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. 2017 Dec 18;41(6):868–880. doi: 10.3906/biy-1704-39

Mg deficiency changes the isoenzyme pattern of reactive oxygen species-related enzymes and regulates NADPH-oxidase-mediated ROS signaling in cotton

Rengin ÖZGÜR UZİLDAY 1, Barış UZİLDAY 1, Tolga YALÇINKAYA 1, İsmail TÜRKAN 1
PMCID: PMC6353280  PMID: 30814852

Abstract

The aim of this work was to investigate changes in isoenzyme patterns of enzymes related to reactive oxygen species (ROS) detoxification such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), and glutathione-S-transferase (GST) in cotton under Mg deficiency. Moreover, we aimed to elucidate how a ROS producer, NADPH oxidase (NOX), responds to changing Mg levels. Cotton plants were grown with different concentrations of MgSO 4 in hydroponic medium to create nutrient deficiency (0, 75, 150, 1000 µM Mg). Gradual decreases in growth and photosynthetic rates were observed with declining Mg concentrations and 0, 75, and 150 µM Mg increased oxidative stress as evidenced by H O and 2 2 lipid peroxidation. Total activities of SOD, CAT, POX, APX, GR, and GST were increased while NOX activity was decreased with Mg deficiency. The activities of GR and GST were highest in plants treated with 0 µM Mg, indicating excess use of glutathione for redox regulation. The most striking results were the changes in isoenzyme patterns of SOD, NOX, POX, and GST. For example, a new Cu/ ZnSOD isoenzyme was induced in plants treated with 0 µM Mg. Cotton plants adapt to Mg deficiency by changing the intensity of existing isoenzymes or inducing new ones.

Keywords: Antioxidant enzymes, cotton, Gossypium hirsutum, magnesium deficiency, magnesium starvation, NADPH oxidase, redox regulation

1. Introduction

Mg deficiency is increasingly becoming an important factor that limits plant growth in agricultural areas, especially for soils that are only fertilized with N, P, and K (Cakmak and Yazici, 2010) . In addition, soil Mg is being removed with harvested parts of the plants, causing depletion of Mg in soils. Mg is the central atom of chlorophyll, which makes Mg an indispensable nutrient for plant life. Mg also acts as a cofactor for many enzymes that take roles in different metabolic processes (Verbruggen and Hermans, 2013) . Mg deficiency influences plant performance in different ways such as decrease in photosynthetic activity due to decreased chlorophyll formation, impairment of phloem loading, and change in partitioning of photoassimilates between roots and shoots (Cakmak and Yazici, 2010) . Mg deficiency prevents the proper function of photosystems and absorption of light energy, which may cause excessive excitation of photosystems leading to enhanced production of reactive oxygen species (ROS) such as superoxide anion radical (O2.-), singlet oxygen (1O ), and hydrogen peroxide (H2O2) (Guo et al., 2016) .

Excess production of these ROS causes oxidative damage in chloroplasts, which eventually leads to cell death and necrosis in the leaves (Apel and Hirt, 2004) . It is known that Mg is also required for fixation of CO 2 during photosynthesis (Cakmak and Yazici, 2010) . For example, the Mg ion needs to be coordinated to the carbamylated residue of RuBisCo to generate its active form before the catalytic cycle (Stec, 2012) . Therefore, Mg deficiency also effects the balance between light and carbon reactions of photosynthesis, which might also result in oxidative stress in chloroplasts. Plants have developed various defensive mechanisms to cope with the harmful effects of ROS. Reduction of O2.- and H2O2 to water is accomplished by enzymatic antioxidants such as superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) and nonenzymatic antioxidants such as ascorbate, glutathione (GSH), carotenoids, and tocopherols (Mittler et al., 2004) . However, besides their damaging effects, it is now widely accepted that ROS might also act as signals at low levels. These signals are oeftn produced by ROS-producing systems such as NADPH oxidases, which are located at the plasma membrane (Mittler et al., 2011) . The effects of Mg nutrition on antioxidant defense and redox regulation was investigated in maize (Tewari et al., 2004) , pepper (Anza et al., 2005) , mulberry (Tewari et al., 2006) , and citrus (Tang et al., 2012) . In maize, Mg deficiency increased CAT, APX, and SOD activities but not POX activity, although H2O2 content was not changed (Tewari et al., 2004) . Anza et al. (2005) investigated the effects of Mg deficiency on antioxidant system in Capsicum annuum and demonstrated that Mg deficiency increased the activities of SOD, GR, APX, and DHAR. Moreover, SOD and APX activities of mulberry plants were increased under Mg deficiency (Tewari et al., 2006) . However, all of these works focused solely on the total activities of enzymatic antioxidants and they did not assess changes in their isoenzyme patterns, which is also an important factor for the adjustment of plant cells to changes in the cellular redox environment. In addition to this, roles of ROS in signaling, especially that mediated by NADPH oxidases, is an unexplored topic under Mg deficiency. Therefore, it is imperative to elucidate changes in isoenzyme patterns of antioxidant enzymes and NADPH oxidase under Mg deficiency.

For this aim, we used cotton (Gossypium hirsutum L.), which is an economically valuable and widely cultivated bfier plant all around the world. In addition to its economic value, the effects of Mg deficiency on cotton have not been studied before in regards to redox regulation and antioxidant defense. During the experiments plants were treated with different concentrations of Mg (0, 75, 150, and 1000 µM MgSO4) and changes in activities and isoenzymes of SOD, CAT, POX, APX, GR, MDHAR, DHAR, and NADPH oxidase, accompanied with markers of oxidative stress such as lipid peroxidation and H2O2, were investigated. In parallel to this biochemical analysis, photosynthetic efficiencies of plants treated with different Mg concentrations were also determined with gas exchange measurements.

2. Materials and methods

2.1. Plant material and growth conditions

Cotton (Gossypium hirsutum L. ‘Nazilli 84-S’) plants were grown hydroponically in Hoagland solution with sufficient (1000 µM MgSO4), low (150 µM MgSO4), and deficient (75 µM MgSO ) Mg levels and in the absence of MgSO4 in a controlled plant growth chamber (light intensity: 450 µmol m–2 s–1; light/dark period: 16/8 h; temperature: 24/20 °C, relative humidity: 60%). These treatment groups are abbreviated as 1000 Mg, 150 Mg, 75 Mg, and 0 Mg throughout the manuscript. after 3 weeks of growth, parameters were measured and leaves were sampled for further biochemical analysis. Adequate concentrations of K2SO4 were supplied for Mg-deficient plants for complementing SO4. Nutrient solutions were changed every 3rd day. Mature leaves were used for elemental and biochemical analysis and leaf samples were flash-frozen in liquid nitrogen and stored at –80 °C until further analysis.

2.2. Growth analysis

Six random plants for each group were used for the growth analyses and were separated into shoot and root fractions. The fresh weight (FW) and dry weight (DW, after the samples were dried at 70 °C for 72 h) of shoots and roots were measured.

2.3. Analysis of nutrients

Shoot concentrations of Mg were determined by inductively coupled plasma optical emission spectrometry (Vista-Pro Axial, Varian Pty. Ltd., Mulgrave, Australia) following acid digestion in a closed vessel microwave system (MARSXpress, CEM Corporation, Matthews, NC, USA).

2.4. Leaf relative water content

after harvest, six leaves were obtained from cotton plants and their FWs were determined. The leaves were floated on deionized water for 6 h under low irradiance and then the weights of the turgid leaves (TW) were measured. DW was determined after leaves were dried in an oven. The relative water content (RWC) was calculated by the following formula: RWC (%) = [(FW – DW) / (TW – DW)] × 100

2.5. Gas exchange and chlorophyll fluorescence measurements

Gas exchange and chlorophyll fluorescence data were measured under constant conditions as CO was 385 ppm, cuvette temperature was 25 °C, relative humidity was 60%, and saturating light intensity was 1500 PAR in the hours (4 h) at the middle of the light period. The WALZ GFS-3000 gas exchange system was used for measurements.

2.6. H2O2 content

H2O2 was measured using eFOX reagent (250 µM ferrous ammonium sulfate, 100 µM xylenol orange, 100 µM sorbitol, 1% ethanol (v/v)) (Cheeseman, 2006) . Ice-cold acetone containing 25 mM H2SO4 was used for extraction. Samples were centrifuged for 5 min at 3000 × g at 4 °C and 950 µL of eFOX reagent was used for 50 µL of supernatant. Reaction mixtures were incubated at room temperature for 30 min and then absorbance at 550 and 800 nm was measured. A standard curve prepared with known concentrations of H2O2 was used for calculations.

2.7. Glutathione contents

Leaf samples (0.1 g) were ground in liquid nitrogen and extracted with 1 mL of 0.2 N HCl at 4 °C according to Queval and Noctor (2007) . Samples were centrifuged at 16,000 × g for 10 min and 0.4 mL of 0.2 M NaOH in the presence of 50 µL of 0.2 M NaH2PO4 (pH 5.6) was added to the supernatant (0.5 mL) for neutralization. GSH content was determined using an enzyme cycling assay with GR by following the rate of NADPH spent by GR at 340 nm.

2.8. Lipid peroxidation

Lipid peroxidation levels were determined as thiobarbituric acid reactive substances (TBARS) and were detected according to Heath and Packer (1968) .

2.9. Enzyme extractions and assays

Enzyme extractions were performed at 4 °C. Leaf samples were ground to a fine powder in liquid nitrogen and then were homogenized in a 5:1 ratio (buffer volume:plant material) of 50 mM Tris-HCl, pH 7.8, containing 0.1 mM EDTA, 0.1% (v/v) Triton-X 100, 1 mM PMSF, and 2% (w/v) PVPP. Ascorbate (5 mM) was added to the homogenization buffer for APX activity assays. Samples were centrifuged at 10,000 × g for 10 min, and supernatants were used for the determination of protein content and enzyme activities. Protein contents were detected according to Bradford (1976) .

SOD (EC 1.15.1.1) activity was assayed by its ability to inhibit photochemical reduction of nitro blue tetrazolium (NBT) at 560 nm (Beauchamp and Fridovich, 1971) . One unit of SOD was defined as the amount of enzyme that inhibits 50% NBT photoreduction. CAT (EC 1.11.1.6) activity was estimated according to the method of Bergmeyer (1970) , which measures the initial rate of decomposition of H O2 at 240 nm (molar extinction coefficient: 43.6 M –1 cm2–1). The decrease in the absorption was followed for 1 min and 1 µmol H2O2 min–1 was defined as one unit of CAT. POX (EC 1.11.1.7) activity was based on the method described by Herzog and Fahimi (1973) . The increase in the absorbance at 465 nm due to oxidation of diaminobenzidine (DAB) was followed for 1 min. One unit of POX activity was defined as 1 µmol H2O2 decomposed in 1 min. GR (EC 1.6.4.2) activity was measured according to Foyer and Halliwell (1976) . NADPH oxidation was followed at 340 nm. Activity was calculated using the extinction coefficient of NADPH (6.2 mM–1 cm–1). One unit of GR was defined as 1 µmol GSSG reduced in 1 min. APX (EC 1.11.1.11) activity was measured according to Nakano and Asada (1981) . The assay depends on the decrease in absorbance at 290 nm as ascorbate is oxidized. The concentration of oxidized ascorbate was calculated using an extinction coefficient of 2.8 mM–1 cm–1. One unit of APX was defined as 1 µmol ascorbate oxidized in 1 min. NOX (EC 1.6.3. 1) activity was measured according to Jiang and Zhang (2002) . The assay medium contained 50 mM Tris-HCl buffer, pH 7.5, 0.5 mM XTT, 100 µM NADPH.Na4, and 20 µg of protein. after the addition of NADPH, XTT reduction was followed at 470 nm. The corrections of background production were determined in the presence of 50 U of SOD. Activity was calculated using the extinction coefficient, 2.16 × 10 4 M–1 cm–1. GST (EC 2.5.1.13) activity was based on the method of Habig et al. (1974) . The reaction mixture contained 0.1 M Na-phosphate (pH 6.5), 1 mM GSH, and 1 mM CDNB. Absorbance was followed at 340 nm. Nonenzymatic activity was corrected by subtracting the spontaneous reaction rate in the absence of sample.

2.10. Identification of antioxidant isoenzymes

Equal amounts of protein were separated with nondenaturing polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970) .

SOD isoenzymes were determined by using 12.5% separating gel. Gels were stained with riboflavin and NBT according to Beauchamp and Fridovich (1973) . A SOD standard from bovine liver (Sigma Chemical Co.) was loaded too for calculation of the unit activity. Inhibitors of SOD before staining, such as 2 mM KCN and 3 mM H2O2, were used to detection of the different types of SOD (Vitória et al., 2001 ).

CAT isoenzymes were determined by using 10% separating gel (Woodbury et al., 1971) . The gels were treated with 0.01% H2O2 for 5 min, and then the gels were rinsed with distilled water and stained with 1% FeCl3 and 1% K3Fe(CN)6.

The electrophoretic separation of POX isoenzymes was performed on 10% separating gel according to Seevers et al. (1971) . The gels were treated with 200 mM Na-acetate buffer (pH 5.0) containing 1.3 mM benzidine and 3% hydrogen peroxide for 30 min at room temperature.

GR isoforms were determined by 7.5% native PAGE according to Hou et al. (2004). Each well contained 50 µg of protein and after separation the gels were incubated in 10 mM Tris-HCl (pH 7.9), 4 mM GSSG, 1.5 mM NADPH.Na4, and 2 mM DTNB for 20 min. GR activity was negatively stained by 1.2 mM MTT and 1.6 mM PMS for 5–10 min.

GST isoenzymes were identified according to Ricci et al. (1984) : 10% separating gels were used and after separation gels were incubated in 0.1 M K-P buffer (pH 6.5). Following this, gels were transferred to dye solution containing 4.5 mM GSH, 1 mM CDNB, and 1 mM NBT in 0.1 M K-P (pH 6.5) buffer. GST bands were stained with a solution containing 3 mM PMS prepared in 0.1 M TrisHCl (pH 9.6).

NOX isoenzymes were detected by NBT reduction method (Sagi and Fluhr, 2001) . Native PAGE was performed with 7.5% gels and each well contained 30 µg of protein. Gels were stained in 50 mM Tris-HCl buffer (pH 7.4), 0.2 mM NBT, 0.1 mM MgCl2, and 1 mM CaCl2 in the dark for 20 min. after that, NADPH.Na 4 at a final concentration of 0.2 mM was added to the dye solution and the appearance of blue formazan bands was observed.

The Vilber Lourmat gel imaging system was used to photograph the gels and then analyses were done with BioCapt software package (Vilber L ourmat). Three independent replicates of gels were performed.

2.11. Statistical analysis

The experiments were repeated two times independently, and each data point was the mean of three replicates (n = 6). All data obtained were subjected to a one-way analysis of variance (ANOVA), while the Tukey posttest was used to compare the statistical significance of different groups. Comparisons with P < 0.05 were considered significantly different and are shown with different letters in the figures. In all of the figures, the spread of values is shown as error bars representing standard errors of the means.

3. Results

3.1. Mg deficiency and Mg starvation decreased growth in cotton

A robust decrease was observed in fresh weights of shoots with decreasing Mg concentrations. The 0 Mg treatment decreased shoot fresh weight by 91%, whereas 75 Mg and 150 Mg decreased it by 26% and 20%, respectively (Figure 1A). Similar trends of decrease were also observed in dry weights of shoots, which were 88%, 34%, and 32% for 0, 75, and 150 Mg treatments, respectively (Figure 1B).

Figure 1.

Figure 1

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Growth and physiological parameters of cotton grown under Mg starvation (0 μM Mg), 75 μM Mg, 150 μM Mg, and 1000 μM Mg treatments: A) shoot fresh weight, B) shoot dry weight, C) root fresh weight, D) root dry weight, E) shoot length, F) root length, G) shoot Mg concentrations, and H) relative water content (RWC).

Root fresh weights also decreased with decreased Mg concentration in the growth medium: 87%, 45%, and 28% decreases were determined with 0 Mg, 75 Mg, and 150 Mg treatments, respectively, as compared to 1000 Mg (Figure 1C). The decreases in root dry weights were 90%, 46%, and 17% for the same conditions (Figure 1D).

The lengths of shoots were significantly decreased by lower concentrations of Mg in the growth medium. The 75 and 150 Mg treatments decreased the shoot length by 10% as compared to 1000 Mg treatment, whereas 0 Mg treatment lowered the length by 68% (Figure 1E). Root lengths were also decreased with Mg starvation by 16% in comparison to 1000 Mg conditions (Figure 1F).

Mg content was decreased by 80% and 68% in the 75 Mg and 150 Mg treatments, respectively. The 0 Mg treatment decreased it by 94% as compared to 1000 Mg (Figure 1G). Leaf RWC of plants was not aefcted by different concentrations of Mg.

3.2. Photosynthesis was inhibited by Mg deficiency in cotton leaves

The 0 Mg treatment decreased the assimilation rate by 91% as compared to 1000 Mg. On the other hand, 75 Mg decreased it by 75%, whereas 150 Mg decreased it by 38% as compared to normal conditions (Figure 2A).

Figure 2.

Figure 2

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Gas exchange and fluorescence parameters of cotton grown under Mg starvation (0 μM Mg), 75 μM Mg, 150 μM Mg, and 1000 μM Mg treatments: A) assimilation rate (A), B) Fv/Fm, and C) electron transport rate (ETR).

Fv/Fm decreased from 0.83 to 0.74 in plants treated with 0 Mg as compared to 1000 Mg and it decreased to 0.76 with 75 Mg and 0.8 with 150 Mg treatment (Figure 2B).

The 0 Mg treatment lowered the ETR by 60% as compared to 1000 Mg, whereas 75 and 150 Mg treatments decreased the ETR by 58% and 33% (Figure 2C).

3.3. Mg deficiency increased both H 2O2 content and TBARS levels

H O content of plants treated with 0 Mg was enhanced by 29%, while 75 Mg treatment increased it by 32%. Moreover, 150 Mg treatment increased H2O2 content by 13% as compared to 1000 Mg (Figure 3A).

Figure 3.

Figure 3

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A) H2O2 content and B) lipid peroxidation (TBARS) content of cotton grown under Mg starvation (0 μM Mg), 75 μM Mg, 150 μM Mg, and 1000 μM Mg treatments.

TBARS levels of cotton plants gradually increased with decreasing concentrations of Mg in the growth medium. The 0 Mg treatment increased TBARS by 64% while 75 Mg and 150 Mg increased it by 32% and 17% as compared to 1000 Mg (Figure 3B).

3.4. Mg deficiency altered superoxide anion radicalrelated metabolism by adjusting the activities and isoenzymes of SOD and NOX, while new isoenzymes appeared under Mg starvation

Significant increase was observed in the activity of SOD with the 0 Mg treatment. SOD activity was enhanced by 3-fold with 0 Mg as compared to 1000 Mg. The 75 Mg treatment did not change SOD activity significantly, while the lowest SOD activity was determined in 150 Mg-treated plants and it was decreased by 31% as compared to 1000 Mg (Figure 4A).

Figure 4.

Figure 4

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Activities and isoenzymes of O2 −-related enzymes of cotton grown under Mg starvation (0 μM Mg), 75 μM Mg, 150 μM Mg, and 1000 μM Mg treatments: A) the activity of SOD enzyme, B) native-PAGE separation of SOD isoenzymes, C) total NOX activity, D) native-PAGE separation of NOX isoenzymes. During densitometric analysis, for each isoenzyme 1000 μM Mg treatment was taken as 100% and other groups were calculated as compared to this.

A total of five SOD isoenzymes were detected in this study. All of the 5 isoenzymes were activated in 0 Mgtreated plants, whereas only MnSOD and FeSOD2 were determined in 75, 150, and 1000 Mg-treated plants. The activity of MnSOD increased with 0 Mg treatment. Moreover, the highest FeSOD2 activity was determined in 75 Mg-treated plants. FeSOD1, Cu/ZnSOD1, and Cu/ ZnSOD2 only appeared in 0 Mg-treated plants (Figure 4B).

The activity of NOX was decreased with 0 and 75 Mg by 53% and 18%, respectively. The 150 Mg treatment did not change the activity of NOX significantly (Figure 4C).

Four different NOX isoenzymes were detected in this study. NOX1 was only determined in 150 Mg and 1000 Mg plants, whereas the activity of NOX1 was decreased by 150 Mg as compared to 1000 Mg. NOX2 and NOX4 were found in all the treatments, while NOX3 only appeared in 0 Mg-treated plants (Figure 4D).

3.5. The activities of H 2O2-related antioxidant enzymes

were regulated with Mg deficiency The highest CAT activity was determined in plants grown with 0 Mg while 75 Mg-treated plants showed the lowest CAT activity. The activity of CAT was enhanced with 0 Mg treatment by 33% as compared to 1000 Mg-treated plants. However, 75 Mg treatment decreased it by 33% as compared to 1000 Mg. The 150 Mg treatment did not cause any significant change in the activity of this enzyme (Figure 5A). Only one CAT isoenzyme was determined during the study (Figure 5B).

Figure 5.

Figure 5

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Activities and isoenzymes of H2O2 scavenging enzymes of cotton grown under Mg starvation (0 μM Mg), 75 μM Mg, 150 μM Mg, and 1000 μM Mg treatments: A) the activity of CAT, B) native-PAGE separation of CAT isoenzymes, C) the activity of POX, D) native-PAGE separation of POX isoenzymes, E) the activity of APX. During densitometric analysis, for each isoenzyme 1000 μM Mg treatment was taken as 100% and other groups were calculated as compared to this.

The POX activity was increased with 0 Mg treatment by 3-fold as compared to 1000 Mg plants, and 75 Mg increased POX activity by 13% whereas no significant change was det1–ected with 150 Mg treatment (Figure 5C). No changes observed in the isoenzyme pattern of POX damong diefrnmen6t0treatment groups; however, the intensity of POX2 incr(Seased with 0 Mg treatment (Figure 5D).

3.6. Glutathione-related metabolism is regulated with Mg deficiency in cotton

Similar to POX activity, the activity of GR was also increased by Mg deficiency. The most significant enhancement of GR activity was determined with the 0 Mg treatment, which was 93% higher than that of 1000 Mg plants. The 75 Mg treatment induced the activity of GR by 33%, while 150 Mg treatment did not change the activity as compared to 1000 Mg conditions (Figure 6A). Two different GR isoenzymes were detected during the study. The activities of both GR1 and GR2 were increased by Mg deficiency (Figure 6B).

Figure 6.

Figure 6

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Activities and isoenzymes of glutathione-related enzymes and GSH content of cotton grown under Mg starvation (0 μM Mg), 75 μM Mg, 150 μM Mg, and 1000 μM Mg treatments: A) the activity of GR, B) native-PAGE separation of GR isoenzymes, C) the activity of GST, D) native-PAGE separation of GST isoenzymes, E) total glutathione content. During densitometric analysis, for each isoenzyme 1000 μM Mg treatment was taken as 100% and other groups were calculated as compared to this.

The 0 Mg treatment enhanced the GST activity by 39% as compared to 1000 Mg. The activity of GST was enhanced in response to Mg deficiency. The 75 Mg treatment increased GST activity by 12% as compared to 1000 Mg plants (Figure 6C). Four GST isoenzymes were determined in this work and activities of all of these isoenzymes were mostly induced by 0 and 75 Mg treatments (Figure 6D).

GSH content was increased with 0 Mg treatment by 60%, while an 80% increase was observed in 75 Mg-treated plants. The 150 Mg treatment did not cause a significant change in GSH content when compared to 1000 Mg (Figure 6E).

4. Discussion

Understanding nutrient deficiency responses and mitigating them is of great importance in improving agricultural production (Mueller et al., 2012) . Mg regulates nucleic acid synthesis, protein synthesis, and activities of ATPases, RuBisCo, and several other enzymes. Moreover, chlorophylls constitute Mg in their porphyrin rings and its deficiency effects the photosynthetic machinery, which also decreases the efficiency of other NADPH-dependent processes in chloroplasts, such as the Calvin cycle. Under these circumstances, ROS production related to the photosynthetic electron transfer system is a major factor that disturbs the cellular redox during Mg deficiency (Guo et al., 2016) . Plants can survive under these conditions only if the ROS scavenging system can cope with this excess ROS production (Apel and Hirt, 2004) .

Previous research demonstrated that oxidative stress occurs in Mg-deficient leaves, especially under high light conditions. In some of these works antioxidant responses of different plant species were examined (Tewari et al., 2004, 2006; Anza et al., 2005; Tang et al., 2012) . However, none of these works included a comprehensive picture of isoenzymatic regulation of antioxidant defense under Mg-deficient conditions. Moreover, the role of NADPH oxidases under nutrient deficiency is still not clear. Hence, in this study, we aimed to reveal the effects of Mg deficiency and starvation on cotton, especially in terms of isoenzyme regulation of antioxidant enzymes and ROS signaling components. Mg starvation was used to exaggerate the physiological and biochemical responses that are related to Mg. Also in conjugation with this, to support the redox-related data with the physiology of chloroplasts, we investigated the implications of Mg deficiency on the photosynthetic performance of cotton with gas exchange measurements.

It is known that Mg deficiency effects plant growth negatively in a wide range of plants, from Arabidopsis thaliana (Hermans and Verbruggen, 2005) to Pinus radiata (Laing et al., 2000) . Research suggests that root growth is inhibited earlier than shoot growth, but eventually shoot growth is impaired if the Mg deficiency is prolonged (Cakmak et al., 1994) . Similarly, in our work, Mg availability strictly restricted the growth of cotton, especially under 0 and 75 Mg conditions, which is evident by decreased shoot and root dry weights. However, although root biomass drastically decreased, the decrease in main root length was not proportional to the loss in dry weight, suggesting that the main root growth was not inhibited as much as that of the lateral roots. Mg deficiency generally does not induce increased surface area of roots (lateral roots, root hairs, etc.), which can be observed during P or N deficiency (Marschener, 1998) . Due to this, resources that would be allocated in lateral roots can be invested to maintain main root growth in cotton.

During Mg deficiency, the superoxide anion radical (O2.-)-related antioxidant system was substantially altered in cotton. SOD scavenges O .- and its activity is very important to mitigate the toxic effects of O 2.- produced during photosynthesis in chloroplasts. In the present work, increased activities of SOD indicated severe oxidative stress with Mg deficiency in cotton. These results are consistent with findings in other plants such as mulberry (Tewari et al., 2006) and maize (Tewari et al. 2004) , which were also subjected to Mg deficiency. Moreover, Anza et al. (2005) also observed that SOD activities were increased in response to Mg deficiency in pepper. In addition, Tang et al. (2012) demonstrated increased SOD activity with Mg deficiency in citrus plants. In the present work, besides increased activity, we also observed the new nutrient deficiency-induced isoenzyme of Cu/ZnSOD in 0 Mgtreated plants. Cu/ZnSOD is located in the cytoplasm and/or chloroplasts (Mittler et al., 2004) . This response demonstrates that besides increasing activities of available isoenzymes, induction of new isoenzymes might be required to adapt to changing redox environments of the cell caused by Mg deficiency.

NADPH oxidases (NOX) produce ROS in the apoplast in response to environmental stresses and due to this reason they are key components of ROS signaling in addition to their role in triggering oxidative burst (Mittler et al., 2011) . Previously, by using DPI, a specific NOX inhibitor, Chao et al. (2012) demonstrated that NOX-mediated ROS production increase within the first 3 h of Mg starvation. However, no direct evidence for NOX activity was provided. In the present study, the lowest activity was determined in 0 Mg-treated plants. This contrasting result might be a consequence of the difference in the durations (3 h vs. 3 weeks) of the experiments. NADPH oxidases are known to be triggered upon environmental changes (Mittler et al., 2011) ; however, NADPH oxidase activity after metabolism approaches a steady state is not well documented. Our results might indicate that due to excess ROS production under Mg deficiency, the capacity for ROS production via NOX in the cell might be decreased. Moreover, Mg is a cofactor of this enzyme; hence, under deficient Mg conditions (Cakmak and Kirkby, 2008) , NOX might not be activated properly to induce ROS signaling. However, the latter possibility seems unlikely because ÖZGÜR UZİLDAY et al. / Turk J Biol in gel the NOX activity assay includes Mg and Ca in the dye solution for maximum staining of the bands. These findings strongly suggest that Mg-deficient plants greatly downregulate NOX activity in their cells.

Excess H2O2 accumulation is a major factor causing oxidative stress in the cell. H2O2 disrupts the functions of enzymes with thiol groups at their active sites or regulatory domains and most of the proteins related to photosynthesis have regulatory thiols that depend on redox regulation (Dietz and Pfannschmidt, 2011) . Therefore, it is necessary to reduce the accumulation of excess H2O2 in the chloroplasts and in the cell under adverse conditions to prevent its interference with redox regulation of enzyme activity. CAT, POX, and APX are major scavengers of H2O2 and their activities were evaluated during the study. Among them, CAT is located in peroxisomes and is responsible for the scavenging of the photorespiratory H2O2 (Noctor et al., 2002) . In maize, CAT activity was increased by 10% during Mg deficiency; however, in mulberry, CAT activity was decreased with Mg deficiency, which was related to a decrease in functional levels of Fe (Tewari et al., 2004, 2006) . Similarly, the activity of CAT in citrus plants was also decreased with Mg deficiency (Tang et al., 2012) . In this study, 0 Mg treatment enhanced the activity of CAT but 75 Mg decreased the CAT activity as compared to controls. These results indicate that the response of CAT activity is variable and depends on Mg levels in the tissue; while very low concentrations of Mg can induce CAT activity, its deficiency might decrease it. CAT activity is strictly related to the rate of photorespiration and photorespiratory H2O2 production. Compatible with this, previously it was shown that Mg deficiency decreased the rate of photorespiration (Terry and Ulrich, 1974) , supporting decreased CAT activity with low levels of Mg in the present study. On the other hand, increase in CAT activity with 0 Mg might be a response to general cellular oxidative stress.

Among the mentioned H2O2 detoxification enzymes, the increase in activity of POX was very notable in 0 Mgtreated plants, indicating the occurrence of oxidative stress. POXs are found in many compartments of the plant cell and the apoplast and they scavenge H2O2 in these compartments (Hiraga et al., 2001) . Similar to cotton, Mg deficiency also increased the activity of POX in mulberry (Tewari et al., 2006) . However, similar to induction of CAT, activity of this enzyme was observed after a certain threshold of Mg in tissue, implying that POXs are induced under severe oxidative stress.

APX is responsible for regulation of H2O2 levels in cytosol and chloroplasts. In chloroplasts, it is the main enzyme used for scavenging of H2O2 during the water– water cycle. It has higher affinity for H2O2 as compared to CAT and POX; hence, it is generally employed by plants to fine-tune H2O2 levels (Shigeoka et al., 2002) . The highest APX activity was detected with Mg starvation conditions, followed by 75 Mg. Similar to our results, in mulberry plants the APX activity was also higher under Mg deficiency (Tewari et al., 2006) . The activity of APX was measured in different leaves of Capsicum annum and the activity of APX was highly induced at the 60th day of Mg deficiency in all leaves (Anza et al., 2005) . Due to its role in the water–water cycle, APX activity is an important component for efficient photosynthesis. In our work, the 150 Mg group had lower activity of APX as compared to 1000 Mg-treated plants. This change might be related to the rate of ROS production in chloroplasts due to an increased rate of photosynthesis. As is evident from gas exchange measurements, 1000 Mg plants had a higher rate of photosynthesis as compared to 150 Mg plants, which also indicates a higher rate of photosynthetic light reactions (Foyer and Shigeoka, 2011) . As ROS are byproducts of normal metabolism, a higher rate of light reactions means a higher rate of chloroplastic ROS, demanding an efficient antioxidant defense (Cakmak and Kirkby, 2008) .

GSH is a nonenzymatic antioxidant and GR regenerates oxidized glutathione (GSSG) to GSH during the Asada– Halliwell–Foyer cycle (Foyer and Noctor, 2011) . On the other hand, GSTs conjugate GSH to different substrates such as toxic xenobiotics and oxidatively produced compounds (Edwards and Dixon, 2005) . Due to the important roles of GSH in the cell as redox buffers, we investigated activities of GR and GST under Mg-deficient conditions. The activities of GR and GST were highest in 0 Mg-treated plants, which indicates an excess use of GSH for redox regulation. In pepper, the activity of GR mostly increased under long-term Mg deficiency, whereas total GSH levels were also induced by long-term Mg deficiency (Anza et al., 2005) . However, the activities of GR and GSH content in citrus plants did not show any differences with Mg deficiency (Tang et al., 2012) .

TBARS and H2O2 contents were gradually increased with decreasing Mg concentrations. This indicates that oxidative stress becomes increasingly damaging as Mg concentrations in plant tissue decrease. Similar results were also obtained in maize under Mg deficiency (Tewari et al., 2004) . Changes in activities and isoenzyme patterns of ROS scavenging enzymes are responses to minimize oxidative stress; however, as is evident from the TBARS and H2O2 data, these responses were not sufficient to completely abolish the damaging effects of ROS.

In conclusion, our data demonstrate that Mg deficiency not only regulates total activities of ROS detoxification enzymes but also changes their isoenzyme patterns. In particular, induction of new isoenzymes or loss of an existing one indicates redox change in a specific compartment. In addition, our data suggest that NOXmediated ROS signals are suppressed under Mg deficiency, probably due to oxidative stress caused by deficiency of this element or interference of nutrient deficiency with activity of this enzyme.

Acknowledgments

We would like to thank Assoc Prof Dr Levent Öztürk from Sabancı University, Turkey, for help with nutrient analysis and Dr Özlem Yılmaz from Sabancı University, Turkey, for her helpful suggestions in the pre-review process.

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