The Effect of Lead on the Glomalin Content of Hypha and Root Reactive with Monoclonal Antibody and Bradford in both in Vitro and Pot Culture Conditions

آب و خاک

Article 3, Volume 30, Issue 2 - Serial Number 46, May 2016, Pages 605-617   

Document Type: Research Article

DOI: 10.22067/jsw.v30i2.47802

Authors

Elham Malekzadeh^{* 1}; Jafar Majidi²; Nasser Aliasgharzad³; Jalal Abdolalizadeh²

¹Tabriz

²Tabriz University of Medical Sciences

³University of Tabriz

Abstract

Introduction: Glomalin is known as a specific fungal glycoprotein belonging to the order Glomerales in phylum Glomeromycota and has been introduced as a heat shock protein. We hypothesised that increasing the level of Pb would lead to increase in glomalin production. Glomalin is usually determined by two methods, the Bradford protein dye-binding assay and the enzyme-linked immunosorbent assay (ELISA). Since many laboratories are not equipped to carry out the ELISA assay, many studies have measured glomalin-related soil protein using the Bradford colorimetric total protein assay. While, the ELISA method specifically measures glomalin by using monoclonal antibody MAb32B11.
Materials and Methods: The pot experiment was conducted in the sterile free-glomalin sand with Trifolium repens L. mycorrhized by Rhizophagus irregularis fungus and treated with the Pb levels of 0, 150, 300 and 450 µM. Thus, in vitro experiment was performed in two-compartments plates containing of the transformed carrot roots (Daucus carota L.) mycorrhized with the same fungus in root compartment and hyphal compartment treated with the Pb levels of 0, 0.01, 0.1 and 1 mM as Pb(NO3)2. For glomalin extraction, hyphal or root samples were autoclaved at 121 ⁰C with 50 mM sodium citrate buffer for 60 min in three cycles. Protein concentrations in the extracted samples were determined using a modified Bradford protein assay. Also, glomalin content in the samples were determined by indirect ELISA using monoclonal antibody MAb32B11. The percentages of the total root length were colonised by mycorrhizal fungi in pot culture and both hyphal and spore densities in the metal-containing hyphal compartment were determined.
Results and Discussion: In the in vitro culture the percentage of total hyphae and spore frequency decreased, while Bradford reactive total hyphal protein (BRHP) and Immunoreactive hyphal protein (IRHP) in hyphal compartment increased as the concentrations of Pb increased. Also, there was positive and significant correlation between immunoreactive hyphal protein (IRHP) and Bradford reactive total hyphal protein (BRHP) in hyphal compartment of in vitro culture (r= 0.941**). In the pot culture, the percentage of the total mycorrhized root length in all the treatments increased compared to the unleaded control as the concentrations of Pb raised. In general, Bradford reactive total protein and Immunoreactive protein in both the hyphal and root compartments of pot culture increased with increasing the Pb levels. Also, there were positive and significant correlations between immunoreactive hyphal protein (IRHP) with Bradford reactive total hyphal protein (BRHP) (r= 0.845 **) and immunoreactive root protein (IRRP) with Bradford reactive total root protein (BRRP) (r= 0.706 **) in pot experiment. Some previously researches had reported correlation between ELISA with Bradford contents, whether, Bradford and ELISA values were nearly the same, this means that the extraction process mostly separates glomalin. The results of non-mycorrhizal roots indicated that a small proportion of root protein is cross-reactive with the MAb32B11 antibody. There are some evidences that MAb32B11 is slightly cross-reactive with plant compounds, non-AMF species, and non-target proteins present in large concentration, such as BSA. Additionally, we found the increasing of BRRP contents of AMF-colonized root compared to the non-mycorhizal roots. This may be as a result of uptake and storage of arginine within AMF intraradical hyphae. Considering IRHP to BRHP ratio indicates that immunoreactivity percentage enhances by rising Pb levels. Immunoreactivity indicates a molecular configuration similar to the configuration of glomalin on hyphae. Our findings are in agreement with previous observations confirming that the toxicity-induced stress by metals may be enhancing glomalin production by AMF. The sequence of the glomalin gene revealed that is likely a 60-KDa heat-shock protein (Hsp) homolog. Glomalin relation with the heat shock proteins clarifies how stress is imposed by heavy metals may rapidly increase glomalin production by AMF and its concentrations in polluted soils.
Conclusion: The high contents of glomalin along with the increasing of Pb concentrations may be explained by the overexpression of this protein. This suggests that under Pb-induced stress and the toxic effect of Pb, the fungus exerts a protective mechanism against toxicant. Therefore, glomalin as a heat shock protein can involve in the reduction of possible cytosolic damages and the transfiguration of proteins under Pb toxicity. We can conclude that glomalin may reduce toxic elements availability via their stabilization and decrease their toxicity risk to other microorganisms and plants in heavy metal polluted sites.

Keywords

Arbuscular mycorrhizal fungi; Bradford; ELISA; Glomalin

References

1- Aguilera P., Borie F., Seguel A., and Cornejo P. 2011. Fluorescence detection of aluminum in arbuscular mycorrhizal fungal structures and glomalin by using confocal laser scanning microscopy. Soil Biology and Biochemistry, 43: 2417–31.

2- Ali H., Khan E., and Sajad M.A. 2013. Phytoremediation of heavy metals—concepts and applications. Chemosphere, 91: 869–81.

3- Arnaud St., Hamel M.C., Vimard B., Caron M., and Fortin J.A. 1996. Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus Glomus intraradices in an in vitro system in absence of host roots. Mycological Research, 100: 328–332.

4- Audet P., and Charest C. 2009. Contribution of arbuscular mycorrhizal symbiosis to in vitro root metal uptake: From trace to toxic metal conditions. Botany, 87(10), 913-921.

5- Bai C., He X., Tang H., Shan B., and Zhao L. 2009. Spatial distribution of arbuscular mycorrhizal fungi, glomalin and soil enzymes under the canopy of Astragalus adsurgens Pall. in the Mu Us sand land, China. Soil Biology and Biochemistry, 41: 941-947.

6- Bedini S., Pellegrino E., Avio L., Pellegrin, S., Bazzoffi P., Argese E., and Giovannetti M. 2009. Changes in soil aggregation and glomalin-related soil protein content as affected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus intraradices. Soil Biology and Biochemistry, 41:1491-1496.

7- Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.

8- Bressano M., Curetti M., Giachero L., Gil S.V., Cabello M., March G., Ducasse D.A., and Luna C.M. 2010. Mycorrhizal fungi symbiosis as a strategy against oxidative stress in soybean plants. Journal of Plant Physiology, 167(18): 1622- 1626.

9- Cornejo P., Meier S., Borie G., Rillig M.C., and Borie F. 2008. Glomalin-related soil protein in a Mediterranean ecosystem affected by a copper smelter and its contribution to Cu and Zn sequestration. Science of the Total Environment, 406: 154-160.

10- Dhala B., Thatoib H.N., Dasc N.N., and Pandeya B.D. 2013. Chemical and microbial remediation of hexavalent chromium from contaminated soil and mining/metallurgical solid waste: a review. Journal of Hazardous Material, 250–251: 272–91.

11- Driver J.D., Holben W.E., and Rillig M.C. 2005. Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi. Soil Biology and Biochemistry, 37: 101-106.

12- Gadkar V., and Rillig M.C. 2006. The arbuscular mycorrhizal fungal protein glomalin is a putative homologof heat shock protein60. FEMS Microbiology Letters, 263: 93-101.

13- Giovannetti M., and Mosse B. 1980. An evaluation of techniques for measuring vesicular-arbuscular infection in roots. New Phytologist, 84: 489-500.

14- Gohre V., and Paszkowski U. 2006. Contribution of the arbuscular mycorrhizal symbiosis to heavy metal phytoremediation. Planta, 223: 1115–1122.

15- Gonzalez-Chavez M.C., Carrillo-Gonzalez R., Wright S.F., and Nichols K.A. 2004. The role of glomalin, a protein produced by arbuscular mycorrhizal fungi, in sequestering potentially toxic elements. Environmental Pollution, 130: 317-323.

16- Hammer E.C., and Rillig M.C. 2011. The Influence of different stresses on glomalin levels in an arbuscular mycorrhizal fungus- salinity increases glomalin content. Public Library of Science, Open-access, 6(21): 1-5 (e28426). www.plosone.org.

17- Harner M.J., Ramsey P.W., and Rillig M.C. 2004. Protein accumulation and distribution in floodplain soils and river foam. Ecology Letters, 7: 829-836.

18- Hildebrandt U., Kaldorf M. and Bothe H. 1999. The zinc violet and its colonization by arbuscular mycorrhizal fungi. Journal of Plant Physiology, 154: 709–717.

19- Janouskova M., Pavlikova D., Macek T., and Vosatka M. 2005. Arbuscular mycorrhiza decreases cadmium phytoextraction by transgenic tobacco with inserted metallothionein. Plant and Soil, 272: 29–40.

20- Janouškova M., and Vosatka M. 2005. Response to cadmium of Daucus carotahairy roots dual cultures with Glomus intraradices or Gigaspora margarita. Mycorrhiza, 15: 217-224.

21- Khan A.G. 2006. Mycorrhizoremediation—an enhanced form of phytoremediation. Journal of Zhejiang University Science B, 7(7): 503–14.

22- Kohler J., Caravaca F., del Mar Alguacil M., and Roldan A. 2009a. Elevated CO2 increases the effect of an arbuscular mycorrhizal fungus and a plant-growthpromoting rhizobacterium on structural stability of a semiarid agricultural soil under drought conditions. Soil Biology and Biochemistry, 41:1710-1716.

23- Kohler J., Caravaca F., and Roldan A. 2009b. Effect of drought on the stability of rhizosphere soil aggregates of Lactuca sativa grown in a degraded soil inoculated with PGPR and AM fungi. Applied Soil Ecology, 42: 160-165.

24- Kormanik P.P., and McGraw A.C. 1982. Quantification of vesicular-arbuscular mycorrhizae in plant roots. p. 37-45. In N.C. Schenck (ed.) Methods and principles of mycorrhizal research. American Phytopathological Society, St. Paul.

25- Leung H., Ye Z., and Wong M. 2007. Survival strategies of plants associated with arbuscular mycorrhizal fungi on toxic mine tailings. Chemosphere, 66: 905-915.

26- Lovelock C.E., Wright S.F., Clark D.A., and Ruess R.W. 2004a. Soil stocks of glomalin produced by arbuscular mycorrhizal fungi across a tropical rain forest landscape. Journal of Ecology, 92:278-287.

27- Lovelock C.E., Wright S.F., and Nichols K.A. 2004b. Using glomalin as an indicator for arbuscular mycorrhizal hyphal growth: an example from a tropical rainforest soil. Soil Biology and Biochemistry, 36: 1009-1012.

28- Lutgen E.R., Muir-Clairmont D., Graham J., and Rillig M.C. 2003. Seasonality of arbuscular mycorrhizal hyphae and glomalin in a western Montana grassland. Plant and Soil, 257:71-83.

29- Meier S., Borie F., Bolan N., and Cornejo P. 2012a. Phytoremediation of metal-polluted soils by Arbuscular Mycorrhizal Fungi. Crest, 42: 744–75.

30- Meier S., Borie F., Curaqueo G., Bolan N., and Cornejo P. 2012b. Effects of arbuscular mycorrhizal inoculation on metallophyte and agricultural plants growing at increasing copper levels. Applied Soil Ecology, 61: 280–7.

31- Millner P.D., and Kitt D.G. 1992. The Beltsville method for soilless production of vesicular arbuscular mycorrhizal fungi. Mycorrhiza, 2: 9-15.

32- Nichols K.A. 1999. Role of iron in the accumulation of glomalin, an arbuscular mycorrhizal fungal glycoprotein. Master’s Thesis, West Virginia University.

33- Nichols K.A., and Wright S.F. 2004. Contributions of fungi to soil organic matter in agroecosystems. P. 179-198. In F. Magdoff and R.R. Weil (eds.) Soil Organic Matter in Sustainable Agriculture, CRC Press, Florida.

34- Pawlowska T.E., and Charvat I. 2004. Heavy-metal stress and developmental patterns of arbuscular mycorrhizal fungi. Applied and Environmental Microbiology, 70: 6643–6649.

35- Rillig M.C. 2004. Arbuscular mycorrhizae, glomalin, and soil quality. Canadian Journal of Soil Science, 84:355-363.

36- Rillig M.C., Ramsey P.W., Morris S., and Paul E.A. 2003. Glomalin, an arbuscular mycorrhizal fungal soil protein, responds to land-use change. Plant and Soil, 253: 293-299.

37- Rillig M.C., and Steinberg P.D. 2002. Glomalin production by an arbuscular mycorrhizal fungu: a mechanism of habitat modification? Soil Biology and Biochemistry, 34: 1371-1374.

38- Roberts P., and Jones D.L. 2008. Critical evaluation of methods for determining total protein in soil solution. Soil Biology and Biochemistry, 40:1485-1495.

39- Rosier C.L., Hoye A.T., and Rillig M.C. 2006. Glomalin-related soil protein: Assessment of current detection and quantification tools. Soil Biology and Biochemistry, 38: 2205-2211.

40- Rosier C.L., Piotrowski J.S., Hoye A.T., and Rillig M.C. 2008. intraradical protein and glomalin as a tool for quantifying arbuscular mycorrhizal root colonization. Pedobiologia, 52: 41-50.

41- Schlesinger M.J. 1990. Heat-shock proteins. Journal of Biological Chemistry, 265: 12111–12114.

42- Shaabani Zenoozagh V., Aliasgharzad N., and Oustan Sh. 2013. Glomalin production by two glomeral fungi in symbiosis with corn plant under different Pb levels. International Journal of Agriculture: Research and Review, 3 (4): 854-863.

43- Steinberg P.D., and Rillig M.C. 2003. Differential decomposition of arbuscular mycorrhizal fungal hyphae and glomalin. Soil Biology and Biochemistry, 35:191-194.

44- Treseder K.K., and Turner K.M. 2007. Glomalin in ecosystems. Soil Science Society of America Journal, 71: 1257-1266.

45- USEPA. 1996. Report: recent Developments for In Situ Treatment of Metals contaminated Soils, U.S. Environmental Protection Agency, Office of Solid Waste and Emergency Response.

46- Vodnik D., Grčman H., Maček I., van Elteren J.T., and Kovačevič M. 2008. The contribution of glomalin-related soil protein to Pb and Zn sequestration in polluted soil. Science of the Total Environment, 392: 130-136.

47- Weissenhorn I., Leyval C., and Berthelin J. 1995b. Bioavailability of heavy metals and abundance of arbuscular mycorrhiza in a soil polluted by atmospheric deposition from a smelter. Biology and Fertility of Soils, 19: 22-28.

48- Whiffen L.K., Midgley D.J., and McGee P.A. 2007. Polyphenolic compounds interfere with quantification of protein in soil extracts using the Bradford method. Soil Biology and Biochemistry, 39:691-694.

49- Wright S.F. 2000. A fluorescent antibody assay for hyphae and glomalin from arbuscular mycorrhizal fungi. Plant and Soil, 226: 171-177.

50- Wright S.F., Franke-Synder M., Morton J.B., and Upadhyaya A. 1996. Time-course study and partial characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots. Plant and Soil, 181: 193-203.

51- Wright S.F., and Upadhyaya A. 1996. Extraction of an abundant and unusual protein from soil and comparison with hyphal protein of arbuscular mycorrhizal fungi. Soil Science, 161: 575-585.

52- Wright S.F., and Upadhyaya A. 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant and Soil, 198: 97-107.

53- Wright S.F., and Upadhyaya A. 1999. Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps. Mycorrhiza, 8: 283–285.