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

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

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Main Author: Wisniewski, Michael.
Other Authors: Webb, Robert., Balsamo, Ron., Close, Timothy., Yu, Xiao-Ming., Griffith, Marilyn.
Language: English
Published: 1998
Online Access: http://ezproxy.villanova.edu/login?url=http://digital.library.villanova.edu/Item/vudl:173979
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