60 ACTA BOT. CROAT. 83 (1), 2024 Acta Bot. Croat. 83 (1), 60–68, 2024 CODEN: ABCRA 25 DOI: 10.37427/botcro-2024-008 ISSN 0365-0588 eISSN 1847-8476 Nitric oxide alleviates mercury toxicity by changing physiological and biochemical pathways in maize (Zea mays L.) seedlings Nevzat Esim1*, Aykut Karaman2, Ökkeş Atıcı2 1 Department of Molecular Biology and Genetics, Bingöl University, 12000 Bingöl, Türkiye 2 Department of Biology, Atatürk University, 25240 Erzurum, Türkiye Abstract – Like all life forms, plants suffer from high levels of mercury (Hg), known as one of the most harmful heavy metals in soil. The present study was performed to explore the effects of exogenous nitric oxide (NO) on Hg toxicity in maize (Zea mays L., cv. Arifiye-2) seedlings. Plants were grown in a hydroponic system containing 1/2 diluted Hoagland at 16 h day length, 25/20 °C (day/night) and 60% relative humidity. Eight day-old maize seedlings were first treated with NO (as 0.1 µM sodium nitroprusside) and then they were exposed to Hg toxicity (as 100 µM HgCl2) after 24 h. The toxic Hg decreased seedling growth, chlorophyll content, proline content, calcium and manganese contents, non- enzymatic antioxidant contents, cell membrane viscosity, and antioxidant enzyme activities (superoxide dismutase, catalase, peroxidases, and glutathione reductase) while it increased the generation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and super oxide anion (O2 .–), and lipid peroxidation (as malondialdehyde, MDA) content and the amount of sodium ion (Na+) in the seedlings. However, NO treatment markedly enhanced the growth parameters (dry and fresh weight, and plant height) and manganese and potassium contents as well as contents of antioxidants and chlorophyll thus alleviating the negative effects caused by the Hg stress. Also, it decreased the genera- tion of ROS and lipid peroxidation level by activating the antioxidant enzymes. These results show that NO in maize seedlings under Hg toxicity may improve stress response and mitigate oxidative stress by stimulating the antioxidant system and modulating ion homeostasis. Keywords: antioxidant, nitric oxide, heavy metal, mercury, oxidative stress Introduction Heavy metals are naturally occurring elements that have a higher atomic weight and density than water (Tchounwou et al. 2012, Wani et al. 2021). Heavy metal pollution is in- creasing due to anthropogenic activities, such as mining, smelting, industrial production and metal-containing com- pounds in household and agriculture (He et al. 2005, Wani et al. 2021). Increasing heavy metal pollution gradually ad- versely affects the ecosystem, with terrestrial plants being particularly susceptible compared to other organisms. Heavy metals are transported to various plant parts after root uptake and then eventually enter the food chain (Ding et al. 2019). An excess of heavy metals, one of which is mer- cury (Hg), leads to damage to cells, tissues, and enzymes in organisms. Mercury is a non-essential element and widely accumu- lates in an ecosystem as an industrial pollutant. Anthropo- genic sources for Hg accumulation in soil include mining, gold smelting, fuel combustion, and the industries involved in manufacturing items such as paint, disinfectants, phar- maceuticals, paper, and antimicrobial drugs (Gontia- Mishra et al. 2016). Hg is one of the most important threats to agricultural areas and causes serious damage to crops at various growth stages. Due to its transitional properties Hg is easily taken up by plants, where it inhibits the root growth and development, and affects the water balance and min- eral nutrition (Chen et al. 2015). Hg toxicity leads to a re- duction in chlorophyll and carotenoid contents due to the peroxidation of thylakoid membranes (Amooaghaie and Enteshari 2017). Also, it induces lipid peroxidation (LPO) and oxidative damage by generating excessive reactive oxy- gen species (ROS) such as hydrogen peroxide (H2O2), hy- droxide radical (OH−) and superoxide anion radical (O2 .–) * Corresponding author e-mail: nesim@bingol.edu.tr THE EFFECTS OF NITRIC OXIDE ON MERCURY TOXICITY ACTA BOT. CROAT. 83 (1), 2024 61 within plant subcellular structures (Zhou et al. 2007, Gao et al. 2010, Sahu et al. 2012). Nitric oxide (NO), which is a bioactive gaseous molecule and acts as a signaling molecule, may enhance the plant’s response to environmental stresses, including heavy metals, by regulating their physiological and biochemical processes (García-Mata and Lamattina 2013). Additionally, NO acts as an antioxidant, effectively preventing and scavenging ROS in cells, thus alleviating oxidative damage (Chen et al. 2018). There are some previous studies about the role of ex- ogenous NO in plants under heavy metal stress. They re- ported that the application of NO donors not only increased chlorophyll content, biomass and root length but it also de- creased lipid peroxidation and ROS production under heavy metal stress such as cadmium (Cd), copper (Cu), nickel (Ni), zinc (Zn), and arsenic (As) (Terrón-Camero et al. 2019). Furthermore, NO may decrease Cu, Cd, lead (Pb) and Pb/ Cd-induced oxidative damage by restraining heavy metal uptake capacity of plant root system in ryegrass, peanut, rice and alfalfa (Mostofa et al. 2014, Bai et al. 2015, Dong et al. 2016, Fang et al. 2019). Moreover, NO could alleviate phy- totoxicity by directly regulating accumulation and translo- cation of mercury in rice (Chen et al. 2015). The detoxifica- tion by NO might be related to the modulation of cell wall components, pectin and hemicelluloses (Chen et al. 2015). However, there is little information regarding the role of NO in regulating Hg-induced stress in maize. Maize (Zea mays L.), one of the oldest field crops culti- vated by humans, is a vital cereal plant from the Poaceae family, widely grown worldwide. Due to its rich nutritional content, maize is very valuable for both human and animal nutrition, with a wide range of uses. Starch, glucose and corn oil obtained from corn grain, which are of great im- portance for the sufficient and economic production of plant-based proteins in the world, are used as raw materials in the economy. The protection of maize under adverse con- ditions is important in terms of both food supply and hu- man health. Based on the above studies, we hypothesized that NO is quite crucial in helping plant tolerance under Hg stress. Therefore, our aim was to show that NO is able to alleviate Hg-induced toxicity by activating the antioxidant system and reducing oxidative stress in maize seedlings. Materials and methods Plant material and growing conditions In this study we utilized maize (Zea mays L. cv. Arifiye-2) as plant material, sodium nitroprusside (SNP) as the source of nitric oxide (NO), and mercury chloride (HgCl2) as the source of mercury (Hg). Maize seeds were subjected to a surface sterilization in 70% ethanol for 1 min, subsequently in 5% sodium hypochlorite for 5 min, and then rinsed 6 times with distilled water. In preliminary experiments surface-sterilized maize seeds were sown on Petri dishes (15 cm diameter) with double-layer filter paper and treated with different HgCl2 concentrations (0, 1, 5, 10, 20, 40, 50, 100, 200, 500 and 1000 µM) for 5 days in a growth chamber at 25 ± 2 °C. The 100 µM HgCl2 was chosen for further experiments because it caused at least a 50% reduction in germination rate and root length. A similar study was conducted to determine the appropriate SNP concentration by sowing maize seeds on Petri dishes with 100 µM HgCl2 and different SNP concentrations, and 0.1 µM SNP was chosen because it markedly alleviated nega- tive growth effects of Hg. For treatments with 100 µM HgCl2 and 0.1 µM SNP, the plants were grown in a hydroponic system containing 1/2 diluted Hoagland solution (Hoagland and Arnon 1950) in conditions of 16 h day length, 25/20 °C (day/night), and 60% relative humidity. When seedlings were 8-days old, initial treatment with 0.1 µM SNP was applied to the hydroponic medium. Half-strength Hoagland’s solution used as control. After 24 h, the seedlings were exposed to 100 µM HgCl2 and then seedlings were harvested after 3 days of Hg treatment. These periods (24 h and 3 days) were determined according to a previous experiment and literature (Chen et al. 2015). Harvested seedlings were stored at –80 °C for subsequent experiments or immediately used for measurement of growth parameters. The purity of all chemicals was more than 98%. Measurement of seedling growth and photosynthetic pigments content Ten randomly selected seedlings were used for morpho- logical measurements. Firstly, fresh weight (FW) and shoot length (SL) were measured using a balance and a scale. Then the seedlings were put in an oven for 72 h at 80 °C to detect dry weight (DW). The chlorophyll pigments content in the leaves was detected in acetone extracts by the spectropho- tometric method according to Lichtenthaler (1987) and was expressed as mg g–1 fresh weight (FW). Determination of reactive oxygen species, lipid peroxidation and electrolyte leakage Hydrogen peroxide (H2O2) level was determined accord- ing to the method of Hu et al. (2005). Briefly, 0.5 g of the samples were homogenized in 10 mL of cold acetone and then centrifuged at 5000 × g for 15 min at + 4 °C. Then, 0.5 mL of the supernatant was combined with 0.15 mL of 5% Ti(SO4)2 and 0.3 mL of 19% NH4OH. The mixture was cen- trifuged at 3000 × g for 10 min at + 4 °C. The pellet was washed twice with cold acetone and dissolved in 3 mL of 1 M H2SO4. After filtration, absorbance measurement of fil- trate was measured at 415 nm using H2SO4 as a blank. The amount of H2O2 was calculated by a constructed standard curve and defined as µg g–1 fresh weight (FW). The production of super oxide anion (O2 .–) was determined by Liu et al. (2007). Fresh tissue (0.5 g) was homogenized in 3 mL of 50 mM phosphate buffer (pH 7.8), and the homo- genate was centrifuged at 10000 × g for 10 min. The super- natant (0.1 mL) was combined with 0.9 mL of 65 mM phos- phate buffer (pH 7.8) and 0.1 mL of 10 mM hydroxylamine ESIM N., KARAMAN A., ATICI O. 62 ACTA BOT. CROAT. 83 (1), 2024 hydrochloride. It was incubated at 25 °C for 15 min, and afterwards 1 mL of the mixture, 1 mL of 17 mM 1-naphthyl- amine and 1 mL of 17 mM anhydrous amino benzene sulfonic acid were combined and again incubated at 25 °C for 20 min. Butyl alcohol (3 mL) was supplemented to the mixture and the absorbance was measured at 530 nm. NaNO2 was used for a standard curve to calculate the con- tent of O2 .– which was defined as µg g–1 fresh weight (FW). The lipid peroxidation (LPO) level was determined by measuring the content of malondialdehyde (MDA). Briefly, 0.5 g of the samples were homogenized in 5 mL of 1% tri- chloroacetic acid (TCA) and then centrifuged at 12000 × g for 20 min. One mL of the supernatant was combined with 4 mL of 0.5% TBA (2-thiobarbituric acid) in 20% TCA. The reaction mixture was incubated for 30 min in a boiling wa- ter bath, and then the reaction was stopped in an ice bath. Then the reaction tubes were centrifuged again at 5000 × g for 10 min. The absorbance of the supernatant was mea- sured at 532 nm and it was corrected by subtracting non- specific absorbance at 600 nm. MDA level was calculated using extinction coefficient of 155 mM–1 cm–1 and expressed as nmol g–1 fresh weight (FW) (Heath and Packer 1968). For electrolyte leakage (EL), the fresh leaves (0.1 g) washed with distilled water were placed in each of the test tubes including 4 mL distilled water and incubated for 4 hours at + 4 °C. Then, the amount of ions that passed into the pure water in the tubes was measured by an electrical conductivity meter (Griffith et al. 1992). Activity of antioxidant enzymes and their isoenzyme profiles The sample (0.2 g) was ground in 2 mL of extraction buf- fer (0.1 M KH2PO4, pH 7.0) including 0.2% polyvinylpyrro- lidone and 1 mM ethylenediaminetetraacetic acid ( EDTA), and the homogenate was centrifuged at 12000 × g for 15 min at + 4 °C. The supernatant was collected for enzyme activity and also used for protein determination by using bicincho- ninic acid assay (BCA) reagent spectrophotometrically at 562 nm (Smith et al. 1985). Activity of antioxidant enzymes was measured accord- ing to the method of Agarwal and Pandey (2004). Superox- ide dismutase (SOD, EC 1.15.1.1) activity was described as one unit (U), the value of enzyme that inhibited 50% of the photoreduction of nitroblue tetrazolium chloride (NBT). Activity of SOD enzyme was recorded as U mg–1 protein. Peroxidase activity (POX, EC 1.11.1.7) was assayed by deter- mining the absorbance increase at 470 nm caused by tetra- guaiacol, which is a product of the reaction in which guaiacol and H2O2 are used as substrates. One unit of POX is identi- fied as the value of enzyme that increases the absorbance at a rate of 0.01 within 1 min at 25 °C, and data are recorded as U mg–1 protein. Catalase (CAT, EC 1.11. 1.6) activity is based on the measurement of the decrease in absorbance at 240 nm when CAT provides the conversion of H2O2 to O2 and H2O. One unit of CAT is determined as the value of en- zyme disrupting 1 mM H2O2 within 1 min at 25 °C, and data are expressed as U mg–1 protein. Glutathione reductase (GR, EC 1.6.4.2) activity was measured by monitoring glutathione dependent oxidation of NADPH at 340 nm. The reaction mixture included 0.2 mM NADPH, 1 mM EDTA, 3 mM MgCl2, 0.5 mM oxidized glutathione (GSSG), and 100 mM Tris-HCl (pH 7.8). Data were expressed as U mg–1 protein. Native proteins were run on polyacrylamide gel electro- phoresis (PAGE) under non-denaturing conditions as sug- gested by Laemmli (1970). For SOD isoenzymes, the gel was incubated in 0.05 M KH2PO4 (pH 7.8) containing 0.24 mM NBT, 33.2 μM riboflavin, 0.2% N,N,N',N' -tetramethylethyl- enediamine (TEMED), and 1 mM EDTA on a shaker in the dark for 30 min at 37 °C. Then, the gel was placed in 0.05 M potassium phosphate buffer (pH 7.8) containing 1 mM EDTA and incubated under white fluorescent light for 10 – 30 min to determine isoenzymes (Beauchamp and Fridovich 1971). POX and CAT isoenzymes were monitored according to Weydert and Cullen (2010). For POX isoenzymes, the activ- ity staining was realized after incubation for 30 min in 0.2 M sodium acetate buffer (pH 5.0) containing 30 mM H2O2 and 10 mM guaiacol. For CAT isoenzymes, the gel was in- cubated in 30 mM H2O2 for 10 min, it was stained with 2% FeCl3 and 2% K3FeCN6 solutions. GR staining was carried out by incubation in a reaction solution including 50 mL of Tris-HCl (pH 7.5) containing 10 mg of 3-(4, 5-dimethylthi- azol-24)-2,5-diphenyl tetrazolium bromide, 10 mg of 2,6-di- chlorophenolindophenol, 3.4 mM GSSG, and 0.5 mM NADPH. Duplicate gels were assayed for GR activity, one with and one without GSSG (Rao et al. 1996). Determination of glutathione, ascorbic acid, and proline contents Reduced glutathione (GSH) content was determined en- zymatically using the method of Griffith (1980) with slight modification. Fresh tissue (0.2 g) was homogenized in 2 mL of 5% meta-phosphoric acid and centrifuged at 12000 × g for 20 min at 4 °C. The reaction mixture consisted of 150 µL of the supernatant and 1850 µL of KH2PO4 (50 mM, pH 7.5) including 2.5 mM EDTA, 1 mM 5,5'-dithiobis-(2-nitroben- zoic acid) (DTNB), 0.3 U glutathione reductase, and 1 mM NADPH. The increase in absorbance at 412 nm was moni- tored for 3 min at 25 °C. The amount of GSH was calculat- ed by a constructed standard curve and data were expressed as nmol g–1 fresh weight (FW). The content of ascorbate (AsA) was determined as de- scribed by Okamura (1980). Fresh tissue (0.2 g) taken from the powder obtained from liquid nitrogen grinding was ho- mogenized in 2 mL of 5 % TCA. The extract was centrifuged at 12000 × g for 10 min at 4 °C. For AsA content, reaction mixture consisted of 1 mL of supernatant and 1.5 mL of KH2PO4 (pH 7.4) including 10 mM dithiothreitol (DTT), 0.014% N-ethylmaleimide, 2.6% TCA, 11.7% H3PO4, 1% 2,2'-dipyridyl, and 0.3% FeCl3. Samples were incubated for 60 min at 37 °C and the absorbance was recorded spectro- photometrically at 525 nm. The amount of AsA was calcu- lated by a constructed standard curve and data were ex- pressed as nmol g–1 fresh weight (FW). THE EFFECTS OF NITRIC OXIDE ON MERCURY TOXICITY ACTA BOT. CROAT. 83 (1), 2024 63 Colorimetric detection of proline was determined based on proline’s reaction with ninhydrin (Bates et al., 1973). The amount of proline was calculated by a constructed standard curve and data were expressed as µg g–1 fresh weight (FW). Determination of nutrient element contents The seedlings (0.5 g) were powdered in a mill and sieved (0.5 mm) and dried at 70 °C and digested with concentrated HNO3 in a microwave system CEM, Mars 5 (CEM Corp., USA). In the seedlings digested, the concentrations of Na, Ca, Mn, and K were measured by inductively coupled plas- ma-mass spectrometry (ICP-MS, Agilent-7800, Agilent Technologies, Tokyo, Japan). Statistical analysis Each experiment was repeated at least three times in three replicates. Analysis of variance (ANOVA) was con- ducted in a one-way ANOVA test using SPSS 17.0 for Mi- crosoft Windows, and means were compared by Duncan test at the 0.05 level of confidence. The data were represent- ed as the mean ± standard error from three experiments. Results Physiological growth parameters The outcomes of the fresh weight (FW), the dry weight (DW), and the shoot length (SL) for the control groups (con- trol and SNP) and mercury (Hg) treated groups (Hg and SNP + Hg) are presented in Tab. 1. Exposure to toxic Hg caused a significant inhibition (P < 0.05) by 36% in FW, 20% in DW, and 18.8% in SL compared to the control (Tab. 1). But, when the SNP (as a donor of NO) was applied to the seedlings exposed to Hg toxicity (SNP + Hg), the same pa- rameters were increased by 24%, 20%, and 6%, respectively, in comparison to Hg application alone (Tab. 1). Chlorophyll content Hg exposure resulted in decreased chlorophyll content in maize seedlings (Tab. 2), with reductions of 9% in chlo- rophyll-a (Chl-a), 6% in chlorophyll-b (Chl-b), and 7.79% in total chlorophyll content compared to the control (Tab. 2). Moreover, the inhibitory effect of Hg on Chl-a content was more pronounced than on Chl-b. However, the application of NO treatment (SNP + Hg) to Hg-exposed plants led to significant increases of 12.7% in Chl-a, 28% in Chl-b, and 19.45% in total chlorophyll content compared to control plants (Tab. 2). Remarkably, the SNP treatment seemed to have a greater impact on Chl-b content in Hg-exposed seed- lings. Interestingly, SNP alone led to a decrease in chloro- phyll content compared to control plants (Tab. 2). Reactive oxygen species, lipid peroxidation and electrolyte leakage Table 2 displays the outcomes of ROS such as H2O2 and O2 .–, lipid peroxidation (MDA) and electrolyte leakage (EL) measurements for the control groups and the Hg-treated groups. NO treatment under normal conditions did not have any effect (P > 0.05) on H2O2 and O2 .– contents. Con- versely, exposure to Hg increased H2O2 content by 44% and the O2 .– content by 8% compared to the control (Tab. 3). Tab. 1. Effects of SNP (as a donor of NO) and Hg on fresh and dry weight, and height of maize seedlings. Treatments: Control – half-strength Hoagland’s solution, Hg – 100 µM HgCl2, SNP + Hg – 0.1 µM SNP + 100 µM HgCl2, SNP 0.1 µM SNP. Values are means of three independent experiments ± standard errors. Dif- ferent letters in the same column indicate statistically significant differences between means (P < 0.05). Treatments Fresh weight (g) Dry weight (g) Height (cm) Control 2.56 ± 0.19a 0.15 ± 0.003b 39.09 ± 0.96a Hg 1.63 ± 0.16c 0.12 ± 0.007c 31.71 ± 0.46c SNP + Hg 2.02 ± 0.21b 0.15 ± 0.005b 33.61 ± 0.51b SNP 2.76 ± 0.32a 0.17 ± 0.001a 39.92 ± 0.85a Tab. 2. Changes in the content of chlorophyll-a (Chl-a), chloro- phyll-b (Chl-b), and total chlorophyll (total chl) of maize seed- lings under Hg treatment with or without SNP (as a donor of NO). Treatments: Control – half-strength Hoagland’s solution, Hg – 100 µM HgCl2, SNP + Hg – 0.1 µM SNP + 100 µM HgCl2, SNP – 0.1 µM SNP. Values are means of three independent experiments ± standard errors. Different letters in the same column indicate statistically significant differences between means (P < 0.05). Treatments Chl-a (mg g–1 FW) Chl-b (mg g–1 FW) Total chl (mg g–1 FW) Control 2.126 ± 0.03a 1.59 ± 0.01ab 3.722 ± 0.01b Hg 1.936 ± 0.04b 1.498 ± 0.05b 3.434 ± 0.02c SNP+Hg 2.182 ± 0.06a 1.916 ± 0.09a 4.102 ± 0.01a SNP 1.99 ± 0.01b 1.464 ± 0.02b 3.462 ± 0.04c Tab. 3. Effects of SNP (as a donor of NO) and Hg on reactive oxygen species (H2O2 and O2 .-), lipid peroxidation (MDA) and electro- lyte leakage (EL) levels of maize seedlings. Treatments: Control – half-strength Hoagland’s solution, Hg – 100 µM HgCl2, SNP + Hg – 0.1 µM SNP + 100 µM HgCl2, SNP – 0.1 µM SNP. Values are means of three independent experiments ± standard errors. Different letters in the same column indicate statistically significant differences between means (P < 0.05). Treatments H2O2 (µg gˉ¹ FW) O2 – (µg gˉ¹ FW) MDA (nmol g–1 FW) EL (%) Control 101.4 ± 0.43c 3.62 ± 0.15c 1.13 ± 0.10c 14.3 ± 0.27c Hg 146.03 ± 0.52a 3.91 ± 0.16b 1.67 ± 0.09a 72.6 ± 0.46a SNP+Hg 115.07 ± 0.51b 3.48 ± 0.08c 1.41 ± 0.01b 45.3 ± 0,19b SNP 97 ± 0.18c 4.25 ± 0.27a 1.15 ± 0.07c 16.5 ± 0.31c ESIM N., KARAMAN A., ATICI O. 64 ACTA BOT. CROAT. 83 (1), 2024 However, in the SNP + Hg seedlings, there was a signifi- cant decrease in generation of H2O2 and O2 .– by 21% and 11%, respectively (Tab. 3). Moreover, toxic Hg increased MDA content by 47% and EL level by 408% indicating oxidative damage to membranes but SNP + Hg significantly reduced MDA content by 15.6% and EL level by 37.6% (Tab. 3). Activities and isozyme profiles of antioxidant enzymes Figure 1 and Figure 2 present the activities and isoen- zyme profiles of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and glutathione reductase (GR). Exposure to Hg led to reduc- tions in the activities of SOD, CAT, POX, and GR by 11.6%, Fig. 1. Effects of SNP (as a donor of NO) and Hg on activities of antioxidant enzymes in maize seedlings. a) Superoxide dismutase (SOD) b) Catalase (CAT) c) Peroxidase (POX) d) Glutathione reductase (GR). Treatments: Control – half-strength Hoagland’s solution, Hg – 100 µM HgCl2, SNP + Hg – 0.1 µM SNP + 100 µM HgCl2, SNP – 0.1 µM SNP. Values are means of three independent experiments; bars indicate standard errors. Different letters show significant differences (P < 0.05) between maize groups. Fig. 2. Effects of SNP (as a donor of NO) and Hg on isoenzyme profiles of maize seedlings a) Superoxide dismutase (SOD), b) Catalase (CAT), c) Peroxidase (POX), d) Glutathione reductase (GR). Treatments: Control – half-strength Hoagland’s solution, Hg – 100 µM HgCl2, S + Hg – 0.1 µM SNP + 100 µM HgCl2, S – 0.1 µM SNP. THE EFFECTS OF NITRIC OXIDE ON MERCURY TOXICITY ACTA BOT. CROAT. 83 (1), 2024 65 unchanged (Tab. 5). Intriguingly, treatment with NO in- creased Mn and K content by 7.3% and 8.7%, respectively, while reducing Na content by 26% in maize seedlings ex- posed to Hg compared to control plants (Tab. 5). SNP alone increased the amount of K, Ca, Na and Mn in maize seed- lings compared to control plants. These findings highlight NO’s potential in modulating essential element balances. Discussion Mercury (Hg), a heavy metal known for its high toxicity despite being non-essential, poses a global threat as it is readily absorbed by plants and accumulates across various plant tissues, jeopardizing crop yield and food safety (Wani et al. 2021). The imperative to counteract heavy metal-in- duced phytotoxicity has driven the search for effective, safe, and economically feasible solutions. Many studies have been carried out on plant signaling molecules that regulate anti- oxidant defense mechanisms in plants to increase their tol- erance to stressful environments including heavy metals such as As, Cd, Al, Hg (Chen et al. 2015, Ahmad et al. 2021, Wani et al. 2021). Especially, previous studies have demon- strated the potential of salicylic acid and carbon monoxide to alleviate Hg toxicity (Zhou et al. 2007), while other re- search has unveiled the detoxification effects of nitric oxide (NO) on heavy metals like As, Cd, Al, and Ni (Singh et al. 2009, Saxena and Shekhawat 2013). The present study in- vestigated the effects of NO treatment on maize seedlings exposed to Hg toxicity. The results revealed significant al- terations in various physiological parameters, ROS levels, antioxidant enzyme activities, antioxidant molecules con- tents, chlorophyll content, and elemental composition. The negative impact of toxic Hg on the growth of maize seedlings was evident from the reduction in fresh weight, dry weight, and shoot length. These findings are in agree- ment with earlier studies that reported the inhibitory effects of heavy metal stress on plant growth (Zhou et al. 2007, Hasanuzzaman et al. 2011). Furthermore, we observed that Hg toxicity in maize induced the generation of excess ROS such as H2O2 and O2 .– along with an increase in lipid per- oxidation and electrolyte leakage. These outcomes reflect the oxidative stress imposed by Hg on the seedlings, thus leading to cellular damage and inhibition of growth (Gao et 9.4%, 13.9%, and 19.5%, respectively. Interestingly, the ad- dition of SNP + Hg significantly stimulated the activities of these enzymes by 9.5%, 20.6%, 31.9%, and 15.2%, respec- tively (Fig. 1). The isoenzyme profile analysis, depicted in Fig. 2, re- vealed distinct bands for SOD (SOD 1–5), CAT (CAT 1–2), POX (POX 1–4), and GR (GR 1–2). Notably, exposure to Hg resulted in increased band densities for some isoenzymes of SOD, POD, and GR compared to the control. Furthermore, some isoenzymes appear thicker in maize treated with SNP + Hg, indicating a potential enhancement of the enzyme activities. AsA, GSH, and proline contents Exposure to Hg significantly elevated the contents of glutathione (GSH) by 21.5%, ascorbate (AsA) by 22.5%, and proline by 31.1% compared to control plants (Tab. 4). Notably, SNP + Hg treatment also increased the contents of AsA (11.4%) and proline (26.4%), although GSH levels re- mained unchanged (Tab. 4). These findings indicate the participation of NO in augmenting non-enzymatic antiox- idant defenses. Some nutrient element contents Exposure to toxic Hg increased sodium (Na) content by 29% compared to the control plants but reduced the content of manganese (Mn) and calcium (Ca) by 39.4% and 13.3%, respectively. The potassium (K) content, however, remained Tab. 5. Effects of SNP (as a donor of NO) and Hg on contents of elements of some nutrients of maize seedlings. Treatments: Control – half-strength Hoagland’s solution, Hg – 100 µM HgCl2, SNP + Hg – 0.1 µM SNP + 100 µM HgCl2, SNP – 0.1 µM SNP. Values are means of three independent experiments ± standard errors. Different letters in the same column indicate statistically significant differences between means (P < 0.05). LOD – limit of detection, LOQ – limit of quantitation. Treatments mg kg-1 dry weight Mn Na K Ca Control 24.35 ± 0.15b 3.519 ± 0.05c 33351.53 ± 12.05c 872.85 ± 5.9b Hg 14.75 ± 0.33d 4.543 ± 0.08b 33222 ± 12.54c 756.23 ± 8.5c SNP+Hg 15.83 ± 0.12c 3.364 ± 0.03d 36127 ± 23.57b 751.21 ± 6.3c SNP 26.04 ± 0.36a 4.710 ± 0.09a 38361.38 ± 31.12a 1026.3 ± 9.7a LOD 0.00051 0.48 2.40 2.98 LOQ 0.01 5.00 5.00 5.00 Tab. 4. Effects of SNP (as a donor of NO) and Hg on glutathione (GSH), ascorbate (AsA) and proline contents of maize seedlings. Treatments: Control – half-strength Hoagland’s solution, Hg – 100 µM HgCl2, SNP + Hg – 0.1 µM SNP + 100 µM HgCl2, SNP – 0.1 µM SNP. Values are means of three independent experi- ments ± standard errors. Different letters in the same column indicate statistically significant differences between means (P < 0.05). Treatments GSH (nmol g–1 FW) AsA (nmol g–1 FW) Proline (µg g–1 FW) Control 3947.5 ± 9.86b 1098.1 ± 5.78c 0.3196 ± 0.02c Hg 4796 ± 7.128a 1342.5 ± 4.56b 0.419 ± 0.01b SNP+Hg 4865 ± 5.82a 1495.6 ± 8.46a 0.53 ± 0.02a SNP 3990 ± 8.92b 1160.5 ± 6.11c 0.338 ± 0.03c ESIM N., KARAMAN A., ATICI O. 66 ACTA BOT. CROAT. 83 (1), 2024 al. 2010, Sahu et al. 2012, Chen et al. 2015). Similar findings have been reported in studies focusing on heavy metal-in- duced oxidative stress (Zhou et al. 2007, D’Souza Myrene and Devaraj 2013). The possible reason for increased ROS despite the observed increase in content of antioxidants (ascorbic acid, glutathione, and proline), could be the re- duced activities of key antioxidant enzymes, including SOD, CAT, POX and GR, which we noticed after exposure to Hg. Prior studies have documented heavy metal-induced sup- pression of antioxidant enzymes (Chen et al. 2018). The application of NO donor SNP to Hg-exposed maize seedlings led to a significant improvement in FW, DW, and SL. This finding aligns with previous research where exog- enous NO application was shown to enhance plant growth and development under stress conditions (Kopyra and Gwóźdź 2003). Moreover, Chen et al. (2015) showed that ex- cessive Hg-induced root growth inhibition and oxidative stress in rice plants can be effectively mitigated through ap- plication of NO released from sodium nitroprusside (SNP). NO at low concentrations is a second messenger in plants. It prompts the plant response system into an unfavorable situation. Previous research has demonstrated the antioxi- dant properties of NO and its ability to attenuate oxidative stress (Kopyra and Gwóźdź 2003, Singh et al. 2009, Kazemi et al. 2010, Cui et al. 2010). In the present study, the applica- tion of NO significantly reduced the levels of H2O2 and O2 .– in SNP + Hg-treated seedlings, confirming its role in ROS scavenging. Additionally, NO treatment attenuated MDA content and EL level, signifying its protective effect against Hg-induced lipid peroxidation and membrane damage and resulting in improved growth. Remarkably, SNP treatment provided enhanced antioxidant enzyme activities. Isozyme analysis revealed alterations in the density and thickness of bands under Hg stress and SNP treatment. This might sig- nify changes in enzyme isoforms or post-translational mod- ifications in response to NO treatment, indicating a poten- tial regulatory role of NO in the antioxidant defense system (Ahmad et al. 2021). Moreover, SNP treatment further en- hanced the contents of AsA and proline indicating its in- volvement in strengthening the adaptive mechanisms that can counteract Hg-induced oxidative stress as has been al- ready reported in plants under As and Cd toxicity (Hsu and Kao 2004). These observations are in line with the results of Ahmad et al. (2021) showing that the correlation between SNP application and the mitigation of Hg toxicity in soy- bean cultivars is reinforced by an enhanced antioxidant re- sponse and improved AsA – GSH cycle (Ahmad et al. 2021). It is also possible that NO may have acted as an antioxidant molecule as NO produces peroxynitrite, less harmful oxidants, by reacting with O2 .– (Saxena and Shekhawat 2013, Chen et al. 2015). One of the most used methods to understand the effects of abiotic stress factors on plants is to determine chlorophyll (Chl) content (Kupper et al. 1996). Hg exposure led to a re- duction in chlorophyll content in maize seedlings, which is consistent with previous reports of heavy metal-induced chlorophyll degradation (Cho and Park 2000, Amooaghaie and Enteshari 2017). Specifically, Chl-a was more adversely affected than Chl-b. This reduction in chlorophyll content indicates impaired photosynthetic capacity, which is crucial for plant growth and development. Strikingly, the addition of NO through SNP treatment reversed this trend, resulting in a significant increase in Chl-a, Chl-b, and total chloro- phyll content in SNP + Hg-treated plants. These findings suggest that NO treatment can mitigate Hg-induced chlo- rophyll degradation, potentially by maintaining chlorophyll synthesis or preventing its breakdown. The disbalance in plant nutrients may disrupt ion ho- meostasis and interfere with various metabolic processes. The altered elemental composition under Hg stress, includ- ing increased sodium (Na) and reduced manganese (Mn) and calcium (Ca) contents, corroborates with established findings on heavy metal-induced disruptions in mineral up- take (Fang et al. 2019). NO treatment led to a rebalancing effect by decreasing Na and enhancing Mn and potassium (K) contents. Nutrient elements such as Mn, K and Ca have an important role as cofactors in cells and it is essential that under stress conditions the cytosol maintain a high K+/Na+ ratio for optimal metabolic functions. NO may protect the maize seedlings against toxic Hg by increasing the K+/Na+ ratio as increase of Na+ in cells disrupts the function of ma- ny proteins. 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