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 5Ј-and 3Ј-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).

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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"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.The breaking of dormancy of Phycomyces blakesleeanus spores by a heat shock is directly followed by a 15 -30-min period, called the critical period [l], during which the spores need to be incubated in the presence of glucose or 6-deoxyglucose in order to germinate eventually. This period is characterized by several transient metabolic changes, including the activation of trehalase [2], the formation of hexose 6-phosphates and of fructose 2,6-bisphosphate [l], the formation of cyclic AMP [3] and the production of about 300 pmol glycerol/g spores [4]. On the other hand, a link between the transport of glucose, the formation of cyclic AMP as well as of fructose 2,6-bisphosphate and the activation of trehalase has been recently established in Saccharomyces cerevisiae [5] suggesting that all these metabolic transformations could be connected with cyclic AMP.The present work was undertaken in order to investigate the respective roles of cyclic AMP and of fructose 2,6-bisphosphate in the formation of glycerol which is known to occur during the first minutes after spore activation. It led to the discovery of an interconvertible glycerol-3-phosphatase, which is a substrate for cyclic-AMP-dependent protein kinase,

When dormant spores of Phycomyces blakesleeanus were activated without concomitant activation of trehalase, breakdown of storage trehalose during early germination was not prevented. Measurement of trehalase activity during early germination of spores activated in this way indicated a subsequent rapid activation of trehalase upon incubation of the spores in germination medium. Trehalase activity reached a maximum after about 10 min of germination; thereafter it declined to values somewhat higher than those found in dormant spores. The same was observed when the activation of trehalase which normally occurs during heat activation of the spores was suppressed by adding long-chain alcohols to the activation medium. These results argue against previous speculation that trehalase is a 'luxury' molecule in the spores and that its activation has no significant role in the induction of germination. They point, on the other hand, to an important role for trehalase in the induction of germination. The main factor in the germination medium responsible for the activation of trehalase was found to be glucose. When spores were incubated under conditions in which they reverted to the dormant state, this subsequent trehalase activation was not seen. The increase in trehalase activity was not dependent on protein synthesis. A less pronounced increase was seen with glucose analogues. In the presence of azide the activation was only retarded, whereas in the presence of azide and salicylhydroxamic acid strong inhibition of trehalase activation was observed.