Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: A dehydrin from peach (Prunus persica)

Dehydrins are glycine-rich, hydrophilic, heat-stable proteins and are gener ally induced in response to a wide array of environmental stresses. In prev ious research (Artlip et al, 1997, Plant Molecular Biology 33: 61-70), a fu ll-length dehydrin gene, ppdhn1, was isolated from peach, and its expressio n was associated with qualitative and quantitative differences in cold hard iness in sibling genotypes of evergreen and deciduous peach, Similar result s were obtained for levels of the corresponding 60 kDa peach dehydrin prote in (PCA60). The objective of the present study was to purify the PCA60, tes t the purified protein for cryoprotective and/or antifreeze activity, and t o determine the cellular localization of PCA60 using immunomicroscopy. PCA6 0 was extracted from winter bark tissues of peach (Prunus persica [L.] Bats ch) and purified in a two-step process. Separation was based on free-soluti on isoelectric focusing followed by size exclusion, Purified PCA60, as well as crude protein extract, preserved the in vitro enzymatic activity of lac tate dehydrogenase after several freeze-thaw cycles in liquid nitrogen. PCA also exhibited distinct antifreeze activity as evidenced by ice crystal mo rphology and thermal hysteresis, This is the first time antifreeze activity has been demonstrated for dehydrins. Immunomicroscopy, utilizing an affini ty-purified, polyclonal antibody developed against a synthetic peptide of t he lysine-rich consensus portion of dehydrins, indicated that PCA60 was fre ely distributed in the cytoplasm, plastids, and nucleus of bark cells and x ylem parenchyma cells. Although the functional role of dehydrins remains sp eculative, the data support the hypothesis that it plays a role in preventi ng denaturation of proteins exposed to dehydrative stresses.