Quantitative Proteomics Reveals Common and Specific Responses of a Marine Diatom Thalassiosira pseudonana to Different Macronutrient Deficiencies

Abstract

Macronutrients such as nitrogen (N), phosphorus (P), and silicon (Si) are essential for the productivity and distribution of diatoms in the ocean. Responses of diatoms to a particular macronutrient deficiency have been investigated, however, we know little about their common or specific responses to different macronutrients. Here, we investigated the physiology and quantitative proteomics of a diatom Thalassiosira pseudonana grown in nutrient-replete, N-, P-, and Si-deficient conditions. Cell growth was ceased in all macronutrient deficient conditions while cell volume and cellular C content under P- and Si-deficiencies increased. Contents of chlorophyll a, protein and cellular N decreased in both N- and P-deficient cells but chlorophyll a and cellular N increased in the Si-deficient cells. Cellular P content increased under N- and Si-deficiencies. Proteins involved in carbon fixation and photorespiration were down-regulated under all macronutrient deficiencies while neutral lipid synthesis and carbohydrate accumulation were enhanced. Photosynthesis, chlorophyll biosynthesis, and protein biosynthesis were down-regulated in both N- and P-deficient cells, while Si transporters, light-harvesting complex proteins, chloroplastic ATP synthase, plastid transcription and protein synthesis were up-regulated in the Si-deficient cells. Our results provided insights into the common and specific responses of T. pseudonana to different macronutrient deficiencies and identified specific proteins potentially indicating a particular macronutrient deficiency.

Keywords: Thalassiosira pseudonana; macronutrient; marine diatom; nitrogen; phosphorus; quantitative proteomics; silicon.

FIGURE 1

Cell density (A), Fv/Fm (B), and cell volume (C) of T. pseudonana grown under different macronutrient deficiencies (N deficiency, P deficiency, Si deficiency, and nutrient replete conditions). Error bars represent the standard deviations of the means generated from triplicates.

FIGURE 2

External nutrient concentration, cellular elemental composition, and biosynthetic compounds content per cell of T. pseudonana in different macronutrient deficient conditions. Error bars represent the standard deviations of the means generated from triplicates. ^∗∗P < 0.01 and ^∗P < 0.05 indicate significant correlation.

FIGURE 3

The Venn diagram (A), the number (B), and the hierarchical cluster (C) of DEPs in T. pseudonana in N-, P-, and Si- deficient cells relative to the nutrient-replete cells.

FIGURE 4

The number of DEPs in T. pseudonana from several crucial KEGG pathways that were significantly enriched with a p-value of less than 0.05 under different macronutrient deficiencies. (A) DEPs under N-deficiency; (B) DEPs under P-deficiency; (C) DEPs under Si-deficiency. Changes are denoted as the percentage of up-regulated (red) and down-regulated (blue) genes within each pathway.

FIGURE 5

Heat maps of DEPs in T. pseudonana from key pathways for different expressions in the N-, P-, and -Si deficient cells relative to the nutrient-replete cells. (A) Photoreaction and chloroplastic ATPase; (B) Carbon fixation; (C) Carbohydrate metabolism; (D) Chlorophyll biosynthesis. Each nutrient condition corresponds to a single column and each protein to a single row. The color chart indicates fold change of protein expression using a base 2-logarithmic scale. The color scale ranges from saturated firebrick for up-regulated proteins to saturated navy for down-regulated proteins; white indicates no significant change.

FIGURE 6

Heat maps of differentially expressed ribosomal proteins in T. pseudonana for different expressions in the N-, P-, and -Si deficient cells relative to the nutrient-replete cells. Each nutrient condition corresponds to a single column and each protein to a single row. The color chart indicates fold change of protein expression using a base 2-logarithmic scale. The color scale ranges from saturated firebrick for up-regulated proteins to saturated navy for down-regulated proteins; white indicates no significant change.

FIGURE 7

Relative transcripts of selected genes from key biological processes in T. pseudonana in the N-, P-, and -Si deficient cells relative to the nutrient-replete cells. (A) N transport and utilization; (B) P transport and utilization; (C) Si transport; (D) Carbon fixation; (E) Photorespiration; (F) Glycolysis; (G) Pyruvate dehydrogenase; (H) TCA cycle; (I) Chlorophyll synthase; (J) Lipid synthesis. Error bars represent the standard deviations of the values generated from three biological replicates. ^∗∗P < 0.01 and ^∗P < 0.05 indicate significant correlation.

FIGURE 8

Cellular pathways and processes affected by different macronutrient deficiencies in T. pseudonana. (A) N-deficiency; (B) P-deficiency; (C) Si-deficiency. Red, blue, and black texts indicate up-regulation, down-regulation and no change of pathways or proteins. OAA, oxaloacetate; RuBP, ribulose-1,5-bisphosphate; Ru5P, ribulose-5P; G3P, glycerate-3P; Chl a, chlorophyll a; chlG, chlorophyll synthase; LHC, light-harvesting chlorophyll protein complex; PPDK, pyruvate phosphate dikinase; PEPC, phosphoenolpyruvate carboxylase; CA, carbonic anhydrase; rbcL, Rubisco large chain; rbcS, Rubisco small chain; PRK, phosphoribulokinase; TKT2, fructose-bisphosphatealdolase; PGK, phosphoglycerate kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SIT, silicic acid transporter; TC.NCS2, xanthine/uracil permease; NRT, nitrate/nitrite transporter; DUR3, urea-proton symporter; SLC14A, urea transporter; NPT, sodium phosphate co-transporter; AP, alkaline phosphatase; uraH, 5-hydroxyisourate hydrolase; URE, urease; alc, allantoicase; NIT2, nitrilase; NIR_2, Ferredoxin-nitrite reductase; GLN, glutamine synthetase; VTC4, vacuolar transporter chaperone 4; SHMT, glycine/serine hydroxymethyltransferase; AGXT, alanine-glyoxylate transaminase; gcvP, glycine decarboxylase P protein; gcvH, glycine decarboxylase H protein; gcvT, glycine decarboxylase T protein; GRHPR, glycerate dehydrogenase/hydroxypyruvatereductase; PDHA1, pyruvate dehydrogenase E1 component subunit alpha-1; PDHB1, pyruvate dehydrogenase E1 component subunit beta-1; LAT2, pyruvate dehydrogenase E2 (dihydrolipoamide s-acetyltransferase); CS, citrate synthase; ACO, aconitasehydratase 2; icd, isocitrate dehydrogenase; SDHA, succinate dehydrogenase flavoprotein subunit; ACACA, acetyl-CoA carboxylase; ACSL, long-chain acyl-CoA synthetases; G6PD, glucose-6-phosphate 1-dehydrogenase; PGLS, 6-phosphogluconolactonase; PGD, 6-phosphogluconate dehydrogenase; talA, ttransaldolase; pgm, phosphoglucomutase; GPI, glucose-6-phosphate isomerase; FBP, fructose-1,6-bisphosphatase; pfkA, 6-phosphofructokinase; ALDO, fructose-bisphosphatealdolase; TPI, triose-phosphate isomerase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; ENO, alpha enolase; and PYK, pyruvate kinase.