Natural variants of pearl millet (Pennisetum typhoides) with altered levels of set II alcohol dehydrogenase activity

Two linked genes, Adh1 and Adh2, specify three sets of ADH isozymes in pearl millet. Set I is a homodimer specified by Adh1, Set III is a homodimer specified by Adh2, and Set II is a heterodimer consisting of one ADH1 subunit and one ADH2 subunit. Dry seeds exhibit only Sets I and II. Anaerobic treatment of seeds greatly increases the activity of Sets I and II and causes the Set III isozymes to be expressed. In the investigation reported here, the ADH zymogram phenotypes of 112 inbred pearl millet lines were analyzed. Two kinds of naturally occurring ADH variant strains were observed: in the low-activity variant, Set II activity is low in the dry seed, and no Set III activity is present upon anaerobic treatment. In the high-activity variant, Set II activity is high and Set III isozymes are expressed in the dry seed. The mutation in the high-activity strain appears to affect the product of Adh2 and not the product of Adh1. Dominance tests show that the mutations in both types of variant strains act in cis. These observations and linkage tests indicate that the mutations are closely linked to or at the Adh2 locus.This work was supported by a PHS National Research Service Award Training Grant in Genetics to the Biology Department of the University of Oregon.     

Genetic analysis of alcohol dehydrogenase isozymes in pearl millet (Pennisetum typhoides)

Pearl millet (Pennisetum typhoides) produces three ADH isozymes, sets I, II, and III, with set III being expressed only in anaerobically treated seeds or seedlings. Variant strains have been identified which produce ADH isozymes with altered electrophoretic mobilities for sets I and II but not for set III activity. Based on genetic analysis of these variants and on dissociation-reassociation experiments, we propose that the three ADH isozymes are dimers of subunits coded by two structural genes, Adh1 and Adh2, with set I being a homodimer specified by Adh1, set III a homodimer specified by Adh2, and set II a heterodimer formed between the products of Adh1 and Adh2.This work was supported by BRSG Grant RR 07080 awarded by the Biomedical Research Grant Program, Division of Research Grants, National Institutes of Health, to D. R. H., and by funds from the Margenroth Endowment to F. B.-B., who is a PHS Research Service Award Trainee in Genetics.     

Genetic relationships between the multiple alcohol dehydrogenases of maize

There are three electrophoretically separable sets of alcohol dehydrogenase isozymes in maize. Previous work has shown that two of these isozymes (Sets I and II) share a subunit in common, since mutations in one of the Adh genes, Adh ₁, alter both isozymes. A mutation in the second Adh gene, Adh ₂, has now been induced and recovered. This mutant allele also alters two of the three isozymes—Sets III and II. Adh ₁ and Adh ₂ appear to segregate independently. Gel filtration data show that all ADH isozymes are indistinguishable in size. These findings support the hypothesis that the two Adh genes specify promoters which homo- and heterodimerize, yielding three types of ADH isozymes.This research was supported by National Science Foundation Grant GB 25594. M.F. is a recipient of Public Health Service Genetics Training Grant GM 82-12.     

Dimerization of multiple maize ADHs studied in vivo and in vitro

Anaerobically induced primary roots simultaneously express two alcohol dehydrogenase (Adh) genes which specify three types of electrophoretically separable dimers: Set I, II, and III ADH. The S inbred line yields a particular activity ratio among these three sets. By use of an Adh ₁ ^null mutant allele and in vitro chemical dissociation and reassociation of ADH dimers, these studies extrapolate from an ADH activity ratio to the actual ratio of ADH protein. Conclusions are that (1) ADH₁ and ADH₂ promoters dimerize randomly in vivo and in vitro, (2) the heterodimeric isozyme (Set II) is approximately the enzymological sum of its subunits under these assay conditions, and (3) ADH-2 subunits are from 10 to 20% as active as ADH₁ subunits under these assay conditions. These conclusions imply that the unlinked Adh genes are coordinately regulated and reconfirm the two-gene-three-dimer model for the maize ADH isozymes.     

Three alcohol dehydrogenase genes in wild and cultivated barley: Characterization of the products of variant alleles

Barley (Hordeum vulgare) and its wild progenitor (H. spontaneum) have three loci for alcohol dehydrogenase (EC 1.1.1.1; ADH). The Adh1 locus is constitutively expressed in seed tissues, whereas expression of the loci Adh2 and Adh3 requires anaerobic induction. The Adh3 gene is well expressed in aleurone and embryo tissues kept under N₂ for 2–3 days. Using N₂-treated embryos, a diverse collection of H. spontaneum was screened in starch gels for electrophoretic variants at the Adh3 locus. Four variants were found: two were conventional mobility variants (Adh3 S, Adh3 V); one was a null variant (Adh3 n); and the fourth (Adh3 I) variant lacked active homodimers and showed reduced heterodimer activity. The ³⁵S-labeled monomers induced under N₂ in the lines homozygous for Adh1, Adh2, or Adh3 variants were immunoprecipitated with antiserum raised against maize ADH. Fluorography after separation by SDS-PAGE and by urea-isoelectric focusing indicated that the Adh3 n allele was CRM- and that the Adh3 I gene product was smaller than normal. The Adh1 and Adh3 variants showed independent segregation.     

Molecular evolutionary characterization of an Activator (Ac)-like transposable element sequence from pearl millet (Pennisetum glaucum) (Poaceae)

We present data on the evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). A defective Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and its entire 4531 bp sequence has been determined. When the pearl millet Ac-like sequence is aligned with the maize Ac sequence, it is found that there is approximately 70% DNA similarity in the central region spanning most of maize Ac exon II and all of exon III. In addition, there are two smaller regions of similarity at the Ac terminii. Besides these three major structural similarities, Pennisetum Ac has two large regions, one 5 and one 3, that show little similarity to Zea Ac. Furthermore, most of the sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between the central region of maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. Conserved DNA and amino acid sequence motifs are also examined. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet and have thus existed in the grasses for at least 25 million years.     

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities

Two unlinked genes, Adh ₁ and Adh ₂, control the production of alcohol dehydrogenase (ADH) in seeds of the annual sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels following D-R of an intergenic isozyme showed that the Adh ₂ isozymes were more than twice as active as those of Adh ₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh ₁ ^F /Adh ₁ ^F , Adh ₂ ^S /Adh ₂ ^S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh ₁, Adh₂, and intergenic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.Supported by Graduate School and BioMed grants and by NSF Grant GB35853.     

Identification of a genetic element that controls the organ-specific expression of adh1 in maize

Allozyme balances serve as markers of quantitative behavior of electrophoretically distinguishable alleles. By the use of ADH Set I allozyme balances, it is demonstrated that all Adh1-S/Adh1-F individuals from more than 20 diverse S/F families exhibit a reciprocal correlation between Adh1 quantitative behavior in two maize organs: the scutellum and primary root. Within an electrophoretic mobility class, the Adh1 allele that is relatively underexpressed in the scutellum is relatively overexpressed in the primary root, and vice versa. Segregation tests prove that this "reciprocal effect" is the property of a cis-acting site that is closely linked to or within the Adh1 structural gene, and it is not affected by diverse genetic backgrounds. Immunological and [(3)H]-leucine incorporation experiments establish that Adh1 quantitative variants differ in ADH1.ADH1 synthetic rates in the anaerobic primary root. The reciprocal-effect phenomenon suggests that the cis-acting loci controlling Adh1 quantitative expression in each respective organ are at least in close proximity, or may share common DNA sequences. We discuss the possibility that the reciprocal-effect locus is a regulatory component of the Adh1 cistron.     

Molecular analysis of a somaclonal mutant of maize alcohol dehydrogenase

Summary Plants regenerated from tissue cultures of maize were screened for variants of ADH1 and ADH2. Root extracts of 645 primary regenerant plants were tested, and one stable mutant of Adh1 was detected. The mutant gene (Adh1-Usv) produces a functional enzyme with a slower electrophoretic mobility than that of the progenitor Adh1-S allele, and is stably transmitted to progeny. The mutant was not present among four other plants derived from the same immature embryo, and therefore arose as a consequence of the culture procedure. The gene of Adh1-Usv was cloned and sequenced. A single base change in exon 6 was the only alteration found in the gene sequence. This would translate in the polypeptide sequence to a valine residue substituting for a glutamic acid residue, resulting in the loss of a negative charge and the production of a protein with slower electrophoretic mobility.Abbreviations kb kilobase pairs - ADH alcohol dehydrogenase     

Alcohol dehydrogenase (ADH) isozymes in the AdhN/AdhN strain ofPeromyscus maniculatus (ADH− deermouse) and a possible role of class III ADH in alcohol metabolism

Although the Adh^N/Adh^N strain ofPeromyscus maniculatus (so-called ADH^? deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the Adh^F/Adh^F strain (so-called ADH⁺ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH^? strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (<100 mM) greatly increased (about sevenfold), to more than 50% that of ADH⁺ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH^? deermouse. It was also found that even the ADH⁺ strain has low liver ADH activity (<40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH^? and ADH⁺ deermouse were different not only from each other but also from that in the ddY mouse; the ADH^? strain was deficient in only Class I ADH, and the ADH⁺ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH^? strain.     

Evolution of Ac and Dsl elements in select grasses (Poaceae)

We present data on evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). An Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and 2387 bp of it have been sequenced. When the pearl millet Ac-like sequence is aligned with the corresponding region of the maize Ac sequence, it is found that all sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we can assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet, and have thus existed in the grasses for at least 25 million years. Ac-like sequences may be widely distributed among the grasses. We also present the first 2 Dsl controlling element sequences from teosinte species: Zea luxurians and Zea perennis. A total of 10 Dsl elements had previously been sequenced from maize and a distant maize relative, Tripsacum. When a maximum likelihood network of genetic relationships is constructed for all 12 sequenced Dsl elements, the 2 teosinte Dsl elements are as distant from most maize Dsl elements and from each other, as the maize Dsl elements are from one another. Our new teosinte sequence data support the previous conclusion that Dsl elements have been accumulating mutations independently since maize and Tripsacum diverged. We present a scenario for the origin of Dsl elements.     

Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers

Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity.

Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O₂ deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O₂ levels were reduced below 5%, and were most intense at 0% O₂.

In vivo³⁵S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O₂, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O₂, the synthesis of all three ADH peptides increased markedly.

Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O₂ deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O₂, and about 100-fold after 1 day at 0% O₂.

We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O₂, that regulation of mRNA level is likely to be a major controlling factor, and that whereas the ADH system of barley has strong similarities to that of maize, it also has some distinctive features.

     

Alcohol dehydrogenase isozymes in grain sorghum (Sorghum bicolor): Evidence for a gene duplication

Two sets of alcohol dehydrogenase (ADH) bands are regularly observed in grain sorghum (Sorghum bicolor): set I is a permanent triplet; set II is variable, as either two or three bands. A faint set III is detected only when extracts from seeds subjected to anerobiosis are run in neutralpH gels. Dissociation-reassociation experiments reveal that the central band of the set I triplet is a heterodimer of the other two. Full-sib progeny analysis from selfed plants shows that the set II bands are doublets, with heterozygotes having only three apparent bands instead of four because of the similar mobilities of the fast-migrating isozyme specified by the slow allele and the slow isozyme specified by the fast allele. We propose a three-locus model as the best explanation of these patterns. Set I consists of the products of two loci and their intergenic heterodimer. Set III is specified by a third locus. Set II isozymes are the intergenic heterodimers of the two set I loci and the set III locus. This explanation is similar to that of Schwartz and Freeling for maize but suggests that the evolution ofSorghum includes a gene duplication of the homologue of theAdh-1 locus inZea. Supported by USDA Grant 59-2063-01522 to NCE and KWF.     

Exploring the Evolutionary History of the Alcohol Dehydrogenase Gene (<Emphasis Type="Italic">Adh</Emphasis>) Duplication in Species of the Family Tephritidae

In the olive fruit fly Bactrocera oleae and the med fly Ceratitis capitata previous studies have shown the existence of two Adh genes in each species. This observation, in combination with the former finding that various Drosophila species of virilis and repleta group encode two isozymes of ADH which are the result of a gene duplication, challenged us to address a scenario dealing with the evolutionary history of the Adh gene duplication in Tephritidae. In our lab we proceeded to the cloning and sequence analysis of Adh genes from more tephritid species, a prerequisite for further study of this issue. Here we show that phylogenetic trees produced from either the nucleotide or the amino acid sequences of 14 tephritid Adh genes consisted of two main clusters, with Adh sequences of the same type grouping together (i.e., Adh1 sequences form a cluster and Adh2 sequences form a second one), as expected if there was one duplication event before speciation within the family Tephritidae. We used the amount of divergence between the two isozymic forms of Adh of the species carrying both Adh1 and Adh2 genes to obtain an estimate of the age of the duplication event. Interestingly, our data again support the hypothesis that the duplication of an ancestral Adh single gene in the family Tephritidae occurred before the emergence of the genera Bactrocera and Ceratitis, thus suggesting that Adh duplication was based on a prespeciation rather than a postspeciation event that might have involved two independent duplication events, one in each of the two genera.     

Selective recruitment of <Emphasis Type="Italic">Adh</Emphasis> genes for distinct enzymatic functions in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alcohol dehydrogenase (ADH) activity in plants is generally associated with glycolytic fermentation, which facilitates cell survival during episodes of low-oxygen stress in water-logged roots as well as chronically hypoxic regions surrounding the vascular core. Work with tobacco and potato has implicated ADH activity in additional metabolic roles, including aerobic fermentation, acetaldehyde detoxification and carbon reutilization. Here a combination of approaches has been used to examine tissue-specific patterns of Adh gene expression in order to provide insight into the potential roles of alcohol dehydrogenases, using Petunia hybrida, a solanaceous species with well-characterized genetics. A reporter-gene study, relying on the promoters of Adh1 and Adh2 to drive expression of the gene for a green fluorescent protein derivative, mgfp5, revealed unexpectedly complex patterns of GFP fluorescence in floral tissues, particularly the stigma, style and nectary. Results of GC-MS analysis suggest the association of ADH with production of aromatic compounds in the nectary. Overall the results demonstrate selective recruitment of Adh gene family members in tissues and organs associated with diverse ADH functions.     

RFLP analysis of mitochondrial DNA from cytoplasmic male-sterile lines of pearl millet

Mitochondrial DNA (mtDNA) from 13 cytoplasmic male-sterile (cms) lines from diverse sources were characterized by Southern blot hybridization to pearl millet and maize mtDNA probes. Hybridization patterns of mtDNA digested with PstI, BamHI, SmaI or XhoI and probed with 13.6-, 10.9-, 9.7- or 4.7-kb pearl millet mtDNA clones revealed similarities among the cms lines 5141 A and ICMA 1 (classified as the S-A1 type of cytoplasm based on fertility restoration patterns), PMC 30A and ICMA 2. The remaining cms lines formed a distinct group, within which three subgroups were evident. Among the maize mitochondiral gene clones used, the coxI probe revealed two distinct groups of cytoplasms similar to the pearl millet mtDNA clones. The atp9 probe differentiated the cms line 81 A4, derived from P. glaucum subsp. monodii, while the coxII gene probe did not detect any polymorphism among the cms lines studied. MtDNA digested with BamHI, PstI or XhoI and hybridized to the atp6 probe revealed distinct differences among the cms lines. The maize atp6 gene clone identified four distinct cytoplasmic groups and four subgroups within a main group. The mtDNA fragments hybridized to the atp6 gene probe with differing intensities, suggesting the presence of more than one copy of the gene in different stoichiometries. Rearrangements involving the coxI and/or rrn18-rrn5 genes (mapped within the pearl millet clones) probably resulted in the S-A1 type of sterility. Rearrangements involving the atp6 gene (probably resulting in chimeric form) may be responsible for male sterility in other cms lines of pearl millet.     

Nucleotide Polymorphism in the Adh1 Locus of Pearl Millet (Pennisetum Glaucum) (Poaceae)

We investigated nucleotide polymorphism in the Adh1 locus of pearl millet (Pennisetum glaucum) (Poaceae) by determining the DNA sequence of 20 alleles from 10 individuals. The individuals were sampled from throughout pearl millet's indigenous range and represent both wild and cultivated accessions. Our results indicated that there is little nucleotide polymorphism in the Adh1 locus. Estimates of per site nucleotide polymorphism did not differ significantly between cultivated and wild millet accessions. We compared nucleotide polymorphism in pearl millet Adh1 with nucleotide polymorphism in maize (Zea mays) Adh1 and conclude that the maize Adh1 sample is more polymorphic. Increased polymorphism in maize Adh1 may be attributable, in part, to faster substitution rates in the maize lineage. Analysis suggests that substitution rates in the maize Adh1 lineage are ~1.7 times faster than substitution rates in the millet Adh1 lineage.     

Structure,expression, chromosomal location and product of the gene encoding ADH1 inPetunia

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated fromPetunia hybrida cv. V30 by screening aPetunia genomic library with a maizeAdh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of twoAdh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay inPetunia protoplasts. We have designated this genePetunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction ofAdh1 mRNA.     

Variation in the biochemical properties of the Drosophila alcohol dehydrogenase allozymes

Thirteen Drosophila Adh variants have been characterized with respect to gene expression, substrate preference, thermostability, and specific activity. The results suggest that the variants may be grouped into two biochemical classes, typified by the properties of the two most common enzyme forms, ADH-F and ADH-S. Membership of these classes cannot be predicted from electrophoretic mobility, nor is any simple classification possible with regard to the characteristics of level of gene expression (in terms of ADH activity or ADH protein) or thermostability of the gene product.