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Nitric oxide is involved in the regulation of melatonin-induced antioxidant responses in Catharanthus roseus roots

under cadmium stress

Journal: Botany

Manuscript ID cjb-2019-0107.R2

Manuscript Type: Article

Date Submitted by the Author: 24-Aug-2019

Complete List of Authors: Nabaei, Masoomeh; Shahrekord University, biologyAmooaghaie, Rayhaneh; Shahrekord University, Biology

Keyword: Antioxidative defense, Cadmium, Catharanthus roseus, Melatonin, Nitric oxide

Is the invited manuscript for consideration in a Special

Issue? :Not applicable (regular submission)

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Nitric oxide is involved in the regulation of melatonin-induced antioxidant responses in

Catharanthus roseus roots under cadmium stress

Masoomeh Nabaei*1 and Rayhaneh Amooaghaie1,2

*,1 Plant Sci. Department, Science Faculty, Shahrekord University, Shahrekord, Iran2 Biotechnology research institute, Shahrekord University, Shahrekord, Iran

Corresponding Author:

Masoomeh Nabaei

Plant Sci. Department, Science Faculty, Shahrekord University, Shahrekord, Iran

e-mail: mahsa.nabaei@gmail.com

Tel No: 0098-9184892855

other Author:

Rayhaneh Amooaghaie1Plant Sci. Department, Science Faculty, Shahrekord University, Shahrekord, Iran

2 Biotechnology research institute, Shahrekord University, Shahrekord, Iran

e-mail: rayhanehamooaghaie@yahoo.com

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Abstract

Nitric oxide (NO) and melatonin are two bio-stimulant molecules in plants that not only

modulate growth and development of plants but also confer enhanced tolerance to abiotic

stresses. Therefore, in the present study, the interactive effect of melatonin and NO on

Catharanthus roseus seedlings was evaluated under both control and Cd stress conditions.

Results showed that both melatonin and sodium nitroprusside (SNP as a NO donor)

significantly improved seedling growth that was associated with the enhanced concentration

of photosynthetic pigments in both control and Cd stress conditions. Impacts of both

melatonin and SNP were more pronounced in Cd-stressed plants than control seedlings. The

Cd stress increased H2O2 and lipid peroxidation levels in roots. Melatonin, as well as SNP,

increased endogenous NO concentration in roots. Both melatonin and SNP enhanced the

concentration of proline and the activities of antioxidant enzymes (SOD, POD, APX, CAT)

and lowered H2O2 and lipid peroxidation levels in roots of C. roseus plants under Cd stress.

These melatonin-induced responses in the roots were suppressed by 4-carboxyphenyl-4,4,5,5-

tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; a specific scavenger of NO), but inhibition of

melatonin biosynthesis by p–chlorophenylalanine, could not reverse the protective effects

conferred by NO. These outcomes offer that NO, as a downstream signal, is implicated in the

melatonin-promoted antioxidant responses in roots of C. roseus plants.

Keywords: Antioxidative defense; Cadmium; Catharanthus roseus; Melatonin; Nitric oxide

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Introduction

Cadmium is an extremely toxic heavy metal that enters agricultural soils, especially in

developing countries via the application of urban composts, phosphate fertilizers, metal-based

pesticides, ripening agents, and wastewater irrigation (Nazar et al. 2012). Cadmium is a very

toxic metal that causes various visible symptoms in plants, e.g. leaf roll, necrosis, chlorosis,

browning of root tips and growth retardation (Choppala et al. 2014). Cadmium is a non-redox

metal that cannot directly produce reactive oxygen species (ROS), but it can indirectly trigger

ROS generation by disrupting metabolic processes (Tran and Popova 2013). Plants combat

ROS-evoked oxidative stress through activation of antioxidant enzymes like superoxide

dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase

(CAT) and production of non-enzymatic antioxidants such as phenolics, glutathione (GSH),

ascorbic acid and proline (Hasan et al. 2009). Moreover, it has been shown that Cd stress

induces expression of genes involved in signaling pathways such as genes encoding enzymes

related to nitric oxide generation, polyamine and ethylene metabolism and mitogen-activated

protein kinase (MAPK) cascades (Chmielowska-Bąk et al. 2013).

Nitric oxide (NO) is a labile gaseous messenger molecule that not only orchestrates a wide

range of physiological processes in plants (Domingos et al. 2015) but also promotes plant

defence responses against pathogens (Amooaghaie and Mardani Korrani 2018) and various

abiotic stresses like cold (Amooaghaie and Nikzad 2013), alkaline stress (Amooaghaie and

Roohollahi 2016), nanoparticle toxicity (Amooaghaie et al. 2018), and toxicity of heavy

metals (Wang et al. 2012; Alemayehu et al. 2015; Amooaghaie et al. 2017; Sun et al. 2018).

Chmielowska-Bąk and Deckert (2013) reported that after short-term exposure to Cd (3 h),

NO enhanced expression of genes related to signaling pathways including transcription

factors of DOF1 and MYBZ2 and MAPKK2. Sahay and Gupta (2017) showed that sodium

nitroprusside (SNP), as a NO donor, could mitigate cadmium stress in plants via the direct

scavenging of ROS or enhancing activities of antioxidant enzymes. Therefore, internal signal

molecules such as NO may be involved in Cd sensing and the activation of signaling

pathways for the enhancement of antioxidant defenses against Cd stress.

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine molecule similar to indole-3-

acetic acid (IAA) that can exhibit auxin-like properties and influence vegetative development

(Hardeland 2016). Furthermore, melatonin is an ubiquitous signal molecule involved in plant

responses to abiotic stresses, such as extreme temperature, drought, salinity, alkaline stress

and heavy metal stress (Zhang et al. 2015). It has been shown that melatonin synthesis

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increases in Cd-exposed plants. Byeon et al. (2015) declared that Cd stress elevated

melatonin synthesis by up-regulating the expression of genes related to melatonin

biosynthesis such as tryptamine 5-hydroxylase (T5H), tryptophan decarboxylase (TDC),

and N-acetylserotonin methyltransferase (ASMT). Tal et al. (2011) found that melatonin

concentration increased in green macroalga Ulva sp. under Cd, zinc and lead.

It has been also documented that exogenous melatonin increases plant tolerance to heavy

metals, such as aluminum (Zhang et al. 2018), cadmium (Ni et al. 2018) and copper (Tan et

al. 2007). Melatonin is an amphiphilic antioxidant molecule of small size, therefore it can

migrate easily between cell compartments, protecting cells against ROS-induced oxidative

stress (Zhang et al. 2015). Moreover, it has been shown that melatonin metabolites, e.g. N1-

acetyl-N2-formyl-5-methoxykynuramine (AFMK) also possess antioxidant properties (Shi et

al. 2016). Although the influence of melatonin on plant growth and stress tolerance is well

known (Hardeland 2016), primary signaling mechanisms by which melatonin helps plants

cope with Cd stress have been not clearly explored. Melatonin-induced stress tolerance might

be associated to cross-talk between melatonin with other signal molecules such as NO (Sharif

et al. 2018).

There is growing interest but limited studies about the interaction between melatonin and NO

in plant responses to stress conditions. Shi et al. (2015) reported that melatonin activates

nitric oxide-induced innate immunity against bacterial pathogen infection in Arabidopsis. Liu

et al. (2015) also showed that melatonin-induced tolerance against alkaline stress in tomato is

mediated by NO. On the other hand, Lee et al. (2017) stated that nitric oxide, hydrogen

peroxide, and light are necessary for cadmium-promoted melatonin synthesis in rice and it

was significantly impaired by treatment with either 2, 4-carboxyphenyl-4,4,5,5-

tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) a NO scavenger or diphenyleneidonium

(DPI) an H2O2 production inhibitor. Recently, Okant and Kaya (2019) revealed the

significance of interplay between NO and melatonin on Pb tolerance in maize. In this study,

we surveyed the interactive effect of NO and melatonin on Cd tolerance in Catharanthus

roseus (L.) G. Don, which is an ornamental and medicinal plant, belonging to the family

Apocynaceae. This plant is a rich source of anticancer alkaloids, including vinblastine and

vincristine (Van Der Heijden et al. 2004). Several studies revealed that C. roseus is a Cd

tolerant plant (Pandy et al. 2007; Srivastava and Srivastava 2010). However, the literature

review showed that there is no report about interactive impacts of NO and melatonin on Cd

tolerance in this medicinal plant. Therefore, in the current investigation, the hypothesis that

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interplay between melatonin and NO may contribute to triggering antioxidant responses in

roots of Catharanthus roseus under Cd stress was examined.

Material and methods

Plant material and experimental treatments

Catharanthus roseus seeds were surface-sterilized with 1% sodium hypochlorite for 5 min

and then completely washed with distilled water. The seeds were sown in pots containing 2

kg perlite and pots were laid in a greenhouse (25/21 ºC, 14/10 days/night and 60 % relative

humidity) and irrigated using half-strength Hoagland solution for 25 days.

Twenty-five day-old seedlings were transferred to a hydroponic system and after 1 day

acclimation, were exposed to 200 μM CdSO4, 100 μM melatonin, 200 μM SNP, 50 μM p-

CPA (p–Chlorophenylalanine, as an inhibitor of melatonin biosynthesis), 100 μM c-PTIO in

the nutrient solution.

These concentrations were selected based on our previous study (Nabaei and Amooaghaie

2019).

Each container consisted of 6 seedlings representing one replicate. Three replicates were used

for each treatment. The Hoagland solutions were renewed daily. The root samples from

above-mentioned treatments were collected after 5 days. The samples were frozen in liquid

nitrogen and after they were kept at -80 °C until the time of biochemical analysis.

Growth analysis

In this experiment 25-day old seedlings were adapted to new growth medium for 1 day and

exposed to the above-mentioned treatments for 5 days. At the end of the experiment, 31-day

old seedlings were harvested. The fresh weight (FW) of roots and shoots were measured

separately and then the samples were dried for 48h in an oven at 75 ºC for determination of

dry weight (DW).

Total chlorophyll and carotenoid concentration

Fresh leaf (0.1 g) samples were homogenized with 5 ml acetone (80%, v/v). The filtrates

were prepared and absorbance was read at 663, 645 and 470 nm in order to calculate the total

chlorophyll and carotenoids concentrations according to the method of Lichtenthaler and

Buschmann (1987).

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Detection of NO concentration in root

NO concentration in C. roseus roots was evaluated using Griess reagent as previously

described by Amooaghaie et al. (2013, 2015).

Measurement of hydrogen peroxide concentration

The H2O2 concentration was measured using KI reagent as described by Amooaghaie et al.

(2018). The frozen roots (0.2 g) from each treatment were homogenized in 4 ml of 5%

trichloroacetic acid (TCA). After centrifugation at 10,000 rpm for 15 min at 4 ºC, 0.5 ml of

the supernatant was mixed with 0.5 mL phosphate buffer (0.1 M, pH= 7.0) and 1 mL KI (1

M) and this mixture was incubated in the dark for 1 h. The absorbance of this mixture was

read at 390 nm and H2O2 concentration was determined using a standard curve.

Measurement of lipid peroxidation

Malondialdehyde (MDA) concentration was measured using the thiobarbituric acid reaction

(Heath and Packer 1968). The frozen roots (0.5 g) were extracted in 5 ml of 5% TCA. After

centrifugation at 6000 rpm for 10 min at 4 ºC, 1 ml of the supernatant was mixed with 0.5%

thiobarbituric acid dissolved in TCA. The mixture was boiled for 35 min and then was

cooled. After centrifugation for 10 min at 6,000 rpm at 4 ºC, the absorbance of the

supernatant was read at 532 and 600 nm. MDA concentration was calculated by using the

extinction coefficient of 155 mM−1cm−1.

Proline determination

The proline concentration in roots was estimated spectrophotometrically using ninhydrin

reagent (Bates et al. 1973). The roots (0.05 g) were ground in 1 ml sulphosalicylic acid

solution and centrifuged at 10,000 rpm for 15 min at 4 ºC. The supernatant was blended by

adding acetic acid and ninhydrin 2.5% and was incubated at 100 °C for 1 h. Then this mixture

was cooled in an ice bath and toluene was added. The proline concentration was determined

after reading the absorbance of upper phase at 520 nm using a standard curve.

Assay of antioxidant enzyme activity

The activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate

peroxidase (APX) were determined as previously described by Amooaghaie et al. (2018). For

analysis of enzyme activities, roots were homogenized in potassium phosphate buffer (100

mM, pH= 7.5) containing 1% (w/v) polyvinylpyrrolidone (PVP) using a mortar and pestle in

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an ice bath. After centrifugation of the homogenate at 12,000 rpm for 10 min at 4 °C, the

supernatant was used for estimation of enzymatic activities. The superoxide dismutase (SOD)

activity was assessed based on the inhibition of photochemical reduction of nitroblue

tetrazolium (NBT). The activity of catalase (CAT) was assayed by monitoring the decrease in

absorbance at 240 nm related to the decomposition of H2O2. The ascorbate peroxidase (APX)

activity was determined by measuring the decline in the absorbance at 290 nm. The activity

of peroxidase (POD) was estimated following the formation of tetraguaiacol by monitoring

the decrease in absorbance at 470 nm.

Statistical analysis

The experiment was carried out as factorial with a randomized complete design with 3

replicates. Values are means ± SE with at least three replicates for each treatment. For all

parameters, analysis of variance (ANOVA) was done using SAS software version 9.

Comparison between mean values was done according to LSD test at significance level

p<0.05.

Results

Effect of SNP and melatonin on plant growth parameters

Under non-stress condition, the exogenous melatonin and SNP did not significantly affect

fresh (Fig. 1A) and dry weights of roots (Fig. 1B) and fresh weight of aerial parts (Fig. 1C).

However, melatonin and SNP increased the dry weight of aerial parts by 34.1 % and 17.73%

respectively (Fig. 1D).

The Cd stress adversely impacted the fresh and dry weights of roots and aboveground parts of

C. roseus. Exogenous SNP or melatonin greatly recovered growth inhibition of root and

aerial part of C. roseus seedlings exposed to 200 μM Cd (Fig. 1A-D).

Application of cPTIO (NO scavenger) inhibited stimulatory effect of melatonin on growth of

roots and aboveground parts whereas p-CPA (as an inhibitor melatonin synthesis) could not

completely block the positive effects of SNP on growth of Cd-stressed plants (Fig. 1A-D).

Effect of SNP and melatonin on the concentration of photosynthetic pigments

Under non-stress condition, exogenous melatonin and SNP increased the concentration of

total Chl in leaves 5.6 and 16.22% respectively, but did not significantly change the level of

carotenoids in leaves of C. roseus (Fig. 2A, B).

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Cd stress decreased total chlorophyll concentration (Chl) in leaves and both melatonin and

SNP improved reduction of total Chl concentrations evoked by Cd stress (Fig. 2A). Results

showed that Cd stress did not change carotenoid concentration in leaves compared to control,

but exposure to melatonin and SNP, significantly increased concentration of carotenoids in

leaves of Cd-stressed plants (Fig. 2B).

The protective effects promoted by both melatonin and SNP on the concentration of total Chl

and carotenoids were completely blocked by cPTIO, as the specific NO scavenger. By

applying p-CPA, as the melatonin inhibitor, the stimulatory effect of melatonin was negated.

It was also observed that cPTIO eliminated the stimulatory effects of melatonin and SNP on

photosynthetic pigments, but p-CPA had a small potency to intervene with NO’s performance

under Cd stress (Fig. 2A, B).

Effect of SNP and melatonin on NO accumulation

As shown in Fig. 3, Cd stress increased NO accumulation in roots by 1.96-fold compared

with control. However, the application of SNP and melatonin increased NO concentration in

roots by 74.69 and 51.96% under normal condition respectively and 4.78 and 4.12 fold under

Cd stress compared to related control. Data showed that the stimulatory effect of melatonin

on NO accumulation was significantly inhibited in both control and Cd-treated roots when

cPTIO was added. Application of p-CPA, as an inhibitor of melatonin biosynthesis,

suppressed NO burst induced by melatonin but could not completely block the impact of SNP

on NO concentration in both control and Cd-exposed roots.

Effect of SNP and melatonin on MDA and H2O2

Applying melatonin and SNP did not change MDA concentrations (Fig. 4A) but slightly

elevated H2O2 concentrations in roots (Fig. 4B) under non-stress condition.

Cd stress significantly elevated levels of H2O2 and MDA (Fig. 4A, B) in roots. Application of

melatonin and SNP significantly reduced the H2O2 and MDA concentrations in Cd-stressed

roots. Application of cPTIO, increased the concentration of H2O2 and MDA compared to

melatonin and SNP treatments in Cd-exposed roots (Fig. 4A, B). Supplementation with p-

CPA weakened the regulatory effect of melatonin, but it could not significantly negate the

impacts of SNP on H2O2 and MDA generation (Fig. 4) in roots of Cd-treated plants.

Effect of SNP and melatonin on proline concentration

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Under non-stress condition, any treatments (except Mel+ p-CPA) did not change proline

concentration in roots. The proline accumulation was significantly increased in roots under

Cd stress (Fig. 5). Both SNP and melatonin considerably enhanced the increase of proline

concentration in roots of Cd-stressed C. roseus plants. However, co-application of cPTIO

with both SNP and melatonin lowered proline concentration in Cd-treated plants. Applying p-

CPA significantly negated the positive impact of melatonin, but it did not attenuate the

effects of SNP on the proline concentration of Cd-exposed plants (Fig. 5).

Effect of SNP and melatonin on the activities of antioxidant enzymes

Under non-stress condition, individual application of SNP and melatonin respectively

increased activities of SOD 18.75, 14.64 % and POD 13.73, 13.08% in roots but these

treatments did not change APX and CAT activities in roots (Fig. 6A-D).

Activities of SOD, POD, and CAT were significantly enhanced in C. roseus roots when

subjected to Cd stress in comparison to ones in roots of the control plants (Figs. 6A, B, C).

However, the activity of APX was markedly inhibited by Cd stress (Fig. 6D). Both melatonin

and SNP treatments significantly enhanced activities of all four antioxidative enzymes in Cd-

stressed roots. Application of cPTIO, decreased the activity of all four antioxidative enzymes

compared melatonin and SNP treatments in Cd-exposed roots whereas; usage of p-CPA did

not considerably suppress SNP–induced enzymatic activities in roots (Fig. 6A- D).

Discussion

Under normal condition, supplementation with melatonin and SNP improved shoot dry

weight (Fig. 1D) and increased concentrations of total Chl (Fig. 2A) in leaves of C. roseus.

These results indicate that melatonin and SNP may act as modulators of growth and

developmental processes in plants under non-stress condition. It is well known that both

melatonin (Hardeland 2015) and SNP (Domingos et al. 2015) can induce auxin-like effects in

plants. For example, Pagnussat et al. (2004) reported that nitric oxide induces indole acetic

acid synthesis and regulates adventitious root development. Verma et al. (2014) also stated

that SNP enhanced shoot multiplication potential, rhizogenesis and chlorophyll concentration

in peanut cultivars. Similarly, Sarropoulou et al. (2012) found that melatonin increased root

regeneration, biomass, photosynthetic pigments, proline concentration and total

carbohydrates level in the cherry rootstock. A significant increase of NO concentration was

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detected in seedlings exposed to SNP (Fig. 3) and growth parameters (Fig. 1A-D) and

photosynthetic pigments were enhanced by SNP (Fig. 2A, B) and these effects were not

observed when seedlings were treated with cPTIO, as a special scavenger of NO. These

findings support the hypothesis that SNP-released NO, accounts for the increase of plant

growth and chlorophyll concentration. Interestingly, melatonin also augmented NO

concentration (Fig. 3) and the positive effect of melatonin on seedling growth and

photosynthetic pigments was negated by cPTIO (Fig.2 A, B). These results suggest that

effects of melatonin on plant growth and development might be mediated through NO.

Our results indicated that 200 μM Cd significantly decreased fresh and dry weights of the

roots and shoots of C. roseus plants (Fig. 1A-D). Similarly, Pandey et al. (2007) stated 180

μM CdCl2 inhibited root length of C. roseus nearly 50%. Srivastava and Srivastava (2010)

reported that C. roseus was extremely tolerant to Cd stress; as 5 mM Cd had no adverse effect

on the biomass of stem, roots, and leaves. In contrast, Chen et al. (2017) stated that 72 h

exposure to 60 μM Cd reduced the biomass of C. roseus seedlings by approximately 50%.

This discrepancy can be related to difference in the duration and condition of experiments or

may be related to using various ecotypes of C. roseus.

As predicted, upon exposure to 200 µM Cd, the concentration of total chlorophyll decreased

compared to the control (Fig. 2A), but the concentration of carotenoids showed no significant

change (Fig. 2B). Reduction of chlorophyll concentration could be related to Cd-induced

oxidation and degradation of chlorophyll or even could be related to an adverse effect of Cd

on enzymes associated with chlorophyll biosynthesis (Parmar et al. 2013). However, Cd

reduced total chlorophyll and likely disrupted photosynthesis and consequently reduced

growth and development of C. roseus seedlings.

Cd stress caused the high concentrations of H2O2 and increased membrane lipid peroxidation

(Fig. 4A, B) in roots. The activities of SOD, CAT, and POD significantly increased in Cd-

stressed roots (Fig. 6A, B, C) to hamper the accumulation of MDA and H2O2 (Fig. 4A, B) in

roots. In addition, proline concentration significantly increased in Cd-treated roots (Fig. 5). It

is well known that the proline can improve metal stress tolerance by chelating metals,

regulation of osmotic pressure, protection of membrane integrity, maintaining suitable ratios

of NADP+/NADPH in a cell, and interaction with free radicals (Hayat et al. 2012).

Exposure to Cd stress increased the NO concentration in roots (Fig. 3), suggesting that NO

may be implicated in promoting Cd tolerance in C. roseus. Similarly, previous studies also

have addressed the elevation of NO concentration in various tissues of plants under Pb

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(Amooaghaie et al. 2017), Ag (Amooaghaie et al. 2018), Cd (Alemayehu et al. 2015) and Al

(Sun et al. 2018) stresses.

SNP supply improved growth parameters (Fig. 1A-D) and the concentration of total

chlorophyll and carotenoids (Fig. 2A, B) in Cd-stressed plants. These results were consistent

with previous reports about the effect of SNP on Pb-stressed sesame plants (Amooaghaie et

al. 2017) and Ag nanoparticle-exposed Brassica nigra plants (Amooaghaie et al. 2018).

Carotenoids prevent the oxidation of chlorophyll under abiotic stresses (Gururani et al. 2015).

It is possible that higher concentrations of carotenoids triggered by NO (Fig. 2B) contributed

to the protection of chlorophyll under Cd stress.

In plants exposed to Cd stress, SNP treatment elevated NO accumulation (Fig. 3) and lowered

the accumulation of lipid peroxidation and H2O2 in roots (Fig. 4A, B). It has been

demonstrated that NO can scavenge lipid peroxyl radicals and ROS; thereby it can directly

inhibit lipid peroxidation and protect membrane integrity (Domingos et al. 2015).

Furthermore, the application of SNP enhanced proline concentration (Fig. 5) and the

activities of SOD, CAT, POD and APX (Fig. 6A-D) in the roots to overcome Cd-evoked

oxidative injuries. The addition of the NO scavenger, cPTIO, to plant growth medium

completely abrogated the effect of SNP on the accumulation of MDA, H2O2 (Fig. 4A, B),

proline (Fig. 5) and the activities of antioxidant enzymes (Fig. 6A-D) in roots. These results

suggest that NO is involved in mitigation of Cd-induced oxidative damages in C. roseus

roots. Our results were in agreement with findings of Liu et al. (2013) who stated that 100

µM SNP accelerated the activities of the antioxidant enzymes in the roots and leaves of C.

roseus under 25 mg Cd/kg soil in a pot experiment. Wang et al. (2016) also reported that SNP

significantly decreased O2.-, H2O2 and MDA accumulation in both shoots and roots and

considerably increased the chlorophyll concentration, the concentrations of proline, soluble

protein and ascorbic acid, as well as SOD and POD activities in ryegrass plants subjected to

Cd toxicity.

Melatonin supply also amplified Cd tolerance; as it increased the fresh and dry weights of

roots and aboveground parts (Fig. 1A-D) and improved concentrations of the total

chlorophyll and carotenoids (Fig. 2A, B) under Cd stress. Nawaz et al. (2018) also noted that

melatonin supply enhanced relative chlorophyll concentration (SPAD index) in watermelon

leaves under vanadium stress through up-regulating genes involved in chlorophyll

biosynthesis and down-regulating genes related to the chlorophyll degradation.

The reduction of H2O2 concentration (Fig. 4B) and MDA accumulation in roots (Fig. 4A)

after melatonin supplementation, suggest that melatonin may reduce Cd-induced oxidative

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stress and membrane injury in roots. Melatonin acts as a strong antioxidant compound, as

each melatonin molecule can quench four or more reactive oxygen species (Allegra et al.

2013; Tan et al. 2000). In addition, it has been documented that melatonin and its precursors

can chelate aluminium, copper, Cd and other metal ions (Limson et al. 1998); thus it can

moderate metal-induced damages in cells. Melatonin supplementation enhanced proline

accumulation (Fig. 5) and increased the activities of antioxidant enzymes (Fig. 6A-D) to

reduce Cd-evoked oxidative stress in C. roseus roots under Cd stress. This assumption was

further confirmed because application of p-CPA, as the inhibitor of melatonin biosynthesis,

suppressed melatonin effects in roots of Cd-stressed C. roseus plants (Figs 4-6). This implies

that under stress condition, melatonin not only can directly scavenge ROS but also may

indirectly suppress ROS accumulation by enhancing the activities of antioxidant enzymes.

Similarly, Ni et al. (2018) explored that melatonin treatment increased primary root growth,

shoot biomass and counterbalanced hydrogen peroxide concentration under Cd stress through

amplifying activities of SOD and APX in roots and shoots of wheat. Zhang et al. (2017) also

declared that melatonin supply improved aluminum tolerance through regulating activities of

antioxidant enzymes in soybean.

In the present study, because both melatonin and NO exhibited similar responses on plant

growth and concentration of the total chlorophyll and carotenoids in C. roseus plants under

both normal and Cd stress conditions (Figs. 1A-D and 2A, B), we examined interactive

effects of melatonin and NO in roots.

Increasing NO generation in both non-stressed and Cd-exposed roots following melatonin

supplementation (Fig. 3) suggests that melatonin might trigger NO biosynthesis in C. roseus

roots. Exposure with the inhibitor of melatonin biosynthesis, p-CPA, substantially reduced

melatonin-induced NO generation which further confirms our hypothesis. However, we did

not evaluate the potential source of NO production induced by melatonin and further

investigations should elucidate this.

Our results provided evidence that interplay between melatonin and NO are pivotal not only

for regulating growth and development in non-stress condition (Fig. 1A-D), but also for

increasing proline concentration (Fig. 5), enhancing antioxidant enzyme activities (Fig. 6A-

D) and consequently decreasing Cd-induced oxidative damage (Fig. 4A, B) in C. roseus roots

under Cd stress. However, since the effects of NO and melatonin on plant growth (Fig. 1A-D)

and chlorophyll concentration (Fig. 2A) were significantly more pronounced in Cd-exposed

plants than control plants, it can be concluded that role of these signal molecules under stress

conditions is more important than normal condition.

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Our data also confirm that NO might act downstream of melatonin in both non-stress and Cd

stress conditions, because scavenging of NO, caused by cPTIO, suppressed the impacts of

melatonin on plant growth and antioxidant responses, but the addition of p-CPA could not

block SNP-mediated responses under both normal and Cd stress conditions (Figs. 1, 2, 4, 5

and 6). Similarly, Wen et al. (2016) also reported that NO, as a downstream signal, is

involved in the melatonin-evoked adventitious root formation in Solanum lycopersicum. Our

results also are consistent with the report of Okant and Kaya (2019) who stated melatonin

increased NO generation and improved Pb tolerance in maize plants in NO-dependent

manner.

In summary, based on the outcomes of our study, we propose that crosstalk between NO and

melatonin is necessary for initiation of Cd resistance in roots of C. roseus. Further studies

using mutant analyses and advanced molecular techniques are needed for better elucidation of

the role of crosstalk between melatonin and NO in the establishment of stress tolerance in

plants.

Acknowledgement

Authors are grateful from Dr Mitra Noori for assistance to providing laboratory equipments.

This study was supported by the financial grant of Shahrekord University, Iran

This study was supported by the financial grant of Shahrekord University, Iran. Authors

thank from co-workers for their critical reading and edit of the manuscript. Special

acknowledgments are given to the editors and reviewers. The authors declare that they have

no conflict of interest.

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Fig. 1. The effects of melatonin (100 μM) and SNP (200 μM) individually or in combination with 100 µM p-

CPA (as a melatonin scavenger), or 100 μM cPTIO (as a NO scavenger), on the fresh weight and dry weight of

roots (A, B) and shoots (C, D) of plants exposed to 200 µM Cd.

Mean ± SE with different letters, statistically differ at significance level P<0.05 according to LSD tests.

Fig. 2. The effects of melatonin (100 μM) and SNP (200 μM) individually or in combination with 100 µM p-

CPA (as a melatonin scavenger), or 100 μM cPTIO (as a NO scavenger), on the concentrations of total

chlorophyll (A) and carotenoids (B) in the leaves of plants exposed to 200 µM Cd.

Mean ± SE with different letters, statistically differ at significance level P<0.05 according to LSD tests.

Fig. 3. The effects of melatonin (100 μM) and SNP (200 μM) individually or in combination with 100 µM p-

CPA (as a melatonin scavenger), or 100 μM cPTIO (as a NO scavenger), on the NO concentration in roots of

plants exposed to 200 µM Cd.

Mean ± SE with different letters, statically differ at significance level P<0.05 according to LSD tests.

Fig. 4. The effects of melatonin (100 μM) and SNP (200 μM) individually or in combination with 100 µM p-

CPA (as a melatonin scavenger), or 100 μM cPTIO (as a NO scavenger), on the concentrations of

malondialdehyde (MDA) (A) and H2O2 (B) in roots of plants exposed to 200 µM Cd.

Mean ± SE with different letters, statistically differ at significance level P<0.05 according to LSD tests.

Fig. 5. The effects of melatonin (100 μM) and SNP (200 μM individually or in combination with 100 µM p-

CPA (as a melatonin scavenger), or 100 μM cPTIO (as a NO scavenger), on the concentration of proline in roots

of plants exposed to 200 µM Cd.

Mean ± SE with different letters, statistically differ at significance level P<0.05 according to LSD tests.

Fig. 6. The effects of melatonin (100 μM) and SNP (200 μM) individually or in combination with 100 µM p-

CPA (as a melatonin scavenger), or 100 μM cPTIO (as a NO scavenger), on the activities of SOD (A), CAT (B),

POD (C) and APX (D) in roots of plants exposed to 200 µM Cd.

Mean ± SE with different letters, statistically differ at significance level P<0.05 according to LSD tests.

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