Six antifreeze proteins, which have the unique ability to adsorb onto the surface of ice and inhibit its growth, have been isolated from the apoplast of winter rye leaves where ice forms at subzero temperatures. The rye antifreeze proteins accumulate during cold acclimation and are similar to plant pathogenesis‐related proteins, including two endoglucanase‐like, two chitinase‐like and two thaumatin‐like proteins. Immunolocalization of the glucanase‐like antifreeze proteins showed that they accumulate in mesophyll cell walls facing intercellular spaces, in pectinaceous regions between adjoining mestome sheath cells, in the secondary cell walls of xylem vessels and in epidermal cell walls. Because the rye antifreeze proteins are located in areas where they could be in contact with ice, they may function as a barrier to the propagation of ice or to inhibit the recrystallization of ice. Antifreeze proteins similar to pathogenesis‐related proteins were also found to accumulate in closely‐related plants within the Triticum group but not in freezing‐tolerant dicotyledonous plants. In winter wheat, the accumulation of antifreeze proteins and the development of freezing tolerance are regulated by chromosome 5. Rye antifreeze proteins may have evolved from pathogenesis‐related proteins, but they retain their catalytic activities and may play a dual role in increasing both freezing and disease resistance in overwintering plants.

During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (CLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are' produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. l h e distributions of pathogenesis-induced and cold-accumulated CLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues.

Freezing-tolerant plants withstand extracellular ice formation at subzero temperatures. Previous studies have sbown tbat winter rye {Secale cereale L.) accumulates proteins in the leaf apoplast during cold acclimation that have antifreeze im>perties and are similar to patbogenesis-related proteins. To determine whether tbe accumulation of these antifreeze proteins is common among herbaceous plants, we assayed antifreeze activity and total protein content in leaf apoplastic extracts from a ntimber of species grown at low temperature, including bodi monocotyledons (winter and spring rye, winter and spring wbeat, winter barley, spring oats, maize) and dicotyledons (spinach, winter and spring oilseed rape Icanola], kale, tobacco). Apoplastic polypeptides were also separated by SDS-PAGE and immunoblotted to determine whether plants generally respond to low temperature by accumulating pathogenesis-related proteins. Our results sbowed that significant levels of antifreeze activity were present only in the £q>oplast of freezing-tolerant monocotyledons after cold acclimsuion at 5/2°C. Moreover, only a closely related group of plants, rye, wbeat and barley, accumulated antifreeze proteins similar to patbogenesis-related proteins during cold acclimation. Tbe results indicate that the accumulation of antifreeze proteins is a specific response that may be important in the freezing tolerance of some plants, rather than a general response of all plants to low temperature stress.

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