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Toxicity and Reductions in Intracellular Calcium Levels Following Uptake of a Tetracycline Antibiotic in Arabidopsis Shaun M. Bowman,† Kathryn E. Drzewiecki,† Elmer-Rico E. Mojica,‡ Amy M. Zielinski,† Alan Siegel,† Diana S. Aga,‡ and James O. Berry*,† † ‡

Department of Biological Sciences, University at Buffalo, Buffalo, New York 14260, United States Chemistry Department, University at Buffalo, Buffalo, New York 14260, United States

bS Supporting Information ABSTRACT: Plant responses to natural stresses have been the focus of numerous studies; however less is known about plant responses to artificial (i.e., man-made) stress. Chlortetracycline (CTC) is widely used in agriculture and becomes an environmental contaminant when introduced into soil from manure used as fertilizer. We show here that in the model plant Arabidopsis (Arabidopsis thaliana), root uptake of CTC leads to toxicity, with growth reductions and other effects. Analysis of protein accumulation and in vivo synthesis revealed numerous changes in soluble and membrane-associated proteins in leaves and roots. Many representative proteins associated with different cellular processes and compartments showed little or no change in response to CTC. However, differences in accumulation and synthesis of NAD-malic enzyme in leaves versus roots suggest potential CTC-associated effects on metabolic respiration may vary in different tissues. Fluorescence resonance energy transfer (FRET) analysis indicated reduced levels of intracellular calcium are associated with CTC uptake and toxicity. These findings support a model in which CTC uptake through roots leads to reductions in levels of intracellular calcium due to chelation. In turn, changes in overall patterns and levels of protein synthesis and accumulation due to reduced calcium ultimately lead to growth reductions and other toxicity effects.

’ INTRODUCTION Veterinary antibiotics are introduced into the environment via cropland application of animal wastes.1 5 These compounds accumulate near animal feeding operations in soil,6,7 groundwater,8 and aquatic environments.8,9 Tetracycline (TC) antibiotics are the most heavily used in the United States, amounting to about 38% of the approximately 27.85 million pounds of antibiotic sold between 2005 and 2007.10 These are highly stable, and accumulation rates in environments receiving yearly applications can exceed the degradation rate,11 increasing their concentration over time. The widespread use of tetracycline antibiotics in agriculture has led to serious concerns about their environmental impact.2,3,5,12 For manure-treated soil in agricultural fields, chlortetracycline (CTC) has been detected at levels as high as 20 mg/kg.7 Concentrations within agricultural lagoons have been shown to be as high as 1000 ug/kg. An example of the distribution potential from agricultural sites was demonstrated in a recent study9 which measured TC concentrations along the length of a Colorado river. While no TCs were detectable in a mountain location upstream of urban or agricultural sites, significant levels of TCs were found in water from sampling locations near or downstream of agricultural areas. The promotion of resistance to TCs and other antibiotics in microorganisms is of major concern and a primary focus of study.13 16 However, few studies have examined the effects of antibiotics on agricultural plants maintained within these r 2011 American Chemical Society

environments. Several plants have been shown to take up CTC from contaminated soil.17 21 Some plants such as pinto beans show significant toxicity effects when grown in the presence of CTC, while others, such as Zea mays (maize) do not.18 22 CTCinsensitive maize plants induce Glutathione s-transferase (GST) when grown in the presence of CTC, and show increased GST activity after CTC uptake. In contrast, CTC-sensitive pinto beans show no increase in GST activity following CTC uptake. CTC is an efficient calcium antagonist,23 25 and our recent findings provide evidence that intracellular calcium chelation by CTC taken up through the roots of pinto bean plants may contribute to toxicity.21 Taken together, these studies support the hypothesis that GST enzymes are induced in response to CTC exposure to detoxify the antibiotic in resistant plants, preventing accumulation of the antibiotic, thereby not enabling calcium chelation. In contrast, the lack of GST detoxification in sensitive plants allows the antibiotic to build up within cells, allowing chelation-related toxicity effects. We show here that Arabidopsis is highly sensitive to CTC exposure. CTC uptake by Arabidopsis roots causes changes throughout the entire plant, including reduced growth rates, Received: March 15, 2011 Accepted: September 1, 2011 Revised: August 29, 2011 Published: September 01, 2011 8958

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’ EXPERIMENTAL SECTION Plant Material, Growth Conditions, Harvesting, and Treatments. Arabidopsis Col-0 seed was obtained from Lehle Seeds.

Arabidopsis expressing the yellow cameleon YC3 calcium sensor protein was obtained from Simon Gilroy (Dept. of Botany, University of Wisconsin, Madison, WI). Plants were germinated and grown in artificial soil in a growth chamber at 24 °C, with 14 h/d illumination at approximately 170 μmol photons m 2 s 2. The pH of treated and untreated soils was approximately 5.5. For soil treatment, plants were grown in untreated soil for seven days, and then watered daily with 20 mg/L CTC (chlortetracycline hydrochloride, Sigma), or water alone (control), and harvested at times indicated in the figures. CTC solutions were prepared fresh immediately before each application. Plants were carefully removed from soil, washed under slow running water to remove soil from roots, and blotted on paper towels prior to weighing. The entire plant was weighed first, and then cut with a razor blade at a position corresponding to the soil line to obtain separate weights for shoots and roots. To compare toxicity during hydroponic treatment, 3-week-old Arabidopsis or 2-week-old maize plants were excised from soil and placed into foil-wrapped containers with water containing freshly prepared 20 mg/L CTC, or water alone, for 24 h. Analysis of Protein Accumulation and Synthesis. Soluble or membrane-bound protein extracts were prepared as described.27,28 Equal amounts of protein were loaded into each lane of an SDS-PAGE gel, electrophoresed, and either silver-stained or transferred to nitrocellulose for immunoblotting. Antisera for Rubisco LSU and SSU, PEPCase, and NAD-Me have been described.28,29 Antisera against plant CoxII was obtained from Agrisera (V€ann€as, Sweden). Antibody reactions were detected using Amersham ABC luminol reagent system, and visualized using a Storm phosphorimager and ImageQuant software (GE Healthcare). In vivo protein synthesis was determined by radioactively labeling Arabidopsis shoots or roots after 12 days growth in CTCtreated or control soil.27,28 For leaves, stems were cut below the lowest leaf under water and placed into a solution containing 100 μCi of [35S]Met/Cys express labeling mix (PerkinElmer NEN Radiochemicals) in 400 μL of water. Roots were washed in water and cut just below the soil line. The cut end was placed into the [35S]-labeling solution; the labeling container was wrapped in foil to achieve darkness. After two hours, proteins were extracted from equal wet weight of material.27,28 Tricarboxylic acid (TCA) precipitation of proteins from each extract was performed as described.27 Protein extracts were used directly for analysis of total protein synthesis by SDS-PAGE, or immunoprecipitated from equal amounts of labeled protein extracts.27 Fluorescence Measurements and LC/MS Analysis. Uptake of TC and CTC was analyzed as described.21 Briefly, Arabidopsis grown in untreated soil for 18 21 days were removed from soil, washed, and roots were submerged in 0.25 mM TC or CTC solution, or water, for 24 h. Leaves were harvested and prepared as described.21 Fluorescence emission spectra of CTC and TC in Arabidopsis extracts and standards were recorded using an SLM model 8100 spectrofluorimeter equipped with a 450 W Xe arc lamp source. Three readings were obtained for each sample and

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standard. Extracts were also analyzed by LC/MS using an LCQ Advantage ion trap mass spectrometer connected to a Surveyor LC system (Thermo Finnigan, San Jose, CA, USA), using electrospray ionization in positive mode. A reversed-phase Thermo Scientific (Fullerton, CA, USA) BetaBasic C18-column (2.1  100 mm, 3 μm particle size) was used for this separation. Details of mobile phase conditions and instrument parameters were described previously.21 Confocal Imaging and Fluorescence Resonance Energy Transfer (FRET) Analysis. FRET analysis was performed using Arabidopsis expressing the YC3.6 cameleon protein.30,31 To maximize uptake of chelation reagents and minimize variation due to soil binding, evaporation, etc., plants grown for 9 or 11 days in untreated soil were transferred to hydroponic growth in water containing freshly prepared 20 mg/L CTC, 5 mM EGTA, or water alone (control), for 24 h, as shown in Supporting Information 6 and 7. Intact plants were then placed onto slides to which had been attached adhesive silicone isolators (19 mm  32 mm  0.5 mm depth, Grace Biolabs, Bend, OR). Plants were first rinsed in H2O, and then placed within the isolator’s chamber, submerged in water, and then covered with a coverslip. Plants were imaged within 30 min. Images were captured from the midsections of the young primary roots; tip and base regions were not imaged. Fluorescence resonance energy transfer (FRET) analysis of the YC3.6-expressing roots was performed according to methods described in ref 30, using an LSM710-InTune Confocal (inverted) Microscope System (Carl Zeiss MicroImaging), with a 20 objective. CFP excitation was done using a 458-nm argon laser line. CFP fluorescence was collected between 472 and 505 nm, and YFP FRET fluorescence was collected between 526 and 536 nm. Combined YFP/CFP images were analyzed for relative amounts of YFP and CFP fluorescence using ImageJ software (http://rsb.info.nih.gov/ij/). Six independent plants were used for each of the three treatments shown in Figure 3, panel D (for a total of 18 independent plants). Three separate images were obtained and analyzed for each of the independently treated plants (thus, a total of 54 independent images were analyzed for YFP/CFP ratios).

’ RESULTS AND DISCUSSION CTC Toxicity in Arabidopsis. For this study plants were exposed to the highest levels detected in contaminated field soils, 20 mg/kg.7 Treated plants began to show toxicity effects after 3 4 days of CTC exposure, including stunted growth, reduced leaf expansion, and yellowing. These effects were not observed in control (untreated) plants. Figure 1 (top two panels) shows a comparison of CTC-treated versus untreated plants after 9 days of CTC watering. The CTC-treated Arabidopsis clearly showed toxicity effects (left panel), with reduced overall weights (right panel) when compared to control plants. For these young plants, separation of shoots and roots was difficult, and we were unable to obtain separate data for these tissues. Figure 1 (bottom panel), and Supporting Information 1, show CTC effects in older Arabidopsis after 13 14 days of CTC watering. For these older plants, separation of shoots and roots was amenable, and accurate wet weight determinations for each tissue were obtained. It is evident that CTC exposure reduced the growth of both shoot and roots. Growth was reduced more than 2.5 fold for Arabidopsis treated with CTC for 9 14 days. Differences between CTC-treated and 8959

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Figure 1. Arabidopsis growth in response to CTC exposure. Col-0 Arabidopsis were germinated and grown in untreated soil for 7 days, and then watered with 20 mg/L CTC, or water (control). Wet weight in grams was recorded; for both graphs standard error is shown. Top left panel: representative soil-grown plants treated with CTC (left row), and control plants (right row) 9 days after initiating CTC watering. Top right panel: graph of total wet weight in grams for 9 day CTC-treated and control plants. Values were based on 10 treated and 11 control plants. Bottom panel: wet weights determined for entire plant, shoots, and roots of treated and untreated plants. Weight wet data were recorded after 13 days of CTC watering, and combined with data for another population after 14 days of CTC watering. Weight determinations were first made for each entire plant, and then for separated shoots and roots. Weights were recorded for 19 treated and 23 control Arabidopsis.

control plants were stronger than those previously reported for CTC-sensitive pinto beans.22,32 With regard to concentrations used in those studies, it was noted that there was considerable adsorption of the antibiotic to some soils, so that effective amounts of CTC taken up by those plants may have been much lower. Taken together, the findings presented here indicate that CTC exposure affects the growth of the entire Arabidopsis plants, and that toxicity effects do not lessen as exposure time to CTC-treated soil increases. In addition, GST activity studies performed using protocols and reagents described in ref 18 with protein extracts from treated and untreated Arabidopsis plants showed no increase in GST activity in response to CTC exposure (not shown). This indicates that like pinto beans, CTC-sensitive Arabidopsis does not increase overall GST activity in response to CTC exposure. Additional experiments, using antisera and probes specific for individual GST isoforms, will be used in future experiments to investigate in more detail the role of GST induction in response to antibiotic exposure in sensitive and resistant plants. Uptake of Tetracycline Antibiotics by Arabidopsis Plants. Uptake of TC and CTC from roots into leaves was investigated by analyzing leaf extracts from plants treated with these

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antibiotics using liquid chromatography/mass spectrometry (LC/ MS). Supporting Information 3 shows the extracted ion chromatograms (m/z 445 for TC and m/z 479 for CTC) of the TC- and CTC-treated Arabidopsis. These chromatograms showed a distinct peak with retention time at 9.10 min, which matched the retention time of the TC standard (not shown). The MS/MS fragmentation of m/z 445 in the TC-treated extract matched that of the TC standard, forming the characteristic fragment ions m/z 427 (loss of NH3) and m/z 410 (loss of NH3 + H2O).21 Similarly, the retention time at 9.63 min observed in CTC-treated plant extract matched that of the CTC standard (not shown). Further fragmentation of m/z 479 produced m/z 462 ([M NH3]+) and m/z 444 ([M (NH3 + H2O)]+) in the CTC-treated extract. This fragmentation pattern is characteristic of the CTC MS/MS spectra.21 These results provide direct evidence of CTC uptake through the roots and translocation into the leaves of Arabidopsis plants, establishing an association between uptake, reduced growth rates, and other observable toxicity effects in this antibioticsensitive plant. CTC Effects on Protein Accumulation and Synthesis. The toxic effects related to the uptake of CTC by Arabidopsis plants could be caused by direct or indirect effects on the accumulation or synthesis of proteins required for normal growth and development. Immunoblot analyses for a representative group of nuclear- and organelle-encoded proteins indicated only minor reductions, or no reductions, in accumulation following 9 days of CTC exposure (Figure 2A, B, C). Among these representative proteins in leaves were the chloroplast-encoded Rubisco large subunit (LSU, plastid localized) (Figure 2A, L, top panel), the nuclear-encoded Rubisco small subunit (SSU, transported into plastids) (Figure 2A, S, middle panel), and the mitochondrialencoded cytochrome oxidase subunit II (CoxII) (Figure 2A, Co, bottom panel). Similarly, phosphoenolpyruvate carboxylase (PEPCase, cytoplasmic) did not show any significant differences in accumulation in CTC-treated leaves or roots (Figure 2B, P, top panel and 2C, P, top panel, respectively). As expected, the Rubisco proteins were not detectable in the roots (not shown). These immunoblots indicate that CTC toxicity was not associated with significant overall reductions in protein accumulation within the cellular cytoplasm or organelles. While substantial changes in protein composition can occur within different cellular compartments under abiotic stress conditions,33 36 the abundance of these specific organelle or cytoplasmic proteins did not occur in response to CTC stress. It is worth noting that we found only slight changes in the Rubisco LSU or SSU proteins in plants displaying CTC toxicity, since significant Rubisco degradation can be associated with many types of plant stress, including heavy metal contaminants.35 Whereas only small differences between treated and untreated Arabidopsis were observed for the representative proteins described above, there were detectable differences in accumulation of the NAD-dependent malic enzyme (NAD-ME). The accumulation of this protein was reduced approximately 2 3 fold (determined using phosphorimager quantification) in the leaves of 9-day treated plants, relative to control plants (Figure 2B, N, middle panel). At the same time, NAD-ME protein levels in roots of CTC-treated plants increased approximately 2 3 fold over control plants (Figure 2C, N, middle panel). In contrast, another mitochondrial enzyme, the organelle-encoded CoxII, did not shown any changes in abundance (Figure 2A, Co, bottom panel). To confirm that changes in NAD-ME production occurred reciprocally in leaves and roots of CTC-treated Arabidopsis 8960

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Figure 2. Accumulation and synthesis of representative proteins in CTC-treated and untreated plants: Soluble protein extracts were prepared from leaves (Columns A, B) or roots (Column C) of CTC-treated (T) and control (C) plants. Extracts were fractionated by SDS-PAGE, blotted to nitrocellulose paper, and reacted with antisera against the Rubisco large and small subunits, and CoxII (Column A; L, S, and Co in top, middle, and bottom panels, respectively), as well as PEPCase and NAD-ME (Columns B and C; P and N in top and middle panels, respectively). Antibody reactions were visualized with a luminol detection system and quantified using a phosphorimager. For the bottom panels of Columns B and C, in vivo synthesis of NAD-ME (N-ip) was determined in leaves and roots of treated and control plants labeled with [35S]Met/Cys as described in the text. NAD-ME was immunoprecipitated from equal amounts of incorporated label, separated by SDSPAGE, visualized, and quantitated using a phosphorimager.

plants, the in vivo synthesis of this protein was examined in leaves and roots of 9-day treated and control plants. NAD-ME was immunoprecipitated from equalized amounts of 35S-labeled leaf or root extracts (Supporting Information Table 1). Relative levels of NAD-ME synthesis were reduced in the leaves of CTC-treated plants (Figure 2B, N-ip, bottom panel), whereas in roots of treated plants NAD-ME synthesis increased (Figure 2C, N-ip, bottom panel). Synthesis in both tissues correlated with changes in the accumulation of this protein, indicating that production/accumulation of this protein was affected differently by CTC exposure in leaves and roots. The mitochondrial NAD-ME has a variety of specialized functions in plants,37,38 including regulation of alternative oxidase (Aox) activity during oxidative stress.33 Hairy root cultures of sunflowers treated with oxytetracycline induced a stress response, where reactive oxygen species (ROS) were produced by enzymes such as NADPH oxidase and peroxidases.39 ROS exuded from the roots into the media caused oxidative root damage. If CTC induced a similar response in Arabidopsis, then associated ROS root damage would induce oxidative stress, leading to the activation of the Aox response pathway (including NAD-ME). In agreement with our findings, such a partial repartitioning of respiration would require the induction of Aoxrelated enzymes and would not likely affect levels of CoxII itself (for example, see ref 40). The reason for lowering of NAD-ME, but not CoxII, in leaves in response to CTC is not clear. Metabolic changes affected by CTC exposure are likely to be complex, and will likely vary in different plant tissues. Because at least one representative protein showed differences in accumulation/synthesis in response to CTC, it was of interest to examine overall levels of protein synthesis. Total synthesis levels (determined from TCA-precipitated 35S-labeled proteins, Supporting Information Table 1) were similar for soluble leaf and root proteins in treated versus control plants. Synthesis levels were slightly lower (2 3 fold) for membrane-bound leaf

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proteins, and slightly higher (2 4 fold) for membrane-bound root proteins. Within these overall levels, there were easily observable differences for several protein bands in treated and control plants (Supporting Information 4). For example, in leaves there were very minor CTC-associated reductions (less than 2) in Rubisco LSU and SSU synthesis rates. In the soluble and membrane-bound root extracts, some protein bands decreased while others increased (beyond overall changes in synthesis), in response to treatment. Alterations in patterns and levels of protein synthesis continued through 12 days post-treatment, in agreement with the continuing of observable toxicity effects through this period (Supporting Information 5). Taken together, these studies of protein synthesis indicate that, while only one identified protein has been specifically shown to change in terms of accumulation/synthesis levels in response to CTC treatment (NAD-ME), there are many changes in protein synthesis occurring within the leaves and roots in response to abiotic stress brought about by CTC uptake in Arabidopsis. Chelation of Intracellular Calcium by CTC. Calcium chelation in plants by TC antibiotics is well-known23 25 and calcium signaling affects many aspects of plant development and gene expression, especially those relating to abiotic stress.41 46 For this study we made use of transgenic Arabidopsis that constitutively expresses a YC3.6 cameleon protein47 from a CaMV 35-S promoter, to measure FRET in response to CTC exposure. To ensure standardization of dosage/uptake and minimize variation of FRET determinations between samples, plants were treated hydroponically with 20 mg/L CTC. As controls, plants were treated with EGTA (to lower intracellular calcium due to chelation) and with H2O alone. Plants were removed from soil after 9 11 days growth, and placed within hole-punched parafilm floating on the treatment media for 24 h (Supporting Information 6). For these studies we used only roots from the intact plants. Arabidopsis leaves (transgenic and wild type) emitted significant fluorescence within the excitation/emission range used here, making it difficult to differentiate true YFP FRET emission. Roots did not show this background. Although CTC itself is capable of fluorescence,48,49 this was not a factor in the FRET determinations shown here. CTC excitation (390 nm) is much lower than that used here, and CTC emission is reduced 50% at room temperature.50 In fact, using wild type (i.e., nontransformed plants that do not express YC3.6) Col0 Arabidopsis, we detected no difference between the background fluorescence in the roots of plants treated with CTC, relative to EGTA- or nontreated plants (not shown). Figure 3A C shows representative images from a control (H2O treated) root region, which is typical of the images used for this FRET study. Figure 3A shows a root region with YFP emission, and Figure 3B shows the same region in cyan emission. Figure 3C shows a color combined image used for analysis of YFP and CFP emission, with the region outlined in red indicating the entire area that was used for ImageJ fluorescence emission analysis. Figure 3D shows a graphical representation of FRET ratios obtained from images of roots from plants treated with 5 mM EGTA, 20 mg/L CTC, and control plants (H2O alone). Analysis of these images revealed significant reductions in YFP/CFP ratios within roots of CTC-treated plants, relative to controls (approximately 25% reduction in the CTC treated). Treatment of plants with the calcium chelator EGTA reduced the YFP/CFP ratios even more (50% reduction), confirming the validity of this FRET assay for determining changes in relative 8961

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Figure 3. Confocal imaging and FRET analysis of Arabidopsis roots expressing YC3 cameleon protein, in response to EGTA, CTC, and water treatments. (A, B) YFP and CFP emission, respectively, both from the 405 nm wavelength for CFP excitation. (C) Combined image used for quantitation of YFP and CFP emission. The drawn red line shows the region analyzed for emission ratios in this root image. The representative images shown were from an H2O-treated control plant. Images were captured using a 20 objective. (D) YFP/CFP ratios for YC3-expressing roots from plants that were exposed to the treatments indicated. Experimental replicates are described in Experimental Section; standard error is shown.

calcium levels within the roots of these YC3.6-expressing Arabidopsis plants. These FRET data clearly indicate a positive correlation between CTC treatment and calcium reductions within roots. CTC and EGTA appear to cause similar toxicity effects in Arabidopsis during overnight hydroponic treatment (yellowing, leaf curling/wilting, stunted leaf growth); however, at the levels used here the EGTA effects appeared to be more severe (see Supporting Information 7 for a visual comparison of the effects of both treatments). At the resolutions used here, we did not observe any significant effects on overall root morphology for either treatment. Both treatments resulted in reductions in intracellular calcium levels relative to control plants, as determined by confocal FRET analysis, but again, the EGTA treatment caused stronger reductions. Based on these findings, we hypothesize that a comparative analysis of proteins and genes affected by CTC treatment and chelation of calcium by EGTA would likely reveal commonalities in these two toxicity processes. As a central regulator in plants, calcium is known to affect many aspects of growth and development.51 Reductions in calcium are known to decrease plant growth and development, including cell wall and membrane development, photosynthesis, and gene regulation, usually through interactions with calmodulin.41 46,51 Modification of multiple regulatory levels such as cellular signaling, transcription, translation, and protein modification can occur in response to changes in calcium fluctuation. It is therefore not surprising that widespread changes in proteomic patterns (synthesis and accumulation) were observed in response to

calcium chelation caused by CTC uptake. Additional studies will be required to identify specific mRNAs and proteins that fluctuate in direct response to CTC-induced chelation, as well as others that might fluctuate as an indirect response due to reduced growth or general stress. Environmental Relevance of Using Model Species for Phytotoxicity Studies. The experiments presented in this study provide supportive evidence for our model of plant toxicity in CTC sensitive plants. This model proposes that uptake through roots leads to reductions in levels of intracellular calcium due to chelation, leading to changes in overall patterns and levels of protein synthesis and accumulation, ultimately leading to growth reductions and other toxicity effects. This model also suggests that resistant plants, such as maize, produce proteins (GSTs and possibly other proteins)18 20 that modify the antibiotic before it can negatively affect the plant cell’s calcium levels. Although Arabidopsis is not a crop plant, its demonstrated sensitivity to the antibiotic as shown here provides a valuable model system for understanding phytotoxic effects resulting from uptake of antibiotic contaminants that may be introduced into the environment through land-application of manure and biosolids. This study has characterized the toxic effects associated with direct CTC exposure/uptake in Arabidopsis, using upper-end contaminant levels that might be encountered within a highly contaminated environment. It should be noted that these experiments do not necessarily replicate actual field conditions; factors other than CTC exposure that have the potential to reduce or enhance the effects described here (such as retention/absorption by different soils, modification by rhizosphere organisms) were 8962

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Environmental Science & Technology not analyzed in this laboratory study. Using well-characterized plant systems such as Arabidopsis and maize under controlled experimental conditions will help us understand how pharmaceutical contaminants released into the environment may affect the growth and development of plant species that are vulnerable to antibiotic exposure. Since reuse of treated wastewater for irrigating crops is expected to increase in arid regions, and the use of manure and biosolids as fertilizer will remain commonplace in agriculture, it is important to understand the mechanisms of phytotoxicity that pharmaceuticals may cause to susceptible plants.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional images and analyses of CTC-treated and control plants (soil grown and hydroponically treated), MS/MS spectra demonstrating CTC and TC uptake, and SDS-PAGE showing in vivo labeling of total proteins in 9-day-old and 12-day-old plants; a table showing incorporation of [35S]Met/Cys into proteins of CTC-treated and control plants. This information is available free of charge via the Internet at http://pubs.acs.org/.

’ AUTHOR INFORMATION Corresponding Author

*Phone: 1-716-645-4997; fax: 1-716-645-3369; e-mail: camjob@ buffalo.edu.

’ ACKNOWLEDGMENT We are grateful to Dennis Pietras for excellent plant care and maintenance, and Jim Stamos for preparing the illustrations. This work was supported by NSF Grants CHE 0750321 and MCB 0544234, USDA/NRI Grant 2008-01070, and by an Interdisciplinary Research Development Fund (IRDF) from the University at Buffalo. The Zeiss LSM 710 “In Tune” Confocal Microscope used for FRET imaging was purchased through NSF Major Research Instrumentation grant DBI 0923133. K.E. D. was supported in part by an NSF research education for undergraduate (REU) supplement to MCB 0544234, and by an undergraduate research grant from the Rochester Academy of Science. ’ REFERENCES (1) Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Barber, L. B.; Buxton, H. T. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999 2000: A national reconnaissance. Environ. Sci. Technol. 2002, 36, 1202–1211. (2) Boxall, A. B. A.; Kolpin, D. W.; Halling-Sorensen, B.; Tolls, J. Are veterinary medicines causing environmental risks? Environ. Sci. Technol. 2003, 37, 286A–294A. (3) Boxall, A. B. A.; Fogg, L. A.; Blackwell, P. A.; Kay, P.; Pemberton, E. J.; Croxford, A. Veterinary medicines in the environment. Rev. Environ. Contam. Toxicol. 2004, 180, 1–91. (4) Hamscher, G.; Pawelzick, H. T.; Hoeper, H.; Nau, H. Antibiotics in soil: Routes of entry, environmental concentrations, fate and possible effects. In Pharmaceuticals in the Environment, 2nd ed.; K€ummerer, K., Ed.; Springer: Berlin, 2004; pp 139 147. (5) Davis, J. G.; Truman, C. C.; Kim, S. C.; Ascough, J. C.; Carlson, K. Antibiotic transport via runoff and soil loss. J. Environ. Qual. 2006, 35, 2250–2260.

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(6) Aga, D.; Goldfish, S. R.; Kulshrestha, P. Application of ELISA in determining the fate of tetracyclines in land-applied livestock wastes. Analyst 2003, 128, 658–662. (7) Aga, D.; O’Connor, S. K.; Ensley, S.; Payero, J. O.; Snow, D.; Tarkalson, D. Determination of the persistence of tetracycline antibiotics and their degradates in manure-amended soil using enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry. J. Agric. Food Chem. 2005, 53, 7165–7171. (8) Batt, A. L.; Snow, D.; Aga, D. S. Occurrence of sulfonamide antimicrobials in private water wells in Washington County, Idaho, USA. Chemosphere 2006, 64, 1963–1971. (9) Yang, S. W.; Carlson, K. Evolution of antibiotic occurrence in a river through pristine, urban, and agricultural landscapes. Water Res. 2003, 37, 4645–4656. (10) Animal Health Institute. Sales of disease-fighting animal medicines rise; 2008; http://www.ahi.org/files/Media%20Center/Antibiotic% 20Use%202007.pdf. (11) Hamscher, G.; Sczesny, S.; Hoper, H.; Nau, H. Determination of persistent tetracycline residues in soil fertilized with liquid manure by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Anal. Chem. 2002, 74, 1509–1518. (12) Park, S.; Choi, K. Hazard assessment of commonly used agricultural antibiotics on aquatic ecosystems. Ecotoxicology 2008, 17, 526–538. (13) National Academy of Sciences Committee on Drug Use in Food Animals. The Use of Drugs in Food Animals: Benefits and Risks; National Academy Press, 1999. (14) Jones, A. H.; Voulvoulis, N.; Lester, J. N. Potential ecological and human health risks associated with the presence of pharmaceutically active compounds in the aquatic environment. Crit. Rev. Toxicol. 2004, 34, 335–350. (15) Schmitt, H.; Stoob, K.; Hamscher, G.; Smit, E.; Seinen, W. Tetracyclines and tetracycline resistance in agricultural soils: Microcosm and field studies. Micro. Ecol. 2006, 51, 267–276. (16) Kim, S.; Aga, D. S. Potential ecological and human health impacts of antibiotics and antibiotic-resistant bacteria from wastewater treatment plants. J. Toxicol. Environ. Health, Part B 2007, 10, 559-573. (17) Kumar, K.; Gupta, S. C.; Baidoo, S. K.; Chander, Y.; Rosen, C. J. Antibiotic uptake by plants from soil fertilized with animal manure. J. Environ. Qual. 2005, 34, 2082–2085. (18) Farkas, M. H.; Berry, J. O.; Aga, D. S. Chlortetracycline detoxification in maize via induction of glutathione s-transferases after antibiotic exposure. Environ. Sci. Technol. 2007, 41, 1450–1456. (19) Farkas, M. H.; Berry, J. O.; Aga, D. S. Determination of enzyme kinetics and glutathione conjugates of chlortetracycline and chloroacetanilides using liquid chromatography/mass spectrometry. Analyst 2007, 132, 664–671. (20) Farkas, M. H.; Berry, J. O.; Aga, D. S. Antibiotic transformation in plants via glutathione conjugation. In Fate of Pharmaceuticals in the Environment and in Water Treatment Systems; Aga, D. S., Ed.; CRC Press: New York, 2008; pp 199 216. (21) Farkas, M. H.; Mojica, E.-R.; Patel, M.; Aga, D. S.; Berry, J. O. Development of a rapid biolistic assay to determine changes in relative levels of intracellular calcium in leaves following tetracycline uptake by pinto bean plants. Analyst 2009, 134, 1594–1600. (22) Batchelder, A. R. Chlortetracycline and oxytetracycline effects on plant growth and development in soil systems. J. Environ. Qual. 1982, 11, 675–678. (23) Jackson, C. K.; Hall, J. L. A fine structure analysis of auxininduced elongation of cucumber hypocotyls, and the effects of calcium antagonists and ionophores. Ann. Bot. 1993, 72, 93–204. (24) Moller, I. M.; Kay, C. J.; Palmer, J. M. Chlortetracycline and the transmembrane potential of the inner membrane of plant mitochondria. Biochem. J. 1986, 237, 765–771. (25) Ohyam, T.; Cowan, J. A. Calorimetric studies of metal binding to tetracycline: Role of solvent structure in defining the selectivity of metal ion-drug interference. Inorgan. Chem. 1995, 34, 3083–3086. (26) Rhee, S. Y.; Beavis, W.; Berardini, T. Z.; et al. The Arabidopsis Information Resource (TAIR): a model organism database providing a 8963

dx.doi.org/10.1021/es200863j |Environ. Sci. Technol. 2011, 45, 8958–8964

Environmental Science & Technology centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res. 2003, 31, 224. (27) Berry, J. O.; Nikolau, B. J.; Carr, J. P.; Klessig, D. F. Transcriptional and posttranscriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons. Mol. Cell. Biol. 1985, 5, 2238–2246. (28) McCormac, D. J.; Boinski, J. J.; Ramsperger, V. C.; Berry, J. O. C4 Gene expression in photosynthetic and non-photosynthetic leaf regions of Amaranthus tricolor. Plant Physiol. 1997, 114, 801–815. (29) Long, J. J.; Wang, J. -L.; Berry, J. O. Cloning and analysis of the C4 photosynthetic NAD-dependent malic enzyme of amaranth mitochondria. J. Biol. Chem. 1994, 269, 2827–2833. (30) Monshausen, G. B.; Messerli, M. A.; Gilroy, S. Imaging of the yellow cameleon 3.6 indicator reveals that elevations in cytosolic Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis. Plant Physiol. 2008, 147, 1690–1698. (31) Monshausen, G. B.; Bibikova, T. N.; Weisenseel, M. H; Gilroy, S. Ca2+ regulates reactive oxygen species production and pH during mechanosensing in Arabidopsis roots. Plant Cell 2009, 21, 2341–2356. (32) Batchelder, A. R. Chlortetracycline and oxytetracycline effects on plant growth and development in liquid cultures. J. Environ. Qual. 1981, 10, 515–518. (33) Sweetlove, L. J.; Heazlewood, J. L.; Herald, V.; Holtzapffel, R.; Day, D. A.; Leaver, C. J.; Millar, A. H. The impact of oxidative stress on Arabidopsis mitochondria. Plant J. 2002, 32, 891–904. (34) Ndimba, B. K.; Chivasa, S.; Simon, W. J.; Slabas, A. R. Identification of Arabidopsis salt and osmotic stress responsive proteins using two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics 2005, 5, 4185–4196. (35) Feller, U.; Anders, I.; Demirevska, K. Degradation of Rubisco and other chloroplast proteins under abiotic stress. Gen. Appl. Plant Physiol. 2008, 34, 5–18. (36) Heo, J. M.; Livnat-Levanon, N.; Taylor, E. B.; Jones, K. T.; Dephoure, N.; Ring, J.; Xie, J.; Brodsky, J. L.; Madeo, F.; Gygi, S. P.; Ashrafi, K.; Glickman, M. H; Rutter, J. A stress-responsive system for mitochondrial protein degradation. Mol. Cell 2010, 40, 465–80. (37) Berry, J. O.; Zielinski, A. M.; Patel, M. Gene expression in mesophyll and bundle sheath cells of C4 plants. In Advances in Photosynthesis and Respiration Vol. 32. C4 Photosynthesis and Related CO2 Concentrating Mechanisms; Raghavendra, A. S., Sage, R. F., Eds.; Springer: Dordrecht, The Netherlands, 2010; pp 221 256. (38) Chivasa, S.; Berry, J. O.; ap Rees, T.; Carr, J. P. Changes in gene expression during development and thermogenesis in Arum. Aust. J. Plant Physiol. 1999, 26, 391–399. (39) Gujarathi, N. P.; Linden, J. C. Oxytetracycline inactivation by putative reactive oxygen species released to nutrient medium of Helianthus annuus hairy root cultures. Biotechnol. Bioeng. 2005, 92, 393–402. (40) Ederli, L.; Morettini, R.; Borgogni, A.; Wasternack, C.; Miersch, O.; Reale, L.; Ferranti, F.; Tosti, N.; Pasqualini, S. Interaction between nitric oxide and ethylene in the induction of alternative oxidase in ozonetreated tobacco plants. Plant Physiol. 2006, 142, 595–608. (41) Snowden, K. C.; Richards, K. D.; Gardner, R. C. Aluminuminduced genes 1. Induction by toxic metals, low calcium, and wounding and pattern of expression in root tips. Plant Physiol. 1995, 107, 341–348. (42) Hirschi, K. D. Expression of Arabidopsis CAX1 in Tobacco: Altered calcium homeostasis and increased stress sensitivity. Plant Cell 1999, 11, 2113–2122. (43) McCormack, E.; Tsaia, Y.-C.; Braam, J. Handling calcium signaling: Arabidopsis CaMs and CMLs. Trends Plant Sci. 2005, 10, 383–389. (44) Galona, Y.; Nave, R.; Boyce, J. M.; Nachmias, D.; Knight, M. R.; Fromm, H. Calmodulin binding transcription activator (CAMTA) 3 mediates biotic defense responses in Arabidopsis. FEBS Lett. 2008, 582, 943–948. (45) Kim, M. C.; Chung, W. S.; Yun, D. -J.; Cho, M. J. Calcium and calmodulin-mediated regulation of gene expression in plants. Mol. Plant 2009, 2, 13–21. (46) Kudla, J.; Batisti, O.; Hashimoto, K. Calcium signals: The lead currency of plant information processing. The Plant Cell 2010, 22, 541–563.

ARTICLE

(47) Nagai, T.; Yamada, S.; Tominaga, T.; Ichikawa, M.; Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10554–10559. (48) Mathew, M. K.; Balaram, P. A reinvestigation of chlortetracycline fluorescence: Effect of pH, metal ions, and environment. J. Inorg. Biochem. 1980, 13, 339–346. (49) Cerella, C.; Mearelli, C.; De Nicola, M.; D’Alessio, M.; Magrini, A.; Bergamaschi, A.; Ghibelli, L. Analysis of calcium changes in endoplasmic reticulum during apoptosis by the fluorescent indicator chlortetracycline. Ann. N.Y. Acad. Sci. 2007, 1099, 490–493. (50) Oliver, A. E.; Baker, G. A.; Fugate, R. D.; Tablin, F.; Crowe, J. H. Effects of temperature on calcium-sensitive fluorescent probes. Biophys. J. 2000, 78, 2116–2126. (51) Hepler, P. K. Calcium: A central regulator of plant growth and development. Plant Cell 2005, 17, 2142–2155.

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