Effect of methionine sulfoximine on asparaginase activity and ammonium levels in pea leaves

In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.     

Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens

The transfer of genetic material into soybean tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens which contained the binary vector pGA482. The method used for transformation requires no tissue culture steps as it involves the inoculation of the plumule, cotyledonary node, and adjacent cotyledon tissues of germinating seeds. The identification of neomycin phosphotransferase (NPT) II enzyme activity in the tissues of 16 (R₀) soybean plants indicated that the plant expressible Nos-NPT II gene, contained within the T-DNA region from pGA482, had been transferred at least into somatic tissues. Putative transformed R₀ soybean plants were advanced to produce R₁ plants which were also assayed for the presence of the transferred Nos-NPT II gene. The combined results of these assays indicated that about 0.7% of the surviving inoculated seeds yielded transformed tissues in the R₀ plant, and that about 1/10 of these plants yielded transformed R₁ plants. The presence of the Nos-NPT II gene in DNAs isolated from both R₀ and R₁ plant was demonstrated by using genomic blot hybridization and polymerase chain reaction methods. Integration of this gene into the soybean genome was demonstrated for three R₁ soybean plants.     

Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex : Does Photorespiratory Ammonium Influence Mitochondrial Carbon Metabolism?

Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na⁺. NH₄⁺, at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K⁺ in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na⁺ was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH₄⁺ is discussed in relation to a possible regulatory interaction between photorespiratory NH₄⁺ production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.     

Kinetic model of hydrocortisone 1-en-dehydrogenation by Arthrobacter globiformis

A kinetic model of the hydrocortisone-to-prednisolone transformation by Arthrobacter globiformis is constructed using the experimental data obtained in studies of this process. Besides adequately describing experimental data, the model allows one to determine the relation between hydrocortisone oxidation and the level of endogenous substrates in bacterial cells, and the relation between the saturating concentration of hydrocortisone in the enzymic system of bacteria and the content of endogenous substrates in their cells, as well as the regulation of the transmembrane potential and the activation by the uncouplers.     

Modifications of extracellular electric and ionic gradients preceding the transition from tip growth to isodiametric expansion in the apical cell of the fern gametophyte

Fern (Onoclea sensibilis L.) gametophytes exposed to blue light are induced to undergo a morphological transition from a tip-growing filament to a planar prothallus. Extracellular measurements of electric currents and localized ion activities around the apical cell of 8 to 10 day-old gametophytes were made with a vibrating probe and ion selective electrodes. In darkness, we observed exit current densities of an average of 75 nanoamperes per square centimeter near the tip and 2 to 15 nanoamperes per square centimeter along the lateral walls of this cell. Measurements with ion selective electrodes for H⁺, K⁺, and Ca²⁺ showed that this cell was bounded by a thin layer of medium that was depleted in K⁺ and Ca²⁺ and exhibited a lower pH than the bulk solution. Both the K⁺ and Ca²⁺ depletion zones and the zone of higher acidity were particularly pronounced at the tip end of the cell; the pH at 2 micrometers from the tip was nearly 0.5 units more acid than the bulk medium at pH 6. Disruption of steady state, external gradients with media that contained lower concentrations of H⁺, K⁺, Ca²⁺, or Cl⁻ produced certain differences in the rates of restoration of particular ion zones, raising the possibility that some of the ion migrations are interdependent. Within 15 minutes after irradiation with blue light, current leaving the tip declined to levels which were indistinguishable from those leaving the lateral walls and there was a rapid lowering in the rates of tip acidification and K⁺ depletion near the tip. The rapid dissipation of both the longitudinally aligned electrical field and the tip-localized asymmetries in external cation distribution in blue light suggest that loss of electrical polarity in this tip growing cell may be an initial step in the chain of events which govern changes in cell shape.     

Rapid Image Analysis Screening Procedure for Identifying Chloroplast Number Mutants in Mesophyll Cells of Arabidopsis thaliana (L.) Heynh

To analyze the genetic control of the process of chloroplast division, a direct image analysis screening procedure has been developed in which mutants of Arabidopsis thaliana (L.) Heynh. var Landsberg erecta are selected on the basis of abnormal chloroplast number. The selection procedure is based on image analysis thresholding after iodine staining, which facilitates the automatic counting of chloroplasts in isolated mesophyll cells. M2 seedlings are screened for significant deviation from the wild type relationship between mesophyll cell size and chloroplast number. Mutants with both abnormally high and abnormally low chloroplast numbers were identified. Of 3500 individual M2 seedlings screened, 18 mutant lines have been isolated and shown to be stably inherited in three subsequent generations. The most extreme phenotypes show an 80% reduction or a 50% increase in chloroplast number per mesophyll cell.     

A comparison of the submergence response of deepwater and non-deepwater rice

Twelve cultivars of rice (Oryza sativa L.), representing deepwater, short-statured, and semidwarf types, were tested for their response to submergence. The magnitude of the response varied between cultivars; however, all cultivars responded to submergence by rapid growth once internodal elongation had started. Three of these cultivars were tested for elongation capacity at four ages. The deepwater rice was capable of rapid internodal elongation in response to submergence at 4 weeks of age. Growth of the short-statured and semidwarf cultivars was not stimulated by submergence until about 10 weeks of age. In air, the internodes of deepwater rice grew slower than did those of the short-statured and semidwarf cultivars. We also investigated the elongation response of stem sections of all 12 cultivars to an atmosphere containing 3% O₂, 6% CO₂, 91% N₂ (all by volume), and 1 microliter per liter ethylene. We found that the response of each of the non-deepwater cultivars was qualitatively and quantitatively similar to that of the deepwater rice.     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.