The small molecule fenpropimorph rapidly converts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii

Abstract

Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels. Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources. However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive. Here, we report that the fungicide fenpropimorph rapidly causes high levels of neutral lipids to accumulate in Chlamydomonas reinhardtii cells. When treated with fenpropimorph (10 μg mL(-1)) for 1 h, Chlamydomonas cells accumulated at least fourfold the amount of triacylglycerols (TAGs) present in the untreated control cells. Furthermore, the quantity of TAGs present after 1 h of fenpropimorph treatment was over twofold higher than that formed after 9 days of nitrogen starvation in medium with no acetate supplement. Biochemical analysis of lipids revealed that the accumulated TAGs were derived mainly from chloroplast polar membrane lipids. Such a conversion of chloroplast polar lipids to TAGs is desirable for biodiesel production, because polar lipids are usually removed during the biodiesel production process. Thus, our data exemplified that a cost and time effective method of producing TAGs is possible using fenpropimorph or similar drugs.

Keywords: Chlamydomonas reinhardtii fenpropimorph; biofuel; membrane lipid recycling; triacylglycerol.

FIGURE 1

Fenpropimorph induces neutral lipid accumulation in Chlamydomonas reinhardtii. (A) Fenpropimorph-induced LD formation occurs in a dose-dependent manner. The fluorescence intensity (FI) of a neutral lipid specific-dye, Nile red, was determined. Late mid-log phase Chlamydomonas cells (N+, acetate+) were treated with ethanol (solvent control) or fenpropimorph (1 h, at RT). Averages from three replicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Fenpropimorph-induced TAGs were extracted and analyzed using biochemical methods. Control cells were treated with the same volume of ethanol used to dissolve fenpropimorph. Averages from triplicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Images of Nile red-stained LD accumulation in fenpropimorph-treated cells. Cells were treated with fenpropimorph for 1 h. Images were obtained using a fluorescence microscope. (D) Time-dependent change in TAG concentration in fenpropimorph-treated Chlamydomonas cells. TAG accumulation induced by fenpropimorph (10 μg mL^-1) treatment was analyzed biochemically. Averages and SE from three replicate experiments are presented. TAG levels shown were converted to μg from nmol values obtained from GC experiment. The original nmol values for each time point (5, 15, 45, 85, and 125 min) were 26.8 ± 4.7, 40.9 ± 0.4, 51.2 ± 5.1, 66.1 ± 24.2, and 27.7 ± 5.7, respectively, for control samples, and 65.4 ± 3.0, 120.7 ± 1.2, 255.0 ± 8.5, 260.7 ± 21.9, and 278.0 ± 1.0, respectively, for fenpropimorph-treated samples. In experiments shown in (A – D), Chlamydomonas cells in late mid-log phase culture in TAP medium (N+, acetate+) were used. (E,F) Comparison of the effect of nitrogen deprivation and fenpropimorph treatment on lipid induction efficiency in Chlamydomonas cells. (E) Nile red fluorescence intensity of control Chlamydomonas cells, and of cells subjected to fenpropimorph treatment (1 h, 25°C), and nitrogen deprivation (for the indicated number of days). Chlamydomonas cells were grown in normal conditions to mid-log phase, and washed to remove acetate and nitrogen from the medium. They were then re-suspended in TAP medium without an acetate or nitrogen source, and then either treated with ethanol (solvent control) or fenpropimorph (10 μg mL^-1) for 1 h, or transferred to the nitrogen-deficient conditions and incubated for 3, 6, or 9 days. The FI value was measured. Averages from three replicate experiments are presented. Bars represent SE. (F) Biochemical analysis of TAG content in cells treated as in (E). Averages from three replicate experiments are presented. Bars represent SE.

FIGURE 2

Biochemical analysis of TAGs in fenpropimorph-treated C. reinhardtii in late mid-log phase culture in TAP medium (N+, acetate). (A) A comparison of the absolute amount of fatty acids in TAGs isolated from fenpropimorph-treated and control cells. Averages from three replicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Comparison of fatty acid mol% in TAGs isolated from fenpropimorph-treated cells and control cells. Averages from three replicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 3

Alterations in polar membrane lipid and TAG profiles of fenpropimorph-treated C. reinhardtii at late mid-log phase cultured in TAP medium (N+, acetate+). (A) Comparison of the total fatty acid amount in polar lipids, TAGs and total acyl lipids. Bars represent SE (N = 2, n = 3). Significant differences, as determined by Student’s t-test, are indicated by asterisks (***p < 0.001). (B) Comparison of fatty acid profiles in total lipids. A representative result of 10 experiments is shown. Bars represent SE (n = 3). (C) Comparison of the concentration of major membrane lipids and TAGs between fenpropimorph-treated and control cells. Averages from three replicate experiments are presented. Bars represent SE (N = 2, n = 6). Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, ***p < 0.001).

FIGURE 4

Time-dependent changes in TAG (A), MGDG (B), and DGDG (C) contents in fenpropimorph-treated C. reinhardtii. The cells were treated with 10 μg mL^-1 fenpropimorph for up to 1 h at 25°C. Chlamydomonas cells in late mid-log phase culture in TAP medium (N+, acetate+) were used. Averages from three replicate experiments are represented. Bars represent SE.

FIGURE 5

Analysis of lipids labeled with [¹⁴C]acetate in fenpropimorph-treated C. reinhardtii. (A) DAG is found to contain 14C-labeled in the fenpropimorph-treated cells. Two-phase TLC was performed as described in Materials and methods. The left image represents the radioactivity of lipids derived from [¹⁴C]acetate. The right image shows primuline staining of the same TLC plate. (B) Disintegration per minute (DPM) values of non-polar and polar lipids of the solvent control (ethanol) and fenpropimorph-treated cells (10 μg mL^-1, 1 h, at 25°C) in TAP medium without acetate. Averages and SE from three replicate experiments are presented. Significant differences, as determined by Student’s t-test, are indicated by asterisks (**p < 0.01, ***p < 0.001).

FIGURE 6

Comparison of total sterol amount and sterol composition between control and fenpropimorph-treated C. reinhardtii. (A) Total sterol amount of control and fenpropimorph-treated (10 μg mL^-1, 1 h, at 25°C) cells. Chlamydomonas cells in late mid-log phase culture in TAP medium (N+, acetate+) were used. Averages from three replicate experiments are presented. Bars represent SE. (B) Sterol compositions of control and fenpropimorph-treated (10 μg mL^-1, 1 h, at 25°C) cells.

Figure A1

Growth phase-dependent effect of treatment with fenpropimorph (10 μg mL^–1) on Nile red fluorescence of Chlamydomonas. The fluorescence values are normalized by optical density at 750 nm. Averages from three experimental replicates are presented. Bars represent SE.

FIGURE A2

Acetate in the medium was not necessary for the fenpropimorph effect on TAG accumulation. Note the absence of difference in Nile red fluorescence intensities of cells treated with fenpropimorph in media with or without acetate. Chlamydomonas cells in late mid-log phase culture in the TAP (N+, acetate+) medium were harvested, and resuspended either in the same TAP medium or in TAP medium without acetate, then treated with fenpropimorph (10 or 15 μg mL^-1) for 1 h at 25°C. Averages from three experimental replicates are presented. Bars represent SE.

FIGURE A3

Chlorophyll a and b and carotenoid contents of fenpropimorph-treated Chlamydomonas strain CC-125 cells. Cells in TAP (N+, acetate+) medium were treated with fenpropimorph (10 μg mL^-1) or ethanol (solvent control) for 1 h at 25°C. Chlorophyll and carotenoid contents were assayed following the method previously described (Lichtenthaler and Babani, 2004). Approximately 6.0 × 10⁶ cells were collected by centrifugation (2000 g; 10 min), and the pellet was resuspended in 95% ethanol and mixed by vortexing. After centrifugation, the supernatant was transferred to a new 96-well plate and optical absorbance at 648 nm and 664 nm were measured using a Safire fluorescence spectrophotometer (TECAN, Switzerland). Each pigment content was calculated as described previously (Lichtenthaler and Babani, 2004). Averages and SE from three replicate experiments are presented.

FIGURE A4

Fenpropimorph treatment results in Chlamydomonas cell death. Cells in TAP (N+, acetate+) medium were treated with fenpropimorph or ethanol (solvent control) for 1 h at 25°C, then stained with propidium iodide (2 μg mL^-1, 15 min). Nuclear staining of cells treated with fenpropimorph suggests that the cells are dead.

FIGURE A5

Change in chloroplast morphology in fenpropimorph-treated Chlamydomonas cells. (A) Chloroplasts are shrunken in fenpropimorph-treated Chlamydomonas cells. (B) Quantification of the chloroplast area per cell surface area in microphotographs of fenpropimorph-treated Chlamydomonas cells. Averages from fifty experimental replicates are presented. Bars represent SE.

FIGURE A6

Effect of 24-h treatment with various concentrations of fenpropimorph on Nile red fluorescence of Chlamydomonas. Cells were incubated till late mid-log phase in the TAP (N+, acetate+) medium, treated with fenpropimorph (1.25, 2.5, or 10 μg mL^-1) for 24 h at 25°C, then stained with Nile red. The fluorescence intensity (FI) was determined from three replicates and their averages are presented. Bars represent SE.