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Global food security is a pressing issue in our society. Maintaining food security in coming years will require improving crop yield, as well as increased resiliency to abiotic and biotic stress. Nanoscale materials have increasingly been proposed as a tool which could be used to meet these challenges. However, much research is needed to optimize nanoparticle design and crop application for this to become a reality. In this study, we investigated the impact of polymer-functionalized carbon dots on tomatoes (Solanum lycopersicum L.). Tomato seeds were vacuum infiltrated with carbon dots and then grown for 3 weeks before collection of phenotypic and transcriptomic data. No changes to fresh biomass or chlorophyll content were observed, indicating that these particles can be applied without overt harm to the plant at early growth stages. In addition, changes in gene expression suggest that polymer-functionalized carbon dots can initiate the expression of biochemical pathways associated with a pathogen resistance response in tomato plants. Specifically, genes involved in ethylene signaling, ethylene production, and camalexin synthesis were upregulated. These findings suggest that seed priming with carbon dots may improve plant tolerance to biotic stress by modulating ethylene signaling pathways. Carbon dots could also be loaded with nutrients or other agrochemicals to create a multifunctional platform. Future work should focus on understanding the mechanisms by which nanoparticles can modulate ethylene signaling, enabling use of this knowledge to develop sustainable and effective nanoparticles for agricultural applications. 

Surface charge is a key characteristic of nanoparticles which has great potential to impact the interactions of nanoparticles and biological systems. Understanding the role charge plays in these interactions is key to determining the ecological risks of nanoparticle exposure and informing sustainable nanoparticle design. In this study, the model freshwater algae Raphidocelis subcapitata was exposed to carbon dots (CDs) functionalized with polymers to have positive, negative, or neutral surface charges to examine the impact of nanoparticle surface charge on nano-algae interactions. Traditional toxicological endpoints of survival and growth inhibition were measured. Additionally, morphological impacts on whole cells, individual organelles, and cellular components were quantified using high-content fluorescence microscopy, demonstrating one of the first uses of high-content imaging in microalgae. Results indicate that PEI functionalized, positively charged CDs are most toxic to green algae (EC50 42.306 μg/L), but that CDs with negative charge induce sublethal impacts on algae. PEI-CD toxicity is hypothesized to be related to electrostatic interactions between CDs and the algal cell wall, which lead to significant cell aggregation. Interestingly, morphological data suggests that exposure to both positively and negatively charged CDs leads to increased neutral lipid droplet formation, a possible indicator of nutrient stress. Further investigation of the mechanisms underlying impacts of nanoparticle surface charge on algae biology can lead to more sustainable nanoparticle design and environmental protections. 

A lack of mechanistic understanding of nanomaterial interactions with plants and algae cell walls limits the advancement of nanotechnology-based tools for sustainable agriculture. We systematically investigated the influence of nanoparticle charge on the interactions with model cell wall surfaces built with cellulose or pectin and performed a comparative analysis with native cell walls of Arabidopsis plants and green algae (Choleochaete). The high affinity of positively charged carbon dots (CDs) (46.0 ± 3.3 mV, 4.3 ± 1.5 nm) to both model and native cell walls was dominated by the strong ionic bonding between the surface amine groups of CDs and the carboxyl groups of pectin. In contrast, these CDs formed weaker hydrogen bonding with the hydroxyl groups of cellulose model surfaces. The CDs of similar size with negative (−46.2 ± 1.1 mV, 6.6 ± 3.8 nm) or neutral (−8.6 ± 1.3 mV, 4.3 ± 1.9 nm) ζ-potentials exhibited negligible interactions with cell walls. Real-time monitoring of CD interactions with model pectin cell walls indicated higher absorption efficiency (3.4 ± 1.3 10−9) and acoustic mass density (313.3 ± 63.3 ng cm–2) for the positively charged CDs than negative and neutral counterparts (p < 0.001 and p < 0.01, respectively). The surface charge density of the positively charged CDs significantly enhanced these electrostatic interactions with cell walls, pointing to approaches to control nanoparticle binding to plant biosurfaces. Ca2+-induced cross-linking of pectin affected the initial absorption efficiency of the positively charged CD on cell wall surfaces (∼3.75 times lower) but not the accumulation of the nanoparticles on cell wall surfaces. This study developed model biosurfaces for elucidating fundamental interactions of nanomaterials with cell walls, a main barrier for nanomaterial translocation in plants and algae in the environment, and for the advancement of nanoenabled agriculture with a reduced environmental impact. 

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. 

Mechanisms of nanomaterial delivery to plant chloroplasts have been explored to improve plant stress tolerance, promote photosynthesis, facilitate genetic engineering, and manufacture self-repairing biomaterials, fuels, and biopharmaceuticals. However, the molecular interactions of nanomaterials with chloroplast membranes are not well understood. In this study, we examine the interactions of an important set of chloroplast membrane lipids including sulfoquinovosyl diacylglycerols with carbon nanodots varying in functional group charge. To accomplish this objective, we constructed a novel model chloroplast membrane and interrogated the influence of carbon nanodot functional group charge, model chloroplast membrane composition, and ionic strength on the carbon nanodot-chloroplast membrane interactions using quartz crystal microbalance with dissipation monitoring. We further examined the interaction of carbon nanodots with native chloroplasts isolated from Arabidopsis thaliana using confocal laser-scanning microscopy. Our results indicate that carbon nanodot–chloroplast membrane interactions are dictated primarily by electrostatics. Despite being the least abundant lipids in chloroplast membranes, we find that the relative abundance of sulfoquinovosyl diacylglycerol in model membranes is a critical factor governing both the affinity and capacity of the membrane for positively charged carbon nanodots. Rates of carbon nanodot attachment to model chloroplast membranes varied with ionic strength in a manner consistent with electrical double layer compression on carbon nanodots. Our findings elucidate chemical interactions between nanomaterials and plant biosurfaces at the molecular level and potentially contribute to establishing structure–property–interaction relationships of sustainable nanomaterials with plant organelle membranes.