Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana

Abstract

Marine diatoms are important primary producers that thrive in diverse and dynamic environments. They do so, in theory, by sensing changing conditions and adapting their physiology accordingly. Using the model species Thalassiosira pseudonana, we conducted a detailed physiological and transcriptomic survey to measure the recurrent transcriptional changes that characterize typical diatom growth in batch culture. Roughly 40% of the transcriptome varied significantly and recurrently, reflecting large, reproducible cell-state transitions between four principal states: (i) "dawn," following 12 h of darkness; (ii) "dusk," following 12 h of light; (iii) exponential growth and nutrient repletion; and (iv) stationary phase and nutrient depletion. Increases in expression of thousands of genes at the end of the reoccurring dark periods (dawn), including those involved in photosynthesis (e.g., ribulose-1,5-bisphosphate carboxylase oxygenase genes rbcS and rbcL), imply large-scale anticipatory circadian mechanisms at the level of gene regulation. Repeated shifts in the transcript levels of hundreds of genes encoding sensory, signaling, and regulatory functions accompanied the four cell-state transitions, providing a preliminary map of the highly coordinated gene regulatory program under varying conditions. Several putative light sensing and signaling proteins were associated with recurrent diel transitions, suggesting that these genes may be involved in light-sensitive and circadian regulation of cell state. These results begin to explain, in comprehensive detail, how the diatom gene regulatory program operates under varying environmental conditions. Detailed knowledge of this dynamic molecular process will be invaluable for new hypothesis generation and the interpretation of genetic, environmental, and metatranscriptomic data from field studies.

Fig. 1.

Four principal genome-wide expression patterns observed over the growth cycle. (A) The mean and quantiles of expression ratios for state-associated genes are shown over time during the course of the experiment. Gene groups consist of genes whose expression changes were significantly associated with dark:light or exponential:stationary transitions by ANOVA at P < 0.01. Dawn-associated genes were significantly associated with the dark:light transition and higher in expression at the end of the 12-h dark periods (gray backgrounds). Dusk-associated genes were significantly associated with the dark:light transition and higher in expression at the end of the 12-h dark light (yellow backgrounds). Exponential and stationary genes were significantly associated with the exponential:stationary transition. Genes labeled exponential were higher in expression during the exponential phase (up to day 3). Genes labeled stationary were higher in expression during the stationary phase (following day 3). (B) Euler diagrams illustrate the numbers of genes associated with each condition. Genes for which associations were not detected are indicated by the ∅ symbol. *Sensing and signaling genes include all photoreceptors and genes with the following InterPro domains: IPR000719 (protein kinase), IPR001019 (G protein alpha subunit), IPR001632 (G protein beta subunit), IPR001789 (response regulator receiver), IPR003018 (GAF), IPR000337 (GPCR, family 3), IPR000276 (GPCR, rhodopsin-like), IPR002182 (NB-ARC), IPR001806 (Small GTPase), IPR002073 (cyclic nucleotide phosphodiesterase), IPR001054 (guanylyl cyclase), IPR000014 (PAS), IPR001680 (WD40), IPR001611 (Leucine-rich repeat (LRR)). WD40 and LRR domains (110 and 81 genes) are multifunctional and may not all represent signaling genes.

Fig. 2.

Schematic of physiological and transcriptional states of T. pseudonana during diurnal growth. Pathways and functions associated with each state are highlighted. (A) Dawn following 12 h of darkness. (B) Dusk following 12 h of light. (C) Exponential growth in replete nutrients. (D) Stationary phase and nutrient depletion. Those pathways highlighted in red contain genes significantly up-regulated, those in blue contain genes significantly down-regulated, and those in black do not display conditional associations. The association of putative transcription factors (TFs) with each state is depicted in pie charts whose sizes are proportional to the total number of associations detected. TFs that were significantly enriched under a condition are marked with an asterisk.

Fig. 3.

Genes with putative light-sensing and signaling functions differentially expressed between dark:light and exponential:stationary conditions. (A) The expression patterns of light-sensing and signaling genes during diurnal growth. Genes are in rows and growth time points are in columns. Yellow and blue cells show higher and lower expression ratios during the experiment. Only genes with gene/condition associations by ANOVA at P < 0.01 or higher are shown (aqua boxes). See Fig. S9 for expression of all known photoreceptors. The multicolored bar indicates the following classes of genes: blue, putative photoreceptors and photolyases and green, PAS/PAC domain-containing genes. (B) Model for putative light-sensitive regulation of cell states by photoreceptors. Cellular states (interior labels) are hypothetically driven by the sensory and regulatory activities of differentially expressed photoreceptors (purple text).

Fig. 4.

Inferred gene regulatory relationships. Shown are potential transcriptional activation pathways that were consistent with all published T. pseudonana microarray data and known conserved transcription factor-DNA binding sequence motifs for five major TF families. (A) Transcription factors (TFs, colored triangles) are proposed as candidate regulators of target genes (circles) if: (i) the TF and its target genes were highly correlated in expression changes over multiple array experiments (bootstrap P < 0.025); (ii) the upstream region of each target gene contains at least one potential DNA binding site that matches a conserved DNA-binding motif for the TF family (motif P < 1 × 10⁻⁴); and (iii) the target genes are enriched in putative TF-DNA binding sites, compared with all genes (hypergeometric P < 0.05). Gray circles represent target genes that were not statistically associated with diel states. (B) Putative DNA-binding motifs for conserved TF families in higher plants. Dashed arrows denote possible inversions in the heat-shock element for this family of transcription factors. Functional enrichment analysis of TF-gene clusters is provided in Dataset S4.