Research advances in the Pyrenophora teres–barley interaction

Pyrenophora teres f. teres and P. teres f. maculata are significant pathogens that cause net blotch of barley. An increased number of loci involved in P. teres resistance or susceptibility responses of barley as well as interacting P. teres virulence effector loci have recently been identified through biparental and association mapping studies of both the pathogen and host. Characterization of the resistance/susceptibility loci in the host and the interacting effector loci in the pathogen will provide a path for targeted gene validation for better-informed release of resistant barley cultivars. This review assembles concise consensus maps for all loci published for both the host and pathogen, providing a useful resource for the community to be used in pathogen characterization and barley breeding for resistance to both forms of P. teres.     

Conjoined mitochondria and plastids in the barley mutant ‘albostrians’

The intercalary meristem and surrounding tissues of the gene induced plastome mutant albostrians of Hordeum vulgare L. were examined in the electron microscope for ultrastructural evidence of membrane continuities between plastids and mitochondria. In well developed tissues the ribosome-deficient plastids were usually in close proximity or appressed to mitochondria of normal appearance. In some sections through the meristemmatic region however the relationship between the two organelles was observed to be of a fused nature. These conjoinings are thought to be similar to those reported in normal living cells using cinephotomicrography but never before observed by transmission electron microscopy.     

The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers

The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA₃). Using [³⁵S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA₃ was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA₃ were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA₃-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA₃ only three peaks of activity were found. In incubation media, four isoenzymes were found after GA₃ treatment and two were found after incubation without GA₃. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA cyclohepataamylose - DEAE-cellulose diethylaminoethyl-cellulose - ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis     

Allelic characteristics of the β-amylase gene of barley varieties in Ukraine

Allelic diversity in a set of 99 spring and winter barley varieties intended for different use (malting, cereal, and valuable) was studied. PCR analysis with the use of the β-amylase DNA marker showed that genotypes of different barley varieties may include different alleles of the β-amy1 gene.     

Seven members of the (1→3)-β-glucanase gene family in barley (Hordeum vulgare) are clustered on the long arm of chromosome 3 (3HL)

Members of the (13)--glucan glucanohydrolase (EC 3.2.1.39) gene family have been mapped on the barley genome using three doubled haploid populations and seven wheat-barley addition lines. Specific probes or polymerase chain reaction (PCR) primers were generated for the seven barley (13)--glucanase genes for which cDNA or genomic clones are currently available. The seven genes are all located on the long arm of chromosome 3 (3HL), and genes encoding isoenzymes GI, GII, GIII, GIV, GV and GVII (ABG2) are clustered in a region less than 20 cM in length. The region is flanked by the RFLP marker MWG2099 on the proximal side and the Barley Yellow Mosaic Virus (BYMV) resistance gene ym4 at the distal end. The gene encoding isoenzyme GVI lies approximately 50 cM outside this cluster, towards the centromere. With the exception of the gene encoding isoenzyme GIV, all of the (13)--glucanase genes are represented by single copies on the barley genome. The probe for the isoenzyme GIV gene hybridized with four DNA bands during Southern blot analysis, only one of which could be incorporated into the consensus linkage map.     

Biopharming the SimpliRED™ HIV diagnostic reagent in barley, potato and tobacco

This is the first report of an antibody-fusion protein expressed intransgenic plants for direct use in a medical diagnostic assay. By the use ofgene constructs with appropriate promoters, high level expression of ananti-glycophorin single-chain antibody fused to an epitope of the HIV virus wasobtained in the leaves and stems of tobacco, tubers of potato and seed ofbarley. This fusion protein replaces the SimpliRED diagnostic reagent,used for detecting the presence of HIV-1 antibodies in human blood. The reagentis expensive and laborious to produce by conventional means since chemicalmodifications to a monoclonal antibody are required. The plant-produced fusionprotein was fully functional (by ELISA) in crude extracts and, for tobacco atleast, could be used without further purification in the HIV agglutinationassay. All three crop species produced sufficient reagent levels to be superiorbioreactors to bacteria or mice, however barley grain was the most attractivebioreactor as it expressed the highest level (150 g of reagentg^-1), is inexpensive to produce and harvest, poses aminuscule gene flow problem in the field, and the activity of the reagent islargely undiminished in stored grain. This work suggests that barley seed willbe an ideal factory for the production of antibodies, diagnosticimmuno-reagents, vaccines and other pharmaceutical proteins.     

Is quantity of protein in barley forms determined by proteins localized in the subaleurone layer?

Barley biotypes from the world collection differ in their storage protein content even till 200 %. This is the first report including results of the research, in which the structure of grains containing different amount of protein was tested to explain this difference. The endosperm was investigated using scanning electron microscopy. The structure of the aleurone layer, storing large quantities of protein, did not differ between the high- and low-protein forms of barley. It has been proven that the large quantities of kernel protein may be stored in some cells of the zone adjacent to the aleurone layer, defined as the subaleurone cells. It has been shown that morphologically uniform kernels of the same plant and even of the same ear can vary greatly with respect to the number of these subaleurone cells. The purpose of the study was an examination of variation in protein structure in single kernels of a fodder, a brewery and in an extra high-protein form of barley as well. Moreover the studies were aimed to detect qualitative differences in the subaleurone protein. Application of mass spectrometry made possible the identification of several kinds of proteins which were present in subaleurne layer of kernels. In the granule-bound protein fraction isolated from the subaleurone type kernels, a much stronger representation of some protein was found, with the molecular mass between 29 and 45 kDa, in comparison with the low-protein kernels. It is supposed, that these protein are isoforms of z-type serpin and B3-hordein.     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline     

The down-regulated expression of 14-3-3σ may activate the other six 14-3-3 isoforms and they may take over the role of 14-3-3σ in the tumor genesis

Seven isoforms of 14-3-3 protein family have different functions in the cancer genesis and progress. It is found that six isoforms were up-regulated expression and inclined to sustain the cancer survival. Conversely, 14-3-3σ strongly promotes cancer apoptosis. Its down-regulated expression was found in many cancer tissues and thought to be an early event in the tumor genesis. Interestingly, no suggestions are made about the possible effect that the down-regulated expression of 14-3-3σ activated the other six 14-3-3 isoforms and they take over the role of 14-3-3σ in the tumor genesis. The inactivation of 14-3-3σ in the early stage of tumor genesis is a clue to trigger the other six 14-3-3 isoforms activation.     

14-3-3 Proteins in the Regulation of Rotenone-Induced Neurotoxicity Might be via Its Isoform 14-3-3Epsilon’s Involvement in Autophagy

14-3-3 proteins have been confirmed to be involved in Parkinson’s disease. It has been reported that an increase of 14-3-3 (theta, epsilon, and gamma) expression has neuroprotective effect in response to rotenone and MPP⁺ in dopaminergic cell culture and transgenic C. elegans with alpha-synuclein overexpression. To further investigate the detail mechanism of 14-3-3 proteins in rotenone-induced dopamine neurotoxicity, we observed the expression of 14-3-3 isoforms, and the influence of 14-3-3epsilon knockdown on autophagic activity and cell function. The results showed that rotenone led to a decrease in expression of 14-3-3 protein and mRNA, and an increase in expression and aggregation of alpha-synuclein protein. Knockdown of 14-3-3epsilon expression in turn further aggravated PC12 cell damage, such as an enhancement of ROS formation, and a reduction of cell viability and ATP production. Further experiments confirmed that the autophagic activity was promoted with 14-3-3epsilon siRNA transfection, including an enhancement of autophagosome formation and the ratio of LC3-II/LC3-I. Therefore, we concluded that the regulation of 14-3-3 proteins in rotenone-induced neurotoxicity might be associated with its isoform 14-3-3epsilon’s involvement in autophagy, which might be considered a mechanism in addition to the currently known function of 14-3-3 proteins in neurodegenerative disease pathogenesis.     

Association of haplotype diversity in the α-amylase gene amy1 with malting quality parameters in barley

Amylases play an essential role in the germination and malting process. Therefore, these genes are interesting candidates for marker development in order to improve malting quality as an important breeding aim. The intervarietal diversity of the α-amylase gene amy1 mapping to chromosome 6H was investigated. A total of six single nucleotide polymorphisms (SNPs) were detected which defined four haplotypes. Associations between SNP-markers and important malting parameters were discovered in a collection of 117 European spring and winter barley cultivars, representing the current commercial germplasm. Haplotype amy1_H2 was significantly associated with a number of malting related traits and explained 19% of the phenotypic variation of the malting quality index (MQI) for all varieties and 35% in a subset of 72 winter barleys. The diagnostic SNP3 was associated with a 45% difference in the MQI. Within the spring barleys, the average value of haplotype amy1_H1 for friability was significantly higher than that of amy1_H4. All discovered SNPs were converted into high-throughput markers for pyrosequencing and can be used for marker assisted selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Division and DNA distribution in ribosome-deficient plastids of the barley mutant “albostrians”

The ribsome-deficient plastids of the albino leaves of the barley mutant albostrians divide at about the same rate as normal plastids and contain similar levels of plastids DNA to the normal plastids. Double-ring structures were observed around the neck of constricting dumbbell-shaped, ribosome-deficient plastids in the basal intercalary meristem of albino leaves. In the distal region of albino leaves the ribosome-deficient plastids contain a rudimentary thylakoid system often closely associated with DNA nucleoids. It is suggested that nuclear coded proteins synthesized within the cytoplasm are responsible for the formation of the double-ring structures and the rudimentary thylakoids of albino plastids.     

Nicotinate, quinolinate and nicotinamide as precursors in the biosynthesis of nicotinamide–adenine dinucleotide in barley

1. The relative efficiencies of nicotinate, quinolinate and nicotinamide as precursors of NAD(+) were measured in the first leaf of barley seedlings. 2. In small amounts, both [(14)C]nicotinate and [(14)C]quinolinate were quickly and efficiently incorporated into NAD(+) and some evidence is presented suggesting that NAD(+) is formed from each via nicotinic acid mononucleotide and deamido-NAD. 3. [(14)C]Nicotinamide served equally well as a precursor of NAD(+) and although significant amounts of [(14)C]NMN were detected, most of the [(14)C]NAD(+) was derived from nicotinate intermediates formed by deamination of [(14)C]nicotinamide. 4. Radioactive NMN was also a product of the metabolism of [(14)C]nicotinate and [(14)C]quinolinate but most probably it arose from the breakdown of [(14)C]NAD(+). 5. In barley leaves where the concentration of NAD(+) is markedly increased by infection with Erysiphe graminis, the pathways of NAD(+) biosynthesis did not appear to be altered after infection. A comparison of the rates of [(14)C]NAD(+) formation in infected and non-infected leaves indicated that the increase in NAD(+) content was not due to an increased rate of synthesis.     

A cell-free system for the study of α-amylase synthesis in barley aleurone layers

1. A cell-free system capable of alpha-amylase synthesis has been obtained from the aleurone layers of germinating barley. 2. This system requires potassium chloride, sucrose and an amino acid mixture in order to function. The crude preparation does not require calcium chloride. Chloramphenicol inhibits alpha-amylase synthesis as indicated both by increase in measurable enzyme activity and incorporation of l-[U-(14)C]glutamic acid.     

RNA complementary to α-amylase mRNA in barley

Two experimental approaches demonstrate that different types of RNA complementary to -amylase mRNA are present in barley. S1 nuclease assays identify an RNA that is complementary to essentially the full length of both the type A and type B -amylase mRNAs. Complementarity, however, is imperfect: the S1 nuclease-resistant products can only be identified if they are electrophoresed as RNA-DNA hybrids. This RNA is present in developing endosperm + aleurone tissue and in mature aleurone tissue cultured in the absence of hormonal treatment or in the presence of abscisic acid, but not in shoot or root tissue. In mature aleurone tissue treated with abscisic acid, its steady-state abundance is similar to that of -amylase mRNA. Northern blot analysis indicated the presence of a second type of antisense RNA. Under conditions of moderate stringency, antisense-specific probes detect discrete hybridizing species of 1.6, 1.4, and 1.0 kilobases in mature aleurone and shoot tissues that do not represent spurious hybridization to rRNA, -amylase mRNA, or the abundant, G+C-rich mRNA for a probable amylase/protease inhibitor. The different results are consistent with the fact that the hybridization assay can tolerate relatively short regions of complementarity separated by large, nonhomologous sequences, while the nuclease protection assay cannot.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

Polymorphisms in the α-amy1 gene of wild and cultivated barley revealed by the polymerase chain reaction

-Amylases are the key enzymes involved in the hydrolysis of starch in plants. The polymerase chain reaction (PCR) was used to detect polymorphisms in the length of amplified sequences between the annealing sites of two primers derived from published -amy1 gene sequences in barley. These two primers (Bsw1 and Bsw7), flanking the promoter region and the first exon, amplified two PCR fragments in barley. One of the amplified products, with the expected length of 820 bp, appeared together with another shorter PCR band of around 750 bp. This 750-bp fragment seems to be derived from an -amylase gene not reported previously. Both of the PCR products could be amplified from the two-rowed barley varieties tested, including cv Himalaya from which the sequence information was obtained. Five of the six-rowed barley varieties also have the two PCR fragments whereas another two have only the long fragment. These two fragments seem to be unique to barley, neither of them could be amplified from other cereals; for example, wheat, rye or sorghum. These two -amylase fragments were mapped to the long arm of 6H, the location of the -amy1 genes, using wheat-barley addition lines. Amplification of genomic DNA from wild barley accessions with primers Bsw1 and Bsw7 indicated that both of the fragments could be present, or the long and short fragments could be present alone. The results also demonstrated that the genes specifying these two fragments could be independent from each other in barley. The conserved banding pattern of these two fragments in the two-rowed barley varieties implies that artificial selection from these genes may have played an important role in the evolution of cultivated barley from wild barley.     

Spatial distribution of β-amylase in germinating barley seeds based on the avidin-biotin-peroxidase complex method

Summary In comparison with two wild type barley cultivars, Sundance and Bomi, biochemical data show that the high-lysine mutant Hiproly contains abundant amounts of lysine-rich -amylase, whereas mutant Risø 1508, also a high-lysine mutant, contains negligible amounts of this enzyme. Immunocytochemical studies of germinating barley seeds, using both mono- and polyclonal antibodies to -amylase, support the biochemical findings of enzyme abundance in developing seeds. Three immunostaining methods for localization of -amylase were tested; of these, the avidin-biotin-peroxidase complex method, a relatively new procedure for study of plant tissues, is by far the most sensitive. -Amylase occurs predominantly in cytoplasm of the endosperm, with a minor, but previously unknown localization in the aleurone of the mid-region of the seeds. A spatial distribution of -amylase is seen. Endosperm in the upper and lower regions has the greatest amount of -amylase, with the amount decreasing toward the mid-region. In the mid-region, a limited aleurone localization of -amylase is found in all four barley strains. The function of this aleurone localization is unclear. In the endosperm, the abundance of -amylase appears to be inversely correlated with number of starch grains per unit area in Hiproly but not in Risø 1508, yet the rate of germination of the two mutants is essentially identical. Whether -amylase has a role in starch metabolism in these germinating barley seeds is unclear.Abbreviations Ab antibodies - ABC avidin-biotin-peroxidase complex - a -Amylase - BSA bovine serum albumin - DAB diaminobenzidine tetrahydrochloride dihydrate - FAA formalin: glacial acetic acid: ethyl alcohol - FITC fluorescein isothiocyanate - mAb mouse monoclonal antibodies - pAb rabbit polyclonal antibodies - PAP peroxidase-anti-peroxidase - PBS phosphate buffer saline - R-1508 Risø 1508     

On the secretion of α-amylase by barley aleurone layers after incubation in gibberellic acid

Summary Gel filtration and centrifugation studies were used to study the distribution of -amylase activity in homogenates of barley (Hordeum vulgare L.) aleurone layers. The results obtained were consistent with the hypothesis that -amylase is secreted via membrane-bound vesicles. The -amylase activity in an homogenate of barley aleurone layers was derived not only from the enzyme retained in the aleurone cells but also from enzyme previously secreted from the cells but apparently retained by the cell walls. The amount of -amylase retained by the cell wall was influenced by factors such as the buffer in which the layers were incubated or the presence of Actinomycin D in the incubation medium.Abbreviations GA₃ gibberellic acid - RER rough endoplasmic reticulum - Act. D Actinomycin D