Astrid Vieler

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

Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in 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.

Systems Biology Approach in Chlamydomonas Reveals Connections between Copper Nutrition and Multiple Metabolic Steps

RNA-seq assessment of the transcriptome of autotrophic and heterotrophic Chlamydomonas as a function of copper nutrition reveals changes in redox metabolism regulated by CRR1, an SBP domain transcription factor. The changes in RNA abundance impact the abundance of specific plastid-localized proteins and the level of saturation of plastid galactolipids. In this work, we query the Chlamydomonas reinhardtii copper regulon at a whole-genome level. Our RNA-Seq data simulation and analysis pipeline validated a 2-fold cutoff and 10 RPKM (reads per kilobase of mappable length per million mapped reads) (~1 mRNA per cell) to reveal 63 CRR1 targets plus another 86 copper-responsive genes. Proteomic and immunoblot analyses captured 25% of the corresponding proteins, whose abundance was also dependent on copper nutrition, validating transcriptional regulation as a major control mechanism for copper signaling in Chlamydomonas. The impact of copper deficiency on the expression of several O2-dependent enzymes included steps in lipid modification pathways. Quantitative lipid profiles indicated increased polyunsaturation of fatty acids on thylakoid membrane digalactosyldiglycerides, indicating a global impact of copper deficiency on the photosynthetic apparatus. Discovery of a putative plastid copper chaperone and a membrane protease in the thylakoid suggest a mechanism for blocking copper utilization in the chloroplast. We also found an example of copper sparing in the N assimilation pathway: the replacement of copper amine oxidase by a flavin-dependent backup enzyme. Forty percent of the targets are previously uncharacterized proteins, indicating considerable potential for new discovery in the biology of copper.

A Lipid Droplet Protein of Nannochloropsis with Functions Partially Analogous to Plant Oleosins

As our understanding of the dynamics of lipid droplets (LDs) in animal, plant, and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstock for the production of biofuels and high-value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic lipid droplet surface protein (LDSP) with unique primary sequence but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount of triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis (Arabidopsis thaliana) OLEOSIN1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.

Triacylglycerol profiling of microalgae Chlamydomonas reinhardtii and Nannochloropsis oceanica

Triacylglycerols (TAGs) from microalgae can serve as feedstock for the production of biofuels. To gain a comprehensive understanding of TAG metabolism in algae through genetic and molecular approaches, and to improve algal biofuel production, efficient and quantitative phenotyping methods focusing on TAGs are required. Towards this end, a facile ultrahigh performance liquid chromatography-mass spectrometry protocol was developed for TAG profiling, achieving identification and quantification of intact TAG molecular species in two algae. TAG profiling was performed in Chlamydomonas reinhardtii and Nannochloropsis oceanica grown in nitrogen (N)-replete or N-depleted medium. For the quantification of algal TAGs and fatty acids, two sets of internal standards were developed by taking advantage of the presence of pheophytin and specific fatty acids in algal samples. Comparison of algal TAG levels was simplified by using these internal standards for TAG analysis, paving the way for high-throughput mutant screening.

Remodeling of Membrane Lipids in Iron-starved Chlamydomonas

Background: Iron starvation triggers lipid droplet and triacylglycerol (TAG) accumulation in Chlamydomonas reinhardtii. Results: The overall lipid profile shows a decrease in the absolute content of monogalactosyldiacylglycerol (MGDG) and an increase in saturated and monounsaturated fatty acids. Conclusion: Iron starvation has an early and distinct effect on membrane lipids, before onset of chlorosis. Significance: Iron deficiency affects distribution of lipid type as well as fatty acid profile. Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24–48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

The lipid dependence of diadinoxanthin de-epoxidation presents new evidence for a macrodomain organization of the diatom thylakoid membrane.

The present study shows that thylakoid membranes of the diatom Cyclotella meneghiniana contain much higher amounts of negatively charged lipids than higher plant or green algal thylakoids. Based on these findings, we examined the influence of SQDG on the de-epoxidation reaction of the diadinoxanthin cycle and compared it with results from the second negatively charged thylakoid lipid PG. SQDG and PG exhibited a lower capacity for the solubilization of the hydrophobic xanthophyll cycle pigment diadinoxanthin than the main membrane lipid MGDG. Although complete pigment solubilization took place at higher concentrations of the negatively charged lipids, SQDG and PG strongly suppressed the de-epoxidation of diadinoxanthin in artificial membrane systems. In in vitro assays employing the isolated diadinoxanthin cycle enzyme diadinoxanthin de-epoxidase, no or only a very weak de-epoxidation reaction was observed in the presence of SQDG or PG, respectively. In binary mixtures of the inverted hexagonal phase forming lipid MGDG with the negatively charged bilayer lipids, comparable suppression took place. This is in contrast to binary mixtures of MGDG with the neutral bilayer lipids DGDG and PC, where rapid and efficient de-epoxidation was observed. In complex lipid mixtures resembling the lipid composition of the native diatom thylakoid membrane, we again found strong suppression of diadinoxanthin de-epoxidation due to the presence of SQDG or PG. We conclude that, in the native thylakoids of diatoms, a strict separation of the MGDG and SQDG domains must occur; otherwise, the rapid diadinoxanthin de-epoxidation observed in intact cells upon illumination would not be possible.

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using (31)P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L beta) to liquid-crystalline (L alpha) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L alpha to the inverted hexagonal (HII) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L alpha phase compared to those in the presence of HII phase forming lipids. Our data furthermore imply that the HII phase is better suited to maintain high VDE activities at lower temperatures.