Interactions among Flavonoid nod Gene Inducers Released from Alfalfa Seeds and Roots

Alfalfa (Medicago sativa L.) seeds and roots can create complex rhizosphere effects by releasing flavonoids that induce nodulation (nod) genes in Rhizobium meliloti. Previous reports identified luteolin and 4,4'-dihydroxy-2'-methoxychalcone as strong inducers that are released from seeds and roots, respectively, and 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone as weaker inducers which are exuded by roots. As a first step toward identifying flavonoid interactions that may occur in the rhizosphere, combinations of these molecules were tested for transcriptional effects on a nodABC-lacZ fusion in R. meliloti. At low concentrations (e.g. 8.4 nanomolar), interactions of the three nod gene inducers from root exudate were additive. When the strong inducers 4,4'-dihydroxy-2'-methoxychalcone and luteolin were present separately at higher concentrations (e.g. 21 nanomolar), their effect could be decreased significantly by the weaker inducers 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone. In contrast, when low concentrations of luteolin from seed rinses and 4,4'-dihydroxy-2'-methoxychalcone from root exudate were present together, they produced synergistic increases in nod gene transcription. Tests with mixtures of the three nod gene inducers from root exudate indicated that alfalfa seedlings might easily decrease the strong inductive effect of the chalcone by releasing modest amounts of the weaker inducers. In addition, mixtures of luteolin and the nod gene inducers in root exudate suggested that interactions between nod gene inducers from seeds and roots may create a zone highly favorable to root nodule formation near the top of the primary root.