Effects of osmotic stress and growth stage on cellular pH and polyphosphate metabolism in Neurospora crassa as studied by 31P nuclear magnetic resonance spectroscopy

Abstract

High-resolution 31P-NMR was employed to investigate the effects of growth stage and environmental osmolarity on changes of polyphosphate metabolism and intracellular pH in intact Neurospora crassa cells. Our study showed that changes of these parameters were growth-dependent. The ratio of polyphosphate to orthophosphate in vacuoles increased from 2.4 to 13.5 in N. crassa as cells grew from early log phase to stationary phase. Cytoplasmic pH and vacuolar pH changed, respectively, from 6.91 and 6.49 in early log phase cells to 7.25 and 6.84 in stationary phase cells. Hypoosmotic shock of N. crassa produced growth-dependent changes including: (i) a rapid hydrolysis of polyphosphate with a concomitant increase in the concentration of the cytoplasmic phosphate, (ii) an increase in cytoplasmic pH, and (iii) an increase in vacuolar pH. Early log phase cells produced the most dramatic response whereas the stationary phase cells appeared to be recalcitrant to the osmotic stress. Thus, 95% and 60% of polyphosphate in the early log phase and mid-log phase cells, respectively, disppeared in response to hypoosmotic shock, but little or no hydrolysis of polyphosphate occurred in stationary cells. The cytoplasmic pH and the vacuolar pH increased in response to hypoosmotic shock by 0.4 and 0.53 unit, respectively, in early log phase cells; and by 0.22 and 0.27 unit, respectively, in the mid-log phase cells. In contrast, hypoosmotic shock of the stationary phase cells did not cause any change in intracellular pH. The osmotic stress-induced polyphosphate hydrolysis and pH changes in early log and mid-log phase cells were reversible, suggesting that these changes were related environment osmolarity. Addition of polyamines or basic amino acids which are known to be sequestered in vacuoles did not affect polyphosphate metabolism.