In vitro simulation of cytoplasmic membrane senescence in cotyledons

The loss of microsomal NADH-cytochrome c reductase activity (EC 1.6.99.3) in cotyledons, known to accompany germination of Phaseolus vulgaris and thought to reflect the progress of cytoplasmic membrane senescence, can be simulated in an in vitro system in which isolated microsomes from 2-day-old tissue are treated with cytosol fractions (microsomal supernatants). Inactivation of the enzyme is comparatively low when the microsomes are treated for 4 hours with cytosol fractions from 1- and 2-day-old tissue, but increases to about 68% upon treatment with a corresponding fraction from 3-day-old cotyledons. This temporal pattern is consistent with the pronounced in situ decline in NADH-cytochrome c reductase detectable between the 2nd and 4th days of germination. Extensive in vitro inactivation was also effected by cytosol fractions prepared from older tissue, including that harvested after 9 days of germination by which time the cotyledons were beginning to abscise.The degree of inactivation in the in vitro system proved to be proportional to the concentration of cytosol present in the incubation mixture, and also increased with time at fixed cytosol concentrations. Moreover, the rate of in vitro inactivation was some 18-fold greater than that occurring in situ. The data have been interpreted as indicating that cytoplasmic membrane senescence in cotyledons is, in part, mediated by one or more factors in the cytosol, extrinsic agents which are sequestered in situ and released gradually during germination as the pressures of senescence become more intense.