Enzymatic Activity of Alfalfa in a Phenanthrene-contaminated Environment

This research was undertaken to study enzymatic activity in the shoots, roots, and rhizosphere of alfalfa (Medicago sativa L.) grown in quartz sand that was uncontaminated and contaminated with phenanthrene at concentrations of 10 and 100 mg kg-1. The higher concentration of phehanthrene had a distinct phytotoxic effect on alfalfa, inhibiting seed germination energy, plant survival, and biomass accumulation. The plant stress response to the environmental pollution was an increase in peroxidase activity. Peroxidases were the predominant enzymes in the alfalfa shoots and roots. The peroxidase profile in the shoots differed from that in the roots and had different isoenzyme numbers. 2,2'-Azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS) peroxidase was predominant in the shoots, and 2,7-diaminofluorene (2,2-DAF) peroxidase was predominant in the roots. Under the influence of phenanthrene, the activity of 2,7-DAF peroxidase increased in the shoots, and the activity of ABTS peroxidase increased in the roots. Alfalfa root peroxidases were the prevalent enzyme systems in the rhizosphere sand. Examination of the activity of alfalfa root peroxidase toward phenanthrene revealed the possibility of involvement of the plant enzyme in rhizosphere degradation of the PAH.

Authors:

• Anna Yu. Muratova
• Vera V. Kapitonova
• Marina P. Chernyshova
• Olga V. Turkovskaya

Keywords:

• Medicago sativa
• enzymatic activity
• peroxidase
• phenanthrene.

References:

[1] S. Criquet, E. Joner, P. Leglize, C. Leyval, "Anthracene and mycorrhiza
affect the activity of oxidoreductases in the roots and the rhizosphere of
lucerne (Medicago sativa L.)", Biotechnol. Lett., vol. 22, no. 21, pp.
1733-1737, Nov.2000.
[2] A.Yu. Muratova, O.V. Turkovskaya, T. Huebner, P. Kuschk, "Study of
the efficacy of alfalfa and reed in the phytoremediation of hydrocarbon polluted soil", Appl. Biochem. Microbiol., vol. 39, no 6, pp. 599-605, Nov.-Dec. 2003.
[3] L.A. Phillips, C.W. Greer, J.J Germida, "Culture-based and cultureindependent
assessment of the impact of mixed and single plant
treatments on rhizosphere microbial communities in hydrocarbon
contaminated flare-pit soil", Soil Biol. Biochem., vol. 38, no 9, pp.
2823-2833, Sep. 2006.
[4] A.Yu. Muratova, T.V. Dmitrieva, L.V. Panchenko, O.V. Turkovskaya,
"Phytoremediation of oil-sludge-contaminated soil", Int. J. Phytorem., vol 10, no 6, pp. 486-502, 2008.
[5] G. Gramss, K-D. Voigt, B. Kirshe, "Oxidoreductase enzymes liberated
by plant roots and their effects on soil humic material", Chemosphere,
vol. 38, no 7, pp. 1481-1494, Mar. 1999
[6] J.J. Kraus, I.Z. Munir, J.P. McEldoon, D.S. Clark, J.S. Dordick,
"Oxidation of polycyclic aromatic hydrocarbons catalyzed by soybean
peroxidase", Appl. Biochem. Biotechnol., vol. 80, no 3, pp. 221-230,
June 1999.
[7] L. Chroma, M. Mackova, P. Kucerova, C. in der Wiesche, J. Burkhard,
T. Macek, "Enzymes in plant metabolism of PCBs and PAHs", Acta
Biotechnol., vol. 22, no 1-2, pp. 35-41, May 2002.
[8] G.S. Smith, C.M Johnston, I.S. Cornforth, ÔÇÿComparison of nutrient
solutions for growth of plants in sand culture-, New Phytol., vol. 94, no
4, pp. 537-548, Aug.1983.
[9] M.M. Bradford, "A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding", Anal. Biochem., vol. 72, no 1-2, pp. 248-254, May 1976.
[10] R Bartha., L. Bordeleau "Cell-free peroxidases in soil", Soil Biol.
Biochem., vol 1, no 2, pp. 139-143, June1969.
[11] M.-L. Niku-Paavola, E. Karhunen, P. Salola, V. Raunio, "Ligninolytic
enzymes of white-rot fungus Phlebia radiate", Biochem. J., vol. 254, no
3, pp. 877-884, Sep.1988.
[12] U.K. Laemmli, "Cleavage of structural protein during the assembly of
the head of bacteriophage", Nature, vol. 227, no 5259, pp. 680-685.
Aug. 1970.
[13] I. Holoubek, A. Koð©an, I. Holoubkovð▒, K. Hilscherovð▒, J. Kohoutek, J.
Falandysz, O. Roots. (May 2000). The study of PAH phytotoxicity. In:
Persistant, Bioaccumulative and Toxic Chemicals in Central and Eastern
European Countries - State-of-the art Report. Chapter 12.2.2 TOCOEN
Report No.150a. Brno, Czech Republic. Available:
http://recetox.chemi.muni.cz/index.php?language=en&id=4394
[14] K.-H. Baek, H.-S. Kim, H.-M. Oh, B.-D. Yoon, J. Kim, I.-S. Lee, "Effects of crude oil, oil components, and bioremediation on plant
growth", J. Environ. Sci. Heal. A, vol. 39, no 9, pp. 2465-2472, 2004.
[15] Y.F. Song, P. Gong, Q.X. Zhou, T.H. Sun, "Phytotoxicity assessment of
phenanthrene, pyrene and their mixtures by a soil-based seedling emergence test", J. Environ. Sci. (China), vol. 17, no 4, pp. 580-583,2005.
[16] R.S. Thygesen, S. Trapp, "Phytotoxicity of polycyclic aromatic
hydrocarbons to willow trees", J. Soils Sedim., vol. 2, no 2, pp. 77-82,June 2002.
[17] M. Alkio, T.M. Tabuchi1, X. Wang, A. Colon-Carmona, "Stress
responses to polycyclic aromatic hydrocarbons in Arabidopsis include
growth inhibition and hypersensitive response-like symptoms", J. Exp.
Bot., vol. 56, no 421, pp. 2983-2994, Nov. 2005.
[18] G. I. Kvesitadze, G.A. Khatisashvili, T.A. Sadunishvili, Z.G.
Evstegneyeva, Metabolizm antropogennyh toksikantov v vysshih rasteniyah (Metabolism of athropogenic toxicants in higher plants),
Moscow: Nauka, 2005. (In Russian)
[19] G. Gramss, O. Rudeschko, "Activities of oxidoreductase enzymes in
tissue extracts and sterile root exudates of three crop plants and some
properties of the peroxidase component", New Phytol., vol. 138, no 3,
pp. 401-409, Mar. 1998.
[20] G. Gramss, "Degradation of aromatic xenobiotics in aerated soils by
enzyme systems of microorganisms and plants", in Bioremediation of
Contaminated Soils, D.L. Wise, D.J. Trantolo, E.J. Cichon, H.I Inyang,
U. Stottmeister, Eds. New York, Basel: Marcel Dekker Inc., 2000, pp.
489-535.
[21] C. Johannes, A. Majcherczyk, "Natural mediators in the oxidation of
polycyclic aromatic hydrocarbons by laccase mediator systems", Appl.
Environ. Microbiol., vol. 66, no 2, pp. 524-528, Feb. 2000.