Nigella sativa L. to Zinc Excess: Focus on Germination, Growth

Feb 8, 2016 - Thabet, 2020 Ariana, Tunisia. ABSTRACT: A comprehensive analysis of the responses of Nigella sativa L. to elevated zinc concentrations w...
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Responses of Nigella sativa L. to Zinc Excess: Focus on Germination, Growth, Yield and Yield Components, Lipids and Terpenes Metabolisms, Total Phenolics and Antioxidant Activities Ahmed Marichali, Sana Dallali, Saloua Ouerghemmi, Houcine Sebei, Hervé Casabianca, and karim Hosni J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00274 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 16, 2016

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Journal of Agricultural and Food Chemistry

Responses of Nigella sativa L. to Zinc Excess: Focus on Germination, Growth, Yield and Yield Components, Lipids and Terpenes Metabolisms, Total Phenolics and Antioxidant Activities

Ahmed Marichali,†,‡ Sana Dallali, † Saloua Ouerghemmi, † Houcine Sebei, † Hervé Casabianca, § Karim Hosni*,£ †

Ecole Supérieure d’Agriculture de Mograne, 1121 Zaghouan, Tunisia.



Institut Supérieur Agronomique de Chott-Mariem, 4042 Sousse, Tunisia.

§

Institut des Sciences Analytiques, Département Service Central d’Analyse, 5 rue de la Doua,

Villeurbanne, 69100 Lyon, France £

Laboratoire des Substances Naturelles, Institut National de Recherche et d’Analyse Physico-

chimique (INRAP), Biotechpôle de Sidi Thabet, 2020Ariana, Tunisia.

*Corresponding Author: Karim Hosni (Telephone: +216 71537666. Fax: +216 71537888. E-mail addresses: [email protected]; [email protected]).

.

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ABSTRACT

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A comprehensive analysis of the responses of Nigella sativa L. to elevated zinc

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concentrations was assessed in pot experiments. Zn excess supply did not affect the

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germination but drastically reduced radicle elongation. A concentration-dependent reduction

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in all growth parameters, yield and yield components was observed. With the increasing Zn

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concentrations, total lipid contents decreased and changes in fatty composition towards the

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production of saturated ones were underscored. Despite the reduction in the seeds essential oil

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yield, a redirection of the terpenes metabolism towards the synthesis of oxygenated

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compounds has been evidenced. A significant increase in the total phenols and flavonoids

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contents concomitant with improved antioxidant activities has also been found. Collectively,

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these results highlight the possible use of N. sativa L. in phytoremediation applications on one

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hand, and that Zn excess could represents an excellent alternative to improve the nutritional

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attributes of this important species on the other hand.

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Keywords: Nigella sativa L., zinc, growth, lipids, secondary metabolites, antioxidant activity.

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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As an essential micronutrient, zinc (Zn) is required for the growth and development of

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plants. It can act as a functional, structural and regulatory co-factor in many enzymes and

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regulatory proteins.1 Zn plays crucial roles in plant physiology and metabolism such as

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photosynthesis, transpiration, nitrogen metabolism, DNA replication, auxin synthesis,

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cytochrome and chlorophyll biosynthesis, among others.2 It has also a stabilizing and

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protective effect on biomembranes during oxidative stress by the preservation of plasma

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membrane integrity and permeability.3

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Besides being a micronutrient, Zn is also a heavy metal and can have detrimental effects

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on many vital processes in plant cells. Geological and/or anthropogenic activities can result in

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Zn accumulation in soil above toxic levels for plants, leading to inhibition of growth and

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alteration of morpho-physiological and biochemical processes.4 These effects are caused by

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changes in carbohydrate metabolism, deficiencies in essential nutrients (e.g. Cu, Fe and Mg),

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reduction of chlorophyll biosynthesis and net photosynthesis, decrease in root biomass and

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leaf water content, and induction of oxidative stress.5 Zinc toxicity effects have been

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exhaustively investigated in model plants (e.g. Arabidopsis thaliana L. Heynh, A. halleri

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O’Kane and Al-Shehbaz ssp. halleri, Medicago truncatula Gaertn, Oryza sativa L., Triticum

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aestivum L., and Lactuca sativa L.) and in some hyperaccumulator species (e.g. Populus

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deltoids x maximowiczii, P. x canadiensis euramericana Mönch, Thlaspi caerulescens J. & C.

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Presl., Thlaspi goesingense Hálácsy, Sedum alfredii Hance and Paulownia tomentosa (Thunb)

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Steud)1-4, whereas the knowledge about Zn toxicity in medicinal and aromatic plants is still

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limited.6

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Nigella sativa L. (Ranunculaceae) commonly known as "black cumin" is a multipurpose crop

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grown for its seeds which have numerous food-related biological properties and multiple

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functional uses.7 Apart from being used as condiment or flavouring agent, the seeds are often

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used in different traditional medicine systems for the treatment of bronchial asthma,

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dysentery, back pain, obesity and headache.8 Along with these properties, seed extracts and

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essential oils have been reported to exhibit, among others antioxidant, antimicrobial, anti-

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inflammatory, anticancer, immunomodulatory, anti-hyperglycemia and anti-hyperlipidemia,

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wound healing, cytotoxic, hepato-and nephroprotective properties.9 Most of these intriguing

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pharmacological effects were attributed to its prominent constituents including essential oils

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(thymoquinone, thymol and carvacrol), fixed oil (linoleic, oleic and palmitic acids), alkaloids

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(nigellamine, nigellicimine, nigellicimine-N-oxide, nigellidine and nigellicine, damascenine,

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norargemonine and magnoflorine), flavonoids (kaempferol and its glycosylated derivatives),

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triterpene saponins (hederagenin, sapindoside), vitamins (thiamin, riboflavin, niacin, folic

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acid and vitamin E) and minerals.7,9,10 The content of these valuable components is somewhat

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prone to qualitative and quantitative variations depending on genotype, origin, season, plant

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part and nutritional status.10 Although the response of N. sativa to salinity has been reported,11

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very little and fragmentary information is available regarding its response to Zn exposure.12

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Therefore, the present work was intended to investigate the effects of Zn supply on

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germination, growth, lipid profile and quality, essential oils, total phenol and flavonoids

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contents as well as the antioxidant activity of the methanolic extracts from different parts of

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the plant. Results of this study could provide useful insights on the plant response to Zn

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toxicity, thus supporting crop production, and promoting a more efficient land use.

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MATERIALS AND METHODS

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Germination assay

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Seeds of the Tunisian variety of N. sativa were obtained from a non contaminated field

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plantation in the region of Haouaria (North-eastern Tunisia). The seeds were surface-sterilized

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in 0.5% sodium hypochlorite for 10 min, and then washed thoroughly with deionized water

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and soaked in distilled water for 24 h. Fifty seeds were uniformly placed in a 9 cm sterile

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Petri dish lined with one layer of filter paper (Whatman No.1), moistened with 2 mL of ¼

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Hoagland's nutrient solution supplied with 0 (25% of Hoagland), 0.1, 1 and 2 mM Zn as

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ZnSO4.7H2O and placed in a germination cabinet at 25 ± 1 °C in the dark.13 Germinated seeds

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with 1 mm radicle length were recorded and radicle elongation was measured 7 days after

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incubation. All assays were replicated three times.

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Plant cultivation and Zn treatments

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Surface sterilized seeds were sown in 10-L plastic pots (20 seeds per pot), filled with

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commercial peat and sand (1:2, v/v), moistened with distilled water and kept in a growth

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chamber at 25 ± 1 °C. Upon the emergence of seedlings, pots were transferred to a

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greenhouse (École Supérieure d’Agriculture de Mograne, Zaghouan, Tunisia; latitude

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36°25′47″(N); longitude 10°05′59″(E); altitude 149 m) naturally lit with sunlight, with a

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temperature range of 20–30 °C, relative humidity range of 50–80% and supplied with 1 L

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distilled water every 5 days. After two weeks, 5 healthy and uniform seedlings were kept in

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each pot and allocated to Zn treatment using ¼ Hoagland's nutrient solution (pH = 6.8)

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supplied with 0 (25% of Hoagland), 0.1, 1 and 2 mM Zn as ZnSO4.7H2O (renewed every 5

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days). Plants were harvested after 12 weeks of Zn treatments for various analyses.

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Determination of growth parameters, seed yield and yield components

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Growth of the plants was determined by measuring the length and fresh weight (FW) of

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the aerial parts and roots immediately before harvest. Dry weight (DW) was recorded after

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drying in a hot air oven 65 ºC till constant weight. The diameters (mm) of primary and

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secondary branches and the number of secondary branches per plant were determined. Seed

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yield per plant, weight of 1000 seeds, number of seeds per plant, number of fruit capsules per

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plant and the number fruit capsules per primary and secondary branches were also

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determined.

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Zinc determination

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The Zn content in different plant organs was determined by using a Philips PU 9100

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model atomic absorption spectrophotometry (Cambridge, England) after digestion of 0.5g

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samples in 5 mL nitric acid (HNO3).

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Determination of photosynthetic pigments

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Photosynthetic pigments were extracted with 80% acetone from fresh leaves (1.5g) and quantified by measuring the absorbance at 663, 644 and 452 nm.14

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Biochemical analysis

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Determination of proline and total sugar contents

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Proline content was determined following the method of Tiwari et al.15 The total sugar

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content was estimated by colorimetric method using pure glucose as standard.16

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Determination of lipid peroxidation and protein content

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The level of lipid peroxidation was measured in term of malondialdehyde (MDA; a

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byproduct of lipid peroxidation) content and expressed as µmolg-1 FW as described by

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Davenport et al.17 Total protein contents were measured using bovine serum albumin as a

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protein standard.

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Determination of total lipids, fatty acid composition and lipid quality

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Lipid content and fatty acid composition

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Plant tissues (roots, shoots, leaves and fruits) of ground powder (1 g) in triplicate were

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weighed and extracted with chloroform: methanol (2:1,v/v) (LabScan, Dublin, Ireland)

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following the procedure of Bligh and Dyer.18 Fatty acid methyl esters (FAMEs) were

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prepared by using sodium methoxide (Sigma–Aldrich, Buchs, Switzerland) according to the

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method of Cecchi et al.19 The FAMEs were analyzed by gas chromatography (GC) using a

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Shimadzu HRGC-2010 apparatus (Shimadzu Corporation, Kyoto, Japan) equipped with flame

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ionization detector (FID), Auto-injector AOC-20i and auto-sampler AOC-20s was used.

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Separation of different FAMEs was performed on a TRB-Wax capillary column (30 m length,

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0.25 mm i.d., 0.25 µm film thickness). The oven temperature was programmed as follows:

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starting from 150 °C (5 min), increasing to 200 °C at a rate of 15 °C/min, and finally held for

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5 min. The injector and detector temperature was maintained at 250 °C and 275 °C,

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respectively. The split ration was 1:100 and the injection volume was 1µL. Identification of

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FAMEs was made by comparing their retention times with those of reference standards

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purchased from Fluka (Steinheim, Germany). The FAMEs compositions (percent) refer to the

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percentage ratio of each component to total FA.

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The double bond index (DBI), iodine values (IV), oleic desaturation ratio (ODR) and

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linoleic desaturation ratio (LDR) were determined as outlined by Gignon et al. 20; Cecchi et al.

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21

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Indexes of lipid quality

and Mondal et al. 22, respectively.

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In addition to the unsaturated fatty acid/saturated fatty acid (UFA/SFA) ratio, the

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artherogenic index (AI), thrombogenic index (TI) as well as the calculated oxidizability value

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(Cox) and the oxidative susceptibility (OS) were used to evaluate the lipid quality of different

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organs of N. sativa. The AI and TI were calculated according to the formulas of Ulbrich and

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Southgate. 23 The Cox and OS values were determined as described by Fatemi and Hammond,

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and Cecchi et al. 21, respectively.

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Isolation and analysis of essential oils

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Due to the insufficient quantities of roots, leaves and shoots, the analysis of essential oils

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was limited to only fruits. The extraction of essential oils and their analysis by gas

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chromatography and gas chromatography-mass spectrometry were performed as described

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earlier by Marichali et al.6

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Determination of total phenolic and total flavonoid contents

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Total phenolics were determined with the Folin–Ciocalteu assay according to the

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procedure reported by Singleton and Rossi,25 and the results were expressed as milligram of

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gallic acid equivalents per g dried extract (mg GAE/g DW). Total flavonoid content was

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determined by the AlCl3 colorimetric method and the results were expressed as mg of

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quercetin equivalents per g dried extract (mg QE/g DW).26

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Antioxidant activity

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The radical scavenging activity of the methanol extracts (prepared by maceration of 1g of

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plant samples in 20 mL methanol for 24 h under gentle agitation) against DPPH free radical

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was measured using the method of Sánchez-Moreno et al.27 The ability of the extracts to

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inhibit the bleaching of the β-carotene-linoleic acid emulsion was determined using the

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method of Kabouche et al.28

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Statistical analysis

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All determinations were conducted in triplicates and results for each measured parameter

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were expressed as mean ± standard deviation (SD). One way analysis of variance (ANOVA)

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followed by Duncan's Multiple Range Test was applied to compare means at the significance

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level p < 0.05. When necessary, transformations were carried out to normalize the data prior

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to analysis.

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RESULTS AND DISCUSSION

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Effects of Zn on seed germination and seedling growth

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Irrespective of Zn supply, the germination percentage of N. sativa seeds remains

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unchanged. In contrast, increasing Zn concentration significantly reduced the radicle length

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(Table 1). These results continue to support the notion that radicle elongation is more

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sensitive to heavy metals than seed germination.2, 6 Possible mechanisms for the suppression

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of radicle growth under elevated Zn concentrations may be the inhibition of cell proliferation

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and elongation as well as loss of cell viability in the root tips and increased root

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lignification.13 In contrast, the ability of N. sativa seeds to maintain a constant germination

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percentage at elevated Zn concentration (up to 2 mM) may be linked with possible adsorption

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of this metal by the seed coat, enabling thus, the protection of the embryo from the phytotoxic

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effects of Zn.29 Another possible explanation is that elevated Zn concentrations did not affect

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imbibition and interfere with water uptake. Support to this assumption is given by Lefèvre et

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al.30 who showed that the seeds of Dorycnium pentaphyllum that failed to germinate after

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imbibition in high Zn concentration did not germinate after rinsing in dionized water.

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Effects of Zn on plant growth, pigments, soluble protein, soluble sugar, proline and

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MDA contents

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Although that all tested plants remained alive and achieved their growth and

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developmental cycle until the end of treatment, toxic effects of Zn excess, namely leaf

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chlorosis were visually observed. Results depicted in table 2 show gradual decrease in all

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growth parameters (plant height, root length, fresh and dry weight of aerial parts and roots,

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number of secondary branches per plant and diameters of primary and secondary branches),

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yield and yield components (number of capsules per plants, number of seeds per plants, 1000

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seeds weight, number of flower per plant, number of flowers per primary and secondary

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branches and number of capsules per plant) with the increase of Zn concentration. The

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decrease in growth and concomitantly, the decrease in yield and yield components under

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increasing Zn concentrations were presumably due to alteration in cell division and

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elongation, net photosynthesis, respiration, ion uptake, and protein synthesis.31 The Zn-

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induced alterations in the fundamental physiological process have been commonly reported in

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plants. Analysis of photosynthetic pigments gives support to these hypotheses. In fact,

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Exposure to elevated Zn concentration resulted in significant decrease in chlorophyll contents

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(total chlorophyll, Chla and Chlb), being more pronounced in Chlb contents as evidenced by

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the ratio Chla/Chlb. The same tendency was also observed for carotenoid contents. The

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decrease in photosynthetic pigments could be regarded as a metal-specific response which

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resulted in inhibition of chlorophyll synthesis possibly by induced iron deficiency and the

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substitution by Mg2+.32 In this context, Fernàndez-Martínez et al.2 linked the decrease in

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cholorophyll content as well as the ratio Chla/Chlb with a reduced proportion of reaction

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center core complexes to light-harvesting antenna complexes in PSI and PSII. The study

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author’s and others also interpreted the Zn-induced inhibition of photosynthesis to the

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reduction in the rate of Rubisco synthesis and/or modification in its activities, as a

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consequence of major decreases in stomatal and mesophyll conductance to CO2.

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possible explanation of pigment reduction includes inhibition of enzymes responsible for

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chlorophyll biosynthesis such as δ-aminolevulinic acid dehydratase and protochlorophyllide

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reductases, enhanced degradation of thylakoids and advanced peroxidation of chloroplast

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membrane lipids by reactive oxygen species (ROS).33

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Other

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Additional biochemical analyses (Table 3) revealed a concentration-dependent reduction

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in soluble protein contents in all organs. Decline in total soluble protein content may be

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attributed to increased activity of protease or other catabolic enzymes, as well as by Zn-

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induced oxidative stress.34 In contrast, the reciprocal trends were observed for total soluble

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sugars, proline and MDA contents. Accumulation of soluble sugar and proline is a general

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response to cope with the deleterious effects of Zn excess which will eventually lead to

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restoration of cellular homeostasis, detoxification, and mitigation of metal-induced lipid

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peroxidation, and therefore survival under stress.35 These results were in accordance with

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those reported in mulberry, tea and citrus.

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indicated that the accumulation of soluble sugar might be due to the degradation of insoluble

33

From biochemical stand point, earlier studies

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carbohydrates and/or the synthesis of sugar in other non-photosynthetic pathways and a

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reduction in growth could be responsible for the increase in soluble sugar content under

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stressful conditions. Moreover, considering the impairment of photosynthetic activity and the

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concomitant increased demand of carbon source and reducing power as NADPH, it seems

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logical to suppose that N. sativa induces sugar metabolism, possibly via upregulation of

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invertase to ensure adequate carbon and NADPH supply as well as a good protection against

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Zn stress.36

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Concerning proline, its accumulation could reflect an increased activity of pyrroline-5-

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carboxylate synthase and γ-glutamyl kinase, the key enzymes in proline biosynthesis, and/or

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inhibition of proline dehydrogenase, an enzyme that catalyzes proline degradation.37

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Whatever the mechanism leading to their accumulation, soluble sugar and proline could play

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significant roles in osmoregulation and osmoprotection. They could also serve as radical

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scavengers and protect bio-membrane from oxidative damages as evidenced by increased

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level of MDA.

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To get a global insight about the extent of lipid peroxidation, the lipid content and fatty acid composition as well as the lipid quality indices were assessed.

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Zinc accumulation

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For all the plant parts, Zn concentration increased with the increase of Zn in the medium

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(Fig. 1). Plants exposed to 2 mM Zn accumulated substantial amounts of Zn in their leaves

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and roots which exhibited 51 and 53% increase in their Zn concentrations, respectively. These

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results indicate that N. sativa has an increased ability to transport Zn to its aerial parts at

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higher Zn concentrations. These results were in line with those reported previously in bean.38

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From nutritional point of view, it appears that Zn treatment may represents an excellent

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alternative for Zn biofortification of N. sativa seeds.

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Effects of Zn on total lipids, fatty acid composition and lipid quality

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With the increasing Zn concentration in the nutrient solution, total lipid content in

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different organs significantly (p