The Plant Dehydrins: Structure and Putative Functions

This review deals with recent data on the structure and biochemical properties of dehydrins, proteins that are normally synthesized in maturating seeds during their desiccation, and also in vegetative tissues of plants treated with abscisic acid or exposed to environmental stress factors that result in cellular dehydration. The dehydrins are considered as stress proteins involved in formation of plant protective reactions against dehydration. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y-, S-, and K-segments. The K-segment representing a highly conserved 15 amino acid motif (EKKGIMDKIKEKLPG) forming amphiphilic -helix has been found in all dehydrins. The pathways of regulation of dehydrin gene expression, putative functions of dehydrins, and molecular mechanisms of their actions are discussed.     

Cryoprotective mechanism of a small intrinsically disordered dehydrin protein

Dehydration proteins (Dehydrins) are expressed during dehydration stress in plants and are thought to protect plant proteins and membranes from the loss of water during drought and at cold temperatures. Several different dehydrins have been shown to protect lactate dehydrogenase (LDH) from damage from being frozen and thawed. We show here that a 48 residue K₂ dehydrin from Vitis riparia protects LDH more effectively than bovine serum albumin, a protein with known cryoprotective function. Light scattering and 8‐anilino‐1‐naphthalene sulfonate fluorescence experiments show that dehydrins prevent aggregation and unfolding of the enzyme. The cryoprotective effects of LDH are reduced by the addition of salt, suggesting that the positively charged K‐segments are attracted to a negatively charged surface but this does not result in binding. Overall K₂ is an intrinsically disordered protein; nuclear magnetic resonance relaxation experiments indicate that the two‐terminal, Lys‐rich K‐segments show a weak propensity for α‐helicity and are flexible, and that the central, polar rich phi‐segment has no secondary structure preference and is highly flexible. We propose that the phi‐segments in dehydrins are important for maintaining the disordered structure so that the protein can act as a molecular shield to prevent partially denatured proteins from interacting with one another, whereas the K‐segments may help to localize the dehydrin near the enzyme surface.     

Sequence composition versus sequence order in the cryoprotective function of an intrinsically disordered stress‐response protein

Intrinsically disordered stress proteins have been shown to act as chaperones, protecting proteins from damage caused by stresses such as freezing and thawing. Dehydration proteins (dehydrins) are intrinsically disordered stress proteins that are found in almost all land plants. They consist of a variable number of the short, semi‐conserved, Y‐, S‐, and K‐segments, with longer stretches of poorly conserved sequences in between. Previous studies have provided conflicting views on the details of the dehydrin cryoprotective mechanism of enzymes. Experiments with polyethylene glycol (PEG) have shown that PEG cryoprotective efficiency is the same as dehydrins of the same hydrodynamic radius, suggesting that the protein's disordered and polar nature is important, rather than the specific order of the residues. To further elucidate the mechanism, we created scrambled variants of the wild grape dehydrins K₂ and YSK₂ and tested their ability to protect lactate dehydrogenase and yeast frataxin homolog‐1 from freeze/thaw damage. The results show that for preventing aggregation, it is the sequence composition and the size of the dehydrin that is the most important factor in protection, while for freeze/thaw damage causing loss of secondary structure, it is the sequence composition that is most significant.     

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.     

Specific and unspecific responses of plants to cold and drought stress

Different environmental stresses to a plant may result in similar responses at the cellular and molecular level. This is due to the fact that the impacts of the stressors trigger similar strains and downstream signal transduction chains. A good example for an unspecific response is the reaction to stressors which induce water deficiency e.g. drought, salinity and cold, especially frost. The stabilizing effect of liquid water on the membrane bilayer can be supported by compatible solutes and special proteins. At the metabolic level, osmotic adjustment by synthesis of low-molecular osmolytes (carbohydrates, betains, proline) can counteract cellular dehydration and turgor loss. Taking the example of Pinus sylvestris, changes at the level of membrane composition, and concomitantly of photosynthetic capacity during frost hardening is shown. Additionally the effect of photoperiod as measured via the phytochrome system and the effect of subfreezing temperatures on the incidence of frost hardening is discussed. Extremely hydrophilic proteins such as dehydrins are common products protecting not only the biomembranes in ripening seeds (late embryogenesis abundant proteins) but accumulate also in the shoots and roots during cold adaptation, especially in drought tolerant plants. Dehydrins are characterized by conserved amino acid motifs, called the K-, Y-or S-segments. Accumulation of dehydrins can be induced not only by drought, but also by cold, salinity, treatment with abscisic acid and methyl jasmonate. Positive effects of the overexpression of a wild chickpea (Cicer pinnatifidum) dehydrin in tobacco plants on the dehydration tolerance is shown. The presentation discusses the perception of cold and drought, the subsequent signal transduction and expression of genes and their products. Differences and similarities between the plant responses to both stressors are also discussed.     

Dehydrins in cold-acclimated apices of birch (Betula pubescens Ehrh.): production, localization and potential role in rescuing enzyme function during dehydration

 Dehydrins accumulate in various plant tissues during dehydration. Their physiological role is not well understood, but it is commonly assumed that they assist cells in tolerating dehydration. Since in perennials the ability of the shoot apex to withstand dehydration is pivotal for survival through winter, we investigated if and how dehydrins may be involved. A first step in assessing such a role is the identification of their subcellular location. We therefore mapped the location of dehydrin homologues, abscisic acid-responsive (RAB 16-like) polypeptides, in the apex of birch (Betula pubescens Ehrh.). In non-cold-acclimated plants a single low-abundant RAB 16-member (a 33-kDa polypeptide) was produced, and localized in the cytoplasm only. During cold acclimation two additional members were produced (24 and 30 kDa) and accumulated in nuclei, storage protein bodies and starch-rich amyloplasts. Western blots of proteins isolated from purified starch granules and from protein bodies revealed the presence of the 24-kDa dehydrin. Since starch and protein reserves are gradually consumed during winter, serving cell maintenance, starch- and protein-degrading enzymes must remain locally active. We therefore investigated the hypothesis that dehydrins might create local pools of water in otherwise dehydrated cells, thereby maintaining enzyme function. In agreement with our hypothesis, enzyme assays showed that under conditions of low water activity a partially purified dehydrin fraction was able to improve the activity of α-amylase (EC 3.2.1.1.) relative to fractions from which dehydrin was removed by immunoprecipitation. The results confirm the general belief that dehydrins serve desiccation tolerance, and suggest that a major function is to rescue the metabolic processes that are required for survival and re-growth. Received: 12 September 1998 / Accepted: 19 April 1999     

Phosphorylation regulated ion-binding is a property shared by the acidic subclass dehydrins

Dehydrins are a family of proteins that accumulate in response to abiotic stresses. Little is known about the biochemical functions of these proteins. It is known that the Arabidopsis dehydrin, ERD14, is activated by phosphorylation to bind calcium and other ions. To begin to categorize the Arabidopsis dehydrins into functional families, we determined whether representative members of the dehydrin sub families share the properties of ERD14. When phosphorylated in vitro with casein kinase II; recombinant COR47, and ERD10 (and ERD14) become activated to bind calcium. ERD14 exhibited the highest calcium-binding activity followed by ERD10 and COR47. These dehydrins, when isolated from cold-treated Arabidopsis plants were also shown to have phosphorylation-dependent, calcium-binding activity. RAB18 showed very little calcium binding activity, even though it was phosphorylated by casein kinase II. XERO2 was not phosphorylated with CKII and did not bind calcium. Competition studies suggest that other divalent cations may bind to the dehydrins COR47, ERD10, and ERD14. Utilizing matrix-assisted laser desorption ionization – time of flight mass spectroscopy (MALDI-TOF), we determined that the poly serine region located in all three calcium-binding family members (COR47, ERD10, and ERD14) is the most likely phosphorylation site responsible for the activation of calcium binding. These results are consistent with a distinct biochemical function for the acidic subclass of dehydrins (COR47, ERD10, and ERD14) as ion (calcium)-interacting proteins.     

Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins

ERD10 and ERD14 (for early response to dehydration) proteins are members of the dehydrin family that accumulate in response to abiotic environmental stresses, such as high salinity, drought, and low temperature, in Arabidopsis (Arabidopsis thaliana). Whereas these proteins protect cells against the consequences of dehydration, the exact mode(s) of their action remains poorly understood. Here, detailed evidence is provided that ERD10 and ERD14 belong to the family of intrinsically disordered proteins, and it is shown in various assays that they act as chaperones in vitro. ERD10 and ERD14 are able to prevent the heat-induced aggregation and/or inactivation of various substrates, such as lysozyme, alcohol dehydrogenase, firefly luciferase, and citrate synthase. It is also demonstrated that ERD10 and ERD14 bind to acidic phospholipid vesicles without significantly affecting membrane fluidity. Membrane binding is strongly influenced by ionic strength. Our results show that these intrinsically disordered proteins have chaperone activity of rather wide substrate specificity and that they interact with phospholipid vesicles through electrostatic forces. We suggest that these findings provide the rationale for the mechanism of how these proteins avert the adverse effects of dehydration stresses.     

Structural investigation of disordered stress proteins. Comparison of full-length dehydrins with isolated peptides of their conserved segments

Dehydrins constitute a class of intrinsically disordered proteins that are expressed under conditions of water-related stress. Characteristic of the dehydrins are some highly conserved stretches of seven to 17 residues that are repetitively scattered in their sequences, the K-, S-, Y-, and Lys-rich segments. In this study, we investigate the putative role of these segments in promoting structure. The analysis is based on comparative analysis of four full-length dehydrins from Arabidopsis (Arabidopsis thaliana; Cor47, Lti29, Lti30, and Rab18) and isolated peptide mimics of the K-, Y-, and Lys-rich segments. In physiological buffer, the circular dichroism spectra of the full-length dehydrins reveal overall disordered structures with a variable content of poly-Pro helices, a type of elongated secondary structure relying on bridging water molecules. Similar disordered structures are observed for the isolated peptides of the conserved segments. Interestingly, neither the full-length dehydrins nor their conserved segments are able to adopt specific structure in response to altered temperature, one of the factors that regulate their expression in vivo. There is also no structural response to the addition of metal ions, increased protein concentration, or the protein-stabilizing salt Na(2)SO(4). Taken together, these observations indicate that the dehydrins are not in equilibrium with high-energy folded structures. The result suggests that the dehydrins are highly evolved proteins, selected to maintain high configurational flexibility and to resist unspecific collapse and aggregation. The role of the conserved segments is thus not to promote tertiary structure, but to exert their biological function more locally upon interaction with specific biological targets, for example, by acting as beads on a string for specific recognition, interaction with membranes, or intermolecular scaffolding. In this perspective, it is notable that the Lys-rich segment in Cor47 and Lti29 shows sequence similarity with the animal chaperone HSP90.     

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.     

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.     

Isolation and characterization of a novel dehydrin gene from Capsella bursa-pastoris

A novel dehydrin gene designated as Cbcor29 was cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE) and genome walker technique. The full-length cDNA of Cbcor29 was 1101 bp long with a 783 bp open reading frame (ORF), encoding a putative protein of 261 amino acids. Like other dehydrin proteins, CbCOR29 contained a high percentage of charged and polar amino acids, in which Cys and Trp amino acids were absent. Besides, predicted CbCOR29 protein possesses three conserved repeats of the characterized Lys-rich domains (K-segments), and a Ser-rich domain (S-segment) prior to the first Lys-rich domain, which presented a typical SK3 structure of dehydrins. Analysis of Cbcor29 genomic DNA revealed that it contained 2 exons and 1 intron, which was a typical character of dehydrin genes. Subsequent bioinformatic analysis also showed that the sequence of CbCOR29 had high homology with other dehydrin proteins, especially with cor47 from Arabidopsis thaliana. Moreover, semi-quantitative RT-PCR revealed that the expression of Cbcor29 could be induced by exposure to drought, low-temperature, NaCl and exogenous ABA treatment respectively. Our study implied that the Cbcor29 gene was a new member of the dehydrin gene family and might exert functions in drought-, cold- and salt- responsiveness in Capsella bursa-pastoris.     

Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2: a solution CD and solid-state ATR-FTIR study

Dehydrins are intrinsically unstructured proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Although their role is not completely understood, it has been suggested that they stabilize proteins and membrane structures during environmental stress and also sequester metals such as zinc. Here, we investigate two dehydrins (denoted as TsDHN-1 and TsDHN-2) from Thellungiella salsuginea. This plant is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We show using circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy that ordered secondary structure is induced and stabilized in these proteins, both in free and vesicle-bound form, by association with zinc. In membrane-associated form, both proteins have an increased proportion of β-strand conformation induced by the cation, in addition to the amphipathic α-helices formed by their constituent K-segments. These results support the hypothesis that dehydrins stabilize plant plasma and organellar membranes in conditions of stress, and further that zinc may be an important co-factor in stabilization. Whereas dehydrins in the cytosol of a plant cell undergoing dehydration or temperature stress form bulk hydrogels and remain primarily disordered, dehydrins with specific membrane- or protein-associations will have induced ordered secondary structures.     

Dehydrins: Emergence of a biochemical role of a family of plant dehydration proteins

A number of proteins have been identified that typically accumulate in plants in response to any environmental stimulus that has a dehydrative component or is temporally associated with dehydration. This includes drought, low temperature, salinity and seed maturation. Among the induced proteins, dehydrins (late embryogenesis abundant [LEA] D-II family) have been the most commonly observed, yet we still have an incomplete knowledge of their fundamental biochemical role in the cell. Current research trends are changing this situation: immunolocalization and in vitro biochemical analyses are, through analogies to other more fully characterized proteins and molecules, shaping our understanding. In brief, dehydrins may be structure stabilizers with detergent and chaperone-like properties and an array of nuclear and cytoplasmic targets. Recent progress on the mapping of dhn genes and the inheritance of freezing tolerance in barley and other Triticeae species tentatively points to dehydrins as key components of dehydration tolerance.     

脱水素研究进展

脱水素(dehydrin)是植物体内的一种LEA蛋白,能够在植物胚胎发育后期以及逆境下大量表达,广泛存在于植物界。它是具有高度热稳定性的亲水性蛋白,有三类非常保守的区域,即K,Y和S片段。依据这三类片段的组成情况,可将脱水素分为5个基本类别。脱水素可通过多种转运方式定位于植物细胞的不同部位,以行使其功能。其基因的表达存在依赖ABA和不依赖ABA两种途径,并且受到多种环境因素的影响,能稳定细胞膜和许多大分子的结构以避免脱水对细胞造成的伤害。近年来,脱水素的结构和组成、在细胞中的定位及转运、基因的表达与调控、功能与作用机理等方面的研究已取得了很大的进展。     

Overexpression of MtCAS31 enhances drought tolerance in transgenic Arabidopsis by reducing stomatal density

? Dehydrins are a type of late embryogenesis abundant protein. Some dehydrins are involved in the response to various abiotic stresses. Accumulation of dehydrins enhances the drought, cold and salt tolerances of transgenic plants, although the underlying mechanism is unclear. MtCAS31 (Medicago Truncatula cold-acclimation specific protein 31) is a Y(2)K(4)-type dehydrin that was isolated from Medicago truncatula. ? We analyzed the subcellular and histochemical localization of MtCAS31, and the expression patterns of MtCAS31 under different stresses. Transgenic Arabidopsis that overexpressed MtCAS31 was used to determine the function of MtCAS31. A yeast two-hybrid assay was used to screen potential proteins that could interact with MtCAS31. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay. ? After a 3-h drought treatment, the expression of MtCAS31 significantly increased 600-fold. MtCAS31 overexpression dramatically reduced stomatal density and markedly enhanced the drought tolerance of transgenic Arabidopsis. MtCAS31 could interact with AtICE1 (inducer of CBF expression 1) and the AtICE1 homologous protein Mt7g083900.1, which was identified from Medicago truncatula both in vitro and in vivo. ? Our findings demonstrate that a dehydrin induces decreased stomatal density. Most importantly, the interaction of MtCAS31 with AtICE1 plays a role in stomatal development. We hypothesize that the interaction of MtCAS31 and AtICE1 caused the decrease in stomatal density to enhance the drought resistance of transgenic Arabidopsis.     

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