Ecotoxicology DOI 10.1007/s10646-014-1364-x

Nano-CuO and interaction with nano-ZnO or soil bacterium provide evidence for the interference of nanoparticles in metal nutrition of plants Christian O. Dimkpa • Joan E. McLean David W. Britt • Anne J. Anderson



Accepted: 4 October 2014 Ó Springer Science+Business Media New York 2014

Abstract The expansion of nanotechnology raises concerns about the consequences of nanomaterials in plants. Here, the effects of nanoparticles (NPs; 100–500 mg/kg) on processes related to micronutrient accumulation were evaluated in bean (Phaseolus vulgaris) exposed to CuO NPs, a mixture of CuO and ZnO (CuO:ZnO) NPs, and in CuO NP-exposed plants colonized by a root bacterium, Pseudomonas chlororaphis O6 (PcO6) in a sand matrix for 7 days. Depending on exposure levels, the inhibition of growth by CuO NPs was more apparent in roots (10–66 %) than shoots (9–25 %). In contrast, CuO:ZnO NPs or root colonization with PcO6 partially mitigated growth inhibition. At 500 mg/kg exposure, CuO NPs increased soluble Cu in the growth matrix by 23-fold, relative to the control, while CuO:ZnO NPs increased soluble Cu (26-fold), Zn (127-fold) and Ca (4.5-fold), but reduced levels of Fe (0.8Electronic supplementary material The online version of this article (doi:10.1007/s10646-014-1364-x) contains supplementary material, which is available to authorized users. C. O. Dimkpa (&)  A. J. Anderson Department of Biology, Utah State University, Logan, UT 84322, USA e-mail: [email protected] Present Address: C. O. Dimkpa Virtual Fertilizer Research Center, International Fertilizer Development Center, 1331 H Street NW, Washington, DC 20005, USA J. E. McLean Utah Water Research Laboratory, Utah State University, Logan, UT 84322, USA D. W. Britt Department of Biological Engineering, Utah State University, Logan, UT 84322, USA

fold) and Mn (0.75-fold). Shoot accumulations of Cu (3.8fold) and Na (1-fold) increased, while those of Fe (0.4fold), Mn (0.2-fold), Zn (0.5-fold) and Ca (0.5-fold) were reduced with CuO NP (500 mg/kg) exposure. CuO:ZnO NPs also increased shoot Cu, Zn and Na levels, while decreasing that of Fe, Mn, Ca and Mg. Root colonization reduced shoot uptake of Cu and Na, 15 and 24 %, respectively. CuO NPs inhibited ferric reductase (up to 49 %) but stimulated cupric (up to 273 %) reductase activity; while CuO:ZnO NPs or root colonization by PcO6 altered levels of ferric, but not copper reductase activity, relative to CuO NPs. Cu ions at the level released from the NPs did not duplicate these effects. Our findings demonstrate that in addition to the apparent phytotoxic effects of NPs, NP exposure may also have subtle impacts on secondary processes such as metal nutrition. Keywords Metal oxide nanoparticles  Plant nutrition  Soil bacteria  Solubility  Bioaccumulation  Reductase

Introduction Engineered metallic nanoparticles (NPs), materials B100 nm in at least one dimension, are used to improve the quality of many domestic, industrial, and medical products (Dimkpa et al. 2012a). Uses of CuO NPs include gas sensing, optoelectronics, catalysis, solar cells, semi conductors, pigments and as fungicides (Meshram et al. 2012; Zhu et al. 2004). Use of CuO NPs in these applications is due to the enhanced reactivity evident in materials at the nano scale. However with such intense use of CuO and other metal-based NPs in a wide array of products, their contamination of the environment and reactivity towards terrestrial organisms is anticipated. In plants,

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the impact of CuO NPs is less studied compared to other NPs but seems to be variable in different crop plant species. Impaired germination and growth of rice by CuO NPs is correlated with oxidative damage (Shaw and Hossain 2013). CuO NPs also have negative impacts on other monocots, including wheat, maize and barley (Dimkpa et al. 2012b, 2013; Shaw et al. 2014; Wang et al. 2012) Similarly, growth of cucumber is reduced by CuO NPs, with accompanying increases in the activities of enzymes involved in metal stress responses (Kim et al. 2012). Negative effects of CuO NPs with radish, rye grass and buckwheat are correlated with changes in DNA integrity (Atha et al. 2012; Lee et al. 2013). On the positive side, there is evidence that CuO NPs act as a plant foliar fungicide with little or no deleterious effect on plant performance (Giannousi et al. 2013). Given the wide range of NP-containing products, it is likely that environmental contamination by NPs will involve mixtures of NPs; yet studies assessing the impact of mixtures of NPs in plant systems are lacking. Like CuO NPs, ZnO NPs have many commercial applications (JuNam and Lead 2008), including a potential for use as plant fertilizers (Gogos et al. 2012). Indeed, Zn deficiency in plants is an issue of global importance (Impa et al. 2013), warranting the potential of use of ZnO NPs in plant fertilization. However, at high levels Zn or ZnO NPs can be phytotoxic (see for example, Dimkpa et al. 2012b, 2013). Although the impact of metallic NPs on plant-associated soil microbes have been well studied (see for e.g., Dimkpa 2014), the influence of such microbes on NP interaction with plants is not as well understood, despite plant roots being in constant interaction with soil microbes. One recent study reveals that soybean exposed to cerium oxide NPs had lowered bacterial nitrogen fixation in their root nodules, leading to reduced plant growth (Priester et al. 2012). Similarly, iron oxide NPs lowered the glomalin content of clover roots with abuscular mycorrhizal fungi, resulting in reduced nutrient acquisition and biomass (Feng et al. 2013). Metallic nutrient elements are essential to plants, many of them being involved in enzymatic processes that direct plant functioning. We recently reported that ZnO NPs reduced the uptake of Fe and Mn in bean (Dimkpa et al. 2014). Viewed more broadly, a negative effect of NPs in crop nutrition could have ramifications for humans or animals who consume food from plants with nutrient imbalance. In addition, root colonization of bean by the plant-associated soil bacterium, Pseudomonas chlororaphis O6 (PcO6) increased the secretion of siderophores but decreased ferric reductase (FRO) activity at the root’s surface when bean is grown with ZnO NPs (Dimkpa et al. 2014). Bean is an example of Strategy I plants that use FRO as a system for obtaining Fe from the environment.

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FRO is embedded in the cytoplasmic membrane, where it reduces Fe3? to Fe2? extracellularly before the ion is internalized in the root epidermal cells. Although the main function of FRO is to reduce external ferric to ferrous ions at the roots’ epidermis, FRO also functions to reduce other metals including Cu (Kim and Guerinot 2007; Mukherjee et al. 2006; Robinson et al. 1999; Welch et al. 1993). The present study explores how CuO NPs impact bean (Phaseolus vulgaris) nutrition when grown with and without root colonization by PcO6 in a sand growth matrix. Sand was used in the study in order to reduce the complexities introduced by soil chemistry. On the basis of what is known about the interaction of ZnO NPs and bean (Dimkpa et al. 2014), the studies included investigations of the potential cross-reactivity of ZnO NPs with CuO NPs. As ions could contribute to the toxicity of metallic NPs (Dimkpa et al. 2012a), the role of Cu ions released from the CuO NPs in the growth responses was assessed by measuring the levels in the aqueous fraction of the growth matrix and by examining how inclusion of the Cu ion chelator, bathocuproine, altered responses. Changes in the solubility and plant uptake of other essential nutrient elements were monitored. Activities of FRO both as an iron and copper reductase were evaluated to assess their function in the plant responses.

Materials and methods Sources of chemicals Commercial CuO NPs (\50 nm) and ZnO NPs (\100 nm) were purchased from Sigma-Aldrich, MO, USA. Details of the characterization of the NPs in double distilled water (dd), and/or in a solid plant growth matrix, including aggregation, shape evolution, dissolution, and surface charge, are contained in prior studies (Dimkpa et al. 2011, 2012b, 2013). These earlier studies showed that the commercial ZnO NP product is not a significant source of metallic impurities, while the CuO NPs contained measurable (lg/g) levels of metallic impurities, including Fe, Mn and Zn. Plant growth conditions Commercial white sand (UNIMIN Corp., ID, USA) was used as the plant growth matrix. Chemical characterization of this sand is provided in Calder et al. (2012) and Dimkpa et al. (2012b). The sand was washed three times in dd-H2O and dried overnight at 80 °C. Subsequently, 300 g of the dry sand was sterilized in closed transparent Magenta boxes by autoclaving, and then amended with different [0, 100 mg/kg (1.6 mol/kg), 250 mg/kg (3.9 mol/kg) and

Nano-CuO and interaction with nano-ZnO

500 mg/kg (7.8 mol/kg)] concentrations of Cu from CuO NPs. These levels of the NPs were used because they cover the range of CuO or ZnO NP concentrations which, dependent on growth media, have shown no, subtle, or toxic effects on plants in prior studies (Dimkpa et al. 2012b; Priester et al. 2012; Wang et al. 2012, 2013). To assess the effect of co-contamination of NPs on the plants, CuO NPs were applied in a 1:1 ratio with ZnO NPs to give final combined NP concentrations of 0, 100, 250 and 500 mg/kg, mimicking the total concentrations of the CuO NP treatments. The NPs were added as dry powders and mixed thoroughly by hand for even distribution. Homogeneity of the NPs in the dry sand is evident from the uniform darkening of the sand, in the case of CuO NPs, and confirmed based on consistent data obtained when samples of the sand were assayed for Cu or Zn contents (Dimkpa et al. 2012b, 2013, 2014). Thereafter, 70 ml dd H2O was added to equilibrate the treated sand; only dd H2O was added for the control studies. Seeds of P. vulgaris cv. pink lady were surface-sterilized in 10 % H2O2 for 10 min and rinsed thoroughly in autoclaved dd-H2O. To evaluate the role of soluble Cu, the sand was augmented with a solution of Cu ions (as CuCl2) providing 2.5 mg/kg, equivalent to the level measured as released into the growth matrix from 500 mg Cu/kg of CuO NPs during 7 days of plant growth. Other amendments included 2.5 and 20 mg/kg of bathocuproine (bathocuproinedisulfonic acid, disodium salt; Acros Organics, NJ, USA) to assess the effect of elimination of soluble Cu in plants exposed to CuO NPs at 500 mg/kg of Cu. Bathocuproine is a Cu ion-specific chelator with stronger efficiency for Cu1? (stability constant, log K = 20) than Cu2? (log K = 7.5; Cherny et al. 2000). To examine the role of soil bacteria in influencing the activity of CuO NPs in the plant, a suspension of PcO6 cells (Loper et al. 2012) was used. The cells were previously stored at -80 °C in 15 % glycerol and then raised in minimal medium (Gajjar et al. 2009) to early logarithmic phase. After centrifugation, the pelleted cells were suspended in sterile H2O to generate an OD 600 nm of 0.1 (31 9 106 cfu/ml). Seeds were soaked for 1 h in this cell suspension, followed by draining to remove excess liquid. Other seeds were soaked in sterile water to raise plants lacking PcO6-root colonization. CuO NPs at 500 mg/kg Cu was used in the plant–microbe studies, being a CuO NP concentration known to exert strong negative effects on PcO6 growth in an aqueous system (Dimkpa et al. 2011). Three bean seeds, with or without PcO6 inoculum, were sown at separate locations per box at a depth of about 0.5 cm. In all plant growth studies, six boxes were established per treatment, generating 18 plants for each treatment. In each of the studies described above, no nutrient solutions were added in the growth microcosms to avoid

speciation of metals from the NPs with nutrient components. Seedlings were grown at 28 °C for 7 days, under fluorescent growth lights that generated a photosynthetic photon flux density of 144 pmol m-2 s-1 at the box surface (Dimkpa et al. 2012b, 2013, 2014). After 7 days, a time at which the effects of the NPs on early growth stage of the plants are manifest, seedlings were harvested and analyzed for growth as shoot and root lengths, FRO activity, and shoot metal contents. Root colonization by PcO6 At harvest, plant roots were cleaned of adhering sand particles. The roots were immediately placed in sterile 50 ml tubes containing 10 ml dd water and the tubes vortexed vigorously for 30 s to detach bacterial cells from the roots. The suspension generated from this process was used to determine colonization of the bean roots by PcO6 (Dimkpa et al. 2014). Dilutions of the suspension was plated onto Luria–Bertani (LB) agar medium, and colonies were counted after 48 to 72 h of incubation at 26 °C. Cell density was normalized per gram root fresh weight (FW). Solubility of CuO and ZnO NPs in the bean rhizosphere Dissolution of the CuO NPs with and without bacteria, as well as the mixed CuO:ZnO NPs in the growth matrix was determined after plant harvest. Aliquots of sand (20 g) were collected from root zones in three boxes of each treatment and transferred to 50 ml centrifuge tubes, followed by addition of 40 ml of sterile dd-H2O. The samples were shaken overnight after which the supernatants were collected and centrifuged (Bian et al. 2011; Dimkpa et al. 2012b, 2013) at 10,0009g for 30 min. The recovered aqueous layer was further centrifuged for 30 min at 10,0009g to completely pelletize the NPs. The supernatants were analyzed by ICP-MS (Agilent 7700) for soluble Cu, Zn and other plant-essential metals. Evaluation of Fe(III) and Cu(II)-chelate reductase activities in bean Plants without and with exposure to CuO NPs (250 and 500 mg/kg), mixed CuO and ZnO NPs (500 mg/kg) and bacterial inoculation were harvested after 7 days, and the roots were rinsed in dd-H2O to remove adhering sand particles and transferred to a reaction mixture to determine FRO activity using a method modified from that of Johnson and Barton (2007). The assay solution (pH 5) for iron reduction contained 20 ml half-strength Hoagland’s solution, 10 mM MES, 300 lM ferrozine (Acros Organics, NJ, USA), and 100 lM of freshly prepared FeEDTA [FeCl3 6H2O:Na2EDTA (1:1)]. Roots were incubated in the

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Determination of accumulation of essential metals in shoots Shoots from the 7 days-old plants were harvested with care to eliminate contamination with the growth matrix. The shoots from the same treatment were pooled in three portions, dried in an oven at 110 °C, and ground into powder in a ceramic mortar before digestion for 4 h with hot HNO3, followed by dilution in dd-H2O. The solutions were analyzed for their Cu, Zn, Fe, Mn, Ca, K, Mg and Na contents using ICP-MS.

B Growth response (cm)

solution at 23 °C for 2 h before spectrophotometric reading was taken at 562 nm after centrifugation of the solution to eliminate plant or NP debris. Additionally, to ascertain whether the effect of NPs on FRO was due to inhibition of the activity of the preformed enzyme, the Fe(III) reductase assay was performed with plants grown without NPs, but subsequently exposed to CuO NPs (250 and 500 mg Cu/l) or mixed NPs (CuO:ZnO 250:250 mg Cu or Zn/l) during the assay incubation period (2 h). The concentration of ferrous in the formed Fe(II)-ferrozine complex was determined using a molar extinction coefficient at 562 nm of 29,800 M-1 cm-1 (Lucena et al. 2006). Cu(II)-chelate reductase activity assay was performed as described previously by Yi and Guerinot (1996). Briefly, roots were transferred to a solution containing CuSO4 (0.2 mM), Na3 citrate [Cu(II) chelator; 0.6 mM], and the Cu(I) chelator, bathocuproine (0.4 mM). The samples were incubated for 2 h, followed by spectrophotometric measurement at 483 nm. The Cu(I)-bathocuproine complex is orange colored; the concentration of Cu(I) in the complex was determined using a molar extinction coefficient of 12.25 mM-1 cm-1 (Welch et al. 1993). To demonstrate that Cu reduction by the plants actually occurs in the presence of CuO NPs, the Cu(II) reductase assay was performed with plants grown without prior exposure to NPs, but with 6 h exposure to suspensions of the NPs (250 and 500 mg/l of Cu) during the assay; the NPs replaced CuSO4 as the source of Cu. Plants with no NP amendment or the NP (250 mg/l Cu) suspension without plants served as biotic and abiotic controls, respectively.

16

12

aA aA aA

8

aA

aA

aA

abB abA

bcA

aA CuO (shoot) CuO:ZnO (shoot) CuO (root) CuO:ZnO (root)

4

0 0

100

bcB

bB 250

aA

cB cA bB 500

CuO or CuO:ZnO NPs (mg/kg )

Fig. 1 Growth response of bean to CuO NPs at 0, 100, 250 and 500 mg Cu/kg sand (a), and effects of CuO NPs (mg Cu/kg) and mixtures of CuO and ZnO NPs (mg Cu and Zn/kg) on the shoot and root growth of bean after 7 days exposure in a sand matrix (b). The mixed NP treatment consisted of 1:1 combinations of CuO and ZnO NPs to generate combined final concentrations of 100 (50:50), 250 (125:125) and 500 (250:250) mg of Cu and Zn/kg. Different small letters on data points represent statistically significant differences among the doses for each NP type (single or mixed), separately for shoot and root, while different large letter denote significant differences between single and mixed NPs at each dose (p = 0.05; n = 18)

A Tukey’s means comparison was performed to further explore the differences with a significant (p B 0.05) ANOVA result. Comparisons between CuO and CuO:ZnO NPs were made on two bases: (i) concentration for concentration equivalency, e.g., effect of 250 mg/kg of Cu from CuO NPs vs effect of 250 mg/kg Cu contained in CuO:ZnO at 500 mg/kg; and (ii), direct mass for mass of NPs, that is, CuO vs CuO:ZnO at each dose.

Statistical analysis Results A one-way analysis of variance (ANOVA) (OriginPro 8.6) was used to determine significant differences in the plant responses to the metal treatments and bacterial inoculation. A two-way ANOVA was used to determine significant differences in the levels of soluble metals in the aqueous sand fractions after plant growth for 7 days, as well as metal uptake in the shoot from CuO NPs and mixed treatment of CuO and ZnO NPs in the growth matrices.

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Inhibition of bean growth by CuO NPs is counteracted by CuO:ZnO NPs Bean shoot and root growth in the sand matrix was not significantly influenced by 100 mg Cu/kg dose of CuO NPs after 7 days. However relative to control plants, significant (p = 0.05) shoot and root growth inhibition was observed

Nano-CuO and interaction with nano-ZnO

Grwoth response (cm)

20

a

a

15

ab

Shoot

a

Root

b

b

10

c d

5 0

No PcO6 Control

PcO6 Control

No PcO6 CuO NPs

PcO6 CuO NPs

Fig. 2 Effects of bacterial inoculation on the growth of bean plants in the presence of CuO NPs at 500 mg Cu/kg. Different letters on bars represent statistically significant effects of treatments on growth, separately for shoot and root (p = 0.05; n = 18)

in plants challenged with CuO NPs at 250 and 500 mg Cu/ kg (Fig. 1). The shoot length reduction by CuO NPs was eliminated upon mixing ZnO NPs with the CuO NPs (1:1), whereupon no statistical differences (p = 0.05) in shoot lengths were observed. While statistically significant reduction in root length still occurred for the ZnO:CuO mixture at 250 and 500 mg/kg mixed concentrations, adding the ZnO NPs reduced the growth-inhibiting effects of CuO NPs at these two higher concentrations (Fig. 1). Inhibition of root elongation by CuO NPs is reduced by PcO6 The influence on plant growth of PcO6 colonizing the bean roots was evaluated with 500 mg Cu/kg of CuO NPs. The bacterium colonized root surfaces to similar levels in the presence and absence of the Cu products: 9.65 ± 0.03 (log10) CFU/g FW of root for the control, 9.03 ± 0.08 CFU/g FW with NPs, and 9.34 ± 0.04 CFU/g FW with Cu ions. Colonization by PcO6 did not influence shoot and root growth after 7 days, relative to the noninoculated control plants. However when the PcO6-colonized plants were exposed to CuO NPs, the inhibition of root, but not shoot growth, was significantly reduced (Fig. 2). Soluble metals in growth matrix with CuO NPs are influenced by ZnO NPs, but not by PcO6 After plant growth a low level of soluble Cu was present and this significantly increased as the dose of CuO NPs was raised (Table 1). However the presence of CuO NPs did not influence the soluble levels of other essential metals, Fe, Mn, Zn, Ca, K, Mg and Na. Relative to the control treatment, co-exposure of the plants to the mixes of CuO and ZnO NPs increased soluble Cu, Zn and Ca, reduced Fe

and Mn, and had no effect on K, Mg and Na (Table 1). When comparing between the CuO and CuO:ZnO NPs treatments, additions of ZnO NPs did not reduce soluble Cu level in terms of Cu dose (i.e., 250 mg Cu from CuO NPs v. 250 mg Cu from CuO:ZnO at 500 mg/kg). Addition of CuO NPs at 500 mg/kg or ZnO NPs at 125 mg/kg and above increased the pH of the sand matrix (Table 1). The thermodynamic chemical equilibrium model (GEOCHEM), predicted Fe and Cu to precipitate as hydroxides with increase in pH from 6.6 to 7.8; the solubility of Zn, Mn, and other cations would not be affected in this pH range. When the growth effect of 2.5 mg Cu/kg ions, matching the level of release from 500 mg Cu/kg of CuO NPs was tested, the soluble Cu in the matrix (pH 7.1 ± 0.05) after growth was 0.2 ± 0.1 mg/kg, compared to the control level, 0.1 ± 0.01 mg/kg. Root colonization by PcO6 had no effect on levels of soluble divalent metals in the presence or absence of CuO NPs. However, soluble K was reduced but Na increased when roots were colonized by PcO6 (Supporting Information, SI Table 1). Soluble Cu from CuO NPs is not involved in bean growth inhibition To deduce the importance of release of Cu ions on the level of phytotoxicity exhibited by CuO NPs, growth studies were conducted with an amendment of Cu ions at 2.5 mg/ kg, equivalent to the soluble Cu level measured in the growth matrices with CuO NPs at 500 mg/kg. It is likely that more Cu was actually released from the NPs than these values because of accumulation of Cu into the biomass. However, the Cu ions did not cause significant inhibition of shoot or root elongation (Fig. 3a). The addition of Cu ions when plants were colonized with PcO6 also had no effect on shoot or root growth (Fig. 3a). The pH of the matrix after plant growth with Cu ions and root colonization was 7.07 ± 0.09. To explore the significance of Cu ion release, bathocuproine, at a dose equivalent to the measured level of released Cu ions, and a second dose that was 8-fold higher, were added to the growth matrix. No effects of the chelator were seen on shoot or root elongation. When 500 mg/kg CuO NPs were added, there also was no effect of the bathocuproine on the extent of inhibition of root or shoot growth (Fig. 3b). Inhibition of Fe reductase activity by CuO NPs is altered by CuO:ZnO NPs and PcO6 The bean roots grown without any amendments had very low ferric reductase activity when no Fe was added as a substrate to the assay mixture (data not shown). When Fe

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C. O. Dimkpa et al. Table 1 Levels of soluble Cu and other plant-essential elements in the growth matrix after 7 days of bean growth with and without exposure to CuO NPs and CuO:ZnO NPs

Treatment (mg/kg)

Nutrient in growth matrix (mg/kg) Cu

Fe

a

a

ab

a

10

0

0.1 ± 0.01c

4.5 ± 0.3a

0.02 ± 0.003a

0.06 ± 0.01c

0









CuO

100

0.8 ± 0.04b

4.2 ± 0.06a

0.02 ± 0.001a

0.04 ± 0.003c

6.8

50:50

0.9 ± 0.5b

1.6 ± 0.04b

0.01 ± 0.001b

0.9 ± 0.05b

6.8

CuO

250

1.1 ± 0.1b

4.2 ± 0.3a

0.02 ± 0.002a

0.04 ± 0.003c

6.8

CuO:ZnO

125:125

1.0 ± 0.6b

1.3 ± 0.4bc

0.01 ± 0.003b

1.6 ± 0.2ab

7.8

CuO

500

2.4 ± 0.2a

4.3 ± 0.1a

0.02 ± 0.002a

0.03 ± 0.003c

7.6

CuO:ZnO

250:250

0.7 ± 0.6b

0.7 ± 0.5c

0.005 ± 0.002b

2.7 ± 1.0a

7.7

Nutrient in growth matrix (mg/kg)

b

a

ab

Control

Cu ions

Na

0

1.6 ± 0.1dc

3.7 ± 0.6a

0.7 ± 0.1a

12.8 ± 0.6a

0









CuO

100

1.4 ± 0.1d

2.3 ± 0.2a

0.7 ± 0.1a

12.3 ± 0.7a

CuO:ZnO

50:50

3.0 ± 0.7bc

4.1 ± 0.8a

0.8 ± 0.1a

11.2 ± 5.0a

CuO

250

1.2 ± 0.1d

2.1 ± 0.1a

0.6 ± 0.04a

13.1 ± 0.3a

CuO:ZnO

125:125

5.0 ± 1.0a

9.5 ± 8.0a

1.2 ± 0.2a

14.6 ± 4.7a

CuO

500

1.2 ± 0.1d

2.1 ± 0.2a

0.6 ± 0.1a

14.3 ± 0.8a

CuO:ZnO

250:250

3.3 ± 0.8b

5.3 ± 06a

0.8 ± 0.6a

12.6 ± 0.9a

a

a a

a

b

b b

b b

b

CuO NPs+chelator (20)

CuO NPs+chelator (2.5)

CuO NPs

Control (20)

Bathocuproine

Fig. 3 Effects of Cu ions (2.5 mg Cu/kg) with and without PcO6 colonization (a), and of the Cu ion chelator, bathocuproine at 2.5 and 20 mg/kg (b) on bean growth in the presence of CuO NPs (500 mg Cu/kg). Different letters on bars represent statistically significant differences among the treatments, separately for shoot and root for Figure a or b (p = 0.05; n = 18)

was added to the reaction mixture enzymatic activity was detected: activities were lower for plants grown with 250 and 500 mg/kg CuO NPs (Fig. 4a). Plants grown with the 250:250 mg/kg mix of CuO and ZnO NPs had an intermediate level of activity in these 2-h incubation assays (Fig. 4a). Inhibition also was seen when plants grown without NPs were assayed in reaction mixtures containing CuO NPs; the mix of CuO and ZnO NPs gave strongest inhibition of ferric reductase (Fig. 4b). The ferric reductase activity in plants colonized by PcO6 was lower than in the noncolonized plants in the 2 h

123

Mg

CuO

Control (2.5)

PcO6

No PcO6

PcO6

0

K

CuO:ZnO

(a) (b)

Root

6.6

CuO:ZnO

5 No PcO6

Growth response (cm)

15

Shoot

a

Zn

CuO

Ca

20

Mn

CuO:ZnO

Treatment (mg/kg)

Data are averages and SDs and different letters after values represent statistically significant effects of the treatments within each column (p = 0.05; n = 3)

pH

assay (Fig. 4c). However activity significantly above control was observed with the roots of PcO6-colonized plants grown with CuO NPs (Fig. 4c). When control roots or roots from PcO6-colonized plants were present in reaction mixes containing NPs all activities were decreased (Fig. 4d). CuO NPs, but not CuO:ZnO NPs nor bacterial colonization enhances Cu(II) reductase activity When the substrate, Cu(II) citrate, was mixed with bathocuproine, no color change to orange measured at 483 nm was seen (data not shown), in agreement with the low stability constant of this chelator with Cu2? ions compared to Cu1? ions (Cherny et al. 2000). When the reductase assay was run without plants but with the CuO NPs (250 mg/L), a low level of Cu(I) was detected (Fig. 5a, the abiotic control). No color change was observed when the assay was run without added Cu(II) but with roots of plants grown without NP amendments (Fig. 5a, the biotic control). Roots of plants grown for 7 days with CuO NPs had significantly higher activity levels over those grown without the NPs, and a dose effect of the NPs was observed (Fig. 5b). Activity level in roots of plants grown with the mix of 250:250 mg/kg CuO:ZnO NP was the same as that of roots grown with 250 mg/kg CuO NPs. The activity levels were not altered by growth of plants colonized by PcO6, and when exposed to CuO NPs, root colonization also had no effect. Exposure to Cu ions at 2.5 mg/kg also did not influence Cu reduction relative to the control treatment (Fig. 5c).

with CuO NPs significantly (p B 0.05) elevated the levels of Cu in the shoot, with the maximum value peaking at 100 mg/kg Cu and lower values at the 250 and 500 mg/kg treatments, suggesting that adaption mechanisms to limit uptake or transport were initiated. Coincident with the elevated Cu levels were decreased Fe, Zn Ca but not Mg levels; with the monovalent metals, K showed little change but Na increased (Table 2). Use of bathocuproine at 2.5 and 20 mg/kg to chelate Cu ions released from CuO NPs (500 mg Cu/kg) in the matrix during plant growth resulted in 14 and 25 % reductions in Cu uptake into shoot, respectively. Each of these reductions was statistically significant (p = 0.05), relative to the CuO NP treatment without bathocuproine. Compared to the control plants, Cu shoot accumulations increased by 10 % upon growth amendment with Cu ions at 2.5 mg/kg. With the combined NP treatment, the Zn from the mixture of CuO:ZnO NPs was bioavailable, causing increased accumulation of Zn but with no dose effect. In contrast, Fe, Mn and Ca accumulation was reduced, while Na levels increased, and there was little affect on K accumulation (Table 2).

a a

c

b

(a) (b)

10

bc

c

5 CuO NPs (250)

Control

(c) (d)

d

b

10

b

b

5 CuO NPs (500)+ PcO6

CuO NPs (500)

Control+PcO6

Control

CuO NPs (500)+ PcO6

Fig. 4 Effects of CuO NPs (250 or 500 mg Cu/kg) and CuO:ZnO NPs (250:250 mg Cu:Zn/kg) and root colonization by PcO6 with and without exposure to CuO NPs on the activity of Fe-chelate reductase in bean grown for 7 days. a plants exposed to NPs during growth; b plants exposed to NPs after growth, c plants exposed to NPs and root colonization by PcO6 during growth; and d plants exposed to NPs after growth, without and with root colonization during growth. Different letters on bars represent statistically significant effects of the treatments (p = 0.05; n = 3), separately for panels a, b, c and d

Root colonization by PcO6 under CuO NPs exposure variably affects bioaccumulation of essential metals Root colonization by PcO6 significantly reduced shoot accumulations of Fe, Zn, Ca and Na, but not of Cu, Mg, Mn and K (Table 3) when plants were grown without amendments. Root colonization for the CuO NP-exposed plants lowered the level of Cu and Na significantly compared to their levels in the plants grown with CuO NPs but without colonization (Table 3). Accumulated levels of other metals were not affected by PcO6 colonization for the CuO NP-exposed plants (Table 3).

Shoot accumulation of essential metals is altered by exposure to CuO and CuO:ZnO NPs The levels of essential metals in the shoots were influenced by NPs during growth (Table 2). Amendment of the matrix

120

(a)

a

(b)

100 80

a

(c)

a

b

b

a

60

Fig. 5 Cu(I) formation by plant roots when a grown without NPs but with CuO NPs at 250 and 500 mg/kg added into the assay reaction mixture, b grown with CuO NPs (250 and 500 mg Cu/kg) or CuO:ZnO NPs (250:250 mg Cu:Zn/kg), and c grown with roots colonized by PcO6 with and without exposure to CuO NPs in the

b

CuO NPs

c

(500)+PcO6

CuO NPs (250)

c

Control

Control (bioc)

0

d

CuO NPs (500)

20

bc

c

b CuO NPs (250)

40

Control (abioc)

Reduced Cu [μM Cu(I)/ g root FW]

140

CuO NPs (500)

CuO NPs (500)

Control+PcO6

0

Control+PcO6

15

a

Control

20

a c

(250:250)

b

CuO:ZnO NPs (250:250)

25

CuO NPs (500)

30

CuO:ZnO NPs (250:250)

CuO NPs (500)

CuO NPs (250)

Control

0

Cu ions (2.5)

b c

15

CuO:ZnO NPs

20

CuO NPs (500)

25

Control

Ferric -chelate reductase acvity [μM Fe(II)/g root]

Ferric -chelate reductase acvity [μM Fe(II)/g root]

Nano-CuO and interaction with nano-ZnO

growth matrix. The biotic control data are for assay mixtures with control roots. The abiotic control data are from assay mixtures with no added Cu(II) chelated with citrate. Different letters on bars represent significant effects of the treatments (p = 0.05; n = 3), separately for panels a, b and c

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C. O. Dimkpa et al. Table 2 Uptake of essential metals from growth matrix by bean exposed to CuO and CuO:ZnO NPs

Treatment (mg/kg)

Cu

Fe

Mn

Zn

CuO

0

26 ± 3e

137 ± 9a

39 ± 2a

93 ± 5c

CuO:ZnO

0









CuO

100

225 ± 7a

105 ± 4ab

36 ± 1ab

55 ± 2d

CuO:ZnO

50:50

60 ± 4d

109 ± 34ab

32 ± 1bc

247 ± 7a

CuO

250

131 ± 6b

83 ± 3b

35 ± 2abc

57 ± 3d

CuO:ZnO

125:125

59 ± 2d

84 ± 2b

33 ± 1bc

223 ± 5b

CuO

500

125 ± 12b

85 ± 11b

30 ± 4c

44 ± 5d

CuO:ZnO

250:250

70 ± 2c

72 ± 3b

31 ± 1bc

233 ± 8ab

Treatment (mg/kg)

Data are averages and SDs and different letters after values represent statistically significant effects of the treatments within each column (p = 0.05; n = 3)

Nutrient in shoot (mg/kg)

Nutrient in shoot (mg/kg) Ca

K

Mg

Na 69 ± 6d

CuO

0

1625 ± 115a

24640 ± 1109ab

2105 ± 121a

CuO:ZnO

0









CuO

100

782 ± 51b

28748 ± 903a

2063 ± 91a

95 ± 9b

CuO:ZnO

50:50

859 ± 65b

13678 ± 917c

1462 ± 78c

110 ± 7b

CuO

250

699 ± 32b

28932 ± 1703a

1991 ± 110ab

99 ± 7bc

CuO:ZnO

125:125

808 ± 24b

26376 ± 93ab

1717 ± 47bc

78 ± 2c

CuO

500

735 ± 42b

28799 ± 3460a

2218 ± 143a

141 ± 9a

CuO:ZnO

250:250

885 ± 13b

23842 ± 568b

1694 ± 39c

103 ± 2b

Table 3 Uptake of Cu and other essential metals from growth matrix by bean grown with and without bacterial inoculation and exposure to CuO NPs (500 mg Cu/kg) Treatment

Nutrient in shoot (mg/kg) Cu

Fe

Mn

Zn

Control

26 ± 3c

137 ± 9a

39 ± 2a

93 ± 5a

Control ? PcO6

18 ± 1c

91 ± 5b

38 ± 3a

54 ± 14b

CuO NPs

125 ± 12a

85 ± 11b

30 ± 4b

44 ± 5b

CuO NPs ? PcO6

106 ± 5b

72 ± 3b

30 ± 1b

39 ± 1b

Treatment

Nutrient in shoot (mg/kg) Ca

K

Mg

Na

Control

1625 ± 115a

24640 ± 1109a

2105 ± 121a

69 ± 6c

Control ? PcO6

892 ± 86b

24941 ± 1325a

1982 ± 180a

40 ± 6d

CuO NPs CuO NPs ? PcO6

735 ± 42b 693 ± 31b

28799 ± 3460a 25734 ± 467a

2218 ± 143a 2068 ± 89a

141 ± 9a 107 ± 5b

Data are averages and SDs and different letters after values represent statistically significant effects of the treatments within each column (p = 0.05; n = 3)

Discussion The dose-dependent inhibition of bean root, and to a lesser extent, shoot elongation, by CuO NPs confirmed studies with other plants, including barley, chickpea, soybean and cabbage (Adhikari et al. 2012; Lei et al. 2011; Shaw et al. 2014). The CuO NPs were highly effective in delivering Cu

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to the shoot which could be involved in the observed growth inhibitions. We lack data on Cu loading into root tissues because we currently do not have a reproducible and conclusive method to separate Cu loading into root due to association/attachment of nanopowders to the root surface versus NPs or ions that have been translocated into plant cells (Dimkpa et al. 2013; Wang et al. 2013). In the

Nano-CuO and interaction with nano-ZnO

shoot, maximum Cu loading was observed with the lowest dose, 100 mg/kg CuO NPs and with lesser levels for the 250 and 500 mg/kg treatments. The accumulations which ranged from 125 to 225 mg/kg were 10- to 20-fold higher than averaged normal levels, 10 mg/kg, for leaf tissues (Yruela 2009). It is possible that a greater degree of aggregation of particles in the root zone at the higher NP concentrations was occurring (Keller et al. 2010), limiting the amount of CuO particles small enough to be taken up into the plant tissue as the dose increased. Prior studies with maize and wheat demonstrated the presence of CuO particles in the shoot (Dimkpa et al. 2012b, 2013; Wang et al. 2012). Other possibilities could be that the plant was deploying mechanisms to restrict Cu levels within its tissues once a toxic maximum was reached (Yruela 2009), or that there was a pH effect whereby increased pH with higher dosing could be precipitating out some of the Cu ions. Treatments with CuO NPs alone had no effect on the soluble levels of other essential metals. However there were declines in the accumulation of Fe and Ca in the shoot tissues. The finding of antagonism between Fe and Cu levels agreed with previous observations of Cu ions competitively inhibiting Fe uptake (Yruela 2009). We speculate that the impaired function of ferric reductase demonstrated in roots exposed to CuO NPs, during growth or assay, was involved in this response. Reduced ferric reductase activity would generate less Fe(II) to be transported into the plant by the Itr-type transporter associated with ferric reductase (Sinclair and Kra¨mer 2012). Indeed, published reports find Cu to reduce plant ferric reductase activity (Barton et al. 2000; Welch et al. 1993), even inhibiting expression of genes encoding the reductase proteins (Kim and Guerinot 2007). Our data revealed that the bean roots reduced Cu(II) chelates to Cu(I), and we suggest that this was due to reductase activity associated with certain members of the FRO family of genes (Lucena et al. 2006; Robinson et al. 1999; Welch et al. 1993) The findings of Welch et al. (1993) that pre-exposure to Cu(II) ions (0.5 lM) lowered Cu reduction in pea contrasted to our observations and also that of Boycheva and Babalakova (2006) which showed stimulated activity in cucumber using 0.2, 2 and 20 lM of chelated Cu(II). Perhaps antagonism between Cu(II) and Fe(III) as a substrate for the FROs in the plant roots when grown on CuO NPs was a mechanism contributing to decreased Fe accumulation in the bean shoots. We suggest that some of the Cu ions released from the NPs in the growth matrix could be chelated by Cu-chelators such as citrate contained in root exudates (Martineau et al. 2014) and reduced to Cu(I) in the root plasma membrane by FRO before uptake by Cu(I) transporters (Ryan et al. 2013; Yruela 2009). Our prior finding that a small portion of the

Cu accumulated within wheat shoots after growth with CuO NPs was associated with sulphur (Dimkpa et al. 2012b, 2013) would be consistent with such a mechanism for Cu(I) accumulation, as also demonstrated in tomato by Ryan et al. (2013), and in bamboo by Collin et al. (2014). The reduction in shoot Cu levels we observed when bathocuproine was present with the CuO NPs suggested that the bathocuproine-Cu(I) complex in the root zone had lower bioavailability than the Cu(I) alone. The presence of ZnO NPs along with the CuO NPs abrogated shoot inhibition of growth by CuO NPs, and reduced the extent of root inhibition. Pandey et al. (2010) reported growth stimulation in chickpea by ZnO NPs, and there was significant stimulation of root elongation in green pea by ZnO NPs (125–500 mg/kg) in soil exposure (Mukherjee et al. 2014). In the current study, improved plant growth was accompanied by reduction in soluble Cu to an extent that was lower than with the CuO NP treatment, but still 2- to 3-fold above the background level. Increased alkalinity of the aqueous fraction when ZnO NPs were present (Dimkpa et al. 2014) could contribute to precipitation of metals (Cu and Fe) as the hydroxides, a possible factor involved in reducing soluble metal levels. Interestingly, the extent of reduction of Fe accumulation was similar whether plants were grown with the mix of NPs or just CuO NPs, in spite of altered ferric reductase activity detected in roots exposed to the mix of ZnO:CuO NPs. The exposure to CuO NPs also reduced Mn, Zn and Ca, but increased Na levels in the shoot tissues, with no effect on Mg and K levels. The lack of change in K levels may indicate the absence of major problems with membrane leakage in the exposed plants. In contrast, Wang et al. (2012) showed that CuO NPs increased the rate of K leakage in root and shoot of maize. With co-exposure to both CuO and ZnO NPs, there was reduction in Cu, Fe and Mn accumulation compared with CuO NP-treatment, which mirrored observations with treatments with ZnO NPs (Dimkpa et al. 2014). Decreased Mg levels in plants exposed to the mixture of NPs differed from results for plants grown with CuO NPs. The processes underlying these changes require further studies. Colonization of the roots by PcO6 was another growth condition that ameliorated root, but not shoot, inhibition by CuO NPs. The extent of PcO6 colonization of the roots was unaffected by the NPs or exposure to Cu ions added at an initial concentration of 2.5 mg/kg (corresponding to 11 mg/l on the basis of 70 ml of water added to the sand growth matrix) although cell death was observed for planktonic PcO6 cells with Cu ions exposure above 1 mg/l (Dimkpa et al. 2011). Thus, there seems to be protection of bacterial culturability at the root surface, perhaps due to growth as biofilms and induction of adaption mechanisms

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C. O. Dimkpa et al.

such as Cu efflux pumps by PcO6 cells. The mitigation of inhibition of root growth was accompanied by reduced accumulation of Cu in the shoots of the colonized plants grown with CuO NPs. Coating of the NPs by organic materials from the root or bacterium also could be involved in these changes (Dimkpa et al. 2011; Martineau et al. 2014). If Cu accumulated in the bean shoots mainly as CuO, as observed for wheat or maize grown with CuO NPs (Dimkpa et al. 2012b, 2013; Wang et al. 2012), then the observation that the PcO6 colonization had no effect on soluble Cu levels in the growth microcosms was not unexpected. Although there was no effect of PcO6 colonization on the soluble levels of essential metals in the growth matrix, colonization decreased shoot accumulations of Fe, Zn, Ca and Na in plants grown under control conditions, and Cu for growth with CuO NPs. The level to which Cu accumulation was reduced by PcO6 colonization during growth with CuO NPs was similar to that observed in growth with the ZnO:CuO mix. The role played by the root surface metal reductases as a Fe- or Cu-reducing system in the roots colonized by PcO6 was not clear. Roots of colonized plants grown with CuO NPs when assayed for reductases showed stimulated Fe reduction but no change in Cu reduction activity. It is possible that bacterial processes such as production of siderophores as PcO6 colonize the root could be influencing these activities (Dimkpa et al. 2014).

Summary We have demonstrated that CuO NPs were phytotoxic to bean when grown in a sand matrix. Growth inhibition was associated with increased shoot accumulations of Cu. However growth upon exposure to both ZnO and CuO NPs or root colonization by a bacterium alleviated the growth inhibition caused by CuO NPs. Our findings demonstrated changes in the levels of other plant-essential metals including Fe, Zn and Ca upon exposure to CuO NPs showing the interconnection between the metabolism of different metals. Zn accumulation increased as Cu accumulation decreased with co-treatments of ZnO and CuO NPs. Cu ions applied at the level of soluble Cu in the growth matrix did not elicit the responses of the CuO NPs. Altered activities of root metal reductases were observed with NP exposure and may contribute to altered nutrient levels and growth inhibition. We acknowledge that the studies described in this paper were conducted both in short-term and in sand. However, the results point to the likely possibility that in long-term field condition, NPs could have subtle consequences in plant nutrition beyond the toxicity effects that are discernible at the organismal

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level. Also, in soil, these outcomes could be different, dependent on the extent of soil chemical complexity. Acknowledgments This work was supported by the United States Department of Agriculture (USDA-CSREES Grant 2011-03581), the Utah Water Research Laboratory, and the Agricultural Experiment Station (AES) Utah State University, and approved as journal paper number 8634. We thank Trevor Hansen and Jacob Stewart for help with growing and measuring plants Conflict of interest

The authors declare no conflict of interest.

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Nano-CuO and interaction with nano-ZnO or soil bacterium provide evidence for the interference of nanoparticles in metal nutrition of plants.

The expansion of nanotechnology raises concerns about the consequences of nanomaterials in plants. Here, the effects of nanoparticles (NPs; 100-500 mg...
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