Abstract
Methods used to estimate the (nonosmotic) bound water fraction (BWF) (i.e. apoplast water) of spinach (Spinacia oleracea L.) leaves were evaluated. Studies using three different methods of pressure/volume (P/V) curve construction all resulted in a similar calculation of BWF; approximately 40%. The theoretically derived BWF, and the water potential (Psi(w))/relative water content relationship established from P/V curves were used to establish the relationship between protoplast (i.e. symplast) volume and Psi(w). Another method of establishing the protoplast volume/Psi(w) relationship in spinach leaves was compared with the results from P/V curve experiments. This second technique involved the vacuum infiltration of solutions at a range of osmotic potentials into discs cut from spinach leaves. These solutions contained radioactively labeled H(2)O and sorbitol. This dual label infiltration technique allowed for simultaneous measurement of the total and apoplast volumes in leaf tissue; the difference yielded the protoplast volume. The dual label infiltration experiments and the P/V curve constructions both showed that below -1 megapascals, protoplast volume decreases sharply with decreasing water potential; with 50% reduction in protoplast volume occurring at -1.8 megapascals leaf water potential.