CHRYSOERIOL AND LUTEOLIN RELEASED FROM ALFALFA SEEDS INDUCE NOD GENES IN RHIZOBIUM-MELILOTI

Flavonoid signals from alfalfa (Medicago sativa L.) seed and root exudates induce transcription of nodulation (nod) genes in Rhizobium meliloti. The flavone luteolin previously was isolated from alfalfa seeds by other workers and identified as the first nod gene inducer for R. meliloti. Our recent study of 'Moapa 69' alfalfa root exudates found no luteolin but did identify three other nod gene inducers: 4,4′-dihydroxy-2′-methoxychalcone, 4′,7-dihydroxyflavone, and 4′,7-dihydroxyflavanone. The goal of the current study was to identify and quantify nod gene-inducing flavonoids that may influence Rhizobium populations around a germinating alfalfa seed. Aqueous rinses of Moapa 69 alfalfa seeds were collected and assayed for induction of a nodABC-lacZ fusion in R. meliloti. During the first 4 hours of imbibition, total nod gene-inducing activity was released from seeds at 100-fold higher rates than from roots of 72-hour-old seedlings. Five flavonoids were purified and identified by spectroscopic analyses (ultraviolet/visible absorbance, proton nuclear magnetic resonance, and mass spectroscopy) and comparison with authentic standards. Two very active nod gene-inducing flavonoids, chrysoeriol (3′-methoxyluteolin) and luteolin, were identified in seed rinses. Luteolin required a higher concentration (18 nanomolar) than chrysoeriol (5 nanomolar) for half-maximum induction of nodABC-lacZ in R meliloti, and both were less active than 4,4′-dihydroxy-2′-methoxychalcone (2 nanomolar) from root exudates. Seeds exuded three other luteolin derivatives: luteolin-7-O-glucoside, 5-methoxyluteolin, and 3′,5-dimethoxyluteolin. Their combined quantities were 24-fold greater than that of luteolin plus chrysoeriol. Most nod gene-inducing activity of these luteolin derivatives apparently is associated with degradation to luteolin and chrysoeriol. However, their presence in large quantities suggests that they may contribute significantly to nod gene-inducing activity in the soil. These results indicate the importance of germinating seeds as a source of nod gene-inducing flavonoids and emphasize the quantitative and qualitative differences in those compounds around the seed and root.