caa a22 4500 46789499X CHVBK 20180406152800.0 cr unu---uuuuu 170328e20061201xx s 000 0 eng 10.1007/s11103-006-9060-8 doi (NATIONALLICENCE)springer-10.1007/s11103-006-9060-8 Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli [Elektronische Daten] [Bala Rathinasabapathi, Shan Wu, Sabarinath Sundaram, Jean Rivoal, Mrittunjai Srivastava, Lena Ma] Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI's role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction. Springer Science+Business Media B.V., 2006 Arsenic tolerance nationallicence Escherichia coli nationallicence Expression cloning nationallicence Glyceraldehyde-3-phosphate nationallicence Glycolysis nationallicence Pteris vittata nationallicence Triosephosphate isomerase nationallicence cTPI : Cytosolic triosephosphate isomerase nationallicence DTT : Dithiothereitol nationallicence EDTA : Ethylene diamine tetraacetic acid nationallicence pTPI : Plastidic triosephosphate isomerase nationallicence GAPDH : Glyceraldehyde-3-phosphate dehydrogenase nationallicence SDS-PAGE : Sodium dodecyl sulfate-polyacrylamide gel electrophoresis nationallicence 3-PGA : 3-Phosphoglycerate nationallicence TPI : Triosephosphate isomerase nationallicence Rathinasabapathi Bala Horticultural Sciences Department, University of Florida, Hull Road, PO Box 110 690, 32611-0690, Gainesville, FL, USA aut Wu Shan Horticultural Sciences Department, University of Florida, Hull Road, PO Box 110 690, 32611-0690, Gainesville, FL, USA aut Sundaram Sabarinath Horticultural Sciences Department, University of Florida, Hull Road, PO Box 110 690, 32611-0690, Gainesville, FL, USA aut Rivoal Jean Institut de Recherche en Biologie Végétale, Départment de Sciences Biologiques, Université de Montréal, H1X 2B2, Montréal, QC, Canada aut Srivastava Mrittunjai Department of Soil and Water Science, University of Florida, 32611-0690, Gainesville, FL, USA aut Ma Lena Department of Soil and Water Science, University of Florida, 32611-0690, Gainesville, FL, USA aut Plant Molecular Biology Kluwer Academic Publishers 62/6(2006-12-01), 845-857 0167-4412 62:6<845 2006 62 11103 https://doi.org/10.1007/s11103-006-9060-8 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11103-006-9060-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Rathinasabapathi Bala Horticultural Sciences Department, University of Florida, Hull Road, PO Box 110 690, 32611-0690, Gainesville, FL, USA aut NATIONALLICENCE 700 1- Wu Shan Horticultural Sciences Department, University of Florida, Hull Road, PO Box 110 690, 32611-0690, Gainesville, FL, USA aut NATIONALLICENCE 700 1- Sundaram Sabarinath Horticultural Sciences Department, University of Florida, Hull Road, PO Box 110 690, 32611-0690, Gainesville, FL, USA aut NATIONALLICENCE 700 1- Rivoal Jean Institut de Recherche en Biologie Végétale, Départment de Sciences Biologiques, Université de Montréal, H1X 2B2, Montréal, QC, Canada aut NATIONALLICENCE 700 1- Srivastava Mrittunjai Department of Soil and Water Science, University of Florida, 32611-0690, Gainesville, FL, USA aut NATIONALLICENCE 700 1- Ma Lena Department of Soil and Water Science, University of Florida, 32611-0690, Gainesville, FL, USA aut NATIONALLICENCE 773 0- Plant Molecular Biology Kluwer Academic Publishers 62/6(2006-12-01), 845-857 0167-4412 62:6<845 2006 62 11103 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer