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1 Soil Science and Plant Nutrition ISSN: (Print) (Online) Journal homepage: O 2 -enhanced induction of photosynthesis in rice leaves: the Mehler-ascorbate peroxidase (MAP) pathway drives cyclic electron flow within PSII and cyclic electron flow around PSI Chikahiro Miyake, Yuji Suzuki, Hiroshi Yamamoto, Katsumi Amako & Amane Makino To cite this article: Chikahiro Miyake, Yuji Suzuki, Hiroshi Yamamoto, Katsumi Amako & Amane Makino (2012) O 2 -enhanced induction of photosynthesis in rice leaves: the Mehler-ascorbate peroxidase (MAP) pathway drives cyclic electron flow within PSII and cyclic electron flow around PSI, Soil Science and Plant Nutrition, 58:6, , DOI: / To link to this article: Published online: 06 Dec Submit your article to this journal Article views: 466 Citing articles: 2 View citing articles Full Terms & Conditions of access and use can be found at

2 Soil Science and Plant Nutrition (2012), 58, ORIGINAL ARTICLE O 2 -enhanced induction of photosynthesis in rice leaves: the Mehler-ascorbate peroxidase (MAP) pathway drives cyclic electron flow within PSII and cyclic electron flow around PSI Chikahiro MIYAKE 1,5, Yuji SUZUKI 2, Hiroshi YAMAMOTO 3, Katsumi AMAKO 4 and Amane MAKINO 2,5 1 Department of Biological and Environmental Science, Faculty of Agriculture, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan, 2 Department of Agriculture, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Japan, 3 Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, , Japan, 4 Faculty of Nutrition, Kobegakuin University, 518 Arise, Iyadani, Nishi, Kobe, Japan and 5 CREST, JST, 7 Gobancho, Chiyoda-ku, Tokyo , Japan Abstract Lowering the oxygen (O 2 ) partial pressure from 21 kpa to 1 kpa delayed the light-dependent increase of the net carbon dioxide (CO 2 ) assimilation rate in rice (Oryza sativa L. cv. Notohikari) leaves. Researching the underlying molecular mechanisms that act before the start of photosynthesis, we established the following facts. First, O 2 at 21 kpa enhanced the quantum yield of PSII [Y(II)] and PSI [Y(I)]. More than 90% of Y(II) and Y(I) were not accounted for by O 2 -dependent electron flow in the Mehler-ascorbate peroxidase (MAP) pathway. Both yields increased further with the start of photosynthesis. Second, O 2 enhanced photochemical quenching of chlorophyll (Chl) fluorescence (ql). ql also increased further with the rate of photosynthesis. Third, O 2 enhanced the photo-oxidation of P700. Fourth, O 2 suppressed the reduction of P700. Fifth, O 2 enhanced non-photochemical quenching of Chl fluorescence (NPQ). These results showed that the MAP pathway triggered cyclic electron flow within PSII (CEF-II) and cyclic electron flow around PSI (CEF-I) by inducing ph across thylakoid membranes and oxidizing the plastoquinone pool, before photosynthesis started. We propose that the photosynthetic electron transport system is controlled by the MAP pathway, which would explain the O 2 -dependent enhancement of the induction of photosynthesis. Key words: cyclic electron flow (CEF), Mehler-ascorbate peroxidase (MAP) pathway, O 2, rice, rubisco. INTRODUCTION In C3 photosynthesis, both the photosynthetic carbon reduction (PCR) cycle and the photorespiratory carbon oxidation (PCO) cycle are operating. The PCR cycle assimilates carbon dioxide (CO 2 ) to produce triose phosphate. The PCO cycle recovers carbon (C) that escaped from the PCR cycle due to the oxygenation of Correspondence: Chikahiro MIYAKE, Department of Biological and Environmental Science, Faculty of Agriculture, Graduate School of Agriculture Science, Kobe University, 1-1 Rokkodai, Nada, Kobe , Japan. Tel: Fax: þ cmiyake@hawk.kobe-u.ac.jp Received 24 June Accepted for publication 29 September ribuose-1,5-bisphosphate (RuBP) by RuBP carboxylase/ oxygenase (Rubisco), and funnels it back into the PCR cycle. The two cycles are driven by chemical energy stored in nicotinamide adenine dinucleotide phosphate (reduced form, NADPH) and adenosine triphosphate (ATP). NADPH is produced by the reduction of nicotinamide adenine dinucleotide phosphate (oxidized form, NADP þ ) catalyzed by ferredoxin (Fd)-NADP oxidoreductase (FNR) at the reducing side of photosystem I (PSI) in thylakoid membranes. Electrons for the reduction of NADP þ are supplied by the photosynthetic linear electron flow (PLEF). ATP is synthesized from adenosine diphosphate (ADP) and phosphate by the chloroplast H þ -ATPase which utilizes ph across thylakoid membranes as its energy source. ß 2012 Japanese Society of Soil Science and Plant Nutrition

3 O 2 triggers alternative electron flow 719 The ph across thylakoid membranes is generated by three electron transport reactions: PLEF, cyclic electron flow (CEF) around PSI (CEF-I; Johnson 2011; Joliot and Johnson 2011), and the Mehler-ascorbate peroxidase (MAP) pathway (the water-water cycle; Schreiber and Neubauer 1990; Asada 2000; Miyake 2010). In CEF-I, photoreduced Fd returns electrons to plastoquinone (PQ) through at least two carriers (Kubo et al. 2011, Yamamoto et al. 2011): Fd-quinone oxidoreductase (FQR) and NADH-dehydrogenase (NDH). The Q cycle in the Cyt b 6 /f-complex contributes to the generation of ph. In the MAP pathway, oxygen (O 2 ) is photoreduced by PSI to superoxide radicals. The superoxide is converted to hydrogen peroxide (H 2 O 2 ) by superoxide dismutase (SOD). H 2 O 2 is reduced to water by ascorbate (Asc) peroxidase (APX), and Asc is regenerated by the photoreduction of the primary oxidation product of Asc, the monodehydroascorbate radical (MDA). The limiting step in the MAP pathway is the photoreduction of O 2, which has a Michaelis constant (Km) for O 2 of about 20 mm (Takahashi and Asada 1982). The electron source for the reduction of O 2 and MDA is water photooxidized in photosystem II (PSII). In the MAP pathway, the Q-cycle in the Cyt b 6 /f-complex generates ph, similarly as in CEF-I. In addition to their physiological function as a source of ATP for the PCR and PCO cycles, the CEF-I and MAP pathways contribute to the induction of non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence (Schreiber and Neubauer 1990; Johnson 2005; Joliot and Joliot 2006; Yamamoto et al. 2006; Hideg et al. 2008). Furthermore, the MAP pathway serves as an electron sink (Park et al. 1996; Ort and Baker 2002). The physiological roles of the CEF-I and MAP pathways have been evaluated based on the magnitude of electron fluxes measured in vivo. For example, Badger et al. (2000), Ruuska et al. (2000), and Driever and Baker (2011) argued that the MAP pathway is no major electron sink as its electron flux was below 10% of the total electron flux in PSII. In contrast, Makino et al. (2002) reported that the MAP pathway carried over 90% of the total PSII electron flux during the induction of photosynthesis, and about 30% at steady state photosynthetic activity. Furthermore, Hirotsu et al. (2004, 2005) found that the electron flux in the MAP pathway corresponded to the total electron flux in PSII at low temperature. These authors supported the idea that the MAP pathway contributed to the induction of NPQ of Chl fluorescence. On the other hand, the electron flux in CEF-I was reported to exceed the total electron flux in PSII (Joliot and Joliot 2002, 2005; Golding and Johnson 2003; Miyake et al. 2005). Joliot et al. (2004) and Miyake et al. (2005) suggested that CEF-I also contributed to the induction of NPQ of Chl fluorescence. However, Kramer et al. (2004), Avenson et al. (2005a, b), and Cruz et al. (2005) demonstrated only minor activities of CEF-I in vivo. Thus, the physiological significances of the MAP pathway and CEF-I remain unclear. A change in ph (ph) across thylakoid membranes is also required for the activation of cyclic electron flow within PSII (CEF-II; Miyake and Yokota 2001). In CEF- II, electrons from a primary electron acceptor (Q A )in PSII and the reduced form of plastoquinone (plastoquinol, PQH 2 ) flow back to the reaction center Chl, P680, through Chl Z þ and Cyt b-559 (Thompson and Brudvig 1988; Whitmarsh and Pakrasi 1996; Shinopoulos and Brudvig 2012). Acidification of the lumenal side suppressed the electron transport from water to P680 þ in PSII, which results in the extension of the lifetime of P680 þ (Krieger et al. 1992, 1993). P680 þ can accept electrons sequentially from the reduced Q A and PQH 2 via Chl Z þ and Cyt b-559 (Thompson and Brudvig 1988; Samson and Fork 1992). CEF-II activity corresponded to PLEF (Miyake and Yokota 2001; Miyake et al. 2002) and oxidized PQH 2, with enhanced photochemical quenching of Chl fluorescence (Miyake and Yokota 2001). Laisk et al. (2006, 2012) showed that CEF-II functioned in intact leaves and that its activity increased with the intensity of actinic light towards the saturation of photosynthesis. The physiological function of CEF-II is to protect PSII from photoinhibition by enhancing the oxidation of PQH 2 (Miyake and Okamura 2003, Laisk et al. 2012). We focused on the fact that O 2 stimulates the induction of photosynthesis (Laisk and Oja 1998). After incubation of plant leaves in the dark, photosynthesis started with a lag after the onset of illumination with actinic light. The lag period was longer in the absence of O 2, compared to control conditions of 21 kpa O 2. Furthermore, in isolated intact chloroplasts, bicarbonate (HCO 3 )-dependent CO 2-fixation does not occur in the absence of O 2 (Heber and French 1968; Ziem-Hanck and Heber 1980). And, 3-phosphoglycerate dependent O 2 -evolution was also suppressed at the low O 2 condition (Takagi et al. 2012). Thus, the requirement of O 2 for the induction of photosynthesis observed in vivo was reproduced in vitro. The facts that O 2 enhanced both the induction and the rate of photosynthesis suggest that the O 2 -dependent MAP pathway cooperates with PLEF to provide the driving force for the synthesis of ATP which then fuels both the PCR and PCO cycles. In other words, PLEF alone is insufficient to fuel the production of enough ATP to satisfy the demands of photosynthesis. In the present work, we aimed to elucidate the molecular mechanisms underlying the O 2 -dependent stimulation of photosynthetic induction. O 2 is the

4 720 C. Miyake et al. substrate for the primary reaction in the MAP pathway (Miyake et al. 1998; Asada 2000) which competes with CEF-I for the electrons produced in PSI. These electrons preferentially flow to the MAP pathway rather than to CEF-I (Hormann et al. 1994). Consequently, the presence of O 2 affects the formation of ph across thylakoid membranes which drives four physiological processes: First, the induction of NPQ of Chl fluorescence, where NPQ contributes to the oxidation of the PQ pool (Miyake et al. 2009); second, the supply of ATP to the PCR and PCO cycles, third, the activation of cyclic electron flow within PSII (CEF-II; Miyake and Yokota 2001; Miyake et al. 2002), and fourth, the suppression of the oxidation of the reduced PQ by the Cyt b 6 /f-complex, resulting in the oxidation of P700 in PSI (Joliot and Johnson 2011). We confirmed the results obtained by Lasik and Oja (1998), using intact leaves of rice (Oryza sativa L. cv. Notohikari) plants. Then, we proceeded to analyze the effects of O 2 on the electron fluxes in PSII and PSI, the redox state of PQ, and NPQ of Chl fluorescence, and characterized the relationships between these parameters during the induction phase of photosynthesis. We found that O 2 triggered CEF-I as well as CEF-II before the start of photosynthesis, which appears to be one of the molecular mechanisms behind the O 2 -dependent acceleration of the induction of photosynthesis. MATERIALS AND METHODS Plant materials Rice plants (Oryza sativa L. cv. Notohikari) were grown hydroponically in an environmentally controlled growth chamber (Makino et al. 1994) with a 14 h photoperiod, 25/20 C day/night temperature, 60% relative humidity, and a photosynthetic photon flux density of 1000 mmol photons m 2 s 1 during the photoperiod. The basal nutrient solution was as previously described by Makino et al. (1988), except that 2 mm instead of 1 mm ammonium nitrate (NH 4 NO 3 ) were used. The measurements were done on the youngest fully expanded leaves of 70- to 80-day-old plants. Before the measurements were taken, all plants were adapted to room light for more than 20 min. Chl fluorescence, P700 þ -absorbance, and gas exchange measurements Chl fluorescence, P700 þ -absorbance, and gas exchange were measured simultaneously with Dual PAM-100 and GFS-3000 (Heintz Walz, Effeltrich, Germany) measuring systems equipped with the 3010-DUAL gas exchange chamber (Heintz Walz, Effeltrich, Germany). The absolute partial pressure of CO 2 was 37 Pa, and that of O 2 was set to 21 or 1 kpa. Leaf temperature was maintained at 25 1 C. The relative humidity of gas entering the leaf chamber was set to 60%. Chl fluorescence parameters were calculated (Baker 2008; Miyake et al. 2009) as follows: maximum quantum efficiency of PSII photochemistry, Fv/Fm ¼ (Fm Fo)/Fm; non-photochemical quenching, NPQ ¼ (Fm/Fm ) 1; quantum yield of photochemical energy conversion in PSII, Y(II) ¼ (Fm Fs)/ Fm ; fraction of PSII centers that are open, ql ¼ [Y(II)/(1 Y(II))] [(1 Fv/Fm)/(Fv/Fm)](NPQþ 1); Fm, maximum fluorescence yield; Fm, maximum variable fluorescence yield; Fs, steady-state fluorescence yield. The oxidation-reduction state of P700 þ was determined according to the methods of Klughammer and Schreiber (2008) and Pfündel et al. (2008), using the following acronyms: Pm, maximum oxidation level of P700 obtained by a saturated pulse of light under far-red illumination; P, oxidation level of P700 under actinic light; Pm, maximum oxidation level of P700 obtained by saturated-pulse light under actinic illumination; Y(I) ¼ (Pm P)/Pm, quantum yield of photochemical energy conversion; Y(ND) ¼ P/Pm, quantum yield of non-photochemical energy dissipation due to donor side limitation; Y(NA) ¼ (Pm Pm )/Pm, quantum yield of non-photochemical energy dissipation due to acceptor side limitation. The three parameters sum up to 1; Y(I) þ Y(NA) þ Y(ND) ¼ 1. Calculation of electron flux in the PCR and PCO cycles The electron flux required by the PCR and PCO cycles, Jg, was calculated from gas-exchange data according to (von Caemmerer and Farquhar 1981): Jg ¼ðA þ RdÞð4Cc þ 8 Þ=ðCc Þ where A is the net CO 2 assimilation rate, Rd is nonphotorespiratory respiration, and Cc is the CO 2 partial pressure in the chloroplast stroma deduced from the assumption that the CO 2 transfer conductance between the intercellular air spaces and the chloroplast stroma is 0.5 mol CO 2 m 2 s 1 (von Caemmerer and Evans 1991 for rice; Makino et al. 1994). * is the partial pressure of CO 2 in the chloroplast at which photorespiratory CO 2 evolution equals the rate of carboxylation: ¼ 0:5VoKcO=VcKo where Vc and Vo denote the maximum Rubisco activity of carboxylation and oxygenation, respectively, Kc and Ko are the Michaelis-Menten constants for CO 2 and O 2 (Makino et al. 1994), respectively, and O is the partial ð1þ ð2þ

5 O 2 triggers alternative electron flow 721 Figure 1 Effects of oxygen (O 2 ) on the net carbon dioxide (CO 2 ) assimilation rate and the electron flux in the photosynthetic carbon reduction (PCR) and photorespiratory carbon oxidation (PCO) cycles (Jg) in intact leaves of rice (Oryza sativa L. cv. Notohikari) plants. The leaves were illuminated with actinic light (AL) of 442 mmol photons m 2 s 1 at 37 Pa CO 2 ; AL was turned on at 30 sec. (a) Comparison of the increase of net CO 2 assimilation rates under 21 kpa O 2 (black line) and 1 kpa O 2 (red line). (b) Dependence of the rate of increase of Jg on O 2 ; measurements were taken under 21 kpa O 2 (black line) and 1 kpa O 2 (red line). Experiments were repeated 3 to 4 times and typical results are shown. Colour online only. pressure of O 2 in the chloroplast (assumed to be the same as in ambient air). The respective Vc and Vo values used here are 17.5 and 5.7 mol (mol Rubisco) 1 s 1, and the respective Kc and Ko values are 24 Pa and 28 kpa (Makino et al. 1994, 1997). RESULTS Effects of O 2 on net CO 2 assimilation rate during the induction of photosynthesis Upon actinic light (AL) illumination at 21 kpa O 2, the net CO 2 assimilation rate in rice leaves started to increase after a lag period of about 5 min, and reached the steadystate rate of 8 mmol CO 2 m 2 s 1 roughly 8 min later (Fig. 1a). This process was greatly retarded under 1 kpa O 2. After a lag of about 14 min, the net CO 2 assimilation rate started to increase to reach a steadystate rate of 13 mmol CO 2 m 2 s 1 some 15 min later. The electron flux in the PCR and PCO cycles, Jg, was calculated from the net CO 2 assimilation rate as described in the Materials and Methods. Similar to net CO 2 assimilation, changes in Jg depended on O 2 (Fig. 1b). These results indicated that regular atmospheric O 2 levels were required for the rapid activation of photosynthesis in intact rice leaves, corroborating the findings of Laisk and Oja (1998). Effects of O 2 on electron fluxes in PSII and PSI during the induction of photosynthesis The electron flux in PSII evaluated as Y(II) increased as soon as the AL was turned on at 21 kpa O 2, and reached the first steady-state value at 5 min (Fig. 2a), the time at which the net CO 2 assimilation rate started to increase more rapidly (Fig. 1a). At about the time when the net CO 2 assimilation rate started to increase to its steadystate (that is, about 8 min after exposure to AL; compare Fig. 1a), Y(II) also started to increase to a second steady-state which was reached at 15 min. These results indicated that the electron flow in PSII occurred before the start of photosynthesis at 21 kpa O 2. Similar to Y(II), the electron flux in PSI evaluated as Y(I) increased immediately when AL was turned on at 21 kpa O 2 (Fig. 2b). However, Y(I) reached the first steady-state value at 3 min, somewhat faster than Y(II). Moreover, the first steady-state value of Y(I) was statistically larger than that of Y(II) [Y(I), ; Y(II), , p < 0.05 (t-test, n ¼ 3)]. Y(I) increased to the second steady-state in parallel with Y(II); the second steady-state values of Y(I) and Y(II) were about equal [Y(I), ; Y(II), , p > 0.05 (t-test, n ¼ 3)]. These results indicated that the electron flow in PSI also occurred before the start of photosynthesis at 21 kpa O 2. Lowering the partial pressure of O 2 to 1 kpa suppressed Y(II) (Fig. 2a), and the response to AL showed a similar lag period as that of the net CO 2 assimilation rate did (compare Figs. 2a and 1a). After about 17 min, Y(II) started to increase to a steady-state value of 0.3. We conclude that the electron flux observed at 21 kpa O 2 in PSII before the accelerated increase of the net CO 2 assimilation rate was suppressed at 1 kpa O 2. The response of Y(I) to AL was changed in a similar manner when the partial pressure of O 2 was reduced to 1 kpa (Fig. 2b). About 14 min after the start of AL illumination, Y(I) began to increase to a steady-state

6 722 C. Miyake et al. Figure 2 Effects of oxygen (O 2 ) on Y(II) and Y(I) in intact rice (Oryza sativa L. cv. Notohikari) leaves illuminated with actinic light (AL; 442 mmol photons m 2 s 1 ) at 37 Pa carbon dioxide (CO 2 ). AL was turned on at 30 sec. (a) Response of Y(II) to AL under 21 kpa O 2 (black line) and 1 kpa O 2 (red line). (b) Response of Y(I) to AL compared between 21 kpa O 2 (black line) and 1 kpa O 2 (red line). Experiments were repeated 3 to 4 times and representative results are shown. Colour online only. value of Evidently, the electron flux in PSI before the accelerated increase of the net CO 2 assimilation rate that we had observed at 21 kpa O 2 was suppressed at 1 kpa O 2. Despite the similarity of the responses, the values of Y(I) were larger than those of Y(II) during the measurements. Effects of O 2 on the reduction-oxidation levels in the photosynthetic electron transport system during the induction of photosynthesis The Chl fluorescence parameter, ql, provides a measure for the reduction-oxidation level of Q A in PSII (Kramer et al. 2004; Miyake et al. 2009). Increases of ql indicate oxidation of Q A, corresponding to the oxidation of the PQ pool. Similar to Y(II), ql increased as soon as the AL was turned on at 21 kpa O 2, and reached the first steadystate value of 0.30 at 5 min (Fig. 3a). After a steady-state phase of about 5 min, ql further increased to a second steady-state value of 0.36 that was reached at 15 min. These results indicated that the turnover of PSII oxidized the PQ pool under 21 kpa O 2. Lowering the O 2 partial pressure to 1 kpa delayed the response of ql to AL (Fig. 3a), with similar kinetics as observed in the net CO 2 assimilation rate (compare Fig. 1a). After about 15 min, ql slowly increased to reach a steady-state value of 0.25 at roughly 30 min. This indicated that O 2 contributed to the oxidation of the PQ pool during the induction of photosynthesis. Y(NA) and Y(ND) correspond to the acceptor-side limiting state and the donor-side limiting state in PSI turnover, respectively (Klughammer and Schreiber 2008; Schreiber and Klughammer 2008). Increases in Y(NA) represent the suppression of P700 photooxidation, while increases in Y(ND) indicate the suppression of P700 reduction. Upon illumination with AL at 21 kpa O 2, Y(NA) rapidly decreased and reached a steady-state value of 0.35 after 5 min (Fig. 3b). This indicates that electrons photoproduced in PSI were rapidly accepted before photosynthesis was induced. On the other hand, Y(ND) increased to 0.4 and subsequently decreased to a steady-state value of 0.22 after about 5 min (Fig. 3c). In other words, following illumination with AL, the reduction of P700 was suppressed and was relieved by the oxidation of PQ, as indicated by the increase in ql (Fig. 3a). In the presence of 1 kpa O 2, the decrease in Y(NA) was delayed (Fig. 3b). Y(NA) started to decrease at 15 min after AL was turned on (Fig. 3b), which corresponds to the time at which the increase in Jg accelerated (compare Fig. 1b). These results indicated that an increase in electron sink capacity stimulated the regeneration of the electron acceptor, NADP þ, for PSI. Moreover, the increase in Y(ND) was suppressed under 1 kpa O 2 compared to 21 kpa O 2. Y(ND) started to increase at 15 min after AL had been turned on (Fig. 3c), corresponding again to the acceleration in the increase of Jg (Fig. 1b). At 26 min, Y(ND) reached a peak value and then decreased. This behavior of Y(ND) resembled that of NPQ, as shown below. Effects of O 2 on NPQ of Chl fluorescence during the induction of photosynthesis NPQ represents the efficiency of heat-dissipation of excess photon energy absorbed by PSII. Its induction requires the formation of ph across thylakoid membranes (Baker 2008). Upon illumination with AL of leaves at 21 kpa O 2, NPQ rapidly increased to a peak value of 2 and decreased to an intermediate value of 1.5 (Fig. 4). At about 8 min, NPQ started to decrease further, which coincided in time with the establishment of the

7 O 2 triggers alternative electron flow 723 Figure 3 Effects of oxygen (O 2 ) on the chlorophyll fluorescence parameter (ql), Y(NA) and Y(ND) in intact leaves of rice (Oryza sativa L. cv. Notohikari) plants. The leaves were illuminated with actinic light (AL; 442 mmol photons m 2 s 1 ) at 37 Pa carbon dioxide (CO 2 ); AL was turned on at 30 sec. (a) Induction of ql by AL under 21 kpa O 2 (black line) and 1 kpa O 2 (Red line). (b) Response of Y(NA) to AL under 21 kpa O 2 (black line) and 1 kpa O 2 (red line). (c) Response of Y(ND) to AL under 21 kpa O 2 (black line) and 1 kpa O 2 (red line). Experiments were repeated 3 to 4 times, and typical results are shown. Colour online only. steady-state net CO 2 assimilation rate (compare Fig. 1a). High rates of CO 2 assimilation require large amounts of ATP, the synthesis of which dissipates ph. Lowering the partial pressure of O 2 to 1 kpa delayed the induction of NPQ (Fig. 4). The rate at which NPQ increased accelerated at about 15 min with the faster increase in Y(I) more than that in Y(II). Figure 4 Effects of oxygen (O 2 ) on non-photochemical quenching (NPQ) in intact rice (Oryza sativa L. cv. Notohikari) leaves. Leaves were illuminated with actinic light (AL; 442 mmol photons m 2 s 1 ) at 37 Pa carbon dioxide (CO 2 ). AL was turned on at 30 sec. The induction of NPQ was delayed under 1 kpa O 2 (red line) compared to 21 kpa O 2 (black line). Experiments were repeated 3 to 4 times, and representative results are shown. Colour online only. DISCUSSION We studied the effects of O 2 on the photosynthetic electron transport (PET) system to elucidate the physiological function of O 2 in the stimulation of the induction of net CO 2 assimilation (Fig. 1). We found that before the start of photosynthesis, O 2 induced, first, electron fluxes in both PSII and PSI (Fig. 2); second, the oxidation of the PET system (Fig. 3); and third, the formation of ph across thylakoid membranes (Fig. 4). From these facts, we propose a regulatory mechanism of the PET system by the MAP pathway.

8 724 C. Miyake et al. O 2 induced the MAP pathway, CEF-II, and CEF-I If the MAP pathway operates, its activity will be reflected by an increase in Y(II). In fact, Y(II) at 21 kpa O 2 was higher than that at 1 kpa O 2 (Fig. 2). Thus, the MAP pathway surely was active. Driever and Baker (2011) reported that in the intact leaf the photoreduction rate of O 2 in the MAP pathway was about 5 mmol e m 2 s 1 at saturating light intensity. Asada and Takahashi (1987) showed that the production rate of superoxide radicals in the MAP pathway ranged from 10 to 40 mmol O 2 (mg Chl) 1 h 1, which corresponds to electron fluxes ranging from 1.7 to 6.5 mmol e m 2 s 1 (600 mg Chl m 2 rice leaf). The electron flux in PSII at 21 kpa O 2 was estimated. MAP pathway cannot function at 1 kpa O 2. Thus, Y(II) reflects Jg. Jg was about 60 mmol e m 2 s 1 at steady-state photosynthesis at 1 kpa O 2 (Fig. 1b). The value of Jg corresponded to 0.35 of Y(II) at 1 kpa O 2.At 21 kpa O 2, Y(II) was 0.35 before the start of photosynthesis (Fig. 1b). That is, the electron flux in PSII was 60 mmol e m 2 s 1. And, the activity of the MAP pathway was about 10% of the electron flux in PSII. Therefore, about 90% of Y(II) at 5 min after AL illumination would have to be ascribed to electron flux in CEF-II (Miyake and Yokota 2001; Laisk et al. 2006, 2012). The electron flux in PSI also was enhanced at 21 kpa O 2 (Fig. 2). The electron flux in PSI fueled by the MAP pathway should be below 10% of Y(I) at 5 min after AL illumination, similar to Y(II). Thus, almost all of Y(I) would reflect the electron flux in CEF-I. Consequently, before the induction of photosynthesis, PSI must have functioned in CEF-I. With the acceleration of the net CO 2 assimilation rate at 21 kpa O 2, both Y(I) and Y(II) further increased (Fig. 2). However, this increase was too small to explain the increased Jg. Thus, electron fluxes in CEF-I and CEF-II seem to have been replaced by fluxes in Jg during the acceleration of the net CO 2 assimilation rate. Especially, the activity of CEF-II depends on the acidification of the thylakoid lumen (Miyake and Yokota 2001). In fact, Y(II) increased with the increase in NPQ (Fig. 2 and 3). With the start of photosynthesis, NPQ decreased, which indicated the dissipation of ph across thylakoid membranes by the increase in both PCR and PCO cycles, presumably resulting in the suppression of CEF-II. O 2 -induced oxidation of the PET system The MAP pathway functions before the start of photosynthesis at 21 kpa O 2. In the MAP pathway, O 2 and MDA act as Hill oxidants (Miyake and Asada 1992). MDA is reduced about 30 times faster than NADP þ at PSI (Miyake and Asada 1994). Upon AL illumination at 21 kpa O 2, Y(NA) rapidly decreased, but not at 1 kpa O 2 Figure 5 Model of the dependence of CEF-I activity on the redox state of plastoquinone (revised from Miyake 2010). CEF- I activity is simulated against the ratio (PQ)/(PQ) t according to the model of Allen (2003), where (PQ) t is the total concentration of PQ and (PQ) is the concentration of oxidized PQ in chloroplasts. In the extremely oxidized and reduced states of PQ, CEF-I activity is negligible. The Mehler-ascorbate peroxidase (MAP) pathway oxidizes PQ and induces CEF-I activity. CEF, cyclic electron flow. (Fig. 3). This appears due to the oxidation of P700 by the effective electron acceptors, O 2 and MDA, in the MAP pathway. In addition to the production of the superior electron acceptors, the MAP pathway induces ph across thylakoid membranes, as observed by the enhanced NPQ of Chl fluorescence at 21 kpa O 2 (Schreiber and Neubauer 1990). The acidification of the lumenal side of the thylakoid membranes drives three processes: first, the induction of NPQ of Chl fluorescence; second, the activation of CEF-II, as described above; and third, the slowing down of the oxidation of plastoquinol by the Cyt b 6 /f-complex (Hope 2000). NPQ suppresses the input of photon energy into the PET system, which contributes to the oxidation of the PQ pool. Furthermore, CEF-II oxidizes plastoquinol (Miyake and Yokota 2001). Consequently, both NPQ and CEF-II increase ql (Fig. 3). The suppression of the turnover of the Cyt b 6 /f-complex oxidizes P700, observable as an increase in Y(ND) (Fig. 3). These processes contribute to the oxidation of the PET system. O 2 -dependent oxidation of the PET system activated CEF-I, resulting in the rapid increase in Y(I) (Fig. 2). The activity of CEF-I depends on the reduction-oxidation level of the PQ in thylakoid membranes (Allen 2003; Miyake 2010; Kubo et al. 2011). The relationship between CEF-I activity and the ratio of oxidized PQ to total PQ, (PQ)/(PQ) t, is shown in Fig. 5. CEF-I activity peaks at intermediate values of (PQ)/(PQ) t and decreases to zero when PQ is oxidized or reduced completely. The oxidation of the PET system, as indicated by the increase in ql at high O 2, enhanced Y(I) (Fig. 3), which mainly represented the activity of CEF-I. From these results, we conclude that the MAP pathway induces CEF-I by oxidizing the PET system.

9 O 2 triggers alternative electron flow 725 Figure 6 The induction mechanism of photosynthesis through the Mehler-ascorbate peroxidase (MAP) pathway. For detailed discussion, see main text. Blue arrows symbolize processes that protect PSII and PSI from photoinhibition; the orange arrow represents the process that activates photosynthesis. O 2, oxygen; ATP, adenosine triphosphate; NPQ, non-photochemical quenching; PCR, photosynthetic carbon reduction; PCO, photorespiratory carbon oxidation. Colour online only. We propose a regulatory mechanism for the PET system based on the activity of the MAP pathway during the induction of photosynthesis (Fig. 6). O 2 drives the MAP pathway (Schreiber and Neubauer 1990), which accepts electrons at PSI, as indicated by the rapid decrease in Y(NA), and induces the formation of ph across thylakoid membranes (a in Fig. 6). ph is the key signal that controls the physiological processes for the protection of PSI and PSII, and for the induction of CO 2 assimilation. First, ph activates CEF-II, which enhances the electron flux in PSII [Y(II)] and oxidizes the PQ pool, which shows as an increase in ql (Fig. 6b). The increase in Y(II) mitigates the photoinhibition of PSII (Fig. 6c) while the oxidation of the PQ pool activates CEF-I (Fig. 6d) (compare Fig. 5). Second, the activated CEF-I accelerates the formation of ph, thus deactivating the turnover of the Cyt b 6 /f-complex, which leads to an accumulation of oxidized P700, P700 þ, through the oxidation of Cyt f and plastocyanin (PC) (Fig. 6e). P700 þ contributes to the dissipation of excess photon energy as heat for the protection of PSI (f). Third, ph drives the formation of ATP to fuel the PCR and PCO cycles, which stimulates the induction of photosynthesis (Fig. 6g), tending to dissipate ph (Fig. 6h). Fourth, ph enhances NPQ of Chl fluorescence to protect PSII from photoinhibition through the dissipation of excess photon energy (Fig. 6i). Induction of net CO 2 assimilation at 1 kpa O 2 We observed a retardation of the induction of photosynthesis at 1 kpa O 2, compared to 21 kpa (Fig. 1). At 1 kpa O 2, NPQ of Chl fluorescence increased only slowly (Fig. 3). This represented the slow formation of ph across thylakoid membranes, and consequently the low rate of ATP production to drive photosynthesis. At 1 kpa O 2, Y(I) was larger than Y(II) (Fig. 2), and the faster increase in Y(I) induced NPQ of Chl fluorescence (Fig. 4). These facts indicated that at low partial pressure of O 2, CEF-I is the main contributor to the induction of NPQ of Chl fluorescence and to the supply of ATP for the PCR cycle. This conclusion is consistent with the results of Hormann et al. (1994). ACKNOWLEDGMENTS This work was supported by the Japan Society for the Promotion of Science (Scientific Research Grant No to CM) and the Ministry of Education, Culture, Sports, Science and Technology, Japan (Scientific Research on Innovative Area No to C.M. and No to AM). REFERENCES Allen JF 2003: Cyclic, pseudocyclic and noncyclic photophosphorylation: New links in the chain. Trends Plant Sci., 8, Asada K 2000: The water-water cycle as alternative photon and electron sink. Phil. Trans. R Soc. Lond. B: Biol. Sci., 355, Asada K, Takahashi M 1987: Production and scavenging of active oxygen in photosynthesis. In Photoinhibition, Eds

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