Abstracts

RESEARCH ARTICLE

Effect of cadmium on growth, micronutrient concentration, and δ-aminolevulinic acid dehydratase and acid phosphatase activities in plants of Pfaffia glomerata

Efeito do cádmio no crescimento, concentração de micronutrientes e atividades da desidratase do ácido δ-aminolevulínico (ALA-D) e fosfatase ácida (AP) em plantas de Pfaffia glomerata

Etiane C. Skrebsky^I,III; Luciane A. Tabaldi^I,III; Luciane B. Pereira^II,IV; Renata Rauber^I; Joseila Maldaner^I,III; Denise Cargnelutti^II,IV; Jamile F. Gonçalves^I,III; Gabriel Y. Castro^I; Maria R.C. ShetingerII,V,^* * , ** Corresponding authors: respectively mariashetinger@gmail.com and ftnicoloso@yahoo.com. ; Fernando T. NicolosoI,III,^** * , ** Corresponding authors: respectively mariashetinger@gmail.com and ftnicoloso@yahoo.com.

^IDepartamento de Biologia

^IIDepartamento de Química

^IIIPrograma de Pós-Graduação em Agronomia

^IVPrograma de Pós-Graduação em Bioquímica Toxicologia. Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brasil

ABSTRACT

Pfaffia glomerata (Spreng.) Pedersen plantlets were grown under different cadmium (Cd) concentrations (0, 20, 40, 60 and 80 μM) in a hydroponic system during 7 d. Plant growth, micronutrient, chlorophyll and carotenoid concentrations, as well as δ-aminolevulinic acid dehydratase (ALA-D; E.C.4.2.1.24) and acid phosphatase (AP; E.C.3.1.3.2) activities were then analysed. Cadmium concentration in both shoots and roots increased with increasing external Cd levels. Metal concentration was on average 12-fold greater in root than in shoot tissues. Root length was unaffected by Cd treatments. In contrast, dry weight of both shoot and roots increased significantly upon addition of 20 and 40 μM Cd. Moreover, shoot and total plant dry weight was only reduced in plants treated with 80 μM Cd. Conversely, root dry weight decreased significantly upon addition of Cd concentrations above 40 μM. A micronutrient- and organ-dependent response to Cd toxicity was observed. Zinc and Cu concentrations in both shoot and roots did not alter upon treatment with Cd. Cadmium stress reduced Mn uptake but not its translocation within the plant. A synergistic effect of Cd on Fe concentration in root at 20 μM and 80 μM Cd levels was observed. The activity of AP, and especially that of ALA-D, was reduced with increasing Cd levels. At those these Cd levels, chlorophyll concentration was also reduced. There was a positive correlation between concentrations of carotenoids and chlorophylls. Our results indicate that P. glomerata seems to have some degree of Cd tolerance.

Key Words: Brazilian ginseng, carotenoid, chlorophyll, heavy metal, micronutrient, phytoremediation.

RESUMO

Plântulas de Pfaffia glomerata (Spreng.) Pedersen foram cultivadas em cinco níveis (0, 20, 40, 60 e 80 μM) de cádmio (Cd) em um sistema hidropônico durante 7 d, visando-se analisar o crescimento, as concentrações de micronutrientes, clorofilas e carotenóides, bem como as atividades da desidratase do ácido δ-aminolevulínico (ALA-D; E.C.4.2.1.24) e fosfatase ácida (AP; E.C.3.1.3.2) nas plantas. A concentração de Cd, na parte aérea e raízes, aumentou com o incremento dos níveis de Cd. A concentração de Cd nas raízes foi, em média, 12 vezes maior do que na parte aérea. O comprimento das raízes não foi afetado pelos tratamentos de Cd. Em contraste, a biomassa seca da parte aérea e raízes aumentou significativamente pela adição de 20 e 40 μM Cd. Além disso, a biomassa seca total das plantas somente diminuiu no nível de 80 μM Cd em relação à das plantas-controle. Por outro lado, a biomassa radicular diminuiu significativamente pela adição de Cd a níveis superiores a 40 μM. Constatou-se resposta dependente do órgão e do micronutriente à toxicidade de Cd. As concentrações de Zn e Cu na parte aérea e raízes não foram alteradas pela presença de Cd. A absorção de Mn foi diminuída pelo estresse de Cd, porém sua translocação não foi alterada. Um efeito sinergístico do Cd na concentração de Fe nas raízes foi observado nos níveis de 20 e 80 μM Cd. As atividades da ALA-D e AP foram diminuídas com o incremento dos níveis de Cd, porém a ALA-D foi mais afetada. Naquelas concentrações de Cd, a concentração de clorofila também foi diminuída. Houve uma correlação positiva entre as concentrações de carotenóides e clorofila. Os resultados indicam que a P. glomerata parece ter algum grau de tolerância ao Cd.

Palavras-Chave: carotenóides, clorofilas, fitorremediação, ginseng brasileiro, micronutrientes, metal pesado.

INTRODUCTION

Heavy metal pollution is of considerable importance and relevant to the present scenario due to the increasing levels of pollution and its obvious impact on human health through the food chain (Hadjiliadis, 1997; Almeida et al., 2007).

It is known that unfavourable effects of heavy metals on plants are manifested, among others, by inhibiting the normal uptake and utilization of mineral nutrients (Jiang et al., 2004; Dong et al., 2006). Cadmium (Cd), for instance, can interfere with mineral nutrition by hampering the uptake and translocation of essential elements (Boussama et al., 1999; Jiang et al., 2004; Dong et al., 2006). Moreover, Cd can inhibit photosynthesis and plant growth (Gallego et al., 1996), in addition to affecting the overall cell metabolism via alterations in (i) the behaviour of key enzymes of important pathways (Verma and Dubey, 2001), (ii) membrane composition and function (Fodor et al., 1995; Quariti et al., 1997) and (iii) by lowering the control of the cell redox state, which ultimately causes oxidative stress (Gratão et al., 2005). Acid phosphatases, AP (ortophosphoric-monoester phosphohydrolases; E.C.3.1.3.2) are widely distributed in plants and significantly differ from their susceptibility to inhibition by various compounds (Penheiter et al., 1997). Acid phosphatases nonspecifically catalyze the hydrolysis of a variety of phosphate esters in an acidic environment (Duff et al., 1994). These enzymes are proposed to function in the maintenance of the phosphorus status of the plant, particularly with respect to a role in accessing phosphorus from the soil (Duff et al., 1994). Several factors have shown to influence AP activity (Duff et al., 1994), but heavy metals effects on AP are poorly understood (Tabaldi et al., 2007).

The enzyme δ-aminolevulinic acid dehydratase (ALA-D; E.C.4.2.1.24), which catalyzes the asymmetric condensation of two molecules of δ-aminolevulinic acid to porphobilinogen, is sensitive to metals due to its sulphydrylic nature (Pereira et al., 2006). The synthesis of porphobilinogen promotes the formation of porphyrins, hemes and chlorophylls, which are essential for adequate aerobic metabolism and for photosynthesis (Jaffe et al., 2000). Furthermore, altered ALA-D activity concomitant with reduced chlorophyll contents has been reported in many terrestrial plants exposed to various metals (Pereira et al., 2006).

The mechanism of action of heavy metals lies in their ability to form strong bonds with bases and phosphates of nucleic acids and with –SH groups of proteins, modifying both their structure and function. They compete with other divalent cations such as Ca, Zn and Mg replacing them in their physiological roles (Pauza et al., 2005; Tabaldi et al., 2007).

The genus Pfaffia belongs to the Amaranthaceae family and it has about 90 species distributed in Central and South America. In Brazil, 27 species have been described (Taniguchi et al., 1997). The roots of Pfaffia glomerata are of special interest due to their popular use as anti-tumoral, anti-diabetic and aphrodisiac tonic (Montanari et al., 1999). Because of such uses and the form of its roots, which are similar to the Asian ginseng, its common name is Brazilian ginseng (Montanari et al., 1999). In a recent study, Carneiro et al. (2002) showed that an undetermined species of the genus Pfaffia exhibited high tolerance to soil contamination, growing quite abundantly in a soil mix with 90 and 1,450 mg kg^-1 of Cd and Zn, respectively. Moreover, this species showed a Cd content higher than 100 mg kg^-1, being considered a Cd hiperacumulator, and possibly contributing to phytoremediation of sites contaminated with heavy metals.

Taking into account these characteristics and the high commercial value of P. glomerata to the pharmaceutical industry, it is important to determine wheter this species accumulates and is tolerant to Cd. If there is a tolerance or an accumulation it is of interest, what mechanisms are involved. The present work was therefore designed to analyze the growth, micronutrient, chlorophyll and carotenoid concentrations, as well as ALA-D and acid phosphatase activies in Pfaffia glomerata (Spreng.) Pedersen plantlets during an extended 7-d period of exposure to different Cd concentrations.

MATERIAL AND METHODS

Experimental design: Tissue culture plantlets of P. glomerata (accession JB/UFSM) were obtained from the Ginseng Germplasm program, Universidade Federal de Santa Maria, State of Rio Grande do Sul, Brazil. Nodal segments (1.0 cm long) were micropropagated in Murashige and Skoog (1962) medium, supplemented with 30 g L^-1 of sucrose, 0.1 g L^-1 of myo-inositol and 6 g L^-1 of agar, according to the protocol established by Nicoloso et al. (2001).

Twenty-five-d-old plantlets from in vitro culture and after 3 d of acclimatization, following the protocol established by Skrebsky et al. (2006), were transferred into plastic boxes (10 L) filled with full nutrient solution of low ionic strength under aeration. The nutrient solution had the following composition (mg L^-1): 85.31 of N; 7.54 of P; 11.54 of S; 97.64 of Ca; 23.68 of Mg; 104.75 of K; 176.76 of Cl; 0.27 of B; 0.05 of Mo; 0.01 of Ni; 0.13 of Zn; 0.03 of Cu; 0.11 of Mn and 2.68 of Fe. The solution pH was adjusted daily to 5.8 ± 0.1 by titration with HCl or NaOH solutions (0.1 M). On the 14^th day after transplanting, cadmium as CdCl₂.H₂O was added to each container to form five concentrations: 0 (control), 20, 40, 60 or 80 μM. The nutrient solution in the growth containers was renewed once a week. Both in vitro and ex vitro cultured plants were grown in a growth chamber at 25 ± 1ºC during 16/8-h light/dark cycle with 35 μmol m^-2 s^-1 of irradiance. Cadmium-treated plantlets remained in each solution for 7 d. Three replicates with 54 plantlets were used for each treatment.

Growth analysis: At harvest, plants were divided into shoots and roots. Roots were rinsed twice with fresh aliquots of distilled water. Subsequently, growth and biochemical parameters were determined. Length of roots was determined according to Tennant (1975), and the length of sprouts was measured with a ruler. To obtain dry weight, the plants were left at 65°C until reaching a constant weight.

Cadmium and micronutrient concentrations: Approximately 0.2 g of roots and shoots were digested with 4 mL HNO₃ using the following stages of heating: a) 50°C for 1 h; b) 80°C for 1 h; and c) 120°C for 1 h in a digester block (Velp, Italy). The samples were then diluted to 50 mL with deionized water. Concentrations of Cd, Zn, Mn, Fe, and Cu were successively measured by atomic absorption spectroscopy (Iyengar et al., 1997).

Acid phosphatase (AP; E.C. 3.1.3.2) activity: Fresh root and shoot extracts were centrifuged at 43200 g for 30 min at 4ºC and the supernatant used for enzyme assay. Acid phosphatase activity was determined according to Tabaldi et al. (2007) in a reaction medium consisting of 3.5 mM sodium azide, 2.5 mM calcium chloride, 100 mM citrate buffer, pH 5.5, in a final volume of 200 μL. A 20 μL aliquot of the enzyme preparation (10-20 μg protein) was added to the reaction mixture and preincubated for 10 min at 35°C. The reaction was started by the addition of substrate and stopped by the addition of 200 μL of 10% trichloroacetic acid (TCA) to a final concentration of 5%. Inorganic phosphate (Pi) was measured at 630 nm using malachite green as the colorimetric reagent and KH₂PO₄ as the standard for the calibration curve. Controls were run to correct for nonenzymatic hydrolysis by adding enzyme preparation after TCA addition. Enzyme specific activities are reported as nmol Pi released min^-1 mg^-1 protein. All assays were performed in triplicate using PPi as substrate at a final concentration of 3.0 mM.

δ-aminolevulinic acid dehydratase (ALA-D; E.C. 4.2.1.24) activity: Shoot tissue was homogenized in 10 mM Tris-HCl buffer, pH 9.0 (1:1, w/v). The homogenate was centrifuged at 12000 g at 4°C for 10 min to yield a supernatant (S1) that was used for the enzyme assay. The S1 was pre-treated with 0.1% Triton X-100 and 0.5 mM DTT. The ALA-D activity was assayed as described by Barbosa et al. (1998) by measuring the rate of porphobilinogen (PBG) formation. The incubation medium for the assays contained 100 mM Tris-HCl buffer, pH 9.0. For the enzyme assay, the final concentration of ALA was 3.6 mM. Incubation was started by adding 100 μL of the tissue preparation to a final volume of 400 μL. The product of the reaction was determined with the Ehrlich reagent at 555 nm using a molar absorption coefficient of 6.1×10⁴ L mol^-1cm^–1 (Sassa, 1982) for the Ehrlich-porphobilinogen salt. Activity of ALA-D was expressed as nmol PBG mg^-1 protein h^-1.

Protein extraction: In all the enzyme preparations, protein was determined by the method of Bradford (1976) using BSA as standard and was expressed in mg mL^-1.

Chlorophyll and carotenoid concentrations: Chlorophyll and carotenoids were extracted following the method of Hiscox and Israelstam (1979) and estimated following Arnon (1949). Briefly, chopped fresh shoot sample (0.1g) was incubated at 65°C in dimethylsulfoxide (DMSO) until tissues were completely bleached. Absorbance of the solution was then measured at 470, 645, and 663 nm in order to determine the concentrations of carotenoids, chlorophyll a, and chlorophyll b, respectively. Chlorophyll and carotenoid concentrations were expressed as mg g^-1 fresh weight.

Statistical analysis: The analyses of variance were computed for statistical significance based on the appropriate F-tests. The results are the means ± SD of at least three independent replicates. Significance was determined at P < 0.05. The mean differences were compared utilizing Duncan's multiple range test.

RESULTS

Cadmium concentration under Cd exposure: Cd concentration in both shoots (Figure 1A) and roots (Figure 1B) increased with increasing Cd levels. It is noteworthy that external Cd concentrations ranging from 20 to 60 μM brought about the same enhancement of Cd concentration in both shoots and roots (36 and 12.5-fold greater than controls, respectively). Cadmium concentration in roots was on average 12-fold greater than in the shoot. The maximum concentration of Cd in shoot and roots was 345 mg kg^-1 DW and 3400 mg kg^-1 DW, at the 80 μM Cd level, respectively.

Characterisation of Cd tolerance: There was no general pattern of plant growth responses to Cd stress. Number of leaves per plant was slightly, but not significantly, reduced by Cd concentrations up to 40 μM, whereas at the 80 μM Cd level it was reduced by 26% compared to control plants (Figure 2A). In addition, length of sprouts (Figure 2B) and length of the root system (Figure 2C) per plant were unaffected by Cd. On the other hand, the root length/shoot length ratio increased significantly at the 20 μM Cd level and this ratio was slightly, but not significantly, increased upon addition of Cd exceeding 20 μM, when compared to control plants (Figure 2D).

Shoot fresh weight was reduced significantly only upon adding 80 μM of Cd (Figure 3A). In contrast, dry weight of both shoot (Figure 3B) and roots (Figure 3C) increased significantly upon addition of 20 and 40 μM Cd. Moreover, shoot and total plant weight were reduced only at the 80 μM Cd level as compared with controls (Figure 3B and 3D, respectively). Conversely, root dry weight decreased significantly upon addition of Cd at levels exceeding 40 μM (Figure 3C).

Effect of Cd on micronutrient concentrations: A nutrient- and organ-dependent response to Cd toxicity was observed (Table 1). Zinc remained unaltered after applying Cd treatments. Conversely, in roots, Mn concentration was reduced significantly over all Cd levels added, Fe concentration increased significantly upon addition of 20 and 80 μM Cd, whereas Zn and Cu levels did not respond to the treatments.

Acid phosphatase activity (AP): Acid phosphatase activity was reduced in shoot (23%) and roots (30%) with increasing Cd levels. No difference in AP activity in both shoot and roots was found for Cd treatments ranging from 20 to 80 μM (Figure 4).

ALA-D activity and concentrations of chlorophyll and carotenoids: Shoot ALA-D activity decreased with increasing Cd levels in the nutrient solution (Figure 5A). A maximum of 89% depletion in ALA-D activity was found at 80 μM Cd. Total chlorophylls were also reduced but only at the 20 and 80 μM Cd levels (Figure 5B). On the other hand, carotenoid concentration was significantly reduced upon addition of 20, 60 and 80 μM Cd (Figure 5C).

DISCUSSION

Cadmium concentration and plant tolerance to Cd: Our data (Figure 1) demonstrate that higher metal exposures led to remarkable Cd accumulation in both root and shoot tissues, hence leading to a high degree of toxicity, which is in agreement with results reported by other authors (e.g., Carneiro et al., 2002; Lima et al., 2006; Mishra et al. 2006). Meanwhile, no significant difference in both shoot and root Cd concentrations was found between the 20 to 60 μM Cd treatments. We also checked Cd concentration in the nutrient solution at 0 and 7 d after applying the treatments, and it was found that external Cd was not significantly depleted during the experiment (data not shown). These data suggest that, up to a certain level of metal concentration, roots of P. glomerata have some mechanism to avoid excess of Cd uptake. Cadmium confinement in the root tissues may be due to an efficient binding and sequestration to the vacuoles by glutathione and phytochelatins, or by imobilization of Cd by cell wall and extracellular carbohydrates (Lima et al., 2006; Mishra et al., 2006, Almeida et al., 2007).

Our data demonstrate that the root length/shoot length ratio increased after treatment with all studied Cd concentrations (Figure 2D), which is not consistent with the results of Guo and Marschner (1995), who reported that usually the inhibition of root elongation of different plant species is the most sensitive parameter of Cd toxicity. In addition, our results also indicate that root elongation was much less affected than the decrease in biomass, which is not consistent with the earlier results of Lima et al. (2006) for Pisum sativum and of Meuwly and Rauser (1992) for Zea mays. According to Meuwly and Rauser (1992), since most of the root elongation is located in the first 10 mm of the root apex, the contribution of the biomass to this portion is probably too small to allow the detection of any toxic symptoms during the first days, but length inhibitions are enough to be detected. Therefore, since root biomass of P. glomerata was significantly reduced upon addition of Cd levels exceeding 40 μM, this result suggests that P. glomerata has some degree of Cd tolerance. Meanwhile, there was an increase in both root and shoot biomass (Figure 3) at lower Cd levels (20 and 40 μM). It should be emphasized that experiments of the present study were carried out three times (with some modifications), and results were almost the same for most parameters analyzed including the increased biomass we found (data not shown). The positive effects of low levels of Cd on plant growth have been poorly discussed in the literature, and the mechanisms are not well understood. This phenomenon is normally related to a so-called hormetic effect that probably represents an "overcompensation" response to a disruption in the homeostasis of the organism (Aina et al., 2007). Khan et al. (2008) observed similar phenomena in sand culture where 10 mM Cd enhanced the activities of leaf superoxide dismutase, ascorbate peroxidase, glutathione reductase and carbonic anhydrase, net photosynthetic rate and plant dry mass of Triticum aestivum at low Zn level. These authors suggested that the synergies among the activities of antioxidative enzymes helped to maintain carbonic anhydrase and thus photosynthesis and plant biomass at low Cd levels under low Zn concentration. Taking into account the fact that the total dry biomass of P. glomerata was only significantly reduced at the 80 μM Cd level and such a concentration is within that observed in highly polluted soils, these results further indicate that P. glomerata seems to have some degree of Cd tolerance, as was found for an undetermined species belonging to the genus Pfaffia, reported by Carneiro et al. (2002). However, additional experiments should be performed in order to allow a better understanding of the mechanism of the effect of Cd toxicity on growth, photosynthesis and antioxidative mechanisms of P. glomerata.

Nutrients concentration under different degrees of Cd stress: It is well known that many toxic effects of Cd action result from interaction with micronutrients, in particular those with the same valence as Cd, such as Zn, Mn, Fe, and Cu. Our results showed that Zn and Cu concentrations in both shoot and roots remained unaltered upon Cd addition. Both synergistic and antagonistic effects of Cd on Zn and Cu were found in other studies (Jiang et al., 2004; Dong et al., 2006). Our data indicate that Mn uptake was affected by Cd stress but not the translocation of Mn within P. glomerata plants. An inhibition in Mn uptake and transport by Cd has been reported by Dong et al. (2006) for Lycopersicon esculentum. In contrast, an increase in Mn uptake and translocation to the shoots was observed in Lactuca sp. exposed to Cd stress and there was a higher Mn accumulation in chloroplasts when Cd was present in the growth medium (Ramos et al., 2002). Some studies showed that there is an antagonistic relationship between Cd and Fe (Sharma et al., 2004). Arabidopsis plants that overexpressed the IRT1 gene, a major transporter responsible for high-affinity iron uptake from the soil, accumulated higher levels of Cd and Zn than the wild type, indicating that IRT1 is responsible for the uptake of these metals (Connolly et al., 2002). Our data demonstrate that a synergistic effect of Cd on Fe concentration in root at the 20 and 80 μM Cd concentrations is likely to occur. Some of these conflicting results found in our study in relation to others might be presumably due to the differences in the culture methods, species, as well as growth conditions including Cd and micronutrient levels in medium, growth period, temperature and light.

Metabolic enzymes, and concentration of chlorophyll and carotenoid in different degrees of Cd stress: Our results showed that under Cd stress, AP activity in both shoot and roots was similarly reduced regardless of the amount of Cd added. Conversely, Tabaldi et al. (2007) found that Cd, Mn and Na did not significantly alter the AP activity in Cucumis sativus. Phosphatases are generally metalloenzymes depending on Ca²⁺ or Mg²⁺. A possible mechanism explaining Cd-toxicity at high concentrations can be the replacement of Ca²⁺ and Mg²⁺ by Cd in the active site of enzyme, or the Cd can be interfering with the PO₄^3- binding sites. Other metals such as Hg and Zn also inhibited AP activity of cucumber, possibly through this mechanism (Tabaldi et al., 2007). Therefore, inhibition of AP activity in P. glomerata caused by Cd stress may impair phosphate mobilization, since this enzyme is involved in P metabolism, an essential element for plant growth and development (Duff et al., 1994).

Altered ALA-D activity concomitantly with reduced chlorophyll contents has been reported in many terrestrial plants exposed to various metals (Pereira et al., 2006). Our data also showed that total chlorophyll concentration was reduced significantly at 20 and 80 μM Cd (Figure 5B), at which greater reduction in ALA-D activity was observed. Moreover, there was a significant positive correlation (r²= 0.73) between concentrations of carotenoids and chlorophylls. Carotenoids play a pivotal role in photoprotection of chlorophylls against photooxidative damage by quenching reactive oxygen species (ROS) such as singlet oxygen (Behera et al., 2002). Similarly, other authors have found a decrease in carotenoid content in Cd-treated plants, which might be interpreted as an overproduction of ROS (Mishra et al., 2006). In addition, ALA-D inhibition could have led to ALA accumulation that within the cell might contribute to enhance ROS production (Noriega et al., 2006). Therefore, in future studies it will be necessary to analyze the effects of Cd on ROS formation and on both enzymatic and non-enzymatic antioxidant systems. These defence systems can remove, neutralise or scavenge oxy-radicals and their intermediates (Gratão et al., 2005).

Acknowledgements: The authors wish to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação e Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) for the research fellowships.

Received: 05 April 2008; Returned for revision: 17 December 2008; Accepted: 20 Febrary 2009

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