Cloning and functional analysis of chicory root fructan1-exohydrolase I (1-FEH I): a vacuolar enzyme derivedfrom a cell-wall invertase ancestor? Massfingerprint of the 1-FEH I enzyme

This paper describes the cloning and functional analysis of chicory (Cichor ium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge i t is the first plant FEH cloned. Full-length cDNA was obtained by a combina tion of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conser ved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was f urther analyzed by in-gel trypsin digestion followed by matrix-assisted las er desorption ionization and electrospray time-of-flight tandem mass spectr ometry. Functionality of the cDNA was demonstrated by heterologous expressi on in potato tubers. 1-FEH I takes a new, distinct position in the phylogen etic tree of plant glycosyl hydrolases being more homologous to cell-wall i nvertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl trans ferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplas tic 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 h ave evolved from a vacuolar invertase, 1-FEH I might have evolved from a ce ll-wall invertase-like ancestor gene that later obtained a vacuolar targeti ng signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.