Cloning and functional analysis of chicory root fructan 1-exohydrolase I (1-FEH I): a vacuolar enzyme derived from a cell-wall invertase ancestor? Mass fingerprint of the 1-FEH I enzyme

• Published in: The Plant journal : for cell and molecular biology 2000-11, Vol.24 (4), p.447-456
• Main Authors: VAN DEN ENDE, Wim, MICHIELS, An, DE ROOVER, Joke, VERHAERT, Peter, VAN LAERE, André
• Format: Article
• Language: English
• Subjects: 1-exohydrolase I; 1-FEH-I gene; Agronomy. Soil science and plant productions; Biological and medical sciences; Cichorium intybus; Classical and quantitative genetics. Population genetics. Molecular genetics; Fundamental and applied biological sciences. Psychology; Generalities. Genetics. Plant material; Genes. Genome; Genetics and breeding of economic plants; Molecular and cellular biology; Molecular genetics
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313X.2000.00890.x

This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall invertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl transferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar invertase, 1-FEH I might have evolved from a cell-wall invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.