Germination, morpho-physiological and biochemical responses of coriander (Coriandrum sativum L.) to zinc excess

Industrial Crops and Products 55 (2014) 248–257

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Germination, morpho-physiological and biochemical responses of coriander (Coriandrum sativum L.) to zinc excess Ahmed Marichali a , Sana Dallali a , Salwa Ouerghemmi a , Houcine Sebei a , Karim Hosni b,∗ a b

Département de Production Végétale, Ecole Supérieure d’Agriculture de Mograne, 1021 Zaghouan, Tunisia Laboratoire des Substances Naturelles, Institut Supérieur de Recherche et d’Analyse Physico-chimique, Biotechpole de Sidi Thabet, 2020 Tunisia

a r t i c l e

i n f o

Article history: Received 25 November 2013 Received in revised form 10 February 2014 Accepted 23 February 2014 Keywords: Coriandrum sativum L Zinc Germination Growth Fatty acids Secondary metabolites

a b s t r a c t A pot experiment was conducted to study the effects of zinc (Zn) on germination, growth, yield, yield components, chlorophyll content, fatty acid composition, total phenol content and fruit essential oil composition of coriander (Coriandrum sativum L.). Application of zinc sulphate (0.1; 1 and 2 mM) in the nutrient solution did not affect the germination, but severely reduced the radicle elongation. In general, there was a decrease in all growth parameters such as plant height, number of secondary branches, diameter of primary and secondary branches, fresh and dry weights of aerial parts and roots. Decrease in seed yield and its components (i.e. number of umbels per primary branches and secondary branches; number of umbel per plant; number of seeds per plant and 1000 seeds weight) were also observed. Zinc treatments reduced the content of total lipids in all plant parts and induced remarkable quantitative changes in fatty acids (FAs) profiles, leading to decrease in their unsaturation, which was indicated by reduction in double bond index (DBI). Exposure to elevated Zn concentrations increased the content of total phenol content in all plant parts. In contrast, Zn excess reduced the yield of essential oil in the fruits and affects negatively the content of oxygenated monoterpenes namely linalool, camphor and geraniol, which were found as the most sensitive compounds. These results are discussed in connection to available data regarding the morpho-physiological and biochemical responses of different plants species to Zn excess and previously tested metals. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Contamination of the environment with heavy metals due to increased industrialization and geochemical activities is a major environmental and human health problem. The heavy metals of most concern are cadmium (Cd) and Zinc (Zn), as they show the greatest mobility in the soil environment (Jiang and Wang, 2008). Zinc, as one of the essential micronutrients in plants is necessary for normal plant growth and development, as it is known to be required in several metabolic processes (Broadley et al., 2007). However, at high concentration, this micronutrient exhibits strong toxicity and hampers plant growth (Ali et al., 1999). It is well documented that excess Zn inhibits plant growth, root and shoot development, and induces leaf chlorosis (Michael and Krishnaswamy, 2011). At the cellular level, Zn excess alters mitotic activity, affects membrane integrity and permeability, disrupts basic physiological processes (e.g., photosynthesis) and

∗ Corresponding author. Tel.: +216 71537666; fax: +216 71537888. E-mail addresses: [email protected], karim [email protected] (K. Hosni). http://dx.doi.org/10.1016/j.indcrop.2014.02.033 0926-6690/© 2014 Elsevier B.V. All rights reserved.

induces oxidative stress via the generation of reactive oxygen species (ROS) and promotes the peroxidation of lipids (Li et al., 2013). While these studies may potentially give insight into the general responses (e.g., germination, photosynthesis, mineral nutrition and whole-plant growth, among others), little attention has been paid to the impact of Zn excess on the secondary metabolism such as essential oils and polyphenols. Assessment of such effects is, however, crucial to achieve a more comprehensive picture of plant responses toward Zn toxicity, and to find possible candidate species for the rehabilitation of polluted environments. Coriandrum sativum L., commonly known as coriander, is a culinary and medicinal plant native to the Mediterranean and Middle Eastern regions. It is a multipurpose herb grown mainly for its foliage and seeds which have numerous food-related biological activities and multiple functional uses (Burdock and Carabin, 2009). Beside their culinary uses as flavoring agent of different dishes including meat sauce, candy, pickles, and alcoholic beverages, among others, the fruits are extensively used for pharmacological purposes due to their stomachic, spasmolytic, carminative, hypoglycemic, lypolytic, antioxidant, antimicrobial, antifungal, anticancerous and antimutagenic activities (Burt, 2004).

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The linalool rich essential oil from coriander fruits, which has received the GRAS (Generally Recognized As Safe) status, is extensively used in perfumery, aromatherapy and the production of soap, creams and lotions (Cooksley, 2003). Another chemical attribute of coriander fruits is their high content of unusual fatty acid; petroselinic acid (C18:1n-12) which is of particular interest for oleochemical raw material since it is used in the production of detergents and nylon polymers (Msaâda et al., 2009a). The content of these valuable components can vary considerably depending on plant variety (Burdock and Carabin, 2009), growing region, season, plant part, fruit maturation, extraction and analytical procedures (Msaâda et al., 2007, 2009a, 2009b, 2012). Compositional changes in response to nutritional constraint namely salinity have previously been reported (Neffati and Marzouk, 2008; Neffati et al., 2012). However, information on the responses of coriander to increasing concentrations of heavy metals is not available. Therefore, the aim of this study was to evaluate the germination, growth, physiological and biochemical responses of coriander to excessive Zn concentrations. This study not only provides a better understanding of the mechanisms involved in metal tolerance/sensitivity, but it also highlights information which will be helpful for the management of this potential economic species. 2. Materials and methods 2.1. Germination assay Seeds of C. sativum used for germination assay were collected from plants cultivated in the region of Haouaria (North-eastern Tunisia). The field of cultivation is not contaminated by Zn. The seeds were surface-sterilized in 0.5% sodium hypochlorite for 10 min, and then washed thoroughly with deionized water and soaked in distilled water for 24 h. Fifty seeds were uniformly placed in a 9 cm sterile Petri dish lined with one layer of filter paper (Whatman No. 1), moistened with 2 mL of ¼ Hoagland’s nutrient solution supplied with 0 (25% of Hoagland), 0.1, 1 and 2 mM Zn as ZnSO4 ·H2 O (Li et al., 2012) and placed in a germination cabinet at 25 ± 1 ◦ C in the dark. Germinated seeds with 1 mm radicle length were recorded and radicle elongation was measured 7 days after incubation. All assays were replicated three times. 2.2. Plant culture and Zn application Surface sterilized seeds of coriander were sown in 10-L plastic pots (20 seeds per pot), filled with commercial peat and sand (1:2, v/v), moistened with distilled water and kept in a growth chamber at 25 ± 1 ◦ C. Upon the emergence of seedlings, pots were transferred to a greenhouse (École Supérieure d’Agriculture de Mograne, Zaghouan, Tunisia; latitude 36◦ 25 47 N; longitude 10◦ 05 59 E; altitude 149 m) naturally lit with sunlight, with a temperature range of 20–30 ◦ C and relative humidity range of 50–80% and supplied with 1 L distilled water every 5 days. After two weeks, 5 healthy and uniform seedlings were kept in each pot and allocated to Zn treatment using ¼ Hoagland’s nutrient solution (pH 6.8) supplied with 0 (25% of Hoagland), 0.1, 1 and 2 mM Zn as ZnSO4 H2 O (renewed every 5 days). Each treatment was replicated five times. At the fruiting stage, 12 weeks after Zn application, coriander plants height was measured, and then harvested, separated into roots, shoots, leaves and fruits. Each plant part was immediately weighed, and then oven dried at 65 ◦ C for 48 h until constant weight. 2.3. Determination of growth parameters, yield and yield components For each treatment, the fresh and dry weight, diameters (mm) of primary and secondary branches and the number of secondary

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branches per plant were determined. Seed yield per plant, weight of 1000 seeds, number of seeds per plant, number of umbels per plant and the number of umbels per primary and secondary branches were also determined. 2.4. Determination of chlorophyll content The pigments (Chla, Chlb and total chlorophyll) were extracted from fresh leaves in 80% acetone at room temperature. The chlorophyll contents were determined spectrophotometrically according to the method of Arnon, 1949 and using the following equations: Chla = 10.05OD663 − 1.97OD645 Chlb = 16.36OD645 − 2.43OD663 Chla+b = 7.62OD663 + 14.39OD645 where Chla = chlorophyll a; Chlb = chlorophyll b; Chla+b = total chlorophyll, OD = optical density (nm). 2.5. Biochemical analysis 2.5.1. Determination of total lipids and fatty acid composition Samples (roots, shoots, leaves and fruits) of ground powder (1 g) in triplicate were weighed and extracted with chloroform: methanol (2:1, v/v) (LabScan, Dublin, Ireland) following the modified procedure of Bligh and Dyer (1959). The mixture was shaken and centrifuged (Eppendorf 5810R, Le Pecq, France) at 3000 × g for 10 min to allow phase separation. The bottom (organic) layer containing total lipids was recovered and dried under a nitrogen stream. Fatty acid methyl esters (FAMEs) were prepared by using sodium methoxide (Sigma–Aldrich, Buchs, Switzerland) according to the method of Cecchi et al., 1985. Methyl nonadecanoate (C19:0) was used as internal standard. The FAMEs were analyzed by gas chromatography (GC) using a Shimadzu HRGC-2010 apparatus (Shimadzu Corporation, Kyoto, Japan) equipped with flame ionization detector (FID), Auto-injector AOC-20i and auto-sampler AOC-20s was used. Separation of different FAMEs was performed on a RT-2560 capillary column (100 m length, 0.25 mm i.d., 0.20 ␮m film thickness). The oven temperature was programmed as follows: starting from 170 ◦ C (2 min), increasing to 240 ◦ C at a rate of 3 ◦ C/min, and finally held for 15 min. The injector and detector temperature was maintained at 225 ◦ C (Msaâda et al., 2009a). Identification of FAMEs was made by comparing their retention times with those of reference standards purchased from Fluka (Steinheim, Germany). The FAMEs compositions (percent) refer to the percentage ratio of each component to total FA. The double bond index (DBI) was calculated as follows: (1 × % monoenoic acids) + (2 × % dienoic acids) + (3 × trienoic acids) (Gignon et al., 2004). 2.5.2. Determination of total phenolic contents Total phenolics were determined with the Folin–Ciocalteu assay according to the procedure reported by Singleton and Rossi, 1965, with some modifications. Briefly, 500 ␮L of appropriately diluted extract was added to 5 mL of freshly diluted 10-fold Folin–Ciocalteu reagent, and the mixture was neutralized with 4 mL of a sodium carbonate solution (7%). The reaction mixture was kept in the dark for 15 min, and its absorbance was measured at 765 nm against a blank, which was prepared according to the procedure described above except that the extract solution was substituted by 500 ␮L of distilled water. Gallic acid was used as the standard, and results were expressed as microgram of gallic acid equivalents (␮g GAE/g).

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2.5.3. Isolation and analysis of essential oils Due to the insufficient quantities of roots, leaves and shoots, the analysis of essential oils was limited to only fruits. To this end, the air dried fruits (50 g) were ground using a Retsch blender mill (Normandie-Labo, Normandy, France), sifted through 0.5 mm mesh screen, and submitted to hydrodistillation for 90 min using a Clevenger type apparatus. The oils obtained were dried over anhydrous sodium sulfate and stored in amber and air-tight sealed vials at 0 ◦ C until analysis. Gas chromatography analyses were carried out on the same apparatus above mentioned. For the separation of volatile components, an apolar capillary column HP 5 (30 m length, 0.25 mm i.d., 0.32 ␮m film thickness) was used. The oven temperature was held at 50 ◦ C for 10 min then programmed at 2 ◦ C/min to 190 ◦ C then held isothermal for 10 min. The injector and detector temperature was maintained at 230 ◦ C. The flow of the carrier gas (N2 ) was 1.6 mL/min and the split ratio was 1:20. Injection volume for all samples was 0.5 ␮L of diluted oils in n-pentane (LabScan Dublin, Ireland). The gas chromatography–mass spectrometry (GC–MS) analyses were performed on a gas chromatograph HP 6890 (II) interfaced with an HP 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with electron impact ionization (70 eV). An HP-5MS capillary column (60 m length, 0.25 mm, 0.25 ␮m film thickness) was used. The column temperature was programmed to rise from 40 to 280 ◦ C at a rate of 5 ◦ C/min. The carrier gas was helium with a flow rate of 1.2 mL/min. Scan time and mass range were 1 s and 50–550 m/z, respectively. The volatile compounds were identified by comparison of their retention indices relative to (C7 –C20 ) n-alkanes with those of literature and/or with those of authentic compounds available in our laboratory. Further identification was made by matching their mass spectral fragmentation patterns with corresponding data (Wiley 275.L library) and other published mass spectra (Adams, 2001) as well as by comparison of their retention indices with data from the Mass Spectral Library “Terpenoids and Related Constituents of Essential oils” (Dr. Detlev Hochmuth, Scientific consulting, Hamburg, Germany) using the MassFinder 3 software (www.massfinder.com). Relative percentage amounts of the identified compounds were obtained from the electronic integration of the FID peak areas without use of the correction factor.

2.6. Statistical analysis All experiments were performed in triplicate, and the data were expressed as mean ± standard deviation (SD). One way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test was applied to compare means at the significance level p < 0.05. Correlation coefficients between quantitative variables were determined by the Pearson’s bivariate correlation analysis. All analyses were performed using SAS v 9.1 software package.

3. Results 3.1. Effects of Zn on seed germination and radicle elongation As shown in Table 1, Zn treatment results in a slight but not statistically significant (p > 0.05) decrease in coriander seed germination. In contrast, it induced remarkable inhibition in the radicle elongation, with the effects being more pronounced at elevated Zn concentration (2 mM). These data strongly suggest that radicle growth is more sensitive to Zn toxicity than seed germination.

Table 1 Effects of different zinc concentrations on seed germination and root length of C. sativum. ZnSO4 concentration (mM)

Germination percentage (%)

Control 0.1 1 2

99.2* 98.3 96.2 95.0

± ± ± ±

0.05a 0.06a 0.04a 0.01a

Root length (cm) 8.9 5.5 3.0 1.2

± ± ± ±

0.33a 0.07b 0.11c 0.01d

Superscripts with different letters within the same column are significantly (p < 0.05) different. * Values given are mean ± standard deviation of triplicate.

3.2. Effects of Zn on morphology, growth parameters, seed yield and its components and chlorophyll content Addition of Zn in the range of 0–0.1 mM did not show apparent morphological changes. However, plant cultivated under 1 and 2 mM Zn developed leaf chlorosis which can progress to purple in the basal leaves (Fig. 1). Such visual symptoms could be ascribed to the loss of chlorophyll and accumulation of anthocyanin on one hand, and to the early senescence on the other hand. To investigate whether these visible symptoms were/or not related to other morphological changes, a quantitative determination of growth parameters, seed yield and its components were undertaken. Analysis of quantitative traits (plant height; number of secondary branches; diameter of primary and secondary branches, fresh and dry weights of aerial parts and roots) revealed that all measured parameters were significantly (p < 0.05) reduced by excess of Zn (Table 2). The same trend was also observed for seed yield and its components (number of umbels per primary branches and secondary branches; number of umbel per plant; number of seeds per plant and 1000 seeds weight). At this point, it appears that the reduction in plant dry weight arose from a reduction of plant height, branching, diameter of branches and root dry weight. Likewise, the decrease in seed yield may be ascribed to the reduction on umbel number per primary and secondary branches and per plant as well as the number of seeds per plant and 1000 seeds weight. Determination of chlorophyll content revealed that this parameter fell to 86.3, 56.3 and 23.9% of the control values when 0.1, 1 and 2 mM Zn were added, respectively. Theses reductions were mainly the results of the drastic decrease in Chl a (17.2, 40.6 and 75.0% for 0.1, 1 and 2 mM Zn, respectively) and Chl b (7.7, 49.0 and 78% for 0.1, 1 and 2 mM Zn, respectively) contents. The remarkable decrease in Chl b and to a lesser extent Chl a contents at higher Zn concentration resulted in an increase of the ratio Chl a/Chl b (13.2 and 16.3% higher than in control plants). At this point, it appears that the biosynthesis of Chl b was more affected than the biosynthesis of Chl a at excessive Zn concentrations. Taken together, it seems that the reduction of growth parameters, seed yield and its components in response to Zn application could be considered as a direct consequence of a limitation of photosynthesis. To shed more light on the observed morphological and physiological changes induced by Zn supply, Pearson’s correlation analysis of all measured parameters was performed (Table 3). Results showed that there were highly significant positive correlations between the height of plant and the four growth parameters (diameter of primary branches, diameter of secondary branches, number of secondary branches and aerial part dry weight) with correlation coefficient (r) values of 0.995 (p = 0.005), 0.999 (p = 0.001), 0.988 (p = 0.012) and 0.972 (p = 0.028), respectively. The height of plant was also positively correlated with the weight of 1000 seeds (r = 0.976; p = 0.024). These results suggest that as plant height increase, the diameter of primary and secondary branches, number

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Fig. 1. Morphological aspects of Zn-treated plants of C. sativum (Left picture: control and plants treated with 0, (25% of Hoagland), 0.1 and 1 mM ZnSO4 ; Right picture: plants treated with 2 mM ZnSO4 ). Table 2 Effects of Zn treatment on growth parameters, yields, yield components and chlorophyll content in C. sativum. Parameters

Zn concentration (mM) Control

Height (cm) Number of secondary branches per plants: NSB Diameter of primary branches: DPB (mm) Diameter of secondary branches: DSB (mm) Dry weight of aerial parts: DWAP (%) Dry weight of roots: DWR (%) Seed yield (g) Number of umbels per plant: NU Pl Number of umbels per primary branches: NU PB Number of umbels per secondary branches: NU SB Number of seeds per plant: NS Pl 1000 seeds weight: 1000 SW (g) Total chlorophyll (mg/g Fw) Chla (mg/g Fw) Chlb (mg/g Fw)

90.7 7.8 76.3 29.4 32.2 26.1 2.4 292.8 23.5 269.3 223.6 15.9 13.8 5.1 8.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.18a 0.25a 3.95a 0.51a 1.09a 0.5a 0.20b 1.10a 0.28a 1.31a 16.82a 0.06a 0.12a 0.02a 0.11a

0.1

1

2

81.8 ± 3.51b 6.5 ± 0.28b 74.7 ± 4.85ab 27.8 ± 1.72ab 18.8 ± 0.69b 17.5 ± 0,96b 3.0 ± 0.19a 201.8 ± 2.28b 18.8 ± 1.54b 183.0 ± 2.78b 229.0 ± 17.12a 13.7 ± 0.20b 11.9 ± 0.01a 4.7 ± 0.01a 7.2 ± 0.01b

59.4 ± 2.89c 4.3 ± 0.62c 66.2 ± 5.14ab 24.5 ± 1.37cb 19.5 ± 0.69b 17.45 ± 0.59b 1.1 ± 0.13c 185.0 ± 2.61b 11.0 ± 0.80c 174.0 ± 2.48b 160.3 ± 10.4b 11.0 ± 0.24c 7.8 ± 0.01b 2.6 ± 0.01b 5.2 ± 0.01c

44.7 ± 1.96d 3.5 ± 0.28c 62.6 ± 2.36b 22.2 ± 1.01b 18.3 ± 0.6b 13.0 ± 0.76c 0.7 ± 0.07c 122.5 ± 3.27c 11.0 ± 0.80c 111.5 ± 3.92c 168.1 ± 10.95b 10.0 ± 0.14d 3.3 ± 0.02c 1.1 ± 0.01c 2.2 ± 0.03d

Values with different superscripts in the same line are significantly different at p < 0.05. Table 3 Pearson correlation coefficients between growth, productive and physiological parameters.

Height NSB DPB DSB DW AP DW R Seed yield NU Pl NU PB NU SB NS Pl 1000 SW Total Chl * **

Height

NSB

DPB

DSB

DW AP

DW R

seed yield

NU Pl

NU PB

NU SB

NS Pl

1000 SW

0.988* 0.995** 0.999** 0.972* 0.789 0.912 0.919 0.949 0.906 0.901 0.976* 0.994**

0.985* 0.989* 0.953* 0.836 0.882 0.925 0.986* 0.909 0.918 0.996** 0.967*

0.991** 0.990* 0.74 0.945 0.877 0.952* 0.861 0.939 0.966* 0.982*

0.964* 0.808 0.899 0.931 0.951* 0.919 0.891 0.980* 0.994**

0.637 0.982* 0.801 0.916 0.782 0.958* 0.922 0.957*

0.481 0.954* 0.846 0.954* 0.579 0.883 0.779

0.676 0.84 0.654 0.954* 0.835 0.896

0.895 0.999** 0.699 0.949 0.924

0.875 0.924 0.987* 0.911

0.67 0.935 0.915

0.885 0.856

0.955*

Significant at p < 0.05. Significant at p < 0.01.

of secondary branches, aerial part dry weight and the 1000 seeds weight increase. These parameters are in turn tightly dependent on the total chlorophyll content as revealed by their high and significant (p < 0.05 and p < 0.01) correlations suggesting that the loss of total chlorophyll resulted in drastic decrease in the plant height, aerial part dry weight, diameter of primary and secondary

branches, number of secondary branches and the weight of 1000 seeds. Concerning the yield and its components, results showed that seed yield was significantly and positively correlated with the number of seeds per plant (r = 0.954, p = 0.046). The latter component was in turn positively correlated with the aerial part dry weight

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(r = 0.958, p = 0.042), revealing that the aerial part dry weight could be assumed as a good indicator for predicting the seed yield and the number of seeds per plant. As commonly happen in many species, the excess of heavy metals was obviously associated with deep changes in all metabolic processes such as photosynthesis, protein and lipid synthesis and the secondary metabolism namely biosynthesis of polyphenols and essential oils. Bearing this in mind, the following section focuses on the effects of excessive Zn concentrations on the variations of the lipidic profile, total phenol content and the essential oil composition of coriander.

induces qualitative changes in the essential oil composition, but it reduced significantly the content of oxygenated monoterpenes which represent the most prominent chemical class of the fruit oil. The major changes in the essential oil from Zn-treated plant were the remarkable decrease in the content of linalool, ␥-terpinene, ␣thujene, camphor and geraniol, while the content of (Z)-␤-ocimene and eugenyl acetate increased. The remaining components showed irregular variations in response to Zn application.

3.3. Effects of Zn on total lipid content and fatty acid (FAs) composition

The present study demonstrated that excessive Zn did not inhibit seed germination, but reduced the root length in coriander seedling, indicating that Zn has low toxic effects on the germination of coriander seeds, and supporting the conclusion that roots are more sensitive than seeds to metal stress. These results were in good agreement with those of Martínez-Fernández et al., 2011, who showed that 150 ␮M Zn did not inhibit seed germination of Bituminaria bituminosa (L.) C.H. Stirton whereas 76 ␮M inhibited root and shoot growth. The authors also concluded that the phytotoxic effects of Zn can occur at concentrations as low as 1.5 ␮M (Martínez-Fernández et al., 2011). From physiological standpoint, the successful seed germination could be inferred to the fact that Zn did not affect imbibition and interfere with water uptake (Kranner and Colville, 2011). Support to this assumption is given by Lefevre et al., 2009, who showed that the seeds of Dorycnium pentaphyllum that failed to germinate after imbibition in high Zn concentrations did not germinate after rinsing in deionised water. The suppression of root elongation by excess Zn may involve inhibition of cell proliferation and subsequent elongation (Michael and Krishnaswamy, 2011). The studies of Finger-Teixeira et al., 2010 and Li et al., 2012 revealed that reduction of root growth is linked to a significant loss of cell viability in the root tips and to an increased level of lignification in wheat seedling exposed to high Zn concentrations. Our results are in partial agreement with Li et al., 2012, Lefevre et al., 2009 and Ozdener and Kutbay, 2009 who have reported similar results in Triticum aestivum, Dorycnium pentaphyllum and Eruca sativa, respectively. In contrast, strong inhibition of germination and drastic reduction in root growth in response to elevated Zn concentrations have previously been reported in other species such as Cucumus sativus (Aydin et al., 2012), Zea mays (Pokhrel and Dubey, 2013) and Eucomis autumnalis (Street et al., 2007). From these contrasting results, it can be argued that the response of germination and root growth to Zn excess is speciesdependent. The first interesting results from the present investigation, is that C. sativum could survive and achieve its growth and development cycle up to 2 mM Zn reflecting hence its relative tolerance to Zn toxicity. However, in response to excessive Zn concentrations, C. sativum plants showed a marked response on their morphological and physiological traits, with affected plant height, number of secondary branches, diameter of primary and secondary branches, fresh and dry weights of aerial parts and roots, number of umbels per primary branches and secondary branches, number of umbel per plant, number of seeds per plant and 1000 seeds weight. Analysis of Pearson’s correlations revealed significant and positive correlations between most of growth parameters, yield and yield components suggesting that the decrease in plant dry weight was likely due to the reduction of plant height, branching, diameter of branches and root dry weight. Similarly, the decrease in seed yield was always associated with to reduction of umbel number per primary and secondary branches and per plant as well as the number of seeds per plant and 1000 seeds weight which are to a very large degree a result

Irrespective to plant part analyzed, increasing Zn concentration in the nutrient solution significantly (p < 0.05) reduced total lipid content (Table 4). The magnitude of reduction of total lipid content was found to be higher in roots (81%) followed by stems (73.0%), leaves (60.6%) and seeds (34.6%). The qualitative and quantitative changes in FAs profile of coriander seeds, leaves, stems and roots were also presented in Table 4. As can be seen, Zn supply resulted in drastic decrease in the content of the main FAs in seed oil such as petroselinic acid (C18:1n-12), while it increased the content of oleic (C18:1n-9), palmitic (C16:0), palmitoleic (C16:1) and to a lesser degree arachidic (C20:0) acids. The other identified FAs remained unchanged. The polyunsaturated FAs mainly ␣-linolenic (C18:3n-3) and linoleic (C18:2n-6) acids were the most affected leaf FAs when exposed to higher Zn concentrations. The decrease in these FAs was concomitant to a remarkable increase in the content of oleic acid (C18:1n-9). No significant changes in the content of the rest of leaf FAs were observed. The major changes in stems FAs in response to elevated Zn concentrations were the significant decrease of palmitoleic (C16:1n-7) and oleic (C18:1n-9) acids, which represent the main FAs, whereas, a remarkable increase in the content of ␣-linolenic (C18:3n-3), erucic (C22:1n-1), palmitic (C16:0) and myristic (C14:0) acids was observed. The root FAs profiles were qualitatively similar to that of leaves and stems but it displayed great quantitative differences. The changes in the content of main FAs such as linoleic, oleic, myristic and palmitic acids were virtually similar to that observed for leaf FAs. In general, Zn treatment induced remarkable increase in the content of saturated FAs in all organs, decreasing thereby the DBI and the unsaturated to saturated FAs ratio. 3.4. Effects of Zn-excess on total phenolic contents (TPC) Table 5 shows the changes in the TPC in coriander plants subjected to elevated Zn concentrations. Under stressful conditions, TPC increased significantly (p < 0.05) in all organs, especially at the level of 2 mM Zn. The fruits recorded the greatest increase (133.3%) followed by leaves (89.4%), stems (78.3%) and roots (57.5%). 3.5. Effects of Zn-excess on yield and chemical composition of the fruit essential oils Data from Table 6 reveal that fruit essential oil yields were drastically reduced in Zn-treated plants. They dropped from 0.5% (w/w, dw) in control samples to 0.2% in plants exposed to high level of Zn (2 mM). The chemical composition of the essential oils was also presented in Table 6, where the identified components are listed in order of elution on the HP5 column along with their retention indices and their percentage. As can be seen, Zn treatment did not

4. Discussion

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Table 4 Effects of Zn treatment on total lipid content and fatty acid composition of different parts of C. sativum. Zn (mM)

Seeds

0 0.1 1 2

85.7 ± 0.33a 81.7 ± 0.88b 64.6 ± 0.58c 56.0 ± 0.58d

0 0.1 1 2

Pentadecanoic acid (C15:0)

Leaves

Stems

Roots

Total lipid contents (mg/g dw) 42.3 ± 0.33a 40.0 ± 0.58b 21.3 ± 0.88c 16.7 ± 0.88d

6.3 ± 0.67a 5.0 ± 0.58ab 3.7 ± 0.33b 1.7 ± 0.33c

0.9 ± 0.03d 2.4 ± 0.05b 3.8 ± 0.06a 2.0 ± 0.10c

2.9 ± 0.47a 1.5 ± 0.23a 2.6 ± 0.82a 2.2 ± 0.76a

2.7 ± 0.09b 3.0 ± 0.21b 3.6 ± 0.29b 9.3 ± 0.51a

6.4 ± 0.25b 13.3 ± 0.86a 14.9 ± 1.73a 15.0 ± 0.95a

0 0.1 1 2

tr tr 0.1 ± 0.03a tr

0.7 ± 0.09a 0.3 ± 0.16a 0.6 ± 0.28a 0.5 ± 0.04a

0.9 ± 0.49a 0.8 ± 0.33a 0.8 ± 0.42a 1.4 ± 0.13a

0.1 ± 0.01a 0.1 ± 0.00a 0.9 ± 0.71a 0.1 ± 0.02a

Palmitic acid (C16:0)

0 0.1 1 2

6.9 ± 0.01d 11.6 ± 0.13c 13.3 ± 0.12b 15.4 ± 0.06a

19.2 ± 0.07a 12.7 ± 4.36a 15.4 ± 0.11a 12.9 ± 0.54a

0.8 ± 0.47b 0.4 ± 0.08b 0.6 ± 0.17b 9.2 ± 0.09a

23.1 ± 0.16d 24.5 ± 0.23c 26.0 ± 0.46b 28.4 ± 0.27a

Palmitoleic acid (C16:1n-7)

0 0.1 1 2

0.8 ± 0.06b 1.2 ± 0.03b 2.1 ± 0.36b 6.3 ± 0.71a

0.2 ± 0.10b 0.6 ± 0.02a 0.2 ± 0.18b 0.2 ± 0.09b

73.4 ± 0.32a 61.2 ± 0.74b 49.7 ± 0.51c 36.4 ± 0.58d

1.6 ± 0.35a 1.0 ± 0.15a 1.5 ± 0.20a 1.4 ± 0.04a

Heptadecanoic acid (C17:0)

0 0.1 1 2

0.2 ± 0.00a 0.4 ± 0.00a 0.8 ± 0.32a 1.6 ± 1.01a

0.3 ± 0.14a 4.4 ± 3.58a 2.0 ± 0.57a 3.4 ± 1.96a

0.6 ± 0.08b 1.7 ± 0.21a 0.8 ± 0.48ba 1.3 ± 0.33ba

1.5 ± 0.18a 3.7 ± 0.31a 4.4 ± 1.82a 2.3 ± 0.18a

Stearic acid (C18:0)

0 0.1 1 2

0.7 ± 0.09a 1.2 ± 0.01a 3.0 ± 0.88a 2.8 ± 1.50a

1.7 ± 0.11a 1.6 ± 0.24a 2.1 ± 0.26a 1.9 ± 0.67a

0.3 ± 0.15a 0.7 ± 0.05a 0.4 ± 0.03a 0.6 ± 0.08a

2.3 ± 0.03ba 2.1 ± 0.12b 4.9 ± 1.51a 2.7 ± 0.38ba

Petroselinic acid (C18:1n-12)

0 0.1 1 2

84.1 ± 0.05a 69.1 ± 0.12b 50.3 ± 0.10c 41.3 ± 0.07d

– – – –

– – – –

– – – –

Oleic acid (C18:1n-9)

0 0.1 1 2

3.4 ± 0.04c 11.2 ± 0.03b 20.9 ± 0.30a 23.4 ± 2.07a

6.0 ± 0.46c 12.6 ± 0.05b 13.5 ± 0.26b 18.3 ± 1.27a

13.7 ± 1.20a 12.4 ± 0.10b 11.2 ± 0.15c 10.8 ± 0.09d

9.5 ± 0.18c 10.6 ± 0.04c 14.4 ± 0.97b 17.1 ± 0.32a

Linoleic acid (C18:2n-6)

0 0.1 1 2

1.6 ± 0.12a 1.4 ± 0.64a 3.5 ± 1.33a 2.5 ± 0.28a

30.1 ± 0.18a 27.6 ± 0.58b 25.6 ± 0.28c 24.2 ± 0.23d

2.2 ± 0.06a 2.6 ± 0.14a 2.1 ± 0.86a 3.1 ± 0.15a

43.1 ± 0.09a 32.1 ± 0.13b 20.3 ± 0.08c 15.8 ± 0.03d

␣-linolenic acid (C18:3n-3)

0 0.1 1 2

0.9 ± 0.01a 0.2 ± 0.02b 0.4 ± 0.04b 0.4 ± 0.22b

36.4 ± 0.50a 34.2 ± 0.14b 30.7 ± 0.15c 28.4 ± 0.84d

3.2 ± 1.48c 6.6 ± 0.79bc 12.8 ± 2.31a 9.4 ± 0.28ba

6.4 ± 0.21a 4.7 ± 0.02b 4.0 ± 0.23c 2.4 ± 0.11d

Arachidic acid (C20:0)

0 0.1 1 2

0.1 ± 0.02b 0.7 ± 0.67b 0.8 ± 0.47b 2.9 ± 0.75a

0.3 ± 0.15b 0.3 ± 0.13b 0.7 ± 0.42a 0.6 ± 0.26a

2.5 ± 0.51b 3.6 ± 2.87b 6.8 ± 2.25a 1.1 ± 0.44c

1.7 ± 0.06b 2.7 ± 0.12a 2.2 ± 0.45ba 2.7 ± 0.16a

0.1 ± 0.00b 0.1 ± 0.00b 0.2 ± 0.04ba 0.6 ± 0.21a

0.7 ± 0.39b 0.3 ± 0.34c 0.7 ± 0.34b 1.2 ± 0.15a

0.2 ± 0.12b 0.5 ± 0.16ba 1.1 ± 0.13a 0.8 ± 0.36ba

0.2 ± 0.06a 0.1 ± 0.05a 0.3 ± 0.11a 0.2 ± 0.03a

1.3 ± 0.26b 1.6 ± 0.68b 2.5 ± 0.50a 3.0 ± 0.58a

0.3 ± 0.09c 3.2 ± 1.80bc 4.4 ± 0.43b 9.4 ± 0.24a

0.2 ± 0.03a 0.2 ± 0.03a 0.5 ± 0.32a 0.2 ± 0.05a

Fatty acid composition (%) Myristic acid (C14:0)

Gadoleic acid (C20:1n-9)

0 0,1 1 2

Erucic acid (C22:1n-9)

0 0.1 1 2

0.1 ± 0.01c 0.4 ± 0.00b 0.7 ± 0.14a 0.8 ± 0.44a

DBI

0 0.1 1 2

95.7a 85.0b 82.5b 78.6c

UFA/SFA

0 0.1 1 2

10.2a 5.1b 3.6c 3.0c

177.4a 172.9a 160.1b 156.3b 2.9ab 3.3a 2.7b 3.0a

tr: <0.05%; (–): not detected. Values with different superscripts in the same column are significantly different at p < 0.05.

9.0 ± 0.58a 7.3 ± 0.67ab 6.0 ± 0.58b 1.7 ± 0.33c

101.5b 102.2b 109.0a 92.0c

117.1a 90.2b 69.2c 57.5d

13.2a 6.4b 4.4c 2.3d

1.6a 1.1b 0.7c 0.6c

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Table 5 Effects of Zn treatment on total phenols content in different parts of C. sativum. Zn (mM)

Seeds

0 0.1 1 2

76.8 125.5 175.3 179.1

Leaves ± ± ± ±

0.14d 0.14c 0.22b 0.44a

± ± ± ±

98.4 160.8 177.4 186.4

Stems 0.55d 0.38c 0.22b 0.73a

72.3 106.2 126.1 128.8

Roots ± ± ± ±

0.80d 0.68c 0.60b 0.68a

104.3 133.5 154.3 164.4

± ± ± ±

0.17d 0.14c 0.17b 0.22a

Values with different superscripts in the same column are significantly different at p < 0.05.

of the direct effects of Zn supply (Movahhedy-Dehnavy et al., 2009). Most of the measured parameters namely plant height, aerial part dry weight, diameter of primary and secondary branches, number of secondary branches and the weight of 1000 seeds are positively correlated with total chlorophyll content which was dramatically reduced by exposure to increased Zn concentrations. Our results also showed that Chl b was more sensitive to Zn excess than Chl a which is in line with the results of Li et al., 2013 who reported that the reduction in the content of Chl b of wheat plants in response to Zn excess was more pronounced than that of Chl a. They ascribed the loss of Chl b to its faster hydrolysis compared with Chl a. In general, the decrease of chlorophyll concentration is considered as

one of the earliest and distinct symptoms of Zn toxicity in plants (Di Baccio et al., 2009). The Zn-mediated reduction of chlorophyll concentration could at least, partially explains the decrease in the aforementioned parameters. The installation of leaf chlorosis which can progress to reddening in older leaves is ascribed to P deficiency, and is regarded as general response to Zn toxicity in plants (Di Baccio et al., 2009; Azzarello et al., 2012). These authors have also showed that Zn excess induces ultrastructural and physiological changes in leaves including significant increase in thickening of the leaf lamina, decrease in the number of chloroplasts and vacuolar bundles, withdrawal of the plasma membrane and perturbation of the conformation of the photosystem II (PSII core complex, thus leading

Table 6 Effects of Zn treatment on the chemical composition (% total peak area) of the essential oil of C. sativum fruits. Table 1. Component

RI

ZnSO4 (mM) Control

Essential oil yield (% dw) Heptanal ␣-Thujene ␣-Pinene Sabinene ␤-Pinene ␦-3-Carene ␣-Terpinene Limonene 1.8-Cineole (Z)-␤-Ocimene ␥-Terpinene cis-Linalool oxide (furanoid) Terpinolene Linalool Camphor Borneol Menthol Terpinene-4-ol p-Cymen-8-ol cis-Hex-3-enyl butyrate ␣-Terpineol cis-Dihydrocarvone Nerol ␤-Citronellol Neral Carvone Geraniol Geranial Anethole Thymol ␦-Elemene Eugenol Geranyl acetate ␤-Caryophyllene ␣-Humulene Germacrene-D Eugenyl acetate Group components Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others

a

904 932 939 972 981 1009 1016 1031 1035 1046 1065 1076 1086 1089 1145 1167 1175 1178 1186 1189 1190 1194 1226 1228 1241 1243 1258 1274 1285 1290 1340 1354 1385 1428 1450 1484 1526

0.1

1 b

2 c

0.51 0.10a 1.66a 0.18a 0.05a 0.36a 1.52a 10.67a 0.01a 0.43a 0.03a 0.02a 0.14a 0.06a 70.94a 1.24a 0.17a 0.08d 0.10c 1.85ab 0.05d 0.06d 0.18c 0.06b 0.05b 0.05a 0.04a 0.20a 0.02a 0.03a 0.61b 0.01a 0.07a 0.04b 0.33a 0.01a 0.01b 0.06a

0.41 0.02b 1.42a 0.05b 0.08a 0.22a 1.06a 7.07ba 0.01a 0.61a 2.29a tr 0.10ba 0.02ba 50.64ba 1.14a 0.10a 0.78b 0.37b 2.01a 0.55b 0.54b 1.02a 0.04b 0.01b 0.05a 0.02a 0.15a 0.02a 0.03a 1.02a 0.67a 0.14a 0.25a 0.01b 0.03a 0.21a tr

0.35 0.13a 1.21b 0.01b 0.02a 0.11a 0.30a 0.84b tr* 0.01b 2.08a 0.03a 0.04b tr 50.96ba 0.24b 0.12a 1.03a 0.71a 1.83ab 0.99a 1.00a 1.41a 0.67a tr 0.03a 0.03a 0.08a 0.01a 0.01a tr 0.34a 0.10a 0.04b 0.14ba 0.01a 0.02b 0.25a

0.22d 0.09a 0.87c 0.11ba 0.11a 0.20a 0.97a 6.79ba tr tr 2.80a 0.01a 0.08ba tr 47.11b 0.16b 0.10a 0.46c 0.27b 1.32b 0.28c 0.26c 0.54b 0.65a 0.11a 0.08a 0.03a 0.08a tr tr tr tr 0.03a 0.02b 0.21ba 0.16a 0.05b 0.25a

14.56a 76.37a 0.36c – 0.15d

12.22b 59.04b 0.92a – 0.57b

4.6c 58.5b 0.51b – 1.12a

11.97b 51.52c 0.42c – 0.37c

RI: Retention indices on the apolar Rtx-1 column; tr: (<0.01%), (–): Not detected. Values with different superscripts in the same column are significantly different at p < 0.05.

A. Marichali et al. / Industrial Crops and Products 55 (2014) 248–257

to reduced photosynthetic activity and a consequent significant decrease in the net CO2 assimilation (Azzarello et al., 2012). This fact has recently been proved by Cambrollé et al., 2013, who have reported that Zn excess decreased the activity of enzymes involved in C fixation namely RuBisCO following the substitution of Mg2+ for metal ions in the active site of RuBisCO subunits. Supportive data on the deleterious effects of excess Zn on the photosynthetic machinery was recently provided by Di Baccio et al., 2011 using transcriptome analyses of Populus x euramericana. In their excellent work, the authors have reported that the majority of gene encoding products which are involved in the chloroplast thylakoids were dawn-regulated by Zn treatment. They belongs to light harvesting complex II (LHCII) proteins, and polypeptide subunits associated with PSII, the cytochrome b6/f complex and ATP synthases associated with PSI, and electron carriers associated with ferredoxin (Fdx). They also found down-regulation of the large subunit of RuBisCO (Di Baccio et al., 2011). This fact might accordingly affect growth, yield and yield components. To test whether these morpho-physiological perturbations were associated with biochemical changes in C. sativum, a set of biochemical analyses including the determination total lipid contents, fatty acid composition, total phenol content and essential oil composition was carried out. These analyses highlighted a clear negative impact of Zn excess on total lipid content in different plant parts. The reduction of total lipid content was a frequently reported effects caused by some heavy metals (le Guédard et al., 2012). At this point, it seems that the decrease in lipid content was primarily attributed to lipid peroxidation induced by oxidative stress (mainly ROS: reactive oxygen species) mediated by heavy metals (Gajewska et al., 2012). According to Wang et al., 2009, ROS (including O2 − , H2 O2 and OH. ) are actively generated under Zn stress as a consequence of the enhanced activities of NADH oxidase (responsible for ROS generation in plant cell), thus resulting in enhanced lipid peroxidation. By using histochemical staining, they also found that the extent of lipid peroxidation was much higher in root than leaves of rapeseed seedlings, which is in concordance with our results. There are numerous papers describing reduction in total lipid content in plant after exposure to Al, Cu (Chaffai et al., 2005, 2007), Cd (Belkhadi et al., 2010), Cu, Zn, Pb (Nesterov et al., 2009), Cr (Rocchetta et al., 2006) and Ni (Gajewska et al., 2012). However, as far as we know, there is no data concerning the biochemical responses of heavy metals in general and excess Zn in particular on C. sativum. Nevertheless, the effect of salinity on lipid profile of coriander has been previously described in two papers (Neffati and Marzouk, 2008, 2009). They have reported marked decrease in the total lipid content in leaves and roots of C. sativum. The qualitative and quantitative analysis of FAs profile revealed that exposure to Zn leads to decrease in the unsaturation level, which was indicated by reduction in DBI and the ratio unsaturated to saturated FAs. Such decrease in the unsaturation level have been reported in Zn-treated Hydrilla verticillata (Nesterov et al., 2009), Ni-treated wheat (Gajewska et al., 2012), Al and Cu-treated maize (Chaffai et al., 2005, 2007), and Cd-treated flax (Belkhadi et al., 2010). Our results showed that in fruits, excess of Zn was associated with drastic decrease in petroselinic acid (C18:1n-12), while it induces marked increase in oleic (C18:1n-9), palmitic (C16:0) and palmitoleic (C16:1n-7) acids. The reciprocal trend between petroselinic (C18:1n-12) and its precursor palmitic (C16:0) suggest a down regulation of the activity of 4 -palmitoyl-acyl carrier protein deasaturase (enzyme responsible for the conversion of palmitic acid to petroselinic acid) (Kim et al., 2005). Thus, it appears that the biosynthetic pathway of petroselinic acid is particularly prone to metal stress.

255

Compared to control plants, leaf FAs profile of Zn-treated plants showed significantly lower content of ␣-linolenic (C18:3n-3) and linoleic (C18:2n-6) acids, and the higher amount of their precursor oleic acid (C18:1n-9). Such trend has been previously described in different plant species subjected to different metal stress. Indeed, it has been reported that the content of ␣-linolenic acid was dramatically reduced in Populus nigra leaves growing in Pb and Cr contaminated landfill (le Guédard et al., 2012). Tomato leaves (Lycopersicum esculentum) subjected to Cd and Cu stress exhibited lower amount of ␣-linolenic acid (Ouariti et al., 1997). The same species subjected to Zn toxicity has revealed reduced amounts of ␣-linolenic and linoleic acids (Verdoni et al., 2001). It appears that the reduction of the content of polyunsaturated FAs (especially ␣linolenic and linoleic acids) is a general response of plant leaves to metal stress. Such changes in the FAs profile in response to metal stress are considered as a sign of oxidative stress (Belkhadi et al., 2010). Evidence has indicated that oxidative stress mediated by ROS was responsible for lipid peroxidation and membrane damages including increased membrane permeability, enhanced likeness, loss of membrane integrity and impairment of membrane function (Gajewska et al., 2012). Membrane destabilization may inevitably reduce the photosynthetic activity, resulting in impairment of plant growth (Cambrollé et al., 2013). Reduction of the content of linoleic and ␣-linolenic acids and the increase of the content of oleic and palmitic acids were the most noticeable changes in the root FAs profile in Zn treated-plants. The decrease in linoleic and ␣-linolenic acids which was concomitant to increase of their precursor oleic acid might suggest the inhibition of 12 and 15 desaturases. These results where partially in accordance with those of Chaffai et al., 2007 who showed that the exposure of maize seedling to Cu excess resulted in remarkable increase of the content of oleic and palmitic acids, while it decreased the content of ␣-linolenic acid. Two years earlier, these authors have reported increased amounts of linoleic acid while the content of linolenic, oleic and palmitic acids were profoundly reduced in maize root subjected to Al toxicity (Chaffai et al., 2005). The same trend was also observed in the roots of Ni-treated wheat (Gajewska et al., 2012). At this point, the reduction in the content of linoleic and ␣-linolenic acids in roots seems to be a widespread event in plants subjected to metal stress. In stem, the major changes include reduction in the content of palmitoleic and oleic acids, while a remarkable increase in the content of palmitic and ␣-linolenic acids was observed in Zn-treated plants. This pattern has been previously described in the shoots of other plant species exposed to different metal stress such as Ni (Gajewska et al., 2012), Al (Chaffai et al., 2005), Cu (Chaffai et al., 2007), Zn and Pb (Nesterov et al., 2009). In general, our results concerning the changes in FAs (especially the decrease of DBI and the reduction of the content of polyunsaturated FAs) in all organs could represent an indirect evidence for the oxidative stress generated by excess Zn as reported in many studies (Wang et al., 2009; Li et al., 2012). To cope with the deleterious effects of oxidative stress, plants have developed two main mechanisms including (i) enzymatic antioxidant system (represented mainly by catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR)) and (ii) non enzymatic systems which include mainly tocopherols, ascorbic acid and secondary metabolites (phenols and volatiles) (Gill and Tuteja, 2010; Wang et al., 2011). In the present study, the non-enzymatic response was evaluated via the determination of total phenols content and the fruits essential oils. Our results revealed that irrespective to plant part, exposure of C. sativum was associated with remarkable increase of total phenol contents. Similar results were observed in Pb-treated Vallisneria natans (Wang et al., 2011), Cu-treated Panax gensing (Ali

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et al., 2006), Ni-treated Matricaria chamomilla (Kováˇcik et al., 2009), Cd-treated Brassica juncea (Irtelli and Navari-Izo, 2006), Al-treated M. chamomilla (Kováˇcik et al., 2012), B-treated Linum usitatissimum (Heidarabadi et al., 2011), and Cr-treated Nicotiana langsdorffii (Del Bubba et al., 2013). Such an increase in the production of phenolics was ascribed to the increased activities of enzymes of secondary pathway namely shikimate dehydrogenase (SKDH: enzyme of the shikimate pathway that links the metabolism of carbohydrate to synthesis of aromatic compounds including l-phenylalanine), phenylalanine ammonialyase (PAL: the first enzyme of the phenylpropanoid pathway that catalyze the elimination of NH3 from L-phenylalanine to produce trans-cinnamate), and polyphenols oxidase (PPO: enzyme catalyzing the oxidation of the diphenol to the corresponding quinine) (Wang et al., 2011). Additionally, considering that the carbon skeletons for phenols synthesis are provided either by the Calvin cycle or by the oxidation of pentose phosphate pathway (OPP), it seems that Zn excess resulted in enhanced activity of OPP leading to an increased substrate supply for the synthesis of phenolic compounds (Müller et al., 2013). Increased content of phenolics can be expected to reduce oxidative stress to C. sativum plant. In fact, increasing evidences have pinpointed that accumulation of phenolics in stressed plant tissue might contribute to the ROS removal and metal chelating (Kováˇcik ´ et al., 2009; Pawlik-Skowronska and Baˇckor, 2011). Parallel to the determination of total phenolic content, the fruit essential oils were extracted and analyzed. Our results clearly show that Zn treatment reduced essential oil content supporting published statements that heavy metals such as Cd (Zheljazkov et al., 2006), Cu (Elzaawely et al., 2007), Cr, Pb and Ni (Prasad et al., 2011) negatively affect the content of essential oils. In contrast, Misra et al., 2005 reported that Zn treatment did not affect the oil content in geranium. With regard to essential oil content, it seems that the effects of heavy metal is species-dependant and depend to the time of exposure and the nature of the metal (Zheljazkov and Nielsen, 1996). Such a decrease in the oil content may be the results of reduced photosynthetic activity, accelerated senescence and activation of catalytic process. Regarding the chemical composition, our results revealed significant dose-dependant changes in the percentage composition of individual compounds, without noticeable qualitative changes. The major variations include the reduction of the content of oxygenated monoterpenes with linalool, camphor and geraniol being the most affected suggesting their higher sensitivity to excess Zn. In general, the reduction in monoterpene concentration has been attributed to a smaller allocation of carbon into metabolism of terpenes. The down-regulation of monoterpenes synthase namely linalool synthase, Bornyl pyrophosphate synthase and geraniol synthase is suggested too (Selmar and Kleinwächter, 2013). These results were in contrast to those of Elzaawely et al., 2007 who found that the application of excessive Cu concentrations to Alpinia zerumbet, resulted in increased amount of 1,8-cineole, linalool, camphor, cryptone, terpinene-4-ol, ␣-terpineol and cuminaldehyde. Two years earlier, Misra et al., 2005 have reported that exposition of Pelargonium graveolens to different Zn concentration remarkably decreased the content of linalool, nerol and citronellol supporting therefore, our present finding and suggest the sensitivity of these components to Zn excess. More recently, Prasad et al., 2011 have showed that the application of Cr, Pb and Ni at elevated concentration reduced the content of linalool, methyl chavicol and methyl eugenol in Ocimum basilicum. The mechanisms underlying the decrease of the above-mentioned compounds in C. sativum under excess Zn remain unclear. However, it seems logical to hypothesize that the biosynthesis of phenolics compounds (recognized as the most potent antioxidant compounds) received relatively high priority than the biosynthesis of terpenes in C. sativum subjected to metal

stress. Certainly, proving such hypothesis requires further investigations. 5. Conclusions The present study provides a basis for understanding the morpho-physiological and biochemical responses of this important medicinal and economical species. Further research will focus on (i) characterizing the main phenolic compounds suspected to be evolved in the tolerance mechanism, (ii) measuring the activities of the key antioxidant enzymes involved in metal stress tolerance, and (iii) determining the distribution of mineral elements in the seeds and leaves, protein and sugar contents in different plant parts to shed more light into their roles in metal stress tolerance. References Adams, 2001 Adams, R., 2001. Identification of essential oil components by gas chromatography/Quadrupole Mass Spectroscopy. Allured, Carol Stream, IL, USA. Ali, G., Srivastava, P.S., Iqbal, M., 1999. Morphogenic and biochemical responses of Bacopa monniera cultures to zinc toxicity. Plant Sci. 143, 187–193. 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