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Home , Electron transport chain, Photoinhibition

Physiologia Plantarum 143: 396–407. 2011 Copyright © Physiologia Plantarum 2011, ISSN 0031-9317

Mitochondrial alternative pathway protects against by alleviating inhibition of the repair of photodamaged PSII through preventing formation of reactive species in Rumex K-1 leaves Li-Tao Zhang, Zi-Shan Zhang, Hui-Yuan Gao∗, Zhong-Cai Xue, Cheng Yang, Xiang-Long Meng and Qing-Wei Meng

State Key Laboratory of Crop , Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an 271018, Shandong, China

Correspondence The purpose of this study was to explore how the mitochondrial AOX *Corresponding author, () pathway alleviates photoinhibition in Rumex K-1 leaves. e-mail: [email protected] Inhibition of the AOX pathway decreased the initial activity of NADP- Received 18 May 2011; malate (EC 1.1.1.82, NADP-MDH) and the pool size of revised 21 July 2011 photosynthetic end electron acceptors, resulting in an over-reduction of the I (PSI) acceptor side. The over-reduction of the PSI acceptor side doi:10.1111/j.1399-3054.2011.01514.x further inhibited electron transport from the photosystem II (PSII) reaction centers to the PSII acceptor side as indicated by an increase in VJ (the relative variable fluorescence at J-step), causing an imbalance between photosynthetic light absorption and energy utilization per active reaction center (RC) under high light, which led to the over-excitation of the PSII reaction centers. The over-reduction of the PSI acceptor side and the over-excitation of the PSII reaction centers enhanced the accumulation of (ROS), which inhibited the repair of the photodamaged PSII. However, the inhibition of the AOX pathway did not change the level of photoinhibition under high light in the presence of the D1 synthesis inhibitor chloramphenicol, indicating that the inhibition of the AOX pathway did not accelerate the photodamage to PSII directly. All these results suggest that the AOX pathway plays an important role in the protection of plants against photoinhibition by minimizing the inhibition of the repair of the photodamaged PSII through preventing the over-production of ROS.

Abbreviations – φEo, the quantum yield of electron transport; φPo, the maximum quantum yield for primary photochemistry; PSII, photosystem II actual photochemical efficiency; 0, the efficiency for electron transport; ABS/RC, light absorption per active reaction center; AOX, alternative oxidase; APX, ascorbate peroxidase; CAT, catalase; CM, chloramphenicol; COX, oxidase; DAB,3, 3-diaminobenzidine; DTT, dithiothreitol; ET0/RC, the electron transport per active reaction   center; FJ, the fluorescence at J-step; Fo,Fm, initial and maximum fluorescence; Fo,Fm, the minimal and maximum fluorescence in the light-adapted state; FS, the steady-state fluorescence; Ft, the fluorescence at any time t; HSD, honestly significant difference; LED, light-emitting diode; LSD, least significant difference; NADP/NADPH, oxidized/reduced form of -adenine dinucleotide ; NADP-MDH, NADP-; NBT, nitrotetrazolium blue chloride; nPG, n-propyl gallate; OAA, oxaloacetate; PFD, photon flux density; PSI, ; PSII, photosystem II; QA, primary of PS II; qP, photochemical quenching; RC, active reaction center; ROS, reactive oxygen species; SHAM, ; SOD, superoxide dismutase; TR0/RC, trapping of excitation energy per active reaction center; VJ, the relative variable fluorescence at the J-step.

396 Physiol. . 143, 2011 Introduction Padmasree and Raghavendra 1999a, 1999c). It has been shown that the AOX pathway is induced to protect the Excess light energy damages the photosynthetic machin- photosynthetic apparatus from photoinhibition in Ara- ery during , causing photoinhibition bidopsis, comprising the cyclic electron flow around (Murata et al. 2007, Takahashi and Murata 2008). It has photosystem I (PSI) (Yoshida et al. 2007). However, it been demonstrated that photodamage is attributable to remains to be elucidated whether the of light absorbed directly by in the oxygen- the mitochondrial AOX pathway is correlated with the evolving complex (Nishiyama et al. 2006, Tyystjarvi¨ suppression of photodamage to PSII and/or minimizing 2008). Subsequent to photodamage to the oxygen- the inhibition of the repair of the photodamaged PSII. evolving complex, the reaction center of photosystem The accumulation of ROS induced by excess light II (PSII) is damaged by light absorbed by the photosyn- energy has been suggested to play an important role thetic pigments (Hakala et al. 2005, Ohnishi et al. 2005). during photoinhibition (Murata et al. 2007, Takahashi The damaged PSII is rapidly and effectively repaired and Murata 2008). A recent study, using plant material by the replacement of the damaged PSII with lacking AOX1a, demonstrated that the AOX pathway newly synthesized proteins, primarily the D1 protein plays a central role in reducing the accumulation of (Aro et al. 2005, Mattoo and Edelman 1987, Nishiyama ROS generated by photosynthesis in plants (Giraud et al. 2011). Thus, photoinhibition occurs only under et al. 2008). However, the generation site of ROS in conditions in which the rate of photodamage exceeds and the role of ROS accumulation during the rate of its repair (Murata et al. 2007, Nishiyama et al. photoinhibition conditions because of the inhibition of 2011, Takahashi and Murata 2008). To prevent photoin- AOX pathway have not yet been clarified. hibition, plants have evolved mechanisms to suppress The aim of this study was to explore the role of ROS photodamage to PSII and/or to minimize the inhibition of accumulation resulting from the inhibition of the AOX the repair of the photodamaged PSII. It has been demon- pathway during photoinhibition and to clarify whether strated that the photorespiratory pathway and antioxi- the photoprotection of the mitochondrial AOX pathway dant systems protect plants by minimizing the inhibition is correlated with the suppression photodamage to PSII of the repair of the photodamaged PSII (Nishiyama et al. and/or with minimizing the inhibition of the repair of the 2001, Takahashi et al. 2007), and chloroplast avoid- photodamaged PSII. In addition, using a a ance movement can help to prevent photoinhibition fluorescence transient combined with the JIP-test (Jia by directly suppressing the photodamage to PSII (Kasa- et al. 2010, Kalachanis and Manetas 2010, Mathur hara et al. 2002). Furthermore, Takahashi et al. (2009) et al. 2011, Oukarroum et al. 2009, Strasser et al. 2000, suggested that photosynthetic cyclic electron flow can 2004), we studied photosynthetic behaviors (including protect plants against photoinhibition by both prevent- the energy fluxes of absorption, energy trapping and ing photodamage to PSII and minimizing the inhibition electron transport) to analyze the generation site of ROS of the repair of the photodamaged PSII. in chloroplasts under high light when the AOX pathway Recent publications have also suggested that the mito- is inhibited. chondrial alternative oxidase (AOX) pathway plays an important role in protecting photosynthetic machinery from photoinhibition (Florez-Sarasa et al. 2011, Noguchi Materials and methods and Yoshida 2008, Raghavendra and Padmasree 2003). Materials and treatments The AOX pathway is an alternative electron transport pathway in addition to the cytochrome oxidase (COX) Rumex K-1 plants (Rumex patientia × Rumex tian- pathway in the mitochondria. This pathway diverts elec- schaious) were grown from seeds under a 14-h photope- ◦ trons from the ubiquinone pool and reduces oxygen riod at 22/18 C (day/night) in a pot containing soil. The to water without any translocation or ATP syn- plants were thinned to one plant per pot 2 weeks after thesis (Rasmusson et al. 2009, Van Aken et al. 2009). sowing. Nutrients and water were supplied sufficiently Apparently, the AOX pathway is non-phosphorylating throughout the study to avoid any potential nutrient and and can oxidize reducing equivalents efficiently without drought stresses. The photon flux density (PFD) dur- being restricted by the proton gradient across the mito- ing growth was approximately 800 μmol m−2 s−1. Fully chondrial inner membrane or the cellular ATP/ADP ratio expanded leaves of 4-week-old plants were used in the (Yoshida et al. 2007). Several recent studies have demon- experiments. strated that the inhibition of the AOX pathway leads to Intact chloroplasts were isolated according to the decreases in photosynthetic rate in leaves (Yoshida et al. protocol of Bartoli et al. (2005). Leaves were ground 2006) and protoplasts (Dinakar et al. 2010a, 2010b, in buffer containing 50 mM HEPES-KOH (pH 7.5),

Physiol. Plant. 143, 2011 397 330 mM sorbitol, 2 mM Na2-EDTA, 1 mM MgCl2, the sample preparation. The activity of superoxide 5mM ascorbic acid and 0.05% (w/v) BSA using a dismutase (EC 1.15.1.1, SOD) was measured according hand-held homogenizer. The homogenate was filtered to Giannopolitis and Ries (1977). The activities of through a 20 μm pore size nylon mesh and centrifuged ascorbate peroxidase (EC 1.11.1.11, APX) and catalase at 3000 g for 5 min. The pellet was suspended in (EC 1.11.1.6, CAT) were measured according to the wash medium (50 mM HEPES-KOH pH 7.5 and method of Mishra et al. (1993). 330 mM sorbitol), loaded in a solution consisting of 35% (v/v) Percoll in wash medium and centrifuged Measurements of at 2500 g for 5 min. The pellet containing intact fluorescence transient chloroplasts was used to measure modulated chlorophyll fluorescence in the absence (control) or presence Chlorophyll a fluorescence (OJIP) transients before of 1 mM salicylhydroxamic acid (SHAM) or n-propyl and after the high light treatment were measured gallate (nPG). The isolated chloroplasts were 75% intact with a Handy PEA fluorometer (Hansatech Instruments, Norfolk, UK). The transients were induced by red light according to the ferricyanide-dependent O2 evolution μ −2 −1 (Walker, 1988), which was measured with a Chlorolab-2 measuring approximately 3000 mol m s provided oxygen electrode (Hansatech Instruments, Norfolk, UK). by an array of three light-emitting diodes (LEDs, peak Leaf discs (0.5 cm2) were punched from fully 650 nm). All measurements were performed with 15-min expanded leaves and infiltrated with 0 (control) or 1 mM dark-adapted leaf discs at room temperature. SHAM or nPG solutions, respectively, for 2 h in the dark OJIP transients were analyzed by the JIP-test (Appen- at room temperature. Then, the discs were exposed to roth et al. 2001, Oukarroum et al. 2009, Strasser and high light measuring 800 μmol m−2 s−1 for 1 h at room Strasser 1995, Strasser et al. 2000, Yusuf et al. 2010), temperature. using the following original data: (1) the fluorescence intensity at 20 μs, considered to be Fo when all PSII reaction centers were open; (2) the maximal fluores- Capacity of AOX pathway measurement cence intensity, Fm (FP, at about 300 ms), assuming that The respiratory rate in the leaf discs treated with high the excitation intensity was high enough to close all of light was measured using an Oxytherm oxygen electrode the PSII RCs; and (3) the fluorescence intensities at 2 ms ◦ (Hansatech Instruments, Norfolk, UK) at 25 C according (J-step, FJ) and 30 ms (I-step, FI). The following param- to Yoshida et al. (2006) and Zhang et al. (2010). The eters were used for the quantification of PSII behavior leaf discs were incubated in the dark for 10 min before referring to time 0: respiratory measurements were taken. The inhibitors 1. The chlorophyll a fluorescence transient from I to 1mM KCN and 20 mM SHAM were used to inhibit P after double normalization between the Fo and the COX and AOX pathways, respectively. The AOX FI points, WOI = (Ft− Fo)/(FI− Fo),whereFt is the pathway capacity was defined as the O2 uptake rate in fluorescence intensity at any time. the presence of KCN that was sensitive to SHAM. 2. The relative variable fluorescence at J-step (VJ), VJ = (FJ− Fo)/(Fm− Fo). assays 3. The specific energy fluxes (per RC) for absorption (ABS/RC), trapping (TR0/RC) and electron transport were extracted from the leaves according to (ET0/RC). 2 Dutilleul et al. (2003). Leaf discs (7.5 cm ) were ground 4. The flux ratios or yields, i.e. the maximal in liquid nitrogen and extracted in 50 mM HEPES-KOH quantum yield of primary photochemistry (φPo = (pH 7.5) buffer containing 10 mM MgCl2,1mM Na2- TR0/ABS = Fv/Fm), the efficiency with which a EDTA, 5 mM dithiothreitol (DTT), a protease inhibitor trapped exciton can move an electron into the tablet, 5% (w/v) insoluble polyvinylpyrrolidone and − further than QA (the 0.05% (v/v) Triton X-100. After centrifugation for 5 min excitation efficiency for electron transport beyond at 14 000 g, the enzyme activity in the supernatant − = QA , 0 ET0/TR0) and the quantum yield of was measured with an UV-2550 spectrophotometer electron transport (φEo = ET0/ABS). (Shimadzu, Kyoto, Japan). The initial activity of NADP- malate dehydrogenase (EC 1.1.1.82, NADP-MDH) Measurements of modulated chlorophyll was measured according to Dutilleul et al. (2003). fluorescence parameters Assays were performed in 40 mM Tricine-KOH (pH 8.3), 150 mM KCl, 1 mM Na2-EDTA, 5 mM DTT, The modulated chlorophyll fluorescence parameters 0.2 mM NADPH and 2 mM OAA (oxaloacetate) and PSII (PSII actual photochemical efficiency) and qP

398 Physiol. Plant. 143, 2011 (photochemical quenching coefficient) of intact Rumex inhibitor, nPG (Yoshida et al. 2006), in this study. The K-1 chloroplasts were measured with a FMS-2 pulse- results showed that the two different inhibitors have modulated fluorometer (Hansatech Instruments, Norfolk, similar effects on the inhibition of the AOX pathway UK) integrated with a Chlorolab-2 oxygen electrode (data not shown). In addition, there were no differences (Hansatech Instruments, Norfolk, UK). The actinic light in photosynthetic behaviors between leaves treated with − − at 800 μmol m 2 s 1 was offered by the light source. SHAM (Figs 3, 5–7) and those treated with nPG (Figs  Following the onset of actinic light irradiation, the FS,Fm S2–S5). Therefore, we only discussed the effects of the  and Fo were recorded when the chlorophyll fluorescence SHAM treatment in the text, and those caused by the nPG = −  = attained steady-state levels; PSII 1 FS/Fm and qP treatment are presented in the Supporting Information.  −  −  (Fm FS)/(Fm Fo) (Chen et al. 2007). Given that some components of the photosynthetic electron transport chain in chloroplasts are similar to Measurement of P700 state those in the respiratory chain of mitochondria, we examined whether the concentrations of SHAM and The redox state of P700 was measured according to nPG used in this study had direct effects on the photo- Schansker et al. (2003) and Kim et al. (2001) using a synthetic electron transport chain. The results showed PEA Senior fluorometer (Hansatech Instruments, Norfolk, that the treatments with 1 mM SHAM or 1 mM nPG − − UK). Red light, measuring 800 μmol m 2 s 1, was pro- had no direct effects on the actual PSII photochemical duced by an array of four 650-nm LEDs (peak, 650 nm), efficiencies (PSII) and photochemical quenching coef- and the modulated (33.3 kHz) far-red measuring light has ficients (qP) in intact chloroplasts isolated from Rumex a wavelength of 820 nm was provided by an OD820 K-1 leaves (Figs 1 and S1), suggesting that the concentra- LED (Opto Diode Corp., Newbury Park, USA). Upon tions of SHAM and nPG used in this study had no direct − − irradiation with a red pulse (800 μmol m 2 s 1),the effects on photosynthetic behaviors. Therefore, all of the transmission at 820 nm in leaves increases gradually, effects caused by the SHAM and nPG treatments were + which is mainly caused by the reduction of P700 . actually because of the inhibition of the mitochondrial AOX pathway. Histochemical detection of superoxide (O−) To determine a working concentration of the inhibitor 2 for this study, we examined the effects of the inhibitor on and hydrogen peroxide (H2O2) the capacities of the AOX pathways in Rumex K-1 leaves 2 Leaf discs (1.3 cm ) treated with 0 (control) or 1 mM in the dark or in the high light. After the high light treat- −1 SHAM or nPG were vacuum-infiltrated with 0.1 mg ml ment, the AOX pathway capacities in the control leaves nitrotetrazolium blue chloride (NBT) or 1 mg ml−1 3, 3- diaminobenzidine (DAB, pH 3.8) and incubated under high light (800 μmol m−2 s−1) conditions for 1 h. After these treatments, leaf discs were decolorized by immer- sion in boiling ethanol (96%) for 10 min. After cooling, the leaf discs were extracted at room temperature with fresh ethanol and photographed (Jabs et al. 1996, Thordal-Christensen et al. 1997, Zeng et al. 2010).

Statistical analysis Least significant difference (LSD) was used to analyze differences between the measurements and Tukey’s honestly significant difference (HSD) was used for the multiple comparison using SPSS 16.

Fig. 1. Photosystem II actual photochemical efficiency (PSII)and photochemical quenching coefficient (qP)inisolatedRumex K-1 Results chloroplasts in the absence (control) or presence of 1 mM SHAM. Chloroplasts were incubated in darkness for 10 min in the presence SHAM has been widely used to inhibit the AOX pathway of 0 or 1 mM SHAM and then irradiated with 800 μmol m−2 s−1. (Bartoli et al. 2005, Padmasree and Raghavendra 1999a, Each value was obtained after 10 min of irradiation when fluorescence 1999b, 1999c). However, to avoid its side effects and intensities had achieved steady levels. Results are the average of three ensure the sufficient inhibition of the AOX pathway, experiments, using different chloroplast preparations. Different letters indicate significant difference between the SHAM treatment and control we compared the effects of SHAM with another AOX at P < 0.05, LSD.

Physiol. Plant. 143, 2011 399 NADP-MDH is the key enzyme of the malate-OAA shuttle that is the major machinery for the transport of excess reducing equivalents from chloroplasts to mitochondria for oxidation by the respiratory electron transport chain (Noguchi and Yoshida 2008, Raghavendra and Padmasree 2003, Scheibe 2004). The initial activity of NADP-MDH increased by approximately 181% after high light treatment in the control leaves. The SHAM treatment did not alter the initial activity of NADP-MDH in the dark, indicating that the 1 mM SHAM treatment had no direct effect on NADP-MDH. However, the 1 mM SHAM treatment decreased the initial activity of NADP-MDH by approximately 31% under high light in Rumex K-1 leaves (Fig. 2B), suggesting that SHAM-treated leaves exported the reducing equivalents from chloroplasts at lower rates. The chlorophyll a transient has become one of the most popular tools in photosynthetic research (Jia et al. 2010, Kalachanis and Manetas 2010, Mathur et al. 2011, Oukarroum et al. 2009). The shape of the OJIP transient is very sensitive to environmental stresses. Strasser and Strasser (1995) developed a procedure to quantify the OJIP transient, known as the JIP-test. With this test, it is possible to calculate several phenomenological and Fig. 2. Capacities of alternative respiration (A) and initial activities of biophysical expressions of the photosynthetic electron NADP-MDH (B) in Rumex K-1 leaves treated with 0 (control) or 1 mM transport chain (Strasser and Strasser 1995, Strasser et al. 2 SHAM in the dark or high light. Leaf discs (0.5 cm ) were infiltrated 2000, 2004). The JIP-test is thus a powerful tool for the in with0or1mM SHAM in the dark for 2 h and then exposed to high light at 800 μmol m−2 s−1 for 1 h. Means ± SE of three replicates are vivo investigation of photosynthetic behaviors, including presented. Different letters indicate significant difference among the the the energy fluxes of absorption, trapping and electron SHAM treatment and control in the dark and high light at P < 0.05, transport (Strasser et al. 2000, 2004). In the present study, Tukey’s HSD. we used the chlorophyll a fluorescence transient (OJIP) to detect changes in photosynthetic behaviors when the increased significantly (Fig. 2A). The 1 mM SHAM treat- AOX pathway was inhibited. In the dark, the SHAM ment inhibited 59% and 55% of the AOX pathway treatment did not alter the shape of the OJIP transient capacities in the leaves in the dark and high light, (Fig. 3A). However, it decreased the fluorescence inten- respectively. sities significantly in both the control and SHAM-treated

Fig. 3. Changes of chlorophyll a fluorescence transient (OJIP transient) in Rumex K-1 leaves treated with 0 (control) or 1 mM SHAM in the dark (A) or in the high light (B). Leaf discs (0.5 cm2) were infiltrated with 0 or 1 mM SHAM in the dark for 2 h and then exposed to high light at 800 μmol m−2 s−1 for 1 h. Each transient represents the average of eight samples.

400 Physiol. Plant. 143, 2011 Fig. 5. Photosynthetic parameters derived from the JIP-test analysis of chlorophyll a fluorescence transients shown in Fig. 3B. Treatments are as same as those described in Fig. 2, where each point represents the average of eight samples. All parameters in the leaves of the control were taken as 1, whereas those in the leaves treated with 1 mM SHAM were taken as a percentage of the control. The original value of VJ, 0, φEo,ABS/RC,TR0/RC and ET0/RC in the control leaves were 0.52 ± 0.03, 0.48 ± 0.03, 0.36 ± 0.04, 2.53 ± 0.05, 1.87 ± 0.01 and 0.90 ± 0.09, respectively.

electron transport chain, resulting in the momentary − Fig. 4. (A) Traces of P700 reduction in Rumex K-1 leaves treated with maximum accumulations of QA (Strasser and Strasser 0 (control) or 1 mM SHAM after exposure to high light at 800 μmol 1995, Li et al. 2009). In this study, the SHAM treatment m−2 s−1. Leaf discs (0.5 cm2) were infiltrated with 0 or 1 mM SHAM in increased the VJ significantly compared with the control the dark for 2 h. Each transient represents the average of five samples. leaves after high light treatment (Fig. 5). Additionally, (B) Chlorophyll a fluorescence transient from I to P under high light compared with the control leaves, the SHAM-treatment after double normalization between the Fo and FI points. Each transient represents the average of eight samples. increased the light absorption per active reaction center (ABS/RC, the average antenna size) and trapping of leaves under high light, and the extent of decrease was excitation energy per active reaction center (TR0/RC), much greater in the SHAM-treated leaves than in the and it decreased the efficiency of electron transport control leaves (Fig. 3B). These results indicate that the (0), the quantum yield of electron transport (φEo) and inhibition of the AOX pathway significantly influenced electron transport per active reaction center (ET0/RC) photosynthetic behaviors. after the high light treatment (Fig. 5), suggesting that the The extent of the reduction of P700 was more balance between photosynthetic light absorption and pronounced in the SHAM-treated leaves than the control energy utilization was disturbed when the AOX pathway leaves (Fig. 4A). According to the JIP-test, the maximal was inhibited. amplitude of the OJIP transient in the I–P phase after To determine the that the Rumex normalization between the O and I phases decreased K-1 leaves suffered when the AOX pathway was significantly when the AOX pathway was inhibited inhibited under high light, we detected the in situ − by 1 mM SHAM under high light (Fig. 4B), suggesting accumulation of O2 and H2O2 using the NBT and that the pool size of the PSI end electron acceptors DAB staining procedures, respectively. They showed decreased (Kalachanis and Manetas 2010, Yusuf et al. that the inhibition of the AOX pathway enhanced the − 2010). The relative variable fluorescence at J-step (VJ) accumulation of ROS (O2 and H2O2) significantly under represents the subsequent kinetic bottlenecks of the high light in Rumex K-1 leaves (Fig. 6A). The SHAM

Physiol. Plant. 143, 2011 401 noticeably increased in Rumex K-1 leaves under high light (Fig. 2B), indicating that the malate-OAA shuttle was activated to transport excess reducing equivalents generated in the chloroplasts to mitochondria. The equivalents transported from the chloroplasts can be oxidized by the respiratory electron transport chain (Noguchi and Yoshida 2008, Yoshida et al. 2007). It was suggested that, in this situation, the mitochondrial non-phosphorylating electron transport pathway, the AOX pathway, would play a role in the dissipation of chloroplast-derived reducing equivalents (Dinakar et al. 2010b, Yoshida et al. 2007, 2008). The fact that the AOX pathway capacity in the leaves of Rumex K-1 significantly increased (Fig. 2A) under high light support this suggestion. The fact that the treatments of intact chloroplasts isolated from Rumex K-1 leaves with 1 mM SHAM and nPG had no effects on PSII and qP (Figs 1 and S1), together with the fact that neither the SHAM nor the nPG treatments altered the OJIP transients in the dark

− (Figs 3A and S2A), demonstrate that the 1 mM SHAM Fig. 6. Production of O2 and H2O2 (A) and activities of SOD, APX and nPG used in this study had no direct effects on and CAT (B) in Rumex K-1 leaves treated with 0 (control) or 1 mM SHAM under high light. Leaf discs (1.3 cm2) treated with 0 (control) photosynthetic behaviors, which was also demonstrated or 1 mM SHAM were vacuum- infiltrated with 0.1 mg ml−1 NBT or by Padmasree and Raghavendra (1999a), Bartoli et al. 1mgml−1 DAB (pH 3.8) and incubated under high light (800 μmol m−2 (2005) and Yoshida et al. (2006) in their studies, using − s 1) conditions for 1 h. Representative images from five independent other plant materials. Therefore, the observed effects of ± experiments are shown in the figure. Means SE of three replicates are the SHAM and nPG treatments on the photosynthetic presented. Different letters indicate significant difference between the SHAM treatment and control at P < 0.05, LSD. apparatus under high light in this study were because of the inhibition of the mitochondrial AOX pathway. treatment also increased the activities of SOD, APX and It was reported that when the reducing equivalents CAT under high light (Fig. 6B). accumulated in the mitochondria, the activity of the Furthermore, compared with control leaves, the malate-OAA shuttle would be reduced by a feed- SHAM treatment significantly accelerated the decrease back mechanism (Padmasree and Raghavendra 2001). in the maximum quantum yield for primary photochem- Thus, it is reasonable to consider that, under high light, the significant decrease in the initial activity istry (φPo), the excitation efficiency for electron transport of NADP-MDH caused by the inhibition of the AOX beyond QA (0) and the quantum yield for electron pathway (Fig. 2B) was because of the accumulation of transport (φEo) under high light in the absence of the chloroplast D1 protein synthesis inhibitor chlorampheni- reducing equivalents in the mitochondria because the col (CM) in Rumex K-1 leaves (Fig. 7A, C, E). However, consumption of reducing equivalents through the AOX in the presence of CM, the SHAM treatment had no pathway was restricted. The significant decrease in the initial activity of NADP-MDH would inevitably limit the significant effects on the levels of φPo, 0 and φEo under high light (Fig. 7B, D, F). export of reducing equivalents from the chloroplasts to mitochondria, resulting in the accumulation of reducing equivalents in the chloroplast stroma. Discussion Furthermore, the SHAM and nPG treatments markedly Under high light, excess reducing equivalents generated altered the OJIP transient under high light (Figs 3B in chloroplasts can be transported to other organelles and S2B), suggesting that the inhibition of the AOX through the malate-OAA shuttle and oxidized in pathway significantly influenced the performance of the metabolic pathways (Noguchi and Yoshida 2008, photosynthetic machinery. The observation that P700 Raghavendra and Padmasree 2003, Scheibe 2004). was more reduced in SHAM-treated leaves under high NADP-MDH is the key enzyme in the malate-OAA light (Fig. 4A) indicates that the inhibition of the AOX shuttle (Noguchi and Yoshida 2008, Scheibe 2004). It pathway caused an over-reduction of the PSI acceptor is noteworthy that the initial activity of NADP-MDH side under high light because of the accumulation of

402 Physiol. Plant. 143, 2011 Fig. 7. Photosynthetic parameters derived from the JIP-test analysis of chlorophyll a fluorescence transients in Rumex K-1 leaves treated with 0 (control) or 1 mM SHAM in the absence (A, C, E) or presence (B, D, F) of 1 mM CM. Leaf discs (0.5 cm2) treated with 0 (control) or 1 mM SHAM were vacuum-infiltrated with 0 or 1 mM CM in the dark and then exposed to high light at 800 μmol m−2 s−1 for 1, 2 and 3 h. Means ± SE of eight replicates are presented. Different letters indicate significant difference between the SHAM treatment and control at P < 0.05, LSD.

excess reducing equivalents in the chloroplasts. The fact (ET0/RC) decreased, but the light absorption per active that the maximal amplitude in the I–P phase of the reaction center (ABS/RC) and trapping of excitation OJIP transient decreased obviously in the SHAM-treated energy per active reaction center (TR0/RC) increased, leaves under high light (Fig. 4B) indicates a decrease and the excitation efficiency for electron transport in the pool size of the PSI end electron acceptors beyond QA (0) and the quantum yield for electron (Kalachanis and Manetas 2010, Yusuf et al. 2010), transport (φEo) decreased significantly in the SHAM- and further supporting the above suggestion. Moreover, the nPG-treated leaves (Figs 5 and S3), indicating that the fact that the relative variable fluorescence at J-step (VJ) balance between photosynthetic light absorption and increased significantly in both the SHAM- and nPG- energy utilization was disturbed by the over-reduction treated leaves compared with the control leaves (Figs 5 of the PSII acceptor side because of the inhibition of the − and S3) suggests that the ratio of QA /QA increased AOX pathway under high light. The imbalance between (Li et al. 2009, Strasser and Strasser 1995) under high photosynthetic light absorption and energy utilization light when the AOX pathway was inhibited. Thus, the will inevitably lead to the over-excitation of the PSII inhibition of the AOX pathway decreased the electron reaction centers (Vass 2011). transport capacity at the acceptor side of PSII, resulting Under photoinhibitory condition, there are two major in an over-reduction of the PSII acceptor side. In sites of ROS generation in chloroplasts: the end of addition, the electron transport per active reaction center photosynthetic electron transport chain (acceptor side

Physiol. Plant. 143, 2011 403 Fig. 8. Schemes of the possible phenomena in the cell when AOX is inhibited under high light. AOX, alternative oxidase; OAA, oxaloacetate; Pre-D1, precursor to D1 protein; PSII/I, photosystem II/I; ROS, reactive oxygen species. of PSI) and the PSII reaction centers (Bartoli et al. 2005, high light (Figs 6A and S4) either damaged PSII directly Niyogi 1999, 2000, Vass 2011). An over-reduction of or inhibited the repair of the photodamaged PSII. To the PSI acceptor side and over-excitation of PSII reaction distinguish the effect of the inhibition of the AOX centers because of the inhibition of the AOX pathway pathway on the photodamage of PSII and the inhibition would inevitably enhance the generation of ROS under of the repair of the photodamaged PSII, we used CM, high light. Our observation that the accumulation of ROS an inhibitor of D1 protein synthesis (Takahashi et al. − (O2 and H2O2) in leaves was increased by the SHAM 2009), to inhibit the repair of the photodamaged PSII in and nPG treatments (Figs 6A and S4) supports the above this study. We observed that SHAM and nPG treatments suggestion. The observation that the activity of the ROS did not accelerate photoinhibition in the presence of scavenging system (SOD, APX and CAT) increased in CM, indicated by decreases in φPo (Figs 7B and S5B), SHAM-treated leaves under high light (Fig. 6B) indicates 0 (Figs 7D and S5D) and φEo(Figs 7F and S5F), but the that the over-accumulation of ROS was not caused by the treatments did accelerate photoinhibition in the absence influence of the SHAM treatment on the activity of ROS of CM, indicating that the AOX pathway protected plant scavenging system directly but by the over-reduction of leaves against photoinhibition, mainly by alleviating the the photosynthetic electron transport chain. inhibition of the repair of the photodamaged PSII rather The SHAM and nPG treatments caused more severe than by preventing photodamage to PSII directly. photoinhibition as they accelerated decreases in φPo In conclusion, the inhibition of the AOX pathway (Figs 7A and S5A), 0 (Figs 7C and S5C) and φEo(Figs 7E resulted in the loss of a sink of excess reducing and S5E). How did the inhibition of the AOX equivalents and accelerated the accumulation of pathway accelerate photoinhibition? Some studies have NADPH in chloroplasts, leading to the over-reduction demonstrated that the accumulation of ROS induced by of the PSI acceptor side and over-excitation of the PSII excess light energy do not accelerate photodamage to reaction centers to induce the over-production of ROS PSII directly but inhibits the repair of the photodamaged in chloroplasts under high light. The over-production PSII at the step of the de novo synthesis of the D1 protein of ROS inhibited the repair of the photodamaged PSII (Murata et al. 2007, Nishiyama et al. 2006, Takahashi (Fig. 8). Therefore, the mitochondrial AOX pathway and Murata 2008). However, Krieger-Liszkay et al. plays an important role in the protection of plants − (2011) recently reported that O2 generated in PSI can against photoinhibition by alleviating the inhibition of damage PSII directly. Therefore, the over-accumulation the repair of the photodamaged PSII through preventing of ROS due to the inhibition of the AOX pathway under the formation of ROS.

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406 Physiol. Plant. 143, 2011 Yoshida K, Terashima I, Noguchi K (2007) Up-regulation Fig. S1. Photosystem II actual photochemical efficien- of mitochondrial alternative oxidase concomitant with cies (PSII) and photochemical quenching coefficients chloroplast over-reduction by excess light. Plant Cell (qP) in isolated Rumex K-1 chloroplasts in the absence Physiol 48: 606–614 (control) or presence of 1 mM nPG. Yoshida K, Watanabe C, Kato Y, Sakamoto W, Noguchi K (2008) Influence of chloroplastic photo-oxidative stress Fig. S2. Changes of chlorophyll a fluorescence transients on mitochondrial alternative oxidase capacity and in Rumex K-1 leaves treated with 0 (control) or 1 mM respiratory properties: a case study with Arabidopsis nPG in the dark (A) or in the high light (B). yellow variegated 2. Plant Cell Physiol 49: 592–603 Yusuf MA, Kumar D, Rajwanshi R, Strasser RJ, Fig. S3. Photosynthetic parameters derived from the JIP- Tsimilli-Michael M, Govindjee Sarin NB (2010) test analysis of chlorophyll a fluorescence transients Overexpression of γ -tocopherol methyl transferase gene shown in Fig. S2B. in transgenic Brassica juncea plants alleviates abiotic − Fig. S4. Production of O and H O in Rumex K-1 stress: physiological and chlorophyll a fluorescence 2 2 2 measurements. Biochim Biophys Acta 1797: leaves treated with 0 (control) or 1 mM nPG under high 1428–1438 light. Zeng XQ, Chow WS, Su LJ, Peng XX, Peng CL (2010) Fig. S5. Photosynthetic parameters derived from the JIP- Protective effect of supplemental on test analysis of chlorophyll a fluorescence transients in Arabidopsis leaves under high light. Physiol Plant 138: Rumex K-1 leaves treated with 0 (control) or 1 mM 215–225 nPG in the absence (A, C, E) or presence (B, D, F) of Zhang DW, Xu F, Zhang ZW, Chen YE, Du JB, Jia SD, 1mM CM. Yuan S, Lin HH (2010) Effects of light on -resistant respiration and alternative oxidase Please note: Wiley-Blackwell are not responsible for function in Arabidopsis seedlings. Plant Cell Environ 33: the content or functionality of any supporting materials 2121–2131 supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author Supporting Information for the article. Additional Supporting Information may be found in the online version of this article:

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