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Plant and Cell Physiology Advance Access originally published online on June 20, 2008
Plant and Cell Physiology 2008 49(8):1185-1195; doi:10.1093/pcp/pcn094
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Cloning, Characterization and Functional Analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby

Amanda Francine Asega1,*, João Roberto O. do Nascimento2, Lindsey Schroeven3, Wim Van den Ende3 and Maria Angela M. Carvalho1

1Seção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica, CP 3005, 01061-970, São Paulo, SP, Brazil
2Laboratório de Química, Bioquímica e Biologia Molecular de Alimentos, Departamento de Alimentos e Nutrição Experimental, Universidade de São Paulo, Avenida Lineu Prestes 580, Bloco 14, CEP 05508-900, São Paulo, SP, Brazil
3Department of Biology, Laboratory for Molecular Plant Physiology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium

*Corresponding author: E-mail, amandaasega{at}yahoo.com.br; Fax, +55-11-5073-3678.


  Abstract

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5°C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly β-2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81% in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.

Keywords: Asteraceae - Brazilian Cerrado - 1-FEH cloning - Functional analysis - Underground reserve organs - Vernonia herbacea

Abbreviations: BMGY, buffered minimal glycerol medium; BMMY, buffered minimal methanol medium; CDR, coding region; DP, degree of polymerization; 1-FEH, 1-fructan exohydrolase; 6-FEH, 6-fructan exohydrolase; HPAEC/PAD, high-performance anion exchange chromatography/pulsed amperometric detection; iPCR, inverse PCR; KEH, kestose exohydrolase; ORF, open reading frame; RNAi, RNA interference; SSC, sodium citrate and sodium chlorate solution; SST, 1-sucrose: sucrose fructosyltransferase; UTR, untranslated region; YPD, yeast extract peptone dextrose medium; YPDS, yeast extract peptone dextrose sorbitol medium; YT, yeast extract tryptone medium.

(Received March 20, 2008; Accepted June 17, 2008)
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