Multiwalled Carbon Nanotubes Dramatically Affect the Fruit

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Multiwalled Carbon Nanotubes Dramatically Affect the Fruit Metabolome of Exposed Tomato Plants Diamond L. McGehee,†,‡ Mohamed H. Lahiani,†,‡ Fahmida Irin,§ Micah J. Green,§ and Mariya V. Khodakovskaya*,‡ ‡

Department of Biology, University of Arkansas at Little Rock, Little Rock, Arkansas 72204, United States Artie McFerrin Department of Chemical Engineering, Texas A&M University, Austin, Texas 77842, United States

§

S Supporting Information *

ABSTRACT: Here, we reported that multiwalled carbon nanotubes (MWCNT) added to hydroponics system can enhance fruit production of exposed tomato plants. We quantified the exact amount of MWCNT accumulated inside of fruits collected by MWCNT-exposed plants using an advanced microwave induced heating technique (MIH). We found that absorption of MWCNT by tomato fruits significantly affected total fruit metabolome as was confirmed by LC-MS. Our data highlight the importance of comprehensive toxicological risk assessment of plants contaminated with carbon nanomaterials.

KEYWORDS: carbon nanomaterials, multiwalled carbon nanotubes, metabolomics, tomato fruits, hydroponics, LC-MS, nanorisk assessment, microwave-induced heating quantification

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MWCNT, exposure.6 It was also demonstrated recently that activation of nodulation by MWCNT in soybean was in direct correlation with up-regulation of genes involved in nodulation process.7 Thus, it is logical to suggest that the uptake of CNMs may significantly affect the metabolism of CNM-exposed plants. Detection and quantification of nanomaterials in exposed plant tissues is a critical step for risk assessment of nanotechnological applications in Plant Biology and Agriculture. It is also possible that CNMs may induce synthesis of metabolites that could be toxic for animals and humans. A detailed metabolomics analysis of CNM-contaminated plant organs is, therefore, a necessary first step toward a risk assessment of CNMs used as growth regulators in agriculture. To the best of our knowledge, a comprehensive metabolomics study of CNM-exposed plants has not to date been performed. Here, we attempt to clarify the impact of a representative type of CNMs (multiwalled carbon nanotubes, MWCNTs) on the total metabolome of hydroponically grown tomato plants. We have correlated metabolomics analysis performed by liquid chromatography−mass spectrometry (LC-MS) with quantification of the amount of MWCNTs inside fruits collected from exposed plants. The delivery of MWCNTs through a hydroponic system was selected to maximize plant absorption of nanomaterials. Tomato plants were cultivated in a hydro-

xposure of plants to carbon nanomaterials can lead to enhancement of plant productivity, but associated risks of use of carbon nanomaterials as growth regulators must be evaluated. Here, we reported quantification of the exact amount of absorbed multiwalled carbon nanotubes inside tomato fruits collected from exposed plants and illustrated the significant effect of applied multiwalled carbon nanotubes on the total metabolome of tomato fruits. Recent discoveries of the effects of carbon nanomaterials (CNMs) on plant and plant tissues are increasing opportunities for nanotechnological applications in Plant Science and Plant Biotechnology. It has been demonstrated that multiwalled carbon nanotubes (MWCNTs) added to growth medium are absorbed by exposed plants and are able to migrate to reproductive organs.1,2 For example, the presence of MWCNTs was detected in tomato leaves and fruits using Raman spectroscopy and photoacoustic/photothermal spectroscopy methods.1,2 Previous research has demonstrated that CNMs can positively affect plant growth, development, and seed germination.1,3 Such significant positive growth regulation has been observed for multiple plant species, including major food crops.4,5 Our prior experiments revealed that different CNMs can be absorbed by plant organs and induce massive changes in gene expression in planta.2,4,6 Genomic profiling of CNM-exposed plants by microarray and confirmation of results by RT-qPCR in our previous work has shown that the regulation of a significant number of genes involved in the major plant metabolic pathways including secondary metabolism was changed after CNM, specifically © XXXX American Chemical Society

Received: July 18, 2017 Accepted: September 13, 2017

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DOI: 10.1021/acsami.7b10511 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Effect of MWCNTs on the reproductive system of tomato plants growing in hydroponic solution. (A) The phenotype of MWCNT exposed plants compared to control plants during week 5 postexposure. (B) Number of fruits per tomato plant collected after 10 weeks in control and MWCNT-exposed plants. Fruits were collected from 6 plants in each treatment. Results of (C) fresh weight and (D) dry weight of fruits in both treatments are shown as the mean values of one fruit. (E) The quantification of MWCNTs inside tomato fruits using the microwave induced heating technique (MIH) is represented as μg of MWCNT per mg of fruit tissue analyzed. n.d. stands for no detection. Error bars represent the standard error values (*, p < 0.05, compared to control plants).

ponic solution supplemented with a final concentration of 50 mg/L of MWCNTs for 10 weeks and used for the phenotypical study, metabolomics analysis, and quantification of absorbed MWCNTs. The dose of MWCNT (50 mg/L) was determined based on our previous work suggesting a concentration of MWCNT that could lead to positive effects in planta including stimulation of the plant reproductive system.1,2,5,6 It was observed that tomato plants exposed to MWCNTs (50 mg/L) for an extended time (10 weeks) did not show signs of toxicity or distress. On the contrary, the phenotypic study revealed that hydroponically supplying MWCNTs to plants significantly affected the development of the reproductive system of exposed plants in a positive manner (Figure 1). During the flowering stage, MWCNT-exposed tomato plants showed early development and an increased number of flowers compared to control plants growing in regular nutrient solution (Figure 1A). As a result of the application of MWCNTs, the total number of fruits in exposed plants was significantly higher, by 65%, compared to control plants (p = 0.0054727) (Figure 1B). Additionally, MWCNT-exposed plants produced fruits with higher fresh and dry weight. The average fresh weight per fruit was higher, with a 15.5% increase compared to control fruits (p = 0.0048) (Figure 1C). The dry weight of fruits from exposed plants was increased by 22.7% compared to the control (p = 0.0357) (Figure 1D). Observed stimulation of the

reproductive system of plants by application of MWCNTs was expected. Previously, the increase of tomato fruit production in soil-grown plants exposed to water containing MWCNTs was documented.1 The exact mechanism of MWCNTs influence on the plant reproductive system is unknown, but could be linked to their previously documented ability to activate genes/ proteins essential for plant growth and stress response.2,3,5 Thus, the expression of aquaporins (water channels) that are major players in plant-water relationships were up-regulated in plant tissues exposed to MWCNTs.5 Fast absorption of CNMs by plants raises a question about the safety of the CNMs once introduced into the food chain by contaminated plants.8−10 There have been numerous studies questioning the potential toxicity of carbon nanotubes to aquatic and terrestrial organisms; however, most of these studies failed to present a reliable experiment that mimics the true exposure to MWCNTs.11−16 Most of these studies faced difficulties finding a reliable method to analyze the concentration of carbon nanotubes inside the biotic or abiotic samples. To properly set up risk assessment studies using appropriate models, the concentration of absorbed nanomaterials by plants must be measured. To overcome the challenges faced by previous studies, we used the Microwave Induced Heating (MIH) technique to measure the accumulation of MWCNTs in the tomato fruits and correlated this concentration with B

DOI: 10.1021/acsami.7b10511 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 2. Visualization of differences in the fruit metabolome caused by the exposure of tomato plants to MWCNTs. (A) PCA comparing exposed (MWCNT) to unexposed (WT) tomato completed by MetaboAnalyst. Each solid dot represents a sample, red for MWCNT and green for WT. Surrounding clouds are the 95% confidence interval. (B) Fold change analysis completed using Metaboanalyst. The threshold was set at 2. Each red feature represents a potential metabolite that has been up-regulated in the MWCNT tomato fruit. Green features are potentially down-regulated metabolites. This analysis allows the visualization of differences in the metabolome caused by the exposure to MWCNTs.

Figure 3. Top 10 metabolic pathways affected in tomato fruits by exposure of plants to MWCNT. (A) Pathways with up-regulated compounds (red) were discovered through comparison of the 93 up-regulated, identified compounds to the KEGG database. Numbers at the end of each bar indicate the number of compounds in that pathway that were dysregulated in relation to wild type. (B) Pathways with down-regulated compounds (green) were likewise discovered via comparison of the 70 down-regulated, identified compounds to the KEGG database. Three of the 70 compounds were not identified in pathways, possibly due to the database being incomplete.

fruit powders from control plants and six others from plants exposed to MWCNTs. Each sample was prepared from a pool of fruits belonging to the same plant and was analyzed in three technical replicates. Control samples showed no signal of carbon nanotubes inside the fruit tissue. As anticipated, fruit samples from plants exposed to MWCNTs showed the presence of nanomaterials (Figure 1E). The level of MWCNTs ranged from 0.041 to 0.137 μg per mg of dry fruit sample. The

potential effects of absorbed nanomaterials on the fruit metabolome. The efficiency of the Microwave Induced Heating (MIH) is based on the high microwave absorption capacity of MWCNTs, which results in a rapid rise in temperature.4,17 Using a short microwave exposure time, we were able to quantify the MWCNT concentration inside fruits using the previously developed calibration curve.4,17 Figure 1E summarizes the results obtained from the analysis of six samples of C

DOI: 10.1021/acsami.7b10511 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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fruits. Figure 3B shows the top 10 pathways containing downregulated compounds. The full list of MWCNT-affected metabolomics pathways is provided in Table S2. We have noticed that many tomato secondary metabolites were affected by the exposure to MWCNTs. The 28 up-regulated metabolites identified by the KEGG database as compounds associated with secondary metabolism are listed in Table S3. It is known that secondary metabolites can have both beneficial or harmful properties, depending on the type of metabolite and their dosage level.27 In our experiment, several compounds derived from the flavone and flavonol biosynthetic pathways were upregulated in MWCNT-exposed fruits (Table S2). It was reported that plant metabolites with flavonoid chemical structures can have medicinal properties but they can also interfere with drug interactions inside the body.27 Thus, it will be important to investigate how the level of such compounds will be affected in specific plant species exposed to CNMs. Analysis of our metabolomics data revealed that not only flavonoid biosynthesis was influenced by the application of MWCNT. Several metabolites representing other branches of secondary metabolism, such as terpenoid and alkaloid biosynthetic pathways, were up-regulated in MWCNTcontaining tomato fruits compared to control (WT) fruits (Tables S2 and S3). For example, gossypol, a product of the terpenoid pathway was described as a compound that may cause permanent male sterility, potassium deficiency, and fatigue.28 This compound was 3.97 times higher in tomato fruits from plants exposed to MWCNTs than wild-type plant fruits. Also, MWCNT-containing fruits produced 2.61 times more alkaloid senecionine, known for phytotoxic activity (Table S3). Considering the significant impact of carbon nanotubes on the tomato metabolome, further studies must be done to establish the risk of consuming food products grown in the presence of MWCNTs because some up-regulated compounds could directly or indirectly cause harm upon ingestion. In conclusion, our study demonstrated that long-term cultivation in a hydroponics system supplemented with MWCNTs led to the stimulation of fruit production in exposed plants. This observation strongly supports the previously documented potential of CNM application in agriculture.1 However, the risk of using CNMs as growth regulators must be investigated. As shown here, even a small amount of MWCNTs absorbed by fruits may significantly impact the overall metabolism of fruits or other organs produced by exposed plants. Because many plant metabolomics pathways affected by MWCNTs can lead to the synthesis of potentially harmful metabolites, there is an urgent need for a risk assessment of CNM-contaminated plant organs for human consumption using available toxicological models and in vivo animal studies.

standard deviation between readings in each sample was below 0.022, confirming the validity of the data obtained. In two plants, MWCNTs were not detected in fruit samples (Figure 1E). These samples could have amounts of MWCNTs below the threshold of detection of the MIH technique. The ability of the MIH technique to quantify CNM materials was previously shown in different tissues such as root, leaves, or tissue culture.4,17 Other methods, such as radioactivity counting, were used by Larue et al. (2012) and have shown that 0.005% of the applied MWCNTs dose is taken up by wheat plant roots and translocated to the leaves.18 To the best of our knowledge, this experiment is the first to report the quantification of nanomaterial absorbance inside plant fruits after long-term exposure (10 weeks). Considering the highest level of MWCNTs detected in fruits from exposed plants (0.137 μg per mg of dry fruit sample), we can conclude that the accumulation of MWCNTs inside the fruit tissue constitutes approximately 0.001% of the fruit dry weight and 0.00003% of the fruit fresh weight. These moderately low percentages show that even after a long exposure to MWCNTs, plants were able to absorb only a small portion of these materials. This level constitutes 2.5 × 10−7% from the total amount of MWCNTs supplemented to plants in the hydroponic system. The quantification of MWCNTs inside fruit samples caused us to question whether the small amount of MWCNTs located in fruits could still have a significant impact on the metabolome of tomato fruits. To clarify this question, we investigated the total metabolome of tomato fruits grown with MWCNTs by applying LC-MS, one of the most widely used techniques due to ease of sample preparation and high sensitivity.19,20 LC-MS followed by statistical data analysis identified many dysregulated metabolites in tomato fruits exposed to MWCNTs. Analysis of LC-MS data leads to the conclusion that the tomato metabolome is dramatically affected by a small absorbed amount of MWCNTs. The initial 10 000 metabolites identified by LC-MS were narrowed to just over 3000 potentially dysregulated compounds by Metaboanalyst,21 more than 1000 of which were up-regulated (Figure S1). Figure 2 illustrates the metabolic similarities and differences between fruit samples observed after statistical analysis by Metaboanalyst. The principle component analysis (PCA) (Figure 2A) demonstrated that MWCNT-exposed fruits clustered more tightly within their group than with unexposed (WT) tomato fruits, as expected. The colored clouds around each cluster represent the 95% confidence interval. This means that the MWCNT-containing fruits were more similar to one another than to the WT fruits and there were significant differences between them. The dendrogram in Figure S2 also illustrates this relationship. Figure 2B shows the fold change analysis completed by Metaboanalyst. Comparison of the dysregulated metabolites to the KEGG database via MZmine22−26 putatively identified over 1,000 unique metabolites. The chromatogram visualization in Figure S3 demonstrated the presence of more up-regulated metabolites than down-regulated in MWCNTexposed fruits. Indeed, pathway analysis of identified, dysregulated metabolites revealed 93 up-regulated and 70 down-regulated metabolites found in tomato according to the KEGG database, as seen in Table S1. The KEGG database search resulted in the discovery of 71 unique pathways affected by dysregulated metabolites in MWCNT exposed tomato fruits (Figure 3; Table S2). Figure 3A represents the top 10 pathways that contained upregulated compounds due to MWCNTs exposure of tomato

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b10511. Detailed experimental procedure: MWCNT exposure, MIH detection of MWCNT, LC-MS sample preparation, running conditions, and data analysis; MWCNT materials, Figure S1−S3, and Tables S1−S3 (PDF) D

DOI: 10.1021/acsami.7b10511 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Micah J. Green: 0000-0001-5691-0861 Mariya V. Khodakovskaya: 0000-0001-6398-4105 Author Contributions †

D.L.M. and M.H.L. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

This project was partially supported by National Space Grant College Fellowship Program (NNXISAR7H) through a Research Infrastructure Award provided by the Arkansas Space Grant Consortium (award to M.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

The authors are grateful to Dr. Stephen Grace (Department of Biology, UA Little Rock) for providing help with the metabolomics study.

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ABBREVIATIONS CNM, carbon nanomaterials MWCNT, multiwalled carbon nanotubes LC-MS, liquid chromatography−mass spectrometry MIH, microwave-induced heating technique

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REFERENCES

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DOI: 10.1021/acsami.7b10511 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX