Figure 1.
Carotenoid biosynthesis in plants and green algae.
Geranylgeranyl pyrophosphate, phytoene, and phytofluene are all colorless compounds. Colored carotenoids include ζ-carotene and all carotenoids downstream. Xanthophylls include zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, lutein, and loroxanthin (found in C. reinhardtii).
Figure 2.
Phenotype of wild-type C. reinhardtii cells grown norflurazon.
A). Growth of wild-type C. reinhardtii cells on 0, 5, and 10 µM norflurazon (NF) in LL (100 µMol photons m−2 sec−1) or in the dark. B). Overlay of HPLC results of carotenoid and chlorophyll pigments detected in dark-grown wild-type cells treated with 0 µM (solid lines), 5 µM (dashed lines), and 10 µM (dotted lines) NF. N+Lor (neoxanthin+loroxanthin); V (violaxanthin); A (antheraxanthin); L (Lutein); Chl a and Chl b (chlorophyll a and b); α-, ß- (α- and ß-carotenes); P (phytoene). Inset shows absorbance spectrum of phytoene peak at 296 nm.
Figure 3.
Light sensitivity of wild-type, lts1 and pds1 mutants.
Cells were spotted onto TAP-agar and grown for 5 days in the dark before being exposed to light. All cells were grown for a total of 19 days. WT (wild-type), lts1-210 (null psy), pds1-1 (leaky pds1), P3-84 (lts1-301 pds1-2), pds1-2, lts1-301 (leaky psy), and pds1-3 (null pds1) are in the left column. In the right column are intragenic suppressors of pds1-2 mutants (pds1-4, pds1-5, pds1-6), all in the lts1-301 genetic background. Light intensities: Dk (dark), vLL (10 µMol photons m−2 sec−1), LL (100 µMol photons m−2 sec−1), HL (500 µMol photons m−2 sec−1).
Figure 4.
Chlorophyll and carotenoid profiles of PDS-activity deficient mutants.
Chlorophylls and carotenoids were detected at 445 nm and phytoene was detected at 296 nm. Absorbance spectra are shown for the 296 nm phytoene peak present in both pds1-1 and pds1-3 mutants and small peak detected in P3-84. Pigments were extracted from a total of 1×108 cells for each sample and analyzed via HPLC coupled with a diode array detector. N+Lor (neoxanthin+loroxanthin); V (violaxanthin); A (antheraxanthin); L (lutein); Z (zeaxanthin); Chl a and Chl b (chlorophyll a and b); α-, ß- (α- and ß-carotenes); P (phytoene).
Table 1.
Quantification of chlorophyll and carotenoid content of dark-grown lts1 and pds1 mutants.
Table 2.
Tetrad analysis of pds1-1 and pds1-3 crossed to wild-type.
Figure 5.
The PDS gene is genetically linked to the pds1-1 mutant phenotype.
A marker located within the PDS locus cosegregated with the light green, phytoene-accumulating mutant phenotype of pds1-1. Amplification and ScrFI digestion of a 268 bp fragment of the PDS gene containing a single nucleotide polymorphism in exon 2 was used to score progeny from crosses between pds1-1 and a polymorphic wild-type strain (S1D2). Seven full and partial tetrads were scored: individual progeny within tetrads are labeled “a, b, c, d”. Solid circles indicate dark green progeny with wild-type carotenoid composition while open circles indicate light green progeny with phytoene accumulation.
Figure 6.
Multiple sequence alignment of PDS protein sequences.
Alignment of PDS amino acid sequences from eukaryotic green algae, Chlamydomonas and Ostreococcus; a plant, Arabidopsis; and a cyanobacterium, Synechocystis sp. PCC6803. Conserved residues were scored using Blosum 62 matrix, with darker shading indicating higher conservation and no shading low conservation. Asterisks mark positions of mutations in pds1 alleles.
Figure 7.
Analysis of enhancer strain P3-84 (lts1-301 pds1-2) and intragenic suppressors of pds1-2 mutants.
A). Color and pigment phenotype of dark-grown wild type (WT), P3-84, and pds1-1 mutants. (−) indicates no accumulation while (+) indicates presence of phytoene or colored carotenoids. Colored carotenoids include all carotenoids downstream of phytofluene. B). Tetratype tetrad phenotype from crosses between wild-type and P3-84 cells. The presence (+) or absence (−) of phytoene is indicated for each progeny along with their corresponding genotypes. C). Structure of the PSY gene in C. reinhardtii. UTRs are indicated by solid boxes, exons by open boxes and four introns by lines. The bracket highlights the eight amino acids and their corresponding nucleotides deleted from the putative chloroplast transit peptide in lts1-301, P3-84, and in pds1-2 suppressor mutants. Subscript numbers note position of the amino acid residue in the wild-type PSY protein. D). Structure of the PDS gene in C. reinhardtii. UTRs are indicated by solid boxes, exons by open boxes and five introns by lines. The brackets highlight the two missense mutations found in P3-84. Subscript numbers note position of the amino acid residue in the wild-type PDS protein. Asterisks mark approximate location of mutations.
Table 3.
Summary of mutants described in this work.
Figure 8.
Analysis of pds1-3 DNA insertional mutant.
A). Schematic of the C. reinhardtii PDS gene showing DNA insertion location (triangle), region amplified in flanking sequence tag (striped bar), and region of transcript not detectable by RT-PCR in pds1-3 (gray bar). UTRs are indicated by black bars and exons by open bars. Genomic DNA spanning the 5′ UTR to the 4th exon could be amplified by PCR in pds1-3. B). No amplification in wild-type (WT) and amplification in pds1-3 with three vector-specific primers (1, 2, 3) and one primer in PDS genomic DNA (4), indicated by arrows in panel A, confirming insert location in pds1-3. C). Successful amplification of genomic DNA and DNA sequencing of PDS on the opposite side of insertion from flanking sequence tag in wild-type and pds1-3. Amplification products were obtained from genomic DNA 2.5 kb (primers MS031A and MS031B, in exon 1) and 500 bp (primers MS041A and MS041B, in exon 4) distant from insertion site. D). Amplification of PDS transcript (gray bar in panel A) from total RNA in wild-type (1), pds1-3 (2), and pds1-1 (3), with the amplification of tubulin as a positive control. E). Relative PDS transcript levels in wild-type cells (light gray bar), pds1-1 (dark gray bar ), and pds1-3 (black bar). Relative quantification (RQ) fold-change values to the calibrator (WT PDS transcript levels) are shown.
Figure 9.
Results of plating assays of wild-type, lts1-210, pds1-3, and pds1-1.
A). Growth rate per day for wild-type, lts1-210 (null psy), pds1-3, and pds1-1. Biological triplicates of each strain were grown in the dark in liquid TAP on a shaker. *Significantly differently from lts1-210 and wild-type values under the same conditions using a two-tailed t test (P<0.05). B). Percent of cells that survived plating with glass beads (% survival ± standard deviation) after 12 days of growth in the dark. Scale bar represents 5 mm. C). ∼1∶1 ratio of carotenoid mutant to wild-type cells after 12 days of growth in the dark. After accounting for plating efficiencies, the expected CFU/plate was 1250 CFU/plate for pds1 and 1320 CFU/plate for wild-type and lts1-210 strains. Scale bar represents 5 mm.
Figure 10.
Analysis of intragenic suppressors of pds1-2 mutants.
A). Schematic depiction of pds1-1, P3-84, pds1-2, and pds1-2 suppressors (pds1-4, pds1-5, and pds1-6). Cartoon of the C. reinhardtii PDS gene showing only exons one and two. Jagged lines indicate a partial depiction of the PDS gene. UTRs are indicated by solid boxes, exons by open boxes and introns by lines. Asterisks mark positions of mutations in pds1-1, pds1-2, P3-84 and pds1-2 suppressor mutants. Black and blue asterisks represent mutations L64P and L64F, respectively. Orange asterisk in exon 1 signifies location of the K90M mutation, and red asterisks in exon 2 signify the E143K mutation. B). 3DLigandSite structural prediction of the C. reinhardtii PDS protein showing positions of amino acid residues mutated in pds1-1 and pds1-6 mutants, E143K and K90M, respectively. L64P and L64F were not mapped because the first 71 amino acids of the N-terminus had no structural prediction. Ligand and wild-type amino acids corresponding to mutated residues were colored as follows: position 90 (spacefilling, orange); position 143 (spacefilling, red); ligand [NAD(P)/FAD] (cyan); predicted ligand binding sites (indigo); start of predicted N-terminus (amino acid residue 72, white); and carotenoid binding site proposed by Armstrong et al. (amino acid residues 492–517, lavender). Three different perspectives of the predicted structure are shown.