ABSTRACT
The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated "Belgian endive" leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5'- and 3'-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation).
Subject(s)
Cichorium intybus/enzymology , Glycoside Hydrolases/genetics , Isoenzymes/genetics , Plant Leaves/physiology , Plant Roots/enzymology , Amino Acid Sequence , Cichorium intybus/genetics , Cloning, Molecular , Exons , Glycoside Hydrolases/isolation & purification , Isoenzymes/isolation & purification , Molecular Sequence Data , Peptide Fragments/metabolism , Sequence Homology, Amino Acid , beta-FructofuranosidaseABSTRACT
The activities of enzymes of both sucrose and fructan metabolism were measured in chicory (Cichorium intybus L. cv. Turbo) plants during early vegetative growth. From 21 to 42 d after sowing (phase I), carbohydrates were used for structural growth and sucrose was predominantly cleaved by acid invertase whereas neutral invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) activities were low. From 49 to 63 d after sowing (phase II) a cambium formed producing secondary tissues, concomitant with induced sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) and fructan:fructan-1-fructosyl transferase (EC 2.4.1.100) activities, and fructan synthesis in the roots. Accumulation of 1-SST mRNA occurred at the onset of thickening, indicating that 1-SST is controlled at a transcriptional level. Acid invertase activity gradually increased during phase I and remained high during early phase II. It subsequently decreased. The pattern of invertase mRNA accumulation correlated with the enzyme activities, indicating that acid invertase is controlled at the transcriptional level. Both acid invertase and 1-SST probably contributed to the sink strength in the root at the beginning of phase II.
Subject(s)
Cichorium intybus/enzymology , Sucrose/metabolism , Blotting, Northern , Carbon Radioisotopes/metabolism , Cichorium intybus/cytology , Cichorium intybus/growth & development , Cichorium intybus/metabolism , Chromatography, High Pressure Liquid , Fructans/metabolism , Fructose/metabolism , Glucose/metabolism , Glucosyltransferases/metabolism , Glycoside Hydrolases/metabolism , Hexosyltransferases/metabolism , In Vitro Techniques , Plant Roots/cytology , Plant Roots/enzymology , Plant Roots/metabolism , RNA, Messenger/metabolism , Trisaccharides/metabolism , beta-FructofuranosidaseABSTRACT
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.
Subject(s)
Cichorium intybus/genetics , Fructans/metabolism , Hydrolases/genetics , Plant Roots/genetics , Amino Acid Sequence , Blotting, Northern , Cell Wall/enzymology , Cichorium intybus/enzymology , Cichorium intybus/growth & development , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Hexosyltransferases/genetics , Hydrolases/isolation & purification , Hydrolases/metabolism , Molecular Sequence Data , Phylogeny , Plant Roots/enzymology , Plants, Genetically Modified , RNA, Plant/genetics , RNA, Plant/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Solanum tuberosum/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue Distribution , Trypsin/metabolism , Vacuoles/enzymology , beta-FructofuranosidaseABSTRACT
Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants.
Subject(s)
Cichorium intybus/metabolism , Fructans/biosynthesis , Hexosyltransferases/metabolism , Plant Leaves/metabolism , Plant Roots/metabolism , Plants/metabolism , Blotting, Northern , Carbohydrate Metabolism , Cichorium intybus/genetics , Cichorium intybus/growth & development , Fructose/metabolism , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Plant/drug effects , Glucose/metabolism , Hexosyltransferases/drug effects , Hexosyltransferases/genetics , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Roots/enzymology , Plant Roots/genetics , Plants/enzymology , Plants/genetics , RNA, Plant/drug effects , RNA, Plant/genetics , RNA, Plant/metabolism , Sucrose/metabolism , Water/pharmacologyABSTRACT
A survey of adult male immates of the major institutions for mental patients in Belgium was undertaken in 1965. Out of 10417 males examined 61 had positive sex chromatin (5.8%); 201 had bilateral small testis (1.9%), 4 hypogonad patients carried a D/D translocation. Out of 857 karyotyped children, 2.9% carried an autosomal or sex-chromosomal anomaly other than trisomy G. This prevalence of chromosomal anomalies in children with low I.Q. (less than 50) emphasizes the need for further studies.