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.     

Cold Acclimation in Genetically Related (Sibling) Deciduous and Evergreen Peach (Prunus persica [L.] Batsch): I. Seasonal Changes in Cold Hardiness and Polypeptides of Bark and Xylem Tissues

Seasonal patterns of proteins and of cold hardiness were characterized in bark and xylem tissues of genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). In contrast with deciduous trees, which entered endodormancy and abscised leaves in the fall, evergreen trees retained their leaves and exhibited shoot elongation under favorable environmental conditions. A successive increase in the cold hardiness of bark and xylem was observed during the fall in both genotypes. This was followed by a subsequent decrease from midwinter to spring. Xylem tissue in both genotypes exhibited deep supercooling and a significant correlation (r = 0.99) between the midpoint of the low-temperature exotherm and the subzero temperature at which 50% injury occurred (assessed by electrolyte leakage) was noted. The maximum hardiness level attained in deciduous trees was more than twofold that of evergreens. Seasonal pattern of proteins from bark and xylem of the sibling genotypes was characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among other qualitative and quantitative changes, accumulation of a 19-kilodalton polypeptide in the bark of both genotypes was observed during fall followed by a decrease in spring. This polypeptide accumulated to higher levels in the deciduous peach compared with the evergreen. Additionally, a 16-kilodalton protein exhibited the same pattern in deciduous trees but not in the evergreen trees. Both the 19- and a 16-kilodalton bark proteins conform to the criteria of a bark storage protein. The relationship of seasonal changes in protein to cold hardiness and dormancy in these genetically related peach genotypes is discussed.     

Seasonal fluctuation of dehydrins is related to osmotic status in Scots pine needles

Seasonal variation in dehydrins and other soluble proteins of Scots pine (Pinus sylvestris L.) needles, buds and bark were analyzed monthly for 1 year from 1998 to 1999. Dehydrin-related proteins of 60 and 56 kDa were identified immunologically in all tissues. The concentration of the 60-kDa dehydrin was highest during the winter (October-February) in buds and bark but increased in early spring (March-May) in needles. Accumulation of the 60-kDa dehydrin in the needles in springtime was related to the decreasing osmotic potentials of the needles. The 56-kDa dehydrin was present only during the growing season, as was a 50-kDa dehydrin, which only appeared in bud and bark tissues. The soluble protein concentration of needles did not differ significantly between seasons, but in bark and bud tissues the protein concentrations were at their lowest level in newly grown tissues (June-August). The level of several polypeptides was higher during the winter-spring period than in the growing season, especially in bark and bud tissues. These proteins may be related to cold hardiness or dormancy in overwintering Scots pine. Dehydrin-related proteins in needles are linked to springtime changes in the osmotic status of needles rather than to their cold acclimation.     

Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L. Batsch)

In the temperate climate of the northern hemisphere, winter survival of woody plants is determined by the ability to acclimate to freezing temperatures and to undergo a period of dormancy. Cold acclimation in many woody plants is initially induced by short photoperiod and low, non-freezing temperatures. These two factors (5°C and short photoperiod) were used to study changes in the proteome of bark tissues of 1-year-old peach trees. Difference in-gel electrophoresis technology, a gel-based approach involving the labeling of proteins with different fluorescent dyes, was used to conduct a quantitative assessment of changes in the peach bark proteome during cold acclimation. Using this approach, we were able to identify differentially expressed proteins and to assign them to a class of either ‘temperature-responsive’ or ‘photoperiod-responsive’ proteins. The most significant factor affecting the proteome appeared to be low temperature, while the combination of low temperature and short photoperiod was shown to act either synergistically or additively on the expression of some proteins. Fifty-seven protein spots on gels were identified by mass spectrometry. They included proteins involved in carbohydrate metabolism (e.g., enolase, malate dehydrogenase, etc), defense or protective mechanisms (e.g., dehydrin, HSPs, and PR-proteins), energy production and electron transport (e.g., adenosine triphosphate synthases and lyases), and cytoskeleton organization (e.g., tubulins and actins). The information derived from the analysis of the proteome is discussed as a function of the two treatment factors: low temperature and short photoperiod. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning and characterization of a novel cold-regulated gene from Capsella bursa-pastoris.

A novel cor gene was cloned from Capsella bursa-pastoris (designated Cbcor15b) by RACE-PCR. The full-length cDNA of Cbcor15b was 652bp and contained a 417bp open reading frame (ORF) encoding a 139-amino acid hydrophilic protein. Multiple alignments showed that Cbcor15b had high similarity with other cold-regulated genes from Arabidopsis thaliana (cor15b, cor15a), Brassica napus (bn115, bn19 and bn26) and genes encoding late embryogenesis abundant (LEA) proteins. The predicted CbCOR15B protein was found to have a potential chloroplast signal sequence cleavage site, two cAMP- and cGMP-dependent protein kinase (PKA and PKG) phosphorylation sites. Cold acclimation assay showed that Cbcor15b was relevant to cold acclimation. Our study implies that Cbcor15b might have similar functions possessed by other cor genes in increasing plants' freezing tolerance.     

Development of Desiccation Tolerance during Embryogenesis in Rice (Oryza sativa) and Wild Rice (Zizania palustris) (Dehydrin Expression,Abscisic Acid Content,and Sucrose Accumulation)

The ability of seeds to withstand desiccation develops during embryogenesis and differs considerably among species. Paddy rice (Oryza sativa L.) grains readily survive dehydration to as low as 2% water content, whereas North American wild rice (Zizania palustris var interior [Fasset] Dore) grains are not tolerant of water contents below 6% and are sensitive to drying and imbibition conditions. During embryogenesis, dehydrin proteins, abscisic acid (ABA), and saccharides are synthesized, and all have been implicated in the development of desiccation tolerance. We examined the accumulation patterns of dehydrin protein, ABA, and soluble saccharides (sucrose and oligosaccharides) of rice embryos and wild rice axes in relation to the development of desiccation tolerance during embryogenesis. Dehydrin protein was detected immunologically with an antibody raised against a conserved dehydrin amino acid sequence. Both rice and wild rice embryos accumulated a 21-kD dehydrin protein during development, and an immunologically related 38-kD protein accumulated similarly in rice. Dehydrin protein synthesis was detected before desiccation tolerance had developed in both rice embryos and wild rice axes. However, the major accumulation of dehydrin occurred after most seeds of both species had become desiccation tolerant. ABA accumulated in wild rice axes to about twice the amount present in rice embryos. There were no obvious relationships between ABA and the temporal expression patterns of dehydrin protein in either rice or wild rice. Wild rice axes accumulated about twice as much sucrose as rice embryos. Oligosaccharides were present at only about one-tenth of the maximum sucrose concentrations in both rice and wild rice. We conclude that the desiccation sensitivity displayed by wild rice grains is not due to an inability to synthesize dehydrin proteins, ABA, or soluble carbohydrates.     

Ultrastructural and protein changes in cell suspension cultures of peach associated with low temperature-induced cold acclimation and abscisic acid treatment

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 M 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.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - Ms Murashige & Skoog - PMSF phenylmethylsulfonyl fluoride - LT₅₀ or Freezing Tolerance temperature that resulted in 50% decrease in TTC reduction - TTC 2,3,5-triphenyltetrazolium chloride     

cDNA sequence and expression of a cold-responsive gene in Citrus unshiu

A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.     

Chill-responsive dehydrins in blueberry: Are they associated with cold hardiness or dormancy transitions?

To survive winters, woody perennials of temperate zones must enter into endodormancy. Resumption of spring growth requires sufficient exposure to low temperature (chill units, CUs) in winter (chilling requirement), which also plays a role in the development of cold hardiness (cold acclimation). Physiological studies on dormancy breaking have focused on identifying markers, such as appearance or disappearance of proteins in response to varying degrees of chill unit accumulation. However, whether these changes are associated with dormancy transitions or cold acclimation is not clear. In the present study, greenhouse-grown blueberry (Vaccinium section Cyanococcus) plants were used to address this question. Three blueberry cultivars, Bluecrop, Tifblue, and Gulfcoast having chilling requirement of approximately 1 200, 900 and 600 CUs, respectively, were first exposed to 4°C for long enough to provide chill units equivalent to one-half of their respective chilling requirement. This treatment was expected to result in cold acclimation. A fraction of plants was then subjected to a 15/12°C (light/dark) regime for 2 weeks, a treatment expected to be “dormancy-neutral” but cause deacclimation. Before and after each treatment, cold hardiness and dormancy status of floral buds were determined; proteins were extracted from the buds collected on the same sampling date, and separated by one-dimensional SDS-PAGE. Dehydrin-like proteins were identified by immunoblotting, using anti-dehydrin antiserum. Results indicate that the chilling treatment resulted in cold acclimation as indicated by increased bud hardiness in all three cultivars. Data also indicate a distinct accumulation of three dehydrin-like proteins of 65, 60, and 14 kDa during cold acclimation. The cold hardiness and levels of dehydrin proteins decreased during the exposure to 15/12°C for 2 weeks. Results also confirmed that this treatment had no negative effect on chill unit accumulation. Densitometric scans of protein gels indicated a close association between the abundance of dehydrins and degree of cold hardiness in these cultivars. In addition, levels of the dehydrin proteins and cold hardiness remained about the same between 100% and >100% satisfaction of chilling requirement. These results suggest that changes in dehydrin expression are more closely associated with cold hardiness than with dormancy transitions.     

A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn

Several cDNAs related to an ABA-induced cDNA from barley aleurone were isolated from barley and corn seedlings that were undergoing dehydration. Four different barley polypeptides with sizes of 22.6, 16.2, 14.4 and 14.2 kDa and a single corn polypeptide with a size of 17.0 kDa were predicted from the nucleotide sequences of the cDNAs. These dehydration-induced proteins (dehydrins) are very similar to each other and to a previously identified rice protein induced by ABA and salt, and have at least some similarity to a previously identified cotton embryo protein. Each dehydrin is extremely hydrophilic, glycine-rich, cysteine- and tryptophan-free and contains repeated units in a conserved linear order. A lysine-rich repeating unit occurs twice in each protein, once at the carboxy terminus and once partway through the polypeptide, adjacent to a succession of serines. This repeating unit and the adjacent flanking run of serines are conserved with minimal variation among all dehydrins. Another repeating unit is flanked by the two copies of the lysine-rich unit, and varies in number from one to five copies. This latter repeating unit is less conserved than the former, varying even within a singly dehydrin. The messenger RNAs corresponding to each cDNA are abundant in dehydrating, but not in well-watered seedlings. The amino acid sequence of tryptic peptides from purified dehydration-induced proteins of corn established that the corn cDNAs correspond to a protein that is produced in abundance during the response of corn seedlings to dehydration.Abbreviations ABA abscisic acid - GA gibberellin - SDS sodium dodecyl sulfate - EDTA ethylenediaminetetraacetic acid - SSC 0.15 M NaCl, 0.015 M Na₃ citrate, pH 7.0 - ddH₂O double-distilled water - TES N-tris [hydroxymethyl]-methyl-2-aminoethanesulfonic acid     

静电场对苜蓿愈伤组织抗寒能力的影响

用正负高压静电场 (HVEF)处理苜蓿叶片来源的愈伤组织后 ,在4℃下暗培养。我们发现静电场处理可不同程度地提高愈伤组织的淀粉酶 ,过氧化物酶 ,和超氧化物岐化酶的活性和可溶性蛋白的含量 ,促进愈伤组织对冷害的防御能力。在冷处理过程中 ,细胞膜中饱和脂肪酸 (棕榈梭和硬脂酸 )和不饱和脂肪梭含量先升高然后降低 ,而高压静电场处理组的不饱和脂肪酸 (油酸、亚油酸、α -亚麻酸、β-亚麻酸 )含量高于对照组 ,脂肪酸不饱和指数 (UIFA)也高于对照组 ,这说明静电场处理可促进愈伤组织细胞抗寒能力提高。此外 ,丙二醛 (MDA)含量在静电场处理组低于对照组的测定结果也验证了前面的结果。     

Programmed accumulation of LEA-like proteins during desiccation and cold acclimation of overwintering grape buds

Molecular investigation of the process of cold acclimation in woody plants has been limited by the superimposition of dormancy-related events on the process of cold tolerance development. To address this limitation, we have used the grape Vitis labruscana L. ev. Concord to develop a system in which the developmental programme of dormancy can be induced seperately from cold acclimation. Using this system we have characterized differential accumulation of several proteins in grape buds during the normally superimposed endodormancy and cold acclimation programmes, and in buds which have entered only the endodormancy programme. A set of 47 kD proteins accumulated during endodormancy without cold acclimation to a level similar to that found in endodormant and cold-acclimated buds, but without any associated increase in bud cold-acclimation level. However, a 27 kD LEA-like protein accumulated only in cold acclimated buds. We conclude that expression of the 47 kD glycoprotein is endodormancy-related, but is not strictly related to the development of cold acclimation, while the 27 kD protein appears to be more specific to cold acclimation. In addition to strengthening the association of LEA-like proteins with cold acclimation, this system allows more specific assessment of cold acclimation-associated phenomena in overwintering buds.     

Chemical and Biophysical Changes in the Plasma Membrane during Cold Acclimation of Mulberry Bark Cells (Morus bombycis Koidz. cv Goroji)

The lipid and protein composition of the plasma membrane isolated from mulberry (Morus bombycis Koidz.) bark cells was analyzed throughout the cold acclimation period under natural and controlled environment conditions. There was a significant increase in phospholipids and unsaturation of their fatty acids during cold acclimation. The ratio of sterols to phospholipids decreased with hardiness, primarily due to the large increase in phospholipids. The fluidity of the plasma membrane, as determined by fluorescent polarization technique, increased with hardiness. Electrophoresis of plasma membrane proteins including glycoproteins revealed change in banding pattern during the early fall to winter period. Some of the protein changes could be related to growth cessation and defoliation. However, minor changes in proteins also occurred during the most active period of hardening. Changes in glycoproteins were coincident both with changes in growth stages and with the development of cold hardiness.