RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in Chlamydomonas reinhardtii

Abstract

Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga Chlamydomonas reinhardtii, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.Triacylglycerols (TAGs) are stored in lipid droplets which are subcellular structures in specialized cells ubiquitous to eukaryotes but have more recently also been identified in some prokaryotes (26). In plants and animals, lipid droplets are surrounded by cytosol and are believed to bud off the endoplasmic reticulum (ER) (15,26). While traditionally considered merely as storage compartments, recent studies suggest that lipid droplets in animals play important additional roles in lipid homeostasis and protein storage (8). In oilseed plants, TAG accumulated in seeds is used as a reservoir of energy and membrane lipid building blocks to support rapid growth after germination (15). Many green algae are capable of accumulating large amounts of TAG in lipid droplets, particularly as a result of abiotic stresses, such as nutrient deprivation or highlight exposure. Although TAG metabolism in algae has not yet been extensively studied at the biochemical or molecular level, it is proposed that TAG turnover contributes primarily to the assembly of membrane lipids to facilitate rapid cell division after the cessation of nutrient limitation (14, 38).The general structure of lipid droplets is conserved in different species with a globular neutral lipid core enclosed by a membrane lipid monolayer (26). In addition, specific proteins are associated with lipid droplets and play important roles in lipid droplet structure and function. A number of recent proteomic studies of lipid droplets from different animals and tissues (8, 40), Saccharomyces cerevisiae (4), and plants (16,17) have revealed that the lipid droplet-associated proteins of these organisms are quite dist...

Scenedesmus sp. NJ-1 isolated from Antarctica: a suitable renewable lipid source for biodiesel production

“…NJ-1 may be suitable for outdoor mass culture during the whole seasons of the year in warm temperate regions due to its relatively wide range of growth temperature and natural settling. Many microorganisms (Gouda et al 2008) and microalgae (such as diatoms and green algae) are capable of accumulating large amounts of TAGs in oil droplets under environmental stress conditions (Moellering and Benning 2010). The oil droplets in Scenedesmus NJ-1 may also serve as a reserve of energy in the form of TAGs.…”

The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants

“…In higher plants, this is usually in the form of starch (Diaz et al, 2005;Wingler et al, 2006), and Peng et al (2007) described increased transcript levels of two genes involved in starch biosynthesis in Arabidopsis (Arabidopsis thaliana) under nitrogen starvation. In the fresh water green alga Chlamydomonas reinhardtii, on the other hand, excess carbon is reportedly directed to fatty acid biosynthesis (Wang et al, 2009;Moellering and Benning, 2010), and Miller et al (2010) found increased expression of genes involved in lipid biosynthesis in C. reinhardtii when nitrogen was not available. Diatoms are known to store carbon as chrysolaminaran (a b-1,3-glucan) or as lipids (Armbrust et al, 2004;Kroth et al, 2008); however, proteins directly related to the synthesis or degradation of these compounds were not identified in this study.…”

Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism

“…N deprivation for 48 h showed the greatest difference in RNA levels compared with the N-replete cultures. Based on this basic analysis and our previous timecourse study of lipid droplet formation and TAG accumulation (Moellering and Benning, 2010), a 48-h period of N deprivation was chosen to compare global transcript levels in N-replete and N-deprived cells.…”

“…At this time, Chlamydomonas reinhardtii is the premier microalgal molecular model for this analysis. As such, the formation of lipid droplets following nitrogen (N) deprivation has recently been documented in detail (Wang et al, 2009;Moellering and Benning, 2010). Although C. reinhardtii is not under direct consideration for the production of biomass as a biofuel feedstock, the analysis of its metabolism and physiology is expected to provide basic insights into mechanisms of TAG accumulation relevant to other microalgae at least of the green algal phylum.…”

“…The goal of this study was to determine major changes in gene expression following N deprivation, the nutrient condition established in our previous analysis of lipid droplet formation and TAG accumulation in C. reinhardtii (Moellering and Benning, 2010). Comparison of the transcript levels of induced, N-deprived C. reinhardtii cultures with those of uninduced, N-replete cultures was expected to reflect the metabolic changes leading to TAG accumulation.…”