Barbara B. Sears

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.

A Galactoglycerolipid Lipase Is Required for Triacylglycerol Accumulation and Survival Following Nitrogen Deprivation in Chlamydomonas reinhardtii

A mutant of Chlamydomonas reinhardtii with impaired oil accumulation is shown to be deficient in a lipase with specificity for newly assembled monogalactolipids. Passage of fatty acids synthesized in the chloroplast through a transient chloroplast membrane lipid pool into triacylglycerols is proposed. A role of oil biosynthesis for survival following nutrient deprivation is demonstrated. Following N deprivation, microalgae accumulate triacylglycerols (TAGs). To gain mechanistic insights into this phenomenon, we identified mutants with reduced TAG content following N deprivation in the model alga Chlamydomonas reinhardtii. In one of the mutants, the disruption of a galactoglycerolipid lipase-encoding gene, designated PLASTID GALACTOGLYCEROLIPID DEGRADATION1 (PGD1), was responsible for the primary phenotype: reduced TAG content, altered TAG composition, and reduced galactoglycerolipid turnover. The recombinant PGD1 protein, which was purified from Escherichia coli extracts, hydrolyzed monogalactosyldiacylglycerol into its lyso-lipid derivative. In vivo pulse-chase labeling identified galactoglycerolipid pools as a major source of fatty acids esterified in TAGs following N deprivation. Moreover, the fatty acid flux from plastid lipids to TAG was decreased in the pgd1 mutant. Apparently, de novo–synthesized fatty acids in Chlamydomonas reinhardtii are, at least partially, first incorporated into plastid lipids before they enter TAG synthesis. As a secondary effect, the pgd1 mutant exhibited a loss of viability following N deprivation, which could be avoided by blocking photosynthetic electron transport. Thus, the pgd1 mutant provides evidence for an important biological function of TAG synthesis following N deprivation, namely, relieving a detrimental overreduction of the photosynthetic electron transport chain.

Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas reinhardtii: Discovery of the Betaine Lipid Synthase BTA1Cr

ABSTRACT Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.

Complete nucleotide sequence of the Oenothera elata plastid chromosome, representing plastome I of the five distinguishable Euoenothera plastomes

Continued sequencing of Oenothera plastid chromosomes in our laboratory has led to the discovery of various errors in the published plastome I sequence (Hupfer et al. 2000). Some of these errors have also been noted by others [Rice DW, Palmer JD (2006) BMC Biol 4:31]. We have now re-sequenced the plastome I chromosome [Greiner S, Wang X, Rauwolf U, Silber MV, Mayer K, Meurer J, Harberer G, Herrmann RG (2008) The complete nucleotide sequences of the Wve genetically distinct plastid genomes of Oenothera subsection Oenothera: I. Sequence evaluation and plastome evolution. Nucleic Acids Res 36:2366–2378] and deposited the revised sequence in GenBank under the accession no. AJ271079.3. This submission supersedes the previously submitted sequences with the accession nos. AJ271079.1 and AJ271079.2. We wish to emphasize that the basic inferences drawn in the original publication (Hupfer et al. 2000) remain unchanged. We sincerely regret any inconvenience caused by the publication of the original data.

Genes and transcripts for the polypeptides of the cytochrome b6/f complex from spinach thylakoid membranes.

Cytochrome b6/f complex was prepared from washed thylakoid membranes by a procedure involving detergent treatment and centrifugation in sucrose gradients. The complex is composed of at least four polypeptide species, cytochrome f which occurs in two variant forms (mol. wt. 34/33 kd), cytochrome b6 (23 kd), the high‐potential Rieske iron‐sulfur protein (19 kd) and a fourth subunit (17 kd) of unknown function. Transcripts for the cytochromes f, b6 and subunit 4 were found in plastid RNA, those for the Rieske iron‐sulfur protein in cytosolic poly(A)+ RNA. Transcripts for cytochrome b6 and subunit 4 are translated in rabbit reticulocyte lysates into products of correct length. The Rieske iron‐sulfur protein and the cytochrome f apoprotein appear to be made as precursors with excess sequences of 7 and 4 kd, respectively. Cytochrome f, cytochrome b6 and subunit 4 are encoded by uninterrupted plastid genes that are located in the large single‐copy region of the circular DNA molecule. Each of these genes is present once per chromosome. Their location and direction of transcription have been determined by hybrid‐selection mapping and by cell‐free transcription/translation of various recombinant DNAs. The genes for cytochrome b6 and for subunit 4 lie near each other, but do not overlap. They are transcribed into a single message. The gene for cytochrome f maps 15 kbp away from this cluster, close to the 3′ end of the gene for the large subunit of ribulosebisphosphate carboxylase/oxygenase, and is transcribed into a separate 4 kb long RNA. All these genes have the same polarities with respect to each other.

The Sulfolipids 2′-O-Acyl-Sulfoquinovosyldiacylglycerol and Sulfoquinovosyldiacylglycerol Are Absent from a Chlamydomonas reinhardtii Mutant Deleted in SQD1

The biosynthesis of thylakoid lipids in eukaryotic photosynthetic organisms often involves enzymes in the endoplasmic reticulum (ER) and the chloroplast envelopes. Two pathways of thylakoid lipid biosynthesis, the ER and the plastid pathways, are present in parallel in many species, including Arabidopsis, but in other plants, e.g. grasses, only the ER pathway is active. The unicellular alga Chlamydomonas reinhardtii diverges from plants like Arabidopsis in a different way because its membranes do not contain phosphatidylcholine, and most thylakoid lipids are derived from the plastid pathway. Here, we describe an acylated derivative of sulfolipid, 2′-O-acyl-sulfoquinovosyldiacylglycerol (ASQD), which is present in C. reinhardtii. Although the fatty acids of sulfoquinovosyldiacylglycerol (SQDG) were mostly saturated, ASQD molecular species carried predominantly unsaturated fatty acids. Moreover, directly attached to the head group of ASQD was preferentially an 18-carbon fatty acid with four double bonds. High-throughput robotic screening led to the isolation of a plasmid disruption mutant of C. reinhardtii, designated Δsqd1, which lacks ASQD as well as SQDG. In this mutant, the SQD1 ortholog was completely deleted and replaced by plasmid sequences. It is proposed that ASQD arises from the sugar nucleotide pathway of sulfolipid biosynthesis by acylation of the 2′-hydroxyl of the sulfoquinovosyl head group. At the physiological level, the mutant showed increased sensitivity to a diuron herbicide and reduced growth under phosphate limitation, suggesting a role for SQDG and/or ASQD in photosynthesis as conducted by C. reinhardtii, particularly under phosphate-limited conditions.

Plastid inheritance in Oenothera: organelle genome modifies the extent of biparental plastid transmission

SummaryThe transmission abilities of four out of the five major plastome types of Oenothera (I–V) were analyzed in a constant nuclear background by assessing both the frequency of biparental inheritance and the extent of variegation in the progeny. Reciprocal crosses were performed between plants carrying one of four wild-type plastomes and plants carrying one of seven white plastid mutants. The frequency of biparental plastid transmission ranged from 0 to 56% depending on the plastid types involved in the crosses. The transmission abilities of the four representative wild-type plastids appear to be in the order of I > III > II > IV in the nuclear background of O. hookeri str. Johansen. In general, variegated seedlings from crosses that produced a higher frequency of biparental plastid transmission also had an increased abundance of tissue containing plastids of paternal origin. Although the transmission abilities of most Oenothera plastid mutants are comparable to the wild-type plastids, three mutant plastids derived from species having different type I plastids show three distinguishable transmission patterns. This study confirms the significant role of the plastome in the process of plastid transmission and possibly in plastid multiplication. However, the hypothesis of differential plastid multiplication rates suggested by earlier studies can explain the results only partially. The initiation of plastid multiplication within the newly formed zygote also seems to be plastome-dependent.

Complementation analysis of the inorganic carbon concentrating mechanism of Chlamydomonas reinhardtii

SummarySix independently isolated mutants of Chlamydomonas reinhardtii that require elevated CO2 for photoautotrophic growth were tested by complementation analysis. These mutants are likely to be defective in some aspect of the algal concentrating mechanism for inorganic carbon as they exhibit CO2 fixation and inorganic carbon accumulation properties different from the wild-type. Four of the six mutants defined a single complementation group and appear to be defective in an intracellular carbonic anhydrase. The other two mutations represent two additional complementation groups.

Recombination between chloroplast DNAs does not occur in sexual crosses of Oenothera.

SummaryCrosses of Oenothera result in the transmission of chloroplasts from both parents to their offspring. In spite of this biparental inheritance, no wild-type recombinants were recovered from crosses between different chloroplast mutants. Since more than 7500 progeny were examined, the results indicate that recombination between the chloroplast DNAs of higher plants must be a very rare event.