Changes in Microsomal Enzymes and Phospholipid during Dehardening in Stem Bark of Black Locust

Upon dehardening of stem bark of black locust (Robinia pseudoacacia), a significant decrease in phospholipid content on a milligram protein basis was observed in various crude particulate cell fractions. To ascertain this with a defined membrane, microsomal preparations were separated into several membrane fractions on a discontinuous sucrose gradient. Based on the distribution of various enzymes on the gradient, Golgi apparatus membranes, tonoplast, and unidentified membranes containing acid protease were separated with less contamination by other membranes. The subfraction, with an apparent density of 1.10 g/cc, which was enriched in fragmented tonoplast, contained the most phospholipid per milligram protein. Dehardening resulted in a significant quantitative reduction in protein and phospholipid in the submicrosomal fractions. Significant decreases in phospholipid content per milligram protein were observed during dehardening in tonoplast, Golgi apparatus, and unidentified membranes containing acid protease as well as other membrane fractions. During dehardening, marked decreases in inosine diphosphatase and NADH cytochrome c reductase activities were observed, suggesting a marked degradation of the membranes containing those enzymes. The transition of cell membranes from a phospholipid-enriched state to a phospholipid depleted state is apparently involved in the dehardening process concomitant with a decrease in tissue hardiness.