IMMUNOLOCALIZATION OF ANTIFREEZE PROTEINS IN WINTER RYE LEAVES, CROWNS, AND ROOTS BY TISSUE PRINTING

During cold acclimation, antifreeze proteins (AFPs) that are similar t o pathogenesis-related proteins accumulate in the apoplast of winter r ye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to m odify the growth of ice. To elucidate the role of AFPs in the Greeting process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from col d-acclimated rye leaves, crowns, and roots exhibited antifreeze activi ty, whereas no antifreeze activity was observed in extracts from nonac climated rye plants. Antibodies raised against three apoplastic rye AF Ps, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinas e-like protein (GLP, 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-ac climated plants. Although GLPs, CLPs, and TLPs were present in nonaccl imated plants, they were found in different locations and did not exhi bit antifreeze activity, which suggests that different isoforms of pat hogenesis-related proteins are produced at low temperature. The locati on of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of path ogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice e nter and propagate through plant tissues.