It should be noted that detection of NPs within plant tissue via TEM is challenging,but based on our stability studies of the 64Cu-radiolabeled NPs along with the short exposure time that the observed uptake was attributed to intact [64Cu]-NP transport through the roots and into the cotyledons. This study has shown that NPs were transported intact into plants and can be tracked non-invasively using a radioactive tag for in vivo imaging by auto radiography and PET/CT and quantification using a gamma counter. This method allows for a highly sensitive method capable of quantifying NP amounts in an individual seedling, a level that would be challenging by the traditional ICP quantification.We found that [64Cu]- NP accumulation in lettuce was size-dependent indicated by the larger [64Cu]-NPs 11 reaching a plateau at a given concentration, while the smaller [64Cu]-NPs 10 increased continually over time . Our study indicates that both sets of [64Cu]-NPs travel intact from the root radicle to the cotyledon with the smaller 10 nm [64Cu]-NP having a maximum accumulation of 7.56 ± 0.85 μg/g in the whole plant, 6.15 ± 0.63 μg/g in the root, and 1.42 ± 0.20 μg/g in the cotyledons . The larger 20 nm [64Cu]-NPs had an accumulation that reached a maximum of 5.66 ± 1.08 μg/g in the whole plant, 4.93 ± 1.03 μg/g in the root, and 0.73 ± 0.05 μg/g in the cotyledon . It is clear from our imaging data, both by autoradiography and PET/CT that the [ 64Cu]-NPs are transported intact from the root to the cotyledons and are present in the lettuce tissue by TEM . However,vertical gardening system different accumulation patterns for the cotyledons were observed for the two different sized [64Cu]- NPs , while the root and whole plant were similar.

Most of the accumulation for the larger [ 64Cu]-NPs was within the first hour, where cotyledon NP amounts were ∼0.35 ± 0.15 μg/g with the only significant increase after 1 h between the 12 and 24 h time point in which accumulation plateaued at around ∼0.7 μg/g . The larger [64Cu]-NPs also had higher accumulation than the [64Cu]-NPs at the early time points up to the 4 h time period. The smaller [64Cu]-NPs had ∼8.8 fold increase in cotyledon accumulation from the 4 h time point to the 24 h time point with an increase of ∼1.6 fold between 12 and 24 h time period appearing to have a linear increase in absorption over time. The differences in cotyledon accumulation between the two sized [ 64Cu]-NPs maybe linked to NP size effects on the lettuce hydraulic conductivity. Our work suggests that [64Cu]-NPs around 20 nm in size appear to clog root cortical cell walls, or pit membrane preventing further uptake, explaining why 11 reaches a plateau, while the smaller [64Cu]- NPs continued to increase in amount over time. Initial studies with duckweed also illustrated [64Cu]-NP accumulation in regions of growth and at the node and apex of the cotyledons , suggesting that [64Cu]-NP transport to the cotyledons could occur via the phloem. The TEM images further shows the appearance of intact NPs in the lettuce tissue within the expected size range for the [64Cu]-NPs , but [64Cu]-NPs had a size that appeared smaller than those administered; suggesting that the plant may filter larger NPs and has a size-threshold for uptake , which may also explain the clogging phenomenon.In summary, the combined analysis of the imaging by auto radiography and PET/CT and TEM suggested that both sized [64Cu]-NPs are transported intact from the root to the cotyledons. The [64Cu]-NP-uptake and accumulation amounts observed within lettuce seedlings were reasonable and comparable to others reports in the literature, reaching the same general conclusions that NP transport and accumulation in plants is species and size dependent.

For example, Ni-NPs had very high NP uptake ranging from ∼13 200−38 983 μg/g in mesquite.The amount found in the leaves varied from 400 to 803 μg/g of mesquite with most the NPs remaining in the roots ranging from 12 835 to 38 183 μg/g.Another study using small CeO2-NPs exhibited NP accumulation ranging from 300 to 6000 μg/g of plant and indicated that NP accumulation was plant species dependent.NP sizes above active transport ranging from 14 to 40 nm had a large variation in uptake ranging from 0.25 to 3750 μg/g of plant, but typically had accumulation ranging from ∼1−1100 μg/g of plant again with the majority of the NPs contained within the root and with 0.5−183 μg/g in the leaves.NPs , had accumulation in mung bean of 8 μg/g and in wheat of 32 μg/ g.When comparing the accumulation of two similarly sized TiO2-NPs of different crystalline structure [22 nm and 25 nm ] in wheat different accumulation amounts were observed, suggesting size was not the only limiting factor for transportation into a plant.In another study using radioactive NPs, Zhang et al.generated 141Ce by neutron bombardment of CeO2NPs synthesized via a precipitation method. The fabrication of [ 141Ce]CeO2-NPs could make controlling the size distribution very difficult and the exact size of the radioactive [141Ce]CeO2- NPs was never determined. In addition, free radioactive 141Cemetal could dissolute and be transported into the plant, making it appear as if the intact-NPs were in the plant because possible leaching of radioactivity was not explored. We aimed to avoid complications of NP-fabrication in which the exact size distribution during the study could not be determined and to improve upon prior radio labeling methods, which gave low specific activity of 2.7 μCi/mg of NP.We were able to generate stable radioactive [ 64Cu]-NPs with high radio chemical purity and a specific activity of 2.2 mCi/mg of NP with a tight size distribution . Zhang et al.’s work also demonstrated a concentration and size dependence of the [141Ce]CeO2-NPs on plant accumulation in cucumber. At the lower concentration [141Ce]CeO2-NPs roots accumulation was ∼370 μg/g for the small 7 nm-NPs and ∼70 μg/g for the 25 nm-NPs. At the highest concentration , [141Ce]CeO2-NP uptake was much higher for both sizes being ∼700 μg/g and ∼500 μg/g of cucumber .In agreement with our study was that smaller NPs have higher accumulation. Zhang et al. also noted that accumulation in the leaves was less affected by the size of the [141Ce]CeO2-NPs with the average uptake in the leaves for the 7 nm-NP being 0.4 μg/g and 0.18 μg/g for the 25 nm-NPs.We observed similar amounts in lettuce cotyledons for the 24 h uptake period with the accumulation of 10 being 1.4 μg/g and 11 being 0.7 μg/g.

However, the observed accumulation in the cotyledons for [ 64Cu]-NPs was lower than the amounts observed by Zhang et al. at the very early time points and it was not until the 4 h time point when accumulation amounts in the cotyledons started becoming larger than 0.4 μg/g of lettuce. These uptake differences may be attributed to the use of a different species and/or the NP solution administered had 2.4-times higher concentration . Auto radiography images showed [ 141Ce]CeO2-NPs movement to the leaves, implying that once NPs entered into the vascular cylinder, they move along with water flow. This was in good agreement with our study. In contrast, we saw no concentration dependence for either sized [ 64Cu]-NP using 48, 96, and 144 mg L−1 over a 2 h period with approximately the same accumulation amount at all concentrations . Similarly, another study using CuO-NPs administered two concentrations 10 mg L−1 and 100 mg L−1 for a period of 14 days in maize also observed no concentration dependence.The use of NPs tagged with radioactivity and tracked by auto radiography and PET/CT has provided a noninvasive analytical tool to spatially visualize and quantify NP uptake and accumulation in plants. We investigated the fate of [64Cu]-NP transport into plants at the largely unexplored early time points,vertical tower for strawberries which would prevent dissolution events. Stability studies concluded that the [64Cu]-NPs were stable during the imaging and quantification time frame from 0.25 to 24 h resulting in intact NP-transport into lettuce seedlings. We further demonstrated that the transport of [64Cu]-NPs into lettuce was not concentration dependent but was size dependent with the 20 nm [64Cu]-NPs reaching a plateau with accumulation at ∼5.7 μg/g of lettuce and the smaller 10 nm NPs accumulation increasing linearly with the maximum amount at 24 h being ∼7.6 μg/g of lettuce. TEM images further substantiated the intact transport of NPs into plants. With the numerous factors that may dictate NP uptake and accumulation, further studies are warranted to fully understand the molecular mechanism of NP transport into plants.To identify genes that respond specifically and rapidly to P deficiency, total RNA was extracted from diagnostic leaves of potato plants of contrasting P-status grown hydroponically in the glasshouse. Predictive genes identified using these samples were then tested using total RNA obtained from diagnostic leaves of plants growing in the field. Total RNA was extracted according to Hammond et al. . Total RNA from all samples was labelled and hybridised to the Potato Oligo Chip Initiative oligonucleotide array, representing 42,034 potato sequences . Labelled cRNA was generated from RNA using the LowRNA Input Fluorescent Linear Amplification Kit . Dye incorporation for labelled cRNA was 17.02 ± 0.43 pmol dye µg-1 cRNA . Hybridisation cocktails were prepared using the In situ Hybridisation Kit and cocktails contained between 0.5 and 3 µg labelled cRNA per sample. Cocktails were hybridised to micro-arrays rotating at 10 rpm in a hybridisation oven at 65°C for 17 hours. Following hybridisation, micro-arrays were washed according to manufacturer’s instructions. Micro-arrays were scanned on an Agilent DNA Micro-array Scanner BA using the Extended Dynamic Range function and data were extracted from the scanned images using the Feature Extraction software package. For further analysis ‘processed’ signal values were used. Micro-array scans were checked for quality using data from the Feature Extraction software and distribution of data in Gene Spring GX analysis software. The processed signal values were imported into Gene Spring GX . Data from individual micro-arrays were subjected to a Lowess normalisation and the signal value for each gene was divided by the median of its measurements in all samples for an experiment. Data were pre-filtered by removing genes whose raw signal value was less than 50 in five of the seven time points; removing genes flagged as absent; and removing genes whose normalised signal value remained between 0.8 and 1.2 at all time points to leave 28,946 genes for further analysis. To identify genes that were significantly differentially expressed between treatments an ANOVA with a Benjamini & Hochberg FDR multiple testing correction was used.

Gene Ontology terms assigned to genes were analysed using the Gene Ontology Browser in Gene Spring GX. For class prediction, the support vector machine implemented in the Class Prediction tool of GeneSpring GX was used to classify the data. Sets of diagnostic genes were selected using the Golub method with the Polynomial Dot Product kernel function. Different kernel functions and sets of diagnostic genes were changed systematically to optimise the classification of samples. The shoot dry weight of P-replete plants growing hydroponically in the glasshouse increased regularly over the experimental period . A significant reduction in shoot dry weight was observed in P-starved plants 15 days after removing P from the nutrient solution, and this was not recovered by subsequently resupplying P . The P concentration in diagnostic leaves of P-replete plants was 8.5 ± 0.14 mg g-1 DM , which is above that considered sufficient for maximal growth of potato plants . A significant reduction in the P concentration of diagnostic leaves of P-starved plants was observed within one day of removing P from the nutrient solution and was restored to concentrations significantly greater than those found in leaves of P-replete plants within 7 days of resupplying P . A total of 1,659 genes were significantly differentially expressed in diagnostic leaves of P-replete and P-starved plants in at least two of the seven time points assayed during the experimental period. Many of these were characteristic of the acclimation of leaf metabolism to P-starvation and included genes involved in re-routeing carbon metabolism to reduce the demand for phosphorylated metabolites, genes encoding enzymes involved in alternative lipid metabolism to reduce the P-demand of cellular membranes, and genes encoding ribonucleases and cellular phosphatases that release P from RNA and vacuolar sources during P starvation .