Pat J. Langston-Unkefer

Enhancement of Symbiotic Dinitrogen Fixation by a Toxin-Releasing Plant Pathogen

An approximate doubling in plant growth, total plant nitrogen, nodulation, and overall dinitrogen fixation of alfalfa are the consequences of the action of a toxin delivered by a Pseudomonas infesting the alfalfa rhizosphere. The toxin, tabtoxinine-β-lactam, inactivates selectively one form of glutamine synthetase in the nodules. Thus, normal glutamine synthetase-catalyzed ammonia assimilation is significantly impaired; yet these plants assimilated about twice the normal amount of nitrogen. How plants regulate dinitrogen fixing symbiotic associations is an important and unresolved question; the current results imply that the glutamine synthetase-catalyzed step in ammonia assimilation, a plant function, strongly influences overall dinitrogen fixation in legumes.

Interactions between Pseudomonas syringae pv. tabaci and Two Rhizosphere Hosts, Medicago sativa and Avena sativa

The interactions between Pseudomonas syringae pv. tabaci and either nodulating alfalfa (Medicago sativa) or oat (Avena sativa) seedlings were examined to further our understanding of this rhizosphere association. P. syringae pv. tabaci produces and releases a toxin, tabtoxinine-β-lactam (TβL), that inactivates glutamine synthetase (GS). Sinorhizobium meliloti grew well in the presence of TβL in culture and on alfalfa roots. The alfalfa symbiont, S. meliloti, and its bacteroids contained TβL-sensitive glutamine synthetases and TβL detoxifying-β-lactamase. The GS of alfalfa leaves is also sensitive to TβL, but GS activity was unaffected in infested plants. Toxin production was apparently suppressed in the alfalfa and nitrate-fed oat rhizospheres since these plants survived and retained significant amounts of leaf GS activity. The water-soluble extracts of these rhizospheres inhibited TPL production in culture and the inhibition was correlated with the amount of reduced nitrogen present. Furthermore, representative mixtures of pure ammonium and amino acids inhibited TβL production in culture in a concentration dependent manner. Thus, a bi-directional interaction occurs between the nitrogen metabolism of alfalfa and oat and TβL production by P. syringae pv. tabaci.

In vivo inactivation of glutamine synthetase by tabtoxinine-β-lactam in Zea mays suspension culture cells

Abstract Inactivation of glutamine synthetase by tabtoxinine-β-lactam was examined in Zea mays cv. Black Mexican suspension culture cells. Tabtoxinine-β-lactam was readily taken up from micromolar solutions of the toxin and, subsequently, rapidly inhibited glutamine synthetase. When cells were treated with 50 μ m tabtoxinine-β-lactam, less than 40% of the initial glutamine synthetase activity remained after 60 min. The kinetics of in vivo glutamine synthetase inactivation by tabtoxinine-β-lactam (50–1000 μ m ) was non-first order, with progressively slower rates of inactivation, perhaps reflecting the complexity of two separate processes, e.g. toxin uptake and subsequent enzyme inhibition. The kinetics of in vitro inactivation of purified glutamine synthetase by tabtoxinine-β-lactam were also non-first order (50–2000 μ m ) with progressively slower rates of inactivation, suggesting enzyme inactivation becomes the rate limiting step in the plant. Since tabtoxinine-β-lactam flux into the cell is a factor in the inactivation of glutamine synthetase in the plant, in vivo inactivation of glutamine synthetase was examined under conditions that might affect toxin uptake. Incubation with 1 m m methionine or alanine inhibited in vivo inactivation of glutamine synthetase in the presence of 300 μ m toxin. The pH optimum of the treatment solution for tabtoxinine-μ-lactam activity was 4·5, and carbonyl cyanide m -chlorophenylhydrazone, a protonophore and metabolic inhibitor, inhibited toxin transport. The results of this study show that tabtoxinine-β-lactam uptake is metabolically linked and that the concentration of toxin in planta need not be very high to effectively disrupt nitrogen metabolism.