Plant dehydrins and stress tolerance: Versatile proteins for complex mechanisms

Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold and freezing stress. 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 forming amphiphilic a-helix is especially important since it has been found in all dehydrins. Since more than 20 y, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made toward the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.Key words: abiotic stress, dehydration stress, drought, cold acclimation, freezing tolerance, LEA proteins, dehydrins     

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.     

Novel conserved segments are associated with differential expression patterns for Pinaceae dehydrins

Dehydrins are thought to play an essential role in the response, acclimation and tolerance to different abiotic stresses, such as cold and drought. These proteins have been classified into five groups according to the presence of conserved and repeated motifs in their amino acid sequence. Due to their putative functions in the response to stress, dehydrins have been often used as candidate genes in studies on population variability and local adaptation to environmental conditions. However, little is still known regarding the differential role played by such groups or the mechanism underlying their function. Based on the sequences corresponding to dehydrins available in public databases we have isolated eight different dehydrins from cDNA of Pinus pinaster. We have obtained also their genomic sequences and identified their intron/exon structure. Quantitative RT-PCR analysis of their expression pattern in needles, stems and roots during a severe and prolonged drought stress, similar to the ones trees must face in nature, is also reported. Additionally, we have identified two amino acid motifs highly conserved and repeated in Pinaceae dehydrins and absent in angiosperms, presumably related to the divergent expression profiles observed.     

Plant dehydrins: shedding light on structure and expression patterns of dehydrin gene family in barley

Dehydrins, an important group of late embryogenesis abundant proteins, accumulate in response to dehydration stresses and play protective roles under stress conditions. Herein, phylogenetic analysis of the dehydrin family was performed using the protein sequences of 108 dehydrins obtained from 14 plant species based on plant taxonomy and protein subclasses. Sub-cellular localization and phosphorylation sites of these proteins were also predicted. The protein features distinguishing these dehydrins categories were identified using various attribute weighting and decision tree analyses. The results revealed that the presence of the S motif preceding the K motif (Y_nSK_n, SK_n, and S_nKS) was more evident and the Y_nSK_n subclass was more frequent in monocots. In barley, as one of the most drought-tolerant crops, there are ten members of Y_nSK_n out of 13 HvDhns. In promoter regions, six types of abiotic stress-responsive elements were identified. Regulatory elements in UTR sequences of HvDhns were infrequent while only four miRNA targets were found. Furthermore, physiological parameters and gene expression levels of HvDhns were studied in tolerant (HV1) and susceptible (HV2) cultivars, and in an Iranian tolerant wild barley genotype (Spontaneum; HS) subjected to gradual water stress and after recovery duration at the vegetative stage. The results showed the significant impact of dehydration on dry matter, relative leaf water, chlorophyll contents, and oxidative damages in HV2 compared with the other studied genotypes, suggesting a poor dehydration tolerance, and incapability of recovering after re-watering in HV2. Under severe drought stress, among the 13 HvDhns genes, 5 and 10 were exclusively induced in HV1 and HS, respectively. The gene and protein structures and the expression patterns of HvDhns as well as the physiological data consistently support the role of dehydrins in survival and recovery of barley plants from drought particularly in HS. Overall, this information would be helpful for functional characterization of the Dhn family in plants.     

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.     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.     

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.     

Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment

Background

Dehydrins are known as Group II late embryogenesis abundant proteins. Their high hydrophilicity and thermostability suggest that they may be structure stabilizers with detergent and chaperone-like properties. They are localised in the nucleus, cytoplasm, and plasma membrane. We have recently found putative dehydrins in the mitochondria of some cereals in response to cold. It is not known whether dehydrin-like proteins accumulate in plant mitochondria in response to stimuli other than cold stress.

Results

We have found five putative dehydrins in the mitochondria of winter wheat, rye and maize seedlings. Two of these polypeptides had the same molecular masses in all three species (63 and 52 kD) and were thermostable. Drought, freezing, cold, and exogenous ABA treatment led to higher accumulation of dehydrin-like protein (dlp) 63 kD in the rye and wheat mitochondria. Protein 52 kD was induced by cold adaptation and ABA. Some accumulation of these proteins in the maize mitochondria was found after cold exposition only. The other three proteins appeared to be heat-sensitive and were either slightly induced or not induced at all by all treatments used.

Conclusions

We have found that, not only cold, but also drought, freezing and exogenous ABA treatment result in accumulation of the thermostable dehydrins in plant mitochondria. Most cryotolerant species such as wheat and rye accumulate more heat-stable dehydrins than cryosensitive species such as maize. It has been supposed that their function is to stabilize proteins in the membrane or in the matrix. Heat-sensitive putative dehydrins probably are not involved in the stress reaction and adaptation of plants.     

Dehydrin stress proteins in <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. needles under conditions of extreme climate of Yakutia

This is the first study to investigate stress proteins dehydrins with the use of specific antibodies in the Scots pine (Pinus sylvestris L.) needles and their changes in the annual cycle under extreme climate of Yakutia. No pronounced polymorphism of major dehydrins (14–15 and 66 kDa) has been found during the winter dormancy period of P. sylvestris. A clear correlation between the seasonal variations in dehydrins and changes in the water content in needles was revealed. Consistently high levels of dehydrins was retained throughout the period of low negative temperatures. It is assumed that dehydrins can participate in the formation of P. sylvestris L. resistance to the permafrost conditions.     

Potato dehydrins present high intrinsic disorder and are differentially expressed under ABA and abiotic stresses

Dehydrins (DHNs) correspond to late embryogenesis abundant proteins (LEA) of group 2, they are known as glycin rich proteins. Despite their expression during the late seed maturation stages, they are also involved in plant response to a number of abiotic stresses such as drought, salinity and cold. In the present study, we identified five full-length cDNAs encoding dehydrins (designated StDHN2a, StDHN1, TAS14, StDHN25 and StLEA27) isolated from potato. These dehydrins were composed of serine amino acids called S domain and lysine-rich segment corresponding to a K domain. Three DHNs (StDHN1, TAS14 and StLEA27) contained Y segments. In silico analysis showed that these StDHN sequences share high homology with other Solanum dehydrin proteins species. The analysis of gene expression using quantitative RT-PCR showed that they were upregulated by dehydration and salinity. Moreover, the search for putative regulatory element in the promoter sequence of dehydrin genes was investigated.     

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.     

Phosphorylation of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 facilitates cation-induced conformational changes and actin assembly

Group 2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperatures. These proteins are characterized by the presence of at least one conserved, lysine-rich K-segment and sometimes by one or more serine-rich S-segments that are phosphorylated. Dehydrins may stabilize proteins and membrane structures during environmental stress and can sequester and scavenge metal ions. Here, we investigate how the conformations of two dehydrins from Thellungiella salsuginea, denoted as TsDHN-1 (acidic) and TsDHN-2 (basic), are affected by pH, interactions with cations and membranes, and phosphorylation. Both TsDHN-1 and TsDHN-2 were expressed as SUMO fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and structural analysis by circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy. We show that the polyproline II conformation can be induced in the dehydrins by their environmental conditions, including changes in the concentration of divalent cations such as Ca(2+). The assembly of actin by these dehydrins was assessed by sedimentation assays and viewed by transmission electron and atomic force microscopy. Phosphorylation allowed both dehydrins to polymerize actin filaments. These results support the hypothesis that dehydrins stabilize the cytoskeleton under stress conditions and further that phosphorylation may be an important feature of this stabilization.     

Water Deficit in Pea Root Tips: Effects on the Cell Cycle and on the Production of Dehydrin-like Proteins

Dehydrin-like proteins have been detected in nuclei and cytoplasmof meristematic root tip cells from pea seedlings subjectedto slow dehydration at 90% relative humidity for 48 h or more.Evidence was gained from Western blotting and immunocytochemicalexperiments using an antibody raised against the conserved domainof dehydrin proteins. Flow cytometer analysis has shown thatcycling cells of root tip meristems from dehydrated seedlingsare mostly arrested in G2 phase. Other stress treatments thoughtto involve water depletion (osmotic stress, cold treatment)or to modulate cell response to water deficit (abscisic acid)gave less clear-cut results with all treatments lowering theproportion of cells entering the S phase, but without a definiteand persistent arrest in any preferential phase of the cycle.Possible interrelationships between G2 arrest and dehydrin productionare discussed. Cell cycle; dehydrins; flow cytometry; nuclei; pea; Pisum sativum L.; water stress     

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     

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.     

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.     

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.     

Stress proteins and the immune response to mycobacteria — Antigens as virulence factors?

Summary The immune response to mycobacterial infection includes pathogenic as well as protective activities. It is possible that different types of immune responses are associated with recognition of different antigenic determinants. Amongst the antigens which are prominent in antibody and T cell recognition of mycobacteria, we have identified members of highly conserved stress protein families. Mapping of antigenic determinants on stress proteins shows that both species-specific and conserved regions of these proteins can take part in immune recognition. Induction of an immune response to conserved, self-like, determinants on stress proteins could play a role in the immunopathology associated with chronic mycobacterial infections.     

Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress

Background  

Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress.