CHARACTERIZATION AND QUANTIFICATION OF INTRINSIC ICE NUCLEATORS IN WINTER RYE (SECALE-CEREALE) LEAVES

Extracellular ice formation in frost-tolerant organisms is often initi ated at specific sites by ice nucleators. In this study, we examined i ce nucleation activity (INA) in the frost-tolerant plant winter rye (S ecale cereale). Plants were grown at 20 degrees C, at 5 degrees C with a long day, and at 5 degrees C with a short day (5 degrees C-SD). The threshold temperature for INA was -5 to -12 degrees C in winter rye l eaves from all three growth treatments. Epiphytic ice nucleation-activ e bacteria could not account for INA observed in the leaves. Therefore , the INA must have been produced endogenously. Intrinsic rye ice nucl eators were quantified and characterized using single mesophyll cell s uspensions obtained by pectolytic degradation of the leaves. The most active ice nucleators in mesophyll cell suspensions exhibited a thresh old ice nucleation temperature of -7 degrees C and occurred infrequent ly at the rate of one nucleator per 10(5) cells. Rye cells were treate d 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 importa nt for activity. Carbohydrates and phospholipids were important compon ents of ice nucleators derived from 20 degrees C leaves, whereas the p rotein component was more important in 5 degrees C-SD leaves. This dif ference in composition or structure of the ice nucleators, combined wi th a tendency for more frequent INA, suggests that more ice nucleators are produced in 5 degrees C-SD leaves. These additional ice nucleator s may be a component of the mechanism for freezing tolerance observed in winter rye.