A newly identified barley gene, Dhn12, encoding a YSK2 DHN, is located on chromosome 6H and has embryo-specific expression

Dehydrins are water-soluble lipid-associating proteins that accumulate during low-temperature or water-deficit conditions, and are thought to play a role in freezing- and drought-tolerance in plants. Dhn genes exist as multi-gene families in plants. Previously, we screened lambda genomic libraries of two barley cultivars in an effort to isolate all of the barley Dhn genes. We identified 11 unique Dhn genes and estimated a total of 13 Dhn genes in the barley genome. To extend the collection, we used an alternative source of clones, a 1.5×Morex barley BAC library. In this library, we found nine Dhn genes that we described previously and one new Dhn gene, Dhn12. The Dhn12 gene encodes an acidic YSK₂ dehydrin. The Dhn12 gene is located on chromosome 6H, and shows a different expression pattern from all other Dhn genes identified previously. RT-PCR results show that Dhn12 expression is embryo-specific. Dhn12 is not expressed in seedling shoots under any of the conditions tested, including non-stressed as well as dehydrated, or cold-, ABA- or NaCl-treated seedlings. Received: 6 June 1999 / Accepted: 3 November 1999     

Map locations of barley Dhn genes determined by gene-specific PCR

We previously identified 11 unique barley Dhn genes and found, using wheat-barley addition lines, that these genes are dispersed on four chromosomes 3H, 4H, 5H, 6H. In the present work, more precise positions of barley Dhn genes were determined using gene-specific PCR and 100 doubled haploid lines developed from a cross of Dicktoo and Morex barley. Dhn10 is located on 3H between saflp106 and ABG4. Dhn6 is at the previously determined position on 4H between SOLPRO and BCD265a. Dhn1 and Dhn2 are at the previously determined position on 5H between mR and saflp172. The Dhn locus previously called Dhn4a on barley 5H or Dhn2.2 on T. monococcum 5A is in fact Dhn9 and maps to a revised position between BCD265b and saflp218. Dhn3, Dhn4, Dhn7 and Dhn5 each map to the same position on chromosome 6H, suggesting that the previously reported separation of Dhn3, Dhn4 and Dhn5 may reflect limitations in the accuracy of Southern blot data. In addition to clarifying the map positions of these important stress-related genes, these results illustrate the advantage of gene-specific probes for the mapping of individual genes in a multi-gene family. Received: 11 August 1999 / Accepted: 16 December 1999     

The barley (Hordeum vulgare L.) dehydrin multigene family: sequences, allele types, chromosome assignments, and expression characteristics of 11 Dhn genes of cv Dicktoo

 Dehydrins (LEA D11 proteins) have been identified in both higher and lower plants, and are associated with tolerance to, or response to the onset of, low temperature or dehydration. Several studies have suggested that specific alleles of Dhn genes may contribute to a number of phenotypic traits, including the emergence of seedlings in cool or saline soils and the frost tolerance of more-mature plants. However, an incomplete collection of the Dhn multigene family in any system and nucleic acid cross-hybridization between Dhn gene-family members have limited the precision of these studies. We attempted to overcome these impediments by determining the nucleotide sequences of the entire Dhn multigene family in barley and by developing gene-specific probes. We identified 11 unique Dicktoo Dhn genes. Seven appear to be alleles of Dhn genes identified previously in other barley cultivars. Another, Dhn9, appears to be orthologous to a Triticum durum Dhn gene. A statistical analysis of the total collection of genomic clones brings the estimated size of the barley Dhn gene family to 13. Allelic differences in the protein-coding regions appear to result principally from duplications of entire Φ-segments or single amino-acid substitutions, suggesting that polypeptide structural constraints have been a strong force in the evolution of Dhn alleles. Chromosome mapping by PCR with wheat-barley addition lines established the presence of Dhn genes in four barley chromosomes (3H, 4H, 5H, 6H). RT-PCR demonstrated that the Dhn genes are differentially regulated under dehydration, low temperature and ABA treatment, consistent with putative regulatory elements located upstream of the respective Dhn coding regions. This whole-genome, gene-specific study unifies what previously seemed to be disparate-mapping, expression, and genetic-variation data for Dhn genes in the Triticeae and other plant systems. Received: 23 September 1998 / Accepted: 13 October 1998     

Rapid upregulation ofDehyrin3 andDehydrin4 in response to dehydration is a characteristic of drought-tolerant genotypes in barley

The identification of molecular markers and marker-aided selection are essential to the efficient breeding of drought-tolerant plants. However, because that characteristic is controlled by many quantitative trait loci, such markers that can screen and trace desirable barley genotypes in a segregating population or germplasm have not yet been determined. Relative water content has been used to estimate drought tolerance in plants because it is highly correlated with the drought index of yield. To develop reliable gene-specific markers for identifying tolerant versus susceptible genotypes, we performed suppression subtractive hybridization to identify candidate genes. We used two domestic barley cultivars, one having the highest RWC (drought-tolerant ‘Chalbori’) and the other having the lowest (drought-susceptible ‘Daebaekbori’). In response to dehydration at the early seedling stage, rapid upregulation ofDehydrin3 (Dhn3) andDhn4 occurred in the drought-tolerant genotypes, but not in the susceptible ones. Similar results were obtained with mature plants growing under frequent drought stress in the greenhouse. In addition,Dhn3 andDhn4 conferred higher drought tolerance when they were over-expressed in transgenicArabidopsis. Thus, in addition to using assessments of RWC, we propose thatDhn3 andDhn4 expressions can serve as drought-induced gene-specific markers to determine drought-tolerant barley genotypes at the seedling stage.     

Adaptive microclimatic evolution of the dehydrin 6 gene in wild barley at ��Evolution Canyon��, Israel

Dehydrins are one of the major stress-induced gene families, and the expression of dehydrin 6 (Dhn6) is strictly related to drought in barley. In order to investigate how the evolution of the Dhn6 gene is associated with adaptation to environmental changes, we examined 48 genotypes of wild barley, Hordeum spontaneum, from "Evolution Canyon" at Mount Carmel, Israel. The Dhn6 sequences of the 48 genotypes were identified, and a recent insertion of 342?bp at 5'UTR was found in the sequences of 11 genotypes. Both nucleotide and haplotype diversity of single nucleotide polymorphism in Dhn6 coding regions were higher on the AS ("African" slope or dry slope) than on the ES ("European" slope or humid slope), and the applied Tajima D and Fu-Li test rejected neutrality of SNP diversity. Expression analysis indicated that the 342?bp insertion at 5'UTR was associated with the earlier up-regulation of Dhn6 after dehydration. The genetic divergence of amino acids sequences indicated significant positive selection of Dhn6 among the wild barley populations. The diversity of Dhn6 in microclimatic divergence slopes suggested that Dhn6 has been subjected to natural selection and adaptively associated with drought resistance of wild barley at "Evolution Canyon".     

Adaptive microclimatic structural and expressional dehydrin 1 evolution in wild barley, Hordeum spontaneum, at 'Evolution Canyon', Mount Carmel, Israel

'Evolution Canyon' (ECI) at Lower Nahal Oren, Mount Carmel, Israel, is an optimal natural microscale model for unravelling evolution in action highlighting the twin evolutionary processes of adaptation and speciation. A major model organism in ECI is wild barley, Hordeum spontaneum , the progenitor of cultivated barley, which displays dramatic interslope adaptive and speciational divergence on the 'African' dry slope (AS) and the 'European' humid slope (ES), separated on average by 200 m. Here we examined interslope single nucleotide polymorphism (SNP) sequences and the expression diversity of the drought resistant dehydrin 1 gene ( Dhn1 ) between the opposite slopes. We analysed 47 plants (genotypes), 4–10 individuals in each of seven stations (populations) in an area of 7000 m², for Dhn1 sequence diversity located in the 5' upstream flanking region of the gene. We found significant levels of Dhn1 genic diversity represented by 29 haplotypes, derived from 45 SNPs in a total of 708 bp sites. Most of the haplotypes, 25 out of 29 (= 86.2%), were represented by one genotype; hence, unique to one population. Only a single haplotype was common to both slopes. Genetic divergence of sequence and haplotype diversity was generally and significantly different among the populations and slopes. Nucleotide diversity was higher on the AS, whereas haplotype diversity was higher on the ES. Interslope divergence was significantly higher than intraslope divergence. The applied Tajima D rejected neutrality of the SNP diversity. The Dhn1 expression under dehydration indicated interslope divergent expression between AS and ES genotypes, reinforcing Dhn1 associated with drought resistance of wild barley at 'Evolution Canyon'. These results are inexplicable by mutation, gene flow, or chance effects, and support adaptive natural microclimatic selection as the major evolutionary divergent driving force.     

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.     

Expression of the barley dehydrin multigene family and the development of freezing tolerance

Dehydrins (DHNs; LEA D11) are one of the typical families of plant proteins that accumulate in response to dehydration, low temperature, osmotic stress or treatment with abscisic acid (ABA), or during seed maturation. We previously found that three genes encoding low-molecular-weight DHNs (Dhn1, Dhn2 and Dhn9) map within a 15-cM region of barley chromosome 5H that overlaps a QTL for winterhardiness, while other Dhn genes encoding low- and high-molecular-weight DHNs are located on chromosomes 3H, 4H and 6H. Here we examine the expression of specific Dhn genes under conditions associated with expression of the winterhardiness phenotype. Plants grown at 4 degrees C or in the field in Riverside, California developed similar, modest levels of freezing tolerance, coinciding with little low-MW Dhn gene activity. Dicktoo (the more tolerant cultivar) and Morex (the less tolerant) grown in Saskatoon, Canada expressed higher levels of expression of genes for low-MW DHNs than did the same cultivars in Riverside, with expression being higher in Dicktoo than Morex. Dehydration or freeze-thaw also evoked expression of genes for low MW DHNs, suggesting that the dehydration component of freeze-thaw in the field induces low expression of genes encoding low-MW DHNs. These observations are consistent with the hypothesis that the major chilling-induced DHNs help to prime plant cells for acclimation to more intense cold, which then involves adaptation to dehydration during freeze-thaw cycling. A role for chromosome 5H-encoded DHNs in acclimation to more intense cold seems possible, even though it is not the basis of the major heritable variation in winterhardiness within the Dicktoo x Morex population.     

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.     

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.     

Genetic basis of barley caryopsis dormancy and seedling desiccation tolerance at the germination stage

The genomic regions controlling caryopsis dormancy and seedling desiccation tolerance were identified using 152 F₄ lines derived from a cross between Mona, a Swedish cultivar, and an Israeli xeric wild barley Hordeum spontaneum genotype collected at Wadi Qilt, Israel. Dormancy, the inability of a viable seed to germinate, and desiccation tolerance, the ability of the desiccated seedlings to revive after rehydration, were characterized by fitting the germination and revival data with growth curves, using three parameters: minimum, maximum, and slope of germination or revival rate derived by the least square method. The genetic map was constructed with 85 genetic markers (SSRs, AFLPs, STSs, and Dhn genes) using the multipoint-mapping algorithm. Quantitative trait loci (QTLs) mapping was conducted with the multiqtl package. Ten genomic regions were detected that affected the target traits, seven of which affected both dormancy and desiccation tolerance traits. Both the wild barley genotype and the Swedish cultivar contributed the favorite alleles for caryopsis dormancy, whereas seedling desiccation tolerance was attributed to alleles descending from the cultivar. The results indicate that some barley dormancy genes are lost during domestication and that dormancy QTLs are associated with abiotic stress tolerance.     

Analysis of the promoter activity of late embryogenesis abundant protein genes in barley seedlings under conditions of water deficit

Promoters of the late embryogenesis abundant protein genes, HVA1s, Dhn8s and Dhn4s from barley and wsi18j and rab16Bj from rice, were analysed in barley seedlings to assess their strength and timing of induction under water deficit conditions using a transient expression system. Of the drought-inducible promoters, Dhn4s exhibited the highest activity, followed by HVA1s, wsi18j and rab16Bj. Dehydration-induced #-glucuronidase expression levels driven by the Dhn4s, HVA1s and wsi18j promoters were higher than or comparable to that induced by a strong constitutive rice Act1 promoter. The expression patterns of wsi18j and Dhn8s differed from those of their homologous genes (wsi18 and Dhn8): the wsi18j promoter was strongly induced by abscisic acid, while the Dhn8s promoter was highly active in hydrated seedlings. HVA1s- and wsi18j-driven green fluorescent protein expression was induced within 1 h of drying under water deficit stress, while Dhn4s promoter activity was not detectable until 3 h, thus showing a difference in the timing of induction.     

The draft genome of a wild barley genotype reveals its enrichment in genes related to biotic and abiotic stresses compared to cultivated barley

Wild barley (Hordeum spontaneum) is the progenitor of cultivated barley (Hordeum vulgare) and provides a rich source of genetic variations for barley improvement. Currently, the genome sequences of wild barley and its differences with cultivated barley remain unclear. In this study, we report a high‐quality draft assembly of wild barley accession (AWCS276; henceforth named as WB1), which consists of 4.28 Gb genome and 36 395 high‐confidence protein‐coding genes. BUSCO analysis revealed that the assembly included full lengths of 95.3% of the 956 single‐copy plant genes, illustrating that the gene‐containing regions have been well assembled. By comparing with the genome of the cultivated genotype Morex, it is inferred that the WB1 genome contains more genes involved in resistance and tolerance to biotic and abiotic stresses. The presence of the numerous WB1‐specific genes indicates that, in addition to enhance allele diversity for genes already existing in the cultigen, exploiting the wild barley taxon in breeding should also allow the incorporation of novel genes. Furthermore, high levels of genetic variation in the pericentromeric regions were detected in chromosomes 3H and 5H between the wild and cultivated genotypes, which may be the results of domestication. This H. spontaneum draft genome assembly will help to accelerate wild barley research and be an invaluable resource for barley improvement and comparative genomics research.     

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.     

Variation of total soluble seminal root proteins of tetraploid wild and cultivated wheat induced at cold acclimation and freezing

The relationship between total soluble seminal root proteins induced at cold acclimation and freezing tolerance in tetraploid wild wheat Aegilops L. (Ae. biuncialis, Ae. cylindrica) and cultivated wheat Triticum turgitum L. (Firat-93, Harran-95) was investigated. Cold acclimation was performed at 0 °C for 7 days. Freezing tolerance was determined with survived roots after freezing treatments at −5 and/or −7 °C for 3, 6, 12 and 24 h. At −5°C, all tetraploid genotypes showed over 60% tolerance for 3 h. This effect was also present in wild wheat for 6 h, but was decreased in cultivated wheat to 30–35% tolerance for 6 h. Only Ae. biuncialis was able to show 52% tolerance just for 3 h freezing period at −7 °C. However, all the genotypes were not survived at −7 °C, for 6, 12 and 24 h. Cold acclimation induced greater amounts of new soluble seminal root proteins in tolerant Ae. biuncialis (29–104 kDa, pI 5.4–7.4) than in sensitive Harran-95 (29–66 kDa, pI 6.1–8.3). Synthesis and accumulation of these proteins may be related to degree of freezing tolerance of these genotypes.