Academic journal article Genetics

White Mutants of Chlamydomonas Reinhardtii Are Defective in Phytoene Synthase

Academic journal article Genetics

White Mutants of Chlamydomonas Reinhardtii Are Defective in Phytoene Synthase

Article excerpt

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.

CAROTENOIDS are a diverse group of C^sub 40^ tetraterpene pigments that are important in many biochemical and biophysical processes of plants and algae (Cunningham and Gantt 1998). Carotenoids are essential for the structure and function of pigment-binding protein complexes and the prevention of photooxidative damage. In the chloroplast, the majority of carotenoids are located in pigment-binding proteins embedded in the thylakoid membrane. Here, carotenoids provide structural support to their associated proteins, participate in light-harvesting processes, absorbing at 450-570 nm, and dissipate excess light energy absorbed by antenna pigments (HERRIN et al. 1992; DEMMIG-ADAMS et al. 1996; BAROLI and NIYOGI 2000). Carotenoids associated with reaction centers and antenna complexes play a critical role in protection of the photosynthetic apparatus from photooxidative damage by quenching triplet chlorophyll and singlet oxygen (BRITTON 1995; DEMMIG-ADAMS et al. 1996; FRANK and COGDELL 1996). In addition, carotis enoids are precursors of the plant growth hormone abscisic acid (DUCKHAM et al. 1991; ROCK and ZEEVAART 1991) and are the pigments responsible for the yellow, orange, and red coloration of many flowers, fruits, vegetables, and roots.

In plants and algae, carotenoids are synthesized via the biosynthetic pathway shown in Figure 1 (CUNNINGHAM and GANTT 1998). The formation of the colorless carotenoid phytoene from the condensation of two molecules of geranylgeranylpyrophosphate (GGPP) is the first committed step in the pathway and is catalyzed by the enzyme phytoene synthase (PSY). The next step, the conversion of phytoene to the first colored carotenoid in the pathway, ζ-carotene, by the enzyme phytoene desaturase (PDS), is a rate-limiting step of carotenogenesis (CHAMOVITZ et al. 1993). PDS activity is inhibited by the bleaching herbicide norflurazon (SANDMANN et al. 1989). ζ-carotene desaturase (ZDS), an enzyme related to PDS (ALBRECHT et al. 1995), converts ζ-carotene to lycopene. The electron carrier plastoquinone has been identified as an essential component for phytoene desaturation in higher plants (NORRIS et al. 1995). In addition, a recently identified carotenoid isomerase is necessary to generate all-trans lycopene (ISAACSON et al. 2002; PARK et al. 2002), which carotis the actual substrate for cyclization reactions leading to β-carotene, ζ-carotene, and xanthophylls. A mutant blocked in PSY, PDS, or plastoquinone biosynthesis would be expected to exhibit altered pigmentation, including a lack of colored carotenoids and reduced levels of chlorophylls.

Four Chlamydomonas reinhardtii mutants lacking carotenoids have been described previously: lts1-30 (CHEMERILOVA 1978), fn68 (FOSTER et al. …

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