Effect of Localized Nitrate Application on Isoflavonoid Concentration and Nodulation in Split-Root Systems of Wild-Type and Nodulation-Mutant Soybean Plants

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Abstract
Although isoflavonoids are known to be inducers of nod genes in Bradyrhizobium japonicum, it was recently proposed that internal root levels of isoflavonoids may be important in nodule development on soybean (Glycine max [L.] Merr.). The hypernodulating soybean mutants were shown to accumulate higher root concentrations of isoflavonoid compounds (daidzein, genistein, and coumestrol) and to be more extensively nodulated than was the Williams parent when inoculated with B. japonicum. The hypernodulating mutants and the parent line, Williams, also showed decreased isoflavonoid concentrations and decreased nodule development if N was applied. The current study evaluated the effect of localized NO3− application on root isoflavonoid concentration and on nodulation in split-root systems of the Williams wild type and a hypernodulating mutant (NOD1-3). Nitrate application markedly decreased isoflavonoid concentrations in non-inoculated soybean roots. When roots were inoculated, nodule number, weight, and nitrogenase activity were markedly suppressed on the root-half receiving 5 millimolar NO3− compared with the other root-half receiving 0 millimolar NO3−. High performance liquid chromatographic analyses of root extracts showed that the root-half receiving 5 millimolar NO3− was markedly lower in isoflavonoid concentrations in both soybean lines. This was partially due to the localized stimulatory effect of NO3− on root growth. The inoculated NOD1-3 mutant had higher isoflavonoid concentrations than did the Williams control in both the presence and absence of NO3−. These results provide evidence that the site of N application primarily controls the site of nodulation inhibition, possibly through decreasing isoflavonoid levels. Although the effect of NO3− on nodule development and root isoflavonoid concentration was strongly localized, there was evidence that NO3− also resulted in a systemic effect on root isoflavonoids. The results are consistent with previous speculation that internal levels of root isoflavonoids may affect nodule development.