Impact of long-term cadmium exposure on mineral content of Solanum lycopersicum plants: Consequences on fruit production Academic research paper on "Biological sciences"

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Impact of long-term cadmium exposure on mineral content of Solanum lycopersicum plants: Consequences on fruit production

Hédia Hédijia,b, Wahbi Djebalia* Aïcha Belkadhia, Cécile Cabasson b,d, Annick Moingd,e, Dominique Rolin b,d, Renaud Brouquissec, Philippe Galluscib, Wided Chaïbia

a Université Tunis-El Manar, UR Biologie Physiologie et Biochimie de la Réponse des Plantes aux Contraintes Abiotiques, Département de Biologie, Faculté des Sciences de Tunis, El Manar, 1060 Tunis, Tunisia

b Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Centre INRA de Bordeaux, IBVM, BP 81, Villenave d'Ornon F-33140, France c UMR INRA 1301 - CNRS 6243 - Université Nice Sophia Antipolis, Interactions Biotiques & Santé Végétale, BP 167, F-06903, Sophia, Antipolis, France d Plateforme Métabolome-Fluxome du Centre de Génomique Fonctionnelle Bordeaux, Centre INRA de Bordeaux, IBVM, BP 81, Villenave d'Ornon F-33140, France e INRA, UMR 1332 Biologie du Fruit et Pathologie, Centre INRA de Bordeaux, IBVM, BP 81, Villenave d'Ornon F-33140, France

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ARTICLE INFO

ABSTRACT

Article history:

Received 11 July 2014

Received in revised form 14 January 2015

Accepted 15 January 2015

Available online 7 February 2015

Edited by RA Street

Keywords:

Cadmium

Mineral elements

Solanum lycopersicum

In young tomato plants, modifications in mineral composition by short-term cadmium (Cd) treatments have been extensively examined. However, long-term Cd treatments have been fewly investigated, and little information about Cd-stress in fruiting plants is available. In the present work, we examined the changes in mineral nutrients of roots, stems, leaves, flowers, seeds and fruit pericarp of tomato plants submitted to a long-term Cd stress. After a 90-day culture period in hydroponic contaminated environment (0, 20 and 100 ^M CdCl2), fruit production was affected by increasing external Cd levels, with the absence of fruit set at 100 ^M Cd. Meanwhile, Cd altered the plant mineral contents with an element- and organ-dependent response. At 20 ^M, Cd triggered a significant increase in Ca content in roots, mature leaves, flowers and developing fruits. However, at 100 ^M Cd, Ca content was reduced in shoots, and enhanced in roots. Cd stress reduced Zn and Cu contents in shoots and increased them in roots. High Cd level led to a significant decrease in K and Mg content in all plant organs. Furthermore, Fe concentration was reduced in roots, stems and leaves but increased in flowers, seeds and red ripe fruits. Our results suggest that tomato plants acclimatize during long-term exposure to 20 ^M Cd, while 100 ^M Cd results in drastic nutritional perturbations leading to fruit set abortion.

© 2015 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction

Tomato, one of the most important horticultural crops in the world, constitutes a considerable source of minerals, vitamins, and antioxi-dants. It is also an important component of the traditional Mediterranean diet, which has been found to be associated with a reduced risk of various cancers and heart diseases (Galgano et al., 2007; Nguyen and Schwartz, 1999). Unfortunately, a large part of this crop is grown in greenhouses, using fertilizers for improving the nutrient supply in soils and pesticides for disease control and crop protection (Mench, 1998; Moral etal., 2002). Consequently, these agricultural practices amplify the risk of elevating soil contamination by heavy metals and of altering the quality of agricultural products (Mench, 1998).

Among heavy metals, cadmium (Cd) is one of the most dangerous elements to plants, since elevated levels in the soil solution cause numerous harmful effects. It has been shown that Cd may cause root

* Corresponding author at: UR Biologie Physiologie et Biochimie de la Réponse des Plantes aux Contraintes Abiotiques, Département de Biologie, Faculté des Sciences de Tunis, El Manar, 1060 Tunis, Tunisia. Tel.: +216 98 531 763. E-mail address: wahbi.djebali@fst.rnu.tn (W. Djebali).

damages, inhibition of photosynthesis and respiration, reduction of chlorophyll content and alteration of the key enzyme activities of various metabolic pathways, leading to lower yield (Dong et al., 2006; Gratao et al., 2012; Irfan et al., 2013; Wu et al., 2007). However, the severity of the above-mentioned disturbances is related to many factors, such as the species (Wu et al., 2007), Cd concentrations (Djebali et al., 2010) and exposure time (Singh et al., 2004).

Physiological disorders caused by Cd, including plant biomass reduction, can be an indirect consequence of nutrient deficiencies. Mineral nutrition disturbances arise from deleterious Cd effects on the metabolism of essential elements, including calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu) (Carvalho Bertoli et al., 2012). In crop plants, Cd could interfere syner-gistically or antagonistically with nutrient uptake (Bachir et al., 2004; Clemens, 2006; Wu et al., 2007). However, there were significant differences among species and varieties, and inconsistencies exist between the results of the experiments. In fact, the mechanism by which Cd inhibits the uptake and utilization of mineral elements is not completely clear to date. It is assumed that Cd may interfere with nutrient uptake by affecting the permeability of plasma membrane (Obata and Umebayashi, 1997) and modify the activity of the nutrient transporters

http://dx.doi.org/10.1016/j.sajb.2015.01.010

0254-6299/© 2015 SAAB. Published by Elsevier B.V. All rights reserved.

(Clemens, 2006), leading to changes in nutrient concentration and composition.

Cd-induced changes in mineral composition have been examined extensively in seedlings using short-term treatments (Boulila-Zoghlami et al., 2006; Lopez-Millan et al., 2009). However, few investigations have been done on the effect of chronic-Cd-stress on plants producing fruit (Carvalho Bertoli et al., 2012). Since fruits are sink organs that import mineral and photo-assimilates from roots and leaves, their growth and development may be impacted by Cd stress as well (Carvalho Bertoli et al., 2012; Gratao et al., 2012; Moral et al., 1994). In a previous study (Hediji et al., 2010), tomato plant exposure to high Cd concentration (i.e. 100 |jM) led to significant disturbances in leaf metabolic profiles, which induced plant growth reduction and fruit set inhibition. When treated with a lower Cd concentration (i.e. 20 ^M), tomato plants produced fruits and presented only moderate changes of their leaf metabolic profiles, suggesting their relative tolerance to long-term Cd treatment, although they showed vegetative growth reduction.

The present study aims at determining the influence of long-term Cd stress on the accumulation of mineral nutrients (i) in all parts of tomato plants and (ii) in fruit pericarp at different developing stages. Consequences on fruit production were also analyzed.

2. Materials and methods

2.1. Plant material and harvest

Tomato seed (Solanum lycopersicum L., cv Thomas) germination was performed on moistened filter paper for six days at 25 °C in the dark. The seedlings were selected for uniformity and transplanted to a 6 L plastic beaker (one plant per beaker) filled with continuously aerated nutrient solution containing: 3.8 mM KNO3, 0.2 mM NaCl, 3.1 mM Ca(NO3)2, 2.0 mM NH4NO3, 0.8 mM KH2PO4, 0.3 mM K2HPO4, 0.75 mM MgSO4, 10 |aM MnSO4, 1.0 |jM CuSO4, 1.0 |aM ZnSO4, 30 |jM H3BO3, 0.04 |jM (NH4)6Mo7O24, and 90 |aM EDTA-Fe-K (Saglio and Pradet, 1980). Twenty days after transplanting, CdCl2 was added to a fresh nutrient solution at 20 or 100 ^M. Culture without Cd was used as a control. Experiments performed on six plants each, were run in duplicate. Plants were grown in a growth chamber with a 16-h-day (25 °C)/8-h-night (18 °C) cycle, an irradiance of about 300 |jmol photons m-2 s-1 and 70-80% humidity. Flowers were tagged at anthesis (flower opening) and the number of fruits per plant was monitored. Flower maturity was determined as fully opened flowers (anthesis). Fruits were harvested at six stages expressed in days post-anthesis (DPA): expansion phase (25 DPA), mature green stage (MG, 43 ± 5 DPA), breaker (B, 46 ± 5 DPA), turning (T, 47 ± 5 DPA), orange (0,48 ± 6 DPA) and red ripe (RR, 51 ± 5 DPA). For each treatment and developmental stage, 6 fruits from different plants were harvested. The equatorial pericarp of fruits was hand dissected. After 116 days of growth, corresponding to 90 days of exposure to CdCd2, the different plant organs were collected and used for further determination of dry weight (DW) or mineral elements as described below.

2.2. Determination of cadmium and mineral nutrient contents

Plant materials (roots, leaves, stems, flowers, seeds and fruit pericarp) were dried at 70 °C until constant weight, weighed and ground to a fine powder. Cadmium and mineral nutrients were analyzed by digestion of dried samples with an acid mixture (HNO3/HClO4 3:1, v/v) as described by Van Assche et al. (1988). Metal ion concentrations were determined by atomic absorption spectrometry (Analyst 300, Perkin-Elmer) using an air-acetylene flame.

2.3. Data analyses

Data presented are the mean values of three biological replicates for mineral element contents, and of six biological replicates for

physiological analyses. Mean comparison between treatments and control was done using Student's t-test (P < 0.05).

3. Results

3.1. Effect of Cd on fruit production

The effects of Cd on flower and fruit number and fruit fresh weight at different development stages (expansion phase (25 DPA), mature green (MG, 43 DPA), breaker (B, 46 DPA), turning (T, 47 DPA), orange (O, 48 DPA) and red ripe (RR, 51 DPA)) are reported in Figs. 1 and 2. Cd exposure had a significant effect on mature flower number (Fig. 1a), which influences fruit production. At 20 |jM Cd, flowers reached maturity but they were 56% lower than in the control after 54 days of Cd treatment (corresponding to 80 days of plant development). Concomitantly, fruit number per plant was 72% lower than in the control after 66 days of Cd treatment (corresponding to 92 days of plant development) (Fig. 1b). This decrease was due to the reduction of developing flower number and the increase of aborted flower number. This reduction in fruit number was accompanied by a significant decrease in fruit fresh weight (a 54% and 33% reduction compared to the control at 25 DPA and RR stages, respectively) (Fig. 2). By contrast, long-term exposure to 100 |aM Cd led to the abortion of all plant flowers at the immature flower bud stage (Fig. 1a).

3.2. Cadmium accumulation in the fruit pericarp

Under 20 |jM Cd treatment, changes in Cd content in the pericarp of fruit at different development stages (25 DPA, MG, B, T, O and RR) revealed that Cd accumulation increased significantly between the early

Fig. 1. Effect of cadmium on mature flower number (a) and fruit formation (b) of tomato plants submitted to different Cd concentrations. Mean of 6 replicates ± SD. Star (*) indicates significant differences between Cd treatment and control (0) at P < 0.05 level (Student's t-test).

Fig. 2. Changes in fruit fresh weight of control and Cd-treated tomato plants at different development stages (expansion phase (25 DPA), mature green (MG, 43 DPA), breaker (B, 46 DPA), turning (t, 47 DPA), orange (0,48 DPA) and red ripe (RR 51 DPA)). Mean of 6 replicates ± SD. Star (*) indicates significant differences between Cd treatment and control (0) atP < 0.05 level (Student's t-test).

development stage (25 DPA) and late ripening (RR) stages (Fig. 3). For example, Cd content showed approximately 108% increase between 25 DPA and MG stages. However, during the ripening phase, metal accumulation increased by 27% only (MG; 10.16 ig g-1 DW to RR; 12.88 ig g-1 DW) (Fig. 3).

3.3. Effect of Cd on root, stem, leaf, flower and seed mineral element contents

Table 1

Macronutrient concentrations (mg g-1 DW) in organs of tomato plants exposed for 90 days to different concentrations of CdCl2.

Cd (|iM)a

0 20 100

Roots 41.41 ±2.55 83.66 ± 7.89* 68.21 ± 3.35*

Stems 40.88 ±9.29 43.99 ± 2.52 19.47 ± 1.70*

Mature leaves 45.39 ±5.63 56.98 ± 4.23* 23.29 ± 3.42*

Young leaves 25.45 ±5.17 31.92 ± 3.74 18.66 ± 3.56

Flowers 44.51 ±2.13 66.09 ± 3.84* 40.64 ± 1.33

Seeds 27.13 ± 2.97 28.84 ± 8.12 NF

K Roots 30.48 ± 7.01 32.47 ± 2.25 18.34 ± 1.20*

Stems 71.95 ± 10.12 44.02 ± 5.01 29.70 ± 3.57*

Mature leaves 39.48 ± 1.67 41.63 ± 2.36 10.15 ±2.23*

Young leaves 41.63 ± 2.36 44.04 ± 0.42 35.90 ± 2.42*

Flowers 40.69 ± 2.02 41.80 ± 0.82 25.17 ± 1.90*

Seeds 7.95 ± 0.61 10.17 ± 2.14 NF

Roots 2.83 ± 0.07 2.99 ± 0.28 0.72 ± 0.21*

Stems 2.56 ± 0.58 2.08 ± 0.36 1.00 ± 0.30*

Mature leaves 4.49 ± 0.24 4.44 ± 0.27 2.58 ± 0.29*

Young leaves 4.46 ± 0.08 4.45 ± 0.08 2.11 ± 0.36*

Flowers 4.78 ± 0.31 4.96 ± 0.15 1.34 ± 0.26*

Seeds 3.76 ± 0.26 4.71 ± 0.17* NF

For each line, means accompanied by stars were significantly different from the control (0) at P < 0.05 level (*) (Student's t-test). NF, no fruits formed. a Mean of 3 replicates ± SD.

to control, in roots, leaves and stems, respectively. Contrarily, Cd enhanced Fe accumulation over 2-fold, compared to control, in flowers and seeds. Zn content significantly increased in roots in a dose

Cd toxicity altered plant contents of several macronutrients (Table 1). Element- and organ-dependent responses to Cd were observed. After exposure to 20 iM Cd, K and Mg tissue-contents did not show significant variations compared to control, except for Mg seed contents which increased by 25% (Table 1). At the same Cd concentration, Ca content significantly increased by 102%, 26% and 48%, compared to control, in roots, mature leaves and flowers, respectively. However, 100 iM Cd significantly increased Ca content in roots but decreased it in stems and mature leaves. High Cd treatment also led to a significant reduction of K and Mg content in all plant organs (Table 1).

Moreover, Cd affected micronutrient distribution in plant tissues (Table 2). Fe content marked a significant reduction in vegetative organs. At 100 |iM Cd, this decrease reached 33%, 78% and 72%, compared

Fig. 3. Cadmium contents (|ig g-1 DW) in pericarp during fruit development (expansion phase (25 DPA), mature green (MG, 43 DPA), breaker (B, 46 DPA), turning (T, 47 DPA), orange (0,48 DPA) and red ripe (RR, 51 DPA)) of tomato plants exposed for 90 days to different concentrations of CdCl2. Mean of 3 replicates ± SD.

Table 2

Micronutrient concentrations (igg-1 DW) in organs of tomato plants exposed for 90 days to different concentrations of CdCl2.

Cd (|iM)a

0 20 100

Roots 5007.74 ± 714.03 2984.52 ± 246.04* 3371.84 ± 96.42'

Stems 272.40 ± 14.71 94.85 ± 8.78* 76.12 ± 21.14*

Mature leaves 282.76 ± 24.50 108.41 ± 5.65* 62.99 ± 18.67*

Young leaves 297.71 ± 9.80 153.28 ±41.31* 65.87 ± 8.49*

Flowers 128.42 ±9.24 290.55 ± 43.99* 339.69 ± 33.3*

Seeds 94.31 ± 0.83 271.26 ± 6.06* NF

Roots 255.87 ± 8.84 100.63 ± 14.20* 522.08 ± 30.51*

Stems 73.90 ± 3.94 58.53 ± 7.45* 40.38 ± 7.88*

Mature leaves 169.37 ±3.64 199.26 ± 14.55* 114.59 ± 9.82*

Young leaves 141.72 ± 6.73 127.81 ± 1.20* 52.54 ± 5.40*

Flowers 137.04 ±0.70 115.39 ±6.21* 52.51 ± 4.66*

Seeds 76.22 ± 7.09 66.74 ± 3.82 NF

Roots 154.37 ± 10.67 179.61 ± 1.84* 277.46 ± 6.00*

Stems 124.54 ± 18.23 73.97 ± 6.47* 131.99 ± 10.63

Mature leaves 159.28 ±11.24 38.79 ± 0.27* 41.98 ± 2.61*

Young leaves 163.96 ± 24.83 66.75 ± 4.78* 58.63 ± 5.03*

Flowers 87.47 ± 4.85 84.91 ± 9.50 47.36 ± 1.44*

Seeds 80.94 ± 3.74 72.59 ± 1.22* NF

Roots 60.97 ± 6.22 99.91 ± 16.61* 224.99 ± 20.98*

Stems 13.19 ± 1.07 19.00 ± 1.08* 7.36 ± 1.40*

Mature leaves 16.64 ± 2.65 12.80 ± 1.19 10.15 ± 1.43*

Young leaves 18.70 ± 2.33 19.28 ± 1.40 15.44 ± 0.74

Flowers 17.76 ±0.97 16.66 ± 1.00 5.52 ±1.17*

Seeds 17.08 ±0.80 12.56 ± 2.63* NF

For each line, means accompanied by stars were significantly different from the control (0) at P < 0.05 level (*) (Student's t-test). NF, no fruits formed. a Mean of 3 replicates ± SD.

dependent manner, whereas it decreased in stems, leaves and seeds at 20 |jM Cd, and in leaves and flowers at 100 |jM Cd. Similarly, 100 |jM Cd significantly enhanced Cu content in roots, and decreased it in stems, mature leaves and flowers. In addition, 20 |jM Cd enhanced Mn accumulation by 18% in mature leaves and decreased it in roots, stems, young leaves and flowers. By contrast, the highest Cd treatment induced an inverse effect.

3.4. Effect ofCd on fruit mineral element contents

The effects of 20 |jM Cd were evaluated on the accumulation of mineral elements in fruits at the developing stage (25 DPA) and different ripening stages (Fig. 4). Under control conditions, Ca (Fig. 4a), Mg (Fig. 4b), K (Fig. 4c) and Zn (Fig. 4e) contents remained relatively constant throughout fruit development and ripening. However, Fe (Fig. 4d) content of control fruit pericarp decreased from the 25 DPA stage to the T stage and then remained constant. Yet, Cu (Fig. 4f) content of control fruit pericarp increased progressively during ripening to reach a maximum content of 21.6 |ag g-1 DWat red ripe (RR, 51 DPA) stage.

Cd effects on fruit mineral contents were more pronounced at 25 DPA than at ripening stages (Fig. 4). Ca content was twice more abundant in 25 DPA fruits of treated plants as compared to control (Fig. 4a). However, Ca content decreased progressively at ripening stages to reach the same concentration of control at the RR stage. Under Cd stress, Mg content was enhanced in fruits at 25 DPA and MG

stages and decreased at ripening stages, a nearly 34% decrease was observed at the RR stage (Fig. 4b). Inversely, K content was reduced to 26% of control at 25 DPA fruit stage, while RR contaminated fruits exhibited similar K content to that of the respective control (Fig. 4c). In the same way, Cd significantly affected micronutrient content especially at 25 DPA fruit stage, with a 77%, 54% and 21% decrease for Cu (Fig. 4f), Fe (Fig. 4d) and Zn (Fig. 4e), respectively as compared to control. By contrast, at ripening stage, Zn content showed few variations under Cd stress, whereas Cu content remained lower in fruits of Cd-treated plants than in control. Furthermore, Fe content in the pericarp decreased from the MG stage to T stage in Cd-stressed plants and then increased at the late ripening stage by 34% compared to control fruits.

4. Discussion

Cd is known to disturb cell growth and plant yield (Bachir et al., 2004; Gratao et al., 2008; Hediji et al., 2010). Consistently, the present work clearly confirms that prolonged Cd exposure significantly reduces the productivity of tomato plant, as defined by its flower and fruit production.

According to our previous results (Hediji et al., 2010), the decrease in growth and fruit production observed in Cd-treated plant coincided with a considerable accumulation of this metal in aerial parts of plants, especially in mature leaves, which are the fundamental sources of sugar for increasing and accumulating reserves. This raw material comes almost exclusively from the phloem's sap and so probably contributes to

Fig. 4. Macronutrient (Ca, Mg and K) and micronutrient (Fe, Zn and Cu) contents in fruit pericarp of control and Cd-treated tomato plants at different development stages (expansion phase (25 DPA), mature green (MG, 43 DPA), breaker (B, 46 DPA), turning (T, 47 DPA), orange (0,48 DPA) and red ripe (RR, 51 DPA)). Mean of 3 replicates ± SD. Star (*) indicates significant differences between Cd treatment and control (0) at P < 0.05 level (Student's t-test).

the fruit Cd content (Carvalho Bertoli et al., 2012). Through sampling of tomato at different fruit stage development, it was seen that the majority of fruit pericap Cd was accumulated during the last period of fruit development (RR). This result is in agreement with those of Cieslieski et al. (1996), who concluded that in durum wheat, the high accumulation of Cd in grain at maturity reflects redistribution of Cd via the phloem pathway. By contrast, Rodda et al. (2011) found that the majority of Cd in rice grain was accumulated during the early period of grain development. This divergence may be due to differences on the mechanisms determining translocation of Cd to the edible plant tissues. Although, it is known that Cd is both phloem and xylem mobile (Clemens et al., 2013; Irfan et al., 2013), direct quantitative measurement of the contribution of xylem and phloem is difficult. Rodda et al. (2011) proposed two possible pathways of root to grain movement: (1) Cd is taken up directly through the xylem to the developing grain; or (2) Cd is taken up to actively transpiring parts such as culms, rachis, flag leaves and external parts of the panicles, and then quickly remobilized via the phloem to the grain.

Cd treatment results in metal-induced nutritional disturbances, as already described in different plant species (Irfan et al., 2013; Sandalio et al., 2001). In fact, Cd has been assumed to be taken up by transporters for essential elements as a consequence of a lack of specificity of the transporters (Clemens, 2006). Although a specific Cd transporter has been proposed to be located in the root plasma membrane (Zhao et al., 2002), interactions of Cd and other elements, such as Zn and Fe, have been frequently reported in plants (Chen et al., 2007; Irfan et al., 2013). In this experiment, we noted that the effect of long-term (90 days) Cd exposure on nutritional status of tomato varied with Cd level, mineral elements and plant organs. At high Cd concentration, K and Mg were strongly reduced, suggesting a deleterious effect of Cd on the uptake and translocation of these elements. Moreover, we noted that Cd decreases Ca, Mn, Zn and Cu accumulation in the photo-synthetic organs, and enhances their accumulation in the roots.

Hydroponic experiment conducted by Wu et al. (2004) showed that in cotton (Gossypium hirsutum), 1 and 10 mM Cd lowered the Zn, Cu, and Fe concentrations in aboveground vegetative organs; however, a significant increase of these mineral concentrations was observed in roots, implying that the translocation of these elements from roots to shoots was prevented by Cd. Earlier reports hypothesized that increases in root mineral concentrations could be partially explained by Cd interference with nutrient uptake by affecting the permeability of plasma membranes, as by altering their specific transporters (Dong et al., 2006; Irfan et al., 2013; Sandalio et al., 2001). In the same way, it has been shown that Cd-induced morphological changes of the conducting xylem tissue may also contribute to limited translocation of nutrients from roots (Carvalho Bertoli et al., 2012; Vollenweider et al., 2006). An inhibition of micro- and macro-nutrient accumulations by Cd in young tomato plants has been previously reported (Boulila-Zoghlami et al., 2006). These authors found that deleterious effects of Cd (20 and 100 |aM CdCl2) on divalent metal accumulations were notable after 10-days of Cd treatment. Nevertheless, in the present investigation, long-term Cd exposure affected only the nutritional status of high-Cd-treated-plants, while those of moderate-Cd-treated-plants (20 |aM) were generally unaltered. These results suggested the strong ability of tomato plant to acquire tolerance to moderate Cd concentration. Drazkiewicz and Baszynski (2005) reported that plant responses to Cd were dependent on age and metal supply. However, these authors pointed out the possible existence, within the leaf, of a protecting mechanism against Cd toxicity in which some nutrients, such as Ca, might be implicated. Indeed, Ca plays an essential role in various processes that preserve the structural and functional integrity of plant membranes (Tuna et al., 2007), the carbohydrate metabolism (Greger and Bertel, 1992) and the photosynthetic mechanism (Drazkiewicz and Baszynski, 2008). So, the Ca accumulation in photosynthetic tissues of 20 |aM Cd treated tomato plants could contribute to limit the negative effects of Cd toxicity.

Cd-induced leaf-Fe-deficiency could partially explain the alteration of chlorophyll content and growth which was previously observed (Hediji et al., 2010). According to Dong et al. (2006), it is well known that many toxic effects of Cd result from its interaction with essential elements, in particular those with the same valence, such as Cu, Fe, Mn and Zn. It has been suggested that this interaction may be mediated either by competition for binding sites or transporters such as NRAMP (natural resistance-associated macrophage protein) and ZIP (zinc-regulated transporter/iron-regulated transporter-related protein) families, which are able to transport Fe, Zn, Mn as well as Cd (Guerinot, 2000; Clemens et al., 2013). Moreover, Cd has been shown to inhibit Fe uptake by IRT1 (gene encoding a high affinity Fe transporter) probably by competition as a substrate (Clemens, 2006).

In our experiment, Cd also affected Zn accumulation in flowers, and consequently could be responsible for depression of the plant productivity. Consistent with this hypothesis, Pandey et al. (2006) demonstrated that Zn deficiency induced impairment in pollen and stigma morphology and function, leading to a drastic decrease in setting of seeds.

During reproductive development, nutrients accumulated in vegetative organs were transferred into the fruit. Fruit growth requires the supply of organic material through the phloem. The mass movement of phloem fluids may also incidentally deliver to the fruit other materials such as certain heavy metals (Patrick, 1997). However, the potential for xylem transport of micronutrients directly to fruit tissues exists (Waters and Sankaran, 2011).

Although biotic and abiotic factors are known to affect the global quality of tomato fruits (Gratao et al., 2008; Horchani et al., 2010), little is known about their effects on the nutrient composition of fruits (Carvalho Bertoli et al., 2012; Moral et al., 1994). In this case, our result showed that Cd changed fruit nutrient composition, depending on elements and developing fruit stage. Growing-fruit stage appeared to be sensitive to Cd with significant decreases in K, Fe and Zn contents and increases in Ca and Mg contents. Decrease on micronutrient contents may be due to the interference of Cd with their moving pathway from roots to fruits. In agreement with this hypothesis, experimental results in Indian mustard support the idea that the pattern of phloem transport and remobilization of Cd from vegetative tissues to the seeds/grains is similar to Zn (Sankaran and Ebbs, 2008). After long-term Cd treatment, Ca accumulation during fruit development was correlated with a decrease in fruit fresh weight, suggesting the deleterious effect of Cd on fruit growth, process which consumes Ca. Furthermore, reduction in K accumulation in fruit pericarp at the early development phase (25 DPA) probably contributed to limited fruit growth. Indeed, it has been shown that fruit development imposes considerable demands for K, which is implicated in metabolic processes, such as the synthesis of proteins, enzyme activation, membrane transport processes, and the generation of turgor pressure (Chapagain and Wiesman, 2004). Yet, along fruit development, Cu, that plays a key role in growth process (Hansch and Mendel, 2009), displayed a lower level, as compared to the control, contributed probably to limited fruit growth, as suggested by Flynn et al. (1987), who reported reduction on grain yield and quality under copper deficiency. Although mineral composition was affected by Cd treatment in different fruit development phases, red fruits exhibited little variations in their nutritional status, as compared to control, suggesting the strong ability of tomato plant to acquire tolerance to moderate Cd concentration.

In the present study it was observed that the influence of Cd on nutrient content in tomato varied according to the level of Cd, essential element, plant organ and growth stage. Based on these results, excessive Cd accumulation may affect the uptake and distribution of certain nutrients, and consequently may be responsible for mineral disturbances and depression of plant growth and productivity. However, further studies are needed to clarify the mechanisms involved between mineral nutrition and Cd uptake and their impact on the productivity and quality of tomato fruit.

Acknowledgments

This work was supported by the Ministry of Higher Education, Scientific Research and Technology in Tunisia (grant no. 03G0911) and the Institut National de la Recherche Agronomique (INRA, France). The authors thank Pr. C. Abdelly and Pr. A. Saadi, for their help in mineral analysis.

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