White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase

Abstract

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or "white" mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene.

Figure 1.—

Early steps of carotenoid biosynthesis in plants and algae. Intermediates labeled in chalkboard letters are colorless. Small arrows show the position of newly formed double bonds. GGPP, geranylgeranyl pyrophosphate; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; PQ, oxidized plastoquinone; PQH₂, reduced plastoquinone (plastoquinol).

Figure 2.—

Growth phenotypes. Liquid cultures were spotted onto acetate-containing (TAP) agar medium and incubated in the dark or low light (∼8 μmol photons m⁻² sec⁻¹) for 1 week.

Figure 3.—

Genetic analysis showing 2:2 segregation of the white phenotype. Tetrad analysis of a cross between lts1-203 and a wild-type strain (17D−) is shown. Progeny from eight tetrads were grown in complete darkness on TAP medium. Each vertical column represents a tetrad (labeled a–d).

Figure 4.—

Pigment analysis of wild type and a representative white mutant. Pigments from the wild-type strain 4A+ (A) and lts1-203 (B) were separated by HPLC. Each profile represents pigments extracted from an equal number of cells grown in complete darkness. N, neoxanthin; Lor, loroxanthin; V, violaxanthin; A, antheraxanthin; Lut, lutein; Cb, chlorophyll b; Ca, chlorophyll a; β, β-carotene.

Figure 5.—

(A) Alignment of the amino acid sequences of the PSY protein from Chlamydomonas and selected algal and plant species. The aligned sequences are from another green alga (Dunaliella bardawil; GenBank accession no. U91900), Arabidopsis (Arabidopsis thaliana; GenBank accession no. BT002084), maize (Zea mays; GenBank accession no. AY455286), and a cyanobacterium (Synechocystis sp. 6803; GenBank accession no. X69172). Residues that are identical in at least three of the sequences are shaded in black. Mutation sites in lts1 alleles are indicated with asterisks. The mutation in lts1-202 causes a frameshift in the coding region, and the mutation in lts1-208 alters a splice site in the primary transcript. (B) Structure of the Chlamydomonas PSY gene and the positions of point mutations. The gene contains five exons (solid rectangles) and four introns (open rectangles). The 3′-untranslated region is indicated by a thinner box (the 5′-untranslated region is not yet determined). The positions of 10 mutant alleles are indicated by arrows.

Figure 6.—

Cosegregation of lts1 with a marker located in the PSY locus. Progeny from eight tetrads were obtained from a cross between lts1-203 and a polymorphic wild-type strain (S1D2), and a single-nucleotide polymorphism marker located in the fourth intron of the PSY gene was scored. A 891-bp fragment of the PSY gene was amplified from progeny, digested with HincII, and subjected to agarose gel electrophoresis. Both parental strains and progeny (labeled a–d) of four tetrads are shown. The pigmentation phenotype of each progeny is indicated by the letters G (green) or W (white).