The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii

Abstract

iWhen 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, ϳ425 genes are upregulated >2-fold and ϳ875 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.

Two Glycerol-3-Phosphate Dehydrogenases from Chlamydomonas Have Distinct Roles in Lipid Metabolism

“…and C. reinhardtii GPDH enzymes has focused on glycerol biosynthesis and osmotic stress tolerance (Gee et al, 1993;He et al, 2007He et al, , 2009Cai et al, 2013;Casais-Molina et al, 2016). However, previous expression data showing the induction of C. reinhardtii GPD2, GPD3, and GPD4 under nutrient starvation (Goodenough et al, 2014), which in turn induces TAG accumulation (Schmollinger et al, 2014), provide indirect evidence to suggest that these three GPDH isoforms are involved in the production of G3P for entry into the Kennedy pathway for DAG and subsequently TAG biosynthesis (Fig. 1).…”

“…As yet, the exact role of microalgae GPDH enzymes in lipid biosynthesis is unclear. Transcriptomic analyses have demonstrated that some of the C. reinhardtii GPDH genes, such as GPD2, GPD3, and GPD4, are induced under conditions that correlate with lipid induction (Goodenough et al, 2014). Overexpression of a GPDH gene in the diatom Phaeodactylum tricornutum gave increased glycerol alongside a subtle increase in total lipid content, which included higher monounsaturated fatty acids but lower polyunsaturated fatty acids compared with the wild type (Yao et al, 2014).…”

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

“…The Nile Red staining results suggested that some or all of the excess lipids in vip1-1 were triacylglycerol (TAG), the major neutral storage lipid form in Chlamydomonas (Wang et al, 2009;Siaut et al, 2011;Merchant et al, 2012;Goodenough et al, 2014). To compare TAG levels in vip1-1 and the wild type, we used thin-layer chromatography (TLC) separation and iodine staining of separated total lipids ( Figure 6A) or quantitative gas chromatography on transesterified purified TAGs from cells grown under three conditions: TAP (exponential phase), TAP with rapamycin (12 h), and TAP under nitrogen starvation (TAP -N; 12 h) ( Figure 6B).…”

The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii

“…In an effort to understand algal TAG production during N deprivation and to guide engineering of higher oil yields, omics-based approaches have been employed (Jamers et al, 2009). Using the annotated C. reinhardtii genome (Merchant et al, 2007), the C. reinhardtii transcriptome was analyzed before and after N deprivation, which revealed multiple changes in gene expression that affect diverse parts of metabolism (Miller et al, 2010;Blaby et al, 2013;Goodenough et al, 2014;Schmollinger et al, 2014). In addition, proteomics have been used to profile the changes in protein expression during N deprivation in Phaeodactylum tricornutum (Yang et al, 2014), C. reinhardtii Longworth et al, 2012;Schmollinger et al, 2014), and Nannochloropsis oceanica (Dong et al, 2013), and metabolomics have been used to assess changes in metabolite pool sizes (Bölling and Fiehn, 2005;Lee et al, 2012).…”