Fe limitation decreases transcriptional regulation over the diel cycle in the model diatom Thalassiosira pseudonana

Abstract

Iron (Fe) is an important growth factor for diatoms and its availability is further restricted by changes in the carbonate chemistry of seawater. We investigated the physiological attributes and transcriptional profiles of the diatom Thalassiosira pseudonana grown on a day: night cycle under different CO2/pH and iron concentrations, that in combination generated available iron (Fe') concentrations of 1160, 233, 58 and 12 pM. We found the light-dark conditions to be the main driver of transcriptional patterns, followed by Fe' concentration and CO2 availability, respectively. At the highest Fe' (1160 pM), 55% of the transcribed genes were differentially expressed between day and night, whereas at the lowest Fe' (12 pM), only 28% of the transcribed genes displayed comparable patterns. While Fe limitation disrupts the diel expression patterns for genes in most central metabolism pathways, the diel expression of light- signaling molecules and glycolytic genes was relatively robust in response to reduced Fe'. Moreover, we identified a non-canonical splicing of transcripts encoding triose-phosphate isomerase, a key-enzyme of glycolysis, generating transcript isoforms that would encode proteins with and without an active site. Transcripts that encoded an active enzyme maintained a diel expression at low Fe', while transcripts that encoded the non-active enzyme lost the diel expression. This work illustrates the interplay between nutrient limitation and transcriptional regulation over the diel cycle. Considering that future ocean conditions will reduce the availability of Fe in many parts of the oceans, our work identifies some of the regulatory mechanisms that may shape future ecological communities.

Fig 1. The impact of Fe’ concentrations (in pM) on different physiological parameters.

(A). Growth rate (day^-1) and (B) nitrate, (C) phosphate and (D) silicate consumption rates (in μmol L^-1 hr^-1), average of Fv/Fm measured during the (E) day or (F) night during exponential growth. Error bars (A-D) represent standard deviation of the triplicates. Error bars for Fv/Fm (E. F) represent the square root of the variances for each measurement used in the average.

Fig 2

Dendrogram representing the hierarchical clustering (A) or multidimensional scaling (MDS) (B) of the transcriptome profiles. (A) Height in the dendrogram reflects both the order the clusters were joined and the distance between them. HF = high Fe, LF = low Fe, 400 and 800 designate the CO₂ levels (in ppm). L and D indicates samples taken in the light (L) or in the dark (D). Red shades in table indicate Fe’ concentration (darker at 1160 pM to lighter at 12 pM), CO₂ (red = 800 ppm; no color = 400 ppm) and sampling time (red = midday; no color = midnight). (B) Distances between samples indicate the leading log2-fold-change or the root-mean-square log2-fold-change between the samples for the top 500 genes that distinguish those samples, in two-dimensional space (dim 1 and dim 2). Circles represent light samples from the highest Fe’ (dark red) to the lowest Fe’ (yellow). Triangles represent dark samples from the highest Fe’ (dark blue) to the lowest Fe’ (light blue).

Fig 3. Percent of genes in KEGG-defined pathways with differential transcription between midday and midnight at different Fe’.

Yellow: genes with significantly higher transcript abundance at midday (p<0.01 and FDR<0.05), blue: genes with significantly higher transcript abundance at midnight (p<0.01 and FDR<0.05), grey: genes with no significant change in transcript abundance between day-night (p>0.01). Numbers in parenthesis represent the total number of genes in a given pathway. List of genes for each pathway is presented in S6 Table.

Fig 4. Heatmap of selected gene expression across all conditions.

(A) Selected genes belonging to carbon related pathways. Each white line separates one pathway to the next. From top to bottom: Light-harvesting and Photosynthesis, Glycolysis, Carbon Fixation, Pentose Phosphate Pathway, and TCA cycle. (B) Selected genes belonging to the Signaling molecules (top) and the Transcription Factors (bottom).

Fig 5. Schematic of intron splicing for gene JGI Thaps3_30380 and associated transcript abundance of the gene and its isoforms.

The schematic illustrates the different splicing outcomes: splicing of the canonical intron leads to transcripts with the active site (Isoforms 1 and 2 both have the active site but differ in length by 71 amino acids); splicing of the non-canonical intron leads to an isoform without the active site. HF = high Fe addition, LF = low Fe addition. 400 and 800 are the CO₂ levels (in ppm). The combination of Fe addition and CO₂ levels result in four Fe’: 1160, 233, 58 and 12 pM. Bar colors indicate samples taken in the light (yellow) or in the dark (blue). Error bars represent standard deviation of the triplicates. Gene abundance is in counts per million or cpm (counts scaled by the number of fragments sequenced times one million) and isoform abundance is in transcript per million or tpm.