Brush R. A., Griffith M., Mlynarz A.
Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Plant Physiol. 1994 Feb;104(2):725-735. doi: 10.1104/pp.104.2.725.
Extracellular ice formation in frost-tolerant organisms is often initiated at specific sites by ice nucleators. In this study, we examined ice nucleation activity (INA) in the frost-tolerant plant winter rye (Secale cereale). Plants were grown at 20[deg]C, at 5[deg]C with a long day, and at 5[deg]C with a short day (5[deg]C-SD). The threshold temperature for INA was -5 to -12[deg]C in winter rye leaves from all three growth treatments. Epiphytic ice nucleation-active bacteria could not account for INA observed in the leaves. Therefore, the INA must have been produced endogenously. Intrinsic rye ice nucleators were quantified and characterized using single mesophyll cell suspensions obtained by pectolytic degradation of the leaves. The most active ice nucleators in mesophyll cell suspensions exhibited a threshold ice nucleation temperature of -7[deg]C and occurred infrequently at the rate of one nucleator per 105 cells. Rye cells were treated with chemicals and enzymes to characterize the ice nucleators, which proved to be complexes of proteins, carbohydrates, and phospholipids, in which both disulfide bonds and free sulfhydryl groups were important for activity. Carbohydrates and phospholipids were important components of ice nucleators derived from 20[deg]C leaves, whereas the protein component was more important in 5[deg]C-SD leaves. This difference in composition or structure of the ice nucleators, combined with a tendency for more frequent INA, suggests that more ice nucleators are produced in 5[deg]C-SD leaves. These additional ice nucleators may be a component of the mechanism for freezing tolerance observed in winter rye.
耐寒生物中的细胞外结冰通常由冰核剂在特定部位引发。在本研究中，我们检测了耐寒植物冬黑麦（Secale cereale）的冰核活性（INA）。将植物分别种植在20℃、5℃长日照以及5℃短日照（5℃ - SD）条件下。在所有三种生长处理的冬黑麦叶片中，INA的阈值温度为 -5至 -12℃。附生的具有冰核活性的细菌无法解释叶片中观察到的INA。因此，INA必定是内源性产生的。使用通过叶片的果胶分解降解获得的单个叶肉细胞悬液对黑麦自身的冰核剂进行了定量和表征。叶肉细胞悬液中最活跃的冰核剂表现出 -7℃的阈值冰核温度，且出现频率较低，每105个细胞中有一个核剂。用化学物质和酶处理黑麦细胞以表征冰核剂，结果证明其是蛋白质、碳水化合物和磷脂的复合物，其中二硫键和游离巯基对活性都很重要。碳水化合物和磷脂是源自20℃叶片的冰核剂的重要成分，而蛋白质成分在5℃ - SD叶片中更为重要。冰核剂在组成或结构上的这种差异，再加上INA更频繁出现的趋势，表明5℃ - SD叶片中产生了更多的冰核剂。这些额外的冰核剂可能是冬黑麦中观察到的抗冻机制的一个组成部分。
