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1.  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
2.  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
3.  Comparative Physiological and Proteomic Analyses of Poplar (Populus yunnanensis) Plantlets Exposed to High Temperature and Drought 
PLoS ONE  2014;9(9):e107605.
Plantlets of Populus yunnanensis Dode were examined in a greenhouse for 48 h to analyze their physiological and proteomic responses to sustained heat, drought, and combined heat and drought. Compared with the application of a single stress, simultaneous treatment with both stresses damaged the plantlets more heavily. The plantlets experienced two apparent response stages under sustained heat and drought. During the first stage, malondialdehyde and reactive oxygen species (ROS) contents were induced by heat, but many protective substances, including antioxidant enzymes, proline, abscisic acid (ABA), dehydrin, and small heat shock proteins (sHSPs), were also stimulated. The plants thus actively defended themselves against stress and exhibited few pathological morphological features, most likely because a new cellular homeostasis was established through the collaborative operation of physiological and proteomic responses. During the second stage, ROS homeostasis was overwhelmed by substantial ROS production and a sharp decline in antioxidant enzyme activities, while the synthesis of some protective elements, such as proline and ABA, was suppressed. As a result, photosynthetic levels in P. yunnanensis decreased sharply and buds began to die, despite continued accumulation of sHSPs and dehydrin. This study supplies important information about the effects of extreme abiotic environments on woody plants.
PMCID: PMC4167240  PMID: 25225913
4.  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
5.  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
6.  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
7.  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
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.  Influence of CSP 310 and CSP 310-like proteins from cereals on mitochondrial energetic activity and lipid peroxidation in vitro and in vivo 
BMC Plant Biology  2001;1:1.
The development of chilling and freezing injury symptoms in plants is known to frequently coincide with peroxidation of free fatty acids. Mitochondria are one of the major sources of reactive oxygen species during cold stress. Recently it has been suggested that uncoupling of oxidation and phosphorylation in mitochondria during oxidative stress can decrease ROS formation by mitochondrial respiratory chain generation. At the same time, it is known that plant uncoupling mitochondrial protein (PUMP) and other UCP-like proteins are not the only uncoupling system in plant mitochondria. All plants have cyanide-resistant oxidase (AOX) whose activation causes an uncoupling of respiration and oxidative phosphorylation. Recently it has been found that in cereals, cold stress protein CSP 310 exists, and that this causes uncoupling of oxidation and phosphorylation in mitochondria.
We studied the effects of CSP 310-like native cytoplasmic proteins from a number of cereal species (winter rye, winter wheat, Elymus and maize) on the energetic activity of winter wheat mitochondria. This showed that only CSP 310 (cold shock protein with molecular weight 310 kD) caused a significant increase of non-phosphorylative respiration. CSP 310-like proteins of other cereals studied did not have any significant influence on mitochondrial energetic activity. It was found that among CSP 310-like proteins only CSP 310 had prooxidant activity. At the same time, Elymus CSP 310-like proteins have antioxidant activity. The study of an influence of infiltration by different plant uncoupling system activators (pyruvate, which activates AOX, and linoleic acid which is a substrate and activator for PUMP and CSP 310) showed that all of these decreased lipid peroxidation during cold stress.
Different influence of CSP 310-like proteins on mitochondrial energetic activity and lipid peroxidation presumably depend on the various subunit combinations in their composition. All the plant cell systems that caused an uncoupling of oxidation and phosphorylation in plant mitochondria can participate in plant defence from oxidative damage during cold stress.
PMCID: PMC58587  PMID: 11667950
10.  Plant dehydrins and stress tolerance 
Plant Signaling & Behavior  2011;6(10):1503-1509.
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.
PMCID: PMC3256378  PMID: 21897131
abiotic stress; dehydration stress; drought; cold acclimation; freezing tolerance; LEA proteins; dehydrins
11.  Light has a specific role in modulating Arabidopsis gene expression at low temperature 
BMC Plant Biology  2008;8:13.
Light and temperature are the key abiotic modulators of plant gene expression. In the present work the effect of light under low temperature treatment was analyzed by using microarrays. Specific attention was paid to the up and down regulated genes by using promoter analysis. This approach revealed putative regulatory networks of transcription factors behind the induction or repression of the genes.
Induction of a few oxidative stress related genes occurred only under the Cold/Light treatment including genes encoding iron superoxide dismutase (FeSOD) and glutathione-dependent hydrogen peroxide peroxidases (GPX). The ascorbate dependent water-water cycle genes showed no response to Cold/Light or Cold/Dark treatments. Cold/Light specifically induced genes encoding protective molecules like phenylpropanoids and photosynthesis-related carotenoids also involved in the biosynthesis of hormone abscisic acid (ABA) crucial for cold acclimation. The enhanced/repressed transcript levels were not always reflected on the respective protein levels as demonstrated by dehydrin proteins.
Cold/Light up regulated twice as many genes as the Cold/Dark treatment and only the combination of light and low temperature enhanced the expression of several genes earlier described as cold-responsive genes. Cold/Light-induced genes included both cold-responsive transcription factors and several novel ones containing zinc-finger, MYB, NAC and AP2 domains. These are likely to function in concert in enhancing gene expression. Similar response elements were found in the promoter regions of both the transcription factors and their target genes implying a possible parallel regulation or amplification of the environmental signals according to the metabolic/redox state in the cells.
PMCID: PMC2253524  PMID: 18230142
12.  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
13.  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
14.  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
15.  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.
16.  Physiological and Proteomic Adaptation of the Alpine Grass Stipa purpurea to a Drought Gradient 
PLoS ONE  2015;10(2):e0117475.
Stipa purpurea, an endemic forage species on the Tibetan Plateau, is highly resistant to cold and drought, but the mechanisms underlying its responses to drought stress remain elusive. An understanding of such mechanisms may be useful for developing cultivars that are adaptable to water deficit. In this study, we analyzed the physiological and proteomic responses of S. purpurea under increasing drought stress. Seedlings of S. purpurea were subjected to a drought gradient in a controlled experiment, and proteins showing changes in abundance under these conditions were identified by two-dimensional electrophoresis followed by mass spectrometry analysis. A western blotting analysis was conducted to confirm the increased abundance of a heat-shock protein, NCED2, and a dehydrin in S. purpurea seedlings under drought conditions. We detected carbonylated proteins to identify oxidation-sensitive proteins in S. purpurea seedlings, and found that ribulose-1, 5-bisphosphate carboxylase oxygenase (RuBisCO) was one of the oxidation-sensitive proteins under drought. Together, these results indicated drought stress might inhibit photosynthesis in S. purpurea by oxidizing RuBisCO, but the plants were able to maintain photosynthetic efficiency by a compensatory upregulation of unoxidized RuBisCO and other photosynthesis-related proteins. Further analyses confirmed that increased abundance of antioxidant enzymes could balance the redox status of the plants to mitigate drought-induced oxidative damage.
PMCID: PMC4315458  PMID: 25646623
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.  A KS-type dehydrin and its related domains reduce Cu-promoted radical generation and the histidine residues contribute to the radical-reducing activities 
Journal of Experimental Botany  2013;64(6):1615-1624.
Dehydrin is a plant disordered protein whose functions are not yet totally understood. Here it is reported that a KS-type dehydrin can reduce the formation of reactive oxygen species (ROS) from Cu. AtHIRD11, which is the Arabidopsis KS-type dehydrin, inhibited generation of hydrogen peroxide and hydroxyl radicals in the Cu–ascorbate system. The radical-reducing activity of AtHIRD11 was stronger than those of radical-silencing peptides such as glutathione and serum albumin. The addition of Cu2+ reduced the disordered state, decreased the trypsin susceptibility, and promoted the self-association of AtHIRD11. Domain analyses indicated that the five domains containing histidine showed ROS-reducing activities. Histidine/alanine substitutions indicated that histidine is a crucial residue for reducing ROS generation. Using the 27 peptides which are related to the KnS-type dehydrins of 14 plant species, it was found that the strengths of ROS-reducing activities can be determined by two factors, namely the histidine contents and the length of the peptides. The degree of ROS-reducing activities of a dehydrin can be predicted using these indices.
PMCID: PMC3617826  PMID: 23382551
Circular dichroism; dehydrin; disordered protein; heavy metal; histidine; reactive oxygen species.
19.  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
20.  Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage 
Journal of Experimental Botany  2009;60(12):3531-3544.
Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also facilitate the genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at the transcriptional level in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme, NADP-ME, and pyruvate dehydrogenase, PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase, CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase,ALDH, ascorbate-dependent oxidoreductase, ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8, HSP17.8, and dehydrin 3, DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were possibly constitutively expressed in drought-tolerant genotypes. Among them, seven known annotated genes might enhance drought tolerance through signalling [such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP)], anti-senescence (G2 pea dark accumulated protein, GDA2), and detoxification (glutathione S-transferase, GST) pathways. In addition, 18 genes, including those encoding Δl-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C), and several chaperones, were differentially expressed in all genotypes under drought; thus they were more likely to be general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley.
PMCID: PMC2724701  PMID: 19561048
Barley; drought stress; drought tolerance; microarray; reproductive stage
21.  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
22.  Sequence Evolution and Expression Regulation of Stress-Responsive Genes in Natural Populations of Wild Tomato 
PLoS ONE  2013;8(10):e78182.
The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced) gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives.
PMCID: PMC3799731  PMID: 24205149
23.  Abscisic acid induced freezing tolerance in chilling-sensitive suspension cultures and seedlings of rice 
BMC Research Notes  2013;6:351.
The role of abscisic acid (ABA) as a possible activator of cold acclimation process was postulated since endogenous levels of ABA increase temporarily or constitutively during cold-hardening. Exogenous application of ABA has been known to induce freezing tolerance at ambient temperatures in in vitro systems derived from cold hardy plants. Yet, some cell cultures acquired much greater freezing tolerance by ABA than by cold whilst maintaining active growth. This raises questions about the relationships among ABA, cold acclimation and growth cessation. To address this question, we attempted to 1) determine whether exogenous ABA can confer freezing tolerance in chilling-sensitive rice suspension cells and seedlings, which obviously lack the mechanisms to acquire freezing tolerance in response to cold; 2) characterize this phenomenon by optimizing the conditions and compare with the case of cold hardy bromegrass cells.
Non-embryogenic suspension cells of rice suffered serious chilling injury when exposed to 4°C. When incubated with ABA at the optimal conditions (0.5-1 g cell inoculum, 75 μM ABA, 25-30°C, 7–10 days), they survived slow freezing (2°C/h) to −9.0 ~ −9.3°C (LT50: 50% killing temperature) while control cells were mostly injured at −3°C (LT50: -0.5 ~ −1.5°C). Ice-inoculation of the cell suspension at −3°C and survival determination by regrowth confirmed that ABA-treated rice cells survived extracellular freezing at −9°C. ABA-induced freezing tolerance did not require any exposure to cold and was best achieved at 25-30°C where the rice cells maintained high growth even in the presence of ABA. ABA treatment also increased tolerance to heat (43°C) as determined by regrowth. ABA-treated cells tended to have more augmented cytoplasm and/or reduced vacuole sizes compared to control cultures with a concomitant increase in osmolarity and a decrease in water content. ABA-treated (2–7 days) in vitro grown seedlings and their leaves survived slow freezing to −3°C with only marginal injury (LT50: -4°C) whereas untreated seedlings were killed at −3°C (LT50: -2°C).
The results indicate that exogenous ABA can induce some levels of freezing tolerance in chilling-sensitive rice cells and seedlings, probably by eliciting mechanisms different from low temperature-induced cold acclimation.
PMCID: PMC3766701  PMID: 24004611
ABA (abscisic acid); Cold hardiness; Cell culture; Freezing injury; Freezing tolerance; Chilling injury; Rice (Oryza sativa)
24.  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
25.  Complex Regulation by Apetala2 Domain-Containing Transcription Factors Revealed through Analysis of the Stress-Responsive TdCor410b Promoter from Durum Wheat 
PLoS ONE  2013;8(3):e58713.
Expression of the wheat dehydrin gene Cor410b is induced several fold above its non-stressed levels upon exposure to stresses such as cold, drought and wounding. Deletion analysis of the TdCor410b promoter revealed a single functional C-repeat (CRT) element. Seven transcription factors (TFs) were shown to bind to this CRT element using yeast one-hybrid screens of wheat and barley cDNA libraries, of which only one belonged to the DREB class of TFs. The remaining six encoded ethylene response factors (ERFs) belong to three separate subfamilies. Analysis of binding selectivity of these TFs indicated that all seven could bind to the CRT element (GCCGAC), and that three of the six ERFs could bind both to the CRT element and the ethylene-responsive GCC-box (GCCGCC). The TaERF4 subfamily members specifically bound the CRT element, and did not bind either the GCC-box or DRE element (ACCGAC). Molecular modeling and site-directed mutagenesis identified a single residue Pro42 in the Apetala2 (AP2) domain of TaERF4-like proteins that is conserved in monocotyledonous plants and is responsible for the recognition selectivity of this subfamily. We suggest that both DREB and ERF proteins regulate expression of the Cor410b gene through a single, critical CRT element. Members of the TaERF4 subfamily are specific, positive regulators of Cor410b gene expression.
PMCID: PMC3602543  PMID: 23527011

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