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.
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DNA delivery by high aspect ratio nanomaterials to algal chloroplasts
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.
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- Award ID(s):
- 2001611
- NSF-PAR ID:
- 10464936
- Date Published:
- Journal Name:
- Environmental Science: Nano
- ISSN:
- 2051-8153
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary Reactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D3EN00268C
