Genetic and Biochemical Characterization of Early Carotenoid Biosynthesis Mutants of Chlamydomonas reinhardtii

Carotenoids are isoprenoid pigment molecules with a long hydrocarbon skeleton distinguished by its system of conjugated double bonds. They are essential to photosynthetic organisms because of their ability to prevent photo-oxidative damage, and they also harvest light energy for photosynthesis. Chlamydomonas reinhardtii carotenoid-deficient mutants were generated using UV and DNA insertional mutagenesis and screened for pigment accumulation. Mutants that accumulate phytoene, zeta-carotene, or prolycopene were isolated and found to be genetically linked to the predicted phytoene desaturase ( PDS1 ), zeta-carotene desaturase ( ZDS1 ), and carotene isomerase ( CRTISO1 ) genes, respectively. The first C. reinhardtii pds1 mutant isolated, pds1-1 , is a weak allele for phytoene desaturation; it is light-green and accumulates phytoene and downstream colored carotenoids. Two white mutants that appear to be null alleles of PDS1 , pds1-2 and pds1-3 , were identified by their phytoene accumulation and complete lack of colored carotenoids. Compared to white psy mutants, which also lack all colored carotenoids, phytoene-accumulating mutants exhibited slower growth rates in the dark and reduced plating efficiencies, indicating that phytoene accumulation may be deleterious. Three classes of pds1-2 suppressor mutants were identified that synthesized elevated levels of colored carotenoids and were less light sensitive than their parental strain. The pds1-2 suppressor mutants had mutations affecting amino acids at positions 64, 90, and 143 in the predicted PDS protein: pds1-4 (L64P), pds1-5 (L64F and E143K), and pds1-6 (L64P, K90M, E143K). Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 64 and 90 help maintain the structural stability and/or activity of the PDS enzyme.Dark-grown C. reinhardtii zds1 and crtiso1 mutants are yellow in the dark and accumulate zeta-carotenes or prolycopene, respectively, but with gradual illumination they can turn green and accumulate higher levels of xanthophylls. In the case of crtiso1 , dark-accumulated prolycopene can be photoisomerized and efficiently converted to downstream photoprotective carotenoids in the light. The presence of xanthophylls in dark-grown zds1 and crtiso1 null mutants provides evidence for an alternative carotene desaturase in C. reinhardtii . A suppressor of the zds1 mutant, zSup63, with a dominant very-green-in-low-light phenotype was isolated. Transcript levels for a putative protein (protein ID 516552) with homology to plant and algal CRTISO proteins were elevated at least ten-fold in light-grown zSup63 cells compared to the parental zds1 strain. A low light-sensitive, dark-green DNA insertional mutant (CAL028.02.09) with a disruption in the gene encoding protein 516552 has been identified and analysis of double mutants will determine if this protein is responsible for xanthophyll synthesis in dark-grown zds1 and crtiso1 mutants. The collection of early carotenoid biosynthetic mutants reported here, which accumulate cis -configured carotenes, conclusively demonstrate that carotenoids in C. reinhardtii are primarily synthesized by a plant-type poly-cis carotene desaturation pathway, making C. reinhardtii ideal for investigating carotenoid biosynthesis and regulation (and the processes that intersect with them).