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.     

Differential effects of abscisic acid and methyl jasmonate on endoproteinases in senescing barley leaves

Endoproteinase activity was analyzed in chloroplasts isolated from barley leaf segments incubated in the dark with various hormonal senescence effectors. As a control, the endoproteinase activity of the supernatant fraction obtained during chloroplast preparation was also analyzed. Measured against azocaseine as substrate, the endoproteinase activity in chloroplasts increased 18 fold during the induction of senescence. This rise in activity was inhibited by kinetin (the activity increased only 10 fold) and very strongly stimulated by abscisic acid (ABA) (117 fold) and methyl jasmonate (Me-JA) (57 fold). Although less so, the endoproteinase activity of the supernatant fraction, mainly vacuolar and with acid pH optimum, was affected in the same way by all three effectors. Among the five endoproteinases (EC) found in chloroplasts, EC2 and EC4 were induced after incubation in water. ABA increased the levels of EC2 and EC4 (5 fold), and induced the development of EC3 and EC5, while Me-JA totally inhibited EC2 and EC4, and induced the development of EC1. At least one of the endoproteinases, EC2, is synthesized in chloroplasts. Among the six endoproteinases found in the supernatant fraction (E), E1, E2, E3 and E5, which are very probably extrachloroplastic endoproteinases, are stimulated by ABA to varying degrees. However, Me-JA stimulates E1 to a greater extent and totally inhibits E3. The differential effects of ABA and Me-JA on chloroplast and supernatant fraction endoproteinases suggest different action mechanisms for both senescence promotors.Abbreviations ABA abscisic acid - DTT dithiothreitol - E supernatant fraction endoproteinase - EC chloroplast endoproteinase - Me-JA methyl jasmonate - PNP p-nitrophenol - SDS-PAGE polyacrylamide gel electrophoresis containing sodium dodecyl sulphate - TCA trichloroacetic acid     

Characterisation and functional analysis of two barley caleosins expressed during barley caryopsis development

Two full-length cDNA sequences homologous to caleosin, a seed-storage oil-body protein from sesame, were identified from a series of barley grain development cDNA libraries and further characterised. The cDNAs, subsequently termed HvClo1 and HvClo2, encode proteins of 34 kDa and 28 kDa, respectively. Real-time RT-PCR indicated that HvClo1 is expressed abundantly during the later stages of embryogenesis and is seed-specific, accumulating in the scutellum of mature embryos. HvClo2 is expressed mainly in the endosperm tissues of the developing grain. We show that HvClo1 and HvClo2 are paralogs that co-segregate on barley chromosome 2HL. Transient expression of HvClo1 in lipid storage and non-storage cells of barley using biolistic particle bombardment indicates that caleosins have different subcellular locations from the structural oil-body protein oleosin, and by inference participate in different sorting pathways. We observe that caleosin sorts via small vesicles, suggesting a likely association with lipid trafficking, membrane expansion and oil-body biogenesis.     

Peroxidase mediated hydrogen peroxide production in barley roots grown under stress conditions

All applied metals (Co, Al, Cu, Cd) and NaCl inhibited barley root growth. No root growth inhibition was caused by drought exposure, in contrast to cold treatment. 0.01 mM H₂O₂ stimulated root growth and GA application did not affect root growth at all. Other activators and inhibitors of H₂O₂ production (SHAM, DTT, 10 mM H₂O₂, 2,4-D) inhibited root growth. Loss of cell viability was most significant after Al treatment, followed by Cd and Cu, but no cell death was induced by Co. Drought led to slight increase in Evans blue uptake, whereas neither NaCl nor cold influenced this parameter. DTT treatment caused slight increase in Evans blue uptake and significant increases were detected after 2,4-D and 10 mM H₂O₂ treatment, but were not induced by others stressors. Metal exposure increased guaiacol-POD activity, which was correlated with oxidation of NADH and production of H₂O₂. Exposure to drought caused a minor change in NADH oxidation, but neither H₂O₂ production nor guaiacol-POD activity was increased. Cold and NaCl application decreased all monitored activities. Increase in NADH oxidation and guaiacol-POD activity was caused by 10 mM H₂O₂ and 0.01 mM 2,4-D treatment, which also caused enhancement of H₂O₂ production. Slight inhibition of all activities was caused by 0.01 mM H₂O₂, GA, DTT; more pronounced inhibition was detected after SHAM treatment. The role of H₂O₂ production mediated by POD activity in relation to root growth and cell viability under exposure to some abiotic stress factors is discussed.     

The impact of salt stress on the water status of barley plants is partially mitigated by elevated CO2

With the changing climate, plants will be facing increasingly harsh environmental conditions marked by elevated salinity in the soils and elevated concentrations of CO₂ in the atmosphere. These two factors have opposite effects on water status in plants. Therefore, our objective was to determine the interaction between these two factors and to determine whether elevated [CO₂] might alleviate the adverse effects of salt stress on water status in two barley cultivars, Alpha and Iranis, by studying their relative water content and their water potential and its components, transpiration rate, hydraulic conductance, and water use efficiency. Both cultivars maintained their water status under salt stress, increasing water use efficiency and conserving a high relative water content by (1) reducing water potential via passive dehydration and active osmotic adjustment and (2) decreasing transpiration through stomatal closure and reducing hydraulic conductance. Iranis showed a greater capacity to achieve osmotic adjustment than Alpha. Under the combined conditions of salt-stress and elevated [CO₂], both cultivars (1) achieved osmotic adjustment to a greater extent than at ambient [CO₂], likely due to elevated rates of photosynthesis, and (2) decreased passive dehydration by stomatal closure, thereby maintaining a greater turgor potential, relative water content, and water use efficiency. Therefore, we found an interaction between salt stress and elevated [CO₂] with regard to water status in plants and found that elevated [CO₂] is associated with improved water status of salt-stressed barley plants.     

Changes of chloroplasts density and heterogeneity during the senescence of barley leaves

The equilibrium density of chloroplasts from barley (Hordeum vulgare L. cv. Hassan) was analyzed by sucrose gradient centrifugation. Natural and detachment-induced leaf senescence were associated with a decrease in density and an increase in heterogeneity of the chloroplast population. Treatments (with growth regulators and light) which retarded or accelerated senescence, respectively, retarded or accelerated chloroplast density decrease. Accelerators as well as retardants of senescence decreased the heterogeneity of the chloroplast population.     

Promotive effect of jasmonates on the senescence of detached maize leaves

Promotion of senescence of detached maize leaves by jasmonates was investigated. Senescence of detached maize leaves was promoted by linolenic acid, the precursor of biosynthesis of jasmonic acid, and retarded by inhibitors of lipoxygenase, the first enzyme in the biosynthetic pathway of jasmonic acid. Results support a role of endogenous jasmonates in the regulation of senescence of detached maize leaves. Silver thiosulfate, an inhibitor of ethylene action, was found to inhibit methyl jasmonate, linolenic acid- and abscisic acid-promoted senescence of detached maize leaves. It seems that jasmonate-promoted senescence is mediated through an increase in ethylene sensitivity in detached maize leaves.Abbreviations ABA abscisic acid - MJ methyl jasmonate - STS silver thiosulfate     

Methyl jasmonate effects on ethylene synthesis and organ-specific senescence in Helianthus annuus seedlings

Both methyl jasmonate (MJ) and ethylene have been implicated in promoting senescence, but the specific roles of each and the mechanisms by which they act are not well known. We tested the possibility that MJ and ethylene interact to promote senescence. In sunflower seedlings, the ability of MJ to affect ethylene metabolism was investigated in hypocotyls, cotyledons, and leaves. 1-aminocylcopropane-1-carboxylic acid (ACC)-dependent ethylene production was promoted to different extents depending on the organ and the age of the tissue. Newly emerged hypocotyls were sensitive to MJ, but became desensitized as the cotyledons emerged. The cotyledons increased and peaked in MJ sensitivity from emergence to the production of the primary leaves. Leaves were found to be somewhat insensitive to MJ treatment compared to cotyledons at all ages tested. In cotyledons, MJ also promoted ACC and ethylene production. However the changes in ACC, and ACC-dependent ethylene production were not directly correlated with those in ethylene production with respect to MJ concentration or tissue age. Moreover, changes in ACC-dependent ethylene production did not correlate with in vitro ACC oxidase activity. We hypothesized that MJ affects ethylene production by increasing the spatial access of ACC to ACC oxidase perhaps through increased membrane permeability. Ethylene was not involved in the MJ-induced loss of chlorophyll. But the breakdown of cell integrity and cell membranes (estimated by monitoring conductivity of the solution that bathed the cotyledons) was greatly and synergistically promoted by the combination of MJ and ethylene. Promotion of membrane breakdown by MJ and ethylene could be inhibited by treatments with ethylene inhibitors (STS or CoCl₂), and neither MJ nor ACC treatment alone could induce as much membrane breakdown as both together. We suggest that MJ and ethylene interact to accelerate some aspects of senescence in specific organs for nutrient remobilization for the benefit of the whole plant.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MJ methyl jasmonate - STS silver thiosulphate     

Exogenous 2-aminoethanol can diminish paraquat induced oxidative stress in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Effects of unfavourable environmental conditions (stresses) induce stressor specific and unspecific short- and long-term responses in plants. Long-term responses depend on intensity and duration of the stress. Short-term effects comprise the accumulation of reactive oxygen species (ROS), membrane damages by the oxidation of fatty acids, and the release of amino alcohols. They can incite higher stress tolerance in plants. In the present study, shoots of barley (Hordeum vulgare) were pre-treated with 2-aminoethanol, and, 2 days later, with the oxidative stress inducing herbicide, paraquat. Pre-treatments with 2-aminoethanol increased the stress tolerance in barley by the stabilization of the cell membranes, the enhanced production of superoxide dismutase and catalase, and the stimulation of glutathione metabolism (GSH, GST). These mechanisms of stress tolerance activation by 2-aminoethanol are discussed.     

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.     

Heavy metal stress and leaf senescence induce the barley gene HvC2d1 encoding a calcium-dependent novel C2 domain-like protein

By comparing cDNA populations derived from chromium-stressed primary leaves of barley (Hordeum vulgare L.) with controls, differentially expressed cDNA fragments could be identified. The deduced amino acid sequence of one of these cDNAs [named 'C2 domain 1' (HvC2d1)] exhibits a motif that is similar to the known C2 domain and a nuclear localization signal (NLS). Expression of this member of a novel class of plant C2 domain-like proteins was studied using real-time PCR, and subcellular localization was investigated using green fluorescent protein (GFP) fusion constructs. Calcium binding was analysed using a (45)Ca(2+) overlay assay. HvC2d1 was transiently induced after exposure to different heavy metals and its mRNA accumulated during the phase of leaf senescence. HvC2d1 expression responded to changes in calcium levels caused by the calcium ionophore A23187 and to treatment with methylviologen resulting in the production of reactive oxygen species (ROS). Using overexpressed and purified HvC2d1, the binding of calcium could be confirmed. Chimeric HvC2d1-GFP protein was localized in onion epidermal cells at the plasma membrane, cytoplasm and the nucleus. After addition of calcium ionophore A23187 green fluorescence was only visible in the nucleus. The data suggest a calcium-dependent translocation of HvC2d1 to the nucleus. A possible role of HvC2d1 in stress- and development-dependent signalling in the nucleus is discussed.     

Cytosolic calcium is involved in the regulation of barley hemoglobin gene expression

Hemoglobin gene expression is upregulated during hypoxia. To determine whether the induction occurs via similar mechanisms that have been proposed for other hypoxically induced proteins, barley (Hordeum vulgare L.) aleurone layers were treated with various agents that interfere with known components of signal transduction. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced hemoglobin (Hb) and alcohol dehydrogenase (EC 1.1.1.1) (Adh) gene expression in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA also dramatically reduced anoxia-induced Hb and Adh expression. Normal induction of Hb by anoxia in EGTA-treated cells was restored by adding exogenous Ca²⁺ but not Mg²⁺, suggesting that cytosolic calcium is involved in Hb and Adh regulation. W-7, a calmodulin antagonist, did not affect anaerobically induced Hb and Adh expression even though it induced Hb under aerobiosis. A3, a protein kinase inhibitor, did not significantly affect anaerobically induced Hb, but did significantly upregulate the gene under aerobic conditions. The results indicate that calmodulin-independent anaerobic alteration in cytosolic Ca²⁺ and protein dephosphorylation are factors in Hb induction.     

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 Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3 end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3 untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.