In the F2 generation, nonviability in air and the lack of chloroplast glutamine synthetase co-segregated, in both the lines tested. These two lines and four others proved to be allelic, we designate them gin 2a-f. The characteristics of these mutants conclusively demonstrate the major role of chloroplast glutamine synthetase in photorespiration and its associated nitrogen recycling.

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SummaryA recent strategy for pest control in plants has involved transformation with genes encoding cysteine proteinase inhibitors (cystatins). Little is known, however, about the effects of constitutive cystatin expression on whole plant physiology. The present study using oryzacystatin I (OC-I) expression in transformed tobacco was designed to resolve this issue and also to test the effects on abiotic stress tolerance. All transformed plants expressing OC-I showed a conditional phenotype. A marked effect on stem elongation was observed in plants grown under low light intensities. After 7 weeks of growth at low light, the plants expressing OC-I were smaller with fewer expanded leaves and a slightly lower total biomass than empty vector controls or wild type plants. Maximal rates of photosynthesis ( A max ) were also decreased, the inhibitory effect being greatest in the plants with highest OC-I expression. After 12 weeks of growth at low light, however, the plants expressing OC-I performed better in terms of shoot biomass production, which was nearly double that of the empty vector or wild type controls. All plants showed similar responses to drought, however photosynthesis was better protected against chilling injury in plants constitutively expressing OC-I. Photosynthetic CO 2 assimilation was decreased in all plants following exposure to 5 ° C, but the inhibition was significantly less in the OC-I expressing plants than in controls. The transformed tobacco plants expressing OC-I therefore show a phenotypeenvironment interaction with important implications for biotechnological applications.

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO2, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO2 at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of (14)CO2 into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of (14)C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [(14)C]glutamate and [(14)C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.

A mutant line of barley, R(othamsted)-Pr 79/4, has been isolated which grows poorly in natural air, but normally in air enriched to 0.2% CO2. Analysis of the products of (14)CO2 fixation showed that there was no major block in photosynthetic or photorespiratory carbon metabolism in the mutant and that rates of CO2 fixation were only slightly lower than those measured in the wild type (c.v. Maris Mink). Leaves of the mutant line contained only 10% of the catalase (EC 1.11.1.6) activity found in the wild type; and the two major bands of catalase activity detected after starch-gel electrophoresis of extracts of normal leaves were missing from similar extracts of RPr 79/4. Peroxisomes isolated from mutant leaves contained negligible catalase activity, but normal levels of other enzymes involved in photorespiration. Genetic analysis has shown that the mutation is recessive and that both air-sensitivity and catalase-deficiency segregate together in F2 plants derived from a cross between the mutant and the cultivar Golden Promise. [1-(14)C]Glycollate was not converted to (14)CO2 faster in the mutant leaves than in the normal leaves. Thus there was no evidence that photorespiratory CO2 may be obtained by the chemical action of H2O2 on glyoxylate or hydroxypyruvate.

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The Hf-translocating inorganic pyrophosphatase (H+-PPasc) of Beta vacuolar membrane (tonoplast) vesicles has been purified by 90-fold from detergent-solubilized membranes and the MgPPi-binding subunit identified by affinity labeling. The purified enzyme has a specific activity of 1100 pmol/mg.h and contains one prevalent M, = 64000 polypeptide which strictly copurifies with activity.

Manipulation of the CO2 concentration of the atmosphere allows the selection of photorespiratory mutants from populations of seeds treated with powerful mutagens such as sodium azide. So far, barley lines deficient in activity of phosphoglycolate phosphatase, catalase, the glycine to serine conversion, glutamine synthetase, glutamate synthase, 2-oxoglutarate uptake and serine: glyoxylate aminotransferase have been isolated. In addition one line of pea lacking glutamate synthase activity and one barley line containing reduced levels of Rubisco are available. The characteristics of these mutations are described and compared with similar mutants isolated from populations of Arabidopsis. As yet, no mutant lacking glutamine synthetase activity has been isolated from Arabidopsis and possible reasons for this difference between barley and Arabidopsis are discussed. The value of these mutant plants in the elucidation of the mechanism of photorespiration and its relationships with CO2 fixation and amino acid metabolism are highlighted.