Plant Physiol. (1991) 95, 435-442 0032-0889/91 /95/0435/08/$01 .00/0 Received for publication July 30, 1990 Accepted October 26, 1990 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 ABSTRACT The isoflavones, daidzein and genistein, have been isolated and identified as the major inducers of nod genes of Bradyrhizo- bium 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 isofla- vonoid 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 concen- tration of these compounds among the three noninoculated hy- pemodulating mutants and the Williams parent. In response to inoculation, the three hypemodulating mutants had higher isofla- vonoid concentrations than did the Williams control at 9 to 12 days after inoculation when grown at 0 millimolar N level. How- ever, the inoculated nonnodulating mutant also had higher isofla- vonoid 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 N03- was most inhibitory to isoflavonoid concentrations, and inhibition by N03- was concentration dependent. These results are con- sistent with a conclusion that differential N03- inhibition of no- dulation 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 neces- Supported in part by the American Soybean Association, Re- search Project 88412 sary for nitrogen fixation (nif and fix genes) and nodulation (nod genes). Common nodulation genes (nodDABC) in rhi- zobia 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 fu- sions, 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 formono- netin (an isoflavone) from white clover root exudates antag- onize 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 soy- bean 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 autoreg- ulatory control of nodulation have been selected by two laboratories, and initial characterization of these mutants has been reported (1, 10). These mutants exhibit increased no- dulation 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 muta- genized Bragg. All three nonnodulating mutants lack root hair curling (18). Another nonnodulating soybean mutant has also been isolated from N-nitroso-N-methylurea mutagenized Wil- liams from our laboratory (13). Mathews et al. (21) compared uninoculated 3-d-old seedling root extracts of a supernodu- 435 https://plantphysiol.org Downloaded on April 19, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Received for publication July 30, 1990Accepted October 26, 1990
Effect of Inoculation and Nitrogen on IsoflavonoidConcentration 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
ABSTRACT
The isoflavones, daidzein and genistein, have been isolatedand identified as the major inducers of nod genes of Bradyrhizo-bium japonicum. The common nod genes of rhizobia are in turnresponsible for stimulating root hair curling and cortical root celldivision, the earliest steps in the host response. This studyevaluated whether there was a relationship between root isofla-vonoid production and the hypemodulation phenotype of selectedsoybean (Glycine max [L.] Merr.) mutants. Three independentlyselected hypernodulating soybean mutants (NOD1-3, NOD2-4,and NOD3-7) and a nonnodulating mutant (NN5) were comparedwith the Williams parent for isoflavonoid concentrations. Highperformance liquid chromatographic analyses of soybean rootextracts showed that all lines increased in daidzein, genistein,and coumestrol concentrations throughout the 12-day growthperiod after transplanting of both inoculated and noninoculatedplants; transplanting and inoculation were done 6 days afterplanting. No significant differences were detected in the concen-tration of these compounds among the three noninoculated hy-pemodulating mutants and the Williams parent. In response toinoculation, the three hypemodulating mutants had higher isofla-vonoid concentrations than did the Williams control at 9 to 12days after inoculation when grown at 0 millimolar N level. How-ever, the inoculated nonnodulating mutant also had higher isofla-vonoid concentrations than did Williams. N application [urea,(NH4)2SO4 and N03-] decreased the concentration of all threeisoflavonoid compounds in all soybean lines. Application of N03-was most inhibitory to isoflavonoid concentrations, and inhibitionby N03- was concentration dependent. These results are con-sistent with a conclusion that differential N03- inhibition of no-dulation may be partially due to changes in isoflavonoid levels,although the similar response of the nonnodulating mutant bringsthis conclusion into question. Altematively, the nodulation controlin the NN5 mutant may be due to factors totally unrelated toisoflavonoids, leaving open the possibility that isoflavonoids playa role in differential nodulation of lines genetically competent tonodulate.
The symbiotic interaction of Bradyrhizobium japonicumand soybean results in the formation of nitrogen-fixing rootnodules. This symbiosis requires many bacterial genes neces-
Supported in part by the American Soybean Association, Re-search Project 88412
sary for nitrogen fixation (nifand fix genes) and nodulation(nod genes). Common nodulation genes (nodDABC) in rhi-zobia are responsible for stimulating root hair curling andcortical cell division, the earliest steps in the host response(24, 27). These genes are induced by plant signal compoundssuch as flavonoids and isoflavonoids exuded from the rootsof plant hosts (7, 17, 24, 26, 28). The isoflavones, daidzeinand genistein, have been isolated and identified as the majorcomponents that stimulate B. japonicum nodABC-lacZ fu-sions, and this activation showed a concentration dependenceup to 5 ,uM (17). In addition, coumestrol (a coumestan) anddaidzein have also been shown to promote the growth of B.japonicum (3). In contrast, Firmin et al. (7) showed thatdaidzein and genistein are potent antagonists of nod geneinduction in Rhizobium leguminosarum. Djordjevic et al. (6)also showed that umbelliferone (a coumarin) and formono-netin (an isoflavone) from white clover root exudates antag-onize the stimulatory activity of daidzein. Kosslak et al. (17)demonstrated that these compounds induce the nodABCgenes ofRhizobiumfredii and have no effect on the inductionof nodABC genes of Rhizobium trifolii. This suggests thatnodD product-flavone interaction is specific for nod geneexpression (26). These compounds and their precursors alsoact as phytoalexins which are synthesized in response tomicrobial infection and are related with plant defense systems(5, 23).
After bacterial infection, the nodulation on a root of soy-bean is influenced by a plant process called autoregulation.In this process, once soybean becomes nodulated, subsequentnodule formation is inhibited (16, 25). Supernodulating andhypemodulating mutants which have in part lost this autoreg-ulatory control of nodulation have been selected by twolaboratories, and initial characterization of these mutants hasbeen reported (1, 10). These mutants exhibit increased no-dulation capabilities in the presence of NO3-, as well as in theabsence of NO3-, when compared with the respective wildtypes (1, 10). In addition, Carroll et al. (2) have reported theselection of three nonnodulating soybean mutants selectedfrom ethyl methanesulfonate, y-ray, or sodium azide muta-genized Bragg. All three nonnodulating mutants lack root haircurling (18). Another nonnodulating soybean mutant has alsobeen isolated from N-nitroso-N-methylurea mutagenized Wil-liams from our laboratory (13). Mathews et al. (21) compareduninoculated 3-d-old seedling root extracts of a supernodu-
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lating mutant (nts382) and a nonnodulating mutant (nod49)for the inducibility ofnodC-lacZ fusions in B. japonicum withthat of the Bragg parent. They found similar levels of induc-tion in all cases, indicating that uninoculated seedlings ofcultivar Bragg, nod49, and nts382 contained similar inducingcompounds.
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 isknown about the mechanism(s) of NO3- inhibition of nodu-lation and N2 fixation. The symbiotic efficiency of soybeanplants grown on NO3- may be slightly affected by B. japoni-cum strain (22), but the host plant is in primary control ofnodulation in the presence of N03- (9). Vigue et al. (31)showed that N03- is more inhibitory to soybean nodulationthan is urea.The objectives of the present study were to determine if
differential nodulation response of soybean nodulation mu-tants is related to isoflavonoid concentrations in soybean rootextracts, and to determine the effect ofinoculation treatmentsand various N sources on isoflavonoid concentrations insoybean roots.
MATERIALS AND METHODS
Isoflavonoid Analysis of Noninoculated and InoculatedWilliams and Hypernodulating Soybean Plants
Plant Culture
Williams soybean (Glycine max [L.] Merr.) and three hy-pernodulating 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 onedrop of Tween 20 for 10 min and washed with deionizeddistilled water. Seeds were planted in sterilized sand in per-forated trays watered from the bottom with distilled waterand grown in growth chambers programmed for 14-h photo-periods at 650 ,mol photons m-2 s-' at 29°C and 10-h darkperiods at 20°C. Six-day-old seedlings were removed from thesand, and roots were inoculated with suspensions (108 cells/mL) of B. japonicum (strain USDA 110). Noninoculatedcontrols and inoculated seedlings were transplanted to sepa-rate trays containing a modified minus N Hoagland nutrientsolution as previously described (10), except for containing0.25 mm K-phosphate (pH 6.5). Seedlings were suspendedthrough holes in Styrofoam lids placed over 1 8-L polypropyl-ene trays. The solution pH was maintained at pH 6.5 ± 0.5with ion exchange resin columns (14). Three replicates ofeach 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 notconstitute a guarantee or warranty of the vendor or product by theU.S. Department of Agriculture, and does not imply its approval tothe exclusion of other vendors or products that may also be suitable.
weighed. Five grams of fresh roots were rinsed with distilledwater and extracted with 25 mL of acetone by grinding withan Omni-mixer (Omni Corp. Intl., Waterbury, CN) in an icebath for two, 30 s intervals. The acetone extract was decantedand centrifuged at 7,000g and 4°C for 7 min. After decanting,acetone was removed under a steam of pure nitrogen, and theresulting aqueous fraction was reextracted twice with 7 mLanhydrous ethyl ether. The ether extract was transferred witha Pasteur pipette and decreased to dryness under a steam ofpure nitrogen. The resulting residue was dissolved in 1.0 mLof HPLC grade methanol, centrifuged, and filtered through a0.45 ,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 genis-tin (5,7,4'-trihydroxyisoflavone-7-O-glucoside), were pur-chased from Plantech (UK). Coumestrol (3,9-dihydroxycou-mestan) was purchased from ICN Biochemicals, Cleveland,OH. These compounds were dissolved in DMSO and storedat -20°C.
HPLC Analysis
A Waters Associates HPLC system was used, composed oftwo model 6000A solvent delivery systems, a model 680automated gradient controller, a Hitachi model 100-40 UV-vis detector, and a reverse phase 250 x 4.6 mm (i.d.) Econ-osphere C,8 column protected by a C,8 guard column. Sampleswere injected using a 20-,uL sample loop. Elution was effectedwith an aqueous methanol gradient consisting ofthe followingsteps: (a) 0 to 10 min, linear gradient from 30 to 80%methanol; (b) 10 to 12 min, isocratic at 80% methanol; (c) 12to 20 min, linear gradient from 80 to 100% methanol; and(d) 20 to 25 min, isocratic at 100% methanol. The flow ratewas 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 rootextracts initially by cochromatography with known standardsand subsequently by retention times established for eachcompound. Detection was achieved at 254 nm, and peakswere quantified with a Nelson analytical 3000 series chro-matography data system (Nelson Analytical Inc., Cupertino,CA) linked to an IBM PC for data reduction.
Effect of N Source on Isoflavonoid Concentration inSoybean Roots
Plant Culture
Williams, three hypernodulating mutants, and a nonnod-ulating mutant (NN5) were used. Seed germination, seedlinginoculation, and transplanting were as described above, exceptthat seedlings were transplanted to 8-L trays. Nitrogen sourceswere 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 9d after transplanting, nutrient solutions were changed. Threereplicates of each soybean line and N treatment wereevaluated.
436 CHO AND HARPER
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INOCULATION AND N EFFECTS ON SOYBEAN ROOT ISOFLAVONOIDS
Isoflavonoid Analysis of Root Extracts
At 12 d after inoculation, plants were harvested, separatedinto shoots and roots, and weighed. Preparation of root ex-tracts and HPLC analyses of prepared samples were con-ducted as described above.
Statistical Analysis
Analysis of variance was performed for each experimentand least significant difference (LSD)o.05 values were calculatedwhen significant F tests occurred.
RESULTS
Isoflavonoid Analysis of Noninoculated and InoculatedWild-Type and Nodulation-Mutant Soybean Plants
Figure 1 shows the separation profile of isoflavonoid stand-ards and ofisoflavonoids from soybean root extracts harvestedat 12 d after inoculation and transplanting. The glycosideseluted first and were followed by the isoflavone aglycones.The elution order of aglycones was daidzein, geinistein, andcoumestrol. These compounds continued to increase in theirconcentration per g root fresh weight throughout the 12-dgrowth period after transplanting, regardless of soybean lineor inoculation treatment (Fig. 2). Differences in isoflavonoidconcentrations among lines were not significant within thenoninoculated treatment. With the inoculated treatment,nodules on the roots of all the lines could be seen 9 to 10 dafter inoculation. The inoculation treatment resulted in anincrease in all three isoflavonoid compounds, relative to non-inoculated controls. By 9 d after inoculation, all mutantsaccumulated greater (not always significant) concentrationsof isoflavonoid compounds than Williams, with NOD 1-3being most notable. At 12 d after inoculation, there weremarked differences in isoflavonoid concentrations betweenWilliams and the two nodulation mutants analyzed, especiallyin the concentration of genistein and coumestrol (Figs. 1, Band C, and 2). Comparisons between the Williams parent andthe mutants showed that at 9 to 12 d after inoculation therange of isoflavonoid concentrations in the three mutants was
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8 to 27%, 24 to 60%, and 14 to 96% greater than in Williamsfor daidzein, genistein, and coumestrol, respectively. NOD3-7 was not sampled on d 12 due to insufficient plant numbers.All isoflavonoid compounds increased more rapidly in theNOD1-3 line in response to inoculation than they did inNOD2-4, while the Williams parent did not respond toinoculation treatment in the concentration of these com-pounds up to 12 d after inoculation (Fig. 2).
Effect of N Source on Growth and IsoflavonoidConcentration of Soybean Lines
Shoot and Root Fresh Weight
The Williams wild type had significantly higher root freshweight than the hypernodulating mutants when grown at 0mM N level, while there was no significant difference in shootfresh weight (Table I). There was no significant difference inshoot and root fresh weight between Williams and NN5.When N was applied in the growth media, shoot fresh weightof all soybean lines markedly increased, but no significantincrease in root fresh weight of the three hypernodulatingmutants was measurable, relative to respective controls with-out N. The shoot-to-root ratio markedly increased in responseto N application in all soybean lines. The shoot-to-root ratioof the hypernodulating mutants was higher than for the NN5line and the Williams parent in both control and N treat-ments, because of more restricted root growth of the hyper-nodulating mutants. In most cases, the Williams wild typehad more rapid shoot and root growth than did the hyper-nodulating mutants when grown in the presence of N.
Isoflavonoid Response to N Treatment
HPLC analyses of soybean root extracts showed that allthree hypernodulating mutants and the NN5 nonnodulatingmutant had markedly higher isoflavonoid concentrations thandid the Williams control when grown at 0 mm N level (Figs.3 and 4). Isoflavonoid concentrations in the hypernodulatingmutants increased 1.15 to 1.48-, 1.65 to 2.63-, and 1.42 to2.12-fold for daidzein, genistein, and coumestrol, respectively,when compared with the Williams control at 0 mM N level.
Retention Time (min) Retention Time (min) Retention Time (min)
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 NODl-3. 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 percentof methanol in the elution solvent gradient is superimposed on each section of the figure.
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Figure 2. Changes in isoflavonoid concentration in root extracts from selected nodulation mutants and the Williams parent. Plants were sampledthrough 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. Valuesrepresent means of three replicates for each soybean line. Means with the same letter are not significantly different among soybean lines withina sampling time at the 0.05 level using an LSD test.
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438 CHO AND HARPER
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INOCULATION AND N EFFECTS ON SOYBEAN ROOT ISOFLAVONOIDS
Table I. Effect of Various N Applications on Shoot and Root Fresh Weight and Shoot-to-Root Ratio from Selected Nodulation Mutants and theWilliams Parent
Seeds were germinated in sterilized sand and transplanted to nutrient solution with various N treatments on d 6. Roots were inoculatedimmediately prior to transplanting. Plants were harvested, separated into shoots and roots, and weighed at 12 d after transplanting. Valuesrepresent means of three replicates for each soybean line.
Soybean LineParameter N Treatment LSDo05
Williams NOD1-3 NOD2-4 NOD3-7 NN5
Shoot fresh wt (g plant-') Control 1.85 1.79 2.07 1.94 1.69 NSa1.5 mM NaNO3 3.24 2.53 2.55 2.78 2.59 0.575.0 mM NaNO3 3.25 2.86 2.75 2.43 3.21 0.422.5 mm (NH2)2CO 3.42 2.45 2.90 2.59 2.73 0.542.5 mm (NH4)2SO4 2.97 2.34 2.57 2.34 2.17 0.54
Root fresh wt (g plant-1) Control 1.40 0.99 1.02 1.06 1.27 0.231.5 mM NaNO3 1.73 1.00 0.93 1.12 1.25 0.225.0 mM NaNO3 1.62 1.08 0.91 0.82 1.51 0.222.5 mm (NH2)2CO 1.78 1.01 1.03 0.92 1.22 0.242.5 mm (NH4)2SO4 1.26 0.89 0.79 0.85 0.86 0.17
Shoot:root ratio Control 1.32 1.82 2.02 1.83 1.33 0.291.5 mM NaNO3 1.87 2.52 2.79 2.49 2.06 0.385.0 mM NaNO3 2.01 2.67 3.05 2.97 2.13 0.282.5 mm (NH2)2CO 1.92 2.42 2.83 2.81 2.27 0.412.5 mm (NH4)2SO4 2.35 2.63 3.31 2.76 2.51 0.67
a The designation NS denotes nonsignificant difference among soybean lines at the 0.05 level.
Out ofthe hypernodulating mutants, NOD 1-3 had the highestconcentration of all isoflavonoid compounds, NOD2-4 thesecond, and NOD3-7 the lowest. The NN5 line also had asignificantly higher isoflavonoid concentration than did theWilliams parent. Comparisons between the Williams parentand NN5 showed that the range of isoflavonoid concentra-tions in NN5 was 26%, 72%, and 50% greater than inWilliams for daidzein, genistein, and coumestrol, respectively.N application decreased the concentration of all three iso-
flavonoid compounds in all soybean lines (Figs. 3 and 4).Among the soybean lines, application of urea and (NH4)2SO4decreased daidzein concentration by 14 to 44%, genisteinconcentration by 33 to 75%, and coumestrol concentrationby 10 to 58%, relative to respective controls without N.Application of NO3- was more inhibitory to isoflavonoidconcentrations than that of ammoniacal N, and the degree ofinhibition by NO3- treatment was concentration dependent(Fig. 4).
DISCUSSION
The observation that there was no significant difference inisoflavonoid levels between Williams and the hypernodulatingmutants when plants were not inoculated (Fig. 2) is consistentwith observations made by Mathews et al. (21) in comparinga supernodulating mutant and a nonnodulating mutant withthe Bragg parent. Those authors compared uninoculated 3-d-old seedling root extracts for ability to stimulate the inductionof the nodC-lacZ fusion in a USDA 1 10 B. japonicum strain.Sutherland et al. (30) also showed that uninoculated 1 2-d-oldseedling root exudates from their supernodulating, nonno-dulating, and the parent (Bragg) lines had similar nod geneinducing ability.The current study extended this approach by evaluating the
isoflavonoid levels in root extracts from inoculated wild-typeand hypernodulating soybean lines up to 12 d after inocula-tion. Root extracts were analyzed for isoflavonoid levels be-cause preliminary studies were unsuccessful in concentratingroot exudate levels sufficiently to measure isoflavonoid con-centrations by HPLC. It was assumed that differences ininternal concentrations of root isoflavonoids would be re-flected in exudate levels, although this assumption remains tobe confirmed for our mutant lines. No differences amonglines were measured in isoflavonoid levels until 6 d afterinoculation (Fig. 2). This is consistent with the conclusion ofSutherland et al. (30) that no change in nod gene inducingability was observed in root extracts from inoculated anduninoculated Bragg plants up to 3 d after inoculation treat-ments. In the current study, the NOD1-3 mutant did accu-mulate greater concentrations of isoflavonoids by 9 d afterinoculation, and both NOD1-3 and NOD2-4 had signifi-cantly greater accumulation of daidzein and genistein by d 12(Fig. 2). Kapulnik et al. (15) reported a similar result withalfalfa where inoculated HP32 roots (resulting from two gen-erations of phenotypic recurrent selection for higher N2 fixa-tion and nodule formation) had a higher concentration of theflavone luteolin to induce the nodgenes ofRhizobium melilotithan did the inoculated HP parent. Whether the difference inroot isoflavonoid concentration between Williams and thetwo hypernodulating lines is sufficient to account for thedifferential nodulation pattern remains to be shown. The factthat significant differences in isoflavonoid concentrations be-tween Williams and the hypernodulating mutants were notdetected in early stages after inoculation indicates that, ifisoflavonoids play a role in differential nodule expressionbetween the hypernodulating mutants and the Williams par-ent, this effect is on nodule development rather than on theinitial infection stages. The observation that similar numbers
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Figure 3. Effect of various N applications on isoflavonoid concentra-tions in root extracts from selected nodulation mutants and theWilliams parent. Seeds were germinated in sterilized sand and trans-planted to nutrient solution with various N treatments on d 6. Rootswere inoculated immediately prior to transplanting. Plants were har-vested, separated onto shoots and roots, and weighed at 12 d aftertransplanting. Values represent means of three replicates for eachsoybean line. Means with the same letter are not significantly differentamong soybean lines within an N source at the 0.05 level.
of infection events occurred for the supernodulating nts382mutant and the Bragg parent, and that a greater proportionof these infection events developed to an advanced stage ofnodule ontogeny in nts382 (19), provides evidence that thecontrol is post infection and is exercised within the plant root.The possibility that the level of isoflavonoids within the rootis important to the control of nodule development is attrac-tive, but requires further evaluation to support or refute thisspeculation.The previous (10) and current (Table 1) results showed that
root growth of the hypernodulating mutants was decreasedrelative to that of the Williams parent when both were inoc-ulated, with root growth being affected more than shootgrowth. In contrast, there were no significant differencesamong soybean lines in shoot and root dry weight when notinoculated and grown at 0 mm N level (data not shown). Ourresults also showed that N application markedly increasedshoot-to-root ratio. This was due to increased shoot freshweight up to 12 d after transplanting (Table I). This indicatesthat relatively more photosynthetic carbon is utilized for shootgrowth in response to N application. This is consistent withearlier work (8) which showed that supply of NO3- to theplant roots decreased '4C02 flux to nodules. This limitationof carbon movement to roots could result in decreased isofla-vonoid concentrations; isoflavonoids or closely related com-pounds are estimated to account for 1.7% of photosyntheti-cally fixed carbon (29). This is consistent with HPLC analysesof soybean root extracts from the present study which showedthat N application inhibited the concentration of all threeisoflavonoid compounds in all soybean lines, and that theinhibition by NO3- was more marked than that by ammonia-cal N (Figs. 3 and 4). This inhibitory effect of N on isoflavo-noid concentration is consistent with previous conclusionsconcerning N inhibition of nodulation and N2 fixation (12)and of the more marked inhibitory effect of NO3- than ofurea on soybean nodulation (31).
It was previously reported that the nonnodulation pheno-type is under root control (4), while the supernodulation andhypernodulation phenotypes are under shoot control (4, 10).The shoot control of nodule initiation in nitrate tolerantsymbiotic (nts) mutants has been reported to be epistaticallysuppressed by the nonnodulation, root expressed mutation(20). Nonnodulating mutants are either insensitive to therecognition of the plant signal or unable to convert the celldivision stimulus to an actual infection after response to thesignal (1 1). Our results showed that the inoculated NN5 alsohad a more rapid response of isoflavonoid concentrationsthan did the Williams control (Fig. 3), and the nonnodulatingmutant did not form any nodules even in response to appli-cation of 0. 1 to 10 uM of daidzein and genistein into growthmedia (data not shown). This indicates that the nonnodulatingcharacteristic in the NN5 line is unrelated to isoflavonoids.
In conclusion, the observations that hypernodulating mu-tants had higher root concentrations of daidzein, genistein,and coumestrol than did the Williams parent when they wereinoculated, and that N application inhibited isoflavonoidconcentrations in all soybean lines, can be interpreted asevidence for involvement of isoflavonoids in nodulation con-trol. In contrast, the higher isoflavonoid concentration of theNN5 line, compared with Williams, may be an argument
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INOCULATION AND N EFFECTS ON SOYBEAN ROOT ISOFLAVONOIDS
0 1 2 3 4 5
60
50
40
30
20 -
10
0-
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60
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Figure 4. Effect of nitrate concentration on isoflavonoid concentra-tion in root extracts from selected nodulation mutants and the Wil-liams parent. Other details as in Figure 3 legend. Values representmeans of three replicates for each soybean line and vertical bars areLSDo.05.
against the conclusion that the hypernodulation trait is relatedto higher isoflavonoid levels. Nodulation control in the NN5line may, however, be due to factors totally unrelated toisoflavonoids, leaving open the possibility that isoflavonoidsplay a role in differential nodulation of soybean lines geneti-cally competent to nodulate.
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19. Mathews A, Carroll BJ, Gresshoff PM (1989) Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill).Protoplasma 150: 40-47
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