Nodule Formation Is Stimulated by the Ethylene Inhibitor Aminoethoxyvinylglycine

Abstract

Previous researchers found that formation and function of nitrogen-fixing nodules on legume roots were severely inhibited by addition of exogenous ethylene. Nodule formation by Rhizobium meliloti on Medicago sativa was stimulated twofold when the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) was added with the inoculum. Stimulation of nodule formation by AVG showed a similar concentration dependence as inhibition of ethylene biosynthesis, suggesting that the primary action of AVG is the inhibition of ethylene biosynthesis. When AVG was added 2 to 3 days after inoculation, the number of nodules formed was still increased. On a per plant basis, however, the average nitrogen fixation was unchanged by AVG treatment and was independent of nodule number. also showed that ethylene decreased the rates of nitrogen fixation to only 10 and 30% ofthe controls for pea and clover, respectively.Given the adverse effects of exogenous ethylene on nodulation, we determined whether inhibition of ethylene biosynthesis would enhance nodulation and nitrogen fixation. To inhibit ethylene biosynthesis, we chose AVG2 (2), an inhibitor of the enzyme 1 -aminocyclopropane-1 -carboxylic acid synthase which converts S-adenosyl methionine to 1 -aminocyclopropane-1 -carboxylic acid-the immediate precursor of ethylene (1, 5). We report that the presence of AVG stimulates nodule formation but does not result in increased nitrogen fixation. MATERIALS AND METHODS Bacterial Strains and CulturesRhizobium meliloti strain 2011 was maintained on and cultured in YEMG (5.0 g mannitol, 5.0 g sodium gluconate, 0.5 g yeast extract, 0.5 g K2HP04, 0.2 g MgS04 7H20, 0.1 g NaCl, 1.6 g CaCl2 2H20 per L at pH 6.8) at 30°C. Inocula for plants were prepared by diluting log phase cultures (A600 = 0.3-0.6) with sterile water to a concentration of 107 cells/ mL. Rhizobia cell numbers were determined by viable cell counts. Plant MaterialMedicago sativa (alfalfa) cv AS-R3 from Asgrow Seed was used in all experiments. Seeds were surface sterilized by treatment in 70% ethanol for 30-60 min followed by 20 min in 10% Ca(OCI)2. Seeds were rinsed thoroughly with several changes of sterile distilled H20, imbibed in distilled H20 over night, and transferred in groups of five to plastic growth pouches (Northrup King Seed Co., Minneapolis, MN). The growth pouches had been previously sterilized with ethylene oxide and wetted with 10 mL of sterile Jensen's nitrogen-free medium (22). Plants were maintained in a growth chamber at 70 to 80% RH 23°C in light for 16 h, and 21°C in dark for 8 h. Pouches were watered with sterile water as required during the incubation period.

The timing of exogenous 2,4-dichlorophenoxyacetate (2,4-D) application affects nodulation of soybeans,glycine max(L.) merr.

“…The nodules produced were, therefore, smaller and less efficient than those produced by the control plants, at least at 24 d after sowing when the experiment was completed. This interpretation is similar to that of Kent-Peters and Crist-Estes (1989) who observed that Medicago sativa (alfalfa) treated with the ethylene inhibitor aminoethoxyvinylglycine (AVG) exhibited a larger number of nodule than the control plants, although the ARA/plant values were similar.…”

Reactive oxygen species and ethylene play a positive role in lateral root base nodulation of a semiaquatic legume

“…Ethylene requirement is a special feature of S. rostrata nodulation. In Medicago sativa or Glycine max, nodulation was promoted or not influenced by ethylene inhibitors, respectively (8,9,36). Molecular data have confirmed the ethylene requirement: clones homologous to genes coding for S-adenosyl-L-methionine synthetases, 1-aminocyclopropane-1-carboxylate synthases and oxidases, which are three enzymes involved in ethylene synthesis, are up-regulated during intercellular invasion (unpublished data).…”

“…Ethylene plays mostly a negative role in legume symbiosis, probably by restricting infection. In Medicago sativa, nodulation is inhibited by exogenous ethylene and stimulated by an ethylene synthesis inhibitor (8), whereas an ethylene-insensitive mutant of Medicago truncatula is hyperinfected by Sinorhizobium meliloti (9). In Pisum sativum, exogenous ethylene inhibited nodulation (10), and the nodulation-defective mutant (R50, sym16) is rescued by inhibitors of ethylene (11).…”

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

“…Genetic and physiological experiments indicate that the gaseous hormone ethylene plays an important role in controlling nodule number in legumes and that application of exogenous ethylene can inhibit nodulation (93,126). Modulation of in planta synthesis of ethylene in M. truncatula by using aminoethoxyvinylglycine (AVG) and ACC (an inhibitor of the ethylene-producing enzyme ACC oxidase and the substrate of ACC oxidase, respectively) has shown that the nodule number is low in the presence of ACC (high in planta levels of ethylene) and higher in the presence of AVG (low in planta levels of ethylene) (119).…”