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Heavy metal uptake of Geosiphon pyriforme
Nuclear Instruments and Methods in Physics Research B 181 (2001) 659±663
www.elsevier.com/locate/nimb
Heavy metal uptake of Geosiphon pyriforme Stefan Scheloske a b
a,*
, Mischa Maetz a, Arthur Sch uûler
b
Max-Planck-Institut fur Kernphysik, P.O. Box 103980, 69029 Heidelberg, Germany Institut fur Botanik, Technische Universitat Darmstadt, 64287 Darmstadt, Germany
Abstract Geosiphon pyriforme represents the only known endosymbiosis between a fungus, belonging to the arbuscular mycorrhizal (AM) fungi, and cyanobacteria (blue-green algae). Therefore we use Geosiphon as a model system for the widespread AM symbiosis and try to answer some basic questions regarding heavy metal uptake or resistance of AM fungi. We present quantitative micro-PIXE measurements of a set of heavy metals (Cu, Cd, Tl, Pb) taken up by Geosiphon-cells. The uptake is studied as a function of the metal concentration in the nutrient solution and of the time Geosiphon spent in the heavy metal enriched medium. The measured heavy metal concentrations range from several ppm to some hundred ppm. Also the in¯uence of the heavy metal uptake on the nutrition transfer of other elements will be discussed. Ó 2001 Elsevier Science B.V. All rights reserved. PACS: 61.15; 78.70; 81.70 Keywords: PIXE; Geosiphon pyriforme; Heavy metals; Arbuscular mycorrhiza
1. Introduction Geosiphon pyriforme (Fig. 1) is the only known endosymbiosis of a fungus with blue-green algae (cyanobacteria). 18s rRNA-gene analyses have shown that the fungal partner of this symbiosis phylogenetically belongs to the arbuscular mycorrhizal (AM) fungi [1]. More than 80% of the land plant species form an AM, showing clearly the importance of this symbiosis for the terrestrial
* Corresponding author. Tel.: +49-6221-516590; fax: +496221-516540. E-mail address: [email protected] (S. Scheloske).
ecosystem. The Geosiphon symbiosis is characterized by the unicellular symbiotic ``bladders'', which are 1±2 mm in size and thus a system easy to investigate. Therefore we use Geosiphon as a model system for the AM, allowing us to study topics dicult, or not possible at all, to investigate in the AM symbiosis. For a long time, plants on heavy metal soils were believed to be non-mycorrhizal, but recently it was shown, that that some of these plants are heavily colonized by AM fungi, and that this fungi are able to confer heavy metal tolerance also to crop plants [2]. This indicates that AM fungi could be used for phytoremediation (use of plants for remediation) of heavy metal polluted soils, which can be performed by the use of
0168-583X/01/$ - see front matter Ó 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 0 1 ) 0 0 5 3 4 - 1
660
S. Scheloske et al. / Nucl. Instr. and Meth. in Phys. Res. B 181 (2001) 659±663
(b) the time Geosiphon was incubated in the heavy metal containing medium. 2. Material and methods 2.1. Preparation
Fig. 1. A schematic drawing of Geosiphon pyriforme. The unicellular bladder measures about 1 mm in height.
(a) hyperaccumulative plants alone, (b) tolerant plants and additional treatments, (c) combinations of plants and mycorrhizal fungi. We now try to answer some basic questions regarding heavy metal uptake or resistance of AM fungi. These are: 1. Which heavy metals are taken up to what extent? 2. Where are heavy metals deposited, in the vacuoles, cell wall, the symbiotic compartment? 3. Confers the symbiosis heavy metal resistance to the photoautotrophic symbiosis partner (the cyanobaceria)? As a ®rst step, we present quantitative micro-PIXE measurements of a set of heavy metals (Cu, Cd, Tl, Pb) taken up by Geosiphon. The uptake is studied as a function of (a) the metal concentration and composition in the culture medium and on
The Geosiphon bladders were harvested from 4to 6-month-old cultures on a sterilized natural substrate and mechanically cleaned by 20 s gentle shaking in nutrient solution (GM32, a nutrientpoor culture medium, representing GM29 [1] with addition of 1 lM KBr) containing a very ®ne analysis quartz-sand. Thereafter the bladders were washed two times in nutrient solution and left for 2 d in fresh solution. The cells which did not survive this isolation procedure (about 10%) can be easily recognized microscopically after 2 d by a color change and were discarded. The remaining cells spent one day more in GM32 and were thereafter transferred into the heavy metal containing GM32. The bladders were incubated in 5 ml nutrient solution at 20°C at a 14/10 h light/dark rhythm in plastic Petri dishes with a photosynthetic photon ¯ux density of 80 lmol m 2 s 1 . Metal concentrations were 1 or 5 lM or mixtures with these concentrations of each metal. Samples were taken after dierent incubation times, washed for 20 s in 5 ml double distilled water by gentle moving, immediately placed onto Pioloform foil and air dried after removing excessive water with a ®lter paper. 2.2. Measurement conditions The specimens were measured at the Heidelberg proton microprobe [3] with a proton beam energy of 2.2 MeV. To correct the absolute element concentrations for possible beam damage eects as described in [4] each sample was measured twice with a beam spot size of 2±2:5 lm at 20 pA and of about 4:5 lm at 200 pA. A Si(Li) X-ray detector was used with an active area of 80 mm. The detector is mounted at 45° and a distance of 16 mm from the target surface. The given concentrations represent bulk values of the single bladders resulting from raster scan areas of 30 30 lm2 . The
S. Scheloske et al. / Nucl. Instr. and Meth. in Phys. Res. B 181 (2001) 659±663
thickness and homogeneity was measured with scanning transmission ion microscopy (STIM) and only homogenous regions were chosen as representative areas. The standard derivation of the median, representing the thickness, was <1% with 1600 pixels measured. 2.3. Specimens Our measurements included 72 bladders representative for the following treatments: 1. dierent heavy metal containing solutions (1 lM of Cd, Cu, Pb or Tl; ``mix'' of these four metals with 1 lM or 5 lM for each); 2. variation of incubation time (8, 24, 29, 122 and 192 h); 3. control bladders not incubated with heavy metals. All bladders were placed onto Pioloform foil and carbon coated to reduce charging eects. 3. Results Geosiphon bladders (Fig. 1), representing single cells of 1±2 mm in size, were analyzed. To investigate the concentrations of macro and trace elements of heavy metal treated and control bladders
661
that have not been incubated, we analyzed three bladders of each treatment. The results are shown in Fig. 2. The element concentrations measured are in the same range for all controls. One of the 192 h control bladders shows signi®cantly higher concentrations of Al, Si, Ti, Mn and Fe compared to the others. Because most of these elements are known as soil-markers [5,6] this bladder was probably contaminated by sand particle(s). These kind of information is important for the selection of the area of measurement. Another problem is the existence of obviously dead bladders. However, dead bladders can be distinguished from alive ones by their elemental content. In the dead bladders, K and Cl concentrations are about a factor 100 lower compared to the alive bladders (Fig. 3). In living cells these elements occur mainly as monovalent ions which are highly soluble and motile in water and therefore rapidly lost by dying cells. If present in the nutrient solution, Cd, Pb and Cu show signi®cantly higher concentrations in dead bladders compared to living ones (Fig. 3). The values shown in Fig. 3 were obtained from bladders incubated 192 h in a 1 lM heavy metal mix. Therefore the existence of extremely high Pb concentrations, caused by accumulation/absorption
Fig. 2. Measurement of control bladders. The arrows indicate the elements which are typical for soil contamination. LOD means the limit of detection.
662
S. Scheloske et al. / Nucl. Instr. and Meth. in Phys. Res. B 181 (2001) 659±663
Fig. 3. Typical characteristics of dead bladders. All dead bladders have signi®cant higher heavy metal and lower K and Cl concentrations than the living ones, as indicated by the ellipses.
within the dead cells, may also be an indication for dead bladders. Tl does not accumulate within the bladders since it behaves like the monovalent ions K and Cl . To investigate the in¯uence of the duration Geosiphon was incubated in dierent heavy metal
solutions we measured a set of bladders incubated for 8±122 h in a 5 or 1 lM heavy metal mix and in nutrient solutions containing a single heavy metal at a concentration of 1 lM. The results are shown in Fig. 4. In general there seem to be two trends: (i) the application of 5 lM leads to a higher heavy
Fig. 4. Heavy metal uptake in dependence of time, metal composition and concentration. Each value plotted represents the mean from three measured bladders.
S. Scheloske et al. / Nucl. Instr. and Meth. in Phys. Res. B 181 (2001) 659±663
metal accumulation of the cells compared to 1 lM, (ii) Pb and Tl are accumulated in higher concentration when applied as single elements (1 lM), compared to the 1 lM mix. This is not the case for Cu and Cd. 4. Discussion At least 70% of the Cd taken up by humans originate from plant food. Mechanisms for heavy metal uptake and translocation are not yet well understood, but recently it became clear that AM fungi play a role in heavy metal tolerance, as well as in heavy metal accumulation of plants. However, dierent heavy metals are transported to dierent extents to the plants by AM fungal hyphae. In non-mycorrhizal, heavy metal stressed plant roots, a higher S content as compared to the control roots can be found [7]. This possibly could be due to a co-localization of Cd and S as it is demanded if phytochelatins (S-rich polypeptides) play a major role in intracellular Cd-detoxi®cation [8]. This was not found in Geosiphon pyriforme. Geosiphon accumulates heavy metals, e.g., the Pb concentration after 8 h in 1 lM Pb is about 50 times higher within the bladder (related to fresh weight), compared to the nutrient solution. We have to perform further experiments to evaluate this accumulation in dependence from the external concentration, and the in¯uence of the total heavy metal content in mixtures on the uptake of single metals. Dierent uptake or reg-
663
ulation mechanisms, for e.g. Cd/Cu and Tl/Pb are indicated by the present data. The latter elements are taken up in signi®cantly lower rates when applied in mixtures with other heavy metals, meaning a higher total heavy metal stress. Possibly an induced resistance mechanism could play a role here. As a next step we will study the in¯uence of other heavy metals like Ni, Co and Mo. Furthermore, freeze dried cryo-sections will be investigated by elemental mappings to analyze the distribution of the heavy metals within the cell. References [1] A. Sch uûler, M. Kluge, in: B. Hock (Ed.), The Mycota IX, Springer, Berlin, 2000, p. 151. [2] U. Hildebrandt, M. Kaldorf, H. Bothe, J. Plant Physiol. 154 (1999) 709. [3] K. Traxel, P. Arndt, J. Bohsung, K.U. Braun-Dullaeus, M. Maetz, D. Reimold, H. Schiebler, A. Wallianos, Nucl. Instr. and Meth. B 104 (1995) 19. [4] M. Maetz, A. Sch uûler, A. Wallianos, K. Traxel, Nucl. Instr. and Meth. B 150 (1999) 200. [5] T. Schneider, B. Povh, O. Strasser, M. Gierth, W. Przybylowicz, J. Mesjasz- Przybylowicz, C. Churms, A. Sch uûler, Int. J. PIXE 9 (1999) 353. [6] A.G. Sangster, M.J. Hodson, in: G. Rapp jr., S.C. Mulholland (Eds.), Phytolith Systematics, Plenum, New York, 1992. [7] T. Schneider, A. Haag-Kerwer, M. Maetz, M. Niecke, B. Povh, T. Rausch, A. Sch uûler, Nucl. Instr. and Meth. B 158 (1999) 329. [8] E. Grill, E.L. Winnacker, M.H. Zenk, Science 230 (1985) 674.
www.elsevier.com/locate/nimb
Heavy metal uptake of Geosiphon pyriforme Stefan Scheloske a b
a,*
, Mischa Maetz a, Arthur Sch uûler
b
Max-Planck-Institut fur Kernphysik, P.O. Box 103980, 69029 Heidelberg, Germany Institut fur Botanik, Technische Universitat Darmstadt, 64287 Darmstadt, Germany
Abstract Geosiphon pyriforme represents the only known endosymbiosis between a fungus, belonging to the arbuscular mycorrhizal (AM) fungi, and cyanobacteria (blue-green algae). Therefore we use Geosiphon as a model system for the widespread AM symbiosis and try to answer some basic questions regarding heavy metal uptake or resistance of AM fungi. We present quantitative micro-PIXE measurements of a set of heavy metals (Cu, Cd, Tl, Pb) taken up by Geosiphon-cells. The uptake is studied as a function of the metal concentration in the nutrient solution and of the time Geosiphon spent in the heavy metal enriched medium. The measured heavy metal concentrations range from several ppm to some hundred ppm. Also the in¯uence of the heavy metal uptake on the nutrition transfer of other elements will be discussed. Ó 2001 Elsevier Science B.V. All rights reserved. PACS: 61.15; 78.70; 81.70 Keywords: PIXE; Geosiphon pyriforme; Heavy metals; Arbuscular mycorrhiza
1. Introduction Geosiphon pyriforme (Fig. 1) is the only known endosymbiosis of a fungus with blue-green algae (cyanobacteria). 18s rRNA-gene analyses have shown that the fungal partner of this symbiosis phylogenetically belongs to the arbuscular mycorrhizal (AM) fungi [1]. More than 80% of the land plant species form an AM, showing clearly the importance of this symbiosis for the terrestrial
* Corresponding author. Tel.: +49-6221-516590; fax: +496221-516540. E-mail address: [email protected] (S. Scheloske).
ecosystem. The Geosiphon symbiosis is characterized by the unicellular symbiotic ``bladders'', which are 1±2 mm in size and thus a system easy to investigate. Therefore we use Geosiphon as a model system for the AM, allowing us to study topics dicult, or not possible at all, to investigate in the AM symbiosis. For a long time, plants on heavy metal soils were believed to be non-mycorrhizal, but recently it was shown, that that some of these plants are heavily colonized by AM fungi, and that this fungi are able to confer heavy metal tolerance also to crop plants [2]. This indicates that AM fungi could be used for phytoremediation (use of plants for remediation) of heavy metal polluted soils, which can be performed by the use of
0168-583X/01/$ - see front matter Ó 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 0 1 ) 0 0 5 3 4 - 1
660
S. Scheloske et al. / Nucl. Instr. and Meth. in Phys. Res. B 181 (2001) 659±663
(b) the time Geosiphon was incubated in the heavy metal containing medium. 2. Material and methods 2.1. Preparation
Fig. 1. A schematic drawing of Geosiphon pyriforme. The unicellular bladder measures about 1 mm in height.
(a) hyperaccumulative plants alone, (b) tolerant plants and additional treatments, (c) combinations of plants and mycorrhizal fungi. We now try to answer some basic questions regarding heavy metal uptake or resistance of AM fungi. These are: 1. Which heavy metals are taken up to what extent? 2. Where are heavy metals deposited, in the vacuoles, cell wall, the symbiotic compartment? 3. Confers the symbiosis heavy metal resistance to the photoautotrophic symbiosis partner (the cyanobaceria)? As a ®rst step, we present quantitative micro-PIXE measurements of a set of heavy metals (Cu, Cd, Tl, Pb) taken up by Geosiphon. The uptake is studied as a function of (a) the metal concentration and composition in the culture medium and on
The Geosiphon bladders were harvested from 4to 6-month-old cultures on a sterilized natural substrate and mechanically cleaned by 20 s gentle shaking in nutrient solution (GM32, a nutrientpoor culture medium, representing GM29 [1] with addition of 1 lM KBr) containing a very ®ne analysis quartz-sand. Thereafter the bladders were washed two times in nutrient solution and left for 2 d in fresh solution. The cells which did not survive this isolation procedure (about 10%) can be easily recognized microscopically after 2 d by a color change and were discarded. The remaining cells spent one day more in GM32 and were thereafter transferred into the heavy metal containing GM32. The bladders were incubated in 5 ml nutrient solution at 20°C at a 14/10 h light/dark rhythm in plastic Petri dishes with a photosynthetic photon ¯ux density of 80 lmol m 2 s 1 . Metal concentrations were 1 or 5 lM or mixtures with these concentrations of each metal. Samples were taken after dierent incubation times, washed for 20 s in 5 ml double distilled water by gentle moving, immediately placed onto Pioloform foil and air dried after removing excessive water with a ®lter paper. 2.2. Measurement conditions The specimens were measured at the Heidelberg proton microprobe [3] with a proton beam energy of 2.2 MeV. To correct the absolute element concentrations for possible beam damage eects as described in [4] each sample was measured twice with a beam spot size of 2±2:5 lm at 20 pA and of about 4:5 lm at 200 pA. A Si(Li) X-ray detector was used with an active area of 80 mm. The detector is mounted at 45° and a distance of 16 mm from the target surface. The given concentrations represent bulk values of the single bladders resulting from raster scan areas of 30 30 lm2 . The
S. Scheloske et al. / Nucl. Instr. and Meth. in Phys. Res. B 181 (2001) 659±663
thickness and homogeneity was measured with scanning transmission ion microscopy (STIM) and only homogenous regions were chosen as representative areas. The standard derivation of the median, representing the thickness, was <1% with 1600 pixels measured. 2.3. Specimens Our measurements included 72 bladders representative for the following treatments: 1. dierent heavy metal containing solutions (1 lM of Cd, Cu, Pb or Tl; ``mix'' of these four metals with 1 lM or 5 lM for each); 2. variation of incubation time (8, 24, 29, 122 and 192 h); 3. control bladders not incubated with heavy metals. All bladders were placed onto Pioloform foil and carbon coated to reduce charging eects. 3. Results Geosiphon bladders (Fig. 1), representing single cells of 1±2 mm in size, were analyzed. To investigate the concentrations of macro and trace elements of heavy metal treated and control bladders
661
that have not been incubated, we analyzed three bladders of each treatment. The results are shown in Fig. 2. The element concentrations measured are in the same range for all controls. One of the 192 h control bladders shows signi®cantly higher concentrations of Al, Si, Ti, Mn and Fe compared to the others. Because most of these elements are known as soil-markers [5,6] this bladder was probably contaminated by sand particle(s). These kind of information is important for the selection of the area of measurement. Another problem is the existence of obviously dead bladders. However, dead bladders can be distinguished from alive ones by their elemental content. In the dead bladders, K and Cl concentrations are about a factor 100 lower compared to the alive bladders (Fig. 3). In living cells these elements occur mainly as monovalent ions which are highly soluble and motile in water and therefore rapidly lost by dying cells. If present in the nutrient solution, Cd, Pb and Cu show signi®cantly higher concentrations in dead bladders compared to living ones (Fig. 3). The values shown in Fig. 3 were obtained from bladders incubated 192 h in a 1 lM heavy metal mix. Therefore the existence of extremely high Pb concentrations, caused by accumulation/absorption
Fig. 2. Measurement of control bladders. The arrows indicate the elements which are typical for soil contamination. LOD means the limit of detection.
662
S. Scheloske et al. / Nucl. Instr. and Meth. in Phys. Res. B 181 (2001) 659±663
Fig. 3. Typical characteristics of dead bladders. All dead bladders have signi®cant higher heavy metal and lower K and Cl concentrations than the living ones, as indicated by the ellipses.
within the dead cells, may also be an indication for dead bladders. Tl does not accumulate within the bladders since it behaves like the monovalent ions K and Cl . To investigate the in¯uence of the duration Geosiphon was incubated in dierent heavy metal
solutions we measured a set of bladders incubated for 8±122 h in a 5 or 1 lM heavy metal mix and in nutrient solutions containing a single heavy metal at a concentration of 1 lM. The results are shown in Fig. 4. In general there seem to be two trends: (i) the application of 5 lM leads to a higher heavy
Fig. 4. Heavy metal uptake in dependence of time, metal composition and concentration. Each value plotted represents the mean from three measured bladders.
S. Scheloske et al. / Nucl. Instr. and Meth. in Phys. Res. B 181 (2001) 659±663
metal accumulation of the cells compared to 1 lM, (ii) Pb and Tl are accumulated in higher concentration when applied as single elements (1 lM), compared to the 1 lM mix. This is not the case for Cu and Cd. 4. Discussion At least 70% of the Cd taken up by humans originate from plant food. Mechanisms for heavy metal uptake and translocation are not yet well understood, but recently it became clear that AM fungi play a role in heavy metal tolerance, as well as in heavy metal accumulation of plants. However, dierent heavy metals are transported to dierent extents to the plants by AM fungal hyphae. In non-mycorrhizal, heavy metal stressed plant roots, a higher S content as compared to the control roots can be found [7]. This possibly could be due to a co-localization of Cd and S as it is demanded if phytochelatins (S-rich polypeptides) play a major role in intracellular Cd-detoxi®cation [8]. This was not found in Geosiphon pyriforme. Geosiphon accumulates heavy metals, e.g., the Pb concentration after 8 h in 1 lM Pb is about 50 times higher within the bladder (related to fresh weight), compared to the nutrient solution. We have to perform further experiments to evaluate this accumulation in dependence from the external concentration, and the in¯uence of the total heavy metal content in mixtures on the uptake of single metals. Dierent uptake or reg-
663
ulation mechanisms, for e.g. Cd/Cu and Tl/Pb are indicated by the present data. The latter elements are taken up in signi®cantly lower rates when applied in mixtures with other heavy metals, meaning a higher total heavy metal stress. Possibly an induced resistance mechanism could play a role here. As a next step we will study the in¯uence of other heavy metals like Ni, Co and Mo. Furthermore, freeze dried cryo-sections will be investigated by elemental mappings to analyze the distribution of the heavy metals within the cell. References [1] A. Sch uûler, M. Kluge, in: B. Hock (Ed.), The Mycota IX, Springer, Berlin, 2000, p. 151. [2] U. Hildebrandt, M. Kaldorf, H. Bothe, J. Plant Physiol. 154 (1999) 709. [3] K. Traxel, P. Arndt, J. Bohsung, K.U. Braun-Dullaeus, M. Maetz, D. Reimold, H. Schiebler, A. Wallianos, Nucl. Instr. and Meth. B 104 (1995) 19. [4] M. Maetz, A. Sch uûler, A. Wallianos, K. Traxel, Nucl. Instr. and Meth. B 150 (1999) 200. [5] T. Schneider, B. Povh, O. Strasser, M. Gierth, W. Przybylowicz, J. Mesjasz- Przybylowicz, C. Churms, A. Sch uûler, Int. J. PIXE 9 (1999) 353. [6] A.G. Sangster, M.J. Hodson, in: G. Rapp jr., S.C. Mulholland (Eds.), Phytolith Systematics, Plenum, New York, 1992. [7] T. Schneider, A. Haag-Kerwer, M. Maetz, M. Niecke, B. Povh, T. Rausch, A. Sch uûler, Nucl. Instr. and Meth. B 158 (1999) 329. [8] E. Grill, E.L. Winnacker, M.H. Zenk, Science 230 (1985) 674.