Light microscopy and deep-etch electron microscopy were used to visualize triacylglyceride (TAG)-filled lipid bodies (LBs) of the green eukaryotic soil alga Chlamydomonas reinhardtii, a model organism for biodiesel production. Cells growing in nitrogen-replete media contain small cytoplasmic lipid bodies (␣-cyto-LBs) and small chloroplast plastoglobules. When starved for N, ␤-cyto-LB formation is massively stimulated. ␤-Cyto-LBs are intimately associated with both the endoplasmic reticulum membrane and the outer membrane of the chloroplast envelope, suggesting a model for the active participation of both organelles in ␤-cyto-LB biosynthesis and packaging. When sta6 mutant cells, blocked in starch biosynthesis, are N starved, they produce ␤-cyto-LBs and also chloroplast LBs (cpst-LBs) that are at least 10 times larger than plastoglobules and eventually engorge the chloroplast stroma. Production of ␤-cyto-LBs and cpst-LBs under the conditions we used is dependent on exogenous 20 mM acetate. We propose that the greater TAG yields reported for N-starved sta6 cells can be attributed to the strain's ability to produce cpst-LBs, a capacity that is lost when the mutant is complemented by a STA6 transgene. Provision of a 20 mM acetate "boost" during N starvation generates sta6 cells that become so engorged with LBs-at the expense of cytoplasm and most organelles-that they float on water even when centrifuged. This property could be a desirable feature for algal harvesting during biodiesel production.There is currently keen interest in cultivating eukaryotic algae as sources of triacylglycerides (TAGs) to be converted into diesel and jet transportation fuel (16,37,43,50). In the past 2 years, several laboratories, including ours, have reported that the unicellular green soil alga Chlamydomonas reinhardtii, in response to nitrogen (N) starvation, produces TAG-filled lipid bodies (LBs) (9,21,23,24,30,31,44,49,51), also called lipid droplets, oil droplets, and oil bodies. Since C. reinhardtii currently boasts the best-developed resources for algal molecular-genetic analysis and manipulation (14), it could serve as an important model organism for algal biodiesel research even if it eventually proves to be unsuitable as a production strain.The structural correlates of LB formation are poorly detailed in algae, in part because algal morphology tends to be poorly preserved when chemical fixatives are used. We therefore undertook an analysis of LB formation in C. reinhardtii using phase-contrast and bright-field light microscopy of living cells and deep-etch electron microscopy (DEEM) of quickfrozen living cells. We compared starch-forming strains, primarily a cw15 strain (herein designated the STA6 strain), with the sta6 strain, a starch-null mutant strain derived from the STA6 strain that has been shown to produce more LBs and TAG than starch-forming strains in several studies (23,49,51). We analyzed cells in log and stationary phases, in various stages of N starvation both in liquid medium and on agar plates, and in maturing z...

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.

f Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quickfreeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/ colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous ␤-1, 4-and/or ␤-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form "drapes" between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.T he trebouxiophyte green fresh-to brackish-water alga Botryococcus braunii has a unique colonial organization: individual cells of the colony are embedded in an extracellular matrix (ECM) composed of polymerized and liquid hydrocarbons (LH) (29,35,(46)(47)(48). Although they may serve additional functions, these hydrocarbons notably allow B. braunii colonies to float, presumably to increase exposure to light for photosynthesis at the surfaces of ponds or lakes (4). The presence of B. braunii oils and ECM fossils in petroleum, coal deposits, and oil shale suggests that their hydrocarbon products once contributed to these reserves (1,2,8,10,16,17,34,36,52,62,63,69,70) and that these hydrocarbons could be used as a source of renewable energy. In fact, B. braunii hydrocarbons can be readily converted into petroleum equivalent, drop-in transportation fuels by conventional petroleum-processing techniques (22, 28).The three races of B. braunii are classified on the basis of the chemical nature of the liquid hydrocarbons they produce. The A race primarily produces alkadienes and alkatrienes derived from fatty acids (38,49,65,67); the B race (the focus of this study) produces two triterpenoids, tetramethylsqualene as a minor comp...

The course of prior episodes of AWS is the most reliable predictor of subsequent episodes. Thrombocytopenia and hypokalemia also correlate with SAWS. We propose further research into drinking patterns, gender, and medical comorbidities.

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