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  • 1
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Herein the cloning and characterization of a defensin gene (PpDfn1) from a cDNA library made from peach (Prunus persica[L.] Batsch) winter bark tissues is described. A partial clone obtained from the library was extended to full length by 5′ Rapid Amplification of cDNA Ends (RACE). The open reading frame of 237 bp codes for a 79 amino acid peptide related to the defensin family of proteins. Sequence comparison of the encoded protein using blast analysis revealed significant homology to defensins from other plant species. RNA gel blot analysis indicated that the gene is seasonally expressed in bark tissues of 1-year-old shoots, and is also expressed in early fruit development. Results of quantitative RT-PCR and protein blot analysis were similar to those of RNA gel blot analyses for the bark tissues. A recombinant version, rDFN1 was expressed in the yeast, Pichia pastoris. It was found that rDFN1 inhibited germination of the fungal pathogens Penicillium expansum and Botrytis cinerea, but not the Gram-negative bacterium Erwinia amylovora. The potential physiological role of PpDFN1 and its antimicrobial properties are discussed.
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  • 2
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: 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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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 96 (1996), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Although considerable effort has been directed at identifying and understanding the function and regulation of stress-induced proteins in herbaceous plants, reports concerning woody plants are limited. Studies with herbaceous crops have revealed similarities in the types of proteins that accumulate in response to a wide array of abiotic stresses and hormonal cues such as the accumulation of abscisic acid. Many of the identified proteins appear to be related to dehydrins (the D-11 subgroup of late-embryogenesis-abundant proteins). The objective of the present study was to determine if seasonal induction of dehydrins is a common feature in woody plants and to see if seasonal patterns existed for other stress-induced proteins. Bark tissues from eight species of woody plants were collected monthly for a period of 1.5 years. The species included: peach (Prunus persica) cv. Loring; apple (Malus domestica) cv. Golden Delicious; thornless blackberry (Rubus sp.) cv. Chester; hybrid poplar (Populus nigra); weeping willow (Salix babylonica); flowering dogwood (Cornus florida); sassafras (Sassafras albidum); and black locust (Robinia pseudo-acacia). Immunoblots of bark proteins were probed with a polyclonal antibody recognizing a conserved region of dehydrin proteins, and monoclonal antibodies directed against members of the HS70 family of heat-shock proteins. Some proteins, immunologically related to dehydrins, appeared to be constitutive; however, distinct seasonal patterns associated with winter acclimation were also observed in all species. The molecular masses of these proteins varied widely, although similarities were observed in related species (willow and poplar). Identification of proteins using the monoclonal antibodies (HSP70, HSC70, BiP) was more definitive because of their inherent specificity, but seasonal patterns were more variable among the eight species examined. This study represents only a precursory examination of several proteins reported to be stress related in herbaceous plants, but the results indicate that these proteins are also common to woody plants and that further research to characterize their regulation and function in relation to stress adaptation and the perennial life cycle of woody plants is warranted.
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  • 4
    ISSN: 1432-2285
    Keywords: Prunus persica ; Deep supercooling ; Xylem ; Pectins ; Glycoproteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Living xylem tissues and floral buds of several species of woody plants survive exposure to freezing temperatures by deep supercooling. A barrier to water loss and the growth of ice crystals into cells is considered necessary for deep supercooling to occur. Pectins, as a constituent of the cell wall, have been implicated in the formation of this barrier. The present study examined the distribution of pectin in xylem and floral bud tissues of peach (Prunus persica). Two monoclonal antibodies (JIM5 and JIM7) that recognize homogalacturonic sequences with varying degrees of esterification were utilized in conjunction with immunogold electron microscopy. Results indicate that highly esterified epitopes of pectin, recognized by JIM7, were the predominant types of pectin in peach and were uniformly distributed throughout the pit membrane and primary cell walls of xylem and floral bud tissues. In contrast, un-esterified epitopes of pectin, recognized by JIM5, were confined to the outer surface of the pit membrane in xylem tissues. In floral buds, these epitopes were localized in middle lamellae, along the outer margin of the cell wall lining empty intercellular spaces, and within filled intercellular spaces. JIM5 labeling was more pronounced in December samples than in July/August samples. Additionally, epitopes of an arabinogalactan protein, recognized by JIM14, were confined to the amorphous layer of the pit membrane. The role of pectins in freezing response is discussed in the context of present theory and it is suggested that pectins may influence both water movement and intrusive growth of ice crystals at freezing temperatures.
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  • 5
    ISSN: 1432-2048
    Keywords: Prunus ; Cornus ; Cold hardiness ; Supercooling ; Xylem parenchyma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Treatment of stem sections of peach (Prunus persica (L.) Batsch) and flowering dogwood (Cornus florida L.) with macerase, an enzyme mixture rich in pectinase, for 24–48 h resulted in a complete flattening of the low-temperature exotherm (LTE) as determined by differential thermal analysis (DTA). Ultrastructural analysis of macerase-treated tissue demonstrated a nearly complete digestion of the pit membrane (black cap and primary cell-wall) of nearly 100% of the xylem-parenchyma cells examined after 48 h of exposure to the enzyme. Additionally, the underlying amorphous layer was partially degraded in up to 57% of the cells examined. The macerase treatment had no visible effect on secondary cell-walls of xylem tissue. In contrast, treatment of stem tissue with cellulysin (mostly cellulase) resulted in a shift of the LTE to warmer temperatures as determined by DTA, and a digestion of only the outermost layer of the pit membrane in nearly 100% of the cells examined, with little or no effect on the underlying layers. Treatment of tissue with 25 mM sodiumphosphate buffer also resulted in a shift of the LTE to warmer temperatures but the shift was not as great as in cellulysin-treated tissue. The shift was associated with a partial degradation of the outermost layer of the pit membrane in dogwood (33–45% of the cells examined) but not in peach (3–7% of the cells). Collectively, the data indicate that pectins may be an integral structural element of the pit membrane and that this portion of the cell-wall, along with the underlying amorphous layer, play a major role in forming a barrier to water movement and growth of ice crystals. This barrier allows xylem parenchyma of some species of woody plants to undergo deep supercooling.
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  • 6
    ISSN: 1432-2048
    Keywords: Prunus ; Cornus ; Cold hardiness ; Supercooling ; Xylem parenchyma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Treatment of stem sections of peach (Prunus persica (L.) Batsch) and flowering dogwood (Cornus florida L.) with macerase, an enzyme mixture rich in pectinase, for 24–48 h resulted in a complete flattening of the low-temperature exotherm (LTE) as determined by differential thermal analysis (DTA). Ultrastructural analysis of macerase-treated tissue demonstrated a nearly complete digestion of the pit membrane (black cap and primary cell-wall) of nearly 100% of the xylem-parenchyma cells examined after 48 h of exposure to the enzyme. Additionally, the underlying amorphous layer was partially degraded in up to 57% of the cells examined. The macerase treatment had no visible effect on secondary cell-walls of xylem tissue. In contrast, treatment of stem tissue with cellulysin (mostly cellulase) resulted in a shift of the LTE to warmer temperatures as determined by DTA, and a digestion of only the outermost layer of the pit membrane in nearly 100% of the cells examined, with little or no effect on the underlying layers. Treatment of tissue with 25 mM sodiumphosphate buffer also resulted in a shift of the LTE to warmer temperatures but the shift was not as great as in cellulysin-treated tissue. The shift was associated with a partial degradation of the outermost layer of the pit membrane in dogwood (33–45% of the cells examined) but not in peach (3–7% of the cells). Collectively, the data indicate that pectins may be an integral structural element of the pit membrane and that this portion of the cell-wall, along with the underlying amorphous layer, play a major role in forming a barrier to water movement and growth of ice crystals. This barrier allows xylem parenchyma of some species of woody plants to undergo deep supercooling.
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  • 7
    ISSN: 1573-5044
    Keywords: freezing tolerance ; LT50 ; nonacclimated ; Prunus persica
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell suspension cultures were initiated from callus derived from xylem tissues of peach [Prunus persica (L.) Batsch]. Cold acclimation was induced (LT50 of-13°C) in cell suspensions at 3°C in the dark for 10 days. Freezing tolerance returned to the level of nonacclimated cells (LT50 of −4.5°C) when cold-acclimated cells were transferred to 24°C (in dark) for 3 days. Addition of 75 μM abscisic acid (ABA) to the growth medium failed to induce cold acclimation after cells were cultured for 5 days at 24°C. Microvacuolation, cytoplasmic augmentation and disappearance of starch grains were observed in cells that were cold-acclimated by exposure to low temperature. Similar ultrastructural alterations were not observed in ABA-treated cells. Several qualitative and quantitative changes in proteins were noted during both cold acclimation and ABA treatment. Both the ultrastructural and protein changes observed during cold acclimation were reversed during deacclimation. The relationship of these changes to cold acclimation in peach cell-cultures is discussed.
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  • 8
    ISSN: 1573-5028
    Keywords: cold-acclimation ; dehydrin ; endodormancy ; LEA ; peach (Prunus persica L.)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA library was created from cold-acclimated bark tissue of peach and selectively probed using an antibody directed against the lysine-rich consensus region of dehydrin proteins. Several clones were thus obtained which had a high degree of sequence similarity to other dehydrin genes. Northern analysis, using clone 5a, indicated that a 1.8 kb transcript was seasonally expressed in sibling deciduous and evergreen genotypes of peach, and also inducible by water deficit in cv. Rio Oso Gem. The evergreen and deciduous genotypes differ significantly in both their ability to cold-acclimate and in the seasonal expression of the dehydrin transcript and protein. In both genotypes, the transcript was maximally expressed during winter and undetectable in May-July. The evergreen genotype (less cold-tolerant), however, displayed transcript accumulation which lagged behind and declined sooner than in the deciduous genotype. Protein expression was similar to transcript expression, however, protein expression in the evergreen genotype lagged considerably behind transcript accumulation in the fall. This indicates that several levels of regulation of dehydrin proteins may exist during cold acclimation. A genomic clone (G10a) was isolated which contained the full-length dehydrin gene, designated ppdhn1. The peach dehydrin gene encodes 472 amino acids with a predicted size of 50 020 Da. The encoded protein (PCA60) contains nine of the lysine-rich repeats characteristic of dehydrins and two DEYGNP motifs at the amino acid terminus. A genomic blot, probed with clone 5a under stringent conditions, indicated that one or two highly homologous genes are present in peach, whereas an additional member was detected under low-stringency conditions. It is suggested that several members of the dehydrin gene family may exist in peach that vary in their relation to ppdhn1.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 15 (1990), S. 318-319 
    ISSN: 0741-0581
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Additional Material: 3 Ill.
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  • 10
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