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. 2014 May;13(5):591-613.
doi: 10.1128/EC.00013-14. Epub 2014 Feb 28.

The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii

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The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii

Ursula Goodenough et al. Eukaryot Cell. 2014 May.

Abstract

When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then "boosted" after 2 days with additional acetate, the cells become "obese" after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes-encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)-are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

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Figures

FIG 1
FIG 1
Samples of a sta6 culture, boosted with 20 mM acetate after 48 h NF and centrifuged at 10,000 × g for 5 min at 2, 3, 4, 5, and 8 days postboost. Micrographs show phase and bright-field (lower right) microscopy. Asterisk, cellular debris. Bars, 10 μm.
FIG 2
FIG 2
Living sta6 cells after 1 to 8 days of N starvation with (+) or without (−) acetate boost (phase microscopy). N, nucleus; V, vacuole; LB, lipid body; refractile blue bodies, eyespots. Bar, 5 μm.
FIG 3
FIG 3
Autophagosomes in 96 h NF nonboosted sta6 cells. M, myelin figures; LB, cytoplasmic lipid bodies. Bars, 500 nm (left) and 100 nm (right).
FIG 4
FIG 4
Medium acetate concentrations in an N-starved sta6 culture preboost and postboost.
FIG 5
FIG 5
Sensitive gene set. RPKM values at 0 h NF (postcentrifugation) were set at 100; subsequent percentiles are at 0.5, 2, 4, 8, 12, 24, and 48 h NF. RPKM data for these and additional sensitive genes appear in Table 7. A drop in expression in the cw15 strain occurs at late time points (blue when the 48-h NF value is <40% of the initial value), and a drop in sta6 expression occurs early, with a spike at 24 h NF (green when the 24-h NF value is at least 50% greater than the 48-h NF value). Numbers in parentheses are fold increases in gene expression in response to the boost (maximum RPKM level during the 2 h postboost divided by RPKM level at 48 h NF).
FIG 6
FIG 6
Proteins encoded by the acetate-sensitive gene set (blue font) and by genes selectively upregulated in the sta6 strain (red font). Abbreviations: ACS, acetyl-CoA synthetase; AST, aspartate transaminase; CIS, citrate synthase; Cre03.g155250 and Cre17.g735600, candidate TAG synthase enzymes; Cre15.g641200, candidate mitochondrial fatty acid carrier; DGTT, diacylglycerol acyltransferase type 2; FBP, fructose-1,6-bisphosphatase; GFY, GPR1/FUN30/YaaH family (candidate acetate transporters); GPD, glycerol phosphate dehydrogenase; ICL, isocitrate lyase; MAS, malate synthase; PCK, phosphoenolpyruvate carboxykinase; TAL, transaldolase.

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