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.     

Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The 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, andrj ₁) 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, andrj ₁) 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.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions     

Differential sensitivity of nodulation to ethylene in soybean cv. Bragg and a supernodulating mutant

We previously found that the ethylene inhibitor Ag⁺ could overcome the inhibitory effect of nitrate on nodulation of soybean ( Glycine max ) cv. Bragg. The same treatment increased nodulation quantitatively under non-inhibitory conditions, strongly suggesting involvement of ethylene in the control of nodulation in this species. Supernodulation mutants that lack internal autoregulation of nodulation, however, had biosynthesis capacity similar to the wild type. In the present work, the effects of ethylene on nodulation of 'Bragg' and two separate, but allelic, supernodulating mutants ( nts382 and nts1007 ) were compared. The nodulation process appeared much more sensitive than plant growth and development to ethylene, which reduced the number of nodules per plant, but nearly twofold more in the wild type than in the supernodulation mutants. The cause–effect relationship is established by the counteracting effect of Ag⁺ and the fact that the stronger the inhibition by ethylene, the higher the recovery of nodulation ability with the ethylene antagonist. This higher tolerance of or lower sensitivity to ethylene in nts382 persists even under low inoculum dose, where nodule number and mass could be decreased to wild-type levels. Differences between the mutant and the wild type in the triple response test do not appear to support differences in ethylene perception on a whole-plant basis. The results suggest that sensitivity of nodulation to ethylene might have been affected in supernodulation mutants.     

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.     

Mutagenesis of soybean (Glycine max (L.) Merr.) and the isolation of non-nodulating mutants

Soybean seeds (cv. Bragg) were mutagenized with either ethyl methanesulphonate (EMS), gamma-rays or sodium azide (NaN₃). EMS was the most efficient in generating chlorophyll-deficient variants in the M₂. NaN₃ was not an effective mutagen. Approximately 25 200 M₂ plants were screened for absence of nodulation after inoculation with Bradyrhizobium japonicum strain CB1809 (=USDA136). Three stable non-nodulation mutants were recovered and designated nod49, nod772 and nod139. Mutant nod49 was isolated from a mixture of M₂ populations and this mutant also failed to nodulate upon inoculation with Rhizobium fredii strain USDA257, which nodulated the parent cultivar. Mutants nod772 and nod139 came from segregating EMS-derived M₂ families, indicating that these mutations were a result of mutagenesis. Inheritance of the non-nodulation character has been demonstrated through to the M₅, M₄ and M₃ generations for nod49, nod772 and nod139, respectively.     

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.     

Effects of Doubled CO2 on Contents of Photosynthetic and on Kinetic Parameters of Fluorescence Induction in Different Genotypes of Soybean

Effects of doubled C02 on photosynthetic characteristics of soybean ( Glycine max L. ) Bragg (wild type) and its different monogene mutation strains--Nts 382 (supemodulation mutant) and Nod 49 (non-nodulation mutant) were studied. The experimental results showed that the contents of chlorophyll and carotenoid of Bragg, Nts 382 and Nod 49 were increased under doubled CO2, respectively, although to different extent. The determination results of fluorescence induction kinetic parameters showed that PS Ⅱ activity, the efficiency of primary conversion of light energy of PS Ⅱ and the efficiency of potential photosynthetic quantum conversion of a leaf from Bragg and its mutants were raised in doubled CO2. Fluorescence photochemical quenching coefficient and the overall photochemical quantum yield of PS Ⅱ were raised and non-photochemical quenching coefficient was reduced with CO2 enrichment; such changes were bigger in Nts 382 than those in Bragg and Ned 49. It might be that atmospheric N2 was more effectively utilized by Nts 382 than by Bragg and Ned 49.     

Inheritance of supernodulation in soybean and estimation of the genetically effective cell number

Summary Provided the nature of inheritance is known, the frequency of homozygous mutant plants in individual M₂ families (derived from M₁ seed) can be used to estimate the genetically effective cell number (GECN). Segregation ratios in M₃ families derived from M₂ wild-type plants indicated that the supernodulation characters nts382, nts1007 and nts183 are inherited as Mendelian recessives. The nature of inheritance was also known or confirmed to be recessive by crossing the wild type to these and several other mutants derived from the same population of M₂ families. Subsequently, using the frequency of mutant plants in individual M₂ families, the GECN for soybean was calculated to be approximately two.     

Two defined alleles of the LRR receptor kinase GmNARK in supernodulating soybean govern differing autoregulation of mycorrhization

Plants regulate the extent of nodulation and root colonization by arbuscular mycorrhizal fungi (AMF), a phenomenon named autoregulation of symbiosis. We tested AMF colonization in split roots of various soybean genotypes [ Glycine max (L.) Merr. cv. Bragg, Enrei, Harosoy and Williams], where precolonization of one side of the split-root system by the AMF Glomus mosseae resulted in reduced mycorrhization of the other. AMF precolonization failed to control secondary mycorrhization in the supernodulating Bragg nonsense mutant nts1007 (Q106*), indicating that the GmNARK gene (predicted to encode a leucine-rich repeats (LRR) receptor kinase related to CLAVATA1 in Arabidopsis ) is involved in autoregulation of the AMF symbiosis. Here, we tested whether the allelic En6500 nonsense supernodulating mutant ( GmNARK K606*, derived from cv. Enrei) and supernodulating mutants of cv. Williams ( Nod1-3 and Nod2-4 ) with yet-undefined genetic lesions exhibit a similar symbiotic phenotype in mycorrhizal split-root systems. Surprisingly, these supernodulating mutants retained their ability to autoregulate AMF. To examine possible differences between two allelic mutants, we determined levels of IAA, abscisic acid, coumestrol, daidzein and genistein in mycorrhizal and uninoculated control roots. Compared with wild-type plants, both mutants showed reduced IAA accumulation in mycorrhizal roots. Roots of cv. Enrei and En6500 exhibited high levels of isoflavonoids not seen in Bragg or nts1007 . Taken together, these findings showed that supernodulation mutants, despite a common nodulation phenotype, differ in their ability to autoregulate AMF root colonization. This suggests either that the GmNARK gene product of some mutants is still partially functional (Q106* vs. K606*) or that varietal differences reflected in altered physiological responses suppress the loss of function.     

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.     

Mutational Analysis of Mating Type Inheritance in Syngen 4 of PARAMECIUM AURELIA

Six genic mutations restricting clones to mating type VII (O) were isolated in syngen 4, Paramecium aurelia. The only three extensively tested were neither allelic nor closely linked. A second type of mutation, allelic to one of the O restricted mutants, was also found. Clones homozygous for this mutant gene were selfers, producing both O and E (VIII) mating types, but only when they were progeny of mating type E parental clones. While all seven mutant genes behaved as recessives in monohybrid crosses, clones heterozygous at two different loci often demonstrated an unanticipated phenotype: selfing. The significance of the findings is discussed in relation to mating type determination and the evolution of mating type systems.     

Primary Analysis of Components in Different Soybean Nodulating Mutants and their Effects on Nitrate Reductase Activity and Nodulation

The water extracts of leaves and roots from supernodulating soybean (Glycine max (L.) Merr. ) nts 382 and nonnodulating soybean Nod 49 have been chromatographed using filtering method through the column (25 cm × 2 cm) Sephadex G25 and 4 fractions, namly, nts 382 (Nod 49) F1, nts 382 (Nod 49) F2, nts 382 (Nod 49) F3, and nts 382 (Nod 49) F4 could be distinguished according to nitrate reductase (NR) activities inhibited by the eluate. The inhibition of NR activity by the noninoculated nts 382 F2 and the nts 382 F4 in vitro were much stronger than that by the inoculated nts 382 F2 and nts 382 F4. On the contrary, the obvious inhibition of NR activity in vitro by the noninoculated Nod 49 F2 and Nod 49 F4 were substantialy strengthed again by the innoculated Nod 49 F2 and Nod 49 F4. The facts indicated that the quantity of NR inhibitors in the leaf cells of soybean nts 382 reduced after the inoculation but was that in the inoculated Nod 49 leaf cells further more accumulated. Both nodulations assays, the nodulation of soybean "Bragg " injected with inoculated nts 382 Fl, nts 382 F2, nts 382 F3 and nts 382 F4 from leaves and roots and the nodulation of soybean nts 382 injected with inoculated Nod 49 F2, Nod 49 F3 and Nod 49 F4 from leaves only showed that nts 382 Fl and nts 382 F2 increased nodules of soybean "Bragg" by 1 to 3 times but nts 382 F3 and nts 382 F4 did not. Inhibition of soybeannts 382 nodulation by inoculated Nod 49 F2 Nod 49 F3 and Nod 49 F4 expressed that the Nod 49 F4 only inhibited the nodulation strongly by one time in the experiments with nts 382 plants with leaves, and by 15 times in the experiments with nts 382 plants without leaves at 10 d of inoculation and injection and this inhibition was nonreversible even after stopping injection from the 11th day to the 15th day after inoculation.     

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.     

Early action of pea symbiotic gene NOD3 is confirmed by adventitious root phenotype

A supernodulating and Nts (nitrate-tolerant symbiosis) symbiotic mutation of pea (Pisum sativum L.) line RisfixC was found to retain its expression in the distant genetic background of pea lines Afghanistan L1268, Zhodino E900, and cv. Arvika. This finding allowed for reliable scoring for the trait in mapping crosses. The RisfixC mutation was localized 8.2 cM apart from SYM2 and cosegregated with molecular markers for SYM2–NOD3 region Psc923 and OA-1. Grafting experiments showed that supernodulation is root-determined, consistently with mutants in the NOD3 locus. Therefore, the mutation of RisfixC can be localized in gene NOD3. Like in other published nod3 alleles, the RisfixC mutation determines supernodulation when it is expressed in the root but not in the shoot. Supernodulated adventitious roots that are spontaneously formed in the wild-type scions on mutant rootstocks indicate that the descending systemic signal, which is inhibitory to nodule formation, is absent in this type of chimeric plants. Since the descending signal production in the wild-type shoot reflects the presence of the ascending root signal, the nod3-associated lesion must be located in the beginning of the systemic circuit regulating nodule number.     

Relationship between gibberellin, ethylene and nodulation in Pisum sativum

? Gibberellin (GA) deficiency resulting from the na mutation in pea (Pisum sativum) causes a reduction in nodulation. Nodules that do form are aberrant, having poorly developed meristems and a lack of enlarged cells. Studies using additional GA-biosynthesis double mutants indicate that this results from severe GA deficiency of the roots rather than simply dwarf shoot stature. ? Double mutants isolated from crosses between na and three supernodulating pea mutants exhibit a supernodulation phenotype, but the nodule structures are aberrant. This suggests that severely reduced GA concentrations are not entirely inhibitory to nodule initiation, but that higher GA concentrations are required for proper nodule development. ? na mutants evolve more than double the amount of ethylene produced by wild-type plants, indicating that low GA concentrations can promote ethylene production. The excess ethylene may contribute to the reduced nodulation of na plants, as application of an ethylene biosynthesis inhibitor increased na nodule numbers. However, these nodules were still aberrant in structure. ? Constitutive GA signalling mutants also form significantly fewer nodules than wild-type plants. This suggests that there is an optimum degree of GA signalling required for nodule formation and that the GA signal, and not the concentration of bioactive GA per se, is important for nodulation.     

Symbiotic Performance of Supernoclulating Soybean (Glycine max (L.) Merrill) Mutants during Development on Different Nitrogen Regimes

Growth and symbiotic performance of soybean (Glycine max (L.)Merrill) cv. Bragg and three of its induced nodulation mutants(nod49, non-nodulating; ntsl 116, intermediate supernodulator;nts1007, extreme supernodulator) were compared throughout developmentunder different nitrogen regimes (0, 2, 5 and 10 mol nitratem^–3). Nitrogen fixation was assessed using ¹⁵N-isotopedilution and xylem sap analysis for ureide content. Both techniquesconfirmed a complete lack of N₂ fixation activity in nod49.Plant reliance on nitrogen fixation by the other genotypes wasdependent on the nitrate regime and the developmental stage.The ntsl007 and ntsl 116 mutants fixed more nitrogen than theparent cultivar in the presence of 10 mol m^–3 nitratein the nutrient solution, but higher input of symbioticallyderived nitrogen was still insufficient to offset the amountof nitrogen removed in the harvested seed. However, the mutantsutilized less nitrate for growth than Bragg. Comparison of estimatesof N₂ fixation derived from the ¹⁵N-dilution technique withthose based on relative ureide content of xylem sap indicatedthat the latter offered a simple and reliable procedure forevaluating the symbiotic performance of supernodulating plants. Key words: ¹⁵N-isotope dilution, supernodulation, ureides     

Nitrogenase activity and ureide levels in a supernodulating soybean mutant: Effects of inoculum dose and nitrate treatment

The effects of nitrate on nitrogenase (EC 1.18.2.1) activity of soybean ( Glycine max [L.] Merr) cv. Bragg and its supernodulating mutant derivative, nts382, were compared. A short-term nitrate treatment was used to allow effects on nitrogenase activity to be studied in the absence of effects on nodule growth and a low inoculum dose, which prevented supernodulation of nts382, was employed to test for any interaction between supernodulation and the magnitude of the effect of nitrate on nitrogenase activity. At the usual inoculum dose, nitrogenase activity, per g nocule, of nts382 was lower than that of Bragg and was proportionally less affected by nitrate. Decreasing the inoculum dose increased nitrogenase activity of nts382 and also the proportional decline in response to nitrate. The decline in the ureide conentration in xylem exudate in response to nitrate was proportionally similar to the decline in nitrogenase activity per plant. However, although nitrogenase activity per plant of nts382 was several-fold less than that of Bragg, the ureide flux rate (ureide concentration x xylem sap exudation rate), was not different. At the usual inodulum dose, the ureide content of the nocules, stems plus petioles and leaves of nts382 was greater than that of Bragg. Decreasing the inoculum dose reduced the ureide content of the nodules of nts382 but not of Bragg. Ureide degradative capacity of the leaves was the same for Bragg and nts382. Low activities of 5-phosphoribosyl pyrophosphate amidotransferase (EC 2.4.2.14) and glutamine synthetase (EC 6.3.1.2) in the nodules reflected the low nitrogenase activity of nts382.     

Two new male-sterile mutants of Zea mays (Poaceae) with abnormal tapetal cell morphology

Two new recessive male-sterile mutants of Zea mays (Poaceae), or maize, were studied to identify the timing of pollen abortion and to examine the involvement of anther wall cell layers. The results of test crosses indicated that these mutants were not allelic with any known male-sterile mutants of maize. Light and transmission electron microscopy were used to compare pollen development in homozygous male-sterile mutants to that in fertile heterozygous siblings. In both mutants, microspores abort soon after release from the meiotic tetrad. However, the two mutations have strikingly different phenotypes. Large lipid bodies accumulate in the tapetal cells as the microspores vacuolate and die in the mutant ms25. Large vacuoles appear in both the tapetal cells and the young microspores as they begin to disintegrate in the mutant ms26. Because abnormal tapetal cell morphology is detected in both mutants, it is possible that both of these mutations affect the expression of genes in tapetal cells.     

Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.