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. 2015 Feb 24;6:54. doi: 10.3389/fmicb.2015.00054

FIGURE 1.

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.