Using natural selection to explore the adaptive potential of Chlamydomonas reinhardtii

Abstract

Improving feedstock is critical to facilitate the commercial utilization of algae, in particular in open pond systems where, due to the presence of competitors and pests, high algal growth rates and stress tolerance are beneficial. Here we raised laboratory cultures of the model alga Chlamydomonas reinhardtii under serial dilution to explore the potential of crop improvement using natural selection. The alga was evolved for 1,880 generations in liquid medium under continuous light (EL population). At the end of the experiment, EL cells had a growth rate that was 35% greater than the progenitor population (PL). The removal of acetate from the medium demonstrated that EL growth enhancement largely relied on efficient usage of this organic carbon source. Genome re-sequencing uncovered 1,937 polymorphic DNA regions in the EL population with 149 single nucleotide polymorphisms resulting in amino acid substitutions. Transcriptome analysis showed, in the EL population, significant up regulation of genes involved in protein synthesis, the cell cycle and cellular respiration, whereas the DNA repair pathway and photosynthesis were down regulated. Like other algae, EL cells accumulated neutral lipids under nitrogen depletion. Our work demonstrates transcriptome and genome-wide impacts of natural selection on algal cells and points to a useful strategy for strain improvement.

Figure 1. Growth rate of an evolved population of C. reinhardti.

(A) Design of the experimental approach used to generate a rapidly growing C. reinhardtii CC-503 cw92 mt+ population. (B) Results after 283 transfers (1,880 generations) of cells grown under continuous light in liquid TAP medium. At the end of the experiment, the evolved C. reinhardtii population grows ca. 35% faster than the progenitor strain. The progenitor growth rate is indicated with the solid green line. (C) Comparison of growth rates of the progenitor (PL, green line) and one population of the evolved (EL, red line) C. reinhardtii cells grown in TAP medium. The growth rate of these populations when raised in acetate-free (TAP) medium is shown with the dashed green line for PL and the dashed red line for EL. These curves are derived from three independent culture replicates.

Figure 2. The distribution of cell sizes of exponential (A) and stationary phase (B) cultures of the PL (green line) and EL (red line) C. reinhardtii populations used in our analysis.

The size at one standard deviation from the mean for each cell population is shown with the light colored lines. Although a broad range of cell sizes is present in each culture, the stationary phase EL population is on average ca. 1 μm smaller than the PL cells.

Figure 3. Transcriptome analysis of the EL population.

(A) The number of significantly differentially expressed genes (not all genes) in the PL-EL comparison of triplicate RNA-seq samples. Only KEGG categories with ≥4 genes are shown. Significantly up and down-regulated genes are shown with the red and blue bars, respectively. The gray boxes include categories with ≥4 genes. (B) Schematic model of DNA replication (based on the KEGG model) showing the gene expression patterns when comparing the PL-EL populations. All protein components that do not show a significant expression difference are shown in gray, proteins that are significantly up regulated are shown in red, and proteins that are significantly down regulated are shown in blue. The Table below the model shows the results of the statistical analysis of gene expression for the significantly differentially expressed proteins in this complex.

Figure 4. The distribution of significantly up and down-regulated genes in the PL-EL comparison when mapped on the KEGG metabolic pathway.

The gray lines indicate the background RNA-seq data in this comparison, whereas the red and blue lines show genes that were significantly up and down-regulated, respectively. The black lines indicate genes that show both types of effects, due to the existence of gene paralogs with differential expression.

Figure 5. Analysis of lipid production in the PL and EL populations of C. reinhardtii when grown under continuous light in liquid TAP medium.

Shown are differential interference contrast images (D), chlorophyll autofluorescence (C), lipid bodies (Bodipy stain; L), and merged (M). Note that like the PL and EL cells show up regulation of lipid production after depletion of nitrogen (-N) in the medium.