Abstract
The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO4 by addition of CuSO4 solutions to soil and (2) to identify potential unintended effects of the genetic engineering on root and shoot biomass, shoot nutrient content, arbuscular mycorrhizal infection and on the metabolic functions of microbial communities in the rhizosphere. In the absence of exogenous CuSO4 additions to soil shoot biomass and the macronutrient (C, P, K, Ca, Mg and N) content of plants expressing hMT were not significantly different from the parental control. In the 0.5 mM and 1.0 mM CuSO4 treatments transgenic plants expressing the commonly used transgenic β-glucuronidase (GUS) marker had significantly higher Fe content than the parental genotype. Significant differences were observed in the carbon substrate utilization patterns of rhizosphere microbial communities among the transgenic plants; no significant differences were observed in the percent mycorrhizal infection of parental and transgenic plants. Shoot biomass increased significantly in all genotypes treated with 0.5 mM CuSO4 and decreased in all genotypes at CuSO4 concentrations of 1.5 mM and 2.0 mM. Root dry weights decreased significantly in all genotypes at concentrations of 1.0 mM, 1.5 mM and 2.0 mM CuSO4. The largest decreases in root dry weight were observed in hMT genotypes grown in soil treated with 1.5 and 2.0 mM CuSO4. In plants treated with 1.5 mM CuSO4, shoots of transgenic plants expressing the hMT gene accumulated nominally, but not statistically significantly higher levels of Cu in shoot tissue. Our results were surprising with regard to lack of sufficient efficacy of the current hMT constructs for significant accumulation of Cu from soil treated with CuSO4. However, our results suggest the utility of applying adverse levels of CuSO4 or other environmental stressors to identify potential unintended effects of genetic engineering that may not be apparent under typically more optimal plant growth test conditions.
Similar content being viewed by others
References
Adriano, D. C.: 1986, Trace elements in terrestrial environments 2nd ed Springer NY 533 p.
Anderson, T. A., Guthrie, E. A. and Walton, B. T.: 1993, ‘Bioremediation in the rhizosphere’, Environ. Sci. Technol. 27, 2630–2636.
Austin, S., Bingham, E. T., Matthews, D. E., Shahan, M. N., Will, J. and Burgess, R. R.: 1995, ‘Production and field performance of transgenic alfalfa expressing alpha-amylase and manganese dependent lignin peroxidase’, Euphytica 85, 381–393.
Baker, A. J. and Brooks, R. R.: 1989, ‘Terrestrial higher plants which hyperaccumulate metallic elements–a review of their distribution, ecology and phytochemistry’, Biorecovery 1, 81–126.
Bolan, N., Adriano, D., Mani, S. and Khan, A.: 2003, ‘Adsorption, complexation and phytoavailability of copper as influenced by organic manure’, Environ. Toxicol. Chem. 22, 450–456.
Brown, P., Bellaloui, N., Hu, H. and Dandekar, A.: 1999, ‘Transgenically enhanced sorbitol synthesis facilitates phloem boron transport and increases tolerance of tobacco to boron deficiency’, Plant Physiol. 119, 17–20.
Catchpole, G. S., Beckmann, M., Enot, D. P., Mondhe, M., Zywicki, B., Taylor, J., Hardy, N., Smith, A., King, R. D., Kell, D. B., Fiehn, O. and Draper, J.: 2005, ‘Hierarchical metabolomics demonstrates substantial compositional similarity between genetically modified and conventional potato crops’, Proc. Natl. Acad. Sci. USA 102, 14458–14462.
Castaldini et~al.: 2005, ‘Impact of Bt corn on rhizospheric and soil eubacterial communities and on mycorrhizal symbiosis in experimental microcosms’, Appl. Environ. Microbiol. 71, 6719–6729.
Cellini, F., Chesson, A., Colquhoun, I., Constable A., Davies, H. V., Engel, K. H., Gatehouse, A. M. R., Karenlampi, S., Kok, E. J., Leguay J.-J., Lehesranta, S., Noteborn, H. P. J. M., Pedersen, J. and Smith, M.: 2004, ‘Unintended effects and their detection in genetically modified crops’, Food Chem. Toxicol. 42, 1089–1125.
Cunningham, S. D. and Ow, D.: 1996, ‘Promises and prospects of phtyoremediation’, Plant Physiol. 110, 715–719.
Chaney, R. L., Brown, S. L., Li, Y. M., Angle, J. S., Homer, F. A. and Green, C. E.: 1995, ‘Potential use of metal hyperaccumulators’, Mining Environ. Management 3, 9–11.
Datla, R. S. S., Bekkaoui, F., Hammerlindl, J. K., Pilate, G., Dunstan, D. I. and Crosby, W. L.: 1993, ‘Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence’, Plant Science 94, 139–149.
De Verno, L. L., Byrne, J. R., Pitel, J. A. and Cheliak, W. M.: 1989, ‘Constructing conifer genomic libraries: a basic guide’, Information report PI-X-88, Petawawa National Forestry Institute, Forestry Canada.
Deak, M., Kiss, G. B., Koncz, C. and Dudits, D.: 1986, ‘Transformation of Medicago by Agrobacterium mediated gene transfer’, Plant Cell reports. 5, 97–100.
Dela Fuente, J. M., Ramirez-Rodriquez, V., Cabrera-Ponce, J. L. and Herrera-Estrella, L.: 1997, ‘Aluminum tolerance in transgenic plants by alteration of citrate synthesis’, Science 276, 566–1568.
Denchev, P., Velcheva, M. and Atanassov, A.: 1991, ‘A new approach to direct somatic embryogenesis in Medicago’, Plant Cell reports. 10, 338–341.
Di Giovanni, G. D. Watrud, L. S. Seidler, R. J. and Widmer, F.: 1999, ‘Comparison of parental and transgenic alfalfa rhizosphere bacterial communities using Biolog GN metabolic fingerprinting and enterobacterial repetitive intergeneric consensus sequence – PCR (ERIC-PCR)’, Microb. Ecol. 37, 129–139.
Donegan, K. K., Palm, C. J., Fieland, V. J., Porteous, Ganio, L. M., Schaller, D. L., Bucao, L. Q. and Seidler, R. J.: 1995, ‘Changes in levels, species and DNA fingerprints of soil microorganisms associated with cotton expressing the Bacillus thuringiensis var. kurstaki endotoxin’, Appl. Soil Ecol. 2, 111–124.
Donegan, K. K., Schaller, D. L., Stone, J. K., Ganio, L. M., Reed, G., Hamm, P. B. and Seidler, R. J.: 1996, ‘Microbial populations, fungal species diversity and plant pathogen levels in field plots of potato plants expressing the Bacillus thuringiensis var. tenebrionis endotoxin’, Transgenic Res. 5, 25–35.
Donegan, K. K., Seidler, R. J., Doyle, J. D., Porteous, L. A., Di Giovanni, G., Widmer, F. and Watrud, L. S.: 1999, ‘A field study with genetically engineered alfalfa inoculated with recombinant Sinorhizobium meliloti: effects on the soil ecosystem’, J. Applied Ecol. 36, 920–936.
Downs, R. J. and Hellmers, H.: 1975, ‘Environmental and the experimental control of plant growth Academic Press London 145p, Table~4, p111, North Carolina State University phytotron nutrient solution.
Entry, J. A. and Watrud, L. S.: 1998, ‘Potential remediation of 137 Cs and 90 Sr contaminated soil by accumulation in Alamo switchgrass’, Water, Air and Soil Pollution. 104, 339–352.
Entry, J. A., Watrud, L. S. and Reeves, M.: 1999, ‘Accumulation of 137 Cs and 90 Sr from contaminated soil by three grass species inoculated with mycorrhizal fungi’, Environ. Pollution 104, 449–457.
Entry, J. A., Watrud, L. S. and Reeves, M.: 2001, ‘Accumulation of 137 Cs and 90 Sr from contaminated soil by three grass species inoculated with organic amendments’, Water, Air and Soil Pollution 12, 385–398.
Evans, K. M., Gatehouse, J. A., Lindsay, W. P., Shi, J., Tommey, A. M. and Robinson, N. J.: 1992, ‘Expression of the pea MT-like PsMTa in E. coli and Arabidopsis thaliana and analysis of trace metal ion accumulation: implications of PsMTa function’, Plant Molecular Biology 20, 1019–1028.
Garland, J. L.: 1996a, ‘Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization’, Soil Biol. Biochem. 28, 213–221.
Garland, J. L.: 1996b, ‘Patterns of potential C source utilization by rhizosphere communities’, Soil Biol. Biochem. 28, 223–230.
Garland, J. L. and Mills, A. L.: 1991, ‘Classification and characteriation of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source-utilization’, Appl. Environ. Microbiol. 57, 2351–2359.
Garret, S. H. Somji, S., Todd, J. H. and Sens, D. A.: 1998, ‘Exposure of human proximal tubule cells to Cd 2+, Zn 2+, and Cu 2+induces metallothionein protein accumulation but not metallothionein isoform 2 mRNA’, Environ Health Perspect 106, 587–595.
Gilissen, L. J. W., Metz, P. L. J., Stiekma, W. J. and Nap, J.-P.: 1998, ‘Biosafety of E. coli β-glucuronidase (GUS) in plants’, Transgenic Research 7, 157–163.
Goyer, R. A., Haust, M. D. and Cherian, M. G.: 1992, ‘Cellular localization of metallothionein in human term placenta’, Placenta 13, 349–355.
Grayston, S. J., Wang, S., Campbell, C. D. and Edwards, A. C.: 1998, ‘Selective influence of plant species on microbial diversity in the rhizosphere’, Soil Biol. Biochem. 30, 369–378.
Hasegawa, I., Terada, E., Sunairi, M., Wakita, H., Shinmachi, F., Noguchi, A., Nakajima, M. and Yazaki, J.: 1997, ‘Genetic improvement of heavy metal tolerance in plants by transfer of the yeast metallothionein gene (CUP1)’, Plant and Soil 196, 277–281.
Innis, M., Gelfand, A. D. H., Sninsky, J. J. and White, T. J.: 1990, ‘PCR Protocols–A Guide to Methods and Applications’, Academic Press, Inc. San Diego, C. A. 482 p.
Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W.: 1987, ‘GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants’, The EMBO J. 6, 3901–3907.
Jefferson, R. A.: 1989, ‘The GUS reporter gene system’, Nature 342, 837–838.
Jin, S., Komari, T., Gordon, M. P. and Nester, E. W.: 1987, ‘Genes responsible for the supervirulent phenotype of Agrobacterium tumefaciens A281’, J. Bacteriol. 169, 4417–4425.
Kondo, Y., Yanagiya, T., Himeno, S., Yamabe, Y., Schwartz, D., Akimoto, M., Lazo, J. S. and Imura, N.: 1999, ‘Simian virus 40-transformed metallothionein null cells showed increased sensitivity to cadmium but not to zinc, copper, mercury or nickel’, Life. Sci. 64, 145–150.
Kormanik, P. P. and McGraw, A. C.: 1982, ‘Quantification of vesicular-arbuscular mycorrhizae in plant roots’, In: Schenck NC Methods and Principles of Mycorrhizal Research. American Phytopathological Society, St. Paul, pp. 37–45.
Krämer, U. and Chardonnens, A. N.: 2001, ‘The use of transgenic plants in the bioremediation of soils contaminated with trace elements’, Appl. Microbiol. Biotechnol. 55, 661–672.
Kumar, N. P. B., Dushenkov, V., Motto, H. and Raskin, I.: 1995, ‘Phytoextraction: the use of plants to remove heavy metals from soils’, Environ. Sci. Technol. 29, 1234–1238.
Latour, X., Corberand, T., Laguerre, G., Allard, F. and Lemanceau, P.: 1996, ‘The composition of flourescent pseudomonad populations associated with roots is influenced by plant and soil type’, Appl. Environ. Microbiol. 62, 2449–2456.
Lehesranta, S. J., Davies, H. V., Sheperd, L. V. T., Nunan, N., McNicol, J. W., Auriola, S., Koistenen, K. M., Suomalainen, S., Kokko, H. I. and Karenlampi, S. O.: 2005, ‘Comparison of tuber proteomes of potato (Solanum sp) varieties, landraces and genetically modified lines’, Plant Physiol. 138, 1690–1699.
Lemanceau, P., Coberand, T., Gardan, L., Latou, R. X., Laguerre, G., Boefgras, J. M. and Alabouvette, C.: 1995, ‘Effects of two plant species, flax, (Linum usitatissinum L.) and tomato (Lycopersicon esculentum Mill.), on the diversity of soilborne populations of flourescent pseudomonads’, Appl. Environ. Microbiol. 61, 1004–1012.
Mahaffee, W. and Kloepper, J.: 1997, ‘Temporal changes in the bacterial communities of soil, rhizosphere, and endorhiza associated with field-grown cucumber (Cucumis sativus L.)’, Microbial. Ecol. 34, 210–223.
McKersie, B. D., Bowley, S. R. and Jones, K. S.: 1999, ‘Winter survival of transgenic alfalfa overexpressing superoxide dismutase’, Plant Physiol. 119, 839–847.
Mertz, S. M. Jr, Heithaus, J. J. III and Bush, R. L.: 1979, ‘Mass production of axenic spores of the endomycorrhiizal fungus Gigaspora margarita’, Trans. Br. Mycol. Soc. 72, 167–169.
Misra, S. and Gedamu, L.: 1989, ‘Heavy metal tolerant transgenic Brassica napus and Nicotiana tabacum L.’, Theor. Appl. Genet. 78, 161–168.
Pedersen, S. N., Pedersen, K. L., Hojrup, P., Knudsen, J. and Depledge, M. H.: 1998, ‘Induction and identification of cadmium-, zinc- and copper-metallothioneins in the shore crab Carcinus maenas (L.)’, Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol 120, 251–259.
Price-Haughey, J., Bonham, K. and Gedamu, L.: 1987, ‘Metallothionein gene expression in fish cell lines: its activation in embryonic cells by 5-azacytidine’, Biochem. Biophys. Acta 908, 158–168.
Raskin, I. R., Smith, D. and Salt, D. E.: 1997, ‘Phytoremediation of metals: plants to remove pollutants from the environment’, Current Opinions in Biotechnology 8, 221–226.
Rugh, C. L., Wilde, H. D., Stack, N. M., Thompson, D. M., Summers, A. O. and Meagher, R. B.: 1996, ‘Mercuric ion reductase and resistance in transgenic A. thaliana plants expressing a modified bacterial mer A gene’, Proc. Natl. Acad. Sci. (USA) 93, 3182–3187.
Salt, D. E., Blaylock, M., Kumar, N. P. B., Dushenkov, V., Ensley, B. D., Chet, I. and Raskin, I.: 1995, ‘Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants’, Biotechnology 13, 468–474.
Sambrook, J. and Russel, D. W.: 2001, Molecular Cloning–A Laboratory Manual 3rd edition Cold Spring Harbor Laboratory Press Cold Spring Harbor NY.
Schnoor, J. L., Light, L. A., McCutcheon, S. C., Wolfe, N. L. and Carreira, L. H.: 1995, ‘Phytoremediation of organic and nutrient contaminants’, Environ. Sci. Technol. 29, 318–323.
Siciliano, S. D., Theoret, C. M., de Freitas, J. R., Hucl, P. J. and Germida, J. J.: 1998, ‘Differences in the microbial communities associated with the roots of different cultivars of canola and wheat’, Can. J. Microbiol. 44, 844–851.
Steffens, J. C.: 1990, ‘The heavy metal binding peptides of plants’, Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 533–575.
Syring, R. A., Hoexum-Brower, T. and Brouwer, M.: 2000, ‘Cloning and sequencing of c-DNAs encoding for a novel copper-specific metallothionein and two cadmium-inducible metallothioneins from the blue crab Callinectes sapidus’, Comp. Biochem. Physiol. C 125, 325–332.
Tesfaye, M., Dufault, N. S., Dornbusch, M. R., Allan, D. L., Vance, C. P. and Samac, D. A.: 2003, ‘Influence of enhanced malate dehydrogenase expression by alfalfa on diversity of rhizobacteria and soil nutrient availability’, Soil Biol. Biochem. 35, 1103–1113.
Uchimia, H. and Murashige, T.: 1974, ‘Evaluation of parameters in the isolation of viale protoplasts from cultured tobacco cells’, Plant Physiol. 54, 936–944.
van Hoof, N. A. L. M., Hassinen, V. H., Hakvoort, H. W. J., Schat, K. H., Verkleij, J. A. C., Ernst, W. H. O. and Tervahauta, S. O. K.: 2001, ‘Enhanced copper tolerance in Silene vulgaris (Moencl) populations from copper mines is associated with increased transcipt levels of a 2b-type metallothionein gene’, Plant. Physiol. 126, 1519–1526.
Varshney, V. and Gedamu, L.: 1984, ‘Human metallothionein MT-I snd MT-II processed genes’, Gene 31, 135–145.
Verwoerd, T. C., Dekker, B. M. and Hoekama, A.: 1989, ‘A small-scale procedure for the rapid isolation of plant RNAs’, Nucleic Acids Res. 17, 2362.
Vierheilig, H., Alt, M., Lange, J., Gut-Rella, M., Wiemken, A. and Boller, T.: 1995, ‘Colonization of transgenic tobacco constitutively expressing pathogenesis-related proteins by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae’, Appl. & Environ. Microbiol. 8, 3031–3034.
Watrud, L. S.: 2000, ‘Genetically Engineered Plants in the Environment–Applications and Issues’, in N.S. Subbarao and Y.R. (eds.), Microbial Interactions in Agriculture and Forestry Vol. 2, Science Publishers Inc Enfield NH, pp 62–81.
Watrud, L. S., Maggard, S., Shiroyama, T., Coleman, G. C., Johnson, M. G., Donegan, K. K., DiGiovanni, G., Porteous, L. A. and Lee, E. H.: 2003, Bracken (Pteridium aquilinum L.) frond biomass and rhizosphere microbial community characteristics are correlated to edaphic factors. Plant and Soil 249, 359–371.
Watrud, L. S. and Seidler, R. J.: 1998, ‘Nontarget ecological effects of plant, microbial, and chemical introductions to terrestrial systems’, in P.M. Huang (ed.)' Soil Chemistry and Ecosystem Health, Special Publication no. 52, 313–340. Soil Science Society of America, Madison, WI.
Zhu, Y. L., Pilon-Smits, E. A. H., Jouanin, L. and Terry, N.: 1999, ‘Over expression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance’, Plant Physiol. 119, 73–79.
Zimeri, A. M., Dhankher, O. P., McCaig, B. and Meagher, R. B.: 2005, ‘The plant MT1 metallothioneins are stabilized by binding cadmium and are required for cadmium tolerance and accumulation’, Plant Molec. Biol. 58, 839–855.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Watrud, L.S., Misra, S., Gedamu, L. et al. Ecological Risk Assessment of Alfalfa Medicago Varia L.) Genetically Engineered to Express a Human Metallothionein (hMT) Gene. Water Air Soil Pollut 176, 329–349 (2006). https://doi.org/10.1007/s11270-006-9171-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11270-006-9171-5