Calcium Transport in Protoplasts Isolated from ml-o Barley Isolines Resistant and Susceptible to Powdery Mildew

Free cytoplasmic calcium has been postulated to play a role in preventing powdery mildew in a series of homozygous ml-o mutants of barley, Hordeum vulgare L. Protoplasts isolated from 7-day-old plants of the ml-o resistant-susceptible (R-S) barley isolines, Riso 5678/3(*) x Carlsberg II R and S, were used to test for differences in fluxes of Ca(2+) across the plasmalemma. Greater influx or lesser efflux might account for a higher free cytosolic Ca(2+) postulated to exist in ml-o R mutants. Uniform patterns of uptake were maintained for 3 hours from solutions of 0.2 and 2 millimolar Ca(2+). Washout curves of (45)Ca(2+) from R and S protoplasts revealed three compartments-presumed to represent release from the vacuole, organelles, and the cytoplasm (which included bound as well as free Ca(2+)). Uptake and washout did not differ between isolines. On the basis of recent determinations of submicromolar levels of free cytoplasmic Ca(2+) and our initial rates of (45)Ca-labeled Ca(2+) uptake, we show that measurement of the unidirectional influx of Ca(2+) across the plasmalemma is not feasible because the specific activity of the pool of free cytoplasmic calcium increases almost instantaneously to a level that would result in a significant, but unknown, efflux of label. Similarly, measurement of the efflux of Ca(2+) across the plasmalemma is not possible since the activity of the pool of free cytoplasmic calcium is a factor of 350 smaller than the most rapid component of the washout experiment. This pool of cytoplasmic free Ca(2+) will wash out too rapidly and be too small to detect under the conditions of these experiments.