Populations of photoinactivated photosystem II reaction centers characterized by chlorophyll a fluorescence lifetime in vivo

Abstract

Photosystem (PS) II centers, which split water into oxygen, protons, and electrons during photosynthesis, require light but are paradoxically inactivated by it. Prolonged light exposure concomitantly decreased both the functional fraction of PSII reaction centers and the integral PSII chlorophyll (Chl) a fluorescence lifetime in leaf segments of Capsicum annuum L. Acceleration of photoinactivation of PSII by a pretreatment with the inhibitors/uncoupler lincomycin, DTT, or nigericin further reduced PSII Chl a fluorescence lifetimes. A global analysis of fluorescence lifetime distributions revealed the presence of at least two distinct populations of photoinactivated PSII centers, one at 1.25 ns, and the other at 0.58 ns. Light treatment first increased the 1.25-ns component, a weak quencher, at the expense of a component at 2.22 ns corresponding to functional PSII centers. The 0.58-ns component, a strong quencher, emerged later than the 1.25-ns component. The strongly quenching PSII reaction centers could serve to avoid further damage to themselves and protect their functional neighbors by acting as strong energy sinks.

Fig. 1.

The time course of some changes in leaf discs in response to high-light treatment. (A) The time course of decline in the functional fraction (f) of PSII in Capsicum leaf segments during light treatment at 900 μmol of photons per m² per s and 25°C. Leaf segments were preincubated with water (control), lincomycin, lincomycin plus nigericin, or lincomycin plus DTT. For calculation of f (see Materials and Methods), Chl a fluorescence measurements were performed after 30 min of dark-adaptation, without treating the samples with DCMU. (B) The de-epoxidation state (antheraxanthin + zeaxanthin)/(violaxanthin + antheraxanthin + zeaxanthin) of leaf samples as a function of preillumination time. After the same light treatment at 900 μmol of photons per m² per s as for A, the samples were vacuum-infiltrated with DCMU and dark-adapted for 30 min for fluorescence lifetime measurement in low light. They were then stored at –80°C until pigment extraction.

Fig. 2.

Changes in the PSII Chl a fluorescence lifetime distributions in the control (water-treated) leaf samples of C. annuum after exposure to high light (900 μmol of photons per m² per s). Samples were kept in the dark for 30 min during the vacuum-infiltration with 30 μM DCMU (20 min) and the temperature adjustment to –2°C (10 min) before the measurements of fluorescence lifetime. The excitation irradiance (635 nm) was 140 μmol of photons per m² per s. (A) Lifetime-weighted fractions were derived by fitting the data of PSII fluorescence decay (in B) to a continuous Lorentzian distribution model assuming six components. Lifetime center values and widths of all components are given in Table 1. A minor fraction of the longest lifetime component at 10.8 ns is not shown. Changes in the lifetime-weighted fractional intensity of the component at 0.58 ns are enlarged in Inset. Dark, dark-adapted samples; 1 h HL, 3 h HL, and 5 h HL, samples exposed to high light for 1, 3, and 5 h. (B) Phase shift (open symbols) and demodulation (solid symbols) measured at 12 logarithmically spaced modulation frequencies raging from 25 to 200 MHz. (C) Residual phase shift and residual demodulation between measured and calculated values fitted by using the Lorentzian model.

Fig. 3.

Correlation of the integral fluorescence lifetime of PSII with the functional fraction of PSII, determined in leaf samples pretreated at 900 μmol of photons per m² per s for various durations, with or without an inhibitor or an uncoupler. Integral fluorescence lifetime was calculated by integration of the function for the lifetime-weighted fractions g(τ) from time 0 to ∞, i.e., ∫ g(τ) dτ. After illumination, leaf samples were dark-treated for 30 min before measurement of F_o and F_m (used to calculate f). Likewise, the samples used for measurement of the PSII Chl a fluorescence lifetime distribution were dark-treated for 30 min during vacuum-infiltration with DCMU (20 min), followed by temperature adjustment to –2°C in the sample holder (10 min). The excitation irradiance (635 nm) was 140 μmol of photons per m² per s. Replicate leaf samples were also measured for F_o and F_m, but these were not used for fluorescence lifetime measurements.

Fig. 4.

A diagram showing three types of PSII units connected via their antennae (dashed envelope). An active PSII (nonhatched circle), presumed to give Chl a fluorescence lifetime of 2.22 ns when measured in the presence of DCMU, passes its excitation energy to, and is photoprotected by, strongly dissipating, photoinactivated PSII centers (hatched with solid lines) that are presumed to give the fluorescence lifetime of 0.58 ns. An intermediate, weakly dissipating type of photoinactivated PSII (hatched with dashed lines) is presumed to give rise to the fluorescence lifetime of 1.25 ns. When most of PSII centers are photoinactivated, high-energy-state quenching in the antennae that depends on ΔpH is expected to be limited, so it is omitted for simplicity.