Rapid regulation of photosynthetic light harvesting in the absence of minor antenna and reaction centre complexes

Abstract

Plants are subject to dramatic fluctuations in the intensity of sunlight throughout the day. When the photosynthetic machinery is exposed to high light, photons are absorbed in excess, potentially leading to oxidative damage of its delicate membrane components. A photoprotective molecular process called non-photochemical quenching (NPQ) is the fastest response carried out in the thylakoid membranes to harmlessly dissipate excess light energy. Despite having been intensely studied, the site and mechanism of this essential regulatory process are still debated. Here, we show that the main NPQ component called energy-dependent quenching (qE) is present in plants with photosynthetic membranes largely enriched in the major trimeric light-harvesting complex (LHC) II, while being deprived of all minor LHCs and most photosystem core proteins. This fast and reversible quenching depends upon thylakoid lumen acidification (ΔpH). Enhancing ΔpH amplifies the extent of the quenching and restores qE in the membranes lacking PSII subunit S protein (PsbS), whereas the carotenoid zeaxanthin modulates the kinetics and amplitude of the quenching. These findings highlight the self-regulatory properties of the photosynthetic light-harvesting membranes in vivo, where the ability to switch reversibly between the harvesting and dissipative states is an intrinsic property of the major LHCII.

Keywords: Chlorophyll fluorescence; LHCII; NPQ; proton gradient; qE; zeaxanthin.

Fig. 1.

Thylakoid membrane composition of NoM after treatment with lincomycin. (A) Representative sucrose density gradients of solubilized thylakoid membranes isolated from NoM and lincomycin-treated NoM plants. Analysed fractions are labelled and their relative chlorophyll content is shown (the same amount of chlorophyll was loaded into each tube). (B) SDS–PAGE of the main sucrose gradient fractions. The gel lanes displayed were cropped from the same picture of the gels for clarity. Equal sample volumes were loaded into each lane. Chl, chlorophyll; FP, free pigment.

Fig. 2.

qE induction in the absence of minor LHCs and photosystem cores. (A) qE induction in WT, NoM, and lincomycin-treated NoM pre-illuminated plant leaves. Data represent averages of 3–5 replicates ±SD. Black and white bars represent periods of darkness and actinic light illumination (900 µmol m^–2 s^–1), respectively. Grey dashed boxes mark the values used in the comparisons shown in (B). (B) Relaxation of qE in the dark measured with NPQ values (calculated from chlorophyll fluorescence traces, symbols ±SD) and absorption changes at 535 nm (ΔA₅₃₅, solid lines) for WT (black trace and open circles) and NoM leaves (red trace and open triangles). Solid lines represent averages of four independent absorption measurements, while shaded areas represent the SD. To compare the kinetics, NPQ values and absorption traces were offset to zero after 5 min of relaxation and normalized to the maximum value.

Fig. 3.

ΔpH dependence of the qE response in protoplasts and chloroplasts isolated from lincomycin-treated NoM plants. (A) Chlorophyll fluorescence induction traces of protoplasts in the presence (grey line) or absence of nigericin (black line, control). Traces are the average of three independent measurements, normalized at the maximum of fluorescence after the onset of actinic light. A saturating pulse was applied every minute to retrieve the NPQ values. (B) Chlorophyll fluorescence induction trace of chloroplasts exposed to bulk pH variations. After illumination, the pH was lowered to 5 by adding HCl. In the dark, the pH was restored to 8 by adding KOH. Black and white bars represent periods of darkness and actinic light illumination (900 µmol m^–2 s^–1), respectively. A saturating concentration of nigericin (500 nM) was added with HCl to ensure complete equilibration of lumen and bulk pH.

Fig. 4.

Zeaxanthin modulation of qE in leaves of lincomycin-treated NoM plants. (A) Fluorescence quenching induction traces of leaves from lincomycin-treated NoM plants, either dark adapted (+violaxanthin, black line) or pre-illuminated (+zeaxanthin, grey line). Each trace is the average of at least four independent measurements. A saturating pulse was applied every minute to retrieve the NPQ value. (B) Chl a fluorescence lifetime measurements of violaxanthin- (black) and zeaxanthin-enriched (grey) leaves of lincomycin-treated NoM plants. Prior to measurements, leaves were infiltrated with 50 µM DCMU, to fix the F_m state. Traces are averages of at least four independent measurements. The instrument response function (IRF) trace is shown in light grey. (C) Intensity-weighted components (%) obtained from chlorophyll fluorescence lifetime traces shown in (B). Lifetime components were calculated after iterative reconvolution of the IRF (see Equation 2 in the Materials and methods). The fitting procedure yielded good results using four lifetime components (0.1, 0.5, 1.25, and 2.6 ns). The fastest component originates from PSI contribution (Chukhutsina et al., 2019), while the other components were attributed to LHCs in different quenching conformations (Krause and Weis, 1991; Belgio et al., 2012). Error bars represent the SD and asterisks indicate statistical significance (two-tailed t-test: *P<0.05, ***P<0.001). Vio, violaxanthin; Zea, zeaxanthin.

Fig. 5.

Effects of enhancing ΔpH with diaminodurene (DAD) on the amplitudes of qE in NoM and NoM npq4 (devoid of PsbS; –PsbS). Representative PAM traces of chlorophyll (Chl) fluorescence and ΔpH induction from NoM (A) and NoM npq4 (B) chloroplasts. ΔpH was measured by the fluorescence quenching of 9-aminoacridine (9-aa) added before each measurement (5 µM). DAD (100 µM) was added to the suspension at the beginning of the illumination period (red lines). Arrows indicate the F_m values assessed by saturating pulses after 3 min of relaxation in the dark.

Fig. 6.

Effect of PsbS on the ΔpH dependence of qE. Relationship between reversible chlorophyll fluorescence quenching [qE, (F_mrelaxed–F_m')/F_mrelaxed] and 9-aa fluorescence quenching in NoM (black dots, +PsbS) and NoM npq4 (–PsbS) chloroplasts. The data range was obtained by changing either the actinic light intensity or the DAD concentration. Data were fitted to the Hill equation provided in the Materials and methods (R²=0.96 and 0.98 for NoM and NoM npq4 chloroplasts, respectively).

Fig. 7.

Effects of enhancing ΔpH with diaminodurene (DAD) on the amplitudes of qE of lincomycin-treated NoM npq4 in the presence of DCMU. (A) Representative PAM traces of chlorophyll (Chl) fluorescence and ΔpH induction from NoM npq4 chloroplasts. ΔpH was measured by the fluorescence quenching of 9-aminoacridine (9-aa) added before each measurement (5 µM). (B) The effect of DAD (red traces) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on chloroplasts of lincomycin-treated NoM npq4 plants. DCMU (50 μM) was added prior to experiments to close the remaining fraction of PSII reaction centres. DAD (100 µM) was added at the beginning of the illumination phase.