J Appl Phycol DOI 10.1007/s10811-017-1166-7

9TH ASIA PACIFIC CONFERENCE ON ALGAL BIOTECHNOLOGY - BANGKOK

Role of autophagy in triacylglycerol biosynthesis in Chlamydomonas reinhardtii revealed by chemical inducer and inhibitors Wanvisa Pugkaew 1,2 & Metha Meetam 1,2 & Marisa Ponpuak 3,4 & Kittisak Yokthongwattana 5,6 & Prayad Pokethitiyook 1,2

Received: 5 January 2017 / Revised and accepted: 3 May 2017 # Springer Science+Business Media Dordrecht 2017

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 Trisacetate 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 Electronic supplementary material The online version of this article (doi:10.1007/s10811-017-1166-7) contains supplementary material, which is available to authorized users. * Prayad Pokethitiyook [email protected] 1

Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

2

Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Bangkok, Thailand

3

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

4

Systems Biology of Diseases Research Unit, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

5

Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

6

Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

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. Keywords Autophagy . Triacylglycerol . Biodiesel . Lipid droplet . Chlamydomonas reinhardtii

Introduction Macroautophagy (hereafter autophagy) is a catabolic process that functions in cellular degradation and nutrient recycling during the time of stress. This process is found to be conserved among eukaryotic organisms including yeasts, mammals, plants, and algae (Díaz-Troya et al. 2008). Autophagy in photosynthetic eukaryotes has been shown to play a major role in the adaptive response to biotic and abiotic stresses such as an oxidative stress, nutrient starvation, pathogen infection, or senescence (Bassham 2007; Díaz-Troya et al. 2008). Upon receiving an autophagy induction signal, cytoplasmic materials are engulfed into a double membrane bound organelle called autophagosome, which is upon its fusion to the lysosome resulting in the breakdown of the engulfed contents (Rubinsztein et al. 2007). Autophagy induction can be initiated by multiple signaling pathways, one of which is the inhibition of the target of rapamycin (TOR) protein (Mizushima et al. 2010). TOR is an essential protein kinase that regulates cell growth by promoting protein and ribosome synthesis and suppressing mRNA degradation and autophagy (Díaz-Troya et al. 2008).

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TOR function is conserved among eukaryotic organisms, but the number of TOR genes in different organisms may differ. For example, algae contain one gene encoding for TOR while yeast and fungi contain two TOR genes (Díaz-Troya et al. 2008). Active TOR can inhibit autophagy through the suppression of ATG1 activity (Scott et al. 2007). However, during nutrient deprivation or a treatment of TOR inhibitor such as a drug named rapamycin, ATG1 activity is no longer inhibited by TOR and autophagy induction signal can ensue (Fingar and Blenis 2004). Rapamycin has been shown to successfully inhibit TOR activity in yeast and mammals, but it fails to inactivate TOR in plants such as Arabidopsis thaliana (Díaz-Troya et al. 2008; Menand et al. 2002). Crespo et al. (2005) showed that TOR can also be inhibited in Chlamydomonas reinhardtii by rapamycin, resulting in a reduction in algal cell growth and an increase in vacuolization. However, Chlamydomonas rap2 mutant, which lacks the rapamycin-binding protein (FKBP12), exhibits a full resistance to the drug (Crespo et al. 2005). After the autophagy induction signal, autophagosome formation is then initiated by the work of multiple ATG proteins (Rubinsztein et al. 2007). The class III phosphatidylinositol-3kinase (PI3KC3) complex which is composed of PI3KC3 and ATG6 plays an essential role in autophagosome formation (Rubinsztein et al. 2007). Inhibition of PI3KC3 activity, therefore, leads to autophagy inhibition (Mizushima et al. 2010; Rubinsztein et al. 2007). Wortmannin and 3-methyladenine are widely used as PI3KC3 inhibitors in mammalian cells (Rubinsztein et al. 2007). Both chemicals have been reported as autophagy inhibitors in plants as well (Inoue et al. 2006; Takatsuka et al. 2004). Takatsuka et al. (2004) showed that the addition of wortmannin or 3-methyladenine in tobacco (Nicotiana tabacum) cell culture blocked accumulation of autophagic vacuoles in treated cells. The inhibition of autophagic vacuole accumulation was also observed in Arabidopsis root tips treated with 3-methyladenine (Inoue et al. 2006). All of these data indicated that 3-methyladenine and wortmannin may be used as inhibitors of autophagosome formation in photosynthetic eukaryotes. Autophagosome formation is followed by fusion with lysosome. Acidification of the vacuolar/lysosomal lumen is necessary for the autophagosome-vacuole/lysosome fusion process (Yamamoto et al. 1998). Bafilomycin A1 is an inhibitor of the vacuolar-type H+-ATPase (V-ATPase) which is located on the lysosomal membrane (Rubinsztein et al. 2007). The binding of bafilomycin A1 to the VATPase leads to proton-translocation inhibition, an increase in lysosomal pH, and subsequently autophagy inhibition (Rubinsztein et al. 2007). The alkalization of vacuole pH caused by bafilomycin A1 was also successfully demonstrated in maize (Brauer et al. 1997), the green alga Micrasterias pinnatifida (Ehara et al. 1996), and the diatom Phaeodactylum tricornutum (Zhang et al. 2016).

Nutrient starvation is a major factor that induces autophagy in algae (Pérez-Pérez et al. 2012). For example, nitrogen starvation in C. reinhardtii was shown to result in an increase in a level of CrATG8, a protein involved in autophagosome formation (Pérez-Pérez et al. 2010). Interestingly, nitrogen starvation has also been shown as one of the most effective conditions that induce a triacylglycerol (TAG) synthesis in many green algae including C. reinhardtii (Wang et al. 2009; Boyle et al. 2012; Merchant et al. 2012). Three acyltransferase genes— DGAT1, DGTT1, and PDAT1—were shown by Boyle et al. (2012) to be important for TAG synthesis in nitrogen-starved C. reinhardtii cells (Boyle et al. 2012). After TAG is synthesized, it is normally stored in lipid droplets localized in cytoplasm and/or chloroplast (Goold et al. 2015). Although it is known that nitrogen starvation can induce both autophagy and TAG synthesis, the relationship between autophagy and TAG synthesis remains unclear. Therefore, this study aims to elucidate the functional relationship between TAG synthesis and autophagy in nitrogen-starved C. reinhardtii cells.

Materials and methods Microalgal strain and culture treatment Chlamydomonas reinhardtii strains CC-124 and CC-2686 (sta1-1) were obtained from the Chlamydomonas Stock Center. The starchless strain sta1-1 was predominantly used throughout the experiments since its enhanced TAG content helped in visualization of the subcellular lipid droplets. Trisacetate phosphate (TAP) and nitrogen-depleted TAP (TAP-N) medium were prepared as described previously (Boyle et al. 2012). Algal inoculum was cultured in 50 mL TAP medium shaken at 120 rpm under continuous fluorescent light approximately 50 μmol photons m−2 s−1 at 25 °C. Rapamycin (LC Laboratories, USA), wortmannin (Calbiochem, USA), and bafilomycin A1 (Calbiochem, USA) were dissolved in dimethyl sulfoxide (DMSO) to make 100× concentrated solutions. The final concentration of DMSO in all treatments was 1% (v/v). 3-Methyladenine (Sigma-Aldrich, USA) was dissolved in water to make 50× concentrated solutions. Chlamydomonas reinhardtii cells were grown in TAP medium for 72 h, harvested, washed once in TAP-N medium, and transferred to fresh TAP, TAP-N, or medium containing 1 μM rapamycin with the initial density at 5 × 105 cells mL−1. The treatments of CC-2686 cells were conducted in 4-mL vial with 2 mL of culture medium, whereas the treatments of CC-124 cells were conducted in 6 mL medium. The cultures were maintained as described above. Growth of culture was monitored daily by cell counting using a hemocytometer.

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Fluorescence microscopy

Statistical analysis

Two hundred fifty microliter of algal culture was collected and stained with 1 μg L−1 Nile red (Sigma-Aldrich, USA), fixed with 4% (w/v) paraformaldehyde, and washed twice with PBS solution before the supernatant was removed by centrifugation at 8000 rpm for 10 min. The stained algal cells were resuspended in 10 μL of Prolong Gold antifade reagent (Invitrogen, USA) and observed under a Nikon Eclipse 80i epi-fluorescent microscope (Japan) using 430 and 690 nm of excitation and emission wavelength, respectively. Fifteen Nile red-stained cells from each treatment were randomly photographed, and the lipid droplet area of each cell was quantified using Image Frame Work software (Tarosoft, Thailand). Nile red fluorescence and chlorophyll autofluorescence of representative cells were photographed and merged using Adobe Photoshop (Adobe Systems, USA).

All experiments were performed using three independent biological replicates. One-way ANOVA analysis followed by Tukey’s comparison test was performed using GraphPad Prism 5 software (GraphPad Software, USA). The effects of inhibitors were analyzed using two-tailed t test.

TAG analysis Lipid was extracted from culture samples containing 4 × 106 or 5 × 106 cells, as indicated, using a mixture of chloroform and methanol (1:2 v/v) and an extraction method modified from that of Bligh and Dyer (1959). The extracted lipids were separated using silica gel thin-layer chromatography (TLC) plate (Merk Millipore, Germany). Fifty microgram of cooking soybean oil (Thai Vegetable Oil Public Company, Thailand) was spotted as a TAG standard. Polar lipids were first separated using a solvent mixture of acetone/toluene/water (91:30:8, v/v/v), followed by separation of non-polar lipids using hexane/diethyl ether/acetic acid (70:30:1, v/v/v) as a solvent mixture according to Wang and Benning (2011). Lipid bands were visualized by iodine vapors. Intensity of TAG bands, which showed equivalent mobility to that of soybean oil, was quantified using ImageJ software (NIH, USA). To quantify fatty acid content of TAG from the CC-124 cells, the TAG bands were removed from TLC plate, mixed with 0.1 mg of glyceryl triheptadecanoate (catalog no. T2151, Sigma) internal standard, and transesterified in 2 mL methanol containing 5% (v/v) H2SO4 at 90 °C for 2 h. The products were washed twice in deionized water and dissolved in 2 mL hexane. The remaining water was removed by addition of anhydrous Na2SO4, followed by evaporation and resuspension in 80 μL hexane. The fatty acid methyl esters were analyzed using Agilent 6890 N gas chromatograph (Agilent Technologies, USA) and Agilent 5973 Network Mass Selective Detector (Agilent Technologies, USA) through a capillary HP-Innowax column (Agilent Technologies, USA; 30 m × 0.25 mm × 0.25 μm) as previously described (Sirikhachornkit et al. 2016). Total fatty acid content was a sum of detectable fatty acid methyl esters in comparison to the internal standard.

Results Rapamycin-induced TAG synthesis and lipid droplet formation in C. reinhardtii CC-2686 Under nitrogen-replete condition, C. reinhardtii utilizes most of its energy toward growth and cell division, while synthesizing a minimal amount of TAG. Consistently with previous reports, nitrogen deprivation ceased algal growth (Fig. 1a) and induced TAG synthesis as evidenced by the accumulation of subcellular lipid droplets within 48 h (Fig. 1b, c). Imamura et al. (2015) have previously demonstrated that induction of autophagy, which is also activated upon nitrogen deprivation, through rapamycin-induced inactivation of TOR under a nitrogen-replete condition led to TAG accumulation in unicellular red alga Cyanidioschyzon merolae as well as in C. reinhardtii CC-125, suggesting that autophagy functions downstream of the nutrient stress and contributes to TAG synthesis. To verify this finding, we investigated whether rapamycin could trigger TAG synthesis in C. reinhardtii CC2686 under nitrogen-replete condition. Since severe stress in general may lead to TAG accumulation, we first tested whether the rapamycin treatment affected growth of C. reinhardtii CC-2686. The results showed that growth of the algal cells in TAP medium containing 1 μM rapamycin was slightly but not significantly retarded (Fig. 1a). The 1 μM rapamycin treatment however led to lipid droplet accumulation in the nitrogen-replete cells within 24 h (Fig. 1b, c), although the abundance of lipid droplets induced by rapamycin was lower than that of nitrogen-depleted cells. To confirm that the observed lipid droplet formation coincided with TAG synthesis, total lipid extracts from the algal cells subjected to the nitrogen-replete condition, nitrogen deprivation, and rapamycin treatment for 72 h were analyzed by TLC. Figure 1d shows that the nitrogen deprivation and rapamycin treatment clearly induced TAG synthesis in C. reinhardtii CC2686. Autophagy inhibitors compromised lipid droplet accumulation induced by rapamycin or nitrogen deprivation To investigate whether autophagy is necessary for TAG synthesis in C. reinhardtii CC-2686 under nitrogen deprivation or

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Fig. 1 Effect of nitrogen deprivation and autophagy inducer rapamycin on a growth, b, c lipid droplet accumulation, and d TAG synthesis. C. reinhardtii CC-2686 was grown in TAP medium for 72 h and transferred to normal TAP medium (+N), TAP-N medium (−N), or TAP medium containing 1 μM rapamycin (N, Rapa(1 μM)) for 0, 24, 48, and 72 h. Lipid droplets in algal cells were stained in Nile red and photographed. Shown are merged images of Nile red fluorescence and chlorophyll autofluorescence in representative cells. Scale bar represents

10 μm. Relative lipid droplet abundance was quantified as mean droplet area ± SEM in 15 cells per replicate in triplicate cultures. Letters above bars indicate statistically significant differences among data within the same treatment group based on one-way ANOVA, followed by post hoc Tukey’s test. Data that do not share the same letter are significantly different (p < 0.05). TAG abundance was observed in TLC analysis of lipids extracted from 4 × 106 cells grown in indicated conditions. Soy oil, 50 μg, was used as TAG standard

rapamycin treatment, the effects of known chemical inhibitors of autophagy, namely 3-methyladenine, wortmannin, and bafilomycin A1, were investigated. To test whether the chemical inhibitors per se are toxic to the algal cells, their growth inhibitory effect was examined in C. reinhardtii CC-2686 cultured in the TAP medium. The result showed that the treatment dosages of 3-methyladenine and bafilomycin A1 mildly affected growth of nitrogen-replete C. reinhardtii CC-2686 and did not result in growth arrest. The wortmannin treatments had no effect on growth or even slightly promoted growth of the algal cells (Fig. 2a). In comparison, growth of all C. reinhardtii CC-2686 cultures was ceased by nitrogen deprivation, as expected (Fig. 2b). Interestingly, growth of C. reinhardtii CC-2686 was severely retarded in response to a combination of rapamycin and any of the inhibitors (Fig. 2c). The 72-h treatments of all three autophagy inhibitors had little or no effect on lipid droplet accumulation in C. reinhardtii CC-2686 cells under nitrogen-replete condition

(Fig. 2d, e). In contrast, 1 μM bafilomycin A1 significantly (p < 0.05) lowered the amounts of lipid droplets formed in nitrogen-depleted (Fig. 2d, f) or rapamycin-treated cells (Fig. 2d, g). The 50 μM wortmannin treatment also blocked lipid droplet formation in rapamycin-treated cells (Fig. 2d, g), and showed a slight inhibitory effect, but statistically significant, in nitrogen-depleted cells (Fig. 2d, f). Compared to wortmannin and bafilomycin A1, the 3-methyladenine treatments were less effective at these tested concentrations. The inhibitory effects of wortmannin and bafilomycin A1 on lipid droplet formation were also more evident in the rapamycintreated cells than in the nitrogen-depleted cells, suggesting that additional mechanisms that are not sensitive to these inhibitors may contribute to the TAG synthesis in cells subjected nitrogen deprivation. In addition, we tested if the rapamycin treatment functions synergistically with nitrogen deprivation to induce TAG synthesis. The result showed that the rapamycin treatment could further improve lipid droplet accumulation in the nitrogen-depleted C. reinhardtii CC-2686

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ƒFig. 2

Effect of autophagy inhibitors in combination with nitrogen deprivation or rapamycin treatment on a–c growth and d–g lipid droplet accumulation. C. reinhardtii CC-2686 was grown in TAP medium for 72 h and transferred to 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. Cell density of the algal cultures was monitored for 0, 24, 48, and 72 h. Lipid droplets in algal cells were stained in Nile red and photographed under epi-fluorescent microscope. Shown are merged images of Nile red fluorescence and chlorophyll autofluorescence in representative cells. Scale bar represents 10 μm. Relative lipid droplet abundance was quantified as mean droplet area ± SEM in 15 cells per replicate. Asterisks above bar indicate statistically significant differences from the control treatment, based on two-tailed t tests (*p < 0.05; ***p < 0.001). All experiments were performed in triplicate cultures

under the nitrogen-replete condition. In contrast, the TAG accumulation in the nitrogen-depleted C. reinhardtii CC-124 cells was not significantly inhibited by the treatment with 0.1 μM bafilomycin A1.

Discussion

cells (Fig. 2d, f), suggesting that early or direct activation of autophagy may impart ability for C. reinhardtii CC-2686 to synthesize more TAG during nitrogen deprivation. The experiments described above were performed using C. reinhardtii CC-2686, which is impaired in starch accumulation (Ball et al. 1991). To confirm the previous results and to ascertain that the observed effect of autophagy inhibition was not influenced by the metabolic alteration, we repeated some of the experiments using C. reinhardtii CC-124, a common wild-type strain. Figure 3 shows that rapamycin could induce TAG accumulation in C. reinhardtii CC-124, although to a much lesser extent compared to C. reinhardtii CC-2686 presumably due to the metabolic competition for starch biosynthesis. Bafilomycin A1 significantly blocked TAG accumulation in the rapamycin-treated C. reinhardtii CC-124 cells

Although autophagy and TAG synthesis in C. reinhardtii are known to be induced by nitrogen starvation (Pérez-Pérez et al. 2010; Boyle et al. 2012), the relationship between these pathways remains unclear. In this study, we provided preliminary evidence supporting a role of autophagy downstream of the nitrogen starvation response en route to TAG synthesis (Fig. 4). First, we showed that the treatment with autophagy inducer rapamycin was sufficient to induce TAG synthesis and lipid droplet accumulation in nitrogen-replete C. reinhardtii CC-2686 (sta1-1) and CC-124 (wild-type) cells. Similar effects were also observed by Imamura et al. (2015) using C. reinhardtii CC-125, a related wild-type strain. Second, we showed that autophagy inhibitors wortmannin and bafilomycin A1 effectively blocked lipid droplet accumulation in the algal cells subjected to rapamycin treatment or nitrogen starvation. The inhibitory effect of bafilomycin A1 was also repeated in the wild-type strain C. reinhardtii CC124 using quantitative TAG analysis. The effectiveness of chemicals that can inhibit the autophagic process has been demonstrated in some algae, but not in Chlamydomonas. A recent study in the diatom P. tricornutum showed that the number and size of oil bodies in the diatom

Fig. 3 Effect of bafilomycin A1 on TAG content of C. reinhardtii CC124. The algal cells were grown in normal TAP medium for 72 h and transferred to 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. a TAG content quantified

by image analysis of TAG band intensity following TLC separation (Fig. S3) of total lipid extracted from culture samples containing 5 × 106 cells. b Total fatty acids derived from the TAG fraction and quantified by GC-MS as fatty acid methyl esters (Fig. S4). Asterisks above bar indicate statistically significant differences, based on twotailed t tests (*p < 0.05; **p < 0.01). Each bar represents mean ± SEM of six replications

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Fig. 4 A proposed model for role of autophagy in TAG biosynthesis in Chlamydomonas reinhardtii under nitrogen starvation. Autophagy is known to be activated upon nitrogen deprivation. This study demonstrated that the autophagic process contributes to TAG synthesis, as wortmannin and bafilomycin A1, which are known to inhibit

autophagy, blocked TAG synthesis in rapamycin-treated cells. In contrast, these autophagy inhibitors only mildly compromise TAG synthesis in nitrogen-depleted cells, suggesting that nitrogen starvation also triggers TAG synthesis through autophagy-independent pathway(s)

were reduced after treatment with 100 nM bafilomycin A1 (Zhang et al. 2016). In this study, the effect of autophagy inhibitors on lipid droplet accumulation in C. reinhardtii was demonstrated especially for wortmannin and bafilomycin A1. However, the primary targets and side effects of these inhibitors in algal cells are much less known compared to those in yeast and mammals. Further studies on the inhibitory mechanisms of these chemical inhibitors, to confirm that they specifically inhibit autophagy, are needed. The ineffectiveness of 3-methyladenine should also be investigated. The relationships between autophagy and nitrogen starvation response toward TAG synthesis in C. reinhardtii remain to be further investigated. Our results suggest that autophagy is both sufficient and important for TAG synthesis. However, additional autophagy-independent pathway(s) may be utilized during nitrogen deprivation and contributes the TAG synthesis in this alga, since wortmannin and bafilomycin A1were less inhibitory in the nitrogen-depleted cells when compared to that of the rapamycin-treated cells (Fig. 4). While the role of autophagy in plants subjected to nutrient starvation or senescence is believed to mediate recycling and remobilization of nutrients (Ren et al. 2014), its function in green algae and its contribution to TAG synthesis remain unclear. It is possible that autophagy may function to provide energy and recycle some intermediates and precursors, e.g., from membrane glycerolipids, for TAG synthesis. In addition to this, the nitrogen starvation response may also trigger autophagyindependent pathways that result in a greater supply of energy and metabolites for TAG synthesis and/or alter cellular structures in a way that better facilitates lipid droplet formation. Even though the TAG accumulated under both nitrogen starvation and rapamycin treatment depends on de novo fatty acid synthesis (Fan et al. 2011; Imamura et al. 2015), the fatty acid compositions of these TAG pools are distinct (Imamura et al. 2015; Fig. S4), and the TAG biosynthesis pathways may involve different enzymes, e.g., CrDGTT1 under nitrogen starvation and CrDGTT2 under rapamycin treatment (Imamura et al. 2015). In the red alga C. merolae, nitrogen depletion was shown to increase transcript abundance of 595 genes,

only 71 of which were also induced by rapamycin treatment (Imamura et al. 2015). These findings clearly indicate differences between the mechanisms that contribute to TAG accumulation in response to autophagy and nitrogen starvation. Further studies especially including use of the algal mutants that are genetically impaired in autophagy and nitrogen starvation response would be helpful to elucidate the exact role and mechanism of these processes in the future. 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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