Dehydrins are glycine-rich, hydrophilic, heat-stable proteins isoelectric focusing followed by size exclusion. Purified PCA60, as well as crude protein extract, preserved the in 7itro and are generally induced in response to a wide array of enzymatic activity of lactate dehydrogenase after several environmental stresses. In previous research (Artlip et al.freeze-thaw cycles in liquid nitrogen. PCA also exhibited 1997, Plant Molecular Biology 33: 61-70), a full-length dehydrin gene, ppdhn1, was isolated from peach, and its distinct antifreeze activity as evidenced by ice crystal morexpression was associated with qualitative and quantitative phology and thermal hysteresis. This is the first time antifreeze activity has been demonstrated for dehydrins. differences in cold hardiness in sibling genotypes of evergreen Immunomicroscopy, utilizing an affinity-purified, polyclonal and deciduous peach. Similar results were obtained for levels of the corresponding 60 kDa peach dehydrin protein (PCA60). antibody developed against a synthetic peptide of the lysinerich consensus portion of dehydrins, indicated that PCA60 The objective of the present study was to purify the PCA60, test the purified protein for cryoprotective and/or antifreeze was freely distributed in the cytoplasm, plastids, and nucleus activity, and to determine the cellular localization of PCA60 of bark cells and xylem parenchyma cells. Although the using immunomicroscopy. PCA60 was extracted from winter functional role of dehydrins remains speculative, the data support the hypothesis that it plays a role in preventing bark tissues of peach (Prunus persica [L.] Batsch) and purified in a two-step process. Separation was based on free-solution denaturation of proteins exposed to dehydrative stresses.Abbre6iations -AFP: antifreeze protein; BSA: bovine serum albumin; LDH: lactate dehydrogenase; PBS: phosphate buffered saline.

Cold acclimation in temperate, woody plants is a complex phenomenon that involves distinct changes in gene activity and protein synthesis. In previous research, a 60-kDa protein (PCA60), belonging to the dehydrin family of stress-related proteins, was identified in peach bark, and its corresponding gene (ppdhn1) was cloned and characterized. Presently, we report on the results of immunolocalization studies and in vitro cryoprotection assays. Seasonal collections of current-year stems were embedded in LR White or epoxy resin and sections of bark were probed with either a polyclonal antibody directed against a 15 amino acid sequence consensus region of dehydrins or a polyclonal antibody directed against partially-purified PCA60. In vitro cryoprotection assays utilized lactose dehydrogenase (LDH), a cold-labile enzyme. Immunolocalization at the light level indicated that the dehydrin was confined to the cytoplasm and absent in organelles. This localization was preliminarily confirmed at the ultrastructural level. LDH assays indicated cryoprotective activity in total protein extracts collected from winter bark tissues but completely absent in extracts of summer bark tissues. Preliminary LDH assays utilizing purified PCA60 also demonstrated cryoprotective activity. In general, the data further support a role for dehydrins in cold acclimation of woody plants.