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

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

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]).

.

1 Environment ACS Paragon Plus


1

ABSTRACT

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

attributes of this important species on the other hand.

14 15

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

16 17 18 19 20 21 22 23 24 25


Page 2 of 42

Page 3 of 42

26


INTRODUCTION

27

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

28

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

29

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

30

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

31

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

32

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

33

membrane integrity and permeability.3

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

limited.6

48

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

49

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

50

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



51

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

52

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

53

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

54

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

55

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

56

pharmacological effects were attributed to its prominent constituents including essential oils

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70 71

MATERIALS AND METHODS

72

Germination assay

73

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

74

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

75

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


Page 4 of 42

Page 5 of 42


76

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

77

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

78

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

79

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

80

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

81

incubation. All assays were replicated three times.

82

Plant cultivation and Zn treatments

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

Determination of growth parameters, seed yield and yield components

94

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

95

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

96

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

97

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

98

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

99

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

100

determined.



101

Zinc determination

102

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

103

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

104

samples in 5 mL nitric acid (HNO3).

105

Determination of photosynthetic pigments

106 107

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

108 109

Biochemical analysis

110

Determination of proline and total sugar contents

111

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

112

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

113

Determination of lipid peroxidation and protein content

114

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

115

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

116

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

117

protein standard.

118

Determination of total lipids, fatty acid composition and lipid quality

119

Lipid content and fatty acid composition

120

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

121

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

122

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

123

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

124

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

125

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


Page 6 of 42

Page 7 of 42


126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

percentage ratio of each component to total FA.

135

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

136

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

137

21

138

Indexes of lipid quality

and Mondal et al. 22, respectively.

139

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

140

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

141

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

142

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

143

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

144

24

and Cecchi et al. 21, respectively.

145 146

Isolation and analysis of essential oils

147

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

148

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

149

chromatography and gas chromatography-mass spectrometry were performed as described

150

earlier by Marichali et al.6



151

Determination of total phenolic and total flavonoid contents

152

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

153

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

154

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

155

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

156

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

157

Antioxidant activity

158

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

159

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

160

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

161

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

162

method of Kabouche et al.28

163

Statistical analysis

164

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

165

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

166

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

167

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

168

to analysis.

169 170

RESULTS AND DISCUSSION

171

Effects of Zn on seed germination and seedling growth

172

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

173

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

174

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

175

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


Page 8 of 42

Page 9 of 42


176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185 186

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

187

MDA contents

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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



Page 10 of 42

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

possible explanation of pigment reduction includes inhibition of enzymes responsible for

213

chlorophyll biosynthesis such as δ-aminolevulinic acid dehydratase and protochlorophyllide

214

reductases, enhanced degradation of thylakoids and advanced peroxidation of chloroplast

215

membrane lipids by reactive oxygen species (ROS).33

2

Other

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

those reported in mulberry, tea and citrus.

225

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

33

From biochemical stand point, earlier studies


Page 11 of 42


226

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

227

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

228

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

229

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

230

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

231

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

232

Zn stress.36

233

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

234

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

235

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

236

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

237

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

238

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

239

level of MDA.

240 241

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.

242 243

Zinc accumulation

244

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

245

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

246

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

247

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

248

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

249

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

250

alternative for Zn biofortification of N. sativa seeds.



251

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

252

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

253

different organs significantly (p

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

Recommend Documents