performance liquid chromatographic analyses of soybean root extracts showed that all lines increased in daidzein, genistein, and coumestrol concentrations ...
Received for publication July 30, 1990 Accepted October 26, 1990
Plant Physiol. (1991) 95, 435-442 0032-0889/91 /95/0435/08/$01 .00/0
Effect of Inoculation and Nitrogen on Isoflavonoid Concentration in Wild-Type and Nodulation-Mutant Soybean Roots1 Myeong-Je Cho and James E. Harper* Department of Agronomy (M-J.C.) and U.S. Department of Agriculture, Agricultural Research Service, Plant Physiology and Genetics Research Unit (J.E.H.), University of Illinois, 1102 S. Goodwin Avenue, Urbana, Illinois 61801 sary for nitrogen fixation (nif and fix genes) and nodulation (nod genes). Common nodulation genes (nodDABC) in rhizobia are responsible for stimulating root hair curling and cortical cell division, the earliest steps in the host response (24, 27). These genes are induced by plant signal compounds such as flavonoids and isoflavonoids exuded from the roots of plant hosts (7, 17, 24, 26, 28). The isoflavones, daidzein and genistein, have been isolated and identified as the major components that stimulate B. japonicum nodABC-lacZ fusions, and this activation showed a concentration dependence up to 5 ,uM (17). In addition, coumestrol (a coumestan) and daidzein have also been shown to promote the growth of B. japonicum (3). In contrast, Firmin et al. (7) showed that daidzein and genistein are potent antagonists of nod gene induction in Rhizobium leguminosarum. Djordjevic et al. (6) also showed that umbelliferone (a coumarin) and formononetin (an isoflavone) from white clover root exudates antagonize the stimulatory activity of daidzein. Kosslak et al. (17) demonstrated that these compounds induce the nodABC genes of Rhizobiumfredii and have no effect on the induction of nodABC genes of Rhizobium trifolii. This suggests that nodD product-flavone interaction is specific for nod gene expression (26). These compounds and their precursors also act as phytoalexins which are synthesized in response to microbial infection and are related with plant defense systems (5, 23). After bacterial infection, the nodulation on a root of soybean is influenced by a plant process called autoregulation. In this process, once soybean becomes nodulated, subsequent nodule formation is inhibited (16, 25). Supernodulating and hypemodulating mutants which have in part lost this autoregulatory control of nodulation have been selected by two laboratories, and initial characterization of these mutants has been reported (1, 10). These mutants exhibit increased nodulation capabilities in the presence of NO3-, as well as in the absence of NO3-, when compared with the respective wild types (1, 10). In addition, Carroll et al. (2) have reported the selection of three nonnodulating soybean mutants selected from ethyl methanesulfonate, y-ray, or sodium azide mutagenized Bragg. All three nonnodulating mutants lack root hair curling (18). Another nonnodulating soybean mutant has also been isolated from N-nitroso-N-methylurea mutagenized Williams from our laboratory (13). Mathews et al. (21) compared uninoculated 3-d-old seedling root extracts of a supernodu-
ABSTRACT The isoflavones, daidzein and genistein, have been isolated and identified as the major inducers of nod genes of Bradyrhizobium japonicum. The common nod genes of rhizobia are in turn responsible for stimulating root hair curling and cortical root cell division, the earliest steps in the host response. This study evaluated whether there was a relationship between root isoflavonoid production and the hypemodulation phenotype of selected soybean (Glycine max [L.] Merr.) mutants. Three independently selected hypernodulating soybean mutants (NOD1-3, NOD2-4, and NOD3-7) and a nonnodulating mutant (NN5) were compared with the Williams parent for isoflavonoid concentrations. High performance liquid chromatographic analyses of soybean root extracts showed that all lines increased in daidzein, genistein, and coumestrol concentrations throughout the 12-day growth period after transplanting of both inoculated and noninoculated plants; transplanting and inoculation were done 6 days after planting. No significant differences were detected in the concentration of these compounds among the three noninoculated hypemodulating mutants and the Williams parent. In response to inoculation, the three hypemodulating mutants had higher isoflavonoid concentrations than did the Williams control at 9 to 12 days after inoculation when grown at 0 millimolar N level. However, the inoculated nonnodulating mutant also had higher isoflavonoid concentrations than did Williams. N application [urea, (NH4)2SO4 and N03-] decreased the concentration of all three isoflavonoid compounds in all soybean lines. Application of N03was most inhibitory to isoflavonoid concentrations, and inhibition by N03- was concentration dependent. These results are consistent with a conclusion that differential N03- inhibition of nodulation may be partially due to changes in isoflavonoid levels, although the similar response of the nonnodulating mutant brings this conclusion into question. Altematively, the nodulation control in the NN5 mutant may be due to factors totally unrelated to isoflavonoids, leaving open the possibility that isoflavonoids play a role in differential nodulation of lines genetically competent to nodulate.
The symbiotic interaction of Bradyrhizobium japonicum and soybean results in the formation of nitrogen-fixing root nodules. This symbiosis requires many bacterial genes necesSupported in part by the American Soybean Association, Research Project 88412
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lating mutant (nts382) and a nonnodulating mutant (nod49) for the inducibility of nodC-lacZ fusions in B. japonicum with that of the Bragg parent. They found similar levels of induction in all cases, indicating that uninoculated seedlings of cultivar Bragg, nod49, and nts382 contained similar inducing compounds. Although it is well known that soil N (primarily NO3-) inhibits all phases of nodulation such as bacterial infection, nodule development, and nitrogenase function (12), little is known about the mechanism(s) of NO3- inhibition of nodulation and N2 fixation. The symbiotic efficiency of soybean plants grown on NO3- may be slightly affected by B. japonicum strain (22), but the host plant is in primary control of nodulation in the presence of N03- (9). Vigue et al. (31) showed that N03- is more inhibitory to soybean nodulation than is urea. The objectives of the present study were to determine if differential nodulation response of soybean nodulation mutants is related to isoflavonoid concentrations in soybean root extracts, and to determine the effect of inoculation treatments and various N sources on isoflavonoid concentrations in soybean roots. MATERIALS AND METHODS
Isoflavonoid Analysis of Noninoculated and Inoculated Williams and Hypernodulating Soybean Plants Plant Culture
Williams soybean (Glycine max [L.] Merr.) and three hypernodulating mutants (NOD 1-3, NOD2-4, and NOD3-7; M8 generation) selected from Williams (10) were evaluated. Seeds were surface-sterilized with 5% (v/v) Clorox2 plus one drop of Tween 20 for 10 min and washed with deionized distilled water. Seeds were planted in sterilized sand in perforated trays watered from the bottom with distilled water and grown in growth chambers programmed for 14-h photoperiods at 650 ,mol photons m-2 s-' at 29°C and 10-h dark periods at 20°C. Six-day-old seedlings were removed from the sand, and roots were inoculated with suspensions (108 cells/ mL) of B. japonicum (strain USDA 110). Noninoculated controls and inoculated seedlings were transplanted to separate trays containing a modified minus N Hoagland nutrient solution as previously described (10), except for containing 0.25 mm K-phosphate (pH 6.5). Seedlings were suspended through holes in Styrofoam lids placed over 1 8-L polypropylene trays. The solution pH was maintained at pH 6.5 ± 0.5 with ion exchange resin columns (14). Three replicates of each soybean line and inoculation treatment were evaluated. Preparation of Root Extracts At 0, 3, 6, 9, and 12 d after inoculation and transplanting, plants were harvested, separated into shoots and roots, and 2 Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the vendor or product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other vendors or products that may also be suitable.
Plant Physiol. Vol. 95, 1991
weighed. Five grams of fresh roots were rinsed with distilled water and extracted with 25 mL of acetone by grinding with an Omni-mixer (Omni Corp. Intl., Waterbury, CN) in an ice bath for two, 30 s intervals. The acetone extract was decanted and centrifuged at 7,000g and 4°C for 7 min. After decanting, acetone was removed under a steam of pure nitrogen, and the resulting aqueous fraction was reextracted twice with 7 mL anhydrous ethyl ether. The ether extract was transferred with a Pasteur pipette and decreased to dryness under a steam of pure nitrogen. The resulting residue was dissolved in 1.0 mL of HPLC grade methanol, centrifuged, and filtered through a 0.45 ,um Millipore filter prior to analysis. Chemicals Daidzein (7,4'-dihydroxyisoflavone) and genistein (5,7,4'trihydroxyisoflavone), and their respective 7-O-glucosides, daidzin (7,4'-dihydroxyisoflavone-7-O-glucoside), and genistin (5,7,4'-trihydroxyisoflavone-7-O-glucoside), were purchased from Plantech (UK). Coumestrol (3,9-dihydroxycoumestan) was purchased from ICN Biochemicals, Cleveland, OH. These compounds were dissolved in DMSO and stored at -20°C. HPLC Analysis A Waters Associates HPLC system was used, composed of two model 6000A solvent delivery systems, a model 680 automated gradient controller, a Hitachi model 100-40 UVvis detector, and a reverse phase 250 x 4.6 mm (i.d.) Econosphere C,8 column protected by a C,8 guard column. Samples were injected using a 20-,uL sample loop. Elution was effected with an aqueous methanol gradient consisting of the following steps: (a) 0 to 10 min, linear gradient from 30 to 80% methanol; (b) 10 to 12 min, isocratic at 80% methanol; (c) 12 to 20 min, linear gradient from 80 to 100% methanol; and (d) 20 to 25 min, isocratic at 100% methanol. The flow rate was 1.0 mL/min. The retention times of daidzein, genistein, and coumestrol were determined for the elution gradient. Daidzein, genistein, and coumestrol were identified in root extracts initially by cochromatography with known standards and subsequently by retention times established for each compound. Detection was achieved at 254 nm, and peaks were quantified with a Nelson analytical 3000 series chromatography data system (Nelson Analytical Inc., Cupertino, CA) linked to an IBM PC for data reduction. Effect of N Source on Isoflavonoid Concentration in Soybean Roots Plant Culture
Williams, three hypernodulating mutants, and a nonnodulating mutant (NN5) were used. Seed germination, seedling inoculation, and transplanting were as described above, except that seedlings were transplanted to 8-L trays. Nitrogen sources were 0, 1.5, and 5.0 mM NaNO3, 2.5 mm urea, and 2.5 mM (NH4)2SO4 imposed at the time of transplanting. At 6 and 9 d after transplanting, nutrient solutions were changed. Three replicates of each soybean line and N treatment were evaluated.
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Isoflavonoid Analysis of Root Extracts
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Statistical Analysis
8 to 27%, 24 to 60%, and 14 to 96% greater than in Williams for daidzein, genistein, and coumestrol, respectively. NOD37 was not sampled on d 12 due to insufficient plant numbers. All isoflavonoid compounds increased more rapidly in the NOD1-3 line in response to inoculation than they did in NOD2-4, while the Williams parent did not respond to inoculation treatment in the concentration of these compounds up to 12 d after inoculation (Fig. 2).
Analysis of variance was performed for each experiment and least significant difference (LSD)o.05 values were calculated when significant F tests occurred.
Effect of N Source on Growth and Isoflavonoid Concentration of Soybean Lines
At 12 d after inoculation, plants were harvested, separated into shoots and roots, and weighed. Preparation of root extracts and HPLC analyses of prepared samples were conducted as described above.
RESULTS
Isoflavonoid Analysis of Noninoculated and Inoculated Wild-Type and Nodulation-Mutant Soybean Plants Figure 1 shows the separation profile of isoflavonoid standards and of isoflavonoids from soybean root extracts harvested at 12 d after inoculation and transplanting. The glycosides eluted first and were followed by the isoflavone aglycones. The elution order of aglycones was daidzein, geinistein, and coumestrol. These compounds continued to increase in their concentration per g root fresh weight throughout the 12-d growth period after transplanting, regardless of soybean line or inoculation treatment (Fig. 2). Differences in isoflavonoid concentrations among lines were not significant within the noninoculated treatment. With the inoculated treatment, nodules on the roots of all the lines could be seen 9 to 10 d after inoculation. The inoculation treatment resulted in an increase in all three isoflavonoid compounds, relative to noninoculated controls. By 9 d after inoculation, all mutants accumulated greater (not always significant) concentrations of isoflavonoid compounds than Williams, with NOD 1-3 being most notable. At 12 d after inoculation, there were marked differences in isoflavonoid concentrations between Williams and the two nodulation mutants analyzed, especially in the concentration of genistein and coumestrol (Figs. 1, B and C, and 2). Comparisons between the Williams parent and the mutants showed that at 9 to 12 d after inoculation the range of isoflavonoid concentrations in the three mutants was
Shoot and Root Fresh Weight The Williams wild type had significantly higher root fresh weight than the hypernodulating mutants when grown at 0 mM N level, while there was no significant difference in shoot fresh weight (Table I). There was no significant difference in shoot and root fresh weight between Williams and NN5. When N was applied in the growth media, shoot fresh weight of all soybean lines markedly increased, but no significant increase in root fresh weight of the three hypernodulating mutants was measurable, relative to respective controls without N. The shoot-to-root ratio markedly increased in response to N application in all soybean lines. The shoot-to-root ratio of the hypernodulating mutants was higher than for the NN5 line and the Williams parent in both control and N treatments, because of more restricted root growth of the hypernodulating mutants. In most cases, the Williams wild type had more rapid shoot and root growth than did the hypernodulating mutants when grown in the presence of N. Isoflavonoid Response to N Treatment HPLC analyses of soybean root extracts showed that all three hypernodulating mutants and the NN5 nonnodulating mutant had markedly higher isoflavonoid concentrations than did the Williams control when grown at 0 mm N level (Figs. 3 and 4). Isoflavonoid concentrations in the hypernodulating mutants increased 1.15 to 1.48-, 1.65 to 2.63-, and 1.42 to 2.12-fold for daidzein, genistein, and coumestrol, respectively, when compared with the Williams control at 0 mM N level.
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Figure 1. HPLC gradient chromatograms of isoflavonoid standards and soybean root extracts using an aqueous methanol gradient: A, isoflavonoid standards: (1) daidzin, (2) genistin, (3) daidzein, (4) genistein, (5) coumestrol; B, inoculated Williams parent; C, inoculated NODl3. Seeds were germinated in sterilized sand and transplanted to nutrient solution on d 6. Roots were inoculated immediately prior to transplanting. Acetone-ether extracts from 5 g of soybean roots at 12 d after transplanting were reduced to dryness and redissolved in 1.0 mL of methanol. Twenty-microliter samples were injected and monitored at 254 nm. The concentration of all isoflavonoid standards was 0.1 mg/mL. The percent of methanol in the elution solvent gradient is superimposed on each section of the figure.
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Days after transplanting Figure 2. Changes in isoflavonoid concentration in root extracts from selected nodulation mutants and the Williams parent. Plants were sampled through a 1 2-d growth period after transplanting, except for NOD3-7 where plants were sampled through 9 d due to inadequate plant material. Plants were separated into shoots and roots, and weighed at 3-d intervals from transplanting. Other details as in Figure 1 legend. Values represent means of three replicates for each soybean line. Means with the same letter are not significantly different among soybean lines within a sampling time at the 0.05 level using an LSD test.
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Table I. Effect of Various N Applications on Shoot and Root Fresh Weight and Shoot-to-Root Ratio from Selected Nodulation Mutants and the Williams Parent Seeds were germinated in sterilized sand and transplanted to nutrient solution with various N treatments on d 6. Roots were inoculated immediately prior to transplanting. Plants were harvested, separated into shoots and roots, and weighed at 12 d after transplanting. Values represent means of three replicates for each soybean line. Soybean Line Parameter
LSDo05
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Williams
Shoot fresh wt (g plant-')
NOD1-3
NOD2-4
1.79 2.07 1.85 Control 3.24 2.53 2.55 1.5 mM NaNO3 3.25 2.86 2.75 5.0 mM NaNO3 3.42 2.45 2.90 2.5 mm (NH2)2CO 2.97 2.34 2.57 2.5 mm (NH4)2SO4 0.99 1.02 1.40 Control Root fresh wt (g plant-1) 1.00 0.93 1.73 1.5 mM NaNO3 1.62 1.08 0.91 5.0 mM NaNO3 1.01 1.03 1.78 2.5 mm (NH2)2CO 0.89 0.79 1.26 2.5 mm (NH4)2SO4 1.82 2.02 1.32 Shoot:root ratio Control 2.79 2.52 1.87 1.5 mM NaNO3 3.05 2.01 2.67 5.0 mM NaNO3 2.42 2.83 1.92 2.5 mm (NH2)2CO 3.31 2.35 2.63 2.5 mm (NH4)2SO4 a The designation NS denotes nonsignificant difference among soybean lines at the 0.05 level.
Out of the hypernodulating mutants, NOD 1-3 had the highest concentration of all isoflavonoid compounds, NOD2-4 the second, and NOD3-7 the lowest. The NN5 line also had a significantly higher isoflavonoid concentration than did the Williams parent. Comparisons between the Williams parent and NN5 showed that the range of isoflavonoid concentrations in NN5 was 26%, 72%, and 50% greater than in Williams for daidzein, genistein, and coumestrol, respectively. N application decreased the concentration of all three isoflavonoid compounds in all soybean lines (Figs. 3 and 4). Among the soybean lines, application of urea and (NH4)2SO4 decreased daidzein concentration by 14 to 44%, genistein concentration by 33 to 75%, and coumestrol concentration by 10 to 58%, relative to respective controls without N. Application of NO3- was more inhibitory to isoflavonoid concentrations than that of ammoniacal N, and the degree of inhibition by NO3- treatment was concentration dependent
(Fig. 4). DISCUSSION The observation that there was no significant difference in isoflavonoid levels between Williams and the hypernodulating mutants when plants were not inoculated (Fig. 2) is consistent with observations made by Mathews et al. (21) in comparing a supernodulating mutant and a nonnodulating mutant with the Bragg parent. Those authors compared uninoculated 3-dold seedling root extracts for ability to stimulate the induction of the nodC-lacZ fusion in a USDA 1 10 B. japonicum strain. Sutherland et al. (30) also showed that uninoculated 1 2-d-old seedling root exudates from their supernodulating, nonnodulating, and the parent (Bragg) lines had similar nod gene inducing ability. The current study extended this approach by evaluating the
NOD3-7
NN5
1.94 2.78 2.43 2.59 2.34 1.06 1.12 0.82 0.92 0.85 1.83 2.49 2.97 2.81 2.76
1.69 2.59 3.21 2.73 2.17 1.27 1.25 1.51 1.22 0.86 1.33 2.06 2.13 2.27 2.51
NSa 0.57 0.42 0.54 0.54 0.23 0.22 0.22 0.24 0.17 0.29 0.38 0.28 0.41 0.67
isoflavonoid levels in root extracts from inoculated wild-type and hypernodulating soybean lines up to 12 d after inoculation. Root extracts were analyzed for isoflavonoid levels because preliminary studies were unsuccessful in concentrating root exudate levels sufficiently to measure isoflavonoid concentrations by HPLC. It was assumed that differences in internal concentrations of root isoflavonoids would be reflected in exudate levels, although this assumption remains to be confirmed for our mutant lines. No differences among lines were measured in isoflavonoid levels until 6 d after inoculation (Fig. 2). This is consistent with the conclusion of Sutherland et al. (30) that no change in nod gene inducing ability was observed in root extracts from inoculated and uninoculated Bragg plants up to 3 d after inoculation treatments. In the current study, the NOD1-3 mutant did accumulate greater concentrations of isoflavonoids by 9 d after inoculation, and both NOD1-3 and NOD2-4 had significantly greater accumulation of daidzein and genistein by d 12 (Fig. 2). Kapulnik et al. (15) reported a similar result with alfalfa where inoculated HP32 roots (resulting from two generations of phenotypic recurrent selection for higher N2 fixation and nodule formation) had a higher concentration of the flavone luteolin to induce the nod genes of Rhizobium meliloti than did the inoculated HP parent. Whether the difference in root isoflavonoid concentration between Williams and the two hypernodulating lines is sufficient to account for the differential nodulation pattern remains to be shown. The fact that significant differences in isoflavonoid concentrations between Williams and the hypernodulating mutants were not detected in early stages after inoculation indicates that, if isoflavonoids play a role in differential nodule expression between the hypernodulating mutants and the Williams parent, this effect is on nodule development rather than on the initial infection stages. The observation that similar numbers
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of infection events occurred for the supernodulating nts382 mutant and the Bragg parent, and that a greater proportion of these infection events developed to an advanced stage of nodule ontogeny in nts382 (19), provides evidence that the control is post infection and is exercised within the plant root. The possibility that the level of isoflavonoids within the root is important to the control of nodule development is attractive, but requires further evaluation to support or refute this
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N Treatment Figure 3. Effect of various N applications on isoflavonoid concentrations in root extracts from selected nodulation mutants and the Williams parent. Seeds were germinated in sterilized sand and transplanted to nutrient solution with various N treatments on d 6. Roots were inoculated immediately prior to transplanting. Plants were harvested, separated onto shoots and roots, and weighed at 12 d after transplanting. Values represent means of three replicates for each soybean line. Means with the same letter are not significantly different among soybean lines within an N source at the 0.05 level.
speculation. The previous (10) and current (Table 1) results showed that root growth of the hypernodulating mutants was decreased relative to that of the Williams parent when both were inoculated, with root growth being affected more than shoot growth. In contrast, there were no significant differences among soybean lines in shoot and root dry weight when not inoculated and grown at 0 mm N level (data not shown). Our results also showed that N application markedly increased shoot-to-root ratio. This was due to increased shoot fresh weight up to 12 d after transplanting (Table I). This indicates that relatively more photosynthetic carbon is utilized for shoot growth in response to N application. This is consistent with earlier work (8) which showed that supply of NO3- to the plant roots decreased '4C02 flux to nodules. This limitation of carbon movement to roots could result in decreased isoflavonoid concentrations; isoflavonoids or closely related compounds are estimated to account for 1.7% of photosynthetically fixed carbon (29). This is consistent with HPLC analyses of soybean root extracts from the present study which showed that N application inhibited the concentration of all three isoflavonoid compounds in all soybean lines, and that the inhibition by NO3- was more marked than that by ammoniacal N (Figs. 3 and 4). This inhibitory effect of N on isoflavonoid concentration is consistent with previous conclusions concerning N inhibition of nodulation and N2 fixation (12) and of the more marked inhibitory effect of NO3- than of urea on soybean nodulation (31). It was previously reported that the nonnodulation phenotype is under root control (4), while the supernodulation and hypernodulation phenotypes are under shoot control (4, 10). The shoot control of nodule initiation in nitrate tolerant symbiotic (nts) mutants has been reported to be epistatically suppressed by the nonnodulation, root expressed mutation (20). Nonnodulating mutants are either insensitive to the recognition of the plant signal or unable to convert the cell division stimulus to an actual infection after response to the signal (1 1). Our results showed that the inoculated NN5 also had a more rapid response of isoflavonoid concentrations than did the Williams control (Fig. 3), and the nonnodulating mutant did not form any nodules even in response to application of 0. 1 to 10 uM of daidzein and genistein into growth media (data not shown). This indicates that the nonnodulating characteristic in the NN5 line is unrelated to isoflavonoids. In conclusion, the observations that hypernodulating mutants had higher root concentrations of daidzein, genistein, and coumestrol than did the Williams parent when they were inoculated, and that N application inhibited isoflavonoid concentrations in all soybean lines, can be interpreted as evidence for involvement of isoflavonoids in nodulation control. In contrast, the higher isoflavonoid concentration of the NN5 line, compared with Williams, may be an argument
INOCULATION AND N EFFECTS ON SOYBEAN ROOT ISOFLAVONOIDS
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against the conclusion that the hypernodulation trait is related to higher isoflavonoid levels. Nodulation control in the NN5 line may, however, be due to factors totally unrelated to
isoflavonoids, leaving open the possibility that isoflavonoids play a role in differential nodulation of soybean lines genetically competent to nodulate. -C
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LITERATURE CITED
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1. Carroll BJ, McNeil DL, Gresshoff PM (1985) A supernodulation and nitrate-tolerant symbiotic (nts) soybean mutant. Plant
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Nitrate Concentration (mM) Figure 4. Effect of nitrate concentration on isoflavonoid concentration in root extracts from selected nodulation mutants and the Williams parent. Other details as in Figure 3 legend. Values represent means of three replicates for each soybean line and vertical bars are LSDo.05.
Physiol 78: 34-40 2. Carroll BJ, McNeil DL, Gresshoff PM (1986) Mutagenesis of soybean (Glycine max (L.) Merr.) and the isolation of nonnodulating mutants. Plant Sci 47: 109-114 3. d'Arcy Lameta A (1987) Study of soybean and lentil root exudates. III. Influence of soybean isoflavonoids on the growth of rhizobia and some rhizospheric microorganisms. Plant Soil 101: 267-272 4. Delves AC, Mathews A, Day DA, Carter AS, Carroll BJ, Gresshoff PM (1986) Regulation ofthe soybean-Rhizobium nodule symbiosis by shoot and root factors. Plant Physiol 82: 588590 5. Dixon RA (1986) The phytoalexin response: elicitation, signalling and control of host gene expression. Biol Rev 61: 239-291 6. Djordjevic MA, Redmond JW, Batley M, Rolfe BG (1987) Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii. EMBO J 6: 1173-1179 7. Firmin JL, Wilson KE, Rossen L, Johnston AWB (1986) Flavonoid activation of nodulation genes in Rhizobium reversed by other compounds present in plants. Nature 324: 90-92 8. Gibson AH (1974) Comparison of the growing legume as a symbiotic association. Proc Indian Natl Sci Acad 40B: 741767 9. Gibson AH, Harper JE (1985) Nitrate effect on nodulation of soybean by Bradyrhizobium japonicum. Crop Sci 25: 497-501 10. Gremaud MF, Harper JE (1989) Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean. Plant Physiol 89: 169-173 11. Gresshoff PM, Mathews A, Krotzky A, Olsson JE, Carroll BJ, Delves AC, Kosslak RM, Appelbaum ER, Day DA (1988) Supernodulation and non-nodulation mutants of soybean. In R Palacios, DPS Verma, eds, Molecular Genetics of PlantMicrobe Interactions 1988. APS Press, St. Paul, MN, pp 364369 12. Harper JE (1987) Nitrogen metabolism. In JR Wilcox, ed, Soybeans: Improvement, Production, and Uses, Ed 2 (Agronomy Monograph No. 16). American Society of Agronomy, Madison, WI, pp 497-533 13. Harper JE (1989) Nitrogen metabolism mutants of soybean. In AJ Pascale, ed, World Soybean Research Conference IV, Vol IV. Buenos Aires, Argentina, pp 212-216 14. Harper JE, Nicholas JC (1976) Control of nutrient solution pH with an ion exchange system: effect on soybean nodulation. Physiol Plant 38: 24-28 15. Kapulnik Y, Joseph CM, Phillips DA (1987) Flavone limitations to root nodulation and symbiotic nitrogen fixation in alfalfa. Plant Physiol 84: 1193-1196 16. Kosslak RM, Bohlool BB (1984) Suppression of nodule development of one side of a split-root system of soybeans caused by prior inoculation of the other side. Plant Physiol 75: 125130 17. Kosslak RM, Bookland R, Barkei J, Paaren HE, Appelbaum ER (1987) Induction of Bradyrhizobiumjaponicum common nod genes by isoflavones isolated from Glycine max. Proc Natl Acad Sci USA 84: 7428-7432 18. Mathews A, Carroll BJ, Gresshoff PM (1987) Characterization of non-nodulation mutants of soybean [Glycine max (L.) Merr]: Bradyrhizobium effects and absence of root hair curling. J Plant Physiol 131: 349-361
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