Fingerprinting of the antioxidant status in Alyssum markgrafii shoots during nickel hyperaccumulation in vitro
*Nemanja Stanisavljevi?1, Jelena Savi?2, Živko Jovanovi?4, Jovanka Miljuš-?uki?1, Jelena Sen?anski3, Mladen Simonovi?3, Svetlana Radovi?4, Dragan Vinterhalter2, Branka Vinterhalter2
1University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, Belgrade, Serbia.
2 University of Belgrade, Institute for Biological Research”Siniša Stankovi?” , Bulevar despota Stefana 142, Belgrade, Serbia.

3 University of Belgrade, Institute of General and Physical Chemistry, Studentski trg 3, Belgrade, Serbia.

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4 University of Belgrade, Faculty of Biology, Studentski trg 3/2, Belgrade, Serbia.

*Corresponding author: Nemanja Stanisavljevi? Tel. +381113976414, Fax. +381113976414,
e-mail: [email protected]
Keywords: Alyssum markgrafii, nickel, hyperaccumulation, antioxidant status.

Abstract
This study investigated the role of antioxidant system of Alyssum markgrafii, during long term exposure to 0.5 mM or 1 mM NiCl2 × 6H2O in vitro. Applied methodology included sample preparation protocol which reduces oxidation of key metabolites along with novel luminescent method and well established photometric procedures. During five-week treatments plants accumulated 1121 and 2470 ppm of Ni2+ respectively followed by severe growth retardation, chlorophyll degradation and peroxidation of lipids. These effects were more pronounced after 1mM Ni2+ treatment and additionally accompanied by increased water loss. Activities of luminol converting peroxidases (LUPO) and glutathione reductase (GR) upon 0.5 mM treatment were increased while catalase (CAT) and superoxide dismutase (SOD) were diminished. The fact that these two groups of enzymes run in antiparallel might suggest functional redistribution between antioxidant enzymes rather than orchestrated action to prevent oxidative damage. Total antioxidant capacity (TAC) was also increased after 0.5 mM treatment which coincided with increased GR activity and elevated glutathione content indicating this low molecular weight antioxidant as an important factor associated with nickel tolerance. This study also emphasizes the possible important role of luminol converting peroxidases in nickel hyperaccumulation, although they are not considered as antioxidant enzymes sensu stricto since some of them can also produce reactive oxygen species as well.

Introduction
Soil contamination with nickel in past decades has become important issue due to significant reduction in agricultural yields and harmful effects on human and animal health which are caused by introduction of this heavy metal into the food chain (Adriano 1986). Generally, maintenance of metal homeostasis, especially in metal rich environments is particularly large challenge for plants (Baker 1981; Ahmad and Ashraf 2011; Hussain et al. 2013). Hyperaccumulating species are able to accumulate heavy metals in concentrations which are toxic for non-accumulator plants, thereby offering sustainable option for treatment of contaminated soil. They are currently in scientific focus, since they represent an interesting example of evolution and can also be exploited for phytoextraction or phytomining on contaminated sites and serpentine soils (Antonkiewicz et al. 2016; Tappero et al. 2007). Nickel itself is not the redox-active, but it could exhibit effects similar to redox-active transition metals such as Cd, Cu or Mn. Excess nickel in plant tissues leads to disruption in delicate balance between production and scavenging of reactive oxygen species (ROS), which was confirmed by several studies (Boomintan and Doran 2002; Chen et al. 2009; Ková?ik et al. 2011). Majority previous studies on several hyperaccumulators from Brassicaceae family have been focused primarily on mechanisms of nickel sequestration, transport and its cellular localization. Few rare studies also examined ROS generating compartments, underlying mechanisms of ROS production mechanisms of ROS production and particular counterparts in the antioxidant system such as various antioxidant enzymes (SOD, CAT, GR and etc.), as well as glutathione and ascorbate metabolism (Schickler and Caspi 1999; Freeman et al. 2004; Gashemi et al. 2009, Agrawal et al. 2013). Recently, in research of Saleh and Plieth (2009), a novel methodology for studying summary parameters of plant antioxidant system have been proposed. ‘Antioxidant fingerprint’ protocol developed by above mentioned authors implies the application of five different assays which yield information about general antioxidant status of plants rather than exact data for single antioxidant species. In the present study approach of Saleh and Plieth was adopted with certain modifications to study antioxidant defense of Alyssum markgrafii shoots grown in vitro during long term stress caused by excess nickel. A. markgrafii is one of the nickel hyperaccumulating species originating from western Balkan which inhabits Dolomite soil types. Together with more than 50 other species of genus Alyssum (Brassicaceae) it belongs to the wide spread heavy metals accumulating plants, achieving extremely high nickel concentrations in their tissues (up to 30 g kg-1 of dry leaf), and are considered as candidates for Ni phytoremediation and phytomining technologies. In vitro culture of A. markgrafii shoots used for this study has been established from the seeds collected in plant populations from the serpentine slopes on the Mountain Go? (Serbia) where soil contained 5.16 – 6.25 µg Ni g-1 DW (Obratov et al 1997). Present research builds on our prior study where we reported remarkable hardiness of A. markgrafii shoots subjected to high nickel concentrations in growth medium (Vinterhalter and Vinterhalter 2005; Stanisavljevi? et al. 2012). Basic principles from the mentioned methodology was adopted, retaining luminol converting peroxidase assay (LUPO) in completely unchanged form, also as the innovative concept of specimen preparation which provides minimized oxidation of extracted compounds and reduces interferences of low molecular weight antioxidants with enzyme assays. To estimate antioxidant capacity of low molecular weight fraction we applied total antioxidant capacity assay (TAC). That was accomplished using improved kinetic DPPH assay (Cheng et al. 2006) which takes into account both kinetic and thermodynamic properties of the reactions between the DPPH radical and various antioxidant compounds present in the extract. This assay allows both fast and slow action antioxidants to react with DPPH radical leading to more accurate estimation of antioxidant capacity (Cheng et al. 2006). Also the well-established assays for catalase, superoxide dismutase and glutathione reductase activity has been applied. Additionally we quantified selected morphological parameters, nickel content as well as the level of lipid peroxidation, glutathione content and two fractions of phenolic compounds, including cell wall-bound and total soluble fraction.
Our main objective was to determine the changes in key groups of enzymes and metabolites which protect Alyssum markgrafii shoots from oxidative damage during long term exposure to excess nickel, using novel methodology for sample preparation which minimizes oxidative changes in samples and improves the accuracy of the obtained results.

Materials and Methods
Plant growth and treatments
In vitro shoot cultures of Alyssum markgrafii O.E. Shulz used in the present study have been established previously (Vinterhalter and Vinterhalter 2005). Aseptic cultures were grown on Murashige and Skoog (1962) medium with addition of benzyladenine (0.2 mg L-1). Experimental treatments with NiCl2 × 6H2O in concentrations of 0, 0.5 and 1 mM were conducted on 15 mm long shoot explants in duration of 35 days. Growth conditions were: 25 ± 2°C, light/dark photoperiod 16h/8h and photon flux density of 45µ mol m2 s-1. All treatments were repeated in three independent biological replicates with 30 explants in each.

Morphological parameters, nickel and chlorophyll content
Determined morphological parameters of A. markgrafii cultures were: fresh and dry mass of the shoots, length of the main shoot and number of formed axillary branches. For the fresh mass determination shoots were rinsed in demineralized water and gently dried using cellulose wool than weighed with accuracy of 10-4 g using analytical balance. For dry mass determination shoots were dried at 65°C until constant mass prior to measurement. Ni2+ content in shoots was determined by atomic absorption spectrometry and following material was prepared by method of Krishnamurty et al. (1976). The determination of the Ni concentration was conducted using the atomic absorption spectrometer SensAA (GBC scientific equipment). Ni was determined by means of the standard solution Fluka Analytical, production number 42242 (Lot BCBD5620). The used reference material was MERCK ICP Multi-Element Standard Solution IX CertiPUR 1,09494-0100 Lot. No.:HC140746. While taking measurements, as to avoid memory effect, a thorough washing out of the equipment was conducted between sample measurements for zero absorbance. Concentration of total chlorophyll was measured according to the previously described protocol (Ritchie 2008). The results were expressed as means ± standard deviations (SD).

Lipid peroxidation
Lipid peroxidation was evaluated using TBARS assay according to the method of Heath and Packer (1986) with modifications described by Du and Bramlage (1992). TBARS content was calculated according to the following formula:
TBARS (nmol mL-1) = (A532nm – A600nm) – (A440nm – A600nm) × 0.0571 / 157 000 × 106
Glutathione content
Glutathione content was assayed according to the previously described method of Noctor et al. (2016) using glutathione reductase and 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) reagent. Absorbance was recorded at 412 nm and results were calculated using glutathione standard curve.

Total soluble phenols and cell-wall bound phenols
Content of total soluble (TSPC) and cell-wall bound phenols (TBPC) was determined according to Sanchez-Rangel et al. (2013). This method allows calculation of corrected values for phenolic content by subtracting the reducing activity of ascorbic and dehydroascorbic acid present in plant extracts. For determination of soluble phenols, 80% methanol was used as a solvent for preparation of extracts. Homogenized shoots (0.5 g) were extracted with 10 mL of ice cold methanol/water mixture (80:20) in dark at 4ºC for 3h with constant shaking. After extraction supernatants were separated by centrifuge at 5000 × g for 5 min and kept. Cell-wall bound fraction of phenols (TBPC) was extracted according to de Ascensao and Dubrey (2003). The residue from the extraction of soluble phenolics (0.2 g) was heated at 60°C for 90 min with twenty volumes of 1M NaOH, and then concentrated HCl was added to achieve pH ; 2.0. After the acidification the mixture was extracted twice with 7.5 mL of diethylether then evaporated to dryness and dissolved in 1 mL of 80% methanol. Prepared extracts (15 µL) were added to microplate wells, mixed with 240 µL of demineralized H2O and 15 µL of Folin-Ciocalteu’s solution, incubated for 3 min and absorbance was red at 765 nm. From the obtained absorbance ascorbic acid (AA) concentration was calculated using ascorbic acid standard curve (0.1 – 3 mM). In the next step 30 µL of 1N Na2CO3 was added to each well, incubated for additional 2h at room temperature and the absorbance was recorded on the same wavelength. Phenolic content (PC) was then calculated using chlorogenic acid (CHA) standard curve. It has been determined that 1mg of ascorbic acid possessed reducing capacity equivalent to 1.43 mg of CHA, and corrected values for phenolic content were obtained by subtracting the values obtained for AA multiplied by 1.43 from the PC values. Corrected phenolic content for both soluble and cell wall-bound phenols was expressed as mg CHA g-1.

Analysis of UV/vis-absorbing compounds
Absorption spectra of plant extracts in UV/vis spectral region was recorded according to the previously described protocol of Szafranska et al. (2012) with minor modification. For this purpose 8-10 plants from each flask (0, 0.5 and 1 mM Ni2+) were pooled and homogenized in liquid nitrogen. Powdered samples (0.5 g) were extracted with 20 mL of 80% methanol in 50 mL tubes filled with argon (Ar), during 2h at +4°C with constant shaking. After extraction supernatants was separated by centrifuge for 10 min at 5000 × g, +4°C and the absorption spectra (200-800nm) were recorded immediately on UV/vis spectrophotometer using quartz cuvettes.

Preparation of extracts for “antioxidant fingerprint”
Frozen shoots were ground into the fine powder with mortar and pestle using liquid nitrogen. One gram of powdered samples was mixed with 10 mL of cold and degassed buffer containing 100 mM Tris-HCl pH 6.8, 2mM calcium chloride and 1mM Triton X-100. The mixture was vigorously shaken during 2 min and then vacum-filtered through cellulose filter under argon atmosphere to minimize the oxidation. This crude extract was divided in two aliquots: one of them was subjected to membrane filtration (intended for TAC assay with DPPH radical) and the other was dialyzed (intended for LUPO, SOD and CAT assay). For TAC assay the crude extract was filtered again using Amicon® Ultra-15 filter with 10 kDa cut off, by at 5000 × g for 20 min, under Ar atmosphere to prevent oxidation. For the enzyme assays (LUPO, CAT and SOD) the other portion of crude extract was dialyzed in dark at 5°C against two hundred-fold volume of cold assay buffer with three exchanges (dialysis membrane with 12.5 kDa cut off). This step is necessary to avoid interference of low molecular weight antioxidants with the enzyme assays.
Total antioxidant capacity (TAC)
We proposed kinetic version of DPPH assay for determination of hydrogen-donating potential of low molecular weight antioxidants. In our assay we chose mixture which does not exceeds 50% of aqueous medium, since 1:1 water-methanol mixture was determined to be optimal for lipophylic and hydrophilic antioxidants in DPPH assay. Additionally, in this water-methanol ratio, the coagulation and precipitation of DPPH is avoided (Magalhaes et al. 2008). In complex plant extracts, which we used, concentration of chlorophylls, carotenoids and xanthophylls was reduced to minimum due to extraction of plant material in Tris-HCl, so the interference of these compounds with DPPH absorbance was avoided. Relative DPPH radical scavenging capacity (RDSC) was determined by the method of Cheng et al. (2006). Reaction mixture consisted of 100 µL plant extract diluted with distilled water in different proportions. Five different concentrations (10, 20, 40, 60 and 80 µL) of extract and standard (Trolox) have been used. Dilutions of extracts and standards were mixed in microplate with 100µL of 208 µM DPPH radical solution. Blank consisted of methanol and control sample contained equal amounts of methanol and DPPH solution. Absorbance was continuously recorded each minute for 1.5 h at 515 nm. The percent of quenched DPPH absorbance in each time point was determined using following equation:
%DPPH quenched = 1- (Asample-Ablank)/(Acontrol-Ablank) × 100
The values of DPPH in percent quenched in different time points for each extract and standard were plotted against reaction time. Area under obtained curve (AUC) was calculated as follows:
AUC = 0.5 f0 + (f1 + f2 + f3 +…+fi-1) + 0.5 fi
Where f0 represents DPPH quenched at the start of the measurement and fi represnts DPPH quenched at reaction time i. Relative radical scavenging capacity (RDSC) was calculated as follows:
RDSC (µM TE g-1FW) = AUCsample/AUCtrolox × molarity trolox/mass sample
Luminol-based peroxidase assay (LUPO)
Assay was conducted according to the method of Saleh and Plieth (2009). Reaction buffer contained the mixure of 0.1 M Tris-HCl bufferpH 8.6, 2 mM calcium chloride and 1mM Triton X-100. After dialysis, the samples were diluted with reaction buffer and 300 µL aliquots of the diluted samples were mixed with 300µL of 1mM luminol in reaction buffer and background luminescence was recorded. Reaction was started by addition of 16mM hydrogen peroxide in reaction buffer. Luminiscence was recorded, as counts per second, during 3 min than the light output was integrated. Obtained light output stood in linear correlation with the amount of luminol converting peroxidase activity.
Superoxide dismutase assay
All extracts were assayed for superoxide dismutase (CAT; EC1.15.1.1) activity photochemically using the assay system consisting of methionine, riboflavin, and NBT reported by Giannopolitis and Ries (1976). The absorbance increase was monitored at 560 nm. Specific activity of superoxide dismutase was calculated by the method of Giannopolitis and Ries (1976) and expressed in IU mg-1 of total protein.

Catalase assay
Catalase (CAT; EC 1.11.1.6) activity was determined by the previously described protocol of Aebi (1984), with the modifications of Noctor et al. (2016). Enzyme activity was expressed in IU mg-1 of total protein.
Glutathione reductase assay
Glutathione reductase activity (GR; EC 1.6.4.2) was measured according to the protocol of Noctor et al. (2016). Reaction solution consisted of 800 µL of 100 µM phosphate buffer with 1mM EDTA, pH 7.5 and 10µL of 10 mM NADPH mixed with 100 µL of dialyzed extract. Reaction was started by addition of 10 µL of 50 mM GSSG. Absorbance decrease was monitored for 2 min at 340 nm. Specific activity of glutathione reductase was determined using NADPH molae extinction coefficient (?=6200M-1cm-1).

Protein content
Protein concentration was measured using Bio-Rad assay kit (Bio-Rad, Hercules, CA, USA) and bovine serum albumin as standard, according to the protocol of Bradford (1976).

Statistical analysis
Experiments in the present study were replicated at least three times and the data were presented as means ± SD. Results were processed using ANOVA and Duncan’s multiple range tests in the SPSS 19 software to determine significant differences at P ? 0.05.
Results
Morphological parameters, nickel and chlorophyll content
Effects of Ni2+ treatment were evaluated by its influence on morphological and biochemical parameters as summarized in Table 1. All applied treatments resulted in healthy looking plants with no observable traces of necrosis, although with significantly reduced biomass and mild chlorosis (Fig. 1). Considering effects of Ni2+ treatments on fresh and dry mass of shoots, it can be observed that 0.5 mM treatment provoked statistically significant decrease in shoot fresh mass which was even more pronounced in 1 mM Ni2+ treatment. On the other hand 0.5 mM Ni2+ also induced a decrease in dry mass but 1 mM treatment did not caused any additional decrease as it can be seen from the Table 1. Changes in main shoot length and number of side branches upon treatments were also observed. Both treatments induced significant decrease in shoot length and number of side branches, but there were any significant differences in plants treated with 1mM and 0.5mM Ni2+, as it can be seen from Table 1. Determination of total chlorophyll content revealed significant decrease upon 0.5 mM and 1 mM treatments which induced 43 and 56% reduction respectively, confirming visually observed chlorosis. Determination of Ni2+ concentration by atomic absorption spectrometry indicated notable accumulation upon 0.5 mM and 1 mM treatments (1121 and 2470 µg g-1 respectively).
Lipid peroxidation, total glutathione and phenolic content
Peroxidation of lipids in A. markgrafii shoots was estimated through evaluation of thiobarbituric reactive compounds (TBARS) (Table 1.). Treatment with 0.5 mM nickel produced consistent increase in TBARS content of about 1.4 fold which was similar to TBARS level observed upon 1 mM treatment where we found no statistical difference from 0.5 mM treatment, although the trend of further increase was observable. Analysis of glutathione content revealed slight elevation upon 0.5 mM Ni2+ treatment while the 1mM treatment showed no significant difference in glutathione content compared to the control group. Results of total soluble phenolic content (TSPC) measured by ascorbate corrected F-C assay were shown in Table 1. Statistically significant decrease in TSPC, compared to control, was recoded in plants uppon 0.5mM Ni2+ as well in plants treated with 1 mM Ni2+. Significant differences in TSPC between two treatments have not been observed. These results were in concordance with spectroscopic quantification of UV/Vis absorbing compounds (Fig. 2) which revealed one representative maximum absorbance peak at 267 nm, most probably reflecting phenolic acids and other phenolic compounds, severely reduced upon Ni2+ treatment in both experimental groups as well as 370 nm peak which most probably originates from the flavonoids. Other observed peaks 410-665 nm originated from the chlorophylls, carotenoids and other pigments as well as their oxidized forms were also reduced upon treatments. On the other hand total bound phenolics (TBPC) were decreased compared to control only after 1 mM Ni2+ treatment (Table 1.), and the ratio between TSPC and TBPC was slightly changed in favor of bound fraction during 0.5 mM Ni2+ treatment.

Antioxidant parameters
As it can be seen from presented values in Table 2. and the polygonal plot on Fig. 3 referred as “antioxidant fingerprint” luminol converting peroxidase (LUPO) activities recorded in A. markgrafii shoots were increased 1.2 fold upon 0.5 mM nickel treatment compared to control while 1 mM treatment provoked a significant reduction of LUPO activity, lowering its value below the values of the control plants. Activities of CAT and SOD also presented as corners of the polygon were lowered significantly in both groups of treated plants compared to control. Observed decrease in activity of these two antioxidant enzymes was more pronounced during 1 mM Ni2+ treatment than in case of 0.5 mM treatment. On the other hand glutathione reductase showed significant variation of activity depended on level of Ni2+ present in medium. While it was increased around 1.3 fold upon 0.5 mM treatment, after 1 mM treatment activity dropped below the level recorded in the control exhibiting similar behavior as LUPO. Total antioxidant capacity (TAC) of low molecular fraction ; 10 kDa, measured by DPPH kinetic assay revealed increased antioxidant capacity of about 20% only in plants treated with 0.5 mM Ni2+, while 1 mM Ni2+ treatment showed no statistical difference compared to control.
Discussion
Observed reduction in fresh mass upon treatment was not accompanied with reduction in dry mass as Ni2+ concentration elevated from 0.5 to 1 mM. Obtained result suggests on water loss in plants treated with 1 mM Ni2+ which might be the consequence of severe membrane damage as it was observed previously by Schickler and Caspi (1999). Observed decrease in ability to retain solutes has been demonstrated in previous studies as one of prominent changes during senescence. Recorded decrease in fresh mass, dry mass, shoot length, side branches length as well as drop of total chlorophyll content and accumulation of Ni2+ in shoots was expected and in concordance with our previous experiments (Stanisavljevi? et al. 2012). This was also similar with the observations of Ingle et al. (2005) who determined that 0.3 mM nickel was near to the maximal concentration which plants could bear without severe reduction in growth. On the other hand, Antonkiewicz et al. (2016), in their experiments on lettuce, observed an increased yield upon nickel treatment, although the applied concentrations in mentioned study were lower than in our experiments. Our previous research revealed that A. markgrafii shoots could survive five week treatment with 1-8 mM Ni2+, accumulating up to 24 900 ppm of Ni2+ (Stanisavljevi? et al. 2012). In mentioned study shoots subjected to five weak treatments were fully recovered upon transfer to growth medium without nickel, resuming normal growth, indicating on reversibility of damaging effects caused by excess nickel. In the present study, enhanced lipid peroxidation upon 0.5 mM Ni2+ treatment indicated by TBARS increase was also expected and previously described by other authors (Boominathan and Doran 2002; Sreekanth et al. 2013). Decrease in superoxide dismutase and catalase activity as well as TBARS increment clearly indicated on leaf senescence (Dhindsa et al. 1981), which was not observable at the first sight by inspecting the plants. On the other hand observed increased glutathione content (GSH) recorded after 0.5 mM Ni 2+ treatment was in collision with the observed level of oxidized lipids showing that glutathione itself did not have the capacity to protect of cellular lipids from oxidation, especially in situations where catalase activity was detrimentally affected by the presence of nickel, which resembles on behavior manifested by non-accumulator plants (Boominathan and Doran 2002). Despite observed behavior, the important role of glutathione in minimizing the effects of oxidation provoked by nickel must not be overlooked, especially knowing the fact that according to the classification of metal ions into weak and hard Lewis acids, nickel belongs to the intermediate class, shifting towards hard Lewis acids, which makes him an appropriate candidate for binding to aromatic nitrogen and SH-groups (Seregin and Kozhevnikova 2006). In the previous reports on nickel hyperaccumulator Thlaspi goesingense, Freeman et al. (2004) observed that changes in cysteine and GSH content were highly related with nickel accumulation and with the ability of plants to overcome its oxidative effects.

The rationale to undertake this study was to reveal the most prominent changes in antioxidant system of A. markgrafii which allows this hyperaccumulator plant to overcome Ni2+ toxic effects. Our current understanding of adaptation to abiotic challenges, such as nickel toxicity, at least considering antioxidant system, assumes genetic induction and mobilization of many counterparts which participates in this elaborate network (Saleh and Plieth 2009). A shift of function which is reflected for instance in down regulation of the selected counterpart in favor of another, was also observed. As it can be seen from Fig. 2, which represents antioxidant “fingerprint”, any corner of a polygonal plot which expands is compensated by a contraction of some other corner of the plot. Thus some of the parameters are changing together often in anti-parallel directions under stress conditions. For instance increase in LUPO activity upon 0.5 mM nickel treatment was accompanied with the simultaneous drop in catalase and superoxide dismutase activity which could be of great importance. Decrease in CAT and SOD activity recorded in the our study are in compliance with previous report of Gashemi et al. (2009), who determined decreased CAT, SOD and APX activity upon 0.35 mM nickel treatment applied to nickel hyperaccumulator Alyssum infantum grown in vitro. They also suggested that Ni2+-mediated alterations in Fe partitioning between root and shoot, showed in mentioned study, might be the cause of decreased activities of SOD, CAT and APX which require Fe for their function. On the other hand there is a possibility that superoxide and hydrogen peroxide formed might be transformed into reactive hydroxyl radical especially in the presence of non-sequestrated nickel in high concentration (Ingle et al. 2005). Boominathan and Doran (2002) also observed reduction of CAT, SOD and APX activity in Alyssum bertolonii roots exposed to 25 ppm Ni2+, which was not a typical response of ROS scavenging enzymes during heavy metal induced stress. Down regulation of antioxidant enzymes could be a consequence of interaction with nickel ions or reactive oxygen species as well as the result of enzyme regulation by stress-related factors (Boominathan and Doran 2002). According to the previously published observations of Schickler and Caspi (1999) Ni2+ tolerance mechanisms of hyperaccumulator plants most probably involve removing of ROS or prevention of their formation which ultimately result in reduced need for enzymatic antioxidant defense. On the other hand Boomintan and Doran (2002) suggested that this might not be the case since they recorded substantial amounts of hydrogen peroxide in nickel treated roots of A. bertolonii. They also hypothesized that the reactions in which ROS are produced could be better compartmentalized in hyperaccumulator plants minimizing their interaction with other biomolecules, but it could not be the situation in case of membrane permeable species such as hydrogen peroxide.

Peroxidases class III, evaluated by LUPO assay are the enzymes which could consume, but also to produce H2O2. A simultaneous decrease in phenolic compounds and increase in peroxidase activity after 0.5 mM nickel treatment could suggest the role of the peroxidase, polyphenols and ascorbate in removal of reactive oxygen species during dehydration (Takahama and Oniki 1997). Increase in LUPO activity could be also explained by other factors such as possible alosteric regulation of peroxidase class III enzymes by nickel, which was documented previously by Pintus et al. (2008). Mentioned study revealed that in vitro incubation of peroxidase from Euphorbia characias, with Ni2+ leads to reversible inhibition of the enzyme, while the mutual presence of nickel and calcium ions increases peroxidase activity. These data could cast new light to mechanisms of peroxides class III activation during nickel hyperaccumulation but this theory need to be confirmed through further elaborate investigations. Glutathione reductase (GR) activity was also increased during the 0.5 mM nickel treatment, coinciding with the observed increase in glutathione content. The similar behavior was also observed in T. goesingense by Freeman et al. (2004) who determined that high GSH level in this hyperaccumulator plant coincide with high activity of GR. Together with our findings this supports the hypothesis that glutathione reductase and glutathione have the pivotal role in Ni2+ hipperaccumulation as members of the wider network which enhances the nickel tolerance in hyperaccumulating species.

Total antioxidant capacity (TAC), determined using kinetic DPPH assay, was slightly increased by following 0.5 mM Ni2+ treatment compared to control, while 1 mM treatment provoked reduced TAC value which was no significantly different from the control. TAC correlated positively with GSH content in case of 0.5 mM treatment which was expected considering the TAC assay specificities. This assay allows both fast and slow action antioxidants (MW ; 10 kDa) to react with DPPH radical which excludes the possibility that some of the slow action antioxidants is not quantified properly. Importantly the fact that TAC runs parallel with GSH and GR might indicate that the major fraction of low molecular weight antioxidants in A. markgrafii plants subjected to nickel treatment is represented by glutathione, as it was observed previously by Saleh and Plieth (2009) on different models of abiotic stress.

Considering quantification of total soluble phenolics (TSPC) in the present study, which was accomplished using two different methods (F-C assay and analysis of UV/Vis absorbing compounds), the two similar outcomes were recorded. Although we applied improved F-C assay which eliminates non-specific absorbance originating from ascorbic acid, the results should be interpreted with great caution since it has been reported previously, by several authors, that many compounds other than phenolics could reduce F-C complex (Prior et al. 2005). Substances, aside from ascorbic acid, which are reactive towards F-C reagent, are: aromatic amines, organic acids as well as some inorganic compounds and ions such as Fe2+, thus the possibility that some other non-phenolic compounds are at least partially responsible for TPC increase in plants treated with 0.5 mM Ni2+ certainly could not be excluded. On the other hand decreased TPC, detected by UV method in treated plants do not necessarily indicate on reduced phenolic synthesis, but could be a consequence of intensified polymerization of phenolic compounds which could not be recorded in extracts due to insolubility of formed complexes in applied organic solvents. Application of nickel treatments also reduced fraction of total bound phenolics (TBPC), but statistically significant decrease was observable only after 1 mM Ni2+ treatment. Slightly changed ratio between TSPC and TBPC, in favor of bound fraction during 0.5 mM Ni2+ treatment might suggest on intensified lignification process, which was previously reported by Ková?ik et al. (2011). This could be supported by the observed increase in activity of LUPO upon 0.5 mM Ni2+ treatment, since this family of enzymes can participate in peroxidative cycle during which phenolic compounds are oxidized into phenoxyl radicals which can build lignin, contributing to the process of cell wall stiffening (Cosio and Dunnand 2009). According to literature the effect of Ni2+ treatment on soluble and bound phenols in hypperaccumulator plants seems to be more pronounced in roots than in other plant organs. During the previous study of Ková?ik et al. (2009) conducted on Matricaria chamomilla plants authors assumed that polyphenols present in the root system could prevent nickel accumulation in shoots. Having in mind that our study was performed on explants without root system it is evident that phenolic compounds could not exhibit similar role, but it can be assumed that they might contribute in the process of cell wall lignification at least during 0.5 mM Ni2+ treatment.

Conclusion
In A. markgrafii major effects of five week exposure to 0.5 mM Ni2+ were decrease in antioxidant enzyme activities with the exception of glutathione reductase and peroxidases class III which could not be considered as a class of antioxidant enzymes sensu stricto since it can also produce H2O2. Elevated total antioxidative capacity during 0.5 mM Ni2+ treatment is most probably the result of glutathione accumulation rather than increased synthesis of phenolic compounds, which was supported by detection of increased glutathione reductase activity and elevated total glutathione content. Peroxidases class III might be responsible for the observed drop of soluble phenol content taking a part in their polymerization which renders them undetectable in tissue extracts using standard colorimetric methods. Never the less, these enzymes play important role in nickel tolerance together with low molecular weight antioxidants among which glutathione has an important integral place.

Author contribution B. Vinterhalter and D. Vinterhalter propagated in vitro culture and designed the experiments J. Savi? conducted the treatments and determined morphological parameters, M. Simonovi?. and J. Sen?anski. determined nickel content of shoots. N. Stanisavljevi?, Ž. Jovanovi?, J. Miljuš-Ðuki? and S. Radovi? performed all other experiments and wrote the manuscript.

Acknowledgments Our study was supported by The Ministry of Education, Science and Technological Development of Republic of Serbia. Grants No. 173015 and 173005.
Conflict of interest The authors of the manuscript have no conflicts of interest to disclose.

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Figure captions
Fig. 1 Shoots of A. markgrafii grown in vitro. a – Control without Ni2+. b – In the presence of 0.5 mM Ni2+. c – In the presence of 1 mM Ni2+.

Fig. 2 UV/vis absorption spectra of A. markgrafii extracts. a) Control b) 0.5 mM Ni2+ treatment. c) 1 mM Ni2+ treatment. Peak centers: 1 – 230.0 nm, 2 – 267.0 nm, 3 – 369.8 nm, 4 – 409.7 nm,
5 – 536.5 nm, 6 – 607.5 nm, 7 – 665.0 nm.

Fig. 3 Fingerprint of the antioxidant status. The changes of five screened parameters in percentage of the control are represented by five radial axes of the polygonal plot. Each corner of the plot represents average value of the marked parameter obtained from three technical replicates run on material pooled from three independent experiments. Standard deviations are given in Table 2.

Table captions
Table 1 Effect of Ni2+ on selected morphological and biochemical parameters. Values are means of three replications (N = 3 ± SD). Values marked by the same small case letter are not significantly different at P ; 0.05 (Duncan’s test).

Table 2 Antioxidant status parameters. Values are means of three replications (N = 3 SD). Values marked by the same small case letter are not significantly different at P < 0.05 (Duncan’s test), * Luminiscence counts

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