Lack of the light-harvesting complex CP24 affects the structure and function of the grana membranes of higher plant chloroplasts

Abstract

The photosystem II (PSII) light-harvesting antenna in higher plants contains a number of highly conserved gene products whose function is unknown. Arabidopsis thaliana plants depleted of one of these, the CP24 light-harvesting complex, have been analyzed. CP24-deficient plants showed a decrease in light-limited photosynthetic rate and growth, but the pigment and protein content of the thylakoid membranes were otherwise almost unchanged. However, there was a major change in the macroorganization of PSII within these membranes; electron microscopy and image analysis revealed the complete absence of the C(2)S(2)M(2) light-harvesting complex II (LHCII)/PSII supercomplex predominant in wild-type plants. Instead, only C(2)S(2) supercomplexes, which are deficient in the LHCIIb M-trimers, were found. Spectroscopic analysis confirmed the disruption of the wild-type macroorganization of PSII. It was found that the functions of the PSII antenna were disturbed: connectivity between PSII centers was reduced, and maximum photochemical yield was lowered; rapidly reversible nonphotochemical quenching was inhibited; and the state transitions were altered kinetically. CP24 is therefore an important factor in determining the structure and function of the PSII light-harvesting antenna, providing the linker for association of the M-trimer into the PSII complex, allowing a specific macroorganization that is necessary both for maximum quantum efficiency and for photoprotective dissipation of excess excitation energy.

Figure 1.

Content of Lhcb6 in Antisense and T-DNA Insertion Mutant Plants. (A) Protein gel blots of wild-type and koLhcb6 plants against antibodies to a range of PSII proteins. The amount of each protein, expressed as percentage of wild type, was determined from densitometry of between three and five replicate blots. Gels were loaded with equal chlorophyll. KO, knockout. (B) Protein gel blots of different plants in the population of the asLhcb6 line against Lhcb6 antibody. Asterisks indicates a wild-type plant included in each gel. (C) Content of Lhcb6 from protein gel blots in the range of antisense plants, expressed as densitometric intensity on an equal chlorophyll basis. Data were obtained from the three gels shown in (B).

Figure 2.

FPLC Gel Filtration Fractionation of Solubilized Wild-Type and Mutant koLhcb6 Thylakoid Membranes. Chromatograms of solubilized thylakoids from the wild type (solid line) and koLhcb6 (dashed line). Major fractions are marked as follows: PSII membrane fragments (I), PSII supercomplexes (II), PSII core complexes (IV), PSI complex (III), and trimeric (V) and monomeric (VI) LHCs. Thylakoids were solubilized with 0.7% α-DM.

Figure 3.

Electron Microscopy of PSII Particles and Grana Membranes. (A) Model of the C₂S₂M₂ supercomplex showing the fitting of the high-resolution structural model of the PSII core dimer, the CP24, CP26, and CP29 minor complexes (pale green), and the M and S LHCIIb trimers (green) (from Dekker and Boekema, 2005). (B) Average projection map of best 500 projections of a set of ∼2000 C₂S₂ supercomplexes from the plants lacking CP24. (C) Average projection map of 10 projections of C₂S₂ supercomplexes lacking one CP26 subunit at the top left tip from the plants lacking CP24. (D) to (F) Projection maps of C₂S₂, C₂S₂M, and C₂S₂M₂ supercomplexes found in wild-type plants (reproduced from Yakushevska et al., 2001) (G) and (H) Partially solubilized grana membranes from plants lacking CP24, showing ordered arrays of C₂S₂ supercomplexes. (I) Partially solubilized grana membranes from wild-type plants, showing ordered arrays of C₂S₂M₂ supercomplexes. Thylakoids were solubilized with 0.6% α-DM. Bars = 10 nm in (B) to (F) and 100 nm in (G) to (I).

Figure 4.

CD spectroscopy of Leaves and Thylakoid Preparations from the Wild Type and Plants Lacking CP24. (A) Wild-type leaves (a), asLhcb6 leaves with different CP24 contents ([b] to [e]), and koLhcb6 leaves (f). (B) Wild-type thylakoids (g), wild-type thylakoids solubilized with 0.01% β-DM (h), asLhcb6 thylakoids (i), and asLhcb6 thylakoids solubilized with 0.01% β-DM (j).

Figure 5.

Relationship between Lhcb6 Protein Content and Spectroscopic Parameters of Thylakoid Structure and Function in asLhcb6 Plants. (A) Amplitude of CD bands at 690 nm (closed circles) and 448 nm (open circles) obtained from the spectra recorded in Figure 4, normalized to the amplitude of the (−)653-nm band and the relative CP24 content, assessed from densitometry of protein gel blots as shown in Figure 1 and normalized to the maximum amount (= 1). (B) Value of reversible NPQ (qE) obtained as in Figure 7 and the CP24 content. (C) Correlation between NPQ obtained as in Figure 7 and amplitude of CD obtained as in Figure 4.

Figure 6.

Low-Temperature Fluorescence Spectroscopy of Thylakoids from Wild-Type and CP24-Depleted Plants. (A) Fluorescence emission spectra excited at 435 nm. (B) Fluorescence excitation spectra for emission at 695 nm. Solid line, wild type; dashed line, koLhcb6. Spectra were normalized to the intensity at 735 nm. In (B), the difference spectrum, wild type-minus-koLhcb6, is displayed (dashed-dotted line).

Figure 7.

Effect of CP24 Depletion on Chlorophyll Fluorescence Quenching. (A) Chlorophyll fluorescence traces recorded for wild-type leaves. (B) Chlorophyll fluorescence traces recorded for asLhcb6 leaves. (C) Chlorophyll fluorescence traces recorded for koLhcb6 leaves. (D) NPQ induction and relaxation calculated from the data in (A) to (C). Wild type, circles; asLhcb6, triangles; and koLhcb6, inverted triangles. Data are means ± se of seven (wild type and asLhcb6) or eight (koLhcb6) replicates from different plants. Actinic light, intensity 800 μmol photons m⁻² s⁻¹, was given for 10 min followed by a 10-min dark period. In (A) to (C), data are normalized to an Fm of 1.0.

Figure 8.

Rapidly Reversible qE Type of NPQ Recorded at Different Actinic Light Intensities. Experiment performed as described in Figure 7. Wild type, circles; asLhcb6, triangles; and koLhcb6, inverted triangles. Data are means ± se of between three and seven replicates using different plants.

Figure 9.

Effect of CP24 Depletion on State Transitions. (A) Wild-type plants. (B) koLhcb6 plants. Dark-adapted leaves were illuminated with PSII light to reach state II. Periods of PSI light were given (upward arrow, on; downward arrow, off). Light saturation pulses were given to record Fm, Fm^I, and Fm^II.

Figure 10.

Model Showing How Lack of CP24 Affects the Macrostructure of the Antenna of PSII and the Regulation of Light Harvesting. In State I, wild-type membranes contain the typical C₂S₂M₂ supercomplexes, which allow formation of large arrays of connected megacomplexes. In the grana of plants lacking CP24, head-to-tail megacomplexes of C₂S₂ supercomplexes are present, together with peripherally associated M-LHCII trimers. These two grana structures differ in their ability to form NPQ. In the transition to State II, there is detachment of M-trimers from PSII and their association with PSI. In the wild type, this results in appearance of some C₂S₂M₁ supercomplexes. In the plant lacking CP24, peripherally associated LHCII can more readily become associated with PSI, whereas in the wild type, the availability of M-trimers is restricted by their location within the supercomplexes. S-LHCII trimers are dark green, M-trimers are olive green, CP26 and CP29 are pale green, and CP24 is turquoise. Reaction center cores are in gray. Also shown are the subunits of LHCI, also in green.