Gibberellins (GAs) are phytohormones required for normal growth and development in higher plants. The Dwarf3 (03) gene of maize encodes an early step in the GA biosynthesis pathway. We transposon-tagged the 0 3 gene using Robertson's Mutator (Mu) and showed that the mutant allele d3-2::Mu8 is linked to a Mu8 element. The DNA flanking the Mu8 element was cloned and shown to be linked to the d3 locus by mapping in a high-resolution population developed by selecting for recombination between d3 and linked genetic marken. To establish unambiguously the identity of the cloned gene as 03, a second mutant allele of 0 3 (d3-4) was also cloned and characterized using the d3-2::MuB sequences as a probe. d3-4 was found to have a nove1 insertion element, named Sleepy, inserted into an exon. A third mutant allele, d3-1, which has the same size 3' restriction fragments as d3-4 but different 5' restriction fragments, was found to contain a Sleepy insertion at the same position as d3-4. On the basis of the pedigree, Sleepy insertion, and restriction map, d3-1 appears to represent a recombinational derivative of d3-4. The 0 3 gene encodes a predicted protein with significant sequence similarity to cytochrome P450 enzymes. Analysis of D3 mRNA showed that the 03 transcript is expressed in roots, developing leaves, the vegetative meristem, and suspension culture cells. We detected reduced 0 3 mRNA levels in the mutant allele d3-5.

Maize (Zea mays L.) DwarJ8-1 (DS-1) is an andromonoecious dwarf mutant proposed to be involved in gibberellin (GA) reception (Fujioka et al. 1988b;Harberd and Freeling 1989). The mutant D8-1 is dominant and GA-nonresponsive (Phinney 1956). We show by map position and similarity of phenotype that five additional dwarf mutants are D8 alleles. We show by map position and similarity of phenotype that a second andromonoecious dwarf mutant, D9-1, defines a duplicate gene. Maize D9-1 and each dominant D8 allele specify a different plant stature, from very mild to very severe dwarfism. Plants of D9-1 and all dominant D8 alleles, except D8-1591, were GA-nonresponsive when treated with 7500 nmol GA 3. The behavior of the mild dwarf D8-1591 was unique in that a small but significant growth response was detected (37% for D8-1591 vs. 130% for the wild type) when treated with 7500 nmol GA 3. These results establish that all dwarf genotypes, except D8-15,91, in one dose set a maximum limit on plant growth and block the normal response to GA. When treated with the GA-synthesis inhibitor paclobutrazol, plants of all dwarf genotypes and wild-type siblings were severely dwarfed. Plants of all dwarf genotypes treated with the GA-synthesis inhibitor paclobutrazol and GA 3 were returned to their normal dwarf phenotype. Dominant dwarfing, delayed flowering, increased tillering, and anther development in the ear are characteristic features of D9-1 and all D8 alleles. The GA-synthesis-deficient dwarfs also have these characteristic features. We discuss the function of the wild-type gene product in the context of the observed results.

Gibberellins (GAs) are phytohormones required for normal growth and development in higher plants. The Dwarf3 (D3) gene of maize encodes an early step in the GA biosynthesis pathway. We transposon-tagged the D3 gene using Robertson's Mutator (Mu) and showed that the mutant allele d3.2::Mu8 is linked to a Mu8 element. The DNA flanking the Mu8 element was cloned and shown to be linked to the d3 locus by mapping in a high-resolution population developed by selecting for recombination between d3 and linked genetic markers. To establish unambiguously the identity of the cloned gene as D3, a second mutant allele of D3 (d3.4) was also cloned and characterized using the d3.2::Mu8 sequences as a probe. d3.4 was found to have a novel insertion element, named Sleepy, inserted into an exon. A third mutant allele, d3.1, which has the same size 3' restriction fragments as d3.4 but different 5' restriction fragments, was found to contain a Sleepy insertion at the same position as d3.4. On the basis of the pedigree, Sleepy insertion, and restriction map, d3.1 appears to represent a recombinational derivative of d3.4. The D3 gene encodes a predicted protein with significant sequence similarity to cytochrome P450 enzymes. Analysis of D3 mRNA showed that the D3 transcript is expressed in roots, developing leaves, the vegetative meristem, and suspension culture cells. We detected reduced D3 mRNA levels in the mutant allele d3.5.

A Mn2-dependent enzymic breakdown of allantoate has been detected in crude and partially purified extracts of developing soybeans. The products detected were CO2, NH3, glyoxylate, labile glyoxylate derivatives, and low levels of urea. Urea is initially produced at less than 10% the rate of urease-independent CO2 release indicating that the activity is not allantoate amidinohydrolase (i.e. urea is not directly cleaved off allantoate). The urease-independent CO2 releasing activity has an apparent K,. of 1.0 nillimolar for allantoate. Ethylenediaminetetraacetate, borate, and acetohydroxamate (all at 10 millimolar) inhibit the enzymic production of NH3, C02, and labile glyoxylate derivatives from allantoate. However, the potent urease inhibitor, phenyl phosphordiamidate does not inhibit CO2 and NH3 release indicating that the action of acetohydrexamate is not due to its inhibition of urease. That the allantoatedegrading activity was more than 5-fold greater in seed coats than in embryos is consistent with the data of Rainbird et al. (Plant Physiol 1984 74: 329-334) which indicate that available ureides are metabolized before reaching the embryo. 2-Ethanolthio, 2'-ureido, acetic acid (NH2COHNCHCO2HSCH2CH20H), the first allantoate-derived product detected by HPLC analysis, is an addition product of mercaptoethanol with an unidentified enzymically produced ureido intermediate that is not derived from ureidoglycolate or oxalurate.

The soybean (Glycinc max L. [Merrilli) var Itachi has 0.2 to 03% 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 (65480% 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 (4748% of total enzyme) from both leaf (Itachi and Prize) and Itachi seed extracts. Under identical conditions, greater than 90% ofPrize 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 ofcycloheximide, 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).The significance of high levels of urease in soybean seeds is still not understood. A recently identified seed urease-negative variety, Itachi (19,23), was found to germinate normally and grow to maturity with symbiotically fixed N2. These observations suggest that the abundant seed urease is not involved in either the mobilization of nitrogen reserves in the germinating seed or in the catabolism of ureides in the developing seed. However, Itachi is not completely urease-negative: its cell cultures can