Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum

Abstract

Diatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids have not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol.

Figure 1.

Preliminary comparison of accessions of P. tricornutum grown in artificial medium depleted in N or P. A, Geographical origin and major morphotypes of Pt accessions. The origin areas of sampling of Pt accessions are shown: Pt1 off Blackpool, United Kingdom; Pt2 and Pt3 off Plymouth, United Kingdom; Pt4 near the island of Segelskå; Pt5 in the Gulf of Maine; Pt6 off Woods Hole, Massachusetts; Pt7 off Long Island, New York; Pt8 near Vancouver, Canada; Pt9, Territory of Guam, Micronesia; PtHK, near Hong Kong; and Pt10, in the Yellow Sea. The genomic strain Pt1 8.6 derives from the Pt1 accession. Pt3 is a stress form deriving from Pt2. Major morphotypes observed for each accession in artificial seawater are indicated (i.e. the triradiate, fusiform, and oval morphotypes; from De Martino et al. [2007]). B, Accumulation of nonpolar lipids in N-limiting conditions. Cells in the exponential phase of growth were harvested by centrifugation and transferred to a fresh replete (1N1P; black bars) or N-depleted (0N1P; red bars) ESAW medium. Nonpolar lipid accumulation was measured after 3 d by Nile Red staining and expressed as fluorescence intensity normalized by cell number. C, Accumulation of nonpolar lipids in P-limiting conditions. Cells in the exponential phase of growth were harvested by centrifugation and transferred to a fresh replete (1N1P; black bars) or P-depleted (1N0P; blue bars) ESAW medium. Nonpolar lipid accumulation was measured after 8 d by Nile Red staining and expressed as fluorescence intensity normalized by cell number. r.f.u., Relative fluorescence units.

Figure 2.

Photosynthetic activity and lipid accumulation in the Pt1 ecotype of P. tricornutum cultivated in replete or N- or P-depleted conditions. A, Time-course evolution of photosynthetic efficiency. The F_v/F_m ratio, representative of the photosynthetic efficiency of the diatom, was measured for Pt1 cells grown either in a replete medium (10N10P; black) or in medium deprived of N (0N10P; blue) or P (10N0P; red). B, Nonpolar lipid accumulation measured at day 13. Nonpolar lipid accumulation was estimated by Nile Red fluorescence normalized to cell number. In 10N10P, the fluorescence signal remained at background level, indicating that the F_v/F_m decrease was not due to N or P starvation. r.f.u., Relative fluorescence units. C, Total glycerolipid accumulation. The total level of glycerolipids (membrane lipids + TAG) was estimated by the total FA content after 5 d of cultivation in the replete condition (black bar) or following N starvation (red bar) or 13 d of P starvation (blue bar). To avoid any N deficiency in 10N10P or 10N0P culture, the media were replaced by fresh ESAW 10N10P medium every 3 d.

Figure 3.

Separation by TLC of the glycerolipids from P. tricornutum. Lipids from Pt1 cells grown in a replete medium (10N10P) were extracted and resolved following the procedures described in “Materials and Methods.” The cross indicates the initial deposit. A, One-dimensional separation of nonpolar lipids (DAG and TAG) and free FA (FFA). Migration was performed in hexane:diethylether:acetic acid (70:30:1, v/v). B, Two-dimensional separation of polar (membrane) lipids. Migration was performed in chloroform:methanol:water (65:25:4, v/v) for the first dimension (arrow 1) and chloroform:acetone:methanol:acetic acid:water (50:20:10:10:5, v/v) for the second migration (arrow 2). Lipids were visualized under UV light, after spraying with 2% 8-anilino-1-naphthalenesulfonic acid in methanol, and scraped off the plate for analyses. Identification of the lipid in each spot was performed by MS2 analyses. The spot circled in white is an unknown compound with a structure that differs from a glycerolipid.

Figure 4.

Quantitative analysis of P. tricornutum glycerolipids. Lipids from Pt1 cells grown in a replete medium (10N10P) were extracted, separated by TLC, and analyzed as described in “Materials and Methods.” A, Global FA profile in a total lipid extract. FA proportions are given in percentages. B, Quantitative analysis of the various glycerolipids identified after TLC separation. Glycerolipids are expressed in nmol 10⁻⁶ cells and not as the summed FA content in each class. Each result is the average of three biological replicates ± sd. ASQ, 20:5-Acyl-SQDG; FFA, free FAs.

Figure 5.

Molar profiles of FAs in PC, DAG, TAG, MGDG, and SQDG. Lipids from Pt1 cells grown in a replete medium (10N10P) were extracted, separated by TLC, and analyzed for their FAs as described in “Materials and Methods.” Note that a cross contamination is possible between SQDG and PC due to the proximity of the TLC spots, leading to moderate enrichment of 20:5 in SQDG and 14:0 in PC. Each result is the average of three biological replicates ± sd.

Figure 6.

Quantitative analysis of FAs and glycerolipids in P. tricornutum grown in nutrient-replete conditions or in medium devoid of either N or P. Lipids from Pt1 cells grown either in a replete medium (10N10P; black) or in medium deprived of N (0N10P; blue) or P (10N0P; red) were extracted, separated by TLC, and quantified as described in “Materials and Methods.” To avoid any N deficiency in 10N10P or 10N0P culture, media were replaced by fresh ESAW 10N10P or 10N0P medium every 3 d. Lipids were analyzed after 5 d of cultivation in replete conditions (black bars), N starvation (red bars), or after 13 d for P starvation (blue bars). A, Changes in glycerolipid content. Note that in the P-depleted condition, phospholipids were not detectable. B, Changes in FA content. C, FA profile in TAG. Each result is the average of three biological replicates ± sd. ASQ, Acyl-SQDG; FFA, free FAs.

Figure 7.

Distribution of 16:1 and 25:0 FAs in the glycerolipid classes of P. tricornutum grown in nutrient-replete conditions or in medium devoid of either N or P. Lipids from Pt1 cells grown in a replete medium (10N10P) were extracted, separated by TLC, and quantified as described in “Materials and Methods.” Lipids were analyzed after 5 d of cultivation in replete condition (black bars), N starvation (red bars), or after 13 d of P starvation (blue bars). A, Quantitative distribution of 16:1 in the major glycerolipid classes. B, Quantitative distribution of 25:0 in the major glycerolipid classes. Each result is the average of three biological repeats ± sd.