Purification and identification of barley (Hordeum vulgare L.) proteins that inhibit the alkaline serine proteinases of Fusarium culmorum

It has been proposed that microbial proteinase inhibitors, which are present in abundance in cereal grains, protect the seed against plant pathogens. So far, however, very little is known about the interactions of those inhibitors with the proteinases of phytopathogenic microbes. The increased alkaline proteinase activities of Fusarium head blight (FHB) diseased wheat and barley grain imply that the Fusarium fungi synthesize those enzymes during the colonization of the kernel. To study which barley proteins can inhibit Fusarium proteinases, and hence, possibly protect the seed from FHB, the proteins of a grain extract have been separated and tested for their abilities to inhibit two alkaline serine proteinases that we previously isolated from F. culmorum. The proteins were separated by size exclusion, ion exchange, and reversed-phase-HPLC chromatographies. The purified inhibitors were identified by their molecular masses and N-terminal amino acid sequences. The proteins that inhibited the subtilisin-like Fusarium proteinase were the chymotrypsin/subtilisin (CI) inhibitors 1A, 1B, and 2A and the barley alpha-amylase/subtilisin inhibitor (BASI). Only one of the purified proteins inhibited the trypsin-like proteinase, the barley Bowman-Birk inhibitor (BBBI). No novel inhibitors were detected.     

The barley (Hordeum vulgare L.) of Sardinia, Italy

Since ancient times, barley has been an important food resource for the people of Sardinia. The oldest traces of its cultivation are from the mid-Neolithic (fourth millennium B.C.). Archaeological, historical and anthropological aspects of barley cultivated in Sardinia are discussed in this paper. We describe the traditional process for making barley bread (orgiathu) in Sardinia, where a special starter called ghimisone was prepared. Today, barley is cultivated only as animal feed, with two uses, grain yield and grazing. Many farmers prefer to grow local populations belonging to landrace locally known as S' orgiu sardu. Local Sardinian populations of barley evolved in diverse environments, being cultivated from sea-level up to 1000 m elevation, on various soil types at different intensities of abiotic stresses, and with climates and environments associated with various agricultural practices, depending both on production strategies and climatic conditions. These barley materials are thought to be valuable genetic and cultural inheritance which must be preserved and used for both productive and research purposes.     

Regrowth in barley (Hordeum vulgare L.) and rye (Secale cereale L.)

Regrowth after cutting at four development stages, from heading to grain maturity, was investigated in a pot experiment containing three rye and four barley varieties. Regrowth in the barley varieties decreased strongly from heading to grain maturity. Rye generally showed stronger regrowth than barley after late cutting, but only the perennial variety ‘Soperta’ regenerated as many tillers at cutting as formed when harvested at the ripe grain stage. In both species, significant differences were found between varieties. The level of soluble carbohydrates reached a maximum between heading and maturity, but differences in regrowth could not be explained by such differences. Total N decreased from heading to maturity, except for perennial rye, where an increase was observed towards ripening. It is, however, uncertain whether this was an effect rather than a cause of the regrowth.     

Interactions of malt and barley (Hordeum vulgare L.) endoproteinases with their endogenous inhibitors.

For producing worts that are optimal for beer production, some, but not all, of the barley proteins must be degraded during malting and mashing. This protein hydrolysis is controlled by endoproteinases, and, in turn, is partially regulated by the presence of low-molecular-weight (LMW) proteinaceous inhibitors. This paper reports studies of the interactions between the proteinases and inhibitors and an "affinity" method for concentrating the inhibitors. The malt inhibitors (I) and proteinases (E) quickly formed strong (E-I) complexes when dissolved together, and all of the I was complexed. Heating at 100 degrees C, but not 70 degrees C, dissociated the complex, even though the enzyme activities were destroyed at 70 degrees C. The released I readily recomplexed with fresh E. Barley, however, contained insufficient E to complex all of its I complement. The E-I complex was treated with salts, detergents, and reducing agents to release active E molecules, but none disrupted the complex. By removing the LMW proteins from a malt E-I extract and dissociating the complex by heating, the concentration of I molecules was greatly increased. This "affinity" method can thus be used to concentrate the I molecules for further purification.     

Endosperm structure affects the malting quality of barley (Hordeum vulgare L.)

Twenty-seven barley (Hordeum vulgare L.) samples collected from growing sites in Scandinavia in 2001 and 2002 were examined to study the effect of endosperm structure on malting behavior. Samples were micromalted, and several malt characteristics were measured. Samples were classified as having a mealier or steelier endosperm on the basis of light transflectance (LTm). Because endosperm structure is greatly dependent on protein content, three barley sample pairs with similar protein contents were chosen for further analysis. During malting, the steelier barley samples produced less root mass, but showed higher respiration losses and higher activities of starch-hydrolyzing enzymes. Malts made from steelier barley had a less friable structure, with more urea-soluble D hordein and more free amino nitrogen and soluble protein. The reason for these differences may lie in the structure or localization of the hordeins as well as the possible effects of endosperm packing on water uptake and movement of enzymes.     

Metabolomic profiling of five hulless barley (Hordeum vulgare L.) with different

Genetic Resources and Crop Evolution - Colored hulless barley (Hordeum vulgare L.) is a high-quality germplasm resource rich in nutrients, such as protein, β-glucan, flavonoids, amino acids,...     

Identification and selection of low-phosphate-tolerant germplasm in barley (Hordeum vulgare L.)

Phosphorus (P) deficiency is one of the major constraints to crop yield worldwide, and genotypes or cultivars with high phosphate use efficiency (PUE) sustain growth when exposed to phosphate stress. Therefore, it is imperative to develop the genotypes or cultivars with high PUE. A pot experiment was conducted to evaluate the PUE among 150 barley (Hordeum vulgare L.) genotypes. Two high-tolerant and -sensitive accessions were selected. These two candidate materials were used to investigate the differences among the root morphology characteristics, antioxidant enzyme activity, inorganic phosphate (Pi) content and gene expression of HvPT5 under P-deficiency and P-sufficiency conditions. The values of these parameters were higher in the low-P-tolerant genotype than in the sensitive one. In pot experiment 1, all genotypes showed a significant difference in low-P tolerance, with variety GN121 achieving the highest tolerance, and GN42 being most sensitive. The results of this study may provide elite genetic germplasms for future work on isolation of P-related genes, and the improvement of PUE in barley.

Abbreviations: PUE: phosphate use efficiency; CAT: catalase; POD: peroxidase; SOD: superoxide dismutase; DMSO: dimethyl sulphoxide; MDA: malondialdehyde; TOPSIS: technique for order preference by similarity to an ideal solution; MCDM/MADM: multi-criteria (or attribute) decision making     

Potassium placement effect on dynamics of barley (Hordeum vulgare L.) nutrition

The effects of local placement of potassium (K) on mineral nutrition dynamics of barley (Hordeum vulgare L.) in fertile Сhernozem were studied. A pot experiment with local K-placement at 4–5 cm soil depth was carried out and the dynamics of nitrogen, phosphorus, potassium (NPK) concentrations in tillers, organs and parts of spring barley was measured. K-placement increased the productivity index from 0.49 to 0.54, despite optimal and slightly varying NPK concentrations during the second half of the vegetation period (60–100 d). This occurs due to partitioning of assimilates, N, K, and especially P in generative organs of primary and secondary tillers forming quality grains. Nutrient concentrations in certain primary tiller parts of a 60-d plant (senescing leaves and the main spike axis) proved to be more sensitive indicators of the K-placement effect than average whole-plant characteristics. While being beneficial, K-placement had little impact on the overall NPK removal in barley, which shows a significant role of factors related to K uptake kinetics. Thus, the chosen parameters in the soil–plant system (the high content of available K in Chernozem, in the second part of the vegetation period) have for the first time allowed the evaluation of the impact of local K-placement on mineral nutrition dynamics in barley.     

Bacteria and fungi on roots of different barley varieties (Hordeum vulgare L.)

Summary We investigated the abundance of bacteria and fungi on roots of different barley varieties grown in soil and in a nutrient solution. Measurements were made on the rhizoplane and, for soil-grown plants, also in the rhizosphere soil. Further, the influence of plant age was investigated. Barley variety, had a significant influence both for plants grown in soil and in the nutrient solution, and the effects were most pronounced on the rhizoplane. There were no significant differences among varieties in fungal hyphal lengths on the roots. Bacterial abundance on the rhizoplane was significantly decreased with increasing plant age. Varietal differences were maintained over different plant ages.     

Biological nitrogen fixation associated with roots of field-grown barley (Hordeum vulgare L.)

Nitrogenase (C₂H₂) activity was measured in microbial media inoculated with barley root segments or corresponding rhizosphere soil. Three different media were used, Döbereiner's malate medium, a modified Ashby medium, and an acid nitrogen-free medium. Only Döbereiner's medium gave consistently positive results, and cultures inoculated with roots showed higher activity than cultures inoculated with corresponding rhizosphere soil. Similar experiments with roots and rhizosphere soil from wheat gave only negligible nitrogenase activity, whereas the tropical grass, Cynodon dactylon, gave higher activity than barley. Measurements on intact soil cores containing barley root systems showed an initial lag phase followed by a rather stable activity level over a period from 12 h to 48 h, and then the activity again decreased. The activity during the stable period corresponded to fixation of about 100 to 200 g N₂ ha^?1 24 h^?1. Measurements on isolated, washed barley roots showed only negligible nitrogenase activity.     

Molecular classification of barley (Hordeum vulgare L.) mutants using derivative NIR spectroscopy

Near-infrared reflectance (NIR) spectroscopy was used in the characterization of grain morphology mutants of barley ( Hordeum vulgare L.) in relation to grain nitrogen (N) content and protein composition. Derivative spectroscopy provided spectra with enhanced resolution, allowing wavelengths to be identified with clear differences in contribution from associated chemical bonds. Comparisons of fourth-derivative spectra of wholemeal flour from high-N grains with flour from low-N grains identified wavelengths at which there were statistically significant differences between the groups. Their importance was independently confirmed by step-up regression using these wavelengths to generate an equation predicting N content (R(2) = 0.98). Fourth-derivative spectral comparisons also allowed novel biochemical differences to be predicted. Visual assessment of the spectra of all mutants revealed a variable region (1470-1520 nm, corresponding to N-H stretch vibrations) that allowed two extreme sets to be defined. The protein extracted from these two sets differed markedly in hordein content.     

Distribution of microbial biomass across the rhizosphere of barley (Hordeum vulgare L.) in soils

Summary The microbial activity at the soil-root interface (rhizosphere) of barley was examined using a rhizobox system. In this system, the soil was placed in several compartments separated from each other by a 500-mesh nylon cloth. Plants were grown in the central compartment and after a 2-month growing period the roots were still confined to this compartment. The soil from each compartment was then analyzed for ATP, NO₃ ^/–, total N, total C and CO₂ production. The increase in ATP concentration was found in a range of 4 mm around the roots. The ATP content and CO₂ production across the rhizosphere were correlated in all the soils used, but changes in NO₃ ^– were not correlated with ATP changes. The range of NO₃ ^– change was wider than that of the ATP change, indicating that NO₃ ^– production is not influenced by the biological activity in the rhizosphere.     

In vivo modeling of beta-glucan degradation in contrasting barley (Hordeum vulgare L.) genotypes

An important determinative of malt quality is the malt beta-glucan content, which in turn depends on the initial barley beta-glucan content as well as the beta-glucan depolymerization by beta-glucanase (EC 3.2.1.73) during malting. Another enzyme, named beta-glucan solubilase, has been suggested to act prior to beta-glucanase; its existence, however, has not been unequivocally proven. We monitored changes in beta-glucan levels and in the development of beta-glucan-degrading enzymes during malting of five lots of contrasting barley genotypes. Two models of in vivo kinetics for beta-glucan degradation were then compared as follows: (i) a biphasic model based on the sequential action of beta-glucan solubilase and beta-glucanase and (ii) a monophasic model assuming that all beta-glucans are depolymerized by beta-glucanase without the previous intervention of another enzyme. Confirmatory regression analysis was used to test the fit of the models to the observed data. Our results show that beta-glucan degradation is mostly monophasic, although some enzyme other than beta-glucanase seems to be required for the early solubilization of a small fraction of insoluble beta-glucans (on average, 7% of total beta-glucans). Furthermore, the genotype-dependent kinetic rate constant (indicating beta-glucan degradability), in addition to beta-glucanase activity, is suggested to play a major role in malting quality.     

Nitrogenase activity (Acetylene reduction) during the growth cycle of spring barley (Hordeum vulgare L.)

The nitrogenase activity (C₂H₂-reduction) was measured during the growth cycle of field grown spring barley in soil cores both with and without barley plants, and at two levels of nitrogen application, 30 and 120 kg N ha^?1 year^?1 respectively. The main purpose of the investigation was to study the effects of the growing barley plants on nitrogenase activity in the soil, and temperature and moisture contents were kept constant in all experiments. Therefore, the results cannot be used to calculate actual amounts of fixed nitrogen in the field, but should be considered rather as potential values. The nitrogenase activity was found to vary during the growth cycle, and seemed to be correlated to the photosynthetic activity of the plants. Relatively low nitrogenase activity was found in the early growth stages, and the activity increased up to a maximum in the late reproductive stage, followed by a rapid decrease during the grain filling stage. The mean values of nitrogenase activity in samples without barley plants and with barley plants were 40 and 78 nmoles C₂H₄ g soil dwt^?1 24 h^?1 respectively. The positive effect of barley plants on nitrogenase activity was stronger at 120 kg N than at 30 kg N fertilization. As a mean of the whole growth cycle the ratio between samples with and without barley plants was 1.7 with 30 kgN and 2.3 with 120 kg N fertilization. The inhibitory effect of nitrogen application on nitrogenase activity was measurable until 6–7 weeks after application, and it was strongest in cores without plants.     

Trait phenotyping and molecular marker characterization of barley (Hordeum vulgare L.) germplasm from Western Himalayas

Genetic Resources and Crop Evolution - Barley (Hordeum vulgare L.) is one of the principal cereal crops grown in Western-Himalayas of India. A set of 105 barley genotypes including 38...     

Antioxidant effects of water extracts from barley (Hordeum vulgare L.) prepared under different roasting temperatures

The antioxidant effects of water extracts of roasted barley (WERB) were investigated under different roasting temperatures and compared with those of the water extracts of unroasted barley (WEUB). It was found that the Maillard reaction products increased upon increasing the roasting temperatures. Both WERB and WEUB exhibited significant antioxidant activities in linoleic acid and liposome model systems. Although WERB and WEUB afforded considerable protection against the damage of deoxyribose and proteins, the antioxidant efficiency of roasted samples was weaker than that of unroasted samples because of the reduction of antioxidant components (catechin, tocopherol, and lutein) with increasing roasting temperature. Unroasted samples were more effective in reducing power, quenching free radical, hydroxyl radical, and chelating iron than the roasted samples. The different antioxidant activity among roasted and unroasted barley samples may be partly attributed to the changes in catechin, tocopherol, and lutein contents.     

Purification and partial characterization of a second cysteine proteinase inhibitor from ungerminated barley (Hordeum vulgare L.)

It was previously shown that ungerminated barley contains inhibitors that suppress the activities of green malt cysteine proteinases. This paper reports the purification and partial characterization of a second barley cysteine endoproteinase inhibitor, a protein called lipid transfer protein 2 (LTP2). The chromatographically purified inhibitor had a molecular mass of 7112. The amino acid composition and sequence data of the purified inhibitor indicated that it was a protein whose gene, but not the protein itself, was isolated earlier from barley aleurone tissue. The purified protein inhibited the activities of electrophoretically separated green malt cysteine proteinases but not the activities of the serine- or metalloproteinases. The purified LTP2 inhibited the same proteases as the LTP1 that was characterized previously but was present in the mature seed in much smaller amounts. Neither LTP1 nor LTP2 has been proven to transport lipids in vivo, and it seems possible that both serve to keep cysteine endoproteinases that are synthesized during barley seed development inactive until the plant needs them. The small amount of LTP2 in the seed made it impossible to determine whether it, like LTP1, is involved in beer foam formation. Because of its proteinase-inhibiting ability and its resistance to heat inactivation, some of the LTP2 may persist in beer.     

Dynamics of microbial biomass and soil fauna in two contrasting soils cropped to barley (Hordeum vulgare L.)

Summary This study compared the dynamics of shoots, roots, microbial biomass and faunal populations in two different soils cropped to barley. The dynamics of microbial C, protozoa, nematodes, acari, collembola, shoot and root mass were measured between July and October under barley at Ellerslie (Black Chernozem, Typic Cryoboroll) and Breton (Gray Luvisol, Typic Cryoboralf) in central Alberta. Very wet soil conditions in early July reduced the barley yield at Breton. The peak shoot mass was greater at Ellerslie (878 g m^–2) compared to Breton (582 g m^–2), but the root mass did not differ significantly between sites. Microbial C at 0–30 cm depth was greater at Ellerslie (127 g m^–2) than Breton (68 g m^–2). The average protozoa population (no. m^–2) did not differ significantly between sites. The average nematode population at 0–20 cm depth was greater at Ellerslie (5.1 × 10⁶ no. m^–2) compared to Breton (1.0 × 10⁶ no. m^–2) Acari and collembola populations at 0–10 cm depth at Ellerslie (43 × 10³ and 43 × 10² no. m^–2), respectively) were greater than at Breton (2 × 10⁴ and 9 × 10² no. m^–2) respectively). Tenday laboratory incubations of 0–10 cm soil samples from Ellerslie evolved more CO₂-C (120 g g^–1 soil) compared to samples from Breton (97 g g^–1 soil), but the CO₂-C evolution did not differ when expressed on an area basis (g m^–2) due to the greater soil bulk density at Breton. The soil from Breton respired twice as much CO₂-C when expressed as a proportion of soil C and 1.5 times as much CO₂-C when expressed as a proportion of microbial C, compared to the soil from Ellerslie. The greater CO₂-C: microbial C ratio, lower flush C:N ratio, and greater protozoa population: soil C ratio at Breton compared to Ellerslie suggest that the food web was relatively more active at Breton and was related to greater C availability and water availability at Breton.     

Identification of flavone C-glycosides including a new flavonoid chromophore from barley leaves (Hordeum vulgare L.) by improved NMR techniques

Six flavone C-glycosides were isolated from young leaves of barley. One of the C-glucosides has a new type of nucleus, a 2',4',5,5', 7-penta-OH-substituted flavone bearing a 6-C-beta-D-glucoside, which has apparently never been isolated before. One mono- and two di-C-glycosyl flavones were isolated for the first time from barley and identified as isoscoparin 7-O-beta-D-glucoside, carlinoside, and shaftoside, respectively. Other flavones were 7-O-beta-D-glucosides of isoorientin and isovitexin. The known problematic NMR structure elucidation of C-glycosyl flavonoids has been solved by using both a temperature close to the freezing point of the solvent (22.5 degrees C in DMSO-d(6)) and a high temperature (70, 90 degrees C) for comparison during NMR measurements. Structural determination of all the compounds was achieved by employing 1D and 2D NMR techniques.     

Analysis and mapping quantitative trait loci for histidine content in barley (Hordeum vulgare L.) using microsatellite markers

Genetic Resources and Crop Evolution - Mining the gene of histidine content in barley grain helps with the breeding of functional barley varieties. The study constructed a recombinant inbred lines...