Sensitive Detection of the Leaching‐out from Membrane‐surrounded Compartments: a New Approach Explored with Cells and Tissues of Nicotiana tabacum L.

Abstract

In search of a simple method for testing the very early events of ozone damage to susceptible plants as well as complete destruction after threshold‐exceeding treatments an over‐all measurement of cell ingredients by their optical density in the UVB and UVC range was investigated. The cell particles were liberated after membrane permeabilization or after cell bursting. Uncontrolled results could be excluded. Furthermore, the results of the developed spectrophotometrical test could be, in the case of tissue samples (leaf discs), very well reproduced with an osmometrical measurement. The latter was less sensitive and not suitable for cellular samples because the protoplasts must be dissolved in a nearly isotonic medium which caused too large a background for this method but not for the UVS test. Contrary to the osmometric measurement, the photometric one cannot be used for determining the absolute amount of cell ingredients but only for relative measurement between samples in a given range of concentrations. Oxidative changes of the liberated ingredients do not influence their detection, which was demonstrated with ascorbate. The developed leaching test was also useful for determining the membrane damage caused by the detergent Triton X‐100, although this was known to have UV absorbance by itself. It was noted that the far UV maximum is not only caused by absorbance and scattering is discussed as an additional explanation.

Illustration of the Genetic Differences in Ozone Sensitivity Between the Varieties Nicotiana tabacum Bel W3 and Bel B Using Various Plant Systems

“…Plant material, growing conditions, media, protoplast isolation, ozone fumigation and membrane leaching measurement with the UVS test are previously described in detail (Stadtler, 1993). Therefore only additionally needed information for the present work is listed as follows: Sabine Stiidtler and H. Ziegler…”

“…It has been suggested that at first cytoplasmic membrane components were oxidized (Moeschlin, 1980), especially unsaturated fatty acids from lipids (Criegee, 1975) and/or thiols from proteins (Bailey, 1958;Tomlinson and Rich, 1968) leading to the loss of semipermeability. The detection of the leaching of cell ingredients by lNS was sensitive enough to also estimate the first stages of damage (Stadtler, 1993), whereas direct evidence of these reactions is difficult to obtain, because even small changes may evoke severe effects: ozone-induced oxidation of 1 % of the total cell lipids is sufficient to kill Chlorella sorokiniana (Frederick and Heath, 1975).…”