Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.

The unicellular green alga Chlamydomonas reinhardtii displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light–dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that Chlamydomonas exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes PSBS , LHCSR1 , and LHCSR3 show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while LHCSR3 genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.

To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall–deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation–induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10²) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicated increased succinate, malate, and Glc-6-P and decreased Fru-1,6-bisphosphate, illustrating the effect of these changes. Comparisons of independent data sets in multiple strains allowed the delineation of a sequence of events in the global N starvation response in C. reinhardtii, starting within minutes with the upregulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual upregulation of genes encoding enzymes of TAG synthesis. Finally, genome resequencing analysis indicated that (1) the deletion in sta6 extends into the neighboring gene encoding respiratory burst oxidase, and (2) a commonly used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.

Significance: The growing human population generates increasing demand for food and energy. Microalgae are a promising source of sustainable bioproducts whose production may not exacerbate worsening environmental problems. The green algaChromochloris zofingiensishas potential as a biofuel feedstock and source of high-value nutraceutical molecules, including the carotenoid astaxanthin. We present a high-quality, chromosome-level assembly of the genome by using a hybrid sequencing approach with independent validation by optical mapping. Our analyses of the genome and transcriptome, in addition to experiments characterizing astaxanthin production, advance understanding of the green lineage and carotenoid production, and enhance prospects for improving commercial production ofC. zofingiensis.

A collection of chemical tools and spectroscopic techniques demonstrate that Zn availability influences Cu+ storage and localization in the green alga Chlamydomonas, with Zn limitation causing the accumulation of Cu+ in lysosome-related organelles. We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano–secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu+ accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu+ became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

Summary: Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal‐based platform for producing biofuels and bio‐products. Its highly repetitive, ~205‐kbp circular chloroplast genome and ~15.8‐kbp linear mitochondrial genome were sequenced prior to the advent of high‐throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA‐Seq was used to quantify their relative abundances across 12 different growth conditions. Forty‐six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA‐Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine‐rich polynucleotide tails were observed at the 3’‐end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.