Photosystem inhibition and protection in tomato leaves under low light

Scientia Horticulturae 217 (2017) 145–155

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Photosystem inhibition and protection in tomato leaves under low light Zhaojuan Meng a,b,c , Tao Lu a,b,c , Guoxian Zhang a,b,c , Mingfang Qi a,b,c , Wan Tang a , Linlin Li d , Yufeng Liu a,b,c,∗ , Tianlai Li a,b,c,∗ a

College of Horticulture, Shenyang Agricultural University, Shenyang, Liaoning Province, China Key Laboratory of Protected Horticulture of Ministry of Education, Shenyang, Liaoning Province, China c Collaborative Innovation Center of Protected Vegetable Surround Bohai Gulf Region, Shenyang, Liaoning Province, China d Key Laboratory of Protected Horticulture of Ministry of Education, Dalian Nationalities University, Dalian, Liaoning Province, China b

a r t i c l e

i n f o

Article history: Received 21 October 2016 Received in revised form 4 January 2017 Accepted 21 January 2017 Available online 4 February 2017 Keywords: Low light PSII and PSI P515 Cyclic electron flow Xanthophyll cycle ROS metabolism

a b s t r a c t In this study, the effect of low light (LL, 340–360 ␮mol m−2 s−1 ) on thylakoid membrane activity, photosystem I and II (PSI and PSII) activities, transient quantum yields, reactive oxygen species (ROS), cyclic electron flow (CEF) and proton motive force of tomato leaves was investigated. Results indicated that LL treatment led to low integrity of the thylakoid membrane, ATPase activity, and photoinhibition of PSII and PSI. The treatment also yielded low electron transport rate [ETR(II) and ETR(I)], high PSI donor side limitation [Y(ND)] and efficient electron transfer between the intermediate carriers to the final acceptors of PSI (␦Ro). Hence, the possible inhibition sites include QA -QB and PSI-Fd . Moreover, LL increased the excitation pressure, ROS scavenge enzyme activities, CEF/Y(II) radio, formation of proton gradient and decreased chlorophyll a/b ratio in the thylakoid membrane, thereby alleviating inhibition of PSII and PSI to a certain extent. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Light is one of the main factors that substantially affect plant growth. Plants in greenhouses in Northern China is readily subjected to damage under low light (LL), which inhibits plant photosynthesis and induces variations in the thylakoid membrane (Dai et al., 2009; Fan et al., 2013; Shao et al., 2014). Tomato (Lycopersicon esculentum Mill) is, a light crop, widely cultivated in northeastern China. Therefore, the effects of LL on tomato photosystem inhibition must be determined through systematic research. Light energy is the driving force for photosynthesis; therefore, inhibition inevitably generates in photosynthetic organisms under LL (Anderson and Chow, 2002; Murata et al., 2007; Tyystjärvi and Aro, 1996). Several mechanisms underlying photosystem inhibition have been proposed (Goh et al., 2011; Noam et al., 2003; Ohnishi et al., 2005). Studies have focused on photosystem under multiple stresses. Generally, photosystem II (PSII) is speculated to be the inhibition site. PSII is generally considered more sensitive than PSI and is easily damaged under temperature and light stresses

∗ Corresponding authors at: College of Horticulture, Shenyang Agricultural University, Shenyang, Liaoning Province, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (T. Li). http://dx.doi.org/10.1016/j.scienta.2017.01.039 0304-4238/© 2017 Elsevier B.V. All rights reserved.

(Zhang et al., 2014). Exposure of leaves to moderate light and chilling temperature led to selective damage to PSII in tropical trees; the damage to PSII activity could be quickly repaired under low light in several hours, whereas PSI activity was minimally affected during stress and recovery treatments (Huang et al., 2010). However, several studies have shown that the damage to PSII is negligible under LL and chilling temperature (< 100 ␮mol m−2 s−1 )in contrast to the serious damage to PSI (Havaux and Davaud, 1994). Terashima et al. (1994) found that PSI was more easily inhibited in the leaves of Cucumis sativus compared with PSII, which showed almost no damage; hence, PSII is believed to be the main site of inhibition (Sonoike, 1996b, 1998). Li et al. (2004) also found that PSI inhibition was the main factor that limits subsequent recovery under irradiation after chilling treatment. LL stress significantly affects PSII and PSI; but the effects likely to be masked by temperature in short period of time. Therefore, the effect of LL alone on inhibition of PSII and PSI must be investigated. Under fluctuating light, plants can generally adapt their photosynthetic characteristics to the light condition in the environment by changing their photosynthetic apparatus for several hours to a week (Kono and Terashima, 2014). Mechanisms, including excitation pressure, PSII/PSI ratio, anti-oxidative scavenging system, Mehler s reaction and cyclic electron flow (CEF) around PSI may

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protect photosystems (Joliot and Johnson, 2011) by eliminating surplus electrons and ROS. ROS are eliminated by conversion to water and heat (Miyake, 2010; Takahashi and Badger, 2011). Nonphotochemical quenching (NPQ) is necessary for dissipating excess energy in the energy imbalance between PSI and PSII; the fastest responses were observed when qE was linked to the xanthophyll cycle (XC) (Roach and Krieger-Liszkay, 2014). PSII/PSI ratio and CEF can also change the redox state of PSI (Shikanai, 2014; Tikkanen et al., 2014). Our previous study indicated that the net photosynthesis in tomato was reduced under LL treatment (Meng et al., 2012). In the present study, we investigated the effect of low light on PSII and PSI. This work aims to (1) determine the inhibition site (whether PSII, PSI, or others) and (2) identify the mechanism by which photosystems are regulated under LL conditions.

Dual-PAM-100 and the automated routines provided by the DUAL-PAM software with minor modifications. After 1 h of dark adaptation, rapid P515 changes induced by saturating single turnover flashes were recorded to evaluate the integrity of the thylakoid membrane and activity in ATPase. Slow dark–light–dark induction transients of the 550 nm to 515 nm signals reflect changes in both the membrane potential (electrochromic pigment absorbance shift) and the zeaxanthin content. The transients were measured when actinic light (AL; 531 ␮mol m−2 s−1 ) was turned on after 30 s and off after 330 s. Before this measurement, the leaf was kept for 2 h in darkness, resulting in low zeaxanthin content. Determination of zeaxanthin content, transmembrane potential and proton gradient using the dark-light-dark induction transients was done as described previously by Schreiber and Klughammer (2008). All measurements were performed at a CO2 concentration of 400 ± 10 ␮mol mol−1 .

2. Materials and methods 2.4. Chlorophyll fluorescence and P700 measurement 2.1. Plant materials and growth conditions A popular tomato variety ‘W’ was used in the experiments. ‘W’ produced high-quality fruits, and the tomato seedlings exhibited good growth performance from 5 April to 28 May 2013 in a greenhouse at the experimental farm of Shenyang Agricultural University (41◦ 82 N, 123◦ 56 E), which is located at the southern boundary of the temperate zone. Six-leaf-old seedlings grown in the matrix were cultivated in a chamber under sunlight climate. The chamber was built by Kooland and had an average relative humidity of 60%. The highest photosynthetic photon flux density (PPFD) at midday reached 1450 ␮mol m−2 s−1 . 2.2. LL treatment and subsequent inhibition In previous studies in our lab (Yang et al., 2007), the intensity of LL (75, 50 and 25% of NL) which significantly reducing photosynthesis were optimized, using the same tomato cultivar as the present study. It found that LL (25% of NL) had remarkable effect on seedling growth and photosynthesis. Therefore, LL treatment (25% of NL) was established in our study to clarify the mechanism of photosynthesis inhibition. The LL experiment and physiological measurements were conducted from 18 May to 28 May in 2013. During this period, the day and night temperatures in the sunlight −climate chamber were 25 ◦ C (from 6:00 a.m. to 5:30 p.m.) and 15 ◦ C (from 6:00 p.m. to 5:30 a.m.), respectively. Seedlings with similar vigor were planted in a nutrient substance in a same room and divided into two groups with 46 pots in each group. One group was placed under natural light (NL) with maximum PPFD in the range 1380–1450 ␮mol m−2 s−1 at noon (sunshine conditions), and the other group was placed under LL with maximum PPFD in the range 340–360 ␮mol m−2 s−1 for 11 days. The means of PPFD under NL and LL throughout the experimental days were shown in Supplementary Fig. S1 in the online version at DOI: http://dx.doi.org/10. 1016/j.scienta.2017.01.039. Insufficient light was compensated by an efficient automatic plant growth sodium lamp under both treatments. Moreover, a set of 18 tomato seedlings from each group was used to separately determine the fast and slow chlorophyll a fluorescence parameters, and 10 tomato seedlings from each group were tested for the physiological indices. The sixth leaf from each plant was used for subsequent experiments. All experiments were conducted using the sixth fully expanded leaves and were repeated three times with three replicates for each analysis. 2.3. P515 relaxation kinetics and transients of 550–515 nm signal The dual-beam 550 nm to 515 nm difference signal was monitored simultaneously by using the P515/535 module of the

The Chlorophyll fluorescence of PSII and the redox state of P700 were simultaneously measured at room temperature with the automated induction and recovery program Dual-PAM-100 fluorometer (Walz, Effeltrich, Germany) and Dual-PAM software. The sixth leaves under NL and LL were dark-adapted for 20 min before measurement. The fluorescence and P700+ signals were recorded with a saturation pulse (300 ms) of saturating light (10,000 ␮mol m−2 s−1 ) to determine the minimum fluorescence of the dark-adapted state (Fo ), the maximum fluorescence (Fm ) in the dark-adapted state and the maximum P700+ (Pm ). The fluorescence parameters included the effective quantum yield of PSII [Y(II)], the quantum yield of non-regulated energy dissipation [Y(NO)], and the quantum yield of regulated energy dissipation [Y(NPQ)]. They were calculated as follows: Fv /Fm = (Fm − Fo )/Fm , Y(II) = (Fm − Fs )/Fm , Y(NO) = Fs /Fm , Y(NPQ) = 1 − Y(II) − Y(NO). Fm  represents maximum fluorescence values upon illumination in the light-adapted state and Fs is steadystate fluorescence in light. Fv /Fm reflects the maximum quantum yield of PSII (Kramer et al., 2004; Lei et al., 2014). Y(NPQ) and Y(NO) reflect the ability of a plant to self-protection. The maximal P700 changes (Pm ) were measured with a dual wavelength (830/875 nm) unit (Huang et al., 2012; Klughammer and Schreiber, 2008) by Dual-PAM-100, which was recorded by applying a saturation pulse after pre-illumination with far-red light and explained the level at P700 fully oxidised the amount of efficient PSI complex. Pm  was determined similarly to Pm , but without far-red pre-illumination. The parameters included PSI donor side limitation [Y(ND)], PSI acceptor side limitation [Y(NA)], the effective quantum yield of PSI [Y(I)]. They were calculated as follows: Y(ND) = 1−P700 red, Y(NA) = (Pm −Pm  )/Pm , Y(I) = 1 − Y(ND) − Y(NA). Y(ND) is the fraction of overall P700 that is oxidised due to a lack of donors. While Y(NA) represents the fraction that cannot be oxidised because of a lack of acceptors. And Y(I) accounted for the fraction of overall P700 in a given state, which is reduced and not limited by the acceptor side. The parameters related to CEF were calculated as follows: Y(CEF) = Y(I) − Y(II), Y(CEF)/Y(II) = [Y(I) − Y(II)]/Y(II). Ten sixth leaves in different tomato seedlings were dark adapted for 20 min in a leaf clip prior to Chlorophyll a fluorescence transient measurement with a Plant Efficiency Analyzer fluorometer (PEA, Hansatech Instruments Ltd, King’s Lynn, Norfolk, UK), according to methods (Jiang et al., 2008; Strasser et al., 2004; Yordanov et al., 2008). The fast chlorophyll a fluorescence of dark-adapted leaves was induced by an array of six red (peak at 650 nm) LEDs of 3000 mmol m−2 s−1 for 2 s. Many biophysical parameters derived from cardinal points in the fluorescence versus time curve were used to calculate the following parameters according to the JIP-test. They reflect photosynthetic efficiencies at the onset of illumination,

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including: (i) the maximum quantum yield of primary photochemistry ␸Po = TRo/ABS, where TR and ABS denotes the trapped and absorbed excitation energy; (ii) the efficiency to conserve trapped excitation energy as redox energy ␺Eo = ETo/TRo; (iii) the quantum yield of electron transfer to intermediate electron carriers (Quantum yield for electron transport) ␸Eo = ETo/ABS; (iv) the efficiency of electron transfer between intermediate carriers to final acceptors of PSI, ␦Ro = REo/ETo; and (v) the quantum yield of reduction of final electron acceptors of PSI per photon absorbed, ␸Ro = REo/ABS = ␸Po* ␺Eo* ␦Ro (Zeliou et al., 2009). 2.5. Situ histochemical localization of O2

•−

and H2 O2

Accumulation of O2 ·− and H2 O2 was detected by a histochemical staining method with nitro blue tetrazolium (NBT) and diaminobenzidine (DAB), according to methods (Romero-Puertas et al., 2004; Shi et al., 2010; Wang et al., 2011). For localization • of O2 − , the leaves from NL and LL treatment were excised and immersed in a 1 mg mL−1 solution of NBT in 10 mM phosphate buffer (pH 7.8), then incubated at room temperature for 1–2 h until blue spots appeared. For H2 O2 detection, another set of samples were immersed in DAB solution (1 mg mL−1 , pH 3.8) made in 10 mM phosphate buffer (pH 7.8), vacuum-infiltrated for 10 min and incubated at room temperature for 8 h in the dark and then 1 h in the light until brown spots were visible. For both staining methods, the samples were bleached in boiling ethanol to present the blue and brown spots, which were kept in 70% ethanol and 5% glycerinum for taking pictures by a scanner. The results were expressed as percentage of spots area versus total leaf area to compensate for differences in leaf size. 2.6. Determination of superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities Frozen leaves samples were homogenized with 8 mL cold extraction solution (50 mM phosphate buffer, pH = 7.0, contained 1.0 mM Ethylene Diamine Tetraacetic Acid (EDTA)). The homogenate was centrifuged at 15 × 103 g for 20 min at 4 ◦ C. The supernatant was used to measure the level of lipid peroxidation, SOD and APX activity, according to the method (Shi et al., 2010). For determination of SOD activity, 25 ␮L extracting enzyme solution was added to 3 mL reaction solution in test tube (50 mM phosphate buffer at pH 7.8, 13 mM methionine, 63 ␮M NBT, 1.3 ␮M riboflavin, 1.0 mM EDTA) in the dim light and this reaction was conducted in 3,000 Lux illumination for 17 min at 25 ◦ C. Absorbance of the solution was measured at 560 nm wavelength. To determinate activities of APX, 200 ␮L extracting enzyme solution was diluted with 3.2 mL reaction solution in test tube (25 mM phosphate buffer at pH 7.0, 0.1 mM EDTA), 200 ␮L H2 O2 (10 mM) and 200 ␮L ascorbic acid (0.25 mM). Absorbances of the solution were measured at 290 nm wavelength in one or two min.

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am to 10 am and 0.5 g fresh leaf was used as a biological repetition. There are three repetitions in each treatment. 2.8. Statistical analysis Experiments were displayed on randomly selected samples with 3–10 independent biological replicates. The data were analyzed by one-way analysis of variance (ANOVA) in SPSS software version 18.0 (SPSS, Chicago, USA), and differences were detected by Duncan’s multiple range test and significance was considered when p < 0.05. Graphs were plotted on Origin 8.0 (Origin Lab, Northampton, MA, USA). 3. Results 3.1. Effect of LL on rapid P515 relaxation kinetics The decay of the P515 signal reflects the relaxation of flash induced electric field (created by charge separation in two photosystems and electrogenic electron transport in Q-cycle at the cytb6 f complex) by H+ efflux via the H+ channel of ATPase. A functionally intact photosynthetic apparatus is characterised by slow decay after dark-adaptation (high membrane integrity) and a fast decay after illumination (high ATPase activity). Our observations indicated that thylakoid membrane was damaged and ATPase activity was reduced after LL treatment, compared with those under NL (Fig. 1). 3.2. Effect of LL on the activities of PSII and PSI Fv /Fm decreased from day 5 after LL stress, and Pm significantly decreased from day 9. Hence, PSII and PSI activities were inhibited, and the former was first inhibited. (Fig. 2). The value of Y(II) under LL was lower than that under NL, whereas the values of Y(NO) and Y(NPQ) were high from day 5 and 7 (Fig. 3A–C). The Y(I) values in the LL treatment groups were significantly lower than those in NL treatment (Fig. 3D). Under LL, we speculate that the low Y(I) values could be due to increase in Y(ND); because Y(ND) remained high in all LL treatment groups at all days than those in NL treatments (Fig. 3E). 3.3. Effect of LL on energy fluxes between PSII and PSI in the electron transport chain The differences in quantum yields increased along the linear electron flow from PSII to PSI under LL. The ␸Po , ␸Eo and ␺Eo (Fig. 4A–C) decreased, thereby reducing the activity of PSII; consequently, an enhanced transformation of active PSII centres to non-QA centres or PSI can be presumed in the leaves under LL. PSI was also negatively affected, as indicated by increased ␦Ro and decreased ␸Ro values (Fig. 4D, E). Accordingly, QA -QB and PSI-Fd may be the inhibition sites.

2.7. Pigment analysis

3.4. Effect of LL on excitation pressure on PSII and PSI

Chlorophyll (Chl a, Chl b, Chl a + b) contents were estimated from 0.5 g fully expanded leaves, which were extracted by grinding leaves in 80% acetone in the dark at room temperature. The absorbance of Chl a and Chl b in acetone solution was measured at 663 nm and 645 nm. The concentrations of Chl a, Chl b per leaf fresh weight unit were determined, using the equations of Mackinney (1941), as described (Porra, 2005). A663 = 82.04Ca (g L−1 ) + 9.27Cb (g L−1 ); A645 = 16.75Ca (g L−1 ) + 45.60Cb (g L−1 ). The contents of chlorophyll (mg g−1 ) = CV/1000W (C, the concentration of chlorophyll (mg L−1 ); V, total volume of extract (ml); W, fresh weight of leaves (g)). In the experiment, sampling was carried out between 9

The 1-qP indicated the excitation pressure on PSII or the proportion of closed PSII reaction centres (Huang et al., 2010). 1-qP under LL was higher than that under NL, indicating increased excitation pressure on PSII under LL. We used ␣ to demonstrate the excitation pressure on PSI. The excitation pressure on PSI under LL was lower than that under NL (Fig. 5). 3.5. Pigment content with PSII inhibition under LL The contents of chlorophyll a, b, and a + b in leaves under LL significantly decreased from day 5. However, the chlorophyll a/b ratio

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Fig. 1. Rapid P515 relaxation kinetics induced by single-turnover saturating flash under low light (LL) for 11 d. Two treatments, namely, natural light (NL, 1380 to 1450 ␮mol m−2 s−1 at noon) and LL (340–360 ␮mol m−2 s−1 ), were examined in the sunshine climate chamber (±S.E.; n = 3). Single recordings of Fast Kinetics (no curve averaging) using Auto ML on and MF-10000. Display with an average of 10 points. A) Dark-adapted for 1 h. B) Preilluminated for 10 min at 531 ␮mol m−2 s−1 followed by 4 min dark.

Fig. 2. Changes in Fv /Fm (A) and Pm (B) under low light (LL) for 11 d. Two treatments, namely, natural light (NL, 1380 to 1450 ␮mol m−2 s−1 at noon) and LL (340–360 ␮mol m−2 s−1 ), were examined in the sunshine climate chamber (±S.E.; n = 3). The asterisks * and ** indicate significant of p < 0.05 and 0.01, respectively.

in the leaves under LL increased obviously (Fig. 6). We suppose this situation could be due to decreased content of PSI relative to that of PSII (Brestic et al., 2015; Pietrzykowska et al., 2014; Yamazaki et al., 1999). Under low PSI/PSII ratio, the acceptor side of PSI and the components between PSI and PSII were reduced. The observed light-induced signal increase in P515 reflects not only the increase in membrane potential (ECS) but also the formation of zeaxanthin (Schreiber and Klughammer, 2008). The relative extent of zeaxanthin formation can be judged from the increase in the ‘dark baseline’ apparently after light-off. The increase in ‘dark baseline’ was significantly lower after LL stress than that of NL indicating reduced in terms of reversible changes in zeaxanthin content (Fig. 7A). The rapid light-off response reflects H+ efflux from the lumen to the stroma of chloroplasts via the thylakoid ATPase. The rapid decline in signal is followed by a biphasic increase in the signal to an apparent ‘dark baseline’. As indicated in Fig. 7A, the characteristic levels observed during the light-off response can be used to estimate the relative amplitudes of transmembrane potential (␺) and pH. The difference between the steady-state signal and the ‘dark baseline’ reflects a substantial ␺ during steady-state illumination. The ‘undershoot’ below the ‘dark baseline’ is considered

a measure of steady-state pH. As shown in Fig. 7B, LL treatment significantly increased pH and decreased ␺. 3.6. ROS metabolic on PSI inhibition under LL •

O2 − and H2 O2 production in LL treatment were considerably enhanced, and reached higher levels than those in NL treatment, in the maximum accumulation of approximately 1.82 and 12.05 ␮mol (g h)−1 , respectively (Fig. 8A). SOD and APX contents notably decreased under LL to the respective minimum values [3.00 U and 33.60 ␮mol (g h)−1 ] on day 7 (Fig. 8B). LL stress induced a significant accumulation of ROS. This finding indicates that such an increase in NPQ in LL was insufficient to dissipate all excess excitation energy and oxidative stress was induced. Meanwhile, LL reduced the activities of SOD and APX to dissipate excess excitation energy. 3.7. CEF between PSII and PSI under LL The electron transport rates in both photosystems [ETR(II) and ETR(I)] were lower under LL than those under NL (Fig. 9A, B), indicating that linear electron transfer decreased under LL. No sig-

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Fig. 3. Changes in Y(II),Y(NO), Y(NPQ) and Y(I), Y(ND), Y(NA) (A-F) under LL for 11 d. Two treatments, namely, natural light (NL, 1380 to 1450 ␮mol m−2 s−1 at noon) and LL (340–360 ␮mol m−2 s−1 ), were examined in the sunshine climate chamber (±S.E.; n = 3). Y(ND) (donor side limitation) represents the fraction of the overall P700 that is oxidised in a given state, which is enhanced by a trans-thylakoid proton gradient (photosynthetic control at the cytb/f complex as well as downregulation of PSII) and photodamage to PSII. Y(NA) (acceptor side limitation) represents the fraction of the overall P700 that cannot be oxidised by a saturation pulse in a given state because of a lack of acceptors, and is enhanced by dark adaptation and damage at the site of CO2 fixation. The asterisks * and ** indicate significant of p < 0.05 and 0.01, respectively.

nificant difference was found in the efficiencies of CEFs [Y(CEF)] under LL and NL treatments on all days. However, the proportion of effective quantum yield in CEF to Y(II) increasingly improved from day 7 under LL (Fig. 9C, D). The results demonstrated that CEF decreased the excessive reduction stress of the acceptor side in PSI; hence, CEF was more important in the electron transport chain under LL than that under NL. 4. Discussion 4.1. Inhibition on PSII and PSI under LL Inhibition has been regarded as a decrease in photosynthetic efficiency under high light conditions, in which the input of photons exceeds the photon requirement for photosynthesis (Barber and Andersson, 1992; Huang et al., 2011). Inhibition may occur under

LL (Shao et al., 2014) as long as the product of light intensity and time reaches a certain photon requirement threshold (Park et al., 1995). The present study indicated that PSII and PSI were inhibited under LL; activity in thylakoid membrane and ATPase decreased compared with that under NL (Figs. 1 and 2). Y(II) was significantly lower and Y(NO) was higher under LL than those under NL (Fig. 3A, B). This finding suggested that the PSII super-complex may have been damaged and (or) the turnover of D1 may have been disturbed by exposure to excess light energy. Similarly, in guayule grown in vitro, Y(II) decreased under LL (100 ␮mol m−2 s−1 ) than that under high light (1,250 ␮mol m−2 s−1 ) (Turan et al., 2014). The decrease in Y(I) in the treated tomato leaves could be due to the increase in the donor side limitation of PSI, as reflected by Y(ND) (Fig. 3E). Hence, LL did not release an excessive amount of light energy to PSI and the donor side limitation of PSI. The proportion of reduced electron carriers on the donor side of PSI that cannot be oxidised. We

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Fig. 4. Changes in energy capture and transformation along linear electron transport (A-E). Quantum yields of energy capture and electron flow as well as probabilities for trapped excitation energy to move electrons under NL and LL treatments (±S.E.; n = 10). Abbreviations: ␸Po , maximum quantum yield of primary photochemistry; ␸Eo , quantum yield of electron transfer to intermediate carriers; ␸Ro , quantum yield for the reduction of end acceptors of PSI per photon absorbed; ␺Eo , efficiency to conserve trapped excitation energy to electron transfer; ␦Ro , efficiency in electron transfer from reduced intermediate carriers to final electron acceptors of PSI. The asterisks * and ** indicate significant of p < 0.05 and 0.01, respectively.

Fig. 5. Changes in 1-qP (A) and ␣ (B) under LL for 11 d. Two treatments, namely, natural light (NL, 1380 to 1450 ␮mol m−2 s−1 at noon) and LL (340–360 ␮mol m−2 s−1 ), were examined in the sunshine climate chamber (±S.E.; n = 3). The asterisks * and ** indicate significant of p < 0.05 and 0.01, respectively.

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Fig. 6. Changes in chlorophyll contents (A-D) under LL. Two treatments, namely, natural light (NL, 1380 to 1450 ␮mol m−2 s−1 at noon) and LL (340–360 ␮mol m−2 s−1 ), were examined in the sunshine climate chamber (±S.E.; n = 3).

Fig. 7. Changes in 550–515 nm signal induced by slow dark-light-dark. Slow P515 changes induced by actinic illumination (10 min at 531 ␮mol m−2 s−1 ) after 30 s and return to darkness after 330 s. Same tomato leaf was used as in experiment of Fig. 7 after dark-adaptation. Use of Auto MF-High, switching from MF 200–2000. A) Complete recording of light-on and light-off responses. B) Enlarged display of light-off response with indication of estimated pH and ␺ components of pmf (±S.E.; n = 3). The asterisks ** indicate significant of p < 0.01.

speculated that this phenomenon might be a result of the electron accumulation in the PSI donor side caused by decreased capacity for PQ (Zhang et al., 2014). By contrast, the conclusion is different from Terashima’s reported that PSI could not be inhibited and PSI complex was not degraded in the cucumber leaves under anaerobic conditions in chilling-light treatments (Terashima et al., 1994). The discrepancy in the results could be due to different treatments employed, that is, chilling-light was used in the study of Terashima,

whereas only LL was investigated in the present study. In addition, under chilling temperature at very LL (<10 ␮mol m−2 s−1 ), the main site of inhibition was located in the H-ATPase (Terashima et al., 1989). Our results confirmed that PSII and PSI were successively inhibited under LL for 11 days. Combined to energy fluxes between PSII and PSI, it showed that QA -QB and PSI-Fd may be the inhibi-

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Fig. 8. Situ histochemical localization of O2 ·− , H2 O2 (A) and activity changes in SOD, APX (B). Two treatments, namely, natural light (NL, 1380 to 1450 ␮mol m−2 s−1 at noon) and LL (340–360 ␮mol m−2 s−1 ), were examined in the sunshine climate chamber (±S.E.; n = 3). The asterisks * and ** indicate significant of p < 0.05 and 0.01, respectively.

Fig. 9. Changes in line electron flow ETR(II), ETR(I) and in cyclic electron flow Y(CEF), Y(CEF)/Y(II) (A-D). Two treatments, namely, natural light (NL, 1380 to 1450 ␮mol m−2 s−1 at noon) and LL (340–360 ␮mol m−2 s−1 ), were examined in the sunshine climate chamber (±S.E.; n = 3). The asterisks * and ** indicate significant of p < 0.05 and 0.01, respectively.

tion sites under LL (Figs. 1–4). Furthermore, light intensity differs among various crops. 4.2. Photoprotection in PSII and PSI under LL 4.2.1. Photoprotection in PSII The extent of PSII inactivation in the leaves could be modulated by various protective strategies evolved by plants to ameliorate the inherent vulnerability of PSII to light stress, wastage by nonradiative dissipation and the utilizsation of absorbed photons in electron transport (Goh et al., 2011). In the present study, the exci-

tation pressure of PSII under LL was higher than that under NL (Fig. 5A), whereas that of PSI was lower. Consequently, we inferred that the excitation pressure could be due to inhibition of PSII, but not PSI (Fig. 5B). Therefore, we supposed that the excitation pressure is a protective mechanism, resulting in increased in Y(NPQ), consistent with the known role of XC in heat dissipation. Thus, state transitions, which can migrate, may have changed the energy deliverance to PSII or PSI via LHCII (data not shown). Moreover, the movement of LHCII away from PSII relieved the excitation pressure (Sonoike, 2011). The migration of LHCII was governed by the plastoquinone pool redox state and the relative activities of PSII

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Fig. 10. Inhibition and protection mechanisms in photosynthetic apparatus under LL. The color depth in solid line or dashed lines represents increasing induction or suppression, and the degree of thickness in arrows stands for increase or decrease. The activity in thylakoid membrane and ATPase declined. The main inhibition sites were photosystems, QA -QB and PSI-Fd (black cross) under LL, compared with those under NL. The repair can be accelerated through NPQ dissipating, CEF and ROS scavenging. Abbreviations: NL, natural light; LL, low light; PS, photosystem; PQ, plastoquinone; CEF, cyclic electron transport; Cytb6 f, cytochrome b6 f complex; Fd , ferredoxin.

and PSI (Roach and Krieger-Liszkay, 2014). We inferred that energy consumption may be assigned as a synergistic action of pH considering that low pH in the thylakoid lumen activated XC (Fig. 7). Our results showed that the ratio pH/␺ was significantly higher under LL than that under NL and this change was accompanied by lower PSII activity and ATP synthesis in the former than those in the compared to that under NL latter (Figs. 1 and 7). The trans-thylakoid proton motive force (pmf) consists of electrical (␺) and osmotic (pH) components. Both components can drive the synthesis of ATP in the chloroplast ATP synthase. In addition, the pH component regulates photosynthesis, down-regulating the efficiency of light capture by photosynthetic antennae via the qE mechanism; which can harmlessly dissipate the excess absorbed light energy as heat (Ioannidis et al., 2012). We predicted that the degradation and resynthesis of PSII occurred even under LL, consistent with previously reported results (Andersson and Aro, 1997). These phenomena led to the decreased efficiency of PSII (Figs. 3A and 9A) because of the imbalance between the two processes. In addition, the decreased efficiency of PSII mitigated the damage induced by stress; thus, such change could be regarded as a regulatory mechanism rather than damage (Sonoike, 2011). 4.2.2. Photoprotection in PSI In this study, Chl a/b ratio significantly increased under LL. There was a compensatory relationship between PSII/PSI ratio and Chl a/b ratio (Haldrup et al., 2000; Pietrzykowska et al., 2014; Wientjes et al., 2013; Yamazaki et al., 1999). The reduction of PSI/PSII ratio, the acceptor side of PSI would be more reduced, and components between PSI and PSII would be further reduced in cyanobacteria and algae (Hihara and Sonoike, 2001). Therefore, PSI inhibition may have been caused by the increase of Chl a/b ratio (Fig. 6D), resulting in the imbalance of the two photosystems (Fig. 5). The variety of morphological structures in the photosystems may be the most primitive reason for PSI inhibition.

Previous studies described several mechanism through which PSI is protected from excess electrons. CEF around PSI was considered an effective protection mechanism in which electrons on the reducing acceptor side of PSI reverted to the PQ pool either from ferredoxin by ferredoxin quinone oxidoreductase pathway or from NADPH by NAD(P)H dehydrogenase pathway (Sonoike, 2011). In the present study, CEF around PSI and the effective quantum yield of photosystem II [Y(CEF)/Y(II)] ratio (Figs. 9 and 10) were more considerably stimulated from day 7 under LL compared with those under NL. This phenomenon relieved the severe injury by high accu• mulation of O2 − and H2 O2 as a result of CEF and protected PSI from inhibition under LL stress. 4.2.3. ROS metabolism Under conditions of excess light, the production of ROS was accelerated in both PSII and PSI in chloroplasts, but different ROS were produced by each photosystem. In PSII, the excitation of oxygen by triplet excited state chlorophyll causes the production of singlet oxygen (1 O2 ); in PSI, electron transfer to oxygen in the acceptor side of PSI causes the production of hydrogen peroxide (H2 O2 ) via the superoxide anion radical (O2 − ) (Asada, 2006)). In the • present study, O2 − and H2 O2 production considerably increased under LL (Fig. 8A). Hence, ROS accumulated in PSI. ROS are highly reactive and accelerate photoinhibition through direct oxidative damage to PSII. However, recent studies have demonstrated that ROS, such as 1 O2 and H2 O2 , accelerate photoinhibition by suppressing the repair of photo-damaged PSII rather than causing direct damage (Nishiyama et al., 2006). We considered that ROS might have participated in expending excess energy (Fig. 5) and slowing down the repair of PSII inhibition; as such, ROS might confer protection to direct damage to PSII transitorily. The light-dependent O2 uptake induced the univalent reduction of O2 to superoxide anion • radical (O2 − ) in chloroplasts. That alleviated excessive reduction to PSI because of the accumulation of electrons in the acceptor side. A

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temporary and covert protection was observed. Although ROS was toxic (Zubair et al., 2013), we speculated that it probably elicited more serious damage to tissue than excess electrons in the electron transport chain. Chloroplasts scavenge ROS effectively by using multiple enzymes to avoid oxidative stress; these enzymes include superoxide dismutase, ascorbate peroxidase and peroxiredoxin; antioxidants, such as water-soluble ascorbate and membrane-bound a-tocopherol; and carotenoids, such as zeaxanthin, neoxanthin and lutein in chloroplasts (Goh et al., 2011). In the present study, the content of SOD and APX in water–water cycle (WWC) significantly decreased (Fig. 8) and the conversion were aggravated in zeaxanthin, neoxanthin and lutein (Fig. 7). Although ROS scavenger alleviates inhibition in photosystem (Sonoike, 1996a), we found the protection to PSII and PSI by XC and WWC to some extent. 5. Conclusions LL treatment initially inhibited QA -QB and PSI-Fd . PSII and PSI inhibition resulted in low energy in line electron transport and increased NPQ; in addition, the remaining excitation energy balanced in LHC state transformation between two photosystems, proton-motive force and ROS metabolism. Chloroplasts scavenge ROS effectively by using multiple enzymes including zeaxanthin, neoxanthin and lutein in XC as well as SOD and APX in WWC to avoid oxidative stress. The ratio of CEF increased in electron transport chain to build the trans-thylakoid membrane proton gradient, thereby promoting the synthesis of ATP and the recovery of PSII. Acknowledgments We are grateful to Y.Q. Li, H.Y. Wang, and W.J. Zhou for expert technical assistance and W.G. Dong, Y.L. Wu, Y.C. Zhou, S.C. Liu, and B. Zhu for assist in the process of experiments, A.D. Wang, B. Wang, Z.Y. Yang, J. Jiang, and Z.H. Shi, for modification of thesis. This work was supported by the China Agriculture Research System (Grant No. CARS-25), the National Natural Science Foundation of China (Grant No. 31301813), the 12th Five-Year Support Project of Liaoning Province (Grant No. 2015103003), the Major Scientific Research Projects of Liaoning Province (Grant No. 2011215003), Specialized Research Fund for the Doctoral Program of Higher Education (20132103120007), the National Natural Science Foundation of China (Grant No. 31301814). References Anderson, J.M., Chow, W.S., 2002. Structural and functional dynamics of plant photosystem II. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 357, 1421–1430, discussion 1469–1470. Andersson, B., Aro, E.-M., 1997. Proteolytic activities and proteases of plant chloroplasts. Physiol. Plant. 100, 780–793. Asada, K., 2006. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol. 141, 391–396. Barber, J., Andersson, B., 1992. Too much of a good thing: light can be bad for photosynthesis. Trends Biochem. Sci. 17, 61–66. Brestic, M., Zivcak, M., Kunderlikova, K., Sytar, O., Shao, H., Kalaji, H.M., Allakhverdiev, S.I., 2015. Low PSI content limits the photoprotection of PSI and PSII in early growth stages of chlorophyll b-deficient wheat mutant lines. Photosynth. Res. 125, 151–166. Dai, Y., Shen, Z., Liu, Y., Wang, L., Hannaway, D., Lu, H., 2009. Effects of shade treatments on the photosynthetic capacity, chlorophyll fluorescence, and chlorophyll content of Tetrastigma hemsleyanum Diels et Gilg. Environ. Exp. Bot. 65, 177–182. Fan, X.-X., Xu, Z.-G., Liu, X.-Y., Tang, C.-M., Wang, L.-W., Han, X.-l., 2013. Effects of light intensity on the growth and leaf development of young tomato plants grown under a combination of red and blue light. Sci. Hortic. 153, 50–55. Goh, C.-H., Ko, S.-M., Koh, S., Kim, Y.-J., Bae, H.-J., 2011. Photosynthesis and environments: photoinhibition and repair mechanisms in plants. J. Plant Biol. 55, 93–101. Haldrup, A., Simpson, D.J., Scheller, H.V., 2000. Down-regulation of the PSI-F subunit of photosystem I (PSI) in Arabidopsis thaliana The PSI-F subunit is

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