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Chloroplast are sites of photosynthesis that have been bioengineered to produce food, biopharmaceuticals, and biomaterials. Current approaches for altering the chloroplast genome rely on inefficient DNA delivery methods, leading to low chloroplast transformation efficiency rates. For algal chloroplasts, there is no modifiable, customizable, and efficient in situ DNA delivery chassis. Herein, we investigated polyethylenimine-coated single-walled carbon nanotubes (PEI-SWCNT) as delivery vehicles for DNA to algal chloroplasts. We examined the impact of PEI-SWCNT charge and PEI polymer size (25k vs 10k) on the uptake into chloroplasts of wildtype and cell wall knockout mutant strains of the green algae Chlamydomonas reinhardtii. To assess the delivery of DNA bound to PEI-SWCNT, we used confocal microscopy and colocalization analysis of chloroplast autofluorescence with fluorophore-labeled single-stranded GT15 DNA. We found that highly charged DNA-PEI25k-SWNCT have a statistically significant higher percentage of DNA colocalization events with algal chloroplasts (22.28% ± 6.42, 1 hr) over 1-3 hours than DNA-PEI10k-SWNCT (7.23% ± 0.68, 1 hr) (P<0.01). We determined the biocompatibility of DNA-PEI-SWCNT through assays for living algae cells, reactive oxygen species (ROS) generation, and in vivo chlorophyll assays. Through these assays, it was shown that algae exposed to DNA-PEI25k-SWCNT (30 fg/cell) and DNA-PEI10k-SWCNT (300 fg/cell) were viable over 4 days and had little impact on oxidative stress levels. DNA coated PEI-SWCNT transiently increased ROS levels within one hour of exposure to nanomaterials (30- 300 fg/cell) both in the wildtype strain and cell-wall knockout strain, followed by ROS decline to normal levels due to reaction with antioxidant glutathione and lipid membranes. PEI-SWCNT can act as biological carriers for delivering biomolecules such as DNA and have the potential to become novel tools for chloroplast biotechnology and synthetic biology. 

The breakdown of symbiotic mutualism between cnidarian hosts and dinoflagellate algae partners (i.e., bleaching) has been linked to an immune-like response pathway brought on by a nitro-oxidative burst, a symptom of thermal stress. Stress induced by reactive oxygen species (ROS)/reactive nitrogen species is a problem common to aerobic systems. In this study, we tested the antioxidant effects of engineered poly(acrylic acid)-coated cerium dioxide nanoparticles (CeO 2 , nanoceria) on free-living Symbiodiniaceae ( Breviolum minutum ), a dinoflagellate alga that forms symbiotic relationships with reef-building corals and anemones. Results show that poly(acrylic acid)-coated CeO 2 with hydrodynamic diameters of ~4 nm are internalized by B. minutum in under 30 min and subsequently localized in the cytosol. Nanoceria exposure does not inhibit cell growth over time, with the treated cultures showing a similar growth trend over the 25-day exposure. Aerobic activity and thermal stress when held at 34°C for 1 h (+6°C above control) led to increased intracellular ROS concentration with time. A clear ROS scavenging effect of the nanoceria was observed, with a 5-fold decrease in intracellular ROS levels during thermal stress. The nitric oxide (NO) concentration decreased by ~17% with thermal stress, suggesting the rapid involvement of NO scavenging enzymes or proteins within 1 h of stress onset. The presence of nanoceria did not appear to influence NO concentration. Furthermore, aposymbiotic anemones ( Exaiptasia diaphana , ex Aiptasia pallida ) were successfully infected with nanoceria-loaded B. minutum , demonstrating that inoculation could serve as a delivery method. The ability of nanoceria to be taken up by Symbiodiniaceae and reduce ROS production could be leveraged as a potential mitigation strategy to reduce coral bleaching. 

Abstract Artificial photosynthesis systems are proposed as an efficient alternative route to capture CO 2 to produce additional food for growing global demand. Here a two-step CO 2 electrolyser system was developed to produce a highly concentrated acetate stream with a 57% carbon selectivity (CO 2 to acetate), allowing its direct use for the heterotrophic cultivation of yeast, mushroom-producing fungus and a photosynthetic green alga, in the dark without inputs from biological photosynthesis. An evaluation of nine crop plants found that carbon from exogenously supplied acetate incorporates into biomass through major metabolic pathways. Coupling this approach to existing photovoltaic systems could increase solar-to-food energy conversion efficiency by about fourfold over biological photosynthesis, reducing the solar footprint required. This technology allows for a reimagination of how food can be produced in controlled environments. 

Photosynthetic organisms are sources of sustainable foods, renewable biofuels, novel biopharmaceuticals, and next-generation biomaterials essential for modern society. Efforts to improve the yield, variety, and sustainability of products dependent on chloroplasts are limited by the need for biotechnological approaches for high-throughput chloroplast transformation, monitoring chloroplast function, and engineering photosynthesis across diverse plant species. The use of nanotechnology has emerged as a novel approach to overcome some of these limitations. Nanotechnology is enabling advances in the targeted delivery of chemicals and genetic elements to chloroplasts, nanosensors for chloroplast biomolecules, and nanotherapeutics for enhancing chloroplast performance. Nanotechnology-mediated delivery of DNA to the chloroplast has the potential to revolutionize chloroplast synthetic biology by allowing transgenes, or even synthesized DNA libraries, to be delivered to a variety of photosynthetic species. Crop yield improvements could be enabled by nanomaterials that enhance photosynthesis, increase tolerance to stresses, and act as nanosensors for biomolecules associated with chloroplast function. Engineering isolated chloroplasts through nanotechnology and synthetic biology approaches are leading to a new generation of plant-based biomaterials able to self-repair using abundant CO 2 and water sources and are powered by renewable sunlight energy. Current knowledge gaps of nanotechnology-enabled approaches for chloroplast biotechnology include precise mechanisms for entry into plant cells and organelles, limited understanding about nanoparticle-based chloroplast transformations, and the translation of lab-based nanotechnology tools to the agricultural field with crop plants. Future research in chloroplast biotechnology mediated by the merging of synthetic biology and nanotechnology approaches can yield tools for precise control and monitoring of chloroplast function in vivo and ex vivo across diverse plant species, allowing increased plant productivity and turning plants into widely available sustainable technologies. 

Abstract In cnidarian-Symbiodiniaceae symbioses, algal endosymbiont population control within the host is needed to sustain a symbiotic relationship. However, the molecular mechanisms that underlie such population control are unclear. Here we show that a cnidarian host uses nitrogen limitation as a primary mechanism to control endosymbiont populations. Nitrogen acquisition and assimilation transcripts become elevated in symbiotic Breviolum minutum algae as they reach high-densities within the sea anemone host Exaiptasia pallida . These same transcripts increase in free-living algae deprived of nitrogen. Symbiotic algae also have an elevated carbon-to-nitrogen ratio and shift metabolism towards scavenging nitrogen from purines relative to free-living algae. Exaiptasia glutamine synthetase and glutamate synthase transcripts concomitantly increase with the algal endosymbiont population, suggesting an increased ability of the host to assimilate ammonium. These results suggest algal growth and replication in hospite is controlled by access to nitrogen, which becomes limiting for the algae as their population within the host increases. 

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.