Soil and Biota of Serpentine: A World View 2009 Northeastern Naturalist 16(Special Issue 5):131–138 Uptake and Accumulation of Cobalt by Alyssum bracteatum, an Endemic Iranian Ni Hyperaccumulator Seyed Majid Ghaderian1,*, Mahsa Movahedi1, and Rasoul Ghasemi1 Abstract - Alyssum bracteatum is the first Ni hyperaccumulator reported from serpentine soils of western Iran. In this study, uptake and accumulation of Co by a serpentine and a non-serpentine population of this species were tested under controlled conditions. Seedlings of A. bracteatum were grown in different concentrations of Co (0, 2, 5, 10, 15, and 30 mg Co L-1) in solution culture (perlite) for 21 days. Tolerance to Co of serpentine population seedlings was significantly greater than the Co tolerance of seedlings from the non-serpentine population. Analysis of shoots and roots showed that the concentration of Co in both populations of A. bracteatum increased with increasing Co in solution culture, but amounts of Co in the shoots of non-serpentine plants were significantly less than those in serpentine plants. Plants of the serpentine population contained as much as 1830 μg Co g-1 dry weight when grown in 15 mg Co L-1 conditions, showing that this species is capable of hyperaccumulating Co under solution culture conditions. Introduction Serpentine soils exist in many parts of the world and are renowned for their specialist plant life. These soils are derived from ultramafic rocks and are characterized by high levels of Ni, Co and Cr, low levels of nutrients (such as N, P, K, Ca), and a high Mg/Ca ratio. Serpentine soils also tend to be shallow and dry (Baker et al. 2000). Due to these environmental stresses, the growth forms and physiognomy of plant species growing in these soils can differ from plant species of non-serpentine soils. Total Ni concentrations of serpentine soils are generally in the 500–8000 μg g-1range, and Ni/Co ratios in serpentine soils typically range from 5 to 10. Concentrations of Ni and Co in most serpentine plants are somewhat elevated, usually to about 10–100 and 15–50 μg g-1, respectively. A number of serpentine plants are able to accumulate extraordinary concentrations of Ni in their aboveground parts, especially the leaves. The term hyperaccumulation has been applied to plants that contain more than 1000 μg g-1 dry weight of elements such as Ni, Co, or Cr in aboveground tissues when growing in their natural habitat (Baker and Brooks 1989). So far, more than 360 Ni hyperaccumulator plant taxa have been reported from serpentine soils (Reeves and Baker 2000). We could find only one recorded case of Co hyperaccumulation on serpentine soils (Reeves 2005). Plants that hyperaccumulate Ni from serpentine soils show a wide variation in their concomitant Co-accumulating ability in their natural habitat, ranging from less than 1 μg g-1 to occasional values in the 10–100 μg g-1 range. Reeves and Baker (1984) discussed the co-tolerance of Ni hyperaccumulators to other 1Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran. *Corresponding author - ghaderian@sci.ui.ac.ir. 132 Northeastern Naturalist Vol. 16, Special Issue 5 metals, as co-tolerance has been reported for a number of heavy metal hyperaccumulators. Baker et al. (1994) reported that Thlaspi caerulescens J. & C. Presl., a hyperaccumulator of Zn, could tolerate and accumulate high levels of Co when it was supplied in the absence of other metals. In the case of Ni hyperaccumulators of the genus Alyssum, accumulation of Ni is accompanied by Co accumulation of up to 100 μg g-1. Homer et al. (1991) found that Alyssum species could accumulate and tolerate high concentrations of Co from artificial rooting media. In temperate regions, most Ni hyperaccumulators belong to the family Brassicaceae, the largest number being in the genus Alyssum, which contains the greatest number of Ni hyperaccumulators (more than 50 taxa) (Reeves and Adigüzel 2008). The distribution of hyperaccumulating Alyssum species is mostly on serpentine soils in southern Europe and Asia Minor, stretching from Portugal in the west to the Iraq/Turkey/Iran border areas in the east (Brooks 1998). Alyssum bracteatum Boiss. and Buhse is a Ni hyperaccumulator which naturally grows in serpentine soils and is endemic to Iran (Ghaderian et al. 2007). Some populations of this species are also found on non-serpentine soils. Populations on serpentine soils contain more than 1000 μg g-1 Ni in their shoots, but the amounts of Co in the same individuals are less than 50 μg g-1. It has been known for many years that Co is an essential element for humans, other animals, and prokaryotes (Marschner 1995). In algae, only one study has indicated a physiological function for Co: the enzyme that catalyses decarboxylation of a fatty aldehyde to a hydrocarbon and carbon monoxide in the green alga Botyrococcus braunii Kützing was suggested to contain a porphyrin moiety with Co in its center (Dennis and Kolattukudy 1992). There is no evidence that Co has a direct role in the metabolism or any other functions of higher plants (Marschner 1995). Normal Co concentration in plant shoots seldom reaches above 2 μg g-1 (ranging from 0.03 to 2 μg g-1) (Reeves and Baker 2000). Research on Co hyperaccumulator plants can be useful for Co phytoremediation of contaminated soils and waters, Co phytomining (Reeves and Baker 2000), and also for using plants with high Co content as feed supplements for domesticated animals (Robinson et al. 1999). There is little information about the ability of A. bracteatum to take up metals under controlled conditions. The aim of this study was to compare the ability of a serpentine and a non-serpentine population of A. bracteatum to take up and accumulate Co under controlled conditions. Methods Seeds of A. bracteatum were collected from serpentine and non-serpentine soils of western and central Iran, respectively. Seeds were kept at 4 ºC for 1 month and then sowed in pots filled with perlite. In each pot, 5–6 seeds were sowed and kept wet with distilled water for 1 week until germination. After germination, seedlings were watered with 10% strength Hoagland solution containing 0.4 mM Ca(NO3)2, 0.5 mM KNO3, 0.2 mM MgSO4, 0.1 mM KH2PO4, 10 μM FeEDDHA (Ferric ethylenediamine–di-2-hydroxyphenylacetate), 10 μM H3BO3, 2 μM MnCl2, 0.2 μM CuSO4, 0.2 μM ZnSO4, and 0.1 μM Na2MoO4. The pots were put in trays, and Hoagland solution was 2009 S.M. Ghaderian, M. Movahedi, and R. Ghasemi 133 poured into the trays. The volume of the Hoagland solution in the trays was kept constant by adding distilled water to prevent an increase in concentrations of nutrient elements in the solution due to evaporation of water. The solution in the trays was replaced every 5 days. Different concentrations (0, 2, 5, 10, 15, 30 mg L-1) of Co were achieved by adding Co(NO3)2 salt to the Hoagland solution. Treatment of plants was started 2 months after germination of seeds and continued for 3 weeks. Controlled growth conditions were 16/8 h light/dark period with 24 ºC for light periods and 18 ºC for dark periods. Light intensity was 200 μmol photon m-2 s-1. To measure Co concentration, the plants were separated into roots and shoots, then washed well with double–distilled water, and dried at 70 ºC for 48 h. Dry weights of plant materials were measured; all dried materials of shoot and root in each pot containing 4–5 plants were mixed and used as one sample for elemental measurements. Each sample was added to a 25-ml beaker and ashed in a muffle furnace for 14 h at 480 ºC. The ash was taken up in 5 ml 10% HNO3, and the digest was finally made up to 20 ml in 10% HNO3. The solutions were analyzed for elemental composition by an atomic absorption spectrophotometer (ASS, Shimadzu 6200). Statistical comparisons of serpentine and non-serpentine plants grown under the same Co concentrations used t-tests. Tukey tests were used for multiple comparisons of values from the same population exposed to different Co treatments. All statistical analyses were performed using SPSS software (version 13). Results Cobalt uptake by serpentine and non-serpentine plants of A. bracteatum is shown in Figure 1. The concentration of Co in root and shoot tissues increased as the concentration of Co increased in the medium. Serpentine plants accumulated a maximum of 2207 μg Co g-1 in shoots and 1971 μg Co g-1 in roots. Shoots and roots of non-serpentine plants had maximum concentrations of 1212 and 835 μg Co g-1, respectively. Maximum Co concentrations in roots and shoots of plants from both populations occurred when grown in 30 mg Co L-1 solutions. In all treatments (2, 5, 10, 15, and 30 mg Co L-1), concentrations of Co in the shoots of non-serpentine plants were significantly lower than those in serpentine plants (P < 0.05). High concentrations of Co in the medium (e.g., 15, 30 mg Co L-1) caused a significant decrease in dry weight of both serpentine and non-serpentine plants (Fig. 2) and led to leaf chlorosis. At a Co concentration of 30 mg L-1 in the medium, shoot relative dry weight diminished by 40% and 24% in serpentine and non-serpentine populations, respectively. Compared to controls, statistically significant decreases in relative shoot dry weight were observed at 5 mg Co L-1 for non-serpentine plants and at 10 mg Co L-1 for serpentine plants (Fig. 2). Discussion Nickel hyperaccumulating plants necessarily have very efficient mechanisms for tolerance to Ni. They have unique properties which enable them to 134 Northeastern Naturalist Vol. 16, Special Issue 5 Figure 1. Concentration of Co (mean ± SE) in: A. the shoots of serpentine and nonserpentine populations of A. bracteatum treated with different concentrations of Co in the medium. The concentrations of Co in control serpentine and non-serpentine plants were 4.3 ± 0.9 and 0.05 ± 0.01, respectively; and B. the roots of serpentine and non-serpentine populations of A. bracteatum treated with different concentrations of Co in the medium. The concentrations of Co in control serpentine and non-serpentine plants were 0.1 ± 0.03 and <0.001, respectively. Different letters in each series (lower case letters for serpentine plants and capital letters for non-serpentine plants) show statistically significant differences between treatments based on a Tukey test (P < 0.05). * indicates a significant difference between serpentine and non-serpentine plants within each treatment based on a t-test (P < 0.05). 2009 S.M. Ghaderian, M. Movahedi, and R. Ghasemi 135 hyperaccumulate Ni in their shoots. Among these properties, the presence of specific chelators and specific mechanisms for transport and compartmentation of excess concentrations of Ni are notable (Clemens et al. 2002, Krämer et al. 1996). Up to now, there was no clear report of a natural Co hyperaccumulator plant from serpentine soils, despite serpentine soils being rich in Co (Reeves and Baker 2000). The low ratio of Co/Ni, low availability of Co and high pH in serpentine soils, and absence of specific mechanisms for Co uptake and accumulation in serpentine Ni hyperaccumulators are considered to explain the absence of co-accumulation of Co and Ni in serpentine Ni hyperaccumulators (Homer et al. 1991, Li et al. 2003, Tappero et al. 2007). In experimental conditions, it has been reported that some Ni hyperaccumulator plants from the genus Alyssum can accumulate Co (Homer et al. 1991, Tappero et al. 2007), but this result might be due to the composition of the rooting medium, such as presence of other metals (e.g., Ni) and organic materials in the soil (Li et al. 2003). The present study showed that serpentine plants of A. bracteatum are not only more tolerant to higher concentrations of Co in the medium (Fig. 2), but also can accumulate more Co in their shoots (Fig. 1). The results suggest that mechanisms that result in higher tolerance and accumulation of Ni in serpentine plants of A. bracteatum also result in Figure 2. Relative shoot dry weight (mean ± SE), defined as percent of control dry weight, of plants from serpentine and non-serpentine populations of A. bracteatum treated with different concentrations of Co in the medium. Different letters in each series (lower case letters for serpentine plants and capital letters for non-serpentine plants) show statistically significant differences between treatments based on a Tukey test (P < 0.05). * indicates a significant difference between serpentine and non-serpentine plants within each treatment based on a t-test (P < 0.05). 136 Northeastern Naturalist Vol. 16, Special Issue 5 higher tolerance and accumulation of Co. A similar association of Ni and Co hyperaccumulation in Alyssum spp. was reported by Homer et al. (1991). Uptake of high concentrations of Co led to decreased biomass production by non-serpentine plants of A. bracteatum, relative to serpentine plants. This effect may be a specific property of plants and populations which are from metalliferous soils and are considered to be metallophyte plants. In general, it has been accepted that metal hyperaccumulating plants are among the most metal-tolerant plants found in nature (Pollard et al. 2002). The greater tolerance of the serpentine population of A. bracteatum to high concentrations of Co appears to correlate with their ability to accumulate greater concentrations of Co. It has been suggested that, on the basis of the physicochemical similarities of Co and Ni, one mechanism controls the uptake of both elements (Homer et al. 1991, Rancelis et al. 2006). Thus, one ligand may bind to Ni or Co in the root, and complexing by another ligand can then affect xylem transport, terminating in detoxification in the leaves. The ligand for transport of Ni in Ni hyperaccumulators from Alyssum may be histidine (Krämer et al. 1996), but it has not been determined if histidine is responsible for Co translocation. Most studies on Ni hyperaccumulators have indicated that the predominant chelated forms of Ni are citrate-Ni and malate-Ni in the root/ stem and leaves, respectively (Chaney et al. 2007, Kersten et al. 1980, Montargès- Pelletier et al. 2008). In addition to decreased growth, chlorosis was also evident, particularly in non-serpentine plants of A. bracteatum growing in media containing high concentrations of Co. Chlorosis can be related to changes in the Co/Fe ratio, and Co toxicity symptoms are in part similar to Fe deficiency symptoms (Palit et al. 1994, Zeid 2001). An interesting question is whether Ni and Co tolerance and accumulation mechanisms are the same in Ni hyperaccumulators of the genus Alyssum. Antagonistic behavior of Co and Ni for uptake by roots has been reported (Homer et al. 1991). Therefore, it is possible that, for initial steps of the process, there is an overlap in the pathways of uptake and chelation of both metals in the root. On the other hand, Tappero et al. (2007) reported that mechanisms for storage of Ni and Co in the shoot of A. murale Waldst. & Kit. (which can hyperaccumulate both metals) are not similar. They have shown that the predominant location for accumulation of Ni in the leaves is intracellular compartments (potentially vacuoles) of epidermal cells, whereas Co accumulates in the apoplast. They proposed that A. murale leaves lack the transport system needed to sequester Co in epidermal cells. Also, it has been reported that Co supplied through the root system tended to accumulate in leaf margins, with higher concentrations in young leaves and higher concentrations in leaves relative to stems and roots (Cataldo et al. 1978, Homer et al. 1991, Page and Feller 2005, Page et al. 2006). Therefore, as Tappero et al. (2007) concluded, the pattern of accumulation of Co in the leaves is due to mass flow of solutes which finally causes deposition of Co in the apoplast and the surface, tip, and margins of leaves. It is not clear which mechanism or transporting system is used for sequestration of Ni in the vacuoles of epidermal cells, but is not used for sequestration of Co. 2009 S.M. Ghaderian, M. Movahedi, and R. Ghasemi 137 In summary, our findings from pot trials show that the Co tolerance and accumulation characteristics of two serpentine and non-serpentine populations of Alyssum bracteatum differ. It appears that non-serpentine populations of this species do not possess the ability to accumulate heavy metals (such as Co) to the same degree as serpentine plants. The most significant finding from our work is the observation that, in this species of Alyssum, hyperaccumulation of Ni is associated with the potential to accumulate Co. Acknowledgment This research was carried out using an M.Sc. grant to M. Movahedi, offered by the Graduate school of the University of Isfahan. Literature Cited Baker, A.J.M., and R.R. Brooks. 1989. Terrestrial higher plants which hyperaccumulate metallic elements: A review of their distribution, ecology, and phytochemistry. Biorecovery 1:81–126. Baker, A.J.M., R.D. Reeves, and A.S.M. Hajar. 1994. 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