Mutation of negative regulatory gene CEHC1 encoding an FBXO3 protein results in normoxic expression of HYDA genes in Chlamydomonas reinhardtii.

Oxygen is known to prevent hydrogen production in Chlamydomonas, both by inhibiting the hydrogenase enzyme and by preventing the accumulation of HYDA-encoding transcripts. We developed a screen for mutants showing constitutive accumulation of HYDA1 transcripts in the presence of oxygen. A reporter gene required for ciliary motility, placed under the control of the HYDA1 promoter, conferred motility only in hypoxic conditions. By selecting for mutants able to swim even in the presence of oxygen we obtained strains that express the reporter gene constitutively. One mutant identified a gene encoding an F-box only protein 3 (FBXO3), known to participate in ubiquitylation and proteasomal degradation pathways in other eukaryotes. Transcriptome profiles revealed that the mutation, termed cehc1-1 , leads to constitutive expression of HYDA1 and other genes regulated by hypoxia, and of many genes known to be targets of CRR1, a transcription factor in the nutritional copper signaling pathway. CRR1 was required for the constitutive expression of the HYDA1 reporter gene in cehc1-1 mutants. The CRR1 protein, which is normally degraded in Cu-supplemented cells, was stabilized in cehc1-1 cells, supporting the conclusion that CEHC1 acts to facilitate the degradation of CRR1. Our results reveal a novel negative regulator in the CRR1 pathway and possibly other pathways leading to complex metabolic changes associated with response to hypoxia.

Optimizing Fe Nutrition for Algal Growth.

Chlamydomonas is an excellent reference system for dissecting the impact of iron (Fe) nutrition on photosynthetic and other metabolisms. The operational definition of four stages of Fe nutrition is described and a guide to the practical use of these stages is offered, specifically the preparation of media and growth of mixotrophic cultures. A key consideration is the impact of carbon metabolism on the expression of Fe-containing enzymes and hence the Fe quota. The absolute concentration of Fe in the medium is less determinative of gene expression than the Fe available on a per-cell basis. In nature, algal cells may transition from Fe-replete to -deficient to -limited during a bloom.

Chlamydomonas Genome Resource for Laboratory Strains Reveals a Mosaic of Sequence Variation, Identifies True Strain Histories, and Enables Strain-Specific Studies.

Chlamydomonas reinhardtii is a widely used reference organism in studies of photosynthesis, cilia, and biofuels. Most research in this field uses a few dozen standard laboratory strains that are reported to share a common ancestry, but exhibit substantial phenotypic differences. In order to facilitate ongoing Chlamydomonas research and explain the phenotypic variation, we mapped the genetic diversity within these strains using whole-genome resequencing. We identified 524,640 single nucleotide variants and 4812 structural variants among 39 commonly used laboratory strains. Nearly all (98.2%) of the total observed genetic diversity was attributable to the presence of two, previously unrecognized, alternate haplotypes that are distributed in a mosaic pattern among the extant laboratory strains. We propose that these two haplotypes are the remnants of an ancestral cross between two strains with ∼2% relative divergence. These haplotype patterns create a fingerprint for each strain that facilitates the positive identification of that strain and reveals its relatedness to other strains. The presence of these alternate haplotype regions affects phenotype scoring and gene expression measurements. Here, we present a rich set of genetic differences as a community resource to allow researchers to more accurately conduct and interpret their experiments with Chlamydomonas.

Systems-level analysis of nitrogen starvation-induced modifications of carbon metabolism in a Chlamydomonas reinhardtii starchless mutant.

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 × 10(2)) 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.

Iron economy in Chlamydomonas reinhardtii.

While research on iron nutrition in plants has largely focused on iron-uptake pathways, photosynthetic microbes such as the unicellular green alga Chlamydomonas reinhardtii provide excellent experimental systems for understanding iron metabolism at the subcellular level. Several paradigms in iron homeostasis have been established in this alga, including photosystem remodeling in the chloroplast and preferential retention of some pathways and key iron-dependent proteins in response to suboptimal iron supply. This review presents our current understanding of iron homeostasis in Chlamydomonas, with specific attention on characterized responses to changes in iron supply, like iron-deficiency. An overview of frequently used methods for the investigation of iron-responsive gene expression, physiology and metabolism is also provided, including preparation of media, the effect of cell size, cell density and strain choice on quantitative measurements and methods for the determination of metal content and assessing the effect of iron supply on photosynthetic performance.