The inheritance of a urease-null trait in soybeans

Summary Four soybean seed urease nulls (lacking both the activity and antigen of the embryo-specific urease) were intermated and the F₁ and F₂ seed examined for urease activity. Both generations were without urease activity, and the nulls were therefore considered noncomplementing. In crosses of each null line to cultivars homozygous for the allelic, codominantly inherited urease slow or fast isozyme, the F₁ seed expressed the embryo-specific urease isozyme of the urease-expressing parent. A 3 1 segregation for presence and absence of urease was observed in progeny from F₁ and heterozygous F₂ plants. The F₂ and F₃ from fastXnull combinations revealed that urease-positive seed were all phenotypically urease fast, while the same seed from slowXnull combinations showed a segregation of one seed containing a fast urease, either exclusively or in a heterozygous state with the slow isozyme, for every 69 phenotypic slows. Data pooled from F₂ plants which segregate for both the presence (Sun) and absence (Sun) of urease and for the fast (Eu1-b) or slow (Eu1-a) urease allele indicate that the null lesion (Sun) is linked to Eu1 by approximately one map unit. The evidence is consistent with two models: (1) sun is an allele at the embryo-specific urease isozyme locus (Eu1) and that a high degree of exchange (and/or conversion) within the locus results in a 1% recombination frequency between the null trait and urease allozyme; (2) sun is at a distinct locus which is separated by one map unit from the embryo-specific urease isozyme locus (Eu1) upon which it acts in the cis position. Polyadenylated embryo RNA from one of the null lines, PI 229324, exhibited no urease template activity in vitro. Thus, the lack of urease antigen is due to lack of accumulation of translatable urease mRNA. The availability of soybeans lacking seed urease should be extremely useful to breeders as a trait for linkage studies and to geneticists as a transformation marker.Portions of this work were funded by the Illinois and Missouri Agricultural Experiment Stations, the SOHIO-University of Illinois Center of Excellence in Crop Molecular Genetics and Genetic Engineering and by grants PCM-8219652 from the National Science Foundation and USDA/SEA-CRCR-1-1374 from the USDA Competitive Grants Office     

Pleiotropic soybean mutants defective in both urease isozymes

Summary Two new soybean [Glycine max (L.) Merr. cv. Williams] loci, designated Eu2 and Eu3, were identified in which ethyl methanesulfonate (EMS)-induced mutation eliminated urease activity. These loci showed no linkage to each other or to the Sun-Eul locus described in the accompanying paper (Meyer-Bothling and Polacco 1987). Unlike sun (seed urease-null) mutations those at Eu2 and Eu3 affected both urease isozymes: the embryo-specific (seed) and the ubiquitous (leaf) urease. The eu2/eu2 mutant had no leaf activity and 0.6% normal seed activity. Two mutant Eu3 alleles were recovered, eu3-e1 and Eu3-e3. The eu3-e1/eu3-e1 genotype lacked both activities while Eu3-e3/Eu3-e3 had coordinately reduced leaf (0.1%) and seed (0.1%) activities. Only the Eu3-e3 mutation showed partial dominance, yielding about 5%–10% normal activity for each urease in the heterozygous state. Each homozygous mutant contained normal levels of embryo-specific urease mRNA and protein subunit, both of normal size. However, urease polymerization was aberrant in all three mutants. In all cases where urease could be measured, it was found to be temperature sensitive and, in addition, the embryospecific urease of Eu3-e3/Eu3-e3 had an altered pH dependence. These mutants may be defective in a urease maturation function common to both isozymes as suggested by the normal levels of urease gene product, coordinately (or nearly so) reduced urease isozyme activities, temperature sensitivity in both ureases (Eu3-e3) and the non-linkage of Eu2 and Eu3 to the locus encoding embryo-specific urease (Sun-Eul). Ubiquitous urease activity is reduced in mutant seed coat and callus culture as well as in leaf and cotyledon tissue. No mutant callus utilized urea (5 to 10 nM) as sole nitrogen source. However, all mutant cell lines tolerated normally toxic levels of urea (25 to 250 mM) added to medium containing KNO₃/NH₄NO₃ as nitrogen source. Urea thus may be used in cell culture as a selection agent for phenotypes either lacking or regaining an active ubiquitous urease.     

A new mutant class of soybean lacks urease in leaves but not in leaf-derived callus or in roots

Summary We reported earlier the recovery of two classes of soybean urease mutants in soybean (Glycine max L. Merr. cv. Williams). Class I mutants lack the embryo-specific urease while class II mutants lack the activities of both urease isozymes, the embryo-specific and the ubiquitous urease, the latter found in all tissues examined. We report here the recovery of a true-breeding mutant, aj3, which represents the third phenotypic class: normal levels of embryo-specific urease and little or no ubiquitous urease. Unlike class II mutant plants which lack urease in all tissue, aj3 lacks urease activity only in leaves (ca. 2% normal activity); its roots have near normal urease activity. Callus derived from leaves of aj3 has 14% to 40% the urease activity of Williams 82 callus. This partial reduction in urease activity in aj3 callus is sufficient to reduce growth with urea as sole nitrogen source and to confer resistance to 50 mM urea added to callus maintenance medium. Leaves of aj3 produce more than 40 times the urease antigen expected from their urease activity. The aj3 trait is due to a single recessive lesion which is not allelic with lesions at theEu2, Eu3 (class II) orEu1 (class I) loci. We designate the aj3 genotype aseu4/eu4.     

The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase

The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, , of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN"), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any constructs; however, PhbN" interacted with a region of (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN" was also screened for interactions against a variety of fragments of the catalytic subunit of PhK; however, no interactions were detected, even with fulllength . Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.     

Female-specific phages and F-minus strains of Escherichia coli K12

Summary The female-specific phages, I, II, W31 and H (but not T3 and T7) show a low efficiency of plating on all F-minus strains of Escherichia coli K12 except for the thr leu thi mutants descended from strain Y53. The locus responsible is linked to the histidine region and was presumably eliminated from the Y53 line of mutants in the course of mutagenesis.     

Soybean Roots Retain the Seed Urease Isozyme Synthesized during Embryo Development

Roots of young soybean (Glycine max [L.] Merr.) plants (up to 25 days old) contain two distinct urease isozymes, which are separable by hydroxyapatite chromatography. These two urease species (URE1 and URE2) differ in: (a) electrophoretic mobility in native gels, (b) pH dependence, and (c) recognition by a monoclonal antibody specific for the seed (“embryo-specific”) urease. By these parameters root URE1 urease is similar to the abundant embryo-specific urease isozyme, while root URE2 resembles the “ubiquitous” urease which has previously been found in all soybean tissues examined (leaf, embryo, seed coat, and cultured cells). The embryo-specific and ubiquitous urease isozymes are products of the Eu1 and Eu4 structural genes, respectively. Roots of the eu1-sun/eu1-sun genotype, which lacks the embryo-specific urease (i.e. `seed urease-null'), contain no URE1 urease activity. Roots of eu4/eu4, which lacks ubiquitous urease, lack the URE2 (leaflike) urease activity. From these genetic and biochemical criteria, then, we conclude that URE1 and URE2 are the embryo-specific and ubiquitous ureases, respectively. Adventitious roots generated from cuttings of any urease genotype lack URE1 activity. In seedling roots the seedlike (URE1) activity declines during development. Roots of 3-week-old plants contain 5% of the total URE1 activity of the radicle of 4-day-old seedlings, which, in turn, has approximately the same urease activity level as the dormant embryonic axis. The embryo-specific urease incorporates label from [³⁵S]methionine during embryo development but not during germination, indicating that there is no de novo synthesis of the embryo-specific (URE1) urease in the germinating root. We conclude that the seedlike urease (URE1) found in roots of young soybean plants is a remnant of the Eu1-encoded, abundant, embryo-specific urease which accumulates in the embryonic root axis during seed development.     

A method for the isolation of phage mutants altered in their response to lysogenic induction

Summary Phage cl ⁺ gives clear plaques whereas phage cIind ^- gives turbid plaques on a lawn of a mutant strain of E. coli K12. This strain, called STS, carries mutation spr in a tif sfi genetic background. I hypothesize that upon temperate phage infection, STS bacteria spontaneously inactivate phage repressor by the same mechanism involved in normal lysogenic induction which results in obligatory lytic growth of ⁺. The use of the STS mutant facilitates the isolation and genetic analysis of phage mutants with an abnormal response to lysogenic induction.     

Modification of the B-ring during flavonoid synthesis in Petunia hybrida: Introduction of the 3′-hydroxyl group regulated by the gene Ht1

The flower-color mutants of Petunia hybrida W37 and W18, which are homozygous recessive for the anthocyanin gene An3, accumulate flavanone glycosides in the flowers. It is concluded that the gene An3 is not directly involved in the synthesis of the C₁₅ skeleton, but that it probably takes part in modifying the skeleton. Complementation experiments with the mutants W18 and M5 show that the hydroxylating gene Ht1, which is reponsible for the introduction of the second hydroxyl group in the B-ring at position 3, is expressed after gene An3. In P. hybrida introduction of the 3-hydroxyl group is therefore not achieved by specific incorporation of caffeic acid during synthesis of the C₁₅ skeleton, but by hydroxylation of a C₁₅ skeleton. When anthocyanin synthesis is blocked by homozygous recessive hydroxylating genes Ht1 and Hf1, as in the mutant M5, dihydrokaempferol-7-glucoside is accumulated. This intermediate is discussed as a possible substrate for B-ring hydroxylation.     

Genetic tests of the roles of the embryonic ureases of soybean

We assayed the in vivo activity of the ureases of soybean (Glycine max) embryos by genetically eliminating the abundant embryo-specific urease, the ubiquitous urease, or a background urease. Mutant embryos accumulated urea (250-fold over progenitor) only when lacking all three ureases and only when developed on plants lacking the ubiquitous urease. Thus, embryo urea is generated in maternal tissue where its accumulation is not mitigated by the background urease. However, the background urease can hydrolyze virtually all urea delivered to the developing embryo. Radicles of 2-day-old germinants accumulated urea in the presence or absence of the embryo-specific urease (2 micromoles per gram dry weight radicle). However, mutants lacking the ubiquitous urease exhibited increased accumulation of urea (to 4-5 micromoles urea per gram dry weight radicle). Thus, the ubiquitous and not the embryo-specific urease hydrolyzes urea generated during germination. In the absence of both of these ureases, the background urease activity (4% of ubiquitous urease) may hydrolyze most of the urea generated. A pleiotropic mutant lacking all urease accumulated 34 micromoles urea per gram dry weight radicle (increasing 2.5-fold at 3 days after germination). Urea (20 millimolar) was toxic to in vitro-cultured cotyledons which contained active embryo-specific urease. Cotyledons lacking the embryo-specific urease accumulated more protein when grown with urea than with no nitrogen source. Among cotyledons lacking the embryo-specific urease, fresh weight increases were virtually unchanged whether grown on urea or on no nitrogen and whether in the presence or absence of the ubiquitous urease. However, elimination of the ubiquitous urease reduced protein deposition on urea-N, and elimination of both the ubiquitous and background ureases further reduced urea-derived protein. The evidence is consistent with the lack of a role in urea hydrolysis for the embryo-specific urease in developing embryos or germinating seeds. Because the embryo-specific urease is deleterious to cotyledons cultured in vitro on urea-N, its role may be to hydrolyze urea in wounded or infected embryos, creating a hostile environment for pest or pathogen. While the ubiquitous urease is operative in leaves and in seedlings, all or most of its function can be assumed by the background urease in embryos and in seedlings.     

Assessment of similarities of pairs and groups of proteins using transformed amino-acid-residue data

Summary Using as a primary standard a representative set of 208 proteins whose amino-acid-residue mole frequencies have been accurately established, a set of standard distributions of mole frequencies is defined for each amino acid, in terms of which percentile values for the observed mole frequencies of the amino-acid residues in any other protein can be determined. Data so transformed have a distribution much closer to Gaussian than untransformed values, and allow meaningful determinations of correlations between the amino-acid-residue compositions of two proteins as well as between pairs of amino-acid-residues within groups of proteins. Of the 153 possible pairs of amino acids (Asx and Glx are used) 39 are significantly correlated atp 0.01 and 22 atp 0.001. A percentile table is included for those wishing to use the method with programmable calculators.The transformed data for amino-acid compositions have been used to perform principal components analyses on groups of proteins in order to determine if meaningful sub-groupings (observable clusters in scatter diagrams) were detectable. Such analyses are shown for the representative set of proteins and for a group of 184 globins. With regard to the globin chains, a correlation is observed for alpha chains in the first principal component projection (PCP), (accounting for 22% of the variance) with respect to the evolutionary time-scale while beta chains show such a correlation in the first and second PCPs (22% and 18% of the variance respectively). Thus, alpha and beta chains appear to diverge from a common progenitor, similar in position to globin chains from primitive forms. Furthermore, globins from primitive forms are nearer to one another than they are to globins from the vertebrates, a finding without a priori reason, suggesting perhaps that once a chain has reached a stable relationship with its environment, strong constraints are placed on the co-existing globin chains so that they maintain appropriate interaction with one another. In addition, positions of the epsilon, gamma and delta chains are in the order: epsilon (embryonal) more primitive than gamma (foetal) more primitive than delta equal to beta (adult).     

Abiotic formation of particles from extracellular organic carbon released by phytoplankton

¹⁴C-labeled extracellular organic carbon (EOC) released by the phytoplankton in a Danish Estuary was shown immediately to form particles (>0.2m) when the products were added to a natural water sample. About 14%–20% of the added activity could be recovered as particles. Any bacterial assimilation of the extracellular products was thus masked. The abiotic origin of the particulate EOC was verified, and it was shown that the particle formation was due to some factors present in the estuarine water with a nominal diameter >0.2m. Precaution must be taken to avoid misinterpretations in studies concerning carbon flow from algae to bacteria.     

Two new species of Isospora (Apicomplexa: Eimeriidae) from passeriform birds of South America

Summary Two new species of Isospora (Apicomplexa: Eimeriidae) are described from the faeces of passeriform birds of South America. I. cyanocoracis n. sp. is described from Cyanocorax chrysops (Passeriformes: Corvidae) and I. paroariae n. sp. from Paroaria coronata (Passeriformes: Emberizidae). I. cyanocoracis oocysts are spherical or subspherical, 28.7×26.8 m (25.0–30.5×24.5–29.0), with bi-layered wall about 2.0 m thick. Micropyle and oocyst residuum are absent; large polar granule present. Sporocysts are ovoid, 19.3×11.4 m (17.0–21.0×10.5–12.2), with smooth, single-layered wall about 0.8 m thick. Stieda and substiedal bodies and sporocyst residuum are present. Sporozoites 12.2×4.2 m (10.5–15.0×3.5–4.5), possess spherical anterior and posterior refractile bodies. I. paroariae oocysts are spherical or subspherical, 22.3×21.4 m (19.5–25.5×18.5–24.0), and have bi-layered wall about 1.8 m thick. Micropyle, polar granule, and oocyst residuum are absent. Sporocysts ovoid, 15.2×10.0 m (14.0–16.5×8.0–11.5), possess smooth, single-layered wall about 0.7 m thick. Stieda and substiedal bodies and sporocyst residuum are present. Sporozoites elongate, 11.3×3.4 m (10.0–13.5×3.2–4.0), have single, large, posterior refractile body. ac]19840712     

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N₂ or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested.In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N₂, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen.In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated feeding with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.     

Vitellogenin concentrations in the haemolymph and ovaries of Ixodes scapularis ticks during vitellogenesis

The vitellogenin and vitellin concentrations in the haemolymph and ovaries of Ixodes scapularis females were determined using a double sandwich enzyme-linked immunosorbent assay. The level of vitellogenin in the haemolymph began to increase just prior to tick detachment from the host and continued to increase until 2 days after detachment. There was a slight decrease in the vitellogenin level 4 days after detachment, but a second peak was observed approximately 5 days after oviposition. Subsequent to oviposition, the vitellogenin levels in the haemolymph quickly decreased. The concentration of vitellogenin in the haemolymph ranged from 1.55 to 11.48 g l^-1 during the period after dropping from the host through oviposition. The concentration of vitellin in the ovaries began to increase as the female began rapid engorgement (0.03 mg per female) and declined after oviposition (0.1 mg per female).     

Phytoferritin accumulations in leaves of diseased coconut palms

Summary A study by electron microscopy of coconut palm (Cocos nucifera L.) leaves from trees infected by the Cape St. Paul wilt (Kaincopé) disease of West Africa was carried out. Samples were obtained during the dry season (Dec.–Jan.) and fixed immediately upon removal from the trees in buffered glutaraldehyde. Further processing for electron microscopy was carried out within a week. No virus particles, mycoplasma-like organisms (MLO), fungi, or bacteria were detected in thin sections. Crystalline or paracrystalline accumulations of electron-opaque granules, approximately 5.5–6 nm in diameter, were observed in disintegrated chloroplasts of mesophyll cells. Based upon their morphological characteristics, formation of the slightly curved, fingerprint arrays or linear rows running parallel, and the visualization of electron-opaque cores in unstained preparations, the granules were identified as phytoferritin particles.This work was sponsored in part by National Science Foundation Grant GB-29280 and by a travel grant from the Food and Agriculture Organization of the United Nations.     

The electrochemical proton gradient and its influence on citrate uptake in tonoplast vesicles of Hevea brasiliensis

The relationship between the electrochemical proton gradient, _H+ ^– , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference () across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a pH of 0.6 units (interior acid) and a of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H⁺ diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m^-3 MgATP the pH was increased to about 1.0 unit and the to about-10 mV. Under these conditions the proton-motive force ( _{p H+} ^– /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg²⁺ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg²⁺ again had no effect. Nigericin greatly increased pH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H⁺-translocating ATPase. The _H+ ^– generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.Abbreviations _H+ ^– electrochemical proton gradient - electrical potential difference across membrane - p proton-motive force ( _H+ ^– /F) - FCCP p-trifluoromethoxycarbonylcyanidephenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Isolation and identification of plant growth inhibitors as candidate(s) for allelopathic substance(s), from aqueous leachate from mesquite (Prosopis juliflora (Sw.) DC.) leaves

As candidate(s) for allelopathic substance(s), two plant growthinhibitors were isolated from aqueous leachate of leaves of mesquite, whichshowa strong allelopathy, and which were identified as syringin and(–)-lariciresinol by their spectral analyses. Syringin inhibited root andshoot growth of lettuce seedlings at concentrations greater than 0.8M, and root and shoot growth of barnyard grass seedlings atconcentrations greater than 2.7 and 26.9 M, respectively. Onthe other hand, (–)-lariciresinol inhibited root and shoot growth oflettuce seedlings at concentrations greater than 2.8 and 0.8M,and root and shoot growth of barnyard grass seedling at concentrations greaterthan 0.8 and 2.8 M, respectively. The contents of syringin and(–)-lariciresinol in the rhizosphere soil of mesquite were 0.34 and 0.38g/g soil, respectively. These results indicate that syringinand (–)-lariciresinol are allelopathic substance(s), and may play rolesinthe allelopathy of mesquite.     

A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells

A strong oxidative stress-inducible peroxidase (POD) promoter was cloned from sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco plants and cultured cells in terms of environmental stress. A POD genomic clone (referred to as SWPA2) consisted of 1824 bp of sequence upstream of the translation start site, two introns (743 bp and 97 bp), and a 1073 bp coding region. SWPA2 had previously been found to encode an anionic POD which was highly expressed in response to oxidative stress. The SWPA2 promoter contained several cis-element sequences implicated in oxidative stress such as GCN-4, AP-1, HSTF, SP-1 reported in animal cells and a plant specific G-box. Employing a transient expression assay in tobacco protoplasts, with five different 5-deletion mutants of the SWPA2 promoter fused to the -glucuronidase (GUS) reporter gene, the 1314 bp mutant deletion mutant showed about 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in transgenic tobacco plants under the control of the –1314 SWPA2 promoter was strongly induced in response to environmental stresses including hydrogen peroxide, wounding and UV treatment. Furthermore, GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the –1314 bp SWPA2 promoter-GUS fusion was strongly expressed after 15 days of subculture compared to other deletion mutants. We anticipate that the –1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.