A mechanism of nonphotochemical energy dissipation, independent from PsbS, revealed by a conformational change in the antenna protein CP26

Abstract

The regulation of light harvesting in higher plant photosynthesis, defined as stress-dependent modulation of the ratio of energy transfer to the reaction centers versus heat dissipation, was studied by means of carotenoid biosynthesis mutants and recombinant light harvesting complexes (LHCs) with modified chromophore binding. The npq2 mutant of Arabidopsis thaliana, blocked in the biosynthesis of violaxanthin and thus accumulating zeaxanthin, was shown to have a lower fluorescence yield of chlorophyll in vivo and, correspondingly, a higher level of energy dissipation, with respect to the wild-type strain and npq1 mutant, the latter of which is incapable of zeaxanthin accumulation. Experiments on purified thylakoid membranes from all three mutants showed that the major source of the difference between the npq2 and wild-type preparations was a change in pigment to protein interactions, which can explain the lower chlorophyll fluorescence yield in the npq2 samples. Analysis of the xanthophyll binding LHC proteins showed that the Lhcb5 photosystem II subunit (also called CP26) undergoes a change in its pI upon binding of zeaxanthin. The same effect was observed in wild-type CP26 upon treatment that leads to the accumulation of zeaxanthin in the membrane and was interpreted as the consequence of a conformational change. This hypothesis was confirmed by the analysis of two recombinant proteins obtained by overexpression of the Lhcb5 apoprotein in Escherichia coli and reconstitution in vitro with either violaxanthin or zeaxanthin. The V and Z containing pigment-protein complexes obtained by this procedure showed different pIs and high and low fluorescence yields, respectively. These results confirm that LHC proteins exist in multiple conformations, an idea suggested by previous spectroscopic measurements (Moya et al., 2001), and imply that the switch between the different LHC protein conformations is activated by the binding of zeaxanthin to the allosteric site L2. The results suggest that the quenching process induced by the accumulation of zeaxanthin contributes to qI, a component of NPQ whose origin was previously poorly understood.

Figure 1.

Effect of Different Light Conditions during Growth of Wild-Type and Mutant Arabidopsis Plants. (A) to (C) Each pot contained five seedlings. Control plants (A) (120 μE m⁻² s⁻¹); high light–grown plants (B) (1300 μE m⁻² s⁻¹); plants grown in limiting light conditions (C) (20 μE m⁻² s⁻¹). In low light, npq2 plants show a lower growth, consistent with sustained dissipation of excitation energy. In high light, npq2 plants appear to be highly resistant with respect to npq1 and npq4. npq1 plants, lacking zeaxanthin, appear to be more severely affected than npq4 plants, lacking PsbS. (D) Fresh weight increase measured on populations of Arabidopsis wild-type and mutant genotypes growth for 3 weeks in different light conditions. During this treatment, the npq2 plant had been daily sprayed with 10 μM abscisic acid. Data are expressed as means ± sd, n = 15. Data sets with a significance level of P < 0.05 according to Student's t test are marked with an asterisk.

Figure 2.

Relative Fluorescence Yield of Leaves from Arabidopsis Wild Type and npq Mutants. Fluorescence amplitude was measured at F_m on intact leaves (A) and on leaves infiltrated (B) with either 5 mM Hepes, pH 7.0, (light gray) or 50 μM nigericin (dark gray). We used a video imaging apparatus for F_m determination (see Methods) and normalized F_m to the chlorophyll content per leaf surface. Values are the average of measurements performed on four leaves. These results have been confirmed using a PAM fluorimeter and leaf discs. Data sets with a significance level of P < 0.05 according to Student's t test are marked with an asterisk. a.u., arbitrary units.

Figure 3.

Kinetics of NPQ of Wild Type Arabidopsis and Mutants. In vivo NPQ of chlorophyll fluorescence was measured with a PAM fluorimeter, and it was induced by saturating actinic light (1200 μE m⁻² s⁻¹, white bar) on wild-type and mutant leaves. Dark-relaxation kinetic was followed (black bar). The values of NPQ were calculated as described in Methods. Values are the average of measurements performed on four distinct leaves.

Figure 4.

Fluorescence Yield of Thylakoid Membranes upon Step Solubilization. (A) Sucrose density gradient profiles of thylakoids solubilized with 0.5% α-DM (right) and 5% Triton X-100 (left). The same level of solubilization and separation into component green bands was obtained using thylakoids from npq1, npq2, and npq4 genotypes. Composition of each band is indicated. (B) Thylakoid membranes were purified from dark-adapted leaves of wild-type, npq1, and npq2 genotypes and from wild-type leaves after light treatment (see Methods for details); their fluorescence yield was measured after solubilization with 0.5% α-DM and 5% Triton X-100. Chlorophyll concentration of isolated thylakoids was set to 0.1 μg/mL. The samples were measured at room temperature under continuous stirring. Data are expressed as means ± sd of four measurements. a.u., arbitrary units.

Figure 5.

Flatbed IEF Fractionation. Profiles obtained after IEF of monomeric Lhcb proteins (A) isolated from npq2 (top) and wild-type (bottom) thylakoids by sucrose gradient fractionation, monomeric Lhcb complexes (B) isolated from light-treated wild-type plants (WT-S), whose thylakoids were isolated in acidic buffer to maintain deepoxidation level, and recombinant Lhcb5 proteins (C) reconstituted in vitro with either zeaxanthin (top, CP26-Z) or violaxanthin (bottom, CP26-V).

Figure 6.

Spectral Properties at Room Temperature of Lhcb5 Isoforms Purified by IEF (See Figure 5B). (A) Comparison of fluorescence yield of CP26-V (solid line) and CP26-Z (dotted line) upon excitation at 625 nm. Data are the average of four independent measurements. a.u., arbitrary units. (B) Absorption spectra of CP26-V (solid line) and CP26-Z (dotted line) normalized to the chlorophyll content. Inset, difference spectrum in the Q_y transition region. (C) Circular dichroism spectra of CP26-V (solid line) and CP26-Z (dotted line). All the spectra were normalized to the same chlorophyll content.

Figure 7.

Fluorescence Analysis of Nigericin-Sensitive and Nigericin-Resistant NPQ Components in Wild-Type and npq1 Genotypes of Arabidopsis. (A) Wild-type and npq1 leaves were either left in the dark or illuminated (1200 μE m⁻² s⁻¹) for 15 min at room temperature to induce NPQ and zeaxanthin accumulation (in the wild type). Leaves were then rapidly infiltrated with either 5 mM Hepes, pH 7.0, or 50 μM nigericin to abolish ΔpH. F_m level was measured using a video imaging fluorescence system as described in Methods. The time required for infiltration and measurement was ∼1 min. a.u., arbitrary units. (B) Chlorophyll fluorescence intensity determinations in Arabidopsis wild-type leaves for F_m and F_m′. After each light window, the F_m′ value was measured on leaves rapidly infiltrated with 50 μM nigericin. (C) Comparison of nigericin-resistant NPQ component in wild-type and lhcb5 leaves, measured as in Figure 7B. Data sets with a significance level of P < 0.05 according to Student's t test are marked with an asterisk.