Plant nanobiotechnology is an emerging field utilizing nanomaterials to study and engineer plant biological functions. The use of nanotechnology on plants can improve the efficacy of plant bioengineering and agriculture tools to improve future food securities. There is immense potential for applying nanomaterials-based tools’ physical and chemical properties to chloroplast biotechnology. The chloroplast prokaryotic-like genome makes them excellent targets for genetic engineering application due to their polycistronic gene structure, lack of silencing mechanisms, and ability to isolate genetic markers in parental lines. Current chloroplast transformation techniques are limited to a handful of plant species (<10) due partly to the absence of efficient gene delivery mechanisms to chloroplasts. If appropriately engineered, nanomaterials can overcome plant cell barriers such as walls and internal organelle compartments, making them ideal systems for chemical and gene delivery tools in plant model systems. In this dissertation, we standardize methods to interface nanomaterials into plant tissues in chapter one. Chapter two; we designed nanomaterials to localize inside chloroplasts using biorecognition motifs and deliver biochemicals to modulate chloroplast function in Arabidopsis thaliana. Chapter three utilized the targeted strategies implemented in chapter two to construct two carbon-based nanomaterials (Carbon dots and single-walled carbon nanotubes) complexes for chemical and gene delivery into chloroplasts. In Chapter three we investigate the biological impact of nanomaterials on Arabidopsis plants using targeted nanomaterials. We found increased localization and confirmed increased chemical and gene delivery into chloroplasts using cell and molecular-based assays. Furthermore, no significant difference in the cell or chloroplast integrity demonstrating low cell damage. However, the targeted nanomaterials affect the levels of oxidative DNA damage in whole plant cell extracts and increase hydrogen peroxide (H2O2) levels at 24 hours of exposure. Photosynthetic measurements showed no significant difference in Fv/Fm dark-adapted photosystem II efficiencies but, a decrease in chlorophyll content and a reduction in photosynthesis in the carboxylation limited region was observed. Together we demonstrate targeted nanomaterials for chemical and genetic material delivery into the chloroplast. With this information, we can improve the development of biocompatible nanomaterials for a broad range of applications, from improving the understanding of plant biology, enhancing crop yields to transforming plants into technology.

Nanotechnology approaches to deliver biomolecules to chloroplasts has been applied to land plants, but not well studied in algae. It was found that little is known about how nanomaterial size, charge, and plant biomolecule coatings influence interactions with green algae and its outer algae matrix. Intellectual merit for this research, explored in Chapter 1, included environmental sustainability of industrial runoff and chloroplast transformation for use in synthetic biology. Broader impacts of nanotechnology apply to chloroplast biotechnology would be increased plant productivity, enhanced lipid production for renewable biofuels, and more sustainable biodegradable materials. These nanotechnology approaches, explored in Chapter 2, can lead to new abilities for plants, or bolster existing abilities, such as dealing with the reactive oxygen species created from abiotic or biotic stressors. To enable chloroplast biotechnology through nanomaterial in situ approaches, cerium oxide nanoparticles, outlined in Chapter 3, were fabricated to scavenge reactive oxygen species within chloroplasts of Arabidopsis and confocal microscopy with colocalization analysis was used for scavenging confirmation.

In Chapter 4, we delivered DNA to the chloroplasts of algae with single-walled carbon nanotubes coated with a polymer. Carbon nanotubes were coated and bound to single-stranded DNA. Varying polymer lengths and mass ratios of DNA:coated-nanotube were characterized for their size and charge, and those characteristics were analyzed against the green algae Chlamydomonas reinhardtii with and without a cell wall. Assays were used to analyze the impact the polymer-coated single-walled carbon nanotubes impact on production of reactive oxygen species, living cell enzymatic activity, and total carotenoid production over four days. To confirm that the DNA biomolecule was being uptaken to the algae chloroplast after a one hour exposure, a dye was bound to the DNA (Dye-DNA). With confocal microscopy, the Dye-DNA was confirmed to colocalize within the chloroplast due to chlorophyll autofluorescence, with a Manders colocalization analysis and ANOVA tests for statistical significance. Our results indicate that the higher charged nanoparticle was able to deliver Dye-DNA at a higher rate than the lower charged nanoparticle, confirming previous hypotheses and models seen in land plants.  Together we demonstrated biomolecule delivery to algal chloroplasts and biocompatibility of DNA and polymer-coated single-walled carbon nanotubes in the model organism green algae Chlamydomonas reinhardtii. With this, we can move forward with applications to chloroplast transformation into algae, furthering our understanding and capabilities of synthetic biology for chloroplast biotechnology advancements.