N-limited due to insufficient root nodule formation (10) and suggests that more ..... Firmin JL, Wilson KE, Rossen L, Johnston AWB (1986) Fla- vonoid activation ...
Received for publication May 10, 1989 and in revised form June 22, 1989
Plant Physiol. (1989) 91, 842-847 0032-0889/89/91 /0842/06/$01 .00/0
A Chalcone and Two Related Flavonoids Released from Alfalfa Roots Induce nod Genes of Rhizobium meliloti' Carl A. Maxwell, Ueli A. Hartwig, Cecillia M. Joseph, and Donald A. Phillips* Department of Agronomy and Range Science, University of California, Davis, California 95616 ABSTRACT
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 of the 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 of this study was to identify, quantify, and characterize the major nod inducers exuded by roots of young, unnodulated alfalfa seedlings.
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',7dihydroxyflavanone.
MATERIALS AND METHODS Plant Culture
One 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 remove seed-derived compounds. At the end of 8 h, seeds were placed on a sterile cheeseclothcovered screen over 300 mL of sterile, aerated, N-free nutrient solution (6; with sodium ferric diethylenetriamine pentaacetate as the iron source, same concentration of iron) in a plastic, 473 mL, 'freezer' container. A sterile clear plastic sheet was positioned over the assembled container and held in place with a rubber band. Containers were maintained under an irradiance of 320 ,uE m22s-' (400-700 nm), 16/8 h light/ dark, 25/20°C, and 50% RH. Solutions containing root exudate were changed every 24 h for 8 d, sampled for nodinducing activity, and stored at -20°C. At each sampling time four replicates consisting of solutions from three containers
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 of the 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), while different, commercially obtained, flavonoids had weaker inducing capabilities (23, 24). Closely related compounds have similar functions with other Rhizobium- and Bradyrhizobium-legume associations (1, 9, 17, 25, 27, 31). The availability of nod-inducing flavonoids in the rhizosphere may limit alfalfa seedling growth. Kapulnik et al. (14) observed significant increases in nodulation, N2 fixation, and seedling growth of 'Hairy Peruvian' alfalfa when the rhizosphere was supplemented with luteolin. This is consistent with
were collected. Biological Activities The nod-inducing capacity of root exudate and specific compounds was assayed as f3-galactosidase activity from transcription of the nodC-lacZ fusion on plasmid pRmM57 in Rhizobium meliloti strain 1021 (22), obtained from Dr. S. R. Long (Stanford University). Cultures were maintained on LMB (29) slants with yeast extract as the nitrogen source and 10.8 ,ug tetracycline- mL-', before transfer to LMB liquid
' Supported in part by U.S. Department of Agriculture CRGO grant 87-CRCR-1-2552 and grant IS-1348-87 from BARD, the U.S.Israel Binational Agricultural Research and Development Fund. U. A. H. was supported by the Swiss National Research Foundation.
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ALFALFA ROOT FLAVONOIDS INDUCE RHIZOBIAL nod GENES
843
media, containing NH4NO3, at least 12 h prior to use in the assay. The i3-galactosidase assay was modified from Miller (21) as previously reported (22). Volumes were adjusted to 100 ,uL with 0.1 M sodium phosphate buffer (pH 7.0), and 375 gL of R. meliloti culture in midlog phase (0.2-0.3 A60o) was added. Samples were incubated at room temperature with shaking for 3 h, and 50 ,uL each of 0.1% (w/v) SDS and chloroform were added.
tions containing nod inducers were identified by spectral analysis (200-500 nm) and dried under N2 gas. Some compounds were purified further by normal phase HPLC with a column (250 x 4.6 mm) containing 5 um Hypersil (Alltech Associates, Inc.) Following isocratic chromatography in 100% chloroform for the first 10 min, a linear gradient to 90:9.5:0.5 (v/v/v) chloroform:methanol:water at 30 min was produced. Compounds of interest were tracked by spectral analysis and dried under N2 gas.
Purification of nod Inducers
Identification of nod Inducers
Root exudate samples were thawed, centrifuged at 6200g for 15 min, and passed through 0.8 and 0.2 ,um polycarbonate filters (Nuclepore Corp., Pleasanton, CA). Subsamples (50 mL) were adsorbed to 900 mg C18 Maxi-Clean cartridges (Alltech Associates, Inc., Deerfield, IL). Flavonoids from each cartridge were eluted with acetone, dried with a N2 gas stream (25°C), and dissolved in 400 ,uL of 50% methanol. Aliquots (100 ,gL) were loaded onto a Waters HPLC system (Millipore Corp., Milford, MA) fitted with a 250 x 4.6 mm Lichrosorb SRP 18 column (Phenomenex, Rancho Palos Verdes, CA) and eluted at 0.5 mL- min-' from 0 to 20 min with an isocratic solvent system of 52.5:45:2.5 (v/v/v) water:methanol:acetic acid. From 20 to 30 min a linear gradient to 60:37.5:2.5 (v/ v/v) methanol:water:acetic acid was applied, and the analysis continued isocratically at that concentration for another 30 min. Eluting compounds were monitored with a Waters 990 photodiode array detector, which measured absorbance (230400 nm) every second with 1.4 nm resolution. Eluant fractions were collected every 30 s, combined when associated with absorbance peaks, dried under N2 (45C), redissolved in 500 )L of 0.1 M sodium phosphate buffer (pH 7.0), and assayed for nod induction. The quantity of nod-inducing activity associated with a subsample of each HPLC absorbance peak was related to the concentration of crude root exudate by assuming the peak represented the total amount of a particular compound injected into the HPLC. Flavonoid nod inducers detected after initial HPLC analyses of root exudate were purified to homogeneity from exudate produced between 48 and 72 h after seeds were exposed to water. Exudate was partitioned against hexane to remove lipid, and flavonoids in the aqueous fraction were adsorbed to C18 cartridges. Acetone eluants from C18 cartridges were combined and dried under N2 gas at 45°C. Flavonoids were solubilized in 500 gL of 50% methanol and separated by HPLC on a 250 x 10 mm Lichrosorb SRP18 semi-preparative column (Alltech Associates, Inc.) with an elution rate of 2 mL.min-'. HPLC solvents and gradients were identical to those described above. Eluant fractions were collected every 30 s, combined when associated with absorbance peaks, and dried either under N2 gas or with an Evapotec (Haake Buchler, Saddle Brook, NJ). Closely eluting contaminants were removed from nod inducers by a second HPLC separation on the semipreparative column with appropriate concentrations of methanol. Fractions of interest were further purified by chromatography in 100% methanol on a column (1.5 x 25 cm) of Sephadex LH20 (Sigma Chemical Co., St. Louis, MO). Frac-
UV/visible spectral shift analyses (19) were done with a Lambda 6 dual beam spectrophotometer (Perkin Elmer, Norwalk, CT). Authentic standards were compared after tentative identifications. Standards were obtained from the following sources: 4',7-dihydroxyflavone, 4',7-dihydroxyflavanone, and luteolin (Spectrum Chemical MFG Corp., Gardena, CA); 2',4,4'-trihydroxychalcone (L. Jurd, USDA, Albany, CA); 4,4'-dihydroxy-2'-methoxychalcone (R. Carlson, Ecochem Research, Inc., Chaska, MN). COSY2 and one-dimensional proton NMR experiments were done in [U-2H]methanol on a Nicolet NT-360 spectrometer at the NMR Facility, University of California, Davis. One study of the 'strong' inducer was done, courtesy of Dr. J. Dallas, on a GE-NMR GN-500 Omega spectrometer at the General Electric Co., Fremont, CA. Spectra were referenced to the methyl peak (3.30 ppm) of methanol. Electron ionization MS data were collected by staff at the Facility for Advanced Instrumentation, University of California, Davis using a solids probe to introduce the samples on a Trio-2 MS (VG Masslab, Altrincham, UK). Flavonoid Concentrations After identities of nod inducers were verified by comparisons with authentic standards, their concentrations in root exudate and in nod-induction assays were determined spectrophotometrically in methanol from the following log e values: 4,4'-dihydroxy-2'-methoxychalcone, 4.25 at 349 nm (4); 4',7-dihydroxyflavone, 4.52 at 328 nm (15); 4',7-dihydroxyflavanone, 4.14 at 275 nm (13); 2',4,4'-trihydroxychalcone 4.20 at 370.5 nm (13). Luteolin concentrations were determined in ethanol from a log c of 4.17 at 350 nm (19). Amounts of the nod inducers in root exudate were determined by HPLC with known quantities ofauthentic standards by applying the integrator function within the Waters 990 software package. Values were corrected for percent germination and flavonoid recovery, as determined with known amounts of 4',7-dihydroxyflavone processed in the normal manner used for root exudate. RESULTS The nod-inducing activity of root exudate reached a maximum during the third day after initiation of imbibition (Fig.
2Abbreviations: COSY, two-dimensional homonuclear shift correlation spectroscopy; 6H, chemical shift of proton; 2H, two protons, etc.; C-1, carbon one, etc.; d, doublet; dd, double doublet; s, singlet; m, multiplet; J, coupling constant; Hz, Hertz; m/z, mass charge ratio.
Plant Physiol. Vol. 91,1989
MAXWELL ET AL.
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