Cucumber plants are generally more sensitive to contaminants and their bio-accumulation is higher than many other plants due to their high transpiration rate. Our previous study showed both CeO2 and ZnO NPs triggered more physiological changes in cucumber plants compared to corn plants, which have lower transpiration rates.Here we hypothesized that nano-Cu would induce physiological responses in cucumber plants. To evaluate this, we selected metabolomics studies as a novel approach to understand plant−nanoparticle interactions. We selected a 1 H nuclear magnetic resonance -based environmental metabolomics platform to detect the induced alteration, because NMR can simultaneously detect a variety of metabolites with simple sample preparation.In addition, compared with other “omics”, metabolomics reveals effects downstream of DNA and simultaneously provides a nonspecific assessment of the end result of multiple biological responses. Therefore, 1 H NMR has been employed to evaluate the toxicity of a large variety of environmental contaminants on different organisms.The toxicity and toxicity mechanism of titanium dioxide NPs to earthworms and rats was evaluated via an environmental metabolomics platform using 1 H NMR. Furthermore, most previous studies of the effect of various NPs to plants have concentrated on toxicity.
It is well known that approximately 30−40% of all photosynthetically fixed carbon will be transferred to the rhizosphere as root exudates,outdoor vertical plant stands including organic acids, amino acids, sugars, proteins, phenolic compounds, and CO2.These compounds play an important role in plant stress tolerance and external exclusion of pollutants.Considerable evidence exists that plants upregulate some organic acids, including amino acids, to chelate or complex toxic metals , to hinder their translocation to above ground plant tissues.Murphy et al.reported copper ions induce citrate production in root secretions. Our hypothesis is that root exudates may play an important role in NP mobility and bioavailability, as well as upor down-regulation of metabolite levels due to toxic effects and to induce detoxification. Therefore, the aims of the present study were to investigate the uptake, translocation, bio-accumulation, and toxicity of nano-Cu in cucumber tissues. We used nontargeted 1 H NMR and GC-MS based metabolomics to evaluate the physiological changes induced by nano-Cu in cucumber plants. Mineral nutrient metabolism was also evaluated by determining the elemental content in different tissues.After 7 days exposure to 10 and 20 mg/L of nano-Cu, cucumber plants exhibited significant decrease in root length compared to the control. Exposure to 10 and 20 mg/L of nano-Cu also resulted in root biomass reduction by 11.7% and 30.2%, respectively , but only the effect at 20 mg/L was statistically significant . Similar results have been reported in zucchini, squash, wheat, lettuce, and alfalfa.Stampoulis et al.observed reduced root elongation of zucchini seeds after exposure to 1000 mg/L Cu NPs.
Musante and White reported that exposure to Cu NPs at 100 and 500 mg/L resulted in significant biomass reduction of squash.Lee et al. also found Cu NPs at 200 mg/L affected wheat root elongation.More recently, Hong et al. reported that Cu NPs at a concentration of 20 mg/L significantly reduced the root length of alfalfa and lettuce. It has been reported that copper toxicity results from inhibition or activation of some enzymes in the root zone.For instance, Kennedy and Gonsalves reported Cu inhibited ATPase activity in the plasma membrane of Zea mays roots.Previous studies showed copper toxicity triggered oxidative damage and increased the antioxidative enzymes.Compared to the roots, nano-Cu had no statistically significant impact on stem and leaf biomass. This is consistent with previous reports that roots are the most vulnerable organ under nano-Cu stress. Lee et al. reported that nano-Cu at 200 mg/L affected the root length of wheat, while the threshold to induce shoot length reduction was 800 mg/L. CuSO4 was observed to inhibit root growth but not shoot growth in rice seedlings.Fernandes and Henriques attributed the differential effect of Cu on root and shoot growth to the fact that Cu is mainly bio-accumulated in roots compared to shoots. However, Hong et al. demonstrated that the Cu toxicity is a species-specific response.They showed that although the Cu concentration was high in alfalfa shoots, compared to lettuce, there was no shoot length reduction.In all tissues including root, stem, and leaves, Cu concentration in plants treated with 10 and 20 mg/L of nano-Cu were significantly higher than that in the control . This indicates that copper was taken up by and transported from root to stem and leaves within 7 days.
The distribution patterns of Cu indifferent tissues in both control and nano-Cu treated plants are similar: Cu was predominant in roots , followed by stems , and leaves . This indicates that Cu was sequestered primarily in the root compartment, and only a small percentage was transported to upper tissues. This is consistent with previous reports showing that Cu is located primarily in roots of alfalfa and lettuce.It is interesting to note that the stem/root translocation factors have the tendency to increase with increasing nano-Cu concentration, while the leaf/ stem translocation factors decrease . This indicates cucumber plants tend to retain/ sequester more Cu in their stems. The mechanism underlying this phenomenon is complex, and still unclear. We hypothesize that this is a detoxification or active protective process for the plants to avoid damage to the more vulnerable tissue of roots and leaves. It has been shown that copper in xylem sap is almost 100% bound to amino acids.The stem may secret more ligands, e.g., carboxyl and amino groups, organic acids, glutathione, cysteine, to complex or chelate with Cu when it is in excess.Liao et al.showed evidence that more than 99.6% of total Cu in the xylem sap of tomato plants was in a complexed form, with the xylem sap producing amino acids to chelate Cu and hinder its translocation to leaves. These findings with Cu are consistent with previous reports that plants are able to minimize the adverse effects of excess metals by regulating their distribution and translocation within their organs or cells.As shown in Table 1, in the presence of nano-Cu, nutrient elements were markedly affected. The concentrations of Na, P, S, K, Mo, and Zn decreased in all cucumber tissues in exposed plants. Except K in roots and leaves , and Na in leaves , all the decreases were statistically significant . In addition, nano-Cu decreased Fe and Mg uptake in roots, but not in stems and leaves. Reduced uptake of P, S, and Fe in lettuce and alfalfa by nano-Cu was also observed in hydroponic systems.Because the applied nanoCu concentrations in the two studies are similar, cucumber seems to be more sensitive to nano-Cu than lettuce or alfalfa, probably because of its higher transpiration rate that carries more Cu to plant cells. Our previous study showed that nCeO2 and nZnO NPs decreased Cu and Mo content in cucumber fruits; however, neither nCeO2 nor nZnO affected mineral element accumulation in corn cobs.The decrease of K concentration in all tissues may be the result of leakage mediated by ion channels. Murphy demonstrated that Cu promotes K+ efflux rather than inhibiting K+ uptake in Arabidopsis seedlings.Several studies have shown that iron uptake is decreased by excess Cu.Hong et al. also found nano-Cu significantly decreased Fe uptake in lettuce. Waters and Armbrust found that high Cu supply inhibited the activity of ferric reductase, which is an indicator of Fe demand. This led to decreased demand for Fe by the plant, which subsequently led to less Fe accumulation. Decreased P is due to the formation of Cu−phosphate complexes at the root surface.However, some studies showed the up-regulation of S in the presence of nano-Cu or other stressors,vertical plant rack which is quite different from our finding. It is possible that nano-Cu damages the root cell membrane and leads to leakage of many ions. These elements participate in respiration and photosynthesis and in some enzyme system functions. The decrease of these minerals may lead to othermetabolic changes. For example, Cu-induced Fe deficiency can contribute to decreased leaf chlorophyll content and reduced photosynthesis.Reichman reported that the chlorotic symptoms on young leaves of plant experiencing Cu toxicity could be an induced Fe-deficiency.Principal component analysis was performed as a first step to provide a general overview of trends, grouping, and outliers in the 1 H NMR data.PCA of the metabolomics data set extracted from 12 cucumber leaf samples produced three principal components which explained more than 66.9% of the total variance .
The score plots from PC1 and PC2 reflect that leaf tissues from the control and nano-Cu treated plants were clearly separated from each other by PC2 reflecting differences in metabolic profiles. However, no difference was found between 10 and 20 ppm treatments, indicating that nano-Cu at either level changed the pattern of metabolites in cucumber leaves. The PCA loading plot identified the regions of the NMR spectra that contribute to the observed differences in PC scores. Further, bins with high weighting score were selected and subjected to one-way ANOVA to identify metabolites significantly regulated due to nano-Cu stress. A total of 25 bins were found to be significantly altered in cucumber leaves in response to nano-Cu exposure .In total, 22 metabolites were identified as significantly altered using Chenomx . Most of the altered metabolites are secondary metabolites. Among the five up-regulated metabolites, 4-aminobutyrate , acetylglucosamine, and phenyllactate have previously been shown to be related to stress response. GABA is a nonprotein amino acid. In plants there are numerous observations of a rapid accumulation of GABA in response to biotic and abiotic stress.Zulak reported that GABA levels were rapidly increased in elicitor-treated opium poppy cell cultures.Acetylglucosamine, an amino-sugar, plays an important role in cell signaling. It has been reported that inflammation induced by bacterial infection can result in increased release of amino sugars from mammalian host cells.Phenyllactate and p-hydroxyphenyllactate were found to decrease ROS production in both mitochondria and neutrophils.Those metabolites were further analyzed with MetaboAnaylst 2.0 to identify the major perturbed metabolic pathways induced by nanoCu. The pathway impact value threshold was set as 0.1.Results showed that none of the pathways were disturbed by nano-Cu in cucumber leaves.Extracts of root exudate from control and nano-Cu treated plants were analyzed by 1 H NMR followed by PCA analysis. PCA analysis revealed clear separation in the root exudate metabolomics profiles collected from control and nano-Cu treated cucumber plants along PC1 . The PCA loadings identify the regions of NMR spectra that contribute to the differences in PC1 . In the region of organic acids , a number of bin areas increased in the presence of nano-Cu, indicating nano-Cu and released Cu2+ increased the level of a number of amino acids or organic acids . 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 ofinterest, 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, 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.