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

Summary The 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.     

Soybean Seedling Extracts Inhibit Supernodulation

When soybean (Glycine max ) nodulation mutant nts 382 was inoculated with Bradyrhizobium japonicum, these plants nodulated significantly more than the parental type Bragg. Nts 382 seedlings displayed wild-type nodulation pattern when aqueous extracts of young Bragg shoots were applied to the cultural medium together with nutrient solution. Application of young nts 382 shoot extracts to Bragg seedlings did not result in any apparent increase in nodule number. In graft experiments, young shoots from mutant nts 382 induced supernodulation on Bragg root stocks, while no supernodulation was observed when Bragg seedlings were used as scion and grafted onto nts 382 root stocks. Further, the effectiveness of Bragg plant extracts to suppress supernodulation on nts 382 seedlings was found to depend on the age of the plant material used, being very ineffective with extracts from 60-day-old plants. The age effect was not observed in graft experiments. These findings suggest that soybean supernodulation phenomenon may be controlled by one or a few unknown chemicals or plant hormones.     

Relationship between autoregulation and nitrate inhibition of nodulation in soybeans

Ten of 11 supernodulating mutants of soybean [ Glycine max (L.) Merr.] cv. Bragg, in which nodulation was far in excess of that in the wild type, showed pronounced tolerance of nodulation to applied nitrate. Mutant nts (nitrate-tolerant symbiosis) 1116 had an intermediate nodulation response and also showed some inhibition by nitrate. Mutant 1029, a revertant of nts382 (an extreme supernodulator), showed a wild-type nodulation pattern and was equally sensitive to nitrate as cv. Bragg. Grafting experiments with cv. Bragg and nts382 indicated that both supernodulation and tolerance of nodulation to nitrate were dependent on shoot factors. Total leaf nitrate reductase (EC 1.6.6.1 and EC 1.6.6.2) activity of the supernodulating mutants was similar to that in cv. Bragg. We conclude from these results that the inhibitory effect of nitrate on nodule initiation and development in soybean depends on an interaction between nitrate and the autoregulation singal. In the supernodulating mutants, the autoregulation signal is either altered or absent and cosequently nodulation in these mutants is not sensitive to nitrate.     

Catabolism of indole-3-acetic acid to indole-3-methanol in a crude enzyme extract and in protoplasts from Scots pine (Pinus sylvestris)

The antagonistic effects of ethylene and Ag⁺ on the metabolism of [1-¹⁴C]indole-3-acetic acid (IAA) and on the rates of ethylene production were studied in tobacco leaf discs ( Nicotiana rustica var. Brasilia ). During the first 10 h of incubation, Ag⁺-pretreated leaf discs contained more free [¹⁴C]IAA than untreated ones due to decreased oxidative decarboxylation, and the discs also produced more ethylene. Exogenously supplied ethylene nullified these effects of Ag⁺. However, the most pronounced effect of Ag⁺ in increasing ethylene production, as well as the strongest antagonistic effect of exogenous ethylene, were found between 24 and 48 h of incubation. During this time span no effect on the level of free IAA and on its decarboxylation could be observed. It is suggested that ethylene exerted its autoinhibitory effect by a feedback control on the IAA-induced ethylene biosynthesis. Possible mechanisms for the autoinhibitory effect of ethylene are discussed.     

Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors

The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts1116) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype.     

Ethylene modulates the susceptibility of the root for nodulation in actinorhizal Discaria trinervis

Ethylene is produced by plants in response to a wide variety of environmental signals and mediates several developmental processes in higher plants. We investigated whether ethylene has a regulatory function in nodulation in the actinorhizal symbiosis between Discaria trinervis and Frankia BCU110501. Roots of axenic D. trinervis seedlings showed aberrant growth and reduced elongation rate in the presence of ethylene donors [i.e. 2-aminocyclopropane carboxylic acid (ACC) and 2-chloroethylphosphonic acid (CEPA)] in growth pouches. By contrast, inhibitors of ethylene synthesis (aminoethoxyvinylglycine, AVG) or perception (Ag⁺) did not modify root growth. This indicates that the development of D. trinervis roots is sensitive to elevated ethylene levels in the absence of symbiotic Frankia . The drastic response to higher ethylene levels did not result in a systemic impairment of root nodule development. Nodulation occurred in seedlings inoculated with Frankia BCU110501 in the presence of ethylene donors or inhibitors. Overall, the ability of the seedlings to shut down nodule formation in the younger portions of the root (i.e. to autoregulate nodulation) was not significantly impaired by a modification of endogenous ethylene levels. In contrast, we detected subtle changes in the nodulation pattern of the taproots. As a result of exposing the roots to CEPA, less nodules developed in older portions of the taproot. In line with this observation, AVG or Ag⁺ caused the opposite effect, i.e. a slight increase in nodulation of the mature regions of the taproot. These results suggest that ethylene is involved in modulating the susceptibility for nodulation of the basal portion of D. trinervis seedling roots.     

Ethylene production and involvement during the first steps of durum wheat (Triticum durum) anther culture

The role of ethylene in anther culture of durum wheat ( Triticum durum Desf. cv. Ardente) was analyzed by testing the effects of 2-chloroethylphosphonic acid (ethrel) silver thiosulfate (Ag⁺), a -aminooxyacetic acid (AOA) and 1-aminocyclopropane-l-carboxylic acid (ACC) on microspore division observed after 21 days of culture and on development of calli estimated at day 45. The use of ethrel and Ag⁺ indicated a positive effect of ethylene on microspore division, whereas the use of AOA, and to a lesser extent ACC, snowed a negative effect. In contrast, the addition of ethrel or Ag⁺ indicated that ethylene inhibits the development of microspore-derived calli. AOA gave contradictory results. Ethylene production by anthers was about 7 pl anther⁻¹h⁻¹ and decreased during culture. ACC content in the anthers was maximal at day 9, whereas malonyl ACC (MACC) increased sharply from day 0 to day 3 and then decreased. The addition of AOA or ACC to the culture medium decreased or increased, respectively, ethylene production of anthers and the ACC and/or MACC content, but at concentrations higher than those that modified the formation of calli. This formation seems to occur in two successive phases: induction and initiation of microspore division, which was promoted by ethylene, followed by callus development, which was inhibited by ethylene.     

Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild-type parent

The growth of a supernodulating, nitrate-tolerant soybean [ Glycine max (L.) Merr.] mutant nts 382 (nitrate-tolerant symbiosis) was compared to that of its wild-type parent, cv. Bragg, over the first 50 days after sowing. Plants were grown either inoculated in the absence of an external nitrogen source or uninoculated in the presence of 5 m M KNO₃. For both treatments, nts 382 growth up to 13 days after planting was faster than that of cv. Bragg. Thereafter, supernodulation of inoculated nts 382 occurred and growth of cv. Bragg was faster; shoot and root dry weight increments and leaf area were greater in cv. Bragg, but the N content of nts 382 was higher. Relative growth and net assimilation rates were lower in nts 382, which had faster shoot and root respiration rates. Shoot growth of uninoculated plants was similar for both mutant and wild-type but roots of nts 382 were slightly smaller than those of cv. Bragg. Total plant N content was similar in uninoculated cv. Bragg and nts 382 but the latter had a higher leaf N content. Early lateral root formation (prior to nodule emergence) was greater in nts 382 regardless of whether rhizobia or KJNO₃ were present. We conclude that nts 382 has some inherent differences from its parent but that supernodulation significantly retards plant growth.     

Effects of growth regulators on light-dependent anthocyanin production in Zea mays seedlings

Rengel, Z. and Kordan, H. A. 1987. Effects of growth regulators on light-dependent anthocyanin production in Zea mays seedlings.
The effects of ethylene, indolyl- and naphthylacetic acids, zeatin, benzyladenine, gib-berellic acid and triiodobenzoic acid on anthocyanin production in seedlings of Zea mays L. cv. Golden Bantam were investigated. Endogenously produced and exogen-ously supplied ethylene, as well as the other growth regulators tested markedly suppressed anthocyanin formation. Except for triiodobenzoic acid, the other growth regulators stimulated ethylene production, the amounts produced in the light being larger than those in the dark. Absorption of ethylene by permanganate as well as inhibition of ethylene production or action by Co²⁺ or Ag⁺ increased anthocyanin formation in maize seedlings above the level found in the control plants. The inhibiting effect of auxins and cytokinins on anthocyanin production was reversed by Co²⁺ or Ag⁺. In contrast, decreased anthocyanin formation caused by gibberellic acid or triiodobenzoic acid seemed unrelated to ethylene and could not be alleviated by Co²⁺ or Ag+.     

Arabidopsis mutants with reduced response to NaCl and osmotic stress

We isolated 6 mutant lines of Arabidopsis thaliana that expressed reduced sensitivity to salt and osmotic stress during germination. All 6 lines cum recessive mutations in a single gene, designated reduced salt sensitivity (rss), linked to the ADH marker on chromosome 1. The rss mutants are less sensitive than wild type for NaCl and osmotic stress inhibition of germination, tolerating approximately 150 mM higher concentrations of NaCl and about 250 mM higher concentrations of sorbitol in the media. Germination assays on media containing various salts indicate that the rss mutations reduce sensitivity lo Na⁺ and Rh⁺ but also, to a much lesser degree, to K⁺ and Cs^s+. However, the rss mutation does not improve plant growth when plantlets are transferred to high salt or high osmotic pressure media after germination. The rss plantlets accumulate praline to a significantly lesser degree than wild type when they are exposed to either salt or osmotic stress. Thus, the rss mutants differ from wild type both at germination and during vegetative growth indicating that the rss mutations are pleiotropic and might affect perception of solutes or some aspect of stress-induced signaling. The rss mutations do not alter ABA sensitivity and therefore probably do not affect ABA-mediated signaling.     

Quantitative relationships between osmotic potential amd epicotyl growth in Vigna radiata as affected by osmotic stress and cotyledon excision

Ethylene biosynthesis in leaf discs of tobacco ( Nicotiana tabacum L. cv. Xanthi), as measured by the conversion of L-[3,4-¹⁴C]-methionine to ¹⁴C₂H₄, was markedly inhibited by exogenous ethylene. This inhibition was accompanied by a decrease in total (free + conjugated) content of 1-aminocyclopropane-1-carboxylic acid (ACC), most of which appeared in its conjugated inactive form. The autoinhibitory effect of ethylene was reversible and could be relieved by Ag⁺. The Ag⁺-treated leaf discs, with or without ethylene, contained only free ACC at an increased level. The results suggest that in tobacco leaves, the autoinhibition of ethylene production resulted from reduction in the availability of free ACC, through both suppression of ACC formation and increased ACC conjugation.     

Effect of 2-chloroethylphosphonic acid on capsule formation and alkaloid content in Papaver somniferum

Application of different concentrations of ethephon (2-chloroethylphosphonic acid) to Papaver somniferum L. at the times of stem elongation, bud, and capsule formation produced different effects. Ethephon (10^-2 M ) retarded growth of the plant and inhibited capsule formation during stem elongation, significantly reduced capsule size during the flowering period, but did not alter capsule development during capsule formation. When applied during the period of stem elongation, ethephon (10^-3 M and 10^-4 M ) reduced capsule size; alkaloid accumulation was reduced by ethephon at a concentration of 10^-3 M , but slightly increased by 10^-4 M . Ethephon (10^-3 M and 10^-4 M ) did not alter capsule development or alkaloid content significantly when applied during bud formation, but stimulated capsule size and alkaloid content when applied during capsule formation. Pretreating the plants with Ag⁺ (silver nitrate) did not reverse the ethephon effect. The results suggest that capsule maturation and alkaloid accumulation in P. somniferum are modified by ethylene, which is produced as a result of exogenous ethephon treatment.     

Mutagenesis of pea (Pisum sativum L.) and the isolation of mutants for nodulation and nitrogen fixation

Pea mutants for nodulation have been obtained by treating seeds with ethyl methane sulfonate (EMS) followed by 2 screening procedures. In one, mutants resistant to nodulation (nod⁻), or with ineffective nodules (nod⁺, fix⁻) were obtained, whilst in the other 4 hypernodulated mutants (nod⁺⁺) with 5–10 times more nodules than cv. Frisson and expressing a character of nitrate tolerant symbiosis (nts) were discovered. All mutations are under the control of single recessive genes. (nod⁻), (nod⁺, fix⁻) and (nod⁺⁺, nts) mutations result from mutation events at 6, 7 and 1 different loci respectively.

Grafting experiments showed the (nod⁻) and (nod⁺, fix⁻) phenotypes are associated with the root genotypes and that (nod⁺⁺, nts) phenotype is associated with the shoot genotype.     

Characterization of phytochelatin synthase from tomato

The enzyme that synthesizes Cd-binding phytochelatins (PCs), PC synthase, has been studied in tomato ( Lycopersicon esculentum ) cell cultures and plants. This enzyme transfers γ-GluCys from GSH or PC to either GSH or an existing polymer of (γ-GluCys)_nGly. PC synthase from tomato requires GSH or PCs as substrates but cannot utilise γ-GluCys or GSSG. PC synthase is activated both in vivo and in vitro by a variety of heavy metal ions, including Cd²⁺, Ag⁺, Cu²⁺, Au⁺, Zn²⁺, Fe²⁺, Hg²⁺ and Pb²⁺. In crude protein extracts from tomato cells the enzyme has an apparent K_m of 7.7 m M for GSH in the presence of 0.5 m M Cd²⁺, and exhibits maximum activity at pH 8.0 and 35°C. PC synthase is present in tomato cells grown in the absence of Cd. The level of enzyme activity is regulated during the cell culture cycle, with the highest activity occurring 3 days after subculture. Cadmium-resistant tomato cells growing in medium containing 6 m M CdCl₂ have a 65% increase in PC synthase activity compared to unselected cells. PC synthase is also present in roots and stems of tomato plants, but not in leaves or fruits. The distribution of the enzyme in tomato plants and regulation of PC synthase activity in tomato cells indicate that PC synthase, and PCs, may have additional functions in plant metabolism that are not directly related to the formation of Cd-PC complexes in response to cadmium.     

1,3-Propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow

Glycerol fermentation and product formation of two product-tolerant mutants of Clostridium butyricum DSM 5431 were investigated in continuous culture at increasing glycerol feed concentrations. Under conditions of glycerol excess (above 55 g l⁻¹ at D = 0·15 h⁻¹), the mutants maintained a constant level of glycerol consumption and product formation, whereas the parent strain exhibited a substantial decrease in substrate conversion, 1,3-propanediol and butyrate formation, and an increase in acetate formation. The activities of the glycerol dehydrogenase, the glycerol dehydratase and the 1,3-propanediol dehydrogenase showed only slight changes with glycerol concentrations in the mutants, but dropped markedly at high concentrations in the wild type. Intracellular concentrations of NADH, NAD ⁺ and acetyl-CoA remained at a relatively constant level in the mutants, but increased sharply with the wild type strain. The NADH content was always higher than the NAD ⁺ content in the mutants as well as in the wild type.     

Nitrogen use efficiency. 3. Nitrogen fixation: genes and costs

The nitrogen use efficiencies (NUE) of N₂ fixation, primary NH ₄⁺ assimilation and NO ₃⁻ assimilation are compared. The photon and water costs of the various biochemical and transport processes involved in plant growth, N-assimilation, pH regulation and osmolarity generation, per unit N assimilated are respectively likely to be around 5 and 7% greater for N₂ fixation than for a combination of NH ₄⁺ and root and shoot NO ₃⁻ assimilation as occurs with most crops. Studies on plant and rhizobial genes involved in nodulation and N₂ fixation may lead to more rapid nodulation, production of more stress-tolerant N₂ fixing systems and transfer of the hydrogenase system to rhizobium/legume symbioses which currently do not have this ability. The activity of an uptake hydrogenase is predicted to decrease the photon cost of diazotrophic plant growth by about 1%.     

Role of ethylene and auxin in regenerative differentiation and orientation of tracheids in Pinus pinea seedlings

A low auxin concentration (0.1% naphthalene acetic acid) induced tracheids with longitudinal polarity parallel to the hypocotyl axis in young Pinus pinea seedlings. Application of 0.1% ethrel laterally and 0.1% naphthalene acetic acid apically disturbed axial tracheid polarity and promoted the differentiation of tracheids with a lateral orientation. Ethrel by itself, with no auxin background, did not affect tracheid differentiation. Apical application of 1% gibberellic acid with the low auxin, reversed the polarity disorder induced by ethrel. Disturbance of axial tracheid polarity was observed under a high auxin concentration (0.5% naphthalene acetic acid) which was similar to the combined effect of ethrel and auxin. The high auxin concentration increased tracheid number significantly. This effect was curtailed following treatment with inhibitors of ethylene formation (Co²⁺; 1-aminoethoxy- vinylglycine) and action (Ag²⁺). The role of ethylene in controlling the differentiation of radial tracheids, which characterize the vascular rays of pines, is discussed.     

Catalytic properties of Na+-translocating NADH:quinone oxidoreductases from Vibrio harveyi, Klebsiella pneumoniae, and Azotobacter vinelandii

The catalytic properties of sodium-translocating NADH:quinone oxidoreductases (Na⁺-NQRs) from the marine bacterium Vibrio harveyi , the enterobacterium Klebsiella pneumoniae , and the soil microorganism Azotobacter vinelandii have been comparatively analyzed. It is shown that these enzymes drastically differ in their affinity to sodium ions. The enzymes also possess different sensitivity to inhibitors. Na⁺-NQR from A. vinelandii is not sensitive to low 2- n -heptyl-4-hydroxyquinoline N-oxide (HQNO) concentrations, while Na⁺-NQR from K. pneumoniae is fully resistant to either Ag⁺ or N-ethylmaleimide. All the Na⁺-NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify the noncovalently bound FAD of the enzyme.     

The genetic locus controlling supernodulation in soybean (Glycine max L.) co-segregates tightly with a cloned molecular marker.

Summary The genetic locus (nts) controlling nitrate-tolerant nodulation, supernodulation, and diminished autoregulation of nodulation of soybean (Glycine max (L.) Merill) was mapped tightly to the pA-132 molecular marker using a restriction fragment length polymorphism (RFLP) detected by subclone pUTG-132a. The nts (nitrate-tolerant symbiotic) locus of soybean was previously detected after its inactivation by chemical mutagenesis. Mutant plant lines were characterized by abundant nodulation (supernodulation) and tolerance to the inhibitory effects of nitrate on nodule cell proliferation and nitrogen fixation. The large number of RFLPs between G. max line nts382 (homozygous for the recessive nts allele) and the more primitive soybean G. soja (P1468.397) allowed the detection of co-segregation of several diagnostic markers with the supernodulation locus in F₂ families. We located the nts locus on the tentative RFLP linkage group E about 10 cM distal to pA-36 and directly next to marker pA-132. This very close linkage of the molecular marker and the nts locus may allow the application of this clone as a diagnostic probe in breeding programs as well as an entry point for the isolation of the nts gene.