Changepoints and half-lives in min.

Based on the transcripts counts following the addition of iron (rec) or the addition of actinomycin D (actD) or actinomycin D and iron (actDFe) a changepoint was determined. The changepoint is the point of time when the slope reaches its plateau. The half-life was calculated based on the slope of the curve before reaching the changepoint used as k in t1/2 = ln2/k. (XLSX)</p

THAOC numbers of all targeted transcripts.

This list includes the identifiers of the targeted transcriptome experiment. The probes were designed based on these sequences. (XLSX)</p

Effect of initial concentrations on the removal of P from solution to the solids.

<p>Data show that increasing concentration leads to an increased mass loading to the solids, but also as slight delay in reaching equilibration (e.g., 12 h at 3 mg/L, but nearly 48 h at 25 mg/L).</p

Structure and evolutionary distance to other CREG proteins of CREGx2_<i>To</i>.

(A) CREGx2_To protein structure. Transmembrane domain (TM) and N-glycosylation sites are indicated as barrels and squares, respectively. (B) Phylogenetic tree of CREG proteins. The T. oceanica sequence is shown in bold (THAOC08512). The maximum likelihood tree was generated in MEGA [44] using the top 100 blastp hits. The sequence alignment is based on a muscle alignment, and the tree was generated with the WAG model and gamma distribution. The black circle represents a bootstrap of > 0.5. Three conserved domains are indicated. Complex I intermediate-associated protein 30 (CIA-30) is in red, Pyrodoxamin-5’phosphate oxidase (Pyr-ox) in blue and histidine-rich region (His-rich) is indicated in green. The signal peptide is abbreviated with SP.</p

Normalized transcript counts for 21 targeted genes (A) and heatmap of Spearman correlation between transcript levels (B).

(A) Normalized transcript counts (counts/100ng of total RNA). Low-iron (blue), high-iron (black), and iron-recovery (rec) (orange) samples are plotted for each gene. The x-axis is plotted in log-scale, with the genes plotted in order of decreasing transcript abundance. (B) Heatmap with clustering based on Spearman correlation. Transcript counts are plotted as log2 values, with yellow and dark blue indicating low and high counts, respectively. The four coloured bars represent treatments, sampling times, iron levels, and experiment type. For the treatment, low-iron samples are shown in blue, dimethyl sulfoxide (DMSO)-treated samples are purple, iron-recovery samples are shown in orange, and high-iron samples are black. Timepoints are separated into Initial, 5–15 min, 30–45 min, 1 h, and >1 h in increasingly darker shades of green, with darker shades indicating the time progression from earlier to later timepoints. Iron levels are indicated in grey for samples without iron and black for samples with iron. Samples from the long-term experiment are shown in dark brown, and samples from the short-term experiment are shown in light brown. The dendrogram above the heatmap indicates the following clusters: a) downregulated genes following iron addition and b) upregulated genes. The dendrogram on the left indicates clusters 1 and 3 with samples from 12 h and 14 h after iron addition. The split region shown as cluster 2 consists of iron-recovery samples from time points with rapid acclimation of the iron-responsive transcripts. The following abbreviations were used: flavodoxin (FLDA1), fructose-bisphosphate aldolase (FBA), plastocyanin (PETE), ferredoxin (PETF), iron starvation-induced protein (ISIP), cellular repressor of EA1-stimulated genes (CREG), heat shock factor (HSF), manganese-superoxide dismutase (Mn-SOD), transcription factor (TF), oligosaccharyltransferase (OST), nuclear import-export receptor house-keeping (nucl_hk), N-acetylglucosaminyltransferase (GnT1), UDP-glucose glucosyltransferase (UGGT), iron reductase (FRE1).</p

Heatmap clusters 1 and 3 with sample-specific names.

Clusters 1 and 3, as outlined in the text and indicated in Fig 8, are shown with sample-specific names. The name is divided into treatment (high/low), experiment-type (LT-long-term experiment), and the point of time after iron addition as experiment-specific identifiers. Twelve h and 14 h are 7 PM and 9 PM, respectively. (TIF)</p

Changes in cellular chlorophyll content.

Box plots (A) and short- and long-term time-courses (B) of chlorophyll. High-iron, low-iron, and iron-recovery samples are shown in black, blue, and orange, respectively. A two-tailed Student’s t-test was used for the statistical analysis of box plots (A). Box plots represent duplicate measurements of three experiments (n = 6) for each time point (0 h, 1 h, 6 h). Line graphs (B) show trends throughout the long-term and short-term experiments. Error bars represent standard errors (S.E.). Statistically significant P values are indicated as * <0.05, **, 0.01, *** < 0.001, **** < 0.0001. A line without stars indicates a test that is not statistically significant.</p

Correlation analysis of FBA3/FBA4 and NanoString probe alignment for possible cross binding activity.

(A) Correlation analysis of all sample points for FBA3 and FBA4. actD (red), DMSO (purple), high-iron (grey), low-iron (blue), iron-recovery (orange). (B) Alignments of FBA3 probe A and probe B onto full-length FBA4 are shown. Dark blue indicates matching nucleotides. The percent identity is shown to the right of the alignments. 75% identity is the threshold for possible false binding (Kane et al., 2000). (C) Alignment of FBA4 Probe A and B onto full-length FBA3 is shown. Dark blue indicates matching nucleotides, and the percent identity of the alignment is shown to the right. (TIF)</p

Changes in photo-physiological parameters throughout 6 h during the short-term (ST) experiment.

High-iron, low-iron, and iron-recovery samples are shown in black, blue, and orange, respectively. The 0 h, 1 h, and 6 h sampling times are indicated as triangles, squares, and circles, respectively. (A) Electron transfer rate (ETR) in μmol e m-2s-1. (B) Bar graphs of ETRmax with Student’s t-test indicate statistically significant differences. (C) Photochemical quantum yield (PS-quantum yield Y(II)), non-photochemical quenching (Y(NPQ)), and non-regulated energy dissipation (Y(NO)). (D) Bar graphs of Y(II), Y(NPQ), and Y(NO) with Student’s t-test. (E) Light-induction curves and the corresponding Fv/Fm values with ‘Student’s t-test showing statistical differences. Statistically significant P values are indicated as * <0.05, ** < 0.01, *** < 0.001, **** < 0.0001. A line without stars indicates a test that is not statistically significant.</p

Percent identity of all probe sequences aligned to each gene.

All target sequences were divided into their respective probe A and probe B, resulting in 50bp length. Each probe was then aligned with all targeted genes that were analyzed in this study. The alignment was done using the Needleman-Wunsch algorithm with a gapopen penalty of 99 and a gapextend penalty of 10 (max), aiming to align the probes without gaps. X-axes are percent identity, and y-axes are Needleman-Wunsch scores. The size of each circle represents the number of alignments included in this circle. The exact hit between the probe and its target gene has 100% identity and a Needleman-Wunsch score of 250. The sequences higher than the 75% identity threshold show very low Needleman-Wunsch scores, which indicated partial alignment of the probes. (TIF)</p