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Plant and Cell Physiology Advance Access originally published online on January 19, 2009
Plant and Cell Physiology 2009 50(3):489-503; doi:10.1093/pcp/pcp008
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Molecular Characterization of a Putative Sucrose:Fructan 6-Fructosyltransferase (6-SFT) of the Cold-Resistant Patagonian Grass Bromus pictus Associated With Fructan Accumulation Under Low Temperatures

F. del Viso1, A. F. Puebla1,2, C. M. Fusari1, A. C. Casabuono3, A. S. Couto3, H. G. Pontis2, H. E. Hopp1,4 and R. A. Heinz1,4,*

1Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA, Castelar), (1686), Hurlingham, Buenos Aires, Argentina
2Centro de Investigaciones Biológicas, Fundación para Investigaciones Biológicas Aplicadas (FIBA), Vieytes 3103 (7600), Mar del Plata, Argentina
3CIHIDECAR, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (1428), C.A. Buenos Aires, Argentina
4Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina

*Corresponding author: E-mail, rheinz{at}cnia.inta.gov.ar; Fax, +54-11-621-0199.


  Abstract

Fructans are fructose polymers synthesized from sucrose in the plant vacuole. They represent short- and long-term carbohydrate reserves and have been associated with abiotic stress tolerance in graminean species. We report the isolation and characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) gene from a Patagonian grass species, Bromus pictus, tolerant to drought and cold temperatures. Structural and functional analyses of this gene were performed by Southern and Northern blot. Sugar content, quality and fructosyltransferase activity were studied using HPAEC-PAD (high-pH anion-exchange chromatography with pulsed amperometric detection), enzymatic and colorimetric assays. The putative 6-SFT gene had all the conserved motifs of fructosyl-transferase and showed 90% identity at the amino acid level with other 6-SFTs from winter cereals. Expression studies, and determination of sugar content and fructosyl-transferase activity were performed on five sections of the leaf. Bp6-SFT was expressed predominantly in leaf bases, where fructosyltransferase activity and fructan content are higher. Bp6-SFT expression and accumulation of fructans showed different patterns in the evaluated leaf sections during a 7 d time course experiment under chilling treatment. The transcriptional pattern suggests that the B. pictus 6-SFT gene is highly expressed in basal leaf sections even under control temperate conditions, in contrast to previous reports in other graminean species. Low temperatures caused an increase in Bp6-SFT expression and fructan accumulation in leaf bases. This is the first study of the isolation and molecular characterization of a fructosyltransferase in a native species from the Patagonian region. Expression in heterologous systems will confirm the functionality, allowing future developments in generation of functional markers for assisted breeding or biotechnological applications.

Keywords: Abiotic stress - Bromus pictus - Fructan - Low temperatures - Patagonia - 6-SFT.

Abbreviations: DMSO, dimethylsulfoxide; DP, degree of polymerization; FEH, fructan exohydrolase; 1-FFT, fructan:fructan 1-fructosyltransferase; FT, fructosyltrans-ferase; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; Indels, insertions/deletions; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; ORF, open reading frame; PAR, photosynthetically active radiation; PMSF, phenylmethylsulfonyl fluoride; RACE, rapid amplification of cDNA ends; RT–PCR, reverse transcription–PCR; 6-SFT, sucrose:fructan 6-fructosyltransferase; SNP, single nucleotide polymorphism; 1-SST, sucrose:sucrose 1-fructosyltransferase; TBA, thiobarbituric acid; TLC, thin-layer chromatography; UTR, untranslated region; WSC, water-soluble carbohydrate.

The nucleotide sequence of Bp6-SFT, reported in this paper has been submitted to GenBank under accession number FJ424612. A list of the accession numbers used for phylogenetic analyses is available on Supplementary Table S2.

(Received November 12, 2008; Accepted January 5, 2009)
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