Effect of Inoculation and Nitrogen on Isoflavonoid Concentration in Wild-Type and Nodulation-Mutant Soybean Roots

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Abstract
The isoflavones, daidzein and genistein, have been isolated and identified as the major inducers of nod genes of Bradyrhizobium japonicum. The common nod genes of rhizobia are in turn responsible for stimulating root hair curling and cortical root cell division, the earliest steps in the host response. This study evaluated whether there was a relationship between root isoflavonoid production and the hypernodulation phenotype of selected soybean (Glycine max [L.] Merr.) mutants. Three independently selected hypernodulating soybean mutants (NOD1-3, NOD2-4, and NOD3-7) and a nonnodulating mutant (NN5) were compared with the Williams parent for isoflavonoid concentrations. High performance liquid chromatographic analyses of soybean root extracts showed that all lines increased in daidzein, genistein, and coumestrol concentrations throughout the 12-day growth period after transplanting of both inoculated and noninoculated plants; transplanting and inoculation were done 6 days after planting. No significant differences were detected in the concentration of these compounds among the three noninoculated hypernodulating mutants and the Williams parent. In response to inoculation, the three hypernodulating mutants had higher isoflavonoid concentrations than did the Williams control at 9 to 12 days after inoculation when grown at 0 millimolar N level. However, the inoculated nonnodulating mutant also had higher isoflavonoid concentrations than did Williams. N application [urea, (NH4)2SO4 and NO3−] decreased the concentration of all three isoflavonoid compounds in all soybean lines. Application of NO3− was most inhibitory to isoflavonoid concentrations, and inhibition by NO3− was concentration dependent. These results are consistent with a conclusion that differential NO3− inhibition of nodulation may be partially due to changes in isoflavonoid levels, although the similar response of the nonnodulating mutant brings this conclusion into question. Alternatively, the nodulation control in the NN5 mutant may be due to factors totally unrelated to isoflavonoids, leaving open the possibility that isoflavonoids play a role in differential nodulation of lines genetically competent to nodulate.