The widespread use of nanoparticles (NPs) raises concern over their potential toxicological effects in humans and ecosystems. Here we used transcriptome sequencing (RNA-seq) to evaluate the effects of exposure to four different metal-based NPs, nano-Ag (nAg), nano-TiO ₂ (nTiO ₂ ), nano-ZnO (nZnO), and CdTe/CdS quantum dots (QDs), in the eukaryotic green alga Chlamydomonas reinhardtii . The transcriptome was characterized before and after exposure to each NP type. Specific toxicological effects were inferred from the functions of genes whose transcripts either increased or decreased. Data analysis resulted in important differences and also similarities among the NPs. Elevated levels of transcripts of several marker genes for stress were observed, suggesting that only nZnO caused nonspecific global stress to the cells under environmentally relevant conditions. Genes with photosynthesis-related functions were decreased drastically during exposure to nTiO ₂ and slightly during exposures to the other NP types. This pattern suggests either toxicological effects in the chloroplast or effects that mimic a transition from low to high light. nAg exposure dramatically elevated the levels of transcripts encoding known or predicted components of the cell wall and the flagella, suggesting that it damages structures exposed to the external milieu. Exposures to nTiO ₂ , nZnO, and QDs elevated the levels of transcripts encoding subunits of the proteasome, suggesting proteasome inhibition, a phenomenon believed to underlie the development and progression of several major diseases, including Alzheimer's disease, and used in chemotherapy against multiple myeloma.

Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the 5′ untranslated region of the chloroplast psbC mRNA of the green alga Chlamydomonas reinhardtii. These analyses revealed that RNA-binding proteins of 30–32, 46, 47, 60, and 80 kD are associated with chloroplast membranes. The buoyant density and the acyl lipid composition of these membranes are compatible with their origin being the inner chloroplast envelope membrane. However, unlike previously characterized inner envelope membranes, these membranes are associated with thylakoids. One of the membrane-associated RNA-binding proteins appears to be RB47, which has been reported to be a specific activator of psbA mRNA translation. These results suggest that translation of chloroplast mRNAs encoding thylakoid proteins occurs at either a subfraction of the chloroplast inner envelope membrane or a previously uncharacterized intra-chloroplast compartment, which is physically associated with thylakoids.

Many proteins of the photosynthesis complexes are encoded by the genome of the chloroplast and synthesized by bacterium-like ribosomes within this organelle. To determine where proteins are synthesized for the de novo assembly and repair of photosystem II (PSII) in the chloroplast of Chlamydomonas reinhardtii, we used fluorescence in situ hybridization, immunofluorescence staining, and confocal microscopy. These locations were defined as having colocalized chloroplast mRNAs encoding PSII subunits and proteins of the chloroplast translation machinery specifically under conditions of PSII subunit synthesis. The results revealed that the synthesis of the D1 subunit for the repair of photodamaged PSII complexes occurs in regions of the chloroplast with thylakoids, consistent with the current model. However, for de novo PSII assembly, PSII subunit synthesis was detected in discrete regions near the pyrenoid, termed T zones (for translation zones). In two PSII assembly mutants, unassembled D1 subunits and incompletely assembled PSII complexes localized around the pyrenoid, where we propose that they mark an intermediate compartment of PSII assembly. These results reveal a novel chloroplast compartment that houses de novo PSII biogenesis and the regulated transport of newly assembled PSII complexes to thylakoid membranes throughout the chloroplast.

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO₂-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically wasteful oxygenase activity of Rubisco, and mitigates limiting CO₂ availability in aquatic environments. Hence, the pyrenoid is functionally analogous to the carboxysomes in cyanobacteria. However, a comprehensive analysis of pyrenoid functions based on its protein composition is lacking. Here we report a proteomic characterization of the pyrenoid in the green alga Chlamydomonas reinhardtii. Pyrenoid-enriched fractions were analyzed by quantitative mass spectrometry. Contaminant proteins were identified by parallel analyses of pyrenoid-deficient mutants. This pyrenoid proteome contains 190 proteins, many of which function in processes that are known or proposed to occur in pyrenoids: e.g. the carbon concentrating mechanism, starch metabolism or RNA metabolism and translation. Using radioisotope pulse labeling experiments, we show that pyrenoid-associated ribosomes could be engaged in the localized synthesis of the large subunit of Rubisco. New pyrenoid functions are supported by proteins in tetrapyrrole and chlorophyll synthesis, carotenoid metabolism or amino acid metabolism. Hence, our results support the long-standing hypothesis that the pyrenoid is a hub for metabolism. The 81 proteins of unknown function reveal candidates for new participants in these processes. Our results provide biochemical evidence of pyrenoid functions and a resource for future research on pyrenoids and their use to enhance agricultural plant productivity. Data are available via ProteomeXchange with identifier PXD004509.