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

Iron (Fe) is an important growth limiting factor for diatoms and its availability is further restricted by changes in the carbonate chemistry of the water. 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 transcriptional patterns for genes in most central metabolism pathways, the diel periodicity 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 pattern at low Fe’, while transcripts that encoded the non-active enzyme lost the diel pattern. 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. Axenic Thalassiosira pseudonana (CCMP 1335) was grown in batch cultures using chelexed Aquil media with the addition of either 1000 nmol L-1 or 50 nmol L-1 iron (EDTA at 100 µmol L-1). Cells were grown at 20˚C under a 12:12 light:dark cycle at 50 µmol photons m-2 sec-1. Prior to start of the experiments, cells were grown at the same light and acclimated to iron-replete (1000 nM) or iron-limiting (50 nM) conditions for at least a month (~20 generations at Fe-limiting, ~40 generations at iron-replete), but not to their respective CO2/pH conditions. The media was bubbled with air containing 400 or 800 ppm CO2 prior to cell inoculation resulting in pH of 8.1 and 7.8 respectively. The starting cell concentration for the three experimental replicates was 30,000 cell/mL. The combination of Fe addition and pH resulted in four concentration of available Fe: 1160, 233, 58 and 12 pM . All experiments were performed in triplicate. Samples were collected in triplicate for each of the experimental replicate for: macronutrients, fluorescence, Fv/Fm, carbonate chemistry measurements, and flow cytometry. All experimental samples were drawn from the bottles via a syringe port 6 hours into the light (midday) and 6 hours into the dark phase (midnight). Samples for transcriptomes were collected over one day during the exponential growth phase at 6 hours into the light and 6 hours into the dark phase.