Anne Mathews

Development of bradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (glycine-max [L] merrill)

SummaryThe early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj1) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj1) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.

Characterization of Non-Nodulation Mutants of Soybean [Glycine max (L.) Merr]: Bradyrhizobium Effects and Absence of Root Hair Curling

Summary The characterization of the non-nodulation mutants nod49, nodl39 and nod772, isolated from mutagenized soybean cv. Bragg seeds, as well as the naturally occurring rj 1 mutant is described. Mutant nod49 has been characterized in greatest detail. Mutants nod49 and nodl39 lack curled root hairs when inoculated with Bradyrhizobium japonicum USDA110 at medium (10 7 –10 8 viable cells/ml) cell numbers. Mutant nod772 shows occasional hair curling. All mutants occasionally formed nodules; nodulation was more frequent at higher concentrations of inoculant. Such nodules are normal in morphology and nitrogenase activity. Bacteria isolated from nodules on nod49 retain the nodulation properties of the original inoculant and do not suppress the non-nodulation phenotype. Field grown nod49 and nodl39 plants are non-nodulated. Co-culture of nod49 or nodl39 with Bragg plants indicate that the non-nodulation mutants like rj 1 do not alter the phenotype of the parent cv. in a positive or negative way. Thus there is no evidence at this stage that the mutants are deficient in a nodulation related root exudate signal. The results indicate that these mutants are blocked in a very early stage of nodulation. The stage of blockage as well as attempts to circumvent non-nodulation are discussed.

Suppression of the Symbiotic Supernodulation Symptoms of Soybean

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 (1) 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.

Plant Host Genetics of Nodulation and Symbiotic Nitrogen Fixation in Pea and Soybean

The need to study the plant functions controlling nodulation and symbiotic nitrogen fixation has received progressive recognition and attention. Comprehensive reviews of recent advances can be found in LaRue et al (1985) as well as Miflin and Cullimore (1984) and Verma and Nadler (1984).

The genetic interaction between non-nodulation and supernodulation in soybean: an example of developmental epistasis

SummaryThe interaction between three non-nodulation mutants (nod49, nod772 and nod139) and a supernodulation mutant (nts382) of soybean was studied by analysing the progeny from crosses between these mutants. Previously it had been shown that the non-nodulation mutants arose from single mutation events and that nod49 and nod772 are allelic, whereas nod139 represents another gene required for nodulation. Analysis of progeny from crosses between nts382 and the wild type showed that this mutant also arose from a single mutation. Complementation tests demonstrated that the mutation responsible for supernodulation in nts382 is not allelic to either of these non-nodulation characters, and that it segregates independently. Progeny were identified that were homozygous for both supernodulation and non-nodulation, and these plants were incapable of nodulation. Thus, non-nodulation is epistatic over supernodulation and this is discussed in terms of the developmental blockage in the two mutant types. The identification and confirmation of these double mutants of the supernodulation and non-nodulation mutations are described. Although the non-nodulation mutations behave as recessive characters in a wild-type background, these mutations are incompletely dominant in a genetic background homozygous for supernodulation. The significance of these results to the understanding of nodule ontogeny is discussed.

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.)

Abstract 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 naturally-occuring mutation rj1 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-nodulation in mutant nod49 is root controlled and supernodulation in mutant nts382 is shoot controlled. Non-nodulation in mutants nod772 and nod139 (the newly found non-nodulation gene in soybean) is also root controlled. In addition, scions of nts382 failed to alter the nodulation phenotype when grafted onto stocks of the non-nodulating lines. Lateral roots developing from the scion of nts382 and Bragg exhibited supernodulation and the wild-type pattern 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 clearly 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.

Plant Host Genetics of Nodulation Initiation in Soybean

Symbiotic nitrogen fixation as exemplified by the legume-Rhizobium (or Bradyrhizobium) root nodule interaction is a well researched phenomenon illustrating plant-microbe interaction. The functional nitrogen fixing symbiosis requires cooperation between the bacterium and the plant. The last decade has witnessed a rapid expansion of the definition of Rhizobium genes that are involved in the symbiosis. The plant’s contribution, although always recognised as being important, recently received more attention through two major developments. The first was the application of DNA technology to the analysis of gene expression of legume symbiotic genes (Verma et al 1985) and the second was the realisation that existant plant variability may be insufficient in many legumes to permit the isolation of symbiotically defective germplasm. For this reason, research with Pisum sativum (Feenstra and Jacobson, 1985) (LaRue et al, 1985), Cicer arietinum (Davies et al, 1985) and Glycine max (our laboratory) has concentrated on the isolation of symbiotic mutants after induced mutagenesis (Carroll et al, 1985a,b).

Rhizosphere colonization by Bradyrhizobium japonicum is related to extent of nodulation of Glycine max CV. Bragg and its supernodulating mutants

Abstract Supernodulating soybean [ Glycine max (L.) Merr.] mutants nts382 and nts1116 supported substantially larger populations of Bradyrhizobium japonicum in their seedling rhizospheres than did the parent cultivar Bragg. The size of rhizosphere populations was significantly correlated with the number of nodules that formed subsequently. More cells (1.55-fold) of B. japonicum attached to the roots of supemodulating mutant nts382 than to the roots of Bragg. However, the extent of the difference was much less than the increase in nodule number for nts382 relative to Bragg which was between 2.40- and 10.10-fold. Thus, the supemodulating character cannot be accounted for by superior attachment of rhizobia to root surfaces. Differences in rhizosphere colonization of, and rhizobial attachment to, the roots between non-nodulation mutants and the parent Bragg were relatively small; this suggested that the genes coding for non-nodulation do not interfere with these early events normally leading to nodulation. Nodule mass for nts382 constituted 7.1% of whole plant dry matter compared with 2.5% for Bragg ( P P > 0.05) between nts382 and Bragg in N accumulation or dry matter production.

Non-Nodulation Mutants of Soybean

Three non-nodulating mutants (nod49, nod139 and nod772) were isolated from mutagenized soybean populations (Carroll et al, 1986). The genetic, anatomical and physiological analysis of these mutants as well as the naturally occurring non-nodulation (rj1) mutant (Williams and Lynch, 1954) was carried out.