High yield isolation of mesophyll protoplasts from wheat, barley and rye

Efficient procedures are described for high-yield isolation of mesophyll protoplasts from spring wheat ( Triticum aestivum L. cv. Glenlea), winter wheat ( Triticum aestivum L. cv. Frederick), barley ( Hordeum vulgare L. cv. Bruce) and rye ( Secale cereale L. cv. Puma). Factors such as plant age, composition of the incubation medium during isolation, purification procedures and culture medium affect protoplast yield, viability and metabolic competence, as measured by light-dependent CO₂ fixation. Optimal osmolarity of the isolation medium was equivalent to 1.8 times that measured in the leaves of all plant material used. The presence of 2 m M ascorbic acid in the preincubation and isolation medium increased the yield by 50% and conserved viability and metabolic competence. The protoplasts were stable for up to 48 h without loss of either viability or of original activity of CO₂ fixation, which was in the order of 100 μmol CO₂ (mg chl)⁻¹h⁻¹.
In our MC-56 liquid medium these protoplasts regenerated cell walls within 72 h and a few divided.     

Cd - tolerance of maize, rye and wheat seedlings

The effect of cadmium on growth parameters of seedlings of maize, rye and wheat as well as the role of phytochelatins in Cd detoxication in these species were studied. Cadmium was found to inhibit root growth and decrease fresh weight and water content in roots and shoots of the studied plants. Although a considerably lower Cd accumulation was shown in maize seedlings than in other species, they were characterized by the highest sensitivity to cadmium. Among γ-Glu-Cys peptides synthetized by plant species, phytochelatins — glutathione derivatives predominated. In maize they were synthetized in amounts sufficient for binding the total pool of the metal taken up, and the detoxication mechanism was localized in their roots. Larger amounts of cadmium were accumulated in roots of wheat and rye, but the quantity of the formed γ-Glu-Cys peptides seems insufficient for detoxication of the metal.     

The protein synthesis inhibitors from wheat, barley, and rye have identical antigenic determinants

We have purified the protein synthesis inhibitors from wheat (tritin) and investigated their antigenic relationship to inhibitors from other grains and more distantly related plants. Three forms of tritin (tritin 1, 2, and 3) indistinguishable with regards to their molecular weight and enzymatic properties were found. Antiserum against the most abundant tritin, tritin 2, cross reacted with tritin 1, tritin 3, and with the translation inhibitors from barley and rye, but not with other plant-derived translational inhibitors. The results indicate that only closely related plants contain antigenically similar protein synthesis inhibitors.     

Subcellular localization of beta-glucosidase in rye, maize and wheat seedlings

The subcellular compartmentation of β -glucosidase was studied in rye, maize and wheat seedlings by immunocytochemical methods. For detection, we used a 10 nm gold-labeled secondary antibody, and results were observed using transmission electron microscopy. In all three species, β -glucosidase was found in plastids, cytoplasm and cell walls. In rye, gold particles were seen on cell walls and cytoplasm in epidermal cells of the root tip and shoot, in bundle sheath cells of the shoot and in all cells, except the vascular bundle cells of the coleoptile. Gold labeling was also observed in plastids of the bundle sheath cells of rye shoot tips and in cortical cells of root tips. In wheat, gold labeling was observed on cell walls and cytoplasm of epidermal cells in the shoot base and coleoptile, and on cell walls and plastids in epidermal cells of the root tip. In maize, gold labeling was mainly found in plastids or proplastids in vascular bundle cells and bundle sheath cells of the shoot, in bundle sheath cells of the coleoptile and in epidermal cells of the root. Some gold particles were also found in cell walls and cytoplasm of stomatal guard cells of the shoot base and vascular bundle cells of the shoot tip and in the cell walls of bundle sheath cells of the shoot tip and root tip epidermal cells. Results are discussed in relation to the role of β -glucosidase in hydroxamic acid release and overall defense mechanism of monocotyledons.     

Molecular evolution of the seed storage proteins of barley, rye and wheat

The major storage proteins (prolamins) of barley, rye and wheat are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. Because of this repetitive structure and their restricted distribution (only in grasses), it has been suggested that the prolamins are of recent origin. Contrary to this hypothesis, we show that parts of the non-repetitive domain of one group of prolamins are homologous with sequences present in a large group of seed proteins from monocotyledonous and dicotyledonous plants; including Bowman-Birk protease inhibitors, cereal inhibitors of alpha-amylase and trypsin, and 2 S globulin storage proteins of castor bean and oil seed rape. This implies an ancient origin for these non-repetitive domains. The origins of the repetitive domains are not known but may lie within the grasses.     

Chromosomal location of the genes for ferredoxin in wheat,barley and rye

Communicated by J.W. Snape     

Identification of guttation fluid proteins: the presence of pathogenesis-related proteins in non-infected barley plants

The main driving force behind water transport in plants is the air's low water potential. In the presence of high humidity, the transpiration process is halted and water transport is mainly sustained by the root pressure. The surplus of water following the removal of essential components (e.g. salts) is excreted by the plant via guttation through the hydathodes. When guttation occurs, the plant surface is wetted. These are the conditions that will allow epiphytic living, motile bacteria to move and to eventually enter the plant's interior via the hydathodes. The question arose as to whether the plant has developed a protection mechanism against motile bacteria in the vicinity of the hydathodes. Such a protection mechanism could use the well known pathogenesis-related (PR) proteins. Indeed, an analysis of the guttation fluid using one- and two-dimensional electrophoresis showed a clustering of approximately 200 proteins, primarily with isoelectric points in the acidic pH. Proteins identified using electrospray ionization mass spectroscopic analysis and western blot analysis belong mostly to the family of PR-proteins suggesting a role in plant protection against invaders. The protein profile of the guttation fluid was remarkably modified by treating plants with methyl jasmonic acid suggesting that the protein composition of the guttation fluid is controlled by internal and/or external stimuli.     

Regeneration potential of different wheat, rye and barley species in leaf explant culture

Regeneration potential of different wheat, rye and barley species in leaf explant culture. Comparative analysis of the induction ability of morphogenetic processes in vitro has been carried out in 16 wheat genotypes, 4 barley species and 6 rye genotypes. It has been shown that tetra- and hexaploid wheat species as well as wild barley species exhibited the highest embryogenic potential in the leaf explant culture while diploid wheat species and rye genotypes showed the lowest one. Genotypic dependence of processes of callus formation, induction of embryogenic calli and regeneration was revealed in the studied species.     

The chromosomal locations of leaf peroxidase genes in hexaploid wheat,rye and barley

Summary Eight leaf peroxidase isozymes were distinguished by IEF in Chinese Spring. Two genes which control the production of three of these isozymes were located on chromosome arms 1BS and 1DS by nullisomic analysis. These loci probably form part of a homoeoallelic series and have been designated Per-B1 and Per-D1 respectively. Analysis of chromosome 1B short arm terminal deletion stocks indicated that the Per-B1 locus is located between the nucleolar organiser region and another isozyme marker, Hk-B1. Two variant leaf peroxidase phenotypes were distinguished in a small sample of hexaploid wheat varieties. Analysis of wheat-alien addition and substitution lines identified homoeologous loci in rye (Per-R1) and barley (Per-H1).     

Tissue localization of β-glucosidase in rye, maize and wheat seedlings

The localization of β -glucosidase was determined at the tissue level in roots and shoots of rye, wheat and maize seedlings, using an immunohistochemical approach with antibodies directed against purified maize β -glucosidase as the primary antibody. In the roots, the β -glucosidase was found in the epidermis and the underlying cell layer. In the leaves, staining was seen in the epidermis (rye and wheat) and nearby vascular tissue (rye, wheat and maize). In all 3 species, β -glucosidase activity was highest in the coleoptile. Here the enzyme was restricted to the epidermis in wheat and to cells near the vascular tissue in maize, but was found in the whole tissue, except the vascular tissue, in rye. Maize, wheat and rye all contain hydroxamic acid glucosides and results are discussed in relation to a proposed role of β -glucosidase as part of a defense system releasing hydroxamic acid aglucone upon herbivore attack, pathogen penetration or aphid infestation.     

The purification and characterization of homologous high molecular weight storage proteins from grain of wheat,rye and barley

Summary Homologous high molecular weight storage prolamins were purified from grain of wheat, rye and barley using combinations of gel filtration, ion-exchange chromatography and preparative isoelectric focusing. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that the components were single bands with apparent mol.wts. of above 100,000. Molecular weights determined by sedimentation equilibrium ultracentrifugation were considerably lower; 54,700, 67,600 and 69,600 for the components from barley, rye and wheat respectively. Amino acid analysis showed the presence of 13.6 to 16.5 mol% glycine, 29.6 to 34.0 mol% glutamate + glutamine, 11.4 to 13.7 mol% proline and a total of 4.0 to 5.7 mol% basic amino acids. Automated N-terminal amino acid sequencing of the component from wheat showed the presence of cysteine residues at positions 5 and 10, and this is discussed in relation to the possible role of these proteins in the visco-elastic gluten network.     

Occurrence and synthesis of lectin in coleoptiles of germinating wheat,rye and rice seedlings

Occurrence, synthesis and localization of lectins in coleoptiles of 3-day old seedlings of wheat, rye, barley and rice were studied by a combination of high resolution ion-exchange chromatography, in vivo labelling with ³⁵S-cysteine and immunocytochemistry. Whereas no lectin can be isolated or localized in barley coleoptile, 1.9 and 40 ng of lectin per coleoptile was obtained from wheat and rye respectively. Wheat germ agglutinin was localized in the outer layer of the wheat coleoptile and both inner and outer layers of rye coleoptile displayed a specific reaction. In rice, 250 ng of lectin is present in the coleoptile and is distributed throughout this organ. Wheat coleoptiles synthesize no lectin and rye coleoptiles synthesize minute amounts while those from developing rice seedlings incorporate reasonable amounts of ³⁵S-cysteine into lectin.Abbreviations FPLC Fast Protein Liquid Chromatography - GlcNAc N-acetylglucosamine - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Constitutive and cold-induced resistance of rye and wheat seedlings to oxidative stress

The influence of cold hardening of rye (Secale cereale L.) and wheat (Triticum aestivum L.) seedlings on their resistance to the oxidative stress (OS) agents, namely, 50 mM hydrogen peroxide or 5 mM iron (II) sulfate was studied. Unhardened rye seedlings were more resistant to hydrogen peroxide than those of wheat, since their growth was less inhibited, and they accumulated lesser amounts of lipid peroxidation products after a treatment with H₂O₂. The interspecific differences in responses to FeSO₄ were less significant. The unhardened seedlings of rye, in comparison with those of wheat, possessed more active guaiacol peroxidase (GPO) and more levels of anthocyanins and proline. In response to the OS agents, the unhardened rye seedlings enhanced activities of superoxide dismutase and catalase, whereas the wheat seedlings enhanced GPO activity and proline content. The cold hardening (6 days at 2°C) increased activities of antioxidant (AO) enzymes, contents of proline, sugars, and anthocyanins in seedlings of both species, and made the seedlings more resistant to the OS agents. After the cold hardening, rye seedlings were more resistant to OS than wheat seedlings. The hardened seedlings of both species activated the AO enzymes in response to H₂O₂ or FeSO₄ greater than the unhardened ones. However, the hardened wheat seedlings, in contrast to the unhardened ones, did not augment the proline content in contact with the OS agents. The conclusion was drawn on different contributions of AO enzymes and low-molecular weight compounds to the basal and induced by the cold—hardening resistances of rye and wheat seedlings to OS.     

Comparative RFLP maps of the homoeologous group-2 chromosomes of wheat,rye and barley

Summary Genetic maps of the homoeologous group-2 chromosomes were constructed, comprising 114 loci in wheat and 34 loci in rye. These include the genes coding for sucrose synthase, sedoheptulose-1,7-bisphosphatase, a bZIP protein (EmBP-1), a peroxidase and an abscisic acid-induced protein (#7). Overall, gene orders are highly conserved in the genomes of wheat, barley and rye, except for the distal ends of chromosome arms 2BS and 2RS, which are involved in interchromosomal, probably evolutionary, translocations. Clustering of loci in the centromeric regions of the maps, resulting from the concentration of recombination events in the distal chromosomal regions, is observed in wheat and rye, but not in barley. Furthermore, loci for which homoeoloci can be detected in rye and barley tend to lie in the centromeric regions of the maps, while non-homoeologous and wheat-specific loci tend to be more evenly distributed over the genetic maps. Mapping of the group-2 chromosomes in the intervarietal Timgalen x RL4137 cross revealed that the T. timopheevi chromosome segment introgressed into chromosome 2B in Timgalen is preferentially transmitted. Recombination is also greatly reduced in that segment.     

Adaptation of membrane fluidity of rye wheat seedlings according to temperature

Order parameters of chloroplast membrane lipids of rye wheat seedlings differing in cold hardiness were compared before after hardening. Seedlings grown at 25° exhibited similar membrane microviscosities. When hardened, the cultivars most resistant to freezing temperatures possessed the most fluid membranes, while those sensitive to cold were unable to alter them. Changes in linolenic acid levels alone cannot be responsible for the observed phenomena.     

The isolation of high molecular weight DNA from wheat,barley and rye for analysis by pulse-field gel electrophoresis

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals-hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6 Mb. DNA samples prepared by this method were shown to be useful for restriction analysis using both frequent and rare cutting enzymes.     

Growth responses of barley and wheat seedlings to lead and cadmium

The responses of barley and wheat seedlings to lead and cadmium ions in relation to the concentration and duration of treatment were studied. Both metals inhibited seed germination and growth of roots and shoots, but the toxic effect of cadmium was observed at lower concentrations. Inhibition of seedling growth was already recorded already within a day after the beginning of the treatment, and then increased further. The sensitivity of the processes studied to both the metals decreased in the order: root growth, shoot growth and seed germination. The resistance of barley and wheat to lead was similar, whereas the resistance to cadmium was higher in barley