Light sensitivity of wild-type, <i>lts1</i> and <i>pds1</i> mutants.

<p>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), <i>lts1-210</i> (null <i>psy</i>), <i>pds1-1</i> (leaky <i>pds1</i>), P3-84 (<i>lts1-301 pds1-2</i>), <i>pds1-2</i>, <i>lts1-301</i> (leaky <i>psy</i>), and <i>pds1-3</i> (null <i>pds1</i>) are in the left column. In the right column are intragenic suppressors of <i>pds1-2</i> mutants (<i>pds1-4</i>, <i>pds1-5</i>, <i>pds1-6</i>), all in the <i>lts1-301</i> 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).</p

Summary of mutants described in this work.

<p>Summary of mutants described in this work.</p

Multiple sequence alignment of PDS protein sequences.

<p>Alignment of PDS amino acid sequences from eukaryotic green algae, <i>Chlamydomonas</i> and <i>Ostreococcus</i>; a plant, <i>Arabidopsis</i>; and a cyanobacterium, <i>Synechocystis sp.</i> 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 <i>pds1</i> alleles.</p

Carotenoid biosynthesis in plants and green algae.

<p>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 <i>C. reinhardtii</i>).</p

Chlorophyll and carotenoid profiles of PDS-activity deficient mutants.

<p>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 <i>pds1-1</i> and <i>pds1-3</i> 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 <i>a</i> and Chl <i>b</i> (chlorophyll <i>a</i> and <i>b</i>); α-, ß- (α- and ß-carotenes); P (phytoene).</p

Analysis of enhancer strain P3-84 (<i>lts1-301 pds1-2</i>) and intragenic suppressors of <i>pds1-2</i> mutants.

<p>A). Color and pigment phenotype of dark-grown wild type (WT), P3-84, and <i>pds1-1</i> 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 <i>PSY</i> gene in <i>C. reinhardtii</i>. 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 <i>lts1-301</i>, P3-84, and in <i>pds1-2</i> suppressor mutants. Subscript numbers note position of the amino acid residue in the wild-type PSY protein. D). Structure of the <i>PDS</i> gene in <i>C. reinhardtii</i>. 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.</p

The <i>PDS</i> gene is genetically linked to the <i>pds1-1</i> mutant phenotype.

<p>A marker located within the <i>PDS</i> locus cosegregated with the light green, phytoene-accumulating mutant phenotype of <i>pds1-1</i>. Amplification and <i>Scr</i>FI digestion of a 268 bp fragment of the <i>PDS</i> gene containing a single nucleotide polymorphism in exon 2 was used to score progeny from crosses between <i>pds1-1</i> 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.</p

Quantification of chlorophyll and carotenoid content of dark-grown <i>lts1</i> and <i>pds1</i> mutants.

<p>Chlorophyll (Chl) and carotenoid quantities (represented as fmol/cell) were extracted from a total of 1×10⁸ cells for each sample with 200 µl of acetone. Colored carotenoids detected in <i>C. reinhardtii</i> include α-carotene, β-carotene, lutein, violaxanthin, antheraxanthin, neoxanthin, loroxanthin, zeaxanthin. Total xanthophylls include lutein, loroxanthin, violaxanthin, antheraxanthin, neoxanthin, and zeaxanthin. Averages and standard deviations are from three independent cultures.</p

Phenotype of wild-type <i>C. reinhardtii</i> cells grown norflurazon.

<p>A). Growth of wild-type <i>C. reinhardtii</i> 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 <i>a</i> and Chl <i>b</i> (chlorophyll <i>a</i> and <i>b</i>); α-, ß- (α- and ß-carotenes); P (phytoene). Inset shows absorbance spectrum of phytoene peak at 296 nm.</p

Analysis of <i>pds1</i>-3 DNA insertional mutant.

<p>A). Schematic of the <i>C. reinhardtii PDS</i> gene showing DNA insertion location (triangle), region amplified in flanking sequence tag (striped bar), and region of transcript not detectable by RT-PCR in <i>pds1-3</i> (gray bar). UTRs are indicated by black bars and exons by open bars. Genomic DNA spanning the 5′ UTR to the 4^th exon could be amplified by PCR in <i>pds1-3</i>. B). No amplification in wild-type (WT) and amplification in <i>pds1-3</i> with three vector-specific primers (1, 2, 3) and one primer in <i>PDS</i> genomic DNA (4), indicated by arrows in panel A, confirming insert location in <i>pds1-3</i>. C). Successful amplification of genomic DNA and DNA sequencing of <i>PDS</i> on the opposite side of insertion from flanking sequence tag in wild-type and <i>pds1-3</i>. 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 <i>PDS</i> transcript (gray bar in panel A) from total RNA in wild-type (1), <i>pds1-3</i> (2), and <i>pds1-1</i> (3), with the amplification of tubulin as a positive control. E). Relative <i>PDS</i> transcript levels in wild-type cells (light gray bar), <i>pds1-1</i> (dark gray bar ), and <i>pds1-3</i> (black bar). Relative quantification (RQ) fold-change values to the calibrator (WT <i>PDS</i> transcript levels) are shown.</p