Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Abstract

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.

Fig 1. Growth and chlorophyll fluorescence screen pipeline.

Mutants were scored for growth on (A) D+ac, (B) LL+ac, (C) HL+ac, (D) LL+ac+zeocin, (E) LL-min, (F) HL-min. F_v/F_m values were measured on cells grown on (G) D+ac, (H) LL-min, (I) HL-min. The false color scale is indicated below the images (G-I). FST, flanking sequence tag. A representative plate spotted from a 96-well plate is shown. D, dark; LL, low light; HL, high light; +ac, added acetate; min, minimal media.

Fig 2. Examples of structural variations and the frequency of mutants with simple or complex insertions in ARC.

Boxes contain schematic examples of mapped reads as seen in IGV. Black box, mapped reads (concordant and discordant) against plasmid and chromosome. Blue box, examples of “Simple insertions”; Gray box, examples of “Complex insertions”. Gray box shows examples of different complex insertions that are intra- or interchromosomal rearrangements. Second from left in gray box shows a possible translocation between two chromosomes. Pie chart shows frequency of “Simple mutants” containing only simple insertions and “Complex mutants” containing complex insertions.

Fig 3. Structural variation accompanying insertions.

(A) Duplication and deletion sizes and (B) number of mutants grouped by the number of genes affected by two-sided insertions. Only two-sided insertions were included in this analysis.

Fig 4. Genes represented by multiple mutant alleles are more likely to be causative genes.

(A) Number of genes affected by plasmid-associated insertions in ARC grouped by the number of mutant alleles that represent the gene. Schematic of mutant alleles disrupted in (B) cpsfl1 mutants and (C) lpa3 mutants and the allele frequencies of surrounding genes. Note that not all genes with multiple mutant alleles are causative; some genes belong to this group because of their physical proximity to the true causative genes. CAL040_01_25 (lpa3-3, Fig 5) is indicated in gray because this mutant is not included in S1 Table or the analysis for panel A but represents the ninth mutant allele of cpsfl1.

Fig 5. Identification of CrLPA3 and CrPSBP4 required for photoautotrophic growth.

(A) Schematic of loci and deletions indicated from whole-genome sequence data in mutants lpa3-1 (CAL028_01_27), lpa3-2 (CAL039_03_42), and lpa3-3 (CAL040_01_25) that share a disruption in Cre03.g184550, gene encoding a predicted ortholog of Arabidopsis LOW PHOTOSYSTEM II ACCUMULATION 3 (LPA3) and mutant psbp4-1 (CAL032_04_48) that had a deletion encompassing Cre08.g362900, a gene encoding a protein predicted as PSBP4. Numbered arrowheads indicate the PCR probes used in testing for deletions shown in the agarose gel photos. WT and lpa3-3 sequences indicate the plasmid insertion site and associated 4 bp-deletion. (B) Growth and chlorophyll fluorescence phenotype of WT, mutants and their complemented lines. Images are representative of an experiment repeated twice. Cells were grown with acetate in the dark or without acetate under 400 μmol photons s^-1 m^-2 and imaged for growth and F_v/F_m measurements (HL-Ac). F_v/F_m value are represented by false colors as shown in the reference bar. (C) F_v/F_m values of each genotype under different growth conditions. Values indicate averages of three biological replicates; error bars represent standard deviations. comp, complemented line. (D) PSII and PSI subunit accumulation shown by immunoblotting against subunits of PSII (D1 and CP43) and PSI (PsaA and PsaD). Mitochondrial ATP synthase (F₁β) was probed as loading control with an antibody that also detects the F₁β subunit of the chloroplast ATP synthase (CF₁β). Each genotype was analyzed in biological duplicates (one per lane). Dilutions of the WT samples are shown in the four left lanes.