The regulation of trehalose breakdown during dormancy and the induction of germination in yeast ascospores was studied both by in vivo high-resolution NMR spectroscopy and in vitro assays of trehalase activity. Natural-abundance 13C NMR spectra taken during the induction ofgermination with glucose and phosphate showed a rapid breakdown ofpart of the trehalose content. The presence of both glucose and phosphate was important for maximal trehalose breakdown. The 13C NMR spectra showed that the externally added glucose and the internal trehalose were metabolized mainly to glycerol and ethanol. Under these conditions of nitrogen deprivation, full germination is not possible and trehalose breakdown stopped after -1 hr. At this moment resynthesis of trehalose occurred while glycerol and ethanol production from the exogenous glucose continued. In complex media where full spore germination can occur, trehalose breakdown was more pronounced. Measurements of trehalase activity in spore extracts made after addition of varying amounts of glucose and phosphate to the spores revealed a sudden 10-fold increase in the activity of trehalase, within the first minutes of spore germination. The activation was transient: after reaching a maximum between 5 and 10 min, the activity declined back to low values during the next hours. The increase in trehalase activity was not inhibited by cycloheximide or by anaerobic conditions. The decline in trehalase activity that occurred after the initial activation could be correlated with the extent of trehalose breakdown as measured by 13CNMR. In addition to the increase in trehalase activity, differences in the control properties were found between the enzymes from dormant and germinating spores. Trehalase from dormant spores was strongly inhibited by ATP at a concentration of -0.5 mM, which corresponds with the ATP concentration found in dormant spores. On the other hand, trehalase from germinating spores was not inhibited by ATP up to the much higher ATP concentrations that are found in germinating spores. It is suggested that the low activity and the stringent ATP feedback inhibition of trehalase from dormant spores are responsible for the very slow mobilization ofthe huge amount oftrehalose in dormant spores. Therefore, dormancy seems to be caused primarily by extreme curtailment of the energy production within the spore at one selective and primary point. The switch towards high activity and low ATP inhibition upon induction of germination is suggested to be responsible for the breaking of dormancy and for the rapid breakdown of trehalose that occurs during the initial phase of germination.In general, dormant fungal spores have very low metabolic rates compared to normal cells (1). An intriguing question in this respect is why the large amount of trehalose present in the spores is not metabolized because trehalase, the enzyme responsible for the breakdown of trehalose, is known to be present in the spores of several species (2)(3)(4)(5) (14). Tetracycline (20 ,ug/ml) was used to ...