Stimulation of cyclic electron flow around photosystem I upon a sudden transition from low to high light in two angiosperms Arabidopsis thaliana and Bletilla striata

Stimulation of cyclic electron flow around photosystem I upon a sudden transition from low to high light in two angiosperms Arabidopsis thaliana and Bletilla striata

Plant Science 287 (2019) 110166 Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Stimulat...

1MB Sizes 0 Downloads 15 Views

Plant Science 287 (2019) 110166

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

Stimulation of cyclic electron flow around photosystem I upon a sudden transition from low to high light in two angiosperms Arabidopsis thaliana and Bletilla striata Ying-Jie Yanga,b,1, Xiao-Xi Dingc,1, Wei Huanga,

T



a

Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, 650201, China University of Chinese Academy of Sciences, Beijing, 100049, China c Kunming Forest Resources Administration, Kunming, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Angiosperms Electron transport Fluctuating light Photoprotection Redox state of P700 Photosystem I

In angiosperms, cyclic electron flow (CEF) around photosystem I (PSI) is more important for photoprotection under fluctuating light than under constant light. However, the underlying mechanism is not well known. In the present study, we measured the CEF activity, P700 redox state and electrochromic shift signal upon a sudden transition from low to high light in wild-type plants of Arabidopsis thaliana and Bletilla striata (Orchidaceae). Within the first 20 s after transition from low to high light, P700 was highly reduced in both species, which was accompanied with a sufficient proton gradient (ΔpH) across the thylakoid membranes. Meanwhile, the level of CEF activation was elevated. After transition from low to high light for 60 s, the plants generated an optimal ΔpH. Under such condition, PSI was highly oxidized and the level of CEF activation decreased to the steady state. Furthermore, the CEF activation was positively correlated to the P700 reduction ratio. These results indicated that upon a sudden transition from low to high light, the insufficient ΔpH led to the over-reduction of PSI electron carriers, which in turn stimulated the CEF around PSI. This transient stimulation of CEF not only favored the rapid ΔpH formation but also accepted electrons from PSI, thus protecting PSI at donor and acceptor sides. These findings provide new insights into the important role of CEF in regulation of photosynthesis under fluctuating light.

1. Introduction Plants use photosynthesis to convert light energy into chemical energy in the forms of ATP and NADPH, which are consumed in the primary metabolism including the Calvin-Benson cycle and photorespiration. The generation of ATP and NADPH is dependent on photosynthetic electron flows. In linear electron flow (LEF), electrons derived from water splitting in PSII are transported to PSI and ultimately to NADP+ via ferredoxin: NADP+ oxidoreductase, which is coupled with the generation of H+ translocation from the stroma to the thylakoid lumen via the quinone (Q) cycle in the Cyt b6/f complex. In cyclic electron flow (CEF) around PSI, electrons from ferredoxin are cycled around PSI into the plastoquinone pool without reducing NADP+, and the Cyt b6/f complex moves H+ into the thylakoid lumen via the Q cycle. Both LEF and CEF contribute to the generation of the proton motive force that is composed of a transthylakoid proton gradient (ΔpH) and a membrane potential (ΔΨ) [1–3]. Both ΔpH and ΔΨ drive

the ATP synthesis via ATP synthase. As a result, LEF contributes to the generation of ATP and NADPH, but CEF contributes to additional ATP production without generation of NADPH. The ATP/NADPH stoichiometry of LEF is 1.29 whereas the ATP/NADPH ratio required by the primary metabolism is 1.6 [4–6]. Such difference between ATP/NADPH supply from LEF and demand from primary metabolism could be balanced by CEF around PSI [6–10]. Therefore, an important physiological function of CEF around PSI is to balance the energy budget to maintain optimal photosynthetic CO2 assimilation under changing environmental conditions [6,11,12]. In addition, some studies reported that CEF contributed to the redox homeostasis in chloroplasts rather than CO2 assimilation in rice [13], especially under fluctuating light [14]. However, the performance of CEF activation under fluctuating light is still not unclear. In nature, plants continuously experience extreme fluctuations of light intensity caused by wind, cloud and shading. A sudden increase in light intensity causes the rapid increases in light absorption and



Corresponding author. E-mail address: [email protected] (W. Huang). 1 These authors contributed equally to this study. https://doi.org/10.1016/j.plantsci.2019.110166 Received 15 April 2019; Received in revised form 20 May 2019; Accepted 6 June 2019 Available online 11 June 2019 0168-9452/ © 2019 Elsevier B.V. All rights reserved.

Plant Science 287 (2019) 110166

Y.-J. Yang, et al.

electron flow in PSII [15,16]. However, the Calvin-Benson cycle has a slower kinetics, leading to the imbalance of electron flow PSII and electron transport to NADP+, thus resulting in a strong acceptor-side limitation in PSI and over-reduction of PSI reaction centers [17–20]. In nonflowering plants from cyanobacteria up to gymnosperms, this overreduction of PSI reaction centers is rapidly relieved by flavodiiron proteins [15,21–23]. As a result, PSI activity is insusceptible to fluctuating light in these evolutionary groups. By comparison, flavodiiron proteins are absent in angiosperms [15,21], and the resulting generation of reactive oxygen species (ROS) with PSI causes photoinhibition of PSI [24–26], even in the wild-type angiosperms such as Arabidopsis thaliana, rice (Oryza sativa) and Bletilla striata (Orchidaceae) [14,20,27,28]. In opposite to PSII, photodamaged PSI cannot be repaired quickly and the accumulated PSI photodamage is fatal for plants [25,29]. Therefore, plants must have feasible mechanisms to protect PSI against photoinhibition under fluctuating light. In fact, CEF around PSI is found to be crucial for PSI photoprotection under fluctuating light in angiosperms such as A. thaliana and rice (Oryza sativa) [14,25,30]. Firstly, Arabidopsis pgr5 mutant dies at the seedlings stage under fluctuating light conditions [25]. Secondly, under fluctuating light, the pgr5 and crr6 mutants showed severe photoinhibition of PSI than the wildtype plants [14,27,28]. Furthermore, in pgr5 and crr6 mutants, PSI were more susceptible to fluctuating light than constant high light [14]. However, the performance of CEF around PSI after transition from low to high light in angiosperms has not yet been clarified, complicating our understanding of the mechanism of CEF in protecting PSI against photoinhibition under fluctuating light. These CEF mutants showed significantly lower ΔpH than the wildtype plants when exposed to high light [25,26,31,32]. As a result, PSI photoprotection by CEF is considered to be linked with the CEF-dependent ΔpH formation [33]. Optimal acidification of the thylakoid lumen not only balances the energy budget (ATP/NADPH ratio) but also down-regulates the electron flow from PSII to PSI at the Cyt b6/f complex (photosynthetic control), preventing the over-reduction of P700 and the generation of ROS within PSI [34–37]. In the pgr5 mutant, PSI photoinhibition under fluctuating light is strongly suppressed when PSII activity is reduced [30,38], indicating that PSI photoprotection by CEF is dependent on the slowdown of electron flow from PSII to PSI by luminal acidification (donor-acceptor regulation) [30,33,39]. However, within the first seconds after transition from low to high light, wild-type plants of A. thaliana cannot build up a sufficient ΔpH, leading to the over-reduction of electron carriers in PSI [20,23]. Interestingly, a recent study suggested that CEF could protect PSI under fluctuating light at acceptor side [28]. Based on these previous studies, we hypothesize that, upon a sudden increase in light intensity, CEF activation is elevated to accept electrons from PSI and to help the rapid ΔpH formation in angiosperms. In the moss Physcomitrella patens, the role of alternative electron flow mediated by flavodiiron proteins is prominent for a few seconds after an increase in light intensity [15]. After transition from low to high light for 1 min, the role of flavodiiron proteins is negligible during prolonged illumination with constant light. Therefore, the time courses of alternative electron flows give important information about the mechanisms of photosynthetic regulation. However, at present, the time course of CEF around PSI after transition from low to high light is little known. In the present study, we studied the change in CEF after transition from low to high light and its relationship to PSI redox state. The aims of the study are: (1) to examine whether CEF is stimulated upon a sudden transition from low to high light in angiosperms; (2) to assess the acceptor-side and donor-side regulation of CEF in protecting PSI against photoinhibition under fluctuating light in angiosperms. In order to address these issues, we measured chlorophyll fluorescence, P700 signal and the electrochromic shift signal during transition from low to high light in two angiosperms A. thaliana (Cruciferae) and Bletilla striata (Orchidaceae). Our results clearly showed that within the first 20 s after transition from low to high light, the level of CEF activation

was significantly elevated. This transient stimulation of CEF not only contributed to the rapid ΔpH formation but also accepted electrons from PSI, thus protecting PSI at donor and acceptor sides. 2. Materials and methods 2.1. Plant materials We used two angiosperms Arabidopsis thaliana and Bletilla striata (Orchidaceae) for experiments. As we known, A. thaliana is a model angiosperm for the study of CEF in photoprotection for PSI. Furthermore, we previously used B. striata to study the photosynthetic regulation under fluctuating light, and found that the mechanism of photosynthetic regulation in B. striata was similar to A. thaliana. As a result, in the present study, we used these two angiosperms A. thaliana and B. striata to test our hypothesis. A. thaliana plants were grown in a greenhouse with high relative air humidity (60%–70%), low light (10% of full sunlight) and night/day temperatures of 18/25 °C. B. striata plants were grown in the same greenhouse but the growth light intensity was changed to 30% full sunlight. All plants were cultivated without water or nutrition stress. After grown for 8 weeks, the intact fully expanded rosette leaves of A. thaliana were used for experiments. For B. striata, mature fully expanded leaves that had flushed threemonth ago were used for measurements. 2.2. P700 and chlorophyll fluorescence measurements We used a Dual PAM-100 (Heinz Walz, Effeltrich, Germany) to simultaneously record PSI and PSII parameters at 25 °C. After dark adaptation for 30 min, a saturating pulse was applied to measure the maximum fluorescence and the maximum change in P700, and then leaves were illuminated at a saturating light of 1178 μmol photons m−2 s−1 for 15 min. Subsequently, the PSI and PSII parameters were recorded after exposure for 3 min to each light intensity (1178, 759, 501, 272, 172 or 59 μmol photons m−2 s−1). Afterwards, leaves were illuminated at 59 μmol photons m−2 s−1 for 5 min, then exposed to a saturating light of 1178 μmol photons m−2 s−1 for 2 min. After this transition from low to high light, PSI and PSII parameters were recorded. We first examined the PSI and PSII parameters under different light levels in the wild-type plants of Arabidopsis thaliana (Fig. 1). The PSI parameters measured included the quantum yield of PSI photochemistry, Y(I), the quantum yield of non-photochemical energy dissipation due to the donor side limitation, Y(ND), and that of energy dissipation due to acceptor side limitation, Y(NA). The PSII parameters measured included the effective quantum yield of PSII photochemistry, Y(II), and the non-photochemical quenching in PSII, NPQ. Y(I) and Y(II) were used to calculate the values of electron transport rate through PSI and PSII (ETRI and ETRII), respectively. Furthermore, the electron transport rate of cyclic electron flow around PSI was estimated as ETRI – ETRII. The PSI photosynthetic parameters were measured according to the method of Klughammer and Schreiber [[40]]. The P700+ signals (P) could vary between a minimum (P700 fully reduced) and a maximum level (P700 fully oxidized). The maximum, Pm, was determined by applying a saturation pulse (300 ms and 10,000 μmol photons m−2 s−1) after pre-illumination with far-red light for 10 s. The Pm' was similarly obtained, except that actinic light was used instead of far-red light. Calculations of PSI parameters included the quantum yield of PSI photochemistry, Y(I) = (Pm' – P)/Pm; the quantum yield of PSI nonphotochemical energy dissipation due to donor side limitation, Y(ND) = P/Pm; the quantum yield of non-photochemical energy dissipation due to acceptor side limitation, Y(NA) = (Pm – Pm')/Pm. The electron transport rate through PSI (ETRI) was calculated as ETRI = PPFD × Y (I) × 0.84 × 0.5. PSII parameters were calculated as follows: Y(II) = (Fm' – Fs)/Fm' 2

Plant Science 287 (2019) 110166

Y.-J. Yang, et al.

group [43,44]. The slow relaxation of the ECS signal was measured to calculate the proton gradient (ΔpH) [4,45]. All ΔpH levels were normalized against the magnitude of ECSST. This normalization accounted for variations in leaf thickness and chloroplast density among the leaf samples [44,46]. 2.4. Statistical analysis The results were displayed as mean values of at least five independent measurements. One-Way ANOVA test was used at α = 0.05 significance level to determine whether significant differences existed between different treatments. 3. Results 3.1. Light intensity dependence of PSI and PSII parameters in A. thaliana When the light intensity was below 300 μmol photons m−2 s−1, the values of Y(NA) were higher than that of Y(ND) (Fig. 1A). However, when exposed to light intensities above this light level, Y(ND) gradually increased and Y(NA) gradually decreased. During steady state photosynthesis at a saturating light of 1178 μmol photons m−2 s−1, values for Y(ND) and Y(NA) were 0.69 and 0.10, respectively (Fig. 1A). ETRII rapidly increased with the increase in light intensity and was saturated at 500 μmol photons m−2 s−1 (Fig. 1B). NPQ was slightly activated under low light (< 200 μmol photons m−2 s−1) and was saturated at approximately 800 μmol photons m−2 s−1 (Fig. 1B). At the low light of 59 μmol photons m−2 s−1, CEF was slightly activated, as indicated by the low values of ETRI/ETRII ratio and ETRI – ETRII (Fig. 1C). With the increase in light intensity, the values of ETRI/ETRII ratio and ETRI – ETRII increased and were saturated at 500 μmol photons m−2 s−1 (Fig. 1C). Under this light intensity, ETRI – ETRII reached 34 μmol m−2 s−1 and ETRI/ETRII increased to 1.5. 3.2. Change in ΔpH, PSI and PSII parameters after transition from low to high light in A. thaliana

Fig. 1. Light-intensity dependence of PSI and PSII parameters in wild-type plants of Arabidopsis thaliana. Dark-adapted leaves were illuminated at 1178 μmol photons m−2 s−1 for 15 min. Subsequently, PSI and PSII parameters were measured under different light levels (1178, 759, 501, 272, 172 and 59 μmol photons m−2 s−1). Y(ND), quantum yield of PSI non-photochemical energy dissipation due to donor side limitation; Y(NA), quantum yield of PSI non-photochemical energy dissipation due to acceptor side limitation; NPQ, non-photochemical quenching in PSII; ETRII, electron transport rate through PSII; ETRI, electron transport rate through PSI. Values are means ± SE (n = 5).

It is well documented that CEF-dependent generation of ΔpH plays a crucial role in regulating PSI redox state at donor-sides. In order to further understand the over-reduction of PSI reaction centers upon a sudden increase in light intensity, we measured ECS signals to analyze the change in ΔpH after transition from 59 to 1178 μmol photons m−2 s−1. The results indicated that in the first 20 s after transition from low to high light, the wild-type plants of Arabidopsis thaliana could not generate a sufficient ΔpH (Fig. 2A). As a result, the CEF-dependent donor-side regulation was weakened upon a sudden increase in light intensity. Next, we measured the changes in PSI and PSII parameters after transition from a low light (59 μmol photons m−2 s−1) to a high light (1178 μmol photons m−2 s−1) (Fig. 2). After light adaption at the low light of 59 μmol photons m−2 s−1, the values of Y(ND) and Y(NA) were 0.02 and 0.19 in the wild-type plants of Arabidopsis thaliana (Fig. 2B). After a sudden transition from this low light to the high light, Y(ND) increased to 0.41 in the first 20 s. Concomitantly, Y(NA) increased to a high level of 0.38, indicating the over-reduction of PSI reaction centers at this moment (Fig. 2B). After this light transition for 40 s, Y(ND) increased to 0.68 and Y(NA) decreased to 0.12, suggesting the over-reduction of PSI reaction centers was relaxed. Upon this light transition, ETRII and NPQ rapidly increased over time (Fig. 2C). Interestingly, ETRI – ETRII and ETRI/ETRII values first increased to a peak in first 20 s after this light transition (Fig. 2D). Subsequently, both parameters gradually deceased and reached steady state over time (Fig. 2D). This result suggested the level of CEF was elevated in the first 20 s upon transition from low to high light. To further confirm this high level of CEF activation, we monitored the reduction kinetics of P700, which were fitted to a single-

[41], NPQ = (Fm – Fm')/Fm'. Fm and Fm′ represent the maximum fluorescence after dark and light adaptation, respectively. Fs is the lightadapted steady state fluorescence. Y(II) represents the quantum yield of PSII photochemistry, NPQ indicates the non-photochemical quenching in PSII. The electron transport rate through PSII (ETRII) was calculated as ETRII = PPFD × Y(II) × 0.84 × 0.5.

2.3. Electrochromic shift (ECS) analysis The ECS signal was monitored as the change in absorbance at 515 nm, using a Dual PAM-100 equipped with a P515/535 emitterdetector module (Heinz Walz) [31,42]. After dark-adaptation for 30 min, the 515-nm absorbance change induced by a single turnover flash (ECSST) was measured. Subsequently, the leaves were illuminated at 1178 μmol photons m−2 s−1 for 15 min. Afterward, the actinic light was changed to 59 μmol photons m−2 s−1 for 5 min, and then the ECS signal was measured after transition to 1178 μmol photons m−2 s−1 for 20 s. Subsequently, leaves were repeatedly acclimated to 59 μmol photons m−2 s−1 for 5 min, and then the ECS signal was measured after transition to 1178 μmol photons m−2 s−1 for 60 s. We analyzed ECS dark interval relaxation kinetics (DIRKECS) as described by Kramer 3

Plant Science 287 (2019) 110166

Y.-J. Yang, et al.

Fig. 3. (A) Time course of the P700+ reduction rate after transition from 59 to 1178 μmol photons m−2 s−1 in Arabidopsis thaliana. (B, C) Relationships between the P700+ reduction rate, ETRI – ETRII and ETRI/ERTII after transition from low to high light in wild-type plants of A. thaliana. Values are means ± SE (n = 5). Fig. 2. (A) Change in the proton gradient (ΔpH) across the thylakoid membranes after transition from 59 to 1178 μmol photons m−2 s−1 for 20 and 60 s in Arabidopsis thaliana. (B, C, D) The time course of PSI and PSII parameters after transition from 59 to 1178 μmol photons m−2 s−1 in A. thaliana. Values are means ± SE (n = 5).

light, the ETRI - ETRII was positively correlated to Y(NA), but negatively correlated to Y(ND) (Fig. 4). The highest activation of CEF was accompanied with the highest value of Y(NA), suggesting that the overreduction of Y(NA) triggered the activation of CEF in A. thaliana upon a sudden increase in light intensity.

exponential-decay curve and the P700+ reduction rate was calculated. In the first 20 s after this light transition, the P700+ reduction rate was 191 s−1 (Fig. 3A). By comparison, the P700+ reduction rate decreased to 126 s−1 after transition from low to high light for 60 s (Fig. 3A). These results further confirmed that the activation of CEF around PSI was highly elevated within the first seconds upon a sudden transition from low to high light. In addition, we compared these two methods that were usually used for measuring the activation of CEF around PSI. Tight positive linear relationships were found between the P700+ reduction rate and values of ETRI – ETRII and ETRI/ERTII (Fig. 3B and C), indicating that both methods are applicable for estimating CEF activation under fluctuating light. Furthermore, we found that, after transition from low to high

3.3. Change in ΔpH, PSI and PSII parameters after transition from low to high light in B. striata In order to confirm the findings in A. thaliana, we further examined the changes in ΔpH, PSI and PSII parameters after transition from low to high light in B. striata. Similarly, B. striata generated an insufficient ΔpH within the first 10 s after transition from 59 to 1178 μmol photons m−2 s−1 (Fig. 5A). At this moment, B. striata showed high level of Y (NA), indicating that PSI was highly reduced (Fig. 5B). After this light transition, ETRII and NPQ rapidly increased over time (Fig. 5C). Interestingly, within the first 20 s after transition from low to high light, CEF was highly stimulated (Fig. 5C), similar to the phenomenon in A. thaliana. Furthermore, the value of ETRI - ETRII was positively 4

Plant Science 287 (2019) 110166

Y.-J. Yang, et al.

Fig. 4. The relationship between CEF stimulation and PSI redox state after transition from low to high light in Arabidopsis thaliana. All data were used from Fig. 2.

correlated to Y(NA) and negatively correlated to Y(ND) (Fig. 6), which was consistent with the findings in A. thaliana, suggesting that CEF was also stimulated by the over-reduction of PSI electron carriers in B. striata. 4. Discussion The ability of photosynthetic organisms to convert light into chemical energy is composed of light reactions (photosynthetic electron transport) and dark reactions (carbon fixation and other primary metabolism). Photosynthetic electron transport involves the formation of instable molecules that must be readily consumed to avoid the formation of reactive oxygen species. Under fluctuating light, a sudden increase in light intensity induces the immediate increases in light absorption and PSII electron transfer. However, the dark reactions have slower kinetics, and thus they cannot immediately consume all the ATP and NADPH produced by the linear electron flow [15,16]. The resulting accumulation of excess excitation energy in PSI causes PSI photoinhibition, even in the wild-type plants of Arabidopsis thaliana [27,28], Oryza sativa [14] and Bletilla striata [20,23]. Once PSI photoinhibition occurs, the light use efficiency and effective photoprotection are depressed, and thus the plant growth is impaired [14,29,47–49]. As a result, all oxygenic photosynthetic organisms must have feasible mechanisms to consume excess electrons from PSI, and thus to protect PSI against photoinhibition under fluctuating light. For example, flavodiiron proteins (Flvs) are the main player enabling algae and mosses growth under fluctuating light [15,22,50,51]. Upon a sudden increase in illumination, Flvs rapidly oxidize P700 by accepting excess electrons to O2, making PSI insusceptible to fluctuating light. However, flv genes are not conserved in angiosperms [15,21]. Although CEF have taken over the Flv function in protecting PSI from over-reduction in angiosperms [15,28], the performance of CEF after transition from low to high light in angiosperms is little known. In angiosperms, CEF around PSI is an important strategy for protecting PSI against photoinhibition under fluctuating light [14,25,28,33]. The activation of CEF is dependent on light intensity [9,27,52,53]. Consistently, the values of ETRI – ETRII and ETRI/ETRII were significantly higher under saturating light when compared with low light in A. thaliana (Fig. 1C). Under constant high light, the coordination among LEF, CEF and chloroplast ATP synthase builds up an optimal ΔpH to maximize light use efficiency and to maintain an effective protection from eventual excess excitation energy [8,19,35,46,54–56]. In pgr5 mutants, both the decreased CEF and

Fig. 5. Photosynthetic parameters under fluctuating light in Bletilla striata. (A) Change in ΔpH after transition from 59 to 1178 μmol photons m−2 s−1 for 10 and 60 s. (B, C, D) The time course of PSI and PSII parameters after transition from 59 to 1178 μmol photons m−2 s−1. Values are means ± SE (n = 7).

increased thylakoid proton conductivity cause the defect in ΔpH formation [7,27,31]. This reduction in ΔpH leads to the lack of ΔpH-dependent down-regulation of plastoquiol oxidation at the Cyt b6/f complexes, resulting in excess electron transport from PSII to PSI and thus severe PSI photoinhibition under high light [25,26,38]. As a result, under constant high light, CEF regulates the redox state of PSI mainly by donor-side regulation via controlling ΔpH formation. By comparison, upon a sudden increase in light intensity, the wild-type plants of A. thaliana and B. striata could not build up a sufficient ΔpH (Figs. 2A and 5 A). This low level of lumen acidification of thylakoid made the ΔpHdependent donor-side regulation to be weakened. As a consequence, the resulting excess electron flow from PSII to PSI caused the over-reduction of PSI after transition from low to high light (Figs. 2B and 5 B). Therefore, CEF-dependent donor-side regulation could not optimize the redox state of PSI upon a sudden increase in light intensity. 5

Plant Science 287 (2019) 110166

Y.-J. Yang, et al.

photoprotection under fluctuating light than under constant high light. However, the underlying mechanisms are not clear. In this study, we found that within the first 20 s after a sudden increase in light intensity, the insufficient ΔpH led to excess electron flow from PSII to PSI, resulting in the over-reduction of PSI. Under such condition, the activation of CEF was elevated to accept electrons from PSI, consuming excess excitation energy, thus alleviating the accumulation of reducing power in PSI. Furthermore, an additional beneficiary effect of this transient enhanced CEF activation was to help the rapid formation of ΔpH. After transition from low to high light for 60 s, plants formed an optimal ΔpH, providing a sufficient photosynthetic control, thus making PSI reaction centers highly oxidized. Meanwhile, the level of CEF activation decreased to the steady state to avoid over-acidification of thylakoid lumen. Taken together, after an increase in light intensity, the transient stimulation of CEF plays a crucial role in photoprotection for PSI in angiosperms. Conflict of interest Fig. 6. The relationship between CEF stimulation and PSI redox state after transition from low to high light in Bletilla striata. All data were used from Fig. 5.

The authors declare no conflict of interest. Acknowledgements

In this article, we demonstrated that in wild-type plants of A. thaliana and B. striata, CEF around PSI was highly activated upon a sudden transition from low to high light (Figs. 2D and 5 C). This significant stimulation of CEF accepted electrons from PSI, consuming a significant fraction of the excess excitation energy, and thus alleviating over-reduction of PSI electron carriers. As a result, CEF could protect PSI under fluctuating light at the acceptor side. The acceptor-side regulation by CEF around PSI is prominent for the first 20 s after an increase in illumination. After transition from low to high light for 1 min, the Y(NA) decreased to a low level and CEF activity decreased to the steady state value. These results suggest that the level of CEF activation under fluctuating light is significantly correlated to the reduction state of PSI (Figs. 4 and 5D). Over-reduction of PSI electron carriers may be an important stimulus for CEF activation in angiosperms, indicating that CEF protects PSI under fluctuating light at acceptor side. By comparison, in algae and mosses, the over-reduction of PSI electron carriers stimulated the alternative electron flow mediated by flavodiiron proteins [15,22]. As a result, CEF protects PSI under fluctuating light in a similar way to flavodiiron proteins. In addition, the transient elevated CEF activation increased the total photosynthetic electron transport and thus helped the fast formation of ΔpH across thylakoid membranes. After transition from low to high light, plants must have feasible mechanisms to rapidly increase the ΔpH across thylakoid membranes. Such increase in ΔpH is essential for plant survival under fluctuating light [25,57,58]. However, within the first 20 s after a sudden increase in light intensity, plants could not generate a sufficient ΔpH (Figs. 2A and 5 A), indicating that the optimal acidification of thylakoid lumen was a time-consuming process that needs more than 20 s. Under such conditions, an important physiological function of the elevated CEF activation was to help the fast formation of ΔpH. After transition from low to high light for 60 s, the ΔpH was formed to an optimal level (Figs. 2A and 5 A), and the level of CEF activation decreased to the steady state (Figs. 2D and 5 C). This decrease in CEF activation was essential for optimizing light use efficiency because over-acidification of thylakoid lumen not only depresses LEF but also results in the photoinhibition of PSII [28,46,54]. As a result, the change in CEF activation after transition from low to high light plays a crucial role in regulating the formation of ΔpH and thus optimizing the tread-off between photoprotection and photosynthesis.

This study was supported by the National Natural Science Foundation of China (Grant 31670343), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (Grant 2016347). References [1] D.M. Kramer, J.A. Cruz, A. Kanazawa, Balancing the central roles of the thylakoid proton gradient, Trends Plant Sci. 8 (2003) 27–32. [2] D.M. Kramer, T.J. Avenson, G.E. Edwards, Dynamic flexibility in the light reactions of photosynthesis governed by both electron and proton transfer reactions, Trends Plant Sci. 9 (2004) 349–357. [3] J.F. Allen, Photosynthesis of ATP-electrons, proton pumps, rotors, and poise, Cell. 110 (2002) 273–276. [4] C.A. Sacksteder, A. Kanazawa, M.E. Jacoby, D.M. Kramer, The proton to electron stoichiometry of steady-state photosynthesis in living plants: a proton-pumping Q cycle is continuously engaged, Proc. Natl. Acad. Sci. 97 (2000) 14283–14288. [5] B.J. Walker, A. VanLoocke, C.J. Bernacchi, D.R. Ort, The costs of photorespiration to food production now and in the future, Annu. Rev. Plant Biol. 67 (2016) 107–129. [6] B.J. Walker, D.D. Strand, D.M. Kramer, A.B. Cousins, The response of cyclic electron flow around photosystem I to changes in photorespiration and nitrate assimilation, Plant Physiol. 165 (2014) 453–462. [7] T.J. Avenson, J. a Cruz, A. Kanazawa, D.M. Kramer, Regulating the proton budget of higher plant photosynthesis, Proc. Natl. Acad. Sci. 102 (2005) 9709–9713. [8] D.M. Kramer, J.R. Evans, The importance of energy balance in improving photosynthetic productivity, Plant Physiol. 155 (2011) 70–78. [9] W. Yamori, N. Sakata, Y. Suzuki, T. Shikanai, A. Makino, Cyclic electron flow around photosystem i via chloroplast NAD(P)H dehydrogenase (NDH) complex performs a significant physiological role during photosynthesis and plant growth at low temperature in rice, Plant J. 68 (2011) 966–976. [10] W. Yamori, T. Shikanai, A. Makino, Photosystem I cyclic electron flow via chloroplast NADH dehydrogenase-like complex performs a physiological role for photosynthesis at low light, Sci. Rep. 5 (2015) 13908. [11] W. Yamori, T. Shikanai, Physiological functions of cyclic Electron transport around photosystem I in sustaining photosynthesis and plant growth, Annu. Rev. Plant Biol. 67 (2016) 81–106. [12] C. Miyake, Alternative electron flows (water-water cycle and cyclic electron flow around PSI) in photosynthesis: molecular mechanisms and physiological functions, Plant Cell Physiol. 51 (2010) 1951–1963. [13] Y. Nishikawa, H. Yamamoto, Y. Okegawa, S. Wada, N. Sato, Y. Taira, K. Sugimoto, A. Makino, T. Shikanai, PGR5-dependent cyclic electron transport around PSI contributes to the redox homeostasis in chloroplasts rather than CO2 fixation and biomass production in rice, Plant Cell Physiol. 53 (2012) 2117–2126. [14] W. Yamori, A. Makino, T. Shikanai, A physiological role of cyclic electron transport around photosystem I in sustaining photosynthesis under fluctuating light in rice, Sci. Rep. 6 (2016) 20147. [15] C. Gerotto, A. Alboresi, A. Meneghesso, M. Jokel, M. Suorsa, E.-M. Aro, T. Morosinotto, Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens, Proc. Natl. Acad. Sci. 113 (2016) 12322–12327. [16] W. Yamori, Photosynthetic response to fluctuating environments and photoprotective strategies under abiotic stress, J. Plant Res. 129 (2016) 379–395. [17] H. Yamamoto, S. Takahashi, M.R. Badger, T. Shikanai, Artificial remodelling of

5. Conclusions It is documented that CEF was more important for PSI 6

Plant Science 287 (2019) 110166

Y.-J. Yang, et al.

[18]

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34] [35]

[36]

[37]

alternative electron flow by flavodiiron proteins in Arabidopsis, Nat. Plants 2 (2016) 16012. S. Wada, H. Yamamoto, Y. Suzuki, W. Yamori, T. Shikanai, A. Makino, Flavodiiron protein substitutes for cyclic electron flow without competing CO2 assimilation in rice, Plant Physiol. 176 (2018) 1509–1518. W. Huang, X. Quan, S.B. Zhang, T. Liu, In vivo regulation of proton motive force during photosynthetic induction, Environ. Exp. Bot. 148 (2018) 109–116. W. Huang, Y.-J. Yang, S.-B. Zhang, Photoinhibition of photosystem I under fluctuating light is linked to the insufficient ΔpH upon a sudden transition from low to high light, Environ. Exp. Bot. 160 (2019) 112–119. P. Ilík, A. Pavlovič, R. Kouřil, A. Alboresi, T. Morosinotto, Y. Allahverdiyeva, E.M. Aro, H. Yamamoto, T. Shikanai, Alternative electron transport mediated by flavodiiron proteins is operational in organisms from cyanobacteria up to gymnosperms, New Phytol. 214 (2017) 967–972. M. Jokel, X. Johnson, G. Peltier, E.M. Aro, Y. Allahverdiyeva, Hunting the main player enabling Chlamydomonas reinhardtii growth under fluctuating light, Plant J. 94 (2018) 822–835. W. Huang, Y.-J. Yang, S.-B. Zhang, The role of water-water cycle in regulating the redox state of photosystem I under fluctuating light, Biochimica et Biophysica Acta (BBA) – Bioenergetics 1860 (2019) 383–390. D. Takagi, S. Takumi, M. Hashiguchi, T. Sejima, C. Miyake, Superoxide and singlet oxygen produced within the thylakoid membranes both cause photosystem I photoinhibition, Plant Physiol. 171 (2016) 1626–1634. M. Suorsa, S. Järvi, M. Grieco, M. Nurmi, M. Pietrzykowska, M. Rantala, S. Kangasjärvi, V. Paakkarinen, M. Tikkanen, S. Jansson, E.-M. Aro, PROTON GRADIENT REGULATION5 is essential for proper acclimation of Arabidopsis photosystem I to naturally and artificially fluctuating light conditions, Plant Cell 24 (2012) 2934–2948. Y. Munekage, M. Hojo, J. Meurer, T. Endo, M. Tasaka, T. Shikanai, PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis, Cell 110 (2002) 361–371. M. Kono, K. Noguchi, I. Terashima, Roles of the cyclic electron flow around PSI (CEF-PSI) and O2-dependent alternative pathways in regulation of the photosynthetic electron flow in short-term fluctuating light in Arabidopsis thaliana, Plant Cell Physiol. 55 (2014) 990–1004. H. Yamamoto, T. Shikanai, PGR5-dependent cyclic electron flow protects photosystem I under fluctuating light at donor and acceptor sides, Plant Physiol. 179 (2019) 588–600. M. Zivcak, M. Brestic, K. Kunderlikova, O. Sytar, S.I. Allakhverdiev, Repetitive light pulse-induced photoinhibition of photosystem I severely affects CO2 assimilation and photoprotection in wheat leaves, Photosyn. Res. 126 (2015) 449–463. M. Suorsa, F. Rossi, L. Tadini, M. Labs, M. Colombo, P. Jahns, M.M.M. Kater, D. Leister, G. Finazzi, E.M. Aro, R. Barbato, P. Pesaresi, PGR5-PGRL1-Dependent cyclic electron transport modulates linear electron transport rate in Arabidopsis thaliana, Mol. Plant 9 (2016) 271–288. C. Wang, H. Yamamoto, T. Shikanai, Role of cyclic electron transport around photosystem I in regulating proton motive force, Biochimica et Biophysica Acta 1847 (2015) 931–938. Y. Munekage, M. Hashimoto, C. Miyake, K. Tomizawa, T. Endo, M. Tasaka, T. Shikanai, Cyclic electron flow around photosystem I is essential for photosynthesis, Nature 429 (2004) 579–582. T. Shikanai, H. Yamamoto, Contribution of cyclic and pseudo-cyclic electron transport to the formation of proton motive force in chloroplasts, Mol. Plant 10 (2017) 20–29. M. Tikkanen, E.M. Aro, Integrative regulatory network of plant thylakoid energy transduction, Trends Plant Sci. 19 (2014) 10–17. D. Takagi, K. Amako, M. Hashiguchi, H. Fukaki, K. Ishizaki, T. Goh, Y. Fukao, R. Sano, T. Kurata, T. Demura, S. Sawa, C. Miyake, Chloroplastic ATP synthase builds up a proton motive force preventing production of reactive oxygen species in photosystem I, Plant J. 91 (2017) 306–324. W. Huang, Y.F. Cai, J.H. Wang, S.B. Zhang, Chloroplastic ATP synthase plays an important role in the regulation of proton motive force in fluctuating light, J. Plant Physiol. 226 (2018) 40–47. W. Huang, M. Suorsa, S.B. Zhang, In vivo regulation of thylakoid proton motive force in immature leaves, Photosyn. Res. 138 (2018) 207–218.

[38] M. Tikkanen, N.R. Mekala, E.-M. Aro, Photosystem II photoinhibition-repair cycle protects Photosystem I from irreversible damage, Biochimica et Biophysica Acta (BBA) - Bioenergetics 1837 (2014) 210–215. [39] M. Tikkanen, S. Rantala, E.-M. Aro, Electron flow from PSII to Psi under high light is controlled by PGR5 but not by PSBS, Front. Plant Sci. 6 (2015) 521. [40] C. Klughammer, U. Schreiber, Saturation Pulse method for assessment of energy conversion in PS I, PAM Appl. Notes (2008) 11–14. [41] B. Genty, J.-M. Briantais, N.R. Baker, The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence, Biochimica et Biophysica Acta (BBA) – Gen. Subj. 990 (1989) 87–92. [42] W. Huang, S.-B. Zhang, J.-C. Xu, T. Liu, Plasticity in roles of cyclic electron flow around photosystem I at contrasting temperatures in the chilling-sensitive plant Calotropis gigantea, Environ. Exp. Bot. 141 (2017) 145–153. [43] C.A. Sacksteder, M.E. Jacoby, D.M. Kramer, A portable, non-focusing optics spectrophotometer (NoFOSpec) for measurements of steady-state absorbance changes in intact plants, Photosyn. Res. 70 (2001) 231–240. [44] K. Takizawa, A. Kanazawa, D.M. Kramer, Depletion of stromal Pi induces high “energy-dependent” antenna exciton quenching (qE) by decreasing proton conductivity at CFO-CF1 ATP synthase, Plant Cell Environ. 31 (2008) 235–243. [45] J.A. Cruz, T.J. Avenson, A. Kanazawa, K. Takizawa, G.E. Edwards, D.M. Kramer, Plasticity in light reactions of photosynthesis for energy production and photoprotection, J. Exp. Bot. 56 (2005) 395–406. [46] A.K. Livingston, J.A. Cruz, K. Kohzuma, A. Dhingra, D.M. Kramer, An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NADPH dehydrogenase complex, Plant Cell 22 (2010) 221–233. [47] Y.N. Munekage, B. Genty, G. Peltier, Effect of PGR5 impairment on photosynthesis and growth in Arabidopsis thaliana, Plant Cell Physiol. 49 (2008) 1688–1698. [48] W. Huang, P.-L. Fu, Y.-J. Jiang, J.-L. Zhang, S.-B. Zhang, H. Hu, K.-F. Cao, Differences in the responses of photosystem I and photosystem II of three tree species Cleistanthus sumatranus, Celtis philippensis and Pistacia weinmannifolia exposed to a prolonged drought in a tropical limestone forest, Tree Physiol. 33 (2013) 211–220. [49] M. Brestic, M. Zivcak, K. Kunderlikova, O. Sytar, H. Shao, H.M. Kalaji, S.I. Allakhverdiev, Low Psi content limits the photoprotection of Psi and PSII in early growth stages of chlorophyll b-deficient wheat mutant lines, Photosyn. Res. 125 (2015) 151–166. [50] Y. Allahverdiyeva, H. Mustila, M. Ermakova, L. Bersanini, P. Richaud, G. Ajlani, N. Battchikova, L. Cournac, E.-M. Aro, Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light, Proc. Natl. Acad. Sci. 110 (2013) 4111–4116. [51] Y. Allahverdiyeva, M. Suorsa, M. Tikkanen, E.M. Aro, Photoprotection of photosystems in fluctuating light intensities, J. Exp. Bot. 66 (2015) 2427–2436. [52] W. Huang, Y.-J. Yang, S.-B. Zhang, Specific roles of cyclic electron flow around photosystem I in photosynthetic regulation in immature and mature leaves, J. Plant Physiol. 209 (2017) 76–83. [53] W. Huang, Y.J. Yang, H. Hu, S.B. Zhang, Seasonal variations in photosystem I compared with photosystem II of three alpine evergreen broad-leaf tree species, J. Photochem. Photobiol. B, Biol. 165 (2016) 71–79. [54] M. Rott, N.F. Martins, W. Thiele, W. Lein, R. Bock, D.M. Kramer, M.A. Schöttler, ATP synthase repression in tobacco restricts photosynthetic electron transport, CO2 assimilation, and plant growth by overacidification of the thylakoid lumen, Plant Cell 23 (2011) 304–321. [55] W. Huang, M. Tikkanen, Y.-F. Cai, J.-H. Wang, S.-B. Zhang, Chloroplastic ATP synthase optimizes the trade-off between photosynthetic CO2 assimilation and photoprotection during leaf maturation, Biochimica et Biophysica Acta (BBA) Bioenergetics 1859 (2018) 1067–1074. [56] M. Zivcak, H.M. Kalaji, H.B. Shao, K. Olsovska, M. Brestic, Photosynthetic proton and electron transport in wheat leaves under prolonged moderate drought stress, J. Photochem. Photobiol. B Biol. 137 (2014) 107–115. [57] M. Tikkanen, M. Grieco, M. Nurmi, M. Rantala, M. Suorsa, E.-M. Aro, Regulation of the photosynthetic apparatus under fluctuating growth light, Philos. Trans. R. Soc. B: Biol. Sci. 367 (2012) 3486–3493. [58] U. Armbruster, V. Correa Galvis, H.H. Kunz, D.D. Strand, The regulation of the chloroplast proton motive force plays a key role for photosynthesis in fluctuating light, Curr. Opin. Plant Biol. 37 (2017) 56–62.

7