Flavonoid signals from alfalfa (Medicago sativa L.) induce transcription of nodulation (nod) genes in Rhizobium melioti. Previous investigations identified the flavone luteolin as an active inducer in alfalfa seed extracts, but the nature of nod inducers released from roots has not been reported. Root exudate from 3-day-old alfalfa seedlings was purified and then assayed for biological activity with a nodABC-IacZ fusion in R. meliloti. Indentities of major nod inducers were established by spectroscopic analyses (ultraviolet/visible, proton nuclear magnetic resonance, and mass spectroscopy) and comparison with authentic standards. Major nod inducers, which were identified as 4',7-dihydroxyflavone, 4'-7-dihydroxyflavanone, and 4,4'-dihydroxy-2'-methoxychalcone, were released from seedling roots at 54, 22, and 20 picomole.plantr'. day-1, respectively. Luteolin was not found in these root exudates. The 4,4'-dihydroxy-2'-methoxychalcone induced nod genes at a concentration one order of magnitude lower than luteolin and is the first naturally released chalcone reported to have this function. Moderate and weak nod-inducing activity was associated, respectively, with 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone.Alfalfa (Medicago sativa L.), an important leguminous forage crop throughout the world, forms N2-fixing root nodules in association with the soil bacterium Rhizobium meliloti. The earliest events of alfalfa nodule formation require expression ofthe nodulation (nod) DABC genes on the megaplasmid (pSym) of R. meliloti (8,16). Transcription of nodABC is induced through the cooperative action of the constitutive nodD product and components of root and seed exudates (22). Luteolin, 3',4',5,7-tetrahydroxyflavone, was isolated from alfalfa seed extracts and shown to participate in nod induction (23) evidence that some N2-dependent alfalfa seedlings are initially N-limited due to insufficient root nodule formation (10) and suggests that more nodules might be formed if more rhizobial cells are induced to initiate the infection process.The presence of active nod inducers in plants does not guarantee release into the rhizosphere. Yelton et al. (30) observed that extracts from some plants induced nod transcription in R. meliloti even when exudates ofthe same plants did not. In contrast, both extracts and exudates of alfalfa induced nod genes, but it is unclear if luteolin was solely responsible for nod induction.In order to better understand the process of nod-inducer release, it is necessary to identify active compounds actually exuded into the rhizosphere. The purpose ofthis study was to identify, quantify, and characterize the major nod inducers exuded by roots of young, unnodulated alfalfa seedlings. MATERIALS AND METHODS Plant CultureOne g of alfalfa (Medicago sativa L.) seed (cv 'Moapa 69') containing about 400 seeds (94% viable) was scarified, surfacesterilized 3 min with 70% ethanol, rinsed with sterile water, and imbibed in sterile, aerated water. Imbibing solutions were changed after 4 and 8 h to rem...
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...
Spectroscopic data (nuclear magnetic resonance, mass spectrometry, ultraviolet-visible) in this study identify trigonelline and stachydrine as major components of alfalfa (Medicago sativa L.) seed rinse. Moreover, biological assays show that these natural products induce nodulation (nod) gene transcription in Rhizobium meliloti by activating the regulatory protein NodD2, but not the homologous NodDl protein. These findings contrast with the fact that the only previously identified NodD2 activator, 4,4'-dihydroxy-2'-methoxychalcone (MCh), also activates NodDl protein. Trigonelline and stachydrine induce nod genes only at much higher concentrations than MCh, but they are released from seeds in correspondingly greater amounts. The existence of these amphoteric, nonflavonoid nod gene inducers broadens our understanding of the biochemical processes and ecological mechanisms that a legume host uses to regulate its microbial symbiont.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.