Biologia 68/2: 223—230, 2013 Section Botany DOI: 10.2478/s11756-013-0005-9
Cadmium effects on mineral nutrition of the Cd-hyperaccumulator Pfaffia glomerata Marcelo Pedrosa Gomes1,3*, Teresa Cristina Lara Lanza Sá e Melo Marques2 & Angela Maria Soares3 1
Université du Québec ` a Montréal, Institut des Sciences de l’environnement, Succ. Centre-Ville, C.P. 8888, H3C 3P8, Montréal, Québec, Canada; e-mail:
[email protected] 2 Universidade Federal de Lavras, Departamento de Ciˆencias do Solo, C.P. 3037, 37200-000, Lavras, MG, Brazil 3 Universidade Federal de Lavras, Departamento de Biologia, Setor de Fisiologia Vegetal, C.P. 3037, 37200-000, Lavras, MG, Brazil; e-mail: author:
[email protected]
Abstract: A plant’s ability to survive in a stressful environment is correlated with its nutritional status, which can be affected by cadmium (Cd) uptake. The present study evaluated the influence of Cd on the concentrations and distributions of nutrients in the roots and shoots of the Cd-hyperaccumulator Pfaffia glomerata (Sprengel) Pedersen. Plantlets were cultivated in nutrient solutions containing increasing Cd concentrations during 20 days under greenhouse conditions, and the concentrations of Cd and essential macro- (N, P, K, Ca, Mg and S) and micro- (Zn, Fe, Mn, Cu) elements in the roots and shoots were subsequently determined. Cd did not affect the plant biomass production. Cd accumulation was found to be higher in roots than in shoots, and influenced the distribution of macro and micro elements in those plants. Despite the high phytotoxicity of this element, our results indicated the existence of Cd-tolerance mechanisms in both nutrient uptake and distribution processes that enabled these plants to survive in Cd-contaminated sites. Key words: phytotoxicity; tolerance mechanisms; translocation; symptoms
Introduction The heavy metal cadmium (Cd) is highly toxic to humans and all other living organisms and it has no known biological function in aquatic or terrestrial organisms (Chen et al. 2007). Recent advances in industry and agriculture have led to increasing Cd levels in agricultural soil environments (Sarwar et al. 2010). Anthropogenic sources including phosphate fertilizers, waste water, Cd-contaminated sewage sludge and manures, metal industries, urban traffic, and cement industries have been associated with increasing environmental levels of Cd and its accumulation in plants due to its high mobility in the soil (Alloway & Steinnes, 1999; Yang et al. 2004). Heavy metals cannot be biodegraded and thus can only be extracted from contaminated sites (Ghnaya et al. 2005). Common physic-chemical techniques used to remediate soils or sediments polluted by metals are usually expensive and unsuitable for situations affecting extensive areas (Dary et al. 2010), but biotechnological approaches have received a great deal of attention in recent years. Phytoremediation (the use of plants for metal reclamation) has emerged as an environmentally friendly proposal and may be the most cost-effective * Corresponding author
c 2013 Institute of Botany, Slovak Academy of Sciences
treatment for metal-polluted soils, especially in cases of extensive contamination (Dary et al. 2010). A strong indicator of the potential of a given plant for use in phytoremediation programs is its natural occurrence in contaminated areas. Natural occurrences of species of the genus Pfaffia (Amaranthaceae) in heavy metal contaminated soil have been reported, and these species have been observed growing quite abundantly in a soil with 90 and 1,450 mg kg−1 of Cd and Zn respectively (Carneiro et al. 2002). These species can also accumulate Cd to concentrations higher than 100 mg kg−1 and is considered as Cd hyperaccumulator (Carneiro et al. 2002). P. glomerata are perennial subshrubs or shrubs usually found growing at edges of woods or rivers and in the Brazilian rupestrian field (Nascimento et al. 2007). From their tuberous roots, several economically important compounds have been isolated and identified (De Paris et al. 2000) which poses this species as has high commercial pharmaceutical value (Montanari et al. 1999). Due to the intense predatory exploitation of natural sources of this species, its cultivation have been increasingly stimulated (Montanari Jr. 1999), so that the use of P. glomerata in environmental recuperation programs could represent an interesting strategy
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224 for both environmental recover and economic development. Growth reductions in plants in response to rising levels of Cd clearly indicates its ecophysiological impact of this heavy metal (Monni et al. 2001), and reductions in assimilation capacity, water balance disturbances, and structural changes have been reported in Cd-exposed plants (Barceló & Poschenreider 1990; Lux et al. 2004; Marques et al. 2000; Wójcik et al. 2005). Negative effect of Cd on photosynthetic processes have been attributed to the ability of this metal to interfere with chlorophyll synthesis (Horváth et al. 1996; Prasad 1995), chloroplast organization (Sandalio et al. 2001), and photosynthetic electron transport by inhibiting water-splitting enzymes located at the oxidizing site of photosystem II (PSII) (Van Assche & Clijsters, 1990). The presence of Cd in the growth media may also cause nutritional disturbances directly competing with nutrient uptake and/or altering essential element distributions and other metabolic processes (G¨othberg et al. 2004; Gussarson et al. 1996; Paiva et al. 2004; Sarwar et al. 2010). Plant responses to Cd contamination are variable and species-specific (Paiva et al. 2004; Vatehová et al. 2012), and the present study investigated the effects of different Cd doses in nutrient solution on the macro- and micronutrient content of P. glomerata. Material and methods Plant growth P. glomerata seeds were germinated in Styrofoam trays in a thermostat-controlled and darkened chamber (70% relative humidity and 25 ◦C). Forty-five-day-old seedlings having similar sizes and weights were transferred to plastic beakers (6 L capacity, two plants per beaker) filled with Clark’s nutrient solution (Clark 1975). After an initial growth period of 15 days, Cd was added (as CdSO4 ) at different concentrations (0, 15, 25, 45 and 90 µmol) to the culture media of plants selected for their apparent vigor. The nutrient solutions were continuously aerated and renewed weekly. The pH of the medium was checked and adjusted on a daily basis to 5.5 ± 0.1. After 20 days of Cd-treatment, the plants were harvested and divided into root and shoot fractions, placed in paper bags and dried in an air circulation oven at 70 ◦C to a constant weight to determine biomass production. Chemical analyses Plant samples (0.1 g) were digested in 5 ml of a strong acid solution (HNO3 /HClO4 , 3:1, v/v) (Van Assche & Clijsters, 1990) and their Ca, Cd, Cu, K, Fe, Mg, Mn and Zn concentrations were determined using a flame atomic absorption spectrometer (Perkin-Elmer Analyst 400, Norwalk, CT). Nitrogen was determinate by titration after Kjeldahl digestion; sulfur by turbidimetry (Malavolta et al. 1989); and phosphorous concentrations in solution were measured by colorimetry following the phosphorus-molybdate method. Statistical analyses The results are expressed as the averages of five replicates. The data were statistically evaluated using analysis of variance run on the SAS software program (SAS Institute Ins., 1996). Regression and correlation analyses were also performed to test for relationships between the variables.
Fig. 1. Total Dry weight (DW) production and Root/Shoot DW ratio of P. glomerata plants cultivated in nutritive solution amended with 0, 15, 25, 45 and 90 µmol Cd. Vertical bars represent standard errors (n = 5).
Results and discussion Plant growth and visual symptoms Total dry weight (DW) production and root/shoot DW ratios were not statistically affected by Cd doses in solution (Fig. 1). Biomass reduction is a typical symptom of metal phytotoxicity (Barceló & Poschenreider 1990), although this negative effect was not seen in our study. The maintenance of biomass production, even when exposed to Cd levels considered phytotoxic (KabataPendias & Adriano 1995), is an indicator of the Cdtolerance of P. glomerata. Furthermore, this species occurs naturally in soils with high levels of this metal, such as in mining areas, and it has been shown to be tolerant of heavy metal stress (Carneiro et al. 2002; Skrebsky et al. 2008). Although dry matter production was not affected, plants growing in the highest Cd doses in solution showed symptoms of heavy metal phytotoxicity. At the end of the experimental period the plants were wilted and had yellowed leaves and blackened roots. Similar results were seen with Eucalyptus urophylla and E. maculate, both of which are considered to be Cd-tolerant at 90 µmol Cd (Soares et al. 2005); and visual symptoms of toxicity occurred at doses of 25 µmol Cd (Lagriffoul et al. 1998) in maize plants. According to Breckle & Kahle (1992), these symptoms may be associated with multiple deficiencies of several nutrients essential to the formation, expansion, and operation of chloroplasts and with Cd-phytotoxic effects on extensibility or synthesis of cell wall materials (Barceló & Poschenrieder 1992). Cadmium content The Cd content of root and shoots increased as the Cd doses in the nutrient solution were increased (Fig. 2). Regression analysis was used to establish the relationship between the Cd contents of the roots (r2 = 0.99) and shoots (r2 = 0.93) and Cd additions to the nutrient
Pfaffia glomerata Cd-phytoremediation ability
Fig. 2. Cd contents of P. glomerata plants cultivated in nutritive solution amended with 0, 15, 25, 45 and 90 µmol Cd. Each point is the mean of five measurements.
solution (Fig. 2). The Cd content of roots was consistently higher than in the shoots, regardless of the Cd doses in solution, and they showed sharp increases as Cd doses increased (Fig. 2). In contrast, the Cd content of shoots showed only small increases as Cd doses increased (Fig. 2). P. glomerata plants increased their natural Cd-absorption with increasing doses of Cd in nutrient solution, but allocated most of the ions to their root systems. In spite of the high Cd content in the roots, they did not show growth restrictions – suggesting that this species is heavy metal-tolerant. One of the mechanisms associated with heavy metal tolerance in plants involves decreasing metal translocation to the shoots (Shi & Cai 2009). Plants can thus avoid negative growth effects by reducing heavy metal interference of photosynthetic processes (including chlorophyll synthesis, chloroplast organization, and PSII activity) (Horváth et al. 1996; Prasad 1995; Sandalio et al. 2001). Macronutrient contents Nitrogen Nitrogen (N) is an essential macronutrient and an important component of many structural, genetic, and metabolic compounds in plants (Hassan et al. 2005). N content was higher in shoots (33.66 to 49.25 g kg−1 DW) than in roots (21.66 to 27.75 g kg−1 DW) for all Cd treatments (Fig. 3). The higher N content seen in the shoots than in the roots was probably related to the maintenance of protein synthesis, electron transference in photosynthesis, and respiration processes (Wiedenhoeft 2006). Additionally, increases in Cd accumulation in the shoots would lead to increased N contents there as N is required to synthesis Cd-detoxifying chelator molecules such as glutathione and phytochelatins (Gojon & Gaymard 2010). A positive correlation (r = 0.999, P = 0.01) was seen for shoot N content and shoot dry weigh production with higher Cd treatments, indicating the contribution of N to the maintenance of biomass increases in these treated plants. No statistical differ-
225 ences (P > 0.05) were seen between root and shoot N contents of Cd treated plants in relation to controls, regardless of the Cd doses used. Previous reports about the relationship between Cd and N accumulation in plants were contradictory. While Mitchel et al. (2000) noted that N and Cd accumulation were positively associated, Bhandal & Kuar (1992) reported the opposite results. Cd concentrations have been found to be related to decreases in nitrate reductase (NR) activity in plants, leading to decreases in photosynthetic rates and chlorophyll contents (Campbell 1999; Hernandez et al. 1997), or to increases in enzymatic break down induced by active oxygen species generated during their exposure to stress (Hassan et al. 2008). Our data, however, indicates that Cd had little or no influence on N nutrition in P. glomerata. Phosphorous Phosphate is an important element for energy transfer and protein metabolism in plants (Marschener 1995), and decreases in phosphorous (P) contents in response to Cd have been reported (Paiva et al. 2000; Paiva et al. 2004; Soares et al. 2005; Yang et al. 1996). Root P content did not statistically differ (P > 0.05) among the different Cd concentrations, except for the control, and the P content was greater in shoots (1.62 to 4.00 g kg−1 DW) than roots (1.72 to 2.28 g kg−1 DW) in all Cd treated plants (Fig. 3). Shoot P contents were higher in Cd treated plants, which could be directly related to their greater Cd contents, and phosphorous may be accumulated to promote plant growth levels that can neutralize the toxic effect of Cd by dilution (Sarwar et al. 2010). Phosphorous is involved in glutathione (GSH) biosynthesis (May et al. 1998) (a compound that is considered a precursor of phytochelatin [PC] synthesis) (Sarwar et al. 2010), and phytochelatins are known to compartmentalize Cd into vacuoles by forming Cd/PC complexes (Salt & Rauser 1995). Additionally, the higher P contents seen in shoots when Cd was present in large amounts was apparently related to the proliferation of antioxidant systems (i.e., superoxide dismutase, ascorbate peroxidase and catalase) that can mitigate oxidative stress and prevent membrane damage (Wang et al. 2009). We saw a positive correlation between P and N in roots (r = 0.976, P = 0.05) and shoots (r = 0.971, P = 0.05) at higher Cd exposures, so that N as well as P can apparently partly alleviate Cd toxicity (Sarwar et al. 2010) – and the mobilization of these elements to the shoots represents a Cd-tolerance mechanism in P. glomerata. Sulfur Sulfur (S) is an important component of amino acids and is a biologically ubiquitous element (Wiedenhoeft 2006); it is also closely linked with phytochelatin metabolism involved in heavy metal-tolerance (HartleyWhitaker et al. 2001). Glutathione (a sulfur-containing tripeptide thiol with the formula γ-glutamate-cysteineglycinesulfur) is a precursor of phytochelatins (Rosen 2002) and is a very important antioxidant involved in cellular defenses against toxicants (Scott et al. 1993). We did not find statistical differences (P > 0.05) in root
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Fig. 3. Macronutrient contents of P. glomerata plants cultivated in nutritive solution amended with 0, 15, 25, 45 and 90 µmol Cd. Vertical bars represent standard errors (n = 5).
S contents among the different Cd treatments (Fig. 3). S contents were greater in shoots that in roots in plants exposed to 15 and 45 than 90 µmol Cd. Changes in N supply affect S demands of plants, and vice versa (Matula 2004). Sulfur is an important structural component of many co-enzymes and prosthetic groups, such as the ferredoxins necessary for N assimilation (Sarwar et al. 2010). As can be seen in Fig. 3, shoot N content was lower in plants exposed to 90 mmol Cd, and the positive correlation (r = 0.965, P = 0.05) found between shoot N and S contents could explain their lower S content. Gomes et al. (2011) reported decreases in the S translocation indexes of Salix humboldtiana plants grown in soils contaminated by heavy metals. According to these authors, these translocation indexes can explain the differences in S content in the shoots, as heavy metals appear to affect S translo-
cation from the roots to the shoots but not S uptake. Potassium The potassium (K) contents of roots were lower in plants exposed to 25 and 45 µmol Cd than in control plants, while shoot K content was higher in the 25 µmol Cd treatment than under control conditions (Fig. 3). This data indicates that K is translocated from roots to shoots at 25 to 45 µmol Cd as Cd accumulates in shoots, leading to lower K contents in the roots. A negative correlation (r = -0.950, P = 0.05) was in fact seen between root K and shoot Cd in 45 µmol Cd treatment plants. The increase seen in the K translocated index could also explain the greater shoot K content observed in 25 µmol Cd treated plants. According to Mengel & Kirkby (1987), K is preferentially transported to the shoots and has close relationships with protein synthe-
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Fig. 4. Micronutrient contents of P. glomerata plants cultivated in nutritive solution amended with 0, 15, 25, 45 and 90 µmol Cd. Vertical bars represent standard errors (n = 5).
sis, cytokinin supplies and plant growth, and also serves as a dominant cation for counterbalancing anions in plants (Marschner 1995). Disturbances of K nutrition with Cd exposure have been reported in several studies (Ghnaya et al. 2005; Zornoza et al. 2002). Changes in shoot translocation of K and other nutrients have also been attributed to vascular system alterations and reductions in the numbers and diameters of xylem elements (Ouzounidou et al. 1994). Calcium and Magnesium No statistical differences between root and shoot calcium (Ca) and magnesium (Mg) contents (P > 0.05) were observed at different Cd concentrations, with shoot concentrations being consistently higher than in roots (Fig. 3). Maintenance of Ca and Mg uptake and distribution may be a tolerance strategy of P. glomerata plants. Studies have shown that Ca can aid in alleviating Cd toxicity and that Cd competes for Ca channels in plants (Clemens et al. 1998; Nelson 1986). The alleviation of Cd toxicity by Ca may be due to the fact that plasma membrane surfaces are usually negatively charged, and high concentrations of Ca2+ would tend to neutralize them and thus minimize the toxic effect of Cd; likewise, high Ca concentrations near ion channels might decrease the influx of Cd (Sarwar et al. 2010). Heavy metals are known to interfere in chlorophyll biosynthesis (Cagno et al. 1999; Chugh & Sawhney 1999; Horváth et al. 1996), and as magnesium serves as the central atom of the chlorophyll molecule and as a co-factor in many enzymes activating phosphorylation processes (Tu & Ma 2005), maintenance of
uptake and Mg transport to shoots may help ensure the maintenance of chlorophyll biosynthesis and avoid further damage to the photosynthetic system. Micronutrient contents Zinc Zinc (Zn) is an important micronutrient essential for plant growth and development as a component of enzyme structures and regulatory transcriptional proteins (Valle & Falchuk, 1993) Zn content was observed to be higher in roots (79.14 to 102.90 mg kg−1 DW) than in shoots (39.24 to 63.60 mg kg−1 DW) in all Cd treatments (Fig. 4). Zn is important in alleviating Cd-phytotoxicity as this heavy metal induces rapid free radical synthesis (ROS) (Ercal et al. 2001; Kawano et al. 2001) that can be reduced by Zn (Aravind et al. 2009). As such, the maintenance of higher Zn levels in roots (where Cd was preferably allocated) will contribute to minimizing toxic Cd effects. As reported above, Zn content was lower in the shoots of all Cd-treated plantlets as compared to control plants (Fig. 4). Phosphorous-zinc interactions have been described in the literature (Cakmak & Marschner 1986; Webb & Loneragan 1988) and, according to Cakmak & Marschener (1986), Zn deficiencies in leaves enhance P uptake rates by roots and its translocation to the shoots. As such, the greater shoot P contents of Cd-treated plants would be associated with their lower shoot Zn contents. The negative correlation observed between Zn and P contents in the shoots of Cd-treated plants (data not showed), especially at 25 mmol Cd L−1
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228 (r = −0.990, P = 0.01), would explain the greater shoot P content in these plants (4.00 mg kg−1 DW). Iron Iron (Fe) is an important plant modulator of redox potentials and a component of two major groups of proteins – heme and Fe-S proteins (R¨omheld & Nikolic 2007). Fe contents were higher in roots (1,228.31 to 2,454.75 mg kg−1 DW) than shoots (61.94 to 116.68 mg kg−1 DW) in all Cd treatments, although root Fe contents did not statically differ (P > 0.05) between Cd-treated and control plants (except at 25 µmol Cd) (Fig. 4). Fe contents were lower in Cd-treated shoots than in control plants (Fig. 4) however, Shao et al. (2007) showed that Cd-induced oxidative stress in rice is alleviated by Fe. Fe is an integral cofactor of antioxidant enzymes such as catalase and ascorbate peroxidase (Sharma et al. 2004). As such, the maintenance of high levels of Fe in the roots when Cd concentrations were highest can increase the activity of these enzymes, and would be an important defense mechanism against ROS generated by Cd-stress (Wang et al. 2013). Our data suggests that Cd did not affect Fe uptake by P. glomerata roots, and that the differences seen in shoot Fe contents could be explained by Cd interference in the translocation of this micronutrients to the shoots (as was reported by Sandalio et al. 2001). Cd can inhibit the translocation of Fe into the aerial portion of a plant by different mechanisms, such as the radial movement of Fe in the roots, Fe loading into xylem vessels, or its absorption by the shoots (Sandalio et al. 2001). Fe translocation is also dependent on the production of phytochelatins that have variable affinities for different metals, and Cd could affect Fe translocation to the shoots (Sandalio et al. 2001). The high Cd contents seen in roots may lead to increased productions of phytochelatins that could sequester Fe as well as Cd in the roots. Manganese Manganese (Mn) is involved in many biochemical functions, as an activator of the enzymes involved in respiration, in redox reactions within the photosynthetic electron transport system, in the Hill reactions in chloroplasts, in amino acid and lignin synthesis, and in regulating hormone concentrations (Humphries et al. 2007). Mn concentrations were higher in shoots (73.20 to 78.89 mg kg−1 DW) than roots (33.63 to 19.86 mg kg−1 DW) with all Cd-treatments (Fig. 4). Mn translocation to shoots may represent a tolerance mechanism that alleviates Cd toxicity effects on the photosynthetic apparatus, as Cd can replace the central Mg2+ ion in the chlorophyll structure and Mn can partially restore these damaged chloroplast structures (Baszynski et al. 1980). Mn content was lower in Cd-treated than in the roots and shoots of control plants as the availability of Mn is known to be partly decreased by Cd (Sarwar et al. 2010). According to Baszynski et al. (1980), Cd and Mn compete for the same membrane transporters, which may have contributed to the lower Mn contents seen in Cd-treated plants. A positive correlation (r = 0.979, P =0.05) between root Mn and shoot N contents was seen
at 90 µmol Cd. According to Balang et al. (2006), Mn is related to NO− 3 uptake, so that root Mn content may improve N nutrition under conditions of Cd toxicity and therefore reduce Cd-phytotoxicity effects. Copper Copper (Cu) contents were higher in roots (18.10 to 42.63 mg kg−1 DW) than shoots (6.81 to 12.37 mg kg−1 DW) under all Cd treatments, and root Cu contents were greater in Cd-treated than in control plants (except at 15 µmol Cd) (Fig. 4). We noted a decrease in Cu content in the shoots in relation to control plants, but this was only statistically significant at 15 µmol Cd (Fig. 4). According to Obata & Umebayashi (1997), Cd increases Cu uptake but restricts its transport to shoots, which was confirmed in the present study. In conclusion, we observed that Cd did not influence the growth of Pfaffia glomerata seedlings. Despite the high phytotoxicity of this element, our results indicated the existence of Cd-tolerance mechanisms in both nutrient uptake and distribution processes that enabled these plants to survive in Cd-contaminated sites. Plants exposed to very high Cd concentrations, however, demonstrated marked symptoms of intoxication that may be related to the developmental age of plants or to other tolerance mechanisms – aspects that will require more detailed investigations.
Acknowledgements Authors are grateful to the Funda¸ca ˜o de Amparo a ` Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support.
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