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Chemosphere 225 (2019) 627e638

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Chemosphere

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

Cengiz Kaya a, Mustafa Okant b, Ferhat Ugurlar a, Mohammed Nasser Alyemeni c,Muhammad Ashraf d, Parvaiz Ahmad c, e, *

a Soil Science and Plant Nutrition Department, Agriculture Faculty, Harran University, Sanliurfa, Turkeyb Field Crops, Agriculture Faculty, Harran University, Sanliurfa, Turkeyc Botany and Microbiology Department, College of Science, King Saud University, P.O. Box. 2460 Riyadh 11451, Saudi Arabiad Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistane Department of Botany, S.P. College Srinagar, Jammu and Kashmir, India

h i g h l i g h t s

� Cd-toxicity induced oxidative stress in wheat by accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2).� Cd-also affects photosynthetic efficiency, chlorophyll and mineral elements.� Melatonin-mediated nitric oxide improves pigments and regulates uptake of essential elements.� cPTIO combined with the MT treatments enhanced the oxidative stress and decreases antioxidant enzymes.

a r t i c l e i n f o

Article history:Received 3 January 2019Received in revised form4 March 2019Accepted 5 March 2019Available online 6 March 2019

Handling Editor: T Cutright

Keywords:Cadmium toxicityMelatoninWheatNitric oxideAntioxidant systemOxidative stress

* Corresponding author.E-mail address: parvaizbot@yahoo.com (P. Ahmad

https://doi.org/10.1016/j.chemosphere.2019.03.0260045-6535/© 2019 Elsevier Ltd. All rights reserved.

a b s t r a c t

Two independent trials were conducted to examine the involvement of nitric oxide (NO) in MT-mediatedtolerance 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 (Kþ) and calcium(Ca2þ). 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 mM) treatments increased plant growth attributes and leaf Ca2þ and Kþ in the leaves,but reduced MDA, H2O2 as well as leaf Cd content compared to those in Cd-stressed plants. A furtherexperiment was designed to understand whether or not NO played a role in alleviation of Cd stress inwheat seedlings by melotonin using a scavenger of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-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 inturn considerably reduced the levels of endogenous NO. The results evidently showed that MT enhancedtolerance of wheat seedlings to Cd toxicity by triggering the endogenous NO. This was reinforced by therise in the levels of MDA and H2O2, and decrease in the activities of superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC. 1.11.1.6) and peroxidase (POD; EC. 1.11.1.7). The cPTO supply along with that ofMT caused growth inhibition and a considerable increase in leaf Cd. So, both MT and NO togetherenhanced Cd tolerance in wheat.

© 2019 Elsevier Ltd. All rights reserved.

1. Introduction

The increasing interest on cadmium (Cd) toxicity is because ofits harmful effects on plant growth as well as potential health risks

).

connected with food chain pollution (Faroon et al., 2012; Hasanet al., 2015). Cadmium toxicity is one of main problems disrupt-ing almost all features of the physiology and biochemistry of plants(Gill and Tuteja, 2011). So a potential approach needs to be exploredimmediately (Lin et al., 2012; Lee and Back, 2017a). Furthermore, Cdtoxicity triggers the over-accumulation of reactive oxygen species(ROS), ultimately resulting in oxidative stress in plants (Anjum

C. Kaya et al. / Chemosphere 225 (2019) 627e638628

et al., 2015; Gupta et al., 2017). In order to alleviate the harmfuleffects of Cd contamination, the cogent method may be of use ofbiostimulators, which can effectively enhance resistance of plantsto harmful stressors.

Melatonin is known as a biopromoter because of its obviousphysiological roles including inhibition of leaf senescence,improved root and shoot growth, improved mineral nutrition andimproved tolerance to heat stress of plants (Llanes et al., 2016; Zuoet al., 2017; Liang et al., 2018; Ahammed et al., 2018a). Recently,physiological role of melatonin has been investigated in plants byplant scientists using synthetic melatonin compounds or plantshaving higher endogenous MT (Nawaz et al., 2015; Erland et al.,2018). Melatonin has also been examined if it can enhance toler-ance to heavy metal stress such as cadmium stress in plants (Hasanet al., 2015; Li et al., 2016a; Cai et al., 2017; Lee and Back, 2017a).Although the mechanism of MT-induced tolerance to Cd stress hasbeen reported to be associated with enhancement of antioxidantsynthesis, activation of associated enzymes, maintenance of poly-amine metabolism and improved scavenging of ROS when plantsare exposed to Cd stress (Shi et al., 2015; Lee and Back, 2017a; Niet al., 2018), there seems to be no report in the literature on thepossible mechanism of endogenous nitric oxide induced by mela-tonin in plants subjected to Cd stress.

Nitric oxide (NO) has been widely reported to play a crucial rolein regulating various physiological events such as photomorpho-genesis, stomatal opening, leaf senescence, plant defense, flower-ing, and pollination, etc. (Wendehenne and Hancock, 2011; Yu et al.,2014; Buet et al., 2015). Nitric oxide also regulates a series oftolerance strategies in plants subjected to extremely harsh condi-tions (Zhao et al., 2007; Fancy et al., 2017). For example, it controlsoverproduction of reactive oxygen species (ROS) through alteringthe activities of several ROS scavenging enzymes (Niu and Guo,2012; Corpas and Palma, 2018).

Wheat is one of the major crops cultivated globally as a staplefood, but it is not much tolerant to several ecological stressesincluding heavy metals, salt and extremes of temperature(Trethowan and Mujeeb-Kazi, 2008). Cadmium contamination haspronounced influence on the plant growth and yield of wheat andso this has become a serious global issue (Ni et al., 2018). However,further studies are still needed to mitigate the cadmium toxicity ordevelop crops tolerant to cadmium toxicity (Ni et al., 2018).Although various research reports show that NO plays a significantrole in numerous hormonal, developmental and ecological re-actions in the plant (Sanz et al., 2015; Castillo et al., 2018), theputative role of NO involved in melatonin-induced oxidativedefence system needs to be clarified. So, the principal objective ofcarrying out the present experiment was to examine whether ornot melatonin-induced NO synthesis/accumulation is involved inalleviation of Cd toxicity in wheat plants.

2. Material and methods

2.1. Plant cultivation and treatments

Two independent trials were conducted using bread wheat(Triticum aestivum L. cv. Pandas) in glasshouse. Before starting theexperiment, seed samples were treated with a 1% solution of NaOClfor surface sterilization. Fifty seeds were sown per pot of 5 L-ca-pacity each consisting of perlite. After the germination of seeds, 15of those were thinned and the rest in the pot were retained tofurther grow for quantifying physiological and growth attributes.The seedlings were watered with a half-strength nutrient solution(NS). More details of the composition of NS for wheat are reportedelsewhere (Steinberg et al., 2000). The photoperiod was kept at 11/14 h light/dark during the entire experiment.

In the first experiment, melatonin (MT) solution (50 or 100 mM)prepared in 0.01% tween-20 was sprayed to the leaves of wheatplants subjected to Cd toxicity every two days for 10 days. The twolevels of MT (50 or 100 mM) chosen in the present experiment werebased on some earlier published reports. For example, 100 mMmelatonin (MT) solution sprayed to Chinese licorice (Afreen et al.,2006) and tomato (Martinez et al., 2018) was found to be effec-tive in enhancing tolerance to different abiotic stressors. The un-stressed plants were sprayed with a solution of 0.01% tween-20prepared in distilled deionized H2O alone without MT as a blanktreatment. Following 10 days of MT treatment, cadmium (Cd)toxicity (100 mM) as cadmium chloride (CdCl2) treatment wasstarted and wheat seedlings grown for a further four weeks. Basedon plant size, 100e1000mL of half-strength NS was provided toeach pot every two days during the trial. Each treatment consistedof 3 replications and each replicate had three pots, so containing 9pots in each treatment.

Plants were harvested after four weeks of stress treatments toevaluate plant fresh and dry weights and endogenous nitric oxide(NO) as well as oxidative stress attributes such as, hydrogenperoxide (H2O2), malondialdehyde (MDA) and electrolyte leakages(EL) along with antioxidant enzyme activity.

In view of the findings of the initial experiment, whereinendogenous NO level increased in the leaves of plants subjected toCd stress as well as a further increase in its concentration due to MTapplication, a further experiment was set up under identical factorsdescribed above to further understand whether or not NO pro-duced due to MT application was involved in alleviation of Cdstress. Therefore, a scavenger of NO (100 mM), i.e., 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxidepotassium salt (cPTIO) was applied to MT treatments each week for4weeks as explained earlier. Following growth and physio-biochemical parameters were measured:

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

2.2. Leaf chlorophyll levels

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

2.3. Electrolyte leakage (EL)

The EL was measured following the procedure of Dionisio-Seseand Tobita (1998) and a detailed description given in a manuscriptby Kaya and Ashraf (2015).

2.4. Chlorophyll fluorescence measurements

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

2.5. Leaf water potential

All water potential measurements were made on a recentlydeveloped leaf of each plant early in the morning, and subjectedspontaneously to a water potential measuring apparatus (PressureChamber; PMS model 600, USA).

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2.6. Analysis of chemical elements

For determination of Cd2þ, Ca2þ and Kþ in the leaf tissue sam-ples, well ground samples were subjected to 500 �C for 6 h in amuffle furnace. To the resulting white ash, 5mL of 2M hot HCl wereadded and final volume raised to 50mL by adding distilled deion-ized H2O. For determining the concentrations of Cd2þ, Ca2þ and Kþ

the samples mixtures were subjected to an ICP (Chapman and Pratt,1982).

2.7. Leaf soluble protein content

Leaf soluble proteins were quantified following Bradford (1976).Fresh leaf tissue (each 50mg) macerated in solution of phosphatebuffer (200 mM, pH 6.2). After centrifugation of the sample mixtureat 2000 rpm for 10min, a 5mL of the Coomassie Brilliant Blue re-agent, were added to 1.0mL of the extract and vortexed for 30 s. Theabsorbance of each treated sample was recorded at 595 nm.

2.8. Nitric oxide (NO) determination

Nitric oxide in the leaf samples was determined using themodified protocol of Zhou et al. (2005). A sample of fresh leaf (each600mg) was triturated in a mortar and pestle containing coldacetic acid buffer (50mM, 3ml, pH 3.6, consisting of 4% zinc diac-etate) and the extract was subjected to centrifugation (10 000 g) at4 �C for 15min. Charcoal (100mg) was added to mixture. Afterfiltration and vortexing the mixture (1ml) and the Greiss reagent(1ml) were kept at room temperature for 30min. The OD of eachtreated sample was noted at 540 nm.

2.9. Leaf malondialdehyde (MDA)

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

2.10. Hydrogen peroxide (H2O2)

Leaf H2O2 quantification was done following Loreto andVelikova (2001). Fresh leaf tissue (each 500mg) was homoge-nised in 3ml of TCA (1%). Following centrifugation of the homog-enate, a 0.75ml of supernatant was reacted with a 0.75ml of10mMK buffer and 1.5ml of 1M KI. The absorbance of the mix-tures was noted at 390 nm.

2.11. Antioxidant enzymes

A half g of fresh leaf tissue was homogenised in sodium-phosphate buffer (50mM) solution consisting of soluble polyvinylpyrolidine (1%). The homogenised solution was subjected tocentrifugation at 20 000 g at 4 �C for ¼ h. The aliquot was used todetermine the activity of CAT following the method of Kraus andFletcher (1994), that of SOD following the method of Beauchampand Fridovich (1971), and that of POD following the method ofChance and Maehly (1955).

2.12. Statistical analysis

The data for plant growth and physiological parameters wereexposed to a two-way analysis of variance by using SPSS statistics20 statistical package. Duncan’s Multiple Range test (P� 0.05) wasused to appraise the significant differences between the meanvalues. Values of all attributes are the average of 3 repli-cates± standard error.

3. Results

3.1. Preliminary experiment

3.1.1. Improvement of plant growth by melatonin (MT)Cadmium (Cd) stress significantly (P� 0.05) suppressed total

fresh weight by 45.41% and dry weight by 48.20% of wheat plantscompared to those in the unstressed plants (Fig. 1A and B). How-ever, foliar spray of both levels of melatonin (50 and 100 mM)enhanced total fresh by 45,13 and 55.75% and dry weights by 40.59and 52.47%, respectively compared to those of the Cd-stressedplants. The treatment of 100 mM (MT2) was slightly better inenhancing those parameters appraised compared with the otherlevel (50 mM) of MT used. Applications of MT were not effective inimproving both total fresh and dry matter of plants grown undercontrol conditions.

3.1.2. Melatonin reverses the effect of Cd stress on chlorophyllcontent and maximum fluorescence yield

The Cd stress significantly (P� 0.05) reduced maximum fluo-rescence yield (Fv/Fm) and total chlorophyll content by 33.58 and27.49%, respectively, but both MT doses (50 and 100 mM) signifi-cantly reversed the harmful effects of Cd stress on Fv/Fm andchlorophyll content by increasing Fv/Fm by 33.51 and 45.67%, andchlorophyll content by 17.10 and 20.39%, respectively compared tothose in the Cd-stressed plants receiving no exogenous treatment.Although there were no marked differences between the effects ofthe two MT doses on these parameters, the MT2 (100 mM) wasslightly more effective in improving the earlier mentioned pa-rameters (Fig. 1C and D). Furthermore, the MT treatments were noteffective in improving both total chlorophyll and Fv/Fm in thecontrol plants.

3.1.3. Ameliorative effect of MT on leaf water potentialThe wheat plants exposed to Cd stress showed reduced leaf

water potential (Jl) by 2.81-fold, but both treatments of MT alle-viated the reduction in Jl by 22.69% and 49.03%, respectivelycompared to that in plants subjected to Cd stress alone. No markeddifferences were found between the two levels of MT in terms ofJlof Cd-stressed plants. Moreover, the MT treatments did not alterthe Jl in the control plants (Fig. 2A).

3.1.4. Melatonin regulates mineral elements in Cd-Stressed wheatplants

In order to understand the role of MT in the regulation of ho-meostasis of mineral nutrients in plants exposed to Cd stress, thecontents of leaf Kþ and Ca2þ were determined. Cadmium stresssignificantly (P� 0.05) lowered the concentrations of Kþ and Ca2þ

by 35.14 and 32.52%, respectively in the leaves compared withthose in the control plants. In contrast, both MT treatments (50 and100 mM) significantly elevated the concentrations of Kþ by 23.49and 32.69%, and Ca2þ by 10.84 and 30.12%, respectively in the leavesof plants exposed to Cd stress alone. The higher level of MT(100 mM) was found to be slightly more effective in elevating theconcentrations of nutrient elements, particularly of Ca2þ in theleaves of plants under Cd stress compared with the other MTtreatment (50 mM). However, both doses of MT did not considerablyalter these nutrients in the unstressed (control) plants (Fig. 2B andC).

The concentration of leaf Cd increased in the wheat plantssubjected to Cd stress, but both MT treatments lowered leaf Cd by50.33 and 57.70%, respectively in the leaves of plants exposed to Cdstress alone. BothMT treatments showed almost a uniform effect inreducing leaf Cd levels in the wheat plants (Fig. 2D).

Fig. 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 MT2:0.10mM) 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 eachparameter differ significantly (P� 0.05) based on Duncan’s multiple range test.

C. Kaya et al. / Chemosphere 225 (2019) 627e638630

3.1.5. MT treatment increases nitric oxideCadmium stress significantly (P� 0.05) improved intrinsic nitric

oxide (NO) in the leaves of wheat plants by 3-fold over that of thecontrol plants. The treatments of MT at both levels significantlycaused a further increase in NO contents by 1.95- and 2.24-fold,respectively in the leaves of wheat plants under Cd stress (Fig. 2 E).

3.1.6. MT-induced mitigation of oxidative stressCadmium toxicity caused a considerable rise in H2O2 and MDA

contents by 5.65- and 3.68-fold, respectively compared with thosein the unstressed plants. Although MT applications along with Cdstress significantly (P� 0.05) reduced the levels of H2O2 by 34.33and 45.45%, and those of MDA by 28.13 and 38.33%, respectivelycompared with those in the Cd-stressed plants not sprayed withMT, they could not totally inhibit the accumulation of H2O2 andMDA in the Cd-stressed plants compared to that in the unstressedplants. There were no significant differences between the twolevels of MT in lowering down the levels of H2O2, but 100 mMMTseemed to be more effective than the other MT dose in reducingMDA content of seedlings of wheat exposed to Cd stress. As ex-pected, MT treatments did not affect the H2O2 andMDA contents inthe unstressed plants (Fig. 3A and B).

Electrolyte leakage (EL) also increased in the Cd-stressed plantsby 2.28-fold compared to that in Cd-stressed plants, however, bothMT levels-sprayed to Cd-stressed plants caused less rise in EL by42.10 and 46.50%, respectively compared to that in the Cd-stressedplants not sprayed with MT (Fig. 3C), showing no significant valuescompared to those in the control plants.

3.2. MT-induced antioxidant defence systems

Cadmium toxicity led to a significant increase in SOD, CAT andPOD activities by 1.96, 3.73 and 3.17-fold, respectively compared

with those in unstressed plants. MT treatments (0.05 and 100 mM)along with Cd stress led to a further increase in SOD activity by29.56 and 31.88%, CAT by 53.73 and 55.21% and POD by 32.81 and35.87%, respectively compared with those in the Cd-stressed plantswithout MT treatment, but they did not alter these enzyme activ-ities in the leaves of unstressed plants (Fig. 3D, E, F).

3.3. Second experiment

3.3.1. The role of MT-Induced generation of NO in improving plantgrowth, 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� 0.05) byCd stress relative to the control (Fig. 4A, B, C, D). Foliar applicationsof both treatments of MTwere effective in enhancing the total freshweight 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 and37.07%, respectively under Cd stress relative to those in the Cd-stressed plants receiving no other treatment; however, MT treat-ments did not alter these attributes in the control plants. However,those positive effects of MT on the plant growth were almostcompletely reversed by combining cPTIO with MT.

3.3.2. The role of MT-Induced generation of NO in improving plantwater potential

As observed in the previous experiment, Cd stress significantly(P� 0.05) reduced leaf water potential (Jl) by 3.64-fold, but bothtreatments of MT alleviated the decrease in Jl. No significant dif-ferences between the MT treatments were found in terms of theireffect onJl in Cd-stressed plants. Moreover, the MT treatments didnot alter the Jl in control plants (Fig. 5A). However, the positiveeffects of MT on plant water potential were almost completely

Fig. 2. Leaf water potential [Jl; (A)], leaf K (B), Ca (C), Cd (D) and nitric oxide [NO; (E)] of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10mM) 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.

C. Kaya et al. / Chemosphere 225 (2019) 627e638 631

reversed when cPTIO was applied in combination with MT.

3.3.3. The role of MT-Induced generation of NO in improvingmineral elements

In order to understand the role of endogenous NO generated byMT in the regulation of homeostasis of mineral nutrients in plantsexposed to Cd stress, the contents of leaf Kþ and Ca2þ were alsodetermined in an additional experiment. Based on the resultsrecorded, cadmium stress significantly (P� 0.05) reduced theconcentrations of Kþ by 37.78% and Ca2þ by 40.90% in the leavesrelative to those in the control plants (Fig. 5B and C). However, bothMT treatments significantly elevated the concentrations of Ca2þ by44.03 and 59.26% and Kþ by 28.63 and 40.92%, respectively in theleaves of plants exposed to Cd stress alone. The higher concentra-tion of MT (100 mM) seemed to be relatively more effective inelevating Ca2þ and Kþ in the leaves of plants under Cd stresscompared with the other MT treatment MT1 (50 mM). However,these nutrients were not altered in the control plants by MTtreatments. The treatment with the scavenger of NO, cPTIO alongwith both MT levels totally reversed the leaf Ca2þ and Kþ to thelevels present in Cd-stressed plants.

3.3.4. The role of MT-Induced generation of NO in reducing leaf Cdcontent

Cadmiumwas determined in the leaves of plants subjected to Cdstress and Cd stress plus MT in order to understand whether or notthe endogenous NO generated by MT played a role in reducingplant Cd content. When wheat plants were subjected to Cd stress,leaf Cd content increased dramatically compared to that (whichwas not detectable) in the unstressed plants. However, both MTtreatments (50 and 100 mM) significantly (P� 0.05) reduced the leafCd by 51.67 and 59.06%, respectively in Cd-stressed plants. Usingthe scavenger of NO, cPTIO, re-increased leaf Cd to the levels ofthose in the Cd-stressed plants (Fig. 5D).

Under unstressed condition, Cd was almost not detectable in theleaves under any of the treatments, because it was not added to thegrowingmedium because of the fact that it is not required by plantsfor maintaining growth and development.

3.3.5. The role of melatonin-induced generation of NO in Cd stresstolerance

In this trial, cPTIO, a scavenger of NO (100 mM), was applied towheat plants subjected to Cd stress along with MT treatments (50and 100 mM) so as to understand whether or not MT-inducedgeneration of NO was involved in mitigation of Cd stress in wheatplants.

Fig. 3. Hydrogen peroxide [H2O2; (A)], malondialdehyde [MDA; (B)], electrolyte leakage [EL; (C)], superoxide dismutase [SOD; (D)], catalase [CAT; (E)] and peroxidase [POD; (F)] inthe leaves of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10mM) and non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of threereplications. Mean values carrying different letters within each parameter differ significantly (P� 0.05) based on Duncan’s multiple range test.

C. Kaya et al. / Chemosphere 225 (2019) 627e638632

Cadmium stress significantly (P� 0.05) improved the NO by200.41% in the leaves of wheat plants as observed in the previousexperiment. The MT supply at both levels significantly caused afurther increase in NO contents by 94.91 and 124.84%, respectivelyin the leaves of wheat plants subjected to Cd stress relative to theCd-stressed plants alone, but MT treatments did not alter NO in thecontrol plants. Application of cPTIO along with MT treatmentssignificantly reversed the NO content in the leaves of Cd-stressedplants by reducing its content to the levels or below those in theCd-stressed plants (Fig. 6A).

3.3.6. The role of MT-Induced generation of NO in reversingoxidative stress

In order to have an insight whether endogenous NO triggered byMT plays a role in oxidative stress induced by Cd stress, we havedetermined the contents of hydrogen peroxide (H2O2), malondial-dehyde (MDA) and electrolyte leakages (EL). Cadmium stress led toremarkable elevations (P� 0.05) in H2O2 and MDA contents as wellas EL by 3.05-, 2.56- and 2.40-fold respectively in the leaves ofwheat plants compared to those in the control plants. However,H2O2, MDA and EL in the leaves of wheat seedlings exposed to Cdstress reduced by 32.73, 36.58 and 46.42% by 50 mMMT and by

41.82, 45.17 and 56.47%, respectively by 100 mMMT relative to thosein the Cd-stressed plants alone, but as expected, the treatments ofMT had been non-significant (P� 0.05) in altering these attributesin the control plants. However, both levels of MTapplied alongwithcPTIO completely reversed the reduction in these oxidative stressattributes to the levels of those in Cd-stressed plants (Fig. 6B, C, D).

3.3.7. The role of MT-Induced generation of NO in improvingantioxidant defence system

To get further evidence on the role of endogenous NO generatedby MT on antioxidant defence system, we determined the activitiesof some key enzymes relating to antioxidant defence system. Sig-nificant increases (P� 0.05) in the antioxidant enzymes’ (SOD, CATand POD) activities by 1.99-, 3.68- and 3.75-fold, respectively wereobserved in the wheat plants subjected to Cd stress compared tothose in the control plants (Fig. 7A, B, C). A further rise in the ac-tivities of all the above mentioned enzymes by 24.45, 46.99 and30.24% in the Cd-stressed plants treated with 50 mMMT and by23.14, 46.77 and 33.17%, respectively with 100 mMMTwas recordedrelative to those in the Cd-stressed plants alone. However, cPTIOalong with MT completely reversed the activities of these antioxi-dant enzymes to the levels of those in the Cd-stressed plants.

Fig. 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: 0.10mM) and sprayed with 0.1mM scavenger of NO, cPTIO or non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of threereplications. Mean values carrying different letters within each parameter differ significantly (P� 0.05) based on Duncan’s multiple range test.

Fig. 5. Leaf water potential [Jl; (A)], leaf K (B), Ca (C) and Cd (D) of wheat plants sprayed with melatonin (MT1: 0.05 and MT2: 0.10mM) and sprayed with 0.1mM 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 parameterdiffer significantly (P� 0.05) based on Duncan’s multiple range test.

C. Kaya et al. / Chemosphere 225 (2019) 627e638 633

Fig. 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: 0.10mM) and sprayed with 0.1mM scavenger of NO, cPTIO or non-sprayed (NS) under control (C) and cadmium toxicity (CdT). Data are means ± S.E of threereplications. Mean values carrying different letters within each parameter differ significantly (P� 0.05) based on Duncan’s multiple range test.

Fig. 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: 0.10mM) andsprayed with 0.1mM 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 carryingdifferent letters within each parameter differ significantly (P� 0.05) based on Duncan’s multiple range test.

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4. Discussion

4.1. The role of MT in improving plant growth under Cd stress

Cadmium (Cd) is a non-required and lethal metal to all existingorganisms, triggering observable symptoms, kidney failure andcancers (Il’yasova and Schwartz, 2005; Gu et al., 2017). In the firstexperiment, wheat seedlings were grown under Cd stress to eval-uate the effects of cadmium toxicity on plant growth. The presentfindings showed that Cd caused harmful effects on plant growth(Fig. 1A and B). A noticeable reduction in plant growth could be dueto harmful effect of Cd on plant physiological processes anddistraction in mineral uptake (Nazar et al., 2012).

To study whether melatonin (MT) can relieve the reduction inthe growth of plants exposed to cadmium stress, MT was appliedalong with cadmium added to the root growing medium of wheatseedlings. The present work showed that the application of bothdoses of melatonin (50 and 100 mM) significantly mitigated Cd-induced inhibition in plant growth. Notably, 100 mM melatoninshowed the highest defensive role, but the effects of both doses ofMT on plant growth inhibition induced by Cd stress did not differsignificantly. These findings exhibited that melatonin could haveinvolved in the response to Cd toxicity in wheat plants, also beingsuggested by Ni et al. (2018). So, exogenous application of mela-tonin could be a very promising strategy to cope with the adverseeffects of environmental stresses on plants by increasing endoge-nous MT levels. Some studies have reported the effect of MT onplants exposed to Cd stress, e.g. in tomato (Hasan et al., 2015), andalfalfa (Gu et al., 2017), but there has been also a sound evidencethat it involves in response of plant to ecological stresses other thanmetal stress (Shi et al., 2016; Ding et al., 2018). It was earlier pro-posed that MT might act as a possible stimulator of plant growthand development, and MT showed more mitigating effect at lowconcentrations rather than high ones in relieving salinity, cold andCd 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 plantsprovides more tolerance to cadmium stress compared to that inwild-species.

4.2. The role of MT in improving chlorophyll content and maximumphotochemical efficiency under Cd stress

Chlorophylls are crucial plant pigments, which absorb lightenergy and transport electrons to the reaction centre during themechanism of photosynthesis. However, the synthesis of chloro-phylls can be disturbed when plants are subjected to variousenvironmental 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 forcomputing photo-oxidative outcome on photosystem II (Sharmaet al., 2015). However, exogenously applied MT improved Fv/Fmand chlorophyll levels of wheat plants subjected to Cd stress (Fig.1Cand D). Szafra�nska et al. (2017) reported that MT conserved chlo-rophyll deficit during paraquat (PQ)-induced oxidative stress in peaby mitigating the leaf senescence process, as observable from leafchlorophylls impairment. Likewise, MT was reported that PS 2 (Fv/Fm) activities were improved in tomato plants exposed to stresscombinations (Martinez et al., 2018). Corresponding improvementsbyMTwere also noticed in ryegrass (Zhang et al., 2017a) under heatstress and inwatermelon subjected salinity stress (Li et al., 2017). Inearlier investigations, it has been shown that the decrease inchlorophyll due to stress is primarily because of the accumulationof H2O2 at high levels in the leaves of plants (Liang et al., 2015; Ni

et al., 2018). The present findings also show that the reducedchlorophyll content can be associated with over-accumulation ofH2O2 in the leaves of wheat plants exposed to Cd stress (Figs. 1Dand 3A). Analogous to that interrelationship between chlorophyllcontent and endogenous H2O2 in the Cd-stressed plants, melatoninlowered the levels of H2O2 and elevated chlorophyll content in theleaves. These findings suggest that MT might play a crucial role inalleviating the harmful effects of Cd stress on chlorophyll, perhapsby lowering the accumulation of H2O2. Another possible role of MT-induced Cd tolerance in thewheat plantsmight have been due to itsrole in enhancing the antioxidant defense systems to scavengeH2O2, leading to increased chlorophyll content. This speculationwas also proved in our second experiment where MT treatmentsfurther increased the activities of antioxidant enzymes (SOD, CATand POD) and chlorophyll contents (Figs. 4 and 7).

4.3. The role of MT in improving water status and mineral nutritionunder Cd stress

Cadmium toxicity has been stated to lead the imbalance ofwater status and suppress the nutrient uptake (Singh and Tewari,2003; Nazar et al., 2012), which could be the cause of decrease inleaf water potential, Ca and K under Cd stress, but these attributeswere found to be enhanced with exogenously applied MT under Cdtoxicity (Fig. 1). Mineral nutrients are needed for several keymetabolic processes, such as plant growth and development, andwater status (Epstein and Bloom, 2005; Lambers et al., 2014; Lianget al., 2018). Adequate accumulation of minerals is vital to safe-guard mechanical integrity of the plant and key physiologicalprocesses, and any changes in mineral uptake may markedly per-turb plant metabolism (Liang et al., 2018). Melatonin has a sub-stantial regulatory influence on the contents of plant mineralnutrients in plants and relieves stress by allocation to sustain thoseelements. For example, application of MT substantially alleviated Kcontent in Malus plants under different stress conditions (Li et al.,2016b) and improved mineral nutrition in cucumber plants undernitrate stress (Zhang et al., 2017b). In view of our findings, it couldbe stated that MT enhanced tolerance of the wheat plants to Cdstress by lowering Cd content and restoring Ca and K levels in theleaves. After absorption of Cd by the roots, it is then transferred tothe above parts of the plants using the same transport systemswithessential mineral nutrients; thus, the absorbed Cd changes plasmamembrane integrity and the ionic stability (Llamas et al., 2000; Hanet al., 2014). The present results show that Cd was dramaticallyaccumulated 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 Liet al. (2016a) who reported that MT reduced Cd content in to-mato plants. Those results obviously showed that one of the crucialroles of MT in improving tolerance to Cd stress is to reduce Cdcontent in leaves. Moreover, it has been stated that MT could play afunctional role in improving tolerance to Cd-stressed tomato plantsby inducing synthesis of phytochelatins and consequent distribu-tion of Cd in cell wall and vacuole (Hasan et al., 2015).

4.4. The role of MT-Induced generation of NO in reversing oxidativestress

A significant rise in H2O2, MDA and EL was observed in thewheat plants subjected to Cd stress in the present experiment(Fig. 3A, B, C). Previous studies have shown that Cd stress triggersthe generation of reactive oxygen species, e.g., H2O2 and superoxideradicals (radical O2

�) accumulate to a great extent in the plant(DalCorso et al., 2008; Sharma and Dietz, 2009). Those changesmight interrupt ion exchange capacity of plasma membrane and all

C. Kaya et al. / Chemosphere 225 (2019) 627e638636

physiological processes associated with cell membrane functioning(Gupta et al., 2015; Mutlu et al., 2016). Overproduction of H2O2 candamage redox potential of the cell and causes the promotion ofantioxidants and the elevation of antioxidant system (Shahid et al.,2014). On the other hand, exogenously applied MT significantlyenhanced Cd-induced oxidative stress as could be evidenced by lowH2O2 production, depressed MDA level and lowered EL values. Thepresent findings showed that melatonin might act as a scavenger ofhydrogen peroxide in reversing the overproduction of hydrogenperoxide in the leaves of wheat plants exposed to Cd stress. Manyinvestigations have shown that MT has a crucial role in the miti-gation of Cd-induced oxidative stress (El-Sokkary et al., 2010;Shagirtha et al., 2011; Hasan et al., 2015; Gu et al., 2017). In to-mato plants, remarkable elevations in antioxidant enzymes’ activityand low ROS contents were linked to Cd tolerance induced by MT(Hasan et al., 2015). Based on the earlier reports, the present studyshows that MT improved antioxidant defence system therebyboosting the activities of SOD, CAT and POD in the leaves of plantsexposed to Cd stress (Fig. 7A, B, C), wherein it may play a role insustaining membrane stability and curtailing EL to confer Cdtolerance (Fig. 3C) by scavenging H2O2 and MDA.

4.5. Melatonin and endogenous NO are jointly responsible fortolerance to Cd stress

One more attribute appraised in the present study was theendogenous nitric oxide (NO) which is believed to be involved inthe mechanism of response to a broad range of abiotic stresses. Anincreased NO level in the wheat plants subjected to Cd stress wasobserved (Fig. 6A), as has previously been reported for other plantsexposed to regimes enriched with different heavy metals (Besson-Bard et al., 2009; Singh et al., 2017). So, these results suggest thatNOmight be involved in some key physiological functions of plantsunder cadmium stress. Furthermore, MT treatment led to a furtherelevation in endogenous NO content in wheat seedlings under Cdtoxicity. MT is one of the numerous biomolecules participatingmore effectively in the production of endogenous NO as observedin Arabidopsis infected by bacterial pathogen (Shi et al., 2015). It isreasonably possible that the MT treatments might generateendogenous NO, perhaps acting as an antioxidant to improve stresstolerance of plants.

Over-accumulation of NO may damage plants (Corpas et al.,2011; Da Silva et al., 2017). Thus, a balanced amount of NO in thecell is essentially required to impart resistance in plants againstdifferent stressful cues. In the present study, MT-mediated syn-thesis/accumulation of NO did not surpass the critical levels, soinjurious effects on plant metabolic processes cannot be antici-pated. Previously, the relationship of melatonin along with NO inplants under stress was reviewed. For example, a parallel statementwas made in the case of Arabidopsis with a strong protectionagainst bacterial pathogens (Shi et al., 2015). Furthermore, it hasbeen shown that application of melatonin promotes NO formationin Arabidopsis thaliana under iron deficiency stress, and it has beensuggested that NO could be a downstream signal involved in theimproved tolerance of Arabidopsis thaliana to iron deficiency stressproduced by melatonin (Zhou et al., 2016). In the present study, inCd-stressed wheat plants, the elevated NO synthesis of SNP wassimulated in response to MT (Fig. 6A), while when plants weretreated with cPTIO the mitigating effect of MT was reversed bylowering endogenous NO. This shows that the positive effect of MTon wheat seedlings grown under Cd toxicity could be interrelatedwith NO synthesis. Analogous results have been reported by Wenet al. (2016), who showed that MT enhanced NO contents in to-mato 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 anew perception of NO as a downstream signal that plays a crucialrole in melatonin-induced tolerance of wheat plants to Cd stress.

4.6. The role of MT-Induced generation of NO in triggeringantioxidant defence system

One of the strategies developed by plants to increase theirability to withstand heavy metal contaminated soil is to scavengeH2O2 by inducing some of enzyme such as those of CAT, SOD andPOD to safeguard cell’s membrane against stress-induced disrup-tion and dysfunction (Carvalho et al., 2017). It is believed that MTcannot directly reverse overproduction of hydrogen peroxide(Bonnefont-Rousselot et al., 2011), but it might improve the anti-oxidant systems to regulate hydrogen peroxide accumulation. So,the present investigation also examined how the antioxidant de-fense systems reacted to MT application under Cd stress. Likewise,in the present experiment, Cd stress improved those enzyme ac-tivities in the wheat plants. In addition, the present findings haveshown that MT improved Cd stress-induced oxidative dysfunctionin the wheat plants, which might have been improved by thereduced ROS accumulation, lipid peroxidation and chlorophyllcontent (Fig. 6B and C), as well as the stimulation antioxidant en-zymes (Fig. 7A, B, C). On the other hand, cPTIO, a NO scavenger,reversed those enhancements, suggesting that endogenous NOplays a role in melatonin-induced tolerance to Cd stress of wheatplants. Similar results were reported in reed plants and Arabidopsisunder saline stress (Zhao et al., 2004, 2007). This was also provedthat exogenously applied melatonin might trigger the synthesis/accumulation of NOwhichmay play a role in increasing antioxidantenzyme activities and reducing H2O2 and MDA to improve Cdtolerance inwheat plants as was observed by the application cPTIO,the scavenger of NO, which effectively reduced endogenous NO andreversed the positive effects of MT-induced NO on all those pa-rameters. So MT and NO are both jointly responsible signallingmolecules which function in conjunction in conferring tolerance inwheat plants to Cd stress.

5. Conclusion

In general, this study revealed that MT-induced improvement inCd stress tolerance in wheat plants was associated with anenhanced functioning of the antioxidant defence machinery, andthe scavenging of H2O2 to alleviate oxidative impairment inducedby Cd stress. Nitric oxide was found to be partially linked to MT-induced antioxidant defence. Furthermore, possible cross-talk be-tween MT and NO might have played a crucial role in Cd stresstolerance of wheat plants. So MT and NO are jointly responsible forimproved tolerance to Cd stress in wheat plants. In addition, thepresent results provide additional insights into MT signal trans-duction in plants subjected to Cd toxicity, but the complex molec-ular system operating during the Cd stress enabled by MT needs tobe further examined explicitly.

Author contributions

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

Competing interests

The authors declare that they have no competing interests.

C. Kaya et al. / Chemosphere 225 (2019) 627e638 637

Acknowledgement

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

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