A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching

Abstract

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

Keywords: LHCII aggregate; LHCII trimer; NPQ; NoM; amphipol A8-35; chlorophyll; fluorescence; light-harvesting complex (antenna complex); lincomycin; nonphotochemical quenching; photosystem II; plant biochemistry.

Figure 1.

Purification and characterization of trimeric and aggregated forms of the major LHCII. A and B, sucrose density gradient of solubilized thylakoid membranes obtained from violaxanthin-enriched (A) and zeaxanthin-enriched (B) NoM plants, devoid of photosystem II and I cores by lincomycin treatment. Dark and NPQ conditions are compared. The experiments were performed on two independent replicates for violaxanthin-enriched thylakoid membranes and three replicates for zeaxanthin-enriched thylakoid membranes (Tables S2 and S3). C, representative chlorophyll (Chl) fluorescence quenching traces measured on violaxanthin-enriched (black line) and zeaxanthin-enriched (red line) chloroplasts. The arrow shows when actinic light was applied (AL-on; 1,380 μmol photons m⁻² s⁻¹, for 30 min). NPQ state was fixed by lowering the pH from 7.6 to 5.0 units adding 2 m HCl to chloroplast suspensions. D, immunodetection of the LHCII trimeric (tr.) and aggregate (ag.) bands from zeaxanthin-enriched NoM thylakoid membrane (Fig. 1B) against the Lhcb1 antibody. The samples were loaded as equal volumes (20 μl). a.u., arbitrary unit(s); Viol, violaxanthin; Zea, zeaxanthin.

Figure 2.

Absorption spectra of LHCII aggregates and assessment of PSI contamination. LHCII aggregates (ag.) enriched in violaxanthin (A and C) and zeaxanthin (B and D) are shown for dark-adapted and NPQ states. Red lines represent normalized absorption spectra of native LHCII aggregates corrected to remove PSI contamination (see Fig. 1 and S2). Black lines represent normalized absorption of uncorrected LHCII aggregates. Dashed blue lines represent the normalized absorption spectra of PSI. Solid blue lines represent the proportion of PSI subtracted from the uncorrected spectra to obtain the corrected LHCII aggregate spectra. In each panel, the corrected relative (rel.) contributions (%) of LHCII aggregates from Fig. 1 are indicated. Viol, violaxanthin; Zea, zeaxanthin.

Figure 3.

Fluorescence lifetime decay kinetics of isolated LHCII complexes. Shown are the time-correlated single-photon counting measurements of the sucrose density gradient bands of zeaxanthin-enriched LHCII trimers and aggregates, under NPQ conditions. Representative traces of LHCII trimers (tr., black trace) and aggregates (ag., red trace) are shown.

Figure 4.

77 K fluorescence emission spectra of LHCII trimers (black lines) and aggregates (red dotted lines). Violaxanthin-enriched (A and C) and zeaxanthin-enriched thylakoid membranes (B and D), in dark-adapted and NPQ states are shown. Emission spectra were recorded from sucrose density gradient bands of the solubilized thylakoid membranes (see Fig. 1). Representative emission spectra are shown for each condition. ag., aggregate; a.u., arbitrary unit(s); tr., trimers; Viol, violaxanthin; Zea, zeaxanthin.

Figure 5.

Purification and characterization of sucrose density-harvested LHCII bands under NPQ and relaxation states. A, isolation of the LHCII trimers (tr.) and aggregates (ag.) from solubilized photosynthetic membranes obtained from protoplasts, by sucrose density gradient. Violaxanthin-enriched NoM plants devoid of photosystem cores, under NPQ and relaxation states, were used. B, SDS-PAGE of samples fractionated by sucrose density gradients, after relaxation in the dark (relaxation in A). Western blotting analysis of PSI (PsaB) and Lhcb1 proteins is shown. C and D, representative PAM fluorescence quenching traces of NoM chloroplasts, obtained from protoplasts, used to induce NPQ (C) and recovery state (D), respectively. NPQ was fixed by lowering the pH from 7.6 to 5.0 adding 2 m HCl. AL-off, actinic light off; AL-on, actinic light on; a.u., arbitrary unit(s); Chl, chlorophyll.

Figure 6.

Absorption spectra of LHCII aggregates obtained from protoplast preparations. PSI contamination was estimated for NPQ (A) and recovery states (B). Red lines represent normalized absorption spectra of natural LHCII aggregates (ag.) corrected to remove PSI contamination (see Fig. 5). Black lines represent normalized absorption of the uncorrected LHCII aggregates. Solid blue lines represent the proportion of PSI subtracted from the uncorrected spectra to obtain the corrected LHCII aggregate spectra. rel., relative.