Chrysoeriol and Luteolin Released from Alfalfa Seeds Induce nod Genes in Rhizobium meliloti

Abstract

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 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-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-7-0-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. 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',5,7-tetrahydroxyflavone, 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 off...

Concurrent Synthesis and Release of nod-Gene-Inducing Flavonoids from Alfalfa Roots

“…Transcription of nodABC is induced through the cooperative action of the protein product of the nodD gene and components of root and seed exudates (21). Luteolin, 3',4',5,7-tetrahydroxyflavone, and chrysoeriol, 3'-methoxy-4',5,7-trihydroxyflavone, are present in alfalfa seed exudates and can induce rhizobial nod genes (9,23). Those compounds are not found in alfalfa root exudates, but other flavonoid nod-gene inducers, 4,4'-dihydroxy-2'-methoxychalcone, 4',7-dihydroxyflavone, and 4',7-dihydroxyflavanone, are present (20).…”

“…This prospect is hypothetically possible for the dominant alfalfa nod-gene inducer, luteolin, because it is present in extracts of unimbibed alfalfa seeds (8,23). However, with the exception of trace amounts of 4',7-dihydroxyflavone, no nod-gene inducer found in root exudates also has been identified in alfalfa seeds (8) or seed exudates (9). As an alternative to complete synthesis in the previous generation, precursor flavonoids stored in seeds may be modified to nod-gene inducers with known reactions (10), transported to roots, and released during imbibition.…”

Effects of alfalfa nod gene-inducing flavonoids on nodABC transcription in Rhizobium meliloti strains containing different nodD genes

“…One well-described transcriptional unit, the nodABC genes, initiates cortical cell division (2, 26), root hair deformation (15), and production of a receptorlike protein (12,13). The nodABC genes in R. meliloti are expressed primarily in the presence of particular flavonoids exuded by alfalfa seeds or roots (7, 18, 21,23).Precisely how plant flavonoids induce transcription of nod genes in R. meliloti is not known, but a family of regulatory genes including nodDI, nodD2, and nodD3 is involved (4,5,9,22,25). Studies of the nodDi gene product (NodDl), nod gene-inducing flavonoids, and the promoter region of nod-ABC suggest that NodDl is a DNA-binding protein (3,8,16).…”

“…The biological significance of this diversity of nod gene inducers is not clear, but at least three points are noteworthy. First, although luteolin probably is the most prevalent nod gene inducer in the soil around a germinating alfalfa seed (7), the methoxychalcone and chrysoeriol induce half-maximum transcriptional rates of nodABC-lacZ fusion at 10 and 25%, respectively, of the concentration required for luteolin (7, 18). Second, the 4',7 substitution pattern (flavone numbering * Corresponding author.…”

“…One well-described transcriptional unit, the nodABC genes, initiates cortical cell division (2, 26), root hair deformation (15), and production of a receptorlike protein (12,13). The nodABC genes in R. meliloti are expressed primarily in the presence of particular flavonoids exuded by alfalfa seeds or roots (7, 18, 21,23).…”