A major light-harvesting polypeptide of photosystem II functions in thermal dissipation

Abstract

Under high-light conditions, photoprotective mechanisms minimize the damaging effects of excess light. A primary photoprotective mechanism is thermal dissipation of excess excitation energy within the light-harvesting complex of photosystem II (LHCII). Although roles for both carotenoids and specific polypeptides in thermal dissipation have been reported, neither the site nor the mechanism of this process has been defined precisely. Here, we describe the physiological and molecular characteristics of the Chlamydomonas reinhardtii npq5 mutant, a strain that exhibits little thermal dissipation. This strain is normal for state transition, high light-induced violaxanthin deepoxidation, and low light growth, but it is more sensitive to photoinhibition than the wild type. Furthermore, both pigment data and measurements of photosynthesis suggest that the photosystem II antenna in the npq5 mutant has one-third fewer light-harvesting trimers than do wild-type cells. The npq5 mutant is null for a gene designated Lhcbm1, which encodes a light-harvesting polypeptide present in the trimers of the photosystem II antennae. Based on sequence data, the Lhcbm1 gene is 1 of 10 genes that encode the major LHCII polypeptides in Chlamydomonas. Amino acid alignments demonstrate that these predicted polypeptides display a high degree of sequence identity but maintain specific differences in their N-terminal regions. Both physiological and molecular characterization of the npq5 mutant suggest that most thermal dissipation within LHCII of Chlamydomonas is dependent on the peripherally associated trimeric LHC polypeptides.

Figure 1.

Modulated Fluorescence and NPQ in Wild-Type and npq5 Mutant Cells. (A) Chlorophyll fluorescence during induction and relaxation of NPQ. (B) NPQ calculated from fluorescence as (F_m − F_m′)/F_m′. (C) Relaxation of NPQ by the addition of nigericin. (D) Light intensity dependence of NPQ. The curves for wild-type cells and the npq5 mutant are shown in black and gray, respectively. The data shown in (B) to (D) are average values of experiments obtained with four separately grown wild-type and mutant cultures. The wild-type cultures were at cell concentrations between 1.6 × 10⁶ and 1.8 × 10⁶ cells/mL [2.8 to 3.2 μg chlorophyll (a + b)/mL], and the mutant cultures were at cell concentrations between 1.7 × 10⁶ and 1.9 × 10⁶ cells/mL [2.4 to 2.7 μg chlorophyll (a + b)/mL]. For (A) and (B), white bars above the graphs indicate illumination with 350 μmol·m⁻²·s⁻¹, and black bars indicate illumination with ≈1 μmol·m⁻²·s⁻¹ far-red light (690 to 710 nm). For (C), the arrowhead at top indicates the time at which nigericin was added. For (D), the level of NPQ attained after 10 min at each light intensity is shown. For (B) to (D), se values are <3%; representative traces are shown in (A).

Figure 2.

Deepoxidation after Exposure of Cells to 1100 μmol· m⁻²·s⁻¹. Deepoxidation state (DPS) was calculated as (zeaxanthin + antheraxanthin)/(zeaxanthin + antheraxanthin + violaxanthin). The wild-type culture used was at a concentration of 1.65 × 10⁶ cells/mL [2.82 μg chlorophyll (a + b)/mL], and the npq5 mutant culture was at 1.7 × 10⁶ cells/mL [2.48 μg chlorophyll (a + b)/mL].

Figure 3.

77K Fluorescence Emission Spectra of Wild-Type and npq5 Mutant Cells Treated with Far-Red Light, Far-Red Light followed by High-Light, or Far-Red Light, High Light, and Then Far-Red Light. (A) Wild type (WT). (B) npq5. Samples were excited at 435 nm, and fluorescence emission was measured between 650 and 750 nm. The wild-type culture used was at a concentration of 1.75 × 10⁶ cells/mL [2.95 μg chlorophyll (a + b)/mL], and the npq5 mutant culture was at 1.83 × 10⁶ cells/mL [2.55 μg chlorophyll (a + b)/mL]. Spectra were measured two times and yielded essentially identical data, and data from one of the two replicates is shown. FR, far-red light; HL, far-red light followed by high light; Rec, far-red light, high light, and then far-red light.

Figure 4.

Photoinhibition in the Wild-Type and npq5 Strains. Photosynthetic efficiency (F_v/F_m) was measured after 10 and 20 min of high-light exposure (1100 μmol·m⁻²·s⁻¹) in the presence of lincomycin. Before measuring F_v/F_m, the cultures were exposed to low-fluence far-red light for 4 min. To calculate the half-time of photosynthetic efficiency in high light, linear trend lines were added (R² > 0.96 for both). The half-time of efficiency for the wild-type and npq5 strains were 14.1 ± 1.1 min and 12.2 ± 0.4 min, respectively. Points are means ± se (n = 4). The wild-type cultures used were at cell concentrations between 1.5 × 10⁶ and 1.7 × 10⁶ cells/mL [2.7 to 3.1 μg chlorophyll (a + b)/mL], and the npq5 mutant cultures were at cell concentrations between 1.6 × 10⁶ and 1.8 × 10⁶ cells/mL [2.3 and 2.6 μg chlorophyll (a + b)/mL].

Figure 5.

Scheme of the Lhcbm1 Locus in the Wild-Type and npq5 Mutant Strains. Lhcbm1 has five exons and four introns. In the npq5 mutant, an incomplete pJD67 plasmid was inserted in the first exon of the gene, generating a 320-nucleotide deletion at the site of insertion. Black boxes represent exons, and the gray box represents genomic DNA within the Lhcbm1 gene of npq5 that was deleted during the insertion of pJD67.

Figure 6.

Alignment of the Amino Acid Sequences of the Chlamydomonas Lhcbm Polypeptides and Lhcb2.1 from Pea (Ps). Residues identical to the consensus sequence are shown in black, and stars mark potential Thr phosphorylation sites. (A) Predicted transit peptides. (B) Predicted mature proteins.

Figure 7.

Transformation with the Genomic Lhcbm1 Sequence Complements the Mutant Phenotype in npq5. (A) Scheme of the constructs used for complementation, and complementation frequencies. (B) False-color image of NPQ in the wild-type strain, the npq5 mutant strain, and a complemented strain. (C) Levels of Lhcbm1 RNA expression in strains used in (B). The CBLP transcript level was used as a loading control.