nitrogenase and potassium-nitrate

Soybean (Glycine max L. Merr.) plants exposed to 10 mM KNO(3) for a 4 d period were used to test the correlation between nitrogenase activity, gene expression and sucrose metabolism. Nitrate caused the down-regulation of sucrose synthase (SS) transcripts within 1 d, although a decline in nodule SS activity and an increase in nodule sucrose content only occurred after 3-4 d. In a second experiment, plants were exposed to (15)N-labelled nitrate for 48 h to determine the time period during which nitrate was taken up, and to relate this to the decline in apparent nitrogenase activity (H(2) production in air) and the reduction in SS gene transcript levels. The peak of nitrate uptake appeared to be between 8 h and 14 h whilst apparent nitrogenase activity began to decline at about 17.5 h. The SS mRNA signal declined markedly between 14 h and 24 h. The correlative association of these factors is clear. However, SS activity per se does not appear to be related to the initial decline in apparent nitrogenase activity as a result of nitrate uptake. These findings, therefore, do not support the hypothesis that the regulation of nodule function is mediated by the regulation of SS activity.

Topics: Blotting, Northern; Down-Regulation; Gene Expression Regulation, Plant; Glucosyltransferases; Glycine max; Leghemoglobin; Nitrates; Nitrogen Fixation; Nitrogen Isotopes; Nitrogenase; Plant Roots; Potassium Compounds; Starch; Sucrose; Urea

Using anti-(Fe protein) antibody raised against the Fe protein of the photosynthetic bacterium Rhodospirillum rubrum, it was found that the Fe protein component of nitrogenase (EC 1.18.2.1) from Azotobacter chroococcum cells subjected to an ammonium shock, and hence with an inactive nitrogenase, appeared as a doublet in Western blot analysis of cell extracts. The Fe protein incorporated [32P]phosphate and [3H]adenine in response to ammonium treatment, and L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2), prevented Fe protein from inhibition and radioisotope labelling. These results support that A. chroococcum Fe protein is most likely ADP-ribosylated in response to ammonium. After ammonium treatment, when in vivo activity was completely inhibited, Fe-protein modification was still increasing. This suggests the existence of another mechanism of nitrogenase inhibition faster than Fe-protein modification. When ammonium was intracellularly generated instead of being externally added, as occurs with the short-term nitrate inhibition of nitrogenase activity observed in A. chroococcum cells simultaneously fixing molecular nitrogen and assimilating nitrate, a covalent modification of the Fe protein was likewise demonstrated.

Topics: Adenosine Diphosphate Ribose; Ammonium Chloride; Azotobacter; Enzyme Inhibitors; Glutamate-Ammonia Ligase; Kinetics; Methionine Sulfoximine; Nitrates; Nitrogenase; Oxidoreductases; Potassium Compounds; Protein Processing, Post-Translational