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     

A Supernodulation and Nitrate-Tolerant Symbiotic (nts) Soybean Mutant

The nodulation characteristics of soybean (Glycine max) mutant nts382 are described. The mutant nodulated significantly more than the parent cultivar Bragg in the presence and absence of several combined nitrogen sources (KNO₃, urea, NH₄Cl, and NH₄NO₃). The number of nodules on the tap root and on lateral roots was increased in the mutant line. In the presence of KNO₃ and urea, nitrogenase activity was considerably higher in nts382 than in Bragg. Mutant plants were generally smaller than wild-type plants. Although nts382 is a supernodulator, inoculation with Rhizobium japonicum was necessary to induce nodule formation and both trial strains CB1809 (= USDA136) and USDA110 elicited the mutant phenotype. Segregation of M₃ progeny derived from a M₂ wild-type plant indicated that the mutant character is inherited as a Mendelian recessive. The mutant is discussed in the context of regulation of nodulation and of hypotheses that have been proposed to explain nitrate inhibition of nodulation.     

Stimulation of respiration and nitrogenase in bacteroids of Siratro (Macroptilium atropurpureum) by plant nodule cytosol

Nitrogenase activity (acetylene reduction) of isolated Siratro (Macroptilium atropurpureum) bacteroids was stimulated by addition of plant cytosol fractions which also preserved activity at high (up to 3%) O₂ tensions. These effects were not due to leghaemoglobin. Boiling removed some, but not all, of the protective capacity of the cytosol. Heat treated cytosol substantially stimulated the respiration of siratro bacteroids. Of a wide variety of compounds tested, only ascorbate could mimic the cytosol. Ascorbate was present in the cytosol fraction, in significant quantities. The effect of ascorbate was evident at low O₂ concentrations and in purified bacteroids, and was inhibited by cyanide. Siratro bacteroids appear to possess an oxidase which could serve a protective role in vivo.     

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.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

DNA amplification fingerprinting of bacteria

Summary We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng·l^–1 and high MgCl₂ levels was especially important for reproductibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.Correspondence to: G. Caetano-Anollés     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.