Functional Genomics of Eukaryotic Photosynthesis Using Insertional Mutagenesis of Chlamydomonas reinhardtii  

Abstract

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover lightsensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.As with other model organisms, the availability of genome sequence data is revolutionizing and revitalizing research into the biology of the unicellular green alga Chlamydomonas reinhardtii Ledford et al., 2005). Over the past four decades, many fundamental insights into the structure, function, assembly, and regulation of the photosynthetic apparatus have come from studies of Chlamydomonas, which offer several advantages for the genetic dissection of eukaryotic photosynthesis (for review, see Davies and Grossman, 1998; Hippler et al., 1998;Grossman, 2000;Rochaix, 2001). First and foremost, photosynthesis is fully dispensable in Chlamydomonas, as cells can grow heterotrophically in the dark using acetate as a sole carbon source. Cells grown in the dark, however, still synthesize and assemble a fully functional photosynthetic apparatus. This allows the isolation and analysis of mutants that are unable to perform photosynthesis, and lightsensitive mutants can be maintained in complete darkness. Because Chlamydomonas is predominantly maintained in a haploid form, it is not necessary to generate homozygous nuclear mutants, and mutants affecting photosynthesis can be screened immediately following mutagenesis. Chlamydomonas has an easily controlled and rapid sexual cycle (approximately 2 weeks) with the possibility of tetrad analysis, which facilitates genetic analysis. Its rapid cell-doubling time (approximately 10 h) and microbial lifestyle mean that it is easy to grow homogeneous cultures on any scale, simplifying physiological and biochemical characterization in comparison to multicellular land plants (Ledford et al., 2005). By way of example, the application of inhibitors and generators of various types of reactive oxygen species results in uniform uptake of the chemical by each cell. In land plants, multicellularity leads to differential ...

Isolation and characterization of pigment deficient insertional mutants in the chlorophyte Chlamydomonas reinhardtii

“…We observed that in 77 % of the transformants analysed, the insertion took place in intragenic regions. This rate is similar to that observed by Dent and coworkers [20] and González-Ballester and coworkers [21] in functional genomic studies of photosynthesis and the regulation of nitrate assimilation, respectively. In all cases the marker insertion has occurred within a predicted gene or very near one, so identification of the gene responsible for the observed phenotype, has been done considering that the gene interrupted is the most probable responsible of the observed phenotype.…”

“…This approach has allowed isolation of mutants affected in different metabolic and physiological aspects [14] and has become a popular method for forward genetics studies because identification of the affected genomic region is generally easier than location of genomic lesions caused by traditional mutagenesis procedures based on chemical or physical agents. In C. reinhardtii, this technique has allowed the isolation of mutants affected in flagellar function [15], phototaxis [16], microtubules [17], carbon assimilation [18], H 2 production [19], photosynthesis [20], nitrogen [21] and sulphur metabolism [22].…”

“…The most complete functional genomic analysis of photosynthesis in Chlamydomonas by insertional mutagenesis was carried out by Dent and Coworkers [20]. They introduced a linearized plasmid carrying the ble gene from Streptoalloteicus hindustanus that confers resistance to bleomicin [23] and generated 2760 insertional mutants that were screened for light sensitive and nonphotosynthetic phenotypes.…”

“…Mutants which have higher violaxanthin content (B20), higher pigments concentration (A7), lower chlorophyll content (C13) and total lack of chlorophylls and carotenoids (C2), together with three mutants that synthesize small quantities of zeaxanthin in response to high light stress (Z7, Z34 and Z198), have been isolated and physiologically characterized. The insertion site in 15 of the selected mutants has been characterized by RESDA PCR (Polymerase Chain Reaction) [25] or inverse-nested PCR [26], contributing to increase the information generated by previous photosynthetic functional genomic analysis in Chlamydomonas [20].…”

Inactivation of genes coding for mitochondrial Nd7 and Nd9 complex I subunits in Chlamydomonas reinhardtii. Impact of complex I loss on respiration and energetic metabolism

“…The transmission pattern of the dark -and Hyg R loci in crosses were determined by random analysis of the meiotic products. Amplification of insertion-linked sequence by thermal asymmetric interlaced (TAIL)-PCR was conducted with degenerate primers as described in (Dent et al, 2005) with APH7 (hygromycine) specific primers: Hygterm1 (5"-CCGCGAACTGCTCGCCTTCACCT -3"), Hygterm2 (5"-TTCGAGGAGACCCCGCTGGATC-3"), and Hygterm3 (5"-CGATCCGGAGGAACTGGCGCA-3").…”