Live-cell imaging of photosystem II antenna dissociation during state transitions

Abstract

Plants and green algae maintain efficient photosynthesis under changing light environments by adjusting their light-harvesting capacity. It has been suggested that energy redistribution is brought about by shuttling the light-harvesting antenna complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) (state transitions), but such molecular remodeling has never been demonstrated in vivo. Here, using chlorophyll fluorescence lifetime imaging microscopy, we visualized phospho-LHCII dissociation from PSII in live cells of the green alga Chlamydomonas reinhardtii. Induction of energy redistribution in wild-type cells led to an increase in, and spreading of, a 250-ps lifetime chlorophyll fluorescence component, which was not observed in the stt7 mutant incapable of state transitions. The 250-ps component was also the dominant component in a mutant containing the light-harvesting antenna complexes but no photosystems. The appearance of the 250-ps component was accompanied by activation of LHCII phosphorylation, supporting the visualization of phospho-LHCII dissociation. Possible implications of the unbound phospho-LHCII on energy dissipation are discussed.

Fig. 1.

Measurement of CFL in live C. reinhardtii WT cells in states 1 and 2. CFL images in state 1 (A) and state 2 (B) cells as indicated by the color scale from 100 to 300 ps. (Scale bars, 10 μm.) (C) The photon count ratio of CFL_{250 ps}/CFL_{170 ps} (±SD, n = 11 cells) in state 1 (S1) and state 2 (S2) cells. (D) Time-lapse CFL images of WT cells during state 2 transitions as indicated by the color scale from 100 to 300 ps. (Scale bar, 10 μm.)

Fig. 2.

Quantitative measurements of the CFL shift observed during state 2 transitions in single WT (A–C), stt7 (D–F), and ΔPSI/II (G–I) cells. The overall CFL distribution measured in single WT (A), stt7 (D), and ΔPSI/II (G) cells during the 5-min state 2 transition. The spatiotemporal distribution of the CFL shift observed in single WT (B), stt7 (E), and ΔPSI/II (H) cells during the same time course shown in A, D, and G, respectively. Colors indicate pixels whose strongest CFL was 170 ± 10 ps (blue) and 250 ± 10 ps (red). These colors are overlaid on the total fluorescence images indicated in gray scale. (Scale bars, 5 μm.) The change in the photon count for CFL_{170 ps} (blue) and CFL_{250 ps} (red) observed in single WT (C), stt7 (F), and ΔPSI/II (I) cells during the same time course shown in A, D, and G, respectively. Phosphorylation of major LHCII detected by immunoblot analysis using an anti-phosphothreonine antibody is shown under the photon count data.

Fig. 3.

Protein composition in the ΔPSI/II mutant. (A) Immunoblot analysis of thylakoid membranes isolated from WT and ΔPSI/II cells using antibodies specific for PSI (PsaA, PsaF), PSII (CP47, D1), cytochrome b₆f complex (Cyt b₆, Cyt f), minor LHCII (CP26, CP29), and major LHCII (LhcbM6). Protein samples were normalized to the amount of major LHCII. (B) Sucrose density gradient centrifugation to separate chlorophyll-protein complexes of thylakoid membranes isolated from ΔPSI/II. (C) SDS/PAGE analysis of the chlorophyll-protein complexes separated in the sucrose density gradient in B. The gel was stained with Coomassie brilliant blue R-250. Arrows and arrowheads indicate the protein bands for LHCII and LHCI, respectively.

Fig. 4.

Biochemical differences between LHCII and phospho-LHCII. (A) Thylakoid membranes isolated from WT cells in state 1 (S1) and state 2 (S2) were subjected to sucrose density gradient ultracentrifugation, which separated LHCII, PSII, and PSI-LHCI protein complexes. (B) Phosphorylated proteins in the LHCII fractions obtained in A are shown by immunoblot analysis using an anti-phosphothreonine antibody. Proteins were normalized with the amount of LHCII protein (α-LHCII). Electron micrographs of the negative-stained LHCII fractions in S1 (C) and S2 (D) obtained in A. (Scale bars, 50 nm.) Energy-dissipative state of phospho-LHCII are shown by mean CFL of LHCII fractions in state 1 (E) and state 2 (F) which were obtained in A. Color scale indicates mean CFL. (Scale bars, 5 μm.)

Fig. 5.

A model for the dissociation of phospho-LHCII from PSII during state 2 transitions. (A) Dissociation of phospho-LHCII (both major and minor LHCII) from PSII. (B) Aggregation of phospho-LHCII. (C) Migration of LHCII from aggregates to PSI-LHCI supercomplex. (D) Association of LHCII (both major and minor LHCII) with PSI-LHCI supercomplex. The reverse process occurs during the transition from state 2 to state 1 (D’–A’). The crystal coordinates were obtained from the Protein Data Bank: PSII core dimer, 2AXT; LHCII, 2NHW; and PSI-LHCI supercomplex, 2O01.