Antioxidative protection in the leaves of dark-senescing intact barley seedlings

In order to elucidate the possibility of in vivo oxidative modification of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) as a triggering mechanism for its preferential degradation early in senescence, some antioxidant compounds, protective enzymes, H₂O₂ and protein carbonylation levels were studied in the leaves during dark-induced senescence of barley (Hordeum vulgare L. cv. “Obzor”) seedlings. Analyses were performed in extracts as well as in purified chloroplasts. Some weakening of the antioxidative protection was detected during the treatment: diminution in the ascorbate and non-protein SH (mainly glutathione) pools, lower activities of superoxide dismutase, guaiacol and ascorbate peroxidases. However, no accumulation of H₂O₂ was found, lower level of protein carbonylation in darkness was measured and the percentage of reduced ascorbate was maintained high. Data concerning antioxidant compounds in chloroplasts revealed some impairment of the ascorbate and glutathione pools under induced senescence - the level of non-protein thiols declined during early senescence whereas the ascorbate pool was not significantly changed. The percentage of reduced ascorbate remained high in the chloroplasts and the activities of superoxide dismutase and of ascorbate peroxidase were conserved. Taken together the results are not in accordance with the possibility of in vivo oxidative modification of Rubisco in the case of dark-induced senescence. Our data bring some support to the view about redox regulation of Rubisco turnover in senescence through the pool of the low-molecular chloroplastic thiols.     

The effect of cadmium on shoot apical meristems of barley

We studied the effects of cadmium acetate at various concentrations (200 to 800 mg/kg substrate) on growth and development of shoot apical meristem of the barley plants (Hordeum vulgare L.) under the conditions of vegetative experiment. It was shown that in the presence of increasing cadmium concentrations in the soil substrate, the apex length and number of inflorescence elements decreased and the rate of organogenesis slowed down, thus affecting the spike potential productivity and morphological parameters of barley plants at the flowering stage. It is possible that the negative effect of cadmium on the shoot apical meristem is associated with its influence on division of the apex cells.     

Effects of chromosome reconstruction of barley genome on tissue culture response using two different regeneration systems

Mature embryos and seedlings from mature embryos of one standard and five reconstructed karyotypes of barley (Hordeum vulgare L.) were cultured in vitro to study the influence of repositioning of particular chromosome segments of barley genome on the regeneration response. A comparative analysis of the regeneration response of a reconstructed karyotype having complete and well characterized rearrangement of the chromosome complement, and its four parental lines were used as experimental material. Depending on the source of explants two systems of in vitro culture were applied. The regeneration ability was found to be significantly influenced by both chromosome reconstruction and protocol applied. Possible reasons underlying the effects of chromosomal reconstruction on the regeneration response of karyotypes are briefly discussed.     

Effects of brassinosteroids on barley root growth,antioxidant system and cell division

Homobrassinolide (HBR), which is one of the most biologically active forms of Brassinosteroids (BRs), was used to examine the potential effects of hormone on root germination, antioxidant system enzymes and cell division of barley (Hordeum vulgare L.). Seeds were germinated between filter papers in 0.1, 0.5 and 1.0 μM HBR-supplemented distilled water for 48 h at dark with their controls. HBR application increased especially the primary root growth significantly with increasing concentrations when compared with the control materials and reached two fold increase in 1.0 μM HBR treated material. Treated and untreated control group roots were fixed in 1:3 aceto-alcohol and aceto-orcein preparations were made. Roots treated with HBR showed more mitotic activity, mitotic abnormalities and significant enlargements at the root tips when compared with control material. HBR application decreased total soluble protein content, superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.11) activities significantly at 1.0 μM HBR concentration. Data presented here is one of the first detailed analyses of HBR effect on barley root development.     

Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars

Effects of cadmium (Cd(2+)) on photosynthetic and antioxidant activities of maize (Zea mays L.) cultivars (3223 and 32D99) were investigated. Fourteen-day-old cultivar seedlings were exposed to different Cd concentrations [0, 0.3, 0.6 and 0.9mM Cd(NO(3))(2).4H(2)O] for 8 days. The results of chlorophyll fluorescence indicated that different levels of Cd affected photochemical efficiency in 3223 much more than that in 32D99. In parallel, the level of Cd at 0.9mM caused oxidative damage but did not indicate cessation of PSII activity of the cultivars; plant death was not observed at highly toxic Cd levels. Additionally, the increase in Cd concentration caused loss of chlorophylls and carotenoid and membrane damage in both cultivars, but greater membrane damage was observed in 3223 than in 32D99. Depending on Cd accumulation, a significant reduction in dry biomass was observed in both cultivars at all Cd concentrations. The accumulation of Cd was higher in roots than in leaves for both cultivars. Nevertheless, cultivar 3223 transferred more Cd from roots to leaves than did 32D99. On the other hand, our results suggest that there were similar responses in SOD, APX and GR activities with increasing Cd concentrations for both cultivars. However, POD activity significantly increased at highly toxic Cd levels in 32D99. This result may be regarded as an indication of better tolerance of the Z. mays L. cultivar 32D99 to Cd contamination.     

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP⁺ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.Abbreviations ABA abscisic acid - DCPIP 2,6-dichlorophenol-indo-phenol - DOC deoxycholate - Me-JA methyl jasmonate - NADH-FeCN-ox NADH ferricyanide oxidoreductase - NADPH-FeCN-ox NADPH ferricyanide oxidoreductase     

The stress- and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms

Abscisic acid (ABA) induces the expression of a battery of genes in mediating plant responses to environmental stresses. Here we report one of the early ABA-inducible genes in barley (Hordeum vulgare L.), HVA22, which shares little homology with other ABA-responsive genes such as LEA (late embryogenesis-abundant) and RAB (responsive to ABA) genes. In grains, the expression of HVA22 gene appears to be correlated with the dormancy status. The level of HVA22 mRNA increases during grain development, and declines to an undetectable level within 12 h after imbibition of non-dormant grains. In contrast, the HVA22 mRNA level remains high in dormant grains even after five days of imbibition. Treatment of dormant grains with gibberellin (GA) effectively breaks dormancy with a concomitant decline of the level of HVA22 mRNA. The expression of HVA22 appears to be tissue-specific with the level of its mRNA readily detectable in aleurone layers and embryos, yet undetectable in the starchy endosperm. The expression of HVA22 in vegetative tissues can be induced by ABA and environmental stresses, such as cold and drought. Apparent homologues of this barley gene are found in phylogenetically divergent eukaryotic organisms, including cereals, Arabidopsis, Caenorhabitis elegans, man, mouse and yeast, but not in any prokaryotes. Interestingly, similar to barley HVA22, the yeast homologue is also stress-inducible. These observations suggest that the HVA22 and its homologues encode a highly conserved stress-inducible protein which may play an important role in protecting cells from damage under stress conditions in many eukaryotic organisms.     

The effects of DNA hypomethylating drugs on androgenesis in barley (Hordeum vulgare L.)

Summary The effects of DNA hypomethylating drugs (azacytidine and ethionine) on induction of microspore-derived calluses and embryos were studied in barley (Hordeum vulgare L.) ev. Igri. The results were as follows: (1) Yield of calluses and embryos pretreated with the different concentrations of azacytidine for 3 d was several-fold higher than that of the control. The highest yield of calluses and embryos in all treatments appeared at a concentration of 3 mg l⁻¹, which reached 11.03 per anther. It was 110-fold higher than the control. (2) There was a significant difference in yield of calluses and embryos between the different days of pretreatment. The highest yield was obtained at a 3-d pretreatment. If the period of pretreatment was shorter or longer than 3 d, yield of calluses and embryos was reduced sharply, and was similar to that of the control. (3) The data obtained with ethionine pretreatment were very similar to those obtained with azacytidine. (4) Tests on the different methods of pretreatment showed that yield of calluses and embryos pretreated with distilled H₂O, mannitol, azacytidine, and ethionine was much higher than other pretreatments and the control, and reached 6.53–11.39 per anther. The yield of calluses and embryos pretreated with DNA hypomethylating drugs was higher than with mannitol. However, pretreatment with hypomethylation drugs supplemented with induction medium was not effective.     

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv Igri) were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.     

Correlation of gibberellin-induced growth, polyamine levels and amine oxidases in epicotyl, root and leaf blade of barley seedlings

The potential role of diamine oxidase (DAO) and polyamine oxidase (PAO) in relation to polyamines was investigated in epicotyls, roots and leaf blades at 3 and 6 days after gibberellic acid (GA) application in barley (Hordeum vulgare L.) seedlings of cvs. Maythorpe (non-mutant parent) and Golden Promise (semi-dwarf mutant). There was a significant increase in epicotyl and leaf-blade elongation rates in GA-treated seedlings of cv. Maythorpe as compared to cv. Golden Promise. DAO and PAO were detectable in all segments of the leaf blade, but the highest activities were present in basal segments. These enzymes, which are thought to have a role in the elimination of cellular polyamines, increased in activity following GA application compared to controls. Application of 10⁻⁶ M GA to the first leaf, significantly increased endogenous bound putrescine (Put) levels in both the epicotyl and leaf blade of cv. Maythorpe. In contrast, there was only a slight increase in cv. Golden Promise. Levels of soluble Put increased in roots and leaf blades of both cultivars following GA treatment but the effect was greatest in leaves of cv. Maythorpe. It is suggested that polyamines may play a role in GA-induced epicotyl and leaf-blade elongation in barley.     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.     

Extraction and genetic control of two new water-soluble proteins of mature barley seed

This paper describes the genetic control of two new water-soluble proteins in barley. Water-soluble proteins (WSPs) of mature barley seed form part of the albumin/globulin class of seed proteins. They can be extracted from hand-milled grain with water, though some WSPs are more efficiently extracted with a solution of 10 mM dithiothreitol. Polymorphisms for WSPs were detected in isoelectric focusing gels incorporating various ampholine combinations. Two new controlling genes (Wsp4 andWsp5) have been identified and located using wheat/barley chromosome addition lines and barley doubled haploids.Wsp4 is located on chromosome 2 (2H), andWsp5 was found to be tightly linked toWsp2 on the long arm of chromosome 7 (5HL). Segregation of a sixth gene (Wsp6) is also described, but this has not been mapped. The results are discussed with respect to other previously mappedWsp loci.This work was funed by the Scottish Office of Agriculture and Fisheries Department and the Agricultural and Food Research Council.     

Response of barley seedlings to UV-B radiation as affected by NaCl

The response of barley seedlings, subjected to 150 mmol/L NaCl for 4 days at different light regimes (4 d in the light, 4 d in darkness and a 12 h light/dark cycle) before UV-B radiation was investigated. NaCl treatment resulted in a decrease of total chlorophyll content and an increase in H2O2, free proline and lipid peroxidation, as quantified by measurement of malondialdehyde. Significantly more proline was accumulated in the light than in darkness. The combination of UV-B and NaCl treatment produced an additive effect on most of the parameters studied. UV-B radiation reduced the chlorophyll/carotenoids ratio and photochemical efficiency of PSII as estimated by chlorophyll fluorescence. NaCl pre-exposure decreased H2O2 generation and lipid peroxidation and alleviated the inhibitory effect of UV-B on PSII activity. Proline accumulated under salt stress conditions might be one of the reasons for the observed tolerance of barley seedlings to UV-B radiation.     

Analysis of genetic diversity of hordein in wild close relatives of barley from Tibet

We analyzed genetic diversity in the storage protein hordein encoded at Hor-1, Hor-2 and Hor-3 loci in seeds from 211 accessions of wild close relatives of barley, Hordeum vulgare ssp. agriocrithon and H. vulgare ssp. spontaneum. Altogether 32, 27 and 13 different phenotypes were found for Hor-1, Hor-2 and Hor-3, respectively. A comparison of our results with those of previous studies indicates that Tibetan samples reflect the highest diverse level of hordein phenotypes when compared to samples from Israel and Jordan. This high degree of polymorphism supports the hypothesis that Tibet is one of the original centers of H. vulgare L.Communicated by H.F. Linskens     

Amino acid substitutions of the limit dextrinase gene in barley are associated with enzyme thermostability

Limit dextrinase (LD) is a key enzyme in determining the malting quality. A survey of 60 barley varieties showed a wide range of variation for the enzyme activity and thermostability. Galleon showed low enzyme activity and high thermostability while Maud showed high activity and low thermostability. Alignment of the LD amino acid sequences of Galleon and Maud identified seven amino acid substitutions Lys/Arg-102, Thr/Ala-233, Ser/Gly-235, Gly/Ala-298, Cys/Arg-415, Ala/Ser-885 and Gly/Cys-888. Genetic diversity of LD was investigated using single strand conformation polymorphism based on the amino acid substitutions. Only limited genetic variation was detected in the current malting barley varieties, although wide variation was observed in the wider barley germplasm. The Thr/Ala-233 and Ala/Ser-885 substitutions were associated with enzyme thermostability (P < 0.0001), but no polymorphism was associated with the enzyme activity. This result was confirmed from further sequence analysis. The results will provide a tool for understanding and selection of high LD thermostability.     

Exogenous treatment with salicylic acid leads to increased antioxidant capacity in leaves of barley plants exposed to paraquat

Our previous study suggests that salicylic acid mediates tolerance in barley plants to paraquat (Ananieva et al. 2002). To further define the role of SA in paraquat induced responses, we analysed the capacity of the antioxidative defence system by measuring the activities of several antioxidative enzymes: superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), dehydroascorbate reductase (DHAR, EC 1.8.5.1), catalase (CAT, EC 1.11.1.6), and guaiacol peroxidase (POX, EC 1.11.1.7). Twelve-day-old barley seedlings were supplied with 500 micromol/L SA or 10 micromol/L Pq via the transpiration stream and kept in the dark for 24 h. Then they were exposed to 100 micromol m(-2) s(-1) PAR and samples were taken 6 h after the light exposure. Treatment of seedlings with 10 micromol/L Pq reduced the activity of APX and GR, did not affect the activity of POX and DHAR but caused over a 40% increase in the activity of CAT. Pre-treatment with 500 micromol/L SA for 24 h in the dark before Pq application increased the activities of the studied enzymes in both the chloroplasts (SOD activity) and the other compartments of the cell (POX, CAT activity). The effect of SA pre-treatment was highly expressed on DHAR and POX activity. The data suggest that SA antagonizes Pq effects, via elicitation of an antioxidative response in barley plants.     

A consensus linkage map of barley

A consensus linkage map of the barley genome was constructed. The map is based on six doubled haploid and one F₂ population. The mapping data for three of the doubled haploid populations was obtained via the GrainGenes database. To allow merger of the maps, only RFLP markers that produce a single scorable band were included. Although this reduced the available markers by about half, the resultant map contains a total of 587 markers including 87 of known function. As expected, gene order was highly conserved between maps and all but two discrepancies were found in closely linked markers and are likely to result from the small population sizes used for some maps. The consensus map allows the rapid localisation of markers between published maps and should facilitate the selection of markers for high-density mapping in defined regions.     

A comparison of Hordeum bulbosum-mediated haploid production efficiency in barley using in vitro floret and tiller culture

Summary A high efficiency of Hordeum bulbosum-mediated haploid production in barley has been achieved using a floret culture technique in which florets pollinated with Hordeum bulbosum are cultured on modified N₆ media containing 0.5 mg/l kinetin and 1.2 mg/l2,4-D. Cultures were maintained at 25 °C with a 16 h photoperiod for 9 days before embryo rescue. In a comparison of haploid production efficiency using five F₁ hybrids from winter x winter and winter x spring barley crosses, 41.6 haploid plants/100 florets pollinated were produced using floret culture. Using detached tiller culture, 13.5 haploid plants/100 florets pollinated were produced. Higher efficiencies achieved with floret culture are attributed to the formation of larger, differentiated embryos. Such embryos lead to higher frequencies of plant regeneration. The F₁ from a winter x winter cross was inferior in haploid production compared to F₁s from winter x spring crosses. No genotype x technique interaction was observed.Oregon Agricultural Experiment Station Technical Paper No. 8653