Discovery of transposable element activity among progeny of tissue culture--derived maize plants.

Tissue culture-derived plants of many species have often been observed to possess both genetic and cytogenetic abnormalities. A high frequency of structurally altered chromosomes in maize (Zea mays L.) plants regenerated from tissue culture led to the prediction that newly activated transposable elements could be detected in regenerated plants. Testcrosses of 1200 progeny from 301 regenerated maize plants confirmed that ten regenerated plants from two independent embryo cell lines contained an active Actransposable element. No active Ac elements were present in the explant sources. Recovery of transposable element activity in regenerated plants indicates that some tissue culture-derived genetic variability may be the result of insertion or excision of transposable elements, or both.

Characterization of Maize Acetyl-Coenzyme A Carboxylase.

Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography. Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 [mu]mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide. The fraction represented 29% of the original activity and was designated ACCase I. A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I. ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA. Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I. Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts. The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms.

Expression of the Acc1 Gene-Encoded Acetyl-Coenzyme A Carboxylase in Developing Maize (Zea mays L.) Kernels.

A mutation (Acc1-S2) in the structural gene for maize (Zea mays L.) acetyl-coenzyme A carboxylase (ACCase) that significantly reduces sethoxydim inhibition of leaf ACCase activity was used to investigate the gene-enzyme relationship regulating ACCase activity during oil deposition in developing kernels. Mutant embryo and endosperm ACCase activities were more than 600-fold less sensitive to sethoxydim inhibition than ACCase in wild-type kernel tissues. Moreover, in vitro cultured mutant kernels developed normally in the presence of sethoxydim concentrations that inhibited wild-type kernel development. The results indicate that the Acc1-encoded ACCase accounts for the majority of ACCase activity in developing maize kernels, suggesting that Acc1-encoded ACCase functions not only during membrane biogenesis in leaves but is also the predominant form of ACCase involved in storage lipid biosynthesis in maize embryos.

Development of maize caryopses resulting from in-vitro pollination.

Intact maize (Zea mays L.) ovaries were excised from unpollinated ears (pistillate inflorescences of field-grown plants and placed on defined, agar-based media in Petri dishes. Application of pollen to the end of silks (styles) positioned outside the Petri dish resulted in fertilization of 46% of the ovaries. The extent of subsequent kernel (caryopsis) development varied. After 40 days some kernels had only embryo development while others had embryo and variable endosperm development. About 5% of the initial ovaries developed into normal kernels; 60% of the kernels with some endosperm germinated under laboratory conditions, and 70% of the embryos excised from the embryo-only kernels germinated on culture media.

Genetic and molecular analysis of tissue-culture-derived Ac elements.

Our previous experiments on maize (Zea mays L.) plants regenerated from tissue culture revealed genetic activity characteristic of the transposable element Activator (Ac) in the progeny of 2-3% of the plants tested, despite the lack of Ac activity in the progenitor plants. The objective of the present study was to determine whether the presence of Ac activity in tissue-culture-derived plants was associated with changes in the number or structure of Ac-homologous DNA sequences. Families segregating for Ac activity were obtained by crossing plants heterozygous for Ac activity onto Ac-responsive tester plants. A DNA probe derived from a previously isolated Ac sequence was used to examine the Ac-homologous sequences within individual progeny seedlings of segregating families and noncultured control materials. All plants tested had six or more Ac-homologous DNA sequences, regardless of whether Ac activity was present. In the segregating progeny of one tissue-culturederived plant, a 30-kb Ac-homologous SstI restriction fragment and a 10-kb Ac-homologous BglII restriction fragment were found to cosegregate with Ac activity. We propose that these fragments contained a previously silent Ac sequence that had been activated during tissue culture. Although one or more Ac sequences were often hypomethylated at internal PvuII and HpaII sites in plants with Ac activity, hypomethylation was not a prerequisite for activity. Reduced methylation at these sites may have been a result rather than a cause of Ac activity.

Kinetic studies of lysine-sensitive aspartate kinase purified from maize suspension cultures.

Steady state substrate kinetics and feedback regulation properties were determined for lysine-sensitive aspartate kinase (AK) purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures. Two AK isoforms (AK Early and AK Late) were separated by two passages through an anion exchange column as the final steps in a procedure giving 1200-fold purification. Kinetic properties were determined for the major AK Late eluting isoform. Assays were conducted at the pH activity maximum (8.0) and with excess Mg(2+) to favor a two-substrate reaction involving aspartate and complexed MgATP. AK catalyzed a sequential reaction in which MgATP and aspartate both bind to the enzyme complex before the ADP and aspartyl-phosphate products are released. The K(m) value calculated for MgATP was 0.43 millimolar and for aspartate was 1.04 millimolar. Cooperativity in substrate binding was not observed and was not induced by lysine. The lysine concentration required for 50% inhibition of AK activity was 7 micromolar. An apparent Hill coefficient of 1.4 indicated a minimum of two lysine-binding sites on the active AK complex. At nonsaturating substrate concentrations, lysine inhibition was characteristic of an S-parabolic, I-parabolic noncompetitive allosteric inhibitor. The parabolic inhibitor replot, Hill coefficients > 1, and the lack of substrate cooperativity were consistent with a model for multiple lysine-binding sites per active AK subunit. Similar kinetic properties were observed for the AK Early isoform.

Tissue culture isolation of a second mutant locus for increased threonine accumulation in maize.

Regenerable maize (Zea mays L.) tissue cultures were selected for ability to grow in the presence of inhibitory (1.0-1.5 mM) concentrations of L-lysine plus L-threonine. Testcross kernels from one regenerated plant (LT20) segregated for wild-type and high free threonine concentration in a 1∶1 ratio consistent with a single dominant gene for high free threonine. Free threonine concentrations (nmol/mg dry weight) increased an average of 29-fold in bulked F2 kernel samples from heterozygous mutant plants, and the total (free plus protein-bound) threonine concentration increased 68%. Increases in protein-bound methionine, lysine and glycine concentrations were also noted, suggesting a possible effect of the mutation on protein concentration and composition. Allelism tests with a previously selected mutant line, Ltr (*)19, showed that two unlinked, codominant genes conditioned the high free threonine phenotype. Based on a separate study of aspartate kinase feedback inhibition characteristics in the two mutant lines, we propose that the mutant alleles [gene and allele designations are according to guidelines for maize genetic nomenclature (Burnham et al. 1975)] be designated Ask-LT19 and Ask2-LT20 for the Ltr (*)19 and LT20 mutants, respectively.

Purification and characterization of lysine-sensitive aspartate kinase from maize cell cultures.

Aspartate kinase is a feedback-regulated enzyme that controls the first step common to the biosynthesis of lysine, threonine, isoleucine, and methionine in plants. Aspartate kinase was purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures for physical and kinetic characterization studies. Partial purification and elution from an anion exchange column resolved two lysine-sensitive aspartate kinase isoforms. Both isoforms were purified >1,200-fold to a minimum specific activity of 18 units/milligram of protein. Both isoforms were sensitive to the lysine analogues S-2-aminoethyl-l-cysteine, l-lysine ethyl ester, and delta-hydroxylysine. No threonine-sensitive form of aspartate kinase was detected at any stage during the purification. Additional purification steps were combined with preparative gel electrophoresis to obtain apparently homogeneous lysine-sensitive aspartate kinase. Aspartate kinase appeared to be a tetramer with a holoenzyme molecular weight of 254,000 and to be composed of 49,000 and 60,000 subunits. The tetramer appeared to disassociate during native gel electrophoresis to 113,000 dalton species that retained aspartate kinase activity.

Recombination is associated with polymorphism of the mitochondrial genomes of maize and sorghum.

Extensive recombination events characterize higher-plant mitochondrial DNAs. Numerous recombination events resulted in the appearance of an unusual mitochondrial open reading frame, urf13-T, which encodes a 13 kDa polypeptide in the male-sterile T cytoplasm of maize. Maize lines with T cytoplasm are unusually susceptible to two fungal pathogens which produce host-selective toxins. Mutants derived from tissue culture expressing male fertility and toxin-insensitivity are characterized by truncation or deletion of urf13-T. These events result from a frameshift associated with a tandem 5 base pair repeat, placing a premature stop codon in frame, or from a recombination event, apparently limited to tissue culture, resulting in the deletion of urf13-T. Neither class of mutants produces the 13 kDa gene product. Repeated sequences that participate in recombination in sorghum appear to be randomly distributed among male-fertile or male-sterile cytoplasms. Processes involved in the evolution of mitochondrial DNAs in higher plants therefore include the generation and deletion of configurations through recombination.

Inheritance of selected pathotoxin resistance in maize plants regenerated from cell cultures.

Texas male-sterile cytoplasm (cms-T) maize is susceptible to Helminthosporium maydis race T and its pathotoxin, whereas nonsterile cytoplasm maize is resistant. Callus cultures initiated from immature embryos of a cms-T genotype, BC(1)A188(T), were susceptible to the toxin and were capable of plant regeneration. Toxin-resistant cell lines were selected by a sublethal enrichment procedure in which cms-T callus was grown for several selection cycles (subculture transfers) in the presence of progressively higher concentrations of toxin. Periodically during the selection process, plants were regenerated from the cms-T cultures to determine their susceptibility or resistance to the toxin. Plants regenerated after four cycles of selection were male-sterile and toxin-susceptible as shown by leaf bioassays. All plants regenerated from cell lines isolated from the fifth selection cycle onward, however, were toxin-resistant and 52 of 65 were fully male-fertile. The remaining 13 "male-sterile" resistant plants did not shed pollen and did not resemble cms-T plants in tassel morphology. Some "male-sterile" plants produced anthers containing a small amount of starch-filled pollen, suggesting that the sterility of these 13 plants was not the result of the cms-T trait. Leaf bioassays on F(1) progeny from regenerated resistant plants indicated that resistance to the toxin was inherited only through the female. The male-fertility trait also was inherited only through the female. After inoculation with H. maydis race T spores, leaf lesion size for progeny from regenerated resistant plants coincided with their reaction to the toxin. This result indicated that plant resistance to the pathogen was closely correlated with the toxin resistance obtained through cell culture selection.

Mutation to male fertility and toxin insensitivity in Texas (T)-cytoplasm maize is associated with a frameshift in a mitochondrial open reading frame.

Tissue culture-derived mutants of male-sterile and disease toxin-sensitive Texas (T)-cytoplasm maize that exhibit male fertility and toxin insensitivity carry numerous alterations in mitochondrial DNA. In these mutants, a 6.7-kilobase Xho I fragment characteristic of parental T cytoplasm has been rearranged. In the mutant T-4, the parental 6.7-kilobase Xho I fragment contains a guanine to adenine transition adjacent to a 5-base-pair insertion not found in T cytoplasm. The insertion, internal to a 345-base-pair open reading frame (T ORF13), generates a frameshift, resulting in a premature stop codon that terminates the open reading frame at base pair 222. In other mutants, the 345-base-pair ORF is part of a 3-kilobase deletion, which extends into a 5-kilobase repeat characteristic of mtDNA from T but not N male-fertile cytoplasm. Clones specific to T ORF13 hybridize to eight transcripts in T and T-4, yet only hybridize to three transcripts in T-7, a deletion mutant. Transcription of the T ORF13 region appears not to be altered in T-4, but the frameshift mutation in the T ORF13 reading frame indicates that a biologically inactive gene product could be associated with the mutational events. The results suggest that cytoplasmic male sterility and disease toxin sensitivity may be associated with presence of T ORF13 in T-cytoplasm maize.

Plastid DNA in developing maize endosperm : genome structure, methylation, and transcript accumulation patterns.

Amyloplasts in storage organs such as maize (Zea mays L.) endosperm are plastid-derived, nonphotosynthetic, starch-accumulating organelles. This study was initiated to characterize the plastid genome in maize endosperm cells containing differentiated amyloplasts and to determine whether plastid genes are transcribed during the period of amyloplast biogenesis in endosperm development. Four cosmid clones representing the total sequence diversity of the maize plastid genome were hybridized to restriction digests of total cellular DNA from isolated 16-day-old endosperms. The hybridization patterns indicated that the plastid DNA present in endosperm tissue was indistinguishable from that in leaf total DNA. Methylation of maize endosperm amyloplast DNA or leaf chloroplast DNA was not detected with the methylation-sensitive enzymes HpaII and EcoRII. Transcripts homologous to the 17 specific plastid DNA BamHI fragments tested were detectable in total RNA prepared from 16-day-old endosperm tissue. Compared with leaf transcripts, the abundance of endosperm transcripts was substantially lower for transcripts detected by 12 different BamHI fragments and was similar or relatively higher for some transcripts homologous to five BamHi fragments. Transcripts homologous to genes for plastid ribosomal small subunit proteins 7 and 12 on fragments 10 and 23 and to an open reading frame on fragment 14 accumulated primarily as unprocessed or partially processed species in endosperm RNA. The demonstration that maize endosperm cells contain an intact, transcriptionally active plastid genome indicates that plastid genes could contribute to amyloplast biogenesis, although no transcripts unique to endosperm were identified.

Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA- auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35,854, predicted from the deduced amino acid sequence, was similar to the 38,000 Mr determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.

Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize.

Aspartate kinase (AK; EC 2.7.2.A) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations,Ask LT19 andAsk2 -LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived amino acids in maize. Kernels of the homozygousAsk2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine, compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 µML-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygousAsk and homozygousAsk2 mutants required 25 and 760 µM lysine for 50% inhibition, respectively, indicating thatAsk andAsk2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the homozygous mutantAsk2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutantAsk2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor ofAsk2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive inhibition of wildtype AK. Reduced lysine sensitivity of theAsk2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygousAsk2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from theAsk andAsk2 structural genes.

Lysine accumulation in maize cell cultures transformed with a lysine-insensitive form of maize dihydrodipicolinate synthase.

Lysine is one of the nutritionally limiting amino acids in food and feed products made from maize (Zea mays L.). Two enzymes in the lysine biosynthesis pathway, aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS), have primary roles in regulating the level of lysine accumulation in plant cells because both enzymes are feedback-inhibited by lysine. An isolated cDNA clone for maize DHPS was modified to encode a DHPS much less sensitive to lysine inhibition. The altered DHPS cDNA was transformed into maize cell suspension cultures to determine the effect on DHPS activity and lysine accumulation. Partially purified DHPS (wildtype plus mutant) from transformed cultures was less sensitive to lysine inhibition than wild-type DHPS from nontransformed cultures. Transformed cultures had cellular free lysine levels as much as four times higher than those of nontransformed controls. Thus, we have shown that reducing the feedback inhibition of DHPS by lysine can lead to increased lysine accumulation in maize cells. Increasing the capacity for lysine synthesis may be an important step in improving the nutritional quality of food and feed products made from maize.

Genetic and amino-acid analysis of two maize threonine-overproducing, lysine-insensitive aspartate kinase mutants.

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37-54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.

Molecular genetics of the maize (Zea mays L.) aspartate kinase-homoserine dehydrogenase gene family.

Aspartate kinase (AK) and homoserine dehydrogenase (HSDH) are enzymes in the aspartate-derived amino acid biosynthetic pathway. Recent biochemical evidence indicates that an AK-HSDH bifunctional enzyme exists in maize (Zea mays L.). In this report, we characterize three genes that encode subunits of AK-HSDH. Two cDNAs, pAKHSDH1 and pAKHSDH2, containing the full-coding sequence, and one partial cDNA, pAKHSDH3, encode amino acid sequences similar to the reported monofunctional AK and HSDH enzymes from prokaryotes and yeast (Saccharomyces cerevisiae) and to AK-HSDH bifunctional enzymes of prokaryotes, yeast, carrot (Daucus carota), and Arabidopsis thaliana. Immunological and biochemical analyses verify that the cDNAs encode AK-HSDH and indicate that both the AK and HSDH activities are feedback inhibited by threonine. RNA blots identify a 3.2-kb transcript in all maize tissues examined. pAKHSDH1 and pAKHSDH2 map to chromosomes 4L and 2S, respectively. This study shows that maize contains AK-HSDH bifunctional enzyme(s) encoded by a small gene family of at least three genes. Maize AK-HSDH has conserved sequences found in communication modules of prokaryotic two-component regulatory systems, which has led us to propose that maize AK-HSDH may be involved in a similar regulatory mechanism.

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase.

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA, as a lacZ fusion protein was selected in a dapA- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.

Mitochondrial DNA variation in maize plants regenerated during tissue culture selection.

Plants resistant to Helminthosporium maydis race T were obtained following selection for H. maydis pathotoxin resistance in tissue cultures of susceptible, Texas male-sterile (T) cytoplasm maize. The selected lines transmitted H. maydis resistance to their sexual progeny as an extranuclear trait. Of 167 resistant, regenerated plants, 97 were male fertile and 70 were classified male sterile for reasons that included abnormal plant, tassel, anther or pollen development. No progeny were obtained from these male-sterile, resistant plants. Male fertility and resistance to the Phyllosticta maydis pathotoxin that specifically affects T cytoplasm maize were co-transmitted with H. maydis resistance to progeny of male-fertile, resistant plants. These three traits previously were associated only with the normal (N) male-fertile cytoplasm condition in maize. Three generations of progeny testing provided no indication that the cytoplasmic association of male sterility and toxin susceptibility had been broken by this selection and regeneration procedure. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) revealed that three selected, resistant lines had distinct mtDNA organization that distinguished them from each other, from T and from N cytoplasm maize. Restriction patterns of the selected resistant lines were similar to those from T cytoplasm mtDNA; these patterns had not been observed in any previous analyses of various sources of T cytoplasm. The mtDNA analyses indicated that the male-fertile, toxin-resistant lines did not originate from selection of N mitochondrial genomes coexisting previously with T genomes in the T cytoplasm line used for selection.

Isolation and characterization of two homoserine dehydrogenases from maize suspension cultures.

Homoserine dehydrogenase in unpurified extracts of maize (Zea mays L.) cell suspensions is inhibited 73% by the feedback regulator threonine; the remaining 27% of the total activity is not affected even by high concentrations of threonine. The threonine-resistant and threonine-sensitive homoserine dehydrogenase activities were separated by affinity chromatography on Blue Sepharose columns, and the two distinct homoserine dehydrogenases were purified. The threonine-resistant enzyme is an Mr = 70,000 dimer of two Mr = 38,000 subunits and the threonine-sensitive enzyme is an Mr = 190,000 dimer containing two apparently different subunits with molecular weights of 89,000 and 93,000. The threonine-resistant enzyme exhibits normal Michaelis-Menten kinetics and its activity is not affected by any of the amino acid end products of the aspartate pathway. The threonine-sensitive enzyme exhibits positive cooperative kinetics with respect to NADPH and is inhibited by threonine and stimulated by isoleucine. All attempts to affect interconversion of the two purified enzymes have been unsuccessful. Because the purified enzymes correspond to activities present in crude extracts of various maize tissues, it is concluded that the two types of homoserine dehydrogenase are natural in vivo constituents of maize.