Higher Reduced State of Fe/S-Signals, with the Suppressed Oxidation of P700, Causes PSI Inactivation in Arabidopsis thaliana

Abstract

Environmental stress increases the risk of electron accumulation in photosystem I (PSI) of chloroplasts, which can cause oxygen (O₂) reduction to superoxide radicals and decreased photosynthetic ability. We used three Arabidopsis thaliana lines: wild-type (WT) and the mutants pgr5^hope1 and paa1-7/pox1. These lines have different reduced states of iron/sulfur (Fe/S) signals, including F_x, F_A/F_B, and ferredoxin, the electron carriers at the acceptor side of PSI. In the dark, short-pulse light was repetitively illuminated to the intact leaves of the plants to provide electrons to the acceptor side of PSI. WT and pgr5^hope1 plants showed full reductions of Fe/S during short-pulse light and PSI inactivation. In contrast, paa1-7/pox1 showed less reduction of Fe/S and its PSI was not inactivated. Under continuous actinic-light illumination, pgr5^hope1 showed no P700 oxidation with higher Fe/S reduction due to the loss of photosynthesis control and PSI inactivation. These results indicate that the accumulation of electrons at the acceptor side of PSI may trigger the production of superoxide radicals. P700 oxidation, responsible for the robustness of photosynthetic organisms, participates in reactive oxygen species suppression by oxidizing the acceptor side of PSI.

Keywords: Fe/S clusters; P700; ferredoxin; photoinhibition; photosynthetic electron transport; photosystem I.

Figure 1

Kinetics of (A) oxidized P700 (P700⁺) and (B) reduced Fe/S signals (Fe/S⁻) in response to short-pulse light for wild-type (WT), pgr5^hope1, and paa1-7 Arabidopsis intact leaves. Short-pulse light (15,000 µmol photons m⁻² s⁻¹, 300 ms) was started at 0 ms. The redox reactions of both P700 and Fe/S were monitored simultaneously. Relative values of both P700⁺ and [Fe/S]⁻ were normalized to the maximum oxidation and reduction levels, respectively, as described in the “Materials and Methods”. The negative values of Fe/S⁻ show the reduction of Fe/S. The data points for WT (gray), pgr5^hope1 (magenta), and paa1-7/pox1 (red) are the means of six biological replicates (darker color) and the shadowed area is the standard deviation (lighter color).

Figure 2

Effects of rSP light illumination on the incident photo-oxidizable P700 (Pm’), photo-oxidizable P700 (Pm), maximum quantum efficiency of PSII (Fv/Fm), and amount of Fe/S in wild-type (WT) and pgr5^hope1 Arabidopsis. The leaves were illuminated every 10 s with short-pulse light (300 ms) of 15,000 μmol photons m⁻² s⁻¹ under the atmospheric conditions (40 Pa CO₂/ 21 kPa O₂) for 30 min. The rSP light illumination started at 0 min. (A) Pm’. Black, WT; magenta, pgr5^hope1. The mean values were normalized to the primary values before the rSP light treatment; error bars represent the standard deviation; data were acquired from six biological replicates. (B) Pm, (C) Fv/Fm, and (D) Fe/S were compared before and after the rSP light illumination treatment in WT and pgr5^hope1. These parameters were evaluated after the illuminated leaves were left for 1 h in the dark. Each value was normalized to the value before the rSP light illumination treatment. ** p < 0.01 (Welch’s t-test).

Figure 3

Effects of rSP light illumination and illumination time on the incident photo-oxidizable P700 (Pm’), photo-oxidizable P700 (Pm), maximum quantum efficiency of PSII (Fv/Fm), and amount of Fe/S in wild-type (WT) and paa1-7/pox1 Arabidopsis. The leaves were illuminated every 10 s with short-pulse light of 15,000 μmol photons m⁻² s⁻¹ under atmospheric conditions (40 Pa CO₂/ 21 kPa O₂) for 30 min. The rSP light illumination started at 0 min. (A) The illumination time was set to the two durations, as indicated by the arrows (I, 100 ms; II, 300 ms), based on the reduction kinetics of Fe/S of both WT and paa1-7/pox1 (redrawn from Figure 1, Black, WT; red, paa1-7/pox1). In experiments I and II, the parameters Pm’, Pm, Fv/Fm, and [Fe/S]⁻ were analyzed. (B) Pm’. Black, WT; magenta, pgr5^hope1. The values were normalized to the primary values before the rSP light illumination treatment and are shown with standard deviations. The data were obtained from six biological replicates. (C) Pm, (D) Fv/Fm, and (E) Fe/S before and after the rSP light illumination treatments in both WT and pgr5^hope1 were compared. These parameters were evaluated after the illuminated leaves were left for 1 h in the dark. Each value was normalized against the value before the rSP light illumination treatment. ** p < 0.01; *** p < 0.001 (Welch’s t-test).

Figure 4

Effect of photon flux density on the gross CO₂ assimilation rate (A + Rd), oxidized P700 (P700⁺), and reduction ratio of Fe/S signals [Fe/S]⁻ in wild-type (WT) and pgr5^hope1 Arabidopsis. (A) The net CO₂ assimilation rates were measured simultaneously with P700⁺ and Fe/S⁻ under atmospheric conditions (40 Pa CO₂, 21 kPa O₂). The dark respiration rates (Rd) were measured before starting actinic light illumination. After the net CO₂ assimilation reached the steady state at the photon flux density of 150 μmol photons m⁻² s⁻¹, the intensity was increased to 550 and 1100 sequentially, after reaching each steady-state CO₂ assimilation. The gross CO₂ assimilation rates are expressed as A + Rd. (B) The oxidized P700 (P700⁺) is plotted against the photon flux density. (C) The reduction ratio of [Fe/S]⁻ is plotted against the photon flux density. The mean of three biological replicates and standard deviation are shown. Gray symbols, WT; Blue symbols, pgr5^hope1.

Figure 5

Effects of the continuous illumination of actinic light on the photo-oxidizable P700 (Pm), maximum quantum efficiency of PSII (Fv/Fm), and amount of Fe/S ([Fe/S]) in wild-type (WT) and pgr5^hope1 Arabidopsis. After reaching the steady state of the net CO₂ assimilation rate induced by the continuous illumination of actinic light (500 μmol photons m⁻² s⁻¹, 30 min) in both WT and pgr5^hope1, the photon flux density was increased to 1100 μmol photons m⁻² s⁻¹, and the illumination was continued for 2 h. The parameters, (A) Pm, (B) Fv/Fm, and (C) [Fe/S] of both WT and pgr5^hope1 treated with continuous illumination were evaluated after the illuminated leaves were left for 1 h in the dark, and values obtained before and after the continuous light illumination treatment were compared. The mean of three biological replicates and standard deviation are shown. * p < 0.05; ** p < 0.01; *** p < 0.001 (Welch’s t-test).

Figure 6

Production and suppression mechanism of ROS in PSI. PSI catalyzes the photosynthetic electron transport through a photo-oxidation reduction cycle in the reaction center of P700. P700 is photo-excited to P700* by the absorbed light energy and donates electrons to the primary electron acceptor chlorophyll a (A₀). P700⁺ accepts electrons from PSII through plastoquinol, Cyt b6/f-complex, and plastocyanin (PC), regenerating P700. The electron in A₀ flows to ferredoxin (Fd) through phylloquinone A₁ and the iron-sulfur (4Fe-4S) clusters F_x and F_A/F_B. The accumulation of P700⁺ under the constant photon flux density is determined by the rate-determining step (RdS) of the production-consumption rate of P700⁺ in the photo-oxidation reduction cycle of P700 in PSI. At the RdS of P700*, oxidation P700⁺ does not accumulate; at the RdS of P700⁺ reduction, P700⁺ accumulates. (A) The RdS of P700* oxidation is caused by the accumulation of electrons at the acceptor-side of PSI, as observed with the reduction of Fe/S clusters (Fe/S⁻), including F_x, F_A/F_B, and Fd, where the possibilities of the reduction of phylloquinone A₁ also increase. These accumulated electrons would flow to O₂ to produce superoxide radical (O₂⁻), and O₂⁻ would degrade F_X and F_A/F_B with the release of Fe and hydrogen peroxide (H₂O₂). Both H₂O₂ and the reduced Fe further react to produce hydroxyl radical (OH) through Fenton reaction. This highly reactive oxygen species (ROS) would oxidatively degrade PSI irreversibly, leading to PSI inactivation. (B) The RdS of P700⁺ reduction is caused by the limitation of photosynthetic electron transport from plastoquinol to P700 through Cyt b6/f-complex and PC [46,48]. Even under low photosynthesis efficiency conditions (drought, high intensity light, low/high temperature, low CO₂, etc.), P700 is oxidized to P700⁺. The RdS of P700⁺ reduction is induced by the acidification in the luminal side of thylakoid membranes, which suppress the oxidation activity of plastoquinol by the Cyt b6/f-complex, with the oxidation of the electron acceptors A₀, A₁, F_X, F_A/F_B, and Fd, leading to the suppression of ROS production. This is the physiological function of P700 oxidation to suppress ROS production in PSI.