Silver Nanoparticle-Mediated Enhancement in Growth and Antioxidant Status of Brassica juncea Priyadarshini Sharma, Deepesh Bhatt, M.G.H.Zaidi, P.

Pardha Saradhi, P.K.Khanna & Sandeep Arora

Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Appl Biochem Biotechnol DOI 10.1007/s12010-012-9759-8

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Appl Biochem Biotechnol DOI 10.1007/s12010-012-9759-8

Silver Nanoparticle-Mediated Enhancement in Growth and Antioxidant Status of Brassica juncea

Priyadarshini Sharma & Deepesh Bhatt & M. G. H. Zaidi & P. Pardha Saradhi & P. K. Khanna & Sandeep Arora

Received: 20 November 2011 / Accepted: 29 May 2012 # Springer Science+Business Media, LLC 2012

Abstract Metal nanoparticles can potentially be used as tools for engineering biological redox reactions. Present study underlines the effect of silver metal nanoparticles (at 0, 25, 50, 100, 200 and 400 ppm) on the growth and antioxidant status of 7-day-old Brassica juncea seedlings. Fresh weight, root and shoot length, and vigor index of seedlings is positively affected by silver nanoparticle treatment. It induced a 326 % increase in root length and 133 % increase in vigor index of the treated seedlings. Improved photosynthetic quantum efficiency and higher chlorophyll contents were recorded in leaves of treated seedlings, as compared to the control seedlings. Levels of malondialdehyde and hydrogen peroxide decreased in the treated seedlings. Nanoparticle treatment induced the activities of specific antioxidant enzymes, resulting in reduced reactive oxygen species levels. Decrease in proline content confirmed the improvement in antioxidant status of the treated seedlings. The observed stimulatory affects of silver nanoparticles are found to be dose dependent, with 50 ppm treatment being optimum for eliciting growth response. Present findings, for the first time indicate that silver nanoparticles promote the growth of B. juncea seedlings by modulating their antioxidant status. Keywords Silver nanoparticles . Growth . Antioxidant status . Redox potential . Quantum efficiency . Reactive oxygen species
P. Sharma : D. Bhatt : S. Arora (*) Department of Molecular Biology & Genetic Engineering, G B Pant University of Agriculture & Technology, Pantnagar, Uttarakhand, India e-mail: plantstress@gmail.com M. G. H. Zaidi Department of Chemistry, G B Pant University of Agriculture & Technology, Pantnagar, Uttarakhand, India P. P. Saradhi Department of Environmental Studies, University of Delhi, Delhi, India P. K. Khanna Defense Institute of Advance Technology, Pune, India

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Introduction Plant growth and development are controlled by internal regulators that respond to environmental conditions. In nature, plants seldom find optimal quantities of essential factors required for maximal growth and productivity. Therefore, most often, the physiologically normal type of plant is an exception in nature. Under sub-optimal environmental conditions, plants show perturbations in their biochemical and physiological processes. Redox reactions, involving electron exchange, are one of the most vulnerable physico-chemical processes affected by fluctuations in the external environment. Therefore, even under seemingly favorable environmental conditions, a plant continuously produces reactive oxygen species (ROS). Accumulation of these ROS is potentially harmful for the growth and development of plants. Thus, occurrence of oxidative stress, through the generation of free radicals, is an inevitable by-product of normal plant metabolism [1]. Generally, these ROS are continuously reduced and detoxified by an extensive antioxidant system. However, the detoxification process consumes essential cellular resources in terms of energy, carbon skeleton and loss of nitrogen as NH3. Therefore, any treatment that can help reduce the ROS load would prove beneficial in improving the overall growth and productivity. Wang et al. [2] have shown that plants produce natural mineralized nanoparticles, which are required for growth. Specific reports are now available on the bio-synthesis of nanoparticles by plants [3]. However, ectopic use of engineered nanoparticles is one of the most recent advances in the field of agricultural biotechnology. Studies indicate that treatment of plants with a mixture of nano SiO2 and TiO2 can enhance activities of specific enzymes and could be used for improving seed germination and seedling growth [4]. Tang and Cao [5] have suggested that nano-SiO2 treatment could be related to increased strength, resistance to disease and thus, increased yields in rice. Zheng et al. [6] have proposed a protective role for nano-anatase in spinach exposed to UV light stress. However, the mode of action for these nanoparticles has not yet been established. Metal nanoparticles, by virtue of having extremely large surface area to volume ratio and an ability to engineer electron exchange, can develop favorable interactions with various bio-molecules in a cell. Silver nanoparticles are among the most potential candidates for modulating the redox status of plants, because of their ability to support electron exchange with Fe2+ and Co3+ [7], the two elements that participate in several biological redox reactions. Silver nanoparticle clusters show efficient catalytic activity in redox reactions by acting as electron relay centers, behaving alternatively as an acceptor and donor of electrons [8]. An effective transfer of electrons is facilitated when the redox potential of the cluster is intermediate between the electron donor and electron acceptor system. In spite of the electron channelizing ability of silver nanoparticles, response of biological systems exposed to silver nanoparticles has been studied mainly with regard to toxicity, and little attention has been paid to the possibility that silver nanoparticles can possibly help improve plants redox status and growth. Therefore, the present work attempts to elucidate the role of silver nanoparticles in plant growth promotion under specified conditions.

Material and Methods Preparation of Silver Nanoparticles Silver nanoparticles were synthesized through chemical reduction of silver nitrate by trisodium citrate salt, as described by Sileikaite et al. [9]. The synthesized silver nanoparticles were characterized by transmission electron microscopy and UVvis spectroscopy.

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Plant Material and Treatment Seeds of Brassica juncea (var. pusa jaikisan), obtained from Indian Agriculture Research Institute, New Delhi (India), were germinated on MS medium [10] supplemented with silver nanoparticles at 0, 25, 50, 100, 200 or 400 ppm concentration. The seedlings were grown at 261 C temperature and relative humidity of 70 %. Illumination was provided by four 40-W florescent tubes having a photon flux density of approximately 16 Wm2 with a 16/8 h day/night cycle. Seven-day-old seedlings were used for the experiments. Growth Profile Measurement Percent seed germination was calculated by dividing the number of seeds germinated by the total number of seeds inoculated. Shoot fresh weight was recorded by weighing individual shoots. Shoot and root length of 7-day-old seedlings was recorded with the help of a standard meter-scale. Vigor index was calculated as: Vigor index root length shoot length %germination Chlorophyll contents were determined by the modified Arnon [11] method. Leaf disks of 7 mm diameter were soaked in acetone and DMSO (1:1 v/v) solution for 12 h. The Chla, Chlb and total chlorophyll concentrations (milligram per gram fw) in the leaf tissues were calculated according to the following equations: Chla 12:7 A663 2:63 A645 =wt:ing 1;000 Chlb 22:9 A645 4:48 A663 =wt:ing 1;000 Totalchlorophyll 20:2 A645 8:02 A663 =wt:ing 1; 000 Chlorophyll fluorescence in untreated and silver nanoparticle treated leaves was measured at room temperature (26 C) and ambient CO2 concentration. Measurements were done using an actinic light of 3,000 mol photons m2 s1, 4-s flashes, after dark adapting the leaves for 20 min. The fluorescence parameters evaluated were: F0, initial fluorescence; Fm, maximal fluorescence; and the Fv/Fm ratio, representing the maximum quantum efficiency of PS-II, where Fv is the variable fluorescence FmF0. Malondialdehyde Content The procedure of Heath and Packer [12] was followed for measuring malondialdehyde (MDA) content. Leaf material was homogenized and extracted in 10 ml of 0.25 % TBA (w/v) prepared in 10 % trichloroacetic acid (TCA). The homogenate was heated at 95 C for 30 min and centrifuged at 10,000g for 30 min. Absorbance of the supernatant was recorded at 532 and 600 nm. Absorbance at 600 nm was subtracted from the absorbance at 532 nm for non-specific absorbance. The concentration of MDA was calculated by using an extinction coefficient of 155 mM1 cm1. Hydrogen Peroxide Content Hydrogen peroxide was measured as per the protocol given by Alexieve et al. [13]. Leaf material was homogenized in 10 ml of 0.1 % (w/v) aqueous TCA and centrifuged at

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10,000g for 30 min at 4 C. 1 ml of supernatant, 1 ml of 0.1 M potassium phosphate buffer and 4 ml of 1 M KI reagent were mixed. The reaction was allowed to develop for 1 h in dark and the absorbance was measured at 390 nm. The amount of hydrogen peroxide (H2O2) was calculated using standard curve of H2O2. Free Proline Content Free proline was determined by the method of Bates and Waldren [14]. Leaf tissue extract homogenized in 3 % sulfosalicylic acid was mixed with equal quantities of glacial acetic acid and acid ninhydrin reagent and incubated for 1 h at 100 C. The reaction was terminated in an ice bath, followed by extraction of the colored chromophore in toluene. The absorbance of the chromophore was measured at 520 nm. Concentration of proline in the samples was computed from a standard curve of L-proline. Antioxidant Enzyme Assay Ascorbate Peroxidase (EC 1.11.1.11) Ascorbate peroxidase activity was determined as described by Joshi et al. [15] with minor modifications. Fresh leaf material was homogenized in 100 mM phosphate buffer (pH 7.0) and 0.1 mM EDTA and centrifuged at 12,000g for 30 min at 4 C. For enzyme assay, 60 l of supernatant was mixed with 1,438 l of assay buffer [50 mM phosphate buffer (pH 6.0), 0.1 M EDTA, 0.5 mM ascorbate] and 2 l of 0.5 M H2O2 was added to start the reaction. The decrease in absorbance was recorded and enzyme activity was calculated by using extinction coefficient 2.8 mM1 cm1 at 290 nm. Specific enzyme activity was expressed as enzyme units per milligram of protein. Guaiacol Peroxidase (EC 1.11.1.7) Guaiacol peroxidase (GPX) activity was determined as described by Joshi et al. [15]. Leaf material was homogenized in 3.0 ml of 100 mM phosphate buffer (pH 7) containing 0.1 mM EDTA and centrifuged at 12,000g for 30 min at 4 C. Two millilitre reaction mixture was prepared by adding 60 l of enzyme extract to 1,790 l of assay buffer [100 mM phosphate buffer (pH 7), 0.1 M EDTA, 5.0 mM Guaiacol, 15.0 mM H2O2]; guaiacol was added in the last to start the reaction. Increase in absorbance was recorded at 470 nm and enzyme activity was quantified using a molar extinction coefficient of 26.6 mM1 cm1. The enzyme specific activity was expressed as enzyme units per milligram protein. Catalase (EC 1.11.1.6) Catalase activity was measured according to Joshi et al. [15] with minor modifications. Leaf material was homogenized in 100 mM phosphate buffer (pH 7) containing 0.1 mM EDTA and centrifuged at 12,000g for 30 min at 4 C. 70 l of enzyme extract was added to 1,370 l of assay buffer (100 mM phosphate buffer pH 7.0, 0.1 M EDTA) and 60 l of 0.5 M H2O2 was added to start the reaction. Decrease in absorbance was recorded at 240 nm and enzyme activity was calculated by using the H2O2 molar extinction coefficient of 36 mM1 cm1 and the enzyme specific activity was expressed as enzyme units per milligram protein.

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Statistical Analysis All experiments were carried out three times, with two replicates each. One-way analysis of variance was carried out to determine significant differences (P 0.05) between the means. The experimental data are expressed as meanSE.

Results and Discussion Chemically synthesized silver nanoparticles were characterized by UVvisible spectrophotometry. Formation of silver nanoparticles was ascertained by profiling the absorption spectra of the synthesized particles from 190 to 1,100 nm. The absorption maximum (max) was recorded at 425 nm (Fig. 1a). The average size of synthesized nanoparticles, as deduced by transmission electron microscopy is 29 nm (Fig. 1b and c). Further the uptake of silver nanoparticles from the media was also confirmed through transmission electron microscopy of tissues from treated seedlings (Fig. 1d). The uptake of silver nanoparticles by B. juncea has also been shown earlier [16]; however, these reports did not explore the biological effects of such an uptake. Growth Profile A significant enhancement in growth of B. juncea seedlings was recorded at 25 and 50 ppm silver nanoparticle treatment. The growth profile was measured in terms of shoot fresh

Fig. 1 Characterization of Silver Nanoparticles: a UVVIS spectrum in aqueous phase, b morphology, c particle size distribution, d presence of silver nanoparticles in B. juncea

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weight, shoot and root length, and vigor index. Above 50 ppm the silver nanoparticle treatment proved detrimental for the growth of seedlings. A maximum of 22 % decline in percent germination was recorded at 400 ppm silver nanoparticle concentration (Fig. 2a). Stampoulis and Sinha [17] have also reported a decrease in seed germination at higher levels of nanoparticle exposure. Silver nanoparticles interfere with the activities of certain hydrolytic enzymes, required during germination process [18], resulting in a reduction of per cent seed germination [17]. Lin and Xing [19] have also highlighted that seed germination is inhibited at higher concentrations of nanoparticles. A 17 % increase in shoot fresh weight was recorded at 50 ppm silver nanoparticle treatment. Shoot and root length increased up to 200 ppm of silver nanoparticle concentration in the growth medium. Sharp increases of 15 and 25 % in shoot length and 167 and 277 % in root length were observed at 25 and 50 ppm, respectively (Fig. 2b). These increases could be mediated via plant growth regulators like cytokinins and gibberellins, which are involved in cell division and cell elongation, respectively [17]. Roots are the first target tissues which come in direct contact with silver nanoparticles in the growth medium by penetrating through seed coat and are responsible for the continuous uptake of mineral nutrients from the medium. Therefore, the effect of silver nanoparticle treatment is more prominent in roots as compared to shoots. A decrease in shoot and root length of seedlings treated with higher silver nanoparticle concentrations, indicates that the recorded phenomenon is dose dependent. Similar observations have also been reported for TiO2 nanoparticle-treated spinach seedlings [6]. Vigor index represents the combined effect of various external and internal growth regulating factors. An increase in vigor index of the treated seedlings was recorded up to 50 ppm silver nanoparticle treatment.

Fig. 2 Response of B. juncea seedlings grown in media supplemented without and with different concentrations of silver nanoparticles: a percent germination and vigor index; b root and shoot length and shoot fresh weight; c Chl-a, Chl-b, total chl and Fv/Fm; d malondialdehyde, H2O2 and proline content. Values represent meanSE

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Thereafter, the decline in percent germination offset the effect of increased shoot and root length on vigor index. Results indicate that the percent seed germination up to 50 ppm is similar to the control values, and after that it declines consistently. An increase in vigor index of the treated seedlings rules out the restricted availability of water as a possible cause for decrease in germination. At all the tested concentrations of silver nanoparticles, the vigor index of treated seedlings was significantly higher than the control seedlings. This indicates that silver nanoparticle treatment improves the overall growth profile of the treated seedlings. An increase in chlorophyll contents was observed at 25, 50 and 100 ppm silver nanoparticle treatment. Maximal increase of 40 % in chlorophyll-a and 25 % in total chlorophyll content was recorded at 100 ppm silver nanoparticle treatment. Maximum photosystem II quantum efficiency, as measured through Fv/Fm ratio, was recorded at 50 ppm silver nanoparticle treatment (Fig. 2c). Fv/Fm ratio determines whether or not the imposed treatment affects the photosystem II efficiency and is a measure of plants photosynthetic performance [20]. Higher quantum efficiency (Fv/Fm), as recorded in the silver nanoparticle-treated seedlings, indicates that more number of reaction centers are in an open state to carry out light reaction. Presence of higher number of open or oxidized electron acceptors in PS-II decreases the probability of generation of reactive radicals [20]. Thus, our observations signify a lower incidence of photo-oxidative damage in the seedlings treated with 25 and 50 ppm silver nanoparticles. Improved quantum efficiency positively correlates with higher chlorophyll contents in the leaves of treated seedlings. Zheng et al. [6] have also reported that nano-TiO2 could promote photosynthesis and improve spinach growth. These observations clearly indicate that silver nanoparticles improve the cellular electron exchange efficiency in the treated seedlings. An efficient electron exchange mechanism arrests electron leakage, reducing the formation of reactive oxygen species [21]. An improvement in PS-II electron transport also positively correlates with the recorded reduction in the levels of MDA and H2O2, in the treated seedlings (Fig. 2d). Antioxidant Status Levels of malondialdehyde and hydrogen peroxide declined significantly at 25 and 50 ppm silver nanoparticle treatment, as compared to the controls. Maximum decline of 28.4 % in

Fig. 3 Specific activity of antioxidant enzymes: guaiacol peroxidase, catalase and ascorbate peroxidase activity in B. juncea seedlings grown in media supplemented without and with different concentrations of silver nanoparticles. Values represent meanSE

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MDA and 64.41 % in H2O2 levels was recorded at 50 ppm silver nanoparticle treatment. Even at 400 ppm silver nanoparticle treatment, the levels of H2O2 and MDA were lower than the control (0 ppm) seedlings (Fig. 2d). Decrease in MDA, an index of ROS production, was significant at 25 and 50 ppm silver nanoparticle treatment, as compared to other concentrations (Fig. 2d). Further, the level of H2O2, one of the most damaging forms of reactive oxygen species, also decreased in the treated seedlings. This decrease in H2O2 is due to the induction of H2O2 metabolizing enzyme like GPX, as noted in our experiments. H2O2 can easily cross the biological membranes, because of its high pKa, and can damage various cellular organelles [15]. A decrease in H2O2 production also indicates that the efficiency of redox reactions has increased in presence of silver nanoparticles. Higher concentration of silver nanoparticles, in the growth medium, significantly increased the activity of H2O2metabolizing enzymes. Specific activity of guaiacol peroxidase increased continuously with increasing concentration of silver nanoparticles in the growth medium. A maximum increase of 335 % in GPX activity was recorded at 400 ppm silver nanoparticle treatment (Fig. 3). Free proline content decreased drastically in the seedlings treated with varying concentrations of silver nanoparticles. Figure 2d shows the decrease in proline content at different nanoparticle treatments, with a maximal of 85 % decrease being recorded at 400 ppm. Several studies attribute an antioxidant property to proline, suggesting ROS scavenging activity and also acting as a singlet oxygen quencher [15]. Proline is an excellent index of the existing stress experienced by the plant, since proline level declines after relief from stress [22]. An unprecedented decline in proline levels recorded in the present experiments is one of the most convincing evidences for improved electron exchange efficiency in the silver nanoparticle-treated seedlings. The results for the first time demonstrate that presence of silver nanoparticle in the growth media can improve the growth of B. juncea seedlings by improving their antioxidant status. It follows that optimized use of silver nanoparticles can modulate oxidative stress in plants.
Acknowledgments The authors are thankful to the Department of Biotechnology, Govt. of India, for financial support. We thank, the Head, Department of Genetics, Indian Agricultural Research Institute, New Delhi for providing seed material.

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