TOR (target of rapamycin) is a key regulator of triacylglycerol accumulation in microalgae

ABSTRACT

Most microalgae abundantly accumulate lipid droplets (LDs) containing triacylglycerols (TAGs) under several stress conditions, but the underlying molecular mechanism of this accumulation remains unclear. In a recent study, we found that inhibition of TOR (target of rapamycin), a highly conserved protein kinase of eukaryotes, by rapamycin resulted in TAG accumulation in microalgae, indicating that TOR negatively regulates TAG accumulation. Here, we show that formation of intracellular LDs and TAG accumulation were also induced in the unicellular green alga Chlamydomonas reinhardtii after exposure to Torin1 or AZD8055, which are novel TOR inhibitors that inhibit TOR activity in a manner different from rapamycin. These results supported quite well our previous conclusion that TOR is a central regulator of TAG accumulation in microalgae.

Abbreviations

DAG

diacylglycerol

DMSO

dimethyl sulfoxide

G3P

glycerol-3-phosphate

PA

phosphatidic acid

PC

phosphatidylcholine

PE

phosphatidylethanolamine

PG

phosphatidylglycerol

PI

phosphatidylinositol

Microalgae have been considered as a promising renewable energy bioresource because of their high photosynthetic activities and growth rates in comparison with higher plants, the absence of competition for resources used in agricultural food production, and high neutral lipid contents.^1,2 Microalgae usually store neutral lipids as cytoplasmic lipid droplets (LDs), mainly in the form of triacylglycerols (TAGs) that can be used for biodiesel production.

Although several key genes for TAG accumulation have been reported in the unicellular green alga Chlamydomonas reinhardtii,^4-6 a signaling pathway that connects environmental sensing to TAG accumulation has remained elusive in microalgae. Recent work in this research group revealed that inhibition of the target of rapamycin (TOR) by rapamycin,⁷ a specific inhibitor for TOR kinase, induces LD and TAG accumulation in Cyanidioschyzon merolae, a unicellular red alga, as well as in C. reinhardtii. TOR is a highly conserved serine/threonine protein kinase that plays pivotal roles in nitrogen and other signaling pathways in eukaryotes.⁷ Based on these findings, it was concluded that the TOR-signaling pathway negatively regulates TAG accumulation in microalgae.⁸

Mammalian TOR (mTOR) inhibitors are generally used as immunosuppressors as well as antineoplastic drugs in various cancer treatments.⁹ Therefore, development of new chemicals that specifically inhibit mTOR activity has been an intensive research area for future medical applications. Besides rapamycin, several new compounds, such as Torin1 and AZD8055, have been thus far identified as ATP-competitive mTOR-specific inhibitors.⁹ Recently, it has been reported that such novel mTOR inhibitors also inhibit TOR kinase activity in higher plants.^10,11 As in microalgae, this study found that C. reinhardtii growth was effectively inhibited and that LDs were detected in cytoplasm in the presence of Torin1 or AZD8055 in a dose-dependent manner (Fig. S1), as has been found in rapamycin treatments.^8,12,13 Effective growth inhibitions, one of the phenotypes of TOR inactivation,^12,13 and abundant LD accumulation were observed in the presence of Torin1 or AZD8055 at a final concentration of 1.0 μM or 2.0 μM, respectively (Fig. 1A and Fig. S1). Under the same conditions, TAG contents clearly increased after Torin1 or AZD8055 treatment, with concentrations being 2.9 or 4.0-fold greater compared with controls, respectively (Fig. 1B). These results indicated that LD and TAG accumulation were induced after TOR-inactivation by Torin1 or AZD8055 and well supported the previous conclusion here that TOR plays a critical role in TAG accumulation as a negative regulator in microalgae. In contrast, it was previously shown by this research group that C. merolae is insensitive to Torin1 and AZD8055. This could have been because of unique characteristics of the C. merolae TOR structure or drug instabilities in the hot, acidic medium used for C. merolae cultivation.¹² It is of note that TAG accumulation and fatty acid composition were variable depending on the applied TOR inhibitor (Figs. 1B and 1C),⁸ which suggested that each inhibitor affected TOR activity differently. This possibility is supported by a recent report that demonstrated that genome-wide gene expression profiles are not identical after rapamycin and AZD8055 treatments.¹¹

Figure 1.

Accumulation of LDs and TAG under TOR-inactivation conditions in C. reinhardtii. (A) BODIPY staining of cells after DMSO, Torin1, or AZD8055 treatment for 24 h. Torin1 and AZD8055 added to cells to a final concentration of 1.0 and 2.0 μM, respectively. DMSO added to cells as controls. Bright field and BODIPY staining (top and bottom, respectively) images indicated. Each BODIPY staining image was merged with relevant chlorophyll fluorescence image. Bar = 5 μm. (B) Intracellular TAG content. TAG contents were quantified 24 h after growth under indicated conditions. Values are averages of three independent experiments and represent percent of dry weight. Error bars indicate standard deviation. (C) Fatty acid composition of purified TAG. TAG fatty acids indicated as percent of total fatty acids. Each fatty acid indicated by number of carbons:number of double bonds. Values are averages of three independent experiments. Error bars indicate standard deviation. Asterisks indicate significant difference compared with DMSO treatment (Student's t-test, p<0.05).

TOR plays a central role in the regulation of nutrient status and cell growth sensing in eukaryotes.⁷ Thus, under nutrient rich conditions, where TOR is active, TOR positively controls synthesis de novo of biomolecules that are essential for cell growth. In contrast, conditions of nutrient depletion or the presence of TOR inhibitors results in cell growth inhibition (Fig. 2).^12,13 Under such conditions, cells do not need to synthesize building blocks but alternatively accumulate energy storage compound, TAGs.^6,8 As for lipid synthesis de novo, lipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, are synthesized in the endoplasmic reticulum to build cell membranes, and TAG synthesis occurs at endoplasmic reticulum in part through the same pathway (Fig. 2). Thus, the following regulation of TAG accumulation is proposed as one possibility. Under nutrient rich conditions, most acyl-CoA is likely used for membrane lipid synthesis and cell membrane construction. Conversely, under TOR-inactivation and nutrient depletion conditions where the cell growth is arrested, it is considered that the main fluxes of acyl-CoA and intermediates of TAG synthesis, such as phosphatidic acid and diacylglycerols, are alternatively directed to TAG formation instead of polar membrane lipids. Here, TOR is likely to function as a “checkpoint kinase” to switch the metabolic flows in response to environmental conditions.(Fig. 2)

Figure 2.

A possible model for TAG accumulation regulation by TOR in microalgae. TOR behaves as a “checkpoint kinase” that switches between growth state (synthesis of building blocks for cell growth) or energy storage state (TAG accumulation). See details in the text. “+” with small circles denotes a positive effect.

While the underlying molecular mechanism regarding how a cell changes metabolic flows remains unclear, one possible mechanism is enhancement of acyl-CoA:diacylglycerol acyltransferase (DGAT) expression (Fig. 2). In C. reinhardtii, transcripts of DGAT gene, DGTT1, DGTT2, DGTT3 and DGTT4, are increased in response to nutrient depletion and TOR-inactivation conditions.^4,6,8 Consistent with these observations, compulsory overexpression of DGTT4, enhances TAG accumulation.⁶ In C. merolae, transcripts encoding glycerol-3-phosphate acyltransferase (GPAT, CMA017C and CMK217C) as well as DGAT (CMB069C) are increased among TAG-synthesis genes under TOR-inactivation conditions.⁸ The GPAT transcripts are also increased in response to nitrogen depletion conditions.⁸ This suggests that either or both of these expressions contribute to TAG accumulation in this alga.(Fig. 2)

It remains unclear how TOR receives environmental cues, such as nitrogen status, and transmits it to downstream regulators in microalgae. Further analyses regarding this issue will provide important information regarding the regulatory mechanism of TAG accumulation and such understanding will be useful for improvement of TAG productivity in microalgae.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid 22681010, 24117521, 25440129, and 26117711 to S.I. and Grants-in-Aid 24248061 to K.T.).