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.     

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.     

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     

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.     

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 effect of selenium on sulfur uptake by barley and rice

Because of their chemical and physical similarities, plant uptake of S and Se are closely related. Barley (Hordeum vulgare L.) and rice (Oryza sativa L.) were grown in greenhouse solution culture to examine the synergistic interactions between SO₄ and Se⁶⁺ in plant uptake. In the presence of low concentrations of solution SO₄, shoot and root yields were decreased with additions of Se⁶⁺. However, when SO₄ was present in elevated concentrations, no Se-induced yield reduction occurred. A synergistic interaction between SO₄ and Se⁶⁺ caused an increase in the shoot S concentrations with increasing concentrations of Se⁶⁺ at low SO₄ solution concentrations. At elevated SO₄ concentrations, no synergism was osberved. Selenium had a lesser effect on the S concentration in plant roots.     

Generative cell division and sperm cell formation in barley

Summary Shortly before and during division, the generative cell of barley (Hordeum vulgare L.) is located near the vegetative nucleus, in the peripheral layer of the highly vacuolated vegetative cell at the aperture pole. This position is also characteristic of the two resulting sperm cells. Conventional mitosis of the generative cell is followed by cytokinesis through cell plate formation. Just after division, the two sperm cells are enclosed together within a common inner vegetative cell plasma membrane, and they gradually separate from each other only during pollen maturation. The space between the generative or sperm cell plasma membrane and the vegetative cell plasma membrane is very thin and appears to be devoid of a cell wall. Both the generative cell and the young sperm cells contain a normal set of organelles; plastids devoid of starch are only sporadically observed. Our data indicate that in Hordeum vulgare the generative cell divides after migrating inside the pollen grain. This follows the pattern of development well established for several species with tricellular pollen.     

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.     

Contact with ballotini (glass spheres) stimulates exudation of iron reducing and iron chelating substances from barley roots

Contact between roots and Fe-containing solid substrate is known to facilitate acquisition of iron by plants, but the actual mechanism of this contact effect is not yet clear. This study was undertaken to evaluate the effect of root contact with ballotini (glass spheres) on exudation of substances capable of reducing or chelating insoluble Fe(III) compounds by the roots of barley (Hordeum vulgare L. cv. Europa) seedlings. Seedlings with roots encountering mechanical impedance (i.e., in contact with ballotini) produced more lateral roots than the seedlings with unimpeded (i.e., freely suspended) roots in the nutrient solution. Nutrient solution bathing roots in contact with ballotini showed higher concentrations of Fe(III)-chelating (83% on day 7) and Fe(III)-reducing (107% on day 12) substances than solutions bathing unimpeded roots. The pH of all solutions rose continuously during the course of the experiment but was always lower (by a nonsignificant degree) in the solutions with roots in contact with ballotini than in those with unimpeded roots. The data indicate that under natural soil conditions the amount of Fe-chelating and Fe-reducing root exudates may be higher than is usually measured from roots of terrestrial plants artificially suspended in nutrient solution.     

大麦与油菜种皮特异启动子表达模式的比较

启动子位于转录起始位点上游并能特异性地结合RNA聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得Gerb和Bntt两个种皮特异性启动子,并对其进行生物信息学分析,构建了Gerb::GUS和Bntt::GUS植物表达载体并转化拟南芥,通过组织化学染色观察了GUS的表达情况。结果表明:两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件;转基因拟南芥幼苗期,大麦Gerb种皮特异启动子驱动GUS全株表达且子叶和下胚轴较真叶和根中表达量高;油菜Bntt种皮特异启动子表达较弱;成株期,Gerb在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性;Bntt仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb表达模式未发生显著变化,而Bntt仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子Gerb和油菜种皮特异性启动子Bntt在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。     

Effects of aluminum and cadmium toxicity on growth and antioxidant enzyme activities of two barley genotypes with different Al resistance

A hydroponic experiment was carried out to study genotypic differences in effect of Al and Cd on growth and antioxidant enzyme activities by using 2 two-row winter barley genotypes (Hordeum vulgare L.) with different Al resistance, the relatively resistant Gebeina and the sensitive Shang 70–119. The seedling growth, presented as shoot height, root length and dry weight of root and shoot, and tillers per plant were inhibited by all stress treatments, including low pH, 100 M Al (pH 4.0) and 1.0 M Cd+100 M Al (pH 4.0), while 1.0 M Cd showed a slight stimulation of growth. The inhibition was more severe in 1.0 M Cd +100 M Al (pH 4.0) than in 100 M Al (pH 4.0), indicating that the effect of Cd and Al is synergistic. Al-sensitive genotype Shang 70–119 was more inhibited than Al-resistant genotype Gebeina. Proline concentration in leaves was significantly increased when plants were exposed to all stress treatments, being more pronounced in Shang 70–119 than in Gebeina. A highly significant increase in malonaldehyde (MDA) concentration, and a stimulation of superoxide dismutase (SOD) and peroxidase (POD) activities were recorded in the plants subjected to low pH, 100 M Al (pH 4.0) and 1.0 M Cd +100 M Al(pH 4.0) treatments, and the extent of the increase varied greatly depending on concentration and time of exposure. Shang 70–119 had a higher MDA concentration, and less increase in SOD activity when first exposed than Gebeina had.     

Mycorrhizal responses of barley cultivars differing in P efficiency

The purpose of this study was to investigate how barley cultivars which are different in dry matter yield at low phosphorus (P) supply (i.e. they differ in agronomic P efficiency) respond to mycorrhizal infection. In a preliminary experiment, six mycorrhizal fungi were tested for their ability to colonize barley (Hordeum vulgare L.) roots at a soil temperature of 15°C.Glomus etunicatum was the most effective species and was used in the main experiment. The main experiment was conducted under glasshouse conditions in which soil temperature was maintained at 15°C. Treatments consisted of a factorial arrangement of 8 barley cultivars, 2 mycorrhiza (inoculated and non-inoculated), and 3 rates of P (0, 10 and 20 mg kg^-1). P utilization efficiency (dry matter yield per unit of P taken up) and agronomic P efficiency among the barley cultivars was significantly negatively correlated with mycorrhizal responses. However, the response to mycorrhizal infection was positively correlated with response to P application. Poor correlation was observed between P concentration when neither mycorrhiza nor P were supplied and the percentage of root length infected. The extent of mycorrhizal infection among the barley cultivars in soil without P amendment varied from 8.6 to 28.6%. Significant interactions between cultivar and P addition, and between mycorrhiza and P addition were observed for shoot dry weight but not root dry weight.     

Sequences variation and classification of B-hordein genes in hull-less barley from the Qinghai-Tibet Plateau

The goal of this study is to understand the evolution relationship of the members of the B-hordein gene family in hull-less barley by analysis of their structure and to explore their utility in grain quality improvement. Six copies of the B-hordein gene (Hn1-Hn3, Hn7-Hn9) were cloned from six hull-less barley cultivars collected from Qinghai-Tibet Plateau and molecularly characterized. Comparison of their predicted polypeptide sequences with the published data suggested that they all share the same basic protein structures. In addition, we found that the C-terminal end sequences of all B-hordeins shared a similar feature. In the six clones and the other three published genes (Hn4, Hn5, and Hn6) from hull-less barley, Hn2 and Hn7 contained the identical C-terminal end sequence DIMPVDFWH. Hn3, Hn4, Hn5, Hn8 and Hn9 also shared the common sequence DIMPPDFWH, which was similar to that of a B-hordein reported previously. Both Hn1 and Hn6 exhibited differences in their C-terminal end sequences, and they clustered into different subgroups. The B-hordeins with identical C-terminal end sequences were clustered into the same subgroup, so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide. Phylogenetic analysis also indicated that there is a relatively weak identity between our predicted B-hordeins and those reported from H. chilense and H. brevisubulatum. All of our nine predicted B-hordeins were clustered together and other B-hordeins formed another cluster. The possible use of these genes in relation to barley quality is discussed. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 1, pp. 63–70. The text was submitted by the authors in English     

Effect of anther orientation on microspore-callus production in barley (Hordeum vulgare L.)

Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium (flat). When anthers were plated with only one loculus in contact with the medium (up), a high proportion of the anthers produced calluses. The top loculus of the up anthers was most productive. Flat anthers, when compared with up anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in flat anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.     

NaCl-induced amoeboid plastids and mitochondria in meristematic cells of barley roots

The barley (Hordeum vulgare L.) seeds were germinated in the non-saline conditions after 12 h imbibition in 2 % of NaCl solution. The results of treatment were: (l) the membrane system in meristematic cells of root tips developed well; (2) many profiles of endoplasmic reticulum and Golgi bodies appeared; and (3) the quantities of amoeboid plastids and amoeboid mitochondria increased. Thus the inhibitory effects of short-term NaCl stress on plants were reversible, and simultaneously NaCl treatment enhanced the metabolic activities in cells. The amoeboid form may be an adaptive form of plastids or mitochondria to an enhanced metabolic activity.     

Membrane- and cell wall-associated heat shock proteins in two genotypes of barley seedlings

ABSTRACT

The heat-shock (HS) response of two genotypes (cv. Onice, winter type, and cv. Georgie, spring type) of barley (Hordeum vulgare L.) was compared. Protein synthesis was markedly reduced by HS in roots and coleoptiles of both genotypes. The reduction in cv. Onice was higher than in cv. Georgie. The pattern of cytosolic, membrane and cell wall HSPs was analysed by SDS-PAGE in coleoptiles and roots of the two genotypes. Differences and similarities in coleoptiles and roots of the same genotype and between the two genotypes were observed. In roots of the genotype Onice, LMW and HMW HSPs isolated from the cytosol, membranes and cell walls were resolved into a diverse array of polypeptides by two-dimensional gel electrophoresis. Present results confirm the de novo synthesis of cytosolic and membrane HSPs, and demonstrate, for the first time, their presence in cell walls.     

Effects of culture conditions on isolated microspore response of barley cultivar Igri

Pretreatment of anthers in mannitol prior to isolation of microspores by glass rod homogenization was effective for in vitro induction of embryogenesis in barley cv. Igri. A procedure for separation of viable microspores using centrifugation on 20% maltose was developed. The concentration of microspores was important and greatly increased the number of developing structures. Initial culture of microspores on FHG medium containing 62 g l^-1 maltose, 4.4 M (1 mg l^-1) BA and 200 g l^-1 Ficoll-400 resulted in high frequencies of plant regeneration. Albino plant frequency was correlated to length of time in culture. Stock plant condition appeared to be a major factor influencing induction frequency. From 868 to 1738 green plants per 100 anthers were produced. The number of calli and embryos obtained and the number of green plantlets regenerated were improved by increasing the Ficoll concentration from 100 g l^-1 to 400 g l^-1 during the culture period compared to continuous culture on FHG Ficoll 200 g l^-1.Abbreviations BA benzyladenine     

Production of a recombinant industrial protein using barley cell cultures

The use of recombinant DNA-based protein production using genetically modified plants could provide a reproducible, consistent quality, safe, animal-component free, origin-traceable, and cost-effective source for industrial proteins required in large amounts (1000s of metric tons) and at low cost (below US$100/Kg). The aim of this work was to demonstrate the feasibility of using barley suspension cell culture to support timely testing of the genetic constructs and early product characterization to detect for example post-translational modifications within the industrial protein caused by the selected recombinant system. For this study the human Collagen I alpha 1 (CIa1) chain gene encoding the complete helical region of CIa1 optimized for monocot expression was fused to its N- and C-terminal telopeptide and to a bacteriophage T4 fibritin foldon peptide encoding sequences. The CIa1 accumulation was targeted to the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide sequence and an ER retention signal HDEL. The construct containing the CIa1 gene was then introduced into immature barley half embryos or barley cells by particle bombardment. Transgenic barley cells resulting from these transformations were grown as suspension cultures in flasks and in a Wave bioreactor producing CIa1 similar to CIa1 purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance, and mass spectroscopy analysis. The barley cell culture derived-CIa1 intracellular accumulation levels ranged from 2 to 9 μg/l illustrating the need for further process improvement in order to use this technology to supply material for product development activities.