Ecotoxicology and Environmental Safety 115 (2015) 174–186

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Morphological, biochemical, molecular and ultrastructural changes induced by Cd toxicity in seedlings of Theobroma cacao L. Andressa V. Castro, Alex-Alan F. de Almeida n, Carlos P. Pirovani, Graciele S.M. Reis, Nicolle M. Almeida, Pedro A.O. Mangabeira Universidade Estadual de Santa Cruz – UESC, Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, 45662-900 Ilhéus, Iran

art ic l e i nf o

a b s t r a c t

Article history: Received 1 September 2014 Received in revised form 2 February 2015 Accepted 4 February 2015

Seeds from Theobroma cacao progenies derived from the self-pollination of ‘Catongo’
‘Catongo’ and the crossing between CCN-10
SCA-6 were immersed for 24 h in different Cd solutions (2; 4; 8; 16 and 32 mg L  1) along with the control treatment (without Cd). Shortly after, the seeds were sown in plastic tubes containing organic substrate and were grown in a greenhouse for 60 days. The treatment with Cd was observed to cause morphological, biochemical, molecular and ultrastructural changes in both progenies of T. cacao. There has been deformation in chloroplasts, nuclear chromatin condensation, and reduction in thickness of the mesophyll. As for ‘Catongo’
‘Catongo’, a decrease in thickness of the epidermis was noted on the abaxial face. There has been increased guaiacol peroxidase activity in the roots of CCN-10
SCA-6, as well as in the’‘Catongo’
‘Catongo’ leaves. In the presence of Cd, CCN-10
SCA-6 showed increased expression of the genes associated with the biosynthesis of phytochelatin (PCS-1) and class III peroxidases (PER-1) in leaves, and metallothionein (MT2b), in roots. In ‘Catongo’
‘Catongo’, there has been an increase in the expression of genes associated with the biosynthesis of PER-1 and cytosolic superoxide dismutase dependent on copper and zinc (Cu–Zn SODCyt) in leaves and from MT2b and PCS-1 and roots. There was higher accumulation of Cd in the aerial parts of seedlings from both progenies, whereas the most pronounced accumulation was seen in’‘Catongo’
‘Catongo’. The increase in Cd concentration has led to lower Zn and Fe levels in both progenies. Hence, one may conclude that the different survival strategies used by CCN-10
SCA-6 made such progeny more tolerant to Cd stress when compared to’‘Catongo’
‘Catongo’. & 2015 Published by Elsevier Inc.

Keywords: Abiotic stress Electron microscopy Gene expression Oxidative stress Plant anatomy Toxicity of cadmium

1. Introduction Cd is considered one of the most toxic heavy metals that exist in nature (Al-Kedhairy et al., 2001) and that can contaminate plants, animals and humans (Almeida et al., 2007). The bioavailability of heavy metals in the soil, including Cd, is regulated by physical, chemical and biological processes and their interactions (Ernest, 1996). Small variations in pH can greatly alter the availability of metals (Pierangeli et al., 2001). During weathering, Cd is easily transferred to the soil solution. Despite its occurrence as Cd2 þ is known, it can form many complex ions such as CdCl þ , CdOH þ , CdHCO3 þ , CdCl42  , CdCl3  , CdCI42  , Cd(OH)3  , Cd(OH)42  and organic chelates (Kabata-Pendias and Pendias, n

Corresponding author. E-mail addresses: [email protected] (A.V. Castro), [email protected] (A.-A. de Almeida), [email protected] (C.P. Pirovani), [email protected] (G.S.M. Reis), [email protected] (N.M. Almeida), [email protected] (P.A.O. Mangabeira). http://dx.doi.org/10.1016/j.ecoenv.2015.02.003 0147-6513/& 2015 Published by Elsevier Inc.

2001). In the soil, Cd is chiefly seen in the available form, which is exchangeable and can be easily absorbed by plants and cause phytotoxicity (Soares et al. 2005). The phosphate rocks used in fertilizer production are the major sources of Cd contamination in agricultural soils (Mortvedt and Beaton, 1995). In recent decades, it has been observed a significant increase of Cd in the environment, mainly as a result of industrial activities such as mining, smelting and refining of zinc, manufacture and use of phosphate fertilizers and fungicides (Al-Kedhairy et al., 2001; Arduini et al., 2004; Benavides et al., 2005). Plants normally absorb Cd present naturally in the soil or from atmospheric depositions, or from that present in organic or phosphate fertilizers (Gallego et al., 2012). Once inside the plant, Cd accumulation promotes morphological and ultrastructural changes, and alterations in physiological, biochemical and molecular processes, modifying metabolic activities (Almeida et al., 2013). In plant cells, Cd triggers a sequence of metabolic reactions and promotes a number of changes in plants, such as induction of differential gene expression; increased activity of antioxidant enzymes, like for example class III peroxidases and superoxide

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dismutase (SOD); induction of phytochelatin production (PCs); and morphological and ultrastructural changes (Almeida et al., 2010; Pietrini et al., 2010; Callahan et al., 2006; Alscher et al., 2002). Class III peroxidases are located in vacuoles and cellular walls and belong to a family of multigenes involved in various physiological processes. Moreover, they act on a wide variety of substrates and show a moderate specificity for phenols (Almagro et al., 2009). SOD enzymes are classified into three groups: Fe SOD, Mn SOD and Cu–Zn SOD, which are located in different cellular compartments (Alscher et al., 2002). Cu–Zn SOD enzymes are divided into two classes of isoforms, one located in the cytoplasm and another located in the chloroplasts (Kurepa et al., 1997). PCs are enzymatically produced from the precursor of GSH tri-peptide (GluCys) n-Gly; where n ¼ 2–11 (Inouhe, 2005). Due to the presence of cysteine thiol groups, PCs chelate Cd and form complexes with various molecular weights, thus protecting the harmful effects of free Cd ions and eventually remove Cd in the vacuole (Clemens, 2006; Cobbett, 2000; Ernst et al., 2008; Schat et al., 2002). The accumulation of PC has been observed as a response to stress by heavy metals (Lee and Korban, 2002; Noctor et al., 1998; Rauser, 1999). For living in environments contaminated with toxic metals, some plant species have developed survival strategies such as the exclusion of metals through the selective absorption of mineral elements; chelation of toxic metal ions by means of a specific high affinity ligand (Patra et al., 2004); accumulation of phytochelatins and metallothioneins ([Pietrini et al., 2010,Schützendübel and Polle, 2002]); retention of toxic metals in the roots, preventing its translocation to the aerial part; compartmentalization of toxic metals for vacuoles, and adsorption to the cell wall; activation of antioxidant enzymes; differential expression of proteins (Gomes et al., 2012); exclusion and accumulation of large amounts of toxic metals in their tissues; and cellular repair mechanisms (Almeida et al., 2010). Theobroma cacao is a tropical evergreen tree species that is Eudicotyledons and diploid (2n ¼20) (Figueira et al., 1992), preferentially allogamous, and belongs to the Malvaceae family; the geographic location of the species, in turn, is South America (Almeida and Valle, 2007). This is one of the most important perennial crops worldwide, with an estimated production exceeding 400,000 ton in 2012–2013 (ICCO, 2013). This species is chiefly exploited for the production of chocolate; but it can also be used in cosmetics, beverages, jellies, creams and juices (Almeida and Valle, 2007). Cd concentrations were observed in cocoa powders and related products (beans, liquor, butter) of different geographical origins (Mounicou et al., 2003). With regard to its morphological and physiological characteristics, T. cacao displays great genetic variability (Daymond et al., 2002). The ‘Catongo’ genotype is self-compatible, midsize, and medium to high productivity. This genotype is the result of a natural mutation for anthocyanins in T. cacao variety 'Common'. Logo is devoid of anthocyanins, and water-soluble vacuolar pigments reddish in color. In normal plants, these pigments are typically more concentrated in young leaf tissue cells as well as young fruits, and seeds ripe. The main characteristic of ‘Catongo’ is the absence of anthocyanins in these organs, although there is fruit seeds showing ocher by cross pollination effect (Bartley, 2005). It is highly susceptible to Moniliophtora perniciosa and has moderate resistance to Phytophthora palmivora. Moreover, SCA-6 is considered as a wild type genotype, low size, self-incompatible, low productivity; with moderate resistance to high to M. perniciosa, moderate to P. palmivora; and susceptible to Moniliophtora roreri (Cervantes-Martinez et al., 2006); presents with leaves young, fruits and seeds slightly red by the presence of anthocyanins (Bartley, 2005). Another study in order to evaluate the effects of flooding on growth and mineral nutrition of six T. cacao clones,

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the authors observed that the CCN-10 genotype reached an intermediate position in relation the flooding tolerance compared to other clones evaluated (Rehem et al., 2009). Therefore, it is expected that the F1 progenies resulting from the crossing of these genotypes (CCN-10
SCA-6) are genetic materials of high resistance to various biotic and abiotic stress, by show a high degree of heterozygosity. On the other hand, the F1 progenies, resulting from self-pollination of ‘Catongo’
‘Catongo’, are thus more homozygous and intolerants to biotic and abiotic stresses, contrast with the CCN-10
SCA-6. This study aimed to describe the main defense mechanisms to stress cadmium used by two progenies of T. cacao. The results may be used in future programs of genetic enhancement.

2. Material and methods 2.1. Plant material and growing conditions The experiment was conducted under greenhouse conditions at Universidade Estadual de Santa Cruz-UESC, Ilhéus, BA, Brazil. The assay has used two T. cacao progenies contrasting for the tolerance to various types of stresses, like flooding, salinity, drought, disease, among others (Silva et al., 2012; Bertolde et al., 2010). The progenies were derived from the crossing between CCN-10
SCA-6 and the self-pollination of ‘Catongo’ [(‘Catongo’
‘Catongo’)]. CCN-10
SCA-6 and ‘Catongo’
‘Catongo’ were obtained via controlled pollination at the Active Germplasm Bank (BAG) from the Centro de Pesquisas do Cacau (CEPEC) of the Comissão Executiva do Plano da Lavoura cacaueira (CEPLAC). SCA-6 is a wild genotype native to Peru, has alleles with resistance to fungal diseases (witches’ broom) caused by the basidiomycete M. perniciosa and by black pod rot, which is caused by Phytophthora spp. Furthermore, its fruit and seeds are small (o1 g) and vary in number between fruit (Bartley, 2005). CCN-10 is a genotype native to Ecuador, also exhibits high resistance to M. pernicisa and has large fruit and seeds (4 1 g) (Silva et al., 2010). On the other hand, ‘Catongo’ is a genotype native from Bahia, Brazil, and derives from the 'Cacau Comum’', is susceptible to M. perniciosa and has medium size fruit and seeds (  1 g); aside from being a natural mutant for anthocyanins, it soon exhibits leaves and young, light-green fruit that will become yellow when ripe, and white seeds (Neto et al., 2005). Seeds from physiologically ripe fruit deriving from both progenies were previously cleaned by rubbing with sawdust to remove the mucilage, followed by removal of seed coat in such a way as to make tissues in contact with the metal when in solution. After that, the seeds were fully immersed in various solutions containing different concentrations of Cd (2, 4, 8, 16 and 32 mg L  1 Cd), with the same volume (500 mL), provided in the form of CdCl2⋅5/2H2O (Sigma, USA) along with the control (deionized H2O). The seeds were soaked in solution for 24 h and, at the moment the root was seen in protrusion (about 2 mm long), seeds were grown in black plastic tubes of 235 cm3 containing Pinus bark and turf þtriturated coconut fiber (1:1) as organic substrate. During the experimental period, the seedlings were daily watered and weekly fertilized with 4 g of NH4H2PO4 (Sigma, USA) 3 g of (NH2)2CO (Sigma, USA) and 3 g of KNO3(Sigma, USA) for each liter of water; 5 mL of this mixture were placed on each tube. 2.2. Collection of plant material for analysis Sixty days after seedlings emerged-same period when cotyledon reserves were totally depleted and cotyledons started to fallprimary roots, stem and 2nd or 3rd mature leaves were collected from the apex of the orthotropic axis of seedlings from both T.

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cacao progenies evaluated. The collected material was used to analyze guaiacol peroxidase (GPX); anatomical and ultrastructural changes in tissues and cells, respectively; gene expression; and concentrations of macro- (P, K, Ca, Mg and S) and micronutrients (Mn, Fe, Cu and Zn) in minerals and Cd.

and the mesophyll) through software for quantitative analysis studies in plant anatomy (Anati Quant, Version 2, UFV, Viçosa, MG, Brazil). The cross-sections of leaves from three plants per treatment and ten fields per replication were measured, totaling thirty measurements per treatment.

2.3. Guaiacol peroxidase (EC 1.11.1.7)

2.5. Transmission Electron Microscopy (TEM)

In total, 10 samples of leaves and 6 samples of roots have been collected from each progeny and treatment. Shortly after, the leaves and roots were immersed in liquid nitrogen (N2), stored in 80 °C and then lyophilized. Samples of lyophilized leaves were macerated in N2 until they reached a consistency of fine powder, and were transferred to 2 mL microtubes. After that, sodium phosphate buffer (Sigma, USA) was added to the macerated leaves (50 mM, pH 6.0) at a 1:20 ratio (macerate:buffer). These samples resuspended with buffer were sonicated for 1 min and 20 s with pulses of 05 s on and 10 s off and amplitude of 60%, using an ultrasonic processor (GEX 130, 130 W, East Bunker Court Vernon Hills, USA), followed by centrifugation at 4 °C for 15 min at 13,000g (microcentrifuge refrigerated model 5415R, Hamburg, Germany). The supernatant (crude extract) was collected and transferred to 1.5 mL microtubes. For assessing the activity of GPX, the extracts were used in the preparation of the activation reaction assay. The basic activation reaction of GPX activity consisted in 140 mL of guaiacol (Sigma, USA) solution (20 mM): H2O2 (Sigma, USA) (0.03% v/v) in phosphate buffer (Sigma, USA) (10 mM, pH 6.0), 90 mL of phosphate buffer (50 mM, pH 6) and 50 mL of crude extract. The samples were placed in 96-well microplates: buffer, crude extract and guaiacol solution: H2O2. The reaction was colorimetrically monitored at 470 nm in a microplate spectrophotometer (VERSAmax, California, USA). Reading was carried out by 100 s and the consumption of guaiacol was expressed in m mol s  1 g  1 of lyophilized tissue, using the formula y¼0.0189 þ0.1284x(R2 ¼ 0.99) derived from the standard curve for GPX-guaiacol (Rehem et al., 2011).

For electron microscopy, samples were extracted from the middle region of mature leaves and primary roots from seedlings of both T. cacao progenies (control) and treated with concentrations of 8 and 32 mg Cd L  1. To this end, the material was prefixed in 3% glutaraldehyde (Merck, Germany) in sodium cacodylate (Merck, Germany) buffer (0.1 M), pH 6.9, under vacuum, and was then washed in cacodylate buffer (0.1 M), pH 6.9; 6
, for 15 min. Post-fixation was carried out with 1% osmium tetroxide (Merck, Germany) prepared in the same buffer, followed by other washes in the same buffer for 6x. Dehydration mas performed with the use of ethanolic (Merck, Germany) series (30, 50, 70, 90 for 15 min and 100% for 30 min). Soaking was performed in ethanol/LR White (Merck, Germany) (3:1, 1:1, 1:3 and neat resin 3
). Inclusion was made using a gelatin capsule filled with pure LR White. Polymerization was carried out at 60 °C for 24 h. After that, ultrathin sections of 70 nm in thickness were obtained using a Leica ultramicrotome (model UC6, Nussloch, Germany) and were then deposited on copper grids of 300-mesh. For each treatment, 3 grids (with three sections on each) have been used. Then, the sections were counterstained with 3% uranyl acetate (Merck, Germany) for 20 min, followed by 0.4% lead citrate (Merck, Germany) for 5 min. Shortly after, the sections were examined in a transmission electron microscope (TEM) (Morgagni ™-model 268 D, Eindhoven, Netherlands), operating at an accelerating voltage of 80 kV, and were micro-processed and controlled using a Windows platform fitted with a CCD camera. The Cu grids containing the samples were afterwards photographed and the images were analyzed. 2.6. Gene expression

2.4. Plant anatomy For light microscopy, tissue samples were taken from the middle region of the 2nd or 3rd mature leaf from the apex of the axis orthotropic seedling progenies of both T. cacao, derived from the seeds of the control (without Cd) and subjected to concentrations of 8 and 32 mg Cd L  1. As these treatments were selected because Souza et al. (2011) in a study of phytotoxicity by Cd, found that the other doses used in this study do not cause changes to the tissue level. Immediately after collection, the plant material was fixed in 3% glutaraldehyde (Merck, Germany) and sodium cacodylate (Merck, Germany) buffer 0.1 M, pH 6.9, for 4 h. Then the samples were transferred to 50% alcohol (Merck, Germany) and stored at 4 °C in refrigerator. Subsequently, the leaf samples were dehydrated in series butanol (Merck, Germany) (70% for one hour, 80%, 90% and 100% for half an hour) and then subjected to tertiary butanol (Merck, Germany) with 100% exchange at 2, 4 and 1 h. Shortly thereafter, were subjected to tertiary butanolþparaffin oil (2 h) paraffin-butanol þ½ exchange (2 h) paraffin-butanol þ ½ exchange (2 h) and paraffin total exchange (2 h). After that the plant material was embedded in paraffin and sectioned in the microtome Leica (model RM 2145, Nussloch, Germany), with a thickness of 10 μm. Then the assembly was held for plant samples on glass slides. Staining was performed with 1% astra blue (Merck, Germany) and safranin (Merck, Germany). Soon after, the blades were observed and photographed under a microscope Leica (model DM 500, Nussloch, Germany) for subsequent measurement of leaf tissues (thickness of the epidermis, the adaxial and abaxial faces; palisade and spongy parenchymas,

The gene expression analyses performed by Real Time qRT-PCR have used gene-specific primers relative to oxidative stress enzymes: (i) PER-1, associated with the biosynthesis of class III peroxidase; (ii) Cu–Zn-SODcyt, associated with the biosynthesis of cytosolic SOD; (iii) MET, associated with the biosynthesis of the metallothionein polypeptide; and (iv) PCS, associated with the biosynthesis of phytochelatin synthase (Table S2). The RNA from lyophilized leaves and roots derived from seedlings of both T. cacao progenies subjected to the control treatments – 8 and 32 mg Cd L  1 – was extracted using RNAqueous kit (Ambions, NY, USA), according to the manufacturer’s instructions. Initially, the purity and integrity of RNA were tested by gel electrophoresis in 1% agarose. Next, RNA samples were used for cDNA synthesis with Revertaid H-Minus Reverse Transcriptase (Fermentas, Pittsburgh, USA), in line with the manufacturer’s instructions, using oligo d(T)18 primers. Reactions were incubated at 65 °C for 5 min, 37 °C for 5 min, 42 °C for 60 min, and 70 °C for 10 min. Real-time quantitative PCR (qPCR) was performed on a Real Time PCR thermocycler (Applied Biosystems, model 7500, Foster City, USA) using a non-specific detection sequence (fluorophore)-SYBR Green I (Life Technologies, NY, USA). The abundance of transcripts was analyzed by means of specific primers (Table S1) designed from the analysis of gene sequences known in the library of T. cacao. In order to test the quality of these primers, as well as the specificity and the identity of reverse transcription products, qPCR products were monitored after each PCR by a curve analysis relative to the reaction products; this curve was able to distinguish products from gene-specific and non-specific PCRs.

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The temperature of PCR products was increased from 55 to 99 °C at a rate of 1 °C/5 s, whereas the resulting data were analyzed using the LightCycler software. Each sample only had a single band with a characteristic melting point, indicating that the qPCR yielded a product that was specific to the used primers. Values regarding the Threshold cycle (CT) were determined using the LightCycler software. The numbers of relative gene expression were calculated as a percentage of the control treatment, using the 2  ΔΔCt method (Livak and Schmittgen, 2001), whereas the β-tubulin gene (Table S1) was used as the endogenous (baseline) so as to detect changes in the abundance of transcripts. 2.7. Analyses of macro- and micro-minerals and Cd In order to examine the mineral nutrients, ten seedlings from each treatment (0, 2, 4, 8, 16 and 32 mg Cd L  1) have been collected. After that, seedlings from both T. cacao progenies were washed in tap water (1
), 3% HCl (Sigma, USA) (1
) and distilled water (2
) and were separated into root, stem and leaf. After washing, all the three organs were separately placed in paper bags and dried at 75 °C in an oven with forced air circulation until reaching constant weight. Next, the dry plant materials were ground on a Wiley (Thomas Scientific, Swedesboro, USA) mill using a 20 mm mesh screen and were chemically analyzed. For determination of mineral nutrients, 200 mg of each sample were weighted with the aid of an analytical balance (Shimadzu, model AUW220D, Kyoto, Japan). Soon after, the samples were placed in glass tubes (50 mL) and were subjected to acid digestion in a block digester (Tecnal-model TE- 007MP, São Paulo, Brazil). For acid digestion, 4 mL of concentrated nitric acid were added so as to heat the digester block while maintaining the temperature at 50 °C for 30 min. Then, the temperature was increased to 80 °C and maintained for 1 h. The temperature was then raised to and maintained at 130 °C for 2 h and 1 mL of hydrogen peroxide (Sigma, USA) (30%) was added within regular intervals of 20 min, totaling another hour of heating. At the end of digestion, the digested material was transferred to15 mL falcon tubes. Milli-Q (Billerica, USA) water was added until reaching a final volume of 15 mL. The next step was to determine the concentration of macro- (P, K, Ca, Mg and S) and micronutrient minerals (Mn, Fe, Cu and Zn) and Cd through the technique of optical emission spectrometry by inductively coupled plasma ( ICP-OES, Varian, model 710 ES, North Carolina, USA).

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3. Reults 3.1. Guaiacol peroxidase (GPX) As a result of the treatment with Cd, the ‘Catongo’
‘Catongo’ progeny showed an increase in the activity of GPX in leaves. In comparison with the control, the significant values (p o0.05) (Fig. 1A). In contrast, leaves of CCN-10
SCA-6 did not exhibit marked differences. On the other hand, roots of CCN-10
SCA-6 displayed increased in GPX activity when compared to the control, at concentrations of 8 mg Cd L  1 (52%) and 16 mg Cd L  1 (58%). With regard to ‘Catongo’
‘Catongo’, there has been a significant reduction (p o0.05) in GPX activity in roots: 58% at a concentration of 16 mg Cd L  1 in comparison with the control (Fig. 1B). 3.2. Anatomical analyses Anatomical analyses of the mesophyll leaf of T. cacao seedlings from both progenies showed significant effects (p o0.05), as well as inter- and intra-progenies with increasing doses of Cd applied via seminal fluid all over the epidermis and the upper (UE) and lower (LE); the layer palisade (PL) and spongy layer (SL); and the mesophyll (M) (Table S1). For a concentration of 8 mg Cd L  1 progeny ‘Catongo’
‘Catongo’ exhibited significant increases (p o0.05) in the

2.8. Statistical analysis The experiment was carried out in a completely randomized design with 12 treatments arranged in a 2
6 factorial design. The treatments corresponded to both T. cacao progenies (‘Catongo’
‘Catongo’) and CCN-10
SCA-6) and 5 Cd concentrations (2, 4, 8, 16 and 32 mg Cd L  1)þcontrol (no Cd), with a variable number of repetitions (four for analysis of enzyme activity, three for anatomical and ultrastructural analyses, nine repetitions aimed to determine the concentration of macro- and micro-minerals, and six repetitions for the analysis of gene expression) and one seedling per experimental unit. Analysis of variances (ANOVA) and comparison of means were carried out to analyze treatments using Tukey test (p o0.05) and t-test (po 0.05). Furthermore, regression analyses were performed for macro- and micro-mineral nutrients. Statistical analysis was performed using the Bioestat 5.0 software (Instituto de Desenvolvimento Sustentável Mamirauá, Tefé, AM, Brazil).

Fig. 1. Activity of guaiacol peroxidase (GPX) in two progenies of T. cacao (‘Catongo’
‘Catongo’ and CCN-10
SCA-6) at 60 days after the emergence of seedlings from seeds soaked for 24 h in different concentrations of Cd in solution. Activities in the leaves (A) and roots (B). The Statistical significance of inter-progenies was determined by ANOVA, followed by t-test. (n) po 0.05; (nn) p o0.01; (nnn) po 0,001; (n.s) non-significant. Intra-progenies followed by the same lowercase letters do not differ by Tukey’s test (p o 0.05). Mean values of four replicates7 standard error of the mean.

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thickness of LE and US (10% and 9%, respectively) and reduced SL and M (45% and 8%, respectively). At the concentration of 32 mg Cd L  1 was observed thickening of the PL (27%). Regards the CCN-10
SCA-6, it has been noted the occurrence of intra-progeny variations in the thickness of the US, LE, PL, SL and M at a concentration of 8 mg L  1 Cd caused an increase in the thickness of the UE. At the concentration of 32 mg L  1 Cd caused the reduction of the thickness of the EU and increased thickness of the LE. The 8 mg Cd L  1 for the 32 mg Cd L  1 observed was a reduction in the thickness of PL, SL and M. Likewise, significant inter-progeny variations (p o0.05) were observed for UE, LE, PL and SL. As compared to ‘Catongo’
‘Catongo’, the percentage reduction for UE was 17% in CCN-10
SCA-6 at a concentration of 32 mg Cd L  1. Yet, the observed reductions in LE were 55% and 20% at concentrations of 8 and 32 mg Cd L  1 in CCN-10
SCA-6, respectively, when compared to ‘Catongo’
‘Catongo’. PL, in turn, showed a reduction of 34% when subjected to the highest concentration of the metal in CCN-10
SCA-6, when compared with ‘Catongo’
‘Catongo’. In contrast, in comparison with ‘Catongo’
‘Catongo’, there was an

increase of 22% in the thickness of LE in CCN-10
SCA-6 at a concentration of 8 mg Cd L  1. 3.3. Transmission Electron Microscopy (TEM) In the absence of Cd (control), the ‘Catongo’
‘Catongo’ and CCN-10
SCA-6 progenies had their leaf mesophyll cells with a normal appearance, keeping the integrity of membranes from the chloroplast and nucleus, without evidence of changes ( Fig. 2A and D, respectively). Nonetheless, in the presence of Cd, both progenies showed changes in the cell ultrastructures of the leaf mesophyll as a function of the applied Cd concentration (Fig. 2B and C and E and F). The ‘Catongo’
‘Catongo’ progeny showed structural changes in the chloroplasts both when subjected to both Cd concentrations: 8 mg Cd L  1 and 32 mg Cd L  1. (Fig. 2B and C). However, as compared to the control, the starch content was found to be higher when it was subjected to the highest Cd concentration. Also, there have been deformation of the chloroplast and expansion of the thylakoid membranes. On the other hand, the leaf mesophyll cells of seedlings from CCN-10
SCA-6 subjected to a concentration of

Fig. 2. Electron micrographs of leaf mesophyll cells from ‘Catongo’
‘Catongo’ in the control [(A) (5 mm)] and subjected to doses of 8 mg Cd L  1 [(B) (2 mm)] and 32 mg Cd L  1 [(C) (2 mm)]; and CCN-10
SCA-6 in the control [(D) (2 mm)] and subjected to doses of 8 mg Cd L  1 [(E) (1 mm)] and 32 mg Cd L  1 [(F) (10 mm)] 60 days after the emergence of seedlings from seeds soaked for 24 h in different concentrations of Cd in solution. In C, arrows denote the increased accumulation of starch and disorganization in the structure of the chloroplast; in F, the arrow indicates the accumulation of electro-dense material. C – chloroplast; m – mitochondrion; n – nucleus; sg – starch; ep – epidermis.

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Fig. 3. Electron micrographs of cells from ‘Catongo’
‘Catongo’ in the control [2 mm; A] and subjected to a dose of 32 mg Cd L  1 [5 and 2 mm, respectively; B and C]; and CCN10
SCA-6 in the control [2 mm; D] and subjected to a dose of 32 mg Cd L  1 [10 and 2 mm, respectively, E and F] 60 days after the emergence of seedlings from seeds soaked for 24 h in different concentrations of Cd in solution. In B, the arrow denotes invagination of the nuclear membrane and disorganization of the nucleus structure; in C, it indicates disappearance of nucleoli; in E, accumulation of electron-dense material in the vacuole and, in F, accumulation of electron-dense material in the cell wall. In cw – cell wall; ep – epidermis; n – nucleus; m – mitochondrion; v – vacuole.

8 mg Cd L  1 did not exhibit changes in the chloroplasts (Fig. 2E). At a concentration of 32 mg Cd L  1, an accumulation of electron-dense material was observed in the wood parenchyma (Fig. 2F). In the absence of Cd in both T. cacao progenies evaluated, the root tissue cells exhibited normal ultrastructural characteristics (Fig. 3A and D). Nonetheless, several ultrastructural changes were observed at the concentration corresponding to 32 mg Cd L  1, as invagination of the nuclear membrane (Fig. 3B) and change the nucleolus showing chromatin condensation (Fig. 3C) and a tendency of disappearing nucleolus. When subjected to the highest Cd dose (32 mg L  1), the ‘Catongo’
‘Catongo’ progeny showed electron-dense materials on the cell wall and the vacuole; in addition, a deformation was observed on the nucleus, as evidenced by the invagination of the membrane, nuclear chromatin condensation, and the disappearance of the nucleolus (Fig. 3B and C ). In contrast, when subjected to the highest dose (32 mg L  1), CCN10
SCA-6 showed accumulation of electron-dense material on the cell wall and the vacuole (Fig. 3E and F).

3.4. Gene expression There was an expression of genes associated with the biosynthesis of PER-1 in both T. cacao progenies. In leaves, the expression of PER-1 gene was greater in CCN-10
SCA-6 at concentrations of 8 mg Cd L  1 and 32 mg Cd L  1, for which increases were of the order of 45% and 26%, respectively, as compared to ‘Catongo’
‘Catongo’ (Fig. 4A). The same was observed in the roots, for which increases were 38% and 57% at concentrations of 8 mg Cd L  1 and 32 mg Cd L  1, respectively (Fig. 4E). Likewise, there have been significant intra-progeny variations (po 0.05 ) relative to the expression of PER-1 in’‘Catongo’
‘Catongo’ leaves as a function of the increased Cd concentrations: 1.5% and 36% for 8 mg Cd L  1 and 32 mg Cd L  1, respectively. The same was observed for CCN-10
SCA-6, where increases were of 44% and 53% for concentrations of 8 mg Cd L  1 and 32 mg Cd L  1, respectively, both compared with the control. Moreover, when compared with the control, the increase in the PER-1 gene expression subjected to a Cd concentration of 8 mg L  1 was 4% in the root system of ‘Catongo’
‘Catongo’. As regards the highest dose of Cd, in contrast,

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Fig. 4. Relative expression of genes encoding for peroxidase (PER-1), cytoplasmic superoxide dismutase (SODCyt), metallothioneins (MET), phytochelatins (PCS) in the leaves (A–D) and roots (E–H ) of two T. cacao progenies 60 days after the emergence of seedlings from seeds soaked for 24 h in different concentrations of Cd in solution. mRNA levels normalized using β-tubulin, and expression relative to the control assumed as 1. The statistical significance of inter-progenies was determined by ANOVA, followed by t-test. (n) p o0.05; (nn) p o0.01; (nnn) p o 0,001; (n.s) non-significant. Intra-progeny averages followed by the same lowercase letters do not differ by Tukey’s test (p o 0.05). Mean values of four replicates7 standard error of the mean.

there was no PER-1 expression in the root cells of this progeny. On the other hand, as compared to the control, the observed increases in PER-1 were of 40% and 53% at concentrations of 8 mg Cd L  1 and 32 mg Cd L  1, respectively. With respect to the expression of genes associated with the biosynthesis of SODcyt, a more pronounced increase was observed for the leaves of ‘Catongo’
‘Catongo’, when subjected to the highest Cd concentration, for which the increase was of 45% when compared with the control (Fig. 4B). No significant differences

(p o0.05) were observed in expression of this gene in roots, though (Fig. 4F). Inter-progeny variations were observed in the production of mRNA from the gene encoding for MET, whereas the highest expression was found in the leaves of CCN-10
SCA-6 (Fig. 4C). As regards the expression of MET, there has been a significant intra-progeny variation (p o0.05) in roots, which were subjected to concentrations of 8 and 32 mg Cd  1 (Fig. 4G). As compared to ‘Catongo’
‘Catongo’, the progeny deriving from the crossing CCN-10
SCA-6 exhibited an increase in the

A.V. Castro et al. / Ecotoxicology and Environmental Safety 115 (2015) 174–186

Fig. 5. Accumulation of Cd in the roots (●), stems (Δ) and leaves (□) of two T. cacao progenies [CCN-10
SCA-6 (A) and ‘Catongo’
‘Catongo’ (B)] 60 days after the emergence of seedlings from seeds soaked for 24 h in different concentrations of Cd in solution. Mean values of nine replicates7 standard error of the mean. The absence of error bars indicates the size of the error did not exceed the size of the symbol. The equations regarding the regression curves for Cd in CCN-10
SCA-6 were Y′¼0.2 þ 0.74x-0.004
2 (R2 ¼ 0.98; leaves), Y′¼ 1.1 þ0.48x – 0.005x2 (R2 ¼ 0.97, stems) and Y′ ¼0.13þ 0.05 þ 0.004x2 (R2 ¼ 0.96; roots); for ‘Catongo’
‘Catongo’ Y′¼ 0.65 þ 0.84x – 0.01
2 (R2 ¼ 0.90; leaves), Y′ ¼ 1.1 þ0.48x-0.01x2 (R2 ¼ 0.77; stems) and Y′¼ 0.008þ 0.05x – 0.0004x2 (R2 ¼ 0.97; roots).

expression of genes associated with the biosynthesis of PCs both in leaves and roots (Fig. 4D and H). The mRNA production encoding for PCS was significantly higher in leaves of CCN-10
SCA-6 both subjected to 8 mg L  1 (9.5%, po 0.01) and 32 mg cd L  1 (56%, p o 0.001) (Fig. 4D). Yet, this difference was not statistically significant (p o0.01) in the roots (Fig. 4H). Furthermore, considering that there was a small increase (0.05%) in leaves from CCN-10
SCA-6, there have been intra-progeny differences relative to the expression of PCs with increasing doses of Cd applied via seminal fluid. 3.5. Analyses of Cd, macro- and micro-mineral nutrients With regard to the absorption of Cd, and macro- and microminerals in response to the increased concentration of Cd applied via seminal fluid, distinct but significant inter-progeny behaviors (p o0.05) were observed (Figs. 5 and S1). In response to increasing Cd concentrations, the CCN-10
SCA-66 progeny showed higher Cd absorption when compared to ‘Catongo’
‘Catongo’. In leaves from CCN-10
SCA-6, a quadratic increase was observed in the Cd content, whereas the control had a 98% increase when subjected to the highest dose of Cd, followed by the stem, with an increase of 90% in relation to the control, and roots, with 99% (Fig. 5A). The same was noted for the leaves of ‘Catongo’
‘Catongo’, where the

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increase of the control in response to the highest dose was 97%, followed by 90% and 99% for shoots and roots, respectively (Fig. 5B). The Cd concentration added via seminal fluid has promoted a differential accumulation of P, K and S in different vegetative organs of seedlings from both T. cacao progenies evaluated. The progenies did not exhibit significant differences (po 0.05) regarding the accumulation of Mg, but the largest observed accumulation was seen in the stem, followed by the leaves, and a lower accumulation in the roots (Fig. S1A and F). In comparison with CCN-10
SCA-6, there was higher accumulation of P in the leaves and stems of’‘Catongo’
‘Catongo’. However, there was a linear reduction of 36% in P content as a function of the increasing concentration of Cd via seminal fluid in the stems of ‘Catongo’
‘Catongo’ (Fig. S1G). CCN-10
SCA-6 exhibited a greater ability to accumulate K in the leaves and stems (Fig. S1C). None of the progenies showed significant differences (p o0.05) regarding the accumulation of Ca, but the progeny of CCN-10
SCA-6 was seen to have accumulated greater Ca levels in the leaves and stems. As compared to CCN10
SCA-6 (Fig. S1J), for which the quadratic increase was of 43% in the leaves, the ‘Catongo’
‘Catongo’ progeny exhibited higher accumulation of S in the leaves and stems. With regard to the concentration of Cu, Fe and Zn, a differential accumulation of these micronutrients was observed with increasing concentration of Cd via seminal fluid. CCN-10
SCA-6 showed a linear increase of 14% in Cu content in the roots, whereas ‘Catongo’
‘Catongo’ had the Cu levels constant in the leaves, roots and stems (Fig. 6A and E). In the same way, it was found that Fe content was significantly (p o0.05) different in the roots from both T. cacao progenies as a function of the increased concentration of Cd via seminal fluid. As for CCN-10
SCA-6, there has been a quadratic decrease of 90% in Fe content in the roots, whereas for ‘‘Catongo’
‘Catongo’ had a quadratic decrease of 78%. Fe concentrations did not show changes in the leaves and stems from both T. cacao progenies, though (Fig. 6B and F). CCN-10
SCA-6 had a linear reduction of 31% in Zn content in the stems, whereas no significant differences relative to this metal element (p o0.05) were observed for ‘Catongo’
‘Catongo’ (Fig. 6D and H).

4. Discussion The contamination of the environment by heavy metals is caused by anthropogenic activities, including phosphate fertilizers, pesticide application, traffic of agricultural machinery, among others, and represents a big concern worldwide, especially because of its devastating and irreversible effects (Li et al., 2004; Vidal et al., 2004). The high incidence of diseases resulting from heavy rainfall and high relative humidity of the air in the cocoa producing regions, as well as the need for high soil fertility for production requires the use of fungicides, pesticides, and fertilizers, which are in most cases phosphate-based. This could lead to accumulation of heavy metals in the soil, especially Cd and Pb. Zarcinas et al. (2004) have associated the high levels of Cd in soils and the over-concentrations of Cd in T. cacao to the use of phosphate fertilizers. In Nigeria, though, the low Cd levels in T. cacao were attributed to earlier reports that over 70% of the Nigerian cocoa producers (Ogunlade et al., 2009) and above 95% of the cocoa farmers in the study area (Agbeniyi et al., 2009) do not use fertilizers in the cultivation of T. cacao. Therefore, there is strong evidence that the use of phosphate fertilizers in T. cacao is responsible for environmental pollution by heavy metals, including Cd. Hence, studies related to changes caused by Cd in T. cacao seedlings are of great importance and will serve as a basis for understanding the mechanisms underlying resistance to this metal

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Fig. 6. Accumulation of mineral micronutrients in the leaves (□), stems (Δ) and roots (●) of T. cacao progenies [CCN-10
SCA-6 (A–D) and ‘Catongo’
‘Catongo’ (E–H)] 60 days after the emergence of seedlings from seeds soaked for 24 h in different concentrations of Cd in solution. Mean values of nine replicates 7 standard error of the mean. The absence of error bars means that the size of the error did not exceed the size of the symbol. The equations relative to the regression curves for Cu in CCCN-10
SCA-6 were Y ′¼ 3.99 (leaves), Y′ ¼4.64 (stems) and Y′¼ 7.42 þ 0.04x (roots, R2 ¼ 0.47); with respect to’‘Catongo’
‘Catongo’, they were Y′ ¼ 4.25 (leaves), Y′¼ 7.10 (stems) and Y′¼ 8.60 (roots). Corcerning Fe in CCN-10
SCA-6, the values were Y’¼ 69.7 (leaves), Y′¼31.92 (stems) and Y′ ¼422-25.14
0.59x2 (roots, R2 ¼ 0.72); as for ‘Catongo’
‘Catongo’, the values were Y’¼78.75 (leaves), Y′¼48.57 (stems) and Y′¼ 451-23x-0.5x2 (roots, R2 ¼0.75). For Mn in CCN-10
SCA-66, the values were Y′ ¼1978 (leaves), Y′¼ 379 (stems) and Y′¼101 (roots); the values regarding ‘‘Catongo’
‘Catongo’ were Y′¼ 2128 (leaves),Y′¼ 309 (stems) and Y′¼ 72.52 (roots). As respects Zn in CCN-10
SCA-6, the observed values were Y′¼ 43.5 (leaves), Y′¼ 70-0.4x (stems, R2 ¼ 0.40) and Y′¼ 30.52 (roots); in ‘‘Catongo’
‘Catongo’, the observed values were Y′¼ 49.25 (leaves), Y′¼ 62.47 (stems) and Y′¼ 17.75 (roots).

stress. In the roots of CCN-10
SCA-6, a hybrid progeny with a high heterozygosity index exhibits increased GPX activity when subjected to higher Cd concentrations, unlike the observed in the selfpollinated ‘‘Catongo’
‘Catongo’. Despite the fact that the latter is

more homozygous, it is observed to be a defense mechanism developed by CCN-10
SCA-6 to fastly detoxify cells against excess reactive oxygen species (ROS) produced in response to Cd stress (Fig. 1B). The higher GPX activity in the roots of CCN-10
SCA-6 can also be linked to other possible defense mechanisms, such as

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preventing the translocation of Cd to the aerial part of the plant. This can prevent damages caused by Cd to the cell organelles responsible for the normal cell metabolism, including nuclei, mitochondria, chloroplasts, peroxisomes, among others. The results of this study corroborate those found in the literature, since different species (Avena sativa, Brassica campestris cv. Chinensis, Lactuca sativa cv. hanson) stressed with Cd have exhibited increased GPX activity in their leaves and roots – including the controls – when compared to the treatments with Cd (Correa et al., 2006). In contrast to the tolerant progeny (CCN-10
SCA-6), it is noteworthy the natural trend of increase in the GPX activity of the intolerant progeny (‘Catongo’
‘Catongo’), since there has been enzymatic activity both in the leaves and the roots of ‘‘Catongo’
‘Catongo’, including the control. The increased GPX activity in the leaves was in line with the increased concentrations of Cd in both T. cacao progenies evaluated. With regard to CCN10
SCA-6, the increased activity of GPX in the leaves of ‘Catongo’
‘Catongo’ shows a delayed response to stress, revealing a signaling system to stress that is less efficient when compared with CCN-10
SCA-6. Hence, both T. cacao progenies showed increased GPX activity as a result of the increased concentration of Cd, being faster in CCN-10
SCA-6. The same was observed in other studies of oxidative stress and enzymatic activity due to biotic and abiotic stresses (Nojosa et al., 2003; Smeets et al., 2007). Results concerning GPX activity were seen to be associated with the expression of the gene PER -1, where an increase in the biosynthesis of class III peroxidases was seen to be more pronounced in CCN-10
SCA-6 when compared to ‘Catongo’
‘Catongo’ both in the leaves and in roots. Then, the existence of a defense mechanism developed by CCN-10
SCA-6 and aimed at eliminating excess ROS produced due to oxidative stresses caused by the phytotoxicity of the metal was confirmed (Xiao et al., 2008). On the other hand, since there was no differential expression of the gene SODcyt in the roots of both T. cacao progenies, it is presumable that this gene was expressed at another moment, that is, prior to the collection of plant material for analysis. In comparison with ‘‘Catongo’
‘Catongo’, CCN-10
SCA-6 was the progeny with the highest expression of mRNA associated with the production of METs, revealing another strategy of this progeny to tolerate the toxicity caused by Cd. MTs are peptides that have promoter regions for the transport of certain metal elements whose genes are responsible for their biosynthesis and are chiefly expressed when the organisms are exposed to toxic metals (Cobbett and Goldsbrough, 2002). These peptides are primarily known for their ability to detoxify organisms contaminated by toxic metals using their ability to bind to free metal ions or metals associated with other ligands (Asselmana et al., 2012; Janssens et al., 2009). A study involving A. thaliana and genetically modified Nicotiana tabacum organisms has revealed that the introduction of MTs genes caused the species to develop tolerance to Cd and the ability to accumulate this metal element (Eapen and D’Souza, 2005). In the presence of Cd, CCN-10
SCA-6 was likewise observed to have a higher production of mRNA related to the biosynthesis of PCS, whereas the production in the roots was even greater. Thus, corroborate by this, and other studies, the expression of genes encoding for PCS was found to be an important mechanism for the detoxification of Cd-stressed cells (Gallego et al., 2007). The increased production of mRNA for the biosynthesis of PCS can be regarded as a genetic tool used by tolerant plant species so as to reduce the phytotoxic effects of Cd on the cells, particularly in the roots, thereby impeding its translocation to the aerial parts through the chelation of Cd and sequestration in the vacuoles in such a way that cellular homeostasis is maintained. Furthermore, this study has verified the absence of expression of PCS in the

183

progeny of T. cacao susceptible to various types of stresses (‘Catongo’
‘Catongo’). It is therefore suggested that this progeny does not have a well-developed signaling system, which would otherwise enable the activation of defense molecules, such as the expression of genes encoding for PCSs. The most pronounced defense strategies against Cd stress in CCN-10
SCA-6 helped to protect plants from damaging effects, as demonstrated via electron micrograph of the roots (Fig. 3). The increase in GPX activity the result of increased gene expression that produces GPX and other genes associated with plant defenses were decisive in protecting cellular organelles. In CCN-10
SCA-6, only accumulation of electron-dense material was observed in the vacuole and the cell wall (Fig. 3E and F). In ‘Catongo’
‘Catongo’, though, the defense mechanisms were not as efficient and damages to the leaves could be easily observed: disorganization of the chloroplasts, which have an important role in photosynthesis, and changes in the size and number of starch grains, which is possibly a survival strategy aimed at accumulating reserves (Fig. 2C). In addition, in the root system was observed nucleus deformation, nuclear membrane invagination and condensation of the nuclear chromatin (Fig. 3B and C) are irreversible changes that can cause the death of the organism. Chloroplasts are extremely susceptible to the oxidative stress resulting from increases in oxygen concentration and electron flow, as well as the presence of metal ions in their microenvironments (Daud et al., 2009). Structural changes in the chloroplasts of the progeny more susceptible (‘Catongo’
‘Catongo’) to stress by metals was probably due to increased ROS, as evidenced by severe damages in the structures of chloroplasts, particularly in thylakoid membranes and the granum (Paramonova et al., 2003). The same was observed in the structure of leaf chloroplasts of Genipa americana subjected to toxicity by Cd (Souza et al., 2011). Furthermore, a study by Daud et al. (2009) involving Gossypium hirsutum cultivars (BR001 and GK30) has found ultra-structural changes resulting from increasing Cd concentrations, as for example the higher number of nucleoli. The present study, on the other hand, has verified a trend of disappearance of nucleoli in the progeny more susceptible to Cd (‘Catongo’
‘Catongo’). Reduction in the thickness of the leaf mesophyll of ‘Catongo’
‘Catongo’ seedlings and CCN-10
SCA-6 derived from seeds subjected to concentrations of 8 and 32 mg Cd L  1 was primarily due to decreased thickening of the palisade layer (PL) and the spongy layer (SL) in both progenies. Particularly at lower Cd concentrations, there has been an increase in the thickness of the leaf upper epidermis (UE) and lower epidermis (LE) of both T. cacao progenies. This fact is in the same way evidence in other woody species (Gomes et al., 2011). Unlike ‘Catongo’
‘Catongo’, though, CCN-10
SCA-6 showed an increase in the thickness of (LE) even when it was subjected to the highest Cd concentration, proving to be one of the survival strategies more tolerant to contamination by Cd. Plant species can respond differently as structural changes in leaf level and are described as specific for each metal (Shi and Caia, 2009). According to Srighar et al. (2005), some plant species develop morphological and anatomical changes in the leaf mesophyll tissue, enabling a wide phenotypic plasticity to different stress conditions. As reported by other authors (Chugh and Sawhney, 1999) in studies involving ‘Catongo’
‘Catongo’, the highest compression of the leaf tissue can lead to decreased photosynthetic capacity in the presence of Cd. Moreover, the results of this study corroborate those found in the literature, reinforcing that the treatment with toxic metals leads to increased thickness of the leaf epidermis (Gomes et al., 2011) and reduced size of leaf mesophyll cells (Malathi et al., 2001; McQuattie and Schier, 1993). The highest concentration of Cd was observed in the plant’s

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aerial parts and, because of this, several ultra-structural changes were observed in the leaf mesophyll, particularly in ‘Catongo’
‘Catongo’. In comparison with ‘Catongo’
‘Catongo’, CCN10
SCA-6 exhibited a higher concentration of Cd in the roots. This study reveals that, during stress by Cd, the concentrations of this metal increase in the roots; yet, this is not due to the increased absorption of this element, but rather by the decreased accumulation of dry matter (Souza et al., 2008). Despite the fact that there is a high concentration of Cd in the roots, this element is also found in the leaves and stems, showing that Cd is not totally immobilized in the roots, but it can be translocated to the aerial parts just as in other plant species (Souza et al., 2008; Unterbrunner et al., 2007). The present study has verified that changes occurred during the absorption of certain nutrients – especially P, S, Cu, Fe and Zn – in the seedlings of both T. cacao progenies derived from seeds exposed to different Cd concentrations via seminal fluid. The toxicity of Cd in plants can influence the concentration of mineral elements in the tissues and thereby impair mineral nutrition (Souza et al., 2008). The presence of Cd in cells can affect the levels of polyvalent cations through competition for binding sites of proteins or transporters (Sandalio et al., 2001). In a study that analyzed the toxicity of Cd in the roots and leaves of 20 genotypes of Oryza sativa grown in the soil, Liu et al. (2003) have observed significant and positive correlations with Fe, Zn, Cu, Mn in the roots and leaves; yet, with respect to Mn, the correlation was seen to be significant and negative, without any significant correlation with Mg. In a study involving G. americana subjected to stress by Cr in nutrient solution, Barbosa et al. (2007) have observed increased concentrations of Cu as a function of stress. In contrast, Fe and K contents were proportional to the increased concentration of Cr. The linear reduction in P content observed in the stems and leaves of ‘Catongo’
‘Catongo’ corroborates the results found in the literature. In a study involving Lupinus albus cv. Multolupa grown at different Cd concentrations in nutrient solution, Zornoza et al. (2002) have found a reduction in P, K, Fe, Mn and Zn contents in the plant’s aerial part with increasing doses of Cd. As observed for CCN-10
SCA-6 in this study, a study conducted by Souza et al. (2011) examined morphological and physiological responses in G. American specimens subjected to Cd stress in nutrient solution (0.5, 1, 2, 4, 8 and 16 mg L  1) and confirmed a higher accumulation of K as a function of increasing doses of Cd. The function of K is of catalytic and osmotic nature, and is essential for various vital functions in the plant. Its catalytic activity refers to enzyme activation, since K activates around 60 enzymes that participate in phosphorylation reactions, protein synthesis, metabolism of N and carbohydrates, carbohydrate transport and the symbiotic fixation of N. The increase in S content observed in the leaves and stems of ‘Catongo’
‘Catongo’ is in line with the results observed in Pisum sativum (Paivoke, 2002), where a high metal concentration has caused the S content to increase in the leaves. According to Sinha et al. (2006), though, increased doses of the metal in Brassica oleracea have led to a decrease in its concentration. It was noted in this study that the roots of CCN-10
SCA-6 showed a linear increase in the absorption of Cu, which is an micronutrient fundamental for the catalytic activity of a number of enzymes whose absorption and transport are regulated and mediated by specific transporters (Luchese et al., 2004). This metal element acts as a mediator in the signaling directed to the production of ethylene (Silva et al., 2012). Excess Cu is identified by associations with transcription factors. Moreover, it activates several defense mechanisms against abiotic stress, as for example the increased expression of metallothioneins, phytochelatins and antioxidants, which help to remove the “free” Cd and restore ionic homeostasis and cellular redox (Souza et al., 2008). These free

metals are potentially dangerous and their absorption and cell concentration should be therefore adjusted. Cu has a high affinity for peptide, carboxylic and phenolic groups. It is believed that the SOD enzyme containing Cu/Zn, Fe or Mn at its reaction center plays a dual role in preventing the toxicity of the metal (Hirt and Shinozaki, 2004). Other studies corroborate the results of this study, revealing a decrease in Fe and Zn contents in species like Betula pendula, Helianthus annuus and P. sativum (Rodriguez-Serrano et al., 2009; Gussarson et al., 1996) with increasing Cd concentration. This reveals the possible competition of these ions for the same active site of Cd in the plasma membrane (Sanitá di Toppi and Gabbrielli, 1999). Leaf chlorosis is usually observed with increasing concentrations of Cd, which are probably associated with decreased Fe translocation to the leaves (Wong et al., 1984). Nonetheless, other studies suggest that Cd induced chlorosis may occur due to changes in the Fe/Zn ratio and not to Fe deficiency, once plants treated with Cd exhibit a high concentration of this micro-nutrient. Thus, depending on the species, Cd can promote the increase (Wong et al., 1984), decrease (Gussarsson, 1994), or simply not affect (Souza et al., 2011) Fe absorption in plants.

5. Conclusions Cd exhibited dynamic behavior in the T. cacao progenies studied, since higher concentrations were found in the shoots, showing that it can be translocated in the plant. The GPX activity can be used as an indicator parameter by Cd stress, with higher benefits when most activity occurs in the root system, as occurred in CCN-10
SCA-6. High content of Cd can cause serious ultrastructural changes in progenies of ‘Catongo’
‘Catongo’, which can lead to death, like damage to the nucleolus. The increase in the expression of SODcyt, GPX, PCS and MET genes were associated with defense mechanisms against the harmful action of metal. Regarding the toxicity of Cd, CCN-10
SCA-6 has been observed to be more tolerant than ‘Catongo’
‘Catongo’.

Acknowledgments We thank MS. Thaíse Almeida of the electron microscopy center-UESC. A. V. Castro was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) (28007018003P6) the second senior author gratefully acknowledges the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (28007018003P6), Brazil for the concession of A scholarship of scientific productivity.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.02. 003.

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