All members of the Arabidopsis DGAT and PDAT acyltransferase families operate during high and low temperatures.

The accumulation of triacylglycerol (TAG) in vegetative tissues is necessary to adapt to changing temperatures. It has been hypothesized that TAG accumulation is required as a storage location for maladaptive membrane lipids. The TAG acyltransferase family has five members (DIACYLGLYCEROL ACYLTRANSFERSE1/2/3 and PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1/2), and their individual roles during temperature challenges have either been described conflictingly or not at all. Therefore, we used Arabidopsis (Arabidopsis thaliana) loss of function mutants in each acyltransferase to investigate the effects of temperature challenge on TAG accumulation, plasma membrane integrity, and temperature tolerance. All mutants were tested under one high- and two low-temperature regimens, during which we quantified lipids, assessed temperature sensitivity, and measured plasma membrane electrolyte leakage. Our findings revealed reduced effectiveness in TAG production during at least one temperature regimen for all acyltransferase mutants compared to the wild type, resolved conflicting roles of pdat1 and dgat1 by demonstrating their distinct temperature-specific actions, and uncovered that plasma membrane integrity and TAG accumulation do not always coincide, suggesting a multifaceted role of TAG beyond its conventional lipid reservoir function during temperature stress.

LIP4 Is Involved in Triacylglycerol Degradation in Chlamydomonas reinhardtii.

Degradation of the storage compound triacylglycerol (TAG) is a crucial process in response to environmental stimuli. In microalgae, this process is important for re-growth when conditions become favorable after cells have experienced stresses. Mobilization of TAG is initiated by actions of lipases causing the release of glycerol and free fatty acids, which can be further broken down for energy production or recycled to synthesize membrane lipids. Although key enzymes in the process, TAG lipases remain to be characterized in the model green alga Chlamydomonas reinhardtii. Here, we describe the functional analysis of a putative TAG lipase, i.e. LIP4, which shares 44% amino acid identity with the major TAG lipase in Arabidopsis (SUGAR DEPENDENT1-SDP1). The LIP4 transcript level was downregulated during nitrogen deprivation when TAG accumulates, but was upregulated during nitrogen resupply (NR) when TAG was degraded. Both artificial microRNA and insertional mutants showed a delay in TAG mobilization during NR. The difference in TAG degradation was more pronounced when the cultures were incubated without acetate in the dark. Furthermore, the lip4 insertional mutants over-accumulated TAG during optimal growth conditions. Taken together, the results suggest to us that LIP4 likely acts as a TAG lipase and plays a role in TAG homeostasis in Chlamydomonas.

Lipid catabolism in microalgae.

Lipid degradation processes are important in microalgae because survival and growth of microalgal cells under fluctuating environmental conditions require permanent remodeling or turnover of membrane lipids as well as rapid mobilization of storage lipids. Lipid catabolism comprises two major spatially and temporarily separated steps, namely lipolysis, which releases fatty acids and head groups and is catalyzed by lipases at membranes or lipid droplets, and degradation of fatty acids to acetyl-CoA, which occurs in peroxisomes through the ß-oxidation pathway in green microalgae, and can sometimes occur in mitochondria in some other algal species. Here we review the current knowledge on the enzymes and regulatory proteins involved in lipolysis and peroxisomal ß-oxidation and highlight gaps in our understanding of lipid degradation pathways in microalgae. Metabolic use of acetyl-CoA products via glyoxylate cycle and gluconeogenesis is also reviewed. We then present the implication of various cellular processes such as vesicle trafficking, cell cycle and autophagy on lipid turnover. Finally, physiological roles and the manipulation of lipid catabolism for biotechnological applications in microalgae are discussed.

Correction: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism.

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue.

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl-constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER-to-chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant.

Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii.

In plants, neutral lipids are frequently synthesized and stored in seed tissues, where the assembly of lipid droplets (LDs) coincides with the accumulation of triacylglycerols (TAGs). In addition, photosynthetic, vegetative cells can form cytosolic LDs and much less information is known about the makeup and biogenesis of these LDs. Here we focus on Chlamydomonas reinhardtii as a reference model for LDs in a photosynthetic cell, because in this unicellular green alga LD dynamics can be readily manipulated by nitrogen availability. Nitrogen deprivation leads to cellular quiescence during which cell divisions cease and TAGs accumulate. The major lipid droplet protein (MLDP) forms a proteinaceous coat surrounding mature LDs. Reducing the amount of MLDP affects LD size and number, TAG breakdown and timely progression out of cellular quiescence following nitrogen resupply. Depending on nitrogen availability, MLDP recruits different proteins to LDs, tubulins in particular. Conversely, depolymerization of microtubules drastically alters the association of MLDP with LDs. LDs also contain select chloroplast envelope membrane proteins hinting at an origin of LDs, at least in part, from chloroplast membranes. Moreover, LD surface lipids are rich in de novo synthesized fatty acids, and are mainly composed of galactolipids which are typical components of chloroplast membranes. The composition of the LD membrane is altered in the absence of MLDP. Collectively, our results suggest a mechanism for LD formation in C. reinhardtii involving chloroplast envelope membranes by which specific proteins are recruited to LDs and a specialized polar lipid monolayer surrounding the LD is formed.

The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas.

Microalgae are prolific photosynthetic organisms that have the potential to sustainably produce high-value chemical feedstocks. However, an industry based on microalgal biomass still is faced with challenges. For example, microalgae tend to accumulate valuable compounds, such as triacylglycerols, only under stress conditions that limit growth. To investigate the fundamental mechanisms at the base of this conundrum--the inverse relationship between biomass production and storage compound accumulation-we applied a combination of cell biological and genetic approaches. Conceptually, nutrient deprivation, which commonly is used to induce the accumulation of triacylglycerol in microalgae, leads to a state of cellular quiescence defined by a halt of cell divisions that is reversible upon nutrient resupply. To identify factors that govern cellular quiescence, we screened for mutants of the model alga Chlamydomonas reinhardtii that, in contrast to wild-type cells placed under conditions of nitrogen deprivation, were unable to degrade triacylglycerols following nitrogen resupply. One of the mutants described here in detail, compromised hydrolysis of triacylglycerols 7 (cht7), was severely impaired in regrowth following removal of different conditions inducing cellular quiescence. The mutant carries a deletion affecting four genes, only one of which rescued the quiescence phenotype when reintroduced. It encodes a protein with similarity to mammalian and plant DNA binding proteins. Comparison of transcriptomes indicated a partial derepression of quiescence-related transcriptional programs in the mutant under conditions favorable to growth. Thus, CHT7 likely is a repressor of cellular quiescence and provides a possible target for the engineering of high-biomass/high-triacylglycerol microalgae.

Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

TAG, you're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation.

Photosynthetic organisms are responsible for converting sunlight into organic matter, and they are therefore seen as a resource for the renewable fuel industry. Ethanol and esterified fatty acids (biodiesel) are the most common fuel products derived from these photosynthetic organisms. The potential of algae as producers of biodiesel precursor (or triacylglycerols (TAGs)) has yet to be realized because of the limited knowledge of the underlying biochemistry, cell biology and genetics. Well-characterized pathways from fungi and land plants have been used to identify algal homologs of key enzymes in TAG synthesis, including diacylglcyerol acyltransferases, phospholipid diacylglycerol acyltransferase and phosphatidate phosphatases. Many laboratories have adopted Chlamydomonas reinhardtii as a reference organism for discovery of algal-specific adaptations of TAG metabolism. Stressed Chlamydomonas cells, grown either photoautotrophically or photoheterotrophically, accumulate TAG in plastid and cytoplasmic lipid bodies, reaching 46-65% of dry weight in starch accumulation (sta) mutants. State of the art genomic technologies including expression profiling and proteomics have identified new proteins, including key components of lipid droplets, candidate regulators and lipid/TAG degrading activities. By analogy with crop plants, it is expected that advances in algal breeding and genome engineering may facilitate realizing the potential in algae.