Zeaxanthin accumulation in the absence of a functional xanthophyll cycle protects Chlamydomonas reinhardtii from photooxidative stress

Abstract

Xanthophylls participate in light harvesting and are essential in protecting the chloroplast from photooxidative damage. To investigate the roles of xanthophylls in photoprotection, we isolated and characterized extragenic suppressors of the npq1 lor1 double mutant of Chlamydomonas reinhardtii, which lacks zeaxanthin and lutein and undergoes irreversible photooxidative bleaching and cell death at moderate to high light intensities. Here, we describe three suppressor strains that carry point mutations in the coding sequence of the zeaxanthin epoxidase gene, resulting in the constitutive accumulation of zeaxanthin in a range of concentrations. The presence of zeaxanthin in these strains was sufficient to prevent photooxidative damage in the npq1 lor1 background. The size of the light-harvesting antenna in the suppressors decreased in high light in a manner that was proportional to the relative content of zeaxanthin, with the strain having the most zeaxanthin showing a severe reduction in levels of the major light-harvesting complex II proteins in high light. We show that the effect of constitutive zeaxanthin on light harvesting is not the main cause of increased photoprotection, because in the absence of zeaxanthin, a strain with a smaller light-harvesting antenna showed only minor protection against photobleaching in high light. Furthermore, the zeaxanthin-accumulating suppressors were able to tolerate higher levels of exogenous reactive oxygen than their parental strain under conditions that did not affect light harvesting. Our results are consistent with an antioxidant role of zeaxanthin in the quenching of singlet oxygen and/or free radicals in the thylakoid membrane in vivo.

Figure 1.

The Xanthophyll Biosynthetic Pathway in Chlamydomonas. The reactions impaired in the npq1, npq2, and lor1 mutants are indicated.

Figure 2.

Growth of Chlamydomonas Strains in Minimal Medium under Continuous Light. (A) Cells grown in LL (50 μmol·m⁻²·s⁻¹) for 7 days. (B) Cells grown in HL (500 μmol·m⁻²·s⁻¹) for 7 days.

Figure 3.

Pigment Characteristics of Chlamydomonas Strains. (A) Relative content of xanthophyll-cycle pigments. (B) Deepoxidation state, (Z+A)/(V+A+Z). (C) Relative content of β-carotene. Cells were grown on plates in minimal medium under either continuous LL or HL. Values shown are means of three to five independent experiments. In (A), the standard deviation was ≤15% of the means. In (B) and (C), the error bars represent standard deviations.

Figure 4.

Genetic Analysis of a Suppressor of the HL Sensitivity of npq1 lor1. An example of tetrad analysis from a backcross between the parental strain npq1 lor1 arg7-1 mt+ and npq2-3 npq1 lor1 arg7-8 mt− (sup). Progeny from four tetrads were grown under continuous LL (50 μmol·m⁻²·s⁻¹) or HL (500 μmol·m⁻²·s⁻¹) in minimal medium for 7 days. Each vertical column represents a tetrad (labeled a–d). Both parental strains grown under the same conditions are shown as controls.

Figure 5.

Alignment of the Deduced Amino Acid Sequences of Zeaxanthin Epoxidase Precursor Proteins from Chlamydomonas and Selected Plant Species. Residues that are identical in at least three of the sequences are shaded in black. The long monooxygenase domain, which contains the putative ADP and flavin adenine dinucleotide binding sites, is shown with a solid underline, the two lipocalin conserved motifs are shown with a dotted underline, and the FHA (Forkhead-associated) domain is shown with a dashed underline. The mutation sites in the npq2-3 and npq2-4 alleles are indicated with asterisks. The mutation in npq2-2 causes a change in the translation initiation codon.

Figure 6.

DNA Gel Blot Analysis of Wild-Type Chlamydomonas, the npq1 lor1 Mutant, and Strains Carrying Mutations in the NPQ2 Gene. Genomic DNA was digested with restriction enzymes and probed with a 1.8-kb fragment corresponding to the 5′ portion of the zeaxanthin epoxidase cDNA. (A) DNA digested with PstI. (B) DNA digested with BamHI and SpeI.

Figure 7.

Structure of the Chlamydomonas Zeaxanthin Epoxidase Gene and Positions of Point Mutations. (A) Scheme of the zeaxanthin epoxidase locus in Chlamydomonas. The gene has 10 exons (indicated in black) and 9 introns (indicated in white). The 5′ and 3′ untranslated regions are indicated by the thinner boxes. The positions of the three point mutations in the npq2 suppressors are indicated by arrows. (B) Sequence of the zeaxanthin epoxidase gene in the regions surrounding the point mutations showing the amino acid changes.

Figure 8.

Lipid Peroxidation in Suppressors of npq1 lor1 after Transfer to High Light. Exponentially growing liquid cultures were transferred from LL to HL conditions at time 0. Lipid peroxides were measured as thiobarbituric acid–reactive substances (TBARS). The results are shown relative to the initial culture content of TBARS, which was similar in all strains tested. Data are from three independent cultures. Error bars represent standard deviations and are not shown if smaller than the symbols.

Figure 9.

Protein Analysis of Chlamydomonas Whole Cell Extracts. Cells were grown for several generations under continuous LL or HL conditions, except for mutants npq1 lor1 and cbn1 npq1 lor1, which were grown only in LL. Lanes were loaded with an equal number of cells. (A) Immunoblot analysis of the LHC of PSII. The major LHC polypeptides of PSII (LHCII) were visualized with the anti-P17 antibody (Bassi and Wollman, 1991). (B) Coomassie blue staining of a protein gel identical to the one used in (A) for immunoblot analysis.

Figure 10.

Effect of Light-Harvesting Antenna Size on the HL Sensitivity of npq1 lor1. The cbn1 mutation, which causes an impairment in Chl b biosynthesis, was introduced in the npq1 lor1 genetic background, and the resulting strain was tested for survival in HL. The wild-type and npq1 lor1 strains shown here are the same strains shown in Figure 1 and are included for comparison. (A) Cells grown in minimal medium under continuous LL (50 μmol· m⁻²·s⁻¹). (B) Cells grown in minimal medium under continuous HL (500 μmol· m⁻²·s⁻¹).

Figure 11.

Response of Wild-Type and Mutant Strains of Chlamydomonas to Exogenously Generated Singlet Oxygen. (A) Cells grown in minimal medium under continuous LL. (B) Cells grown in minimal medium containing 3.8 μM rose bengal under continuous LL. Because of strain variability in the response to rose bengal, each of the zeaxanthin-accumulating suppressors is shown with the corresponding parental strain as a control. Strain npq1 lor1 arg7-8 is the parent of the suppressor carrying the npq2-2 allele, and strain npq1 lor1 arg7-1 is the parent of the suppressors carrying the npq2-3 and npq2-4 alleles. Each row represents a 10-fold serial dilution of cells at the time of plating. The total number of cells plated is indicated at left.