Soybean Leaf Urease: A Seed Enzyme?

The soybean (Glycine max L. [Merrill]) var Itachi has 0.2 to 0.3% the urease activity found in developing embryos of a normal line, Prize. The hydroxyurea sensitivity and pH preference of this basal seed urease indicate that it represents a unique enzyme rather than an unusually low level of the normal seed urease. Itachi's seed urease is less sensitive to hydroxyurea inhibition (65-80% inhibition) than Prize seed urease (85-95% inhibition) and is more active at pH 6.1 and 8.8 than at 7.4, whereas the normal seed urease is least active at pH 8.8. Both properties of the basal seed urease are in agreement with the behavior of the leaf urease in extracts of Prize and Itachi leaves.

Neither the leaf urease nor the Itachi seed urease is immuneprecipitated by affinity-purified seed urease antibodies. However, when antibody is in excess, Staphylococcus aureus (Cowan) cell walls containing protein A can precipitate soluble antibody-urease complexes (47-68% of total enzyme) from both leaf (Itachi and Prize) and Itachi seed extracts. Under identical conditions, greater than 90% of Prize seed urease is precipitated. At a 100-fold dilution of antibody, 60% of Prize seed urease is still antibody-complexed while the antibody recognition of the leaf or Itachi seed urease is reduced to 2 to 24%.

The cell culture urease also resembles leaf urease by the criteria of pH preference, hydroxyurea sensitivity, and recognition by seed urease antibodies. In the presence of cycloheximide, nickel stimulates cell culture urease levels (14- or 35-fold depending on assay pH) indicating that cell cultures make a preponderance of apourease under nickel-limiting conditions.

Inasmuch as the ureases of leaf, cell culture, and Itachi seeds are more closely related to each other than they are to the abundant (Prize) seed urease, suggests that the three tissues either contain an identical urease or related tissue-specific isozymes. This second form of urease may have an assimilatory role since it is found in both leaf and seed sink tissues and is required for urea assimilation in cell culture (Polacco 1977 Plant Physiol 59: 827-830).

     

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.

     

Nickel is not required for apourease synthesis in soybean seeds

Soybeans (Glycine max L. Merr. cv `Maple Presto') harvested from plants cultured in nickel-free medium had <0.005% the activity of nickel-sufficient beans and only 15% the activity of a urease-null variety, Itachi. However, whereas Itachi has no detectable urease protein, nickel-free beans of the variety Maple Presto exhibit normal or near normal levels of urease apoprotein. Thus, nickel isn't necessary for urease apoprotein synthesis. The apoprotein wasn't activated by nickel in vitro but, upon seed imbibition of nickel, urease was partially activated. This in vivo activation was not inhibited by cychoheximide.     

Patterns of urease synthesis in developing soybeans

An examination of in vivo polysome-bound activity indicates that soybean (Glycine max, cv. Prize) seed urease is synthesized on large polysomes (n ≥ 15). In vitro urease synthesis is directed by a large RNA (3,000-3,300 nucleotides). Urease synthesis occurs throughout the normal protein biosynthetic phase of the developing seed. Surprisingly, the activity/antigen ratios of urease increase throughout development. Urease appears to be in a more highly polymerized state in mature beans versus beans in early development.     

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     

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.     

Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein

During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [³H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat.     

Soybean lectin and related proteins in seeds and roots of le and le soybean varieties

The localizations of soybean lectin (SBL) and antigenically related proteins in cotyledons and roots of lectin positive (Le⁺) and lectin negative (Le⁻) soybean cultivars were compared by light level immunocytochemistry using antibodies produced against the 120 kilodalton (kD) native seed lectin tetramer or its subunits. Lectin is present in the protein bodies of cotyledons cells as are two other seed proteins, the Kunitz trypsin inhibitor and the storage protein glycinin. Analysis of single seed extracts by immunoblotting of sodium dodecyl sulfate-polyacrylamide gels using the same antibodies, reveals up to 4 milligrams of the 30 kD seed lectin protein is present per seed in the Le⁺ varieties. There is no detectable lectin in the protein bodies of Le⁻ cotyledons as determined by immunocytochemistry and immunoblotting. Enzyme-linked immunosorbent assay confirmed this result to a sensitivity of less than 20 nanograms per seed. In contrast, the roots of both Le⁺ and Le⁻ plants bind the seed lectin antibody during immunocytochemistry, with fluorescence mainly localized in vacuole-like bodies in the epidermis. Root extracts contain a 33 kD polypeptide that binds anti-SBL antibody at an estimated minimal level of 20 nanograms per 4-day seedling, or 2.0 nanograms per primary root tip. This polypeptide is also present in the embryo axis and in leaves. The latter also contain a 26 kD species that binds seed lectin antibody. The 30 kD seed lectin subunit, however, is not detectable in roots or leaves.     

Soybean leaf urease: Comparison with seed urease

Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide-derived urea to NH₃ and CO₂. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 m M ). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent K_m (0.8 m M ), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross-reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.     

Biochemical, electrophoretic and immunological characterization of peroxisomal enzymes in three soybean organs

Biochemical, electrophoretic and immunological studies were made among peroxisomal enzymes in three organs of soybean [Glycine max (L.) Merr. cv. Centennial] to compare the enzyme distribution and characteristics of specialized peroxisomes in one species. Leaves, nodules and etiolated cotyledons were compared with regard to several enzymes localized solely in their peroxisomes: catalase (EC 1.11.1.6), malate synthase (EC 4.1.3.2), glycolate oxidase (EC 1.1.3.1), and urate oxidase (EC 1.7.3.3). Catalase activity was found in all tissue extracts. Electrophoresis on native polyacrylamide gels indicated that leaf catalase migrated more anodally than nodule or cotyledon catalase as shown by both activity staining and Western blotting. Malate synthase activity and immunologically detectable protein were present only in the cotyledon extracts. Western blots of denaturing (lithium dodecyl sulfate) gels probed with anti-cotton malate synthase antiserum, reveal a single subunit of 63 kDa in both cotton and soybean cotyledons. Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.     

The chemical ionization mass spectrometric analysis of phenylthiohydantoin and 2-anilino-5-thiazolinone amino acids obtained from the Edman degradation of proteins and peptides

Using the ability of ammonia to form complexes with several compounds, we have developed a method for visualization and fixation of urease (EC 3.5.1.5) activity in polyacrylamide gels. In this method lead acetate was used alone or in combination with a pH-sensitive indicator to detect the enzyme activity. Results indicated a fairly linear relationship between the urease concentration (from 0.6 to 3.0 IU) and the amount of lead precipitate formed. Further, this method appears to be more sensitive than staining with pH indicator alone. It can also be effectively used to detect the enzyme activity in crude protein extracts. In addition, lead acetate-stained gels can be stored either wet or dry indefinitely with no visual loss in the activity profiles.     

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     

Arginase isozymes and their detection by catalytic staining in starch gel

A method for the separation by starch gel and the in situ detection of the isozymes of arginase is presented. The method is capable of detecting 0.2 units of activity; it is specific, giving no staining in the absence of either arginine or urease from the developing solution. For purposes of the present study all parameters have been optimized for the separation and localization of arginase isozymes in tissue extracts. The method ought to be applicable, however, with appropriate modifications, to other enzymes yielding either alkaline reaction products (detectable in the presence of excess thiol) or yielding free thiol (such as reduced Coenzyme A) in alkaline medium.     

Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants

Exogenous applications of gibberellins (GAs) increased the extractable activity of leaf sucrose phosphate synthase (SPS) in soybean (Glycine max [L.]) and spinach (Spinacia oleracea [L.]). The response to GA applications was detectable within 2 h postapplication and was still observed 6 h, 24 h, and 7 d after treatment. When paclobutrazol, a GA biosynthesis inhibitor, was applied to intact soybean and spinach plants, decreased extractable SPS activity resulted within 24 h following the treatment. Different methods of GA application (spray, injection, capillary wick, and excised leaf systems) produced similar effects on SPS activity of soybean leaves. Protein synthesis in soybean leaves appeared to be necessary for GA-promoted SPS activity because gibberellic acid only partially reversed the inhibitory effect of pretreatment with cycloheximide. Levels of SPS protein from crude extracts of spinach plants were measured by a dot blot technique using monoclonal antibodies against SPS. Application of gibberellic acid to spinach leaves increased levels of SPS protein 2 h, 24 h, and 7 d after treatment. The results suggest that, in both soybean and spinach, GA is one of the endogenous hormonal factors that regulate the steady-state level of SPS protein and, hence, its activity.     

One-step affinity purification of urease from jack beans

Jack bean (Canivalia ensiformis) urease (EC3.5.1.5) was purified in one-step by ligand affinity chromatography using epoxy-activated Sepharose 6B-urea. The yield of the purified enzyme was about 80% with a specific activity of about 500 U/mg of protein. The enzyme was apparently homogeneous when analyzed by SDS-PAGE and native PAGE. The protein band on native PAGE coincided with the stained band of urease activity. The affinity column could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either acetamide or semicarbazide as affinity ligands were also found to be useful for the isolation of urease.     

Nitrogen metabolism in soybean tissue culture: I. Assimilation of urea

Cultured soybean (Glycine max, Kanrich variety) cells grow with 25 mm urea as the sole nitrogen source but at a slower rate than with the Murashige and Skoog (MS) (Physiol. Plant. 15: 473-497, 1962) nitrogen source of 18.8 mm KNO(3) and 20.6 mm NH(4)NO(3). Growth with urea is restricted by 18.8 mm NO(3) (-), 50 mm methylammonia, 10 mm citrate or 100 mum hydroxyurea, substances which are much less restrictive or nonrestrictive in the presence of ammonia nitrogen source. The restrictive conditions of urea assimilation were examined as possible bases for selection schemes to recover urease-overproducing mutants. Since urease has higher methionine levels than the soybean seed proteins among which it is found, such selections may be a model for improving seed protein quality by plant cell culture techniques.Callus will not grow with 1 mm urea plus 18.8 mm KNO(3). Urease levels decrease 80% within two divisions after transfer from MS nitrogen source to 1 mm urea plus 18.8 mm KNO(3). Hydroxyurea is a potent inhibitor of soybean urease and this appears to be the basis for its inhibition of urea utilization by callus cells.Stationary phase suspension cultures grown with MS nitrogen source exhibit trace or zero urease levels. Soon after transfer to fresh medium (24 hours after escape from lag), urease levels increase in the presence of both MS or urea nitrogen source. However, the increase is 10 to 20 times greater in the presence of urea. NH(4)Cl (50 mm) lowers urease induction by 50% whereas 50 mm methylammonium chloride results in more drastic reductions in urea-stimulated urease levels. Citrate (10 mm) completely blocks urease synthesis in the presence of urea.Ammonia and methylammonia do not inhibit soybean urease nor do they appreciably inhibit urea uptake by suspension cultures. It appears likely that methylammonia inhibits urea utilization in cultured soybean cells primarily due to its "repressive" effect on urease synthesis.Citrate does not inhibit urease activity in vitro and exhibits only a partial inhibition (0-50% in several experiments) of urea uptake. It appears likely that the citrate elimination of urease production by cultured soybean cells is due to its chelation of trace Ni(2+) in the growth medium. Dixon et al. (J. Am. Chem. Soc. 97: 4131-4133, 1975) have reported that jack bean (Canavalia ensiformis) urease contains nickel at the active site.     

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.     

Ribosome inactivating protein-like activity in seeds of diverse Cucurbitaceae plants

• 1.1. Seed extracts of 20 plants species belonging to the family Cucurbitaceae were examined for their ability to inhibit protein synthesis in rabbit reticulocyte lysate and induce mid-term abortion in mice.
• 2.2. Eleven extracts were found to inhibit protein synthesis by about or over 90%, seven extracts produced about 80% inhibition, one caused about 70% inhibition and one brought about approx. 40% inhibition, when the extracts were tested at a final concentration of 10 μg per ml.
• 3.3. All of the seed extracts possessed potent mid-term abortifacient activity.
• 4.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the seed extracts disclosed the existence of a Coomassie Blue-stainable band with a mol. wt of ca 30,000 Da. This band probably accounts for the protein synthesis inhibiting and mid-term abortifacient activities.
• 5.5. There was a similarity in the electrophoretograms of seed extracts of plants belonging to the same genus.

     

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.