The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale 1.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-p-l,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-p-l,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice.
During cold acclimation, winter rye (Secale cerealeL.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold‐acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold‐acclimated leaves than in unextracted cold‐acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation
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 (Secak cereale). Plants were grown at 20°C, at 5°C with a long day, and at 5°C with a short day (5'C-SD). The threshold temperature for INA was -5 to -12°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. lntrinsic 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°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 adivity. Carbohydrates and phospholipids were important components of ice nucleators derived from 20°C leaves, whereas the protein component was more important in 5°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'C-SD leaves. These additional ice nucleators may be a component of the mechanism for freezing tolerance observed in winter rye.
1993. Proteins accumulate in the apoplast of winter rye leaves during cold acchmation. -Physiol. Plant. 87: 499-507.During cold acclimation, winter rye {Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacctimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acciimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exeit a direct influence on the growth of ice. in addition, freezing injury was greater in extracted cold-acciimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoptast are an important component of the mechanism by which winter rye leaves tolerate ice formation.
12When winter settles upon the landscape and the soil and lakes have frozen, it is important to realize that many biological organisms have frozen too. After a period ofacclimatization to cold autumn temperatures and short daylengths, multicellular organisms ranging from fungi to perennial plants to frogs develop the ability to survive subzero temperatures by forming ice within their tissues. Freezing does not occur throughout these organisms; instead, ice forms only outside the cells in specific locations within the tissues.
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