A Chalcone and Two Related Flavonoids Released from Alfalfa Roots Induce nod Genes of Rhizobium meliloti

Abstract

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...

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

“…Rhizobium-free alfalfa (Medicago sativa L, cv 6Moapa 69') seedlings were grown as previously described (20) in a hydroponic system with sterile N-free nutrient solution (4) …”

“…Exudate solutions were supplemented with known quantities of 4',7-dihydroxyflavanone, 4,4'-dihydroxy-2'-methoxychalcone, and 4',7-dihydroxyflavone, centrifuged at 6500g, and processed with C18 cartridges as previously described (20). Dried acetone eluants of C18 cartridges were stored at -80°C, then solubilized in 50% (v/v) methanol in water, and fractionated by analytical HPLC.…”

“…No C18 cartridges were employed; the complete extract was processed on the analytical HPLC. HPLC fractions were collected every 30 s, combined when associated with absorbance peaks, and assayed for nod-gene-inducing activity (below (20) were identified in HPLC eluants by retention time and spectral traits. Quantification and bioassays for nod-gene-inducing activity with a nodC-lacZ fusion on plasmid pRmM57 in Rhizobium meliloti strain 1021 were done as described previously (20).…”

“…The predominant flavonoid in alfalfa roots, later recognized as formononetin, was isolated from extracts and identified by UV/visible spectroscopy with shift reagents (18,20), one-dimensional proton NMR (20), and MS (20 …”

“…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). Related flavonoids can induce nodgene transcription in other Rhizobium and Bradyrhizobium species (2,7,16,25,26,30).…”