Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare)

The least ambiguous genetic markers are those based on completely characterized DNA sequence polymorphisms. Unfortunately, assaying allele states by allele sequencing is slow and cumbersome. The most desirable type of genetic marker would be unambiguous, inexpensive to assay and would be assayable singly or in parallel with hundreds of other markers (multiplexable). In this report we sequenced alleles at 54 barley (Hordeum vulgare ssp. vulgare) loci, 38 of which contained single-nucleotide polymorphisms (SNPs). Many of these 38 loci contained multiple polymorphisms, and a total of 112 polymorphisms were scored in five barley genotypes. The polymorphism data set was analyzed both by using the individual mutations as cladistic characters and by reducing data for each locus to haplotypes. We compared the informativeness of these two approaches by consensus tree construction and bootstrap analysis. Both approaches provided similar results. Since some of the loci sequenced contained insertion/deletion events and multiple point mutations, we thought that these multiple-mutated loci might represent old alleles that predated the divergence of barley from H. spontaneum. We evaluated sequences from a sample of H. spontaneum accessions from the Eastern Mediterranean, and observed similar alleles present in both cultivated barley and H. spontaneum, suggesting either multiple domestication events or multiple transfers of genes between barley and its wild ancestor.     

Effects of barley straw (Hordeum vulgare) on freshwater and brackish phytoplankton and cyanobacteria

A short-term laboratory study was conductedto investigate the effect of barley strawin controlling several common phytoplanktonand cyanobacterial species. Following aone-month incubation of barley straw incoarsely filtered fresh Potomac River andbrackish Patuxent River waters, the growthof six autotrophic taxa was followed inculture. Barley straw slurry reduced theyield of three taxa (Ankistrodesmusfalcatus, Chlorella capsulata, Isochrysis sp.) in comparison withcultures not receiving the slurry. Although no significant changes in growthwere detected with three other taxa (Cyclotella sp., Prorocentrumminimum, freshwater Pseudanabaenasp.), some patterns indicated potentialimpacts of the barley straw. First, ahigher addition of straw to Cyclotella sp. resulted in a lower biomassaccumulation than in cultures receivinglower levels. Second, the bloom-formingdinoflagellate Prorcentrum minimumwas apparently stimulated at low barleystraw levels, perhaps suggesting conditionsassociated with the straw(metals-chelation, bacterial-producednutrients) might stimulate dinoflagellategrowth. Third, species shifts wereobserved in two of the cultures, withbarley straw favoring shifts from Isochrysis to a Cyclotella sp. –Thalassiosira sp. mixture and shiftsfrom Pseudanabaena to a Pseudanabaena – Scenedesmus mixture. These results provide new records for thesusceptibility of freshwater and brackishphytoplankton taxa to barley strawexposure, including species-specificresponses and shifts in species dominancein mixed assemblages.     

Properties of phenylalanine ammonia-lyase extracted from Cucumis sativus hypocotyls

Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two K_m values were determined, these were K_m^H, 2·9 × 10^?4 M and K_m^L, 4·3 × 10^?5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.     

The characterization of phenylalanine ammonia-lyase from several plant species

Inhibition of the enzyme phenylalanine ammonia-lyase is considered as a target for the design of herbicides. A reliable and simple assay for the enzyme has been used and the kinetics of the enzyme from several sources compared. Purification of the enzyme from the grass green foxtail (Setaria glauca) did not change its kinetic behavior. The distribution of phenylalanine ammonia-lyase and tyrosine ammonia-lyase activity in various plant species was determined.     

Differential expression of phenylalanine ammonia-lyase genes in barley induced by fungal infection or elicitors

Gene-specific probes were used to assess the expression patterns of four different phenylalanine ammonia-lyase ( pal ) genes in infected or elicitor-treated leaves and suspension-cultured cells of barley. Genes corresponding to hpal2 , hpal3 , hpal4 , and hpal6 were all induced by mercuric chloride and fungal infection by Bipolaris sorokiniana Sacc. (Shoem.) in barley ( Hordeum vulgare L. cv. Pokko) leaves, but with considerable variation in their expression level and timing. The expression patterns of hpal2 and hpal6 were similar, both showing a rapid, strong induction after treatment with mercuric chloride and a slower induction after fungal inoculation, whereas the more divergent hpal3 was induced at a later time and at a lower level after both treatments. Hpal4 was expressed with timing like that of hpal2 and hpal6 in infected or mercuric chloride-treated leaves but its expression was much weaker. Hpal2 and hpal4 were induced in elicitor-treated, suspension-cultured barley cells, whereas the expression of hpal3 was nearly undetectable, and hpal6 was strongly and constitutively present. All pal genes except hpal4 were developmentally regulated, but differentially expressed in various barley tissues. The results suggest that the four pal genes studied here might be responsible for the activation of different branches in the phenylpropanoid biosynthesis of barley.     

Isolation of sucrose: sucrose 1-fructosyltransferase (1-SST) from barley (Hordeum vulgare)

The enzyme sucrose: sucrose 1-fructosyltransferase was partially purified from barley leaf growth zones. Four steps (ammonium sulphate precipitation and polyethylene glycol precipitation, followed by chromatography on Concanavalin A-sepharose and hydroxylapatite) yielded a 35-fold purification. The resulting preparation of 1-SST which still contained a number of different activities related to fructan metabolism, was subjected to preparative isoelectric focusing, and sections of the gel were analysed individually for 1-SST and related activities, using sucrose and 1-kestose as substrates. This procedure yielded a 196-fold purification and revealed the presence of two isozymes of 1-SST with pI values of 4.93 and 4.99, as determined by analytical isoelectric focusing of the corresponding fractions. Both isozymes produced glucose and 1-kestose when incubated with sucrose. In addition, small amounts of 6-kestose and tetrasaccharides were formed. In particular, one of the two 1-SST isozymes yielded fructose when incubated with 1-kestose, indicating that it also acts as a fructan exohydrolase. The other isozyme exhibited less fructan exohydrolase activity. Nystose was also degraded by the fructan exohydrolase activity but less than 1-kestose, whereas 6-kestose was not a substrate for the enzyme. Incubation of both 1-SSTs with different concentrations of sucrose showed that the enzyme was not saturated even at 500 mM. As for the barley sucrose: fructan 6-fructosyltransferase, both isozymes of 1-SST yielded two polypeptide bands of molecular weight 50 and 22 kDa upon sodium dodecylsulphate polyacrylamide gel electrophoresis, suggesting their close relationship to invertase (composed of two subunits of similar size), as previously reported for other plants.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Action spectra for phenylalanine ammonia lyase in Hordeum vulgare

5-Day-old etiolated barley shoots were illuminated with monochromatic light (10 nm bandpass) and assayed for PAL 5 hr later. Action spectra for enhance     

Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (Hordeum vulgare)

The in vitro plant regeneration frequencies for immature scutella, leaf-bases/apical meristems (LB/AM) and mature embryos of four commercially important barley genotypes were compared. Production of shoots from mature embryos or calluses of LB/AM incubated on media containing 1.0 or 2.0 mg l^–1 6-benzylaminopurine (BA) were comparable to regeneration frequencies obtained for scutella-derived calluses of the same genotypes. Incubation of excised mature embryos and LB/AM on media containing the plant growth regulator, thidiazuron (TDZ), resulted in an increased shoot production. However, TDZ treatment did not stimulate plant regeneration from calluses derived from scutella or LB/AM. Shoots formed from TDZ-treated mature embryos and LB/AM were induced without a callus interphase and the in vitro culture system gave a three- to eight-fold higher regeneration frequency than recorded for scutella-derived calluses on BA medium. The simplicity and rapid development of shoots using the mature embryo system could potentially be used for the regeneration and genetic transformation of barley over alternative regeneration systems.     

Inhibition of dinoflagellate growth by extracts of barley straw (Hordeum vulgare)

Growth of dinoflagellates representing three orders, the Gymnodiniales,Peridiniales, and the Prorocentrales was examined following treatment withbarley straw extract. Selected dinoflagellate taxa showed growth responsessimilar to those reported for freshwater algae including: inhibition(Gyrodinium galatheanum, Gymnodiniumsanguineum, Heterocapsa triquetra andH. pygmaea); stimulation (Gyrodinium instriatum,Prorocentrum minimum and P. micans); and no effect(Gyrodinium estuariale, G. uncatenum,Ceratium furca, Peridinium sp.).Although barley straw extracts do not appear to have value as a universalmanagement tool for dinoflagellates, they may have potential in management ofspecific taxa and possibly taxonomic groups.     

Agmatine coumaroyltransferase from barley seedlings

4-Coumaroyl-CoA: agmatine N-4-coumaroyltransferase (agmatine coumaroyltransferase; ACT) from barley seedlings was purified with a 100-fold increase     

The competitive effects of volunteer barley (Hordeum vulgare) on the growth of oilseed rape (Brassica napus)

The effects of competition from volunteer barley (Hordeum vulgare) on the growth and yield of oilseed rape (Brassica napus) were investigated in four experiments over three seasons. The growth of rape in the autumn was reduced by 50 - 91 % by competition from 400 barley plants m^-2. A lower barley density of 200 plants m^-2 had less effect but still reduced growth of rape by 65 - 81% in two of the experiments and 25 - 40% in the other two. During winter and spring the barley decreased in vigour and in the spring the rape started to recover, especially on the early drilled (23 - 30 August) plots. The rape sown in mid-September recovered less quickly. In Experiment 3, herbicides applied in November to control barley did not result in increased growth of rape in winter but led to greater recovery in spring. The barley died during the winter in Experiments 2 and 4, even in the absence of herbicides. Despite the marked effects of barley on the growth of rape in the autumn, yields on plots that had previously contained 200 barley plants m^-2 were reduced by a maximum of only 16% in three of the experiments. In Experiment 3, where the barley was most competitive, this density and 400 plants m^-2 lowered yields by 39% and 78%, respectively. Where a herbicide was used in November to control the barley these yield losses were reduced to 5%. In many rape crops the cost of herbicide treatment would be greater than the financial returns from the expected increase in yield resulting from the control of weeds. Possible reasons for the small loss in yield of rape from barley densities that had substantial effects on the growth of rape in the autumn are discussed.     

Polyamines in barley seedlings

Polyamine levels in barley seedlings grown in the dark or in diurnal illumination have been determined, by direct dansylation, 3, 6 and 12 days after g     

Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors

Phenylalanine ammonia-lyase (PAL) catalyzes the beta-elimination of ammonia from L-phenylalanine to trans-cinnamic acid. A study of inhibition of PAL by phenol, ortho-cresol, and meta-cresol gave mixed inhibition; para-cresol is not an inhibitor. The calculated values of K(i) and alphaK(i) are phenol, K(i)=2.1+/-0.5 mM and alphaK(i)=3.45+/-0.95 mM; ortho-cresol, K(i)=0.8+/-0.2 mM and alphaK(i)=3.4+/-0.2 mM; meta-cresol, K(i)=2.85+/-0.15 mM and alphaK(i)=18.5+/-1.5 mM. The synergistic inhibition of the same inhibitors with glycine showed a lack of inhibition with the para-cresol/glycine pair, while mixed inhibition was observed with the ortho-cresol/glycine pair (K(i)=0.038+/-0.008 mM, alphaK(i)=0.13+/-0.04 mM) and phenol/glycine pair (K(i)=0.014+/-0.003 mM, alphaK(i)=0.058+/-0.01 M). The meta-cresol/glycine pair gave competitive inhibition (K(i)=0.36+/-0.076 mM). The strong synergistic inhibition observed implies that the inhibitors bind at the active site: in fact, the inhibitors used imitate the structure of the substrate. The order of synergistic inhibition is the same for the sites related to K(i) and alphaK(i). These results are in agreement with the inhibitors entering two active sites located in two different subunits.     

An arabinogalacto(4-O-methylglucurono)xylan from the leaves of Hordeum vulgare

An arabinogalacto(4-O-methylglucurono)xylan with a $\text{DP}$_n of ca. 96 has been isolated from the leaves of barley. Based on structural studies it is proposed that the hemicellulose consists of a main chain of β (1→4)-linked d-xylopyranosyl residues to which are attached an average of 8·1 l-arabinofuranosyl residues, 3·8 galactopyranosyl-(1→4)-d-xylopyranosyl-(1→2)-l-arabinofuranosyl residues and 4·4 4-O-methyl-d-glucopyranuronosyl residues.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Isolation of ribosomes from cell-wall preparations of barley (Hordeum vulgare)

Ribosomes can be released from highly purified preparations of the cell walls of barley shoots by passing the suspension of cell walls through a French press. The sedimentation coefficients and base composition of the cell-wall ribosomes are identical with those of cytoplasmic ribosomes. It is suggested that the ribosomes isolated from the walls were originally incorporated into the wall structure during formation of the primary wall.