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.     

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.     

Soybean genes involved in nickel insertion into urease

In soybean, mutation in the Eu2 or in the Eu3 gene eliminates the activities, but not the proteins, of the embryo-specific and the ubiquitous ureases, encoded by Eu1 and Eu4, respectively. This pleiotropic urease-negative phenotype is consistent with accessory gene functions encoded by Eu2 and Eu3, i.e. correct insertion of Ni into the metallocentre of each urease. To test an accessory gene function an examination was made of segregation of alleles at Eu2 and Eu3 with segregation of an RFLP revealed by a plant homologue of the bacterial urease accessory gene, ureG. The eu3-e1/eu3-e1 mutant, which has a urease activity-null phenotype, lacked a 1.4 kb EcoRV genomic fragment found in progenitor cultivar Williams, cv Williams 82 and in two mutants at the Eu2 locus, eu2/eu2 and EN24, the latter described here for the first time. The lack of the 1.4 kb band segregated with eu3-e1 in a cross of eu3-e1/eu3-e1 x EN24. The second approach was to attempt partial correction of the urease-negative trait by Ni supplementation in vitro. First a small, reproducible stimulation of activity in mixed extracts of mutants which complement genetically, namely {eu2/eu2 plus eu3-e1/eu3-e1} and {EN24 plus eu3-e1/eu3-e1} was observed. Activation proceeded for several hours in these extracts containing endogenous Ni. In mixed extracts from Ni-free embryos, activation was dependent on added Ni; Ni had no effect on individual mutant extracts. By genetic and biochemical criteria the ubiquitous urease was the sole or major species activated, an activation which approached 10% normal activity.Key words: Nickel, urease, soybean, UreG, UreE.      

Urease-null and hydrogenase-null phenotypes of a phylloplane bacterium reveal altered nickel metabolism in two soybean mutants

Mutation at either of two genetic loci (Eu2 or Eu3) in soybean (Glycine max [L.] Merr.) results in a pleiotropic elimination of the activity of both major urease isozymes. Surprisingly, the phenotype of a phylloplane bacterium, Methylobacterium mesophilicum, living on the leaves of eu2/eu2 or eu3-e1/eu3-e1 mutants is also affected by these plant mutations. The bacteria isolated from leaves of these soybean mutants have transient urease- and hydrogenase-deficient phenotypes that can be corrected by the addition of nickel to free-living cultures. The same bacterium growing on wild-type soybeans or on urease mutants eu1-sun/eu1-sun or eu4/eu4, each deficient in only one urease isozyme, are urease-positive. These results suggest that the bacterium living on the eu2/eu2 or eu3-e1/eu3-e1 mutant is unable to produce an active urease or hydrogenase because it is effectively starved for nickel. We infer that mutations at Eu2 or Eu3 result in defects in nickel metabolism but not in Ni²⁺ uptake or transport, because eu2/eu2 and eu3-e1/eu3-e1 mutants exhibit normal uptake of ⁶³NiCl₂. Moreover, wild-type plants grafted on mutant rootstocks produce seeds with fully active urease, indicating unimpeded transport of nickel through mutant roots and stems.     

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.     

Mutational analysis of the embryo-specific urease locus of soybean

Summary By a non-destructive urease screen of M₂ soybean (Glycine max [L.] Merr. cv. Williams) seeds, four truebreeding mutants (n4, n6, n7 and n8) were recovered which lack most (n6, n8) or all (n4, n7) embryo-specific urease activity. This trait was due to a single, recessive lesion at the Sun (seed urease-null) locus identified earlier in an exotic germplasm (PI 229324, Itachi). All sun mutants produced normal ubiquitous urease, the low abundance isozyme found in all soybean tissues examined. Tight mutants n4 and n7 accumulated no detectable embryo-specific urease protein or mRNA; n6 and n8 accumulated normal or near normal levels of urease mRNA but had seed urease protein levels approximately 5% and 0.5%, respectively, of the progenitor. Mutant n8 appeared to produce a low level of fully active urease (approximately 0.7% activity level, approximately 0.5% protein level) while n6 produced a higher level of an altered, nearly inactive urease (0.09% activity level, approximately 5% protein level). Urease alterations in n6 were manifested by its increased temperature sensitivity and variation in aggregation state and pH preference. Thus, mutations in the Sun locus affected both the level and the nature of the embryo-specific urease gene products indicating that Sun encodes the embryo-specific urease. We reported earliet that the Eul locus, which controls the aggregation state of the embryo-specific urease, is one map unit from Sun and that the Eul allele cis to sun is not expressed (Kloth et al. 1987). That the level of urease gene product, its aggregation state and other enzyme properties can be affected by induced sun mutations, suggests that the Eul and sun alleles are at the same locus.Abbreviations ME -mercaptoethanol - NMU N-nitroso-N-methyl urea - TM Tris-maleate     

Structure and possible ureide degrading function of the ubiquitous urease of soybean

Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 800-804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease.

The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection (“Western Blot”). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-0.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric.

The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid.

     

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     

Soybean ubiquitous urease with purification facilitator: An addition to the moonlighting studies toolbox

Ureases are nickel-dependent enzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. In soybean (Glycine max), the embryo-specific urease (eSBU), the ubiquitous urease (uSBU), and a third isoform (SBU-III) are synthesized. Our group has previously demonstrated that eSBU, purified from seeds, has antifungal properties against phytopathogenic fungi, entomotoxicity against Dysdercus peruvianus, the ability to induce blood platelet aggregation, and these properties are independent of its enzymatic activity. Here we describe the biological properties of apo-uSBU fused to glutathione S-transferase (GST) produced in Escherichia coli. Removal of GST affected apo-uSBU stability. We performed a Response Surface Methodology to optimize GST-uSBU production to 5 mg per liter and then bioassays were carried out. The recombinant protein exhibited inhibitory effects on filamentous fungi and affected fungal secondary metabolism. Candida albicans and C. tropicalis were also susceptible to GST-uSBU and formed pseudo-hyphae. The fusion protein was toxic against Rhodnius prolixus, with the toxicity being accompanied by in vivo and in vitro hemocyte aggregation. Rabbit platelet also aggregated in the presence of GST-uSBU. Thus, uSBU displayed similar biological properties as previously described for eSBU even when fused to GST, reinforcing the proposed role of ureases in plant defense.     

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO₄) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.     

Host-Parasite Relationships of Hoplolaimus columbus on Cotton and Soybean

Hoplolaimus columbus suppressed growth and pod yield of soybean in greenhouse tests. Although populations of H. columbus decreased in short-term experiments, increases occurred in long-term studies. The nematode caused extensive damage to the cortical parenchyma and occasionally to the endodermal-vascular region of both cotton and soybean roots. The nematode frequently entered secondary root primordia. Roots of soybean parasitized by H. columbus at high inoculum levels were severely damaged. The relationship of populations of H. columbus and stunting of soybean and cotton is discussed.     

Prion Variants and Species Barriers Among Saccharomyces Ure2 Proteins

As hamster scrapie cannot infect mice, due to sequence differences in their PrP proteins, we find “species barriers” to transmission of the [URE3] prion in Saccharomyces cerevisiae among Ure2 proteins of S. cerevisiae, paradoxus, bayanus, cariocanus, and mikatae on the basis of differences among their Ure2p prion domain sequences. The rapid variation of the N-terminal Ure2p prion domains results in protection against the detrimental effects of infection by a prion, just as the PrP residue 129 Met/Val polymorphism may have arisen to protect humans from the effects of cannibalism. Just as spread of bovine spongiform encephalopathy prion variant is less impaired by species barriers than is sheep scrapie, we find that some [URE3] prion variants are infectious to another yeast species while other variants (with the identical amino acid sequence) are not. The species barrier is thus prion variant dependent as in mammals. [URE3] prion variant characteristics are maintained even on passage through the Ure2p of another species. Ure2p of Saccharomyces castelli has an N-terminal Q/N-rich “prion domain” but does not form prions (in S. cerevisiae) and is not infected with [URE3] from Ure2p of other Saccharomyces. This implies that conservation of its prion domain is not for the purpose of forming prions. Indeed the Ure2p prion domain has been shown to be important, though not essential, for the nitrogen catabolism regulatory role of the protein.     

Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture

The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2′,4′-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4-dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site.     

Aluminum Partitioning in Intact Roots of Aluminum-Tolerant and Aluminum-Sensitive Wheat (Triticum aestivum L.) Cultivars

Aluminum (Al) partitioning in intact roots of wheat (Triticum aestivum L.) cultivars that differ in sensitivity to Al was investigated. Roots of intact seedlings were exposed to Al for up to 24 hours and distribution of Al was assessed visually by hematoxylin staining or by direct measurement of concentration of Al by atomic absorption spectrophotometry or ion chromatography. Major differences in Al accumulation between Al-tolerant (Atlas 66) and Al-sensitive (Tam 105) cultivars were found in the growing regions 0 to 2 and 2 to 5 millimeters from the root apex. Al content was 9 to 13 times greater in the 0 to 2 millimeters root tips of cv Tam 105 than in the tips of cv Atlas 66 when exposed to 50 micromolar Al for 19 to 24 hours. The oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazone and the protein synthesis inhibitor cycloheximide increased Al uptake by intact root tips of cv Atlas 66. Also, loss of Al from the roots of both cultivars was measured after the roots were “pulsed” with 50 micromolar Al for 2 hours and then placed in an Al-free nutrient solution for 6 hours. The 0 to 2 millimeter root tips of cv Tam 105 lost 30% of the absorbed Al, whereas the tips of cv Atlas 66 lost 60%. In light of these results, we conclude that the differential Al sensitivity in wheat correlates with the concentration of Al in the root meristems. The data support the hypothesis that part of the mechanism for Al tolerance in wheat is based on a metabolism-dependent exclusion of Al from the sensitive meristems.     

Evidence for the existence of an intracellular root-lectin in soybeans

Soybean (Glycine max (L.) Merr.) root lectin, identified as extractable agglutination activity, was shown to reappear following 15-h incubations of roots that had previously been stripped of all extractable lectin activity. Additional lectin activity was released following disruption of the root tissues and cellular fractionation. These lectin activities were shown to have binding specificity an antibody cross-ractivity similar to soybean seed and root lectins previously described. Thus, it is possible that this intracellular lectin represents the source of extracellular root lectins.