Cloning and functional analysis of chicory root fructan1-exohydrolase I (1-FEH I): a vacuolar enzyme derivedfrom a cell-wall invertase ancestor? Mass fingerprint of the 1-FEH I enzyme.
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.
Van den Ende
W
W
K.U. Leuven, Department of Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001 Leuven, Belgium. wim.vandenende@bio.kuleven.ac.be
Michiels
A
A
De Roover
J
J
Verhaert
P
P
Van Laere
A
A
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D002473:Cell Wall / Q000201:enzymology; D018651:Cichorium intybus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005630:Fructans / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006602:Hexosyltransferases / Q000235:genetics; D006867:Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics*; D030821:Plants, Genetically Modified; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution; D014357:Trypsin / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology; D043324:beta-Fructofuranosidase; D001426:Bacterial Proteins
D018076:DNA, Complementary; D005630:Fructans; D006026:Glycoside Hydrolases; D006602:Hexosyltransferases; D006867:Hydrolases; D018749:RNA, Plant; D014357:Trypsin; D043324:beta-Fructofuranosidase; C021451:inulosucrase; D001426:Bacterial Proteins