Higher plant photosystem II light-harvesting antenna, not the reaction center, determines the excited-state lifetime-both the maximum and the nonphotochemically quenched

Abstract

The maximum chlorophyll fluorescence lifetime in isolated photosystem II (PSII) light-harvesting complex (LHCII) antenna is 4 ns; however, it is quenched to 2 ns in intact thylakoid membranes when PSII reaction centers (RCIIs) are closed (Fm). It has been proposed that the closed state of RCIIs is responsible for the quenching. We investigated this proposal using a new, to our knowledge, model system in which the concentration of RCIIs was highly reduced within the thylakoid membrane. The system was developed in Arabidopsis thaliana plants under long-term treatment with lincomycin, a chloroplast protein synthesis inhibitor. The treatment led to 1), a decreased concentration of RCIIs to 10% of the control level and, interestingly, an increased antenna component; 2), an average reduction in the yield of photochemistry to 0.2; and 3), an increased nonphotochemical chlorophyll fluorescence quenching (NPQ). Despite these changes, the average fluorescence lifetimes measured in Fm and Fm' (with NPQ) states were nearly identical to those obtained from the control. A 77 K fluorescence spectrum analysis of treated PSII membranes showed the typical features of preaggregation of LHCII, indicating that the state of LHCII antenna in the dark-adapted photosynthetic membrane is sufficient to determine the 2 ns Fm lifetime. Therefore, we conclude that the closed RCs do not cause quenching of excitation in the PSII antenna, and play no role in the formation of NPQ.

Figure 1

Representative Western blot result showing the polypeptide composition of Arabidopsis plants grown with lincomycin. Total protein extracts from leaves were loaded in equal amounts (100% = 10 μg of total protein per lane), detected using designated antibodies, and analyzed as described in Materials and Methods.

Figure 2

FPLC gel filtration fractionation of solubilized control (solid line) and treated (dashed line) thylakoid membranes. Major fractions are marked as follows: PSII membrane fragments (I), PSII supercomplexes (II), PSI complex (III), PSII core complexes (IV), trimeric LHCII (V), and monomeric minor LHCIIs (VI). Thylakoids were solubilized with 1% α-DM. The top-right inset shows the absorption spectrum of fraction V (solid line) compared with the spectrum of the isoelectric focusing-prepared major LHCII complex (dashed line).

Figure 3

PAM chlorophyll fluorescence analysis of untreated (A) and treated (B) Arabidopsis leaves. (C) NPQ measured with 200 μmol photons m⁻² s⁻¹ illumination (gray bar) or 700 μmol photons m⁻² s⁻¹(white bar) in control and treated plants after the second illumination cycle. Data are means ± SE from three replicates.

Figure 4

Induction of chlorophyll fluorescence at room temperature from untreated (continuous line) and lincomycin-treated (dashed line) Arabidopsis leaves. (A) Original fluorescence induction traces. (B) Normalized fluorescence induction traces to Fv = 1. The area above the induction curve is reciprocal to the antenna cross-section size. This area is larger for the control than for the treated sample. The difference corresponds to the PSII antenna cross-section increase in the treated samples by ∼30%. Fluorescence induction kinetic traces were measured with PAM 100 in fast kinetics mode, providing a flash of light of 10 μmol m⁻²s⁻¹ intensity after infiltration of the leaf with 30 μM DCMU, 150 mM sorbitol, and 10 mM Hepes, pH 7.5.

Figure 5

TCSPC analysis of the chlorophyll fluorescence lifetime in Arabidopsis leaves in harvesting and photoprotective states. (A) Chlorophyll fluorescence lifetime decay traces of control (black) and treated (red) Arabidopsis leaves. The light-harvesting (Fm) state (vacuum-infiltrated with 50 μM nigericin) is shown in comparison with the photoprotective (Fm′) state measured in the presence of 200 and 700 μmol photons m⁻² s⁻¹ of light, for treated and untreated plants, respectively. (B and C) Relative time-resolved fluorescence lifetime component amplitudes of control (black) and treated (dashed) leaves in the Fm (B) or Fm′ (C) states. Average intensity-weighted lifetimes: control Fm 2.0 ± 0.1 ns, lincomycin Fm 2.2 ± 0.1 ns, ct NPQ 0.9 ± 0.1 ns, lincomycin NPQ 0.8 ± 0.1 ns. Fluorescence was detected at 683 nm using a 470 nm excitation wavelength. Data are means ± SE from four replicates. IRF, instrument response function.

Figure 6

Fluorescence spectroscopy analysis of the changes in the state of the photosynthetic membrane induced by lincomycin. (A) The 77 K fluorescence spectrum of leaf homogenate from untreated (solid line) and treated (dash-dotted line) leaves. A PSI spectrum is shown for comparison (dotted line). (B) The second derivative of untreated (solid line) and treated (dash-dotted line) 77 K fluorescence spectra. (C) Room-temperature spectra of diluted leaf homogenates of the control (solid line) and lincomycin-grown (dashed line) plants. (D) 77 K excitation fluorescence spectra for 680 nm (solid line) and 700 nm (dashed line) bands of leaf homogenates from treated plants.

Figure 7

Low-temperature fluorescence spectral analysis of leaf homogenates. (A) Comparison between the fluorescence spectrums of treated leaf homogenate from which PSI spectrum was subtracted (upper curve) and the spectrum of aggregated LHCII with an average 2 ns lifetime. (B) The 77 K fluorescence spectra of control membranes (ct), aggregated LHCII with ∼2 ns fluorescence lifetime (LHCII), isolated PSI (PSI), and a difference ct-minus-LHCII-minus-PSI spectrum (ct-LHCII-PSI).