Photochemical Modulation of Biosafe Manganese Nanoparticles on

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Photochemical Modulation of Biosafe Manganese Nanoparticles on Vigna radiata: A Detailed Molecular, Biochemical, and Biophysical Study Saheli Pradhan,*,† Prasun Patra,† Sumistha Das,† Sourov Chandra,† Shouvik Mitra,† Kushal Kumar Dey,† Shirin Akbar,† Pratip Palit,‡ and Arunava Goswami† †

Biological Sciences Division, Indian Statistical Institute, 203 B.T. Road, Kolkata 700108, India Plant Physiology Section, Central Research Institute for Jute and Allied Fibres, Indian Council of Agricultural Research, Barrackpore, Kolkata 700 120, India

‡

S Supporting Information *

ABSTRACT: Manganese (Mn) is an essential element for plants which intervenes mainly in photosynthesis. In this study we establish that manganese nanoparticles (MnNP) work as a better micronutrient than commercially available manganese salt, MnSO4 (MS) at recommended doses on leguminous plant mung bean (Vigna radiata) under laboratory condition. At higher doses it does not impart toxicity to the plant unlike MS. MnNP-treated chloroplasts show greater photophosphorylation, oxygen evolution with respect to control and MS-treated chloroplasts as determined by biophysical and biochemical techniques. Water splitting by an oxygen evolving complex is enhanced by MnNP in isolated chloroplast as confirmed by polarographic and spectroscopic techniques. Enhanced activity of the CP43 protein of a photosystem II (PS II) Mn4Ca complex influenced better phosphorylation in the electron transport chain in the case of MnNP-treated chloroplast, which is evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and corresponding Western blot analysis. To the best of our knowledge this is the first report to augment photosynthesis using MnNP and its detailed correlation with different molecular, biochemical and biophysical parameters of photosynthetic pathways. At effective dosage, MnNP is found to be biosafe both in plant and animal model systems. Therefore MnNP would be a novel potential nanomodulator of photochemistry in the agricultural sector.

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INTRODUCTION

seed’s vigor and chlorophyll biosynthesis of spinach, particularly rubisco activity and photosynthetic efficiency.8 Racuciu and Creanga have addressed the influence of tetramethylammonium hydroxide-coated magnetic nanoparticles on the growth of Zea mays plant in early ontogenic stages.9 The chemical as well as magnetic influence of these nanoparticles can affect the enzymatic structures involved in the different stages of photosynthesis. While Dimpka et al. reported that metal ions released from NP cause inhibition of wheat plant growth, uptake of these ions and their translocation in plants create

Manganese (Mn) is an essential micronutrient for growth regulation and development of plants. It plays a pivotal role in oxygenic photosynthesis both directly and indirectly; and is considered to be an integral component of the catalytic center that carries out water oxidation at PS II.1 It also shuttles electrons to the thylakoid bound electron transport chain (ETC) which generates reducing power and ATP for carbon dioxide assimilation.2 However the major drawbacks associated with Mn deficiency are plant nutritional disorders.3,4 To circumvent this nutritional disorder of plants, nanoparticlemediated crop management has of late found potential applications.5,6 Hong et al. has shown that nano-titanium dioxide (TiO2) can promote photosynthesis and simultaneously improve spinach growth.7 It can also promote aged © 2013 American Chemical Society

Received: Revised: Accepted: Published: 13122

June 18, 2013 September 18, 2013 October 21, 2013 October 21, 2013 dx.doi.org/10.1021/es402659t | Environ. Sci. Technol. 2013, 47, 13122−13131

Environmental Science & Technology

Article

both particle-specific and ion release related challenges.10−12 Therefore the nanobiotechnological platform aids in the study of the nanoparticles−plant interaction and promises to deliver beneficial response in floral systems.13−16 Herein we proposed manganese nanoparticles (MnNP) as a suitable alternative to commercially used manganese salts MnSO4 (MS) for nanobiotechnology based crop management studies. MnNP is a less explored nanomaterial; its interaction with the plant system and toxicity study is yet to be analyzed. We demonstrated the photosynthetic efficacy of stable MnNP on leguminous plant:mung bean (Vigna radiata) and compared it with commercially available bulk counterpart MS salts physiologically as well as biochemically. To outline the reason behind high photosynthetic efficacy, in vitro plant−MnNP interaction was studied by the entire ETC of photosynthesis using isolated chloroplasts from mung bean and compared with MS treatments as well. Microscopic analysis confirmed that MnNP did not alter the structural organization of chloroplasts or anatomy of plant tissues. Bioavailability and uptake of MnNP was established by inductively coupled plasma optical emission spectroscopy (ICP-OES) and fluorescence microscopic studies. In continuation, the underlying reason behind high photosynthetic efficacy had been correlated with gel electrophoresis, Western blot, and spectroscopic analysis. Reactive oxygen species (ROS) is well-known to alter cellular and molecular network in animal and plant cells.17,18 Therefore biosafety studies were performed on a plant model as well as a murine model system for toxicological evaluation. Our results indicate the promising aspect of MnNP against commercially available bulk counterpart Mn-salts for the development of a nanobiotechnology-based platform for crop management and will enhance the study of nanoparticle−plant interaction in near future.

shoot length, fresh and dry weight of plant, and rootlet numbers per plant. Dry weight was recorded by drying the plants at 80 °C for 24 h. Estimation of Photosynthetic Pigment Content. Chlorophyll Content, Carotenoid Content, Chloroplast Isolation. Chlorophyll content was estimated spectrophotometrically at 645 nm and 663 nm according to Arnon’s Formula.19 Carotenoid was estimated at 425 nm and 450 nm according to the method of Davies with little modification.20 Chloroplast was isolated by standard method;21 leaves were ground in mannitol-isolation buffer, centrifuged to isolate chloroplasts and stored for maximum of 7days. Details were described in Supporting Information. Estimation of Fluorescence of Chloroplast by Photoluminescence (PL) Spectra. The chloroplast suspension contained 100 μM potassium phosphate buffer (pH7.8), 2 μM NaCN, 1.25 mM sucrose. The fluorescence spectra of the purified control and treated chloroplasts were recorded at 273 K under 440 nm excitation wavelength (Perkin-Elmer LS55). Photoreduction Activities. Whole Chain Electron Transport, Oxygen Evolution Measurement, Hill reaction, Photophosrylation, ATP Synthesis Measurement. The electron transport through the whole chain of photosynthesis, that is, from water to methyl viologen (MV) (oxygen uptake) was measured polarographically with an Oxygraph oxygen electrode (Hansatech Instruments, UK).22 Oxygen evolution was assayed in a medium containing chloroplasts equivalent to 378 μg/mL of chlorophyll.23 Hill activity was assayed with 2,6-dichlorophenol indophenol according to the method of Vishniac.24 Ferricyanide and NADP reduction were determined by the spectrophotometric methods of Trebst with different electron acceptors, like ferricyanide, ferredoxin, NADP.25 Light-induced ATP content of chloroplasts was measured by comparing the ATP level in the dark and 1 min illumination.26 Detailed protocols were described in the Supporting Information. Electron Spin Resonance Study. Chloroplast samples were isolated following the procedure as mentioned above. Isolated chloroplast was then suspended in sucrose buffer followed by the addition of MnNP of a concentration of 0.05 mg/L under steady state illumination.27 The samples were then transferred to an EPR tube, frozen at liquid nitrogen temperature (77 K), and then subjected to EPR measurement. SDS PAGE and Western Blot of Isolated CP43 Protein. CP43 was purified from mung bean chloroplast as described elsewhere.28 CP43 was dissolved in a buffer containing 0.09% (w/v) β-DM (n-decyl-β-D-maltopyranoside), 20 mM NaCl, 70% (v/v) glycerol, and 20 mM HEPES (pH 7.5). After isolation of CP43, 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was performed using Biorad protein gel apparatus, and a subsequent Western blot analysis was also carried out by using the CP43 antibody (Agrisera). Uptake of MnNP by Fluorescence Microscopy Study. MnNP was converted to its fluorescent counterpart by amine functionalization followed by FITC (fluorescein-B-isothiocyanate)-conjugation (detailed in Supporting Information) and used for uptake study for 6 h in the dark. An ultrathin cross section of treated plant samples was fixed in 2% glutaraldehyde followed by washing in graded ethanol and observed under confocal microscope. In contrast, a similar procedure was repeated for the control samples using MnNP instead of MnNP-FITC. Electron Micrographic Study. TEM and FESEM of the treated and untreated chloroplasts were performed to under-

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EXPERIMENTAL SECTION Physicochemical Characterization of MnNP. Custommade MnNPs were purchased from MK Implex, Canada. Physico-chemical characterizations of MnNP were carried out by field emission scanning electron microscope (FESEM), transmission electron microscope (TEM), powder X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-ray analysis (EDX), thermogravimetric analysis (TGA), dynamic light scattering (DLS), zeta potential, inductively coupled plasma optical emission spectroscopy (ICP-OES), electron paramagnetic resonance spectroscopy (EPR), and confocal and light microscopy. Other experimental details are described in the Supporting Information (SI). Plant Material and Growth Conditions. Seeds of mung bean (Vigna radiate var. Sonali) were purchased from Berhampur Pulse and Oil Research Centre, West Bengal. Seeds (20seeds/replicate; total of three replicates were taken for analysis) were rinsed with deionized double distilled water and soaked in 5% sodium hypochloride solution for surface sterilization for 20 min. After that seeds were thoroughly washed with deionized double distilled water and imbibed with the treatment solutions (control; MnNP at 0.05 mg/L, 0.1 mg/ L, 0.5 mg/L, 1 mg/L; MS at 0.05 mg/L, 0.1 mg/L, 0.5 mg/L, 1 mg/L) in dark for minimum 4−6 h, prior to the germination. Detailed growth conditions of plants were described in Supporting Information. Morphological Parameters. The effect of MnNP versus MS on growth parameters were studied in terms of root and 13123

dx.doi.org/10.1021/es402659t | Environ. Sci. Technol. 2013, 47, 13122−13131


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Figure 1. (a) FESEM image, (b) TEM image, (c) XRD pattern, and (d) FTIR spectra of MnNP.

Malik and Singh.36 Detailed procedures of all these assays were described in the Supporting Information. Estimation of Total Amino Acid (AA), Lipid, Protein and Carbohydrate Content. AA content in plant sample was estimated according to the ninhydrin method of Lee and Takahashi with some modifications.37 Protein content of the leaves and roots of treated plants were estimated according to Lowry’s method.38 Lipid content was estimated according to Bligh and Dyer’s protocol.39 Total carbohydrate content was estimated following Anthrone method.40 Biosafety Study in Mice Model. The Biosafety study was carried out using young female nulliparous, nonpregnant mice weighing 20−22 g each, following OECD guideline;41,42 details in Supporting Information.

stand the interaction between chloroplast and MnNP in photosynthesis. Chloroplasts were fixed with glutaraldehyde, stained with osmium tetraoxide, and washed in graded ethanol to observe under electron microscope. Details were described in Supporting Information. Mn2+ Release Study by ICP-OES. Mn release from MnNP was monitored by using inductively coupled plasma optical emission spectroscopy (ICP-OES) at pH 7, that is, the pH of plant growth medium. A 100 mL aliquot of the MnNP dispersion of the aforementioned concentrations in milli-Q water was allowed to stir for 24 h. The resultant dispersion was centrifuged and subjected to digestion in the presence of ultrapure nitric acid followed by estimation against a standard (details in Supporting Information). Enzyme Assays Related to Oxidative Stress. Plant tissues were homogenized in Tris-HCl buffer (pH 7.5) containing EDTA and polyvinylpyrrolidone followed by centrifugation at 0 °C. The supernatant was used for enzymatic assays. Super oxide dismutase (SOD) activity was estimated following the standard procedure.29 The enzymatic activity of peroxidase (POD) was assayed according to Chance and Maehly.30 The total peroxide content of plant tissue was estimated according to the method of Thurman et al.31 Glutathione reductase (GR) was assayed according to Foyer and Halliwell.32 Catalase (CAT) activity was assayed according to Beers and Sizer.33 Activity of polyphenol oxidase (PPO) was assayed according to the standard methods.34 Estimation of proline was done according to the method of Bates et al.35 Phenol content was estimated according to the method of

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RESULTS AND DISCUSSION Physicochemical Characterization of MnNP. The morphology of MnNP was determined using FESEM which showed its distinct square morphology (Figure 1a), and the squares were distributed homogeneously on the surface of glass slide. A transmission electron microscopic (TEM) image result justified the same, the square-shaped MnNPs were nearly 20 nm in size (Figure 1b) and selected area electron diffraction (SAED) pattern (Figure 1b inset) indicated its crystalline nature. Meanwhile the XRD pattern confirmed its crystalline structure; seven distinct peaks were obtained with 2θ = 35.16°, 40.76°, 42.98°, 45.33°, 47.80°, 52.36°, and 58.89° indexing (211), (220), (221), (310), (311), (320), and (410) diffraction planes of Mn (JCPDS card no. 32-0887) (Figure 1c) with a β 13124



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Table 1. Effect of MnNP and MS on root and shoot length, fresh and dry weight and rootlet numbers of 15 days treated mung bean plantsa treatment root length (cm) MnNP MS shoot length (cm) MnNP MS fresh weight (g) MnNP MS dry weight (g) MnNP MS rootlet no. MnNP MS

control

0.05 mg/L

0.1 mg/L

3.98 ± 0.477 Aa 3.98 ± 0.477 Aa

6.06 ± 0.447 Ba 5.96 ± 0.014 Ba

5.95 ± 0.496 Ba 5.21 ± 0.267 Cb

5.65 + 0.288 Ca 4.58 ± 0.356 Db

5.48 ± 0.349 Da 3.44 ± 0.288 Eb

5.6 ± 0.294 Aa 5.6 ± 0.294 Aa

7.74 ± 0.702 Aa 7.03 ± 0.418 Ab

7.63 ± 0.599 Aa 6.7 ± 0.141 Bb

6.98 ± 0.446 Ba 6.28 ± 0.192 Cb

6.51 ± 0.203 Ca 5.82 ± 0.192 Db

0.29 ± 0.004 Aa 0.29 ± 0.004 Aa

0.40 ± 0.008 Ba 0.37 ± 0.006 Bb

0.37 ± 0.001 Ca 0.35 ± 0.011 Cb

0.34 ± 0.007 Da 0.32 ± 0.006 Db

0.33 ± 0.0121 Ea 0.30 ± 0.005 Eb

0.02 ± 0.004 Aa 0.02 ± 0.004 Aa

0.04 ± 0.008 Aa 0.02 ± 0.002 Ab

0.03 ± 0.003 Ba 0.02 ± 0.0001 Bb

0.02 ± 0.003 Ba 0.02 ± 0.0001 Cb

0.03 ± 0.002 Ca 0.02 ± 0.0001 Db

13.22 ± 0.006 Aa 13.22 ± 0.006 Aa

22.62 ± 1.40 Ba 17.62 ± 2.87 Bb

18.81 ± 1.81 Ca 15.75 ± 0.886 Cb

0.5 mg/L

18.5 ± 1.87 Da 12 ± 2 Cb

1 mg/L

15.6 ± 1.34 Da 9.67 ± 1.033 Db

Data represents mean ± standard errors (no. of samples = 25). Capital letter changes along the row owing to significance of respective pairwise treatments, while the small letter changes along the column.

a

Figure 2. (a) Effect of MnNP and MS on chlorophyll contents (chlorophyll a and chlorophyll b) of 15 days treated mung bean plants. Chlorophyll a, F = 580.86, P < 0.00001; chlorophyll b, F = 633.13, P < 0.00001. (b) Effect of MnNP and MS on carotenoid content of 15 days treated mung bean plants. Data represent mean ± SE (n, no. of samples = 3). Carotene, F = 580.86, P < 0.00001, xanthophyll, F = 52.78, P < 0.00001. Within each type of treatment mean pigment content (±SE, n = 3) followed by the same upper case letter is not significantly different for a particular dose, within each dose mean pigment content followed by the same lower case letter is not significantly different; Tukey-Kramer HSD test.

were ionized to −CO2− and −O− to give negative zeta potential. Prior to application we checked the stability of MnNP with the aid of their hydrodynamic radius measurements which were found to be around 100 nm (Supporting Information Figure S2). The hydrodynamic radius justifies that MnNP produced a stable dispersion, and the size of the dispersed particles remains well within the nanosize range. MnNP was thermally stable up to 55 °C; only 6.28% weight loss was observed due to the loss of adsorbed water molecules on its surface (Supporting Information Figure S3). But at higher temperature a sharp weight loss was noted due to higher reactivity and oxophilicity of Mn as mentioned in the literature.43 However the stability of MnNP up to 55 °C signified that it could be used under laboratory conditions or field applications for crop management studies. Effects of MnNP in Plant Growth Parameters. MS was used as the conventional source of manganese at a recommended dose of 0.05 mg/L for increased crop yield and productivity.44 At and above the concentration of 0.5 mg/ L, MS is known to exhibit a toxic effect on plant morphology

crystalline structure. Additional peaks for the impurities were avoided signifying its chemical purity; even no evidence of corresponding oxides was detected. The chemical purity of MnNP was further supported by EDX analysis shown in the Supporting Information Figure S1 in which Mn was present as the main chemical constituents of these nanoparticles. The surface functionality of MnNP was confirmed by the FTIR spectrum shown in Figure 1d. The surface of the MnNP was covered with hydroxyl groups which were signified by O−H stretching at 3400 cm−1.41 C−H asymmetric and symmetric stretching was noted at 2925 and 2851 cm−1, while the carboxyl group and CO stretching was observed at 1737 cm−1 and 1623 cm−1, respectively.41 O−H bending and C−O stretching was assigned to 1395 cm−1 and 1091 cm−1. The absence of characteristic stretching at 948 cm−1 in FTIR spectra indicated the absence of manganese oxide in the powdered sample; meanwhile the lower band observed at around 620 cm−1 was assigned to the Mn−O counterpart of MnNP. A small but negative zeta potential of −4.3 mV at pH 7 corroborated its surface functionality as well. Carboxyl and hydroxyl groups 13125



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Figure 3. Polarographic and biochemical changes in photosynthetic pathway in MnNP- and MS-treated chloroplasts. (a) Polarographic changes in whole chain electron transport; F = 100.46, P < 0.00001; (b) polarographic changes in oxygen evolution; F = 258.32, P < 0.00001; (c) Hill reaction in treated chloroplasts; F = 78.82, P < 0.00001; (d) ferricyanide reduction; F = 126.11, P < 0.00001; (e) NADP reduction; F = 56.69, P < 0.00001; (f) activity of ATPase; F = 39.84, P < 0.00001. Within each type of treatment, mean data (±SE, n = 3) followed by the same upper case letter is not significantly different for a particular dose, within each dose, mean followed by the same lower case letter is not significantly different; Tukey-Kramer HSD test.

higher concentration; even all the plants were healthy. Meanwhile plants dosed at 0.5 mg/L or above of MS showed severe toxicity symptoms like necrotic leaves, brown roots, and gradual disappearance of the rootlet after 15 days of treatment (Supporting Information Figure S4). Chlorophyll a molecules are the chemically active pigment at the reaction center, that is, they are used to take part in the photochemical reaction. Figure 2a indicates increase in both chlorophyll a and b content in MnNP and MS treatments. Chlorophyll b was found to be

and physiology. As shown in Table 1, 0.05 mg/L concentration of MnNP was found to be the most effective among all the dosages of MnNP as well as MS treatments. At 0.05 mg/L dose MnNP significantly increased root and shoot length of mung bean plants by 52.26% and 38.29%, respectively, with respect to control. Fresh and dry weight of MnNP-treated plant at 0.05 mg/L concentration was also increased by 38.97% and 53.6%, respectively, with respect to control. MnNP-treated plants did not exhibit any toxicity symptoms neither in leaf nor in root at 13126



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ferredoxin can be used to monitor the entire ETC. In all the cases reductions of electron acceptors signify more efficient electron transport from one photosystem to another.48 Ferricyanide reduction assay with control and treated mung bean chloroplasts confirmed better performance of MnNP over MS from the oxygen evolving complex (OEC) to PS II (Figure 3d). Highest efficacy was observed at 0.05 mg/L of MnNP concentration, and reduction was decreased gradually at higher concentrations of MnNP. NADP along with ferricyanide and ferridoxin was further used to monitor the MnNP mode of action in the reaction center of PS I. A similar gesture was noted in each case (Figure 3e) which indicated better activity of MnNP in ETC during cyclic and/or noncyclic photophosphorylation in contrast to the MS. In addition to oxygen and NADPH, the noncyclic ETC is coupled to the synthesis of ATP. ATP production is another significant process which was utilized in carbon assimilation by “dark biochemistry” in photosynthesis. Therefore production of more ATP signifies more photophosphorylation activity of ETC.49 A similar trend was observed which indicated the probable modulation of MnNP in noncyclic photosynthetic activity (Figure 3f). In all the experiments, 0.05 mg/L and 0.1 mg/L of MS concentrations were found to be more effective with respect to control, but MnNP worked better than the MS in all their respective dosages. At the higher doses of MS (0.5 mg/L and 1 mg/L) photosynthetic activities were decreased resulting in toxicity to photosynthetic machinery. Hence, MnNP might amplify photosynthetic capacity by transferring it to the reaction center of photosystems, thereby increasing the oxygen evolution in chloroplasts of mung bean. Morphological Analysis of MnNPs-Treated Plants. Structural alteration of chloroplast was checked in in vitro experiments by using FESEM and TEM. Interestingly FESEM (Supporting Information, Figure S6a) and TEM (Supporting Information Figure S6b) image showed deposition of MnNP on the surface of treated chloroplasts. Chloroplasts are particularly sensitive to oxidative stress and the presence of MnNP might dissemble the thylakoid.50 But the treated chloroplast was round in shape, and no ultrastructural abnormalities were observed. The presence of MnNP on the isolated chloroplast proved that MnNP influenced the functionality of the chloroplast and it did not disrupt the structural integrity of the system. Especially, the high resolution TEM image (Supporting Information Figure S6c) of the treated chloroplast illustrated the distinct deposition of MnNP on its surface which in fact played the pivotal role for such an overwhelming response. Meanwhile control chloroplasts were spherical without the presence of particles as revealed from low and high resolution TEM images (Supporting Information, Figure S7). As conclusive evidence we carried out EDX analysis associated with TEM (Supporting Information, Figure S8) and FESEM (Supporting Information, Figure S9) which also revealed that Mn content was present in the treated samples but absent in the control chloroplast. Control samples did not exhibit the presence of MnNP (characteristic Mn peak was absent), while the treated samples justified the presence of MnNP showing Mn content. It was well documented that manganese deficiency could affect PS II first, but excess manganese was also detrimental to the system.51 The light microscopic studies of sections of MnNP-treated mung bean leaves showed no anatomical changes (Supporting Information, Figure S10a and S10b). Mesophyll tissues along with palisade and spongy parenchyma

decreased in the control and at the higher doses of MS treatments. Carotenoids are known as accessory light harvesting pigments absorbing and transferring light energy to chlorophyll molecules. Carotenoids also play an important structural role in the assembly of the light harvesting complex and has an indispensible function in protecting the photosynthetic apparatus from photo-oxidative damage.45 Enhanced carotenoid content after MnNP treatment (Figure 2b) would not only improve the activity of ETC but also provided protection from photo-oxidative damage. MnNP Enhanced Activity of ETC in Photosynthesis. Metal nanoparticle (e.g., gold and silver nanoparticle) mediated photosynthesis enhancement due to chemical energy transfer was already established.48 We took the charge to assess the PL property of a light harvesting system during interaction with MnNP. PL properties of untreated and treated chloroplasts were measured at 440 nm excitation wavelength;46 interestingly quenching was noted for all of the treated samples with maximum quenching noted at 0.05 mg/L MnNP treatment (Supporting Information, Figure S5). This quenching was due to energy transfer from the light harvesting system to metal nanoparticles resulting in a decrease in the quantum yield of the corresponding light harvesting system. When a metal nanoparticle interacts with the light harvesting system, two competing counterparts control the photosynthetic efficiency; the first one is the plasmon enhancement of chlorophyll molecules yielding high photosynthetic efficiency, and the second one is the energy transfer from chlorophyll to metal nanoparticles.47 The latter one had been established by PL measurements, and we had a hunch that the former could enhance photosynthetic activity by modulating the electrochemical pathway of the photosynthesis. Therefore we carried out a series of biochemical reactions to follow the photosynthetic efficiency. Oxygen is the byproduct of photosynthesis during water oxidation in light reaction. The entire ETC of treated chloroplast in photosynthesis was monitored polarographically in the presence of an electron acceptor MV. Oxygen uptake in ETC was maximized at 0.05 mg/L MnNP concentration (Figure 3a), which was decreased gradually, but each concentration of MnNP treatment executed improved uptake with respect to the control as well as MS. Identical conditions were used for both treated and control sets to study oxygen evolution in the presence of dichlorophenol indophenol (DCPIP) as oxidant (Figure 3b). In the Hill reaction (Figure 3c), both Mn-salt and MnNP signified an elevated oxygen evolution rate, interestingly MnNP showed better results in contrast to its bulk salt counterpart. Meanwhile maximum efficacy was found to be at 0.05 mg/L MnNP concentration. This result corroborated the polarographic study that used the oxygen electrode in which we found maximum oxygen evolution at the aforementioned MnNP concentration. This MnNP concentration was decreased later on in a dosedependent manner and it confirmed the influence of MnNP in the photosynthetic ETC. During electron transfer to the reaction centers of PS II and PS I, electrons are raised to a higher energy level thereby generating holes which can accept electrons from water. In PS II, electrons are sprinted downhill through a series of electron carriers to PS I. Incoming photons boost the electrons on their way to primary acceptors of PS I, NADP, and eventually to CO2. Manganese is known to modulate activity of PS II; several artificial electron acceptors like ferricyanide, NADP, and 13127



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Figure 4. Confocal microscopic images showing uptake of MnNP (a) in root and (b) in leaf (scale bar 10 μm).

Figure 5. (a) SDS-PAGE analysis of isolated CP43 protein from control and treated chloroplasts, (b) Western blot images of isolated CP43 protein of control and treated chloroplasts.

tissues were normal in shape and sizes, stomata were normal in position and were not even shrunken. There were no abnormalities found in leaf vascular bundles as well. MnNPtreated root sections (Supporting Information, Figure S11a and S11b) showed normal anatomical structures with no abnormalities in epidermal, cortical tissues or vascular bundles. Uptake of MnNP by Plant. To address the bioavailability issue of MnNP by plants we deliberately functionalized MnNP by FITC by a two-step synthetic approach (detailed in the Supporting Information). FITC coupled MnNP exhibited a strong PL property at 488 nm excitation wavelength shown in Supporting Information, Figure S12; these fluorescent MnNP dispersions were then used for the uptake study. When root (Figure 4a) and leaf (Figure 4b) cross section were observed under a confocal microscope at 488 nm excitation, very distinct green coloration was observed in individual root and leaf sections. Even cortical and stellar structure in the root and stomata and mesophyll tissues in leaves were distinctly visible since MnNP was translocated through the xylem from roots to shoots. Meanwhile no green fluorescent coloration was noted either in the root or in the leaf of the control samples (Supporting Information, Figure S13). This phenomenon confirmed the uptake and translocation of FITC coupled MnNP in the root and leaf sample. Elemental mapping associated with EDX analysis was used to verify the biodistribution of nanoparticles 43,52 which revealed the presence of Mn content in treated plant leaf samples during uptake study; while corresponding mapping analysis showed the distribution of MnNP on leaf (Supporting Information, Figure S14a and S14b). On the contrary no trace of Mn was detected in the control leaf samples (Supporting Information,

Figure S15). This bioavailabity of Mn on plant samples corroborated the electron microscopic experiments mentioned earlier. Mn Release from MnNPs. Mn released from MnNP was monitored by ICP-OES at pH 7, which was the pH of the MnNP dispersion sprayed on the plant samples and also the pH of the plant growth medium. We found that very small amounts of 0.001 ppm, 0.0056 ppm, 0.0061 ppm, 0.008 ppm, and 0.0098 ppm Mn were released after 24 h of the experiment from five different concentrations of MnNP as expected. This released Mn together with MnNP was carried through to the leaf of the plant samples which, in contrast to the control samples, interacted with chloroplast and augmented photosynthesis by higher oxygen splitting and generating a large number of electrons. Biomass distribution of MnNP on plant samples was also studied using ICP-OES. The 0.05 mg/L MnNPtreated plant samples showed a very small enhancement in Mn content in the leaf with respect to control (Supporting Information, Table 1) which was expected at low concentrations of the nanoparticle. Mechanistic Interpretation. We carried out EPR analysis to verify the stoichiometric binding of Mn2+ with thylakoid membrane of chloroplast. It was well documented that a Mn4Ca complex resides within the thylakoid membrane of PS II. A nearly Mn2+ content of 4.0/400 chloroplast is responsible for higher oxygen evolution in PS II; somewhat of an excess stoichiometric Mn2+ content (4.6/400 chloroplast) of that aforementioned value can produce EPR detectable six line spectra, otherwise a single line EPR spectra is expected.27 Interestingly the control chloroplast exhibited a single line EPR signal as expected27 at around g = 1.99 value (Supporting 13128



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conditions.56 There were no significant changes in POD (Supporting Information, Figure S17a), SOD (Figure S17b); CAT (Figure S17c) and GR (Figure 17d) or proteins like proline (Figure S18a), total peroxide (Figure S18b), polyphenol oxidase (Figure S18c) and phenol (Figure S18d) after MnNP treatment. Comprehensive analyses of these enzymatic assays were detailed in the Supporting Information. Biosafety Study. Nanoparticles harbor their specific properties not only by their chemical nature, but also with their small size. Triggered by a few reports on the phytotoxicity of some nanoparticles,57,58 considerable attention on the adverse effects of nanoparticles on the plant system have been raised. When plants are exposed to nanoparticles treatment it is essential to evaluate the status of the basic biochemical component of the plant system. There were no significant changes found in protein (Supporting Information, Figure S19), lipid (Figure S20) contents in both treated leaf and root, though sugar content was high in treated leaves (Figure S21), and AA level was little high in treated roots (Figure S22). Therefore MnNP underscored its biosafety issue in the plant model; detailed discussions were summarized in the Supporting Information. MnNPs were biosafe toward beneficial soil microorganism (Trichoderma viride); when treated with MnNP of different concentrations, the microbial system did not exhibit growth retardation (Supporting Information, Figure S23). In acute oral toxicity tests in the mice model, no death was recorded in the 15 days of observation period in all control and treated mice. The animals did not show any significant changes in the general appearance or the percentage of weight gain between the control and treatment groups of both male and female mice during the observation period. No significant changes in blood biochemical parameters (Supporting Information, Table 2) and histopathology (Supporting Information, Figure S24) was recorded after 15 days observation which justified its biocompatibility (detailed in Supporting Information). In summary, stable square shaped MnNPs were used as a suitable alternative of commercially available bulk MS salts for crop management. These MnNP promoted plant growth as well as augmented photosynthesis; at higher concentration the MnNP did not exhibit any toxicity, but commercially available manganese salt did even at a lower concentration. Interestingly MnNP was found to be relatively nontoxic even at a higher concentration while MS underscored the toxic effect at a much lower concentration than MnNP treatment. The small size of MnNP probably helped plants to uptake these particles more readily and they were translocated well in the leaves via xylem, which was confirmed by confocal microscopy. The MnNP was the star player in the reaction center of PS II where water molecules were converted to oxygen. The MnNP also transferred electrons to the thylakoid-bound ETC which generated reducing power and ATP for carbon dioxide assimilation. A series of biochemical reactions in the presence of an artificial electron acceptor justified that MnNP enhanced the total ETC of photosynthesis in contrast to control; even its efficacy was higher in contrast to commercially available MS. Bioavailability study by ICP-OES revealed a very small amount of Mn release from MnNP while availability on plant system was also established. Finally SDS-PAGE, Western blot, and EPR analysis revealed the reason behind such augmentation of photosynthesis which corroborated biochemical measurements. Moreover MnNP maintained a balanced antioxidative-ROS network in the plant system. An acute oral toxicity study of

Information, Figure S16a). Even in our treated samples with MnNP a more or less similar EPR spectral pattern was noted at that aforementioned g value (Supporting Information, Figure S16b). Usually Mn3+ is considered to be EPR silent with large zero-field splitting, while Mn2+ and Mn4+ both are EPR active and a significant amount can produce a sextet EPR spectral pattern.53 Therefore we speculated that during MnNP treatment the stoichiometric binding of available Mn2+:chloroplast was well within the limit of 4.0/400 which was sufficient to result in higher oxygen evolution, and a single line EPR spectrum was obtained.27 MnNP was stable and a very small amount of Mn release was noted from the ICP-OES results; therefore, the availability of Mn2+ toward the chloroplast was small which also corroborated EPR studies and contributed to higher oxygen splitting in PS II as observed from biochemical parameters. PS II is organized within the stacked grana regions of the thylakoid membranes as a dimer which is composed of two copies of reaction center core proteins and the core antenna chlorophyll binding proteins CP43 and CP47. The D subunit is supposed to play the pivotal role during higher electron transport which is subsequently coupled with protein chain CP43 and CP47. CP43 is known to play the significant role with manganese stabilizing protein (MSP), which is responsible for water oxidation, oxygen evolution, and also energy transfer from a light harvesting complex to the reaction center core. CP43 protein has two forms or protein domain that actively helps in the process of photophosphorylation.54 Our SDSPAGE analysis (Figure 5a) and Western blot analysis (Figure 5b) justified that the activity of CP43 protein chain was enhanced during MnNP treatment which in turn modulated the activity of the D subunit contributing toward higher electron transport. The isolated and purified CP43 from MnNP-treated chloroplasts showed an intense band of CP43 with respect to the control in SDS-PAGE gel and was also reflected in the Western blot analysis. Therefore as a whole EPR analysis signified higher oxygen evolution through water splitting, while the excess electron generated was cycled to PSII by the D subunit whose enhanced activity was interlinked with enhanced activity of the CP43 protein domain. The entire process was summarized in the Supporting Information, schematic representation 1 . Furthermore when MnNP-treated chloroplasts reduced more NADP during NADPH reduction assay, it confirmed the efficacious activity of MnNP in the process of the noncyclic photophosphorylation pathway. All together the mechanistic pathway elucidation revealed the reason behind augmentation of photosynthesis which corroborated with all other biochemical parameters. MnNP-Treated Plants Did Not Trigger Oxidative Stress. Manganese works in redox reactions as one of the important cofactors for many enzymes in a plant system; in particular, it is the metal component of superoxide dismutase. The toxic effects of manganese are due to the production of free radicals which are the side product of normal biological reactions, but their lifespan and diffusion into the cell space are closely controlled by the cell antioxidative system. To withstand such damages low molecular weight antioxidants and protective enzymes work simultaneously in plant cells.54 Superoxide radicals are scavenged by SOD while hydrogen peroxide (H2O2) is detoxified in the ascorbate glutathione cycle by GR and Fenton reaction (CAT, POD).55 However under stress conditions, formation of these radicals in excess amount triggers oxidative stress contrary to normal plant physiological 13129



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MnNP on a murine model system ensured that MnNPs were biosafe. This work highlighted a new direction in photochemistry, bringing a new idea that MnNP could be used as a plant micronutrient thereby enhancing growth and photosynthesis without disturbing ROS-antioxidative equilibrium. It has a promising future as a nanofertilizer in the agricultural sector for nanobiotechnology-based applications and in the study of nanoparticle plant interactions.

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ASSOCIATED CONTENT

S Supporting Information *

Figures, tables, and experimental details as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +91-332577-3227. Fax: +91-332577-3049. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was generously supported by four major grants from the Department of Biotechnology, Government of India (DBT) (Grant No: BT/PR9050/NNT/28/21/2007-2013, BT/PR8931/NNT/28/07/2007-2013, T/PR15217/NNT/28/ 506/2011-2014 and BT/BIPP0439/11/10/2011−2014). We are grateful to the NAIP (ICAR) Grant No. NAIP/Comp-4/ C3004/2008-2014), the National Fund (ICAR) (Grant No. NFBSFARA/GB-2019/2011−2015) and ISI plan project funds (2008−2013) for providing financial support. We acknowledge Mr. Ashim Dhar for his assistance during plant experiments and Mr. Sanjib Naskar (Tech. Supdt. CSS, IACS) for his help during EPR spectroscopy. The author acknowledges SGS India Pvt. Ltd. (Kolkata) for providing the ICP-OES facility. S.M. is thankful to CSIR, New Delhi for SRF.

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REFERENCES

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Photochemical Modulation of Biosafe Manganese Nanoparticles on

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