Fig 1.
Flow cytometry analysis indicates that the DNA content of evolved cultures is similar to that of ancestral cells.
The upper panel shows flow cytomery data of the DNA content in standard haploid, diploid, triploid and tetraploid populations. The mean fluorescence intensity (in arbitrary units) of G1 cells in each strain was used as the reference points (1N, 2N, 3N, and 4N shown in the lower panel). The lower panel shows the DNA content of ancestral and evolved cultures. Newly-constructed isogenic diploid (grey) and tetraploid (orange) cells were propagated in YPD medium at 23°C with daily dilutions. Cell cultures at generation zero (anc2N and anc4N) and 1000 (evo2N and evo4N) were analyzed using flow cytometry.
Fig 2.
Array-based comparative genome hybridization (aCGH) analysis reveals that most evolved tetraploid clones remain euploid.
Total genomic DNA was isolated from ancestral clones and the representative clones from individual evolved cultures and subjected to aCGH. Genomic DNA of a standard haploid strain was used as the reference. The ratios of signal intensities between tetraploid and reference genomic DNA were log2-transformed. Each dot represents a gene, and all genes of the 16 yeast chromosomes were plotted. Of the evolved clones, only evo4N-2 carried a 154-kb deletion on chromosome VII (indicated by a green arrowhead) and evo4N-7 contained an extra chromosome II (indicated by a red arrowhead). The remaining clones did not have large-scale deletions or duplications.
Fig 3.
Evolved tetraploid clones have improved fitness even under stress conditions.
(A) Evolved tetraploids have increased growth rates at 28°C. The growth rates of individual clones were measured using a flow cytometry-based competitive assay (see Materials and Methods). The relative fitness increase indicates the difference between the evolved and ancestral clones. Three replicates of the fitness measurement were performed. Error bars represent the standard deviation. (B) Polyploid cells are sensitive to high temperature. Cell cultures of different ploidy were serially-diluted and spotted onto YPD plates. The plates were then incubated at 23°C or 36°C until colonies were easily observed. (C) Robust growth at 36°C is detected in the evolved tetraploid clones, but not in the ancestral clones. (D) Evolved tetraploids display various levels of improvement in fitness when grown on plates containing the spindle formation inhibitor benomyl (7.5 μg/ml) at 23°C.
Fig 4.
Transcriptome data reveals that protein homeostasis machineries are adjusted in the evolved clone.
(A) Biological processes enriched in the down-regulated and up-regulated genes of the evo4N-4 clone at 36°C. Whole-genome gene expression levels of anc4N-4 and evo4N-4 were measured using microarrays and differentially-regulated genes were analyzed using GO analysis. The enrichment score was calculated by dividing the proportion of differentially-regulated genes classified into the indicated category by the proportion of those in the genome. Down-regulated genes are predominantly involved in rRNA processing, ribosome biogenesis and translation. Up-regulated genes are enriched in the processes of protein folding and mRNA processing. (B) The tetraploid cells have become physiologically diploid-like in their gene expression after evolution. Genes with at least 2-fold changes between anc4N-4 and evo4N-4 were selected for the comparison, and a higher correlation in expression levels was observed between ancestral diploids and evolved tetraploids (Fisher’s z-transformation, Z-value = 7.435, p-value = 1.178 × 10−13). A comparison using all gene expression data is shown in S2 Fig.
Fig 5.
Tetraploid cells increase the protein abundance of Sch9 and resistance to an inhibitor of TORC1 networks after evolution.
(A) Polyploid cells are more sensitive to rapamycin. Growth rates of cells with different ploidy were measured in liquid YPD medium with or without 10 ng/ml of rapamycin. Relative growth rates of each strain in rapamycin-containing medium were normalized to that of medium lacking the drug. Three replicates of the fitness measurement were performed. Error bars represent the standard deviation. (B) Most evolved tetraploid clones increase their resistance to rapamycin. Ancestral and evolved cells were serially diluted and spotted onto YPD plates containing different concentrations of rapamycin (0, 5, 10 and 20 ng/ml). The plates were then incubated at 23°C until colonies were easily observed. (C) Total protein was extracted from cells with or without HA-tagged SCH9. Immunoblotting analysis showed that the abundance of Sch9 protein was increased more than 1.5-fold in most evolved clones. G6PDH was used as the internal control. For the HA-tagged construct, three independent transformants from ancestral or evolved clones were tested and consistent patterns were observed between clones. (D) The relative abundance of Sch9 in ancestral tetraploids is about 1.8-fold lower than that in ancestral diploids. The ratio has been corrected for the difference in copy number (i.e., only one of four copies of SCH9 is tagged with HA in tetraploids, but half of the copies are tagged in diploids). (E) The protein stability of Sch9 is enhanced in the evolved tetraploid cells. Log-phase cells were treated with cycloheximide to stop protein synthesis and collected at different time points. Total cell protein was extracted and examined by Western blot. The right panel shows quantitative data of the Western blot. The 50-min time point was not included since the signal in ancestral cells was already undetectable. All the Sch9/G6PDH ratios were normalized to the first time point data (0 min) of the same strain. Protein half-life was calculated using the change of relative protein intensity after the cycloheximide treatment (see Materials and Methods). The asterisk indicates non-specific hybridization that appears in both HA-tagged and non-tagged strains.
Fig 6.
Sch9 plays a key role in the evolved phenotype.
(A) Overexpressing SCH9, but not TOR1, enhanced the growth of anc4N-4 cells to a level near evo4N-4 cells. Multicopy plasmids carrying either TOR1 or SCH9 (OE-TOR1 and OE-SCH9) were introduced into the anc4N-4 and evo4N-4 strains and tested for their effects on cell growth at 36°C. Overexpression of TOR1 had no effect on ancestral cells and negative effects on evolved cells. Overexpression of SCH9 had no effect on evolved cells but enhanced the growth of ancestral cells. (B) Ancestral tetraploids with SCH9 overexpression exhibited improved fitness on plates containing the microtubule depolymerizing drug benomyl. (C) The enhanced survival of evolved tetraploids at 36°C was completed abolished in the absence of Sch9. Two premature stop codons were introduced into all genomic copies of SCH9 to generate the sch9 null mutants (see Materials and Methods). For each strain, we performed phenotypic assays for at least three sch9 null mutant clones and observed consistent phenotypes between clones. (D) Both activity and regulation of the kinase are important for the evolved phenotype. Overexpression of kinase-dead (SCH9k.d.) or constitutively active (SCH92D3E) mutants showed no effect at 23°C, but reduced the growth of evolved cells at 36°C. The correct ploidy was confirmed for all strains used in this figure using flow cytometry.
Fig 7.
Evolved tetraploids can stably maintain their genomes when growing under non-optimal conditions.
Eight ancestral tetraploid clones (anc4N-1 to anc4N-8) and eight evolved tetraploid clones (evo4N-1 to evo4N-8) were propagated in regular YPD medium at 30°C for 200 generations and the evolved cultures were analyzed using flow cytometry. Half of the cell lines derived from ancestral clones became aneuploid or diploid, but all cell lines derived from evolved clones remained tetraploid.