Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction

Plant Physiology and Biochemistry xxx (2014) 1e7

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Research article

Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction Ryoichi Sato a, Hiroyuki Ohta b, c, Shinji Masuda b, c, * a b c

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan Earth-Life Science Institute, Tokyo Institute of Technology, Tokyo 152-8550, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 October 2013 Accepted 18 March 2014 Available online xxx

In chloroplasts, regulated formation of the proton gradient across the thylakoid membrane (DpH) is important for controlling non-photochemical quenching (NPQ), which is crucial for plants to perform photosynthesis under fluctuating light conditions. The DpH is generated by two electron flows: the linear electron flow (LEF) and the cyclic electron flow (CEF). The Arabidopsis CEF mutant, pgr5, showed significantly lower NPQ values than those observed in WT, indicating that DpH, generated by the PGR5dependent CEF, has a crucial role in controlling NPQ. However, the respective significance of LEF and CEF for DpH formation is largely unknown. Here we applied computer simulation to reproduce NPQ induction kinetics and estimate the respective contribution of LEF and PGR5-dependent CEF to the dynamics of DpH formation. The results indicate that the contribution of CEF to total DpH formation for induction of NPQ varies from 60e80%. The simulation also suggested a role of the PGR5-dependent CEF in accelerating electron transfer in the cytochrome b6f complex. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Regulation of photosynthesis NPQ induction Cyclic electron flow PGR5 Chlorophyll fluorescence

1. Introduction Plants convert sunlight energy to biochemical energy by photosynthesis, a process that sustains most biological activity on Earth. Therefore, controlling photosynthesis is critically important for almost all living organisms. On the Earth, sunlight is changeable in its intensity and spectral quality during the day, and plants have to perform photosynthesis with this fluctuating light (Li et al., 2009). Under high light conditions, plants switch on a photoprotective state in the photosynthetic machinery that safely dissipates the excess absorbed energy as heat (Horton et al., 1996). This dissipation of the excess absorbed energy can be monitored by a chlorophyll fluorescence parameter, non-photochemical quenching

Abbreviations: PSI, photosystem I; PSII, photosystem II; b6f, cytochrome b6f complex; DpH, proton gradient across thylakoid membrane; NPQ, non-photochemical quenching; LEF, linear electron flow; CEF, cyclic electron flow; PAM, pulseamplitude modulation; PQ, plastoquinone; PC, plastocyanin; Fd, ferredoxin; NADPH, nicotinamide adenine dinucleotide phosphate; ATP, adenosine triphosphate. * Corresponding author. Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan. Tel.: þ81 45 924 5737; fax: þ81 45 924 5823. E-mail address: [email protected] (S. Masuda).

(NPQ) (Holt et al., 2004). NPQ has four components, which are termed qE (Johnson and Ruban, 2011), qZ (Nilkens et al., 2010), qT (Quick and Stitt, 1989), and qI (Müller et al., 2001). These components have different time scales for induction, indicating that acclimation to high light through NPQ is regulated in a stepwise manner (Nilkens et al., 2010). Among the four NPQ components, qE makes the greatest contribution to total NPQ induction, which is rapidly increased upon light illumination, and reversibly disappears in the dark (Johnson and Ruban, 2011). In the qE-dependent NPQ, chlorophyll fluorescence is quenched in LHCII antenna, although exact mechanisms of this quenching are still being debated (Ruban et al., 2012). qE is activated by an increment in the proton gradient across thylakoid membranes (DpH), and is regulated by PsbS, a subunit of photosystem II (PSII) (Johnson and Ruban, 2011). Specifically, acidification of the lumen side of the thylakoid membranes, by the increment in DpH formation, results in the protonation of several key amino-acid residues of PsbS that leads to induction of qE-dependent NPQ (Li et al., 2000). An Arabidopsis PsbS mutant (npq4) shows complete loss of the rapidly forming component of DpH-dependent NPQ induction (Li et al., 2000). The induction of qE is also affected by zeaxanthin concentration (Johnson and Ruban, 2011). The maximum qE induction requires not only high DpH but also accumulation of zeaxanthin

http://dx.doi.org/10.1016/j.plaphy.2014.03.017 0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Sato, R., et al., Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.03.017

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(Niyogi et al., 1998). Zeaxanthin is generated and located in LHCII antenna under high-light condition; thus this carotenoid is considered to be quencher of qE-dependent NPQ (Ruban et al., 2012). In fact, accumulation level of zeaxanthin has relation to qE-dependent NPQ induction level (Johnson and Ruban, 2011). The accumulation level of zeaxanthin is regulated by violaxanthin deepoxidase (Niyogi et al., 1998). This enzyme is located in thylakoid lumen and activated by lowering pH (Bratt et al., 1995). Hence, qE induction largely depends on the DpH formation. The DpH is generated by photosynthetic electron flow. There are two types of photosynthetic electron flow in chloroplasts: linear electron flow (LEF) and cyclic electron flow (CEF) (Johnson, 2011). In the LEF, electrons are transferred from water to NADPþ via PSII, plastoquinone (PQ), the cytochrome b6f complex (b6f), plastocyanin (PC), photosystem I (PSI), and ferredoxin (Fd) (Fig. 1) (Nelson and Ben-Shem, 2004). Water oxidation in PSII as well as electron transfer in b6f result in generation of the DpH that is used for ATP synthesis. Electrons, passed through LEF, finally reduce NADPþ to generate NADPH. The DpH is also generated by CEF, in which electrons go back to PQ from Fd. Thus, CEF contributes to ATP synthesis without NADPþ reduction; therefore, a certain extent of CEF is important for maintaining the correct ATP/NADPH ratio for control of CO2 fixation (Munekage and Shikanai, 2005). There are two principal routes for CEF: the NADH dehydrogenase-like complex dependent pathway and the PGR5PGRL1 complex dependent pathway (Hertle et al., 2013). The Arabidopsis pgr5 mutant showed significant reduction in NPQ, indicating that the PGR5-dependent CEF has crucial roles for NPQ control through DpH generation (Munekage et al., 2002). In fact, the pgr5 mutant is very sensitive to fluctuating light conditions (Suorsa et al., 2012). On the other hand, Arabidopsis mutants lacking NADPH dehydrogenase dependent CEF activity show normal induction of NPQ (Ishikawa et al., 2008). These results indicate that NPQ induction by CEF is mostly due to PGR5 activity, not NADPH dehydrogenase activity. Although many components involved in NPQ have been elucidated, the respective contribution and dynamics of LEF and CEF activity during NPQ induction remain largely unknown. In this paper, we estimate the respective reaction dynamics of LEF and PGR5dependent CEF for qE-dependent NPQ induction. We combine PAM analysis and computer simulation to calculate the induction dynamics of the two electron flows in generating the DpH.

different light intensities (Fig. 2). Under low light conditions, WT showed high NPQ induction at an early phase (w1 min after light illumination) (Fig. 2A), which rapidly decreased to low levels after w2 min. In contrast, the pgr5 mutant did not show such a high NPQ induction at the early phase (Fig. 2A) as shown previously (Munekage et al., 2002). The kinetics of NPQ induction in the pgr5 mutant was similar to that in the npq4 mutant. Given that the npq4 mutant lacks the rapidly forming component of DpH-dependent NPQ induction (Johnson, 2011), the observed small NPQ increments in the pgr5 and npq4 mutants during the early phase reflect NPQ components other than DpH-dependent NPQ induction. The xanthophyll cycle carotenoids, such as zeaxanthin, do not accumulate in dark-adapted leaves (Johnson and Ruban, 2011), indicating that the increment of NPQ in WT during the first 1 min is due to DpH-dependent NPQ induction. Low NPQ induction in the pgr5 mutant at the early phase suggests that the DpH generated by LEF is not enough to induce qE under low light conditions (Munekage et al., 2002). Under high light conditions, WT showed high NPQ induction at the early phase (w1 min), which did not decrease, at least for 10 min (Fig. 2B). Under the high light conditions, the pgr5 mutant showed small but significant NPQ induction at the early phase (w1 min) (Fig. 2B). The NPQ induction in the pgr5 mutant indicates that LEF can induce the rapidly forming component of DpHdependent NPQ induction to some extent under high light conditions. The NPQ induction in the pgr5 mutant decreased within 2 min after illumination, and showed levels similar to the npq4 mutant (Fig. 2B), indicating that some mechanisms relaxing DpH formation are activated in this time range. To estimate the contribution of CEF and LEF to NPQ induction, we measured NPQ induction kinetics in WT and the pgr5 mutant at the early phase in more detail (Fig. 3). In this experiment, NPQ was expressed as qE to directly show the extent of DpH formation at the early phase. To directly reflect the DpH-dependent NPQ induction at the early phase, qE was calculated by subtraction of the NPQ value of the npq4 mutant from the NPQ value of the WT or the pgr5

2. Results & discussion 2.1. Kinetics of NPQ induction in wild type (WT) and the pgr5 mutant To estimate the contribution of CEF to NPQ induction, we compared NPQ induction kinetics in WT and the pgr5 mutant under

Fig. 1. A schematic representation of electron flow and DpH generation in the model. Proton and electron flows are represented by dotted and dashed lines, respectively.

Fig. 2. Induction kinetics of NPQ in WT and pgr5 and npq4 mutants under (A) low light conditions (80 mmol m2 s1) and (B) high light conditions (1200 mmol m2 s1).

Please cite this article in press as: Sato, R., et al., Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.03.017

R. Sato et al. / Plant Physiology and Biochemistry xxx (2014) 1e7

mutant (Zaks et al., 2012). In both WT and the pgr5 mutant, qE induction increased depending on time and light intensity (Fig. 3A and B), although it did not show linear increments. The values of qE in the pgr5 mutant were significantly (w0.2-fold) lower than those in WT under all light conditions tested. Because LEF, but not CEF, is functional in the prg5 mutant, the contribution of LEF to qE induction may be very low even in WT. The induced qE values in WT and the pgr5 mutant decreased within w30 s after illumination, indicating that some mechanisms decreasing DpH formation are activated in this time range. 2.2. Simulation reproducing NPQ induction kinetics To estimate the contribution of LEF and CEF to qE-dependent induction of NPQ, we next performed a computer simulation to reproduce the experimentally obtained qE induction kinetics in WT and the pgr5 mutant. We calculated the respective reaction kinetics of all photosynthetic electron transfer components (Fig. 1), and fitted the values to reproduce the experimental data shown in Fig. 3. All parameters used for the simulation are listed in Table 1. Fig. 4 shows the experimental and simulated values of qE induction. The experimental data, shown in gray symbols, are the

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same as those in Fig. 3. The experimental qE values in the pgr5 mutant (Fig. 4B, D, F, H) increased to 0.03e0.12 in 20 s after illumination. Within the next 20 s, these values relaxed to a smaller extent at light intensities of 9 and 31 mmol photons m2 s1 (Fig. 4B, D). At higher light intensities (104 and 135 mmol photons m2 s1), the qE values increased slowly within the next 10 s, and the relaxation of qE occurred only after 30 s (Fig. 4 F, H). The simulated qE values (black solid lines) for the pgr5 mutant reproduced the experimental data very well (Fig. 4B, D, F, and H). The qE values obtained experimentally in WT (Fig. 4A, C, E, G) increased to 0.3e0.6 in the first 20 s. Interestingly, we could not reproduce the WT kinetics by simulation when applying the same parameters used for simulation of the prg5 mutant, (Supplemental Fig. 1). Thus, we set up different Vmax values for b6f activity to reproduce the WT kinetics. We found that experimental data for 9, 31, 104 and 135 mmol photons m2 s1 could be reproduced in simulations using 1.8-, 2.0-, 3.3-, and 3.8-fold higher b6f Vmax values, respectively, than those used for fitting pgr5 mutant kinetics (Fig. 4A, C, E, G). This suggests that the electron transfer rate in b6f in WT is higher than in the pgr5 mutant, which is variable depending on light intensity. Recently, the principal protein complex for electron transfer in the PGR5-dependent CEF was reported (Hertle et al., 2013). This complex is composed of PGR5 and PGRL1, and transfers electrons from Fd to PQ through direct interaction with b6f (Hertle et al., 2013). One possibility is that the complex formation of PGR5, PGRL1 and b6f may result in the acceleration of electron transfer from b6f to PC, as simulated here, although the mechanism is not known. The experimental qE values of WT relaxed within 20e30 s after illumination (Fig. 4A, B, C and D). Our simulation for WT qE reproduced the relaxation kinetics of qE quite well. 2.3. Estimation of significance of CEF to total electron transfer

Fig. 3. Induction kinetics of qE in WT (A) and the pgr5 mutant (B) under various light conditions. qE was calculated by subtracting the NPQ value of the npq4 mutant from each corresponding value. The experiments were performed under 135 mmol m2 s1 (squares), 104 mmol m2 s1 (diamonds), 31 mmol m2 s1 (triangles), and 9 mmol m2 s1 (circles).

We next estimated the significance of CEF to total electron flow by our model (Fig. 5). Relative Fd oxidation ratios from PRG5dependent CEF and FNR were used for prediction. The contribution of CEF rapidly increased within a few seconds after illumination (Fig. 5). Then the contribution decreased depending on light intensity. For a few seconds after illumination, the contribution of CEF was higher than 90% (Fig. 5). Previous studies showed that the CEF activity, monitored by a change in P700 absorbance, was more than 9-fold higher than the LEF activity (Joliot and Joliot, 2006). Our simulation values in the time range fit these previous results well. However, the steady state values for the CEF contribution determined in this study differed from those in previous reports (Joliot and Joliot, 2005, 2006; Breyton et al., 2006). Specifically, our simulation indicated that the ratio of Fd oxidation by CEF to total Fd oxidation was higher than 60% at any light intensity (Fig. 5). On the other hand, in a light adapted leaf, the contribution of CEF activity to total electron flow was reported to be w20% (Joliot and Joliot, 2005, 2006; Breyton et al., 2006). The higher values for the CEF contribution, calculated here (Fig. 5), indicate either that the LEF ratio is too low in our model or the estimation of the ratio in previous reports was too high. Previous determination of the CEF ratio by P700 absorbance change used plants exposed to a lightedark transition or electron transport inhibitors such as 3-(3,4dichlorophenyl)-1,1-dimethylurea (DCMU). One possibility is that these LEF inhibitory conditions may affect CEF activity, which would influence the enhancement of CEF-dependent b6f activity predicted by our model (see above). Another possibility is that our simulation model contains inappropriate assumptions in some extent. In our calculation model, the DpH-dependent qE induction was based on the presupposition that WT and the prg5 mutant have the same zeaxanthin levels in the dark. The accumulation level of

Please cite this article in press as: Sato, R., et al., Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.03.017

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Table 1 Parameters of simulation analysis. Parameter

Value

Source

Chlorophyll content Lumen volume Stroma volume Thylakoid membrane volume PSII Number of PSII Km for PQ reduction kcat Plastoquinone Nunmer of PQ Cytochrome Number of b6f Km for PQ oxidation kcat for PQ oxidation Km for PC redaction kcat for PC redaction Plastocyanin Number of PC PSI Number of PSI Km for PC oxidation kcat for PC oxidation Km for Fd redaction kcat for Fd redaction Ferredoxin Number of Fd FNR Km for Fd oxidation Vmax of Fd oxidation Km for NADPþ reduction Vmax of NADPþ reduction NADPþ Number of NADPþ and NADPH CalvineBenson cycle Km for NADPH oxidation Vmax of NADPH oxidation KgR KR ATP synthase Vmax KA1 Km KgADP KADP ns Ks nred Kt C a for WT b for WT a for pgr5 b for pgr5 CEF Vmax Km Non-photochemical reduction Vmax Km Non-photochemical oxidation Vmax Km

1 mol 44.8 L 89.6 L 5.15 L

(Schuldiner et al., 1972) (Winter et al., 1994) (Kirchhoff et al., 2002)

1.09 mmol 10 mM 104 s1

(Kirchhoff et al., 2002) (Robinson and Crofts, 1983) (Robinson and Crofts, 1983)

8.72 mmol

(Kruk and Karpinski, 2009)

1.4 mmol 4.1 mM 71.8 s1 4.0 mM 70 s1

(Kirchhoff et al., 2002) (Clark and Hind, 1983) fitted (Hope et al., 1994) (Hope, 2000) (Hope, 2000)

3.33 mmol

(Katoh et al., 1961)

2.25 mmol 100 mM 2  104 s1 54 mM 1.08  104 s1

(Kirchhoff et al., 2002) (Bottin and Mathis, 1985) (Bottin and Mathis, 1985) (Sétif, 2001) (Sétif, 2001)

4 mmol

(BÖHME, 1978)

0.57 mM 114 M s1 0.05 mM 40 M s1

(Hanke et al., 2004) (Hanke et al., 2004) fitted (Corneille et al., 1998) (Corneille et al., 1998) fitted

9.6 mmol

(Hanke et al., 2008)

0.5 mM 0.2 M s1 0.3 8.5

Fitted Fitted Fitted Fitted

20 mmol s1 2.4 1.3 0.135 53 1.5 12 0.7 50 0.06 4.26 20.5 0.421 16.9

Fitted (Pänke and Rumberg, 1996) (Pänke and Rumberg, 1996) (Kohzuma et al., 2013) (Kohzuma et al., 2013) (Konno et al., 2012) (Konno et al., 2012) fitted (Konno et al., 2012) (Konno et al., 2012) (Kohzuma et al., 2013) Fitted Fitted Fitted Fitted

1.58 mmol s1 5 nM

Fitted Fitted

quantification of zeaxanthin is required to evaluate the errors range, zeaxanthin is not detectable in dark-adapted leafs (Johnson and Ruban, 2011)[data not shown]. Thus, the error, if it exists, seems to be minor. In addition, our simulation analysis was based on NPQ induction observed within 1 min after illumination where influence of slowly forming NPQ components (qT, qI etc.) is considered to be little (Zaks et al., 2012). If these slowly forming NPQ were considerably overlapped in our experimental data of qE induction kinetics, we overestimate the qE induction and LEF activity. 3. Conclusion We reproduced qE-dependent NPQ induction kinetics by computer simulation using values for parameters that were mostly taken from the literature. Our simulation suggests that the CEF contribution to total Fd oxidation is especially high, and that the contribution of CEF to total electron flow is 60e80%. Our data also suggest that CEF itself enhances the electron transfer reaction in b6f by 1.8- to 3.8-fold in response to increased light intensity. This prediction gives new perspectives to the role of CEF in controlling photosynthesis. 4. Materials and methods 4.1. Plant materials and growth conditions Plants used were the Columbia ecotype of Arabidopsis thaliana. The pgr5 (Munekage et al., 2002) and npq4 (Li et al., 2000) mutants were kindly provided by Dr. Toshiharu Shikanai at Kyoto University. All plants were grown on Murashige and Skoog medium (without sucrose and hormones) with 0.8% (w/v) agar. The plants were incubated at 23  C under continuous light condition (40 mmol photons m2 s1) for 3e4 weeks. 4.2. Analysis of chlorophyll fluorescence Before the all measurements, samples were acclimated to darkness for 10 min. Chlorophyll fluorescence parameters were measured using a Dual-PAM system (Walz, Effeltrich, Germany). The minimum chlorophyll fluorescence at the open PSII center (Fo) was determined by measuring light (655 nm) at an intensity of 0.05e0.15 mmol m2 s1. A saturating pulse of white light (800 ms) was applied to determine the maximum chlorophyll fluorescence at closed PSII centers in the dark (Fm) and during actinic light illumination (Fm0 ). Fv/Fm was calculated as (Fm  Fo)/Fm. NPQ was calculated as ðFm  Fm0 Þ=Fm0 . qE was calculated by subtraction of the NPQ value of the npq4 mutant from the NPQ value of the WT or the pgr5 mutant (Zaks et al., 2012). 4.3. Simulation analysis of photosynthetic electron flow

1

5 mmol s 0.1 mM

Fitted Fitted

5 mmol s1 0.15 mM

Fitted Fitted

zeaxanthin in the dark affects relationship between DpH and qE induction level upon light illumination (Johnson and Ruban, 2011). Therefore, if there are some differences in zeaxanthin concentration between dark-adapted WT and the pgr5 mutant, our calculation potentially has errors. Specifically, if the zeaxanthin accumulation in the pgr5 mutant was lower than that in WT under dark condition, we underestimate the LEF activity. Although the

The activities of PSII, ferredoxin-NADPþ reductase (FNR), CEF and the CalvineBenson cycle were calculated based on Michaelise Menten kinetics:

v ¼

Vmax ½S Km þ ½S

(1)

v ¼

Vmax ½S 1 $ Km þ ½S 1 þ eKgR ðtKR Þ

(2)

[S] is the concentration of a substrate for each photosynthetic component. Vmax and Km were obtained from previous reports (Table 1). Because FNR has two substrates, Fd and NADPþ, we

Please cite this article in press as: Sato, R., et al., Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.03.017

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Fig. 4. Comparison of experimental and simulated kinetics of qE induction in WT (A, C, E, G) and the pgr5 mutant (B, D, F, H) under various light conditions. Experimental values were same as in Fig. 3. Solid lines indicate simulated values. The light conditions were 9 mmol m2 s1 (A, B), 31 mmol m2 s1 (C, D), 104 mmol m2 s1 (E, F), and 135 mmol m2 s1 (G, H).

calculated FNR activity with each respective substrate, and adopted the lower one as the rate-limiting FNR activity. The CalvineBenson cycle activity was calculated with Equation (2). Given that Rubisco, a rate-limiting enzyme in the CalvineBenson cycle, is activated by low stromal pH and Rubisco activases (Portis, 2003), Equation (2) includes the effects of the activation processes as a timedependent reaction. The activities of b6f and PSI are conspicuously pH dependent (Hope et al., 1994); thus, the activities of b6f and PSI were calculated as:

v ¼

Vmax ½S Kb $10pH 
 $
Km þ ½S 1 þ Ka $10pH 1 þ Kb $10pH

(3)

This Equation (3) was proposed previously, and could be applied to calculate activities of b6f and PSI with their conspicuous pH dependence (Hope et al., 1994). The Vmax, Km, Ka and Kb were also obtained from (Hope et al., 1994). Because b6f and PSI use two substrates, we calculated the respective activity of the components for each substrate, and adopted the lower one as the rate-limiting

Please cite this article in press as: Sato, R., et al., Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.03.017

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to the experimental data. We also considered activity of nonphotochemical PQ reduction (Feild et al., 1998). PQ is a substrate for various enzymes, and even in dark conditions, some enzymes reduce the PQ pool (Feild et al., 1998) (Bondarava et al., 2003). Thus, we calculated the effects of non-photochemical PQ reduction as:

vnpp ¼ vnpr  vnpo Where vnpp accounts for the effects of non-photochemical PQ redox change. vnpr is the effect of non-photochemical PQ reduction. vnpo is the effect of PQ oxidation other than that dependent on b6f. vnpo and vnpo were calculated based on MichaeliseMenten kinetics. The parameters for the equations Vmax and Km were modified to allow fitting to experimental data (Table 1). Acknowledgments

Fig. 5. The kinetics of the CEF ratio to the total photosynthetic electron transfer flow. The values were calculated as the ratio of the CEF-dependent oxidation of Fd to the total Fd oxidation.

activity. The relationship between DpH and qE has been well studied (Johnson and Ruban, 2011), and the qE value was calculated as:

DpHnqE qE ¼ qEmax DpHnqE þ DpHn0qE

(4)

This Equation (4) was proposed previously (Johnson and Ruban, 2011). Parameters for this equation, qEmax, n, and DH0 were modified to allow fitting to the experiment data. The activity of ATP synthase was calculated as:

vHþ ¼ Vmax

½Hþ $106 KA1

½Hþ $106

KA1

vADP ¼ Vmax

Km þ KA1

1 1 þ eKgADP ðXðtÞKADP Þ

(5)

(6)

Where vþ H is the pH-dependent activity of ATP synthesis and vADP is the substrate-dependent activity of ATP synthesis. Equations (5) and (6) were derived from a previous report (Pänke and Rumberg, 1996). We adopted the lower activity as the ratelimiting ATP synthase activity. ATP synthase activity is regulated by photoreduction of a specific disulfide bond of the g subunit of ATP synthase (Kohzuma et al., 2013). X[t] is an equation for ratio (%) of the photoreduced ATP synthase to total ATP synthase, calculated as:

XðtÞ ¼

t ns

! t ns f ðlÞ $ þ C $100 þ Ksns f ðlÞ þ Ktnred

(7)

This Equation (7) was built from a previous report (Konno et al., 2012). The level of photoreduction depends on light intensity (Konno et al., 2012). f(l) is an equation for ratio of the photoreduced disulfide to total disulfide in the steady-state, which depends on light intensity. f(l) was calculated as:

f ðlÞ ¼ a lnðLÞ þ b

(8)

In Equation (8), L is the light intensity (in mmol photons m2 s1). a and b are constants, and were modified to allow fitting

We thank Dr. Toshiharu Shikanai at Kyoto University and Dr. Krishna K. Niyogi at the University of California, Berkeley for kindly providing plant materials. R.S. also thanks Dr. Koichi Hori and Ms. Miyu Kanamori for fruitful discussions. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (25120709) to S.M. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.03.017. References BÖHME, H., 1978. Quantitative determination of ferredoxin, ferredoxin-NADPþ reductase and plastocyanin in spinach chloroplasts. Euro. J. Biochem. 83, 137e 141. Bondarava, N., De Pascalis, L., Al-Babili, S., Goussias, C., Golecki, J.R., Beyer, P., Krieger-Liszkay, A., 2003. Evidence that cytochrome b 559 mediates the oxidation of reduced plastoquinone in the dark. J. Biol. Chem. 278, 13554e 13560. Bottin, H., Mathis, P., 1985. Interaction of plastocyanin with the photosystem I reaction center: a kinetic study by flash absorption spectroscopy. Biochemistry 24, 6453e6460. Bratt, C.E., Arvidsson, P.O., Carlsson, M., Åkerlund, H.E., 1995. Regulation of violaxanthin de-epoxidase activity by pH and ascorbate concentration. Photosyn. Res. 45, 169e175. Breyton, C., Nandha, B., Johnson, G.N., Joliot, P., Finazzi, G., 2006. Redox modulation of cyclic electron flow around photosystem I in C3 plants. Biochemistry 45, 13465e13475. Clark, R.D., Hind, G., 1983. Isolation of a five-polypeptide cytochrome bf complex from spinach chloroplasts. J. Biol. Chem. 258, 10348e10354. Corneille, S., Cournac, L., Guedeney, G., Havaux, M., Peltier, G., 1998. Reduction of the plastoquinone pool by exogenous NADH and NADPH in higher plant chloroplasts: characterization of a NAD (P) Heplastoquinone oxidoreductase activity. Biochim. Biophys. Acta 1363, 59e69. Feild, T.S., Nedbal, L., Ort, D.R., 1998. Nonphotochemical reduction of the plastoquinone pool in sunflower leaves originates from chlororespiration. Plant Physiol. 116, 1209e1218. Hanke, G.T., Kimata-Ariga, Y., Taniguchi, I., Hase, T., 2004. A post genomic characterization of Arabidopsis ferredoxins. Plant Physiol. 134, 255e264. Hanke, G.T., Endo, T., Satoh, F., Hase, T., 2008. Altered photosynthetic electron channelling into cyclic electron flow and nitrite assimilation in a mutant of ferredoxin: NADP (H) reductase. Plant Cell. Environ. 31, 1017e1028. Hertle, A.P., Blunder, T., Wunder, T., Pesaresi, P., Pribil, M., Armbruster, U., Leister, D., 2013. PGRL1 is the elusive ferredoxin-plastoquinone reductase in photosynthetic cyclic electron flow. Mol. Cell. 49, 511e623. Holt, N.E., Fleming, G.R., Niyogi, K.K., 2004. Toward an understanding of the mechanism of nonphotochemical quenching in green plants. Biochemistry 43, 8281e8289. Hope, A.B., 2000. Electron transfers amongst cytochrome f, plastocyanin and photosystem I: kinetics and mechanisms. Biochim. Biophys. Acta 1456, 5e26. Hope, A.B., Valente, P., Matthews, D.B., 1994. Effects of pH on the kinetics of redox reactions in and around the cytochrome bf complex in an isolated system. Photosynth. Res. 42, 111e120. Horton, P., Ruban, A.V., Walters, R.G., 1996. Regulation of light harvesting in green plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 655e684.

Please cite this article in press as: Sato, R., et al., Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.03.017

R. Sato et al. / Plant Physiology and Biochemistry xxx (2014) 1e7 Ishikawa, N., Takabayashi, A., Ishida, S., Hano, Y., Endo, T., Sato, F., 2008. NDF6: a thylakoid protein specific to terrestrial plants is essential for activity of chloroplastic NAD (P) H dehydrogenase in Arabidopsis. Plant Cell. Physiol. 49,1066e1073. Johnson, G.N., 2011. Reprint of: physiology of PSI cyclic electron transport in higher plants. Biochim. Biophys. Acta 1807, 906e911. Johnson, M.P., Ruban, A.V., 2011. Restoration of rapidly reversible photoprotective energy dissipation in the absence of PsbS protein by enhanced DpH. J. Biol. Chem. 286, 19973e19981. Joliot, P., Joliot, A., 2005. Quantification of cyclic and linear flows in plants. Proc. Natl. Acad. Sci. USA 102, 4913e4918. Joliot, P., Joliot, A., 2006. Cyclic electron flow in C3 plants. Biochim. Biophys. Acta 1757, 362e368. Katoh, S., Suga, I., Shiratori, I., Takamiya, A., 1961. Distribution of plastocyanin in plants, with special reference to its localization in chloroplasts. Arch. Biochem. Biophys. 94, 136e141. Kirchhoff, H., Mukherjee, U., Galla, H.J., 2002. Molecular architecture of the thylakoid membrane: lipid diffusion space for plastoquinone. Biochemistry 41, 4872e4882. Kohzuma, K., Dal Bosco, C., Meurer, J., Kramer, D.M., 2013. Light-and metabolismrelated regulation of the chloroplast ATP synthase has distinct mechanisms and functions. J. Biol. Chem. 288, 13156e13163. Konno, H., Nakane, T., Yoshida, M., Ueoka-Nakanishi, H., Hara, S., Hisabori, T., 2012. Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes. Plant Cell. Physiol. 53, 626e634. Kruk, J., Karpinski, S., 2009. An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis. Biochim. Biophys. Acta 1757, 1669e1675. Li, X.P., Björkman, O., Shih, C., Grossman, A.R., Rosenquist, M., Jansson, S., Niyogi, K.K., 2000. A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403, 391e395. Li, Z., Wakao, S., Fischer, B.B., Niyogi, K.K., 2009. Sensing and responding to excess light. Ann. Rev. Plant Biol. 60, 239e260. Müller, P., Li, X.P., Niyogi, K.K., 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiol. 125, 1558e1566. Munekage, Y., Shikanai, T., 2005. Cyclic electron transport through photosystem I. Plant Biotechnol. 22, 361e369.

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Munekage, Y., Hojo, M., Meurer, J., Endo, T., Tasaka, M., Shikanai, T., 2002. PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis. Cell 110, 361e371. Nelson, N., Ben-Shem, A., 2004. The complex architecture of oxygenic photosynthesis. Nat. Rev. Mol. Cell. Biol. 5, 971e982. Nilkens, M., Kress, E., Lambrev, P., Miloslavina, Y., Müller, M., Holzwarth, A.R., Jahns, P., 2010. Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis. Biochim. Biophys. Acta 1797, 466e475. Niyogi, K.K., Grossman, A.R., Björkman, O., 1998. Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. Plant Cell. 10, 1121e1134. Pänke, O., Rumberg, B., 1996. Kinetic modelling of the proton translocating CF0 CF1ATP synthase from spinach. FEBS Lett. 383, 196e200. Portis Jr., A.R., 2003. Rubisco activaseeRubisco’s catalytic chaperone. Photosynth. Res. 75, 11e27. Quick, W.P., Stitt, M., 1989. An examination of factors contributing to nonphotochemical quenching of chlorophyll fluorescence in barley leaves. Biochim. Biophys. Acta 977, 287e296. Robinson, H.H., Crofts, A.R., 1983. Kinetics of the oxidationdreduction reactions of the photosystem II quinone acceptor complex, and the pathway for deactivation. FEBS Lett. 153, 221e226. Ruban, A.V., Johnson, M.P., Duffy, C.D., 2012. The photoprotective molecular switch in the photosystem II antenna. Biochim. Biophys. Acta 1817, 167e181. Schuldiner, S., Rottenberg, H., Avron, M., 1972. Determination of DpH in chloroplasts. Eur. J. Biochem. 25, 64e70. Sétif, P., 2001. Ferredoxin and flavodoxin reduction by photosystem I. Biochim. Biophys. Acta 1507, 161e179. Suorsa, M., Järvi, S., Grieco, M., Nurmi, M., Pietrzykowska, M., Rantala, M., Aro, E.M., 2012. PROTON GRADIENT REGULATION5 is essential for proper acclimation of Arabidopsis photosystem I to naturally and artificially fluctuating light conditions. Plant Cell. 24, 2934e2948. Winter, H., Robinson, D.G., Heldt, H.W., 1994. Subcellular volumes and metabolite concentrations in spinach leaves. Planta 193, 530e535. Zaks, J., Amarnath, K., Kramer, D.M., Niyogi, K.K., Fleming, G.R., 2012. A kinetic model of rapidly reversible nonphotochemical quenching. Proc. Natl. Acad. Sci. USA 109, 15757e15762.

Please cite this article in press as: Sato, R., et al., Prediction of respective contribution of linear electron flow and PGR5-dependent cyclic electron flow to non-photochemical quenching induction, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.03.017