Vacuolar Membrane Lesions Induced by a Freeze-Thaw Cycle in Protoplasts Isolated from Deacclimated Tubers of Jerusalem Artichoke(Helianthus tuberosus L.)

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The processes of freezing injury in Jerusalem artichoke (Helianthus tuberosus L.)tubers were studied using protoplasts isolated from cold-acclimated and deacclimated tubers. Prior to freezing, protoplasts were preloaded with 10 μM fluorescein diacetate (FDA) in an isotonic sorbitol solution. After freeze-thawing at various temperatures, cell viability was evaluated under a fluorescence microscope. In cold-acclimated tubers, more than 80% of protoplasts survived freezing to -20℃. By contrast, in deacclimated tubers, the cell survival abruptly declined after freezing to temperatures below -5℃. Thus, freezing tolerance differed significantly between protoplasts isolated from cold-acclimated and deacclimated tubers. Two distinct types of cell injury, which were caused by either damage to plasma membrane (cell-lysis type) or by damage to the vacuolar membrane (abnormal-staining type), were observed. depending on the cold hardiness and freezing temperature. In the cells of the abnormal-staining type, shrinkage of the central vacuolar space and simultaneous acidification of the cytoplasmic space were characteristically observed immediately before complete cell-rehydration during thawing. The decrease in freezing tolerance of protoplasts after deacclimation was suggested to be due mainly to destabilization of the vacuolar membrane by freeze-induced dehydration stress.
日本植物生理学会の論文