Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance |
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Authors: | Florencia del VisoAdriana C. Casabuono Alicia S. CoutoH. Esteban Hopp Andrea F. Puebla Ruth A. Heinz |
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Affiliation: | a Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA Castelar) (1686), Hurlingham, Provincia de Buenos Aires, Argentina b CIHIDECAR, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (1428), C.A. Buenos Aires, Argentina c Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina |
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Abstract: | We have previously reported the molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of Bromus pictus, a graminean species from Patagonia, tolerant to cold and drought. Here, this enzyme was functionally characterized by heterologous expression in Pichia pastoris and Nicotiana tabacum. Recombinant P. pastoris Bp6-SFT showed comparable characteristics to barley 6-SFT and an evident fructosyltransferase activity synthesizing bifurcose from sucrose and 1-kestotriose. Transgenic tobacco plants expressing Bp6-SFT, showed fructosyltransferase activity and fructan accumulation in leaves. Bp6-SFT plants exposed to freezing conditions showed a significantly lower electrolyte leakage in leaves compared to control plants, indicating less membrane damage. Concomitantly these transgenic plants resumed growth more rapidly than control ones. These results indicate that Bp6-SFT transgenic tobacco plants that accumulate fructan showed enhanced freezing tolerance compared to control plants. |
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Keywords: | Bif, bifurcose EL, electrolyte leakage EV, empty vector 1-FFT, fructan:fructan 1-fructosyltransferase FW, fresh weight F, fructose FT, fructosyltransferase G, glucose HPAEC-PAD, high performance anion exchange chromatography with pulsed amperometric detection 1-K, 1-kestotriose 6-K, 6-kestotriose NeoK, Neo-kestose Nys, Nystose S, sucrose 6-SFT, sucrose:fructan 6-fructosyltransferase 1-SST, sucrose:sucrose 1-fructosyltransferase TBA, thiobarbituric acid TLC, thin layer chromatography wt, wild type |
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