Murai M, Yoshida S
Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan.
Plant Cell Physiol. 1998 Jan;39(1):87-96. doi: 10.1093/oxfordjournals.pcp.a029294.
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 microM 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 degrees C. By contrast, in deacclimated tubers, the cell survival abruptly declined after freezing to temperatures below -5 degrees C. 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.
利用从冷驯化和脱驯化块茎中分离的原生质体，研究了菊芋（Helianthus tuberosus L.）块茎的冻害过程。在冷冻前，将原生质体在等渗山梨醇溶液中用10微摩尔荧光素二乙酸酯（FDA）预加载。在不同温度下进行冻融后，在荧光显微镜下评估细胞活力。在冷驯化的块茎中，超过80%的原生质体在冷冻至-20℃时存活。相比之下，在脱驯化的块茎中，冷冻至低于-5℃后细胞存活率急剧下降。因此，从冷驯化和脱驯化块茎中分离的原生质体的耐冻性存在显著差异。根据抗寒性和冷冻温度，观察到两种不同类型的细胞损伤，一种是由质膜损伤引起的（细胞裂解型），另一种是由液泡膜损伤引起的（异常染色型）。在异常染色型细胞中，在解冻过程中完全细胞复水之前，特征性地观察到中央液泡空间收缩和细胞质空间同时酸化。脱驯化后原生质体耐冻性的降低主要是由于冷冻诱导的脱水胁迫导致液泡膜不稳定。
