Fig 1.
Cell growth (abundance, left vertical axis) and photosynthetic efficiency (Fv/Fm, right vertical axis) of N-limited (A) and P-limited (B) T. pseudonana cultures (mean ± SD of triplicate measurements).
Fig 2.
Concentrations of N-NO3− (A) and P-PO43− (B) in media when T. pseudonana cells were cultured under control, N-limited, and P-limited conditions from days 0 to 8. Concentrations of intracellular phosphorus indicated as particulate phosphorus (C) in control and P-limited T. pseudonana cells from days 0 to 6. The error bars represent the standard errors from triplicate measurements (mean±SD).
Table 1.
General physiological and biochemical characteristics of N-limited, P-limited, and control T. pseudonana cells on day 4.
Fig 3.
Examination of ROS production, biochemical characteristics of programmed cell death (PCD), and cell death in T. pseudonana cells grown under different conditions.
In vivo detection of the ROS level (CM-H2DCFDA; A, E), caspase activity (CaspACE; B, F), externalization of phosphatidylserine (Annexin V; C, G), and dead cells (SYTOX; D, H) in T. pseudonana using flow cytometry. Cells were grown in nutrient replete (control), N-limited (A-D), or P-limited (E-H) media on days 2–7. Numbers above columns represent ratios of positive cells under limited: control conditions.
Fig 4.
Good reproducibility between two biological replicates for each cultural condition, which indicated by the data that 95% of the proteins had differences with a delta error of less than 0.5 between two samples in all three conditions.
(A) Control conditions; (B) N-limited culture conditions; (C) P-limited culture conditions.
Table 2.
Summary of numbers of identified proteins under different conditions on day 4.
Fig 5.
Functional category (COG) coverage of the significantly regulated proteins identified in T. pseudonana under N-limited (A) and P-limited (B) conditions based on iTRAQ-LC-MS/MS analysis. One protein may be assigned to more than one functional category. Significantly regulated proteins without COG description (58 and 35 proteins in N- and P-limited cells, respectively) are not shown.
Fig 6.
Comparison of mRNA and protein levels of selected genes.
Genes include TpDSP1 (Thaps3_11118), TPR (tetratricopeptide repeat protein, Thaps3_22882), NTPase (Thaps3_24162), RRM domain (RNA-binding proteins, Thaps3_21534), PDCE2 (pyruvate/2–oxoglutarate dehydrogenase complex, dihydrolipoamide acyltransferase (E2) component, Thaps3_14235), AP (alkaline phosphatase protein, Thaps3_1179), AP-like protein (Thaps3_1669), CDK (cyclin-dependent kinase, Thaps3_20362), PSrp-1 (ribosome-associated protein Y, Thaps3_255232), AOX1_2 (Thaps3_38428), AOX2 (Thaps3_42992), and TpDSP2 (Thaps3_11117). Translation elongation factor-1α (Thaps3_29435) and ubiquitin-conjugating enzyme (Thaps3_27711) were used as housekeeping markers. Fold changes in the transcript expression for individual genes in N-limited (A) or P-limited (B) cells relative to control are normalized by the geometric mean of the housekeeping markers. The expression of genes unidentified in the present iTRAQ data are indicated in the right of the vertical dotted lines. Left and right vertical axes show relative mRNA and relative protein levels, respectively (mean log2 x-fold expression ratio ± SD from triplicate measurements).
Fig 7.
Hypothetical cellular pathways and processes in the diatom T. pseudonana under N-limited growth conditions.
Red words and arrows represent proteins with increased abundance and enhanced pathways, respectively. Green words and arrows represent proteins with decreased abundance and inhibited pathways, respectively. Fluorescence micrograph shows the neutral lipid accumulation in N-limited cells. HemA: porphobilinogen deaminase; BchP: dehydrogenases (flavoproteins); Psb27: photosystem II Psb27 protein; PSII AF: uncharacterized protein related to plant photosystem II stability/assembly factor; Fd: ferredoxin; FNR: ferredoxin-NADP reductase; Cyto c2: cytochrome c2; COX6B1: cytochrome c oxidase subunit 6B1; ROS: reactive oxygen species; Msr: peptide methionine sulfoxide reductase; PCD: programmed cell death; TpDSP: T. pseudonana death-specific protein; FKBP PPIase 1: FKBP-type peptidyl-prolyl cis–trans isomerase 1; GlcN6P deaminase: glucosamine-6-phosphate isomerase; MenG: demethylmenaquinone methyltransferase; PDCE2: pyruvate/2-oxoglutarate dehydrogenase complex, dihydrolipoamide acyltransferase (E2) component; TCA: tricarboxylic acid cycle; GSIII: uncharacterized protein related to glutamine synthetase (GSIII).
Fig 8.
Neutral lipid accumulation in N-limited, P-limited and control T. pseudonana cells at sampled time points for proteomic analysis (day 4).
Fluorescence micrographs (B, D, F) and the corresponding bright-field images (A, C, E) of oil-containing lipid bodies stained with green fluorescent dye (BODIPY 505/515) in cells cultured in control (A, B), N-limited (C, D), and P-limited (E, F) conditions on day 4. Bars: 5 μm.