What Quantity of Photosystem I Is Optimum for Safe Photosynthesis? (2024)

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Abstract

Plants carry out photosynthesis, at the risk of photo-oxidative damage, by converting the energy from sunlight to reducing power in the photosynthetic electron transport system, which begins with photo-excitation of the reaction-center chlorophylls in PSII and PSI, i.e. P680 and P700, respectively, in the thylakoid membrane in chloroplasts. Electrons originating from the oxidation of water on the luminal side of PSII are transported into PSI via the plastoquinone (PQ) pool, the cytochrome b₆/f complex (Cyt b₆/f), and plastocyanin, accompanied by the formation of a proton gradient across the thylakoid membrane, to produce ATP. Photo-excited P700 gives electrons to the acceptor side of PSI and finally to NADP⁺. Both ATP and NADPH are consumed in the Calvin-Benson cycle for photosynthetic CO₂ assimilation. However, the production and consumption of reducing power is easily unbalanced because of various environmental fluctuations. If photo-excitation energy originating from P700 is more than the demand for NADPH production, it can cause reactive oxygen species–derived photo-oxidative damage to PSI (Terashima et al., 1994; Sejima et al., 2014). Once reactive oxygen species are generated, PSI is rapidly inactivated and thereafter takes days or even weeks to recover completely, thus inhibiting photosynthesis and growth of oxygenic photoautotrophs (Zivcak et al., 2015a; Shimakawa et al., 2016). This photo-oxidative damage to PSI is termed PSI photoinhibition and is recognized as a lethal event for oxygenic photoautotrophs.

Generally, PSI photoinhibition rarely occurs because plants suppress excessive photo-excitation of P700 by keeping it in its oxidized form that functions as the quencher of light energy (Foyer et al., 1990; Klughammer and Schreiber, 1994; Trissl, 1997; Bukhov and Carpentier, 2003; Golding and Johnson, 2003; Miyake et al., 2005). This is the mechanism by which oxygenic photoautotrophs safely carry out photosynthesis under the sun. Oxidation of P700 is controlled by various molecular mechanisms, known collectively as the P700 oxidation system (Shimakawa et al., 2017; Takagi et al., 2017; Shimakawa and Miyake, 2018). Conversely, it is supposed that P700 can be easily kept oxidized, even without any regulatory mechanisms, due to the stoichiometric quantities of photosynthetic apparatus, including PSII, Cyt b₆/f, and PSI. The stoichiometric quantity (mol per leaf area) of Cyt b₆/f is modulated and kept lower than those of PSII and PSI in plant leaves (Yamori et al., 2011; Schöttler and Tóth, 2014), which is one of the reasons why the electron transport in Cyt b₆/f is usually recognized as the limiting step in the photosynthetic electron transport system (Anderson, 1992; Schöttler et al., 2015). Oxidized P700 is reduced with electrons predominantly from PSII, which clearly suggests that PSII quantity larger than that of PSI potentially impairs the maintenance of P700 in an oxidized state. Indeed, Bukhov et al. (2004) observed a faster reduction rate of oxidized P700 in those plant leaves in which PSI photoinhibition occurred under chilling stress (Bukhov et al., 2004). Additionally, a barley mutant having a decreased amount of PSII showed higher P700 oxidation than that of the wild type (Pfündel et al., 2008), whereas a chlorophyll b–deficient mutant showing a low PSI/PSII ratio was impaired in P700 oxidation (Brestic et al., 2015). Furthermore, PSI photoinhibition was reported in a mutant that contained a lower quantity of PSI than that of PSII (Ivanov et al., 2015). These facts imply that the stoichiometric quantity of PSI is optimized to induce P700 oxidation to protect PSI from photoinhibition and ensure robust photosynthesis. In this study, we sought to characterize the effects of quantitative decrease of the photochemically active PSI on a variety of photosynthetic parameters using the intact leaves of the Arabidopsis wild type.

RESULTS

Relationship between the Quantity of the Photochemically Active PSI and Photosynthetic Parameters of PSI in Arabidopsis

For experimentally modifying the quantity of photochemically active PSI in the plant leaves, we used the method “repetitive short-pulse (rSP) illumination,” which facilitates rapid and selective induction of PSI photoinhibition in the plant leaves in vivo. For this method, rSP (300 ms, 20,000 µmol photon m⁻² s⁻¹, every 10 s) is applied to the plant leaves of angiosperms in darkness, which instantaneously fills the photosynthetic electron transport system with electrons in the absence of stimulus for any regulatory mechanism of P700 oxidation (Sejima et al., 2014; Zivcak et al., 2015a, 2015b; Kono et al., 2017; Takagi et al., 2017; Shimakawa et al., 2019; Tikkanen and Grebe, 2018). Using rSP illumination, we created plant leaves partially impaired in PSI and evaluated the relationship between the quantity of photochemically active PSI with various photosynthetic parameters in Arabidopsis, as follows.

In Arabidopsis, rSP illumination caused PSI photoinhibition (Fig. 1). The quantity of photochemically active PSI was spectroscopically evaluated in vivo as the total quantity of photo-oxidizable P700 (P_m) before and after rSP illumination (Fig. 1A; Klughammer and Schreiber, 1994). In the leaves of Arabidopsis wild-type plants, P_m decreased to ∼20% of the intact value after rSP illumination in the dark (300 ms, 20,000 μmol photons m⁻² s⁻¹, every 10 s) for 1 h in ambient air (Fig. 1B). In contrast, maximum quantum efficiency of PSII photochemistry (F_v/F_m) was sustained, i.e. ∼80% of the intact value, even after rSP illumination (Fig. 1C), indicating that the method selectively induces inactivation of PSI in Arabidopsis like in other angiosperms (Sejima et al., 2014). After rSP illumination, we incubated the plants in darkness for 30 min so as to relax qE quenching and state transition (Baker, 2008) before the determination of P_m and F_v/F_m (Fig. 1A).

Photosynthetic parameters of PSI were measured in Arabidopsis at a steady state of photosynthesis under red actinic light (820 μmol photons m⁻² s⁻¹) after the determination of P_m and F_v/F_m (Fig. 1A). Commonly in C₃ plants, photorespiration functions as a large alternative electron sink (Badger et al., 2000; Driever and Baker, 2011; Hanawa et al., 2017), likely affecting a variety of photosynthetic parameters. In this study, the measurements of photosynthetic parameters were performed under high CO₂ conditions (21 kPa O₂, 100 Pa CO₂) to exclude the effects of photorespiration on these parameters. With a 80% loss of PSI photochemical activity, effective quantum yield of PSI [Y(I)] increased from 0.4 to 0.9 (Fig. 2A). The photochemical reaction in PSI is limited at both electron donor and acceptor-sides. Quantum yield of nonphotochemical energy dissipation due to donor-side limitation [Y(ND)] indicates the ratio of oxidized P700 to total photo-oxidizable P700; that is, Y(ND) is the indicator of P700 oxidation. At the steady state of photosynthesis, in intact leaves of Arabidopsis under actinic light, Y(ND) was calculated as 0.5, which reflects that 50% of P700 remained oxidized (Fig. 2B). However, Y(ND) dramatically decreased from 0.5 to 0.1 with a decrease in P_m from 100% to 60% (Fig. 2B). These data suggest that PSI photoinhibition in this range prevented oxidation of P700. Additionally, we plotted quantum yield of nonphotochemical energy dissipation due to acceptor-side limitation [Y(NA)] against P_m. When P_m decreased from 100% to 60%, Y(NA) increased with the inactivation of PSI (Fig. 2C). However, when the amount of photochemically active PSI was less than 20% of the intact value, we could not detect Y(NA) at all (Fig. 2C).

Relationship between the Quantity of the Photochemically Active PSI and Photosynthetic Parameters of PSII in Arabidopsis

Next, we assessed the impact of the quantity of photochemically active PSI on the photosynthetic parameters of PSII in Arabidopsis. Effective quantum yield of PSII [Y(II)] showed an approximately linear relationship with P_m (Fig. 3A), which suggests that the operating efficiency of the photochemical reaction in PSII is correlated to the quantity of the photochemically active PSI in plant leaves. Similarly, nonphotochemical quenching of chlorophyll fluorescence in PSII (non-photochemical quenching), the indicator of a thermal dissipation of excess light energy in PSII, was proportional to P_m (Fig. 3B). Furthermore, photochemical quenching of chlorophyll fluorescence based on the lake model of the antenna system in PSII (represented as qL), reflecting the oxidation level of Q_A and possibly the PQ pool, showed a trend similar to that of Y(II) (Fig. 3C).

Relationship between the Quantity of the Photochemically Active PSI and Photosynthetic CO₂ Assimilation in Arabidopsis

We measured photosynthetic CO₂ assimilation rate, calculated as the sum of net CO₂ assimilation rate (A) and dark respiration rate, in intact and damaged leaves of Arabidopsis. When compared with how Y(II), NPQ, and qL responded to PSI photoinhibition, a less linear correlation was observed in the inactivation of photosynthetic CO₂ assimilation when P_m decreased (Fig. 4A). The inhibition of CO₂ assimilation rate might be suppressed until the residual P_m was less than 60%, and thereafter, it started to decrease with the inactivation of PSI (Fig. 4A). However, ∼50% of the normal CO₂ assimilation rate was maintained even with an 80% reduction in PSI photochemical activity in the leaves of Arabidopsis wild-type plants (Fig. 4A). Additionally, when Y(II) was plotted against the CO₂ assimilation rate (Fig. 4B), the resultant relationship indicated that the effective quantum yield of PSII can relatively reflect the photosynthetic CO₂ assimilation rate when PSI photoinhibition occurs.

DISCUSSION

Sufficient Quantity of PSI Supports P700 Oxidation in Plant Leaves

In the current study, we investigated the relationship between the stoichiometric quantity of the photochemically active PSI and P700 oxidation in vivo during steady-state photosynthesis in Arabidopsis. Although diverse regulatory mechanisms for the oxidation of P700 have been reported in oxygenic photoautotrophs (Shimakawa and Miyake, 2018), the effect of the quantity of PSI on P700 oxidation was poorly understood. rSP illumination is a useful tool to rapidly and selectively induce PSI photoinhibition in plant leaves (Sejima et al., 2014; Zivcak et al., 2015a, 2015b; Kono et al., 2017; Takagi et al., 2017; Shimakawa et al., 2019; Tikkanen and Grebe, 2018). This method enabled us to decrease the quantity of active PSI in the leaves of Arabidopsis wild-type plants. With PSI inactivated, Y(I) in principle increased (Fig. 2A); that is, the residual photochemically active PSI efficiently drives the photooxidation/reduction cycle, which is reasonable considering that the electron donor (i.e. PSII) and acceptor (i.e. the Calvin-Benson cycle) of PSI are relatively larger in these situations. In contrast, when P_m was decreased from 100% to 60%, Y(ND) decreased from 0.5 to 0.1 (Fig. 2B), indicating that plant leaves cannot keep P700 oxidized when 40% of PSI is inactivated even in the presence of a variety of regulatory mechanisms (i.e. P700 oxidation system). In other words, the leaves of Arabidopsis contain a quantity of PSI that is likely sufficient to enable P700 oxidation in excess light conditions, which may be regulated at the genetic level and modulated to suit any variation in environmental conditions. In the illustrated model of the photosynthetic electron transport system in the processes of PSI photoinhibition in Arabidopsis leaves (Fig. 5), we summarized the effects of a decrease in the stoichiometric quantity of PSI on P700 oxidation as well as on the other physiological states as follows.

Capacity of Remaining PSI Can Suppress the Inhibition of Photosynthesis

When P_m decreased to 60% of that present in untreated leaves, the photosynthetic CO₂ assimilation rate was likely to show a smaller decrease than that in the phase whereby P_m further decreased from 60% to 20% of normal levels, as supported by the low linear correlation for the relationship between P_m and CO₂ assimilation rate (Fig. 4A). The former phase of PSI photoinhibition, termed as “first-degree” in Figure 5, corresponds to the phase in which oxidized P700 can be still observed as reflected in Y(ND). In the latter phase (“second-degree” in Fig. 5), P700 oxidation is no longer maintained in the steady-state photosynthesis (Fig. 2B). In principle, oxidized P700 should not cause charge separation in PSI, and rather it should engage in thermal dissipation of light energy (Trissl, 1997; Bukhov and Carpentier, 2003). This may be the reason why the decrease in photosynthetic CO₂ assimilation rate was alleviated when P_m ranged from 100% to 60% (Fig. 4A); that is, the fractions of PSI with oxidized P700 reflect the capacity of remaining PSI complexes following PSI photoinhibition and can be recognized as surplus PSI for photosynthesis in intact leaves (Fig. 5).

Oxidized P700 Functions as a Quencher of Light Energy to Balance the Photo-Excitation between PSII and PSI

Theoretically, oxidized P700 thermally dissipates the light energy in PSI, which can mitigate the pressure of photo-excitation in PSII. As PSI photoinhibition advanced and P_m ranged from 100% to 40%, Y(NA) slightly increased; and thereafter, it disappeared (Fig. 2C). As mentioned above, with a decrease in photochemically active PSI, the limitation on the electron acceptor side should in principle be relieved. Therefore, the slight increase in Y(NA; Fig. 2C) suggests that the electron influx into PSI on the donor side was enhanced. Light energy absorbed by chloroplasts in the plant leaves is distributed into PSII and PSI (Fig. 5), which suggests that a decrease in the quantity of the photochemically active PSI can cause an increase in the light energy flow into PSII. Indeed, Bukhov et al. (2004) reported accelerated rereduction of oxidized P700 in PSI-damaged cucumber leaves. In this study, the observed increase in Y(NA) when P_m ranged from 100% to 40% (Fig. 2C) supports the theory that oxidized P700 functions as the quencher of light energy in PSI. In PSII, Y(II) decreased with PSI photoinhibition (Fig. 3A), but did not show a linear relationship with photosynthetic CO₂ assimilation rate passing through the graph origin, different from that in intact C₃ plant leaves (Fig. 4A; Genty et al., 1989). This can be explained by an increase in the absorbance of PSII fraction (Genty et al., 1989; Baker et al., 2007). These data also demonstrate the effects of the change in the distribution of light energy between PSII and PSI on the chlorophyll fluorescence parameters. Simultaneously, the decrease in qL is assumed to reflect two effects: a decrease in the electron acceptor (i.e. PSI) and an increase in the photo-excitation pressure (Fig. 3C). Evidence for the hypothesis that oxidized P700 suppresses the charge separation of PSII by quenching the light energy in PSII through light harvesting complex and/or spillover-dependent mechanisms (Ueno et al., 2018; Yokono and Akimoto, 2018) might be also present within the results of the current study.

Decrease in PSI Is Correlated with a Decrease in NPQ

NPQ showed a linear inverse correlation with the extent of PSI photoinhibition in Arabidopsis (Fig. 3B). In the chlorophyll fluorescence measurement, NPQ reflects a thermal dissipation of excess light energy in PSII to protect PSII against photoinhibition (Li et al., 2002). Thus, a reduction of the electron acceptor side of PSII at steady-state photosynthesis, following PSI photoinhibition, as reflected in a decrease in qL (Fig. 3C), might be contributed also by the decrease in NPQ. These results suggest that PSI photoinhibition possibly causes PSII photoinhibition as a secondary effect under illuminated conditions. To our knowledge, the linear relationship between PSI/PSII ratio and NPQ was first characterized by Brestic and coworkers in early growth stages of chlorophyll b–deficient wheat mutant lines (Brestic et al., 2015). In C₃ plants, NPQ is mainly composed of qE quenching, which is believed to correlate with the lumen acidification produced by photosynthetic linear and possibly cyclic electron flow, both of which depend on PSI (Kanazawa and Kramer, 2002; Li et al., 2002). Therefore, a loss of PSI can decrease the thylakoid membrane potential to lower qE quenching. In fact, PSI photoinhibition causes a decrease in the proton gradient across the thylakoid membrane in wheat leaves (Brestic et al., 2015; Zivcak et al., 2015a). On the other hand, the thylakoid membrane potential is modulated also by the narrowing proton conductance of chloroplast ATP synthase and thylakoid ion transporters (Rott et al., 2011; Armbruster et al., 2014). However, proton conductance is not affected by PSI photoinhibition (Zivcak et al., 2015a). In this study, we present a correlation between the amount of photochemically active PSI and NPQ in the wild-type plant leaves (Fig. 3B). The contribution of PSI to NPQ should be further quantitatively characterized in future works.

MATERIALS AND METHODS

Arabidopsis Growth Conditions

Arabidopsis (Arabidopsis thaliana) wild type plant (Columbia ecotype) was grown under a long-day condition (16-h light, 23°C, 100 µmol photons m⁻² s⁻¹, white fluorescent lamp; 8-h dark, 21°C) with a relative humidity of 60% ± 10%. Seeds were planted in pots that contained a 1:1 mixture of vermiculite and Metro-Mix 350 (Sun Gro Horticulture); 1000-fold diluted Hyponex solution (Hyponex) was used as a watering solution.

Measurements of Gas Exchange, Chlorophyll Fluorescence, and P700

Gas exchange (CO₂ and water) was measured using a LI-7000 gas analyzer (Li-COR) equipped with a 3010 DUAL gas exchange leaf chamber (Walz, Effeltrich, Germany), in which the flowing gas was saturated with water vapor at 18.0 ± 0.1°C and the leaf temperature was maintained at 25°C. Chlorophyll fluorescence and oxidized P700 were simultaneously measured using a Dual-PAM-100 fluorometer (Walz).

The photosynthetic parameters of PSII were calculated using chlorophyll fluorescence parameters as follows (Baker, 2008): PSII operating efficiency (quantum yield of photochemical energy conversion in PSII), Y(II) = (F_mʹ – Fʹ)/F_mʹ; nonphotochemical quenching, NPQ = (F_m – F_mʹ)/F_mʹ; fraction of “open” PSII centers (with Q_A oxidized), on the basis of a lake model, for the PSII photosynthetic apparatus, qL = [(F_mʹ – Fʹ)/(F_mʹ – F_oʹ)] × (F_oʹ/Fʹ), where, F_o represents minimum fluorescence from a dark-adapted leaf; F_oʹ, minimum fluorescence from a light-adapted leaf; F_m, maximum fluorescence from a dark-adapted leaf; F_mʹ, maximum fluorescence from a light-adapted leaf; and Fʹ, fluorescence emission from a light-adapted leaf. Pulse-amplitude modulated red measuring light (620 nm, 0.1 µmol photons m⁻² s⁻¹) was used to determine F_o. Red actinic light was supplied using a chip-on-board light emitting diode (LED) array (635 nm). Short-saturation pulse light (8,000 µmol photons m⁻² s⁻¹, 300 ms) was also provided by the LED array for the determinations of F_m and F_mʹ.

The photosynthetic parameters of PSI were calculated from the redox state of P700 as follows (Klughammer and Schreiber, 1994; Schreiber and Klughammer, 2008): quantum yield of photochemical energy conversion in PSI, Y(I) = (P_mʹ − P)/P_m; quantum yield of nonphotochemical energy dissipation due to donor-side limitation, Y(ND) = P/P_m; quantum yield of nonphotochemical energy dissipation due to acceptor-side limitation, Y(NA) = (P_m − P_mʹ)/P_m, where, P_m represents total quantity of photo-oxidizable P700; P_mʹ, maximum quantity of photo-oxidized P700 by a saturation pulse; and P, quantity of photo-oxidized P700 at steady state. Pulse-amplitude modulated near-infrared measuring lights (830 and 870 nm) were applied to measure the transmittance of oxidized P700.

Statistical Analysis

All statistical analyses were performed using Origin 2017 (LightStone).

ACKNOWLEDGMENTS

The authors thank Editage (www.editage.jp) for providing English corrections.

LITERATURE CITED

Author notes

2

Senior author.

3

Present address: Institute for Integrative Biology of the Cell, Centre National de la Recherche Scientifique, Comissariat à l'Energie Atomique et aux Energies Alternatives Saclay, Institut de Biologie et de Technologie de Saclay, Université Paris-Sud, Gif-sur-Yvette, France.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Chikahiro Miyake (cmiyake@hawk.kobe-u.ac.jp).

G.S. performed all experiments and wrote the article; C.M. conceived the original screening and research plans; G.S. and C.M. designed the experiments and analyzed the data.

© 2019 American Society of Plant Biologists. All Rights Reserved.

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