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

Abstract

Flavonoid signals from alfalfa (Medicago sativa L.) induce transcription of nodulation (nod) genes in Rhizobium meliloti. Alfalfa roots release three major nod-gene inducers: 4',7-dihydroxyflavanone, 4',7-dihydroxyflavone, and 4,4'-dihydroxy-2'-methoxychalcone. The objective of the present study was to define temporal relationships between synthesis and exudation for those flavonoids. Requirements for concurrent flavonoid biosynthesis were assessed by treating roots of intact alfalfa seedlings with [U-14C]-L-phenylalanine in the presence or absence of the phenylalanine ammonia-lyase inhibitor L-2-aminoxy-3-phenylpropionic acid (AOPP). In the absence of AOPP, each of the three flavonoids in exudates contained 14C. In the presence of AOPP, 14C labeling and release of all the exuded nod-gene inducers were reduced significantly. AOPP inhibited labeling and release of the strongest nod-gene inducer, methoxychalcone, by more than 90%. Experiments with excised cotyledons, hypocotyls, and roots incubated in solution showed that the flavonoids could be synthesized in and released from each organ. However, the ratio of the three flavonoids in exudates from intact plants was most similar to the ratio recently synthesized and released from excised roots. A portion of recentiy synthesized flavonoid aglycones was found conjugated, presumably as glycosides, in root extracts and may have been involved in the release process. Data from root extracts showed that formononetin, an isoflavonoid which does not induce nod genes, was present in conjugated and aglycone forms but was not released by normal intact roots. In contrast, roots stressed with CuCI2 did release the aglycone formononetin. Thus, the release process responsible for exudation of nod-gene inducers appears to be specific rather than a general phenomenon such as a sloughing off of cells during root growth. The synthesis and specific concurrent release of flavonoid nod-gene inducers in this study is consistent with the physiological requirement for nodule formation of the 3-day-old seedlings used.Alfalfa (Medicago sativa L.), an important leguminous forage crop, forms N2-fixing root nodules in association with the soil bacterium Rhizobium meliloti. Early events of alfalfa nodule formation require expression of nodulation genes including nodDABC on the megaplasmid of R. meliloti (6,15). 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, '

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

“…4; Table II). In contrast, 5-deoxyflavonoids (flavone numbering system) from alfalfa root exudate (17,18) did not promote growth ofR. meliloti (Tables I and IV) promote polysaccharide formation by rhizobia (3), that phenomenon did not have major effects on A600 data in these experiments.…”

“…Culture and Treatment of Organisms 'Moapa 69' alfalfa (Medicago sativa) seeds were imbibed and rinsed with water to collect seed exudate as described previously (10 (10,17,18).…”

“…1) 4,4'-dihydroxy-2'-methoxychalcone, 4',7-dihydroxyflavone, and 4',7-dihydroxyflavanone (17). An additional flavonoid, formononetin (7-hydroxy-4'-methoxyisoflavone), also is released from seedling roots under stress (18). Genetic and biochemical evidence indicates that flavonoids induce rhizobial nod genes by activating a regulatory nodD gene product (15).…”

Suppression of defence responses in mycorrhizal alfalfa and tobacco roots

“…It is interesting to note that the concentration of formononetin increases when an AM fungus was present in the rhizosphere, even in P-fertilized plants that were not yet colonized by the fungus (Volpin & Kapulnik, 1994), This implies that formononetin production in the roots is induced merely by the presence of the fungus in the host rhizosphere, or by appressoria formation, but that visible colonization of the tissue is not required, Formononetin has not been shown to have antimicrobial activities, but is a precursor of the isoflavonoid phytoalexins produced in alfalfa in response to stresses and microbial infections (Dalkin et al, 1990a), Formononetin is usually not found in exudates of young (8-d-old) alfalfa roots, but is secreted from stressed roots (Maxwell & Phillips, 1990) and is produced in leaves of alfalfa inoculated with Ascockyta imperfecti (Olah & Sherwood, 1971), Formononetin precursors (2',4,4'-trihydroxychalcone, 4',7-dihydroxyflavanone and daidzein) and medicarpin precursors (2'-hydroxyformononetin and vestitone) were all below detectable levels (Volpin et aL, 1995), However, in well-colonized roots, 20-35 d after germination, the concentrations of two other isoflavonoids, genistein and biochanin A, were both elevated. This response might be specific to the G. intraradices-Medicago sativa interaction, Harbison & Dixon (1993) reported elevated amounts of medicarpin, medicarpinmalonylglucoside, daidzein, coumestrol, formononetin, formononetin-malonylglucoside and 4',7-dihydroxyBavone in roots of M. truncatula 7^0 d after Glomus inoculation, but HPLC profiles of the uninoculated M. truncatula roots were also very different from those obtained from M. sativa.…”

Induction of Microbial Genes for Pathogenesis and Symbiosis by Chemicals from Root Border Cells

“…It has been known for nearly a century that root exudates-the chemicals released from root systems into the soil-have profound effects on microorganisms in the rhizosphere (for review, see Curl and Truelove, 1986;Rovira, 1991). In recent years, specific sugars and phenolic compounds from plant exudates have been shown to induce microbial genes required for pathogenesis and symbiosis (Rossen et al, 1985;Stachel et al, 1985;Shearman et al, 1986;Peters and Long, 1988), and the cellular sources of such chemicals are now being examined (Maxwell and Phillips, 1990;Graham, 1991;Oommen et al, 1994;McKhann et al, 1997).…”