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.     

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.     

Cold-Specific Induction of a Dehydrin Gene Family Member in Barley

An interval on barley (Hordeum vulgare L.) chromosome 7 accounting for significant quantitative trait locus effects for winter hardiness were detected in a winter (Dicktoo) x spring (Morex) barley population (P.M. Hayes, T. Blake, T.H.H. Chen, S. Tragoonrung, F. Chen, A. Pan, and B. Liu [1993] Genome 36: 66-71). Two members of the barley dehydrin gene family, Dhn1 and Dhn2, were located within the region defining the winter hardiness quantitative trait locus effect (A. Pan, P.M. Hayes, F. Chen, T. Blake, T.H.H. Chen, T.T.S. Wright, I. Karsai, Z. Bedo [1994] Theor Appl Genet 89: 900-910). To investigate the possible role of Dhn1 and Dhn2 in winter hardiness, we examined the expression pattern of six barley dehydrin gene family members in shoot tissue in response to cold temperature. Incubation of 3-week-old barley plants at 2[deg]C resulted in a rapid induction of a single 86-kD polypeptide that was recognized by an antiserum against a peptide conserved in the dehydrin gene family. Northern blot analysis confirmed the induction of an mRNA corresponding to Dhn5. The expression patterns of cold-induced dehydrins in shoot tissue for Dicktoo and Morex were identical under the conditions studied, in spite of the known phenotypic differences in their winter hardiness. These results, together with the allelic structure of selected high- and low-survival lines, suggest that the Dicktoo alleles at the Dhn1 and Dhn2 may not be the primary determinants of winter hardiness in barley.     

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.     

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     

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.     

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     

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.     

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".     

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.     

Differential expression of dehydrin genes in wild barley, Hordeum spontaneum, associated with resistance to water deficit

Dehydrin gene (Dhn) expression is associated with plant response to dehydration. The aim of the present study was to investigate the association of differential expression of Dhn genes (Dhn 1, 3, 5, 6, and 9) with drought tolerance found in wild barley (Hordeum spontaneum). Tolerant and sensitive genotypes were identified from Israeli (Tabigha microsite) and Jordanian (Jarash and Waddi Hassa) populations (based on scoring of water loss rate of 390 genotypes). The five Dhn genes were up‐regulated by dehydration in resistant and sensitive wild barley genotypes. Notably, differences between resistant and sensitive genotypes were detected, mainly in the expression of Dhn1 and Dhn6 genes, depending on the duration of dehydration stress. Dhn1 tended to react earlier (after 3 h) and higher (12 h and 24 h) in resistant compared to sensitive genotypes. The level of expression of Dhn6 was significantly higher in the resistant genotypes at the earlier stages after stress. However, after 12 and 24 h Dhn6 expression was relatively higher in sensitive genotypes. The present results may indicate that these genes have some functional role in the dehydration tolerance in wild barley. The authors suggest that the observed differences of Dhn expression in wild barley, originating from different micro‐ and macro ecogeographic locations, may be the result of adaptive edaphic and climatic selective pressures.     

Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana

Levels of endogenous glycine betaine in the leaves were measured in response to cold acclimation, water stress and exogenous ABA application in Arabidopsis thaliana. The endogenous glycine betaine level in the leaves increased sharply during cold acclimation treatment as plants gained freezing tolerance. When glycine betaine (10 mM) was applied exogenously to the plants as a foliar spray, the freezing tolerance increased from -3.1 to -4.5 degrees C. In addition, when ABA (1 mM) was applied exogenously, the endogenous glycine betaine level and the freezing tolerance in the leaves increased. However, the increase in the leaf glycine betaine level induced by ABA was only about half of that by the cold acclimation treatment. Furthermore, when plants were subjected to water stress (leaf water potential of approximately -1.6 MPa), the endogenous leaf glycine betaine level increased by about 18-fold over that in the control plants. Water stress lead to significant increase in the freezing tolerance, which was slightly less than that induced by the cold acclimation treatment. The results suggest that glycine betaine is involved in the induction of freezing tolerance in response to cold acclimation, ABA, and water stress in Arabidopsis plants.     

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。     

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.     

Genetic variation in pea (Pisum) dehydrins: sequence elements responsible for length differences between dehydrin alleles and between dehydrin loci in Pisum sativum L.

 The electrophoretic patterns of dehydrins extracted from mature seeds of a range of pea (Pisum) species revealed extensive variation in dehydrin polypeptide mobility. Variation was also observed among lines of P. sativum. Crosses between lines with different dehydrin electrophoretic patterns produced F₁ seeds with additive patterns, and segregation in the F₂ generation was consistent with a 1 : 2 : 1 ratio, indicating allelic variation at each of two dehydrin loci (Dhn2, Dhn3). Genetic linkage was observed between Dhn2 and Dhn3, and the segregation ratios indicated preferential transmission of one allele at the Dhn3 locus. Dehydrin cDNA clones were characterised that encoded the allelic variants at Dhn2 and Dhn3. Their deduced amino-acid sequences were very similar to each other as well as to the product of the Dhn1 locus reported previously. Comparisons were made between the sequences of allelic variants at a single locus, and between the products of different loci. Differences in the electrophoretic mobilities between allelic variants at Dhn2 and Dhn3 were associated with differences in polypeptide length resulting principally from tandem duplications of 21 (Dhn2) or 24 (Dhn3) amino-acid residues. These duplications accounted for much of the difference in length between dehydrins encoded by the different loci. The conserved core of one of the duplicated regions varied in copy number, and small insertions/deletions of amino acids near this core also contributed to length variation both between allelic forms and between loci. Dehydrins possess characteristic highly conserved amino-acid sequence motifs, yet vary considerably in length. Mechanisms involving sequence duplication appear to be responsible for generating the length differences observed between allelic variants as well as between the products of different loci. Received: 12 June 1997 / Accepted: 29 October 1997     

The Use of Microwaves to Monitor the Freezing and Thawing of Water in Plants

Harbinson, J. and Woodward, F. I. 1987. The use of microwavesto monitor the freezing and thawing of water in plants.—J.exp. Bot. 38: 1325–1335. The use of radiation with a frequency of 10·687 GHz tomeasure the freezing and thawing of water in plants is discussed.Results showing the freezing and thawing of plant material arepresented. In its simplest form the technique offers a methodfor investigating the freezing and thawing of water in plants. Key words: Freezing, microwaves