Abstract
Autophagy mediates degradation and recycling of cellular components and plays an important role in senescence and adaptive responses to biotic and abiotic stresses. Nutrient deprivation has been shown to trigger triacylglycerol (TAG) accumulation and also induces autophagy in various green algae. However, the functional relationship between TAG metabolism and autophagy remains unclear. To gain preliminary evidence supporting a role of autophagy in TAG synthesis, Chlamydomonas reinhardtii CC-2686 was grown in Tris-acetate phosphate medium with or without nitrogen and treated with an autophagy inducer (rapamycin) or inhibitors (wortmannin, 3-methyladenine, and bafilomycin A1). Fluorescence microscopic analysis of Nile red-stained cells following 72-h treatments showed that rapamycin induced accumulation of subcellular lipid droplets which are storage sites of TAG. Rapamycin treatment in combination with nitrogen starvation led to a greater abundance of lipid droplets. Wortmannin and bafilomycin A1, but not 3-methyladenine, inhibited lipid droplet accumulation in rapamycin-treated cells and to a less extent in nitrogen-depleted cells. These results suggested that autophagy contributes to TAG synthesis in C. reinhardtii, but is not a necessary process. Autophagy induction may also be used to further enhance TAG accumulation in microalgae under nutrient deprivation.
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Acknowledgements
This research was supported by the PTT Research and Technology Institute, the Royal Golden Jubilee Ph.D. Program of Thailand (Thailand Research Fund), the Center of Excellence on Environmental Health and Toxicology (EHT), the Science & Technology Postgraduate Education and Research Development Office (PERDO), and the Ministry of Education, Thailand.
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Fig. S1
Appearance of C. reinhardtii CC-2686 cultures grown for 72 h in normal TAP medium (+N), TAP-N (-N), and TAP medium containing 1 μM rapamycin (+N, Rapa(1 μM)) in which autophagy inhibitors wortmannin (WM), 3-methyladenine (3-MA), and bafilomycin A1 (Baf) were added to the final concentration as indicated. (JPEG 169 kb)
Fig. S2
Representative images of Nile red-stained cells of C. reinhardtii CC-2686. The alga was grown in TAP-N medium, TAP-N medium containing inhibitors, and TAP-N medium containing 1 μM rapamycin for 72 h. Scale bar represents 10 μm. Three images are shown for each treatment. (JPEG 139 kb)
Fig. S3
TLC analysis of total lipids from C. reinhardtii CC-124 cells grown in normal TAP medium (+N), TAP medium containing 1 μM rapamycin (+N, Rapa(1 μM)), TAP medium containing 1 μM rapamycin together with 0.1 μM bafilomycin A1 (+N, Rapa(1 μM)-Baf(0.1 μM)), TAP-N medium (-N), and TAP-N medium containing 0.1 μM bafilomycin A1 (-N, Baf(0.1 μM) for 72 h. Equal amounts of cells, 5×106 cells, were used for each lane. Representative images are shown. (JPEG 56 kb)
Fig. S4
Fatty acid composition of TAG derived from TLC separation (Fig. S3) of total lipids from 5×106 cells C. reinhardtii CC-124 cells grown in normal TAP medium (+N), TAP medium containing 1 μM rapamycin (+N, Rapa(1 μM)), TAP medium containing 1 μM rapamycin together with 0.1 μM bafilomycin A1 (+N, Rapa(1 μM)-Baf(0.1 μM)), TAP-N medium (-N), and TAP-N medium containing 0.1 μM bafilomycin A1 (-N, Baf(0.1 μM) for 72 h. Bands corresponding to TAG were removed from TLC plates, transesterified with methanol, and quantified using GC-MS as described in materials and methods. Each bar represents mean ± SEM of 6 replications. (JPEG 52 kb)
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Pugkaew, W., Meetam, M., Ponpuak, M. et al. Role of autophagy in triacylglycerol biosynthesis in Chlamydomonas reinhardtii revealed by chemical inducer and inhibitors. J Appl Phycol 30, 15–22 (2018). https://doi.org/10.1007/s10811-017-1166-7
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DOI: https://doi.org/10.1007/s10811-017-1166-7