Interestingly, the B.napus plants grown on fritted clay exhibited odd growing patterns, with certain leaves exhibiting a shriveled curled leaf phenotype in both well-watered and drought conditions. Furthermore, the sand-like quality ofthe substrate caused compaction post-watering, leading to decreased root growth as compared to soil. Replicating the low moisture assay done for our A.thaliana mutants would be more appropriate to look at drought-tolerant phenotypes in our B.napus. Evaluation of PP2C levels at the end of the drought assay would allow us to have better insight on gene expression of these negative regulators, and the effectiveness of our knockdown. Similarly as proposed for A.thaliana mutants, assessment of stomatal conductance through petiole feeding of ABA, and stomatal density would be important to give us insight on whether our plants are more ABA-responsive when fully grown, and if there are any stomatal development was affected by the knockdown. The Fritted clay approach towards looking into drought tolerance of B.napus plants was shown to give a uniform stress to the rd29a-mediated PP2C knockdown lines, however, issues of both high seedling mortality rates and lack of real-world applications lead me to believe that the drought assay developed for A.thaliana plants is a better method. We were able to generate ABA responsive B.napus rd29a-mediated PP2C knockdown lines, however the expression levels ofthe knockdown between lines stiil needs to be assessed. Finally, testing the same ABA responsive lines in the drought assay we developed for A.thaliana plants would allow us to observe if the enhanced ABA response would lead to increased drought tolerance.The use of copper-based nanoparticles in agriculture as fungicides and bactericides is increasing rapidly due to their relatively low toxicity and higher efficiency in delivering the active component .

There are numerous copper containing pesticides on the market, e.g. copper sulfate , cuprous oxide , copper hydroxide ,hydroponic channel and nano copper . The U.S. Department of Agriculture maintains an official list of synthetic substances that can be used for organic farming. According to this list, copper-based materials are allowed for use in organic crop production.However, more and more evidence indicates that copper-based nanoparticles induce phytotoxicity in various plants, such as bean,lettuce,alfalfa,cilantro,cucumber.The adverse impact include decreased root and shoot elongation, disturbed mineral nutrients homeostasis, decreased photosynthesis rate, inhibited antioxidant enzyme activities.So far the molecular mechanism underlying those physiological changes is not well understood. In recently years, “omics” have become a promising methodology for studying plant responses to abiotic and biotic stress.Transcriptomics-based gene expression and proteomics-based protein production have been applied to evaluate the changes of plants to external stressors at the molecular level.Unlike transcriptomics and proteomics, which reveal what might be happening in plant tissues, metabolomics profiling can tell what already happened. Metabolites are the end product of gene expression, and the changes of metabolites are regarded as ultimate responses of plant to stress.Thus, environmental metabolomics is becoming a powerful tool to investigate the response of plants to various stressors, e.g., water, light, temperature, and high levels of metals.Recently, Pidatala et al. employed LC-MS/ MS based metabolomics to elucidate the stress response mechanism of lettuce to lead. They observed several key metabolic pathways, including sugar and amino acid metabolism, that were disturbed by lead.Our recent study,applying GC-TOF-MS based metabolomics and PLS-DA multivariate analysis, also revealed the profile of metabolites in root exudates was significantly altered by nCu.More recently, we determined that nCu altered the nutritional supply of cucumber fruit,using 1 H NMR and GC-MS based metabolomics. Those studies demonstrate that metabolomics is a powerful tool to investigate the stress response of plant to contaminants.

Therefore, the present study was designed to generate a mechanistic understanding of the effects of nCu on cucumber plants by applying untargeted GC-TOF-MS based metabolomics. Fruit metabolic profiling provides information on low molecular weight metabolites, which not only directly reflects fruit nutrient levels, but also generates a more comprehensive understanding of the metabolic network and the biological pathways impacted by NPs.ICP-MS based metallomics were also performed to supply the elemental changes under nCu stress. All those techniques provide a comprehensive insight into the overall stress response mechanism of cucumber plants to nCu, which helps understand their long-term impact in terrestrial environments.Cucumber seeds were purchased from Seed Savers Exchange . The soil was collected from the Natural Reserve System of UC Santa Barbara . The soil composition is shown in Table S1.NCu was suspended in nanopure water and sonicated for 30 minutes before being applied to the soil. The final concentration of nCu in soil was 0 , 200 , 400 and 800 mg kg−1 . These concentrations are within the range of those predicted for biosolids applied to soils or due to the application of copper-based nanopesticides.Each treatment had four replicates. In each replicate, pairs of cucumber seedlings were grown in 3.0 L Poly-Tainer containers. The cucumber plants were grown from April 2, 2015 until harvest on June 28, 2015. The temperature in the greenhouse was controlled to be 25.5–30.0 °C during the day and 17.7–18.9 °C at night. At harvest, the fresh weight of root, stem, leaf and fruit from each treatment was recorded. In addition, the weight of each mature fruit from each treatment was recorded. Only the matured fruits were selected for metallomics and metabolomics analysis. The number of matured cucumber fruits from each treatment were 6 , 5 , 3 and 6 ; fruits from each treatment were split in two parts, to use for metabolomics and for metallomics analyses.At harvest, the cucumber tissues were oven-dried for 7 days at 60 °C. The oven-dried tissues were ground to powder and digested with HNO3 and H2O2 using a microwave oven system .

The digestion method was based on EPA 3051.Standard reference materials, NIST 1547 and 1570a , were also digested and analyzed as samples. The recoveries for all elements were between 90% and 99%.7 The mineral nutrient elements were analyzed using inductively coupled plasma-mass spectrometry .At harvest, the fresh cucumber fruits were immediately placed in liquid N2, and lyophilized . The freeze-dried fruit samples were delivered to the Genome Center Core Services at University of California Davis to analyze metabolites via gas chromatography time-of-flight mass spectrometer-mass spectrometry . A description of the analytical method has been reported previously.Sample pretreatment was done similar to Fiehn et al.Partial least-squares discriminant analysis was run based on GC-TOF-MS data using online resources . PLS-DA is a supervised clustering method, which uses a multiple linear regression technique to maximize the separation between groups and help to understand which variables carry the class separating information.More details regarding data acquisition, data processing and data reporting are provided in the ESI.†Biomass, and mineral nutrients data were analyzed by oneway ANOVA, and group means were compared by conducting Tukey’s Honestly Significant Difference test using IBM SPSS Statistics 22. A probability of p ≤ 0.05 was considered to be significant. Photosynthesis rate, transpiration rate, stomatal conductance, and water use efficiency were analyzed with anested ANCOVA in which the independent factors were treatment; with replicates nested within a treatment,hydroponic dutch buckets and leaf temperature as a covariate. Leaf temperature was used as a covariate because gas exchange is often very sensitive to air temperature.Photosynthesis is one of the physiological processes most sensitive to environmental stresses.In order to monitor the physiological changes during development without damaging the plant tissue, photosynthetic rate , transpiration , stomatal conductance and instantaneous water use efficiency of cucumber leaves were measured 30 days after sowing. Fig. 1 shows that photosynthetic rate decreased in all nCu treatments compared to the control, but only the low and medium group were statistically significant . Previous studies showed that nano copper depleted PSII action centers, leading to photoinhibition25 and disruption of the repair cycle.In addition, stomatal conductance and transpiration rates tended to increase in nCu treatments compared to the control. The actual mechanism for Cu to increase stomatal conductance and transpiration rate is still unknown. One possible explanation is that Cu can trigger the formation of reactive oxygen species through redox process cycling between Cu+ and Cu2+.It is not known if nCu by itself can trigger ROS. Previous work has shown that ROS open the stomata, increasing stomatal conductance and therefore the transpiration rate.WUEi is defined as the ratio of the rate of carbon assimilation to the rate of transpiration. The decline in carbon assimilation rate and the increase in transpiration rates in response to exposure to nCu resulted in a statistically significant decline in WUEi .Previous studies demonstrated that nCu has comparatively higher solubility than its oxidized forms, and is sensitive to aqueous matrix pH.Therefore, we hypothesized that Cu would bio-accumulate in cucumber tissues, including fruits. As shown in Fig. 2 and Table S2,† Cu concentrations in all tissues in nCu treatment groups were significantly higher than that in the control , which indicates Cu is taken up by the roots and translocated even to the fruits. In both control and nCu treated plants, Cu was most abundant in the roots, although there was significantly more bioaccumulation in the nCu exposed plants.

This is consistent with previous reports that copper is sequestered primarily in the root compartment.It is noteworthy that the translocation factors of nCu/Cu ions in control plants is 0.33, but it decreased to 0.21, 0.22 and 0.23 in the plants exposed to 200, 400 and 800 mg kg−1 nCu, respectively. In the control treatments, plants uptake ionized Cu from the soil. One would expect a similar translocation factor in the nCu treated group if ionized Cu was translocated in a similar manner. The reduced translocation rate suggests that cucumber plants grown in nCu treated soils not only took up Cu ions, but also nCu. But we believe that most of the nCu just adsorbed on the root surface. Confocal microscopy and μ-XRF evidenced that ZnO and CeO2 NPs are mainly distributed on the outer layer of epidermis cells or trapped in epidermis cell walls; only a small portion of NPs could penetrate the epidermis cell wall and translocate to upper plant tissues via the transpiration stream.In addition, high ionic strength in the soil aqueous matrix probably accelerated the aggregation of nCu.Moreover, nCu or released Cu ions may complex with soil clay minerals or organic matter.All these factors reduced the possibility of small sized and free nCu existing in soil. Thus, Cu in the stems, leaves and fruits is likely from solubilized Cu leached from nCu. In order to evaluate the ability of nCu for releasing Cu ions into soil, additional experiments were conducted : soil was spiked with 200 mg kg−1 of CuCl2·2H2O and nCu separately. Cucumber seedlings were allowed to grow in these spiked soil samples for 10 days, and water soluble Cu 42 was determined via ICP-MS after removing the plants on day 10. After 10 days, the water-extractable Cu in the control , 200 mg kg−1 CuCl2 and 200 mg kg−1 nCu treatment was 1.8, 43 and 24 mg kg−1 , respectively.Thus, within 10 days nCu released a considerable amount of ionized Cu into the soil for plant uptake. These significant differences in bioavailable Cu between exposure to copper salts or nCu highlight the challenge in conducting comparisons. Simply exposing plants to “equivalent” amounts of copper may lead to very different results.Root, stem, and leaves supply and transport mineral nutrients. Results showed that nCu did not disturb any mineral nutrient homeostasis in these tissues except Fe. The Fe concentration in root and leaves were significantly decreased by nCu at all concentrations . In roots, Fe decreased by 67%, 54%, and 66% compared to the control, when exposed to 200, 400 and 800 mg kg−1 nCu, respectively. In leaves, Fe decreased by 56%, 43% and 54% compared to the control. In addition, Fe concentrations in stems also tended to decrease when exposed to nCu, but this was not statistically significant. Previous studies demonstrated a competitive relationship between Cu and Fe uptake: it was observed that in Arabidopsis thaliana and Cucumis sativus, a low Fe supply led to increased Cu concentration in leaves, while high Cu supply lowered leaf Fe concentration.The authors found that the presence of Cu affected the activity of ferric reductase. At high Cu supply, ferric reductase activity was inhibited. This led to decreased demand for Fe by the plant and subsequently less Fe accumulation.