Suppression of the Symbiotic Supernodulation Symptoms of Soybean

J.PlantPhysiol. Vol. 132.pp. 417-423(1988)

Suppression of the Symbiotic Supernodulation Symptoms of Soybean1,2 PETER M. GRESSHOFF\ ARNO KROTZKY\ ANNE MATHEWS, DAVID A. DAY, KATHRYN A. SCHULLER, JANE OLSSON, ANGELA

C.

DELVES,

and

BERNARD

J. CARROLL

Botany Department, Australian National University, Canberra ACT 2600, Australia * New Address: Plant Molecular Genetics (OHLD), College of Agriculture, University of Tennessee, Knoxville TN37901, USA Received June 3,1987' Accepted July 9,1987

Summary Supernodulation mutants of soybean (Glycine max L. Merr.) produce very high numbers of nodules and increased nodule mass compared to the parent cultivar Bragg in the absence or presence of nitrate. All (12 were tested) mutants also display a nitrate tolerant symbiotic (nts) as well as a supernodulation phenotype suggesting that nitrate inhibition of nodulation and endogenous autoregulation are at least in part jointly controlled by plant genes. Genetic analysis suggests that single recessive mendelian alleles at a single locus are involved. Supernodulation of nts mutants was suppressed by a variety of means. These include (a) suppression by low inoculum, (b) suppression by grafting, (c) suppression by another gene (epistatic), and (d) suppression by wild type vascular sap or methanol extract refeeding. These methods show that (i) nitrate tolerance of nodulation can be expressed even if supernodulation phenotype is not expressed, (ii) non-nodulation mutants epistatically suppress supernodulation, (iii) shoots of wild-type or non-nodulation mutant soybeans, or Glycine soja Sieb. and Zucc., suppress supernodulation and (iv) methanol extracts from inoculated wild type plants (but not uninoculated wild type or mutant nts382 plants) suppress supernodulation by 60 to 80 %. We are presently using these tools and the relevant material to investigate further the genetic basis of autoregulation of nodulation in legumes.

Key words: Nitrogen fixation, Glycine max, host genetics, nodulation, Bradyrhizobium. Introduction The root nodule symbiosis between Rhizobium or Brady· rhizobium and legumes has immense agronomic and academic importance. Agronomically this is caused by the ability of legumes to contribute to the nitrogen balance of a crop and the ecosystem as a whole through the process of biological nitrogen fixation. Legumes are important as forage, oil and grain seeds and forest species (Gress hoff and Rao, 1987). Legumes can introduce nitrogen into the plant product without the reliance on nitrate derived nitrogen. Such Dedicated to the 75th birthday of Professor William Hayes. Paper presented at a workshop in Amalfi (April 1987) supported by the «OECD Co-operative Research Project on Food Production and Preservation». 1

2

© 1988 by Gustav Fischer Verlag, Stuttgart

moves away from a high nitrate utilising agriculture (as is presently being used in most developed countries) must be seen as a benefit from the ecological as well as economic perspective. Legumes just like other plants can be seen as a producer of renewable industrial resources, not only as the provider of food stuffs. The genetic analysis of the symbiosis has focussed on the bacterial side, understandably, as the prokaryote provides a better experimental system. Over the last few years, however, it has become increasingly evident that a large number of symbiotic functions are under the direct control of the plant. For example, the plant secretes the flavone/isoflavone «cocktaih>, the plant controls the site of infection and the number of developing nodules, and the amount of substrates (oxygen and carbohydrates) that the engulfed bacterium receives. Finally the plant, through its own senescence

418

P. M. GRESSHOFF, A. KROTZKY, A. MATHEWS, D. A. DAY, K. A. SCHULLER,]. OLSSON, A. C. DELVES, and B. ]. CARROLL

mechanism, can terminate the symbiosis (see Rolfe and Gresshoff, 1988 for review). In plant analysis several approaches have been used . One focused on the molecular identification of macromolecular difference (nodulins etc.). Another looked at functional differences through the isolation of genetic diversity. At present a relatively large number of plant mutants (mainly in Pisum sativum and Glycine max) affecting the nodulation phenotypes have been isolated and partially characterized. This report continues the analysis of supernodulation, nitrate tolerant symbiotic (nts) mutants of soybean (Glycine max 1. Merr.), which have the ability to form large numbers of nodules. The report emphasizes our ability to suppress the supernodulation phenotype by a number of techniques. The comparison of plants which show a normal phenotype, with plants being suppressed, allows the development of hypotheses to explain the genetics and biochemistry of the observed mutation.

Materials and Methods Plant material: all experiments deal with soybean Glycine max cultivar Bragg and mutants derived therefrom. The isolation and characterization of the supernodulation (nts) and non-nodulation (nod-) mutants is described (Carroll et aI., 1985a,b,c, 1986, 1988; Gresshoff and Delves, 1986; Day et al., 1986, 1987; Delves et aI., 1986, 1987 a, b, 1988; Gresshoff et aI., 1987; Mathews et al., 1987 a, b, 1988). Plants were cultured in a variety of ways, either in pots or Leonard jars. Growth media were sand: vermiculite mixtures irrigated with diluted Herridge's nutrient solution (d. Delves et al., 1986) which contained differing amounts of KN0 3 • Bradyrhizobium japonicum strain USDAllO or CB1809 were used as inoculant where required. Plants were harvested and scored over a range of maturities although most experiments were terminated 4 - 8 weeks after sowing. Biochemical and genetical procedures and assay conditions are described in the above references and, where essential, will be referred to in detail below.

Results and Discussion The following is a synthesis of a large number of observations dealing with the common concept of suppression of the supernodulation phenotype. «Suppression» has different biological and genetical connotations. The phenomenon in any case serves to illustrate whether certain associated phenotypic characteristics are a direct consequence of the final ontogenetic expression of a particular developmental gene (phenotype) or whether they are a direct consequence of the primary alteration in that gene. Fig. 1 presents a scheme setting out the possible interactions in phenotype. Basically what is required is to ascertain whether mode A or B is effective and the relative contribution of both. Several «side-effects» associated with supernodulation were noted in the nts mutants. Some of these were segregating mutations and now we have seed stocks free from these conditions. For example, dwarfism (in ntsl007), male sterility (in nts183) and leaf shape mutations segregated out in

MUTATED GENE

"

.

ALTERED SECONDARY PRODUCT i.e. metabolite

....

ALTERED PRIMARY PRODUCT i.e. enzyme

n/ SECONDARY MUTANT PHENOTYPES or PLEIOTROPIC CHANGES

i.e.

shortened root

... ~

•

'r PRIMARY MUTANT PHENOTYPE i.e.

supernodulation

B Fig. 1: Altered DNA sequences may affect the actIVity of biosynthetic or regulatory polypeptides, which in turn may affect the timing, tissue location or level of certain metabolites. Such alterations at the biochemical level may have a direct phenotypic effect, such as lack of autoregulation and associated supernodulation. The same metabolic alteration may have pleiotropic effects, which are seen as secondary phenotypes. Alternatively the mutant phenotype may cause physiological changes, which result in the associated effects and cause pleiotropic changes. Double arrows between the phenotype boxes indicate the reciprocal possibilities of interaction at different levels of penetrance.

some nts and nod lines (A. Mathews, unpubl. data; B. ]. Carroll, unpubl. data). These were most likely the result of initial mutagenic damage, as the EMS mutagenesis regime was chosen because of its high mutagenic frequency (Carroll et aI., 1986). However, there were other phenotypes, which were not easily explained, as they seemed to be connected to the originally selected nodulation phenotype. For example, mutant nts382 nodules showed an altered morphology as well as a lowered specific nitrogenase activity (Carroll et aI., 1985 b; Day et al., 1987). Mutant nts382 also has reduced root growth and an altered shoot: root ratio compared to parent cultivar Bragg (Day et al., 1986) (see Fig. 2), and it has altered elemental composition in the leaves (R. Boerma, pers. comm.). Were these changes the result(s) of other, yet undefined, mutations in the nts382 background or were they the direct effect of the supernodulation phenotype? Or were they the result of the change in the physiology of the plant, which occurred because of the physiological alterations caused by the expression of the mutant allele? The first pairing of phenotypes of interest was that of nitrate-tolerant nodulation and supernodulation. Some researchers expressed the opinion that these are caused by two separate mutations. We found that only one locus is involved, because of mendelian segregation in the F2 in backcrosses with wild-type soybean cultivars Clark and Williams. Furthermore the F3 progeny of F2 nts plants showed 100 %

Suppression of supernodulation

419

L Suppression by low inoculum dose

Fig. 2: Root system of a supernodulating soybean. - Mutant nts382 was inoculated with B. japonicum strain USDA110 at a high inoculant level (about 109 cells per plant) and grown in a sand: vermiculite (2: 1) mixture for 4 weeks. Watering was with 0.5 mM KN0 3 in Herridge's nutrient solution (quotation in Delves et al., 1986). Root systems of up to 4000 nodules have been recorded (D. Day, unpublished data).

stability of the phenotype (Carroll et ai., 1988; Delves et ai., 1988). Secondly, we characterised twelve independent mutants for nitrate tolerance. All showed the supernodulation phenotype as well (B. Carroll data, in Gresshoff and Delves, 1986), suggesting that in all cases the unselected phenotype was co-inherited.

Day et ai. (1986) reported growth analyses of cultivar Bragg and mutant nts382 grown either symbiotically or in the presence of nitrate (but uninoculated). Analyses proceeded for 50 days. It was clear that supernodulated plants had smaller shoots and roots (see Table 1). In contrast, when plants were grown on nitrate (asymbiotically), shoot growth of the parent and the mutant were identical, while root growth still lagged behind. This suggests that nts382 has a reduced root system only in part due to excessive nodulation. The mutant also had higher numbers of lateral roots. Thus it is likely that either another root-affecting mutation (presumably closely linked to the nts382 locus) or a secondary mutant phenotype effect (mode A, Fig. I) are the cause. We are presently analysing backcross material for these growth parameters. Similar morphological changes are associated with supernodulation in Pisum sativum mutants described by E. Jacobsen (see Postma et al., this volume). One also has to consider that all possible mechanisms are operative and that no clear single causative component can be isolated quickly. Day et al. (1987) noted that the lower specific nitrogenase activity found in nts382 nodules (as compared to Bragg nodules) grown symbiotically (no nitrate) was mainly caused by the reduced amount of symbiotic tissue per nodule. In general they noted that the nts382 nodule was seemingly «arrested» at an early stage of nodule development, with smaller nodule cells (lack of hypertrophy), fewer bacteroids per peribacteroid membrane vesicle and lowered haem content per nodule. The question arose as to whether those phenomena are the consequence of supernodulation or the direct effect of the mutated gene. Day et al. (1987) and Schuller (1986) used a low inoculum treatment to answer that question (see Table2). When nts382 plants were inoculated with 103 viable bacteria per pot (instead of the normal 109 cells per pot), nodule number was reduced to a near wild-type level. Such pseudo-wild-type plants «reverted» to wild-type not only in their nodule number and mass, but also mimicked wild-type for biochemical parameters such as haem content, specific nitrogenase activity and sensitivity to short term nitrate shock. In other words, the observed pleiotropic effects were a direct consequence of supernodulation, and not of the mutated gene, per se. Most interestingly, when high or low inoculum Bragg plants were grown continuously on nitrate sufficient to inhibit nodule formation, similar degrees of nodulation inhibition were observed; low inoculum nts382 plants (pseudo wild-types) on the other hand showed a similar tolerance to

Table 1: Growth analysis of Bragg and nts382"). Genotype

Bragg Bragg nts382 nts382

Inoculation/nitrate treatment +USDAllO unmoc. +USDAllO umnoc.

(0 mM) (SmM) (0 mM) (S mM)

Root and nodule weight (g plant -1) 2SDAP SODAP

Shoot weight (g plant-I) 2SDAP SODAP

Shoot/Root ratio SO DAP

0.13 (0.01) 0.18 (O.OOS) 0.16 (0.01) 0.14 (0.01)

0.29 (0.02) 0.61 (0.01) 0.27 (0.02) 0.S4 (0.01)

2.52 3.2S 2.23 4.SS

0.92 (0.07) 1.80 (0.12) 0.47 (O.OS) 1.24 (0.04)

*) Data were extracted from Day et al. (1986). Data in parentheses are standard errors.

2.32 (0.10) S.86 (0.17) LOS (0.11) S.6S (0.12)

420

P. M. GRESSHOFF, A. KROTZKY, A. MATHEWS, D. A. DAY, K. A. SCHULLER, J. OLSSON, A. C. DELVES, and B. J. CARROLL

Table 2: Pseudo-wild-type nodulation I) caused by low inoculum level. Specific nitrogenase activity Control 2d N0 3 shocP) Nodule mass Nodule number /-tmoles hr - 1 • /-tmoles hr - 1 • g. plant- I plant- I gNFW gNFW 9 Bragg 10 13.5±2.5 3.3±0.3 1.07±0.17 107± 17 Bragg 103 13.9±2.9 0.93±0.29 4.3±1.0 46± 19 nts382 109 4.4±1.2 2.9±0.7 1.91±0.55 608±302 nts382 103 8.9±0.8 3.0±0.8 0.97±0.22 99± 8 I) Data are amalgamated from Day et al. (1987) and Schuller (1986). 2) Plants were exposed for 2 days to 10 mM KN0 3 and nitrogenase activity was determined by acetylene reduction using 20 min incubation of nodulated root systems. Genotype

Inoculum dose

nitrate as did high inoculum, supernodulated, nts382 plants (Day, unpublished data). We noted, however, that the nodulation pattern of low-inoculant nts382 (scattered) was not similar to that of low-inoculant Bragg (crown nodulation). Also the degree of nodulation may be different if nodule number is expressed per plant weight, as nts382 plants were slightly smaller. Likewise the low nodule number of nts382 may still be the «early form» of supernodulation, which was delayed because of low Bradyrhizobium numbers. This shows that the ability to display nitrate tolerant nodulation is independent of the phenotypic expression of supernodulation, suggesting that primary and secondary gene products per se affected by the nts382 gene can directly lead to nitrate tolerance.

II Suppression by grafting Nine independent supernodulation and one hypernodulation (ntsl116) mutants have been tested by reciprocal grafting for an evaluation of the site of regulation of supernodulation (Delves et aI., 1986, 1987 a, b). In all cases the genotype of the shoot controlled the phenotype of the root (i.e. supernodulation was shoot controlled). In parallel experiments two independent non-nodulation genes (characterised by nod49 and nod139) acted entirely through the root (Mathews, 1987; Mathews et aI., 1987 b, 1988). In reciprocal grafts with the non-nodulation mutants and wild-type plants, it is always the genotype of the root which governs the phenotype of the root. Thus supernodulation can be suppressed by the grafting of a wild-type Bragg or nod49/ nod139 shoot onto an nts root. In all cases, wild-type nodulation is observed (Delves et aI., 1986; Mathews, 1987). This phenomenon can even be extended to other species such as Glycine soja (Delves et aI., 1987 a). Similar tissue interactions as described here were observed in pea supernodulation and non-nodulation mutants (see Postma et aI., 1988; this volume), although the nod3 mutation of pea seems to act through the root in contrast to the soybean mutants. An interesting side question relates to root size. It is generally observed that reduced root size of the supernodulation mutant, is also shoot controlled. Another form of suppression occurs when an nts shoot is grafted onto a nod - root and a non-nodulation phenotype is produced (Delves et aI., 1986). In the chimaeric plant the supernodulation status cannot be expressed because of an earlier mutational block in the root dealing with infection and nodule establishment (see

Mathews et aI., 1987 a, b, 1988; Mathews, 1987 for a detailed description of the nod- mutants). Interestingly, root size is reduced in such chimaeric plants confirming earlier suggestions (Day et aI., 1986) (see above) that a non-nodulated nts382 plant (now even if in a chimaeric configuration) controls the formation of a reduced root system. Thus, the site of control for supernodulation, increased lateral root number and reduced root growth are common, pointing towards a common causality of the three phenomena.

III Epistatic suppression Mutant nod49 and nod139 are two separate non-nodulation mutants of cultivar Bragg (Mathews et aI., 1988). They are single, mendelian recessives and most likely are linked (statistically the data fit a segregation ratio of 1: 1 rather than a 9: 7 ratio) (Mathews 1987; Mathews et aI., 1988). Following the promising results from the grafting work, crosses were carried out to select a double mutant between nts382 and nod49 or nod139 (Mathews, 1987). Results are identical for reciprocal crosses with either nod - parent. Crosses between mutants nts382 and nod49 (or nod139) gave wild-type progeny in the Flo Upon selfing, the F2 segregated in a ratio of 9 wild-type : 3 nts mutants : 3 non-nodulation mutants: 1 double mutant. Thus the two loci are unlinked and behave as recessives. The double mutant category fell into the non-nodulation category giving a final phenotypic ratio of 9: 3 : 4, providing an example of epistatic suppression. F2 nts plants (homozygous recessive for the nts allele) were grown to maturity and selfed F3 progeny was screened. Non-nodulated plants in the F3 were detected (presumably because of their double mutant genotype). The presence of a homozygous recessive nts condition was verified by using grafts onto Bragg roots (Mathews, 1987), under which condition supernodulated plants were detected. Double mutants in the F3 (ex nts lineage) had a somewhat (10-20%) decreased root mass. Growth analysis of the double mutant suggests that reduced root growth is partially attributable to an inherent function of the nts locus. The composite of all these findings provides evidence which suggests that the primary (or secondary) gene products of the nts382 gene result in (a) supernodulation (lack of autoregulation), (b) nitrate tolerance to nodulation and (c) reduced root growth. As nitrate inhibition of nodulation appears to be non-systemic (Hinson, 1975; Carroll and Gresshoff, 1983), plant

421

Suppression of supernodulation

tissues locally exposed to nitrate are «potentiated» to accentuate the autoregulation signal normally found in wildtype legumes. The nts mutants, by virtue of the lack (or decrease) of this signal, cannot «nitrate-regulate» effectively. Likewise the above findings imply that reduced root growth may be a direct consequence of the lack of the shoot signal in nts382 plants. Growth analysis (Krotzky, unpubl. results) demonstrated that inoculation of Bragg plants caused a temporary decrease of shoot gtowth as indicated by leaf expansion, internodal lengths and shoot weights. This response did not occur in nts382. In parallel with these growth phenomena we noted that mevalonic acid incorporation profiles are altered in inoculated Bragg (on nitrate) as compared to un inoculated Bragg (on nitrate) plants. Epicotyls of nitrate (5.5mM) grown plants (16 days after planting and inoculation) were thrice injected with 14C-Iabelled mevalonic acid once per day (see Fig. 3). Extraction occurred after 3 days and partitioned fractions were separated by HPLC prior to scintillation counting. The uninoculated nts382 (on nitrate) incorporation profile is very similar to that of uninoculated Bragg (grown on nitrate) plants, showing a similar distribution of

gibberellic acid pathway intermediates and other, as yet unknown, metabolites. However, the nts382 profile is maintained upon inoculation resulting in distinct differences in the potential phytohormone profile of inoculated Bragg and nts382 plants (Fig. 3). IV. Suppression by plant extracts

The recognition of shoot control of supernodulation led to a range of experiments designed to ascertain whether the Table 3: Plant extract effect on nts382 nodulation. Extract source Nodule number plant - I Nodulation pattern supernodulation control (water injected) 391 nts382 (inoculated) 380 supernodulation nts382 (uninoculated) 371 supernodulation Bragg (inoculated) 106 delayed nodulation Bragg (uninoculated) 373 supernodulation Plants were inoculated with strain USDAll0 at the time of sowing. Extract source plants were grown in parallel with rhizotrons in sand/vermiculite (2: 1) mix. Nodules were counted on 16 replicate plants after 23-25 days.

(a)

(e)

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a:

100

,

A

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30

40

50

60

20

10

Fraction no.

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,

400

Bragg

E

+inoc

,

,

K

F

,

F

,

?:

.S; :;:;

-inoc

,

B

~

F

nt5382

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ECo

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30 Fraction no.

(d) K

,

50

40

E

60

nt5382 +inoc

~

?:

:~

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L

OJ 0

'0

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a:

Fraction no.

Fraction no.

Fig. 3: HPLC elution profiles of leaf extracts after 14C-mevalonic acid injections in soybean stems. - Mevalonic acid was injected for three successive days into soybean plants of 16 to 18 days of age after sowing and inoculation with B. japonicum USDAll0 (where required). HPLC eluate was fraction-collected and evaluated by liquid scintillation counting. All source plants were irrigated with 5.5 mM KN0 3 to facilitate similar growth rates and physiological conditions. This level of nitrate limits, but not restricts, nodulation in cultivar Bragg. Uninoculated plants, as expected, were non-nodulated and inoculated plants showed nodules. Panel A: Bragg, uninoculated; panel B: Bragg, inoculated; panel C: nts382, uninoculated; panel D: nts382, inoculated. Peak M: mevalonic acid; peak D: possibly GA4 ; peak lIC: possibly G~4/ 19. Further peak characterisation is not completed. Fraction B is suppressive of nodulation, if injected into nts382.

422

P. M. GRESSHOFF, A. KROTZKY, A. MATHEWS, D. A. DAY, K. A. SCHULLER, J. OLSSON, A. C. DELVES, and B. J. CARROLL

«signal» is an absence of an inhibitor or the presence of an activator in the mutant. Numerous experimental designs were tried and in general these pointed towards the presence of an inhibitor in the wild-type plants (Olsson, 1988). Gresshoff and Delves (1986) proposed a model to explain the temporal aspects of the establishment of an autoregulation circuit, which is based on the assumption of an inhibitor of nodulation being induced after an initial round of infection and nodule initiation has occurred in the wild type. Direct evidence for an inhibitor came from work by Krotzky et al. (unpublished) who used vacuum extracted shoot sap preparations from plants grown either with or without Bradyrhizobium inoculation and injected these into nts382 and Bragg test plants. The experimental procedure was simplified by the inclusion of a methanol extraction step, which replaced the vacuum extraction and allowed the analysis of individual plant parts such as leaves, stems and cotyledons. Table3 gives data from an experiment using methanol extracts from leaves. Methanol extracts (80 %) did not contain nitrate or nitrite, were rotary-evaporated for concentration and pH 3 vs. pH 8 extracted into ethylacetate. Injections occurred over a 20 day period with up to 10 to 20 Itl extract injected every second day. Volumes were adjusted to feed about one plant equivalent per injection per plant. Plant growth was significantly affected only with extracts from inoculated Bragg injected into nts382. However, the differences in nodule number per plant weight were still highly significant. Reciprocal injections into Bragg test plants gave no significant alteration of nodule number (being around 20 in all cases, as plants were grown in the presence of nitrate). Further expansion of this protocol to use size-fractionated plant extracts, extracts from other species, reconstruction experiments with verified compounds under plus and minus nitrate growth conditions are planned or in progress. Preliminary results suggest that the inhibitor is found in the aqueous fraction. The significance of the findings stated in Table 3 is that a factor can be extracted from nodulated wild type shoots which is absent (or reduced) in uninoculated wild-type or nts382 shoots. This factor may result in the observed autoregulation in wild-type soybean but it may also cause the growth related phenomena (root and nodule morphology) and changes in mevalonic acid incorporation. Acknowledgements Agrigenetics Research Associates, the Australian National University, CSIRO (Canberra), The Australian Government (through DITEC, ARGS and Rural Credits Scheme) and the CPRA scheme are thanked for support in this research. We thank our technical staff and research students as well as other colleagues for perpetual help. Joanne Perks and Tessa Raath are thanked for help in preparation of the manuscript.

References CARROLL, B. J. and P. M. GRESSHOFF: Nitrate inhibition of nodulation and nitrogen fixation in white clover. Z. Pflanzenphysiol. 110,77 -88 (1983). CARROLL, B. J., D. L. McNEIL, and P. M. GRESSHOFF: A supernodulation and nitrate tolerant symbiotic (nts) soybean mutant. Plant Physiol. 78, 34-40 (1985 a).

- - Isolation and properties of soybean [Glycine max (L.) Merr.] mutants that nodulate in the presence of high nitrate concentrations. Proc. Nat. Acad. Sci. (USA) 82, 4162-4166 (1985 b). CARROLL, B. J., D. L. McNEIL, D. WHITMORE-SMITH, and P. M. GRESSHOFF: Host genetics and physiological studies of nitrate inhibition of nodulation and nitrogen fixation in soybean. In: Nitrogen Fixation Research Progress. EVANS, H. J., P. BOTTOMLEY, and W. NEWTON (eds.). M. Nijhoff Publ. Dordrecht, The Netherlands p. 39 (1985 c). CARROLL, B. J., D. L. McNEIL, and P. M. GRESSHOFF: Mutagenesis of soybean [Glycine max (L.) Merr.] and the isolation of non-nodulating mutants. Plant Science 47, 109-114 (1986). CARROLL, B. J., P. M. GRESSHOFF, and A. C. DELVES: Inheritance of supernodulation in soybean and estimation of the genetically effective cell number. Theor. Appl. Genet. (submitted) (1988). DAY, D. A., H. LAMBERS, J. BATEMAN, B. J. CARROLL, and P. M. GRESSHOFF: Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild-type parent. Physiol. Plant. 68, 375-382 (1986). DAY, D. A., G. D. PRICE, K. A. SCHULLER, and P. M. GRESSHOFF: Nodule physiology of a supernodulating soybean (Glycine max) mutant. Aust. J. Plant Physiol. 14, 527 - 538 (1987). DELVES, A. c., A. MATHEWS, D. A. DAY, A. S. CARTER, B. J. CARROLL, and P. M. GRESSHOFF: Regulation of the soybean-Rhizobium symbiosis by shoot and root factors. Plant Physiol. 82, 588-590 (1986). DELVES, A. c., A. HIGGINS, and P. M. GRESSHOFF: Supernodulation in interspecific grafIs between Glycine max (soybean) and Glycine soja. J. Plant Physio!. 128, 473 - 478 (1987 a). - - - A common shoot control of supernodulation in a number of mutant soybeans Glycine max (L.) Merr. Aust. J. Plant Physio!. 14, 689-694 (1987b). DELVES, A. c., B. J. CARROLL, and P. M. GRESSHOFF: Genetic analysis and complementation studies on a number of mutant supernodulating soybean lines. Journal of Heredity (in press) (1988). GRESSHOFF, P. M. and A. C. DELVES: Plant genetic approaches to symbiotic nodulation and nitrogen fixation in legumes. In: Plant Gene Research III. A genetical approach to plant biochemistry, BLONSTEIN, A. D. and P. J. KING (eds.). pp. 159-206. Springer Verlag, Wien (1986). GRESSHOFF, P. M., J. E. OLSSON, D. A. DAY, K. A. SCHULLER, A. MATHEWS, A. C. DELVES, A. J. KROTZKY, G. D. PRICE, and B. J. CARROLL: Plant host genetics of nodulation initiation in soybean. In: Molecular Genetics of Plant-Microbe Interactions, VERMA, D. P. S. and N. BRISSON (eds.). pp. 85-90. M.Ninhoff, Dordrecht, Pub!. (1987). GRESSHOFF, P. M. and A. N. RAo: Symbiotic nitrogen fixation, genetic engineering and food production. Proc. Asean Science Congress COSTED, Madras, India (MOHAN RAM, H. J. and A. N. RAo eds.) (in press) (1987). HINSON, K.: Nodulation response from nitrate applied to soybean half-root systems. Agron. Journal 67, 799-804 (1975). MATHEWS, A.: The host contribution to nodule initiation in the Bradyrhizobium-soybean symbiosis. Ph. D. Thesis, Botany Department, Australian National University, Canberra (1987). MATHEWS, A., B. J. CARROLL, and P. M. GRESSHOFF: Characterization of non-nodulation mutants of soybean [Glycine max (L.) Merr.]: Bradyrhizobium effects and absence of root hair curling. J. Plant Physio!. 131, 349-361 (1987 a). - - - Non-nodulation mutants of soybean. In: Molecular Genetics of Plant-Microbe Interactions, VERMA, D. P. S. and N. BRISSON (eds.). pp. 94 - 95. M. Nijhoff, Pub!., Dordrecht (1987 b). - - - A new recessive gene conditioning non-nodulation in soybean. Genetics (submitted) (1988). OLSSON, J. E.: Genetic control of soybean (Glycine max) nodule autoregulation. Ph. D. Thesis, Botany Department, Australian National University, Canberra (1988). -

Suppression of supernodulation POSTMA, J. G., E. JACOBSEN, and W. J. FEENSTRA: Three pea mutants with an altered nodulation studied by genetic analysis and grafting. J. Plant Physiol. (in press) (1988). ROLFE, B. G. and P. M. GRESSHOFF: Genetic analysis of legume nodule initiation. Ann. Rev. Plant Physiol. Plant Mol. BioI. 39, in press (1988).

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SCHULLER, K. A.: Nitrogen Fixation and Nitrate Assimilation in Soybean. Ph. D. Thesis, Botany Department, Australian National University, Canberra (1986). SCHULLER, K. A., D. A. DAY, A. H. GIBSON, and P. M. GRESSHOFF: Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate. Plant Physiol. 80, 646 - 650 (1986).