<i>Neochloris oleoabundans</i> is worth its salt: Transcriptomic analysis under salt and nitrogen stress

<div><p><i>Neochloris oleoabundans</i> is an oleaginous microalgal species that can be cultivated in fresh water as well as salt water. Using salt water gives the opportunity to reduce production costs and the fresh water footprint for large scale cultivation. Production of triacylglycerols (TAG) usually includes a biomass growth phase in nitrogen-replete conditions followed by a TAG accumulation phase under nitrogen-deplete conditions. This is the first report that provides insight in the saline resistance mechanism of a fresh water oleaginous microalgae. To better understand the osmoregulatory mechanism of <i>N</i>. <i>oleoabundans</i> during growth and TAG accumulating conditions, the transcriptome was sequenced under four different conditions: fresh water nitrogen-replete and -deplete conditions, and salt water (525 mM dissolved salts, 448mM extra NaCl) nitrogen-replete and -deplete conditions. In this study, several pathways are identified to be responsible for salt water adaptation of <i>N</i>. <i>oleoabundans</i> under both nitrogen-replete and -deplete conditions. Proline and the ascorbate-glutathione cycle seem to be of importance for successful osmoregulation in <i>N</i>. <i>oleoabundans</i>. Genes involved in Proline biosynthesis were found to be upregulated in salt water. This was supported by Nuclear magnetic resonance (NMR) spectroscopy, which indicated an increase in proline content in the salt water nitrogen-replete condition. Additionally, the lipid accumulation pathway was studied to gain insight in the gene regulation in the first 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen-deplete conditions in a comparable way in both fresh and salt water. The mechanism behind the biosynthesis of compatible osmolytes can be used to improve <i>N</i>. <i>oleoabundans</i> and other industrially relevant microalgal strains to create a more robust and sustainable production platform for microalgae derived products in the future.</p></div

Carbon metabolism in <i>N</i>. <i>oleoabundans</i>.

<p>For the figure legend refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194834#pone.0194834.g002" target="_blank">Fig 2</a>. Codes in the white boxes represent the corresponding EC numbers. Abbreviations: PEP phosphoenol pyruvate; LysoPA Lysophosphatidic acid; PA Phosphatidic acid; DAG 1,2-Diacylglycerol; TAG Triacylglycerol. EC:2.3.1.12 could not be annotated from the transcriptome of <i>N</i>. <i>oleoabundans</i>.</p

Biomass composition of <i>N</i>. <i>oleoabundans</i> in the four tested conditions.

<p>Upper panel: Confocal laser scanning microscope images of <i>N</i>. <i>oleoabundans</i> under four different cultivation conditions. (A) Nitrogen-replete fresh water (FN+). (B) Nitrogen-deplete fresh water conditions (FN-). (C) Nitrogen-replete salt water conditions (SN+). (D) Nitrogen-deplete salt water conditions (SN-). Chlorophyll autofluorescence is shown in red and the BODIPY stain is shown in yellow. The bar represents 20 μm. Lower panel: (E) Lipid and starch content. TFA (dark grey), TAG (light grey), and starch (white) 24 hours after medium replacement. The height of the bars represents the average of the two independent measurements. Error bars represent distance of the sample values to the average value. TAG, TFA and starch are given as a percentage of total dry weight. (F) Fatty acid profile under the different conditions expressed as percentage of TFA.</p

Proline and GABA biosynthesis pathway.

<p>For the figure legend refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194834#pone.0194834.g002" target="_blank">Fig 2</a>. Abbreviations: GSA: glutamate-semialdehyde. P5C: L-1-Pyrroline-5-carboxylate. GABA: 4-aminobutanoate. P5CR: P5C reductase. P5CS1: glutamate-5-kinase. P5CS2: GSA dehydrogenase. GDH: glutamate dehydrogenase (EC:1.4.1.3/4). GST: glutamate synthase (EC:1.4.1.13/14). GSTT: glutamine synthetase. GDC: glutamate decarboxylase. OAT: ornithine aminotransferase. PRODH: Proline dehydrogenase. *This reaction occurs non-enzymatically.</p

Glutathione biosynthesis pathway and glutathione-ascorbate cycle.

<p>For the figure legend refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194834#pone.0194834.g002" target="_blank">Fig 2</a>. Abbreviations: SOD Superoxide dismutase; CAT Catalase; APX Ascorbate peroxidase (not annotated); MDHA monodehydroascorbate; MDHAR monodehydroascorbate reductase; DHA dehydroascorbate; DHAR dehydroascorbate reductase; GSH (reduced) Glutathione; GSSG oxidised glutathione; GSHR Glutathione reductase; Glu Glutamate; Cys Cysteine; Gly Glyceine; γ-GCSTT γ-glutamylcystein Synthetase; γ-Glu-Cys γ-glutamylcystein; GSHSTT Glutathione Synthetase; GSH-S-T glutathione S-transferase; PCST Phytochelatin synthase. * This reaction can occur enzymatically or non-enzymatically.</p

Biosynthesis pathway of sucrose and starch.

<p>The values that are shown in the following figures refer to the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for each condition. The white left-most box represents the FPKM value of the respective gene in the fresh water nitrogen-replete reference condition. This is followed by three colored boxes that represent the log2 fold change (LFC) of the other conditions compared to the reference condition. The order of the remaining three boxes are from left to right, FN-, SN+, SN- respectively. Abbreviations: G1PUT: glucose-1-phosphate uridylyltransferase; UTP: Uridine triphosphate. SucPS: sucrose-phosphate synthase. SucST: sucrose synthase. G1PAT: glucose-1-phosphate adenylyltransferase. αTrPS: alpha,alpha-trehalose-phosphate synthase. αTrS: alpha,alpha-trehalose synthase. TrP: trehalose-phosphatase. StS:starch synthase. GlBE: glycogen branching enzyme. SucPP: sucrose-phosphate phosphatase. SucGH: sucrose glucohydrolase. β-am: β-amylase. D-enzyme: 4-alpha-glucanotransferase. StarchP: Starch phosphorylase.</p