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Absorption and circular dichroism spectra of chloroplast membrane fragments from spinach, barley and a barley mutant at room temperature and liquid nitrogen temperature.

The absorption and CD spectra of chloroplast fragments from spinach, barley and a barley mutant (chlorophyll b-minus) were studied at temperatures of 23 degrees C and --196 degrees C. The CD spectrum of wild type barley and spinach at --196 degrees C showed troughs at 640, 653, 676 and 695 nm and a maximum at 667 nm. The CD spectrum of the barley mutant at --196 degrees C consisted of a large trough at 684 nm, a small trough at 695 nm and a positive peak at 670 nm. A new feature observed at --196 degrees C but not at 23 degrees C is the trough at 640 nm. This 640 nm CD signal is missing in the CD spectrum of the barley mutant. It is attributable to the light-harvesting chlorophyll a/b protein which appears to be missing in the mutant. Another new feature, the trough at 695 nm, was observed in the CD spectra of spinach, barley and the barley mutant at --196 degrees C. The 695 nm trough appears to be sensitive to detergents and it may be due to a labile chlorophyll a.protein complex. Possible interpretations of the data are discussed.

Chloroplasts↗

Rice-barley synteny and its application to saturation mapping of the barley Rpg1 region.

In order to facilitate the map-based cloning of the barley stem rust resistance gene Rpg1, we have demonstrated a high degree of synteny at a micro level between the telomeric region of barley chromosome 1P and rice chromosome 6. We have also developed and applied a simple and efficient method for selecting useful probes from large insert genomic YAC and cosmid clones. The gene order within the most terminal 6.5 cM of barley chromosome 1P was compared with the most terminal 2.7 cM of rice chromosome 6. Nine rice probes, previously mapped in rice or isolated from YAC or cosmid clones from this region, were mapped in barley. All, except one, were in synteny with the rice gene order. The exception, probe Y617R, was duplicated in barley. One copy was located on a different chromosome and the other in a non-syntenic position on barley chromosome 1P. The barley probes from this region could not be mapped to rice, but two of them were inferred to be in a syntenic location based on their position on a rice YAC. This work demonstrates the utility of applying the results of genetic and physical mapping of the small genome cereal rice to map-based cloning of interesting genes from large genome relatives.

Chromosome Mapping↗

Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.

Chimera↗

Hydroxycinnamic acids and ferulic acid dehydrodimers in barley and processed barley.

Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley varieties after alkaline hydrolysis. Ferulic acid (FA) was the most abundant hydroxycinnamate with concentrations ranging from 359 to 624 microg/g dry weight. p-Coumaric acid (PCA) levels ranged from 79 to 260 microg/g dry weight, and caffeic acid was present at concentrations of <19 microg/g dry weight. Among the ferulic acid dehydrodimers that were identified, 8-O-4'-diFA was the most abundant (73-118 microg/g dry weight), followed by 5,5'-diFA (26-47 microg/g dry weight), the 8,5'-diFA benzofuran form (22-45 microg/g dry weight), and the 8,5'-diFA open form (10-23 microg/g dry weight). Significant variations (p < 0.05) among the different barley varieties were observed for all the compounds that were quantified. Barley grains were mechanically fractionated into three fractions: F1, fraction consisting mainly of the husk and outer layers; F2, intermediate fraction; and F3, fraction consisting mainly of the endosperm. Fraction F1 contained the highest concentration for ferulic acid (from 77.7 to 82.3% of the total amount in barley grain), p-coumaric acid (from 78.0 to 86.3%), and ferulic acid dehydrodimers (from 79.2 to 86.8%). Lower contents were found in fraction F2, whereas fraction F3 exhibited the lowest percentages (from 1.2 to 1.9% for ferulic acid, from 0.9 to 1.7% for p-coumaric acid, and <0.02% for ferulic acid dehydrodimers). The solid barley residue from the brewing process (brewer's spent grain) was approximately 5-fold richer in ferulic acid, p-coumaric acid, and ferulic acid dehydrodimers than barley grains.

Chemical Fractionation↗

The barley apoptosis suppressor homologue BAX inhibitor-1 compromises nonhost penetration resistance of barley to the inappropriate pathogen Blumeria graminis f. sp. tritici.

BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, overexpression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.

Apoptosis↗

Identification of wheat-barley translocations by sequential GISH and two-colour FISH in combination with the use of genetically mapped barley SSR markers.

Five wheat-barley translocations in a wheat background were characterized through the combination of cytogenetic and molecular genetic approaches. The wheat chromosome segments involved in the translocations were identified using sequential GISH and two-colour FISH with the probes pSc119.2 and pAs1. The barley chromatin in these lines was identified using SSR markers. A total of 45 markers distributed over the total barley genome were selected from a recently published linkage map of barley and tested on the translocation lines. The following translocations were identified: 2DS.2DL-1HS, 3HS.3BL, 6BS.6BL-4HL, 4D-5HS, and 7DL.7DS-5HS. Wheat-barley disomic and ditelosomic addition lines for the chromosomes 3HS, 4H, 4HL, 5H, 5HL, and 6HS were used to determine the correct location of 21 markers and the position of the centromere. An intragenomic translocation breakpoint was detected on the short arm of the barley chromosome 5H with the help of SSR marker analysis. Physical mapping of the SSR markers on chromosomes 1H and 5H was carried out using the intragenomic and the interspecific translocation breakpoints, as well as the centromere, as physical landmarks.

Genetic Markers↗

Identification of individual barley chromosomes based on repetitive sequences: conservative distribution of Afa-family repetitive sequences on the chromosomes of barley and wheat.

The Afa-family repetitive sequences were isolated from barley (Hordeum vulgare, 2n = 14) and cloned as pHvA14. This sequence distinguished each barely chromosome by in situ hybridization. Double color fluorescence in situ hybridization using pHvA14 and 5S rDNA or HvRT-family sequence (subtelomeric sequence of barley) allocated individual barley chromosomes showing a specific pattern of pHvA14 to chromosome 1H to 7H. As the case of the D genome chromosomes of Aegilops squarrosa and common wheat (Triticum aestivum) hybridized by its Afa-family sequences, the signals of pHvA14 in barley chromosomes tended to appear in the distal regions that do not carry many chromosome band markers. In the telomeric regions these signals always placed in more proximal portions than those of HvRT-family. Based on the distribution patterns of Afa-family sequences in the chromosomes of barley and D genome chromosomes of wheat, we discuss a possible mechanism of amplification of the repetitive sequences during the evolution of Triticeae. In addition, we show here that HvRT-family also could be used to distinguish individual barley chromosomes from the patterns of in situ hybridization.

Biological Evolution↗

Barley chromosome addition lines of wheat for screening of AFLP markers on barley chromosomes.

We conducted AFLP (Amplified Fragment Length Polymorphism) analysis with the six wheat-barley chromosome addition lines of common wheat cultivar Chinese Spring. We analyzed the AFLP fingerprints generated by 36 combinations of selective-amplification primers to find 103 markers specific to the barley chromosomes (2.9 markers per combination on average). The numbers of AFLP markers mapped to the barley chromosomes varied (one to 16) depending of the primer combinations. Each barley chromosome had 10 to 27 AFLP markers (17.2 markers on average). We identified the chromosome arms in which these markers are located using the barley telocentric addition lines (one to 20 markers per chromosome arm). The AFLP markers were not distributed evenly among chromosomes and chromosome arms. We could not determine the chromosome-arm locations for some of the barley-specific markers, either because such markers were found in both the short- and long-arm telocentric lines, or in neither line.

Chromosomes↗

Isolation of gibberellin metabolic pathway genes from barley and comparative mapping in barley, wheat and rice.

Gene sequences encoding gibberellin (GA) biosynthetic and catabolic enzymes were isolated from "Himalaya" barley. These genes account for most of the enzymes required for the core pathway of GA biosynthesis as well as for the first major catabolic enzyme. By means of DNA gel blot analysis, we mapped coding sequences to chromosome arms in barley and wheat using barley-wheat chromosome addition lines, nulli-tetrasomic substitution and ditelosomic lines of wheat. These same sequences were used to identify closely related sequences from rice, which were mapped in silico, thereby allowing their syntenic relationship with map locations in barley and wheat to be investigated. Determination of the chromosome arm locations for GA metabolic genes provides a framework for future studies investigating possible identity between GA metabolic genes and dwarfing genes in barley and wheat.

Amino Acid Sequence↗

High-resolution mapping of the Rym4/Rym5 locus conferring resistance to the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2) in barley (Hordeum vulgare ssp. vulgare L.).

Soil-borne barley yellow mosaic virus disease--caused by a complex of at least three viruses, i.e. Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus (BaYMV) and BaYMV-2--is one of the most important diseases of winter barley in Europe. The two genes rym4, effective against BaMMV and BaYMV, and rym5, additionally effective against BaYMV-2, comprise a complex locus on chromosome 3HL, which is of special importance to European barley breeding. To provide the genetic basis for positional cloning of the Rym4/Rym5 locus, two high-resolution maps were constructed based on co-dominant flanking markers (MWG838/Y57c10 - MWG010/Bmac29). Mapping at a resolution of about 0.05% rec., rym4 has been located 1.07% recombination distal of marker MWG838 and 1.21% recombination proximal to marker MWG010. Based on a population size of 3,884 F2 plants (0.013% recombination) the interval harbouring rym5 was delimited to 1.49+/-0.14% recombination. By testing segmental recombinant inbred lines (RILs) for reaction to the different viruses at a resolution of 0.05% rec. (rym4) and 0.019% rec. (rym5), no segregation concerning the reaction to the different viruses could be observed. AFLP-based marker saturation for rym4, using 932 PstI+2/MseI+3 primer combinations only resulted in three markers with the closest one linked at 0.9% recombination to the gene. Two of these markers detected epialleles arising from the differential cytosine methylation of PstI sites. Regarding rym5, profiling of 1,200 RAPD primers (about 18,000 loci) and 2,048 EcoRI+3/MseI+3 AFLP primer combinations (about 205,000 loci) resulted in one RAPD marker and seven AFLP markers tightly linked to the resistance gene. Flanking markers with the closest linkage to rym5 (0.05% and 0.88% recombination) were converted into STS markers. These markers provide a starting point for chromosomal walking and may be exploited in marker-assisted selection for virus resistance based on rym5.

Base Sequence↗

Accumulation of genes for susceptibility to rust fungi for which barley is nearly a nonhost results in two barley lines with extreme multiple susceptibility.

Nonhost resistance is the most common type of resistance in plants. Understanding the factors that make plants susceptible or resistant may help to achieve durably effective resistance in crop plants. Screening of 109 barley (Hordeum vulgare L.) accessions in the seedling stage indicated that barley is a complete nonhost to most of the heterologous rust fungi studied, while it showed an intermediate status with respect to Puccinia triticina, P. hordei-murini, P. hordei-secalini, P. graminis f. sp. lolii and P. coronata ff. spp. avenae and holci. Accessions that were susceptible to a heterologous rust in the seedling stage were much more or completely resistant at adult plant stage. Differential interaction between barley accessions and heterologous rust fungi was found, suggesting the existence of rust-species-specific resistance. In particular, many landrace accessions from Ethiopia and Asia, and naked-seeded accessions, tended to be susceptible to several heterologous rusts, suggesting that some resistance genes in barley are effective against more than one heterologous rust fungal species. Some barley accessions had race-specific resistance against P. hordei-murini. We accumulated genes for susceptibility to P. triticina and P. hordei-murini in two genotypes called SusPtrit and SusPmur, respectively. In the seedling stage, these accessions were as susceptible as the host species to the target rusts. They also showed unusual susceptibility to other heterologous rusts. These two lines are a valuable asset to further experimental work on the genetics of resistance to heterologous rust fungi.

Basidiomycota↗

Haplotype structure at seven barley genes: relevance to gene pool bottlenecks, phylogeny of ear type and site of barley domestication.

Archaeological remains indicate that the origin of western agriculture occurred in a brief period about 10,500 years ago in a region of the Middle East known as the Fertile Crescent, where the wild progenitors of several key agricultural cereal species are endemic. Domestication entailed the appearance of agronomic traits such as seed size and threshability. For a representative sample of 20 domesticated barley (Hordeum vulgare) lines, including 13 two-rowed and 7 six-rowed varieties, we determined the haplotypes at seven loci-Adh2, Adh3, Amy1, Dhn9, GAPDH, PEPC and WAXY encompassing 5,616 bases per line-and compared them to the haplotypes at the same loci for 25 wild forms (Hordeum spontaneum) collected within and outside the Fertile Crescent. In comparisons of wild versus domesticated barley, the number of haplotypes (70 vs. 17), average nucleotide diversity, pi, (0.0077 vs. 0.0028), and Watterson's theta at silent sites (0.0104 vs. 0.0028) was reduced in domesticated lines. Two loci, Amy1 and PEPC, were monomorphic in domesticated lines; Amy1 and GAPDH produced significant values of Tajima's D. At GAPDH, pi was slightly higher in domesticated than wild forms, due to divergent high-frequency haplotypes; for the remaining six loci, 87% of nucleotide diversity has been lost in the domesticated forms. Bottlenecks acting on neutrally evolving loci either during the domestication process, during subsequent breeding, or both, are sufficient to account for reduced diversity and the results of Tajima's test, without the need to evoke selection at these loci. Phylogenetic networks data uncover distinct wild and domesticated barley genotypes and suggest that barley may have been domesticated in the Jordan valley. Because, based on AFLP data, the domesticated Turkish cultivars had a genetic basis as large as that present in large germplasm collections, all comparisons provided in this paper are of general value more than being restricted to the Turkish barley germplasm.

Agriculture↗

Purification of malted-barley endo-beta-D-glucanases by ion-exchange chromatography: some properties of an endo-barley-beta-D-glucanase.

Two endo-beta-D-glucanases which act, respectively, on (1 leads to 3)-beta-D-glucans and barley beta-D-glucan have been isolated from malted barley, and purified by ion-exchange chromatography. The latter enzyme is highly specific for barley beta-D-glucan, and has no action on either (1 leads to 3)- or (1 leads to 4)-beta-D-glucans. It will also act on dyed barley-beta-D-glucan. Certain group-specific reagents inhibit the endo-barley-beta-D-glucanase and the endo-(1 leads to 3)-beta-D-glucanase to similar extents.

Chromatography, Ion Exchange↗

Effect of water activity and temperature on growth and ochratoxin production by three strains of Aspergillus ochraceus on a barley extract medium and on barley grains.

Aspergillus ochraceus Wilhelm is a widespread storage fungus that has been isolated from grains such as barley. The objective of this study was to determine the effects of water activity (a(w)), temperature, time and their interactions on (a) growth on a barley extract agar medium, (b) growth on barley grains and (c) ochratoxin production on barley grains by three strains of A. ochracieus. For the three A. ochreceus isolates examined (NRRL 3174, 3.113 and 3.38), optimal a(w) levels for growth on agar media were in the range 0.98-0.96, with temperature optima of 30 degrees C for two of the isolates and 25-30 degrees C for the other isolate. Growth was observed at 10 and 37 degrees C, but only at higher a(w) levels assayed. Two dimensional profiles were constructed for the range of a(w) and temperature conditions that allow growth of the three isolates. Maximum growth on barley grains was reached at 30 degrees C, at both a(w) levels assayed (0.96 and 0.98), with fungal growth rates in the 4-5 mm day(-1) range. Maximum amounts of ochratoxins were produced at the highest a(w) treatment (0.98 a(w)) and after a three-week incubation time at 25-30 degrees C. The range of ochratoxin concentrations varied considerably, from 1.7 to 12,949 ppm, depending on the a(w) and temperature interactions.

Aspergillus ochraceus↗

Characterization and quantification of free and esterified 9- and 13-hydroxyoctadecadienoic acids (HODE) in barley, germinating barley, and finished malt.

The analysis of (R)-9- and (S)-9-hydroxy-10E,12Z-octadecadienoic acid as well as (R)-13- and (S)-13-hydroxy-9Z,11E-octadecadienoic acid (HODE) as free acids, esterified in triacylglycerols (storage lipids), and esterified in polar lipids (phospholipids, glycolipids, etc.) in barley, germinating barley, and finished malt was performed using [13-(18)O(1)]-(S)-13-HODE isotope dilution assays with GC-MS and straight- and chiral-phase HPLC. 9- and 13- HODE occur approximately racemically in barley, indicating an autoxidation. The enantiomeric excesses increase to 78% S for free 9-HODE and to 58% S for free 13-HODE in germinating barley as a result of lipoxygenase-2 (LOX-2) catalysis, but free HODEs are at low concentration. More than 90% of HODEs in barley and malt are esterified. In the storage lipids of green malt 53 mg/kg 9-HODE and 147 mg/kg 13-HODE were detected. This ratio of 30:70 reflects the regioselectivity of the LOX-2 enzyme in malt. In the polar lipids 45 mg/kg 9-HODE and 44 mg/kg 13-HODE were characterized. The latter indicate a hitherto unknown 9-lipoxygenase activity with polar lipids as substrates. During kilning the contents of most HODEs decreased significantly due to chemical and enzymatic degradation, whereas polar-esterified (R)-13-HODE increased (43%) in the finished malt.

Chromatography, High Pressure Liquid↗

An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm.

A cDNA encoding a DNA-binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin-box (P-box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST-fusion binds a P-box 5'-TGTAAAG-3' containing oligonucleotide derived from the promoter region of an Hor2 gene. Binding was prevented when the P-box motif was mutated to 5'-TGTAgAc-3'. A P-box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10-phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the P-box element of a native Hor2 promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA-binding domain or in the P-box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a Pbf homologue having similar DNA-binding properties to that of BPBF. These results strongly implicate this endosperm-specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the Pbf gene function among small grain cereals.

Amino Acid Sequence↗

Barley BLZ2, a seed-specific bZIP protein that interacts with BLZ1 in vivo and activates transcription from the GCN4-like motif of B-hordein promoters in barley endosperm.

A barley endosperm cDNA, encoding a DNA-binding protein of the bZIP class of transcription factors, BLZ2, has been characterized. The Blz2 mRNA expression is restricted to the endosperm, where it precedes that of the hordein genes. BLZ2, expressed in bacteria, binds specifically to the GCN4-like motif (GLM; 5'-GTGAGTCAT-3') in a 43-base pair oligonucleotide derived from the promoter region of a Hor-2 gene (B1-hordein). This oligonucleotide also includes the prolamin box (PB; 5'-TGTAAAG-3'). Binding by BLZ2 is prevented when the GLM is mutated to 5'-GTGctTCtc-3' but not when mutations affect the PB. The BLZ2 protein is a potent transcriptional activator in a yeast two-hybrid system where it dimerizes with BLZ1, a barley bZIP protein encoded by the ubiquitously expressed Blz1 gene. Transient expression experiments in co-bombarded developing barley endosperms demonstrate that BLZ2 transactivates transcription from the GLM of the Hor-2 gene promoter and that this activation is also partially dependent on the presence of an intact PB. A drastic decrease in GUS activity is observed in co-bombarded barley endosperms when using as effectors equimolar mixtures of Blz2 and Blz1 in antisense constructs. These results strongly implicate the endosperm-specific BLZ2 protein from barley, either as a homodimer or as a heterodimer with BLZ1, as an important transcriptional activator of seed storage protein genes containing the GLM in their promoters.

Amino Acid Sequence↗

[Barley chromosome identification using genomic in situ hybridization in the genome of backcrossed progeny of barley-wheat amphiploids [H. geniculatum All. (2n = 28) x T. aestivum L. (2n = 42)] (2n = 70)].

Genomic in situ hybridization (GISH) has been used to study characteristics of the formation of alloplasmic lines detected among self-pollinated backcrossed progeny (BC1F5-BC1F8) of barley--wheat amphiploids [Hordeum geniculatum All. (2n = 28) x Triticum aestivum L. (2n = 42)] (2n = 70). The chromosome material of the wild barley H. geniculatum has been shown to contribute to these lines. For example, fifth-generation plants (BC1F5) had genotypes (2n = 42w + 2g), (2n = 42w + 1g + 1tg), and (2n = 41w + 1g), where w is common wheat chromosomes, g is barley (H. geniculatum) chromosomes, and tg is the telocentric chromosome of wild barley. Beginning from the BC1F6 generation, alloplasmic telocentric addition lines (2n = 42 + 2tg) and (2n = 42 + 1tg) appear. This lines has been found cytogenetically unstable. The progeny of each of these cytological types include not only the (2n = 42 + 2tg) and (2n = 42 + 2tg) addition plants, but also plants with the monosomic (2n = 41 + 1tg) and the disomic (2n = 40 + 2tg) substitutions, as well as the (2n = 41 + 2tg) plants, which lack one wheat chromosome and have two telocentric barley chromosomes. It has been demonstrated that the selection for well-filled grains favors the segregation of telocentric addition lines (2n = 42 = 2tg) and (2n = = 42 + 1tg).

Breeding↗