The regulation of the biosynthesis of glutathione in leaves of barley (Hordeum vulgare L.)

Between 50 and 65% of the glutathione in barley leaves was present in the chloroplasts depending upon the light regime. However, only 66–76% of the chloroplast glutathione was present in the reduced state (GSH) as opposed to 97–98% of that in the cytoplasm. In shoots treated with the catalase inhibitor aminotriazole and in shoots of the catalase deficient barley mutant RPr 79/4 exposed to air, the glutathione level increased 3-fold in 8 h in the light. The increase was accounted for by a rise in both the chloroplast and cytoplasm level of oxidised glutathione (GSSG), the GSH concentration remained relatively constant in both compartments. Only 2–3% of applied ³⁵SO₄ was metabolised to glutathione by wild-type shoots. In aminotriazole-treated plants this value rose to 17.9% and in the mutant RPr 79/4 exposed to air to 32%.     

The effects of solar ultraviolet-B radiation on the growth and yield of barley are accompanied by increased DNA damage and antioxidant responses

There is limited information on the impacts of present-day solar ultraviolet-B radiation (UV-B) on biomass and grain yield of field crops and on the mechanisms that confer tolerance to UV-B radiation under field conditions. We investigated the effects of solar UV-B on aspects of the biochemistry, growth and yield of barley crops using replicated field plots and two barley strains, a catalase (CAT)-deficient mutant (RPr 79/4) and its wild-type mother line (Maris Mink). Solar UV-B reduced biomass accumulation and grain yield in both strains. The effects on crop biomass accumulation tended to be more severe in RPr 79/4 (≈ 32% reduction) than in the mother line (≈ 20% reduction). Solar UV-B caused measurable DNA damage in leaf tissue, in spite of inducing a significant increase in UV-absorbing sunscreens in the two lines. Maris Mink responded to solar UV-B with increased CAT and ascorbate peroxidase (APx) activity. No effects of UV-B on total superoxide dismutase (SOD) activity were detected. Compared with the wild type, RPr 79/4 had lower CAT activity, as expected, but higher APx activity. Neither of these activities increased in response to UV-B in RPr 79/4. These results suggest that growth inhibition by solar UV-B involves DNA damage and oxidative stress, and that constitutive and UV-B-induced antioxidant capacity may play an important role in UV-B tolerance.     

Molecular cloning,characterization and expression analysis of two catalase isozyme genes in barley

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and-3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).     

Response of antioxidant enzymes to transfer from elevated carbon dioxide to air and ozone fumigation, in the leaves and roots of wild‐type and a catalase‐deficient mutant of barley

A catalase-deficient mutant (RPr 79/4) and the wild-type (cv. Maris Mink) barley (Hordeum vulgare L.) counterpart, were grown for 3 weeks in high CO₂ (0.7%) and then transferred to air and ozone (120 nl 1^?1) in the light and shade for a period of 4 days. Leaves and roots were analysed for catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and glutathione reductase (GR, EC 1.6.4.2) activities. CAT activity in the leaves of the RPr 79/4 catalase-deficient mutant was around 5-10% of that determined in Maris Mink, but in the roots, both genotypes contained approximately the same levels of activity. CAT activity in Maris Mink increased in the leaves after transferring plants from 0.7% CO₂ to air or ozone, reaching a maximum of 5-fold, after 4 days in shade and ozone. For the catalase-deficient mutant, only small increases in CAT activity were observed in light/air and light/ozone treatments. In the roots, CAT activity decreased consistently in both genotypes, after plants were transferred from 0.7% CO₂. The total soluble SOD activity in the leaves and roots of both genotypes increased after plants were transferred from 0.7% CO₂. The analysis of SOD isolated from leaves following non-denaturing PAGE, revealed the presence of up to eight SOD isoenzymes classified as Mn-SOD or Cu/Zn-SODs; Fe-SOD was not detected. Significant changes in Mn- and Cu/Zn-SOD isoenzymes were observed; however, they could not account for the increase in total SOD activity. In leaves, GR activity also increased in Maris Mink and RPr 79/4, following transfer from 0.7% CO₂; however, no constant pattern could be established, while in roots, GR activity was reduced after 4 days of the treatments. The data suggest that elevated CO₂ decreases oxidative stress in barley leaves and that soluble CAT and SOD activities increased rapidly after plants were transferred from elevated CO₂, irrespective of the treatment (light, shade, air or ozone).     

Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of ¹⁴C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of ¹⁴CO₂, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO₂-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O₂ to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH₃+2-oxoglutarate)-dependent O₂ evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.     

Dehydrophylloquinone, α-dehydrotocopherolquinone and dehydrotocopherols from etiolated maize and barley shoots

Dehydrophylloquinone, α-dehydrotocopherolquinone, α-dehydrotocopherol and γ-dehydrotocopherol have been isolated from etiolated maize and barley shoots and excised etiolated maize shoots that have been exposed to light. They are not present, however, in green maize shoots, spinach leaves and etiolated bean leaves. Demethylphylloquinone was not detected in any of the tissues analysed.     

Desaturation of Oleic and Linoleic Acids by Leaves of Dark- and Light-grown Maize Seedlings

Oleate and linoleate desaturation in leaves of maize seedlings was largely independent of previous light treatment of the seedlings; there was no evidence of light-induced desaturase activities. These results are in sharp contrast to those observed with developing cucumber cotyledons in which pronounced increase in desaturation occurs after exposure of tissue to light. The rates of desaturation of oleate were about four times those of linoleate in both etiolated and 16-hour greened maize leaves. In both etiolated and greened tissues, about two-thirds of the label from oleate was esterified after 4 hours, half of which was in phosphatidylcholine. Phosphatidylcholine and diglyceride contained large proportions of [¹⁴C]linoleate formed from [¹⁴C]oleate but not [¹⁴C]linolenate. In monogalactolipid, about two-thirds of the labeled fatty acids were linolenate. In vivo desaturase activity was present in tissue of widely different levels of differentiation and chlorophyll content obtained from light-grown maize seedlings.     

Expression of Early Light-Inducible Proteins in Flag Leaves of Field-Grown Barley

Early light-inducible protein (ELIP) mRNA and protein levels were analyzed during maturation and senescence of barley (Hordeum vulgare L.) flag leaves under field conditions. The data clearly demonstrate that ELIP mRNA levels are related to the sunlight intensity before sample collection. Levels of mRNAs encoding both low and high molecular mass ELIPs fluctuate in parallel. Changes in mRNA levels are accompanied by corresponding changes in protein levels except for days when average temperatures are high. Comparison of flag leaves at different stages of development in spring and winter barley varieties suggests that light-stress-regulated ELIP gene expression is independent of the developmental stage of the leaves. Although chlorophyll content, photosystem II (PSII) efficiency, and 32-kD herbicide-binding protein of PSII levels decrease drastically after the onset of senescence, ELIP mRNA and protein still accumulate to high levels on bright days.     

Inhibitory Effect of Gabaculine on the Biosynthesis of 5-Aminolevulinate in the Tigrina d12 Mutant of Barley

Six-day-old, dark-grown, seedlings of barley homozygous forthe recessive mutation tigrina d¹² accumulated 5-aminolevulinicacid (ALA) and protochlorophyll (ide) in amounts exceeding thewild type level. Transferring the etiolated mutant to lightresulted in the destruction of pigments and the deteriorationof the ALA forming system. Such deleterious effects did notoccurusing light-grown mutant or etiolated and greened wildtype seedlings. Gabaculine (GAB) at 50 µM inhibited ALAsynthesis by about 85% when etiolated wild type seedlings wereexposed to light. In light-grown leaves of either wild typeor mutant strain, ALA production was also sharply (ca. 75%)inhibited by GAB. During dark incubation, however, the inhibitionof ALA accumulation did not exceed 50% in all types of tissues.The results give further evidence for the operation of the C₅pathway in such seedlings since GAB decreased the biosynthesisof ALA to the same extent in both tigrina d¹² mutant and wildtype of barley. (Received July 2, 1990; Accepted May 7, 1991)     

Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme

The NH₂-terminal sequences of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) purified from barley (Hordeum vulgare L.) and Chlamydomonas reinhardtii (Dangeard), and of a barley peptide, were determined and the barley sequences were used to design oligonucleotide primers for the polymerase chain reaction. A specific 1.3-kilobase (kb) cDNA fragment specifying the NH₂-terminal one-third of the mature barley polypeptide, was amplified, cloned and sequenced. The NH₂-terminus of plant Fd-GOGAT is highly conserved and homologous to the NH₂-terminus of the heavy subunit of Escherichia coli NADPH-GOGAT. Based on sequence homologies, we tentatively identified the NH₂-terminal region of Fd-GOGAT as the glutamine-amidotransferase domain, which is related to the corresponding domain of the purF-type amidotransferases. The Fd-GOGAT cDNA clone, and polyclonal antibodies raised against the barley enzyme, were used to analyse four Fd-GOGAT-deficient photorespiratory mutants. Three mutants (RPr 82/1, RPr 82/9 and RPr 84/82) had no detectable Fd-GOGAT protein in leaves, while the fourth (RPr 84/42) had a small amount of cross-reacting material. Hybridization to Northern blots of total leaf RNA revealed that both RPr 82/9 and RPr 84/82 were indistinguishable from the parental line (Maris Mink), having normal amounts of a 5.7-kb mRNA species. On the other hand, RPr 82/2 and RPr 84/42 each contained two distinct hybridizing RNA species, one of which was larger than 5.7 kb, the other smaller. Using a set of wheat-barley telosomic addition lines we have assigned the Fd-GOGAT structural locus to the short arm of chromosome 2.Abbreviations bp kbase pairs - cDNA copy DNA - Fd ferredoxin - GOGAT glutamate synthase - GAT glutamine amidotransferase - kb kilobase - PCR polymerase chain reaction C.A. was the holder of a Fleming award from the British Council and the Spanish Ministry of Education and Science. A.J.M. was for part of the work the recipient of a European Molecular Biology Organization postdoctoral fellowship. The research was also partly supported by contract no. BAP/O354/E of the Biotechnology Action Programme of the E.C., by an Acciones Integradas award (no. 40/125) from the British Council and the Spanish Ministry of Education and Science, by the Junta de Andalucia (to Group 3263) and by project PB91-0613 from DGICYT (Spain). We thank Daryl Pappin (Department of Biochemistry, University of Leeds) for amino-acid sequencing, and Martin Cornelius (Rothamsted Experimental Station, Harpenden, Herts., UK) for synthesis of oligonuleotides.     

Characterization of protoheme levels in etiolated and greening plant tissues

The protoheme content of etiolated, greening, and fully greened bean (Phaseolus vulgaris L. var. Light Red Kidney) leaves has been studied. The protoheme level in etiolated and fully greened leaf tissue stays relatively constant from age 7 to 14 days. In agreement with the studies reported for barley (Castelfranco and Jones 1975 Plant Physiol 55: 485-490), the protoheme content of greening bean and barley (Hordeum vulgare var. Larker) leaves does not change appreciably during the first 9 hours of illumination, but the level rises significantly by the 24th hour of illumination (cf. Hendry and Stobart 1977 Phytochemistry 16: 1545-1548). This increase also occurs in seedlings returned to the dark for 24 to 48 hours following a 10-minute pulse of light. These results demonstrate a limited correlation with previous studies on the development of b-type cytochromes during greening of these tissues (Gregory and Bradbeer 1973; Planta 109: 317-326).