Plant Science, 83 (1992) 35-43 35 Elsevier Scientific Publishers Ireland Ltd.

Studies on the root control of non-nodulation and plant growth of non-nodulating mutants and a supernodulating mutant of soybean

(Glycine max (L.) Merr.)

A n n e M a t h e w s * , B e r n a r d J. C a r r o l l * * a n d P e t e r M. G r e s s h o f f * * *

Botany Department, The Australian National University, GPO Box 4. Canberra. A C T 2601 (Australia)

(Received September 27th, 1991; revision received January 17th, 1991; accepted January 17th, 1991)

Ethyl methanesulphonate mutagensis of soybean cv. Bragg led to the isolation of three non-nodulating mutants: nod49, nod772 and nod139, as well as the supernodulation mutant nts382. Mutants nod49 and nod772 are allelic to the naturally-occurring mutation rjt while nod139 is non-allelic and represents a newly found gene conditioning non-nodulation in soybean. Using the standard reciprocal wedge grafting technique it was shown earlier that non-nodulat ion in mutant nod49 is root controlled and supernodulation in mutant nts382 is shoot controlled. Non-nodulat ion in mutants nod772 and nod139 (the newly found non-nodulation gene in soy- bean) is also root controlled. In addition, scions of nts382 failed to alter the nodulation phenotype when grafted onto stocks of the non-nodulat ing lines. Lateral roots developing from the scion of nts382 and Bragg exhibited supernodulation and the wild-type pat- tern of nodulation, respectively, indicating that the lesion(s) conditioning non-nodulation only affect the root in a localized manner. Physiological studies conducted on the mutants indicate that they are not altered in the assimilation of nitrogen and carbon when grown on nitrate, and a study of their nitrogen contents indicate that the anomaly in the non-nodulation mutants is dearly specific for the nodulation process. This was confirmed by assaying nitrate reductase (NR) under a range of conditions; it was observed that the non-nodulation mutants expressed inducible NR and constitutive NR activity in a manner similar to the wild type.

Key words. non-nodulation; supcr~aodvlation; soybean: nitrate reductase: plant growth; root control of non-nodulation

introduction

Legumc root m~dule initiatu:,:,., amd development a:~ g,~:'.rncd by sig~.t!s ~,r :.":.~,:tc, r~ i::oth external and internal to ti~e two ~,,,~,:cttc partners. The study ot these processes has been facilitated by the isolation and characterization ~qa range of genetic variants of both p:~r.'.,,cJ ~. Mutants altered in sym- biosis are available in many legume species. In soy- beans, host genes conditioning non-nodulation

Correspondence to." Anne Mathews, Plant Molecular Biology Laboratory, C.S . IR.O. , Division of Plant Industry, GPO Box

1600, Canberra, ACT 2601, Australia. *Present address: Plant Molecular Biology Laboratory, C.S.I.R.O., Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. **Present address: John Innes Institute, Colney Lane, Norwich, NR4 7UH, U.K. ***Present address: Plant Molecular Genetics, College of Agriculture, University of Tennessee, Knoxville, U.S.A.

[1,2], ineffective nodulation [3-5] and super- nodulation [6,7] have been reported.

In the legume-Rhizobium symbiosis, the effect of the scion and rootstock on nodulation, has been studied using various grafting techniques [8,9]. Earlier studies in the soybean system have im- plicated root and shoot factors in the control of non-nodulation and supernodulation, respectively [9]. Here, we extend the previous studies and show that the non-nodulation response of a newly discovered non-nodulation gene (characterized as nod139) [1] exhibits identical tissue specificity to that governed by the previously recognized rjl locus in the newly induced mutants nod49 and nod772. Most importantly, we show that non- nodulation is restricted to the root tissue, and shows no systemic effect on nodulation of either supernodulating or normally nodulating root tissue. Similarly, we show that the non-nodulation response cannot be cross-fed to wild type and supernodulating root tissue systemically.

0168-9452/92/$05.00 @ 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

36

Materials and Methods

Plant material and inoculant cultures Soybean (Glycine max (L.) Merr.) cv. Bragg and

its derived non-nodulation mutants nod49, nod772 and nod139 [10], a supernodulation mutant nts382 [6, 7] and the naturally-occurring mutant rjl [2] in the genetic background of the cv. Lee were used in these studies. The Bradyrhizobium japonicum strain U S D A l l 0 peat culture containing about 108 viable cells per gram was used in these experiments.

Plant growth conditions and the grafting technique Plants used for grafting studies were grown in

25 cm diameter pots filled with a 2:1 (v/v) sand/ vermiculite mixture at the rate of one plant per pot. Care was taken to keep the pots sterile and uninoculated prior to grafting the seedlings. Glasshouse temperatures were maintained bet- ween 14°C and 30°C and incandescent 100 W bulbs were used to extend the photoperiod to 16 h per day.

Ten-day-old seedlings were grafted using the standard reciprocal wedge graft [9]. These grafts were made to study the effect of the scion of the supernodulation mutant nts382 and the wild-type Bragg on the nodulation of the non-nodulation mutants as well as to determine whether the non- nodulation mutant scions would cause an increase or decrease in nodulation on nts382 and Bragg roots. The approach grafting technique was used to determine whether the lesion(s) conditioning non-nodulation exert their influence in a localized manner within the root or are systemic in the plants. Here two seedlings to be grafted were grown in each pot. Ten days after germination, a 1.5-cm long slice of the stem below the cotyledon was removed from the adjoining plants. This was deep enough to expose the vascular tissue (approx- imately 50% of the stem was removed in the exci- sion region). The cut regions of the stems were then firmly held together and securely taped with a teflon tape [11]. The goal of these experiments was to determine whether any substance (stimu- latory or inhibitory) formed in the scions of the plants could alter the susceptibility of the rootstocks to rhizobial infection.

Grafted plants were placed in a mist room under automatic intermittent misting for 7 days to pre- vent desiccation before the grafts had functionally joined. Plants were then moved to the glasshouse and inoculated with Bradyrhizobium japonicum strain USDA110 at approximately 108 viable cells per ml by making a slurry of the bacteria, peat and water. The pots were watered with nitrogen-tYee (nitrate absent) nutrient solution as described in Delves et al. [9] twice a week and with a 5 mM KNO3-supplemented plant nutrient solution once a week. The plants were harvested 45 days after planting and examined for nodulation and plant growth characteristics.

Growth studies Plants were grown in the glasshouse in 15-cm

pots filled with a 2:1 (v/v) mixture of sand: ver- miculite with one plant per pot for a period of 4 weeks after germination. At the time of planting the seeds were inoculated with a peat culture of B. japonicum strain USDA110 (10 s cells per pot) and the seedlings were reinoculated 1 week later. Seeds of similar size of all the genotypes were used in the experiments and only seedlings that germinated at the same time were used for growth comparisons. Either nitrogen free plant nutrient solution or 6 mM KNO3-supplemented nutrient solutions were used. The composition of the plant nutrient solu- tion was the same as that used by Herridge [12]. Plants received half strength of all the nutrients (except CaC12 which was administered at full strength) for the first 2 weeks after planting and full strength of all the nutrients in the subsequent 2 weeks before harvest. Each pot was watered with 1000 ml plant nutrient solution daily which was sufficient to flush out the residual nutrients from the previous watering. The glasshouse tempera- tures were maintained between 14°C and 30°C. Incandescent bulbs were used to extend the photoperiod to 16 h per day and to supplement the natural light. Plants were harvested 4 weeks after germination whereupon their root and shoot dry weights and their nitrogen and carbon contents were determined.

In vivo nitrate reductase (NR) assay In addition to noting the ability of the soybean

mutants to grow on nitrate, their nitrate reductase (NR) activity was assessed to confirm that the mutations were specific to the symbiotic process. In vivo NR activity was determined as described by Carroll and Gresshoff [13] on fully developed unifoliolate leaf discs of 10-day-old plants that had been treated either with or without nitrate from planting using NR assay solution containing 0-50 mM KNO 3. The minus NO 3- (no KNO3) assay was used to approximate the in situ activity where the reaction was dependent upon the endogenous pool of metabolically available nitrate within the leaf tissue. The plus NO 3- (50 mM KNO3) assay measured the maximum NR enzyme activity under nitrate-saturated conditions. In both cases, the reactions are dependent upon the in situ concen- trations of cofactors and reducing equivalence [14,15]. The data were expressed as nmol NO2- produced (leaf disc) h -1.

Nitrogen and carbon analysis The percentage of total nitrogen and carbon in

the roots and shoots of the plants used for growth comparisons was determined using a Carlo Erba Carbon and Nitrogen analyser model NA 1500. The plant material was oven dried at 80°C and then ground into a fine powder. Appropriate amounts of plant material (20 mg for roots, 10 mg for shoots and 5 mg for seeds) were accurately weighed for the determination of the carbon and

37

nitrogen content in the samples. Seeds of all the genotypes were collected from plants that had been watered with 5 mM KNO3-supplemented plant nutrient solution three times a week. The nitrogen and carbon contents of these seeds were also determined.

Statistical analysis Data for the grafting experiments were analysed

using the GENSTAT statistical package [16].

Results

Non-nodulation in soybean is root controlled and non-systemic

Non-nodulation in nod49 is controlled by the root as shown in earlier studies [9]. When shoots of either the wild-type parent cv. Bragg or nts382 were grafted onto a nod49 rootstock, the root- stocks were non-nodulated. On the other hand, grafting nod49 onto Bragg did not alter nodula- tion significantly; instead the wild-type pattern of nodulation was observed. Similarly, it has been shown that supernodulation in nts382 is shoot controlled. In other words, irrespective of whether the scion was from a normal nodulation plant or a supernodulation plant, the nod49 rootstock always remained non-nodulated [9]. Non- nodulation of nod139 and nod772 are also root controlled (Tables I and II). The nod139

Table I. Root control of non-nodulat ion in nod139. Ten-day-old seedlings were grafted using the wedge grafting technique and inoculated with B. japonicum strain USDA110. Data are means of 6 plants. Means and L.S.D. of transformed data are shown in paren- theses.

Graft Nodule no. Nodule dry Nodule no. Plant dry (scion/rootstock) per plant wt. per plant per gram wt. (g)

(g) plant dry wt.

Bragg/Bragg 110 (10.42) 0.124 (0.347) 33.3 (5.74) 3.3 nts382/nts382 1541 (39.17) 0.575 (0.757) 509.9 (22.55) 3.0

nod139/Bragg 194 (13.67) 0.135 (0.363) 48.8 (6.82) 4.1 Bragg/nod 139 0 0 0 3.8 nod 139/nod 139 0 0 0 3.0 nod139/nts382 265 (15.92) 0.128 (0.356) 57.4 (7.42) 4.6 nts382/nod 139 0 0 0 3.4 L.S.D. (0.05) (2.8) a (0.064) a (1.41) a 0.7

aRaw data required square root t ransformation to satisfy assumptions for an analysis of variance.

38

Table I1. Root control of non-nodulation in nod772. Ten-day- old seedlings were grafted and inoculated with B. japonicum strain USDA110. Data are means of 6-8 plants. Means and L.S.D. of transformed data are shown in parentheses.

Graft Nodule no. Nodule mass (scion/rootstock) per plant (mg)

Bragg/Bragg I 15.8 (4.723) 152.7 nts382/nts382 510.3 (6.223) 233.3 nod772/Bragg 107.9 (4.658) 125.8 nod772/nts382 121.1 (4.789) [ 19.5 nod772/nod772 0 0 nts382/nod772 0 0 Bragg/nod772 0 0 L.S.D. (0.199) a 43.019

aRaw data for nodule number per plant required log transfor- mation to satisfy the assumptions for an analysis of variance for nodule number for nodulating grafts.

rootstocks remained n o n - n o d u l a t i n g irrespective

of whether the grafted scion was that of the wild- type Bragg or the supernodu la t ion mu tan t nts382. There were no significant differences in the nodule number per gram of plant dry weight of the graft

of nod139 on either Bragg or nts382 compared to the Bragg controls (49 and 57 nodules per p lant compared with 33 nodules per plant on the Bragg controls). Similarly, n o n - n o d u l a t i o n of nod772 is

strictly determined by its root. Graf ts using nod772 as the scion and either Bragg or nts382 as the rootstock resulted in the wild-type pat tern of nodula t ion (108 and 121 nodules per plant, respec-

tively) and were not significantly different from the Bragg controls (116 nodules per plant). Nodule dry weight in all cases followed a similar pattern.

Nodula t ion of the lateral roots arising from the

scion was observed in order to determine whether a non -nodu la t i on lesion(s) exerts a negative in- fluence through the graft from the root to the shoot or whether non -nodu la t i on is localized in

the root. Data in Table lII indicate that non- nodula t ion mutan ts nod49 and nod772 permitted the format ion of nodules on the lateral roots aris- ing adventi t iously from the Bragg and nts382

scions. These laterals exhibited the wild-type nodula t ion pat tern with Bragg as the scion and had the supernodula t ion pat tern with nts382 as the scion. In all cases, the rootstock of the non-

nodula t ion mutan ts remained non-nodula ted . When a Bragg scion was grafted onto a nod49 rootstock, the lateral roots arising from the Bragg scion had the wild-type nodula t ion pattern (Table

III) with 39 nodules per lateral root and a nodula- tion interval of 9.5 cm on the 29-cm total lateral root length. With nts382 as the scion and nod49 as the rootstock, the adventi t ious roots arising from

nts382 had a nodula t ion interval of 10 cm on the total 11.3 cm lateral root length. The lateral root arising from nts382 when grafted onto nod772 had 284 nodules per lateral root and a nodula t ion in-

terval of 18 cm on the 21.5-cm total lateral root length indicating the characteristic supernodula- t ion character where nodules are seen at a higher density on the root. To ascertain that the lesion(s)

control l ing non -nodu la t i on in nod139 is localized in the root, an experiment was conducted using the approach grafting technique. As outl ined in Table IV, when nod139 was approach grafted to Bragg,

the Bragg root had 86 nodules per plant compared to 83 on the Bragg autograft . Similarly, the nts382

Table II1. Non-nodulation in soybean is localized to the root. Results were obtained from adventitious roots arising from the scion portion of the graft. Each entry in the table is the mean ± S.D.

Graft Total lateral Nodulation Nodule no. Percentage of (scion/rootstock) root length interval per lateral root nodulated

(cm) (cm) root C/,,)

Bragg/nod49 a 28.8 + 1.1 9.5 + 2.7 39.3 ± 6.7 32.9 nts382/nod49 b 11.3 ± 4.5 10.0 + 4.6 62.1 + 13.0 88.5 nts382/nod772 ~ 21.5 + 5.3 18.0 + 2.1 283.5 ± 14.9 83.7

aMean of 3 lateral roots. bMean of 6 lateral roots. CMean of 2 lateral roots.

Table IV. Non-nodulation in nod 139 is not systemic. Approach grafts were made on nod139 with either the supernodulation mu- tant nts382 or the wild-type Bragg. Seedlings were grafted 10 days after planting. The grafted plants were inoculated with B.

japonicum strain USDA110. Data are means of 8 plants ± S.D. (a) and (b) in each case designated the plant on which the measuements were made.

Graft Nodule no. Nodule dry per plant weight per

plant (mg)

nts382-nts382 283 ± 62 160 ± 77 Bragg-Bragg 83 ± 32 7t ± 24

Bragg-nod139 (a)Bragg 86 ± 32 88 ± 31 (b) nod139 0 0

nts382-nod139 (a)nts382 290 ± 99 157 ± 61 (b) nod139 0 0

nod139-nod139 0 0

39

Table V. Nitrogen, carbon and protein analysis of the seeds of Bragg, nod49, nod139, nod772, nts382 and rjl (Lee). Plants were cultured with 5 mM KNO3-supplemented plant nutrient solution administered three times a week. Each entry in the table

is the mean of three replications ± S.D. Seeds were obtained from plants grown in an abundant supply of nitrate.

Genotype '% Nitrogen '% Carbon % Protein a

Bragg 6.5 ± 0.1 51.9 ± 0.1 40.5 + 0.6 nts382 6.9 ± 0.1 51.2 ± 0.2 43.1 ± 0.6

nod49 5.5 + 0.3 51.1 ± 0.1 34.6 ± 2.0 nod772 6.5 ± 0.1 50.6 ± 0.0 41.0 ± 0.5 nod139 5.5 + 0.2 51.8 4- 0.1 34.8 4- 1.0 rj/(Lee) 6.2 ± 0.1 50.4 4- 0.3 38.6 4- 0.6

aEstimated from the percentage nitrogen column (conversion factor: % protein estimate = 6.28 × % N).

grafted with nod139 had 290 nodules per plant compared to 283 on the nts382 autograft. The nodule dry weight followed a similar pattern. This indicates that non-nodulation in nod139 is root controlled and non-systemic.

Mutations in nod49, nod772, nod139, rj: and nts382 are nodulation specific

Seeds of the soybean genotypes used in these ex- periments were obtained from plants grown on abundant nitrate but Bragg and particularly nts382 did develop some nodules. Under the assumption of a constant nitrogen to protein ratio, the nitrogen content of the seeds of the mutants were used to calculate an estimated protein con- tent (Table V). The results obtained indicate that genotypes Bragg, nts382, rjl (Lee) and nod;772 had similar nitrogen and carbon content whereas nod49 and nod 139 had a slightly lower content of nitrogen. The highest nitrogen content was measured in the seeds of the supernodulating mu- tant nts382. The percentage of carbon in all the genotypes was similar.

In the absence of nitrate, that is, when the plants were totally dependent on symbiotic nitrogen fixa- tion, the plant dry weight of all the mutants was essentially the same as the wild type (Fig. 1). However, the per cent plant nitrogen content in

8(X)

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nod772 n o d 1 3 9

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Fig. 1. Plant dry weight (PDW) of Bragg, nod49, nod772, nod139, rjl (Lee) and nts382. Plants were cultured with either 6 mM KNO 3- supplemented plant nutrient solution (+N) or in nitrogen free nutrient solution (-N). Bradyrhizobiumjaponicum strain USDA110 (108 viable cells per pot) was used as the in- oculant strain. Plants were harvested 4 weeks after planting. The data are means of five plants + S.D.

40

14 -

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Fig. 2. (a) Total plant nitrogen analysis and, (b) total pianl carbon analysis for nod49, nod772, nod139, rjl (Lee), Bragg and nts382. Plants were cultured with either 6 mM KNO3-supplemented plant nutrient solution (+N) or nitrogen free nutrient solution (-N). Data are means of five plants + S.D.

the n o n - n o d u l a t i n g m u t a n t s w a s l o w e r than in b o t h the n o d u l a t i n g w i l d - t y p e B r a g g a n d the

s u p e r n o d u l a t i o n m u t a n t n t s 3 8 2 (F ig . 2a). F i g u r e 2b s h o w s that the c a r b o n c o n t e n t o f all the g e n o t y p e s w a s s imi lar , t h u s i n d i c a t i n g the a b s e n c e o f an a n o m a l y in c a r b o n a s s i m i l a t i o n in the

m u t a n t s . W h e n the g e n o t y p e s were g r o w n on nitrate ,

there w a s a c o n s i d e r a b l e increase in the p lant dry w e i g h t (Fig . 1) a n d p e r c e n t a g e p lant n i t r o g e n in

all the l ines (F ig . 2a) . M o r e o v e r , the m u t a n t g e n o t y p e s s h o w e d n o d i f f e r e n c e s in p e r c e n t a g e

n i t rogen . T h i s w a s in c o n t r a s t to the p lant s d e p e n - d e n t o n n i t r o g e n f i x a t i o n , g r o w n w i t h o u t nitrate .

F u r t h e r m o r e , there w a s a l m o s t a 2 - f o l d increase in p lant dry m a t t e r in all the g e n o t y p e s , T h e s e p lant g r o w t h c o m p a r i s o n s a n d the a c c u m u l a t i o n o f n i t r o g e n a n d c a r b o n in b o t h the p r e s e n c e a n d

a b s e n c e o f n i tra te i n d i c a t e that the n o n - n o d u l a t i o n m u t a t i o n s are n o d u l a t i o n spec i f i c a n d that their in- ab i l i ty to n o d u l a t e is n o t d u e to l o w e r p lant

v igour .

Table VI. In vivo nitrate reductase activity of Bragg, nod49, nod772, nod139, rjl (Lee) and nts382. Plants were cultured either without nitrate (for constitutive NR assay) or with 3.0 mM KNO3-supplemented plant nutrient solution (for constitutive plus nitrate-induced NR). Leaf tissue was assayed either without nitrate or with 50 mM KNO 3 in the assay solution 10 days after planting. NR activity is expressed as nmol NO z- produced (leaf disc) -l h -1. Each entry in the table is the mean of 6 replications 4- S.D.

Genotype Constitutive Inducible plus constitutive NR NR activity b (nmol NO 2 activity a,° (leaf disc) 1 h-I

- N O 3- +NO 3- assay c assay d

Bragg 21.4 4- 3.6 14.7 4- 4.0 32.4 4- 0.8 nod49 13.6 4- 2.0 13.4 4- 2.0 28.2 4- 5.3 nod772 21.7 4- 4.6 15.3 4- 1.2 29.4 4- 1.2 nod139 17.7 4- 0.1 20.1 4- 1.1 28.7 4- 5.3 rjl (Lee) 15.4 4- 2.0 20.8 4- 9.8 21.1 4- 14.0 nts382 20.3 + 8.7 20.9 + 2.0 30.6 + 5.8

aplants were cultured in the absence of nitrate prior to the assay. bplants were cultured on nitrate prior to the assay, CThe NR assay contained 0 mM added nitrate. dThe NR assay contained 50 mM added nitrate.

The above conclusion was substantiated by analysis of a biochemical character, nitrate reduc- tase (NR) activity. In young soybean leaves, there are both constitutive and substrate inducible forms of NR designated as cNR and iNR, respectively [17]. Table VI indicates the in vivo NR activity of Bragg, nod49, nod772, rjl (Lee) and nts382 grown either without nitrate or with 3.0 mM KNO 3- supplemented plant nutrient solution. In the case of plants grown in the absence of nitrate, the cNR activities of Bragg and the mutants were similar. Lines nod49, nod139 and rjl (Lee) had a slightly lower NR activity. This may have been due to nitrogen stress in these mutants grown in the absence of added combined nitrogen. The NR ac- tivity of plants grown with nitrate was determined either without nitrate or with 50 mM KNO3 in the assay solution. It is clear from Table VI that all the mutants expressed iNR activity irrespective of the presence of nitrate in the assay. Bragg and the mutants had essentially the same activity. When 50 mM KNO3 was added to the assay solution, all the genotypes except rjl (in the genetic background of Lee) had similar NR activity.

Discussion

The results presented in this paper indicate that the non-nodulation phenotype in the non- nodulation mutants nod772 and nod139 is strictly controlled by the root and is determined exclusive- ly by the genotype of the rootstock. Previous studies have shown that the induced mutants nod49 and nod772 are allelic to rjl [1]. The non- nodulation response in these newly described non- nodulation mutants namely nod49 [9] and nod772 (Table II) as well as in the naturally-occurring rj~ mutant in the genetic background of the cultivar Lee is root controlled (data not shown). Further- more, a lesion(s) conditioning non-nodulation af- fects the rootstock in a localized manner, thus permitting normal nodulation on the laterals from the wild-type or supernodulation scions of the grafts. These results are in agreement with those of Clark [18] and Tanner and Anderson [8] for the naturally-occurring non-nodulating rj~ mutant.

Mutant nod139, on the other hand, is not allelic with rj~. Complementation tests conducted on this

41

mutant as well as the other non-nodulation mutants nod49, nod772 and rj) (Lee) indicate that nod139 is in a different complementation group from that of rjl [1]. These other non-nodulating mutants are characterized by the absence of curled root hairs and nod139 also has an erratic distribu- tion of root hairs on the root [20]. However, the results obtained from the present grafting studies show that non-nodulation in nod139 is also root controlled and localized in the root (Table I).

The supernodulation of mutant nts382 is con- trolled by the shoot since grafted nts382 shoots in- duced supernodulation on the wild-type rootstock. This mutant exhibited a normal autoregulatory response (a regulatory mechanism that controls the number of nodules on the plant root system) when grafted to the wild-type Bragg shoots, in- dicating that it is not altered in the root factors that affect autoregulation. On the other hand, non-nodulation is determined by the root and the lesion(s) conditioning non-nodulation only affects the root in a localized fashion. This effect is not translocated to the shoot in such a manner as to af- fect nodulation since if this was not the case, the nodulation of the laterals from the Bragg or nts382 scion would have been suppressed (Table 1II). The laterals arising from nts382 scion when grafted on- to nod49 and nod772 rootstocks showed the characteristic supernodulation pattern of nodula- tion wherein the nodules cover a large length of the root as indicated in the percentage of the root covered by nodules in Table III. The lateral roots arising from nts382 scion grafted onto nod49 rootstock were much younger and smaller (total root length of 11.3 cm) when compared to the laterals arising from the nts382 scions grafted onto nod772 rootstock (total root length of 21.5 cm) as indicated in Table III. This accounts for the dif- ferences in the nodule number per lateral root aris- ing from the nts382 scion grafted onto the nod49 or nod772 rootstocks. Like nod49 [9], nod139 and nod772 are able to autoregulate nodulation on the wild-type roots and the root expresses a mutation that prevents bacterial invasion and nodulation.

The induced non-nodulation mutants nod49, nod139 and nod772 along with the naturally- occurring non-nodulation mutation Ul in the genetic background of the cv. Lee are not altered

42

in the assimilation of nitrogen and carbon when grown with nitrate (Figs. 2a and 2b). Weber [19] obtained similar results for field tests of rjl plants, where increased applied nitrogen produced in- creased seed and dry matter yields as well as in- creased seed size and protein content. The supernodulation mutant nts382, which is a nitrate tolerant symbiosis mutant and nodulates profusely in the presence of nitrate [6,7] is also unaltered in the assimilation of nitrate. A similar conclusion was drawn by Carroll et al. [6,7] and Day et al. [21]. Nitrogen contents were only substantially lower in the non-nodulating mutants in the ab- sence of combined nitrogen (Fig. 2a); therefore, the anomaly in the non-nodulation mutants is clearly specific to the nodulation process. This was confirmed by assaying NR (an enzyme not directly related to nodulation) under a wide range of con- ditions. The non-nodulating mutants expressed cNR and iNR activity similar to the wild-type Bragg (Table VI). In a more general context, in- creases in nitrogen content in Bragg and nts382 in the presence of nitrate indicate that seed reserves and symbiotic nitrogen fixation (that is dependent on nodule formation) in these genotypes could not provide all of the nitrogen required by these plants, thus emphasizing the importance of a com- bined source of nitrogen during early development [22].

Acknowledgements

This research was supported by Agrigenetics Research Associates and a postgraduate scholar- ship to Anne Mathews by The Australian National University. The authors thank Geoffrey Thomas for editorial comments.

References

1 A. Mathews, B.J. Carroll and P.M. Gresshoff, A new non-nodulat ion gene in soybean. J. Hered., 80 (1989) 357-360.

2 L.F. Williams and D.L. Lynch, Inheritance of a non- nodulat ing character in the soybean. Agron. J., 46 (19541 28-29.

3 B.E. Caldwell, Inheritance of a strain-specific ineffective nodulation in soybean. Crop Sci., 6 (1966) 427-428.

4 G. Vest, Rj3 -A gene conditioning ineffective nodulation in soybean. Crop Sci., 10 (1970) 34-35.

5 G. Vest and B.E. Caldwetl, Rj4 - A gene conditioning in- effective nodulation ir~ soybean. Crop Sci., 12 ~19721 692-693.

6 B.J. Carroll, D.L. McNeil and P.M. Gresshoff, A super- nodulation and nitrate tolerant symbiotic (nts) soybean mutant. Plant Physiol., 78 (1985) 34-40.

7 B.J. Carroll, D L . McNeil and P.M. Gresshoff, Isolation and properties of soybean (Glvcine max (L.) Merr.) mutants that nodula~e in the presence of high nitrate con- centrations. P~,,~ >~,tl. Acad. Sci. ITS;.,\., 82 (19851 4162-4166

8 J.W. Tanue+ and L~,. ,\nder~;on +~vestigatit~qs o~ mm- nodulating ar,,<i n~>dui~!!mg sr~.~ca~ strain>, ( 'an. ,I. Plant Sei., 43 (19620 5.{2;*. 54(,

9 A.C. Delves, A. Mathews, D.,,\ l)a3,, A.S. Ca~tcr. t ~, : Carroll and P.M. Gresshoff, Regulation of ihe soybean-Rh:zohium '~ymbiosis by shoot and r,~ot factors Plant Physiol., 82 (1986) 588-590.

10 B.J. Carroll, D.L. McNeil and P.M. Gresshoff, Mutagenesis of soybean (G/ycine ma.x ( I,. ) Merr.) and Ihe isolation of non-nodulat ing mutants. Pkmt Sci., 47 (1986) 109-114.

11 A. Mathews, Host involvement in nodule initiation in the soybean-Bro~/vrh;zohium symbiosis. Ph.D. l)issertation, Australian National University, (1987),

12 D.F. Herridge, Carbon and nitrogen nutrition of two an- nual legumes. P h D . Dissertation, University of Western Australia, Perth, Australia ( 1977/.

13 B..I. Carroll and P.M. Gresshoff, Isolation and initial characterizatum ol constitutive nitrate reductase-deficient mutants NR328 and NR345 of soybean {GIt'ciHe max). Plant Physi~1., 81 (19861 572-$76.

14 J.E. Harper, ( 'onlr ibutions of dinitrogen and soil or fer- tilizer nitrogen to soybean (Glvcine max (L.) Merr.) pro- duction, in: L,D. Hill, (Ed,), World Soybean Research, Interstate Printers and Publishers, Danville, Illinois, 1976, pp. 1(11-107.

15 P. Robin, 1,. Slreit, W.H. Campbell and J.E. Harper, lm-

munochemical characterization of nitrate reductase l\~rms from wild-type (cv. Williams) and nr 1 mutant soybean. Plant Physiol., 77 (1985) 232-236.

16 N. Alvey, C. Banfield, R. Baxter, J. Gower, W. Krzanowski, P. Lane, P. Leech, J. Nelder, R. Payne, K. Phelps, C. Rogers, G. Ross, H. Simpson, A. Tood, R. Wedderburn and G. Wilkinson, G E N S T A T - A general statistical program, in: Lawes Agricultural Trust,

Rothamsted Experimental Station, 1977. 17 J.E. Harper, R,S. Nelson and L. Streit, Nitrate

metabolism of soybean-physiology and genetics, in: R.M. Shibles (Ed.I, World Soybean Research Conference. 111

Proceedings, Westview Press, Botdder, Colorado, 1985, pp. 476.

18 F.E. Clark, Nodulation response of two near isogenic lines of the soybean. Can. J. Microbiol., 3 {19571 113-123.

19 C.R. Weber, Nodulating and non-nodulat ing soybean isolines: 1. Agronomic and chemical attributes. Agron J., 58 (1966) 43-.46.

20 A. Mathews, B.J. Carroll and P.M. Gresshoff, Characterization of non-nodulation mutants of soybean (Glycine max (L.) Merr.): Bradyrhicobium effects and the absence of root hair curling. J. Plant Physiol., 131 (1987) 349-361.

21 D.A. Day, H. Lambers, J. Bateman, B.J. Carroll and P.M.

43

22

Gresshoff, Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild-type parent. Physiol. Plant., 68 (1986) 375-382. J.E. Harper, Soil and symbiotic nitrogen requirements for optimum soybean production. Crop Sci., 14 (1974) 255-260.