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Fluorescence Reports Intact Quantum Dot Uptake into Roots and Translocation to Leaves of Arabidopsis thaliana and Subsequent Ingestion by Insect Herbivores Yeonjong Koo, Jing Wang, Qingbo Zhang, Huiguang Zhu, E. Wassim Chehab, Vicki L. Colvin, Pedro J. J. Alvarez, and Janet Braam Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5050562 • Publication Date (Web): 01 Dec 2014 Downloaded from http://pubs.acs.org on December 10, 2014
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Fluorescence Reports Intact Quantum Dot Uptake into Roots and Translocation to Leaves of Arabidopsis thaliana and Subsequent Ingestion by Insect Herbivores
Yeonjong Koo†,*, Jing Wang‡, Qingbo Zhang§, Huiguang Zhu§, E. Wassim Chehab†, Vicki L. Colvin§, Pedro J. J. Alvarez‡, and Janet Braam†,*
†
Department of BioSciences, Rice University, Houston, TX 77005, USA.
‡
Department of Civil & Environmental Engineering, Rice University, Houston, TX 77005, USA.
§
Department of Chemistry, Rice University, Houston, TX 77005, USA
*
To whom correspondence should be addressed. E-mail:
[email protected], Tel: (713)348-4277
(J.B.); E-mail:
[email protected], Tel: (713)348-6963 (Y.K.).
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ABSTRACT
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We explored the impact of quantum dot (QD) coat characteristics on NP stability, uptake and
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translocation in Arabidopsis thaliana, and subsequent transfer to primary consumers,
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Trichoplusia ni (T. ni). Arabidopsis was exposed to CdSe/CdZnS QDs with three different
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coatings: poly(acrylic acid-ethylene glycol) (PAA-EG), polyethylenimine (PEI) and poly(maleic
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anhydride-alt-1-octadecene)-poly(ethylene glycol) (PMAO-PEG), which are cationic, anionic,
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and relatively neutral, respectively. PAA-EG-coated QDs were relatively stable and taken up
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from a hydroponic medium through both Arabidopsis leaf petioles and roots, without apparent
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aggregation, and showed generally uniform distribution in leaves. In contrast, PEI- and PMAO-
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PEG-coated QDs displayed destabilization in the hydroponic medium, and generated particulate
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fluorescence plant tissues, suggesting aggregation. PAA-EG QDs moved faster than PEI QDs
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was through leaf petioles, however 8-fold more cadmium accumulated in PEI QD-treated than in
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those exposed to PAA-EG QDs, possibly due to PEI QD dissolution and direct metal uptake. T.
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ni caterpillars that fed on Arabidopsis exposed to QDs had reduced performance, and QD
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fluorescence was detected in both T. ni bodies and frass, demonstrating trophic transfer of intact
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QDs from plants to insects. Overall, this paper demonstrates that QD coat properties influence
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plant nanoparticle uptake and translocation and can impact transfer to herbivores.
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Keywords: quantum dots, nanomaterials, surface coating, translocation, root absorption,
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food web, plant-insect interactions
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INTRODUCTION
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Possible uptake and accumulation of nanoparticles (NPs) in plants, especially in the
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edible parts, has raised great concern over human exposure from food web contamination.1
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However, very few studies have investigated the possibility of NP transfer from plant uptake into
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a potential food web, with those published focused on aquatic2-5 and microbial6, 7 systems. For
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example, poly(acrylic acid)-octylamine-coated CdSe/ZnS was found to be transferred from
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zooplankton to zebrafish (Danio rerio) with a biomagnification factor (BMF) of 0.04.3 Over 5-
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fold biomagnification of uncoated CdSe quantum dots (QDs) was also observed in protozoa
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(Tetrahymena thermophila) when QDs were transferred from bacteria (Pseudomonas
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aeruginosa).7 Transfer of carboxylated and biotinylated QDs from ciliated protozoan to rotifers
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was also reported.6 Nevertheless, studies on NP transfer in a terrestrial ecosystem, from primary
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producers (e.g., plants) to primary consumers (e.g., pests), are very limited,8, 9 with one report
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showing transfer of Au NPs of different sizes from tobacco (Nicotiana tabacum L. cv Xanathi) to
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tobacco hornworm (Manduca sexta) with 6.2, 11.6 and 9.6 BMF for 5, 10, and 15 nm Au NPs,
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respectively.8 However, there remains a critical knowledge gap in trophic transfer of NPs in
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food chains more closely relevant to human consumption.
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Furthermore, the mechanism(s) underlying NP uptake and translocation by plants
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remains poorly understood. NP solutions are dynamic, being able to aggregate and dissolve. In
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addition, plants can take up various ions from the growth medium or soil and reductively
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precipitate them as particles.10-13 Therefore, the presence of particles in a plant is not always
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definitive proof for the plant ability to take up and translocate intact metal NPs. QDs are
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particularly suited for studying intact NP uptake in plants. In addition to be useful for tracking of
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particle uptake and movement in variety of organisms,14-17 fluorescence is only detected when
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intact QDs are present. Furthermore, breakdown components are very unlikely to reconstitute
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into fluorescent QDs in plants, especially with the original narrow light spectrum.18, 19 Another
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issue to consider in NP uptake and translocation by plants is the effect of NP surface coatings.
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Many types of metallic NPs, including the core ions of QDs, are coated by a polymer surface to
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increase stability, water-solubility and usefulness (e.g., enhancement of biocompatibility for bio-
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imaging applications).14, 15, 20-23 Once released into the environment, the coatings could affect NP
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interactions with plants. The effect of such NP surface coatings has been explored in a limited
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number of studies.10, 15, 22 It was previously demonstrated that QDs with NH2 groups composing
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the surface coat were able to translocate to plant aerial tissues,24 and QDs conjugated with two
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different polymers also showed different uptake rates by poplar.25 Zhai et al. reported the
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transport of Au NPs through poplar plasmodesmata,26 although it is unknown whether this
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transport pattern also applies to other NPs, such as QDs, and whether NP surface coats affect
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their translocation pattern throughout the whole plant.27, 28
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In this report, we report on the effect of QD coats on particle stability and plant uptake
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and translocation, and trophic transfer to a generalist herbivore. We chose QDs for this study
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because, as nano-scale fluorescent semiconductors, they enable tracking of intact particle uptake
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and movement. In addition, we chose to use Arabidopsis thaliana, a member of Brassicaceae
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family; therefore the findings may be relevant to diverse and popular vegetables, such as cabbage,
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kale, and broccoli. Furthermore, the extensive tools and resources available for this model plant
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will facilitate future investigations of the underlying molecular genetic and genomic bases for
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differential NP uptake, translocation, and toxicity.
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MATERIALS AND METHODS
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QD Synthesis and Characterization
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QDs were synthesized as described in Zhu et al. (2010). For surface modification of QDs,
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the as-synthesized QD core was transferred to aqueous solution using ligand exchange or
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encapsulation methods.29, 30 PAA-EG30 and PMAO-PEG31 were synthesized using procedures
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described previously32 and PEI (MW = 25000) (408727, Sigma-Aldrich, St. Louis, MO) was
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purchased from Sigma-Aldrich. 0.1 mL of polymer solution was added to 1 mL of ethyl ether
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solution of QDs under stirring. 1 mL of water was then added to the solution and sonicated for 2
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minutes. The mixture was stirred overnight to evaporate ethyl ether. The nanoparticles were
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purified by ultracentrifugation at 183,000 x g and 0.2 µm syringe filtration. Purified
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nanoparticles were redispersed in deionized water. Uniform cores and core sizes were
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characterized with TEM imaging.25 QD concentrations were determined by chemical digestion
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with ICP-MS (Elan 9000, PerkinElmer, MA), as described below. The zeta potential of each QD
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solutions was determined with Zen 3600 Zetasizer Nano (Malvern Instruments, United Kingdom)
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at 10 µg/mL concentration. Differential stability of QD fluorescence, indicating particle
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structural integrity and QD tendency to aggregate in plant growth media and water are reported
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in the Supplementary Information (Figure S1).
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Plant Growth and QD Treatments
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QDs were dispersed in water or 1/4, 1/8 or 1/16-strength of Hoagland solution33 at 10
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µg/mL Cd concentration. Arabidopsis were grown in soil or hydroponically in Hoagland solution,
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as described in text, under cycles of 12-hour light (100 µmole/m-2s-1)/12-hour dark at 23oC, 75% 4 ACS Paragon Plus Environment
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relative humidity. For detached leaf petiole assays, 4-week-old soil-grown Arabidopsis plants
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were used as sources of leaves. Leaf petioles were cut 1 mm from the main stem and the cut edge
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was placed immediately into a drop of media with or without QDs, as indicated, on a glass slide
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up to 1-hour (Figure S2A). The detached leaves were placed under 25 µmole/m-2s-1 of light to
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maintain transpiration. For whole plant hydroponics, seed holder components of Araponic
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hydroponic culture system (Kit 140 HD, Araponics, Belgium) were used. Arabidopsis seeds
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were sown on 0.8% agar, with 1/16 strength of Hoagland solution, within the seed holder. After
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germination, single Arabidopsis plants were grown in 15-mL conical tubes for 4 weeks and then
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transferred to 1/16-strength Hoagland solution containing either PAA-EG or PEI QDs at 10
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µg/mL for 7 days before analysis.
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Confocal Microscopy and Fluorescence Measurement
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A confocal microscope, LSM 710 (Zeiss, Germany) equipped with 514 nm laser for
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excitation, was set at 580 nm to 620 nm for harvesting spectra for QD detection and avoiding
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endogenous fluorescence of plant tissues. The laser strength was set at 3000 and gain values set
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at 900 in the operating software Zeiss ZEN 2010 (Zeiss, Germany). QD fluorescence strengths
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were measured with Infinte 200 pro (Tecan, Switzerland) plate reader at 514 nm excitation and
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600 nm emission.
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The whole plant photographs were taken with Nikon D80 digital camera using UV365nm
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light exposure with a UVGL-58 (UVP, Upland, CA) handheld UV lamp.
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ICP-MS Analysis
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Quantitative cadmium (Cd) and selenium (Se) analyses were done following the method described previously.34 QD-treated Arabidopsis were harvested and frozen in liquid nitrogen 5 ACS Paragon Plus Environment
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immediately. Harvested samples were ground using TissueLyser II (Qiagen, Germantown, MD)
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and dried completely at 100℃ for 48 hours before determining dry weight. After overnight
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digestion with 70% HNO3 (A509, Fisher Scientific, PA) and 30% of H2O2 (H325, Fisher
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Scientific, PA) samples were filtered with 0.2 µm of syringe filters (28145, VWR International,
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Radnor, PA). Cd and Se elements were analyzed with Elan 9000 apparatus ICP-MS
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(PerkinElmer, Waltham, MA). The plant extraction method was verified by comparing the Cd
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and Se recovery from direct solubilization of QDs to Cd and Se to plant leaves injected with QDs;
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no significant differences in recovery were found between the samples. Germanium was used for
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the internal standard for recovery and injection control. A standard curve was obtained from 1, 2,
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4, 8, 16 and 32 ng/mL of Ge, Cd and Se ion injection and regression coefficient was over 0.99.
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Trichoplusia ni Treatment
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For cabbage looper Trichoplusia ni (T. ni) treatment, Arabidopsis were grown in 1/4-
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strength Hoagland solution for 4 weeks and 16 Arabidopsis plants were transferred to media
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containing QDs in 1/16-strength Hoagland solution. Plants were grown for 3 days in the presence
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of 10 µg/mL of QDs, at which point no signs of toxicity were apparent, before T. ni treatment. T.
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ni handling was performed as previously described.35, 36 Eggs of the T. ni were purchased from
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Benzon Research, Inc (Carlisle, PA). Fifteen newly hatched T. ni larvae were transferred with a
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fine brush onto the 16 Arabidopsis plants for each treatment group. Arabidopsis and T. ni
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samples were harvested 7 days after T. ni transfer. Harvested T. ni were analyzed with both
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confocal microscopy and ICP-MS, and the remaining Arabidopsis leaves were also analyzed
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with ICP-MS. Arabidopsis growth and T. ni treatment were carried out at 23oC, 50% relative
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humidity under 24-hour continuous light at 25 µmole/m-2s-1 light intensity. 6 ACS Paragon Plus Environment
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Statistical Analyses
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Standard deviations are indicated as error bars for all data sets. Statistical significances between
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data were calculated by the student t test with two-tailed distribution. Data with p values below
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0.05 or 0.01 are indicated as significantly different.
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RESULTS AND DISCUSSION
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QD Coat Characteristics Affect Particle Uptake Rate and Translocation Through
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Arabidopsis Leaf Petioles
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To first assess QD translocation through Arabidopsis leaf tissue in the absence of
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complications or restrictions that could arise from root uptake, we examined movement of QD
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through freshly cut leaf petioles. Petioles were immersed into 1/16-strength Hoagland solution
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containing 10 µg/mL of QDs with PAA-EG, PEI, or PMAO-PEG coats (Figure S2A). All three
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types of QDs were readily visible at the cut petiole base under confocal analysis within one hour
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of leaf exposure to the QDs (Figure 1A). However, only the PAA-EG and PEI QDs were
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detectable as multiple elongated fluorescence traces beyond 15 mm from the cut petiole base
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(Figure 1A-G), suggesting efficient translocation. The QDs appeared to travel through
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contiguous cells, likely major vascular tissues, but with some leakage occurring to neighboring
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cells (Figure 1B, 1D, 1F, Figure S2B). The smooth appearance of the PAA-EG QD-treated plant
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cells suggests that the negatively charged coat may enable relatively fluidic movement through
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the plant vascular tissue (Figure 1, Figure S2B). Fluorescence became less uniform as the
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translocation distance from the cut site increased (Figure 1A-G). PAA-EG QD fluorescence at
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distal sites was strongest along the edges of plant cells (Figure 1C), as if adhering against cell 7 ACS Paragon Plus Environment
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membranes or walls. Whereas the in planta PAA-EG QD fluorescence appeared smooth and
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uniform, the PEI and PMAO-PEG QD fluorescence had a particulate appearance (Figure 1,
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Figure S2B), suggesting particle aggregation. Only minimal fluorescence could be detected in
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distal petiole regions from PMAO-PEG QD-exposed leaves despite strong fluorescence at the
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petiole base. The strong PMAO-PEG QDs fluorescence at the petiole base and minimal
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fluorescence detected more distally are findings consistent with the interpretation of in vitro
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behaviors that the PMAO-PEG QD particles may readily aggregate (Figure S1). Therefore
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PMAO-PEG QDs may form particles of sizes and/or structures that are incapable of efficient
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translocation through plant tissues. These data demonstrate that once introduced directly into the
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vascular tissue of Arabidopsis leaf petioles, PAA-EG and PEI QD particles can move through
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the petiole, whereas PMAO-PEG particles fail to translocate readily, possibly because of PMAO-
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PEG QDs aggregation tendencies. These findings are consistent with previous work that showed
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that nanomaterial coats can influence plant uptake and translocation.21, 23, 25, 37
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To assess the role of Arabidopsis leaf age in QD movement, we compared translocation
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rates of PAA-EG and PEI QDs in younger leaves to that of older leaves. Petioles of younger
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rosette leaves, that is, the 7th, 8th, and 9th leaves to form during rosette development, transported
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both PAA-EG and PEI QDs faster on average than the petioles of rosette leaves that developed
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1st, 2nd, and 3rd (Figure 1I; a and b, p
