Melatonin-mediated nitric oxide improves tolerance to cadmium toxicity by reducing oxidative stress in wheat plants

Accepted Manuscript Melatonin-mediated nitric oxide improves tolerance to cadmium toxicity by reducing oxidative stress in wheat plants

Cengiz Kaya, Mustafa Okant, Ferhat Ugurlar, Mohammed Nasser Alyemeni, Muhammad Ashraf, Parvaiz Ahmad PII:

S0045-6535(19)30462-X

DOI:

10.1016/j.chemosphere.2019.03.026

Reference:

CHEM 23340

To appear in:

Chemosphere

Received Date:

03 January 2019

Accepted Date:

05 March 2019

Please cite this article as: Cengiz Kaya, Mustafa Okant, Ferhat Ugurlar, Mohammed Nasser Alyemeni, Muhammad Ashraf, Parvaiz Ahmad, Melatonin-mediated nitric oxide improves tolerance to cadmium toxicity by reducing oxidative stress in wheat plants, Chemosphere (2019), doi: 10.1016 /j.chemosphere.2019.03.026

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ACCEPTED MANUSCRIPT

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Melatonin-mediated nitric oxide improves tolerance to cadmium toxicity by reducing oxidative stress in wheat plants

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Cengiz Kaya1, Mustafa Okant2, Ferhat Ugurlar1, Mohammed Nasser Alyemeni3, Muhammad Ashraf4, Parvaiz Ahmad5*,

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1Soil

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2Field

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3Botany

Science and Plant Nutrition Department, Agriculture Faculty, Harran University, Sanliurfa, Turkey Crops, Agriculture Faculty, Harran University, Sanliurfa, Turkey

and Microbiology Department, College of Science, King Saud University, P.O. Box. 2460 Riyadh 11451, Saudi Arabia

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4Institute

of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan

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5Department

of Botany, S.P. College Srinagar, Jammu and Kashmir, India

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*

Correspondence: Parvaiz Ahmad: [email protected]

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Abstract

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Two independent trials were conducted to examine the involvement of nitric oxide (NO) in MT-mediated tolerance to Cd toxicity in wheat plants. Cadmium toxicity considerably led to a decrease in plant growth, total chlorophyll, PSII maximum efficiency (Fv/Fm), leaf water potential, potassium (K+) and calcium (Ca2+). Simultaneously, it caused an increase in levels of leaf malondialdehyde (MDA), hydrogen peroxide (H2O2), electron leakage (EL), cadmium (Cd) and nitric oxide (NO) compared to those in control plants. Both MT (50 or 100 µM) treatments increased plant growth attributes and leaf Ca2+ and K+ in the leaves, but reduced MDA, H2O2 as well as leaf Cd content compared to those in Cd-stressed plants. A further experiment was designed to understand whether or not NO played a role in alleviation of Cd stress in wheat seedlings by melotonin using a scavenger of NO, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) combined with the MT treatments. Melatonin-enhanced tolerance to Cd stress was completely reversed by the supply of cPTIO, which in turn considerably reduced the levels of endogenous NO. The results evidently showed that MT enhanced tolerance of wheat seedlings to Cd toxicity by triggering the endogenous NO. This was reinforced by the rise in the levels of MDA and H2O2, and decrease in the activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC. 1.11.1.6) and peroxidase (POD; EC. 1.11.1.7). The cPTO supply along with that of MT caused growth inhibition and a considerable increase in leaf Cd. So, both MT and NO together enhanced Cd tolerance in wheat. Key words: cadmium toxicity, melatonin, wheat, nitric oxide, antioxidant system, oxidative stress

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Introduction

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The increasing interest on cadmium (Cd) toxicity is because of its harmful effects on plant growth as well as potential health risks connected with food chain pollution (Faroon et al., 2012; Hasan et al., 2015). Cadmium toxicity is one of main problems disrupting almost all features of the physiology and biochemistry of plants (Gill and Tuteja 2011). So a potential approach needs to be explored immediately (Lin et al., 2012; Lee and Back 2017a). 1

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Furthermore, Cd toxicity triggers the over-accumulation of reactive oxygen species (ROS), ultimately resulting in oxidative stress in plants (Anjum et al. 2015; Gupta et al. 2017). In order to alleviate the harmful effects of Cd contamination, the cogent method may be of use of biostimulators, which can effectively enhance resistance of plants to harmful stressors.

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Melatonin is known as a biopromoter because of its obvious physiological roles including inhibition of leaf senescence, improved root and shoot growth, improved mineral nutrition and improved tolerance to heat stress of plants (Llanes et al. 2016; Zuo et al. 2017; Liang et al. 2018; Ahammed et al. 2018a). Recently, physiological role of melatonin has been investigated in plants by plant scientists using synthetic melatonin compounds or plants having higher endogenous MT (Nawaz et al. 2015; Erland et al. 2018). Melatonin has also been examined if it can enhance tolerance to heavy metal stress such as cadmium stress in plants (Hasan et al. 2015; Li et al. 2016a; Cai et al. 2017; Lee and Back 2017a). Although the mechanism of MT-induced tolerance to Cd stress has been reported to be associated with enhancement of antioxidant synthesis, activation of associated enzymes, maintenance of polyamine metabolism and improved scavenging of ROS when plants are exposed to Cd stress (Shi et al. 2015; Lee and Back, 2017a; Ni et al. 2018), there seems to be no report in the literature on the possible mechanism of endogenous nitric oxide induced by melatonin in plants subjected to Cd stress.

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Nitric oxide (NO) has been widely reported to play a crucial role in regulating various physiological events such as photomorphogenesis, stomatal opening, leaf senescence, plant defense, flowering, and pollination, etc. (Wendehenne and Hancock, 2011; Yu et al., 2014; Buet and Simontacchi, 2015). Nitric oxide also regulates a series of tolerance strategies in plants subjected to extremely harsh conditions (Zhao et al., 2007; Fancy et al., 2017). For example, it controls overproduction of reactive oxygen species (ROS) through altering the activities of several ROS scavenging enzymes (Niu and Guo, 2012; Groß et al., 2013; Corpas and Palma, 2018).

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Wheat is one of the major crops cultivated globally as a staple food, but it is not much tolerant to several ecological stresses including heavy metals, salt and extremes of temperature (Trethowan and Mujeeb-Kazi, 2008). Cadmium contamination has pronounced influence on the plant growth and yield of wheat and so this has become a serious global issue (Ni et al., 2018). However, further studies are still needed to mitigate the cadmium toxicity or develop crops tolerant to cadmium toxicity (Ni et al. 2018). Although various research reports show that NO plays a significant role in numerous hormonal, developmental and ecological reactions in the plant (Sanz et al., 2015; Castillo et al., 2018), the putative role of NO involved in melatonin-induced oxidative defence system needs to be clarified. So, the principal objective of carrying out the present experiment was to examine whether or not melatonininduced NO synthesis/accumulation is involved in alleviation of Cd toxicity in wheat plants.

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MATERIAL AND METHODS

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Plant Cultivation and Treatments

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Two independent trials were conducted using bread wheat (Triticum aestivum L. cv. Pandas) in glasshouse. Before starting the experiment, seed samples were treated with a 1% solution of NaOCl for surface sterilization. Fifty seeds were sown per pot of 5 L-capacity each consisting of perlite. After the germination of seeds, 15 of those were thinned and the rest in the pot were retained to further grow for quantifying physiological and growth attributes. The seedlings were watered with a half-strength nutrient solution (NS). More details of the

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composition of NS for wheat are reported elsewhere (Steinberg et al. 2000). The photoperiod was kept at 11/14 h light/dark during the entire experiment.

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In the first experiment, melatonin (MT) solution (50 or 100 µM) prepared in 0.01% tween-20 was sprayed to the leaves of wheat plants subjected to Cd toxicity every two days for 10 days. The two levels of MT (50 or 100 µM) chosen in the present experiment were based on some earlier published reports. For example, 100 µM melatonin (MT) solution sprayed to Chinese licorice (Afreen et al., 2006) and tomato (Martinez et al., 2018) was found to be effective in enhancing tolerance to different abiotic stressors. The unstressed plants were sprayed with a solution of 0.01% tween-20 prepared in distilled deionized H2O alone without MT as a blank treatment. Following 10 days of MT treatment, cadmium (Cd) toxicity (100 µM) as cadmium chloride (CdCl2) treatment was started and wheat seedlings grown for a further four weeks. Based on plant size, 100-1000 mL of half-strength NS was provided to each pot every two days during the trial. Each treatment consisted of 3 replications and each replicate had three pots, so containing 9 pots in each treatment. Plants were harvested after four weeks of stress treatments to evaluate plant fresh and dry weights and endogenous nitric oxide (NO) as well as oxidative stress attributes such as, hydrogen peroxide (H2O2), malondialdehyde (MDA) and electrolyte leakages (EL) along with antioxidant enzyme activity.

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In view of the findings of the initial experiment, wherein endogenous NO level increased in the leaves of plants subjected to Cd stress as well as a further increase in its concentration due to MT application, a further experiment was set up under identical factors described above to further understand whether or not NO produced due to MT application was involved in alleviation of Cd stress. Therefore, a scavenger of NO (100 µM), i.e., 2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) was applied to MT treatments each week for four weeks as explained earlier. Following growth and physio-biochemical parameters were measured:

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◦C

After measuring fresh mass of shoots and roots, they were subjected to an oven at 75 for one day and dry mass recorded.

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Leaf Chlorophyll Levels

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A one g of fresh leaf sample from each replicate was homogenized in acetone (90%), and then filtered and the absorbance was read on a UV-visible spectrophotometer (Shimadzu UV-1201, Japan) for quantification of chlorophyll contents of each sample following the equation developed by Strain and Svec (1966).

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Electrolyte Leakage (EL) The EL was measured following the procedure of Dionisio-Sese and Tobita (1998) and a detailed description given in a manuscript by Kaya and Ashraf (2015).

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Chlorophyll Fluorescence Measurements

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The maximum quantum yield (Fv/Fm) of dark adapted leaves (30 min) was determined using the Photosynthesis Yield Analyzer Mini-PAM (Walz, Germany).

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Leaf water potential 3

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All water potential measurements were made on a recently developed leaf of each plant early in the morning, and subjected spontaneously to a water potential measuring apparatus (Pressure Chamber; PMS model 600, USA).

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Analysis of Chemical Elements

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For determination of Cd2+, Ca2+ and K+ in the leaf tissue samples, well ground samples were subjected to 500◦C for 6 h in a muffle furnace. To the resulting white ash, 5 mL of 2 M hot HCl were added and final volume raised to 50 mL by adding distilled deionized H2O. For determining the concentrations of Cd2+, Ca2+ and K+ the samples mixtures were subjected to an ICP (Chapman and Pratt, 1982).

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Leaf Soluble Protein Content

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Leaf soluble proteins were quantified following Bradford (1976). Fresh leaf tissue (each 50 mg) macerated in solution of phosphate buffer (200 µM, pH 6.2). After centrifugation of the sample mixture at 2000 rpm for 10 min, a 5 mL of the Coomassie Brilliant Blue reagent, were added to 1.0 mL of the extract and vortexed for 30 s. The absorbance of each treated sample was recorded at 595 nm.

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Nitric Oxide (NO) Determination

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Nitric oxide in the leaf samples was determined using the modified protocol of Zhou et al. (2005). A sample of fresh leaf (each 600 mg) was triturated in a mortar and pestle containing cold acetic acid buffer (50 mM, 3 ml, pH 3.6, consisting of 4% zinc diacetate) and the extract was subjected to centrifugation (10 000 g) at 4°C for 15 min. Charcoal (100 mg) was added to mixture. After filtration and vortexing the mixture (1 ml) and the Greiss reagent (1 ml) were kept at room temperature for 30 min. The OD of each treated sample was noted at 540 nm.

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Leaf Malondialdehyde (MDA)

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Leaf MDA, a product of lipid peroxidation, was quantified following Weisany et al. (2012).

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Hydrogen Peroxide (H2O2)

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Leaf H2O2 quantification was done following Loreto and Velikova (2001). Fresh leaf tissue (each 500 mg) was homogenised in 3 ml of TCA (1%). Following centrifugation of the homogenate, a 0.75 ml of supernatant was reacted with a 0.75 ml of 10 mM K buffer and 1.5 ml of 1M KI. The absorbance of the mixtures was noted at 390 nm.

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Antioxidant Enzymes

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A half g of fresh leaf tissue was homogenised in sodium-phosphate buffer (50 mM) solution consisting of soluble polyvinyl pyrolidine (1%). The homogenised solution was subjected to centrifugation at 20,000 g at 4° C for ¼ h. The aliquot was used to determine the activity of CAT following the method of Kraus and Fletcher (1994), that of SOD following the method of Beauchamp and Fridovich (1971), and that of POD following the method of Chance and Maehly (1955). 4

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Statistical Analysis

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The data for plant growth and physiological parameters were exposed to a two-way analysis of variance by using SPSS statistics 20 statistical package. Duncan's Multiple Range test (P ≤ 0.05) was used to appraise the significant differences between the mean values. Values of all attributes are the average of 3 replicates ± standard error.

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RESULTS

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Preliminary Experiment

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Improvement of Plant Growth by Melatonin (MT)

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Cadmium (Cd) stress significantly (P ≤ 0.05) suppressed total fresh weight by 45.41% and dry weight by 48.20% of wheat plants compared to those in the unstressed plants (Figure 1A, B). However, foliar spray of both levels of melatonin (50 and 100 µM) enhanced total fresh by 45,13 and 55.75% and dry weights by 40.59 and 52.47%, respectively compared to those of the Cd-stressed plants. The treatment of 100 µM (MT2) was slightly better in enhancing those parameters appraised compared with the other level (50 µM) of MT used. Applications of MT were not effective in improving both total fresh and dry matter of plants grown under control conditions.

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Melatonin Reverses the Effect of Cd Stress on Chlorophyll Content and Maximum Fluorescence Yield

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The Cd stress significantly (P ≤ 0.05) reduced maximum fluorescence yield (Fv/Fm) and total chlorophyll content by 33.58 and 27.49 %, respectively, but both MT doses (50 and 100 µM) significantly reversed the harmful effects of Cd stress on Fv/Fm and chlorophyll content by increasing Fv/Fm by 33.51 and 45.67%, and chlorophyll content by 17.10 and 20.39%, respectively compared to those in the Cd-stressed plants receiving no exogenous treatment. Although there were no marked differences between the effects of the two MT doses on these parameters, the MT2 (100 µM) was slightly more effective in improving the earlier mentioned parameters (Figure 1C, D). Furthermore, the MT treatments were not effective in improving both total chlorophyll and Fv/Fm in the control plants.

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Ameliorative Effect of MT on Leaf Water Potential

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The wheat plants exposed to Cd stress showed reduced leaf water potential (Ψl) by 2.81-fold, but both treatments of MT alleviated the reduction in Ψl by 22.69% and 49.03%,, respectively compared to that in plants subjected to Cd stress alone. No marked differences were found between the two levels of MT in terms of Ψl of Cd-stressed plants. Moreover, the MT treatments did not alter the Ψl in the control plants (Figure 2A).

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Melatonin Regulates Mineral Elements in Cd-Stressed Wheat Plants

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In order to understand the role of MT in the regulation of homeostasis of mineral nutrients in plants exposed to Cd stress, the contents of leaf K+ and Ca2+ were determined. Cadmium stress significantly (P ≤ 0.05) lowered the concentrations of K+ and Ca2+ by 35.14 and 32.52%, respectively in the leaves compared with those in the control plants. In contrast, both 5

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MT treatments (50 and 100µM) significantly elevated the concentrations of K+ by 23.49 and 32.69%, and Ca2+ by 10.84 and 30.12%, respectively in the leaves of plants exposed to Cd stress alone. The higher level of MT (100 µM) was found to be slightly more effective in elevating the concentrations of nutrient elements, particularly of Ca2+ in the leaves of plants under Cd stress compared with the other MT treatment (50 µM). However, both doses of MT did not considerably alter these nutrients in the unstressed (control) plants (Figure 2B, C).

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The concentration of leaf Cd increased in the wheat plants subjected to Cd stress, but both MT treatments lowered leaf Cd by 50.33 and 57.70%, respectively in the leaves of plants exposed to Cd stress alone. Both MT treatments showed almost a uniform effect in reducing leaf Cd levels in the wheat plants (Figure 2D).

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MT Treatment Increases Nitric Oxide

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Cadmium stress significantly (P ≤ 0.05) improved intrinsic nitric oxide (NO) in the leaves of wheat plants by 3-fold over that of the control plants. The treatments of MT at both levels significantly caused a further increase in NO contents by 1.95- and 2.24-fold, respectively in the leaves of wheat plants under Cd stress (Figure 2 E).

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MT-Induced Mitigation of Oxidative Stress

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Cadmium toxicity caused a considerable rise in H2O2 and MDA contents by 5.65- and 3.68fold, respectively compared with those in the unstressed plants. Although MT applications along with Cd stress significantly (P ≤ 0.05) reduced the levels of H2O2 by 34.33 and 45.45%, and those of MDA by 28.13 and 38.33%, respectively compared with those in the Cd-stressed plants not sprayed with MT, they could not totally inhibit the accumulation of H2O2 and MDA in the Cd-stressed plants compared to that in the unstressed plants. There were no significant differences between the two levels of MT in lowering down the levels of H2O2, but 100 µM MT seemed to be more effective than the other MT dose in reducing MDA content of seedlings of wheat exposed to Cd stress. As expected, MT treatments did not affect the H2O2 and MDA contents in the unstressed plants (Figure 3A, B).

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Electrolyte leakage (EL) also increased in the Cd-stressed plants by 2.28-fold compared to that in Cd-stressed plants, however, both MT levels-sprayed to Cd-stressed plants caused less rise in EL by 42.10 and 46.50%, respectively compared to that in the Cdstressed plants not sprayed with MT (Figure 3C), showing no significant values compared to those in the control plants.

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MT-Induced Antioxidant Defence Systems

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Cadmium toxicity led to a significant increase in SOD, CAT and POD activities by 1.96, 3.73 and 3.17-fold, respectively compared with those in unstressed plants. MT treatments (0.05 and 100 µM) along with Cd stress led to a further increase in SOD activity by 29.56 and 31.88%, CAT by 53.73 and 55.21% and POD by 32.81 and 35.87%, respectively compared with those in the Cd-stressed plants without MT treatment, but they did not alter these enzyme activities in the leaves of unstressed plants (Figure 3D, E, F).

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Second Experiment

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The Role of MT-Induced Generation of NO in Improving Plant Growth, Chlorophyll Content and Maximum Fluorescence Yield Total fresh weight by 32.1%, total dry mass production by 38.2%, total chlorophyll contents by 33.98% and Fv/Fm by 29.55%, respectively of wheat plants decreased significantly (P 6

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≤ 0.05) by Cd stress relative to the control (Figure. 4A, B, C, D). Foliar applications of both treatments of MT were effective in enhancing the total fresh weight by 27.53 and 35.03%, total dry mass by 23.47 and 33.04%, total chlorophyll by 24.45 and 33.95% and Fv/Fm by 22.96 and 37.07%, respectively under Cd stress relative to those in the Cd-stressed plants receiving no other treatment; however, MT treatments did not alter these attributes in the control plants. However, those positive effects of MT on the plant growth were almost completely reversed by combining cPTIO with MT

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The Role of MT-Induced Generation of NO in Improving Mineral Elements In order to understand the role of endogenous NO generated by MT in the regulation of homeostasis of mineral nutrients in plants exposed to Cd stress, the contents of leaf K+ and Ca2+ were also determined in an additional experiment. Based on the results recorded, cadmium stress significantly (P ≤ 0.05) reduced the concentrations of K+ by 37.78% and Ca2+ by 40.90% in the leaves relative to those in the control plants (Figure 5B, C). However, both MT treatments significantly elevated the concentrations of Ca2+ by 44.03 and 59.26% and K+ by 28.63 and 40.92%, respectively in the leaves of plants exposed to Cd stress alone. The higher concentration of MT (100 µM) seemed to be relatively more effective in elevating Ca2+ and K+ in the leaves of plants under Cd stress compared with the other MT treatment MT1 (50 µM). However, these nutrients were not altered in the control plants by MT treatments. The treatment with the scavenger of NO, cPTIO along with both MT levels totally reversed the leaf Ca2+ and K+ to the levels present in Cd-stressed plants.

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Under unstressed condition, Cd was almost not detectable in the leaves under any of the treatments, because it was not added to the growing medium because of the fact that it is not required by plants for maintaining growth and development.

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The Role of Melatonin-Induced Generation of NO in Cd Stress Tolerance

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In this trial, cPTIO, a scavenger of NO (100 µM), was applied to wheat plants subjected to Cd stress along with MT treatments (50 and 100 µM) so as to understand whether or not MTinduced generation of NO was involved in mitigation of Cd stress in wheat plants.

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Cadmium stress significantly (P ≤ 0.05) improved the NO by 200.41% in the leaves of wheat plants as observed in the previous experiment. The MT supply at both levels

The Role of MT-Induced Generation of NO in Improving Plant Water Potential As observed in the previous experiment, Cd stress significantly (P ≤ 0.05) reduced leaf water potential (Ψl) by 3.64-fold, but both treatments of MT alleviated the decrease in Ψl. No significant differences between the MT treatments were found in terms of their effect on Ψl in Cd-stressed plants. Moreover, the MT treatments did not alter the Ψl in control plants (Figure 5A). However, the positive effects of MT on plant water potential were almost completely reversed when cPTIO was applied in combination with MT.

The Role of MT-Induced Generation of NO in Reducing Leaf Cd Content Cadmium was determined in the leaves of plants subjected to Cd stress and Cd stress plus MT in order to understand whether or not the endogenous NO generated by MT played a role in reducing plant Cd content. When wheat plants were subjected to Cd stress, leaf Cd content increased dramatically compared to that (which was not detectable) in the unstressed plants. However, both MT treatments (50 and 100 µM) significantly (P ≤ 0.05) reduced the leaf Cd by 51.67 and 59.06%, respectively in Cd-stressed plants. Using the scavenger of NO, cPTIO, re-increased leaf Cd to the levels of those in the Cd-stressed plants (Figure 5D).

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significantly caused a further increase in NO contents by 94.91 and 124.84%, respectively in the leaves of wheat plants subjected to Cd stress relative to the Cd-stressed plants alone, but MT treatments did not alter NO in the control plants. Application of cPTIO along with MT treatments significantly reversed the NO content in the leaves of Cd-stressed plants by reducing its content to the levels or below those in the Cd-stressed plants (Figure 6A).

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The Role of MT-Induced Generation of NO in Reversing Oxidative Stress

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In order to have an insight whether endogenous NO triggered by MT plays a role in oxidative stress induced by Cd stress, we have determined the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA) and electrolyte leakages (EL). Cadmium stress led to remarkable elevations (P ≤ 0.05) in H2O2 and MDA contents as well as EL by 3.05-, 2.56- and 2.40-fold respectively in the leaves of wheat plants compared to those in the control plants. However, H2O2, MDA and EL in the leaves of wheat seedlings exposed to Cd stress reduced by 32.73, 36.58 and 46.42% by 50 µM MT and by 41.82, 45.17 and 56.47%, respectively by 100 µM MT relative to those in the Cd-stressed plants alone, but as expected, the treatments of MT had been non-significant (P ≤ 0.05) in altering these attributes in the control plants. However, both levels of MT applied along with cPTIO completely reversed the reduction in these oxidative stress attributes to the levels of those in Cd-stressed plants (Figure 6B, C, D).

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The Role of MT-Induced Generation of NO in Improving Antioxidant Defence System

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To get further evidence on the role of endogenous NO generated by MT on antioxidant defence system, we determined the activities of some key enzymes relating to antioxidant defence system. Significant increases (P ≤ 0.05) in the antioxidant enzymes’ (SOD, CAT and POD) activities by 1.99-, 3.68- and 3.75-fold, respectively were observed in the wheat plants subjected to Cd stress compared to those in the control plants (Figure 7A, B, C). A further rise in the activities of all the above mentioned enzymes by 24.45, 46.99 and 30.24% in the Cd-stressed plants treated with 50 µM MT and by 23.14, 46.77 and 33.17%, respectively with 100 µM MT was recorded relative to those in the Cd-stressed plants alone. However, cPTIO along with MT completely reversed the activities of these antioxidant enzymes to the levels of those in the Cd-stressed plants.

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DISCUSSION

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The Role of MT in Improving Plant Growth under Cd Stress Cadmium (Cd) is a non-required and lethal metal to all existing organisms, triggering observable symptoms, kidney failure and cancers (Il’yasova and Schwartz, 2005; Gu et al., 2017). In the first experiment, wheat seedlings were grown under Cd stress to evaluate the effects of cadmium toxicity on plant growth. The present findings showed that Cd caused harmful effects on plant growth (Figure 1A, B). A noticeable reduction in plant growth could be due to harmful effect of Cd on plant physiological processes and distraction in mineral uptake (Nazar et al., 2012).

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To study whether melatonin (MT) can relieve the reduction in the growth of plants exposed to cadmium stress, MT was applied along with cadmium added to the root growing medium of wheat seedlings. The present work showed that the application of both doses of melatonin (50 and 100 µM) significantly mitigated Cd-induced inhibition in plant growth. Notably, 100 µM melatonin showed the highest defensive role, but the effects of both doses of MT on plant growth inhibition induced by Cd stress did not differ significantly. These 8

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findings exhibited that melatonin could have involved in the response to Cd toxicity in wheat plants, also being suggested by Ni et al. (2018). So, exogenous application of melatonin could be a very promising strategy to cope with the adverse effects of environmental stresses on plants by increasing endogenous MT levels. Some studies have reported the effect of MT on plants exposed to Cd stress, e.g. in tomato (Hasan et al., 2015), and alfalfa (Gu et al., 2017), but there has been also a sound evidence that it involves in response of plant to ecological stresses other than metal stress (Shi et al., 2016; Ding et al., 2018). It was earlier proposed that MT might act as a possible stimulator of plant growth and development, and MT showed more mitigating effect at low concentrations rather than high ones in relieving salinity, cold and Cd stress (Zhang et al., 2014; Bajwa et al., 2014; Gu et al., 2017). However, conflicting results reported by Lee and Back (2017b) revealed that higher endogenous MT in transgenic rice plants provides more tolerance to cadmium stress compared to that in wild-species.

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The Role of MT in Improving Chlorophyll Content and Maximum Photochemical Efficiency under Cd Stress Chlorophylls are crucial plant pigments, which absorb light energy and transport electrons to the reaction centre during the mechanism of photosynthesis. However, the synthesis of chlorophylls can be disturbed when plants are subjected to various environmental stresses (Kalaji et al., 2016; Ahammed et al. 2018b), Correspondingly, the maximum photochemical efficiency (Fv/Fm) in dark-adapted leaves is recognised as a potential attribute for computing photo-oxidative outcome on photosystem II (Sharma et al. 2015). However, exogenously applied MT improved Fv/Fm and chlorophyll levels of wheat plants subjected to Cd stress (Figure 1C, D). Szafrańska et al. (2017) reported that MT conserved chlorophyll deficit during paraquat (PQ)-induced oxidative stress in pea by mitigating the leaf senescence process, as observable from leaf chlorophylls impairment. Likewise, MT was reported that PS 2 (Fv/Fm) activities were improved in tomato plants exposed to stress combinations (Martinez et al. 2018). Corresponding improvements by MT were also noticed in ryegrass (Zhang et al. 2017a) under heat stress and in watermelon subjected salinity stress (Li et al. 2017). In earlier investigations, it has been shown that the decrease in chlorophyll due to stress is primarily because of the accumulation of H2O2 at high levels in the leaves of plants (Liang et al., 2015; Ni et al., 2018). The present findings also show that the reduced chlorophyll content can be associated with over-accumulation of H2O2 in the leaves of wheat plants exposed to Cd stress (Figure 1D, 3A). Analogous to that interrelationship between chlorophyll content and endogenous H2O2 in the Cd-stressed plants, melatonin lowered the levels of H2O2 and elevated chlorophyll content in the leaves. These findings suggest that MT might play a crucial role in alleviating the harmful effects of Cd stress on chlorophyll, perhaps by lowering the accumulation of H2O2. Another possible role of MT-induced Cd tolerance in the wheat plants might have been due to its role in enhancing the antioxidant defense systems to scavenge H2O2, leading to increased chlorophyll content. This speculation was also proved in our second experiment where MT treatments further increased the activities of antioxidant enzymes (SOD, CAT and POD) and chlorophyll contents (Figure 4, 7).

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The Role of MT in Improving Water Status and Mineral Nutrition under Cd Stress Cadmium toxicity has been stated to lead the imbalance of water status and suppress the nutrient uptake (Singh and Tewari, 2003; Nazar et al., 2012), which could be the cause of decrease in leaf water potential, Ca and K under Cd stress, but these attributes were found to be enhanced with exogenously applied MT under Cd toxicity (Figure 1). Mineral nutrients are needed for several key metabolic processes, such as plant growth and development, and water status (Epstein and Bloom, 2005; Lambers et al., 2014; Liang et al., 2018). Adequate accumulation of minerals is vital to safeguard mechanical integrity of the plant and key 9

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physiological processes, and any changes in mineral uptake may markedly perturb plant metabolism (Liang et al., 2018). Melatonin has a substantial regulatory influence on the contents of plant mineral nutrients in plants and relieves stress by allocation to sustain those elements. For example, application of MT substantially alleviated K content in Malus plants under different stress conditions (Li et al. 2016b) and improved mineral nutrition in cucumber plants under nitrate stress (Zhang et al. 2017b). In view of our findings, it could be stated that MT enhanced tolerance of the wheat plants to Cd stress by lowering Cd content and restoring Ca and K levels in the leaves. After absorption of Cd by the roots, it is then transferred to the above parts of the plants using the same transport systems with essential mineral nutrients; thus, the absorbed Cd changes plasma membrane integrity and the ionic stability (Llamas et al., 2000; Han et al., 2014). The present results show that Cd was dramatically accumulated in the leaves of wheat plants exposed to Cd stress. However, MT treatments reduced the leaf Cd content by about 50% in the wheat plants. Our results were also in line with those of Li et al. (2016a) who reported that MT reduced Cd content in tomato plants. Those results obviously showed that one of the crucial roles of MT in improving tolerance to Cd stress is to reduce Cd content in leaves. Moreover, it has been stated that MT could play a functional role in improving tolerance to Cd-stressed tomato plants by inducing synthesis of phytochelatins and consequent distribution of Cd in cell wall and vacuole (Hasan et al., 2015). The Role of MT-Induced Generation of NO in Reversing Oxidative Stress

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A significant rise in H2O2, MDA and EL was observed in the wheat plants subjected to Cd stress in the present experiment (Figure 3A, B, C). Previous studies have shown that Cd stress triggers the generation of reactive oxygen species, e.g., H2O2 and superoxide radicals (radical O2-) accumulate to a great extent in the plant (DalCorso et al., 2008; Sharma and Dietz, 2009). Those changes might interrupt ion exchange capacity of plasma membrane and all physiological processes associated with cell membrane functioning (Gupta, et al., 2015; Mutlu et al., 2016). Overproduction of H2O2 can damage redox potential of the cell and causes the promotion of antioxidants and the elevation of antioxidant system (Shahid et al., 2014). On the other hand, exogenously applied MT significantly enhanced Cd-induced oxidative stress as could be evidenced by low H2O2 production, depressed MDA level and lowered EL values. The present findings showed that melatonin might act as a scavenger of hydrogen peroxide in reversing the overproduction of hydrogen peroxide in the leaves of wheat plants exposed to Cd stress. Many investigations have shown that MT has a crucial role in the mitigation of Cd-induced oxidative stress (El-Sokkary et al., 2010; Shagirtha et al., 2011; Hasan et al., 2015; Gu et al., 2017). In tomato plants, remarkable elevations in antioxidant enzymes’ activity and low ROS contents were linked to Cd tolerance induced by MT (Hasan et al., 2015). Based on the earlier reports, the present study shows that MT improved antioxidant defence system thereby boosting the activities of SOD, CAT and POD in the leaves of plants exposed to Cd stress (Figure 7A, B, C), wherein it may play a role in sustaining membrane stability and curtailing EL to confer Cd tolerance (Figure 3C) by scavenging H2O2 and MDA.

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Melatonin and Endogenous NO are Jointly Responsible for Tolerance to Cd Stress

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One more attribute appraised in the present study was the endogenous nitric oxide (NO) which is believed to be involved in the mechanism of response to a broad range of abiotic stresses. An increased NO level in the wheat plants subjected to Cd stress was observed (Figure 6A), as has previously been reported for other plants exposed to regimes enriched with different heavy metals (Besson-Bard et al., 2009; Singh et al., 2017). So, these results suggest that NO might be involved in some key physiological functions of plants under cadmium stress. Furthermore, MT treatment led to a further elevation in endogenous NO 10

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content in wheat seedlings under Cd toxicity. MT is one of the numerous biomolecules participating more effectively in the production of endogenous NO as observed in Arabidopsis infected by bacterial pathogen (Shi et al. 2015). It is reasonably possible that the MT treatments might generate endogenous NO, perhaps acting as an antioxidant to improve stress tolerance of plants.

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Over-accumulation of NO may damage plants (Corpas et al., 2011; Da Silva et al., 2017). Thus, a balanced amount of NO in the cell is essentially required to impart resistance in plants against different stressful cues. In the present study, MT-mediated synthesis/accumulation of NO did not surpass the critical levels, so injurious effects on plant metabolic processes cannot be anticipated. Previously, the relationship of melatonin along with NO in plants under stress was reviewed. For example, a parallel statement was made in the case of Arabidopsis with a strong protection against bacterial pathogens (Shi et al., 2015). Furthermore, it has been shown that application of melatonin promotes NO formation in Arabidopsis thaliana under iron deficiency stress, and it has been suggested that NO could be a downstream signal involved in the improved tolerance of Arabidopsis thaliana to iron deficiency stress produced by melatonin (Zhou et al., 2016). In the present study, in Cdstressed wheat plants, the elevated NO synthesis of SNP was simulated in response to MT (Figure 6A), while when plants were treated with cPTIO the mitigating effect of MT was reversed by lowering endogenous NO. This shows that the positive effect of MT on wheat seedlings grown under Cd toxicity could be interrelated with NO synthesis. Analogous results have been reported by Wen et al. (2016), who showed that MT enhanced NO contents in tomato plants. By assessing the regulatory role of MT on Cd stress-induced plant growth suppression, oxidative stress and antioxidant defense system, the present investigation provides a new perception of NO as a downstream signal that plays a crucial role in melatonin-induced tolerance of wheat plants to Cd stress.

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The Role of MT-Induced Generation of NO in Triggering Antioxidant Defence System

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One of the strategies developed by plants to increase their ability to withstand heavy metal contaminated soil is to scavenge H2O2 by inducing some of enzyme such as those of CAT, SOD and POD to safeguard cell’s membrane against stress-induced disruption and dysfunction (Carvalho et al., 2017). It is believed that MT cannot directly reverse overproduction of hydrogen peroxide (Bonnefont-Rousselot et al., 2011), but it might improve the antioxidant systems to regulate hydrogen peroxide accumulation. So, the present investigation also examined how the antioxidant defense systems reacted to MT application under Cd stress. Likewise, in the present experiment, Cd stress improved those enzyme activities in the wheat plants. In addition, the present findings have shown that MT improved Cd stress-induced oxidative dysfunction in the wheat plants, which might have been improved by the reduced ROS accumulation, lipid peroxidation and chlorophyll content (Figure 6B, C), as well as the stimulation antioxidant enzymes (Figure 7A, B, C). On the other hand, cPTIO, a NO scavenger, reversed those enhancements, suggesting that endogenous NO plays a role in melatonin-induced tolerance to Cd stress of wheat plants. Similar results were reported in reed plants and Arabidopsis under saline stress (Zhao et al., 2004; Zhao et al., 2007). This was also proved that exogenously applied melatonin might trigger the synthesis/accumulation of NO which may play a role in increasing antioxidant enzyme activities and reducing H2O2 and MDA to improve Cd tolerance in wheat plants as was observed by the application cPTIO, the scavenger of NO, which effectively reduced endogenous NO and reversed the positive effects of MT-induced NO on all those parameters. So MT and NO are both jointly responsible signalling molecules which function in conjunction in conferring tolerance in wheat plants to Cd stress. 11

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CONCLUSION

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In general, this study revealed that MT-induced improvement in Cd stress tolerance in wheat plants was associated with an enhanced functioning of the antioxidant defence machinery, and the scavenging of H2O2 to alleviate oxidative impairment induced by Cd stress. Nitric oxide was found to be partially linked to MT-induced antioxidant defence. Furthermore, possible cross-talk between MT and NO might have played a crucial role in Cd stress tolerance of wheat plants. So MT and NO are jointly responsible for improved tolerance to Cd stress in wheat plants. In addition, the present results provide additional insights into MT signal transduction in plants subjected to Cd toxicity, but the complex molecular system operating during the Cd stress enabled by MT needs to be further examined explicitly.

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ACKNOWLEDGEMENT

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This study was funded by University of Harran (HUBAK-18221) and this is thankfully acknowledged. Furthermore, the authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding to the Research Group number (RG-1438-039).

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AUTHOR CONTRIBUTIONS

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CK, MO and FU conducted the experimentation and carried out data analysis. CK also wrote up the initial manuscript. PA, MNA and MA helped in designing the study and critically edited the whole manuscript. All authors read and approved the final manuscript.

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COMPETING INTERESTS

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The authors declare that they have no competing interests.

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Figure captions Figure 1: Total plant fresh weight (A), dry matter (B), maximum fluorescence yield (Fv/Fm; C) and total chlorophyll (D) of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10 mM) and non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of three replications. Mean values carrying different letters within each parameter differ significantly (P ≤ 0.05) based on Duncan’s multiple range test. Figure 2: Leaf water potential [Ψl; (A)], leaf K (B), Ca (C), Cd (D) and nitric oxide [NO; (E)] of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10 mM) and non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of three replications. Mean values carrying different letters within each parameter differ significantly (P ≤ 0.05) based on Duncan’s multiple range test. Figure 3: Hydrogen peroxide [H2O2; (A)], malondialdehyde [MDA; (B)], electrolyte leakage [EL; (C)], superoxide dismutase [SOD; (D)], catalase [CAT; (E)] and peroxidase [POD; (F)] in the leaves of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10 mM) and non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of three replications. Mean values carrying different letters within each parameter differ significantly (P ≤ 0.05) based on Duncan’s multiple range test. Figure 4: Total plant fresh weight (A), dry matter (B), maximum fluorescence yield [Fv/Fm; C)] and total chlorophyll (D) in the leaves of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10 mM) and sprayed with 0.1 mM scavenger of NO, cPTIO or non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of three replications. Mean values carrying different letters within each parameter differ significantly (P ≤ 0.05) based on Duncan’s multiple range test. Figure 5: Leaf water potential [Ψl; (A)], leaf K (B), Ca (C) and Cd (D) of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10 mM) and sprayed with 0.1 mM scavenger of NO, cPTIO or non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of three replications. Mean values carrying different letters within each parameter differ significantly (P ≤ 0.05) based on Duncan’s multiple range test. Figure 6: Nitric oxide [NO; (A)], Hydrogen peroxide [H2O2; (B)], malondialdehyde [MDA; (C)] and

electrolyte leakage [EL; (D)] in the leaves of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10 mM) and sprayed with 0.1 mM scavenger of NO, cPTIO or non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of three replications. Mean values carrying different letters within each parameter differ significantly (P ≤ 0.05) based on Duncan’s multiple range test. Figure 7: Superoxide dismutase [SOD; (D)], catalase [CAT; (E)] and peroxidase [POD; (F)] in the leaves of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10 mM) and sprayed with 0.1

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mM scavenger of NO, cPTIO or non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of three replications. Mean values carrying different letters within each parameter differ significantly (P ≤ 0.05) based on Duncan’s multiple range test.

ACCEPTED MANUSCRIPT Highlights 

Cd- toxicity induced oxidative stress in wheat by accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2).

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Cd- also affects photosynthetic efficiency, chlorophyll and mineral elements.

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Melatonin-mediated nitric oxide improves pigments and regulates uptake of essential elements.

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cPTIO combined with the MT treatments enhanced the oxidative stress and decreases antioxidant enzymes.