Dehydrin Protein Research Articles

Biochemical characterization of the Arabidopsis KS-type dehydrin protein, whose gene expression is constitutively abundant rather than stress dependent

Dehydrins are known as plant stress-responsive genes. Arabidopsis thaliana has 10 dehydrin genes. Among them, one of the highly expressed genes is a KS-type dehydrin (At1g54410). However, the gene product, which is a histidine-rich dehydrin whose molecular mass is 11 kDa (AtHIRD11), has not been studied. Thus, we report the biochemical characterization of the AtHIRD11 protein. Although the AtHIRD11 protein was detected in all organs of Arabidopsis, the bolting stem and the flower showed higher accumulation than the other organs, with the AtHIRD11 protein detected in the cambial zone of the stem vasculature. Most of the AtHIRD11 protein was found to be a bound form. The bound AtHIRD11 was solubilized by 1 M NaCl solution. The extracted AtHIRD11 was retained in immobilized metal-affinity chromatography, and eluted by an imidazole gradient. The native AtHIRD11 prepared from Arabidopsis was partially phosphorylated, but further phosphorylated by casein kinase 2 in vitro. Metal-binding assays indicated that Zn2+ may be the best metal for AtHIRD11 binding. These results suggest that AtHIRD11 is a metal-binding dehydrin that shows a house-keeping expression in Arabidopsis.

Cryoprotective mechanism of a small intrinsically disordered dehydrin protein

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

Comparative expression analysis of dehydrins between two barley varieties, wild barley and Tibetan hulless barley associated with different stress resistance

Drought, salinity and cold are the major environmental factors impacting on survival and productivity of Tibetan hulless barley in Tibetan Plateau of China. Tibetan hulless barley cultivar, Tibetan Heiqingke No. 1, has developed a strong tolerance and adaptation to stresses in relation to the wild barley. The differences of dehydrin gene transcription and translation between Tibetan Heiqingke No. 1 and the wild barley under drought, salinity and low temperature stresses were investigated in the present study to figure out the putative mechanism of stress tolerance of Tibetan Heiqingke No. 1. The leaf relative water contents (RWCs) decreased more slowly in Tibetan hulless barley than the wild barley under osmotic and low temperature conditions. Electrolyte leakage, malondialdehyde and H2O2 contents increased faster in wild barley than those of Tibetan hulless barley, which indicated that cells of wild barley received more damages than Tibetan hulless barley. Furthermore, the expression of several dehydrin genes, belonging to four different classifications respectively, was also investigated. Polyclonal antibodies against dehydrins were obtained from rabbit after prokaryotic expression and purification of TDHN4, a dehydrin protein from Tibetan hulless barley. With these antibodies and dehydrin gene fragments, western blotting analysis and RT-PCR showed that Tibetan Heiqingke No. 1 accumulated higher abundance of dehydrins than stress-sensitive wild barley under all stress conditions.

Wheat Dehydrin DHN-5 Exerts a Heat-Protective Effect on β-Glucosidase and Glucose Oxidase Activities

Group-2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are claimed to stabilize macromolecules against damage caused by freezing, dehydration, ionic or osmotic stresses. However, their precise function remains unknown. Here, we investigated the effect of wheat dehydrin (DHN-5) protein on the activity and thermostability of two distinct enzymes, beta-glucosidase (bglG) and glucose oxidase/peroxidase (GOD/POD) in vitro. The purified DHN-5 protein had the capacity to preserve and stabilize the activity of bglG subjected to heat treatment. In addition, DHN-5 stabilized oxidizing enzymes, as it improved reliability in measuring glucose concentrations with a glucose oxidase/peroxidase (GOD/POD) kit while the temperature increased from 37 to 70 degrees C. All together the data presented provide evidence that DHN-5 is a dehydrin able to preserve enzyme activities in vitro from adverse effects induced by heating.

Differential accumulation of dehydrins in response to water stress for hybrid and common bermudagrass genotypes differing in drought tolerance

Expression of dehydrin proteins may be induced or enhanced by environmental stresses that lead to cell dehydration. The objective of the this study was to investigate genetic variation in dehydrin protein accumulation in response to drought stress of whole-plants or dehydration of detached leaves and to identify dehydrins differentially expressed in bermudagrass ( Cynodon spp.) genotypes differing in drought tolerance. Plants of four hybrid bermudagrass ( Cynodondactylon L. × Cynodontransvaalensis L.) (‘Tifway’, ‘Tifdwarf’, ‘Tifeagle’, ‘Kan1’) and four common bermudagrass ( Cynodon dactylon) (‘C299’, ‘Sportbermuda’, ‘H10’, and ‘H19’) genotypes were subjected to 14 d of drought stress and detached leaves of two genotypes were exposed to dehydration in growth chambers. Turf quality and leaf relative water content (RWC) decreased while electrolyte leakage (EL) increased during whole-plant drought stress for all genotypes, with more pronounced changes in each parameter for ‘C299’ and ‘Tifeagle’ than those for other genotypes (‘Tifway’, ‘Kan 1’, ‘Sportbermuda’, ‘H10’, and H19’), suggesting that the former two genotypes were more sensitive to drought stress than the other genotypes. During dehydration of detached leaves, relative water loss rate (RWL) was significantly lower in drought-tolerant ‘Tifway’ than in drought-sensitive ‘C299’. Immunoblotting analysis indicated that no dehydrin polypeptides were detected in all genotypes under well-watered conditions. A 24-kDa polypeptide was detected in ‘C299’ at 6 d of drought, but not in the other genotypes. The dehydrin polypeptides of about 14–74 kDa accumulated at 10 d of drought stress and in a range of RWL for detached leaves, and two dehydrins (31 and 40 kDa) exhibited differential accumulation in the drought-sensitive ‘C299’ and tolerant ‘Tifway’, as demonstrated by the whole-plant drought responses. The 31-kDa dehydrin polypeptide was present only in ‘Tifway’ and ‘H19’ at 10 d of drought stress, and accumulated with the increasing RWL in detached leaves of ‘Tifway’. The expression level of 40-kDa dehydrin polypeptides was greater in ‘Tifway’’ than in ‘C299’ at the same level of water deficit (from 10% to 65% RWL). These results indicated that the accumulation of 31- and 40-kDa dehydrins may contribute to drought or dehydration tolerance in warm-season bermudagrass.

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

Dehydrins are a group of plant proteins that usually accumulate in response to environmental stresses. They are proposed to play specific protective roles in plant cells. Present study showed that the accumulation of dehydrins in water-stressed barley (Hordeum vulgare L.) seedlings was influenced by their treatment with salicylic acid (SA). The level of dehydrin proteins was increased by 0.20 mM SA, but decreased by 0.50 mM SA treatment. Both mRNA expression and protein accumulation of a typical barley dehydrin, DHN5, were enhanced by SA treatment when SA concentrations were lower than 0.25 mM. However, the higher SA concentrations significantly decreased the protein level of DHN5 despite of a stable mRNA level. Our results also showed that low SA concentrations (less than 0.25 mM) decreased the electrolyte leakage and malondialdehyde (MDA) and H2O2 contents in water-stressed barley seedlings. But high SA concentrations (more than 0.25 mM) enhanced H2O2 accumulation, tended to cause more electrolyte leakage, and increase MDA content. These data indicated that SA could up-regulate the dehydrin gene expression and protein accumulation. Since the protective role of dehydrins in plant cells, such effect could be an important reason for the SA-mediated alleviation on water stress injury. But excessive SA could suppress the accumulation of dehydrin proteins and aggravate the oxidative damage.

Desiccation tolerance acquisition in developing beech (Fagus sylvatica L.) seeds: the contribution of dehydrin-like protein

The acquisition of desiccation tolerance (DT) in developing beech (Fagus sylvatica L.) seeds and the role of a dehydrin protein in this process were investigated. DT was determined by measurement of electrolyte leakage and germination capacity after drying to 10–12% moisture content (MC). In addition to mass maturity, the presence of heat-stable proteins, dehydrin accumulation and the peak of ABA content were measured in relation to the acquisition of DT. Mass maturity was achieved at 16 weeks after flowering (WAF). The germination capacity increased from 8% at 12 WAF to 80–90% after 16 WAF. Cell membrane integrity, measured as a decrease in electrolyte leakage after desiccation, was acquired at 16 WAF. Additionally, the ratio of heat-stable to soluble proteins was the highest at 16 WAF. One dehydrin-like protein with a molecular mass 44 kDa, named DHN44, was detected in embryonic axes at 16 WAF and in cotyledons at 17 WAF, and its gradual accumulation was observed in mature seeds. With regard to the acquisition of DT, the strongest correlations were detected between electrolyte leakage, DHN44 accumulation, and the percentage of heat-stable proteins. These results suggest that developing beech seeds become tolerant to desiccation at 16 WAF. The effect of desiccation and ABA treatment on DHN44 synthesis was tested before (14 WAF) and after the DT acquisition (18 WAF). Depending on the maturation stage desiccation and ABA treatment can induce or enlarge DHN44 expression.

Overexpression of an abiotic-stress inducible plant protein in the bacteria Escherichia coli

The aim of our work was the overexpression of the abiotic stress-inducible dehydrin protein, namely RAB16A, from rice in the BL21 strain of Escherichia coli. The Rab16A transcript of 0.5 Kbp was amplified from the total RNA of the salt-tolerant indica rice cultivar Nonabokra by RT-PCR and cloned into the expression vector pGEX-3X. The 47 kDa protein, expressed as GST: RAB16A fusion protein, after 2 mM IPTG-mediated induction, was collected as S10 fraction and purified through glutathionesepharose affinity resin. Immunoblot analysis with the maize dehydrin antiserum showed crossreaction with the above band, but not with GST protein alone, showing functional expression of the heterologous RAB16A protein in the bacterial system.

Bermudagrass Freezing Tolerance Associated with Abscisic Acid Metabolism and Dehydrin Expression during Cold Acclimation

Recent advances in bermudagrass [Cynodon dactylon (L.) Pers. var. dactylon] breeding and cultural management practices have enabled its use as a sports surface in U.S. Department of Agriculture cold hardiness zones 5 and 6. Use of these more cold-hardy bermudagrass cultivars further into transition- and cool-season zones increases the probability of freezing injury and increases the need for an improved understanding of physiological responses to chilling and freezing temperatures. Abscisic acid (ABA) has been shown to increase during cold acclimation (CA) and play a role in dehydration tolerance. This study investigated changes in ABA metabolism and dehydrin expression during CA and their association with freezing tolerance in four bermudagrass cultivars. Two cold-tolerant (‘Patriot’ and ‘Riviera’) and two relatively cold-sensitive (‘Tifway’ and ‘Princess’) cultivars were either subjected to CA at 8 °C day/4 °C night with a light intensity of 250 μmol·m−2·s−1 over a 10-h photoperiod for 21 days or maintained at 28 °C day/24 °C night over a 12-h photoperiod. In a separate study, exogenous ABA at 0, 50, 100, and 150 μm was applied to ‘Patriot’ bermudagrass without CA. ABA content in leaf and stolon tissues increased substantially during the first week of CA and remained relatively stable thereafter. ‘Patriot’ and ‘Riviera’ had greater ABA content and less stolon electrolyte leakage (EL) relative to ‘Tifway’ and ‘Princess’. Expression of a 25 kDa dehydrin protein increased during CA in all four cultivars. A significant correlation was found between ABA content and freezing tolerance. Exogenously applying ABA to ‘Patriot’ at 50, 100, and 150 μm significantly increased endogenous ABA content and the 25 kDa dehydrin expression and reduced stolon EL. The results suggest that alteration of ABA metabolism during CA is closely associated with freezing tolerance. Selection and use of cultivars with substantial accumulation of ABA and certain dehydrins during CA or in response to exogenous ABA could improve bermudagrass persistence in transition zone climates.

A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates

Most signaling networks are regulated by reversible protein phosphorylation. The specificity of this regulation depends in part on the capacity of protein kinases to recognize and efficiently phosphorylate particular sequence motifs in their substrates. Sequenced plant genomes potentially encode over than 1000 protein kinases, representing 4% of the proteins, twice the proportion found in humans. This plethora of plant kinases requires the development of high-throughput strategies to identify their substrates. In this study, we have implemented a semi-degenerate peptide array screen to define the phosphorylation preferences of four kinases from Arabidopsis thaliana that are representative of the plant calcium-dependent protein kinase and Snf1-related kinase superfamily. We converted these quantitative data into position-specific scoring matrices to identify putative substrates of these kinases in silico in protein sequence databases. Our data show that these kinases display related but nevertheless distinct phosphorylation motif preferences, suggesting that they might share common targets but are likely to have specific substrates. Our analysis also reveals that a conserved motif found in the stress-related dehydrin protein family may be targeted by the SnRK2-10 kinase. Our results indicate that semi-degenerate peptide array screening is a versatile strategy that can be used on numerous plant kinases to facilitate identification of their substrates, and therefore represents a valuable tool to decipher phosphorylation-regulated signaling networks in plants.

A comparative study of water distribution and dehydrin protein localization in maturing pea seeds

In this study, the distribution of water in pea seeds after harvesting at different seed stages was traced by magnetic resonance imaging (MRI). MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside seeds. The intensity of the signal coming from water declined from the inner to the outer part of cotyledon tissue. This spatial inhomogeneity of water signals inside cotyledons may be correlated with the gradient of storage substances accumulation within cotyledons. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of protovascular tissue of both the embryo axis and cotyledons. The temporal accumulation of two dehydrin proteins with molecular masses of 30 and 35kDa correlated well with seed desiccation. The pattern of dehydrin localization reflected the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles.

Changes in water status and water distribution in maturing lupin seeds studied by MR imaging and NMR spectroscopy

The changes in water distribution in maturing lupin (Lupinus luteus L.) seeds were visualized with magnetic resonance imaging (MRI). MRI data showed local inhomogeneities of water distribution inside the seed. At the late seed-filling stage the most intense signal was detected in the seed coat and the outer parts of cotyledons in the hilum area, but during maturation drying the decline in MR image intensity was faster in the outer part of the seed than in the central part. The changes in water status were characterized by NMR spectroscopy. Analyses of T(2) relaxation times revealed a three-component water proton system in maturing lupin seeds. Three populations of protons found during seed maturation, each with a different magnetic environment causing a different relaxation rate, were correlated with three fractions of water (structural, intracellular, and extracellular) that were observed during seed germination. This study provides evidence that lupin seeds have similar states of the different water components with regard to seed moisture content at two distinct physiological stages, seed maturation and germination. The unique feature of maturing lupin seeds is the presence of the high (1)H-NMR signal in areas corresponding to the vascular bundles. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of vascular tissue. An anti-dehydrin antibody detected three polypeptides in lupin embryos with molecular masses of 73, 43 and 28 kDa, respectively. The temporal pattern of dehydrin protein accumulation correlates well with seed desiccation.

Response to heat stress in warm season and cool season turf grass cultivars

In this study, we investigated differences between cool-season and warm season grasses when exposed to heat stress. The results show that Kentucky blue seed germination rate was the highest at 25oC (40-50%), decreased significantly at 30oC (23-30%) and dropped to 0 at 40oC. For the Bermuda cultivars, seed germination rate was two fold higher at 30oC then 25oC, and reached the highest at 40oC. The cool season perennial rye and Bent grass seeds germinated well between 25 and 30oC. Their germination rate was significantly lower at 40oC. After exposure to 38oC for 2 d, the chlorophyll content decreased significantly for Bermuda cultivars, remained stable for Bent grass and perennial rye, and increased to the greatest extentfor Kentucky blue. Leaf soluble protein contents increased significantly for Kentucky Blue, but remained stable for most of the other cultivars. Western blot with Hsp70 and dehydrin antibodies identified that the Bermuda cultivars had a dynamic changes in the profile of the heat and dehydration stress-related proteins. No similar changes were detected in Kenblue cultivars. High temperature induced dehydrin homolog proteins, but not the Hsp 70 in Perennial rye and Bent grass cultivars. Findings from this research can be used to differentiate cool season and warm season grass cultivars in terms of germination and survival ability under high temperature. Key words: seed germination, chlorophyll content, leaf soluble protein, heat shock protein, dehydrin protein.

Differential expression of dehydrin in flower buds of two Japanese apricot cultivars requiring different chilling requirements for bud break

In this study, we investigated seasonal changes in protein profiles in dormant flower buds of Japanese apricot (Prunus mume Siebold Zucc.) cultivars 'Ellching', from subtropical Taiwan, and 'Nanko', from temperate Japan. One protein, isolated by two-dimensional polyacrylamide gel electrophoresis of flower bud extracts, was shown by peptide sequencing to be a dehydrin (the group of D-11 LEA (late embryogenesis-abundant) proteins). Patterns of dehydrin protein and transcript accumulation differed between the cultivars, with greater accumulations and longer persistence in 'Nanko' than in 'Ellching'. These differences correspond with the greater requirement for chilling to break flower bud dormancy in 'Nanko' than in 'Ellching'. Our study supports the findings of earlier work comparing dehydrin expression in the bark tissue of the evergreen and deciduous peach (Prunus persica (L.) Batsch) genotypes, and suggests that the role of dehydrin during the dormant season is common to all Prunus species.

Expression of SK3-type dehydrin in transporting organs is associated with cold acclimation in Solanum species

The expression of a gene, encoding a dehydrin protein designated as DHN24 was analyzed at the protein level in two groups of Solanum species differing in cold acclimation ability. The DHN24 protein displays consensus amino acid sequences of dehydrins, termed K- and S-segments. The S-segment precedes three K-segments, classifying the protein into SK3-type dehydrins. A group of Solanum species able to cold acclimation constituted by S. sogarandinum and S. tuberosum, cv. Aster, and a second one composed of a S. sogarandinum line, that lost ability to cold acclimation, and of S. tuberosum, cv. Irga, displaying low ability to cold acclimation were studied. Under control conditions, noticeable levels of the DHN24 protein was observed in stems, tubers, and roots of Solanum species. No protein was detected in leaves. During low temperature treatment the DHN24 protein level substantially increased in tubers, in transporting organs and in apical parts, and only a small increase was observed in leaves. The increase in protein abundance was only observed in the plants able to cold acclimate and was found to parallel the acclimation capacity. Upon drought stress, the DHN24 level decreased in stems and in leaves, but increased in apical parts. These results suggest that Dhn24 expression is regulated by organ specific factors in the absence of stress and by factors related to cold acclimation processes during low temperature treatment in collaboration with organ-specific factors. A putative function of the SK3-type dehydrin proteins during plant growth and in the tolerance to low temperature is discussed.

Cloning and expression of a dehydrin-like protein from Pistacia vera L.

A full-length dehydrin-like cDNA clone was obtained from a Pistacia vera L. cDNA library. The clone was sequenced and it was found that the deduced protein consists of 230 amino acids with a molecular weight of 25.87 kDa. The glycine rich protein is highly hydrophilic, contains 47.4% charged amino acids and belongs to the Kn-type of dehydrins. It comprises five repetitive units, each containing the K-segment diagnostic of dehydrins, but no serine tract (S-segment) and no Y-segment. Temporally, accumulation of the dehydrin transcript coincides with accumulation of the protein, which remains high in the bud scales of the inflorescence throughout winter. The dehydrin protein accumulates in the outer leaves (scales) of the inflorescence bud and in the bark of 1-year-old stems. In the buds it is mostly concentrated in the palisade-like cells underneath the epidermis, in the bud and in the inner bark parenchyma. Immunogold labeling revealed that it is a cytoplasmic protein with no specific organellar localization. It is suggested that the dehydrin of pistachio may have a dual function, a role in drought and cold tolerances, as well as serving as a storage protein.

Breeding for adaptation to drought and heat in cowpea

Accomplishments in breeding for adaptation to drought and heat are reviewed based upon work with the indeterminate grain legume species cowpea. Plant traits and some crop management methods are examined that influence adaptation to rainfed production in the drought-prone, semiarid tropical Sahelian zone of Africa. Drought escape, drought resistance, delayed-leaf-senescence, and varietal intercrops are examined. In addition, adaptation to the heat that can detrimentally impact irrigated production in the hot, subtropical arid zone of California is evaluated. Heat tolerance during reproductive development, electrolyte leakage, membrane thermostability, some aspects of crop management including date of sowing, and chilling tolerance during emergence including the beneficial effects of a dehydrin protein are considered. Methods for breeding cowpeas with adaptation to drought and heat are described that have been effective.

Accumulation of dehydrin transcripts and proteins in response to abiotic stresses in Deschampsia antarctica

Deschampsia antarctica Desv. is one of two vascular plants from the Maritime Antarctic. It is usually exposed to cold, salt, and desiccating winds. We hypothesize that D. antarctica has genes that encode dehydrin proteins and their expression is regulated by low temperature, salt or osmotic stress. To test this hypothesis a fragment of a dehydrin gene from D. antarctica was identified and used as a probe to study dehydrin expression under low temperature, salt, and osmotic stress, and exogenous ABA (abscisic acid) treatments. An anti-dehydrin antibody was also used to study dehydrin protein accumulation under the same treatments. Southern analysis of genomic DNA treated with different endonucleases showed more than four bands recognized by the probe, suggesting that D. antarctica has several dehydrin genes. Northern analysis showed two putative dehydrin transcripts of 1.0 kb accumulated only under exogenous ABA and 1.6 kb under osmotic and salt treatments, suggesting that D. antarctica would have ABA-dependent and - independent pathways for regulation of dehydrin expression. Western analysis showed seven dehydrin proteins (58, 57, 55, 53, 48, 30 and 27 kDa) under the different stress treatments. Cold-accumulated dehydrin proteins were immunolocalized, showing that they are associated with vascular and epidermal tissue, which are preferential ice nucleation zones.

Differential accumulation of a 24-kd dehydrin protein in wheat seedlings correlates with drought stress tolerance at grain filling.

The Pacific Northwest (PNW), an important region for wheat production in the USA, is often subject to water deficits during sowing and grain filling. These deficits reduce the quality and yield of the crop. As a consequence, an important objective of breeding programs in the region is improving the genetic adaptation of wheat cultivars to drought stress. One response to dehydrative stresses is the accumulation of proteins called dehydrins, which are believed to protect membranes and macromolecules against denaturation. We characterized dehydrin accumulation in seedlings during drought stress and its correlation with stress tolerance during grain filling in seven wheat cultivars, 'Connie', 'Gene', 'TAM105', 'Rod', 'Hiller', 'Rhode', and 'Stephens'. A 24-kd dehydrin accumulated in seedlings under stress, but not in irrigated control plants. Connie, TAM105, and Gene started to accumulate dehydrins at the fourth day of stress, while the other cultivars showed dehydrins after twelve days of stress. This differential accumulation in seedlings was associated with stress tolerance at grain filling, characterized by a lower reduction in yield and in the rate of decrease in leaf water potential per day of stress. Connie, TAM105, Gene and Rod where the most tolerant cultivars. The results indicate that expression of this 24-kd dehydrin might serve as a rapid and non-destructive screening technique at the seedling stage. Even though the results are promising, selection experiments using a population segregating for stress tolerance are needed to test more conclusively whether this dehydrin can serve as a genetic marker for cultivars with tolerance to drought stress.

Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco.

Citrus ( Citrus unshiu Marcov.) dehydrin in response to chilling stress was overexpressed in tobacco ( Nicotiana tabacum L.), and the cold stress tolerance of transgenics at low temperature was analyzed. The freezing at -4 degrees C for 3 h of 24 independent lines indicated that a phenotype expressing citrus dehydrin showed less electrolyte leakage than the control. Dehydrin protein content was correlated with freezing tolerance in transgenics. Dehydrin-expressing tobacco exhibited earlier germination and better seedling growth than the control at 15 degrees C. Cell fractionation experiments suggested that the protein was predominantly expressed in mitochondria and the soluble fraction. Malondialdehyde production enhanced by chilling stress was lower in tobacco plants expressing citrus dehydrin than in control phenotypes. Dehydrin protein, purified from Escherichia coli expressing citrus dehydrin cDNA, prevented peroxidation of soybean ( Glycine max L.) liposomes in vitro. The inhibitory activity of dehydrin against liposome oxidation was stronger than that of albumin, glutathione, proline, glycine betaine, and sucrose. These results suggest that dehydrin facilitates plant cold acclimation by acting as a radical-scavenging protein to protect membrane systems under cold stress.