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Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102
1 Corresponding author: Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720-3102.
E-mail: niyogi{at}nature.berkeley.edu
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ABSTRACT |
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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, 
-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). -carotene desaturase (ZDS), an enzyme related to PDS (ALBRECHT et al. 1995), converts -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 is the actual substrate for cyclization reactions leading to ß-carotene, 
-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.
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Although mutants lacking carotenoids have been used in numerous studies of Chlamydomonas biology, the biochemical and molecular basis for their phenotype has not been characterized. In this work, a collection of UV light-induced and chemically induced Chlamydomonas mutants that lack carotenoids, designated "white" because of their colorless appearance, were characterized for their growth phenotypes under the dark and various light conditions, pigment and tocopherol composition, and the genetic basis for their white mutant phenotype. Included in the collection are lts1-30, fn68, and w7, in addition to eight newly isolated mutants (Table 1).
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MATERIALS AND METHODS |
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Mutagenesis:
Liquid cultures of the wild-type strain were grown to a density of 
5 x 106 cells ml–1. For chemical mutagenesis, cells were washed with and resuspended in 70 mM potassium phosphate (pH 6.9) at a concentration of 5 x 107 cells ml–1. Cells were incubated in 0.23, 0.27, or 0.30 M EMS (diluted with 70 mM potassium phosphate) in the dark at room temperature for 60 min with constant shaking. Samples were then washed once with 5% (w/v) sodium thiosulfate, twice with 70 mM potassium phosphate, and once with liquid TAP. For UV mutagenesis, 20 ml of cells (5 x 106 cells ml–1) in a 14-cm-diameter glass petri dish bottom were exposed to UV light ranging from 4 to 6 x 104 µJ cm–2 (Stratalinker; Stratagene, La Jolla, CA) and then incubated in the dark overnight with constant shaking. A series of dilutions of mutagenized cells was plated onto TAP agar medium, and the plates were kept in complete darkness until visible colonies formed (2 weeks). Colonies with a visible alteration in color were picked and restreaked, and single white mutant colonies were selected for further analysis.
Pigment and tocopherol analysis:
Liquid cultures were grown to a density of 5 x 106 cells ml–1 in complete darkness. Normalized quantities of cells were harvested by centrifugation at 20,000 x g for 5 min and the resulting pellet was extracted with 100 µl of acetone by vortexing for 30 sec. The extract was centrifuged at 20,000 x g for 1 min, and the supernatant was filtered (0.45-µm nylon filter) and saved. The pellet was extracted with a second 100 µl of acetone. The two extracts were pooled and immediately transferred to the dark. Twenty-five microliters of the filtered extract was subjected to HPLC and separated on a Spherisorb S5 ODS1 4.6- x 250-mm cartridge column (Waters, Milford, MA) at 30° using a method described previously (MüLLER-MOULé et al. 2003). Pigments were detected by absorbance at 445 nm (550 nm reference) by a diode array detector. Tocopherols were detected via fluorescence with excitation at 295 nm and emission at 325 nm. The concentrations of chlorophyll a and -tocopherol were determined using standard curves of the purified compounds at known concentrations.
Genetic analysis:
Genetic crosses and tetrad analysis were performed according to established methods (HARRIS 1989). In crosses of white mutants to one another, homozygous white mutant zygospores were exposed to very low light (8 µmol photons m–2 sec–1) for 1–3 hr (instead of overnight) to initiate germination. The allelic arg7-1 and arg7-8 mutations exhibit intragenic complementation, allowing the selection of vegetative diploid strains on TAP medium lacking arginine in low light (80 µmol photons m–2 sec–1) for dominance tests (HARRIS 1989). For cosegregation analysis, an 891-bp DNA fragment of the PSY gene was amplified from progeny of crosses to the polymorphic wild-type strain S1D2 (mt–; GROSS et al. 1988) using the primers PSYF2-2 (5'-AGGTCTGTGGTCCAACTGCT-3') and PSYR2 (5'-GTGTCAGAAGGCCACCAAAAC-3'), and the PCR products were digested with HincII overnight.
Isolation of nucleic acids and DNA sequencing:
Nucleic acids were isolated from cells grown on TAP agar medium by lysing the cells in SDS-EB buffer containing 1% (w/v) SDS, 200 mM NaCl, 20 mM EDTA, and 50 mM Tris-HCl, pH 8.0. The solution was extracted twice with phenol:chlorofom:isoamyl alcohol (25:24:1) and once with chlorofom:isoamyl alcohol (24:1), and the DNA was precipitated with ethanol. Putative Chlamydomonas PSY and PDS cDNAs were identified by database searching of expressed sequence tags (ESTs; ASAMIZU et al. 2000). The cDNAs were obtained from the Kazusa DNA Research Institute (Kisarazu, Chiba, Japan) and completely sequenced. The cDNA sequences were used to identify the corresponding PSY and PDS genes in the Chlamydomonas nuclear genome sequence (version 1.0) on scaffolds 682 and 104, respectively (http://genome.jgi-psf.org/chlre1/chlre1.home.html). To sequence the PSY gene from the Chlamydomonas wild-type and mutant alleles, a series of synthetic primers was designed on the basis of both genomic and cDNA sequences. A 2576-bp fragment containing the PSY coding region was amplified with the primers PSYF1-1 (5'-CCAAGAGCATCTCCACCTTC-3') and PSYR2. The same primer pair was used to amplify DNA from the polymorphic wild-type strain S1D2 to identify polymorphic markers for linkage tests. Sequencing of the PCR fragments on both strands was performed with the Big-Dye Terminator Version 3.0 kit (Applied Biosystems, Foster City, CA) and an ABI 3100 automated DNA sequencer (Applied Biosystems). The Lasergene MegAlign software package (DNASTAR, Madison, WI) was used to assemble DNA sequences into contigs and to align the PSY protein sequences.
Complementation with the wild-type phytoene synthase gene:
The plasmid (pSM1) used for transformation (KINDLE 1990) contained a 6-kb fragment of genomic DNA (amplified with primer pairs T-PSYF1 5'-CCTTAATGCAGCGAATCCTT-3' and PSYR2, and T-PSYF2 5'-GAGCGTGTGGGTTATCGTTC-3' and T-PSYR1 5'-ATGCAGCTGTAGCAATGCAG-3') inserted into the 3-kb pGEM-T Easy cloning vector (Promega, Madison, Wisconsin). The insert contained the 2262-bp PSY coding region, 500 bp upstream of the start codon, the 2021-bp 3'-UTR, and 1214 bp downstream of the 3'-UTR. Liquid cultures of lts1-210 were grown to a density of 5 x 106 cells ml–1 in complete darkness. Cells were harvested by centrifugation at 3000 x g for 3 min, resuspended in gamete autolysin (HARRIS 1989; 1/25 the original volume of the culture), and incubated for 90 min at room temperature. Autolysin-treated cells were collected by centrifugation at 1250 x g for 3 min and resuspended in liquid TAP at a density of 1.7 x 108 cells ml–1. The linearized pSM1 plasmid (300 ng), 300 µl (0.5 x 108) cells, and 100 µl of 20% (w/v) polyethylene glycol (PEG) were mixed and vortexed on high for 30 sec with 300 µg of glass beads (150–212 µm diameter). Cells were transferred to 10 ml liquid TAP and incubated overnight in the dark with constant shaking. Cells were then harvested by centrifugation at 1250 x g for 3 min, resuspended in 200 µl liquid TAP, and plated onto TAP agar medium. Plates were kept in the dark overnight and then gradually acclimated to very low light (8 µmol photons m–2 sec–1). Plates were scored for green transformants 2 weeks after exposure to low light.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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8 µmol photons m–2 sec–1) (Figure 2).
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Insight into the number of genes defined by the white mutants in this collection was obtained by crossing white mutants to one another and scoring the resulting progeny for wild-type recombinants. White mutant diploid zygospores did not germinate (undergo meiosis) when subjected to low light (8 µmol photons m–2 sec–1) overnight. Shorter exposure to low light (1–3 hr of 8 µmol photons m–2 sec–1) resulted in a marked increase in germination rates (data not shown). In crosses between independently isolated white mutants, all resulting progeny were white, and no wild-type recombinants were observed (Table 3). These results show that lts1-30 and all eight new white mutants are alleles of a single gene, LTS1, and the new mutants have been designated lts1-203–lts1-210 (Table 1). Crosses to mutant strains fn68 and w7 did not yield zygospores; consequently recombination tests could not be performed on these mutants. Mutants lts1-30 and w7, however, have been shown previously to be alleles of the LTS1 locus on chromosome XI (SPREITZER and METS 1981; IROSHNIKOVA and KVITKO 1986). As described below, w7 and fn68 were both shown to contain mutations in the same gene as all the other lts1 alleles, so they were designated as lts1-201 and lts1-202, respectively.
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-tocopherol per cell as the wild type (lts1-207 contained only 1.6 times more -tocopherol than the wild type; Table 4). Although it was reported previously that phytoene accumulates in lts1-202 (fn68; HERMAN et al. 1999), HPLC analysis (with detection by absorbance at 298 nm) did not show phytoene accumulation in any of the white mutants (data not shown). The absence of colored carotenoids and lack of phytoene accumulation suggested a defect in PSY rather than in PDS or plastoquinone biosynthesis.
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50 amino acids (Figure 5A). Comparison of the cDNA and genomic DNA sequences revealed that the PSY gene is 4.3 kb in length and contains five exons (varying in length from 152 to 426 bp) and four introns (varying in length from 80 to 449 bp), with a 3'-UTR of 2021 bases (Figure 5B).
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200 bp of the PSY 5'-coding region could not be amplified, possibly because of a deletion, insertion, or rearrangement in this region of the gene that affected priming or amplification. Nine mutants generated by EMS, UV, or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis contained a single-base-pair change from G/C to A/T, which corresponded to an amino acid change in the PSY protein (three alleles), a premature stop codon (five alleles), or a mutation affecting a splice site (one allele). One UV-induced mutant, lts1-202, contained both a base pair change and a base deletion in its PSY gene, the latter causing a frameshift in the open reading frame. The amino acid changes in the missense alleles (lts1-203, lts1-205, and lts1-209) are located in highly conserved regions and are likely to affect the function of the mature PSY protein (Figure 5A). Four out of five alleles with premature stop codons are located in the first half of the transcript, which would generate a severely truncated protein product (Figure 5A).
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8 µmol photons m–2 sec–1), green colonies were visible on the transformation plates, whereas no green revertants were observed on negative control plates (following mock transformation with no DNA). Growth phenotypes of the complemented line (lts1-210/PSY) in the dark and very low light (8 µmol photons m–2 sec–1) are shown in Figure 2. The presence of the wild-type PSY gene in the lts1-210/PSY strain was confirmed by PCR of genomic DNA with primers specific to the pSM1 plasmid (data not shown). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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There appears to be some overlap in the spectrum of mutations caused by EMS and UV mutagenesis in Chlamydomonas. The chemical mutagen EMS alkylates guanine leading to the mispairing of guanine with thymidine, instead of cytosine. The resulting point mutations are mainly G/C to A/T transitions. Studies in yeast have shown that G/C to A/T transitions at dipyrimidine sites are the most common UV-induced mutation (MOSHINSKY and WOGAN 1997). This is consistent with our findings, as four out of five UV-induced mutations in the PSY gene were G/C to A/T transitions, and all five mutations occurred at dipyrimidine sites (data not shown).
GGPP, the substrate of PSY, is also a precursor for the biosynthesis of several important molecules in algae and plants, including tocopherols, chlorophylls, and quinones. Therefore, GGPP may not accumulate in an lts1 mutant lacking PSY but instead may be diverted to one or more of these other biosynthetic pathways. In the fn68 mutant, it has been shown that chlorophyll is still synthesized, but it is rapidly turned over in the cell (HERRIN et al. 1992). Therefore, chlorophyll biosynthesis could act as a sink for GGPP in lts1 mutants. HPLC analysis showed that the white lts1 mutants have on average approximately twice as much -tocopherol per cell as wild type does. The increased tocopherol levels in the white lts1 mutants may be due to the diversion of excess GGPP into the tocopherol pathway, or, alternatively, to an upregulation of tocopherol biosynthesis in response to some other stimulus such as increased oxidative stress in the absence of carotenoids.
Chlamydomonas mutants defective in PDS or plastoquinone biosynthesis would be expected to exhibit a white phenotype similar to that of the lts1 mutants, so it is somewhat surprising that neither class of mutants was identified in our white mutant collection. Mutants affecting PDS or plastoquinone biosynthesis should accumulate phytoene, but phytoene accumulation was not detected in any of the white mutants, and the white phenotype did not cosegregate with a marker located in the PDS locus (data not shown). In Arabidopsis thaliana, two nonallelic albino mutations, pds1 and pds2, affecting PDS activity have been identified (NORRIS et al. 1995). The pds1 and pds2 mutants accumulate phytoene and lack plastoquinone, demonstrating that plastoquinone is an essential component of carotenoid biosynthesis in plants (NORRIS et al. 1995). The molecular basis for pds1, but not yet pds2, has been determined, and it was found that pds1 is a mutation in the gene encoding p-hydroxyphenylpyruvate dioxygenase (HPD) in Arabidopsis (NORRIS et al. 1998). Searching for an HPD homolog in the current draft of the Chlamydomonas nuclear genome revealed the presence of two tandemly linked HPD genes. Thus, the lack of a Chlamydomonas white mutant analogous to Arabidopsis pds1 is likely explained by genetic redundancy. Similarly, the presence of more than one functional PDS gene in the Chlamydomonas genome might provide an explanation for the inability to isolate a mutant affecting PDS; however, besides the putative PDS gene that we identified and a putative ZDS gene there does not appear to be an obvious additional homolog of PDS in Chlamydomonas. Alternatively, toxicity of phytoene accumulation or some other mechanism of lethality may prevent the isolation of mutants defective in PDS. However, the accumulation of phytoene in the pds1 and pds2 mutants of Arabidopsis (NORRIS et al. 1995) demonstrates that phytoene concentrations much higher than normal cellular levels are not lethal, at least in Arabidopsis. Isolation of a Chlamydomonas pds mutant by reverse genetics would provide insight into the viability of a mutant impaired in PDS, in addition to the possible redundancy of genes encoding enzymes that are capable of catalyzing phytoene desaturation.
The lts1 mutants grew heterotrophically in the dark using acetate as a carbon source, but they were unable to grow in the light, even under very low light conditions on acetate-containing medium (Figure 2). In contrast, the pds1 and pds2 mutants of Arabidopsis are able to grow to near maturity in the light on agar medium containing sucrose (NORRIS et al. 1995). A likely explanation for this difference in light sensitivity is a difference in chlorophyll accumulation. The Arabidopsis mutants completely lack colored carotenoids and chlorophylls (NORRIS et al. 1995), whereas the Chlamydomonas mutants are not actually completely white. The lts1 mutants accumulated easily detectable amounts of chlorophylls a and b (Figure 2, Figure 4, Table 4), which would act as potent photosensitizers of singlet oxygen formation in the absence of carotenoids (DEMMIG-ADAMS et al. 1996; FRANK and COGDELL 1996; BAROLI and NIYOGI 2000), thereby leading to severe photooxidative stress.
In summary, we have characterized 11 lts1 mutations that affect the PSY gene of Chlamydomonas. The phenotype of lts1 mutants is easily scored, making it an ideal marker for mapping genes to linkage group XI. The lts1 mutants will also be useful in studying the relationships between the pathways downstream of GGPP, including the carotenoid, chlorophyll, tocopherol, and quinone biosynthetic pathways.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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