Proteus mirabilis alleviates zinc toxicity by preventing oxidative stress in maize (Zea mays) plants

Ecotoxicology and Environmental Safety 110 (2014) 143–152

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Proteus mirabilis alleviates zinc toxicity by preventing oxidative stress in maize (Zea mays) plants Faisal Islam a, Tahira Yasmeen a,n, Muhammad Riaz a, Muhammad Saleem Arif a, Shafaqat Ali a, Syed Hammad Raza b a b

Department of Environmental Sciences, Government College University, Faisalabad-38000, Pakistan Department of Botany, Government College University, Faisalabad 38000, Pakistan

art ic l e i nf o

a b s t r a c t

Article history: Received 13 May 2014 Received in revised form 13 August 2014 Accepted 15 August 2014

Plant-associated bacteria can have beneficial effects on the growth and health of their host. However, the role of plant growth promoting bacteria (PGPR), under metal stress, has not been widely investigated. The present study investigated the possible mandatory role of plant growth promoting rhizobacteria in protecting plants from zinc (Zn) toxicity. The exposure of maize plants to 50 mM zinc inhibited biomass production, decreased chlorophyll, total soluble protein and strongly increased accumulation of Zn in both root and shoot. Similarly, Zn enhanced hydrogen peroxide, electrolyte leakage and lipid peroxidation as indicated by malondaldehyde accumulation. Pre-soaking with novel Zn tolerant bacterial strain Proteus mirabilis (ZK1) isolated zinc (Zn) contaminated soil, alleviated the negative effect of Zn on growth and led to a decrease in oxidative injuries caused by Zn. Furthermore, strain ZK1 significantly enhanced the activities of catalase, guaiacol peroxidase, superoxide dismutase and ascorbic acid but lowered the Proline accumulation in Zn stressed plants. The results suggested that the inoculation of Zea mays plants with P. mirabilis during an earlier growth period could be related to its plant growth promoting activities and avoidance of cumulative damage upon exposure to Zn, thus reducing the negative consequences of oxidative stress caused by heavy metal toxicity. & 2014 Elsevier Inc. All rights reserved.

Keywords: PGPR Proteus mirabilis Oxidative damages Antioxidants Zn Zea mays

1. Introduction Heavy metal contamination is becoming a widespread environmental issue because they are persistent in nature and their concentrations are being increased consistently by natural and anthropogenic sources including the use of fertilizers, pesticides, agricultural runoff, streams from mines smelters and industries (Shikazono et al., 2008). Among the heavy metals, Zn is an essential micronutrient for biological metabolic functions in plants and animals. Elevated levels of Zn in agricultural fields originate from various sources including application of industrial effluent for irrigation, animal manure and/or sewage sludge. Toxic concentrations of Zn in soil inhibit plant growth, cause nutrient imbalance, leaf chlorosis and loss of plasma membrane integrity, alter bio-membranes permeability, result in photosynthesis impairment and produce reactive oxygen species (ROS) (Cambrollé et al., 2012; Upadhyaya et al., 2010). Metal induced oxidative stress results in the generation of ROS and leads to increase in Superoxide dismutase (SOD) and lipoxygenase activity

n

Corresponding author. Fax: þ 92 41 9200671. E-mail address: [email protected] (T. Yasmeen).

http://dx.doi.org/10.1016/j.ecoenv.2014.08.020 0147-6513/& 2014 Elsevier Inc. All rights reserved.

(Baisak et al., 1994). These enzymes are extremely harmful to plant cells and cause senescence. In addition, decrease in the iron sequestration and synthesis of stress ethylene leads to a reduction in plant growth under metal toxicity. Under such conditions, plant growth promoting rhizobacteria (PGPR) plays an important role in the rhizosphere. They tend to alleviate metal toxicity to plants and improve plant growth and nutrient uptake. They reduce indigenous ethylene concentrations by metabolizing 1-aminocyclopropane-1carboxylate (ACC), an immediate precursor of ethylene in plants, into a-ketobutyric acid and ammonia in metal stress plants by utilizing their ACC deaminase activity, and encourage development of longer roots. Resultantly, the plants are better adapted to metal toxic concentrations by increasing nutrient uptake and diluting metal concentrations. PGPRs are also known to possess intrinsic abilities to produce different plant growth hormones including indole acetic acid (IAA) and siderophores (He et al., 2010; Sheng et al., 2008). Siderophores are low molecular weight and high-affinity Fe(III)-complexing compounds, and have potential to increase soluble iron availability in soil for uptake by plants. A large number of studies have advocated that PGPR enhance mineral nutrition absorption, especially the nutritional status of phosphorus, promote plant growth and thus improve the resistance of plants to adverse conditions of metal toxicity (Gajewska

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et al., 2006b; Tang et al., 2009). PGPR also affect the production of reactive oxygen directly, and can improve plant nutrition absorption and enhance plant growth. Many PGPR are tolerant to metals and play significant role in metal mobilization and immobilization, however, a limited attempts have been made to study their role in amelioration toxic effects of high metal concentrations in plants. Microbe assisted phytoremediation is environmental friendly and promising technique to remove metals from contaminated environment compared to the chemical phytoremediation since the addition of different compounds like EDTA, for metal chelation, also results in environmental pollution. In the present study, maize was used because the crop has commercially available seeds, good cultivation techniques, fast growth rates and high biomass production. In addition, recently, Vamerali et al. (2010) reviewed the application of field crops for phytoremediation during the last fourteen years and ranked maize the third most widely used crop species. Maize has been shown to tolerate higher copper concentrations and, therefore, has been used for phytoremediation of copper (Chiu et al., 2005; Li and Ramakrishna, 2011; Murakami and Ae, 2009). However, studies are scant on whether there are bacterial isolates to tolerate higher Zn concentrations. Therefore, this study was designed to isolate and characterize Zn resistant PGPR, and to apply highly resistant Zn resistant PGPR to maize under laboratory conditions to:

 elucidate the effect of the Zn-resistant bacterial isolate on growth, development and Zn uptake of the maize plants;

 explore amelioration effects of Zn resistant bacterial isolate on Zn induced oxidative stress;

 establish the activity of enzymatic antioxidants (CAT, SOD, GPX)



and accumulation of non-enzymatic antioxidants (Proline and ascorbic acid) in maize plants inoculated with Zn-resistant bacterial isolate; and measure changes in maize growth, development and biochemical responses at different time periods after germination.

2. Materials and methods 2.1. Description of sampling area For isolation and screening of Zn resistant bacteria, soil samples were collected from an agricultural field irrigated with industrial effluents along Paharang drain in Faisalabad (31.41801N, 73.07901E), a heavily industrialized city of Pakistan. The soil in the sampling area has been reported to receive wastewater for irrigation and has higher concentrations of Zn, 668.80 mg kg  1 (Kahlown et al., 2006).

2.2. Soil sampling, isolation and screening of Zn resistant bacteria Surface soils (0–15 cm) were collected with a stainless steel shovel from five sampling points. The soils were immediately handed-sorted to remove any stones and any live and dead plant materials. The samples were transported to the laboratory on ice and mixed gently to make a composite sample in the laboratory. The bacterial isolation was carried out on at the same day by the pore plate method (McLellan et al., 2009). Briefly, 100 μL of 106 times serially diluted soil solution (1 g soilþ 9 mL saline buffer, 85% NaCl) was spread on Luria Bertani (LB) media plates and incubated for 24 h at 30 1C. Colonies of different morphological appearances were selected and transferred to LB media plates for purification through streak plate method (Harley and Prescott, 2002).

2.2.1. Determination of minimum inhibitory concentration (MIC) MIC of all the bacterial isolates was determined by gradual increase of Zn concentration (ZnSO4) in LB media until the bacterial isolate failed to grow on plates over 7 days of incubation following the method described by Islam et al. (2014). This concentration was considered as MIC of respective isolates.

2.3. Evaluation for PGPR characteristics Plant growth promoting traits of selected bacterial isolate were evaluated by growing the Zn-resistant bacterial strain in LB broth containing control 2 mM Zn (control conditions) and under 50 mM Zn concentrations (Zn-stress conditions). For indole acetic acid (IAA) production assay, Zn resistant bacterial isolates were grown in LB broth with the addition of 0.5 mg L  1 tryptophan. One mL of overnight grown culture (108 CFU mL  1) was added in 20 mL of LB broth and incubated at 150  g for 96 h. The Salkowski’s reagent was prepared by dissolving 4.5 g of FeCl3 in 10.8 mol of H2SO4 L  1 (Gordon and Weber, 1951). After incubation, the broth was centrifuged at 6000  g for 10 min. The pellet was discarded and supernatant was collected. One mL of supernatant and 2 mL of Salkowski’s reagent were mixed in sterilized glass tube at room temperature (25 1C) and left for 25 min. The absorbance of the developed pink color was measured at 530 nm with Halo DB-20 UV–vis double beam spectrophotometer. The IAA concentration in the culture was determined using a calibration curve of pure IAA as a standard following linear regression analysis. The siderophore production of isolated bacteria was determined by chrome azurol-S (CAS) method of Schwyn and Neilands (1987). Siderophore production was measured by orange halo zone production around the bacterial isolates on blue agar. The production of α-ketobutyrate (α-KB) by the enzymatic cleavage of ACC was determined by measuring its absorbance in bacterial culture at 540 nm and comparing with the absorbance of known concentration of pure α-KB (Belimov et al., 2005). Protein concentration in bacterial culture was estimated by the method of Bradford (1976) and then the enzymatic activity was expressed as 1 M αKB mg  1 h  1 (He et al., 2010; Jalili et al., 2009). For the quantitative measurement of P solubilization of Zn resistant bacterial isolate, the freshly prepared bacterial culture (108 CFU mL  1) was inoculated in Pikovskaya’s broth containing 2.5 g of TCP (tri-calcium phosphate). The culture was incubated at 30 1C with a constant shaking of 200  g for 7 days. The supernatant was obtained by centrifugation at 6000  g and used to determine P-solubilization through colorimetric method of Fiske and Subbarow (1925). Colour reagent, prepared by mixing 0.5 mL of 1.5% (w/v) ammonium molybdate in a 5.5% (v/v) sulfuric acid solution and 125 mL of a 2.7% (w/v) ferrous sulfate solution, was added to the sample solution and the production of phosphomolybdate was measured at 700 nm with a Halo DB-20 UV–vis double beam spectrophotometer.

2.4. Bacterial identification Highly resistant bacterial isolate ZK1 having high plant growth promoting abilities under 50 mM of Zn was selected from a pool of bacterial isolates and identified through 16S rRNA gene sequencing. Crude DNA of the isolate was extracted following the method as described by Cheneby et al. (2004). The PCR reaction was performed in a total volume of 25 μL containing 2.5 μL buffer (10  ), 0.5 μL of dNTPs (12.5 mM), 1 μL of each primers 27F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and 1492R (50 - GGT TAC CTT GTT ACG ACT T-30 ) of 10 pmoles μL  1 each, 0.1 μL of Taq DNA polymerase, 1 μL MgSO4 (50 mM), 17.23 μL of water and 1 μL of template. Amplification was performed in a PCR machine programmed as initial denaturation at 94 1C for 4.5 min followed by 30 cycles consisting of denaturation at 94 1C for 30 s, primer annealing at 57 1C for 30 s and primer extension at 68 1C for 90 s and final extension at 68 1C for 10 min. Amplified PCR products of 16S ribosomal gene were separated on 1% agarose gel in 1  TBE (Trisborate-EDTA) buffer. A 100 bp DNA ladder (Promega) was run as a size marker. The gel was viewed under UV light and photographed using Eagle Eye gel documentation system (Modle 3). Amplified PCR product of 16S rRNA gene of selected isolate ZK1 was purified using QIA quick PCR purification kit (QIAGEN) following the standard protocol recommended by the manufacturer, to prepare for sequencing. 16S rRNA gene sequence of bacterial isolate ZK1 was compared with the known nucleotide sequences in the GenBank database using BlastN (http://www.ncbi.nlm.nih.gov/ BLAST). ZK1 showed 99% resemblance with Proteus mirabilis. On the basis of resemblance with specific group of bacteria, we conclude isolate ZK1 as P. mirabilis. The sequences were deposited in the GenBank database under the Accession number KF471515.

2.5. Pot experiment 2.5.1. Plant material, growth and treatment conditions Maize seeds were used in the inoculation experiment. Pot experiment was conducted in plastic pots filled with 350 g pot  1 autoclaved (121 1C for 1 h) acidwashed sand. The seeds were surface sterilized with mixture of ethanol and 30% H2O2 for twenty minutes and washed twice with double distill water. The seeds were separated in two groups; the first group of seeds was dipped in sterile water (un-inoculated control) and other in bacterial suspension (108 CFU mL  1) for two hours in petri plates. The seeds were allowed to germinate on filter paper for 5 days in dark. After germination, un-inoculated seed was soaked in sterile water and inoculated seeds in bacterial suspension (108 CFU mL  1) for 2 h, and then placed in each pot. Five seeds were planted, thinned to three plants per pot, in each pot and

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12 replicates were used for each treatment to facilitate removal of three replicate pots per treatment at 10, 20 and 30 days of germination. Half strength Hoagland nutrient solution with 2 and 50 mM concentration of Zn was used; chemical forms of Zn are summarized in supplementary Table I. The experiment was laid out in complete randomized design (CRD) with four treatments: Control (Zn 2 mM); Zn treatment (50 mM); P. mirabilis (Zn 2 mM) and Zn þP. mirabilis (50 mM). In silico estimations of the concentrations of Zn ionic species in the different nutrient solutions were carried out with MINTEQA2 for Windows (Version 1.50, Allison Geoscience Consultants, USA). All the pots were placed in growth chamber with average temperature of 30 1C, relative humidity of 65% and a 16/8 h under light intensity of 250–300 mmol photons m  2 s  1 (Lasat and Kochian, 2000).

2.5.6. Measurement of non-enzymatic antioxidants Ascorbic acid (AsA) contents of maize leaves were estimated following the method described by Mukherjee and Choudhuri (1983). Fresh leaf material (0.25 g) was homogenised in 10 mL solution of 6% TCA. Four ml of the extract were reacted with 2 mL of 2% dinitrophenyl hydrazine solution in the acidic medium. One drop of 10% thiourea solution (prepared in 70% ethanol) was added to the mixture. The mixture was boiled for 20 min in a water bath. After cooling the mixture at 25 1C, 5 mL of 80% H2SO4 (v/v) were added to the mixture. The absorbance was measured at 530 nm. The amount of AsA in the extracted leaf samples was worked out from a standard curve prepared using varying AsA standards procured from. Proline content was measured by the method of Bates et al. (1973).

2.5.2. Plant biomass and zinc contents Plants were harvested at three time point (10, 20, 30 days after sowing) and roots were washed with deionized water and blot dried. Each time leaf area was measured, roots and shoots were separated and biomass (dry weight) was recorded after drying in an electric heating oven at 70 1C for five days. Oven dried 100 mg plant material was taken into a digestion flask and digested with nitric acid following Allen et al. (1986) method. The Zn concentration in plant samples was measured using atomic absorption spectrophotometer in flame mode (air– acetylene).

2.6. Statistical analysis

2.5.3. Biochemical analysis Leaf photosynthetic pigments (total chlorophyll) and total soluble protein were estimated according to the method of Arnon (1949) and Bradford (1976), respectively. The measurements were performed on plant sampled after 10, 20 and 30 days of germination.

2.5.4. Assays of antioxidant enzymes For the analysis of antioxidant enzymes after 10, 20 and 30 days, 0.5 g fresh leaves were homogenized in 10 mL of ice cold potassium phosphate buffer of pH 7.0 in an ice bath by grinding with a mortar and pestle. The mixture was centrifuged at 12,000  g for 20 min at 4 1C. The supernatants were stored at 4 1C until used for the determination of various antioxidant enzymes. Superoxide dismutase (SOD) activity was measured through the photoreduction of nitro blue tetrazolium chloride (NBT) (Dhindsa and Matowe, 1981). The reaction mixture contained 50 mL of 33 mM NBT, 100 mL of 10 mM L-methionine, 50 mL of 0.0033 mM riboflavin in 250 mL of 50 mM sodium phosphate buffer. The reaction mixture was placed under lamp below 15 W for 25 min before the reaction was stopped by switching off the light. Non-illuminated and illuminated reactions without supernatant served as control. The absorbance was measured at 560 nm. One unit of SOD activity was defined as the quantity of SOD required to produce a 50% inhibition of NBT. For the measurement of catalase (CAT), modified method of Aebi (1974) was followed. In 3 mL of reaction mixture of 1.9 mL of 50 mM phosphate buffer (pH 7), 100 μL of enzyme extract was added and incubated for 25 min. The absorbance of mixture was measured at 240 nm by the addition of 1 mL of 6 mM H2O2 in reaction mixture for 2 min. A 0.1 unit min  1 change in absorbance was defined as 1 unit of CAT activity, and CAT activity was expressed as units mg  1 protein. Guaiacol peroxidase (GPX) activity was measured following the method of Rao et al. (1996) with slight modification. Three mL of reaction mixture was formed by the addition of 2.2 mL of 50 mM phosphate buffer of pH 7, 300 μL of 20 mM of guaiacol with 200 μL of enzyme extract. The mixture was incubated for 5 min at 25 1C and then 6 mM of H2O2 (300 μL) was added in reaction mixture to start the reaction. Change in the absorbance for 2 min was recorded for the calculation of POD activity. One unit of POD activity was defined as an absorbance change of 0.01 unit min  1 and POD activity was expressed as units mg  1 protein.

2.5.5. Determination of MDA and H2O2 contents Zn-induced oxidative damage on membranes was evaluated by measuring changes in lipid peroxidation by quantifying the malondialdehyde (MDA) formation in the plant leaves. Briefly, 1.0 g of fresh leaves were homogenized in 20 mL of 0.1% trichloroacetic acid (TCA) solution and centrifuged for 10 min at 12,000  g. One mL of the supernatant was mixed with 4 mL of trichloroacetic acid (TCA) containing 5% thiobarbituric acid (TBA) and heated for 30 min at 95 1C and cooled on ice. The mixture was centrifuged at 12,000  g for 10 min and absorbance of the supernatant was measured at 532 and 660 nm (Demiral and Türkan, 2005). Concentration of H2O2 was determined using methods of Velikova et al. (2000). Fresh leaf tissues (0.5 g) was homogenized with 5 mL of 0.1% (w/v) TCA in a prechilled pestle and mortar and the mixture was then centrifuged at 12,000  g for 15 min. To 0.5 mL of the supernatant, 0.5 mL of 10 mM potassium phosphate buffer of pH 7.0 and 1 mL of 1 M KI (potassium iodide) were added. The mixture was vortexed and the absorbance was measured at 390 nm (Velikova et al., 2000). The concentration of H2O2 was calculated using a standard curve prepared with known concentrations of H2O2. Electrolyte leakage was measured using electrical conductivity meter as described by Lutts et al. (1996). The electrolyte leakage (EL) was expressed following the formula EL¼ EC1/EC2  100.

Figure and tables contain means of three plants per replicate (n¼ 9). Statistical analysis was performed using SPSS for Windows Software (Version 19.0). Analysis of variance test (ANOVA) was employed to test the significance of treatment effects. Duncan’s multiple range tests was used for multiple mean comparison technique. Significance of differences between plant growth promoting characteristics of bacterial isolate at control (2 mM Zn) and stressed (50 mM Zn) conditions were computed using independent sample t-test technique.

3. Results and discussion 3.1. P. mirabilis characteristics under stressed conditions Plant growth promoting characteristics of P. mirabilis changed significantly when the bacterial isolate was grown with 50 mM Zn compared to 2 mM Zn concentrations (Supplementary Table II). Bacterial isolate ZK1 maintained higher plant growth and expressed maximum value of MIC (1400 mg kg  1 of Zn þ 2) So, the isolate was selected for further studies. 3.2. Effect of Zn and P. mirabilis on growth parameters The chlorophyll contents in Zn treated plants were gradually decreased during the study period while the plants treated with Znþ P. mirabilis were noted for the remarkable increase in chlorophyll concentrations and the increase was 22 and 38% after 10 and 30 d compared to the Zn stressed plants, respectively (Table 1). The highest total chlorophyll contents were observed for the plants treated with bacterial strain P. mirabilis and grown for 30 days. The Zn metal might interfere with synthesis of chlorophyll that results in decreased photosynthesis and inhibition in plant growth. Decline in the level of photosynthetic pigments in Zn-stressed plants may also be attributed to the zinc-induced inhibition of chlorophyll synthesis through inhibition of the chlorophyll synthesizing enzymes and induced nutrient deficiency e.g. that of iron and by the substitution of Mg2 þ (Parlak and Table 1 Effect of P. mirabilis inoculation under Zn stress on total chlorophyll contents (mg g  1 FW) and leaf area (cm2) of maize shoot sampled at 10, 20 and 30 days of growth. Treatments

10 Days

20 Days

30 Days

FW) Total chlorophyll contents (mg g Control 1.45 7 0.14 ef Zn 50 mM 1.50 7 0.27 def P. mirabilis 1.60 7 0.11 def P. mirabilis þ Zn 50 mM 1.52 7 0.11 def

2.107 0.08 ab 1.357 0.21 ef 2.05 7 0.16 ab 1.667 0.17 abc

2.137 0.12 ab 1.247 0.17 f 2.30 7 0.15 a 1.727 0.10 bc

Leaf area (cm2) Control Zn 50 mM P. mirabilis P. mirabilis þ Zn 50 mM

15.107 0.58 g 40.007 1.00 c 33.707 1.91 d 63.80 7 4.19 a

27.20 7 0.70 e 27.107 1.15 e 57.40 74.75 b 45.107 3.11 b

1

22.107 0.97 f 18.40 70.78 g 46.40 7 2.15 b 39.20 7 2.53 c

Values are means of three plants per replicate followed by7 standard error of means (n¼ 9). Values with different letters differ significantly from each other at p o0.05.

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Yilmaz, 2012). Different studies have found that plant inoculation with rhizobacterial isolates under metal stress may improve chlorophyll synthesis. Moreover, the bacterial inoculation under stress might increase the chlorophyll by improving chlorophyll synthesis or slowing the process of chlorophyll degradation. The inhibitory effect of Zn was manifested in the overall growth of the root and shoot system of maize plants; the roots of Zn treated plants were shorter as compared to the control plants (Table 2), and they were yellow after 20 d and became yellow brown after 30 d as compared to white roots of the control plants. These color changes were visible mainly in the apical parts of roots. The roots from ZnþP. mirabilis treatment were considerably longer than the roots of Zn treated plants, and they remained yellow throughout the study period. Roots from P. mirabilis treatment were significantly longer than that from the control treatment but both were of white color (Table 2). The most common response of PGPR inoculation is the improvement of the root system as observed in many inoculated plants (Lucy et al., 2004). For the shoot and root length of maize plants, ZnþP. mirabilis treated plants showed significant increase in root and shoot growth as compared to the Zn treated plants where a slight increase in shoot length was found after 20 d. Trends in the shoot lengths, after treatment application, were very similar to those for the root length; shoot length was significantly lower when Zn concentration was 50 mM. A significant decrease in the dry weight of maize shoot and root was observed under Zn stress (Table 2). The effect was more pronounced after 30 d where significant decrease in dry weight of the shoot and root was recorded. The results also indicated that P. mirabilis inoculation significantly increased dry weight of above and below ground parts in both absence and presence of Zn stress. Plant exposure to the high Zn concentrations resulted in reduction in shoot and root dry biomass which were associated with the stunted root and shoot growth (Table 2). Rhizobacterial inoculation has been shown to facilitate plant growth under heavy metal stress including Zn and arsenic (Nie et al., 2002; Shilev et al., 2006). Regarding leaf area, a significant variation was recorded among treatments. The highest leaf area was obtained under sole bacterial inoculation as compare to control. Leaf area of Zn þP. mirabilis inoculated plants was found 21, 24 and 54% higher at 10, 20 and 30 d of growth as compared to Zn stressed maize plants (Supplementary Table I).

Total soluble proteins were found to be lower in maize plants grown at higher Zn concentrations (Table 2). Protein contents were significantly lower in shoot and root parts of the maize plants at 50 mM Zn concentration when compared to plants grown with P. mirabilis and P. mirabilis þ50 mM Zn concentration. Furthermore, total protein contents in the shoots and roots decreased consistently along the length of plant exposure to high Zn concentrations. The decrease in protein content after prolonged Zn exposure may be due to increased activity of protease or other catabolic enzymes which are activated by heavy metals. A similar result was reported by John et al. (2008) with Lemna polyrrhiza grown under Cd and Pb stress. The decline in plant protein contents under Zn stress has also been linked to higher ROS production due to high Zn load in plants and, as a result, more oxidative stress which resulted in the decline of protein content caused by oxidative damage (Parlak and Yilmaz, 2012). In this study, P. mirabilis enhanced the total protein contents in shoot and root parts of maize under Zn stress showing that P. mirabilis may delay protein degradation and maintain steady protein metabolism, thereby, reducing the stress of ammonia-like substances and increased the ability of plants to withstand stress conditions (Tang et al., 2009). Plant-associated bacteria may also exudate osmolytes such as proline, glycinebetaine and trehalose in response to the stress, which along with other plant growth promoting attributes can act synergistically with plant-produced osmolytes and stimulate the plant growth even under stressed conditions (Paul and Nair, 2008). In our study, the better growth under Zn stress could be due to potential of P. mirabilis to produce growth hormones which is considered a plausible mechanism, controlling growth of stressed plants (see Supplementary Table II). The bacterial hormone indole acetic acid (IAA) is produced in the rhizosphere by using tryptophan as precursor. Bacterial IAA influences the root architecture and increases growth and total surface area of root that help in improving plant nutrition and growth under stress conditions (Somers et al., 2004). Increased ethylene hormone production under heavy metal stress results in reduced root and shoot growth; the decrease in the root and shoot length under Zn stress might be due to the ethylene hormone production. Although ethylene concentrations were not determined in our study, however, the increase in root and shoot length in P. mirabilis inoculated plants under Zn stress may be due to the bacterial ACC deaminase

Table 2 Effect of P. mirabilis inoculation under Zn stress on plant length (cm), plant dry weight (g plant  1) and total soluble protein (mg g  1 FW ) of maize shoot sampled at 10, 20 and 30 days of growth. Treatments

Shoot

Root

10 Days

20 Days

30 Days

10 Days

20 Days

30 Days

Plant length (cm) Control Zn 50 mM P. mirabilis P. mirabilisþ Zn 50 mM

30.0 7 1.53 d 23.0 7 0.58 e 33.07 0.98 d 25.8 7 0.90 e

39.0 7 1.15 d 30.0 7 1.15 d 41.0 7 2.08 cd 31.7 7 1.27 d

43.0 70.58 ab 31.0 71.15 d 45.2 70.99 a 33.771.20 d

10.8 7 1.29 hi 7.0 7 0.58 i 14.6 7 0.79 h 9.3 7 0.75 hi

20.7 71.33 cd 13.2 70.54 gh 22.6 71.18 bc 15.1 70.61 ig

25.0 7 0.58 b 17.4 7 0.85 ef 27.9 7 1.07 a 18.7 7 0.85 de

Dry weight (g plant  1) Control Zn 50 mM P. mirabilis P. mirabilisþ Zn 50 mM

0.08 7 0.02 0.077 0.03 0.09 7 0.03 0.08 7 0.08

0.157 0.04 0.147 0.04 0.187 0.07 0.157 0.08

0.22 70.11 a 0.17 70.05 b 0.25 70.06 a 0.19 70.05 b

0.217 0.01 0.187 0.02 0.23 7 0.01 0.20 7 0.01

0.29 70.01 e 0.23 70.00 e 0.30 70.02 d 0.25 70.01 e

0.357 0.01 0.277 0.01 0.36 7 0.01 0.29 7 0.01

b d a c

1.73 70.06 1.05 70.08 1.88 70.09 1.53 70.05

1.137 0.06 abc 0.94 7 0.07 bcde 1.117 0.11 abc 1.02 7 0.07 bc

1.06 70.04 bc 0.71 70.03 fg 1.30 70.17 a 0.71 70.05 fg

1.187 0.06 0.497 0.03 1.157 0.08 0.88 7 0.05

ab g ab cdf

ef f cd ef

Total soluble protein (mg g  1 FW) Control 2.107 0.10 ab Zn 50 mM 1.86 7 0.06 bcd P. mirabilis 2.39 7 0.10 a P. mirabilisþ Zn 50 mM 2.05 7 0.06 b

b de b bc

1.90 7 0.11 bc 1.58 7 0.14 df 2.157 0.12 ab 1.707 0.10 def

def g bcd f

fg fg f g

Values are means of three plants per replicate followed by 7 standard error of means (n¼ 9). For each parameter, under shoot and root columns, values with different letters differ significantly from each other at po 0.05.

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Fig. 1. Effect of P. mirabilis inoculation under Zn stress on (a) malondialdehyde (MDA), (b) hydrogen peroxide (H2O2) and (c) electrolyte leakages (%) in shoot and root parts of maize plants sampled at 10, 20 and 30 days after treatment application. Bars are means of three plants per replicates. Error bars are 7standard error of means (n¼ 9). Bars with different letters differ significantly from each other at p o 0.05).

activity of P. mirabilis (Supplementary Table II) that degraded ACC precursor of ethylene in Zn stressed plant. Rhizobacteria with ACC deaminase activity have been found to decrease plant ethylene production under different stress conditions such as heavy metal and salinity stress (Mayak et al., 2004; Zahir et al., 2009). 3.3. Effects of P. mirabilis on Zn induced oxidative stress In the present study, MDA contents of maize leaves showed a significant increase of 110, 132 and 204% after 10, 20 and 30 d under Zn stress compared to the control treatment (Fig. 1a). However, for Znþ P. mirabilis treated plants, MDA contents were decreased by 21, 18 and 29% after 10, 20 and 30 d of growth, respectively, when compared with the Zn-stressed plants. Similar results were found in the root part of the plant where Zn þP. mirabilis treatment significantly reduces MDA contents compared to the Zn treated plants and reduction was 16, 5 and 18% at 10, 20 and 30 d, respectively. The increased MDA contents in the Zn treated plants indicate the presence of oxidative stress and, perhaps, this may be one of the possible mechanisms exhibiting Zn toxicity in the plant tissues. However, MDA contents were lower in roots than those in shoots. This could be attributed to low Zn load in roots that probably resulted in low oxidative stress and membrane damage (Gupta et al., 2011). The plasma membranes of

the cells are the first physiological barriers for the entrance of heavy metals into the plant. The interactions of metal ions with the plasma membrane and their toxic effects influence many physiological parameters. These include the induction of lipid peroxidation and the loss of highly mobile essential ions, leading to serious ion imbalances in plant (Liptáková et al., 2013). Lipid peroxidation resulting from ROS exposure (such as high levels of H2O2) has been shown to disrupt membrane organization which leads to functional losses and modifications of proteins and DNA bases (Pitzschke et al., 2006). H2O2 accumulation in plants may also lead to uncontrolled oxidation and free-radical chain reactions leading to oxidative stress in plants (Srivastava et al., 2005). Both redox-active (such as Cu and Fe) and non-redox-active (such as Zn and Cd) metal ions have been reported to increase lipid peroxidation via ROS generation in plants (Chaoui et al., 1997). Increased lipid peroxidation in bean plants exposed to toxic Zn levels has been attributed to the increased activity of membrane bound lipoxygenase, which is known to oxidize polyunsaturated fatty acids and produce free radicals (Chaoui et al., 1997). Under Zn stress, H2O2 contents of the maize shoot were 80, 71 and 90% higher compared to the control treatment whereas H2O2 contents in roots were 36, 50, 56% higher than those of H2O2 contents of the roots of the control treatment after 10, 20 and 30 days of treatment application, and indicated the extent of

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oxidative stress (Fig. 1b). However, in Zn þP. mirabilis treated plants, the level of H2O2 was significantly reduced, reflecting a considerable alleviation of Zn induced oxidative stress by P. mirabilis as compare to Zinc stress plants. Relatively higher levels of H2O2 in Zn stress plants may be a consequence of disturbance in the balance between production and utilisation/scavenging of H2O2. The elevated levels of MDA and H2O2 in Zn stressed plants suggested that antioxidative enzymes were not sufficient to scavenge the overproduction of ROS. In contrast, decrease in H2O2 and MDA contents in Zn þP. mirabilis plants compared to the Zn stressed plants proving that bacterial inoculation helped the plant to lower H2O2 and MDA contents by increasing its scavenging. However, compared to the un-inoculated control treatment, no significant differences were observed when the maize plants were inoculated with P. mirabilis alone (Fig. 1b). Under Zn stress, significant leakage of electrolyte was noticeable in maize plants, however inoculation of P. mirabilis under Zn stress resulted remarkable alleviation in electrolyte leakage (Fig. 1c). The present data suggested that Zn toxicity in maize plants were due to Zn-induced oxidative stress through hydrogen peroxide accumulation and related lipid peroxidation. Similar to results reported in this study, Baranwal et al. (2012) and Haneef et al. (2014) also reported decreases in MDA and ROS due to the greater protection from oxidative damage attributed to a more efficient antioxidative system of rhizobacterial inoculated plants.

Antioxidant enzymatic activities in metal-stressed plants are highly variable and depend on the plant species, metal ion, concentration and exposure duration (Sharma and Dietz, 2008). Comparing the activities of antioxidants enzymes (SOD, CAT and GPX) in this study, it is evident that P. mirabilis induced a strong anti-oxidative response, especially in Zn stressed inoculated plants. The general trend shown by plants was stimulation of enzymes at shorter duration (10 d) followed by decrease at higher duration (20 and 30 d) compared to the control treatment. This decrease in antioxidant enzyme activities might be due to increased accumulation of Zn metal (Fig. 4) in plant organs which subsequently increased oxidative stress (H2O2 and MDA) (Fig. 1) in plants that resulted decrease in the activity of antioxidant enzymes. In contrast, Znþ P. mirabilis inoculated plants maintained higher enzymatic activities as compared to the Zn stressed plants. Therefore, our results suggested that the ability of inoculated plants to induce antioxidant enzymes (CAT, SOD and GPX) appears to be crucial strategy to combat Zn stress (Li et al., 2014). In addition, changes in GPX activity have been suggested as indicators for biotic and abiotic stresses. Karthikeyan et al. (2007) also found increase in CAT, GPX and SOD activities in plant inoculated with Azospirillum, Azotobacter and Achromobacter xylosoxidans under both normal and stressed conditions.

3.4. Antioxidant enzymatic activities under P. mirabilis inoculation and Zn stress

It was observed that bacterial inoculation significantly increased the production of ascorbic acid (AsA) in the maize plants under Zn stress (Fig. 3a). About 37, 42 and 30% augmented contents of ascorbic acid in shoot whereas 34, 30 and 25% increase was observed in roots of P. mirabilis inoculated Zn stressed plants at 10, 20 and 30 d of growth period, respectively. Ascorbic acid is an important secondary metabolite and ROS scavenger responsible for many physiological processes in plants. Moreover, ascorbic acid acts as a co-factor for many enzymes thereby affecting the expression of genes involved in defense and hormone signaling pathways (Uadhyaya et al., 2011). The bacterial inoculation of the maize plants, under Zn stress, resulted in enhanced ascorbic acid production which enabled the plants to minimize oxidative stress induced damages (Islam et al., 2014). Uadhyaya et al. (2011) observed enhanced activity of ascorbic acid in transgenic potato plant under stressed conditions. Ascorbic acid can also directly scavenge 1O2, O  12 and OH  and regenerate tocopherol from tocopheroxyl radical, thus providing membrane protection (Du Laing et al., 2007) as observed in Zn þP. mirabilis plants, where less membrane peroxidation was noted due to higher concentration of ascorbic acid (AsA). The Proline concentrations in Zn treatment plants increased with the time. The highest increase in plant Proline contents was observed after the 30 days of growth (Fig. 3b). However, the increase in Proline content was also noticed in Zn þP. mirabilis treated plants compared to the control plants but this increase in Proline concentration was not as high as in the case of Zn treatment. Comparatively, the Proline concentration of shoots was higher than that of the roots of the maize plants. The reduced contents of Proline might be due to the mitigation abilities of P. mirabilis strain that decreased the metal toxicity and stress in inoculated plants than to the Zn stressed plants. Oves et al. (2013) observed similar Proline content trends in the Pseudomonas inoculated chick pea plant under chromium stress. We found that Zn treatments increased the accumulation of Proline regardless of the fact that soluble protein content declined in response to Zn. In addition to acting as a metal chelator and osmolyte, Proline has been found to depart hydroxyl radicals and singlet oxygen, thus providing conservation against ROS-induced cell damage. Increase in both Proline and lipid peroxidation levels

Differential response of CAT activity at different time points in different treatments was observed. We recorded reduced CAT activity in shoot and root of Zn stressed maize plants as compared to P. mirabilis inoculation under Zn stress (Fig. 2a). It was observed that CAT activity increased at each time interval of 10 d in both root and shoot under P. mirabilis inoculation and control condition, whereas CAT activity gradually decreased at each time point under Zn stress in maiz plants. An increase in the GPX activity over control was recorded in all treatments irrespective of bacterial inoculation (Fig. 2b). However, this increase was significant in the case of Znþ P. mirabilis treatment. Compared to the Zn treated plants, the GPX activity in shoots after 10, 20 and 30 d were 79, 42 and 47% reduced whereas in the roots the reduction were 31, 18 and 24% in Znþ P. mirabilis treated plants. It seems that GPX played a crucial role in scavenging H2O2 helping to minimize excessive ROS in Zn stressed plants. The induction in GPX activity has also been reported under Zn stress (Shah et al., 2001). According to Khatun et al. (2008), increase in antioxidant enzymes appears to be an essential form of protection from Zn induce oxidative stress. Catalase (CAT) is the enzyme that dismutates hydrogen peroxide produced by SOD. In the present study, SOD activity was significantly increased after Zn þP. mirabilis inoculation and the increased was upto 40, 23 and 32% higher in a shoot while 36, 21 and 27% higher in root than the Zn stressed plants after the 10, 20, 30 d, respectively (Fig. 2c). This transition produced more H2O2 catalyzed by SOD but the catalase enzyme due to its decrease activity was unable to eliminate H2O2 in both roots and shoots. The decreased activity of CAT in Zn stressed plants led to increased H2O2 contents of plant in this study. Gajewska et al. (2006a) and Ali et al. (2013) found a decrease in the CAT activity in wheat and Brassica roots under nickel (Ni) and Cadmium (Cd) stress, respectively. We suggest that the lack of induction of CAT activity is responsible for the accumulation of H2O2 in Zn stressed root and shoot. Due to this reason maize plants experienced oxidative damage under Zn stress. On the other hand, the CAT activity was stimulated by the inoculation of bacteria and significant increase was observed in inoculated plants.

3.5. Antioxidants

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Fig. 2. Effect of P. mirabilis inoculation under Zn stress on (a) catalase (CAT), (b) guaiacol peroxidase (GPX) and (c) superoxide dismutase (SOD) in shoot and root parts of maize plants sampled at 10, 20 and 30 days after treatment application. Bars are means of three plants per replicates. Error bars are 7standard error of means (n¼ 9). Bars with different letters differ significantly from each other at p o 0.05).

with increasing Zn accumulation are indicative of a relationship between ROS generation (hydroxyl radicals mostly) and ROS scavenging by Proline (Parlak and Yilmaz, 2012). Accumulation of Proline in response to higher concentration of metal such as copper, cadmium, zinc, and nickel has been reported in several plants (Matysik et al., 2002). Similar to our study, Hamdia et al. (2004) and Nadeem et al. (2007) reported an increase in Proline contents under salt stress and found PGPR inoculation decreased Proline. The decrease in Proline contents after PGPR inoculation indicates positive correlation between Proline accumulation and plant adaptation to stress (Cruz-Tapias et al., 2012). 3.6. Accumulation of Zn in maize plants When maize plants were grown under 50 mM Zn concentration, plants accumulated significant Zn concentrations in shoot parts which were strongly dependent on length of plant exposure (Fig. 4). The maximum Zn concentration of 225 mg g  1 DW was recorded in the shoot, while 160 in mg g  1 DW Zn concentration were found in the roots after 30 d grown Zn þP. mirabilis plants. Hence translocation of Zn from root to shoot was very high. Baker and Walker (1990) suggested that uptake, translocation and accumulation mechanisms were heavy metal and plant species specific. The Zn concentrations were significantly higher in treating with Znþ P. mirabilis than plants growing only in Zn. The increased Zn accumulation in the above ground plant parts corresponded to the high concentration in the nutrient solution

indicating translocation from the roots and accumulation within the shoots (Kabata-Pendias and Pendias, 2001). Similarly, MateosNaranjo et al. (2014) reported Zn accumulation in aerial parts of the Juncus acutus. Langer et al. (2012) also found higher Zn accumulation in plant shoots inoculated with Populus tremula. The increase in metal accumulation under Zn þP. mirabilis treatment might be related to P. mirabilis plant growth promoting activities such as siderophore production and phosphorous solubilization. There has been a large number of studies reporting increased metal concentrations and reduced metal stress in PGPR inoculated plants (Ma et al., 2011; Sheng et al., 2008; Zaidi et al., 2006). In our study, this effect may be associated with Zn solubilization by P. mirabilis which increased the Zn bioavailability in plant rhizosphere by producing organic acids that help in heavy metal uptake. Previously, a PGPR, Gluconacetobacter diazotrophicus, was reported to solubilize Zn by the secretion of organic acid (Saravanan et al., 2007). Solubilization of metals by bacteria would be a more efficient and environment-friendly approach than the application of synthetic chelators such as EDTA with slow degradation rate and long persistence in soil (Li and Ramakrishna, 2011). The significant increase in Zn accumulation of plants treated with P. mirabilis could be attributed to the production of siderophore and the solubilization of P. It seemed that inoculated plants accumulated more toxic metals in their aerial parts. The increased biomass and total Zn uptake of maize plants revealed the important role of PGPR in heavy metal phytoremediation. Furthermore, P. mirabilis inoculation

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Fig. 3. Effect of P. mirabilis inoculation under Zn stress on (a) ascorbic acid (AsA) and (b) Proline accumulation in shoot and root parts of maize plants sampled at 10, 20 and 30 days after treatment application. Bars are means of three plants per replicates. Error bars are 7 standard error of means (n¼9). Bars with different letters differ significantly from each other at po 0.05).

Fig. 4. Effect of P. mirabilis inoculation under Zn stress on Zn contents (mg g  1 DW) of shoot and root parts of maize plants sampled at 10, 20 and 30 days after treatment application. Bars are means of three plants per replicates. Error bars are 7 standard error of means (n¼ 9). Bars with different letters differ significantly from each other at p o0.05).

influenced the translocation and distribution of metal in maize plants and increased metal transportation from soil into aerial plant parts which offered increased phytoremediation efficiency. However, the results reported here are from controlled environment experiments, therefore, inoculation and phytoremediation efficiency of P. mirabilis maize need to be evaluated in field studies.

4. Conclusions Plant-associated bacteria can have beneficial effects on the growth and health of their host. Nevertheless, the role of plant growth promoting bacteria, in terms of plant metal stress tolerance, has not been investigated in depth. A novel multi-metal tolerant bacteria strain ZK1 was isolated from genus Proteus from zinc (Zn) contaminated soil. In addition to its high tolerance to Zn, the isolated bacterial strain ZK1 also produces indole acetic acid (IAA), siderophores, solubilize phosphorous and 1-aminocyclopropane-1carboxylate deaminase activity (ACC) [under control and Zn stress],

demonstrating its potential to contribute to beneficial plant– microbe interactions under metal contaminated site. When interacting with Zea mays plants exposed to Zn, the bacterium depressed Zn induces oxidative stress (MDA, H2O2) by upregulating antioxidant enzymes (CAT, SOD, GPX), promoted relative plant growth and decreased the root absorption of Zn, resulting in increased plant tolerance to Zn. The results indicated that the inoculation of Z. mays plants with P. mirabilis ZK1 promoted better growth in plants cultivated in the presence of Zn. This phenomenon appears to be attributed to a mechanism that decreases Zn concentrations in the roots via a beneficial interaction between the bacteria and the plant roots.

Acknowledgment The authors are thankful to Mam Razia from University of Wah, Wah Cantt for English correction of this manuscript. This work is

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supported financially by Higher Education Commission (HEC), Pakistan (Project No: PM-IPFP/HRD/HEC/2011/0582).

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