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.     

Evaluation of salinity tolerance and analysis of allelic function of <Emphasis Type="Italic">HvHKT1</Emphasis> and <Emphasis Type="Italic">HvHKT2</Emphasis> in Tibetan wild barley

Tibetan wild barley is rich in genetic diversity with potential allelic variation useful for salinity-tolerant improvement of the crop. The objectives of this study were to evaluate salinity tolerance and analysis of the allelic function of HvHKT1 and HvHKT2 in Tibetan wild barley. Salinity tolerance of 189 Tibetan wild barley accessions was evaluated in terms of reduced dry biomass under salinity stress. In addition, Na⁺ and K⁺ concentrations of 48 representative accessions differing in salinity tolerance were determined. Furthermore, the allelic and functional diversity of HvHKT1 and HvHKT2 was determined by association analysis as well as gene expression assay. There was a wide variation among wild barley genotypes in salt tolerance, with some accessions being higher in tolerance than cultivated barley CM 72, and salinity tolerance was significantly associated with K⁺/Na⁺ ratio. Association analysis revealed that HvHKT1 and HvHKT2 mainly control Na⁺ and K⁺ transporting under salinity stress, respectively, which was validated by further analysis of gene expression. The present results indicated that Tibetan wild barley offers elite alleles of HvHKT1 and HvHKT2 conferring salinity tolerance.     

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

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

Expression analysis of dehydrin multigene family across tolerant and susceptible barley (Hordeum vulgare L.) genotypes in response to terminal drought stress

Dehydrins are one of the characteristic families of plant proteins that usually accumulate in response to drought. In the present study, gene expressions of dehydrin multigene family (13 genes) were examined in flag leaves of tolerant (Yousef) and susceptible (Moroco) barley varieties under terminal drought to characterize the involvement of dehydrins in the adaptive processes. The stomatal conductance, RWC, and Chl a, b contents had more reduction in Moroco than the Yousef which has more elevated osmotic adjustment. Drought stress increased significantly MDA and electrolyte leakage levels, but greater in Moroco, indicating a poor protection of cell and cytoplasmic membrane in this variety. Yousef variety had no reduction in grain yield under drought condition. Five genes (Dhn1, Dhn3, Dhn5, Dhn7 and Dhn9) were exclusively induced in Yousef under drought stress. In the stress condition, relative gene expression of Dhn3, Dhn9 had the direct correlations (P < 0.05) with Chl a, b contents, osmotic adjustment, stomatal conductance, plant biomass and grain yield, and the negative correlations (P < 0.05) with MDA and electrolyte leakage levels. The results supported the impending functional roles of dehydrin K_n and particularly Y_nSK_n types in dehydration tolerance of barley during the reproductive stage.     

Identification of proteins associated with ion homeostasis and salt tolerance in barley

Identification and characterization of proteins involved in salt tolerance are imperative for revealing its genetic mechanisms. In this study, ionic and proteomic responses of a Tibetan wild barley XZ16 and a well‐known salt‐tolerant barley cv. CM72 were analyzed using inductively coupled plasma‐optical emission spectrometer, 2DE, and MALDI‐TOF/TOF MS techniques to determine salt‐induced differences in element and protein profiles between the two genotypes. In total, 41 differentially expressed proteins were identified in roots and leaves, and they were associated with ion homeostasis, cell redox homeostasis, metabolic process, and photosynthesis. Under salinity stress, calmodulin, Na/K transporters, and H⁺‐ATPases were involved in establishment of ion homeostasis for barley plants. Moreover, ribulose‐1,5‐bisphosphate carboxylase/oxygenase activase and oxygen‐evolving enhancer proteins were significantly upregulated under salinity stress, indicating the great impact of salinity on photosynthesis. In comparison with CM72, XZ16 had greater relative dry weight and lower Na accumulation in the shoots under salinity stress. A higher expression of HvNHX1 in the roots, and some specific proteins responsible for ion homeostasis and cell redox homeostasis, was also found in XZ16 exposed to salt stress. The current results showed that Tibetan wild barley XZ16 and cultivated barley cultivar CM72 differ in the mechanism of salt tolerance.     

Dehydrin genes and their expression in recalcitrant oak (<Emphasis Type="Italic">Quercus robur</Emphasis>) embryos

In this work, three dehydrin genes, QrDhn1, QrDhn2, QrDhn3, were isolated from recalcitrant oak (Quercus robur). Their expression pattern was analyzed in both zygotic and somatic embryos as well as in vegetative tissues exposed to different kinds of abiotic stresses including desiccation, osmotic stress, and chilling. The QrDhn1 gene encoding for Y_nSK_n type dehydrin was expressed during later stages of zygotic embryo development but in somatic embryos only when exposed to osmotic or desiccation stress. In contrast, the other two oak dehydrin genes encoding for putative K_n type dehydrins were expressed only in somatic embryos (both not-treated and osmotically stressed) and leaves of oak seedlings exposed to desiccation. Behavior of these genes suggests that different dehydrins are involved in processes of seed maturation and response to altered osmotic (water status) conditions in somatic embryos. Revealing further members of dehydrin gene family in recalcitrant oak might contribute to clarify non-orthodox seed behavior as well as identify mechanisms contributing to desiccation tolerance in plants.     

Virus-induced silencing of genes encoding LEA protein in Tibetan hulless barley (Hordeum vulgare ssp. vulgare) and their relationship to drought tolerance

Expression of the late embryogenesis abundant (LEA) gene is usually associated with plant response to dehydration. In this study, a drought-tolerant genotype was screened from 48 accessions of Tibetan hulless barley (Hordeum vulgare ssp. vulgare). By using virus-induced gene silencing, the influence of two LEA genes (HVA1 and Dhn6) on drought tolerance of Tibetan hulless barley was investigated. Results of quantitative real-time PCR indicated that the relative expression levels of HVA1 and Dhn6 in silenced plants were significantly reduced compared with control plants. Both HVA1-silenced and Dhn6-silenced plants showed a consequently lower survival rate than control plants under drought stress. However, only HVA1-silenced plants exhibited a significantly higher water loss rate (WLR). These results suggested that HVA1 and Dhn6 might participate in adaptive responses to water deficit in different ways. Vegetative growth of HVA1-silenced plants was significantly retarded even under optimal growth conditions, and their biomass accumulation was also much lower than that of the controls. These results indicate that HVA1 might play a role in vegetative growth of Tibetan hulless barley.     

Salicylic acid decreases the levels of dehydrin-like proteins in Tibetan hulless barley leaves under water stress

The effects of salicylic acid (SA) on the accumulation of dehydrins in leaves of Tibetan hulless barley seedlings under water stress were investigated. The results indicated that SA decreased the levels of the four dehydrin-like proteins induced by water stress. The concentrations of these dehydrin-like proteins increased under water stress. However, their levels in SA-pretreated seedlings were always lower than in those receiving only water stress. Our results also indicated that the levels of dehydrin-like proteins decreased as the SA concentration increased. In SA-pretreated seedlings, electrolyte leakage, MDA and H2O2 content were rather higher than in seedlings receiving only water stress. By these results, we suggest that lower levels of dehydrin-like proteins in seedlings with SA treatment may be due to the greater accumulation of H2O2 induced by SA, which causes more oxidative injury under water stress.     

Genetic studies of Triticeae dehydrins: assignment of seed proteins and a regulatory factor to map positions

 A collection of 200 wheat (Triticum aestivum L. cv ‘Chinese Spring’) cytogenetic stocks (nullisomic, tetrasomic, nulli-tetrasomic, ditelosomic and deletion lines, addition and substitution stocks from intra- and inter-specific crosses) was utilized to determine the proteins encoded by some of the wheat and barley dehydrin genes, using a western blot procedure. Proteins extracted from seeds were reacted with antibodies that recognize dehydrins in a wide range of plants, including wheat and barley. Proteins encoded by dehydrin loci in chromosome arms 4DS, 5BL and 6AL of ‘Chinese Spring’ wheat were assigned by this method. There was also evidence of a regulatory factor on 5B in the vicinity of the dhn genes, and on 5H in wheat-barley addition lines, that is required for a normal level of expression of seed dehydrins in hexaploid wheat. Further understanding of this putative regulatory factor would be helpful for the interpretation of linkage studies that may relate dehydrin gene expression to phenotypes such as dehydration, salinity or low-temperature tolerance. Received: 27 August 1997 / Accepted: 4 February 1998     

Time‐course analysis of salicylic acid effects on ROS regulation and antioxidant defense in roots of hulled and hulless barley under combined stress of drought,heat and salinity

Greater crop losses can result from simultaneous exposure to a combination of drought, heat and salinity in the field. Salicylic acid (SA), a phenolic phytohormone, can affect a range of physiological and biochemical processes in plants and significantly impacts their resistance to these abiotic stresses. Despite numerous reports involving the positive effects of SA by applying each abiotic stress separately, the mechanism of SA‐mediated adaptation to combined stresses remains elusive. This study, via a time‐course analysis, investigated the role of SA on the roots of hulled and hulless (naked) barley (Hordeum vulgare L. ‘Tarm’ and ‘Özen’, respectively), which differed in salt tolerance, under the combined stress of drought, heat and salt. The combined stress caused marked reductions in root length and increases in proline content in both genotypes; however, Tarm exhibited better adaptation to the triple stress. Under the first 24 h of stress, superoxide dismutase (SOD; EC.1.15.1.1) and peroxidase (POX; EC.1.11.1.7) activity in the Tarm roots increased remarkably, while decreasing in the Özen roots. Furthermore, the Tarm roots showed higher catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) activity than the Özen during the combined stresses. The sensitivity of hulless barley roots may be related to decreasing SOD, POX, CAT and GR activity under stress. Over 72 h of stress, the SA pretreatment improved the APX and GR activity in Tarm and that of POX and CAT in Özen, demonstrating that exogenously applied SA regulates antioxidant defense enzymes in order to detoxify reactive oxygen species. The results of this study suggest that SA treatment may improve the triple‐stress combination tolerance in hulled and hulless barley cultivars by increasing the level of antioxidant enzyme activity and promoting the accumulation of proline. Thus, SA alleviated the damaging effects of the triple stress by improving the antioxidant system, although these effects differed depending on characteristic of the hull of the grain.     

Accumulation of WCS120 and DHN5 proteins in differently frost-tolerant wheat and barley cultivars grown under a broad temperature scale

Proteins WCS120 and DHN5 are known as the major cold-inducible dehydrins in wheat and barley plants, respectively. WCS120 and DHN5 relative accumulation increased exponentially along with a growth temperature decline in the range from optimum to cold temperatures. Even at optimum growth temperatures, the most frost-tolerant wheat and barley cultivars can be distinguished from the remaining ones according to dehydrin relative accumulation. The highly tolerant wheat and barley cultivars started accumulating dehydrins at higher growth temperatures and reached higher dehydrin amounts than the less tolerant ones. Statistically significant correlations between lethal temperature for 50 % of the samples (LT50) and dehydrin relative accumulation have been found at all growth temperatures (5, 10, 15 and 20 °C) for WCS120 in wheats and at 5 and 10 °C for DHN5 in barleys. Analogous relationships between dehydrin relative accumulation at different growth temperatures and plant acquired frost tolerance have been proved for wheat WCS120 and barley DHN5.     

Cryoprotective activity of a cold-induced dehydrin purified from barley

Dehydrins are a family of proteins associated with cell dehydration. Drought, salinity, and high and low temperature may cause water loss from cells. Cold‐induced dehydrins have been reported in several species. P‐80 is a cold‐induced 80 kDa dehydrin in barley. This protein has the same apparent molecular mass as Dhn5, previously described for barley cv Himalaya. P‐80 was localized in the vicinity of vascular cylinders and in the epidermis of leaves and stems. Both tissues have been reported to be sites of early ice nucleation during controlled freezing. The present authors have proposed that this protein cryoprotects macromolecules and frost‐sensitive structures. In the present study, P‐80 and Dhn5 were purified with the purposes of demonstrating their cryoprotective activity in vitro, and comparing both proteins. More than 95% purity was obtained combining heat treatment, cationic exchange chromatography, preparative denaturant electrophoresis and band electroelution. Western blots showed that P‐80 was the major cold‐induced dehydrin in the cultivars examined in the present study. There was a major band of mRNA that showed expression kinetics consistent with P‐80 accumulation. The RT‐PCR picked one major band when using Dhn5‐specific primers in four cold‐acclimated barley cultivars. Both proteins have a similar amino acid composition, with differences in Arg, Asn + Asp, Glu + Gln, His, and Lys. The analysis of proteolytic fragments of Dhn5 and P‐80 by reverse phase chromatography showed a similar pattern. Furthermore, both proteins were able to cryoprotect lactate dehydrogenase (LDH, EC 1.1.1.27) against freeze/thaw inactivation, showing a similar shape dependence on concentration and almost the same protein dosage that renders 50% of cryoprotection (PD₅₀). Thus, P‐80 and Dhn‐5 share more similarities than expected for two different proteins. Their identities, though, remain to be firmly established. Further research is necessary to establish if the observed in vitro cryoprotective activity of these dehydrins is important for cryoprotection in vivo. The association of cryoprotective activity with K repeats of dehydrins is discussed.     

Sequence composition versus sequence order in the cryoprotective function of an intrinsically disordered stress‐response protein

Intrinsically disordered stress proteins have been shown to act as chaperones, protecting proteins from damage caused by stresses such as freezing and thawing. Dehydration proteins (dehydrins) are intrinsically disordered stress proteins that are found in almost all land plants. They consist of a variable number of the short, semi‐conserved, Y‐, S‐, and K‐segments, with longer stretches of poorly conserved sequences in between. Previous studies have provided conflicting views on the details of the dehydrin cryoprotective mechanism of enzymes. Experiments with polyethylene glycol (PEG) have shown that PEG cryoprotective efficiency is the same as dehydrins of the same hydrodynamic radius, suggesting that the protein's disordered and polar nature is important, rather than the specific order of the residues. To further elucidate the mechanism, we created scrambled variants of the wild grape dehydrins K₂ and YSK₂ and tested their ability to protect lactate dehydrogenase and yeast frataxin homolog‐1 from freeze/thaw damage. The results show that for preventing aggregation, it is the sequence composition and the size of the dehydrin that is the most important factor in protection, while for freeze/thaw damage causing loss of secondary structure, it is the sequence composition that is most significant.     

A barley mutant with improved salt tolerance through ion homeostasis and ROS scavenging under salt stress

To investigate key regulatory components and genes with great impact on salt tolerance, near isogenic or mutant lines with distinct salinity tolerance are suitable genetic materials to simplify and dissect the complex genes networks. In this study, we evaluated responses of a barley mutant genotype (73-M4-30), in comparison with its wild-type background (Zarjou) under salt stress. Although the root growth of both genotypes was significantly decreased by exposure to sodium chloride (NaCl), the effect was greater in the wild type. The chlorophyll content decreased under salt stress for the wild type, but no change occurred in the mutant. The mutant maintained the steady-state level of [K⁺] and significantly lower [Na⁺] concentrations in roots and higher [K⁺]/[Na⁺] ratio in shoots under salt conditions. The catalase (CAT), peroxidase (POD) activity, and proline content were higher in the mutant than those in the wild type under controlled conditions. The soluble proline was higher after 24 h of salt stress in roots of the mutant but was higher after 96 h of salt stress in the wild type. The CAT and POD activity of the mutant increased under salt stress which was as a coincidence to lower levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents. The ratio of dry-to-fresh weight of the roots increased for the mutant under salt stress which was as a result of the higher phenylalanine ammonia-lyase (PAL) gene expression and peroxidase activity and involved in cell wall lignification. Consequently, it seems that ion homeostasis and increased peroxidase activity have led to salt tolerance in the mutant’s genotype.     

The relationship between proline content, the expression level of P5CS (Δ1-pyrroline-5-carboxylate synthetase), and drought tolerance in Tibetan hulless barley (Hordeum vulgare var. nudum)

Many plants accumulate proline (Pro) when suffered from drought; thus, the relationship between Pro accumulation and plant drought tolerance becomes an increasing concern. Pro is synthesized from either glutamine or ornithine, and the former pathway dominates under osmotic stress conditions. In this study, the dynamic accumulation of free Pro under drought stress in 10 genotypes of Tibetan hulless barley (Hordeum vulgare var. nudum) with water lose rate (WLR) of 0.3304 to 0.5839 g/(h g dry wt) was investigated. However, no correlation between Pro accumulation and drought tolerance was found. Furthermore, the barley stripe mosaic virus establisheding virus-induced gene silencing was employed to suppress the expression of the encoding gene Δ¹-Pyrroline-5-Carboxylate Synthetase (P5CS), which catalyzes the ratelimiting step of Glu pathway in Pro biosynthesis. By the quantitative real-time polymerase chain reaction, the decrease of the P5CS expression was found, and a consequent Pro degradation was also detected in P5CS-silenced plants. However, neither increased WLR of detached leaves nor decreased survival rate under drought stress was found compared with control plants. These results suggested that the repressed expression of P5CS and decreased content of free Pro may not interfere with the drought tolerance of Tibetan hulless barley.     

青稞HvtAGO1基因的克隆及其在条纹病胁迫下的表达

AGO蛋白是RNA诱导沉默复合体的核心分子,在植物的生长、发育及胁迫响应中起重要作用.为探索青稞AGO基因在青稞抗条纹病病原菌过程中的作用机制,该研究以抗条纹病青稞品种‘昆仑14号’和感病品种‘Z1141’为材料,利用条纹病原菌侵染两品种,从感病前后的转录组测序结果中获得一个差异表达基因,克隆验证了该基因为HvtAGO...     

大麦Dhn6基因的克隆、蛋白质结构预测与干旱胁迫表达模式

脱水素(Dehydrins,DHNs)是高等植物胚胎发育晚期产生的一类特异多肽,其表达累积程度与植物的发育阶段、低温、ABA和脱水信号调节等因素密切相关。为了解脱水素的结构与干旱胁迫表达累积反应,文章从六棱大麦分离到序列全长为1 767 bp的Dhn6基因,序列分析结果表明,该基因含一个92 bp内含子,90~1 759 bp为一个开放阅读框,与裸大麦Dhn6基因(GenBank登录号:AF043091)的同源性最高,达93.18%,编码523个氨基酸残基的多肽,预测蛋白质的分子量为49.68 kDa,理论等电点为8.04。结构分析发现,蛋白质具有3个螺旋区,无规则卷曲构成二级结构的主要组分,亲水氨基酸比例超过83%;三维结构预测发现,多肽链自身反向平行排列成松散的亲水索链,K-片段参与兼性?-螺旋结构域的形成,意味着该脱水素具有束缚自由水、稳定细胞膜相结构的功能。实时定量RT-PCR检测结果表明,Dhn6基因的相对表达水平在干旱处理8 h快速累积,推测DHN6在大麦对干旱胁迫的早期响应中发挥重要功能。     

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.