Interactive effects of aluminum and cadmium on phenolic compounds, antioxidant enzyme activity and oxidative stress in blueberry (Vaccinium corymbosum L.) plantlets cultivated in vitro

Ecotoxicology and Environmental Safety 150 (2018) 320–326

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Interactive effects of aluminum and cadmium on phenolic compounds, antioxidant enzyme activity and oxidative stress in blueberry (Vaccinium corymbosum L.) plantlets cultivated in vitro

T

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K. Manquián-Cerdaa, , E. Crucesb, M. Escudeya,c, G. Zúñigaa, R. Calderónd a

Facultad de Química y Biología, Universidad de Santiago de Chile, Av. B. O′Higgins, 3363 Santiago, Chile Centro Interactivo de Biología y Química Aplicada (CIBQA), Universidad Bernardo O′Higgins, General Gana 1780, 8370854 Santiago, Chile c Center for the Development of Nanoscience and Nanotechnology, CEDENNA, 9170124 Santiago, Chile d Centro de Investigación en Recursos Naturales y Sustentabilidad, Universidad Bernardo O′Higgins, Fabrica 1990, Segundo Piso, Santiago, Chile b

A R T I C L E I N F O

A B S T R A C T

Keywords: Aluminum Cadmium Phenolic compounds Oxidative stress HPLC Chlorogenic acid

To evaluate the potential role of phenolic compounds in Al and Cd stress tolerance mechanisms, Vaccinium corymbosum cv. Legacy plantlets were exposed to different metal concentrations. The present study used an in vitro plant model to test the effects of the following treatments: 100 μM Al; 100 μMAl + 50 μM Cd; and 100 μMAl + 100 μM Cd during periods of 7, 14, 21 and 30 days. The oxidative damage was determined by the accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2). The antioxidant activity values were determined using 1,1-diphenyl-2-picrylhydrazine (DPPH) and the ferric reducing antioxidant power test (FRAP). Additionally, the phenolic compound concentrations were determined using HPLC-DAD. The exposure to Al and Cd increased the MDA and H2O2 contents differentially, while the antioxidant capacity values showed differences between DPPH and FRAP with the largest changes in FRAP relative to Cd. SOD had the highest activity in the first 7 days, leading to a significant increase in phenolic compounds observed after 14 days, and chlorogenic acid was the major compound identified. Our results revealed that phenolic compounds seem to play an important role in the response to ROS. Therefore, the mechanisms of tolerance to Al and Cd in V. corymbosum will be determined by the type of metal and time of exposure.

1. Introduction

superoxide anion conversion to H2O2. Different studies have shown that the SOD activity depends on both the plant species and the type of stress to which it is subjected (Posmyka et al., 2009; Zouari et al., 2016). For example, decreases in the activity of some antioxidant enzymes in the presence of elements such as Cd have been observed (Fidalgo et al., 2011; He et al., 2011; Huang et al., 2005). Cd and Al are among the elements that cause high toxicity in plants, Cd due to its high mobility in natural systems and Al because of its high concentrations in acid soils, limiting the productivity of crops (Sanita di Toppi and Gabbrielli, 1999; Khan et al., 2017). As result of regular agricultural practices, both Al and Cd can be simultaneously present in the soil solution, causing oxidative stress resulting from the generation of ROS. In plants, exposure to cadmium induces phytotoxicity symptoms, such as chlorosis, reduction of biomass, inhibition of root elongation and even cell death (Lux et al., 2011; Rodojčić Redovniković et al., 2017). It has been observed that the presence of Cd indirectly generates free radicals by substituting for Fe in several proteins, increasing the free content of Fe, resulting in the

The accumulation of phenolic compounds in plants is a response to stress caused by toxic elements, either through the reduction of oxidative stress or the ability to chelate metal ions (Michalak, 2010). The antioxidant activity of phenolic compounds is determined by their chemical structure, type, position, and number of functional groups, which finally determine their electron-donating activity, deprotonation equilibrium and radical stability (Cao et al., 1997; Kanski et al., 2002; Cheng et al., 2002). Through their functional groups, both hydroxyl (-OH) and carboxylic acid (-COOH), phenolic compounds are able to bind metals, reducing their harmful effects on a plant (Winkel-Shirley, 2002; Michalak, 2010). Plants exposed to heavy metal stress have been observed to exude high levels of phenolic compounds (Tolrà et al., 2005; Kováčik et al., 2008, 2010; Mongkhonsin et al., 2016). The action of phenolic compounds complements the activity of antioxidant enzymes, the main ROS-scavenging mechanism, especially the superoxide dismutase (SOD) enzyme, which prevents cell damage by catalyzing the

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Corresponding authors. E-mail address: [email protected] (K. Manquián-Cerda).

https://doi.org/10.1016/j.ecoenv.2017.12.050 Received 26 September 2017; Received in revised form 22 December 2017; Accepted 24 December 2017 Available online 04 January 2018 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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(3) pH = 5.2 and 100 µM of Al and 50 µM Cd (Al100/Cd50); and (4) pH = 5.2 and 100 µM of Al and 100 µM Cd (Al100/Cd100). The Al and Cd concentrations were supplied from 0.01 M AlCl3 and CdCl2·2H2O stock solutions. The effects of Al and Cd in V. corymbosum were measured at 7, 14, 21 and 30 days. We used a randomized design with three replicates, and an experimental unit corresponded to 6 clones of blueberry plantlets (6 plantlets: 1 sample).

production of hydroxyl radicals through the Fenton reaction, which finally damages plant tissues (Liu et al., 2007; Cuypers et al., 2011). A recent study from our laboratory suggests that blueberry plantlets produce phenolic compounds with reducing capacity as a protective mechanism against stress caused by Cd (Manquián-Cerda et al., 2016). In the case of aluminum, it has been reported that micromolar concentrations in the soil solution can inhibit root elongation, a consequence that can affect the incorporation of water and nutrients (Barceló and Poschenrieder, 2002; Giannakoula et al., 2010) and increase the production of reactive oxygen species (ROS) (Bontempo et al., 2013; Feng et al., 2013; Zhao et al., 2017). It is estimated that Al3+ induces oxidative stress due to its high affinity for ligands with phosphate and carboxylic groups that possess donor oxygen atoms that bind with the phospholipid membrane causing its rigidification (Ryan et al., 2001). In Rumex acetosa L., Al induced high levels of phenolic compounds (i.e., chlorogenic acid, catechin, and rutin) in shoots that may bind Al, decreasing its toxicity (Tolrá et al., 2005). Vaccinium corymbosum L. is a species with nutraceutical properties due to its high content of antioxidant molecules in its fruits and leaves (Giovanelli and Buratti, 2009; Dragović-Uzelac et al., 2010). It grows mainly in acid soils with a pH between 4.8 and 5.5, which are usually found in Chilean volcanic ash-derived soils. However, among the problems presented by growing this crop in acidic soils, such as those of volcanic origin, is the low concentration of exchange bases (Ca2+, Mg2+, K+, Na+), high rainfall and the use of fertilizers (urea and ammonium phosphate), causing soil acidification, which increases the phytotoxicity of aluminum (Mora et al., 2009). On the other hand, phosphate rock and triple super phosphate are intensively used as fertilizers abd provide Cd (up to 288 mg per kg of P) (Bonomelli et al., 2002; Molina et al., 2009). Cadmium accumulates in agricultural soils because, in general, the inputs (fertilization, irrigation water and atmospheric deposition) to the soil are larger than the outputs (plant uptake and leaching) from this medium to the biosphere. For example, studies in highly intensive maize (Zea mays L.) production systems indicate that the use of phosphate fertilizers over extended periods of time could increase the Cd levels in maize-cultivated soils (Molina et al., 2010). This manifests itself in the first half of the growing season during which the Cd absorption rate is higher than that during the rest of the season (Milone et al., 2003). In the case of blueberries, it has been observed that although they have a lower requirement than most crops, the use of phosphate fertilizers is commonly employed (Bryla and Strik, 2015). Thus, plants growing in the presence of elements such as Al and Cd should regulate the synthesis, accumulation, and profile of secondary metabolites that are produced as a result of the stress induced by these elements (Hawrylak et al., 2007; Nasim and Dhir, 2010, Okem et al., 2015). Therefore, to determine the effects of Al and Cd, i.e., two metals present in acid soils, V. corymbosum cv. Legacy was used as a test subject. The oxidative stress induced in the plants by the metals was measured by the accumulation of malondialdehyde and hydrogen peroxide. Along with the measurement of the antioxidant activity using the DPPH and FRAP assays, the changes in the profiles of phenolic compounds were determined. The purpose of this work was to identify the importance and possible detoxification role of phenolic compounds in the management of oxidative stress in V. corymbosum cultivated in vitro and exposed to Al and Cd for up to a maximum of 30 days.

2.2. Chemical speciation of the Lloyd-McCown nutrient medium The chemical speciation of the Lloyd-McCown nutrient medium (Lloyd and McCown, 1980) was carried out with the computer program Geochem-PC (Parker et al., 1995). The computed speciation considered not only the nutrient medium composition but also the different levels of Al and Cd used in the study and the potential presence of short chain organic acids exuded by plants roots, specifically citric and oxalic acids, which have been reported as the most significant species in in vitro cultures of V. corymbosum (Suzuki et al., 1999). 2.3. Determination of lipid peroxidation and hydrogen peroxide (H2O2) Fresh materials were used in the determinations of thiobarbituric acid reacting substance (TBARS) and H2O2. The lipid peroxidation level was determined in terms of the malondialdehyde (MDA) concentration according to the Heath and Packer (1968) method as modified by Du and Bramlage (1992). The content of MDA was determined using an extinction coefficient of 155 nmol L−1 cm and was expressed as nmol g−1 DW. The hydrogen peroxide content was determined using an RQflex 10 Plus Reflectoquant analyzer (Merck) and applying a sensitivity range between 0.2–20 mg L−1. The results were expressed in mg g−1 of Dry Weight (DW). 2.4. Superoxide dismutase (SOD) activity To determine the enzymatic activity, 0.1 g of fresh leaves were homogenized with 2 mL of 50 mM pH 7.0 potassium phosphate buffer. The samples were centrifuged at 4 °C for 15 min at 12,000 g, and the supernatant was used for the determination of this activity. The activity of superoxide dismutase (SOD; EC. 1.15.1.11) was determined by measuring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm. One SOD unit was defined as the amount of enzyme corresponding to 50% inhibition of the NBT reduction, and the SOD activity was calculated on a protein basis (Donahue et al., 1997). The protein content of the enzyme extracts was determined using the method described by Bradford (1976). The protein concentration was calculated using a calibration curve made with bovine serum albumin (BSA). 2.5. Extract preparation The preparation of the extracts for the determination of the antioxidant activity, total phenol content and HPLC analyses was performed as described by Adam et al. (2009) and as modified by Manquián-Cerda et al. (2016). 2.6. Antioxidant activity

2. Materials and methods 2.6.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenger spectrophotometric assay The free radical scavenger capacity of each extract was evaluated using the radical DPPH assay (Brand-Williams et al., 1995) as modified by Manquián-Cerda et al. (2016). The radical DPPH• has an absorption band at 517 nm. The results are expressed as % of consumed DPPH.

2.1. Growth conditions and treatments The growth of the blueberry plantlets followed the procedure described by Manquián-Cerda et al. (2016). These treatments were considered: (1) pH = 5.2 (Control); (2) pH = 5.2 and 100 µM of Al (Al100); 321

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Cd (p ≤ 0.05). A significant decrease in the DPPH values was observed in the blueberry plantlets treated only with aluminum (Al100) from the second week of study onward (Fig. 2). The measurement of the reducing capacity of the ethanolic extracts expressed as FRAP values showed that the Al100 treatment results decreased with respect to the control from 14 days onward (Fig. 2). The application of Al + Cd increased the reducing capacity of the ethanolic extracts with respect to the control, especially in the Al100/Cd100 treatment, and its greatest increase occurred at 21 days. At 30 days, it was observed that the FRAP values decreased, although they remained significantly different from the control.

2.6.2. Ferric reducing antioxidant power (FRAP) The FRAP activity of the extracts was measured according to the previously described method of Benzie and Strain (1996) as modified by Manquián-Cerda et al. (2016). This assay measures the ability of the extract to reduce Fe3+ to Fe2+ using the blue complex formed with tripyridyltriazine (TPTZ) and measured at 593 nm. The results are expressed in µmol of ascorbic acid equivalents (AAE) g−1 DW. 2.7. Total phenolic content (TPC) The total phenolic content was measured at 765 nm according to the Folin-Ciocalteu method as proposed by Singleton and Rossi (1965) using gallic acid as the standard. The reaction was measured spectrophotometrically using an Agilent 8453 UV–VIS spectrophotometer (Palo Alto, CA, USA). The results are expressed as milligrams of gallic acid equivalent (GAE) g−1 DW.

3.3. Total phenolic content (TPC) and identification of the HPLC-DAD phenolic compounds The total phenolic compound values are shown in Fig. 3. Compared to the control, the Al100 treatments increased their TPC concentration only during the first seven days, and from the second week onward, decreases of the TPC levels were observed (p ≤ 0.05). The different doses of Al + Cd significantly increased their TPC concentrations (p ≤ 0.05) from the second week of exposure onward. The levels of TPC remained constant until the end of the experiment, and there were no significant differences among the combined treatments. The HPLC-DAD analyses of the ethanolic extracts showed that the predominant compound in the controls was chlorogenic acid (Fig. 3b), and it can reach approximately 80% of the total of the phenolic compounds in V. corymbosum (Ribera et al., 2010; Manquián-Cerda et al., 2016). It was also possible to identify compounds such as ellagic acid and gallic acid in significantly lower concentrations (p ≤ 0.05) compared to chlorogenic acid. (Fig. 3c, d). Regarding the individual quantitative analyses of gallic, chlorogenic and ellagic phenolic acids, which are three of the most important phenolic compounds due to their high antioxidant capacity, in general, the increases in these compounds were related to the increases of the doses of Al and Cd. The combination of the metals was the factor that initiated the highest concentrations of these compounds throughout the study. In the case of chlorogenic acid, an increase in the concentration occurred especially in the second and third weeks of the study (up to a 2-fold increase with respect to the control value). The concentration of ellagic acid increased from the second week onward, maintaining a constant value (~ 0.9 mg g−1 DW) until the end of the experiment, while the gallic acid concentration increased in all treatments after 7 days. The combined treatments ofAl + Cd presented the highest levels of gallic acid, especially the treatment of Al100/Cd100, in which, after 14 days, its value increased up to 5-fold compared to the control value.

2.8. HPLC-DAD analysis of the extracts High-performance liquid chromatography equipped with a diode array detector (HPLC-DAD) was used to separate and determine the phenolic compounds in the ethanolic extracts of blueberry tissues. The ethanolic extracts were passed through a 0.45-μm membrane filter and analyzed by HPLC-DAD. An Agilent HPLC-DAD 1100 series chromatograph equipped with an RP-C18 column at 25 °C was used. The mobile phase was the gradient type containing acetonitrile (A) and 1% phosphoric acid (B) with the following program: time = 0–5 min 10% of A; 5–8 min 25% of A; 8–15 min 35% of A; 15–17 min 60% of A; 17–20 min 35% of A; and 20–30 min 10% of A. The initial pressure was approximately 120 bar, flow 1 mL/min and an injection volume of 20 μL using a Reodyne valve. UV-detection was performed at 254, 280, 314 and 340 nm. 2.9. Statistical analysis The effects of the factors (time and concentration) were determined using two-way analysis of variance (two-way ANOVA). The ANOVA assumptions of the homogeneity of the variances and normal distributions were examined using the Levene and Shapiro-Wilk W-tests. Comparisons among the mean treatments were performed using Tukey's multiple comparison test at p < 0.05. Each measurement was performed in sextuple for all evaluated parameters. 3. Results 3.1. Oxidative stress indicator In the first week no significant differences in the MDA content were observed in relation to the control. From the second week onward, a substantial increase in the oxidative stress was observed, especially in the Al100 treatment (approximately 3-fold with respect to the control value) (Fig. 1a). The treatments of Al + Cd showed an increasing accumulation of MDA from the second week of treatment onward, reaching their highest values at 30 days of treatment (p ≤ 0.05). The hydrogen peroxide content (Fig. 1b) increased from 14 days onward in all treatments with respect to the control; however, no significant differences in H2O2 accumulation were observed between the treatments. The Al100/Cd100 treatment showed the highest accumulation at 21 days.

3.4. Effect of Al and Cd on the superoxide dismutase activity During the first week of treatment, the highest SOD activities were observed, and the Al100/Cd100 treatment presenting the highest increase (approximately 2-fold compared to the control) (Fig. 4). From the second week onward, no differences were observed through the end of the study. In general, the SOD activities were slightly higher for the plants exposed to the presence of metals than those in the control. 4. Discussion The mobility and availability of Cd and Al are not only related to their total concentrations but also to the concentrations of the different species associated with the chemical speciation of both cations in the nutrient solution. This involves both organic and inorganic complexes and the presence of free and precipitated species. From the chemical speciation obtained using the GEOCHEM-PC program (Table 1), it was established that the presence of Cd resulted in an increase in the concentration of free Al (Al3+) in the solutions,

3.2. Antioxidant activity The results obtained from the DPPH assays showed that only the application of combined doses of Al + Cd increased the antioxidant capacity after the second week of treatment (Fig. 2). However, no significant differences were observed between the different doses of Al + 322

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Fig. 1. Effect of the Al and Cd concentrations on oxidative stress in V. corymbosum measured as the MDA (a) and H2O2 (b) contents at the different treatment times. Data are mean ± sd; n = 6. aDifferent lowercase letters indicate statistically significant differences (Tukey's HSD at P ≤ 0.05) between Al-Cd treatments for the same exposure time. ADifferent uppercase letters indicate other differences (Tukey's HSD at P ≤ 0.05) between exposure times for the same Al-Cd treatment.

reaching almost twice the free concentration that was observed in the absence of Cd. A short chain organic acid exudate by the roots (Tolrà et al., 2005) may act through complexation as a defensive mechanism. A significant fraction of the short chain organic acids is complexed with Al (99.8% of the citric acid and 18.4% of the oxalic acid), but this has a very low impact in relation to the total amount of Al (0.46% complexed with citric acid and 0.62% with oxalic acid at the Al levels considered in this study). The Al3+ increased because the addition of Cd resulted in the displacement of Al from complexes formed with EDTA and phosphate. As a consequence of the contamination, the blueberry plants can be stressed due to Cd and by the Cd-induced incremental release of Al3+ in the nutrient solution. The effects of Cd and Al on plants still pose a series of questions that need to be answered. In this context, our study shows that, in general, there is an increase in the oxidative stress associated with the action of Al and Cd over time. The direct action of Al on the plasmatic membrane due to its high degree of affinity for the heads of phospholipids can produce process rigidity, which has been reported in multiple studies. (Rout et al., 2001; Barceló and Poschenrieder et al., 2002; Maejima et al., 2016). Although the plasma membrane appears to be impermeable to trivalent cations, low concentrations of Al3+ can enter the cell via Ca2+

channels, inducing lipid peroxidation, and increasing the concentration of MDA (Clemens et al., 1998; Gallego et al., 2012; Zhao et al., 2017). In this respect, the chemical speciation performed on the culture medium used determined that the Al3+ increased with the addition of Cd2+ to the medium. The presence of Cd is mainly as free Cd, and it formed complexes to a lesser extent in both treatments; thus, its action would be reduced, as our results have shown. The presence of Al3+ and Cd2+ and their effects on H2O2 accumulation have been reported (Giannakoula et al., 2010; Zouari et al., 2016; Zhou et al., 2017). In this regard, our results have shown that the accumulation of H2O2 occurs at later stages (14 days), and the greatest increase was observed with the highest concentrations of Cd2+. This effect would be mediated by the fact that higher concentrations of Cd2+ may lead to the inhibition of antioxidant enzymes such as catalase (CAT) and ascorbate peroxidase (APX) through the substitution of Cd for Fe in the active center of the enzymes (He et al., 2011; Karam et al., 2017). In addition, Cd2+ can induce the activation of NADPH oxidase in the membrane, increasing the production of H2O2 involved in the processes of the lignification of the cellular wall, which could act as metal barrier. (Smeets et al., 2005; Rahoui et al., 2016). Plants can synthesize phenolic compounds as a protective mechanism against metal stress to avoid the deleterious effects these

Fig. 2. Effect of the Al and Cd concentrations on the antioxidant activity of the ethanolic extracts. (a) DPPH free radical scavenging (DPPH consumed), (b) FRAP test (ascorbic acid equivalents). Data are mean ± sd; n = 6. aDifferent lowercase letters indicate statistically significant differences (Tukey's HSD at P ≤ 0.05) between Al-Cd treatments for the same exposure time. ADifferent uppercase letters indicate other differences (Tukey's HSD at P ≤ 0.05) between exposure times for the same Al-Cd treatment.

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Fig. 3. Effect of different Al and Cd concentrations on the phenolic content variation in plantlets of V. corymbosum L. (a) TPC, (b) Chlorogenic acid concentration, (c) Gallic acid concentration, (d) Ellagic acid concentration. Values are mean ± sd; n = 6. aDifferent lowercase letters indicate statistically significant differences (Tukey's HSD at P ≤ 0.05) between AlCd treatments for the same exposure time. ADifferent uppercase letters indicate other differences (Tukey's HSD at P ≤ 0.05) between exposure times for the same Al-Cd treatment.

Table 1 Relative distribution of Al and Cd in the Woody-Plant nutritive solution for the experimental conditions considered (GEOCHEM-PC, Parker et al., 1995). Condition

Fig. 4. Total activity of superoxide dismutase (SOD) in V. corymbosum L. plantlets exposed to different Al-Cd concentrations. Data are means ± sd; = 6. aDifferent lowercase letters indicate statistically significant differences (Tukey's HSD at P ≤ 0.05) between AlCd treatments for the same exposure time. ADifferent uppercase letters indicate other differences (Tukey's HSD at P ≤ 0.05) between exposure times for the same Al-Cd treatment.

Speciation (%) 100Al

100Al/50 Cd

100Al/100 Cd

Species

Al

Al

Cd

Al

Cd

Free metal Complexed with Sulfate Borate EDTA Phosphate Cloride Nitrate Hydroxides Citrate Oxalate

0.66

1.05

8.63

1.24

20.72

2.05 0.12 51.5 44.8 0 0 0.79 0,46 0,41

3.20 0.19 23.40 69.95 0 0 1.24 0,44 0,50

4.19 0 86.23 0.07 0.79 0.08 0 0 0033

3.84 0.22 9.00 84.15 0 0 1.47 0,46 0,62

10.05 0 66.82 0.17 1.9 0.2 0 0 0,04

et al., 2011). According to our results, the amount of total phenolic compounds and their production caused by the presence of metals varied according to the treatment applied. An increase in the total phenolic compounds was observed in theAl + Cd treatments over time (Fig. 3a). It was seen that the Al treatment caused an increase in the total phenolic content during the first 7 days. After this period, the total phenolic content decreased and remained stable during the rest of the study. The effect of Al on the induction of phenolic compounds will depend on the degree of tolerance of the plant to this metal. For example, Rumex acetosa L seedlings at concentrations of 50 μM of Al showed a 50% increase in the concentration of phenolic compounds

metals may produce at a cellular level, such as membrane damage, which may disrupt the electron transport due to an induction of oxidative stress or eventually cell death (Tolrá et al., 2009, Okem et al., 2015, Zouari et al., 2016). In this case, the defense strategy against environmental stress will depend on the ontogenetic stage of development and genotypic plasticity of the plant that will ultimately determine the tolerance level (Darkó et al., 2004; Inostroza-Blancheteau 324

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(Tolrà et al., 2005). In this context, the concentration of Al3 + did not induce the synthesis of total phenolic compounds in response to stress in late stages (14 days onward), indicating that the production of these compounds would be triggered by the specific action of Cd2+ and its concentration. The identification and specific increases of some phenolic compounds (viz. chlorogenic, ellagic, and gallic acids) have been recognized by different authors (Irtelli and Navari-Izzo, 2006; Kováčik et al., 2009; Manquián-Cerda et al., 2016). These acids are found in berry fruits such as strawberry, raspberry, blackberry and blueberry (Sellappan et al., 2002; Da Silva et al., 2008; Ribera et al., 2010; Jimenez-García et al., 2013). Likewise, the concentrations of each compound are specific to each species. For example, Manquián-Cerda et al. (2016) observed that the presence of chlorogenic acid in blueberry plantlets represents approximately 80% of the total phenolic compounds in contrast to strawberry where ellagic acid may correspond to 51% of the total phenolic compounds found (Aiyer et al., 2008). This study showed that the concentrations of three phenolic acids (chlorogenic, ellagic, gallic) present in blueberry plantlets exposed to combined applications ofAl + Cd increased over time and were up to 2–3 times higher than those treated only with Al. Our results suggest that the synthesis of these compounds is conditioned by the presence of Cd and to a lesser degree by the Al concentration; thus, the defense mechanisms against metal stress will be conditioned by the type of metal, the time of exposure and the heavy metal concentration in the system (Mongkhonsin et al., 2016). The antioxidant capacity shown in blueberry plantlets appears to be mainly based on these compounds. For example, it has been observed that tomato seedlings in the presence of paraquat, a strong oxidant, showed an improved tolerance to oxidative stress due to the accumulation of chlorogenic acid (Niggeweg et al., 2004). In this sense, the increase in the levels of phenolic compounds at 14 days suggests a threshold of stress (H2O2 accumulation) at which the phenolic acids (i.e., chlorogenic and ellagic acid) are induced and act via chelation to quench the ROS. The antioxidant capacity of phenolic compounds is a consequence of their chemical stability due to their aromatic pi cloud electrons; thus, a radical electron can be delocalized (Cao et al., 1997; Michalak, 2010). In this context, the use of DPPH assays directly determines the ability of phenolic compounds to stabilize free radicals, while the FRAP assay is a complementary technique based on the redox potential of the compounds (Müller et al., 2011). The effects observed in the plantlets with Al compared to theAl + Cd treatments and their availability in the medium suggest that the non-enzymatic antioxidant defense mechanism is differentially activated according to the Al3+ concentration. The tolerance exhibited by cv. Legacy at moderate concentrations of Al (100 µM) (Reyes-Díaz et al., 2011; Manquián et al., 2013) was diminished with the addition of Cd2+ because the increase of Al3+ generates an antioxidant response and an increase in the DPPH values. Recently, studies of the effects of Cd2+ on blueberry suggested that the presence of the metal induces the synthesis of phenolic compounds with reducing abilities (Manquián-Cerda et al., 2016), which was shown in our results, and the increase in the concentration of phenolic compounds was supported by the values from the FRAP. Therefore, a non-enzymatic antioxidant response would be triggered by the type and concentration of the metal. The increase in oxidative damage due to the combined effect of Al and Cd on V. corymbosum was not related to a sustained increase in the SOD since the process that regulates the activity of this enzyme occurred during the first 7 days of the experiment and acted as the first defensive line against the increase in ROS. In this regard, it has been noted that the degree of response in the enzymatic activity of SOD is determined by the usual stress conditions to which plants are subjected in their natural habitat (Rautenberger et al., 2013; Cruces et al., 2017). According to our results, the increase in the lipid peroxidation levels and the activity of SOD against the action of Al suggest that the SOD activity is mainly determined by the higher production of ROS in the

presence of this element. The action of SOD would, therefore, act in the first days of stress, whereas the detoxification processes after two weeks would be modulated mainly by the action of the phenolic compounds. 5. Conclusions In conclusion, the increase in phenolic compounds and antioxidant activity after 14 days in theAl + Cd treatments together with the weak induction of SOD suggest that ROS removal in blueberry plants is mediated mainly by phenolic compounds, specifically chlorogenic and ellagic acids. Likewise, the response to damage by Al and Cd produces reducing and antioxidant molecules. Finally, the induction of different mechanisms of response in V. corymbosum cv. Legacy plantlets to heavy metals will be conditioned by the type of metal and time of exposure. Acknowledgments This work was supported by Funding for Scientific and Technological Centers under project FB0807 (CEDENNA). We are especially grateful to M. Iumius and P. Hiticlin for technical assistance. 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