Chrysoeriol and Luteolin Released from Alfalfa Seeds

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 nodABClacZ fusion in R. meliloti. During the first 4 hours of imbibition, total nod gene-inducing activity was released from seeds at 100fold 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-IacZ 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-70-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. Establishment of the N2-fixing symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa L.) requires a number of interactions between the two organisms. After the plant releases a signal that induces transcription of the bacte'Funded in part by U.S. Department of Agriculture Competitive Research Grants Office grant 87-CRCR-1-2552 and grant IS-134887 from the U.S.-Israel Binational Agricultural Research and Development Fund (BARD). U. A. H. was supported by the Swiss National Research Foundation. rial nodulation genes nodABC (22), rhizobial products induce root hair curling (17) and cortical cell divisions (8). The first molecule showing nod gene-inducing activity in R. meliloti was isolated from alfalfa seeds and identified as 3',4',5,7tetrahydroxyflavone, a compound known as luteolin (23). Recent studies of root exudates from 72-h-old 'Moapa 69' alfalfa seedlings identified 4,4'-dihydroxy-2'-methoxychalcone, 4',7-dihydroxyflavone, and 4',7-dihydroxyflavanone as active nod gene inducers, but no luteolin was detected (20). Numerous flavonoids from various legumes have been reported as active nod gene inducers (4, 9, 18, 25, 26, 29), but qualitative and quantitative differences between compounds actually released from seeds and roots have not been described. The presence of particular flavonoids inside plants cannot be taken as evidence of their release into exudates. Yelton et al. (28) observed that extracts, but not exudates, ofsome plant species induced nod genes in R. meliloti. Presumably, host plant factors controlling the synthesis and release offlavonoids can affect how rhizobia and legumes interact to form root nodules. Thus, the long list of flavonoids extracted from 'Medicago x varia Martyn' seeds (10) (apparently a hybrid of M. sativa L. x M. falcata L. [14]) does not indicate which compounds will be available to rhizobial cells near a seed in the soil. To understand how alfalfa and R. meliloti interact, a more complete identification ofnod gene-inducing flavonoids released from alfalfa seeds is required. The present study was designed to identify nod gene-inducing flavonoids released from Moapa 69 alfalfa seeds and to quantify their importance relative to other nod gene inducers released from roots (20). MATERIALS AND METHODS Preparation of Exudates Alfalfa (Medicago sativa L., cv 'Moapa 69') seeds (1.0 g, about 400 seeds) were scarified, surface-sterilized for 3 min in 70% ethanol, washed three times with sterile water, and imbibed in aerated, sterile water. After 4 h, seeds were removed, and the remaining solution, termed seed exudate, was diluted 1:1 with methanol for quantitative HPLC measurements and assays or dried under vacuum for large-scale purification studies. Root exudate, described previously (20), consisted of an aerated, hydroponic solution that had bathed roots of seedlings developing from the seeds after rinsing for 24 h. The hydroponic containers, over which seedlings were suspended (20), were kept at 25°C in a laminar-flow hood with irradiance of 60 ,iE * m-2 * s-' and a 12-h photoperiod.