VACUOLAR MEMBRANE LESIONS INDUCED BY A FREEZE-THAW CYCLE IN PROTOPLASTS ISOLATED FROM DEACCLIMATED TUBERS OF JERUSALEM-ARTICHOKE (HELIANTHUS-TUBEROSUS L.)

The processes of freezing injury in Jerusalem artichoke (Helianthus tu berosus L.) tubers were studied using protoplasts isolated from cold-a cclimated and deacclimated tubers. Prior to freezing, protoplasts were preloaded with 10 mu M fluorescein diacetate (FDA) in an isotonic sor bitol solution. After freeze-thawing at various temperatures, cell via bility was evaluated under a fluorescence microscope. In cold-acclimat ed tubers, more than 80% of protoplasts survived freezing to -20 degre es C. By contrast, in deacclimated tubers, the cell survival abruptly declined after freezing to temperatures below -5 degrees C. Thus, free zing tolerance differed significantly between protoplasts isolated fro m cold-acclimated and deacclimated tubers. Two distinct types of cell injury, which were caused by either damage to plasma membrane (cell-ly sis type) or by damage to the vacuolar membrane (abnormal-staining typ e), were observed. depending on the cold hardiness and freezing temper ature. In the cells of the abnormal-staining type, shrinkage of the ce ntral vacuolar space and simultaneous acidification of the cytoplasmic space were characteristically observed immediately before complete ce ll-rehydration during thawing. The decrease in freezing tolerance of p rotoplasts after deacclimation was suggested to be due mainly to desta bilization of the vacuolar membrane by freeze-induced dehydration stre ss.