Induction of 1-FEH in mature chicory roots appears to be related to low temperatures rather than to leaf damage.

Large-scale inulin production from chicory roots (Cichorium intybus L.) is hampered by the induction of 1-FEH activity (fructan 1-exohydrolase) and concomitant fructose production in autumn, coincident with a period with low night temperatures that cause leaf damage. To understand whether leaf damage per se is sufficient for 1-FEH induction and fructan breakdown, we defoliated mature chicory plants at a preharvest stage (September 10) and investigated the changes in carbohydrate levels and 1-FEH activities. Also, the activities of 1-SST (sucrose:sucrose 1-fructosyl transferase, EC 2.4.1.99), 1-FFT (fructan:fructan 1-fructosyl transferase, EC 2.4.1.100), and acid invertase (EC 3.2.1.26) were determined. Defoliation did not result in a prompt fructan breakdown and increase in 1-FEH activity, but after 10 days fructan breakdown and 1-FEH activities became higher in the defoliated plants. Defoliation resulted in a sharp decrease in 1-SST activity over the first 24 h. Afterwards, root 1-SST activities of defoliated plants remained at a lower level than in control plants. 1-FFT and invertase activities were not affected by defoliation. It can be concluded that defoliation of plants at the preharvest stage by itself did not induce the same rapid changes as observed in naturally induced October roots by low temperature (harvest stage). Taken together with our finding that 1-FEH is not induced in chicory roots when plants are transferred to the greenhouse early autumn (minimal temperature 14 degrees C), we conclude that low temperatures might be essential for 1-FEH induction.

Defoliation induces fructan 1-exohydrolase II in Witloof chicory roots. Cloning and purification of two isoforms, fructan 1-exohydrolase IIa and fructan 1-exohydrolase IIb. Mass fingerprint of the fructan 1-exohydrolase II enzymes.

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).

Sucrose assimilation during early developmental stages of chicory (Cichorium intybus L.) plants.

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.

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.

The role of fructan in flowering of Campanula rapunculoides.

Inulin type fructan was detected in all vegetative organs of Campanula rapunculoides L. plants. All flower parts contained fructan at some developmental stage. A steady decrease was found in sepals during development. Petals, however, stored fructan in the bud stage. A rapid breakdown during opening of the flower resulted in high concentrations of glucose and especially fructose that may contribute to the osmotic driving force involved in petal expansion. Before complete shrivelling, the hexoses were apparently exported from flower parts. Fructans were hydrolysed and exported from the stamen and style tissue upon flower opening. Similarly, the major fructan reserves in the ovary were broken down almost simultaneously with those in other flower parts. Hexoses did not reach high levels in the ovary, probably because they were rapidly metabolized and/or incorporated by developing seeds.

Cloning, developmental, and tissue-specific expression of sucrose:sucrose 1-fructosyl transferase from Taraxacum officinale. Fructan localization in roots.

Sucrose:sucrose 1-fructosyl transferase (1-SST) is the key enzyme initiating fructan synthesis in Asteraceae. Using reverse transcriptase-PCR, we isolated the cDNA for 1-SST from Taraxacum officinale. The cDNA-derived amino acid sequence showed very high homology to other Asteracean 1-SSTs (Cichorium intybus 86%, Cynara scolymus 82%, Helianthus tuberosus 80%), but homology to 1-SST from Allium cepa (46%) and Aspergillus foetidus (18%) was much lower. Fructan concentrations, 1-SST activities, 1-SST protein, and mRNA concentrations were compared in different organs during vegetative and generative development of T. officinale plants. Expression of 1-SST was abundant in young roots but very low in leaves. 1-SST was also expressed at the flowering stages in roots, stalks, and receptacles. A good correlation was found between northern and western blots showing transcriptional regulation of 1-SST. At the pre-flowering stage, 1-SST mRNA concentrations and 1-SST activities were higher in the root phloem than in the xylem, resulting in the higher fructan concentrations in the phloem. Fructan localization studies indicated that fructan is preferentially stored in phloem parenchyma cells in the vicinity of the secondary sieve tube elements. However, inulin-like crystals occasionally appeared in xylem vessels.

Drought induces fructan synthesis and 1-SST (sucrose:sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.).

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.

[Prevention of recurrent atrial fibrillation and bi-atrial resynchronization]. / Prévention de la fibrillation auriculaire récidivante et resynchronisation bi-atriale.

After conversion of atrial fibrillation, it is important to maintain sinus rhythm. In addition antiarrhythmic drugs, biatrial resynchronization seems to prevent recurrences of atrial fibrillation in patients with interatrial conduction block: local experience.

Carbon and nitrogen allocation in ectomycorrhizal and non-mycorrhizal Pinus sylvestris L. seedlings.

We studied carbon and nitrogen allocation in mycorrhizal and non-mycorrhizal Scots pine (Pinus sylvestris L.) seedlings grown in a semi-hydroponic system with nitrogen as the growth limiting factor. Three ectomycorrhizal fungi were compared: one pioneer species (Thelephora terrestris Ehrh.: Fr.) and two late-stage fungi (Suillus bovinus (L.: Fr.) O. Kuntze, and Scleroderma citrinum Pers.). By giving all plants in each treatment the same amount of readily available nitrogen, we ensured that the external mycelium could not increase the total nitrogen content of the plants, thereby guaranteeing that any change in carbon or nitrogen partitioning was a direct effect of the mycorrhizal infection itself. Carbon and nitrogen partitioning were measured at an early and a late stage of mycorrhizal development, and at a low and a high N addition rate. Although mycorrhizal seedlings had a higher net assimilation rate and a higher shoot/root ratio than non-mycorrhizal seedlings, they had a lower rate of shoot growth. The high carbon demand of the mycobionts was consistent with the large biomass of external mycelia and the increased belowground respiration of the mycorrhizal plants. The carbon cost to the host was similar for pioneer and late-stage fungi. Above- and belowground partitioning of nitrogen was also affected by mycorrhizal infection. The external mycelia of Scleroderma citrinum retained 32% of the nitrogen supplied to the plants, thus significantly reducing nitrogen assimilation by the host plants and consequently reducing their growth rate. By contrast, the external mycelia of T. terrestris and Suillus bovinus retained less nitrogen than the mycelia of Scleroderma citrinum, hence we attributed the decreased growth rates of their host plants to a carbon drain rather than a nitrogen deficiency.

Purification and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Petunia hybrida.

We report isolation and N-terminal amino acid sequencing of three style glycoproteins, which segregate with three S (self-incompatibility) alleles of Petunia hybrida. The S-glycoproteins were expressed mainly in the upper part of the pistil and showed an increasing concentration during flower development. The glycoproteins were purified by a combination of ConA-Sepharose and cation exchange fast protein liquid chromatography. The amount of S-glycoproteins recovered from style extracts varied from 0.5 to 1.6 micrograms per style, which was 40-60% of the amount recovered by a simplified analytical method. N-terminal amino acid sequences of S1-, S2- and S3-glycoprotein showed homology within the fifteen amino terminal residues. These amino acid sequences were compared with the previously published sequences of S-glycoproteins from Nicotiana alata and Lycopersicon peruvianum.

Purification and properties of NAD-dependent glutamate dehydrogenase from Phycomyces spores.

The NAD-dependent glutamate dehydrogenase from Phycomyces spores was purified more than 300-fold. Estimation of Mr by gel filtration gave a value of 98,000 whereas after SDS-PAGE one major band of Mr 54,000 was found, suggesting that the enzyme is a dimer. The enzyme was virtually dependent on the presence of AMP for activity and showed half-maximal activation at 9.5 and 43 microM-AMP in the direction of animation and deamination respectively. ADP was nearly as effective at 20-fold higher concentrations. Other nucleotide monophosphates were ineffective and nucleoside triphosphates were slightly inhibitory. Hyperbolic kinetics were found for all substrates yielding Km values of about 10 mM for ammonium, 1 mM for 2-oxoglutarate and 0.1 mM for NADH in the direction of amination, and 10 mM for glutamate and 0.7 mM for NAD in the direction of deamination.

Glycerol formation after the breaking of dormancy of Phycomyces blakesleeanus spores. Role of an interconvertible glycerol-3-phosphatase.

The breaking of dormancy of Phycomyces blakesleeanus spores by a heat shock was followed by a transient production of glycerol, which culminated within 5-10 min and was terminated at 20 min. Extracts of spores contained a magnesium-dependent glycerol-3-phosphatase active on both L-glycerol 3-phosphate and dihydroxyacetone phosphate but having more affinity for the first substrate than for the second. In extracts from dormant spores, the phosphatase was profoundly inhibited by physiological concentrations of inorganic phosphate, which induced cooperativity for the substrate, whereas the enzyme from heat-activated spores was much less inhibited and this difference in kinetic properties persisted after gel filtration of the enzymic preparation. When measured at 1 mM phosphate and 0.1 mM glycerol 3-phosphate, the phosphatase activity was undetectable in dormant spores, increased sharply during the heat treatment and the following 5 min at 25 degrees C, then fell again to a low value by 20 min. A similar transient activation of the enzyme was observed following the breaking of dormancy by incubation of the spores in the presence of 0.1 M ammonium acetate. Incubation of a cell-free extract or of the partially purified glycerol-3-phosphatase in the presence of ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase released the enzyme from inhibition by phosphate and endowed it with the same kinetic properties as did the heat treatment of the spores. It appears therefore most likely that phosphorylation of glycerol-3-phosphatase by cyclic-AMP-dependent protein kinase causes its activation and that this transient process explains the equally transient formation of glycerol by the spores after the heat shock.

Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores.

Incubation of Mucor rouxii sporangiospores in complex medium under aerobic conditions resulted in a transient 20-fold increase in trehalase activity. Maximum activity was reached after 15 min. Simultaneously, the cyclic AMP (cAMP) content increased approximately eightfold, reaching a maximum within 10 min. Increases in trehalase activity and cAMP content were also observed under anaerobic conditions (CO2). The extent of trehalase activation and the changes in cAMP content, during both aerobic and anaerobic incubation, varied with the medium used. Trehalase was activated in vitro by a cAMP- and ATP-dependent process. An even faster activation was obtained when cAMP was replaced by the catalytic subunit of beef heart protein kinase. The coincidence of, and the correlation between, increased cAMP contents and trehalase activities support the involvement of a cAMP-dependent phosphorylation in the in vivo regulation of trehalase activity.

Fructose 2,6-bisphosphate and germination of fungal spores.

Induction of germination of Phycomyces blakesleeanus spores by a heat shock and subsequent incubation at 25 degrees C in a glucose- or 6-deoxyglucose-containing culture medium resulted in an intense (20-40 times the initial value) rise in the concentration of fructose 2,6-bisphosphate and hexose 6-phosphates. The increase in the concentration of fructose 2,6-bisphosphate but not that of hexose 6-phosphates was restricted to a 25-min period during which the spores acquired an irreversible capacity to germinate. Incubation of the spores in water for any period of time during this critical period resulted in a parallel decrease in their ability to form hexose phosphates and to germinate. A similar rise in hexose phosphate concentration was also observed when P. blakesleeanus spores were activated by incubation in the presence of acetate and also after induction of germination of other dormant (Neurospora tetrasperma) or nondormant (Mucor racemosus) fungal spores. Extracts of dormant and heat-activated P. blakesleeanus spores contain a fructose, 1,6-bisphosphatase that is inhibited by fructose 2,6-bisphosphate and AMP in a synergistic manner. They also contain a 6-phosphofructo-2-kinase and a fructose-2,6-bisphosphatase.

Trehalose metabolism in dormant and activated spores of Phycomyces blakesleeanus Burgeff.

Evidence is obtained for the existence of two different localizations of trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) in Phycomyces spores: one inside the cell, and one in the periplasmic region. The latter enzyme is sensitive to 0.1 mol l(-1) HCl treatment and its activity can be regulated by external pH changes. The periplasmic form of the enzyme is involved in the metabolism of added labelled trehalose. This sugar is hydrolyzed externally to glucose which is found mainly in the incubation medium and which is partly absorbed by the spores. During incubation trehalose leaks out from both dormant and activated spores and is subsequently hydrolyzed to glucose. The intracellular trehalase is probably involved in the breakdown of endogenous trehalose in spores. After heat activation the hydrolysis of endogenous trehalose is stimulated even without an important increase in activity of intracellular trehalase. Additional treatments which break dormancy of spores without a significant activation of trehalase are the following: heating of HCl-treated spores and treatment of spores with reducing substances (e.g. Na2S2O4 and NaHSO3).

Cell wall carbohydrates in Phycomyces blakeslesanus Burgeff.

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fructose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosurgars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-14C]-glucose into wall materials. Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but only by 5-fluorouracil.

Effect of 5-fluorouracil and cycloheximide on the early development of Phycomyces blakesleeanus spores and the activity of N-acetylglucosamine synthesizing enzymes.

The development of germinating Phycomyces spores was not inhibited by 5-fluorouracil (1mM) until the emergence of the germination tube. Fluorouracil was incorporated into RNA as efficiently as uracil; it did not inhibit the synthesis of proteins and the increase in respiratory activity during early development. Cycloheximide inhibited development as well as the increase in respiration and protein synthesis. This suggested that protein synthesis or some other cycloheximide dependent process, but no mRNA synthesis, was needed for the first developmental stages. The activity of two enzymes involved in the synthesis of N-acetylglucosamine increased markedly during germination. THis increase was inhibited by both 5-fluorouracil and cycloheximide; this suggested that those enzymes were synthesized on mRNA formed during germination.