Multiomics resolution of molecular events during a day in the life of Chlamydomonas

Abstract

The unicellular green alga Chlamydomonas reinhardtii displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that Chlamydomonas exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes PSBS, LHCSR1, and LHCSR3 show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while LHCSR3 genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.

Keywords: cell division; chloroplast; histone expression; photobioreactor; systems biology.

Fig. 1.

How to obtain exactly two daughter cells during the Chlamydomonas cell division cycle. (A) Schematic illustration of the flat-panel photobioreactor design. (B–F) Experimental parameters measured under our growth conditions. Real-time profiles of the growth medium temperature (B), pH (C), and optical density at 680 nm (D). (E) Distribution of cell size over the diurnal cycle, shown as a box plot. (F) Number of cells in the culture as a function of diurnal time. Data (except E) are shown as average ± SD (n = 3–9, specified in each panel). The green triangles indicate the timing of cell division.

Fig. 2.

High synchrony across cells illustrated by the expression of genes involved in DNA replication. (A) Heatmap showing transcript abundance for genes involved in DNA RNR (3 genes, brown), DNA helicase (MCM, 6 genes, purple), DNA polymerase (POLD1), histone chaperones (HC, three genes, magenta), histone H1 (2 genes, gray), histone H2A (22 genes, yellow), histone H2B (20 genes, red), histone H3 (26 genes, blue), and histone H4 (26 genes, green). Histone variants (HX.v) are shown in a lighter version of the color used to depict their respective histone groups. In all cases, expression was normalized to 1 for the highest value. (B) Dynamic expression range of replication genes. Minimum and maximum RNA abundance (in FPKM) from the average of three independent experiments is shown. The green vertical lines indicate the timing of cell division.

Fig. 3.

The Chlamydomonas transcriptome is highly dynamic and rhythmic over one diurnal cycle. (A) PCA of the Chlamydomonas transcriptome over the diurnal cycle: samples collected at night (●), samples collected during the day (○). The time of sample collection is indicated next to the corresponding circle. (B) Heatmap representation of gene expression for 10,465 differentially expressed genes over the diurnal cycle. Genes were included if at least 1,000 counts were detected in the experiment, and there was a Benjamini–Hochberg-adjusted P value < 0.01 for differential expression. Genes were grouped into 16 clusters of nuclear genes, 3 clusters of chloroplast genes, and 1 mitochondrial cluster. For easier visualization, the vertical scale of the organellar transcriptomes (chloroplast transcriptome, Middle; mitochondrial transcriptome, Bottom) is enlarged 50 times relative to the nuclear transcriptome. (C) Heatmap representation of gene expression for 6,916 genes based on peak time (phase) of expression, as determined by the algorithm JTK_CYCLE, with minimal expression of 1 FPKM for at least one time-point. For easier visualization, the vertical scale of the organellar transcriptomes (chloroplast transcriptome, Middle; mitochondrial transcriptome, Bottom) is enlarged 25 times relative to the nuclear transcriptome. The green vertical lines indicate the timing of cell division.

Fig. 4.

Genes with similar function are coexpressed. (A) Examples of synchronous gene-expression profiles for histone genes, nucleus-encoded plastid RPGs, and genes involved in the chloroplast ETC. All expression estimates are normalized to 1 for the highest value. The green triangles indicate the timing of cell division. (B and C) Phase distributions over the diurnal cycle for genes belonging to chosen gene categories. Phase values are based on the algorithm JTK_CYCLE, with a cut-off rate BH.Q of 1 × 10⁻⁵. Functional categories shown are: histones, DNA replication, FAPs, RPGs, mitochondrial and chloroplast ETC, chlorophyll biosynthesis, CCM, cilia (flagellar proteome, including all FAPs).

Fig. 5.

Chlamydomonas cells do not use respiration to full capacity in the dark. Total nonpurgeable organic carbon (A) and starch content (B) of Chlamydomonas cells over the diurnal cycle. Data shown on a per cell basis. The green triangles indicate the timing of cell division. (C) Oxygen consumption (blue line) and respiration (resp) capacity of cells treated with the mitochondrial uncoupling agent FCCP (red line). Oxygen consumption was measured on the same samples before and after addition of FCCP. Data shown on a per cell basis. (D) Relative contribution of cytochrome c oxidase and alternative oxidases in oxygen consumption. Potassium cyanide (cyanide in figure) inhibits cytochrome c oxidase, while SHAM and propyl gallate target mitochondrial and plastid terminal oxidases (AOX and PTOX, respectively). Data shown on a per cell basis.

Fig. 6.

Chlamydomonas cells use anaerobic routes for handling pyruvate. (A) Key pyruvate metabolism pathways according to refs. and . Final fermentation products are shown in boxes and enzymes in gray ellipses. Abbreviations: ACK1, acetate kinase 1; ADH1, acetaldehyde/alcohol dehydrogenase; HYDA, [Fe–Fe]-hydrogenase; LDH1, d-lactate dehydrogenase; PAT2, phosphate acetyltransferase 2; PFL1, pyruvate formate lyase; PFR1, pyruvate ferredoxin oxidoreductase. N⁺, NAD⁺; NH, NADH. (B) Normalized expression of fermentation genes listed, shown as a heatmap. Numbers on the right side indicate maximum FPKM values in our samples (diurnal) and in cells grown under dark hypoxia for 6 h (34). (C) Changes in water-soluble metabolites over the diurnal cycle, analyzed by GC-MS. Results are shown as a heatmap of z-score normalized abundance for metabolites that changed significantly over the diurnal cycle. (D) Fermentation enzymes are more abundant in the dark. Total protein samples were separated by denaturing SDS/PAGE, followed by immune-detection with antibodies raised against PFR1, PFL, ADH, and HYDA1+2. Equal protein amounts were loaded and confirmed with Ponceau S stain. All data are shown as average ± SD (n = 3). The immune-detection was performed at least twice on independent samples. The green vertical lines indicate the timing of cell division.

Fig. 7.

Chlamydomonas cells integrate two light inputs to cope with light stress. (A–C) Photosystem II capacity, as determined by Fv/Fm values over the diurnal cycle, in cultures exposed to 200 µmol photons/m²/s with abrupt (A) or gradual 2-h transition at dawn (B), or exposed to 60 µmol photons/m²/s with abrupt transition at dawn (C). Relative (rel) mRNA abundance for LHCSR3.1 (D–F) and PSBS2 (G–I) by quantitative RT-PCR, in samples collected from cells grown under diurnal conditions with 200 µmol photons/m²/s with abrupt transition at dawn (D and G), 200 µmol photons/m²/s with gradual transition at dawn (E and H), or 60 µmol photons/m²/s with abrupt transition at dawn (F and I). All data are shown as average ± SD (n = 3). rel, relative.