Myeong-Je Cho and James E. Harper*. Department of Agronomy ...... Rossen L, Davis EO, Johnston AWB (1987) Plant-induced expression ofRhizobium genes ...
Plant Physiol. (1991) 95, 1106-1112
Received for publication August 27, 1990 Accepted December 11, 1990
0032-0889/91/95/1106/07/$01 .00/0
Effect of Localized Nitrate Application on Isoflavonoid Concentration and Nodulation in Split-Root Systems of Wild-Type and Nodulation-Mutant Soybean Plants1 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 South Goodwin Ave., Urbana, Illinois 61801 ABSTRACT
of NO3- in the soil in contact with the nodulation zone was more inhibitory to soybean nodulation than when NO3- was placed below the nodulation zone (11). Gibson (7) showed a decreased 14C02 flux to nodules following supply of NO3- to the plant roots. In contrast, a split-root study with soybean showed that the root-half receiving a N source (either inoculated or supplied NH4NO3) received the majority of photosynthate compared with the root-half receiving no N (24). It has been recently reported that hypernodulating soybean mutants (NOD 1-3, NOD2-4, and NOD3-7) exhibit increased nodulation capability in either the absence or presence of NO3- (9). These mutants appear to be altered in autoregulatory control of nodule number normally shown by soybean. In another study involving nodulation mutants, Olsson et al. (19) used a split-root experiment to show that a 7-d prior inoculation of one-half of a root system totally suppressed nodulation on the other side for the Bragg parent, but
Although isoflavonoids are known to be inducers of nod genes in Bradyrhizobium japonicum, it was recently proposed that internal root levels of isoflavonoids may be important in nodule development on soybean (Glycine max [L.] Merr.). The hypemodulating soybean mutants were shown to accumulate higher root concentrations of isoflavonoid compounds (daidzein, genistein, and coumestrol) and to be more extensively nodulated than was the Williams parent when inoculated with B. japonicum. The hypernodulating mutants and the parent line, Williams, also showed decreased isoflavonoid concentrations and decreased nodule development if N was applied. The current study evaluated the effect of localized N03 application on root isoflavonoid concentration and on nodulation in split-root systems of the Williams wild type and a hypemodulating mutant (NODl-3). Nitrate application markedly decreased isoflavonoid concentrations in noninoculated soybean roots. When roots were inoculated, nodule number, weight, and nitrogenase activity were markedly suppressed on the root-half receiving 5 millimolar N03- compared with the other root-half receiving 0 millimolar N03-. High performance liquid chromatographic analyses of root extracts showed that the root-half receiving 5 millimolar N03- was markedly lower in isoflavonoid concentrations in both soybean lines. This was partially due to the localized stimulatory effect of N03- on root growth. The inoculated NODl-3 mutant had higher isoflavonoid concentrations than did the Williams control in both the presence and absence of N03-. These results provide evidence that the site of N application primarily controls the site of nodulation inhibition, possibly through decreasing isoflavonoid levels. Although the effect of N03- on nodule development and root isoflavonoid concentration was strongly localized, there was evidence that NO3- also resulted in a systemic effect on root isoflavonoids. The results are consistent with previous speculation that intemal levels of root isoflavonoids may affect nodule development.
not the supernodulation mutant (nts382). It is known that the nod genes in Rhizobium are activated by plant signal compounds (flavonoids and isoflavonoids) exuded from the roots of host plants (6, 15, 20, 22). The isoflavones, daidzein and genistein, have been isolated and identified as the major components which stimulate a Bradyrhizobium japonicum nodABC-lacZ fusion (15). nod gene induction studies (17, 27) showed that there was no significant difference in inducibility of the nod gene-lacZ fusion among noninoculated seedling root extracts or exudates from a supernodulating mutant, a nonnodulating mutant, and the Bragg parent. In addition, coumestrol (a coumestan) and daidzein have also been shown to promote the growth of B. japonicum (5). Isoflavonoids may, however, play other roles in the nodulation process beyond the role in nod gene induction and bacterial growth. Our recent study (4) showed that hypernodulating mutants had higher concentrations of isoflavonoid compounds than did the Williams parent, and that N application markedly decreased isoflavonoid concentrations in nodulation mutants as well as in Williams. It was further speculated that nodule development may be related to root levels of isoflavonoid compounds. The current study was carried out (a) to determine the effect of localized NO3- application on root isoflavonoid concentration and on nodulation of the Williams wild type and hypernodulating soybean mutant (NOD1-3) using
Nitrate inhibition of nodulation is a common regulatory feature of legume root nodule symbiosis which is primarily controlled by the host plant (3, 8). Split-root studies have demonstrated a localized NO3- effect on nodulation in soybean (1 3) and clover (2). It was also shown that incorporation ' Supported in part by the American Soybean Association, Research Project 88412.
1106
N03- EFFECT ON ISOFLAVONOIDS OF SOYBEAN SPLIT-ROOTS
split-root systems, and (b) to test whether NO3- itself also affects isoflavonoid concentrations in noninoculated plants. MATERIALS AND METHODS Split-Root System A modification of the split-root system described by Singleton (23) and Singleton and van Kessel (24) was used. A
planting hole (0.95 cm diameter) was drilled into the joint of each elbow of a 1.6-cm diameter (i.d.) 900 PVC2'3 elbow. Two 3.0-cm lengths of 1.2-cm diameter (i.d.) PVC pipe served as connectors between the 90° elbow and two 450 PVC elbows which extended through holes in the Styrofoam lid. The PVC split-root assemblies were sterilized in 5% (v/v) Clorox for 2 h. After rinsing several times with distilled H20, PVC elbows were packed with autoclaved vermiculite. A string wick was used to facilitate the movement of the solution into the vermiculite for seedling growth. Planting and Plant Culture
Seeds of Williams soybean (Glycine max [L.] Merr.) and of a hypernodulating mutant (NOD 1-3) selected from Williams were surface-sterilized in 5% Clorox (v/v) plus one drop of Tween 20 for 10 min, rinsed, and planted hilum down in autoclaved moist vermiculite. The root tips of 48-h-old seedlings were cut to induce branching, and seedlings were then inserted into the planting holes of the PVC elbow unit assemblies. The seedlings in the assemblies were covered with autoclaved vermiculite and watered with distilled H20 to prevent desiccation of the cotyledons. Plants were grown in a growth chamber maintained at day/night temperature of 29°C/20°C and a 14-h photoperiod at 650 ,mol photons m-2 s '. After 5 d growth, the split-root assemblies with plants were transferred to test tubes supplied with a modified minus N Hoagland nutrient solution (9) to observe and select seedlings with uniform root formation. Three days later, plant roots were inoculated with suspensions (108 cells/mL) of Bradyrhizobiumjaponicum strain USDA 110 for 10 min and transferred to Brute double buckets (Rubbermaid Commercial Products, Inc., Winchester, VA) in which each side contained 8 L of a modified Hoagland nutrient solution. The split-root assemblies with plants were fitted through holes in Styrofoam lids, and each bucket contained eight plants, consisting of four plants of each soybean line. Three N03(NaNO3) treatments were applied: (a) both sides receiving 0 mM N03-, (b) one side receiving 0 mM NO3- and the other side receiving 5 mM NO3-, and (c) both sides receiving 5 mM NO3- at the time of transplanting. At 12 and 18 d after inoculation and transplanting, nutrient solutions were completely changed. The solution pH was maintained at pH 6.5 + 0.5 by ion exchange resin columns (12). These columns were also used to provide aeration to the nutrient containers. 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. 3Abbreviation: PVC, polyvinyl chloride. 2
1107
Reproducibility of the data was confirmed by preliminary runs in which experimental detail was established. Preparation of Root Extracts for HPLC Analysis
After 9 and 12 d growth in the double buckets, two halfroots of each soybean line from each bucket were harvested at each sampling time and prepared for HPLC analysis of isoflavonoid compounds by a modification of the method previously described (4). From 2 to 4 g of fresh roots were weighed and extracted with 20 mL of acetone by grinding with an Omni-mixer (Omni Corp. International, Waterbury, CT) in an ice bath for 1 min. The acetone extract was centrifuged at l0,000g and 4°C for 10 min. The acetone supernatant was decanted and evaporated under a stream of N2 gas. The resulting aqueous fraction was re-extracted three times with 4 mL of anhydrous ethyl ether. The ether fraction was transferred with a Pasteur pipette and evaporated to dryness under a stream of N2 gas. The resulting residue was resuspended in 0.7 mL of HPLC-grade methanol, centrifuged, and filtered through a 0.45 ,um Millipore filter prior to analysis. Recovery percent was estimated by comparing the samples with or without spiking with a known amount of standard compounds at the beginning of the extraction procedure. Isoflavonoid assay by HPLC was performed as described (4). Four replicates of each soybean line and NO3- treatment were evaluated. C2H2 Reduction Assay
Twenty-four days after inoculation, plants from another set of the split-root system were harvested for in vivo assay of C2H2 reduction and measurement of nodules as previously reported (9). Four half-roots of each soybean line from each bucket were cut from the shoot and placed in 500-mL gastight jars. The jars were sealed and 50 mL of C2H2 was injected. Following a 30 min incubation at 30°C, 0.5 mL subsamples were analyzed for C2H4 production by flame ionization gas chromatography (Hewlett Packard 5890A Gas Chromatograph). Use of a closed system C2H2 reduction assay was justified by previous results (our unpublished results) that C2H4 production was linear through 30 min, except for a 3 to 5 min temperature equilibration lag, for hydroponically grown nodulated soybean roots. After the assay, the nodules were removed from the roots and counted. Plant roots and nodules were then dried for dry matter determinations. Four replicates of each soybean line and NO3- treatment were evaluated.
Isoflavonoid Analysis of Noninoculated Plants with or without N03A separate experiment which did not involve a split-root treatment was conducted to evaluate changes in isoflavonoid compounds when roots were not inoculated. Soybean plants (Williams and NOD1-3) were grown in growth chambers as previously described (4), without inoculation treatment. Seeds were germinated in sterilized sand and transplanted on d 6 to 18-L polypropylene trays containing a modified Hoagland nutrient solution with either 0 or 5 mm NaNO3. Plants were
Plant Physiol. Vol. 95, 1991
CHO AND HARPER
1108
sampled at 3-d intervals from transplanting throughout a 12d growth period after transplanting. Daidzein, genistein, and coumestrol were assayed by HPLC as previously described (4). Three replicates of each soybean line and NO3- treatment were evaluated. RESULTS
Nodulation and Growth Characteristics in Split-Root Systems
Five millimolar N03- application to one side of the splitroot system resulted in inhibition of nodulation and N2 fixation (number, dry weight, and C2H2 reduction) in both the Williams wild type and a hypernodulating mutant (NOD13), compared with the other side receiving 0 mm NO3- (Table I). The inhibitory effect of N03- on nodule number, dry weight, and C2H2 reduction activity was more marked in Williams than in NOD 1-3. Nodulation was completely inhibited in the presence of 5 mm NO3- on Williams while some nodules were still formed on NOD 1-3. Nodule numbers and dry weights of both soybean lines were also markedly suppressed where both root-halves received 5 mm NO3- compared with the treatment where both root halves received 0 mM NO3-. Shoot dry weight in both soybean lines markedly increased in the presence of NO3-, especially in Williams, compared with that in the absence of NO3-. The Williams wild type had more rapid root growth than did the NOD 1-3 line in the presence of NO3-. NOD1-3 showed a significant increase in nodule dry weight on the 0 mm NO3- side of the split-root system compared with the other side receiving 5 mM NO3-, or compared with both sides receiving either 0 or 5 mM NO3-. Williams did not show this response pattern for nodule dry weight. Root growth ofboth Williams and NOD 13 was more rapid on the 0 mm NO3- side of the split-root system when the other side received 5 mm NO3- than when both sides received 0 mm NO3;. This may be due to N deficiency at the time of harvest when neither side received NO3-.
Isoflavonoid Analysis of Root Extracts in Split-Root Systems Table II shows isoflavonoid concentrations and contents of root extracts from split-root systems of the Williams wild type and a hypernodulating mutant (NOD1-3) as affected by NO3-, harvested at 9 and 12 d after inoculation. Higher isoflavonoid concentrations and contents were observed at 12 d after inoculation in both soybean lines, relative to respective treatments at 9 d after inoculation, except for isoflavonoid concentrations of Williams roots when both root-halves received 5 mm NO3. There were more marked increases in isoflavonoid contents than in isoflavonoid concentrations with time because of the increased root growth of the plants. The root-half receiving 5 mm NO3- was markedly decreased in isoflavonoid concentrations, compared with the other roothalf receiving 0 mM NO3- (Table II). More marked decreases in isoflavonoid concentrations were detected when both sides received 5 mm NO3- compared with when only one side received 5 mm NO3;. The NOD 1-3 hypernodulating mutant had a higher isoflavonoid concentration than did the Williams parent in all treatments. However, similar or higher isoflavonoid contents were measured in the root-half of Williams receiving 5 mM NO3- compared to the root-half receiving 0 mM NO3-. In NOD1-3, both content and concentration of isoflavonoids were lower in the root-half receiving 5 mM NO3-. In addition to a localized effect of NO3- on root isoflavonoid concentration, there was evidence for a systemic effect when comparing the root-halves receiving 0 mm NO3for the 0/0 and 0/5 mm NO3- treatments. NOD1-3 had higher total contents (sum ofboth root-halves) of isoflavonoid compounds than did the Williams parent, except for coumestrol, when only one side received NO3- treatment. The differences between soybean lines were smaller when only one side received N03- treatment, compared with when both sides received either 0 or 5 mm NO3-. The sum of isoflavonoid contents of both root-halves of Williams decreased when both root-halves received 5 mm NO3-, while the total isoflavonoid contents of NOD 1-3 roots decreased when either one or both root-halves received 5 mm NO3-, compared with the treat-
Table I. Effect of Localized Nitrate Application on Nodulation and Growth in Split-Root Systems of cv Williams and NOD1-3 Seeds were germinated in sterilized vermiculite. The tips of 48-h-old seedlings were cut and then seedlings were inserted into the planting holes of the split-root assemblies. Five days later, the assemblies were transferred to test tubes. After 3 d of growth, roots were inoculated immediately prior to transplanting to the double buckets containing a modified Hoagland nutrient solution with either 0 or 5 mm NO3-. Plants were harvested after 24 d of growth in the double buckets. Data are expressed as means ± SD (n = 4). Nodule Treatment Shoot Dry Root Dry Split-Root C2H2 Side N03Genotype Reduction Weight Weight Number Dry weight Treatment Sampled
mM NO3Williams
NODl-3 Williams Williams
NOD1-3 NOD1-3 Williams
NOD1-3
0/0 0/0 0/5 0/5 0/5 0/5 5/5 5/5
mM NO30 0 0 5 0 5 5 5
No.
half-root-'
50 ± 4 138 ± 19 28 ± 8 0±0 171 ± 34 33 ± 12 0±0 16 ± 5
mol C2H4
mg half-root-'
half-root-' h-1
29 ± 9 34 ± 4 21 ± 4 0±0 94 ± 26
1.11 ± 0.33 1.00 ± 0.34 0.66 ± 0.18 0.00 ± 0.00 2.85 ± 0.57 0.69 ± 0.40 0.00 ± 0.00 0.28 ± 0.13
17 ± 7 0±0 11 ± 4
ghalf-root1 ghlf-oF 0.09 ± 0.01 0.06 ± 0.03 0.20 ± 0.05 0.39 ± 0.12 0.14 ± 0.04 0.22 ± 0.05 0.34 ± 0.04 0.18 ± 0.06
gplant-' gpaF 0.60 ± 0.06 0.42 ± 0.05
3.37 ± 0.84
2.34 ± 0.44 4.08 ± 0.13 2.84 ± 0.48
NO3- EFFECT ON ISOFLAVONOIDS OF SOYBEAN SPLIT-ROOTS
1109
Table II. Effect of Localized Nitrate Application on Isoflavonoid Concentration and Content in SplitRoot Systems of cv Williams and NOD1-3 At 9 and 12 d after inoculation, plant roots were harvested for isoflavonoid analysis. Other details as in Table I legend. Values represent means of four replicates for each treatment at 9 and 12 d after inoculation. Isoflavonoid Split-Root Treatment Total content Isoflavonoid Content Concentration Side (both sides) Genotype N03-1 Compound (both___ ____sides)______ Treatment Sampled 9d 12d 9d 12d 9d 12d
Csompavounod
Ag g'1 root fresh wt
Daidzein
Williams NOD1-3 Williams Williams NOD1-3 NOD1-3 Williams
NOD1-3 Genistein
Williams
NOD1-3 Williams Williams NOD1-3 NOD1-3 Williams NOD1-3
Coumestrol
Williams NOD1-3 Williams Williams NOD1-3 NOD1-3 Williams NOD1-3
0/0 0/0 0/5 0/5 0/5 0/5 5/5 5/5 LSDo.05 0/0 0/0 0/5 0/5 0/5 0/5 5/5 5/5 LSDo.05 0/0 0/0 0/5 0/5 0/5 0/5 5/5 5/5 LSDo.05
0 0 0 5 0 5 5 5 0
0 0 5 0 5 5 5
0 0 0 5 0 5 5 5
97 204 79 57 173 67 40 83 20.1 12.4 35.4 10.9 6.2 21.5 8.3 3.7
8.0 4.5 77 99 50 27 68 33 17 32 11.4
116 229 113 64 186 108 44 136 22.9 15.9 36.8 14.1 6.6 24.2 10.9 2.8 13.5 4.6 104 125 71 45 92 54 15 60 11.1
gg plant-'
Ag half-roor' 121 203 79 100 161 89 61
182 369 193
99
328
23.9 15.5 35.1 10.7 10.8 20.1 11.2 5.7 9.5 5.0 97 99 49 47 64 44 26 38 17.5
236 291 249 120 67.8 24.6 59.4 24.1 24.0 38.6
25.4 7.7 32.1 10.6 160 202 120 164 143 125 41 146 34.4
241 406 178
364 739 429
250
539
122 240 657 199 46.4 112 31.0 49.2 70.3 119 21.5 481
31.3
63.9
11.3 19.0 10.2 195 198 96
15.5 64.3 14.1 320 404 284
108
268
52 75 37.1
81 292 57.6
ment where both root-halves received 0 mm NO3-. The relationship between root isoflavonoid concentration and nodule number was positive and significant for all three isoflavonoids measured in the various split-root, NO3- treatment, and soybean line combinations (Fig. 1). This relationship is strongly influenced by the concomitant inhibitory effect of NO3- on root isoflavonoid concentrations and visible root nodule development. Whether this is a causal relationship or merely an associated response to N03- remains to be determined.
edly decreased the concentration of all three isoflavonoid compounds in both soybean lines relative to respective controls without NO3-. The differences in isoflavonoid concentrations between NO3- treatments became larger with time. Within an NO3- treatment, no significant differences were detected in root isoflavonoid concentrations between soybean lines under the noninoculated growth condition.
Isoflavonoid Analysis of Noninoculated Plants with or without N03- Treatment
It is well documented that N application inhibits all phases of nodulation, including bacterial infection, nodule development, and nitrogenase function (3, 10). It has also been reported that symbiotic efficiency of soybean plants grown on NO3- is slightly affected by B. japonicum strain (18), but it appears that the host plant is in primary control of nodulation in the presence of NO3 (3, 8). Once nodulation has occurred, subsequent nodulation on new roots of normal plants is suppressed by a plant process called autoregulation (14, 21). Partially NO3- tolerant mutants of soybean (NOD 1-3,
Root isoflavonoid concentrations continued to increase throughout the initial 12-d growth period after N03- treatment (18-d growth period after planting), regardless of soybean line or NO3- treatment (Fig. 2). There were no significant differences in root isoflavonoid concentrations between NO3treatments of both soybean lines up to 3 d after NO3- treatment. After that time, however, 5 mm NO3- treatment mark-
DISCUSSION
1110
NOD2-4, and NOD3-7) which have altered autoregulatory
200 0 0
*
4-
*
150 I
A
A
L.0
E
#
100
x
z
_O
50
a)
O
y = - 62.019 + 0.937x R = 0.88** Williams (0/0) 0 w NOD 1-3 (0/0) 0 Williams (0/5) 0 Williams (0/5)5 NOD 1-3 (0/5) 0 NOD 1-3 (0/5) 5 Williams (5/5) 5 NOD1-3 (5/5) 5>A 0
control of nodulation have been recently isolated from the
*
0
z
o 200
0 0100 5 05 1 00
Daidzein (tg g9Y
y
0
0
150 200 root fresh wt.) 1
50
250
2 0 0
25 0
no significant difference in the concentration of isoflavonoid
- 27.439 + 5.251x R = 0.87** /
.
150
/ *
100
In order to know whether NO3- directly affects isoflavonoid concentrations in soybean roots or whether the effect is mediated by interaction of N03- with B. japonicum, noninoculated plants grown on either 0 or 5 mM NO3- were harvested
a)
50
z
A_
and assayed for isoflavonoids in this study. Although 5 mM
0
10
0
20
Genistein (.tgg9-l
40
30 root fresh wt.)
concentration
y= -43.538 +1.386x R=0.76* N 150
*/
a)
100
z *
50
0
,/, ^0.. ,A,
z
.
,
*
|
*
|
0
0
20
40
60
80
1 00
1 20
1 40
Coumestrol(,ug g-1root fresh wt.) Relationship between isoflavonoid concentration and nodin split-root systems of Williams and NODl-3. Data of isoflavono id concentrations are from 12 d after inoculation (TableII) and data (of nodule number are from 24 d after inoculation (TableI). Each date point represents a mean of four replicates. The NO3treatment concentrations of the two root-halves are given in parentheses folllowed by the NO3- concentration of the specific root-half sampled.vel ICorrelation coefficients were significant at the 0.05 level (*) Figure 1.
ule numbEor
or
0.01 leN
of all three isoflavonoid
compounds
(Fig. 2).
NO3-.
I
a)
NO3- application did not affect isoflavonoid concentrations of either Williams or NODl-3 soybean line up to 3 d after NO3- treatment, after that time NO3- markedly decreased the This result supports the previous conclusion (3, 8) that the host plant primarily controls nodulation in the presence of
200
E
compounds (daidzein, genistein, and coumestrol) among the noninoculated hypernodulating mutant(s) and the Williams parent up to 18 d after planting. However, the previous (4) and current (Table II) results showed that in response to inoculation, hypernodulating mutant(s) had higher isoflavonoid concentrations than did the Williams parent, and that N application (especially NO3-) markedly inhibited isoflavonoid accumulation.
z
.0
Williams background, and initial characterization of these mutants has been reported (9). These mutants have greater nodulation capabilities and higher nitrogenase activities in the presence of NO3-, as well as in the absence of NO3-, compared with the wild type, and provide opportunity to evaluate whether isoflavonoids play a role in differential nodulation. Although the hypernodulating mutants developed greater nodule numbers and nodule mass than Williams in the presence of NO3-, both this and a previous study (9) showed that nodulation inhibited by NO3- in both Williams and the nodulation mutant(s) relative to respective without NO3; ~~~controls (4) and current (Fig. 2) results also showed cOur previous was
0
I
Plant Physiol. Vol. 95, 1991
CHO AND HARPER
(**)
Split-root studies (2, 13) showed a direct inhibitory effect of NO3- and indicated that the site of NO3- application controls the site of nodulation inhibition. Our results also showed that the root-half receiving 5 mM NO3- was markedly inhibited in nodulation and N2 fixation compared with the other root-half receiving 0 mM NO3- (Table I). This may be causally related to the decreased concentration of isoflavonoid compounds (Table II). Although 5 mM NO3- application markedly decreased isoflavonoid concentrations in both lines when only one root-half received 5 mM NO3- treatment, similar or higher isoflavonoid contents were detected in the root-half of Williams receiving 5 mM NO3- compared with the root-half receiving 0 mM NO3-. Similar or slightly lower isoflavonoid contents were, however, measured in NOD1-3 (Table II). This may be from a localized stimulatory effect on root development of both soybean lines in response to N03application, especially in the Williams wild type (Table I). The split-root study by Singleton and a similar result. The soybean root
van
Kessel (24) showed
receiving NO3- received
most of the labeled carbon with uninoculated root systems. Furthermore,
dry
matter
preferentially allocated
to
and
current
photosynthate
nodulated roots when only
one
were
side
peeetal loae ondltdroswe nyoesd 23). However, Gibson (7) reported that of NO3- to 14CO2 flux to nodules decreased following supply
was inoculated
(19,
NO3- EFFECT ON ISOFLAVONOIDS OF SOYBEAN SPLIT-ROOTS
250
_
Cl)
,a)
Williams (0 mM) NOD1-3 (0 mM)
....... .......
200
Williams (5mM) NOD1-3 (5 mM)
.-
-0
-
0 0 150 L-
0) 100 C
c
0) 50
in I
I
s
C.I
0)
0
Cl)
7C)
0 1
2
3
4
5
6
7
8
9 10 11 12
0
1
2
3 4
5
6
7
8
9 10 11 12
0
1
2
3 4
5
6
7
8
9 10 11 12
60 50
40 0-
C/) C
30
20 .-
cD0
10
0
0
cn L.
c
40
-0-
CD)
30
U)
cn
E E 0
0
20
10
0
1111
the plant roots. It was proposed earlier that growth conditions which decreased C availability to nodules (i.e. conditions favorable to NO3- uptake, metabolism, and subsequent synthesis of organic N compounds) would result in minimal symbiotic N2 fixation (26, 28). This response to an altered C to N ratio in the plant has been considered as an indirect effect of NO3-. Our previous study (4) showed that N treatment increased the shoot-to-root ratio, primarily through increased shoot weight in soybean plants, indicating that relatively more carbohydrates are utilized for shoot growth in response to N application. The previous (4, 9) and current (Table I) results also showed that shoot and root growth of hypernodulating mutant(s) was decreased, relative to that of the Williams parent, with root growth being affected more than shoot growth. This might be due to higher isoflavonoid concentrations of hypernodulating mutants in response to inoculation. Exogenous introduction of isoflavonoid phytoalexins can retard root growth (25), and our hypernodulating mutants do have less root growth than the Williams parent. The strong positive relationship between root isoflavonoid concentrations and nodule number (Fig. 1) supports the conclusion (4) that isoflavonoids may play a role in differential nodulation of hypernodulated and wild type soybean lines and the partial tolerance of nodulation to NO3- in hypernodulating mutants. This must, however, be viewed with caution, since the hypernodulating mutants and Williams have similar levels of isoflavonoid compounds when roots are not inoculated (Fig. 2), and since the appearance of difference in isoflavonoid concentrations between lines which are inoculated is past the point in time when initial bacterial infection occurs (4). Mathews et al. (16) observed that similar numbers of infection events occurred for the nts382 supernodulating mutant and the Bragg parent, but that a greater proportion of these infection events developed to advanced stage of nodule ontogeny in nts382. Calvert et al. (1) also reported that the host autoregulatory response in Williams acted primarily during the subsequent development of infections into nodules rather than during the initiation of infections. Therefore, the difference in isoflavonoid levels inside the plant root may be of importance in controlling nodule development at the time of post infection, and the control is exercised within the root. This speculation must, however, be further evaluated. The possibility exists that greater root isoflavonoid levels of inoculated hypernodulating mutants in this study results in enhanced nodulation susceptibility of the root, thereby allowing greater numbers of nodules to develop. Alternatively, nodule development in Williams may be limited by inadequate root isoflavonoids.
Days after Nitrate Treatment Figure 2. Effect of NO3- application on isoflavonoid concentration in noninoculated Williams and NOD1 -3. Seeds were germinated in sterilized sand and transplanted on d 6 to nutrient solution with either 0 or 5 mM NO3-. Plants were sampled through a 1 2-d growth period after transplanting. Acetone-ether extracts from 5 g of soybean roots at 3-d intervals from transplanting were reduced to dryness and redissolved in 1.0 mL of methanol. Twenty-microliter samples were injected and monitored at 254 nm for HPLC analysis of isoflavonoid compounds. Values represent means of three replicates for each treatment and vertical bars are LSDo 05. The designation n.s. denotes nonsignificant differences among treatments at the 0.05 level.
LITERATURE CITED 1. Calvert HE, Pence MK, Pierce M, Malik NSA, Bauer WD (1984) Anatomical analysis of the development and distribution of Rhizobium infections in soybean roots. Can J Bot 62: 2375-2384 2. Carroll BJ, Gresshoff PM (1983) Nitrate inhibition of nodulation and nitrogen fixation in white clover. Z Pflanzenphysiol 110: 77-88 3. Carroll BJ, Mathews A (1990) Nitrate inhibition of nodulation in legumes. In PM Gresshoff, ed, Molecular Biology of Symbiotic Nitrogen Fixation. CRC Press, Boca Raton, FL, pp 159180
1112
CHO AND HARPER
4. Cho M-J, Harper JE (1991) Effect of inoculation and nitrogen on isoflavonoid concentration in wild-type and nodulationmutant soybean roots. Plant Physiol 95: 435-442 5. 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 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. EMBOJ6: 1173-1179 7. Gibson AH (1974) Consideration of the growing legume as a symbiotic association. Proc Indian Natl Sci Acad 40B: 741767 8. Gibson AH, Harper JE (1985) Nitrate effect on nodulation of soybean by Bradyrhizobium japonicum. Crop Sci 25: 497-501 9. Gremaud MF, Harper JE (1989) Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean. Plant Physiol 89: 169-173 10. 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 11. Harper JE, Cooper RL (1971) Nodulation response of soybeans [Glycine max (L.) Merr.] to application rate and placement of combined nitrogen. Crop Sci 11: 438-440 12. Harper JE, Nicholas JC (1976) Control of nutrient solution pH with an ion exchange system: effect on soybean nodulation. Physiol Plant 38: 24-28 13. Hinson K (1975) Nodulation responses from nitrogen applied to soybean half-root systems. Agron J 67: 799-804 14. 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 15. 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 16. Mathews A, Carroll BJ, Gresshoff PM (1989) Development of Bradyrhizobium infections in supernodulating and non-no-
17.
18. 19.
20.
21. 22.
23.
24.
25.
26. 27.
28.
Plant Physiol. Vol. 95, 1991
dulating mutants of soybean (Glycine max. [L.] Merrill). Protoplasma 150: 40-47 Mathews A, Kosslak RM, Sengupta-Gopalan C, Appelbaum ER, Carroll BJ, Gresshoff PM (1989) Biological characterization of root exudates and extracts from nonnodulating and supernodulating soybean mutants. Mol Plant Microbe Interact 2: 283-290 McNeil DL (1982) Variations in ability ofRhizobiumjaponicum strains to nodulate soybeans and maintain fixation in the presence of nitrate. Appl Environ Microbiol 44: 647-652 Olsson JE, Nakao P, Bohlool BB, Gresshoff PM (1989) Lack of systemic suppression of nodulation in split root systems of supernodulating soybean (Glycine max [L.] Merr.) mutants. Plant Physiol 90: 1347-1352 Peters NK, Frost JW, Long SR (1986) A plant flavone, luteolin, induces expression of Rhizobium meliloti nodulation genes. Science 233: 977-980 Pierce M, Bauer WD (1983) A rapid regulatory response governing nodulation in soybean. Plant Physiol 73: 286-290 Rossen L, Davis EO, Johnston AWB (1987) Plant-induced expression of Rhizobium genes involved in host specificity and early stages of nodulation. Trends Biochem Sci 12: 430-433 Singleton PW (1983) A split-root growth system for evaluating the effect of salinity on components of the soybean-Rhizobium symbiosis. Crop Sci 23: 259-262 Singleton PW, van Kessel C (1987) Effect of localized nitrogen availability to soybean half-root systems on photosynthate partitioning to roots and nodules. Plant Physiol 83: 552-556 Smith DA, Banks SW (1986) Formation and biological properties of isoflavonoid phytoalexins. In V Cody, E Middleton, JB Harborne, eds, Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity Relationships, Alan R Liss, New York, pp 113-124 Strowd WH (1920) The relation of nitrates to nodule production. Soil Sci 10: 343-356 Sutherland TD, Bassam BJ, Schuller LJ, Gresshoff PM (1990) Early nodulation signals of the wild type and symbiotic mutants of soybean (Glycine max). Mol Plant Microbe Interact 3: 122-128 Wilson PW (1940) The Biochemistry of Symbiotic Nitrogen Fixation. University of Wisconsin Press, Madison, WI
