Luteolin is a phenolic compound from plants that acts as a potent and specific inducer of nodABC gene expression in Rhizobium meliloti. We have found that R. meliloti RCR2011 exhibits positive chemotaxis towards luteolin. A maximum chemotactic response was observed at 10-8 M. Two closely related flavonoids, naringenin and apigenin, were not chemoattractants. The presence of naringenin but not apigenin abolished chemotaxis of R. meliloti towards luteolin. A large deletion in the nif-nod region of the symbiotic megaplasmid eliminated all chemotactic response to luteolin but did not affect general chemotaxis, as indicated by swarm size on semisoft agar plates and chemotaxis towards proline in capillary tubes. Transposon TnS mutations in nodD, nodA, or nodC selectively abolished the chemotactic response of R. meliloti to luteolin. Agrobacterium tumefaciens GMI9050, a derivative of the C58 wild type lacking a Ti plasmid, responded chemotactically to 10-8 M luteolin. The introduction of a 290-kilobase nif-nod-containing sequence of DNA from R. melioti into A. tumefaciens GM19050 enabled the recipient to respond to luteolin at concentrations peaking at 10-6 M as well as at concentrations peaking at 10-8 M. The response of A. tumefaciens GM19050 to luteolin was also abolished by the presence of naringenin.
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.
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