Accumulation of Dehydrins and ABA-Inducible Proteins in Wheat Seedlings during Low-Temperature Acclimation

Using one-dimensional SDS-PAGE and immunochemical methods, we detected the presence and estimated the content of dehydrins and ABA-inducible (RAB) polypeptides in etiolated seedlings of four spring and three winter wheat (Triticum aestivum L.) cultivars differing in frost hardiness. We hardened three-day-old seedlings at 4°C for nine days or grew them at 22°C for a day (control seedlings). We established that heat-stable cold-regulated (COR) polypeptides with mol wts of 209, 196, 169, 66, 50, and 41 kD, which are characteristic of hardened wheat seedlings, were homologous to polypeptides from a dehydrin family and polypeptides with mol wts of 209, 196, 66, 50, and 41 kD were immunologically related to RAB-proteins. We supposed that these COR polypeptides were involved in the prevention of local protein dehydration and denaturation during hypothermia. Analysis of the relative content of COR proteins revealed a close correlation between the cultivar frost hardiness and the concentration of these proteins. It seems evident that different accumulation of dehydrins and RAB polypeptides in different cultivars of a single species is one of the causes for different plant frost hardiness.     

The dual effects of salicylic acid on dehydrin accumulation in water-stressed barley seedlings

Dehydrins are a group of plant proteins that usually accumulate in response to environmental stresses. They are proposed to play specific protective roles in plant cells. Present study showed that the accumulation of dehydrins in water-stressed barley (Hordeum vulgare L.) seedlings was influenced by their treatment with salicylic acid (SA). The level of dehydrin proteins was increased by 0.20 mM SA, but decreased by 0.50 mM SA treatment. Both mRNA expression and protein accumulation of a typical barley dehydrin, DHN5, were enhanced by SA treatment when SA concentrations were lower than 0.25 mM. However, the higher SA concentrations significantly decreased the protein level of DHN5 despite of a stable mRNA level. Our results also showed that low SA concentrations (less than 0.25 mM) decreased the electrolyte leakage and malondialdehyde (MDA) and H₂O₂ contents in water-stressed barley seedlings. But high SA concentrations (more than 0.25 mM) enhanced H₂O₂ accumulation, tended to cause more electrolyte leakage, and increase MDA content. These data indicated that SA could up-regulate the dehydrin gene expression and protein accumulation. Since the protective role of dehydrins in plant cells, such effect could be an important reason for the SA-mediated alleviation on water stress injury. But excessive SA could suppress the accumulation of dehydrin proteins and aggravate the oxidative damage. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 3, pp. 388–394. This text was submitted by the authors in English.     

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.     

Genetic studies of Triticeae dehydrins: assignment of seed proteins and a regulatory factor to map positions

 A collection of 200 wheat (Triticum aestivum L. cv ‘Chinese Spring’) cytogenetic stocks (nullisomic, tetrasomic, nulli-tetrasomic, ditelosomic and deletion lines, addition and substitution stocks from intra- and inter-specific crosses) was utilized to determine the proteins encoded by some of the wheat and barley dehydrin genes, using a western blot procedure. Proteins extracted from seeds were reacted with antibodies that recognize dehydrins in a wide range of plants, including wheat and barley. Proteins encoded by dehydrin loci in chromosome arms 4DS, 5BL and 6AL of ‘Chinese Spring’ wheat were assigned by this method. There was also evidence of a regulatory factor on 5B in the vicinity of the dhn genes, and on 5H in wheat-barley addition lines, that is required for a normal level of expression of seed dehydrins in hexaploid wheat. Further understanding of this putative regulatory factor would be helpful for the interpretation of linkage studies that may relate dehydrin gene expression to phenotypes such as dehydration, salinity or low-temperature tolerance. Received: 27 August 1997 / Accepted: 4 February 1998     

Overexpression of Multiple Dehydrin Genes Enhances Tolerance to Freezing Stress in Arabidopsis

To elucidate the contribution of dehydrins (DHNs) to freezing stress tolerance in Arabidopsis, transgenic plants overexpressing multiple DHN genes were generated. Chimeric double constructs for expression of RAB18 and COR47 (pTP9) or LTI29 and LTI30 (pTP10) were made by fusing the coding sequences of the respective DHN genes to the cauliflower mosaic virus 35S promoter. Overexpression of the chimeric genes in Arabidopsis resulted in accumulation of the corresponding dehydrins to levels similar or higher than in cold-acclimated wild-type plants. Transgenic plants exhibited lower LT50 values and improved survival when exposed to freezing stress compared to the control plants. Post-embedding immuno electron microscopy of high-pressure frozen, freeze-substituted samples revealed partial intracellular translocation from cytosol to the vicinity of the membranes of the acidic dehydrin LTI29 during cold acclimation in transgenic plants. This study provides evidence that dehydrins contribute to freezing stress tolerance in plants and suggests that this could be partly due to their protective effect on membranes.     

Immunoanalysis of dehydrins in Araucaria angustifolia embryos

The aim of this study was to describe the dehydrin content of mature Araucaria angustifolia embryos, a species of endangered and economically important conifers, native to southern Brazil, northeastern Argentina, and eastern Paraguay. The A. angustifolia seeds have been categorized as recalcitrant. Dehydrins were studied by western blot analysis and in situ immunolocalization microscopy using antibodies raised against the K segment, a highly conserved lysine-rich 15-amino acid sequence extensively used to recognize proteins immunologically related to the dehydrin family. Western blot analysis of the heat-stable protein fraction, as estimated by 15 % SDS-PAGE, revealed three main bands of approximately 20-, 26-, and 29-kDa; when 17.5 % SDS-PAGE was used, each band resolved into two other bands. Two thermosensitive dehydrin bands of around 16 and 35 kDa were common to the axis and cotyledons, and another thermosensitive band, with molecular mass of approximately 10 kDa, was present in the cotyledons only. Following alkaline phosphatase (AP) treatment, a gel mobility shift was detected for each one of the four main bands that can be due to phosphorylation. Dehydrins were detected in all axis and cotyledon tissues using in situ immunolocalization microscopy. At the subcellular level, dehydrins were immunolocalized in the nuclei, protein bodies, and microbodies. In the nucleus, dehydrins were found to be associated with chromatin. We concluded that the gel mobility shift for the four main bands (probably due to phosphorylation), the presence of thermosensitive bands, and the specific localizations in nuclei and protein bodies provide key starting points to understand the function of dehydrins in the embryo cells of this species.     

Temporal accumulation and ultrastructural localization of dehydrins in Zea mays

Immunolocalization using polyclonal antibodies raised against a conserved dehydrin amino acid sequence was used to establish the temporal and spatial patterns of dehydrin accumulation in embryo tissue of Zea mays L. (var. Ohio 43) kernels imbibed in the presence of abscisic acid. The temporal pattern of accumulation indicated an increase in dehydrins over time (particularly between 15 and 30 h) and with maximum levels detected 48 h after the onset of imbibition. Dehydrins were first evident, and also the most concentrated, in the cytosol throughout the accumulation period suggesting that the primary function of dehydrins involves the cytosol and the structures contained therein. Only after an accumulation of dehydrins in the cytosol was there an increase in the abundance of nuclear dehydrins. In addition, dehydrins were also observed in association with the proteinaceous matrix of protein bodies and membranes of protein and lipid bodies; these findings have not been reported previously. The observed localization at a number of sites indicates that the specific biochemical roles of dehydrins are likely to be diverse.     

PROTEINS IMMUNOLOGICALLY RELATED TO DEHYDRINS IN FUCOID ALGAE

Adult fucoid algae on Atlantic shores have well-characterized, species-specific tolerances to the varying levels of desiccation that occur from the low to high intertidal zones; however, less is known about embryonic tolerances and their mechanistic basis. We investigated this by 1) exposing embryos of Fucus evanescens C. Agardh, F. spiralis L., and F. vesiculosus L. from the Maine shore to osmotic desiccation in hypersaline seawater and 2) examining whether these embryos contain species-specific dehydrins, proteins first identified in higher plants that are hypothesized to confer tolerance to dehydration. Embryonic survival when cultured in hypersaline seawater >100 practical salinity units (psu) correlated with the position of these species in the intertidal zone (F. spiralis > F. vesiculosus > F. evanescens), but all 1-day-old embryos of these species tolerated treatment with 100 psu or lower seawater. Proteins (17–105 kDa) immunologically related to dehydrins were detected on western blots with dehydrin antibodies raised against a synthetic peptide representing the conserved motif of dehydrins in higher plants. These proteins were constitutive and unstable when subjected to prolonged (>15 min) temperatures above 55° C, unlike most higher plant dehydrins, which are inducible and remain soluble at 75°–100° C. The presence of these proteins was species- and stage-specific. Sperm of F. vesiculosus had a characteristic protein of 76 kDa, whereas eggs and embryos (6 h to 3 days old) had a 92-kDa protein. By 1 week of age, expression of the 92-kDa protein decreased, and the 35-kDa protein of adults was present. Embryos of A. nodosum L. and Pelvetia compressa J. Agardh DeToni contained an 85-kDa protein rather than the 92-kDa protein of Fucus embryos (F. distichus L., F. evanescens, F. spiralis, and F. vesiculosus). The 92-kDa protein became more abundant in embryos exposed to hyperosmotic seawater at 50 psu (F. evanescens and F. vesiculosus) or 150 psu (F. spiralis); however, dehydrin-like proteins of some molecular masses decreased in abundance simultaneously. Further characterization of these proteins is required to establish whether they protect embryos against intertidal desiccation.     

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.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Dehydrin stress proteins in birch buds in regions with contrasting climate

Dehydrin stress proteins were identified in buds of silver birch (Betula pendula Roth) grown in regions with contrasting climate, Karelia and Central Yakutia, using specific antibodies. Two types of dehydrins present in the plant buds, proteins with average (56–73 kDa) and low (14–21 kDa) molecular weight, as well as 17-kDa dehydrin, were detected in all studied plants. The most sensitive to seasonal changes are 14- to 21-kDa dehydrins, the level of which, regardless of the region where the birch grows, significantly increased during the autumn–winter period. The intraspecific polymorphism of dehydrins was more pronounced in B. pendula grown under the sharply continental climate of Yakutia, which is probably due to the peculiarities of the adaptation of woody plants to the extremely low temperatures of the cryolitic zone.     

GTP Promotes the Formation of Early-Import Intermediates But Is Not Required during the Translocation Step of Protein Import into Chloroplasts

Dehydrins are a family of proteins (LEA [late-embryogenesis abundant] D11) commonly induced by environmental stresses associated with low temperature or dehydration and during seed maturation drying. Our previous genetic studies suggested an association of an approximately 35-kD protein (by immunological evidence a dehydrin) with chilling tolerance during emergence of seedlings of cowpea (Vigna unguiculata) line 1393-2-11. In the present study we found that the accumulation of this protein in developing cowpea seeds is coordinated with the start of the dehydration phase of embryo development. We purified this protein from dry seeds of cowpea line 1393-2-11 by using the characteristic high-temperature solubility of dehydrins as an initial enrichment step, which was followed by three chromatography steps involving cation exchange, hydrophobic interaction, and anion exchange. Various characteristics of this protein confirmed that indeed it is a dehydrin, including total amino acid composition, partial amino acid sequencing, and the adoption of alpha-helical structure in the presence of sodium dodecyl sulfate. The propensity of dehydrins to adopt alpha-helical structure in the presence of sodium dodecyl sulfate, together with the apparent polypeptide adhesion property of this cowpea dehydrin, suggests a role in stabilizing other proteins or membranes. Taken together, the genetic, physiological, and physicochemical data are at this stage consistent with a cause-and-effect relationship between the presence in mature seeds of the approximately 35-kD dehydrin, which is the product of a single member of a multigene family, and an increment of chilling tolerance during emergence of cowpea seedlings.     

Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana

Stress-induced accumulation of five (COR47, LTI29, ERD14, LTI30 and RAB18) and tissue localization of four (LTI29, ERD14, LTI30 and RAB18) dehydrins in Arabidopsis were characterized immunologically with protein-specific antibodies. The five dehydrins exhibited clear differences in their accumulation patterns in response to low temperature, ABA and salinity. ERD14 accumulated in unstressed plants, although the protein level was up-regulated by ABA, salinity and low temperature. LTI29 mainly accumulated in response to low temperature, but was also found in ABA- and salt-treated plants. LTI30 and COR47 accumulated primarily in response to low temperature, whereas RAB18 was only found in ABA-treated plants and was the only dehydrin in this study that accumulated in dry seeds.Immunohistochemical localization of LTI29, ERD14 and RAB18 demonstrated tissue and cell type specificity in unstressed plants. ERD14 was present in the vascular tissue and bordering parenchymal cells, LTI29 and ERD14 accumulated in the root tip, and RAB18 was localized to stomatal guard cells. LTI30 was not detected in unstressed plants. The localization of LTI29, ERD14 and RAB18 in stress-treated plants was not restricted to certain tissues or cell types. Instead these proteins accumulated in most cells, although cells within and surrounding the vascular tissue showed more intense staining. LTI30 accumulated primarily in vascular tissue and anthers of cold-treated plants.This study supports a physiological function for dehydrins in certain plant cells during optimal growth conditions and in most cell types during ABA or cold treatment. The differences in stress specificity and spatial distribution of dehydrins in Arabidopsis suggest a functional specialization for the members of this protein family.     

Rice dehydrin K-segments have <Emphasis Type="Italic">in vitro</Emphasis> antibacterial activity

Dehydrins are groups of plant proteins that have been shown to response to various environmental stimuli such as dehydration, elevated salinity, and low temperature. However, their roles in plant defense against microbes have not been demonstrated. In an attempt to discover plant antimicrobial proteins, we have screened a rice cDNA library and isolated several cDNAs coding for dehydrins. Protein extracts from Escherichia coli expressing these cDNAs were tested for their activity against Gram-positive bacteria (Bacillus pumilus, B. subtilis, Staphylococcus aureus, and Sarcina lutea) and Gramnegative bacteria (Escherichia coli and Xanthomonas oryzae pv. oryzae). The results indicate that the crude protein extracts exhibited antibacterial activities against the Gram-positive bacteria. However, dehydrins purified by immunoaffinity chromatography were not active against the bacteria. To pinpoint the dehydrin peptides that were responsible for the bactericidal activity, we expressed DNA sequences coding for truncated dehydrins containing either K- or S-segment and found that K-segment peptides, and not S-segment, were responsible for the antibacterial activities against Gram-positive bacteria. Antibacterial assay with synthetic K-segments indicated that the peptides inhibited growth of B. pumilus with minimum inhibition concentration and minimum bactericidal concentration of 130 and 400 μg/ml, respectively.     

Peptide mapping and assessment of cryoprotective activity of 26/27-kDa dehydrin from soybean seeds

To characterize the molecular weight diversity of seed dehydrin among soybean cultivars, 26/27-kDa soybean dehydrins were purified and compared in peptide mapping patterns, partial amino acid sequences, and cryoprotective activity on enzyme. In reverse phase chromatograms of their trypsin digests, we detected several distinctive peaks, one of which was attributed to a part of the internal glycine-rich region. Partial amino acid sequences of peptide fragments from trypsin and S. aureus V8 protease cleavage were found to be identical to the Mat9 translation. The CP50 of purified 26/27-kDa dehydrins were estimated to be 0.30 and 0.11 microM, respectively.     

Mimicking the plant cell interior under water stress by macromolecular crowding: disordered dehydrin proteins are highly resistant to structural collapse

The dehydrins are a class of drought-induced proteins in plants that lack a fixed three-dimensional structure. Their specific molecular action, as well as the reason for their disordered character, is as yet poorly understood. It has been speculated, however, that the dehydrins are tuned to acquire a biologically active structure only under the conditions in which they normally function (i.e. upon dehydration). To test this hypothesis, we here investigate the effect of reduced water content and macromolecular crowding on three dehydrins from Arabidopsis (Arabidopsis thaliana). As a simplistic model for mimicking cellular dehydration, we used polyethylene glycol, glycerol, and sugars that plants naturally employ as compatible solutes (i.e. sucrose and glucose). Macromolecular crowding was induced by the large polysaccharides Ficoll and dextran. The results show that the dehydrins are remarkably stable in their disordered state and are only modestly affected by the solvent alterations. A notable exception is the dehydrin Cor47, which shows a small, intrinsic increase in helical structure at high concentrations of osmolytes. We also examined the effect of phosphorylation but found no evidence that such posttranslational modifications of the dehydrin sequences modulate their structural response to osmolytes and crowding agents. These results suggest that the dehydrins are highly specialized proteins that have evolved to maintain their disordered character under conditions in which unfolded states of several globular proteins would tend to collapse.     

Genetic variation in pea (Pisum) dehydrins: sequence elements responsible for length differences between dehydrin alleles and between dehydrin loci in Pisum sativum L.

 The electrophoretic patterns of dehydrins extracted from mature seeds of a range of pea (Pisum) species revealed extensive variation in dehydrin polypeptide mobility. Variation was also observed among lines of P. sativum. Crosses between lines with different dehydrin electrophoretic patterns produced F₁ seeds with additive patterns, and segregation in the F₂ generation was consistent with a 1 : 2 : 1 ratio, indicating allelic variation at each of two dehydrin loci (Dhn2, Dhn3). Genetic linkage was observed between Dhn2 and Dhn3, and the segregation ratios indicated preferential transmission of one allele at the Dhn3 locus. Dehydrin cDNA clones were characterised that encoded the allelic variants at Dhn2 and Dhn3. Their deduced amino-acid sequences were very similar to each other as well as to the product of the Dhn1 locus reported previously. Comparisons were made between the sequences of allelic variants at a single locus, and between the products of different loci. Differences in the electrophoretic mobilities between allelic variants at Dhn2 and Dhn3 were associated with differences in polypeptide length resulting principally from tandem duplications of 21 (Dhn2) or 24 (Dhn3) amino-acid residues. These duplications accounted for much of the difference in length between dehydrins encoded by the different loci. The conserved core of one of the duplicated regions varied in copy number, and small insertions/deletions of amino acids near this core also contributed to length variation both between allelic forms and between loci. Dehydrins possess characteristic highly conserved amino-acid sequence motifs, yet vary considerably in length. Mechanisms involving sequence duplication appear to be responsible for generating the length differences observed between allelic variants as well as between the products of different loci. Received: 12 June 1997 / Accepted: 29 October 1997     

Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography

In this study we describe a novel method for purification of Arabidopsis thaliana dehydrins overproduced in Escherichia coli. The cDNAs corresponding to the four dehydrin genes RAB18, LTI29, LTI30, and COR47 were inserted into a bacterial expression vector under an isopropyl beta-d-thiogalactopyranoside (IPTG) inducible bacterial promoter. After IPTG induction all four proteins accumulated in high amounts. The recombinant proteins were efficiently purified to over 95% purity with a three-step purification scheme: heat fractionation, immobilized metal ion affinity chromatography (IMAC), and ion exchange chromatography. In this study we introduce the novel use of IMAC as an efficient purification method for native dehydrins. Characterization of the purified proteins was done by Edman degradation, mass spectrometry, reverse-phase chromatography, and analytical gel filtration under native and denaturing conditions. Yields of purified proteins were between 2.8 and 12.5 mg per liter of bacterial culture, sufficient for further biochemical studies.