Phosphorus-Induced Lipid Class Alteration Revealed by Lipidomic and Transcriptomic Profiling in Oleaginous Microalga Nannochloropsis sp. PJ12

Abstract

Phytoplankton are primary producers in the marine ecosystem, where phosphorus is often a limiting factor of their growth. Hence, they have evolved strategies to recycle phosphorus by replacing membrane phospholipids with phosphorus-free lipids. However, mechanisms for replacement of lipid classes remain poorly understood. To improve our understanding, we performed the lipidomic and transcriptomic profiling analyses of an oleaginous marine microalga Nannochloropsis sp. PJ12 in response to phosphorus depletion (PD) and replenishing. In this study, by using (liquid chromatography couple with tandem mass spectrometry) LC-MS/MS-based lipidomic analysis, we show that membrane phospholipid levels are significantly reduced upon PD, while phosphorus-free betaine lipid levels are increased. However, levels of phosphorus-free photosynthetic galactolipid and sulfolipid are not increased upon PD, consistent with the reduced photosynthetic activity. RNA-seq-based transcriptomic analysis indicates that enzymes involved in phospholipid recycling and phosphorus-free lipid synthesis are upregulated, supporting the lipidomic analysis. Furthermore, enzymes involved in FASII (type II fatty acid synthesis) elongation cycle upon PD are transcriptionally downregulated. EPA (eicosapentaenoic acid) level decrease upon PD is revealed by both GC-MS (gas chromatography coupled with mass spectrometry) and LC-MS/MS-based lipidomic analyses. PD-induced alteration is reversed after phosphorus replenishing. Taken together, our results suggest that the alteration of lipid classes upon environmental change of phosphorus is a result of remodeling rather than de novo synthesis in Nannochloropsis sp. PJ12.

Keywords: Nannochloropsis; lipid class; lipidomics; phosphate depletion; transcriptomics.

Figure 1

Characteristics of cell growth, photosynthetic efficiency, light harvesting pigments, and intra and extracellular phosphorus of PJ12 cultures upon phosphate depletion and restoration. (A) Cell growth and photosynthetic efficiency. Cell growth is determined gravimetrically. CDW stands for cell dry weight. Photosynthetic efficiency (Fv/Fm) is estimated based on PAM fluorescence. PR3, PD3, and PDR3 indicate the sampling points for cells under phosphate replete, phosphate depleted, and phosphate restored samples. (B) Level of light harvesting pigments chlorophyll a and carotene. Pigments are determined using colorimetric methods. (C) Level of intracellular and extracellular phosphorus. Phosphorus concentration is determined colorimetrically.

Figure 2

Characteristics of lipid composition in PJ12 cells prior to and after phosphate depletion and restoration. (A) TLC analysis of total lipid extracted from PJ12 cells. Samples in bold PR3, PD3, and PDR3 are subjected to transcriptomic and lipidomic analysis in triplicate. (B) Level of total acyl lipids. Asterisk sign (*) indicates the significance of level difference between PD3 and PR3, and between PDR3 and PD3. (C) GC analysis of total acyl lipids as FAME (fatty acid methyl esters). Asterisk sign (*) indicates the significance of level difference between PD3 and PR3, and between PDR3 and PD3. (D) Transmission electron microscope. LD and Ch stand for lipid droplet and chloroplast, respectively. A scale bar of 1 μm is indicated.

Figure 3

Lipidomic profiles of PR3, PD3, and PDR3 samples. (A) Level and ratio of 95 lipid molecules found in JP12 in triplicate. Average level and ratio of each molecule as indicated are shown. Asterisk (*) indicate the significant change of levels (level change > 2-fold, p-value < 0.05, n = 3). (B) Level of 15 lipid classes as indicated. Asterisk (*) indicate the significant change of levels (level change > 2-fold, p-value < 0.05, n = 3).

Figure 4

Common DE (differentially expressed) genes are phosphate-specific response genes. (A) Venn diagram showing the common DE genes between the DE gene upon phosphate depletion (PD/PR) and the DE genes upon phosphate restoration (PDR/PD). (B) K-mean 10-group clustering analysis of the common DE genes. (C) K-mean groups containing DE genes whose transcription level change inversely correlated with that of phosphate concentrations. (D) K-mean groups containing DE genes whose transcription level change positively correlated with that of phosphate concentrations.

Figure 5

Transcriptional profiling of enzymes involved in Calvin cycle, glycolysis, and TCA cycle. (A) A schematic drawing of the Calvin cycle, glycolysis, and TCA cycle. EC number of enzymes and metabolites are indicated. Differentially increased and decreased (level change > 1.5-fold, p-value < 0.05) enzymes whose EC number are shown in red and green, respectively. Moderately increased and decreased (level change > 10%) enzymes whose rectangle number box are shown in red and green, respectively. Enzymes not present in the transcriptome are shown without box. Left and right portions of EC numbers indicate the response to phosphate depletion (PD/PR) and phosphate restoration (PDR/PD), respectively. (B) Transcriptional response of enzymes involved in Calvin cycle. Level increase and decrease are indicated by magenta and cyan, respectively. Copy number (Cp#) of enzyme-coding genes are indicated. Asterisk (*) indicate the significant level change (level change > 1.5-fold, p-value < 0.05). (C) Transcriptional response of enzymes involved in glycolysis. The display is identical to (B). (D) Transcriptional response of enzymes involved in TCA cycle. The display is identical to (B).

Figure 6

Transcriptional profiling of enzymes involved in type II fatty acid synthesis (FASII), glycerolipid synthesis in plastid (GLSp) and cytosol glycolysis (GLSc), and beta-oxidation. (A) A schematic drawing of the FASII, GLSp, GLSc, and beta-oxidation. Metabolites whose level increase or decrease is shown in red and green, respectively. Significant (level change > 1.5-fold, p-value < 0.05) and moderate (level change > 10%) changes are indicated by fill and border, respectively. Left and right portions present the change upon phosphate depletion (PD/PR) and phosphate restoration (PDR/PD), respectively. The display is identical to Figure 5A. (B) Transcriptional response of enzymes involved in FASII. The display is identical to Figure 5B. (C) Transcriptional response of enzymes involved in GLSp. The display is identical to Figure 5B. (D) Transcriptional response of enzymes involved in beta-oxidation. The display is identical to Figure 5B. (E) Transcriptional response of enzymes involved in GLSc. The display is identical to Figure 5B. (F) Level change of metabolites involved in the GLSp. It is synthesized through the “prokaryotic” pathway. (G) Level change of metabolites involved in the GLSc. It is synthesized through the “eukaryotic” pathway.

Figure 7

Transcriptional profiling of enzymes involved in PUFA synthesis. (A) A schematic drawing of the PUFA synthetic pathway. The display is identical to Figure 6A. (B) Transcriptional response of enzymes involved in PUFA synthetic pathway. The display is identical to Figure 5B. (C) Level change of metabolites involved in PUFA synthetic pathway. The display is identical to Figure 6F.