LHCSR1-dependent fluorescence quenching is mediated by excitation energy transfer from LHCII to photosystem I in Chlamydomonas reinhardtii

Abstract

Photosynthetic organisms are frequently exposed to light intensities that surpass the photosynthetic electron transport capacity. Under these conditions, the excess absorbed energy can be transferred from excited chlorophyll in the triplet state (3Chl*) to molecular O₂, which leads to the production of harmful reactive oxygen species. To avoid this photooxidative stress, photosynthetic organisms must respond to excess light. In the green alga Chlamydomonas reinhardtii, the fastest response to high light is nonphotochemical quenching, a process that allows safe dissipation of the excess energy as heat. The two proteins, UV-inducible LHCSR1 and blue light-inducible LHCSR3, appear to be responsible for this function. While the LHCSR3 protein has been intensively studied, the role of LHCSR1 has been only partially elucidated. To investigate the molecular functions of LHCSR1 in C. reinhardtii, we performed biochemical and spectroscopic experiments and found that the protein mediates excitation energy transfer from light-harvesting complexes for Photosystem II (LHCII) to Photosystem I (PSI), rather than Photosystem II, at a low pH. This altered excitation transfer allows remarkable fluorescence quenching under high light. Our findings suggest that there is a PSI-dependent photoprotection mechanism that is facilitated by LHCSR1.

Keywords: algae; fluorescence; light; photosynthesis; stress.

Fig. 1.

Time-resolved fluorescence analysis of the isolated thylakoid membranes. (A) Immunoblotting analysis of purified thylakoid membranes from UV-treated cells using antibodies against either ATPB or LHCSRs, or PSBS. (B) Thylakoid membrane samples were analyzed by SDS/PAGE stained by Coomassie Brilliant Blue G-250. The LHCII bands were indicated as CP26 (Lhcb5), CP29 (Lhcb4), LHCII type I (LhcbM3, LhcbM 4, LhcbM 6, LhcbM 8, LhcbM 9); LHCII type III (LhcbM2, -7), or LHCII Type IV (LhcbM1). (C and D) The time-correlated single-photon counting of fluorescence for the thylakoids of (C) npq4 and (D) npq4/lhcsr1 were recorded at 682 nm (slit = 8 nm) at pH 5.5 (red) and 7.5 (blue). Instrumental response function (IRF) is shown as gray line in C. The samples, normalized to 1 μg Chl/mL, were excited at 463 nm.

Fig. 2.

Low-temperature absolute fluorescence spectra of isolated thylakoid membranes. Fluorescence spectra of the isolated thylakoid membranes from npq4 (solid line) and npq4/lhcsr1 (dashed line). The membranes were treated with either pH 7.5 (black) or pH 5.5 (red) buffers. The fluorescence spectra were recorded with an integration sphere to obtain the absolute fluorescence photon counts for the samples. Samples normalized to 8 μg Chl/mL were excited at 480 nm.

Fig. 3.

Time-resolved fluorescence decay-associated spectrum analysis of isolated thylakoid membranes at 77 K. FDAS were derived from the time-resolved fluorescence profiles of thylakoid membranes obtained via excitation at 459 nm. The colored lines represent npq4 at pH 7.5 (green) and pH 5.5 (blue) and npq4/lhcsr1 at pH 7.5 (yellow) and pH 5.5 (red). The spectra were normalized to the maximum intensity of the slowest component (∼2.2 ns).

Fig. 4.

In vivo characterization of photosystem mutants. (A−C) The time-correlated single-photon counting of the fluorescence of (A) ΔPSI, (B) ΔPSII, and (C) ΔPSI/II cells after 6 h of UV treatment were recorded at 682 nm (slit = 8 nm) at pH 5.5 (red) and 7.5 (blue). The samples, normalized to 2 μg Chl/mL, were excited at 480 nm. (D) UV-treated cells (2 μg Chl) were subjected to immunoblotting analysis with antibodies specific to ATPB, PsaA/B (PSI), PsbA (D1), and LHCSR1.

Fig. 5.

Tentative model of LHCSR1-dependent energy quenching in C. reinhardtii. The light captured by LHCIIs is transferred to the PSII−LHCII or PSI−LHCI supercomplexes under LL (representing neutral pH in the lumen, blue arrow) or HL (representing acidic pH in the lumen, red arrow), respectively. When the lumen is acidified, LHCSR1 (process A) mediates excitation energy transfer from LHCIIs in the pool to PSI-LHCI (this study) and/or (process B) triggers energy-dependent quenching in the LHCII pool (18). Icons surrounded by a red line represent LHCSR1.