, Volume 27, Issue 3, pp 591–599 | Cite as

Characterization of Hg-phytochelatins complexes in vines (Vitis vinifera cv Malbec) as defense mechanism against metal stress

  • Adrian A. Spisso
  • Soledad Cerutti
  • Fernanda Silva
  • Pablo H. PachecoEmail author
  • Luis D. MartinezEmail author


An approach to understand vines (Vitis vinifera) defense mechanism against heavy metal stress by isolation and determination of Hg-phytochelatins (PCs) complexes was performed. PCs are important molecules involved in the control of metal concentration in plants. PCs complex toxic metals through −SH groups and stores them inside cells vacuole avoiding any toxic effect of free metals in the cytosol. The Hg-PCs identification was achieved by determination of Hg and S as hetero-tagged atoms. A method involving two-dimensional chromatographic analysis coupled to atomic spectrometry and confirmation by tandem mass spectrometry is proposed. An approach involving size exclusion chromatography coupled to inductively coupled plasma mass spectrometry on roots, stems, and leaves extracts describing Hg distribution according to molecular weight and sulfur associations is proposed for the first time. Medium–low molecular weight Hg–S associations of 29–100 kDa were found, suggesting PCs presence. A second approach employing reversed-phase chromatography coupled to atomic fluorescence spectrometry analysis allowed the determination of Hg-PCs complexes within the mentioned fractions. Chromatograms showed Hg-PC2, Hg-PC3 and Hg-PC4 presence only in roots. Hg-PCs presence in roots was confirmed by ESI–MS/MS analysis.


Vitis vinifera Mercury Phytochelatins Metal stress 



This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (FONCYT) (PICTBID), Universidad Nacional de San Luis (Argentina), Universidad Nacional de Cuyo (Argentina), and Instituto Nacional de Técnicas Agropecuarias.


  1. Afton SE, Catron B, Caruso JA (2009) Elucidating the selenium and arsenic metabolic pathways following exposure to the non-hyperaccumulating Chlorophytum comosum, spider plant. J Exp Bot 60:1289–1297PubMedCrossRefPubMedCentralGoogle Scholar
  2. Ahner BA, Price NM, Morel FMM (1994) Phytochelatin production by marine phytoplankton at low free metal ion concentrations: laboratory studies and field data from Massachusetts Bay. Proc Natl Acad Sci USA 91:8433–8436PubMedCrossRefPubMedCentralGoogle Scholar
  3. Carrasco-Gil S, Álvarez-Fernández A, Sobrino-Plata J, Millán R, Carpena-Ruiz RO, Leduc DL, Andrews JC, Abadía J, Hernández LE (2011) Complexation of Hg with phytochelatins is important for plant Hg tolerance. Plant Cell Environ 34:778–791PubMedCrossRefGoogle Scholar
  4. Chen L, Yang L, Wang Q (2009) In vivo phytochelatins and Hg–phytochelatin complexes in Hg-stressed Brassica chinensis L. Metallomics 1:101–106CrossRefGoogle Scholar
  5. Chen J, Yang ZM (2012) Mercury toxicity, molecular response and tolerance in higher plants. Biometals 25:847–857PubMedCrossRefGoogle Scholar
  6. Chopin EIB, Marin B, Mkoungafoko R, Rigaux A, Hopgood MJ, Delannoy E, Cancès B, Laurain M (2008) Factors affecting distribution and mobility of trace elements (Cu, Pb, Zn) in a perennial grapevine (Vitis vinifera L.) in the Champagne region of France. Environ Pollut 156:1092–1098PubMedCrossRefGoogle Scholar
  7. Dago A, González-García O, Ariño C, Díaz-Cruz JM, Esteban M (2011) Characterization of Hg(II) binding with different length phytochelatins using liquid chromatography and amperometric detection. Anal Chim Acta 695:51–57PubMedCrossRefGoogle Scholar
  8. Dai S, Ren D, Chou CL, Finkelman RB, Seredin VV, Zhou Y (2012) Geochemistry of trace elements in Chinese coals: a review of abundances, genetic types, impacts on human health, and industrial utilization. Int J Coal Geol 94:3–21CrossRefGoogle Scholar
  9. de Knecht JA, van Dillen M, Koevoets PLM, Schat H, Verkleij JAC, Ernst WHO (1994) Phytochelatins in cadmium-sensitive and cadmium-tolerant Silene vulgaris. Chain length distribution and sulfide incorporation. Plant Physiol 104:255–261PubMedPubMedCentralGoogle Scholar
  10. Domagalski JL, Alpers CN, Slotton DG, Suchanek TH, Ayers SM (2004) Mercury and methylmercury concentrations and loads in the Cache Creek watershed, California. Sci Total Environ 327:215–237PubMedCrossRefGoogle Scholar
  11. Fabani MP, Toro ME, Vázquez F, Díaz MP, Wunderlin DA (2009) Differential absorption of metals from soil to diverse vine varieties from the valley of tulum (Argentina): consequences to evaluate wine provenance. J Agric Food Chem 57:7409–7416PubMedCrossRefGoogle Scholar
  12. Fergusson JE (1990) The heavy elements: chemistry environmental impact and health effects. Pergamon, OxfordGoogle Scholar
  13. Finkelman RB (2004) Potential health impacts of burning coal beds and waste banks. Intern J Coal Geol 59:19–24CrossRefGoogle Scholar
  14. Finkelman RB, Orem W, Castranova V, Tatu CA, Belkin HE, Zheng B, Lerch HE, Maharaj SV, Bates AL (2002) Health impacts of coal and coal use: possible solutions. Int J Coal Geol 50:425–443CrossRefGoogle Scholar
  15. Gekeler W, Grill E, Winnacker EL, Zenk MH (1988) Algae sequester heavy metals via synthesis of phytochelatin complexes. Arch Microbiol 150:197–202CrossRefGoogle Scholar
  16. Grill E, Winnacker EL, Zenk MH (1987) Phytochelatins, a class of heavy-metal-binding peptides from plants, are functionally analogous to metallothioneins. Proc Natl Acad Sci USA 84:439–443PubMedCrossRefPubMedCentralGoogle Scholar
  17. Habeeb AF (1972) Reaction of protein sulfhydryl groups with Ellman’s reagent. Methods Enzymol 25:457–464PubMedCrossRefGoogle Scholar
  18. Hirata K, Tsujimoto Y, Namba T, Ohta T, Hirayanagi N, Miyasaka H, Zenk MH, Miyamoto K (2001) Strong induction of phytochelatin synthesis by zinc in marine green alga, Dunaliella tertiolecta. J Biosci Bioeng 92:24–29PubMedGoogle Scholar
  19. Iglesia-Turiño S, Febrero A, Jauregui O, Caldelas C, Araus JL, Bort J (2006) Detection and quantification of unbound phytochelatin 2 in plant extracts of Brassica napus grown with different levels of mercury. Plant Physiol 142:742–749PubMedCrossRefPubMedCentralGoogle Scholar
  20. Kawakami SK, Gledhill M, Achterberg EP (2006) Determination of phytochelatins and glutathione in phytoplankton from natural waters using HPLC with fluorescence detection. TrAC, Trends Anal Chem 25:133–142CrossRefGoogle Scholar
  21. Komárek M, Čadková E, Chrastný V, Bordas F, Bollinger JC (2010) Contamination of vineyard soils with fungicides: a review of environmental and toxicological aspects. Environ Int 36:138–151PubMedCrossRefGoogle Scholar
  22. Kondo N, Isobe M, Imai K, Goto T (1985) Synthesis of metallothionein-like peptides cadystin A and B occurring in a fission yeast, and their isomers. Agric Biol Chem 49:71–83CrossRefGoogle Scholar
  23. Leita L, Mondini C, Nobili M, Simoni A, Sequi P (1998) Heavy metal content in xylem sap (Vitis vinifera) from mining and smelting areas. Environ Monit Assess 50:189–200CrossRefGoogle Scholar
  24. Leopold I, Gunther D (1997) Investigation of the binding properties of heavy-metal-peptide complexes in plant cell cultures using HPLC-ICP-MS. Fresenius J Anal Chem 359:364–370CrossRefGoogle Scholar
  25. Marczenko Z, Lobinski R (1996) Spectrochemical trace analysis for metals and metalloids. Elsevier, AmsterdamGoogle Scholar
  26. Minocha R, Thangavel P, Dhankher OP, Long S (2008) Separation and quantification of monothiols and phytochelatins from a wide variety of cell cultures and tissues of trees and other plants using high performance liquid chromatography. J Chromatogr A 1207:72–83PubMedCrossRefGoogle Scholar
  27. Mounicou S, Meija J, Caruso J (2004) Preliminary studies on selenium-containing proteins in Brassica juncea by size exclusion chromatography and fast protein liquid chromatography coupled to ICP-MS. Analyst 129:116–123PubMedCrossRefGoogle Scholar
  28. Newton GL, Dorian R, Fahey RC (1981) Analysis of biological thiols: derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatography. Anal Biochem 114:383–387PubMedCrossRefGoogle Scholar
  29. Rellán-Álvarez R, Ortega-Villasante C, Álvarez-Fernández A, del Campo FFD, Hernández LE (2006) Stress responses of Zea mays to cadmium and mercury. Plant Soil 279:41–50CrossRefGoogle Scholar
  30. Rijstenbil JW, Wijnholds JA (1996) HPLC analysis of nonprotein thiols in planktonic diatoms: pool size, redox state and response to copper and cadmium exposure. Mar Biol 127:45–54CrossRefGoogle Scholar
  31. Saleem-Saif M, Midrar-Ul-Haq RA, Memon KS (2005) Heavy metals contamination through industrial effluent to irrigation water and soil in Korangi area of Karachi (Pakistan). Int J Agr Biol 7:646–648Google Scholar
  32. Sanitá di Toppi LP, Gabbrielli R (1999) Response to cadmium in higher plants. Environ Exp Bot 41:105–130CrossRefGoogle Scholar
  33. Sobrino-Plata J, Ortega-Villasante C, Flores-Caceres ML, Escobar C, Del Campo FF, Hernandez LE (2009) Differential alterations of antioxidant defenses as bioindicators of mercury and cadmium toxicity in alfalfa. Chemosphere 77:946–954PubMedCrossRefGoogle Scholar
  34. Sobrino-Plata J, Herrero J, Carrasco-Gil S, Pérez-Sanz A, Lobo C, Escobar C, Millán R, Hernández LE (2013) Specific stress responses to cadmium, arsenic and mercury appear in the metallophyte Silene vulgaris when grown hydroponically. RSC Adv 3:4736CrossRefGoogle Scholar
  35. Sobrino-Plata J, Carrasco-Gil S, Abadía J, Escobar C, Álvarez-Fernández A, Hernández LE (2014) The role of glutathione in mercury tolerance resembles its function under cadmium stress in Arabidopsis. Metallomics 6:356–366PubMedCrossRefGoogle Scholar
  36. Spisso A, Pacheco PH, Gómez FJV, Silva MF, Martinez LD (2013) Risk assessment on irrigation of vitis vinifera L. cv malbec with Hg contaminated waters. Environ Sci Technol 47:6606–6613PubMedGoogle Scholar
  37. Thapa G, Sadhukhan A, Panda SK, Sahoo L (2012) Molecular mechanistic model of plant heavy metal tolerance. Biometals 25:489–505PubMedCrossRefGoogle Scholar
  38. Todic S, Beslic Z, Lakic N, Tesic D (2006) Lead, mercury, and nickel in grapevine, Vitis vinifera L., in polluted and nonpolluted regions. Bull Environ Contam Toxicol 77:665–670PubMedCrossRefGoogle Scholar
  39. Vacchina V, Chassaigne H, Oven M, Zenk MH, Łobiński R (1999) Characterisation and determination of phytochelatins in plant extracts by electrospray tandem mass spectrometry. Analyst 124:1425–1430CrossRefGoogle Scholar
  40. Wilcox J, Rupp E, Ying SC, Lim DH, Negreira AS, Kirchofer A, Feng F, Lee K (2012) Mercury adsorption and oxidation in coal combustion and gasification processes. Int J Coal Geol 90–91:4–20CrossRefGoogle Scholar
  41. Wood BA, Feldmann J (2012) Quantification of phytochelatins and their metal (loid) complexes: critical assessment of current analytical methodology. Anal Bioanal Chem 402:3299–3309PubMedCrossRefGoogle Scholar
  42. Wu GH, Cao SS (2010) Mercury and cadmium contamination of irrigation water, sediment, soil and shallow groundwater in a wastewater-irrigated field in Tianjin, China. Bull Environ Contam Toxicol 84:336–341PubMedCrossRefGoogle Scholar
  43. Zeng X, Ma LQ, Qiu R, Tang Y (2009) Responses of non-protein thiols to Cd exposure in Cd hyperaccumulator Arabis paniculata Franch. Environ Exp Bot 66:242–248CrossRefGoogle Scholar
  44. Zenk MH (1996) Heavy metal detoxification in higher plants—a review. Gene 179:21–30PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Instituto de Química de San Luis (INQUISAL–CONICET)San LuisArgentina
  2. 2.Instituto de Biología Agrícola de Mendoza (IBAM–CONICET)MendozaArgentina

Personalised recommendations