IMMUNOGOLD LOCALIZATION OF GLUCANASE-LIKE ANTIFREEZE PROTEIN IN COLD-ACCLIMATED WINTER RYE

Apoplastic antifreeze proteins (AFPs) accumulate in winter rye (Secale cereale L. cv. Musketeer) leaves during cold acclimation. Two of the rye AFPs with molecular masses of 32 and 35 kDa are similar in their a mino acid sequences and epitopes to beta-1,3-endoglucanase. Localizati on of these AFPs, which we refer to as glucanase-like proteins (GLPs), was carried out with antiserum raised against the 32 kDa AFP. Specime ns from leaves and roots of non-acclimated (NA) plants and cold acclim ated (CA) plants were prepared by freeze-substitution for high resolut ion immunoelectron microscopy. In CA leaves, high levels of GLPs were observed in cell walls of mesophyll cells adjacent to intercellular sp aces and in secondary thickenings of xylem vessels. Taken together wit h the absence of GLPs in vacuoles, these results confirm the apoplasti c accumulation of AFPs in CA winter rye. Within the cells of CA leaves , GLPs were localized in cisternae of the rough endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which indicates that GLP s are secreted via an exocytic bulk-flow pathway. The occurrence of hi gh levels of GLPs in CA leaves, their low presence in NA leaves and th e lack of GLPs in roots all suggest that there is a correlation betwee n increased accumulation of GLPs and increased freezing tolerance of t hese plant materials. Furthermore, the localization of GLPs in the imm ediate vicinity of pathways for free water within the tissues supports the view that these proteins have an important role in the crystalliz ation and/or recrystallization of water when the leaves of CA winter r ye are exposed to freezing temperatures.