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1.  Disorder and function: a review of the dehydrin protein family 
Dehydration proteins (dehydrins) are group 2 members of the late embryogenesis abundant (LEA) protein family. The protein architecture of dehydrins can be described by the presence of three types of conserved sequence motifs that have been named the K-, Y-, and S-segments. By definition, a dehydrin must contain at least one copy of the lysine-rich K-segment. Abiotic stresses such as drought, cold, and salinity cause the upregulation of dehydrin mRNA and protein levels. Despite the large body of genetic and protein evidence of the importance of these proteins in stress response, the in vivo protective mechanism is not fully known. In vitro experimental evidence from biochemical assays and localization experiments suggests multiple roles for dehydrins, including membrane protection, cryoprotection of enzymes, and protection from reactive oxygen species. Membrane binding by dehydrins is likely to be as a peripheral membrane protein, since the protein sequences are highly hydrophilic and contain many charged amino acids. Because of this, dehydrins in solution are intrinsically disordered proteins, that is, they have no well-defined secondary or tertiary structure. Despite their disorder, dehydrins have been shown to gain structure when bound to ligands such as membranes, and to possibly change their oligomeric state when bound to ions. We review what is currently known about dehydrin sequences and their structures, and examine the various ligands that have been shown to bind to this family of proteins.
PMCID: PMC4215689  PMID: 25400646
abiotic stress; cold; dehydration; dehydrins; intrinsically disordered proteins; late embryogenesis abundant; localization; structure
2.  Isolation and Characterization of cDNA Encoding Three Dehydrins Expressed During Coffea canephora (Robusta) Grain Development 
Annals of Botany  2006;97(5):755-765.
• Background and Aims Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized.
• Methods The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT–PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2).
• Key Results The CcDH1 and CcDH2 genes encode Y3SK2 dehydrins and the CcDH3 gene encodes an SK3 dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized.
• Conclusions cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.
PMCID: PMC2803416  PMID: 16504969
Dehydrins; late embryogenic abundant protein (LEA); seed development; Coffea; C. canephora; C. arabica; Rubiaceae
3.  Novel dehydrins lacking complete K-segments in Pinaceae. The exception rather than the rule 
Dehydrins are thought to play an essential role in the plant response, acclimation and tolerance to different abiotic stresses, such as cold and drought. These proteins contain conserved and repeated segments in their amino acid sequence, used for their classification. Thus, dehydrins from angiosperms present different repetitions of the segments Y, S, and K, while gymnosperm dehydrins show A, E, S, and K segments. The only fragment present in all the dehydrins described to date is the K-segment. Different works suggest the K-segment is involved in key protective functions during dehydration stress, mainly stabilizing membranes. In this work, we describe for the first time two Pinus pinaster proteins with truncated K-segments and a third one completely lacking K-segments, but whose sequence homology leads us to consider them still as dehydrins. qRT-PCR expression analysis show a significant induction of these dehydrins during a severe and prolonged drought stress. By in silico analysis we confirmed the presence of these dehydrins in other Pinaceae species, breaking the convention regarding the compulsory presence of K-segments in these proteins. The way of action of these unusual dehydrins remains unrevealed.
PMCID: PMC4251312  PMID: 25520734
dehydrins; K-segments; drought; gene expression; qRT-PCR; Pinus
4.  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 
BMC Plant Biology  2010;10:181.
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.
All the analyzed plant species showed constitutive accumulation of thermostable mitochondrial putative dehydrins ranging from 50 to 70 kDa. The mitochondrial dehydrin-like proteins observed in cauliflower and Arabidopsis ranged from 10 to 100 kDa and in lupin imbibed seeds and hypocotyls - from 20 to 90 kDa. Cold treatment increased mainly the accumulation of 10-100 kDa cauliflower and Arabidopsis dehydrin-like proteins, in the patterns different in cauliflower leaf and inflorescence mitochondria. However, in lupin mitochondria, cold affected mainly 25-50 kDa proteins and seemed to induce the appearance of some novel dehydrin-like proteins. The influence of frost stress on cauliflower leaf mitochondrial dehydrin- like proteins was less significant. The impact of heat stress was less significant in lupin and Arabidopsis than in cauliflower inflorescence mitochondria. Cauliflower mitochondrial dehydrin-like proteins are localized mostly in the mitochondrial matrix; it seems that some of them may interact with mitochondrial membranes.
All the results reveal an unexpectedly broad spectrum of dehydrin-like proteins accumulated during some abiotic stress in the mitochondria of the plant species analyzed. They display only limited similarity in size to those reported previously in maize, wheat and rye mitochondria. Some small thermolabile dehydrin-like proteins were induced under stress conditions applied and therefore they are likely to be involved in stress response.
PMCID: PMC3095311  PMID: 20718974
5.  Wheat and barley dehydrins under cold, drought, and salinity – what can LEA-II proteins tell us about plant stress response? 
Dehydrins as a group of late embryogenesis abundant II proteins represent important dehydration-inducible proteins whose accumulation is induced by developmental processes (embryo maturation) as well as by several abiotic stress factors (low temperatures, drought, salinity). In the review, an overview of studies aimed at investigation of dehydrin accumulation patterns at transcript and protein levels as well as their possible functions in common wheat (Triticum aestivum), durum wheat (T. durum), and barley (Hordeum vulgare) plants exposed to various abiotic stress factors (cold, frost, drought, salinity) is provided. Possible roles of dehydrin proteins in an acquisition and maintenance of an enhanced frost tolerance are analyzed in the context of plant developmental processes (vernalization). Quantitative and qualitative differences as well as post-translational modifications in accumulated dehydrin proteins between barley cultivars revealing differential tolerance to drought and salinity are also discussed. Current knowledge on dehydrin role in wheat and barley response to major dehydrative stresses is summarized and the major challenges in dehydrin research are outlined.
PMCID: PMC4089117  PMID: 25071816
dehydrin dynamics; proteins; transcripts; abiotic stress; barley; wheat
6.  A dehydrin-dehydrin interaction: the case of SK3 from Opuntia streptacantha 
Dehydrins belongs to a large group of highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins. It is well known that dehydrins are intrinsically disordered plant proteins that accumulate during the late stages of embryogenesis and in response to abiotic stresses; however, the molecular mechanisms by which their functions are carried out are still unclear. We have previously reported that transgenic Arabidopsis plants overexpressing an Opuntia streptacantha SK3 dehydrin (OpsDHN1) show enhanced tolerance to freezing stress. Herein, we show using a split-ubiquitin yeast two-hybrid system that OpsDHN1 dimerizes. We found that the deletion of regions containing K-segments and the histidine-rich region in the OpsDHN1 protein affects dimer formation. Not surprisingly, in silico protein sequence analysis suggests that OpsDHN1 is an intrinsically disordered protein, an observation that was confirmed by circular dichroism and gel filtration of the recombinantly expressed protein. The addition of zinc triggered the association of recombinantly expressed OpsDHN1 protein, likely through its histidine-rich motif. These data brings new insights about the molecular mechanism of the OpsDHN1 SK3-dehydrin.
PMCID: PMC4193212  PMID: 25346739
yeast two-hybrid; SK3-dehydrin; K-segments; homodimer; histidine-rich region; intrinsically disordered proteins
7.  Identification of salt gland-associated genes and characterization of a dehydrin from the salt secretor mangrove Avicennia officinalis 
BMC Plant Biology  2014;14(1):291.
Salt stress is a major challenge for growth and development of plants. The mangrove tree Avicennia officinalis has evolved salt tolerance mechanisms such as salt secretion through specialized glands on its leaves. Although a number of structural studies on salt glands have been done, the molecular mechanism of salt secretion is not clearly understood. Also, studies to identify salt gland-specific genes in mangroves have been scarce.
By subtractive hybridization (SH) of cDNA from salt gland-rich cell layers (tester) with mesophyll tissues as the driver, several Expressed Sequence Tags (ESTs) were identified. The major classes of ESTs identified include those known to be involved in regulating metabolic processes (37%), stress response (17%), transcription (17%), signal transduction (17%) and transport functions (12%). A visual interactive map generated based on predicted functional gene interactions of the identified ESTs suggested altered activities of hydrolase, transmembrane transport and kinases. Quantitative Real-Time PCR (qRT-PCR) was carried out to validate the expression specificity of the ESTs identified by SH. A Dehydrin gene was chosen for further experimental analysis, because it is significantly highly expressed in salt gland cells, and dehydrins are known to be involved in stress remediation in other plants. Full-length Avicennia officinalis Dehydrin1 (AoDHN1) cDNA was obtained by Rapid Amplification of cDNA Ends. Phylogenetic analysis and further characterization of this gene suggested that AoDHN1 belongs to group II Late Embryogenesis Abundant proteins. qRT-PCR analysis of Avicennia showed up-regulation of AoDHN1 in response to salt and drought treatments. Furthermore, some functional insights were obtained by growing E. coli cells expressing AoDHN1. Growth of E. coli cells expressing AoDHN1 was significantly higher than that of the control cells without AoDHN1 under salinity and drought stresses, suggesting that the mangrove dehydrin protein helps to mitigate the abiotic stresses.
Thirty-four ESTs were identified to be enriched in salt gland-rich tissues of A. officinalis leaves. qRT-PCR analysis showed that 10 of these were specifically enriched in the salt gland-rich tissues. Our data suggest that one of the selected genes, namely, AoDHN1 plays an important role to mitigate salt and drought stress responses.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0291-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4247641  PMID: 25404140
Avicennia officinalis; Salinity; Dehydrin; Subtractive hybridization; Leaf salt glands; Drought stress
8.  Involvement of Multiple Types of Dehydrins in the Freezing Response in Loquat (Eriobotrya japonica) 
PLoS ONE  2014;9(1):e87575.
Dehydrins (DHNs) are a family of plant proteins typically induced in response to stress conditions that cause cellular dehydration, such as low temperatures, high salinity, and drought. Loquat (Eriobotrya japonica) is a perennial fruit crop that blossoms during winter. Loquat fruitlets are frequently injured by freezing. To evaluate the role of the EjDHNs in freezing resistance in loquat fruitlets, two cultivars of loquat, the freezing-sensitive ‘Ninghaibai’ (FS-NHB) and the freezing-tolerant ‘Jiajiao’ (FT-JJ), were analyzed under induced freezing stress. Freezing stress led to obvious accumulation of reactive oxygen species and considerable lipid peroxidation in membranes during the treatment period. Both these phenomena were more pronounced in ‘FS-NHB’ than in ‘FS-JJ.’ Immunogold labeling of dehydrin protein was performed. DHN proteins were found to be concentrated mainly in the vicinity of the plasma membrane, and the density of the immunogold labeling was significantly higher after freezing treatment, especially in the more freezing-tolerant cultivar ‘FT-JJ.’ Seven DHNs, showing four different structure types, were obtained from loquat fruitlets and used to study the characteristics of different EjDHN proteins. These DHN proteins are all highly hydrophilic, but they differ significantly in size, ranging from 188 to 475 amino acids, and in biochemical properties, such as theoretical pI, aliphatic index, and instability index. Freezing treatment resulted in up-regulation of the expression levels of all seven EjDHNs, regardless of structure type. The accumulation of the transcripts of these EjDHN genes was much more pronounced in ‘FT-JJ’ than in ‘FS-NHB.’ Altogether, this study provides evidence that EjDHNs are involved in the cryoprotection of the plasma membrane during freeze-induced dehydration in loquat fruitlets.
PMCID: PMC3909202  PMID: 24498141
9.  Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment 
BMC Plant Biology  2002;2:5.
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.
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.
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.
PMCID: PMC116594  PMID: 12057012
10.  Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress 
BMC Plant Biology  2012;12:140.
Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.
Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.
The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.
PMCID: PMC3460772  PMID: 22882870
Grapevine; Dehydrin; Stress-induced expression; Powdery mildew; Promoter
11.  Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon 
BMC Plant Biology  2014;14:15.
Adverse environmental conditions severely influence various aspects of plant growth and developmental processes, causing worldwide reduction of crop yields. The C-repeat binding factors (CBFs) are critical transcription factors constituting the gene regulatory network that mediates the acclimation process to low temperatures. They regulate a large number of cold-responsive genes, including COLD-REGULATED (COR) genes, via the CBF-COR regulon. Recent studies have shown that the CBF transcription factors also play a role in plant responses to drought and salt stresses. Putative CBF gene homologues and their downstream genes are also present in the genome of Brachypodium distachyon, which is perceived as a monocot model in recent years. However, they have not been functionally characterized at the molecular level.
Three CBF genes that are responsive to cold were identified from Brachypodium, designated BdCBF1, BdCBF2, and BdCBF3, and they were functionally characterized by molecular biological and transgenic approaches in Brachypodium and Arabidopsis thaliana. Our results demonstrate that the BdCBF genes contribute to the tolerance response of Brachypodium to cold, drought, and salt stresses by regulating downstream targets, such as DEHYDRIN5.1 (Dhn5.1) and COR genes. The BdCBF genes are induced under the environmental stress conditions. The BdCBF proteins possess transcriptional activation activity and bind directly to the promoters of the target genes. Transgenic Brachypodium plants overexpressing the BdCBF genes exhibited enhanced resistance to drought and salt stresses as well as low temperatures, and accordingly endogenous contents of proline and soluble sugars were significantly elevated in the transgenic plants. The BdCBF transcription factors are also functional in the heterologous system Arabidopsis. Transgenic Arabidopsis plants overexpressing the BdCBF genes were also tolerant to freezing, drought, and salt stresses, and a set of stress-responsive genes was upregulated in the transgenic Arabidopsis plants.
Taken together, our results strongly support that the BdCBF transcription factors are key regulators of cold stress responses in Brachypodium and the CBF-mediated cold stress signaling pathway is conserved in this plant species. We believe that this study would confer great impact on stress biology in monocot species and could be applied to engineer abiotic stress tolerance of bioenergy grass species.
PMCID: PMC3898008  PMID: 24405987
Brachypodium distachyon; C-repeat binding factor (CBF); COLD-REGULATED (COR); Abiotic stress tolerance; Arabidopsis thaliana
12.  Dehydrin-like Proteins in the Necrotrophic Fungus Alternaria brassicicola Have a Role in Plant Pathogenesis and Stress Response 
PLoS ONE  2013;8(10):e75143.
In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease.
PMCID: PMC3788798  PMID: 24098369
13.  A dehydration-inducible gene in the truffle Tuber borchii identifies a novel group of dehydrins 
BMC Genomics  2006;7:39.
The expressed sequence tag M6G10 was originally isolated from a screening for differentially expressed transcripts during the reproductive stage of the white truffle Tuber borchii. mRNA levels for M6G10 increased dramatically during fruiting body maturation compared to the vegetative mycelial stage.
Bioinformatics tools, phylogenetic analysis and expression studies were used to support the hypothesis that this sequence, named TbDHN1, is the first dehydrin (DHN)-like coding gene isolated in fungi. Homologs of this gene, all defined as "coding for hypothetical proteins" in public databases, were exclusively found in ascomycetous fungi and in plants. Although complete (or almost complete) fungal genomes and EST collections of some Basidiomycota and Glomeromycota are already available, DHN-like proteins appear to be represented only in Ascomycota. A new and previously uncharacterized conserved signature pattern was identified and proposed to Uniprot database as the main distinguishing feature of this new group of DHNs. Expression studies provide experimental evidence of a transcript induction of TbDHN1 during cellular dehydration.
Expression pattern and sequence similarities to known plant DHNs indicate that TbDHN1 is the first characterized DHN-like protein in fungi. The high similarity of TbDHN1 with homolog coding sequences implies the existence of a novel fungal/plant group of LEA Class II proteins characterized by a previously undescribed signature pattern.
PMCID: PMC1550403  PMID: 16512918
14.  Comparison of Amino Acids Physico-Chemical Properties and Usage of Late Embryogenesis Abundant Proteins, Hydrophilins and WHy Domain 
PLoS ONE  2014;9(10):e109570.
Late Embryogenesis Abundant proteins (LEAPs) comprise several diverse protein families and are mostly involved in stress tolerance. Most of LEAPs are intrinsically disordered and thus poorly functionally characterized. LEAPs have been classified and a large number of their physico-chemical properties have been statistically analyzed. LEAPs were previously proposed to be a subset of a very wide family of proteins called hydrophilins, while a domain called WHy (Water stress and Hypersensitive response) was found in LEAP class 8 (according to our previous classification). Since little is known about hydrophilins and WHy domain, the cross-analysis of their amino acids physico-chemical properties and amino acids usage together with those of LEAPs helps to describe some of their structural features and to make hypothesis about their function. Physico-chemical properties of hydrophilins and WHy domain strongly suggest their role in dehydration tolerance, probably by interacting with water and small polar molecules. The computational analysis reveals that LEAP class 8 and hydrophilins are distinct protein families and that not all LEAPs are a protein subset of hydrophilins family as proposed earlier. Hydrophilins seem related to LEAP class 2 (also called dehydrins) and to Heat Shock Proteins 12 (HSP12). Hydrophilins are likely unstructured proteins while WHy domain is structured. LEAP class 2, hydrophilins and WHy domain are thus proposed to share a common physiological role by interacting with water or other polar/charged small molecules, hence contributing to dehydration tolerance.
PMCID: PMC4190154  PMID: 25296175
15.  Natural antisense transcripts of Trifolium repens dehydrins 
Plant Signaling & Behavior  2013;8(12):e27674.
The recently described complex nature of some dehydrin-coding sequences in Trifolium repens could explain the considerable variability among transcripts originating from a single gene.1 For some of the sequences the existence of natural antisense transcripts (NATs), which could form sense-antisense (SAS) pairs, was predicted. The present study demonstrates that cis-natural antisense transcripts of 2 dehydrin types (YnKn and YnSKn) accumulate in white clover plants subjected to treatments with polyethylene glycol (PEG), abscisic acid (ABA), and high salt concentration. The isolated YnKn cis-NATs mapped to sequence site enriched in alternative start codons. Some of the sense-antisense pairs exhibited inverse expression with differing profiles which depended on the applied stress. A natural antisense transcript coding for an ABC F family protein (a trans-NAT) which shares short sequence homology with YnSKn dehydrin was identified in plants subjected to salt stress. Forthcoming experiments will evaluate the impact of NATs on transcript abundances, elucidating the role of transcriptional and post-transcriptional interferences in the regulation of dehydrin levels under various abiotic stresses.
PMCID: PMC4091226  PMID: 24390012
Dehydrins; natural antisense transcripts; sense-antisense pairs; splice variants; Trifolium repens
16.  PR-10, defensin and cold dehydrin genes are among those over expressed in Oxytropis (Fabaceae) species adapted to the arctic 
Functional & Integrative Genomics  2011;11(3):497-505.
In many studied plants, typical responses to cold treatment include up-regulating the hydrophilic COR/LEA genes and down-regulating photosynthesis-related genes, carbohydrate metabolism, GDSL-motif lipase, hormone metabolism and oxidative regulation genes. However, next to nothing is known about gene expression in arctic plants, which are actually adapted to a harsh, cold environment. The molecular mechanisms behind the many specific adaptations of arctic plants, such as slow growth, well-developed root systems and short stature, are not well understood. In this study, we examine whole plantlet transcriptome differences between two arctic and two temperate Oxytropis (Fabaceae) species, grown under their respective controlled environmental conditions. Gene expression differences are analyzed using cDNA library subtraction followed by expressed sequence tags sequencing and annotation. Sequences from a total of nearly 2,000 clones cluster into 121 and 368 unique genes from the arctic and from the temperate plants, respectively. The predominant biological process for genes from the arctic-enriched library is “response to stimulus”. A concurrent overexpression of pathogenesis-related class 10 proteins (PR-10), plant defensin and cold dehydrin genes is a novel feature for species adapted to stressful growth environment. The temperate-enriched genes are involved in photosynthesis, translation and nucleosome assembly. Interestingly, both arctic and temperate-enriched libraries also contain genes involved in ribosome biogenesis and assembly, however of different types. Real-time reverse transcription PCR of cold dehydrin and two PR-10 genes, as well as the light harvesting complex b1 genes demonstrates that the gene expression is dependent on species and growth conditions.
Electronic supplementary material
The online version of this article (doi:10.1007/s10142-011-0223-6) contains supplementary material, which is available to authorized users.
PMCID: PMC3156302  PMID: 21499864
Arctic; Plant; Gene expression; Oxytropis; Library subtraction; Defence response
17.  Seasonal Patterns of Growth, Dehydrins and Water-soluble Carbohydrates in Genotypes of Dactylis glomerata Varying in Summer Dormancy 
Annals of Botany  2005;95(6):981-990.
• Background and Aims Summer dormancy in perennial grasses has been studied inadequately, despite its potential to enhance plant survival and persistence in Mediterranean areas. The aim of the present work was to characterize summer dormancy and dehydration tolerance in two cultivars of Dactylis glomerata (dormant ‘Kasbah’, non-dormant ‘Oasis’) and their hybrid using physiological indicators associated with these traits.
• Methods Dehydration tolerance was assessed in a glasshouse experiment, while seasonal metabolic changes which produce putative protectants for drought, such as carbohydrates and dehydrins that might be associated with summer dormancy, were analysed in the field.
• Key Results The genotypes differed in their ability to survive increasing soil water deficit: lethal soil water potential (Ψs) was −3·4 MPa for ‘Kasbah’ (although non-dormant), −1·3 MPa for ‘Oasis’, and −1·6 MPa for their hybrid. In contrast, lethal water content of apices was similar for all genotypes (approx. 0·45 g H2O g d. wt−1), and hence the greater survival of ‘Kasbah’ can be ascribed to better drought avoidance rather than dehydration tolerance. In autumn-sown plants, ‘Kasbah’ had greatest dormancy, the hybrid was intermediate and ‘Oasis’ had none. The more dormant the genotype, the lower the metabolic activity during summer, and the earlier the activity declined in spring. Decreased monosaccharide content was an early indicator of dormancy induction. Accumulation of dehydrins did not correlate with stress tolerance, but dehydrin content was a function of the water status of the tissues, irrespective of the soil moisture. A protein of approx. 55 kDa occurred in leaf bases of the most dormant cultivar even in winter.
• Conclusions Drought avoidance and summer dormancy are correlated but can be independently expressed. These traits are heritable, allowing selection in breeding programmes.
PMCID: PMC4246749  PMID: 15760915
Orchard grass; drought tolerance; avoidance; dehydration; dehydrins; carbohydrates; Dactylis glomerata, summer dormancy
18.  Transcriptome profiling in conifers and the PiceaGenExpress database show patterns of diversification within gene families and interspecific conservation in vascular gene expression 
BMC Genomics  2012;13:434.
Conifers have very large genomes (13 to 30 Gigabases) that are mostly uncharacterized although extensive cDNA resources have recently become available. This report presents a global overview of transcriptome variation in a conifer tree and documents conservation and diversity of gene expression patterns among major vegetative tissues.
An oligonucleotide microarray was developed from Picea glauca and P. sitchensis cDNA datasets. It represents 23,853 unique genes and was shown to be suitable for transcriptome profiling in several species. A comparison of secondary xylem and phelloderm tissues showed that preferential expression in these vascular tissues was highly conserved among Picea spp. RNA-Sequencing strongly confirmed tissue preferential expression and provided a robust validation of the microarray design. A small database of transcription profiles called PiceaGenExpress was developed from over 150 hybridizations spanning eight major tissue types. In total, transcripts were detected for 92% of the genes on the microarray, in at least one tissue. Non-annotated genes were predominantly expressed at low levels in fewer tissues than genes of known or predicted function. Diversity of expression within gene families may be rapidly assessed from PiceaGenExpress. In conifer trees, dehydrins and late embryogenesis abundant (LEA) osmotic regulation proteins occur in large gene families compared to angiosperms. Strong contrasts and low diversity was observed in the dehydrin family, while diverse patterns suggested a greater degree of diversification among LEAs.
Together, the oligonucleotide microarray and the PiceaGenExpress database represent the first resource of this kind for gymnosperm plants. The spruce transcriptome analysis reported here is expected to accelerate genetic studies in the large and important group comprised of conifer trees.
PMCID: PMC3534630  PMID: 22931377
19.  Dissecting the cryoprotection mechanisms for dehydrins 
One of the common responses of plants to water deficit is the accumulation of the so-called late embryogenesis abundant (LEA) proteins. In vitro studies suggest that these proteins can protect other macromolecules and cellular structural components from the impairments caused by water limitation. Their binding to phospholipids, nucleic acids and/or to divalent cations has suggested multi-functionality. Genetic analyses indicate that these proteins are required for an optimal adjustment of plants to this insult. This diverse information has conducted to propose different models for LEA proteins action mechanisms. Many of these properties are shared by group 2 LEA proteins or dehydrins (DHNs), one of the LEA protein families for which large amount of data is available. This manuscript focuses on the different mechanisms proposed for this LEA protein group by analyzing published data derived from in vitro cryoprotection assays. We compared the molar ratio of protectant:enzyme needed to preserve 50% of the initial activity per enzyme monomer to assess different mechanisms of action. Our results add evidence for protein–protein interaction as a protection mechanism but also indicate that some DHNs might protect by different means. The strength and weakness of the proposed protection mechanisms are discussed.
PMCID: PMC4212605  PMID: 25400649
dehydrins; late embryogenesis abundant proteins; cryoprotection; water deficit; abiotic stress; intrinsically disordered proteins
20.  Identification of Cis-Acting Promoter Elements in Cold- and Dehydration-Induced Transcriptional Pathways in Arabidopsis, Rice, and Soybean 
The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (46 = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species.
PMCID: PMC3276264  PMID: 22184637
plant genome; cis-acting promoter elements; cold; dehydration; microarray
21.  LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana 
BMC Genomics  2008;9:118.
LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown.
We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE) and/or low temperature response (LTRE) elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded.
The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for future efforts to elucidate the functional role of these enigmatic proteins.
PMCID: PMC2292704  PMID: 18318901
22.  Giving drought the cold shoulder: a relationship between drought tolerance and fall dormancy in an agriculturally important crop 
AoB Plants  2014;6:plu012.
Fall dormant/freezing tolerant plants often also exhibit superior tolerance to drought conditions compared to their non-fall dormant/freezing intolerant counterparts. This experiment aimed to investigate this phenomenon in an agriculturally important crop. Seven alfalfa cultivars with varying levels of fall dormancy/freezing tolerance were exposed to a water deficit. The more fall dormant cultivars had superior tolerance to a mild water deficit. Two genes, CAS18 (encodes for a dehydrin like protein) and CorF (encodes for a galactinol synthase), were up regulated in association with this drought tolerance. Both these genes are early response genes, providing clues to the stress signalling pathways involved.
The growth of fall dormant/freezing tolerant plants often surpasses the growth of non-fall dormant/non-freezing tolerant types of the same species under water-limited conditions, while under irrigated conditions non-fall dormant types exhibit superior yield performance. To investigate the mechanism behind this phenomenon, we exposed seven diverse alfalfa (Medicago sativa) cultivars to water-limited and fully watered conditions and measured their shoot growth, shoot water potential and gas exchange parameters and the relative abundance of taproot RNA transcripts associated with chilling stress/freezing tolerance. Fall dormant cultivars had greater shoot growth relative to the fully watered controls under a mild water deficit (a cumulative water deficit of 625 mL pot−1) and did not close their stomata until lower shoot water potentials compared with the more non-fall dormant cultivars. Several gene transcripts previously associated with freezing tolerance increased in abundance when plants were exposed to a mild water deficit. Two transcripts, corF (encodes galactinol synthase) and cas18 (encodes a dehydrin-like protein), increased in abundance in fall dormant cultivars only. Once water deficit stress became severe (a cumulative water deficit of 2530 mL pot−1), the difference between fall dormancy groups disappeared with the exception of the expression of a type 1 sucrose synthase gene, which decreased in fall dormant cultivars. The specific adaptation of fall dormant cultivars to mild water deficit conditions and the increase in abundance of specific genes typically associated with freezing tolerance in these cultivars is further evidence of a link between freezing tolerance/fall dormancy and adaption to drought conditions in this species.
PMCID: PMC4038438  PMID: 24790133
Alfalfa; forage legumes; gene expression; lucerne; moisture stress.
23.  SiLEA14, a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet 
BMC Plant Biology  2014;14(1):290.
Late embryogenesis abundant (LEA) proteins are involved in protecting higher plants from damage caused by environmental stresses. Foxtail millet (Setaria italica) is an important cereal crop for food and feed in semi-arid areas. However, the molecular mechanisms underlying tolerance to these conditions are not well defined.
Here, we characterized a novel atypical LEA gene named SiLEA14 from foxtail millet. It contains two exons separated by one intron. SiLEA14 was expressed in roots, stems, leaves, inflorescences and seeds at different levels under normal growth conditions. In addition, SiLEA14 was dramatically induced by osmotic stress, NaCl and exogenous abscisic acid. The SiLEA14 protein was localized in the nucleus and the cytoplasm. Overexpression of SiLEA14 improved Escherichia coli growth performance compared with the control under salt stress. To further assess the function of SiLEA14 in plants, transgenic Arabidopsis and foxtail millet plants that overexpressed SiLEA14 were obtained. The transgenic Arabidopsis seedlings showed higher tolerance to salt and osmotic stress than the wild type (WT). Similarly, the transgenic foxtail millet showed improved growth under salt and drought stresses compared with the WT. Taken together, our results indicated that SiLEA14 is a novel atypical LEA protein and plays important roles in resistance to abiotic stresses in plants.
We characterized a novel atypical LEA gene SiLEA14 from foxtail millet, which plays important roles in plant abiotic stress resistance. Modification of SiLEA14 expression may improve abiotic stress resistance in agricultural crops.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0290-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4243736  PMID: 25404037
24.  Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.) 
Journal of Experimental Botany  2013;64(18):5569-5586.
Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.
PMCID: PMC3871817  PMID: 24123251
Apoplast; cell wall; common bean; dehydrin; phosphoproteomics; proteomics; root tips.
25.  A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley 
Journal of Experimental Botany  2008;59(12):3359-3369.
Drought is a serious, worldwide problem for crop production and also affects yields of barley and wheat, together with other stressors such as frost, viral diseases, or fungal pathogens. Although a number of candidate genes have been identified by transcriptome approaches in recent years, only very few have been tested in functional assays for a beneficial effect on drought tolerance. Here, a transient assay system in microprojectile-bombarded barley leaves is described that allows the functional testing of dehydration stress-related candidate genes by RNA interference (RNAi) or overexpression. Cellular stress or damage in dedydrated leaves is reported by a reduced accumulation of slowly maturing, native red-fluorescing protein DsRed that is known to be sensitive to denaturing conditions. After a dehydration-stress period of 4 d during which the relative fresh weight of leaves was kept at 60–66% of initial fresh weight, a reproducible reduction of normalized DsRed fluorescence was observed. In order to obtain proof of concept, a number of barley mRNAs homologous to drought response genes were selected and targeted by transient induced gene silencing (TIGS). TIGS of four tested genes resulted in a significantly stronger decrease of normalized DsRed fluorescence in dehydration-stressed leaves, whereas they had no effect in fully turgescent control leaves. These genes encode barley drought-responsive factor HvDRF1 (DREB2-like), dehydrin 6, late embryogenesis-abundant protein HVA1, and the vacuolar sodium/proton antiporter HvHNX1. The four targeted transcripts were also found to accumulate rapidly in dehydration-stressed barley leaf segments. The results suggest a value of the TIGS system for functional pre-screening of larger numbers of drought or dehydration stress-related candidate genes in barley.
PMCID: PMC2529241  PMID: 18641397
DsRed; particle bombardment; RNAi; single cell

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