Fractionation and characterization of histones from barley (Hordeum vulgare) leaves

Histones were extracted from purified nuclei isolated from four cereals,viz. barley (Hordeum vulgare), wheat(Triticum aestivum), Aegilops squarrosa and corn (Zea mais), and from tobacco (Nicotiana tabacum). Analysis of the histones on SDS gels showed complex electrophoretic patterns with one species of both H3 and H4, one to three species of H1 and two to five species of H2. Judged from the electrophoretic patterns, the histones from barley, wheat and Aegilops are identical but different from those of corn with respect to H2. Like tobacco, corn showed two H2 components, whereas barley, wheat and Aegilops showed five H2 components. SDS gel electrophoresis of histones extracted from buds and roots of germinating seeds at different steps of germination and from different parts of ten-day-old leaves revealed that the existence in barley of multiple histone 2 variants is not restricted to any particular stage of differentiation of barley. Histones from barley leaves were resolved into four fractions by Biogel P-100 gel filtration and histones 2 were further fractionated by their differential solubility in HCl-ethanol. Each of these five fractions (H1, H3, H4, H2A and H2B, respectively) were characterized by electrophoresis on SDS or Triton-acid-urea gels and by their amino acid compositions as compared with the homologous histones of calf thymus and chicken erythrocytes. This revealed the following:

1. H3 and H4 are strictly analogous to their animal counterparts. However, H4 has an unexplained lower electrophoretic mobility in Triton-containing acid-urea gels.
2. H1 contains three components with lower electrophoretic mobilities than H1 from erythrocytes, contains more alanine than lysine and has a lower ratio of basic to acidic residues.
3. Both H2A and H2B contain at least four variants each, with higher molecular weights than in animals and higher lysine to arginine ratios. H2A variants comigrate in acid-urea-Triton gels with chicken erythrocytes H2A, whereas H2B migrate much slower.

It was concluded that the presence of multiple major variants of H2A and H2B is a frequent but not universal feature in cereals. The existence of these variants is not restricted to the embryonic stage as previously suggested for wheat (31).     

Ring chromosomes in barley (Hordeum vulgare L.)

A plant with 2n = 14 + 1 ring chromosomes was obtained in the progeny of a primary trisomie for chromosome 7 of a two-rowed cultivar, Shin Ebisu 16. The morphological characteristics of the trisomic plants with an extra ring chromosome were similar to the primary trisomic for chromosome 7 (Semierect), which suggests that it originated from this chromosome. The ring chromosomes were not completely stable in mitotic cells because of abnormal behavior. Chromosome complements varied in different plants and in different roots within a plant. Root tip cells and spikes with 2n = 14 and 14 + 2 ring chromosomes were observed on plants with 14 + 1 ring chromosomes. Breakage-fusion-bridge cycle was inferred. The ring chromosome was associated with two normal homologues forming a trivalent in 17.6% sporocytes at metaphase I. The transmission of the extra ring chromosome was 23.1% in the progeny of the plant with 14 + 1 ring chromosomes. Trivalent formation may have been much higher at early prophase stages which were difficult to analyze in barley; only 4 of 120 sporocytes analyzed showed an isolated ring at pachytene. The ring chromosome moved to one pole without separation in 24.7% of the sporocytes at AI, and divided in 27.1% sporocytes giving rise to 8-8 separation. Only 10% of the sporocytes showed bridge formation at AI.     

Aldehyde oxidase in roots, leaves and seeds of barley (Hordeum vulgare L.)

Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves, roots and seeds of barley (Hordeum vulgare L.) plants were studied. Differences in substrate specificity and mobility in native PAGE between AO proteins extracted from roots, leaves and seeds have been observed. Four clear bands of AO reacting proteins were detected in barley plants capable of oxidizing a number of aliphatic and aromatic aldehydes such as indole-3-aldehyde, acetaldehyde, heptaldehyde, and benzaldehyde. Mouse polyclonal antibodies raised against purified maize AO cross-reacted with barley AO proteins extracted from roots, leaves and seeds. At least three different AO proteins were detected in roots on the basis of their mobility during PAGE after native Western blot analysis while in leaves and seeds only one polypeptide cross-reacted with the antibody. SDS-immunoblot analysis showed marked differences in molecular weight between subunits of the AO bands extracted from roots, leaves and seeds. Two distinct subunit bands were observed in roots, with relatively close molecular weights (160 kDa and 145 kDa), while a single subunit with a molecular weight of 150 kDa was observed in leaf and seed extracts.Menadione, a specific and potent inhibitor of animal AO did not affect barley AO proteins. Root and leaf AO differed in their thermostability and susceptibility to exogenous tungstate. The AO proteins in plants may be a group of enzymes with different substrate specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ.     

Purification and properties of the assimilatory nitrite reductase from barley Hordeum vulgare leaves.

The magnetic-circular-dichroism (m.c.d.) spectra of oxidized 'resting' bovine cytochrome c oxidase and the cyanide-inhibited form are reported at 5.15 T and at 4.2 K along with m.c.d. magnetization curves plotted at selected wavelengths. In both spectra there are features at 790nm and 1564nm due to Cua and haem a respectively, the e.p.r.-detectable components of the enzyme. There is a new peak at 1946nm only in the spectrum of the cyanide-inhibited enzyme. Arguments are advanced that assign this to low-spin ferric haem a3 bridged to Cua3, thereby forming a ferromagnetically coupled pair of metal ions.     

5-n-Alkylresorcinols from grains of winter barley (Hordeum vulgare L.)

The resorcinolic lipid content and homologue composition of winter barley grains harvested at two field locations were evaluated. Depending on the crop location, the predominant alkylresorcinols identified were 1,3-dihydroxy-5-n-heneicosylbenzene or 1,3-dihydroxy-5-n-pentacosylbenzene. Both resorcinol concentration and their homologue profiles were diverse in samples harvested at different fields indicating a prevailing role of the environment upon the alkylresorcinol biosynthesis in cereals.     

Triple hybridization with cultivated barley (Hordeum vulgare L.)

Summary A crossing programme for trispecific hybridization including cultivated barley (Hordeum vulgare L.) as the third parent was carried out. The primary hybrids comprised 11 interspecific combinations, each of which had either H. jubatum or H. lechleri as one of the parents. The second parent represented species closely or distantly related to H. jubatum and H. lechleri. In trispecific crosses with diploid barley, the seed set was 5.7%. Crosses with tetraploid barley were highly unsuccessful (0.2% seed set). Three lines of diploid barley were used in the crosses, i.e. Gull, Golden Promise and Vada. Generally, cv Gull had high crossability in crosses with related species in the primary hybrid. It is suggested that Gull has a genetic factor for crossability not present in cv Vada and cv Golden Promise. One accession of H. brachyantherum used in the primary hybrid had a very high crossability (seed set 54.7%) in combination with cv Vada but no viable offspring was produced. In all, two trispecific hybrids were raised, viz. (H. lechleri x H. brevisubulatum) x Gull (2n=7–30) and (H. jubatum x H. lechleri) x Gull (2n=20–22). The first combination invariably had a full complement of seven barley chromosomes plus an additional chromosome no. 7, but a varying number of chromosomes (19–22) of the wild-species hybrid. The second combination had a full set of barley chromosomes. The meiotic pairing was low in both combinations.     

Isolation and characterization of aleurone protoplasts from a malting variety of barley (Hordeum vulgare L.)

Summary A procedure has been developed to isolate protoplasts from mature aleurone layers of the malting variety Alexis and four other barley genotypes. It combines induction of endogenous cell wall degrading enzymes together with use of Onuzuka cellulase R 10 and driselase and results in better yields for two varieties than can be obtained with the huskless variety Himalaya. The viability of the freshly isolated protoplasts is greater than 90% and in spite of the presence of gibberellic acid during isolation procedures, most of the protoplasts are at an early developmental stage, as judged by ultrastructure. Gibberellic acid-induced changes in protoplast structure resemble those reported for Himalaya protoplasts. The protoplasts secrete both -amylase (EC 3.2.1.1) and (1-3, 1-4)--glucanase (EC 3.2.1.73) into the surrounding medium. Transfection studies using a low pI -amylase promoter to direct chloramphenicol acetyltransferase expression in aleurone protoplasts from Alexis and Himalaya revealed significant differences in their hormone responsiveness. In the absence of hormones, low levels of expression of the reporter enzyme were obtained in Alexis protoplasts, while high levels were characteristic for Himalaya protoplasts. An 8-fold increase in the expression of the reporter gene was induced by supplying the transfected Alexis protoplasts with gibberellin A₃, whereas expression in Himalaya protoplasts remained unchanged. When Himalaya protoplasts were isolated from aleurone layers that had not been incubated with GA₃ during the initial stages of protoplasting (the classical procedure), the hormone response of the promoter was 2.5-fold. It is thus possible to optimize the aleurone protoplast isolation procedure for different barley genotypes and mutants of interest in studies of transgenic gene expression and hormone induced secretion of proteins from this unique secretory plant tissue.Abbreviations ABA abscisic acid - APIM aleurone protoplast isolation medium - CAT chloramphenicol acetyltransferase - EDTA ethylenediamine tetraacetic acid - ER endoplasmic reticulum - GA₃ gibberellin A₃ - IgG immunoglobulin G - MES 2-(N-morpholino)-ethanesulfonic acid - PAGE polyacrylamide gel electrophoresis - PEG polyethylene glycol - pI isoelectric point - PIPES piperazine N,N-bis-(diethanesulfonic acid) - SDS sodium dodecyl sulfate     

The regulation of the biosynthesis of glutathione in leaves of barley (Hordeum vulgare L.)

Between 50 and 65% of the glutathione in barley leaves was present in the chloroplasts depending upon the light regime. However, only 66–76% of the chloroplast glutathione was present in the reduced state (GSH) as opposed to 97–98% of that in the cytoplasm. In shoots treated with the catalase inhibitor aminotriazole and in shoots of the catalase deficient barley mutant RPr 79/4 exposed to air, the glutathione level increased 3-fold in 8 h in the light. The increase was accounted for by a rise in both the chloroplast and cytoplasm level of oxidised glutathione (GSSG), the GSH concentration remained relatively constant in both compartments. Only 2–3% of applied ³⁵SO₄ was metabolised to glutathione by wild-type shoots. In aminotriazole-treated plants this value rose to 17.9% and in the mutant RPr 79/4 exposed to air to 32%.     

Transgenic barley (Hordeum vulgare L.) by electroporation of protoplasts

Summary Protoplasts isolated from calli derived from cultured microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar) were transformed with the neomycin phosphotransferase marker gene (nptII) by electroporation. Screening of the regenerated plants for the NPTII activity by gel assay resulted in three positive signals. Southern blot analysis and NPTII assays of second and third generation plants confirmed the genomic integration of the transferred gene and that the new trait was inherited by the progeny.     

Production and diurnal utilization of assimilates in leaves of spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.)

Rates of CO₂ fixation during the light period and the rates of CO₂ release during the night period were measured using mature leaves from 39- to 49-d-old spinach (Spinacia oleracea L., US Hybrid 424; grown in 9 h light, 15 h darkness, daily) and mature leaves from 21-d-old barley (Hordeum vulgare L., cv. Apex; grown in 14 h light, 10 h darkness, daily). At certain times during the light and dark periods leaves were harvested for assay of their contents of soluble carbohydrates, starch, malate and the various amino acids. Evaluation of the results of these measurements shows that in spinach and barley leaves 46% and 26%, respectively, of the carbon assimilated during the light period is deposited in the leaves for export during the night period. Taking into account the carbon consumption in the source leaves by dark respiration, it is evaluated that rates of assimilate export during the light period from spinach and barley leaves [38 and 42 atom C · (mg Chl)^–1 · h^–1] are reduced in the dark period to 16 atom C · (mg Chl)^–1 · h^–1 in both species. The calculated C/N ratios of the photoassimilates exported during the dark period were 0.029 and 0.015 for spinach and barley leaves, respectively.This work was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. Dieter Heineke for stimulating discussions and Mrs. Petra Hoferichter and Mrs. Marita Feldkämper for their technical assistance.     

Estimation of genetic parameters in barley (Hordeum vulgare L.)

Summary Four different sets of partial diallels were analysed for their relative efficiencies for estimating the genetic parameters in barley: (1) partial diallel with 12 parents, each involved in only 5 crosses; (2) partial diallel with 12 parents, each involved in only 3 crosses; (3) partial diallel with 8 parents, each involved in only 5 crosses; and (4) partial diallel with 8 parents, each involved in only 3 crosses. In partial diallel experiments, the estimates of gca effects were higher than in those of full diallel. Ranking pattern of the parents on the basis of gca effects in partial diallels deviated considerably from the ranking in full diallel. With decreasing s per parent, the deviation in ranking was also more. This clearly suggests the unsuitability of partial diallel analysis for screening high general combiners. Selection of best cross combinations is also not possible because only a sample of crosses (s out of n) is analysed under partial diallel so that there is every possibility of the best cross being excluded from the sample. In general, overdominance was exhibited, indicating that there is ample scope for heterosis breeding in barley.     

Mapping of QTL associated with nitrogen storage and remobilization in barley (Hordeum vulgare L.) leaves

Nitrogen uptake and metabolism are central for vegetative and reproductive plant growth. This is reflected by the fact that nitrogen can be remobilized and reused within a plant, and this process is crucial for yield in most annual crops. A population of 146 recombinant inbred barley lines (F(8) and F(9) plants, grown in 2000 and 2001), derived from a cross between two varieties differing markedly in grain protein concentration, was used to compare the location of QTL associated with nitrogen uptake, storage and remobilization in flag leaves relative to QTL controlling developmental parameters and grain protein accumulation. Overlaps of support intervals for such QTL were found on several chromosomes, with chromosomes 3 and 6 being especially important. For QTL on these chromosomes, alleles associated with inefficient N remobilization were associated with depressed yield and higher levels of total or soluble organic nitrogen during grain filling and vice versa; therefore, genes directly involved in N recycling or genes regulating N recycling may be located on these chromosomes. Interestingly, the most prominent QTL for grain protein concentration (on chromosome 6) did not co-localize with QTL for nitrogen remobilization. However, QTL peaks for nitrate and soluble organic nitrogen were detected at this locus for plants grown in 2001 (but not in 2000). For these, alleles associated with low grain protein concentration were associated with higher soluble nitrogen levels in leaves during grain filling; therefore, gene(s) found at this locus might influence the nitrogen sink strength of developing barley grains.     

Estimation of genetic parameters in barley (Hordeum vulgare L.)

Summary Four exotic and four indigenous strains of barley were used for making diallel crosses. The sets of parents and crosses making full, half and quarter diallel were analysed in a randomized block design for plant height, number of effective tillers, ear length, grain yield per plant, 100 grain weight and number of grains per ear.The three alternatives of diallel were similar with respect to the estimates of degree of dominance, general combining ability and specific combining ability, indicating that all these three methods of diallel were equally efficient. However, as the number of entries are minimum in quarter diallel, it would be economical in terms of cost, time and labour to estimate genetic parameters by this method. Average degree of dominance was found in the range of overdominance. The ranking of parents on the basis of their array mean was similar to the ranking based on gca effects. Similarly, the ranking of crosses on the basis of per se performance was similar to the ranking based on sca effects. This suggests that the selection of best general combiner or best cross combinations may be easier and more effective through array mean for per se performance rather than through high gca and sea effects, respectively. From among 56 crosses, IB-226 X X C-164 was the one which showed superiority for maximum number of characters followed by AB-12/59 X PTS-57. High sea effect for plant height, ear length, grain yield, 100 grain weight and number of grains per ear was the result of cross between parents having high X low general combining ability, indicating additive X dominance type of gene interaction. For number of effective tillers, high sca was produced by low x low general combiners, indicating dominance x dominance gene interaction.Part of a Ph. D. thesis submitted by senior author to Haryana Agricultural University, Hissar.     

Chromosomal instabilities in callus tissue from haploid barley (Hordeum vulgare L.)

Callus culture was derived from haploid barley embryos after crossing withHordeum bulbosum. The callus tissue is cytologically heterogeneous, containing haploid, diploid and polyploid cells. Aneuploidy and karyokinetic irregularities were also observed. Some problems of chromosomal instabilities in plant tissue cultures are discussed.     

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.     

Advanced backcross QTL analysis in barley (Hordeum vulgare L.)

This paper reports on the first advanced backcross-QTL (quantitative trait locus) project which utilizes spring barley as a model. A BC(2)F(2) population was derived from the initial cross Apex ( Hordeum vulgare ssp. vulgare, hereafter abbreviated with Hv) x ISR101-23 ( H. v. ssp. spontaneum, hereafter abbreviated with Hsp). Altogether 136 BC(2)F(2) individuals were genotyped with 45 SSR (simple sequence repeat) markers. Subsequently, field data for 136 BC(2)F(2) families were collected for 13 quantitative traits measured in a maximum of six environments. QTLs were detected by means of a two-factorial ANOVA with a significance level of P < 0.01 for a marker main effect and a marker x environment (M x E) interaction, respectively. Among 585 marker x trait combinations tested, 86 putative QTLs were identified. At 64 putative QTLs, the marker main effect and at 27 putative QTLs, the M x E interaction were significant. In five cases, both effects were significant. Among the putative QTLs, 29 (34%) favorable effects were identified from the exotic parent. At these marker loci the homozygous Hsp genotype was associated with an improvement of the trait compared to the homozygous Hv genotype. In one case, the Hsp allele was associated with a yield increase of 7.7% averaged across the six environments tested. A yield QTL in the same chromosomal region was already reported in earlier barley QTL studies.     

Aquaporin-facilitated water uptake in barley (Hordeum vulgare L.) roots

It is not known to what degree aquaporin-facilitated water uptake differs between root developmental regions and types of root. The aim of this study was to measure aquaporin-dependent water flow in the main types of root and root developmental regions of 14- to 17-d-old barley plants and to identify candidate aquaporins which mediate this flow. Water flow at root level was related to flow at cell and plant level. Plants were grown hydroponically. Hydraulic conductivity of cells and roots was determined with a pressure probe and through exudation, respectively, and whole-plant water flow (transpiration) determined gravimetrically in response to the commonly used aquaporin inhibitor HgCl(2). Expression of aquaporins was analysed by real-time PCR and in situ hybridization. Hydraulic conductivity of cortical cells in seminal roots was largest in lateral roots; it was smallest in the fully mature zone and intermediate in the not fully mature 'transition' zone along the main root axis. Adventitious roots displayed an even higher (3- to 4-fold) cortical cell hydraulic conductivity in the transition zone. This coincided with 3- to 4-fold higher expression of three aquaporins (HvPIP2;2, HvPIP2;5, HvTIP1:1). These were expressed (also) in cortical tissue. The largest inhibition of water flow (83-95%) in response to HgCl(2) was observed in cortical cells. Water flow through roots and plants was reduced less (40-74%). It is concluded that aquaporins contribute substantially to root water uptake in 14- to 17-d-old barley plants. Most water uptake occurs through lateral roots. HvPIP2;5, HvPIP2;2, and HvTIP1;1 are prime candidates to mediate water flow in cortical tissue.     

Isolation and characterization of an endonuclease synthesized by barley (Hordeum vulgare L.) uninucleate microspores

Few biochemical and molecular details are available on microspore growth and development. In this work, a nuclease was partially purified from diffusates of barley (Hordeum vulgare L.) microspores by using concanavalin-A as ligand. The chromatographic preparation contained a 34-kDa protein with nucleolytic activity; the enzyme (called BMN: barley microspore nuclease) was very stable at pH > 8.0 and temperatures below 50 degrees C. Activity was highest at pH 5.6 and increased almost exponentially with temperature until a breakpoint between activity and stability was reached at 70 degrees C. Although BMN was able to cleave RNA, the enzyme showed a remarkable preference for DNA, especially in the single-stranded form. The best homopolymeric substrates were poly(dA) and poly(A), whereas poly(dC), poly(G) and poly(I) were almost completely uncleaved. When incubated with intact nuclei, BMN caused a nucleosomal DNA ladder of approximately 200 bp. On the basis of DNA laddering, substrate specificity, Mg2+ -dependence and best performance at apoplastic pH, BMN can be referred to as a putative apoptotic nuclease involved in pollen development.     

Chromium uptake and transport in barley seedlings (Hordeum vulgare L.)

Summary Potassium chromate is more toxic to the growth of barley in solution culture than chromic chloride, though apparent uptake of the latter is much faster. Inhibitor studies indicate that CrO₄ ^2- uptake is active whereas Cr³⁺ uptake is passive, demonstrating that the two forms do not share a common uptake mechanism. Studies on the form of Cr inside root cells show that in plants fed CrO₄ ^2- the Cr remains largely unchanged whereas in plants fed Cr³⁺ a little CrO₄ ^2- (0.5 per cent) is produced. This conversion is dependent on the presence of living material and is probably enzymatic. Chromate uptake follows Michaelis-Menten kinetics at low concentration and is competitively inhibited by sulphate. Transport of chromium up the root is very slow, accounting for the low levels of Cr in the shoots. Chromate is transported better than Cr³⁺ though still to a very limited extent. These experiments provide a physiological basis for previous observations.