Isolation of auxotrophic mutants in support of ammonia assimilation via glutamine synthetase in Methanobacterium ivanovii

Various enzymes involved in the initial metabolic pathway for ammonia assimilation by Methanobacterium ivanovii were examined. M. ivanovii showed significant activity of glutamine synthetase (GS). Glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) were present, wheras, glutamate dehydrogenase (GDH) was not detected. When M. ivanovii was grown with different levels of NH ₊ ⁴ (i.e. 2, 20 or 200 mM), GS, GOGAT and ADH activities varied in response to NH ₊ ⁴ concentration. ADH was not detected at 2 mM level, but its activity increased with increased levels of NH ₊ ⁴ in the medium. Both GS and GOGAT activities increased with decreasing concentrations of NH ₊ ⁴ and were maximum when ammonia was limiting, suggesting that at low NH ₊ ⁴ levels, GS and GOGAT are responsible for ammonia assimilation and at higher NH ₊ ⁴ levels, ADH might play a role. Metabolic mutants of M. ivanovii that were auxotrophic for glutamine were obtained and analyzed for GS activity. Results indicate two categories of mutants: i) GS-deficient auxotrophic mutants and ii) GS-impaired auxotrophic mutants.Abbreviations GS Glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

The effect of oxygen on nitrate and nitrite assimilation in leaves of Zea mays L. under dark conditions

The assimilation of nitrate under dark-N₂ and dark-O₂ conditions in Zea mays leaf tissue was investigated using colourimetric and ¹⁵N techniques for the determination of organic and inorganic nitrogen. Studies using ¹⁵N indicated that nitrate was assimilated under dark conditions. However, the rate of nitrate assimilation in the dark was only 28% of the rate under non-saturating light conditions. No nitrite accumulated under dark aerobiosis, even though nitrate reduction occurred under these conditions. The pattern of nitrite accumulation in leaf tissue in response to dark-N₂ conditions consisted of three phases: an initial lag phase, followed by a period of rapid nitrite accumulation and finally a phase during which the rate of nitrite accumulation declined. After a 1-h period of dark-anaerobiosis, both nitrate reduction and nitrite accumulation declined considerably. However, when O₂ was supplied, nitrate reduction was stimulated and the accumulated nitrite was rapidly reduced. Anaerobic conditions stimulated nitrate reduction in leaf tissue after a period of dark-aerobic pretreatment.     

Specific photoperiodic stimulation of dry matter production in a high-latitude cultivar of Poa pratensis

Vegetative plants of Poa pratensis L. cv. Holt (origin 69°N) raised in short days gave large and significant increases in plant dry weight, plant height and leaf area upon exposure to continuous light, compared with 8-h short days, at essentially identical daily inputs of radiant energy (8-h summer daylight ± low intensity extension). For example, by the fourth harvest (after 26, 34 and 46 days at 21, 15 and 9°C, respectively), the dry weights of plants in long days were 81, 163 and 195% greater than those of the corresponding short-day controls at the respective temperatures. Plant leaf areas in long days were between two and four times as large as control values by the end of the experiment. This was mainly due to increased leaf length caused by long-day stimulation of cell extension and division. However, the photoperiod did not affect the partitioning of assimilates amongst leaves, culms and stolons. Most of these effects could also be brought about by exogenous gibberellin application to plants in short days. However, in contrast to the effect of long days, gibberellin treatment also induced stem internode elongation even in these vegetative plants. Examination by standard growth analysis procedures revealed that the observed increases in relative growth rate were due primarily to increased net assimilation rate followed, several days later, by increases in leaf area ratio when newly-emerged leaves began to constitute a significant proportion of the leaf area. It is concluded that these reactions are of great adaptive significance for growth at the marginal temperatures prevailing at high latitudes.     

Soybean leaf urease: Comparison with seed urease

Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide-derived urea to NH₃ and CO₂. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 m M ). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent K_m (0.8 m M ), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross-reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.     

Agrobacterium-mediated gene delivery and the biology of hostrange limitations

Insertion of foreign DNA into Ti plasmid-derived vectors in Agrobacterium tumefaciens is currently the most frequently used strategy for generating transgenic plants in a wide variety of species. Limitations of the host range of Agrobacterium restrict its usefulness in many cases, particularly when dealing with monocotyledonous plants. The objective of this presentation is to briefly discuss the efficiency of the transformation process utilized by Agrobacterium tumefaciens , potential barriers to efficient transformation by Agrobacterium that result in limitation of its useful host range, and how an understanding of the successful Agrobacterium /plant cell interaction might lead to advances in a variety of DNA delivery methodologies.     

Influence of salt stress on primary metabolism of Zea mays L. seedlings of model genotypes

Short-term studies for comparing some primary metabolic and growth-responses to salt stress in seedlings of two maize genotypes differing in drought resistance were carried out under controlled conditions. Both genotypes revealed high yielding ability in favourable environments. Treatments: Control (Hoagland-Arnon No 1 solution) and salt stress (Hoagland-Arnon solution plus NaCl, _s = –0.84MPa). It was found that in both genotypes the activity of the principal metabolic pathway supplying reduced nitrogen (¹⁵N) for the synthesis of amino acids and proteins as well as the assimulatory number (¹⁴CO₂—assimilation relation rate per chlorophyll unit) were decreased under the effect of the stress. These effects were more marked in the resistant genotype. In this genotype the stress induced metabolic activity decline was accompanied by a corresonding reduction of the relative growth rate. Conversely, continuing growth, resulting probably from accumulation of solutes, was observed in the susceptible genotype.On the basis of these and other observations it is assumed that the resistant genotype manifests short-term energy saving stress reactions.     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Molecular genetics of sulfate assimilation in plants

The sulfate assimilation pathway is the primary route by which higher plants obtain the sulfur necessary for growth. Sulfur is involved in a myriad of processes of central importance in metabolism. In the past few years much has been learned about this pathway and its regulation through analysis'of the genes encoding the enzymes and proteins that make up the sulfate assimilation pathway. The recent molecular genetic analysis builds on the biochemical and physiological groundwork of past studies. Further, gene analysis has provided the opportunity to compare directly the evolution of sulfate assimilation in plants and other organisms.,     

Purification and characterization of ferredoxin-sulfite reductases from leek (Allium tuberosum) leaves

Ferredoxin-sulfite reductases (Fd-SiRs) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from leek leaves have been purified to homogeneity. The enzymes (SiR 1, SiR 2 and SiR 3) were separated by Mono Q chromatography. The collective molecular mass of the enzymes was estimated to be 65 kDa by gel filtration. In all three cases, subunit analysis by SDS-PAGE yielded a single protein band corresponding to a molecular mass of 64 kDa, indicating that the enzymes each exist as a monomer. In the oxidized forms, SiR 1, SiR 2 and SiR 3 all exhibited nearly identical absorption maxima at 279∼280, 389∼390, 588 and 714 nm, indicating that siroheme is involved in the catalysis of sulfite reduction. On enzymatic properties, SiR 1, SiR 2 and SiR 3 could only react with the physiological electron donor, feriedoxin. The enzymes exhibited different heat stabilities. The pH active curve obtained from SiR 2 was different from the others. Moreover, SiR 1 exhibited a lower Km value for ferredoxin than SiR 2. Although the N-terminal sequence was the same, the results of some enzymatic properties, amino acid analysis, and peptide mapping suggested the presence of the Fd-SiR isozymes in leek leaves.