Journal of Inorganic Biochemistry 134 (2014) 39–48

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Differential effects of aluminum on in vitro primary root growth, nutrient content and phospholipase C activity in coffee seedlings (Coffea arabica) Jesús E. de A. Bojórquez-Quintal a,1, Lucila A. Sánchez-Cach a,1, Ángela Ku-González a, Cesar de los Santos-Briones b, María de Fátima Medina-Lara a, Ileana Echevarría-Machado a, José A. Muñoz-Sánchez a, S.M. Teresa Hernández Sotomayor a, Manuel Martínez Estévez a,⁎ a b

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Calle 43 # 130, Col. Chuburná de Hidalgo, 97200 Mérida, Yucatán, Mexico Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Calle 43 # 130, Col. Chuburná de Hidalgo, 97200 Mérida, Yucatán, Mexico

a r t i c l e

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Article history: Received 12 August 2013 Received in revised form 21 January 2014 Accepted 23 January 2014 Available online 3 February 2014 Keywords: Aluminum PLC activity Coffee Primary root growth Nutrient contents

a b s t r a c t Coffea arabica is a woody species that grows in acid soils, where aluminum is available and may affect growth and productivity. To determine the effect of aluminum on primary root growth of C. arabica cv. Typica, seedlings were exposed over 30 days to different concentrations of AlCl3 (0, 100, 300 and 500 μM) in vitro. The aluminum effect on primary root growth was dose-dependent: low aluminum concentrations (100 and 300 μM) stimulated primary root growth (6.98 ± 0.15 and 6.45 ± 0.17 cm, respectively) compared to the control (0 μM; 5.24 ± 0.17 cm), while high concentrations (500 μM) induced damage to the root tips and inhibition of primary root growth (2.96 ± 0.28 cm). Aluminum (100 μM) also increased the K and Ca contents around 33% and 35% in the coffee roots. It is possible that aluminum toxicity resides in its association with cell nuclei in the meristematic region of the root. Additionally, after 30 days of treatment with aluminum, two different effects could be observed on phospholipase C (PLC) activity. In shoots, aluminum concentrations ≥300 μM inhibited more than 50% of PLC activity. In contrast, in roots a contrasting behavior was determined: low (100 μM) and toxic concentrations (500 μM) increased the activity of PLC (100%). These results suggest the possible involvement of the phosphoinositide signal transduction pathway, with the phospholipase C enzyme participating in the beneficial and toxic effects of aluminum in plants. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Acid soils limit crop production worldwide and constrain the growth of edible plants in many developing countries. These soils are characterized by having a pH of 5.5 or below and are widely distributed in tropical and subtropical areas, representing between 30 and 40% of the arable soils in the world and approximately 70% of the potentially arable soils [1,3]. Aluminum (Al) toxicity is considered the main factor limiting agricultural production in these soils. Aluminum is the most abundant metal and the third most abundant element in earth's crust, after oxygen and silicon, representing approximately 8% of content by weight [4]. Al is found naturally in an insoluble state as aluminosilicates and aluminum oxides, but in aqueous solution, this ion hydrolyzes water molecules to form hydroxide, a process that depends on the pH of the soil [2,5]. The concentration of Al in acid soils can vary in a range from micromolar (μM) to millimolar (mM) [6]. At pH 4.3, trivalent Al (Al3+) is the most available form of this metal and the most toxic to plants [5]. Al3+ affects plants by inhibiting the growth of their roots. Generally, the root apex is the most sensitive ⁎ Corresponding author. Tel.: +52 999 9428330; fax: +52 999 9813900. E-mail address: [email protected] (M.M. Estévez). 1 Both authors contributed equally to this work. 0162-0134/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2014.01.018

zone because it is in direct contact with Al [7]. In fact, the inhibition of root growth has been widely used to evaluate the toxicity of Al [8]. The initial site of Al attachment is most likely the cell wall; this binding is due to the high affinity for the negatively charged carboxyl groups of pectins [9,10]. High Al accumulation causes rigidity, low extensibility of the cell wall, and callous accumulation [11]. Additionally, Al induces structural changes at the level of the plasma membrane that affect the fluidity and integrity of the membrane, the membrane potential, and the absorption of K and Ca [12,13]. Al has other effects at the cellular, tissue and organ levels, interfering with a wide range of physiological and molecular processes. This metal causes cytoskeletal structure disruption, lipid peroxidation, and enzymatic disorders, interferes with signal transduction mechanisms, and inhibits DNA synthesis by binding to this macromolecule [2,14–17]. Conversely, Al has a beneficial effect in some taxa at low concentrations, especially in those species that are endemic to tropical and subtropical regions that can challenge and even thrive in acidic soil conditions [18]. The growth of these species can be stimulated by Al [18], and some members of the families Rubiaceae and Melastomaceae can accumulate high concentrations of Al in their leaves [19–21]. The beneficial effects of Al include the stimulation of root growth [22,23] and the prevention of the toxic effects of excess protons, iron and phosphorus [18,24,25]. In addition, this metal can enhance nutrient uptake [22,26],

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induce antioxidant systems [23] and has also been attributed a role as an insecticide [27]. Melastoma malabathricum, Camellia sinensis and Quercus serrata are woody species that grow in tropical regions, and the growth of their roots has been reported to be stimulated by low concentrations of Al, and a metal hyperaccumulation in their leaves [22,23,26]. Furthermore, it is known that the woody species are relatively more tolerant to Al than agricultural species [26,28]. Coffee is one of the most important agricultural products in the international market and one of the largest tropical crops cultivated in the world. It is a member of the family Rubiaceae and belongs to the genus Coffea. This genus includes about a hundred species; Coffea arabica L. and Coffea canephora P. are the most commercially important. Seventy percent of coffee grown is arabica, and 30% is robusta (C. canephora) [29]. Coffee is a plant, which due to its agronomic requirements, grows in acid soils with high organic matter and is thus affected by the presence of Al. In the past two decades, attention has been given to the effect of Al on coffee; studies primarily addressing the physiological and biochemical processes have been conducted at the cellular and whole plant levels. The growth of cell suspensions of C. arabica was shown to be inhibited by 50% and 90% at 25 μM and 100 μM of Al, respectively [30]. Additionally, in cell suspensions, the Al toxicity has been associated with the signal transduction pathway mediated by phosphoinositides, specifically a reduction of the activity of phospholipase C (PLC), thus inhibiting the formation of inositol-1,4,5-triphosphate (IP 3 ) [30,32]. PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce IP3 and diacylglycerol (DAG) [33,34]. In plants, PLC is involved in physiological processes, such as growth, development and differentiation, due to its role in the regulation of Ca, in kinase enzyme activation and the regulation of cation and anion channels [35]. Additionally, PLC has been associated with DNA synthesis and signaling processes in response to biotic and abiotic stresses [36–38]. Al also inhibits the formation of phosphatidic acid (PA) by blocking or inhibiting the PLC activity in coffee cell suspensions. PA is a lipid second messenger as the IP3 that regulates many signaling processes involved in physiological responses to stress [39]. Furthermore, studies to select genotypes tolerant to the effects of Al have been performed using coffee seedlings grown in nutrient solutions in a greenhouse, mainly by evaluating the root growth. These studies classified the genotypes of C. arabica and C. canephora as tolerant, moderately tolerant, moderately susceptible (intermediate sensitivity) and sensitive to the toxic effect of Al, with the genotypes of C. arabica more tolerant than the corresponding genotypes of C. canephora [40,41,44,47]. Surprisingly, some tolerant genotypes had an increase in root growth at low concentrations of Al [42,43,47]. Other studies have reported both the stimulation and the inhibition of root growth in coffee [46,48]. It has been reported that coffee, low Al concentrations can stimulate early plant development without causing toxic effects by acting directly on the primary root elongation [47,49]. Despite the numerous studies on Al and coffee, it is unclear what the effect of Al is on the root growth of this species. Therefore, the objective of this work was to determine the effect of different concentrations of Al on the growth of the primary root (PR) of C. arabica seedlings in an in vitro model, using morphological and biochemical studies. We report a differential effect on the PR growth of coffee that is dependent on the concentration of Al, and we demonstrated the possible involvement of the phosphoinositide signal transduction pathway.

of Arabidopsis thaliana (Col-0) and the commercial seeds of Capsicum chinense Jacq. ‘Orange’ (Seminis Vegetable Seeds, Inc.). Coffee seeds were disinfected in a solution of 30% (v/v) sodium hypochlorite (Cloralex, 5% NaOCl) for 2 h and 70% ethanol (v/v) for 5 min; after both steps, washes with sterile water were performed. Seeds were stratified in sterile water for 48–72 h until the removal of the zygotic embryos. A. thaliana seeds were surface sterilized with 25% (v/v) sodium hypochlorite (Cloralex, 5% NaOCl) and 5 μL of Triton X-100 and were subsequently stratified by incubating for three days at 4 °C in 0.15% soft agar (w/v). C. chinense seeds were rinsed in 80% ethanol (v/v) for 5 min, and washes were performed with sterile distilled water. The seeds were then incubated with 30% (v/v) sodium hypochlorite (Cloralex, 5% NaOCl) and Tween (1 drop) for 15 min, and washed and soaked with sterile distilled water (dd) for 48 h at 4 °C under dark conditions. 2.2. Germination and growth conditions The embryos of C. arabica were extracted by carefully cutting the endosperm of seeds with a scalpel to locate the embryo. Zygotic embryos were then placed in petri dishes with B5 medium [50] at half its ionic strength (B5/2) at pH 5.8 with 3% sucrose (w/v) to obtain seedlings. The embryos were kept in darkness for eight days at 25 °C, and the petri dishes were placed in a vertical position to allow the growth of the PR. Subsequently, the embryos were transferred to photoperiod conditions (16 h light/8 h dark). A. thaliana seeds were germinated in B5/2 media under the same conditions as the embryos of C. arabica. C. chinense seeds were incubated in petri dishes with sterile water-moistened filter paper in the dark until radicle emergence and then transferred to B5/2 medium under the same conditions of germination for C. arabica and A. thaliana. 2.3. Treatments with aluminum and primary root growth To evaluate the effects of Al, C. arabica seedlings with a 1.6–1.8 cmlong PR were transferred to petri dishes containing B5/2 medium at pH 4.3. The culture medium in the dish was divided into two segments separated by a distance of approximately 3–5 mm; AlCl3 was not added to the top segment, while the bottom segment contained different concentrations of AlCl3 (0, 100, 300, 500 μM). The seedlings were placed on the surface of the agar, so that approximately 2–3 mm from the apex was in contact with the medium that contained the Al. Treatments were incubated in a photoperiod of 16 h light/8 h dark at 25 °C. The PR length was measured during 30 days of treatment with Al at fiveday intervals. Three seedlings were placed on petri dishes, and 15 seedlings were used per treatment. Treatment without AlCl3 in the top and bottom segments was used as control. To evaluate the effects of Al on A. thaliana, seedlings with a 1.9– 2.4 cm-long PR were used. Root growth was measured at day 0 (start of experiment) and on day 5 of treatment. For C. chinense, seedlings with an average PR length of 1.6–1.8 cm were transferred to Al treatment, and root growth was measured on day 0 and daily until day 7 of the treatment, when the root of the seedlings reached the edge of the petri dish. The experiment in both species was conducted in segmented petri dishes, in similar conditions to C. arabica. The PR length was measured with a millimeter ruler from end of experiment. Additionally, the images of roots were taken with Leica IM50 software MZFLIII coupled to a DFC320 camera.

2. Materials and methods

2.4. Scanning electron microscopy (SEM)

2.1. Plant material

C. arabica seedlings treated with Al or untreated for 30 days were used for SEM studies. Seedlings were washed with 0.5 mM CaCl2 and then fixed in FAA (50% formaldehyde, 30% ethanol and 5% acetic acid, v/v/v). After fixed, they were dehydrated in a gradient of 50, 70, 80, 90 and

We used certified seeds of C. arabica cv. Typica, which were donated by Mr. Jose Roberto Martinez Villegas. Furthermore, we used the seeds

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96% ethanol (v/v) at 60-min intervals and then immersed in absolute ethanol three times at intervals of 60 min. Subsequently, PR tips were excised and dried at critical point in a dryer (Tousimis Samdri-795®, Maryland, USA.), exchanging ethanol with liquid CO2 and then with gaseous CO2. Finally, the samples were mounted on carbon adhesive film and coated with gold (21-nm particles) using a metallizer (Denton Vacuum Desk II®, South Carolina, USA). Metallized samples were observed on a scanning electron microscope (JEOL model JSM-6360LV®, Tokyo, Japan). The figures are representative images of three roots per treatment. 2.5. Histochemical analysis of Al on roots To assess the localization and accumulation of Al in the roots of C. arabica, plant roots were stained with hematoxylin and morin. For hematoxylin staining, the protocol of Polle et al. [51] was followed. In short, control and AlCl3 treated seedling roots were transferred to 10-mL test tubes after 30 days and were washed with distilled water. The hematoxylin solution [0.2% hematoxylin in 0.02% potassium iodide (KI), w/v] was added, and samples were kept under constant stirring for 12 h in darkness. The dye was decanted, and several washings were performed with distilled water to remove excess hematoxylin. Finally, the root tips were cut and observed under a Leica stereoscope with MZFLIII IM50 DFC software coupled to a 320 camera. At least three roots per treatment were stained. To visualize the accumulation of Al in root tips, morin dye was employed [52]. In short, PRs of control and Al treated seedlings (30 days of treatment) were washed with distilled water and a solution of 0.5 mM CaCl2. A solution of morin (50 μM) was added, and the roots were incubated for 60 min in the dark and then washed with 0.5 mM CaCl2 to remove excess dye. Subsequently, PR tips were cut and mounted on slides using 10 μL of Vectashield (Vector Labs) for observation. The fluorescence of the Al–morin complex was observed with a FluoViewTM FV1000 confocal microscope (Olympus, Tokyo, Japan). The configuration of the microscope was as follows: Lens UPLFLN 40 × 0 (oil, NA: 1.3), scan speed: 2 μs/pixel, an excitation laser at 405 nm to 5% (Al–morin complex has a wavelength of excitation and emission of 420 and 515 nm, respectively). Images were collected at 1.0 μm Z position, and approximately 30 sections of 512 × 512 pixels per image were obtained; all sections were projected into a single image using the FV10ASW software version 3.01b. Additionally, C. arabica roots (30 days of treatment with 500 μM of AlCl3) were stained with morin (Sigma), Vectashield-DAPI (Vector Labs) for nuclei staining and FM4-64 (Invitrogen) for membrane staining. A sequential scanning in two phases was performed to avoid background. We used a 40 × 0 UPLFLN lens (oil, NA: 1.3), scan rate of 10 μs/pixel, a 405-nm excitation laser for morin and DAPI, and a 543-nm excitation laser for FM4-64. Morin, DAPI and FM4-64 have wavelengths of excitation and emission of 420,515; 358,461; and 515,670 nm, respectively. All images have a resolution of 512 × 512 pixels, and sections in Z for each fluorophore were projected into a single image. A merge of the three fluorophores was created with FV10-ASW software 3.01b. 2.6. Mineral analysis in shoots and roots Seedlings were harvested at the end of treatment and were washed with distilled and deionized water and then separated into shoots and roots. To determine the content of Al, K and Ca, each sample (control and Al treatments) was dried in an electric oven at 60 °C for 72 h; they were then weighed and stored for subsequent analysis. For the preparation, a known weight of the sample was used, and the digestion was conducted in a microwave after adding HNO3:H2O2 at a ratio 5:1. The digestion program used was as follows: a run of 1200 W, with a ramp of 15 min at 200 °C, 10 min at 200 °C, and 5 min at 170 °C. After the digestion, the samples were adjusted to a volume of 25 mL with

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water (Milli Q) and Al content was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES; Thermo IRIS Intrepid II XDL, New York, USA) [53]. Standard curves were used for each element. Experiment was replicated three folds, and 15 seedlings were used for each treatment. 2.7. Protein extraction and PLC activity Roots and shoots of coffee seedlings either untreated or treated with AlCl3 (100, 300 and 500 mM) were rapidly frozen in liquid nitrogen and macerated and homogenized with a polytron in buffer A (1 g tissue in 2.5 mL). Buffer A contained 50 mM NaCl, 1 mM ethylene glycol tetraacetic acid (EGTA), 50 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium pyrophosphate, and 0.2 mM sodium orthovanadate. The extracts were centrifuged at 13,000 ×g for 30 min at 4 °C, and the recovered supernatant was subsequently frozen in liquid nitrogen and stored at −80 °C for measurement of the activity of the PLC. The protein content of each sample was determined with bicinchoninic acid (PIERCE), using bovine serum albumin (BSA) as the standard. The hydrolysis of [3H] PIP2 was measured in a reaction mixture (50 μL) containing 35 mM NaH2PO4 (pH 6.8), 70 mM KCl, 0.8 mM EGTA, 0.8 mM CaCl2 (final concentration 25 mM), 200 μM PIP2 (approximately 333 Bq) and 0.08% deoxycholate. After incubation at 30 °C for 10 min, the reaction was stopped with 100 μL of 1% (w/v) BSA and 250 μL of 10% (w/v) trichloroacetic acid (TCA). The precipitate was removed by centrifugation (13,500 ×g for 10 min), and the supernatant was collected to quantify the [3H]-IP3 released by liquid scintillation counting (ACS by Amersham Ltd.). 2.8. Statistical analysis Data were analyzed with an analysis of variance (ANOVA) of two ways (Sigma Stat Version 3.1). The treatment means were compared using Tukey's multiple range. 3. Results 3.1. Al modified the growth of the primary root (PR) of C. arabica L. seedlings in a concentration-dependent manner C. arabica is the primary species cultivated for the production of coffee and includes a large number of varieties that differ in production, yield, quality, disease resistance and the environmental conditions in which they are cultivated. Regardless of the species or variety of coffee, these plants are normally grown in soils with pH values less than 5.5. These acidic conditions increase the availability of Al, which can provide either a toxic or a beneficial effect on the early stages of growth and root development of the plants. We previously observed an increased PR length in the presence of Al, regardless of the variety of coffee or AlCl3 concentration used [53]. In this study, we report the effect of 30 days of culture with Al (0, 100, 300 and 500 μM AlCl3) on the PR growth of C. arabica cv. Typica, specifically evaluating the root length and root apex morphology. We used an in vitro model in which only the apex was in contact with Al. Al differentially affected PR growth, and this effect was concentration dependent; Al concentrations between 100 and 300 μM stimulated PR growth, whereas higher concentrations inhibited PR length (Fig. 1A). From day 10 until the end of treatment, differences between treatments were observed. Shorter PR lengths were observed at day 20 in the presence of 500 μM AlCl3, while root length was greater at 100 and 300 μM with respect to control (0 μM) and was significant according to Tukey's test (p b 0.05) (Fig. 1B). In Fig. 1C, the increase in the length of the PR is evident at Al concentrations of 100 and 300 μM (6.98 ± 0.15 cm and 6.46 ± 0.17 cm, respectively) compared to control (5.24 ± 0.17 cm), while reduced growth (2.97 ± 0.28 cm) is observed at 500 μM AlCl3.

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Fig. 1. Al induces the growth and inhibition of the primary root (PR) in coffee seedlings. A) PR growth of C. arabica cv. Typica on day 15 and 30 in B5/2 medium at 0, 100, 300 and 500 μM of AlCl3 at pH 4.3. White arrows indicate the position of the apex of the root in the culture medium. B) Time course of the effect of AlCl3 on the PR growth of coffee seedlings. The red line indicates the control treatment (0 μM AlCl3, at pH 4.3). C) PR growth at days 0 and 30 of treatment at different concentrations of AlCl3. Points and bars represent the mean total length at different days ± SE (n = 15 seedlings). Different lowercase letters indicate significance at p b 0.001 level (Tukey's test).

3.2. Effect of aluminum on the PR in other species To corroborate the increased PR growth in coffee seedlings by Al treatments and eliminate the possibility that Al is not available in these conditions, it was suggested to use A. thaliana (sensitive) [54] and C. chinense (tolerant, unpublished data) seedlings as controls grown under the same conditions. The PR growth of A. thaliana was inhibited after five days of treatment with Al, and this inhibition was dependent on the concentration of the metal (Fig. 2A and B). The PR growth was slightly reduced at 100 μM AlCl3 (6.25 ± 0.19 cm), however, it was not statistically significant compared with the control (6.74 ± 0.18 cm; Tukey, p b 0.05). The root growth was statistically reduced at 300 μM (4.6 ± 0.36 cm) and 500 μM (2.84 ± 0.15 cm) compared to control roots. In contrast, in C. chinense, there are no significant differences between treatments at pH 4.3 in the absence or presence of AlCl3 (Fig. 2C and D). We observed that in our in vitro model, the three species had different responses to Al. In C. arabica, we observed an increase and an inhibition of PR growth; A. thaliana was sensitive, and PR inhibition was dependent on the concentration of AlCl3; C. chinense was not affected by the presence of AlCl3. These data suggest that these species utilize different mechanisms in response to the presence of Al.

3.3. Root apex morphology from C. arabica treated with aluminum The apex of the root is the most sensitive portion, and contact with Al affects its morphology [7]; it is the major site of accumulation of Al [55]. In coffee, after 30 days of treatment, root apex damage was

observed only at 500 μM AlCl3 (Fig. 3A). Images acquired by SEM revealed the detachment and disruption of the cells of the root apex at 500 μM AlCl3. In contrast, damage to the morphology of the apex PR was not observed at 100 or 300 μM Al (Fig. 3B).

3.4. Localization of aluminum in coffee roots Several methods have been used to determine the location and accumulation of Al in plant tissues, including the use of Al-specific dyes. To identify the location of Al in the roots of coffee seedlings, we used two different methods of staining: hematoxylin staining to identify Al on the root surface and morin staining to determine the subcellular localization of Al in the PR tips. Fig. S1 shows that in the control samples, Al was not detected at the root by hematoxylin staining. When the roots were exposed to 100, 300 or 500 μM of AlCl3, hematoxylin staining was observed, indicating the presence of Al on the surface of the root. To determine the localization and accumulation of Al in the PR apexes of C. arabica cv. Typica, seedlings were treated with AlCl3 for 30 days. Roots were then incubated for 60 min with the fluorochrome morin, placed on a microscope slide, and fluorescence was observed by confocal microscopy as described in Section 2.5. The green fluorescence characteristic of the Al–morin complex was not observed on the roots of control samples; in contrast, from 100 μM AlCl3, florescence intensity increased in a concentration-dependent manner (Fig. 4A). At the highest concentration of AlCl3 (500 μM), higher fluorescence intensity was observed, and a section of the PR apex indicated the presence of the Al–morin complex in and around nucleus-like structures (white arrows,

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Fig. 2. Effect of Al on primary root growth in A. thaliana and C. chinense. Total length of RP of A. thaliana at 5 days (A and B) and C. chinense after 7 days of culture (C and D) on segmented B5/2 medium at pH 4.3 with 0, 100, 300 and 500 μM AlCl3. White arrows indicate the position of the apex of the root in the culture medium (A and C). Bars represent the mean total length of the root in the different treatments ± SE (n = 14 seedlings). Different lowercase letters indicate significance at p b 0.001 level (Tukey's test).

Fig. 4A). Additionally, an association of the complex with vesicle-like structures was observed (white arrow head, Fig. 4A). To analyze the association of the AlCl3 with the nucleus, roots pretreated with 500 μM were incubated with morin (Al–morin complex), DAPI (nuclei staining) and FM4-64 (membrane staining), and the fluorescence was observed by confocal microscopy in two phases as described in Section 2.5. Fig. 4B shows the fluorescence of each dye individually or the overlap of two or three of them. The association of Al with nuclei was demonstrated by overlapping the morin and DAPI images. Fig. 4B clearly shows the presence of Al in the nuclei of the meristematic zone of the root apex. 3.5. Mineral analysis For mineral analysis, the seedlings of C. arabica treated with different concentrations of AlCl3 were harvested at day 30 of culture, and the samples were separated into shoots and roots. The content of Al, K and Ca was measured by ICP-AES (Table 1). The Al content increased in roots and shoots as the AlCl3 concentration increased. However, there is an anomaly to this behavior at 300 μM in both organs. Furthermore, we observed greater Al association with the roots than the shoots. Our results (Table 1) show an increase in the K content in the roots of C. arabica at 100 and 300 μM AlCl3, concentrations at which there was an increase in PR growth. In shoots, the K content was decremented at concentrations ≥300 μM AlCl3. At the highest concentration of AlCl3, the K content was the lowest in both roots and shoots compared with the control. Furthermore, the Ca content of C. arabica decreased after treatment with Al in both roots and shoots, and this reduction was dependent on the concentration of AlCl3, except at 100 μM, where a greater quantity of Ca was observed in both sites of seedlings (Table 1).

3.6. Aluminum effect on the activity of PLC in shoots and roots Previously, PLC enzymatic activity in coffee cell suspensions was reported to be inhibited by Al [30,32]. In this paper, we evaluated the PLC activity in coffee roots and shoots exposed for 30 days to different concentrations of AlCl3 (0, 100, 300 and 500 μM). In shoots, PLC activity was inhibited by approximately 50% at concentrations ≥300 μM AlCl3 compared with the control (Fig. 5). In the roots, PLC activity increased in the presence of Al, primarily at 100 and 500 μM AlCl3. Both concentrations show a differential effect on PR growth; at 100 μM, there is an increase in the length of the PR, and at 500 μM, PR growth decreases. Coincidentally, both concentrations showed an approximately 100% increase in PLC activity with respect to the controls. Surprisingly, PLC activity in roots treated with 300 μM AlCl3 was the same as in the control.

4. Discussion Aluminum is the most abundant metal in the earth's crust, but its availability depends on soil pH [1]. Despite its abundance and availability, Al is not considered essential, and there is no experimental evidence of a biological role [56,57]. At high concentrations, Al is toxic to plants and animals, which is why most of the research in plants has focused on its toxic effect and especially on the mechanisms of tolerance to this metal. Recent studies have reported some beneficial effects of Al, defined as a beneficial element to those elements that are not required by all plants, but can promote growth and to be essential for certain taxa under certain conditions [18]. Other beneficial elements are sodium (Na), silicon (Si), selenium (Se) and cobalt (Co) [18]. In this paper, we report the effect of Al on the growth of the PR of C. arabica cv. Typica. We observed two different responses to Al on RP

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Fig. 3. Effect of Al on the apex of the primary root (PR) of C. arabica. A) Coffee root images after 30 days of culture in 0, 100, 300 and 500 μM of AlCl3 at acidic pH. The bar corresponds to the first millimeter (mm) of the PR. B) SEM micrographs of the apexes of the PR at the different concentrations of AlCl3. The images of the apexes of PR are at day 30 of treatment with Al stress. The images are displayed on the analysis of three roots per treatment.

growth, which depended on the concentration. Low concentrations (100 and 300 μM) stimulated the growth of the PR, and high concentration (500 μM) inhibited the growth (Fig. 1). In previous studies, we found that the length of the PR for the three varieties of C. arabica was not affected in the presence of ≥100 μM concentrations AlCl3 after 20 days of culture [53]. Other authors previously observed the stimulation of the coffee root system under hydroponic conditions [42,47,49]. Furthermore, to corroborate our results on coffee in our in vitro model, we used as controls A. thaliana (Al sensitive specie) and C. chinense (sensitive species unknown) (Fig. 2). Our results in A. thaliana were similar to those reported by Illes et al. [54]; PR inhibition was concentration dependent. In C. chinense, root growth was not affected by Al. In Capsicum annuum, the destruction of the root apex, and therefore the inhibition of growth by Al, was reported [58]. Apparently, within the genus Capsicum, C. chinense is tolerant to high concentrations of AlCl3. These data showed that Al has a differential effect on the three species and that the stimulation of root growth on coffee is not a unique effect of the in vitro model. The stimulation of root growth has been observed in woody species growing in acid soils [18], which could be the case for coffee roots. Osaki et al. [22] reported the stimulation of root growth and nutrient uptake in woody species, such as M. malabathicum and Melaleuca cajuputi, when they were grown hydroponically with 600 μM Al [22]. In C. sinensis

(tea), an Al hyperaccumulating plant, the stimulation of root growth has also been observed in nutrient solution with 400–500 μM Al [23]. This effect was attributed to increased activity of the antioxidant system, perhaps as an indirect effect of Al [23]. Q. serrata, another plant adapted to acid soils, increased its root growth and nutrient uptake when it was exposed to 250 μM and up to 5 mM of Al [26]. The stimulation of root growth has been reported in other species adapted to acid soils, both in Al-accumulating and non-Al-accumulating species [26]. It has been proposed recently the term “hormesis” to describe a stimulatory effect of low concentrations of toxic elements on any organisms [59]. Studies on the germination and vigor of coffee seeds under Al stress have reported the stimulation of PR growth primarily in varieties of C. arabica [42,46,48]. It was observed that pretreatment with Al during germination increases the root length of coffee varieties [49]. These data has led one to suspect that Al can have a positive effect in the early stages of development on root growth. Other authors have suggested that the presence of Al in the root can trigger a signal to accelerate its growth; however, this hypothesis is only speculative [26]. As mentioned, the woody species adapted to acid soils are more tolerant to Al than the corresponding species of agricultural importance [60]; the presence of Al can stimulate their growth [26]. A mechanism related to the beneficial effect of this metal has been primarily

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Fig. 4. Location of Al on the apex of the primary root of C. arabica cv. Typica. A) Al–morin complex detection in the apexes of PR at different concentrations of AlCl3 after 30 days of culture. The white arrows indicate the possible location of the Al in the nucleus and other subcellular compartments at 500 μM AlCl3. The white bar represents 50 μm and 10 μm for 0–500 μM AlCl3 and zoom 500 μM, respectively. B) Al localization by fluorescence in the nucleus. Coffee roots exposed for 30 days to 500 μM AlCl3 were stained with the fluorochromes morin (Al detection), DAPI (nuclei staining) and FM4-64 (membrane staining). Images are representative of three samples per concentration of AlCl3. The white bar represents 10 μm.

attributed to limiting the toxicity of H+ [59]. Other mechanisms that have been described in which the Al may be involved directly or indirectly as a beneficial element include the avoidance of the toxicity of other elements, such as Fe [18]. Also, it has been proposed an increase in the uptake of elements such as K and Ca [22], both essential macronutrients for a plant to complete its life cycle [61]. In plants, K is the major cationic inorganic nutrient, whose function involves activation of enzymatic reactions, charge balancing and osmoregulation [62]. Cellular functions of Ca are both structural and as a secondary messenger. Removal of membrane Ca or its replacement with other cations rapidly compromises membrane integrity [61].

In Coffea roots, an increase in the content of K was observed at 100 and 300 μM AlCl3 (Table 1); coincidentally, at these concentrations, the growth of coffee roots was stimulated by the presence of Al (Fig. 1). Ca content also increased in both roots and shoots at 100 μM of AlCl3, but decreased with higher concentrations, mainly at 500 μM. These data suggest that just as reported for other woody species, in coffee the stimulation of root growth is related to an increased nutrient uptake. Although there are no biochemical or molecular evidences, we hypothesize that Al may induce the expression and/or activity of transmembrane proteins, such as pumps, channels and ion exchangers that promote the influx of nutrients. Electrophysiological studies have

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Table 1 Mineral analysis of shoots and roots of C. arabica cv. Typica after 30 days in vitro culture with different AlCl3 concentrations. Tissue/organ

Roots

Shoots

Treatment (AlCl3 μM)

Al

0 100 300 500 0 100 300 500

0.64 11.96 6.82 18.41 0.00 3.20 1.31 4.97

K

Ca

(μmol g−1 DW) ± ± ± ± ± ± ± ±

0.07a 0.13b 0.01c 0.80d 0.00a 0.24b 0.06c 0.24d

1213.91 1624.59 1522.38 1085.92 1518.09 1446.32 1271.04 1133.78

± ± ± ± ± ± ± ±

18.69b 96.33a 44.42a 49.18b 34.85a 18.21ab 11.71bc 77.89c

33.37 45.13 35.31 27.76 56.02 66.28 43.61 41.10

± ± ± ± ± ± ± ±

1.78b 2.46a 1.92b 0.77bc 2.16ab 2.57a 6.15b 1.27b

Each treatment was used 15 seedlings (three replicates each). As a control, treatment without AlCl3 was used. Elements in roots and shoots were quantified as micromoles per gram dry weight (μmol g−1 DW). The different lowercase letters in each column (roots or shoots) indicate significant differences between treatments (with or without AlCl3) at p b 0.05 level (Tukey's test).

suggested that Al provokes membrane depolarization and hyperpolarization, inducing both K efflux and influx in roots. K influx could occur through hyperpolarization-activated channels (KIRC's) [63]. Recently, Al-resistant mutants of A. thaliana (alr-140) showed an increase in Mg influx and intracellular content when exposed to 25–100 μM of Al. However, an Mg efflux was observed at high concentrations of Al (500 μM). Therefore, it has been suggested that Al can activate transporters and permeable channels for Mg in resistant genotypes [64]. Magnesium is an essential macronutrient, its involvement in preventing the toxic effect of Al has been reported [65]. In our work, K and Ca contents increased in coffee roots when exposed to low concentrations of AlCl3, which may contribute to prevent its toxic effects and/or even stimulate growth. In future studies it would be interesting to determine ion fluxes in response to Al treatment from woody plants, using coffee roots as a model. The toxicity mechanisms of Al are not clearly understood, since this metal interacts with a number of extracellular and intracellular structures. The principal symptom of Al toxicity is the inhibition of root growth and damage to the root apex. Proposed mechanisms include cell wall and cytoskeletal modifications, disruptions of the plasma membrane, ion transport processes, and signaling pathways, and the binding of Al to DNA [2].

Fig. 5. PLC activity in shoots and roots of C. arabica cv. Typica seedlings exposed to different concentrations of Al. Seedlings were grown for 30 days on agar plates containing segmented B5/2 medium and supplemented with 100, 300 and 500 μM AlCl3; medium without AlCl3 was used as control. Closed circles and open triangles represent the activity of PLC in shoots and roots, respectively. Each point represents the mean of five replicates with three seedlings per treatment. Asterisks indicate statistically significant differences with respect to control (*p b 0.05; **p b 0.005; Tukey's test).

In our results, at 500 μM AlCl3, the growth of the PR was inhibited in coffee seedlings (Fig. 1), and damage was observed in the root apex of AlCl3 at this concentration (Fig. 3). Hydroponically grown coffee showed root growth inhibition at Al concentrations greater than 700 μM; the main observations were short roots and thicker and fewer lateral roots [66]. The difference in the concentration of Al may be due to growing conditions, as well as coffee varieties and seedlings' age. In fact, there are differences in tolerance to Al among species and varieties of coffee [42,45,47]. In studies in cell suspensions of coffee, the mean lethal dose has been reported as 25 μM AlCl3 in a sensitive line and 75 μM in a tolerant line; it has also been observed that Al completely inhibited cell growth of suspensions of C. arabica when they were treated with ≥ 100 μM AlCl3 [30]. It is noteworthy that the cell suspensions can provide greater sensitivity compared to that which would be observed when using a whole organism, as these cells are undifferentiated and may be more likely to exhibit Al toxicity. Furthermore, in this type of model, the components of systemic regulation and tolerance mechanisms that may exist in a whole plant are absent. Another important point is that the plant material (callus) used to obtain cell suspensions was the cotyledonary leaves of the zygotic embryos of C. arabica cv. Catuai [67]; the variety Catuai has previously been characterized as sensitive to Al toxicity [45]. In fact, as mentioned above, the effect of aluminum in plants depends on many factors; in cell suspensions, contrasting responses were observed depending on the source of plant material (leaves or roots) [68,69]. Since the apex of the root is one of the regions most sensitive to the effects of Al in plants, we evaluated the morphology and the accumulation of Al in this area of the coffee PR. In our results, morphological damage was observed only in the root tips exposed to higher concentrations of AlCl3 (Fig. 3B), which corresponds to the growth inhibition of the coffee PR at this dose (Fig. 1). Aluminum binding to the cell wall has been suggested as one of the principal mechanisms of toxicity for this metal. Pectin-bound Ca is displaced by Al, causing rigidity in the cell wall, and hence, less cell elongation [11,70]. Interestingly, Al was present in coffee's root surface when roots exposed to the highest concentration of AlCl3 (Fig. S1), suggesting a possible interaction with the cell wall and/or plasma membrane. Furthermore, higher Al content and lower Ca and K contents were detected in roots under this condition (Table 1). Potassium is also involved in cell expansion processes, and the maintenance of a hydrated state under stress. Therefore, these factors may contribute to PR growth inhibition in coffee. Additionally, we evaluated the accumulation of Al in the apexes of the PR by fluorescent Al–morin complex formation. The fluorescence intensity increased concentration wise, and the highest concentration (500 μM) caused an accumulation of Al in the apex and the association of the Al with nucleus-type structures (Fig. 4A). The possible association of Al to the nucleus has been reported in C. arabica protoplasts [71]. Al association was found in the nucleus by the overlap of DAPI fluorescence and morin, as shown in Fig. 4B. Based on these results, we suggest that the accumulation of Al in the nucleus was possibly by metal binding to the DNA, potentially inhibiting cell division in the growth region of the apex and therefore reducing root growth at the higher Al concentrations used in this work. Previously, it has been reported that Al interferes with cell division in roots, and Al binding to the DNA in the roots of Catharanthus roseus and in cell suspensions of C. arabica has also been reported to reduce the synthesis of DNA [72–74]. In C. arabica, Al concentration in the root and shoots was significantly augmented after 30 days of treatment with different concentrations of AlCl3 (Table 1). Aluminum accumulated mainly in the roots and only small amounts were transported to the shoots. Increased association or accumulation of Al in the roots could prevent the harmful effects of this element in other plant organs; however, it also inhibited root growth as was observed at the highest concentration of AlCl3 (Fig. 1). Surprisingly, the Al content in roots and shoots did not follow a clear

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cut trend in response to applied Al doses, since these values were much lower at 300 μM than at 100 and 500 μM of AlCl3 treatments. One possible explanation may be that, particularly at this Al concentration, a greater efflux of organic acids (citrate, malate or oxalate), phenolic compounds or other substances is being released by roots. This behavior has been previously reported in others woody species, such as Populus tremula [75,76]. The efflux of these compounds may contribute to chelate exogenous Al and so, to prevent its accumulation in the root. Previously, the toxic effect of Al has been associated with the signal transduction pathway mediated by phosphoinositides, mainly by reducing the activity of the PLC. Similarly, it has been speculated that the stimulation of root growth by Al is because this metal triggers a signal transduction pathway [26]. Based on this possibility, we evaluated the effect of Al on the PLC activity in shoots and roots under the conditions described above. We observed that the effect of Al on the activity of PLC was different between roots and shoots (Fig. 5). In cell suspensions of Nicotiana tabacum, it has been found that cell cycle regulation may be in contrast depending on the stimulus and the plant material (leaves and roots) [68,69]. In shoots, PLC activity was inhibited from 300 μM AlCl3 (Fig. 5); previously, PLC activity in leaves was already observed [77]. In coffee cell suspensions (leaf callus) [67], the growth inhibition at different concentrations of AlCl3 has been associated with the inhibition of PLC activity and other enzymes and components of the signal transduction pathway, such as phospholipase D (PLD) [31,32,39]. This is consistent with our results on PLC activity observed in shoots. In roots, an increase in the activity of the PLC was observed at 100 and 500 μM AlCl3 with respect to the control, while 300 μM AlCl3 did not affect root PLC activity (Fig. 5). These results contrast with those reported in wheat roots, where Al caused a dramatic inhibition of PLC activity in a concentrationdependent manner [78]. However, these differences may be due to the Al toxicity threshold between woody and edible species, such as cereals [60,78]. Furthermore, the biphasic behavior observed in coffee roots, possibly attributable to the existence of two or more PLC isoforms, as has been suggested in different study models [31,79]. In C. roseus transformed roots, there are at least two PLC activities, one soluble and other membrane associated [80]. Furthermore, it seems that there are at least two forms of membraneassociated PLC in transformed roots of C. roseus [79]. Coffee roots may present at least two forms of PLC, which can be activated at different concentrations of AlCl3 (100 and 500 μM), as seen in Fig. 5. In fact, PLC is an enzyme involved in physiological processes, such as growth, development and differentiation, through the regulation of Ca levels, enzyme activation, DNA synthesis, both cationic and anionic channels and signaling processes as a result of biotic and abiotic stresses [35,38]. Therefore, we suggest that PLC may participate both in stimulating root growth and in its inhibition in C. arabica seedlings, depending on the plant organ and the dose of Al. In conclusion, in this paper we report a differential or biphasic effect of Al on the growth of coffee seedlings in vitro. Al concentrations between 100 and 300 μM stimulate PR growth, and higher concentrations (500 μM) inhibit it. We also report that the increase in growth could be related to a higher content of K in the roots, and the toxic effect on roots may be due to the inhibition of cell division by the accumulation of Al in the nucleus in the meristematic zone of the root apex. Moreover, we report the potential involvement of a signal transduction pathway in the root growth stimulation and inhibition by aluminum. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2014.01.018.

Acknowledgment We appreciated the technical assistance of Dra. Laura Hernandez Terrones and the grant of CONACYT (166621).

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