The aromatic region comprises many characteristic signals of secondary metabolites,which play a crucial role in plant defense to environmental stress.As shown in Figure S5 C, nano-Cu/Cu ions altered the pattern of some secondary metabolites. We further identified those bins to corresponding compounds. However, due to the extremely low concentration and high baseline, it was difficult to link those bin areas to specific compounds using the Chenomx NMR Suite. Thus, GC-MS was used for identification and quantification. A total of 156 metabolites in root exudates were identified by GC-MS. To visualize the general differences between control and nano-Cu treated plants, the 156 identified metabolites were normalized and analyzed by Partial Least Squares Discriminant Analysis using online resources,which is a supervised clustering method to maximize the separation between groups. The score plot shows that cucumber root exudate exposed to different concentrations of nano-Cu are clearly separated along the first principal axis , which explained 30.4% of the total variability. This indicates nano-Cu considerably altered the metabolic profiles of cucumber root exudate, which is consistent with the NMR data. Using parameters of variable importance in projection score,a total of 56 metabolites were found to be responsible for this separation . For the metabolites of interest, hierarchical clusters analysis was performed by grouping the samples into clusters based on the similarity of their metabolite abundance profiles. Figure 3 presents the resulting heat map for the selected metabolites, which indicates that some metabolites,vertical grow tables including lysine, threonine, phenylalanine, glycine, serine, proline, isoleucine, alanine, valine, leucine, beta-alanine, 4-hydroxybenzoate, benzoic acid, 2- hydroxyvaleric acid, pelargonic acid, salicylic acid, lactic acid, were up-regulated by nano-Cu.

However, some metabolites were down-regulated by nano-Cu, including parabanic acid, 3- propionic acid, N-acetylmannosamine, erythritol, pimelic acid, dehydroascorbic acid, N-acetyl-Dgalactosamine, fructose-6-phosphate, hexonic acid, vanillic acid, and citric acid. In the following section, we categorized those metabolites into five groups: amino acids, organic acids, fatty acid, phenolic and other compounds, and discuss them separately.Eleven amino acids, including Alanine , beta alanine , Glycine , Isoline , Leucine , Lysine , Phenylalanine , Proline , Serine , Threonine , and Valine , were significantly up-regulated in response to nano-Cu in a dose-dependent way .The up-regulated amino acids can provide many binding sites for copper, hindering the translocation from the root cell membrane. Previous studies showed that amino acids play an important role in chelating Cu2+. It is reported that amino acid complexes formed with other metals, e.g. Ni, are much more stable than those with carboxylic acids.Cu in the roots of Cu-tolerant A. maritima exists as a Cu−proline complex.EXAFS spectra demonstrated Cu complexation with the nonproteogenic amino acid nicotianamine, which shows very high affinity for Cu.Kupper et al. assumed that plants keep dissolved metals out of the cytoplasm and sequester/ complex them into the vacuole or cell wall.The upregulated amino acids may also reflect an attempt by the cucumber plants to sequester Cu in stems. Although stem metabolites were not analyzed, it is possible that amino acids are also secreted in the xylem sap to bind with Cu2+ in the transpiration stream. It is reported that more than 99% Cu in tomato and chicory xylem sap was in a bound form with histidine and nicotianamine .Amino acids in root exudate not only bind metals, but also serve as signaling molecules and have an antioxidant defense function.If the upregulation of these amino acids is an active defense of cucumber plant to excess nano-Cu/Cu ions, the increased amino acids possibly will decrease the uptake of Cu by cucumber plants. In order to verify this hypothesis, two-weekold cucumber plants were cultivated in 20 mg/L nano-Cu nutrient solution with different levels of Ser for 48 h. Interestingly, Cu accumulation in roots decreased with increasing Ser concentration, even though the free Cu ions in nutrient solution were much higher in the presence of Ser. After the plants were removed, the pH in nutrient solutions was determined. As shown in Figure 5, the pH decreased from 6.37 to 6.01 as the Ser concentration increased from 0 to 25 mM, which may explain why more Cu ions were released in the presence of Ser compared to the control .

Those results strongly indicate that amino acids are possibly released to detoxify nano-Cu by binding with Cu ions. However, we cannot rule out the possibility that up-regulation of some amino acids was due to membrane damage, which caused the leakage.Organic acids are strong cation chelators, which play an important role in facilitating mineral element uptake and sequester or exclude toxic metals. Several studies have shown that citric, oxalic, and succinic acids are involved in the detoxification of various dissolved metals.GC-MS results showed citric, succinic, malic, and fumaric acids were the most abundant low molecular weight organic acids in cucumber root exudates, which is consistent with previous reports on organic acid composition in cucumber root exudates.Surprisingly, patterns of succinic, malic, and fumaric acids were not changed by nano-Cu , which indicates those organic acids did not respond to Cu stress. In contrast, citric acid, the most abundant organic acid, was down-regulated by nano-Cu . Citric acid in root exudate decreased 5× at 10 ppm nano-Cu and 60× at 20 ppm in treated cucumber plants compared to the control. It is known that organic acids play an important role in restricting the passage of metals across the root, due to their strong affinity to form stable extracellular complexes with Cu, Al, and Cd.Other reports showed organic acids in the rhizosphere help solubilize minerals and facilitate their uptake by the plant.Citric acid has been shown to assist copper uptake in a moderate-accumulator plant .The down-regulation of citric acid is possibly an active process to decrease the dissolution, uptake, and translocation of Cu into cucumber tissues, or it could represent a shift in metabolism in the tricarboxylic acid cycle . As mentioned before, citric acid can play a dual role: it can either mobilize metals to accelerate uptake in plants that are deficient in some elements,or it can complex with metals to hinder their translocation. In this study, we hypothesized that citric acid played a role in mobilizing Cu ions release from nano-Cu, increasing Cu accumulation in cucumber plants. To confirm this hypothesis, we conducted an additional experiment in which we exposed cucumber plants to 20 mg/L nano-Cu at different concentrations of citric acid . We found that the pH of the hydroponic system decreased from 6.37 to 5.28 . This decrease in pH led to increased dissolved Cu ions to concentrations that were 8 times higher than that in the control . This result indicates that citric acid has a strong ability to dissolve nano-Cu by decreasing the system pH. Therefore, it is not surprising that Cu levels in cucumber plants grown in 20 mg/L nano-Cu in the presence of 6.25 mM citric acid were 4 times higher than that in plants grown in 20 ppm nano-Cu without additional citric acid . However, it is noteworthy that citric acid at this concentration may damage biological membranes and lead to passive uptake of Cu through leaky membranes. At least, these additional experiments demonstrate that citric acid increased nano-Cu dissolution in cucumber plants. Taken together, the up-regulation of amino acids and down-regulation of citric acid are likely plant strategies to hinder the uptake of Cu and detoxify the Cu toxicity.The release of fatty acids from the membrane is involved in plant tolerance to biotic and abiotic stresses.Pelargonic acid was up-regulated in root exudates after exposure to nano-Cu . Pelargonic acid is a natural non-selective herbicide, which can disrupt intercellular pH and membrane integrity causing rapid cell death.Evidence has shown that pelargonic acid participates in stress response and can be an indicator of root membrane damage.

The increased pelargonic acids in nano-Cu treated root exudates may be an indicator of membrane damage. Copper also interferes with fatty acid metabolism.Phenolics are secondary metabolites with an important role in stress response.The concentrations of salicylic and benzoic acids were up-regulated in the presence of nano-Cu/Cu, especially salicylic acid . Both compounds are reported to have antioxidant and anti-fungal activities and play a crucial role in plant defense against a variety of biotic and abiotic stressors.The concentrations of salicylic acid in root exudate of 10 and 20 ppm nano-Cu treated plants were 13 and 26 times higher than that in the control. It is reported that salicylic acid serves as an internal signaling molecule in the activation of plant defense after pathogen attack.Previous studies also suggested salicylic acid treatment significantly reduced malondialdehyde and H2O2 concentrations in the roots and leaves of rice under Cu stress, therefore alleviating Cu toxicity.In addition, 4-hydroxybenzoate, a phenolic derivative of benzoic acid, which is a secondary metabolite and also plays a key role in stress response, was up-regulated by exposure to nano-Cu. The up-regulation of these three phenolics is possibly a self-protection mechanism of cucumber to nano-Cu exposure at these levels.As shown in Figure 3,flower pot most of the down-regulated compounds are sugar-related metabolites, which might explain why citric acid exudation decreased as it was preserved for the TCA cycle. It is interesting to find that the level of dehydroascorbic acid in nanoCu treated root exudates is apparently down-regulated . DHA is known to participate in the plant defense against oxidative stress,and it is also an oxidized form of ascorbic acid. Dehydroascorbate is an intermediate product of the reaction between ascorbic acid and ROS, and can be oxidized to threonic acid. Thus, the down-regulation of DHA is an indicator that ROS was triggered by nano-Cu in the rhizosphere and a response to oxidative stress occurred. It is possible that the membrane damage occurred due to lipid peroxidation induced by ROS.Because the nutritional sources of microbes in the rhizosphere come from root exudates,the altered pattern of amino acids, carboxylic acids, phenolics, and sugar metabolites may possibly affect microbial activities and communities in the rhizosphere. Yuan et al.revealed that n-CeO2 did not affect soil bacterial communities in the absence of soybean plants, but did affect them in the presence of soybean plants, likely through the change of quantity and composition of root exudates. Our results indicate that nano-Cu altered the metabolite profile in cucumber root exudates, and this change may alter the mutually beneficial feedback interactions between plant and microbes in the rhizosphere. Root exudate as a hidden part of plant defense system, has been neglected or underestimated in previous studies investigating plant-related nanotoxicity. In this study, the profiling analysis of root exudate metabolites revealed important detoxification mechanisms of cucumber plants to nano-Cu induced stress. In addition, 1 H NMR and GC-MS based metabolomics provided a comprehensive understanding of the changes in metabolites induced by exposure to nano-Cu, and this powerful combination of analytical techniques may be very useful in revealing effects of other NPs on different plant tissues, even at sublethal NP concentrations. The findings reflect the situation in hydroponic media, which is quite relevant for cucumber production in many parts of the world. However, the results may be different in soil media. Further studies are clearly needed to elucidate the contribution of membrane damage to up-regulated metabolites in root exudate.Chemical speciation programs, such as GEOCHEM , GEOCHEM-PC , PHREEQC, MINEQL+, and MINTEQA2 have been excellent tools for scientists to use in designing appropriate solutions for their experiments. Programs of this type allow the user to estimate the interactions between metals and ligands and to calculate the free activities of the ions of interest. In doing so, the scientist can make a solution in which requisite conditions are satisfied and the design is intelligent. For many years we have used GEOCHEM-PC to formulate hydroponic solutions for plant growth, including those employing a variety of metal-chelate systems to control Fe , Fe , and Zn status. In addition, this program has been an important tool in creating test solutions for plant aluminum tolerance experiments. Thus, we are able to estimate Al3+ activities and to create solutions without significantly lowering available phosphate or sulfate. Many of the Al-containing nutrient solutions that are presently in use contain very high Al levels in order to achieve the desired root growth inhibition. These solutions may be redesigned with GEOCHEM-EZ to avoid these problems. As helpful as GEOCHEM-PC has been to a number of scientists in their work, the consensus among users was that the program was not very user friendly and suffered from several functional weaknesses.