Fluorescence lifetime snapshots reveal two rapidly reversible mechanisms of photoprotection in live cells of Chlamydomonas reinhardtii

Abstract

Photosynthetic organisms avoid photodamage to photosystem II (PSII) in variable light conditions via a suite of photoprotective mechanisms called nonphotochemical quenching (NPQ), in which excess absorbed light is dissipated harmlessly. To quantify the contributions of different quenching mechanisms to NPQ, we have devised a technique to measure the changes in chlorophyll fluorescence lifetime as photosynthetic organisms adapt to varying light conditions. We applied this technique to measure the fluorescence lifetimes responsible for the predominant, rapidly reversible component of NPQ, qE, in living cells of Chlamydomonas reinhardtii. Application of high light to dark-adapted cells of C. reinhardtii led to an increase in the amplitudes of 65 ps and 305 ps chlorophyll fluorescence lifetime components that was reversed after the high light was turned off. Removal of the pH gradient across the thylakoid membrane linked the changes in the amplitudes of the two components to qE quenching. The rise times of the amplitudes of the two components were significantly different, suggesting that the changes are due to two different qE mechanisms. We tentatively suggest that the changes in the 65 ps component are due to charge-transfer quenching in the minor light-harvesting complexes and that the changes in the 305 ps component are due to aggregated light-harvesting complex II trimers that have detached from PSII. We anticipate that this technique will be useful for resolving the various mechanisms of NPQ and for quantifying the timescales associated with these mechanisms.

Fig. 1.

PAM fluorescence trace of wild-type C. reinhardtii. The actinic light intensity during the light induction period between T = 0 s and T = 20 s was 1,000 μmol photons m^-2 s^-1. Inset shows the fluorescence yield during the first 3 s after the actinic light is applied. The time resolution was 0.11 s.

Fig. 2.

Schematic of measurement. (A) In the actinic light measurement, the sample remained in the dark until time T = 0 s, when the actinic light treatment began. Measurement of the fluorescence lifetime occurred periodically by closing the shutter to the actinic light beam and opening those in front of the pulsed laser and the detector (thin rectangles). The actinic light shutter was closed at T = 20 s and a measurement was made a time T_relax later. (B) In the laser light treatment, laser light was applied for 0 s < T < 20 s. Fluorescence acquisition periods were ΔT = 0.08 s long and spaced 0.2 s apart. The first F(t,T) was collected at T = 0.1 s to avoid loss of counts for the first measurement due to the opening time for the shutter. A measurement was made at a time T_relax as in the actinic light measurement. A representative fluorescence lifetime trace corresponding to a collection period at the beginning of the 20 s induction time is shown. The data were smoothed with a moving average filter with a span of 25 bins for visual clarity.

Fig. 3.

Normalized fluorescence decays from emission at 680 nm from the measurement described in Fig. 1B. The data were smoothed with a moving average filter with a span of 20 bins for visual clarity. The direction of the black arrow indicates increasing T. Blue represents early T and red represents late T within the timespan specified in each panel.

Fig. 4.

Changes in the amplitudes of the fluorescence lifetime components over T. (A) Amplitude of the 65 ps component [A_65 ps(T), green circles], (B) amplitude of the 305 ps component [A_30 5ps(T), violet circles], and (C) Amplitude of the 1 ns component [A_1 ns(T), blue circles] and the sum of the 65 ps and 305 ps components [A_65 ps(T) + A_305 ps(T), red circles]. The error bars indicate 1 standard deviation in the uncertainty of that parameter and are shown for T = 0.3 s, 1.9 s, 7.9 s, 17.7 s, and 50 s. Insets in A and B show the A_65 ps(T) and A_305 ps(T) for 0.3 s < T < 3 s. The lines in Insets are shown to indicate the changes in the amplitude.