A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Pea dehydrins: identification,characterisation and expression

An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species.The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI=6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum.A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins.B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water.During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.     

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. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 3, pp. 388–394. This text was submitted by the authors in English.     

Accumulation pattern of dehydrins during sugarcane (var. SP80.3280) somatic embryogenesis

The objective of the present study was to determine dehydrin protein levels in sugarcane var. SP80-3280 during somatic embryogenesis. Dehydrins from embryogenic and non-embryogenic cell cultures were analyzed using western blot and in situ immunolocalization microscopy. Both techniques employ antibodies raised against a highly conserved lysine-rich 15-amino acid sequence termed the K-domain, which is extensively used to recognize proteins immunologically related to the dehydrin family. In embryogenic cultures, western blot analysis of the heat-stable protein fraction revealed eleven major bands ranging from 52 to 17?kDa. They were already visible on the first days, gradually increasing until reaching peak values around day 14, when organogenesis begins, to later decrease in concurrence with the appearance of green plantlets (around day 28). These fluctuations indicate that this pattern of accumulation is under developmental control. Dehydrins were mainly immunolocalized in the nuclei. A phosphatase treatment of protein extracts caused a mobility shift of the 52, 49, and 43?kDa dehydrin bands suggesting a putative modulation mechanism based on protein phosphorylation. In sugarcane embryogenic cultures, presence of dehydrins is a novel finding. Dehydrins were absent in non-embryogenic cultures. The novel findings regarding accumulation, nuclear localization, and phosphorylation of dehydrins provide a starting point for further research on the role of these proteins in the induction and/or maintenance of embryogenesis. Key message The novel findings regarding accumulation, nuclear localization, and phosphorylation of dehydrins provide a starting point for further research on the role of these proteins in the induction and/or maintenance of embryogenesis.     

Dehydrin-like proteins in castor bean seeds and seedlings are differentially produced in response to ABA and water-deficit-related stresses

The stress inducibility of dehydrin protein production in seedlingsof castor bean was analysed by subjecting them to ABA and variouswater-deficit-related treatments including desiccation, waterstress, high salt, high osmolarity, and low temperature. A furthergoal was to determine whether the immature seed (at stages priorto major dehydrin synthesis) would respond in a similar mannerto these stresses. A number of dehydrin-like proteins increasedin seedlings subjected to the various stress treatments. Inthe endosperm, these appear to be different from the dehydrin-relatedpolypeptides that are induced during late seed development andwhich persist following germination/growth of mature seeds.In the endosperm of seedlings, ABA, water stress and desiccationinduced the same dehydrin polypeptides, while high osmolarity,high salt and low temperature induced a different set. Stress-specificdifferences in dehydrin synthesis were also found in the cotyledonsand radicle of castor bean seedlings; however, dehydrins indu-cibleby exogenous ABA were consistently produced. Immature seedstreated with ABA or subjected to stress responded by producingdehydrin-like proteins associated with late development; however,the same proteins were induced following detachment of immatureseeds from the parent plant and maintenance on water. When seedlingswere exposed simultaneously to GA and either ABA, high salt,or low temperature, dehydrin production was suppressed. It isconcluded that dehydrin production in castor bean is tissue-specificand is dependent upon the physiological stage of the seed. Inthe endosperm, the response to different stresses may rely uponmore than one signal trans-duction pathway. Key words: Dehydrin, castor bean, ABA, desiccation     

Immunoanalysis of dehydrins in Araucaria angustifolia embryos

The aim of this study was to describe the dehydrin content of mature Araucaria angustifolia embryos, a species of endangered and economically important conifers, native to southern Brazil, northeastern Argentina, and eastern Paraguay. The A. angustifolia seeds have been categorized as recalcitrant. Dehydrins were studied by western blot analysis and in situ immunolocalization microscopy using antibodies raised against the K segment, a highly conserved lysine-rich 15-amino acid sequence extensively used to recognize proteins immunologically related to the dehydrin family. Western blot analysis of the heat-stable protein fraction, as estimated by 15 % SDS-PAGE, revealed three main bands of approximately 20-, 26-, and 29-kDa; when 17.5 % SDS-PAGE was used, each band resolved into two other bands. Two thermosensitive dehydrin bands of around 16 and 35 kDa were common to the axis and cotyledons, and another thermosensitive band, with molecular mass of approximately 10 kDa, was present in the cotyledons only. Following alkaline phosphatase (AP) treatment, a gel mobility shift was detected for each one of the four main bands that can be due to phosphorylation. Dehydrins were detected in all axis and cotyledon tissues using in situ immunolocalization microscopy. At the subcellular level, dehydrins were immunolocalized in the nuclei, protein bodies, and microbodies. In the nucleus, dehydrins were found to be associated with chromatin. We concluded that the gel mobility shift for the four main bands (probably due to phosphorylation), the presence of thermosensitive bands, and the specific localizations in nuclei and protein bodies provide key starting points to understand the function of dehydrins in the embryo cells of this species.     

The recalcitrant plant species, Castanospermum australe and Trichilia dregeana, differ in their ability to produce dehydrin-related polypeptides during seed maturation and in response to ABA or water-deficit-related stresses

In constrast to seeds of orthodox species, those of recalcitrantspecies do not acquire desiccation tolerance during their developmentand are shed from the parent plant at high water contents. Dehydrinproduction in seeds of recalcitrant species was examined duringdevelopment and germination, in response to abscisic acid (ABA),and following the imposition of various water-deficit-relatedstresses, including desiccation, water stress, high salt, highosmolarity, and low temperature. Two tropical species exhibiteda differential capacity to produce dehydrin-related proteinsduring seed maturation. Dehydrins were present in axes and cotyledonsof Castanospermum australe seeds during mid-maturation and atmaturity. In Trichilia dregeana, no dehydrin-related polypeptideswere detected in the mature seed. During the development ofC. australe seeds, the nature of the dehydrin related polypeptidesaccumulated in the cotyledons and axis changed and new polypeptideswere detected in the mature seeds that were not present duringmid-maturation. The dehydrins present in cotyledons of matureseeds (31, 37 and 40 kDa) were still detectable after germination(i.e. in untreated seedlings). These dehydrins became less abundantin the cotyledons of C. australe seedlings following ABA andall stress treatments except cold, although most of the dehydrinswere still detectable. An exception was the desiccation-treatedseedlings, in which no dehydrins were detected. In the rootsof C. australe seedlings, no dehydrins were found after germinationnor were they induced in the root by ABA or any of the stresstreatments imposed on seedlings. Seedlings of Trichilia dregeanadid not produce dehydrins in the roots or cotyledons when exposedto ABA or water-deficit-related stresses. Key words: Dehydrin, ABA, desiccation, recalcitrant, seed     

Modulation of Dehydration Tolerance in Soybean Seedlings (Dehydrin Mat1 Is Induced by Dehydration but Not by Abscisic Acid)

Germinated soybean (Glycine max L. cv Williams 82) seedlings subjected to rapid dehydration begin to lose the ability to recover when the relative water content of the plant decreases below 60%. The expanded cells of the hypocotyl appear more susceptible to dehydration-induced damage than do cells in the hypocotyl zone of cell growth. Pretreatment of seedlings prior to rapid dehydration with nonlethal water deficit or exogenous abscisic acid (ABA) shifts this viability threshold to progressively lower relative water contents, indicating the acquisition of increased dehydration tolerance. Increased tolerance is associated with osmotic adjustment in the hypocotyl zone of cell growth and with increases in soybean dehydrin Mat1 mRNA levels. The accumulation of Mat1 mRNA is dehydration dependent but insensitive to ABA. Induction of Mat1 mRNA accumulation by dehydration but not by ABA makes it an unusual member of the dehydrin family.     

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and coppper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H₂O₂). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.     

Temporal accumulation and ultrastructural localization of dehydrins in Zea mays

Immunolocalization using polyclonal antibodies raised against a conserved dehydrin amino acid sequence was used to establish the temporal and spatial patterns of dehydrin accumulation in embryo tissue of Zea mays L. (var. Ohio 43) kernels imbibed in the presence of abscisic acid. The temporal pattern of accumulation indicated an increase in dehydrins over time (particularly between 15 and 30 h) and with maximum levels detected 48 h after the onset of imbibition. Dehydrins were first evident, and also the most concentrated, in the cytosol throughout the accumulation period suggesting that the primary function of dehydrins involves the cytosol and the structures contained therein. Only after an accumulation of dehydrins in the cytosol was there an increase in the abundance of nuclear dehydrins. In addition, dehydrins were also observed in association with the proteinaceous matrix of protein bodies and membranes of protein and lipid bodies; these findings have not been reported previously. The observed localization at a number of sites indicates that the specific biochemical roles of dehydrins are likely to be diverse.     

GTP Promotes the Formation of Early-Import Intermediates But Is Not Required during the Translocation Step of Protein Import into Chloroplasts

Dehydrins are a family of proteins (LEA [late-embryogenesis abundant] D11) commonly induced by environmental stresses associated with low temperature or dehydration and during seed maturation drying. Our previous genetic studies suggested an association of an approximately 35-kD protein (by immunological evidence a dehydrin) with chilling tolerance during emergence of seedlings of cowpea (Vigna unguiculata) line 1393-2-11. In the present study we found that the accumulation of this protein in developing cowpea seeds is coordinated with the start of the dehydration phase of embryo development. We purified this protein from dry seeds of cowpea line 1393-2-11 by using the characteristic high-temperature solubility of dehydrins as an initial enrichment step, which was followed by three chromatography steps involving cation exchange, hydrophobic interaction, and anion exchange. Various characteristics of this protein confirmed that indeed it is a dehydrin, including total amino acid composition, partial amino acid sequencing, and the adoption of alpha-helical structure in the presence of sodium dodecyl sulfate. The propensity of dehydrins to adopt alpha-helical structure in the presence of sodium dodecyl sulfate, together with the apparent polypeptide adhesion property of this cowpea dehydrin, suggests a role in stabilizing other proteins or membranes. Taken together, the genetic, physiological, and physicochemical data are at this stage consistent with a cause-and-effect relationship between the presence in mature seeds of the approximately 35-kD dehydrin, which is the product of a single member of a multigene family, and an increment of chilling tolerance during emergence of cowpea seedlings.     

脱水素研究进展

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

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and copper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H2O2). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.     

High salinity induces dehydrin accumulation in Chenopodium quinoa Willd. cv. Hualhuas embryos

Background and Aims

Chenopodium quinoa can grow at altitudes of 3,600–4,000 masl and is adapted to the highly arid conditions typical of the salty soils in the South American Altiplano, with less than 250?mm of annual rain and temperatures below 0°C. The aim of the study was to investigate the effect of salinity on the dehydrin content of mature embryos harvested from salt-stressed Chenopodium quinoa cv. Hualhuas plants grown at 100 to 500?mM NaCl. To date, no studies exist on the dehydrins of seeds from salt-stressed plants, although dehydrins in the root, stems and leaves have been reported as an adaptation to water deficit produced by salinity.

Methods

Dehydrin-like protein detection was carried out with an antiserum raised against a highly-conserved lysine-rich 15-amino acid sequence known as the K-segment, which is capable of recognizing proteins immunologically related to the dehydrin family.

Results

Dehydrins were analyzed in embryos by both western blot and in situ immunolocalization. Western blot analysis detected at least four dehydrins (55, 50, 34, and 30?kDa) in seeds harvested from quinoa salt-stressed plants treated under a wide range of salinities. The 30?kDa dehydrin increased its accumulation in both 300 and 500?mM NaCl growth conditions as revealed by densitometric analyses. Dehydrin subcellular localization was mostly nuclear at 500?mM of NaCl. A phosphatase treatment of protein extracts caused a mobility shift of the 34 and 30?kDa dehydrin bands suggesting a putative modulation mechanism based on protein phosphorylation.

Conclusions

We propose that these novel observations regarding dehydrin accumulation, subcellular localization and phosphorylation state are related to the high salt stress tolerant phenotype previously reported on this cultivar.