Betaine Lipid Is Crucial for Adapting to Low Temperature and Phosphate Deficiency in Nannochloropsis

Abstract

Diacylglyceryl-N,N,N-trimethylhomo-Ser (DGTS) is a nonphosphorous, polar glycerolipid that is regarded as analogous to the phosphatidylcholine in bacteria, fungi, algae, and basal land plants. In some species of algae, including the stramenopile microalga Nannochloropsis oceanica, DGTS contains an abundance of eicosapentaenoic acid (EPA), which is relatively scarce in phosphatidylcholine, implying that DGTS has a unique physiological role. In this study, we addressed the role of DGTS in N. oceanica We identified two DGTS biosynthetic enzymes that have distinct domain configurations compared to previously identified DGTS synthases. Mutants lacking DGTS showed growth retardation under phosphate starvation, demonstrating a pivotal role for DGTS in the adaptation to this condition. Under normal conditions, DGTS deficiency led to an increase in the relative amount of monogalactosyldiacylglycerol, a major plastid membrane lipid with high EPA content, whereas excessive production of DGTS induced by gene overexpression led to a decrease in monogalactosyldiacylglycerol. Meanwhile, lipid analysis of partial phospholipid-deficient mutants revealed a role for phosphatidylcholine and phosphatidylethanolamine in EPA biosynthesis. These results suggest that DGTS and monogalactosyldiacylglycerol may constitute the two major pools of EPA in extraplastidic and plastidic membranes, partially competing to acquire EPA or its precursors derived from phospholipids. The mutant lacking DGTS also displayed impaired growth and a lower proportion of EPA in extraplastidic compartments at low temperatures. Our results indicate that DGTS is involved in the adaptation to low temperatures through a mechanism that is distinct from the DGTS-dependent adaptation to phosphate starvation in N. oceanica.

Figure 1.

Biosynthetic pathway for DGTS. A, DGTS is synthesized from DAG via two step reactions. DGHS, Diacylglycerylhomo-Ser. B, Biosynthetic enzymes for DGTS with their catalytic domains. RsBtaA and the C-terminal domain of CrBTA1 catalyze the first step, and RsBtaB and the N-terminal domain of CrBTA1 catalyze the second step. Domains of 3-amino-3-carboxypropyltransferase and N-methyltransferase predicted with Pfam 30.0 are indicated by red and blue boxes, respectively. The start and end point of the domains are indicated above each construct, and the total number of amino acid residues (AA) is shown to the right. Rs, Rhodoobacter sphaeroides; Cr, Chlamydomonas reinhardtii.

Figure 2.

Changes in membrane lipid composition relative expression level of BTA1L and BTA1S genes under stress. A, Membrane lipid composition of N. oceanica wild-type strain grown under normal, nitrogen-starved, Pi-starved, or 15°C conditions for 3 d. B and C, Relative expression of BTA1L (B) and BTA1S (C) under the four conditions. Expression was normalized to that of the gene encoding NADH dehydrogenase subunit 11. Data represent the means ± sd of three biologically independent experiments. PG, Phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol.

Figure 3.

DGTS synthesis in bta1l and the BTA1L- or BTA1LΔC-complemented mutant as assessed with TLC. Total lipid of EV, bta1l, bta1l;BTA1LΔC, bta1s;bta1l, bta1s;bta1l;BTA1LΔC, and bta1s;bta1l;BTA1L cultivated under Pi-starved conditions for 4 d were separated by one-dimensional TLC using solvent system chloroform/methanol/acetic acid/water (170:30:15:3, v/v/v/v). The asterisk denotes spots of DGTS.

Figure 4.

Phenotypes of mutants bta1l, bta1s, and bta1s;bta1l. A and B, Growth curve for EV, bta1l, bta1s, and bta1s;bta1l under Pi-sufficient (A) and Pi-deficient (B) conditions. Each cell culture was diluted with medium to an initial density of 10⁷cells mL^–1. Data represent the mean ± sd of four (A) and three (B) biologically independent experiments. C, Comparison of relative polar lipid composition. The EV line (control) and mutant lines were cultivated under normal conditions for 3 d and then harvested for lipid analysis. Data represent the mean ± sd of three biologically independent experiments. Statistical significance was determined with Dunnett’s test (compared with EV): #, P < 0.1; *, P < 0.05; **, P < 0.01. SQDG, Sulfoquinovosyldiacylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; N.D., not detected.

Figure 5.

Membrane lipid analysis of the BTA1L overexpression line. A, Comparison of membrane lipid composition between EV and OEBTA1L. B to D, Fatty acid profiles for DGTS (B), MGDG (C), and DGDG (D). Data represent the mean ± sd of three biologically independent experiments. Statistical significance was determined with Tukey’s test and is indicated by letters at the top. E, Distribution of 20:5 within the eight classes of membrane lipid after 2 d of culture. Data represent the mean ± sd of three biologically independent experiments. Statistical significance was determined with the two-tailed Student’s t test: *, P < 0.05; **, P < 0.01. SQDG, Sulfoquinovosyldiacylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol.

Figure 6.

Membrane lipid composition of mutants cct1 and pect1 grown under normal conditions. A, Membrane lipid composition compared with EV (control). EV and mutant lines were cultured for 3 d under normal conditions before lipid analysis. B to F, Fatty acid profile of PC (B), PE (C), MGDG (D), DGDG (E), and DGTS (F) in EV, cct1, and pect1. Data represent the mean ± sd of four biologically independent experiments. Statistical significance was determined with Dunnett’s test (compared with EV): #, P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001. SQDG, Sulfoquinovosyldiacylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol.

Figure 7.

Phenotypes of the mutants bta1l, cct1, and pect1 grown at low temperature. A, Number of cells for EV and the indicated mutants after 3 d of culture. B to E, Comparison of relative polar lipid composition (B) and fatty acid profile of PC (C), PE (D), and DGTS (E) in EV, bta1l, cct1, and pect1. F and G, Molar ratio of 20:5 in extraplastidic (F) and plastidic (G) membrane lipids. Data represent the mean ± sd of three biologically independent experiments. Statistical significance was determined with Dunnett’s test (compared with EV): *, P < 0.05; **, P < 0.01; ***, P < 0.001. SQDG, Sulfoquinovosyldiacylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol.

Figure 8.

Schematic model for the DGTS biosynthesis in Nannochloropsis. We proposed that 20:5-containing DGTS and 20:5-free DGTS are formed by different pathways with different physiological relevance. The 20:5-enriched DAG derived from phospholipids would diverge to use for DGTS or MGDG biosynthesis catalyzed by BTA1L and BTA1S or MGD1, respectively. Blue represents enzymes and black represents metabolites.G3P, Glycerol 3-phosphate; MGD1, MGDG synthase 1.