Ammonia Production and Assimilation in Glutamate Synthase Mutants of Arabidopsis thaliana

Ammonia production and assimilation¹ were examined in photorespiratory mutants of Arabidopsis thaliana L. lacking ferredoxin-dependent glutamate synthase (Fd-GluS) activity. Although photosynthesis was rapidly inhibited in these mutants in normal air, NH₄⁺ continued to accumulate. The accumulation of NH₄⁺ was also seen after an initial lag of 30 minutes in 2% O₂, 350 microliters per liter of CO₂ and after 90 minutes in 2% O₂, 900 microliters per liter of CO₂. The accumulation of NH₄⁺ in normal air and low O₂ was also associated with an increase in the total pool of amino acid-N and glutamine, and a decrease in the pools of glutamate, aspartate, alanine, and serine. Upon return to dark conditions, or to 21% O₂, 1% CO₂ in the light, the NH₄⁺ which had accumulated in the leaves was reassimilated into amino acids. The addition of methionine sulfoximine (MSO) resulted in higher accumulations of NH₄⁺ in glutamate synthase mutants and prevented the reassimilation of NH₄⁺ upon return to the dark. The addition of MSO also resulted in the accumulation of NH₄⁺ in glutamate synthase mutants in the light and in 21% O₂, 1% CO₂. These results indicate that glutamine synthetase is essential for the reassimilation of photorespiratory NH₄⁺ and for primary N assimilation in the leaves and strongly suggest that glutamate dehydrogenase plays only a minimal role in the assimilation of ammonia. Levels of NADH-dependent glutamate synthase (NADH-GluS) appear to be sufficient to account for the assimilation of NH₄⁺ by a GS/NADH-GluS cycle.     

Comparison of Triazine-Resistant and -Susceptible Biotypes of Senecio vulgaris and Their F(1) Hybrids

The relationship of triazine resistance to decreased plant productivity was investigated in Senecio vulgaris L. F₁ reciprocal hybrids were developed from pure-breeding susceptible (S) and resistant (R) lines. The four biotypes (S, S × R, R, R × S) were compared in terms of atrazine response, electron transport, carbon fixation, and biomass production. Atrazine response, carbon fixation rate, and PSII and whole-chain electron transport rates of hybrids were nearly identical to those of their respective maternal parents. Significant differences occurred between the two susceptible (S, S × R) and two resistant (R, R × S) biotypes in atrazine response (I₅₀), carbon fixation rate, and PSII and whole-chain electron transport rates; PSI rates were identical in all four biotypes. Coupled and uncoupled, whole-chain electron transport rates of thylakoids of the two susceptible biotypes were approximately 50% greater than those of the two resistant biotypes at photon flux densities greater than 215 micromoles per square meter per second. Carbon exchange rates of the two susceptible biotypes were 23% greater than those of the two resistant biotypes. Hybrid biotypes (S × R, R × S) were not identical to their maternal parents in biomass production. The S, S × R, and R × S plants all achieved greater biomass than R plants. These results suggest that while the resistance mutation influences thylakoid performance, reduced productivity of triazine-resistant plants cannot be ascribed solely to decreases in electron transport or carbon assimilation rates brought about by the altered binding protein. Since the F₁ hybrids differed from their maternal parents only in nuclear genes, it appears that the detrimental effects of the triazine resistance mutation on plant growth may be attenuated by interactions of the plastid and nuclear genomes.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

Indole-3-acetic Acid oxidation and crocin bleaching by horseradish peroxidase

During indoleacetic acid (IAA) oxidation by horseradish peroxidase the water soluble model polyene, crocin, is bleached. IAA-oxidation and crocin bleaching are stimulated at acidic pH as well as by the monophenol p-hydroxyacetophenone. IAA oxidation and crocin bleaching are neither influenced by catalase or superoxide dismutase nor by different OH-radical scavengers, whereas both ascorbate and propylgallate are inhibitory.     

A Mutant of Nicotiana sylvestris Lacking Serine:Glyoxylate Aminotransferase: Substrate Specificity of the Enzyme and Fate of [2-C]Glycolate in Plants with Genetically Altered Enzyme Levels

The photorespiratory mutant of Nicotiana sylvestris, NS 349, lacking serine:glyoxylate aminotransferase (SGAT) grows in 1% CO₂ but not in normal air (NA McHale, EA Havir, I Zelitch 1988 Theor Appl Genet. In press). Alanine:hydroxypyruvate and asparagine:hydroxypyruvate aminotransferase activities were also lacking in the mutant, and plants heterozygous with respect to SGAT which grow in normal air had 50% of the activities present in homozygous plants. Therefore, all these activities are associated with the same enzyme. On feeding [2-¹⁴C]glycolate to leaf discs in the light, NS 349 showed reduced incorporation of radioactivity into the neutral and organic acid fractions and increased incorporation into the amino acid fraction, principally into serine. The effect of reducing SGAT by 50% in heterozygous plants produced little change in the metabolism of [2-¹⁴C]glycolate, showing there is a large excess of this enzyme in wild-type plants.     

Lipid peroxidation is a consequence of elicitor activity

Elicitor-active preparations from the fungal pathogen of bean Colletotrichum lindemuthianum stimulated the accumulation of products characteristic of lipid peroxidation in treated bean tissues. Bean suspension cells treated with crude and purified elicitors accumulated `lipofuscin-like pigment' (LEP) and malondialdehyde. The accumulation of LFP after about 6 h of treatment coincided with the onset of visible browning and production of the bean phytoalexins kievitone, phaseollin, and phaseollinisoflavan. The induction of phytoalexins and accumulation of LFP were also triggered by treatments with generators of activated oxygen species, xanthine:xanthine oxidase and Fe:ethylenediaminedi-o-hydroxyphenylacetic acid. These data suggest that generation of active oxygen species may be involved in lipid peroxidation triggered by elicitors.     

Biochemical Studies of Paraquat-Tolerant Mutants of the Fern Ceratopteris richardii

Enzymes and metabolites associated with mitigation of paraquat toxicity were compared in two paraquat-tolerant mutants and a sensitive wild-type strain of the fern Ceratopteris richardii Brongn. In 21-day-old gametophytes, the specific activities of superoxide dismutase, catalase, peroxidase, glutathione reductase, dehydroascorbate reductase, and ascorbate peroxidase showed no differences that would explain mutant tolerance. Constitutive levels of ascorbate and glutathione also did not differ significantly in the three strains. An experiment testing the inducibility of paraquat tolerance revealed no change in the dose response of mutant or wild type gametophytes after exposure to sublethal concentrations of the herbicide. Uptake of paraquat by whole gametophytes was also equivalent in mutants and wild type. These data suggest that the physiological basis for tolerance in these mutants, unlike several other tolerant biotypes reported, does not lie in the oxygen radical scavenging system, in an inducible stress response, or in a block to whole-plant uptake.     

Evaluation of selectable markers for obtaining stable transformants in the gramineae

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10⁻⁵ to 2 × 10⁻⁶) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.     

Ion Homeostasis in Chloroplasts under Salinity and Mineral Deficiency: II. Solute Distribution between Chloroplasts and Extrachloroplastic Space under Excess or Deficiency of Sulfate, Phosphate, or Magnesium

Spinach (Spinacia oleracea var “Yates”) plants grown hydroponically were exposed to an excess or deficiency of various mineral ions. Solutes were measured in leaf extracts and in isolated intact chloroplasts. Under phosphate (120 millimoles per liter NaH₂ PO₄), sulfate (200 millimolar per liter (Na₂ SO₄), or magnesium excess (150 millimolar per liter MgCl₂), concentrations of these ions in leaf extracts increased, but in chloroplasts, concentrations of all ions remained constant. Concentrations of quarternary ammonium compounds in chloroplasts increased. Under mild phosphate or magnesium deficiency, concentrations of these ions decreased in chloroplasts less than in whole leaf extracts. Under severe sulfate deficiency causing chlorosis in younger leaves, sulfate concentrations in chloroplasts remained even unchanged, despite a drastic decrease of sulfate concentrations both in green and in chlorotic leaves. Together with results from a companion study (G Schröppel-Meier, WM Kaiser 1988 Plant Physiol 87: 822-827) our data demonstrate that leaf cells are able to keep the concentrations of several mineral ions rather constant in metabolically active compartments even at extremely large variations of ion concentrations in the culture solution and in the leaves.     

Gene Expression in Developing Zea mays Embryos: Regulation by Abscisic Acid of a Highly Phosphorylated 23- to 25-kD Group of Proteins

We have earlier identified a set of proteins of 23 to 25 kilodaltons (kD), covering an isoelectric point (pI) range of 6.2 to 8.2, which accumulate gradually during normal embryogenesis of Zea mays and disappear in early germination. These polypeptides can be induced prematurely in immature embryos by abscisic acid (ABA) treatment. We report here that the more acidic protein forms are due to post-translational phosphorylation of at least two polypeptides of 23 kD, pI 8.2 and 25 kD, pI 8.0. A polyclonal antiserum was obtained which recognizes all forms of both the 23-kD and 25-kD polypeptides. Recovery of cDNA clones corresponding to these proteins was accomplished by hybridization with cDNA made from size-selected mRNA enriched for these sequences. Hybrid selection experiments demonstrate that clone MA12 specifically hybridizes with mRNAs encoding the 23-kD and 25-kD protein set which are recognized by the antiserum. By Northern hybridization analysis, the RNA encoded by clone MA12 is shown to accumulate in mature embryos and to be induced in young embryos upon ABA incubation.