A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J and C. Presl) F.K. Meyer

Plant Journal

Volumn 73, Issue 1, 2013, Pages 131-142

  Schneider, Thomas ^a   Persson, Daniel Pergament ^b   Husted, Soren ^b   Schellenberg, Maja ^a   Gehrig, Peter ^c   Lee, Youngsook ^d   Martinoia, Enrico ^a,d   Schjoerring, Jan K ^b   Meyer, Stefan ^a  

^a UNIVERSITY OF ZURICH   (Switzerland)

^b UNIVERSITY OF COPENHAGEN   (Denmark)

^c ETH ZURICH   (Switzerland)

^d Pohang University of Science and Technology (POSTECH)   (South Korea)

Author keywords

Nicotianamine; Noccaea caerulescens; Proteomics; Zinc; Zn hyperaccumulation; Zn speciation

Indexed keywords

2D-LC-MS/MS; COUPLED PLASMA; EPIDERMAL CELLS; EPIDERMAL TISSUES; GLUTATHIONE-S-TRANSFERASE; HIGH CONCENTRATION; HYPERACCUMULATION; LEAF EPIDERMIS; LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY; LIVING ORGANISMS; MESOPHYLL TISSUES; MOLECULAR MECHANISM; NICOTIANAMINE; NOCCAEA CAERULESCENS; PLANT SPECIES; PROTEOMICS; TOXICITY SYMPTOMS; ZN SPECIATIONS;

CHROMATOGRAPHY; INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY; MOLECULAR BIOLOGY; ORGANIC ACIDS; PROTEINS; TISSUE; BIOLOGY; LIQUID CHROMATOGRAPHY; MASS SPECTROMETERS; MASS SPECTROMETRY; SIZE EXCLUSION CHROMATOGRAPHY; TRACE ELEMENTS;

ZINC;

NOCCAEA CAERULESCENS;

PLANT PROTEIN; ZINC;

BRASSICACEAE; MASS FRAGMENTOGRAPHY; MESOPHYLL CELL; METABOLISM; PHYSIOLOGY; PLANT EPIDERMIS; PLANT LEAF; PROCEDURES; PROTEOMICS;

BRASSICACEAE; GAS CHROMATOGRAPHY-MASS SPECTROMETRY; MESOPHYLL CELLS; PLANT EPIDERMIS; PLANT LEAVES; PLANT PROTEINS; PROTEOMICS; ZINC;

EID: 84871980380     PISSN: 09607412     EISSN: 1365313X     Source Type: Journal    
DOI: 10.1111/tpj.12022     Document Type: Article

References (89)

1. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-410.
2. Andreini, C., Banci, L., Bertini, I. and Rosato, A. (2006) Counting the zincproteins encoded in the human genome. J. Proteome Res. 5, 196-201.
3. Assunçao, A.G.L., Martins, P.D.C., De Folter, S., Vooijs, R., Schat, H. and Aarts, M.G.M. (2001) Elevated expression of metal transporter genes in three accessions of the metal hyperaccumulator Thlaspi caerulescens. Plant, Cell Environ. 24, 217-226.
4. Bantscheff, M., Schirle, M., Sweetman, G., Rick, J. and Kuster, B. (2007) Quantitative mass spectrometry in proteomics: A critical review. Anal. Bioanal. Chem. 389, 1017-1031.
5. Becher, M., Talke, I.N., Krall, L. and Kramer, U. (2004) Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. Plant J. 37, 251-268.
6. Bernard, C., Roosens, N., Czernic, P., Lebrun, M. and Verbruggen, N. (2004) A novel CPx-ATPase from the cadmium hyperaccumulator Thlaspi caerulescens. FEBS Lett. 569, 140-148.
7. Bouchereau, A., Aziz, A., Larher, F. and Martin-Tanguy, J. (1999) Polyamines and environmental challenges: Recent development. Plant Sci. 140, 103-125.
8. Broadley, M.R., White, P.J., Hammond, J.P., Zelko, I. and Lux, A. (2007) Zinc in plants. New Phytol. 173, 677-702.
9. Callahan, D.L., Baker, A.J., Kolev, S.D. and Wedd, A.G. (2006) Metal ion ligands in hyperaccumulating plants. J. Biol. Inorg. Chem. 11, 2-12.
10. Callahan, D.L., Kolev, S.D., O'Hair, R.A., Salt, D.E. and Baker, A.J. (2007) Relationships of nicotianamine and other amino acids with nickel, zinc and iron in Thlaspi hyperaccumulators. New Phytol. 176, 836-848.
11. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K. and Madden, T.L. (2009) BLAST+: Architecture and applications. BMC Bioinformatics, 10, 421.
12. Chao, Y. and Fu, D. (2004) Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB. J. Biol. Chem. 279, 12043-12050.
13. Clemens, S. (2006) Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie, 88, 1707-1719.
14. Cosio, C., Martinoia, E. and Keller, C. (2004) Hyperaccumulation of cadmium and zinc in Thlaspi caerulescens and Arabidopsis halleri at the leaf cellular level. Plant Physiol. 134, 716-725.
15. Cui, S., Huang, F., Wang, J., Ma, X., Cheng, Y. and Liu, J. (2005) A proteomic analysis of cold stress responses in rice seedlings. Proteomics, 5, 3162-3172.
16. Cuypers, A., Vangronsveld, J. and Clijsters, H. (2001) The redox status of plant cells (AsA and GSH) is sensitive to zinc imposed oxidative stress in roots and primary leaves of Phaseolus vulgaris. Plant Physiol. Biochem. 39, 657-664.
17. Desbrosses-Fonrouge, A.G., Voigt, K., Schröder, A., Arrivault, S., Thomine, S. and Krämer, U. (2005) Arabidopsis thaliana MTP1 is a Zn transporter in the vacuolar membrane which mediates Zn detoxification and drives leaf Zn accumulation. FEBS Lett. 579, 4165-4174.
18. DiDonato, R.J. Jr, Roberts, L.A., Sanderson, T., Eisley, R.B. and Walker, E.L. (2004) Arabidopsis Yellow Stripe-Like2 (YSL2): A metal-regulated gene encoding a plasma membrane transporter of nicotianamine-metal complexes. Plant J. 39, 403-414.
19. Dietz, K.J., Schramm, M., Betz, M., Busch, H., Dürr, C. and Martinoia, E. (1992) Characterization of the epidermis from barley primary leaves. Planta, 187, 425-430.
20. Dixon, D.P., Davis, B.G. and Edwards, R. (2002) Functional divergence in the glutathione transferase superfamily in plants. Identification of two classes with putative functions in redox homeostasis in Arabidopsis thaliana. J. Biol. Chem. 277, 30859-30869.
21. Dixon, D.P., Hawkins, T., Hussey, P.J. and Edwards, R. (2009) Enzyme activities and subcellular localization of members of the Arabidopsis glutathione transferase superfamily. J. Exp. Bot. 60, 1207-1218.
22. Dräger, D.B., Desbrosses-Fonrouge, A.G., Krach, C., Chardonnens, A.N., Meyer, R.C., Saumitou-Laprade, P. and Krämer, U. (2004) Two genes encoding Arabidopsis halleri MTP1 metal transport proteins co-segregate with zinc tolerance and account for high MTP1 transcript levels. Plant J. 39, 425-439.
23. Elias, J.E. and Gygi, S.P. (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods, 4, 207-214.
24. Espartero, J., Pintor-Toro, J.A. and Pardo, J.M. (1994) Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress. Plant Mol. Biol. 25, 217-227.
25. Freeman, J.L., Persans, M.W., Nieman, K., Albrecht, C., Peer, W., Pickering, I.J. and Salt, D.E. (2004) Increased glutathione biosynthesis plays a role in nickel tolerance in Thlaspi nickel hyperaccumulators. Plant Cell, 16, 2176-2191.
26. Gendre, D., Czernic, P., Conejero, G., Pianelli, K., Briat, J.F., Lebrun, M. and Mari, S. (2007) TcYSL3, a member of the YSL gene family from the hyper-accumulator Thlaspi caerulescens, encodes a nicotianamine-Ni/Fe transporter. Plant J. 49, 1-15.
27. Grubb, C.D. and Abel, S. (2006) Glucosinolate metabolism and its control. Trends Plant Sci. 11, 89-100.
28. Guffanti, A.A., Wei, Y., Rood, S.V. and Krulwich, T.A. (2002) An antiport mechanism for a member of the cation diffusion facilitator family: Divalent cations efflux in exchange for K+ and H+. Mol. Microbiol. 45, 145-153.
29. Gustin, J.L., Loureiro, M.E., Kim, D., Na, G., Tikhonova, M. and Salt, D.E. (2009) MTP1-dependent Zn sequestration into shoot vacuoles suggests dual roles in Zn tolerance and accumulation in Zn-hyperaccumulating plants. Plant J. 57, 1116-1127.
30. Hammond, J.P., Bowen, H.C., White, P.J., Mills, V., Pyke, K.A., Baker, A.J., Whiting, S.N., May, S.T. and Broadley, M.R. (2006) A comparison of the Thlaspi caerulescens and Thlaspi arvense shoot transcriptomes. New Phytol. 170, 239-260.
31. Hanikenne, M. and Nouet, C. (2011) Metal hyperaccumulation and hypertolerance: A model for plant evolutionary genomics. Curr. Opin. Plant Biol. 14, 252-259.
32. Hanikenne, M., Talke, I.N., Haydon, M.J., Lanz, C., Nolte, A., Motte, P., Kroymann, J., Weigel, D. and Krämer, U. (2008) Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4. Nature, 453, 391-395.
33. Haydon, M.J. and Cobbett, C.S. (2007a) Transporters of ligands for essential metal ions in plants. New Phytol. 174, 499-506.
34. Haydon, M.J. and Cobbett, C.S. (2007b) A novel major facilitator superfamily protein at the tonoplast influences zinc tolerance and accumulation in Arabidopsis. Plant Physiol. 143, 1705-1719.
35. Haydon, M.J., Kawachi, M., Wirth, M., Hillmer, S., Hell, R. and Krämer, U. (2012) Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis. Plant Cell, 24, 724-737.
36. Hussain, D., Haydon, M.J., Wang, Y., Wong, E., Sherson, S.M., Young, J., Camakaris, J., Harper, J.F. and Cobbett, C.S. (2004) P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis. Plant Cell, 16, 1327-1339.
37. Husted, S., Persson, D.P., Laursen, K.H., Hansen, T.H., Pedas, P., Schiller, M., Hegelund, J.N. and Schjoerring, J.K. (2011) The role of atomic spectrometry in plant science. J. Anal. At. Spectrom. 26, 52-79.
38. Kawachi, M., Kobae, Y., Mimura, T. and Maeshima, M. (2008) Deletion of a histidine-rich loop of AtMTP1, a vacuolar Zn2+/H+ antiporter of Arabidopsis thaliana, stimulates the transport activity. J. Biol. Chem. 283, 8374-8383.
39. Keller, A., Nesvizhskii, A.I., Kolker, E. and Aebersold, R. (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383-5392.
40. Klatte, M., Schuler, M., Wirtz, M., Fink-Straube, C., Hell, R. and Bauer, P. (2009) The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses. Plant Physiol. 150, 257-271.
41. Kobae, Y., Uemura, T., Sato, M.H., Ohnishi, M., Mimura, T., Nakagawa, T. and Maeshima, M. (2004) Zinc transporter of Arabidopsis thaliana AtMTP1 is localized to vacuolar membranes and implicated in zinc homeostasis. Plant Cell Physiol. 45, 1749-1758.
42. Krämer, U. (2010) Metal hyperaccumulation in plants. Annu. Rev. Plant Biol. 61, 517-534.
43. Krämer, U., Pickering, I.J., Prince, R.C., Raskin, I. and Salt, D.E. (2000) Subcellular localization and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species. Plant Physiol. 122, 1343-1353.
44. Krämer, U., Talke, I.N. and Hanikenne, M. (2007) Transition metal transport. FEBS Lett. 581, 2263-2272.
45. Küpper, H. and Kochian, L.V. (2010) Transcriptional regulation of metal transport genes and mineral nutrition during acclimatization to cadmium and zinc in the Cd/Zn hyperaccumulator, Thlaspi caerulescens (Ganges population). New Phytol. 185, 114-129.
46. Küpper, H., Jie Zhao, F. and McGrath, S.P. (1999) Cellular compartmentation of zinc in leaves of the hyperaccumulator Thlaspi caerulescens. Plant Physiol. 119, 305-312.
47. Lasat, M.M., Baker, A. and Kochian, L.V. (1996) Physiological characterization of root Zn2+ absorption and translocation to shoots in Zn hyperaccumulator and nonaccumulator species of Thlaspi. Plant Physiol. 112, 1715-1722.
48. Lasat, M.M., Baker, A.J. and Kochian, L.V. (1998) Altered Zn compartmentation in the root symplasm and stimulated Zn absorption into the leaf as mechanisms involved in Zn hyperaccumulation in Thlaspi caerulescens. Plant Physiol. 118, 875-883.
49. Lee, J., Reeves, R.D., Brookes, R.R. and Jaffre, T. (1978) The relation between nickel and citric acid in some nickel-accumulating plants. Phytochemistry, 17, 1033-1035.
50. Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2-DDCT method. Methods, 25, 402-408.
51. Ma, B., Zhang, K., Hendrie, C., Liang, C., Li, M., Doherty-Kirby, A. and Lajoie, G. (2003) PEAKS: Powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2337-2342.
52. Ma, J.F., Ueno, D., Zhao, F.J. and McGrath, S.P. (2005) Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of Thlaspi caerulescens. Planta, 220, 731-736.
53. Mari, S., Gendre, D., Pianelli, K., Ouerdane, L., Lobinski, R., Briat, J.F., Lebrun, M. and Czernic, P. (2006) Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens. J. Exp. Bot. 57, 4111-4122.
54. Marschner, H. (1995) Mineral Nutrition of Higher Plants. London: Academic Press.
55. Mills, R.F., Krijger, G.C., Baccarini, P.J., Hall, J.L. and Williams, L.E. (2003) Functional expression of AtHMA4, a P1B-type ATPase of the Zn/Co/Cd/Pb subclass. Plant J. 35, 164-176.
56. Milner, M.J. and Kochian, L.V. (2008) Investigating heavy-metal hyperaccumulation using Thlaspi caerulescens as a model system. Ann. Bot. 102, 3-13.
57. Montarges-Pelletier, E., Chardot, V., Echevarria, G., Michot, L.J., Bauer, A. and Morel, J.L. (2008) Identification of nickel chelators in three hyperaccumulating plants: An X-ray spectroscopic study. Phytochemistry, 69, 1695-1709.
58. van de Mortel, J.E., Almar Villanueva, L., Schat, H., Kwekkeboom, J., Coughlan, S., Moerland, P.D., Ver Loren van Themaat, E., Koornneef, M. and Aarts, M.G. (2006) Large expression differences in genes for iron and zinc homeostasis, stress response, and lignin biosynthesis distinguish roots of Arabidopsis thaliana and the related metal hyperaccumulator Thlaspi caerulescens. Plant Physiol. 142, 1127-1147.
59. van de Mortel, J.E., Schat, H., Moerland, P.D., Ver Loren van Themaat, E., van der Ent, S., Blankestijn, H., Ghandilyan, A., Tsiatsiani, S. and Aarts, M.G. (2008) Expression differences for genes involved in lignin, glutathione and sulphate metabolism in response to cadmium in Arabidopsis thaliana and the related Zn/Cd-hyperaccumulator Thlaspi caerulescens. Plant, Cell Environ. 31, 301-324.
60. Narindrasorasak, S., Yao, P. and Sarkar, B. (2003) Protein disulfide isomerase, a multifunctional protein chaperone, shows copper-binding activity. Biochem. Biophys. Res. Commun. 311, 405-414.
61. Nesvizhskii, A.I., Keller, A., Kolker, E. and Aebersold, R. (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646-4658.
62. O'Lochlainn, S.O., Bowen, H.C., Fray, R.G., Hammond, J.P., King, G.J., White, P.J., Graham, N.S. and Broadley, M.R. (2011) Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens. PLoS ONE, 6, e17814.
63. Papoyan, A. and Kochian, L.V. (2004) Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase. Plant Physiol. 136, 3814-3823.
64. Pence, N.S., Larsen, P.B., Ebbs, S.D., Letham, D.L., Lasat, M.M., Garvin, D. F., Eide, D. and Kochian, L.V. (2000) The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proc. Natl Acad. Sci. USA, 97, 4956-4960.
65. Persson, D.P., Hansen, T.H., Laursen, K.H., Schjoerring, J.K. and Husted, S. (2009) Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley grain tissues using SEC-ICP-MS and IP-ICP-MS. Metallomics, 1, 418-426.
66. Rensing, C., Mitra, B. and Rosen, B.P. (1997) Insertional inactivation of dsbA produces sensitivity to cadmium and zinc in Escherichia coli. J. Bacteriol. 179, 2769-2771.
67. Rigola, D., Fiers, M., Vurro, E. and Aarts, M.G. (2006) The heavy metal hyperaccumulator Thlaspi caerulescens expresses many species-specific genes, as identified by comparative expressed sequence tag analysis. New Phytol. 170, 753-765.
68. Roth, U., von Roepenack-Lahaye, E. and Clemens, S. (2006) Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+. J. Exp. Bot. 57, 4003-4013.
69. Salt, D.E., Prince, R.C., Baker, A.J.M., Raskin, I. and Pickering, I.J. (1999) Zinc ligands in the metal hyperaccumulator Thlaspi caerulescens as determined using X-ray absorption spectroscopy. Environ. Sci. Technol. 33, 713-717.
70. Sappl, P.G., Carroll, A.J., Clifton, R., Lister, R., Whelan, J., Harvey Millar, A. and Singh, K.B. (2009) The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing multiple genes results in altered metabolic sensitivity to oxidative stress. Plant J. 58, 53-68.
71. Sarret, G., Saumitou-Laprade, P., Bert, V., Proux, O., Hazemann, J.L., Traverse, A., Marcus, M.A. and Manceau, A. (2002) Forms of zinc accumulated in the hyperaccumulator Arabidopsis halleri. Plant Physiol. 130, 1815-1826.
72. Schaaf, G., Ludewig, U., Erenoglu, B.E., Mori, S., Kitahara, T. and von Wiren, N. (2004) ZmYS1 functions as a proton-coupled symporter for phytosiderophore-and nicotianamine-chelated metals. J. Biol. Chem. 279, 9091-9096.
73. Schat, H., Llugany, M., Vooijs, R., Hartley-Whitaker, J. and Bleeker, P.M. (2002) The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and non-hyperaccumulator metallophytes. J. Exp. Bot. 53, 2381-2392.
74. Song, W.Y., Choi, K.S., Kim do, Y. et al. (2010) Arabidopsis PCR2 is a zinc exporter involved in both zinc extrusion and long-distance zinc transport. Plant Cell, 22, 2237-2252.
75. Suh, M.C., Samuels, A.L., Jetter, R., Kunst, L., Pollard, M., Ohlrogge, J. and Beisson, F. (2005) Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol. 139, 1649-1665.
76. Takahashi, M., Terada, Y., Nakai, I., Nakanishi, H., Yoshimura, E., Mori, S. and Nishizawa, N.K. (2003) Role of nicotianamine in the intracellular delivery of metals and plant reproductive development. Plant Cell, 15, 1263-1280.
77. Talke, I.N., Hanikenne, M. and Kramer, U. (2006) Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant Physiol. 142, 148-167.
78. Tolra, R.P., Poschenrieder, C., Alonso, R., Barcelo, D. and Barcelo, J. (2001) Influence of zinc hyperaccumulation on glucosinolates in Thlaspi caerulescens. New Phytol. 151, 621-626.
79. Ueno, D., Ma, J.F., Iwashita, T., Zhao, F.J. and McGrath, S.P. (2005) Identification of the form of Cd in the leaves of a superior Cd-accumulating ecotype of Thlaspi caerulescens using 113Cd-NMR. Planta, 221, 928-936.
80. Ueno, D., Milner, M.J., Yamaji, N., Yokosho, K., Koyama, E., Clemencia Zambrano, M., Kaskie, M., Ebbs, S., Kochian, L.V. and Ma, J.F. (2011) Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of Thlaspi caerulescens. Plant J. 66, 852-862.
81. Vallee, B.L. and Auld, D.S. (1990) Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry, 29, 5647-5659.
82. Verbruggen, N., Hermans, C. and Schat, H. (2009) Molecular mechanisms of metal hyperaccumulation in plants. New Phytol. 181, 759-776.
83. Verret, F., Gravot, A., Auroy, P., Leonhardt, N., David, P., Nussaume, L., Vavasseur, A. and Richaud, P. (2004) Overexpression of AtHMA4 enhances root-to-shoot translocation of zinc and cadmium and plant metal tolerance. FEBS Lett. 576, 306-312.
84. Wang, K.L., Li, H. and Ecker, J.R. (2002) Ethylene biosynthesis and signaling networks. Plant Cell, 14(Suppl.), S131-S151.
85. Weber, M., Harada, E., Vess, C., Roepenack-Lahaye, E. and Clemens, S. (2004) Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors. Plant J. 37, 269-281.
86. Weber, M., Trampczynska, A. and Clemens, S. (2006) Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+-hypertolerant facultative metallophyte Arabidopsis halleri. Plant, Cell Environ. 29, 950-963.
87. Weckx, J.E.J. and Clijsters, H.M.M. (1997) Zn phytotoxicity induces oxidative stress in primary leaves of Phaseolus vulgaris. Plant Physiol. Biochem. 35, 405-410.
88. Yuan, J.S., Reed, A., Chen, F. and Steward, C.N. (2006) Statistical analysis of real-time PCR. BMC Bioinformatics, 7, 85.
89. van der Zaal, B., Neuteboom, L., Pinas, J., Chardonnens, A., Schat, H., Verkleij, J. and Hooykaas, P. (1999) Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animals can lead to enhanced zinc resistance and accumulation. Plant Physiol. 119, 1047-1055.