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Plant Biology ISSN 1435-8603 Tetracycline accumulates in Iberis sempervirens L. throughapoplastic transport inducing oxidative stress and growthinhibitionG. Di Marco1, A. Gismondi1, L. Canuti1, M. Scimeca2, A. Volpe2 & A. Canini11 Department of Biology, University of Rome ‘Tor Vergata', Rome, Italy2 Department of Biomedicine and Prevention, University of Rome ‘Tor Vergata', Rome, Italy Antibiotic bioaccumulation; apoplastictransport; confocal microscopy; environmental Environmental antibiotic contamination is due mainly to improper and illegal contamination; oxidative stress; tetracycline; disposal of these molecules that, yet pharmacologically active, are excreted by humans transmission electron microscopy.
and animals. These compounds contaminate soil, water and plants. Many studies havereported the bioaccumulation of antibiotics in plants and their negative effects on photosynthesis, cell growth and oxidative balance. Therefore, the principal objective A. Canini, Department of Biology, University of this paper was the study of antibiotic accumulation sites in plants and its uptake of Rome ‘Tor Vergata', Via della Ricerca modality. Iberis sempervirens L., grown in soil and in agar in the presence or absence Scientifica 1, 00133 Roma, Italy.
of tetracycline, were used as a model system. Using confocal and transmission electron microscopy, we demonstrated that tetracycline was absorbed and propagated in plantsthrough apoplastic transport and also accumulated in intercellular spaces. Tetracy- cline was rarely detected inside cells (in cytoplasm and mitochondria where, coherent to its pharmacological activity, it probably affected ribosomes), except in stomata.
Moreover, we verified and clarified further the phytotoxic effects of tetracycline on Received: 16 July 2013; Accepted: 23 July plants. We observed that the antibiotic induced a large reduction in plant growth and development and inhibition of photosynthetic activity. As tetracycline may lead tooxidative stress in plants, plant cells tried to balance this disequilibrium by increasing the amount and activity of some endogenous enzyme antioxidant agents (superoxidedismutase 1 and catalase) and levels of antiradical secondary metabolites.
causing problems to aquatic organisms, animals, microorgan- isms, humans and plants (Kumar et al. 2005b). In fact, antibi- Over the last 50 years, public awareness of the long-term otics can induce development of bacterial resistance (mutation adverse effects of antibiotics on human health and ecology has in genes of ‘resistance', transfer of resistance genes among greatly increased, despite scientific evidence (Halling-Sørensen microorganisms, increase in selection pressure for resistant et al. 1998; Zuccato et al. 2006; Cox & Wright 2013; Xue et al.
organisms) and alterations in microbial communities, with 2013). Tetracycline (TC) is a broad-spectrum antibiotic largely consequent alteration of nutrient cycles and decomposition of employed in veterinary and human medicine for the treatment organic matter (Jensen 2001). In humans, antibiotics can cause and control of a wide variety of bacterial infections (Baguer chronic or acute toxic effects or allergic reactions, e.g. intoler- et al. 2000). Environmental contamination with pharmaceuti- ance, urticaria, anaphylactic shock, bronchial asthma and cal residues is determined in different ways, but essentially is angioedema, because of overdose or low-level prolonged expo- due to wastewater discharge and agricultural application of sure (Sungpyo & Aga 2007). Finally, antibiotics can affect sewage sludge and animal manure (Hirsch et al. 1999). In par- plants through inhibition of photosynthesis, consistent ticular, intensive animal farming implies considerable use of decreases in growth, reduction in leaf length, reduction in root antimicrobial drugs. These compounds are given to livestock as number and elongation and an increase in levels of reactive ‘medicated' feed additives in order to prevent diseases (Kolpin oxygen species (ROS) (Kapustka 1997). It is reported that et al. 2002). Since only a small percentage of antibiotics are plants grown on contaminated soils absorb antibiotics and absorbed or degraded through metabolism, they are excreted accumulate them in different organs, but the uptake modality, with urine and faeces, as a mixture of bioactive molecules, in the storage sites and the processing of these drugs in cells are sewage systems and consequently in the environment (Carballa not yet documented. Moreover, the major concern about anti- et al. 2004; Glassmeyer et al. 2005). However, the introduction biotic pollution in plants is relative contamination of the food of such chemicals in the environment can be also associated supply and consequent health risks (Migliore et al. 1996, 2010; with animal waste application in field manuring because anti- Kumar et al. 2005a; Dolliver et al. 2007). The aim of this work biotics generally remain stable for a long time during storage was to study the interaction between Iberis sempervirens L. and (Halling-Sørensen et al. 1998; Jørgensen & Halling-Sørensen TC. We chose this plant as a model system because it belongs 2000). These molecules contaminate the soil, plants and waters, to Cruciferae, like Arabidopsis thaliana (L.) Heynh, the most Plant Biology 2013 German Botanical Society and The Royal Botanical Society of the Netherlands Tetracycline uptake and effects in plants Di Marco, Gismondi, Canuti, Scimeca, Volpe & Canini studied plant in plant biology, and because in the Botanical size; Reprosil, Germany). Chromatographic separation was Garden of Rome ‘Tor Vergata' we are also monitoring environ- carried out with a mobile phase consisting of 0.1% formic acid mental contamination, as in previous studies (Di Marco et al.
(v/v) (phase A) and acetonitrile (phase B) at a flow rate of 2012), through the detection of antibiotics in hive products of 0.3 mlmin 1. Elution started at 3% A solvent for 3 min and honeybees (Apis mellifera ligustica Spinola) in foraging of this then was linearly increased up to 95% over 16 min. Finally, Mediterranean species. TC was chosen because it is the cheap- the total run time, including cleaning and pre-equilibration est and most easily available antibiotic for therapy in human steps, was 30 min. Injection volume was 20 ll and column and animal diseases, including bee treatment against Paeniba- temperature was set at 30 °C. Mass spectrometry was carried cillus larvae White (American foulbrood). In particular, we out using a LCMS 2020 single quadrupole mass spectrometer evaluated: (i) antibiotic absorption capacity from soil (detec- (Shimadzu). The instrument was operated using electro-spray tion and quantification); (ii) TC bioaccumulation sites; (iii) ionisation (ESI) in positive ion mode. Data acquisition was uptake mode (physiological and molecular); and (iv) plant performed using LAB-SOLUTION software (Shimadzu). Ionisation response to TC.
source parameters were: capillary voltage 3.0 kV, interfacevoltage 4.5 kV, heat block 200 °C, DL temperature 250 °C,nebulising gas 1.5 lmin 1 and drying gas flow 15 l 1 (N2). The MATERIAL AND METHODS TC standard was diluted in methanol and infused at flow rateof 0.3 mmin 1 in order to establish the pseudo-molecular ion in a positive ESI mode and check for presence of potential Iberis sempervirens was grown and treated with TC (Sigma- impurity or adduct ions. Only protonated species [M+H]+ Aldrich, Milan, Italy) in the Botanical Garden of Rome ‘Tor were observed for all the compounds. Data are expressed as lg Vergata'. TC was dissolved in soil (100 and 200 mgl 1) and of TC per kg of sample fresh weight (lgTCkg 1 FW).
adult plants were monitored for 30 days. Commercial seedswere sterilised with 5% sodium hypochlorite for 30 min. After Confocal microscopy (CM) many vigorous washings in sterile distilled water, seeds weretransferred into transparent plastic flasks (tissue culture flasks Six plants for each treatment, grown on agar for 30 days, were 175 cm2, BD Falcon, Franklin Lakes, New Jersey, USA) con- washed thoroughly with distilled water to remove soil from the taining sterile agarised Murashige and Skoog culture medium roots. Whole seedling was positioned on a microscope slide (Sigma-Aldrich), with or without TC. TC was added to the and observed, according to a modified method of Pautke et al.
medium at nominal concentrations of 100 and 200 mgl 1.
(2010), with a confocal laser scanning FV 1000 system with an Plants were cultivated indoors under a natural photoperiod and Olympus IX81 microscope (Olympus, Shinjuku-ku, Tokyo, temperature (Rome, Italy, March–October). All procedures Japan). TC (visible as yellow) was detected by capturing the were performed in a biohazard cabinet and plants were main- natural emission at 527 nm after excitation performed with a tained in sealed flasks in sterile conditions throughout the laser at 405 nm. For chlorophyll (detectable as red), the excita- experiment. After 30 days, seedlings were harvested, weighed tion was 635 nm and emission 668 nm. Iso-surface and 3-D 80 °C until chemical analysis. TC 100 and TC volume digital images were captured at 209 enlargement and 200 are the code used in this work to identify TC treatments analysed using IMARIS 7.4 (Bitplane, Zurich, Switzerland) soft- with 100 and 200 mgl 1, respectively.
Tetracycline detection in plants Transmission electron microscopy (TEM) Tetracycline extraction was carried out according to Brambilla Root portions and leaf samples were cut on and soaked in a et al. (2007), with some modifications. A total of 2 g plant was drop of 3% paraformaldehyde in 0.06 M Sørensen phosphate finely homogenised and 5 ml citrate buffer, pH 4.7, added.
buffer (pH 7.2). Samples were then transferred into glass vials Samples were cleaned-up with metal chelate affinity chroma- and fixed for 90 min at room temperature (RT). Specimens tography (MCAC): empty SPE cartridge (5 ml) was stoppered were then rinsed in the same buffer (four times for 15 min) at the base with round glass fibres, filled with 1.0 ml chelating and then dehydrated with increasing concentrations of acetone sepharose resin and placed on a SPE vacuum extractor. After (50%, 70%, 90%) twice for 10 min each. Subsequently, samples washing with 5 ml HPLC grade water, the column was condi- were gradually infiltrated with increasing concentrations of LR- tioned with 1 ml 0.1 M copper sulphate solution and then with White resin (30%, 60%, 100%; London Resin Co., Berkshire, 1 ml MeOH/H2O (1:1, v/v). Sample extract was then loaded UK) mixed with acetone (90%) for a minimum of 3 h per step.
into the cartridge that was then washed with 3 ml MeOH/H2O Samples were finally embedded in pure and fresh LR-White and 2 ml H2O. Elution of analyte was achieved with 2 ml resin and polymerised at 50 °C for 48 h under anaerobic condi- McIlvaine solution, pH 4.5. The extract was dried under a tions. Ultrathin sections (80 nm) were cut with a Reichert stream of N2 and finally resuspended in 1 ml MeOH. HPLC- Ultracut S ultramicrotome (Leica Microsystems, Vienna, MS analysis was performed following the Dasenaki & Thomai- Austria) and samples observed with transmission electron dis (2010) modified method. Chromatographic analyses were microscopy (CEM Zeiss 902) at 80 kV.
performed using an HPLC system with a LC-20AD pump, aCBM-20A controller, a SIL-20a HT auto-sampler and a diode Immuno-gold labelling array SPD –M20A (Shimadzu, Tokyo, Japan) with a mass spec-trometer. Separations were achieved using a Dr. Maisch For TC subcellular localisation, immuno-gold labelling was UHPLC reprosil-pr-ods-3 column (100 9 2 mm, 3 lm particle performed according to Canini et al. (2004). Briefly, ultrathin Plant Biology 2013 German Botanical Society and The Royal Botanical Society of the Netherlands Di Marco, Gismondi, Canuti, Scimeca, Volpe & Canini Tetracycline uptake and effects in plants sections (70–80 nm) were included in LR-White resin, as and then absorbance was measured at 415 nm with spectro- described before, and then assembled on nickel with formvar photometer (Uvikon 860 UV-visible; Kontron). A calibration grids. Samples were pre-incubated for 5 min in 1% H2O2, washed for 5 min in 0.1% PBS Tween and incubated 30 min in (10–250 lgml 1) in methanol. Total flavonoid amount is 5% BSA in 0.1% PBS Tween. Then, sections were rinsing in expressed in milligram quercetin equivalent per gram sample 0.1% PBS Tween and incubated overnight with a primary anti- fresh weight (mgQEg 1 FW). Total phenols were determined body (polyclonal from host rabbit anti-TC, conjugated with with the Folin Ciocalteu reagent (McDonald et al. 2001; 10-nm gold particles, 1:20 dilution in 1% BSA; Antibodies on Gismondi et al. 2012). Plant extract of 0.5 ml was mixed with line, Georgia, USA). Observations were carried out with a Zeiss 5 ml Folin Ciocalteu reagent (1:10 v/v in distilled water) and CEM 902 electron microscope at 80 kV. The labelling was car- 4 ml1 M Na2CO3. The mixture was incubated for 60 min and ried out according to the method described in Canini et al.
total phenols determined with colorimetric at 765 nm (Uvikon (1992) and Taylor et al. (1994).
860 UV-visible; Kontron). A standard curve was preparedusing 0–250 mgl 1 solutions of gallic acid in water. Totalphenols were expressed in milligram gallic acid equivalent per Biometric analysis gram sample fresh weight (mgGAEg 1 FW).
Control and TC-treated plants (20 for each treatment) wereanalysed for germination, root and shoot length, leaf length Western blot analysis and width and total length. Percentage germination was calcu-lated as the number of seeds that germinated per flask with Plant protein extraction was performed according to Wang respect to the total number of sown seeds 9 100. Primary root et al. (2005). Sample of 500 mg was powdered with a mortar length was measured from the tip of the primary root to the and pestle under liquid N2. All steps were performed on ice hypocotyl, while secondary root length was measured from the and solvents were used cold. The sample was resuspended in tip of the secondary root to the beginning of the primary root.
2 ml 100% acetone vortexed and centrifuged at 16,000 g for Leaf length was determined from the beginning of the leafstalk 3 min. The pellet was washed consecutively with: 2 ml 100% to the tip of the leaf, and width from the upper to the lower acetone, 2 ml 10% acetone, twice with 2 ml 10% TCA and side. Total length was measured from the tip of the primary twice with 2 ml 80% acetone. Finally, it was resuspended with root to the tip of the shoot. All measurements were obtained 0.8 ml phenol (Tris-buffered, pH 8.0) and 0.8 ml SDS buffer using a gauge and are indicated in cm. Shoot length was deter- (30% sucrose, 2% SDS, 0.1 M Tris-HCl pH 8.0, 5% 2-mercap- mined by subtracting root length from total length. On the toethanol). After centrifugation at 16,000 g for 3 min, the phe- basis of these results, for each sample was also estimated effec- nolic portion was recovered and added to 4 ml 100% methanol tive concentration (EC) for EC 25 and EC 50: the minimum and 0.1 M ammonium acetate. Sample was frozen 30 min at amount of antibiotic (mgl 1) able to inhibit plant growth of 20 °C and then centrifuged at 16,000 g for 15 min. The new 50% and 25%, with respect to the control, as reported in pellet was washed twice with 2 ml 0.1 M ammonium acetate Baguer et al. (2000).
and twice with 2 ml 80% acetone. The pellet was dried, dis-solved in 25 ll lysis buffer (7 M urea, 2 M thiourea, 4% 3-[(3-cholamido propyl)-dimethylammonio]-1-propane sulphonate Photosynthetic pigment content (CHAPS), 0.8% IPG-buffer pI 3–10 NL, 1% DTT) and centri- Photosynthetic pigments were extracted from plant material in fuged at 16,000 g for 30 min at 18 °C. The supernatant was 80% acetone for 24 h in darkness at 4 °C. The resulting suspen- recovered and stored at 20 °C. Protein concentration was sion was centrifuged for 5 min at 3000 g and absorbance of quantified by the Bradford method (Bradford 1976), using the supernatant measured at 470, 645 and 663 nm with an bovine serum albumin as standard. Samples were then sepa- UV/Vis spectrophotometer (Uvikon 860 UV-visible; Kontron, rated on 12% SDS polyacrylamide gel, transferred onto nitro- Eching, Germany). Pigment levels were calculated from an cellulose Protran membrane (Schleicher and Schuell) and equation allowing a simultaneous determination, in the same incubated with the following primary antibodies: rabbit poly- sample, of chlorophyll a (Chl a), b (Chl b) and carotenoids clonal anti-b-actin, goat polyclonal anti-superoxide dismutase (Car), as previously described (Lichtenthaler 1987). Results 1 (SOD1) and mouse monoclonal anti-catalase (CAT) (Sigma- were expressed in microgram pigment per gram sample fresh Aldrich). Primary antibodies were revealed using horseradish weight (lgg 1 FW).
peroxidase-conjugated donkey anti-goat, goat anti-rabbit andanti-mouse antibodies (Jackson Immunoresearch) and the ECLchemiluminescence detection system (Pierce). Molecular Flavonoid and total phenol content weight (MW1 Pierce, MW2 Fermentas) was obtained by white Total flavonoid and phenol concentrations were measured fol- light scanning. SOD1 (18 kDa) and CAT (64 kDa) standards lowing a modified Pourmorad et al. (2006) method. A total of were purchased from Sigma-Aldrich. Quantifications were per- 1 g of each sample was powdered and dissolved in 10 ml meth- formed eith the LAS3000 Image System (Fuji) and IMAGE QUANT anol for 48 h. After that, the solvent was removed under a software (GE Healthcare, Milan, Italy).
stream of N2 and finally resuspended in 1 ml methanol. For fla-vonoid determination was used the aluminium chloride colori- metric method (Chang et al. 2002): 0.5 ml plant extract wasadded to 0.1 ml 10% aluminium chloride, 0.1 ml1 M potas- Crude protein solutions were extracted by homogenising plants sium acetate, 1.5 ml methanol and 2.8 ml distilled water. The with a mortar and pestle under liquid N2. A total of 1 g crushed reaction mixture remained at room temperature for 30 min sample was dissolved in 10 ml phosphate buffer (0.05 M PBS, Plant Biology 2013 German Botanical Society and The Royal Botanical Society of the Netherlands

Tetracycline uptake and effects in plants Di Marco, Gismondi, Canuti, Scimeca, Volpe & Canini 1% polyvinylpyrollidone, pH 7.8). After filtration through four 17.00 lgkg 1 in leaves, and 40.00 and 9.50 lgkg 1 in flowers.
layers of cheesecloth to remove debris, the homogenate was In contrast, seedlings grown in agar had higher levels of TC: centrifuged at 10,000 g for 10 min. All operations were 107.00 lgkg 1 and 226.50 lgkg 1, respectively, for 100 and performed at 4 °C. The supernatant was used for enzyme activ- 200 mgl 1 TC treatments.
ity, as described in Polle et al. (1997). SOD1 activity was mea-sured on the basis of ability to inhibit photochemical reduction Confocal microscopy analysis of nitro-blue tetrazolium (Masia 1998). The reaction mixturecontained 50 mM phosphate buffer (pH 7.8), 13 mM methio- Tetracycline-treated plant samples grown in agar were stud- nine, 75 lM nitro-blue tetrazolium, 2 lM riboflavin, 100 mM ied with CM to investigate and localise antibiotic accumula- EDTA and 0–200 ll plant extract. The reaction mixture was tion sites. Figure 2 contains images of root (A and D), stem read at 560 nm using a UV-Vis spectrophotometer (Uvikon (B and E) and leaf (C and F) portions of treated plants with 860 UV-visible; Kontron). One unit of SOD1 activity was TC 100 mgl 1 (A, B and C) and 200 mgl 1 (D, E and F) defined as the amount of enzyme that caused 50% inhibition for 30 days. The clear presence of yellow colour denotes the of the initial rate of the reaction in the absence of enzyme. The presence of TC throughout the plant. More TC was detected activity of SOD1 was expressed as unit per gram sample fresh after the higher dose than the lower dose. In all cases, TC weight (Ug 1 FW). CAT activity was calculated as reported in was everywhere in the sections, but especially around the Aebi (1984). The reaction mixture contained 10 mM H2O2, cells. Special hotspots were stomatal cells with 200 mgl 1 50 mM K3PO4 buffer, pH 7, and 800 ll extract in a total TC (F). Figure S1 shows a 3-D reconstruction of TC (yellow) volume of 1.2 ml. The principle of this test was based on and Chl (red) disposition in stems (A) and leaves (B) of a hydrolysis of H2O2 and the relative decrease in absorbance at plant exposed to high doses of TC. In particular, in leaves 240 nm. A reduction in absorbance of 0.01 at 240 nm in 1 min (B) it was clear that Chl dissipated inside cells with respect was considered as one unit enzyme activity. CAT activity to TC, which accumulated in apoplastic spaces and in stoma- was expressed as unit per minute per gram fresh weight tal cells (Fig. 2F). In control plants no TC signal was detected (Umin 1 g 1 FW).
(data not shown).
Statistical analysis Transmission electron microscopy observations All experiments were repeated in triplicate. Results are shown Transmission electron microscope analysis was performed as mean  SD of three independent measurements. Analysis of to examine whether TC distribution in seedlings was not variance was conducted using one-way ANOVA with the statisti- confined to the apoplastic space, as demonstrated in CM cal software PAST 0.45 (PAlaeontological STatistics).
observations. As shown in Fig. 3, gold particles associatedwith the TC antibody were clearly visible as black spots,both in cell cytoplasm and in mitochondria, even if at low Tetracycline uptake Biometric analysis Iberis sempervirens were grown in soil and in agar in the pres-ence of TC 100 and 200 mgl 1, as described in Material and To study the effects of TC on germination and development, Methods, in order to determine their antibiotic absorption I. sempervirens seeds were grown under controlled conditions capacity. With respect to the control, where we did not find in agar, with or without TC (TC 100 mgl 1, TC 200 mgl 1 antibiotic traces, the extracts of all treated plants showed TC and control). Figure S2 shows percentage germination of con- presence. The amount of TC detected in each sample is trol and treated seeds. TC affected seed germination in a con- reported in Fig. 1. We separately analysed the level of TC in centration-dependent manner: after 10, 20 and 30 days of flowers and leaves and also in whole seedlings grown in agar.
treatment, compared to the control, TC 100 mgl 1 inhibited In soil, for both treatments (TC 100 and 200 mgl 1), TC accu- germination of 20.1, 8.5 and 6.1%, while TC 200 mgl 1 mulation in adult plants was, respectively, 10.00 and decreased this percentage to 15.1, 35.8 and 23.8%. The mor-phology of seedlings grown in agar for 30 days in the absenceof antibiotic or in the presence of TC 100 mgl 1 and TC200 mgl 1 are shown, respectively, in Figure S3 A, B and C.
With respect to the control, TC treatments altered the growthand development of seedlings: total length of control, TC 100and 1.45  0.10 cm and 1.50  0.10 cm; primary root length was5.80  0.25, 0.25  0.10 and 0.27  0.05 cm; shoot length was5.67  0.60, 1.20  0.10 and 1.23  0.10 cm, cotyledon lengthwas 0.73  0.10, 0.50  0.05 and 0.57  0.10 cm; and cotyle-don width was 0.53  0.10, 0.42  0.05 and 0.40  0.05 cm.
Fig. 1. Detection of TC in different parts (leaves and flowers) of plants Secondary roots and secondary leaves did not develop in trea- grown in soil and in seedlings cultivated in agar. Results are expressed as ted plants (Table 1). Effective antibiotic concentrations able to lgTCkg 1 FW. Data are reported as mean of three different experi- inhibit growth 25% and 50% (EC 25 and EC 50), with respect to the control, were determined (Table 2).
Plant Biology 2013 German Botanical Society and The Royal Botanical Society of the Netherlands

Di Marco, Gismondi, Canuti, Scimeca, Volpe & Canini Tetracycline uptake and effects in plants Fig. 2. Apoplastic TC distribution in seedlings grown in agar. CM analysis showed root (A), stem (B), and leaf (C) images of plants treated with 100 mg/l ofautoflorescent antibiotic. Root (D), stem (E) and leaf (F) districts of seedlings exposed to 200 mgl of TC were also reported. White bars below images corre-spond to 70 lm.
Fig. 3. Observations with TEM of TC subcellular localisa-tion in treated plant cotyledons (A and C). Detectionwas performed with an immunogold-labelled antibodydirected against TC. Arrows indicate position of someTC molecules (black spots) in cells. Black bars indicateunit of the images in micrometer and in white squaresare reported the magnification. B and D are, respec-tively, the enlargements of A and C. Cy, cytoplasm, Mt,mitochondrion, CW, cell wall.
biotic treatment (Table 3). In comparison to the control, TC Primary and secondary metabolite content 100 and TC 200 mgl 1 samples, respectively, presented a The influence of TC on plant production of photosynthetic reduction of 23.6% and 44.5% Chl a, 30.4% and 47.7% Chl pigments and secondary metabolites was analysed. The con- b and a loss in Car of 18.6% and 42.2%. Levels of flavo- tent of Chl a, b and Car was measured in plants after anti- noids evaluated in leaf extracts (Fig. 4A), with respect to the Plant Biology 2013 German Botanical Society and The Royal Botanical Society of the Netherlands

Tetracycline uptake and effects in plants Di Marco, Gismondi, Canuti, Scimeca, Volpe & Canini Table 1. Biometric analysis: total length (TL), primary (PRL) and secondary (SRL) root length, shoot length (SL), secondary leaf number (SLN), cotyledon length(CL) and width (CW) are reported for control (CNTR) and treated (TC 100 and TC 200) seedlings grown in agar. Results were expressed in cm as the mean  sdof three different measurements. All observed differences were statistically significant (ANOVA, TC 100: F = 5.79, P < 0.05; TC 200: F = 5.71, P < 0.05).
biometric parameter Table 2. Effective TC concentrations (EC) that cause 25% and 50% plant growth reduction compared to the control. Data are reported in mgl 1.
Results are expressed in cm as mean  SD of different results.
TC concentration (mgl 1) Table 3. Photosynthetic pigment content, quantified in Iberis sempervirensplants treated with TC (TC 100 and TC 200) or not (CNTR) for 30 days,expressed in lgg 1 FW. Results are reported as mean  SD of three inde-pendent experiments. All differences were statistically significant (ANOVA,F = 6.59, P < 0.01).
pigments (lgg 1 FW) Fig. 4. (A) Flavonoid and phenol content in leaf extracts of Iberis sempervi-rens grown in soil. Results are mg standard per g sample fresh weight(mgQEg 1 FW; mgGAEg 1 FW). Data are as means  SD of three differ- control, increased by 32.5% and 6.5%, respectively, after ent experiments. All values are significantly different at P < 0.01, F = 18.31.
treatment with low (TC 100) and high (TC 200) doses of (B) Flavonoid and phenol content in flower extracts of Iberis sempervirens antibiotic for 30 days. However, phenols decreased following grown in soil. Results are mg standard per g sample fresh weight both TC treatments: 17.9% (TC 100) and 15.6% (TC 200), (mgQEg 1 FW; mgGAEg 1 FW). Data are means  SD of three different compared to the control. Secondary metabolite content was experiments. All values were significantly different at P < 0.01, F = 14.45.
also measured in flower samples (Fig. 4B): flavonoids wereenhanced 90.2% (TC 100) and 51.4% (TC 200), with respectively, but TC 200 extracts had lower enhancement of respect to the control, while total phenolic synthesis was these enzymes of, respectively, 96.26% and 36.03%, with inhibited 57.7% (TC 100) and 25.3% (TC 200), compared respect to the control (Fig. 5D). Although the antibodies to control extracts.
used in Western blotting were extracted from animals theyalso showed high specificity for plant protein extracts. Inparticular, full gels are reported in Figure S4 to confirm Antioxidant enzyme level and activity the specificity and molecular size of the detected proteins Cell antioxidant enzymes, SOD and CAT, were quantified and their standards. To support these data, SOD1 and CAT with Western blot analysis (Fig. 5A, B). In leaf extracts, activity were also measured in leaf extracts using spectro- with respect to the control, SOD1 and CAT increased of photometry (Fig. 6). After treatment with low doses of TC 266.07 and 148.9%, respectively, in the TC 100 sample; (100 mgl 1), SOD1 and CAT had increased activity, respec- while in TC 200 plants only CAT was up-regulated of 156.07  3.90 Umg 1 FW and 17.93  1.15 (30.77%) but SOD1 decreased to 31.87%, compared to Umin 1 g 1 FW. At high doses of TC (200 mgl 1), SOD control plants (Fig. 5C). In flowers, TC 100-treated plants activity was 98.26  2.46 Umg 1 FW and CAT 13.45  increased SOD1 and CAT levels of 202.78% and 38.61%, 0.52 Umin 1 g 1 FW.
Plant Biology 2013 German Botanical Society and The Royal Botanical Society of the Netherlands

Di Marco, Gismondi, Canuti, Scimeca, Volpe & Canini Tetracycline uptake and effects in plants Fig. 5. Immunoblot analysis of CAT and SOD1 protein levels. A (leaf protein extract) and B (flower proteinextract) are representative immunoblots of the threeindependent experiments. b-actin was used as loadingcontrol. Results are expressed as fold changes withrespect to the control (C, leaf extract; D, flower extract).
Data are mean of three different experiments  SD (inFigure S4 full gels are also reported with MW and con-trol standards).
sent an alarm for human health: in fact, through this route notonly animal origin foods but also vegetables can be consideredantibiotic sources (Dolliver et al. 2007). To investigate antibi-otic diffusion modality and to localise the principal accumula-tion sites in plants, we studied TC-treated seedlings grown inagar using CM and TEM. The CM analysis was possible thanksto TC natural fluorescence. In all plant portions (root, stemand leaf; Fig. 2, Figure S1), we detected antibiotic moleculeseverywhere in the apoplastic space. TC signal was rarely presentin cells, except in particular hotspots such as inside stomatalcells (Fig. 2F). In these cells the high turnover of water, due toconservation of turgor pressure, might cause accumulation of Fig. 6. Activity of CAT and SOD1 in leaf extracts of plants grown in soil, the antibiotic. These observations clearly demonstrated that TC with or without TC, in units per minute per g sample fresh weight entered and propagated in plants through apoplastic transport.
(Uming 1 FW) and units per mg sample fresh (Umg 1 FW), respectively.
Therefore, the present work represents an important study in Results are means of three different experiments  SD.
plant biology, as many other studies only verified and demon-strated antibiotic uptake in plants but none clarified themodality (Migliore et al. 2003; Boonsaner & Hawker 2012). InTEM analysis (Fig. 3), however, TC labelled with a golden anti- body was also detected within cells, supporting the possibility Recently, antibiotics were considered an emerging group of that a small amount of TC molecules were transported into the environmental contaminants because of their negative poten- cytoplasm and even inside mitochondria.
tial effects on human health and ecological communities Tetracycline is a bacteriostatic agent that limits the growth (Dirany et al. 2011). Antibiotics are widely used to treat infec- of bacteria by interfering with protein synthesis; it binds the tious diseases in both humans and animals, and to promote prokaryote small ribosomal subunit (30S) and inhibits amino- livestock farming, aquaculture and agriculture (Gao et al.
acyl-tRNA access to the ribosomal acceptor A site (Orth et al.
2012). Many antibiotics cannot be completely absorbed or me- 2000; Pioletti et al. 2001). For these reasons, the TC amount tabolised and most are introduced, through excretion, into the accumulated in mitochondria could be explained by activity of sewage system, which has become an important source of anti- this molecule against 70S ribosomes that they contain. New biotics in the environment (Leung et al. 2012). It has reported studies should be performed to clarify further TC transport that plants grown on contaminated soil absorb antibiotics (Hil- inside cells but, according to its action in bacteria (Nelson et al.
lis et al. 2010); therefore, aim of this work was the study antibi- 2003), we supposed that TC crossed the membrane through otic uptake in I. sempervirens, principal bioaccumulation sites active transport, not through the aquaporins (Kong et al.
and plant responses to high (200 mgl 1) and low (100 mgl 1) 2007). This research also suggested the suitability of TC for doses of TC.
study of antibiotic absorption in plants because of its fluores- Antibiotic uptake in plants, grown on contaminated soil or cent properties and efficient uptake and transport from roots agar, was investigated through chromatographic analysis (Fig. 1). In all parts (leaves and flowers) of treated plants we The last part of our work was focused on the TC treatment found TC traces, in a dose-dependent manner. Absorption was effects on plant phenotype and physiology. We observed that very significant in plants grown in agar and not negligible in TC inhibits seed germination (Figure S2). Biometric analysis samples from treated soil. These differences might be due to on seedlings grown in agar revealed that TC-treated plants had the biotic soil component that is able both to degrade and to a large reduction in growth, they lacked secondary roots, and make TC less available to plants (Jensen 2001). Our results leaves had decreased cotyledon length and width (Table 1).
confirmed plant ability to take up TC but, above all, they repre- The EC50 and EC25 indicated that the development of treated Plant Biology 2013 German Botanical Society and The Royal Botanical Society of the Netherlands Tetracycline uptake and effects in plants Di Marco, Gismondi, Canuti, Scimeca, Volpe & Canini plant roots and stems was inhibited even at low doses of TC, In conclusion, this research demonstrated that: (i) I. semper- while cotyledons were less sensitive (Table 2), probably because virens plants absorb TC from soil and agar via apoplastic trans- roots were the direct organs in contact with the polluted med- port; (ii) only a little amount of TC is internalised in cells, ium (Migliore et al. 2003). Inhibition of mitochondrial and where it probably inhibits mitochondrial ribosome activity; plastid protein synthesis by TC is the principal cause of phyto- (iii) TC caused a reduction of plant growth and photosynthetic toxicity in treated plants (Bradel et al. 2000; Kasai et al. 2004); activity; and (iv) plant cells try to balance the oxidative stress therefore, we analysed if the stress induced by TC could modify of TC by increasing the amount of antioxidant compounds, plant photosynthetic activity. Chlorophyll a, b and carotenoid such as flavonoids, and the levels and activity of some endoge- content decreased in all treated plants (Table 3), suggesting a nous antioxidant enzymes.
decrease in chloroplast photosynthetic efficiency, as easilyobserved in the chlorotic phenotype of some treated plant leaves. We also observed that seedlings grown in agar in thepresence of TC were etiolated (Figure S3); the low levels of We thank Roberto Targa for technical support, the Center of photosynthetic pigments indicated the toxic nature of TC for Advanced Microscopy ‘P.B. Albertano' of the Department of plants (Murata et al. 2007).
Biology, University of Rome ‘Tor Vergata' and Dr. Elena Secondary metabolites are a group of phenolic compounds Romano for assistance in confocal microscopy analysis.
with known properties, which include free radical scavenging,inhibition of hydrolytic and oxidative enzymes and anti-inflam- SUPPORTING INFORMATION matory action (Gryglewski et al. 1987; Frankel 1995; Cook &Samman 1996). The amount of these compounds was reported Additional Supporting Information may be found in the online to increase in plants under stress in order to protect cell vitality version of this article: (Huseynova 2012). As expected, we found that TC treatment Figure S1. Merged image of tetracycline (yellow) and chlo- increased flavonoid levels in all parts of the plants but, surpris- rophyll (red) content revealed with CM in seedling stems trea- ingly, it reduced the simple phenol content (Fig. 4). This differ- ted with 200 mgl 1 (A). 3-D reconstruction of tetracycline ence might be associated with a specific inhibitory effect that TC (yellow) and chlorophyll (red) disposition in a leaf of a seedling could exercise on secondary metabolite synthetic pathways or grown in 200 mgl 1 TC. White bar below the picture corre- on their degradation (Arbona et al. 2013). Oxidations and sponds to 70 lm.
reductions are essential processes for cell survival, but when Figure S2. Percentage germination at 10, 20 and 30 days of their equilibrium is modified, cellular stability is damaged; this control and treated (TC 100 and 200 mgl 1) seed with respect condition is known as oxidative stress. Plant exposure to envi- to total planted seeds in agar.
ronmental contaminants (e.g. salts, pesticides, drugs, drought) Figure S3. Images of seedlings grown in agar for 30 days in leads to this altered condition (Schwanz & Polle 2001; Shigeoka the absence (A) or with TC 100 mgl 1 (B) and 200 mgl 1 et al. 2002; Vranova et al. 2002); therefore, the last step of our (C). Black bar corresponds to 1 cm.
work was the study of oxidative stress that TC induces in plant Figure S4. Representative cells. Western blot analysis was performed on leaf and flower chemiluminescence) reported in full form. The specificity of protein extracts in order to determine SOD1 and CAT quantity, anti-CAT (64 kDa), anti-SOD1 (18 kDa) and anti-b-actin as the principal cellular endogenous antioxidant enzymes (42 kDa) antibodies in all protein extracts (leaf in upper (Fig. 5, Figure S4). The activity of these proteins was also evalu- panel and flower in lower panel) were confirmed from the ated using spectrophotometry (Fig. 6). The results obtained molecular weights. (acquired from white light scanning).
showed that both enzymes increased in level and activity, espe- Commercial CAT and SOD1 standards were also loaded cially after low doses of TC. All these data confirm that TC into a polyacrylamide gel and detected with the same anti- causes oxidative stress in plants and that cells try to limit it using bodies as plant extracts to confirm their specificity (on right antioxidant enzymes and molecules.
in lower panel).
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Plant Biology 2013 German Botanical Society and The Royal Botanical Society of the Netherlands



IEEE TRANSACTIONS ON INFORMATION THEORY, VOL. 56, NO. 9, SEPTEMBER 2010 Thinning, Entropy, and the Law of Thin Numbers Peter Harremoës, Member, IEEE, Oliver Johnson, and Ioannis Kontoyiannis, Senior Member, IEEE Abstract—Rényi's thinning operation on a discrete random vari- where the random variables are independent and identi-

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