Effects of short-term soil flooding on stomata behaviour and leaf gas exchange in barley plants

Exposure of barley plants (Hordeum vulgare L.) to soil flooding for 2 to 24 h reduced the net photosynthetic rate and transpiration rate. Stomatal conductance also decreased in flooded plants. Stomatal closure started within 2 – 6 h and stomata remained closed up to 24 h of treatment.     

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 simple diffusion test for soil phosphorus availability

The availability of phosphorus to plants was estimated by a new method based on the diffusion of P from a thin layer of water-saturated stationary soil to a disc of iron oxide coated filter paper. Twenty-one acid Finnish soil samples were tested using four modifications of the method which differed in the duration of the diffusion time and in the thickness of the soil layer. The new diffusion tests were compared with other soil P testing methods and evaluated as indicators of the availability of P to pot-grown barley and oats. Close linear correlations were found between the initial (18 or 42 h) P diffusion rate and the water extractable P contents determined at the extraction ratios of 1:10 and 1:60, respectively (r=0.97–0.99). The amounts of P obtained by the new method were small when the diffusion time was short, but after prolonged diffusion (1 week) the test values were close to the amounts of P taken up by plants. The relationships of plant yield and P uptake with soil P test values were affected by soil acidity. Although the original soil P test values were significantly correlated, the accuracy of all methods drastically improved when the interfering effect of soil acidity was taken into account using a simple pH-correction model. The correlation coefficients (r) between the test values of diffusible P in soil and the uptake of P by plants increased up to 0.97.     

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.     

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.     

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as -amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

Comparative analysis of the low molecular weight and enzymatic antioxidants in response to the phototoxicity of accumulating uroporphyrin and protochlorophyllide in barley leaves treated with cesium chloride

Cesium chloride treatment of illuminated barley leaves leads to accumulation of uroporphyrinogen which is subsequently either oxidised to uroporphyrin in continuous light or converted to protochlorophyllide in darkness [Shalygo et al. (1998) J Photochem Photobiol 42: 151–158]. We were interested to elucidate the differences in the phototoxicity of uroporphyrin and protochlorophyllide in the CsCI-treated leaves. Photosensitization and the induction of oxidative stress responses in the barley leaves occurred much faster upon protochlorophyllide than upon uroporphyrin accumulation. We compared the time resolved changes in the pool sizes of low molecular weight antioxidants, such as ascorbate, glutathione and tocopherol, as well as of the enzymatic activities of catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase in illuminated barley leaves which accumulated uroporphyrin or protochlorophyllide. A rapid loss of the antioxidant levels correlated with the accumulation of reactive oxygen species. The contents of low molecular weight antioxidants and the activities of most of the antioxidative enzymes declined more rapidly in the presence of protochlorophyllide than of uroporphyrin. Due to its high lipophilicity, free protochlorophyllide is associated with biomembranes. Therefore, it is assumed that it exerts its phototoxic effects to membranes more rapidly than uroporphyrin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic diversity among wild and cultivated barley as revealed by RFLP

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp. spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns was estimated and the barley lines clustered into five groups reflecting geographical origin. The geographical groups of land-race lines showed less intragroup variation than the geographical groups of spontaneum lines. The group of European varieties, representing large variation in agronomic traits, showed an intermediate level. The proportion of gene diversity residing among geographical groups (F_ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups.     

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     

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     

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.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

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.     

Fertilizer and soil nitrogen accumulation by irrigated barley grown on a red-brown earth in south-eastern Australia

¹⁵N labelled (NH₄)₂SO₄ was applied to barley at 5 g N m⁻² (50 kg N ha⁻¹) in microplots at sowing to study the timing of the N losses and the contribution of soil and fertilizer N to the plant. Water treatments included rainfed and irrigation at 45–50 mm deficit beginning in the spring. Recovery of¹⁵N in the plant increased to a maximum of about 20% within 91 days after sowing (DAS 91) and then remained constant. Approximately 16% (0.8 g N m⁻²) of the fertilizer was in the stem and leaves at DAS 91 and this N was subsequently redistributed to the head. At maturity, approximately 75% of the¹⁵N assimilated by the tops was recovered in the grain. Soil N contributed 3.6 g N m⁻² to the head; 2.2 g N m⁻² was remobilized from the stem and leaves, and the balance, approximately 1.4 g N m⁻², was taken up from the soil between DAS 69 to 91. Effects of irrigation treatments on N accumulation were not significant. Residual¹⁵N fertilizer in the soil decreased with time from sowing, and at maturity 40% of the applied N was recovered in the surface 0.15 m.¹⁵N movement to depth was limited and less than 5% of the fertilizer was recovered below 0.15 m. Irrigation had no effect on the¹⁵N recovery at depth. Total recovery of the¹⁵N varied between 60 and 67% and implies that 33–40% was lost from the soil-plant system. The total recovery in the soil and plant was not affected by time or irrigation in the interval DAS 39 to 134. Losses occurred before DAS 39 when crop uptake of N was small and soil mineral N content was high. There was an apparent loss of 1.9 g fertilizer N m⁻² (i.e. 38% of that applied) between DAS 1 and 15. This loss occurred before crop emergence when rainfall provided conditions suitable for denitrification.     

Gravimorphism in rice and barley: Promotion of leaf elongation by vertical inversion in agravitropically growing plants

We have compared shoot responses of agravitropic rice and barley plants to vertical inversion with those of normal ones. When rice plants were vertically inverted, the main stems of a japonica type of rice, cv. Kamenoo, showed negative gravitropism at nodes 2–15 of both elongated and non-elongated intermodes. However, shoots of lazy line of rice, lazy-Kamenoo, bent gravitropically at nodes 11–15 only elongated internodes but not at nodes 2–10 of non-elongated ones. Thus, shoots of Kamenoo responded gravitropically at all stages of growth, whereas shoots of lazy-Kamenoo did not show gravitropic response before heading. In Kamenoo plants, lengths of both leaf-sheath and leaf-blade were shortened by vertical inversion, but those of the vertically inverted plants of lazy-Kamenoo were significantly longer than the plants in an upright position. When agravitropic and normal plants of barley were vertically inverted, the same results as in rice were obtained; elongation of both leaf-sheath and leaf-blade was inhibited in normal barley plants, Chikurin-Ibaragi No. 1, but significantly stimulated in agravitropic plants ofserpentina barley. These results suggest that vertical inversion of rice and barley plants enhances the elongation growth of leaves in the absence of tropistic response.     

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.     

Correlation between hordatine accumulation, environmental factors and genetic diversity in wild barley (Hordeum spontaneum C. Koch) accessions from the Near East Fertile Crescent

Wild barley shows a large morphological and phenotypic variation, which is associated with ecogeographical factors and correlates with genotypic differences. Diversity of defense related genes and their expression in wild barley has been recognized and has led to attempts to exploit genes from H. spontaneum in breeding programs. The aim of this study was to determine the variation in the accumulation of hordatines, which are Hordeum-specific preformed secondary metabolites with strong and broad antimicrobial activity in vitro, in 50 accessions of H. spontaneum from different habitats in Israel. Differences in the accumulation of hordatines in the seedling stage were significant between different H. spontaneum genotypes from different regional locations and micro-sites. Variation in the hordatine accumulation within genotypes was between 9% and 45%, between genotypes from the same location between 13% and 38%, and between genotypes from different locations up to 121%. Principal component analysis showed that water related factors explain 39%, temperature related factors explain 33% and edaphic factors account for 11% of the observed variation between the populations of H. spontaneum. Genetic analysis of the tested accessions with LP-PCR primers that are specific for genes involved in the biosynthetic pathway of hordatines showed tight correlations between hordatine abundance and genetic diversity of these markers. Multiple regression analyses indicated associations between genetic diversity of genes directly involved in hordatine biosynthesis, ecogeographical factors and the accumulation of hordatines.