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

Abstract

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.

The Importance of Size and Disorder in the Cryoprotective Effects of Dehydrins

“…Previously, we showed that V. riparia K 2 and YSK 2 do not have any AFP activity (Livernois et al, 2009). In contrast, two other studies showed that PCA60 (a peach Y 2 K 9 dehydrin; Wisniewski et al, 1999) and a putative dehydrin from F. suspensa (Simpson et al, 2005) have AFP activity. At the time, we thought that the two K-segments may have been insufficient to bring about AFP activity.…”

“…Two previous reports suggested that dehydrins also have the ability to shape ice crystals and prevent their growth at moderate subzero temperatures (Wisniewski et al, 1999;Simpson et al, 2005). This activity was suggested to come from the dehydrin present in peach (Prunus persica) bark (PCA60; Wisniewski et al, 1999) and from a partially purified and characterized Forsythia suspensa protein that may contain K-segments (Simpson et al, 2005).…”

“…In vitro, dehydrins have been shown to protect enzymes from freeze-thaw damage (Lin and Thomashow, 1992;Kazuoka and Oeda, 1994;Momma et al, 2003;Goyal et al, 2005;Hughes and Graether, 2011) and heat denaturation (Kovacs et al, 2008), interact with and protect membranes from cold and dehydrative stresses (Rahman et al, 2010;, and bind water (Tompa et al, 2006), ions (Alsheikh et al, 2003), and nucleic acids (Hara et al, 2009). Dehydrins have also been suggested to prevent the growth of ice crystals by functioning in a manner similar to antifreeze proteins (AFPs; Wisniewski et al, 1999;Simpson et al, 2005).…”

Genetic approaches towards overcoming water deficit in plants - special emphasis on LEAs

“…DHNs localize primarily to the nucleus or cytoplasm (Houde et al, 1995;Bracale et al, 1997), but studies also corroborate their presence either in the plasma membrane (Danyluk et al, 1998), endoplasmic reticulum (Neven et al, 1993), chloroplast (Wisniewski et al, 1999), mitochondria (Borovskii et al, 2002) or even in the vacuole (Heyen et al, 2002). DHNs may thus be expressed either constitutively [e.g.…”

“…Arabidopsis DHNX] or confined to the root tip (ERD14 and LTI29), vascular tissues (ERD14, LTI29 and RAB18) and stomatal guard cells (RAB18). DHNs accumulate within vascular tissues (Godoy et al, 1994;Houde et al, 1995;Danyluk et al, 1998;Nylander et al, 2001), pollen sacs, guard cells and root meristems (Nylander et al, 2001), endosperm and zygotic embryos of mature seeds, developing root primordia (Godoy et al, 1994;Bracale et al, 1997), wheat crown tissues (Houde et al, 1995) and peach shoots (Wisniewski et al, 1999). DHNs found only in the cytosol include those of C. plantagineum DSP16 (Schneider et al, 1993) and rice RAB21 (Mundy and Chua, 1988).…”