Mechanism of photosystem II photoinactivation and D1 protein degradation at low light: the role of back electron flow

Abstract

Light intensities that limit electron flow induce rapid degradation of the photosystem II (PSII) reaction center D1 protein. The mechanism of this phenomenon is not known. We propose that at low excitation rates back electron flow and charge recombination between the QB*- or QA*- semiquinone acceptors and the oxidized S(2,3) states of the PSII donor side may cause oxidative damage via generation of active oxygen species. Therefore, damage per photochemical event should increase with decreasing rates of PSII excitation. To test this hypothesis, the effect of the dark interval between single turnover flashes on the inactivation of water oxidation, charge separation and recombination, and the degradation of D1 protein were determined in spinach thylakoids. PSII inactivation per flash increases as the dark interval between the flashes increases, and a plateau is reached at dark intervals, allowing complete charge recombination of the QB*-/S2,3 or QA*-/S2 states (about 200 and 40 s, respectively). At these excitation rates: (i) 0.7% and 0.4% of PSII is inactivated and 0.4% and 0.2% of the D1 protein is degraded per flash, respectively, and (ii) the damage per flash is about 2 orders of magnitude higher than that induced by equal amount of energy delivered by excess continuous light. No PSII damage occurs if flashes are given in anaerobic conditions. These results demonstrate that charge recombination in active PSII is promoted by low rates of excitation and may account for a the high quantum efficiency of the rapid turnover of the D1 protein induced by limiting light.

Figure 1

Effect of the interval between flash excitations and presence of oxygen on the loss of PSII activity and degradation of the D1 protein. Spinach thylakoids were exposed for 4 h to 360 laser flashes at 40-s dark intervals under aerobic (FL) or anaerobic (FL + N₂) conditions or to 4 h of continuous low light (LL, 20 μmol m⁻²·s⁻¹). Alternatively, a total of 360 flashes were given at 100-ms intervals followed by 4 h of further incubation in the dark (FF). PSII activity was measured by TL (A and B, dotted column) and DCIP reduction (B, horizontal line column). D1 protein content was determined by Western blot analysis (B, vertical line column).

Figure 2

Lifetime of Q_B^•− or Q_A^•−/S_2,3 charge recombination in spinach thylakoids and leaf disks. Samples were exposed to one saturating light flash at 20°C followed by dark interval periods from 0 to 120 s. At the end of the dark period the samples were rapidly cooled (less than 5 s to reach 5°C) and frozen to −40°C after which the residual TL signal was measured. A total of 40 μg Chl/sample or three pea leaf disks, 0.6 cm diameter, were used per measurement. ▴, Leaf disks; ▪, thylakoids; •, thylakoids with addition of DCMU. ñ

Figure 3

Kinetics of the PSII inactivation and D1 protein degradation induced by single turnover flashes. Thylakoids were exposed to flashes delivered at 40-s dark interval (•), to continuous white light (○, 30 μmol m⁻²·s⁻¹) or as a control were kept in the dark for the same period (▵). Lane To, D1 protein level before light exposure. All incubations were at room temperature. Sample where taken at times as indicated and assayed for the D1 protein content (A and B) and TL signal (C). (C Inset) Recorded TL measurements indicating loss of the Q_B^•−/S_2,3 emission (22) as a function of increasing exposure time to the flashes. No block in electron flow from Q_A^•− to Q_B or loss of the oxygen evolving complex activity occurred as evidenced by the absence of the Q_A^•−/S₂ band (emission at 7–10°C).

Figure 4

Relation between the flash induced photoinactivation and degradation of the D1 protein and increasing the dark interval between the flashes. Thylakoids were exposed for 4 h to light flashes in absence or presence of DCMU. The dark intervals between the flashes varied from 8 to 320 s, corresponding to a total of 1800 to 45 flashes delivered per sample, respectively. The samples were analyzed for D1 protein content (A) and TL signal (B). • and ○, Thylakoids with or without addition of DCMU, respectively. The data represent percent of a dark control.

Figure 5

Increase in the efficiency of damage per flash as a function of the charge recombination extent. Calculated loss of PSII activity/flash (open symbols) and degradation of D1 protein (solid symbols) as a function of the extent of charge recombination during the dark interval expressed as t½ units (Fig. 2) using the data of Fig. 4. Thylakoids were exposed to light flashes in the absence (A) or presence (B) of DCMU, respectively.