron flow around PSI branes. At present, cyclic electron flow around PSI (CEF-PSI) is proposed to function in the induction of NPQ of Chl fluorescence (Heber and Walker 1992, Heber et al. 1992, Heber et al. 1995, Miyake et al. 1995, Cornic et al. 2000, Heber 2002). CEF-PSI is mainly composed of two pathways, one ferredoxin (Fd)-quinone oxidoreductase (FQR) dependent (Arnon et al. 1954, Arnon 1959, Arnon et al. 1967, Arnon and Chain 1975, Miyake et al. 1995, Munekage et al. 2002, Munekage et al. 2004) and the other NAD(P)H-dehydrogenase (NDH) dependent (Mi et al. 1992a, Mi et al. 1992b, Asada et al. 1993, Mi et al. 1995). We need to define the relationship between CEF-PSI activity and NPQ of Chl fluorescence to elucidate how CEF- PSI contributes to the dissipation of excess photon energy under steady-state photosynthesis conditions. In our previous report (Miyake et al. 2004), we found that both CEF-PSI and NPQ were induced under high light/co 2 -saturated conditions, and that their activities were positively correlated. Under natural conditions, plants are exposed to many abiotic stresses which suppress photosynthesis, including drought, chilling and high light (Asada 1996). In these situations, LEF is limited by the supply of CO 2 to Rubisco. Therefore, it is expected that CEF-PSI would be enhanced and would induce NPQ of Chl fluorescence. In the present work, we further tested the hypothesis that CEF-PSI functions in the induction of NPQ of Chl fluorescence when LEF is limited by the availability of electron acceptor. We estimated the activity of CEF- PSI in tobacco leaves from a simultaneous analysis of PSII and PSI quantum yields at several partial pressures of CO 2 and several light intensities. At low CO 2, we found that electron flux through PSI [Je(PSI)] remained higher than that through PSII [Je(PSII)], which resulted in a high Je(PSI)/Je(PSII). Furthermore, Je(PSI)/Je(PSII) showed a strong, positive relationship with NPQ of Chl fluorescence at low CO 2. These results strongly support our hypothesis. Results Fig. 1 Influence of light intensity on the net CO 2 assimilation rate as a function of partial pressure of both ambient CO 2 (Ca) and intercellular CO 2 (Ci) in a tobacco leaf. The net CO 2 assimilation rates were measured at 21 kpa O 2 and the indicated Ca, and at light intensities of 150 (open circle), 250 (open square) and 1,100 (open diamond) µmol photons m 2 s 1, and plotted against Ca (A) and Ci (B). In (A), data are the average of 3 6 experiments using two leaves attached to tobacco plants. Vertical bars represent the SDs of the measurements. Effects of CO 2 and light intensity on photosynthesis in tobacco leaves To study the effect of limiting the supply of electron acceptor for LEF on the activity of CEF-PSI, the CO 2 assimilation rate of tobacco leaves was measured at several partial pressures of ambient CO 2 (Ca) and light intensities (Fig. 1). At light intensities of 150, 250 and 1,100 µmol photons m 2 s 1 and a partial pressure of ambient O 2 of 21 kpa, an increase in Ca stimulated photosynthesis (Fig. 1A). In the range of Ca we set, each increase in light intensity increased the CO 2 assimilation rate. CO 2 assimilation rates showed a similar dependence on intercellular partial pressure of CO 2 (Ci) (Fig. 1B). Effects of CO 2 and light intensity on the electron fluxes in both PSI and PSII To study the effects of both Ca and light intensity on the activity of CEF-PSI, the quantum yields of both PSI and PSII, Φ(PSI) and Φ(PSII), were measured simultaneously. From these measurements, Je(PSI) and Je(PSII) were estimated at each light intensity (see Materials and Methods) and plotted against Ca (Fig. 2A, B, C). To determine the activity of CEF- PSI, Je(PSI)/Je(PSII) was plotted against Ca (Fig. 2A, B, C). In the ranges of light intensity and Ca we set, it was found that Je(PSI) was larger than Je(PSII). These results indicated that CEF-PSI was functioning. Both Je(PSI) and Je(PSII) increased

3 CO 2 response of cyclic electron flow around PSI 631 Fig. 2 The effect of light intensity on the electron fluxes through both PSI and PSII, and on Je(PSI)/Je(PSII) as a function of Ca. The electron fluxes through both PSI [Je(PSI)]and PSII [Je(PSII)], and Je(PSI)/Je(PSII) were determined simultaneously with the net CO 2 assimilation rate (see Materials and Methods) at 21 kpa O 2 and the indicated Ca, and at light intensities of 150 (A), 250 (B) and 1,100 (C) µmol photons m 2 s 1, and plotted against Ca. The left axis shows both Je(PSI) (open circles) and Je(PSII) (open squares), and the right axis, Je(PSI)/Je(PSII) (open diamonds). Data are the average of 3 6 experiments using two leaves attached to tobacco plants. Vertical bars represent the SDs of the measurements. with increasing light intensity, and Je(PSI)/Je(PSII) also increased. However, at light intensities of 150 and 250 µmol photons m 2 s 1, these electron fluxes did not show any dependence on Ca (Fig. 2A, B), indicating that LEF was limited by the supply of photons to the photosynthetic electron transport system (Makino and Mae 1999). On the other hand, at 1,100 µmol photons m 2 s 1, both Je(PSI) and Je(PSII) increased further, compared with the lower light intensities, giving a high Je(PSI)/Je(PSII) (Fig. 2C). Lowering Ca below 60 Pa decreased both Je(PSI) and Je(PSII), indicating that LEF was limited by the efficiency of RuBP carboxylation by Rubisco (Makino and Mae 1999). Je(PSI) and Je(PSII) decreased to almost the same extent (Fig. 2C). Even at low Ca, the activity of CEF-PSI [Je(CEF-PSI) = Je(PSI) Je(PSII)] was maintained. As a result, Je(PSI)/Je(PSII) increased from 1.4 to 1.6 as Ca was decreased. These results indicated that CEF-PSI was induced at high light and functioned even at low Ca (Fig. 2C). Relationship between NPQ of Chl fluorescence and CEF-PSI To elucidate the contribution of CEF-PSI to the induction of NPQ of Chl fluorescence, NPQ and the oxidation state of P700 were measured at several light intensities and Cas (Fig. 3A, B). At light intensities of 150 and 250 µmol photons m 2 s 1, NPQ of Chl fluorescence was low and it showed little dependence on Ca. On the other hand, at 1,100 µmol photons m 2 s 1, NPQ was higher than at lower light intensities and showed Ca dependency, similar to Je(PSI)/Je(PSII). When Ca was decreased from 100 to 10 Pa, NPQ of Chl fluorescence increased from 1.4 to >2.5. Previously, we showed that under CO 2 -saturated conditions, increased light intensity induced NPQ and oxidation of P700 in PSI (Miyake et al. 2004). The oxidation of P700 showed a positive relationship with NPQ. In the present study, we plotted both [P700 + ] and [reduced P700] (see Materials and Methods) against Ca at 1,100 µmol photons m 2 s 1, where NPQ was induced (Fig. 3B). A decrease in Ca induced the oxi-

4 632 CO 2 response of cyclic electron flow around PSI Fig. 3 (A) The effect of light intensity on NPQ of Chl fluorescence as a function of Ca. Values for NPQ of Chl fluorescence were calculated from the data in Fig. 2 (see Materials and Methods) and plotted against Ca. (B) The effect of Ca on the oxidation state of P700 at 1,100 µmol photons m 2 s 1. [reduced P700], [P700 + ] and [reduced P700] + [P700 + ] were obtained in the same experiments shown in Fig. 2 (see Materials and Methods) and plotted against Ca. [P700] total = 221 ± 13 (rel. val.) (n = 3 6). (C) NPQ of Chl fluorescence determined in Fig. 2 was plotted against Je(PSI)/Je(PSII). Symbols are 150 (open circle), 250 (open square) and 1,100 (open diamond) µmol photons m 2 s 1. Data are the average of 3 6 experiments using two leaves attached to tobacco plants. Vertical and horizontal bars represent the SDs of the measurements. dation of P700. On the other hand, reduced P700 decreased, indicating that Je(PSI) decreased (Fig. 2C). The sum of both [P700 + ] and [reduced P700], [reduced P700] + [P700 + ], was constant against the change in Ca, showing that the total amount of P700 did not change. Similar results were also obtained in cucumber, pea, sunflower and spinach (data not shown). We conclude that, in these leaves, PSI functioning in LEF would also participate in CEF-PSI. However, Golding and Johnson (2003) reported that at lower Ca, the total amount of P700 increased and claimed that the additional P700 mainly functioned in CEF-PSI. For now, the reason for these discrepancies is unknown, and remains to be clarified in the future. When NPQ of Chl fluorescence was plotted against Je(PSI)/Je(PSII) (Fig. 3C), NPQ showed a biphasic dependence on Je(PSI)/Je(PSII). At Je(PSI)/Je(PSII) <1.3, NPQ showed little dependence on Je(PSI)/Je(PSII). However, above this value, NPQ drastically increased. These results coincided well with the results of Miyake et al. (2004). In our previous work, we suggested that the biphasic dependence of NPQ on Je(PSI)/Je(PSII) reflects the requirement for CEF-PSI to decrease the luminal ph to a value low enough for NPQ to operate (Miyake et al. 2004). For example, ascorbate, the electron donor for violaxanthin de-epoxidase, has a pka of 4.1 and must be in the acid form to support NPQ (Bratt et al. 1995, Jahns and Heyde 1999, Cornell and Northwood 2000). Furthermore, violaxanthin de-epoxidase localized in the lumen must bind to the thylakoid membranes to catalyze the conversion of violaxanthin to zeaxanthin. For this binding to occur, luminal ph must be <6 to allow protonation of the many acidic amino acids in the C-terminal region of the protein (Bratt et al. 1995, Jahns and Heyde 1999, Cornell and Northwood 2000, Eskling et al. 2001). However, we now con-

5 CO 2 response of cyclic electron flow around PSI 633 Fig. 4 Theoretical ratio of Je(PSI) to Je(PSII) required for net CO 2 assimilation at different φ values. To calculate Je(PSI)/Je(PSII), 4H + / ATP and Q cycle activity were assumed (see Discussion). Je(PSI)/ Je(PSII) was expressed as ( φ)/(2 + 2φ) 1/2 (Equation 5). The value of φ is the ratio of the rate of RuBP oxygenation to that of RuBP carboxylation by Rubisco. At high CO 2, where photorespiration does not function, φ is zero. Under such conditions, Je(PSI) is equal to Je(PSII) and, theoretically, the activity of CEF-PSI is not required. An increase in φ means that the proportion of the photosynthetic linear electron flux diverted to the PCO cycle increases and Je(PSI)/Je(PSII) increases. At φ = 2, and where the net CO 2 assimilation rate is zero, i.e. the CO 2 compensation point, Je(PSI)/Je(PSII) reaches In the present work, measured Je(PSI)/Je(PSII) was always larger than that calculated from Equation 5, at all Ca and light intensities (Fig. 2). Furthermore, Je(PSI)/Je(PSII) increased with increasing light intensity and decreasing Ca. Under these conditions, photosynthetic linear electron flow was limited by the regeneration of NADP +, and NPQ of Chl fluorescence was induced. Therefore, CEF-PSI would function in the induction of NPQ of Chl fluorescence, in addition to the supply of ATP for net CO 2 assimilation. sider that sufficient CEF-PSI-dependent acidification occurs at Je(PSI)/Je(PSII) below 1.3 to drive the xanthophyll cycle. This is because Demmig-Adams and Adams (1996) have already reported that NPQ of Chl fluorescence showed a biphasic dependence on the de-epoxidation state of the xanthophyll cycle. These results implied that the de-epoxidation state had a linear relationship with the activity of CEF-PSI, i.e. the biphasic dependence of NPQ of Chl fluorescence on the activity of CEF-PSI reflects the efficiency with which the excess energy of excited Chl a in LHCII is dissipated as heat by zeaxanthin produced in the xanthophyll cycle. Discussion We found a strong, positive relationship between Je(PSI)/ Je(PSII) and NPQ of Chl fluorescence (Fig. 3C). When the net rate of CO 2 assimilation was limited by the rate of RuBP carboxylation by Rubisco in high light, each decrease in Ca lowered the net CO 2 assimilation rate, Je(PSI) and Je(PSII). However, decreased Ca enhanced Je(PSI)/Je(PSII), i.e. the activity of CEF-PSI increased, compared with that of LEF. As a result, CEF-PSI would acidify the thylakoid lumen and stimulate the xanthophyll cycle. In conclusion, these observations implied that the limited turnover of LEF enhanced the activity of CEF-PSI and led to induction of NPQ of Chl fluorescence. The enhancement of CEF-PSI activity at high light/low Ca would presumably be due to the shortage of electron acceptor for LEF (Fig. 2, 3). An increase in CEF-PSI activity was also observed previously when light intensity was increased under CO 2 -saturated conditions (Miyake et al. 2004). In this situation, increased light intensity stimulated the relative electron flux through PSII. However, further increases in light intensity led to saturation of the relative Je(PSII) but not Je(PSI), i.e. Je(PSI)/Je(PSII) increased even under CO 2 -saturated conditions, as was observed in the present work at Ci >60 Pa (Fig. 2). Under CO 2 -saturated conditions, NPQ of Chl fluorescence was dramatically induced after the relative Je(PSII) became saturated against light intensity, and its activity showed a positive relationship with Φ(PSI)/Φ(PSII) (Miyake et al. 2004). Under these conditions, the efficiency of RuBP regeneration decreased, leading to a decrease in the NADP + supply for LEF (Makino et al. 2002). As a result, reduced ferredoxin would accumulate at the acceptor side of PSI. Ferredoxin is a mediator for CEF-PSI. Addition of ferredoxin to thylakoid membranes stimulated the reduction of plastoquinone and the activity of CEF-PSI (Mano et al. 1995, Miyake et al. 1995). Therefore, in high light and/or low Ca, where the supply of electron acceptor to PSI limits LEF, CEF-PSI would be induced. Two physiological functions of CEF-PSI in higher plants have been proposed (Heber and Walker 1992). First, CEF-PSI would dissipate excess photon energy by inducing NPQ of Chl fluorescence (Niyogi 2000, Makino et al. 2002). In C3-plants, this physiological function of CEF-PSI has been verified in the present and previous works (Miyake et al. 2004). Secondly, CEF-PSI would supply ATP to drive net CO 2 assimilation (Farquhar and von Caemmerer 1982, von Caemmerer 2000). In net CO 2 assimilation, both photosynthesis and photorespiration occur. Under photorespiratory conditions, the amount of ATP produced in LEF does not meet the ATP requirement for driving net CO 2 assimilation (von Caemmerer 2000), i.e. CEF-PSI activity would be required as an electron flow that can produce a ph across thylakoid membranes and allow the synthesis of more ATP (von Caemmerer 2000). In both the photosynthetic carbon reduction (PCR) and photorespiratory carbon oxidation (PCO) cycles, the ratio of ATP to NADPH required for fixation of 1 mol of CO 2 is expressed as ( φ)/(2 + 2φ), where φ is the ratio of RuBP oxygenation rate to RuBP carboxylation rate by Rubisco (φ = Vo/Vc) (Farquhar and von Caemmerer 1982). When net CO 2 assimilation is at steady state, the rate of proton production at

6 634 CO 2 response of cyclic electron flow around PSI the lumen side [Vp(H + )lumen] equals the rate at which protons are consumed at the lumen side by the utilization of ATP in both the PCR and PCO cycles [Vc(H + )lumen] (Equation 1). That is, Vp(H + )lumen = Vc(H + )lumen (1) If only LEF produced ATP, both Vp(H + )lumen and Vc(H + )lumen could be represented as follows. Here, it is assumed that the Q cycle operates in photosynthetic electron transport and that four protons are required for the production of 1 mol ATP by chloroplastic ATP synthase (H + /ATP = 4) (Ruuska et al. 2000). Then, Vp(H + )lumen = n1 Je(PSII) (2) and Vc(H+)lumen = n2 Jg (3) where n1 (=3) is the ratio of H + production rate to electron flux in LEF with the Q cycle, and n2 [= 2( φ)/(2 + 2φ)] is the ratio of H + consumption rate to electron consumption rate in both the PCR and PCO cycles (Farquhar and von Caemmerer 1982, von Caemmerer 2000). Furthermore, the electron flux (Jg) to both the PCR and PCO cycles is equal to that through photosynthetic LEF, i.e. Je(PSII) (Cornic and Briantais 1991, Ruuska et al. 2000, von Caemmerer 2000). Therefore, Vp(H + )lumen is equal to Vc(H + )lumen only under non-photorespiratory conditions (φ = 0). On the other hand, under photorespiratory conditions (φ > 0), Vp(H + )lumen is lower than Vc(H + )lumen. These considerations suggest the requirement for an electron flow other than LEF, which produces ph across thylakoid membranes for net CO 2 assimilation. We studied whether CEF-PSI, along with LEF, could supply the ATP required for driving net CO 2 assimilation under photorespiratory conditions. If CEF-PSI functioned with LEF, Vp(H + )lumen can be shown as follows: Vp(H + )lumen = n1 Je(PSII) + n3 Je(CEF) (4) where n3 (=2) is the ratio of H + production rate to electron flux through CEF-PSI (von Caemmerer 2000) and Je(CEF-PSI) is the electron flux through CEF-PSI [= Je(PSI) Je(PSII)] (see Materials and Methods). From Equations 3 and 4, the ratio of Je(PSI) to Je(PSII) [Je(PSI)/Je(PSII)] required for maintaining net CO 2 assimilation is shown as follows (Equation 5): Je(PSI)/Je(PSII) = ( φ)/(2 + 2φ) 1/2 (5) We plotted Je(PSI)/Je(PSII) against φ (Fig. 4). An increase in φ means an enhancement of photorespiration. Under nonphotorespiratory conditions (φ = 0), for example at high CO 2, Je(PSI) is equal to Je(PSII). Under such conditions, no extra electron flux through PSI beyond that through LEF is required for net CO 2 assimilation, i.e. CEF-PSI activity is not required. As φ increases, Je(PSI)/Je(PSII) rises above 1, indicating the requirement for CEF-PSI activity for net CO 2 assimilation. At the CO 2 compensation point (φ = 2), where the rate of net CO 2 assimilation is zero, Je(PSI)/Je(PSII) reaches 1.16 (Fig. 4). In the present work, all measured values for Je(PSI)/ Je(PSII) exceeded the theoretical values calculated from Equation 5 (Fig. 2, 4). Even at high Ca (>80 Pa), where no photorespiration occurred (φ = 0), Je(PSI)/Je(PSII) was larger than about 1.2 at all light intensities (Fig. 1, 2). At about 10 Pa Ca, where the net CO 2 assimilation rate approached zero, Je(PSI)/ Je(PSII) was also larger than about 1.2, even at low light intensity (Fig. 1, 2). Furthermore, increasing the light intensity to 1,100 µmol photons m 2 s 1 enhanced Je(PSI)/Je(PSII) (Fig. 2). These new results support the hypothesis that CEF-PSI functions in the supply of ATP for net CO 2 assimilation. In addition, we wish to emphasize that CEF-PSI always had a higher activity than that required for net CO 2 assimilation, especially at high light intensity and low Ca (Fig. 2C). These results indicated that CEF-PSI contributed to the formation of ph across thylakoid membranes, leading to strong induction of NPQ of Chl fluorescence (Fig. 3A). We have analyzed the response of CEF-PSI to changes in partial pressure of CO 2 (in the present work), light intensity and temperature (Miyake et al. 2004). In all these cases, the activity of CEF-PSI was correlated with the induction of NPQ of Chl fluorescence. The value of NPQ depends largely on the growth conditions of the plants (Niyogi 2000). For example, leaves of plants grown at high light intensity showed a large NPQ of Chl fluorescence. The contribution of CEF-PSI to this large NPQ, i.e. the plasticity of CEF-PSI in response to the environment, should be examined to increase our understanding of the molecular mechanisms used by plants to recognize abiotic stress. Materials and Methods Plant growth conditions Wild-type tobacco plants (Nicotiana tabacum cv Xanthi) were grown from seed under standard air-equilibrated conditions with 30/ 25 C, light (16 h)/dark (8 h) cycles, 50 60% relative humidity and 700 µmol photons m 2 s 1 photon flux density. Seeds were sown in a 0.5 dm 3 pot containing commercial peat-based compost. Plants were watered daily and fertilized (Hyponex ; Hyponex Japan, Osaka, Japan) once a week. All measurements were made using the 11th fully expanded leaf 4 weeks after sowing. Measurements of photosynthetic parameters and collection of leaves were initiated 4 h after the start of the light period. CO 2 fixation, Chl fluorescence and P700 + absorbance CO 2 fixation and Chl fluorescence were measured simultaneously. P700 + absorbance was measured sequentially after Chl fluorescence measurement. All measurements were repeated at least three times using three different plants. The measurements were done over a leaf area of 6 cm 2 on leaves attached to the plant. The basal system of

7 CO 2 response of cyclic electron flow around PSI 635 gas exchange was adopted as previously detailed by Miyake and Yokota (2000), except that a LI-6400 (Li-Cor Inc., Lincoln, NE, U.S.A) was used as the IRGA. Leaf temperature was adjusted to 30.0 ± 0.5 C. The mixture of gases was saturated with water vapor at 18 ± 0.1 C, which corresponded to kpa. Irradiance from a halogen lamp (KL-1500; Walz, Effeltrich, Germany) was provided to the leaf chamber through glass-fiber optics that were linked to a PAM Chl fluorometer that is described below. Chl fluorescence was measured with the PAM Chl fluorometer through the same fiber-optics. The steady-state fluorescence yield (F s ) was monitored continuously and a 1,000 ms pulse of saturating light was supplied at intervals of 60 s to determine maximum variable fluorescence (F m ). The Φ(PSII) at the steady state was defined as (F m F s )/F m, as proposed by Genty et al. (1989). NPQ of Chl fluorescence was calculated as (F m /F m 1) according to Bilger and Bjorkman (1994). The absorbance of P700 + was measured with the same PAM Chl fluorometer by exchanging the Chl fluorescence detector unit for a ED-P700DW-E emitter-detector unit (Walz, Effeltrich, Germany) (Backhausen et al. 1998, Holtgrefe et al. 2003). The amplitude of full P700 oxidation was measured in the dark for each leaf before the illumination was started. In darkness, P700 is in its reduced state, and full oxidation of P700, [P700] total, was achieved by illumination with farred light (>700 nm), which excites only PSI. The oxidation of P700, [P700 + ], was monitored by the change in the A During illumination, the same amount of oxidizable P700 should be available, unless the PSI electron acceptors are already in their reduced state and cannot accept more electrons. During illumination, the fraction of reduced [reduced P700] or PSI acceptor (A ) is determined by short saturating light pulses, which give full oxidation of P700, followed by a dark pulse, which yields fully reduced P700. The difference between the P700 amplitude in the light- and the far-red-induced amplitude determined in the dark-adapted leaf must be attributed to A. The relative Φ(PSI) was calculated as described by Klughammer and Schreiber (1994), Φ(PSI) = [reduced P700]/[P700] total. Estimation of electron fluxes through both PSI and PSII Quantum yields of PSI and PSII, Φ(PSI) and Φ(PSII), were defined as follows: Φ(PSI) = Je(PSI)/(αI PFD) and Φ(PSII) = Je(PSII)/(αII PFD). Je(PSI) and Je(PSII) were the electron fluxes through PSI and PSII, respectively, PFD was the photosynthetically active photon flux density of the light illuminating the leaf, and αi and αii were the distribution ratio of that light to PSI and PSII, respectively. If the intensity of light illuminating the leaf was 1, then 1 = p + β where p was the fraction of the light absorbed by chloroplasts and β was the fraction not absorbed by the leaf. β included the reflectance and transmission of light by the leaf, which may be affected by chloroplast movement. Furthermore, if the intensity of light absorbed by chloroplasts was 1, then 1 = di + dii where di and dii were the distribution ratio of the light absorbed by the chloroplasts to PSI and PSII, respectively. Therefore, αi and αii were expressed, as follows: αi = p di and aii = p dii. The value of αii was determined under non-photorespiratory conditions, where Je(PSII) was derived from the stoichiometry of the Calvin cycle, as follows: Je(PSII) = αii Φ(PSII) PFD = 4 (A + Rd), where A was the net CO 2 assimilation rate and Rd was the day-respiration rate (Genty et al. 1989, Miyake and Yokota 2000, Ruuska et al. 2000, von Caemmerer 2000, Makino et al. 2002, Miyake et al. 2004). At 2 kpa O 2, we obtained a constant ratio of Φ(PSII) PFD to 4 (A+Rd) under all light intensities and Ca, and calculated the value of αii to be 0.44 ± 0.02 (n = 4) (Miyake et al. 2004). The fraction of light absorbed by chloroplasts in the tobacco leaves, p, was determined with an LI-1800 spectroradiometer and an S integrating sphere attachment (Li-Cor Inc.). For each leaf, both a reference scan and a sample scan of reflectance or transmittance were made from 400 to 700 nm at 1 nm intervals. Each sample scan was divided by its corresponding reference scan, and the results integrated over the wavelength range to obtain the average reflectance or transmittance (Chen and Cheng 2003). The value for p was calculated as: 1 reflectance transmittance, and equaled 0.86 ± 0.02 (n = 4). From the values of αii and p, and the above equation (αi = p αii), αi was calculated to be 0.42 ± 0.03 (n = 4). Then, using the following equations, we estimated both Je(PSI) and Je(PSII) from the measured values of Φ(PSI) and Φ(PSII) in tobacco leaves. Je(PSI) = 0.42 Φ(PSI) PFD. Je(PSII) = 0.44 Φ(PSII) PFD. If CEF-PSI functioned, Je(PSI) was larger than Je(PSII). In this case, the activity of CEF-PSI, Je(CEF-PSI), was calculated from Je(PSI) Je(PSII). Furthermore, Je(PSI)/Je(PSII) was also correlated with Je(CEF-PSI). A Je(PSI)/Je(PSII) ratio exceeding 1 showed that CEF-PSI was functioning. The constant value of αii indicated that αi was also constant. In turn, the constant ratio of αi/αii showed that di/dii was constant. From these results, we conclude that any change in di/dii by state transition was negligible. During state transition, reduction of the plastoquinone pool increased di/dii (Allen 1992, Finazzi et al. 1999, Haldrup et al. 2001, Wollman 2001, Finnazi et al. 2002). Such a change would enhance the absorption of light by PSI. 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