ORCID IDs: 0000-0002-7487-8014 (S.S.); 0000-0002-2594-509X (S.S.M.)Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency.
SignificanceChlamydomonas reinhardtii is the premier reference organism for understanding unicellular green algae. Chlamydomonas is an important model for photosynthesis as well as fermentation and other anaerobic pathways under dark anoxic conditions. We have produced a diurnal transcriptome, validated by subproteomic analyses, and matched with measurements of pigments, select metabolites, and physiological parameters. We report that the majority of the algal genome is differentially expressed over the course of the day and the timing of specific genes is dictated by their biological function. We also discovered that fermentation rather than respiration is the preferred metabolic fate of starch-derived glycolytic pyruvate. We offer our rich dataset to the algal and plant communities.
Nuclear pore complexes (NPCs) span the nuclear envelope and mediate nucleocytoplasmic exchange. They are a hallmark of eukaryotes and deeply rooted in the evolutionary origin of cellular compartmentalization. NPCs have an elaborate architecture that has been well studied in vertebrates. Whether this architecture is unique or varies significantly in other eukaryotic kingdoms remains unknown, predominantly due to missing in situ structural data. Here, we report the architecture of the algal NPC from the early branching eukaryote Chlamydomonas reinhardtii and compare it to the human NPC. We find that the inner ring of the Chlamydomonas NPC has an unexpectedly large diameter, and the outer rings exhibit an asymmetric oligomeric state that has not been observed or predicted previously. Our study provides evidence that the NPC is subject to substantial structural variation between species. The divergent and conserved features of NPC architecture provide insights into the evolution of the nucleocytoplasmic transport machinery.
Summary The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high-level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.
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.
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