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.     

Structure, Expression, Chromosomal Location and Product of the Gene Encoding Adh2 in Petunia

In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.     

Cytosolic calcium is involved in the regulation of barley hemoglobin gene expression

Hemoglobin gene expression is upregulated during hypoxia. To determine whether the induction occurs via similar mechanisms that have been proposed for other hypoxically induced proteins, barley (Hordeum vulgare L.) aleurone layers were treated with various agents that interfere with known components of signal transduction. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced hemoglobin (Hb) and alcohol dehydrogenase (EC 1.1.1.1) (Adh) gene expression in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA also dramatically reduced anoxia-induced Hb and Adh expression. Normal induction of Hb by anoxia in EGTA-treated cells was restored by adding exogenous Ca²⁺ but not Mg²⁺, suggesting that cytosolic calcium is involved in Hb and Adh regulation. W-7, a calmodulin antagonist, did not affect anaerobically induced Hb and Adh expression even though it induced Hb under aerobiosis. A3, a protein kinase inhibitor, did not significantly affect anaerobically induced Hb, but did significantly upregulate the gene under aerobic conditions. The results indicate that calmodulin-independent anaerobic alteration in cytosolic Ca²⁺ and protein dephosphorylation are factors in Hb induction.     

Isolation of a 6.2 kb genomic fragment carrying the Adh1 gene of tomato and its expression in transgenic tobacco

An 11 kb Eco RI genomic fragment containing the alcohol dehydrogenase (Adh1) gene was cloned. Cross-hybridization with three Adh2 cDNA clones suggested that the entire coding region of the Adh1 gene was contained on a 6.2 kb Xba I/Hind III subfragment. Using RFLP linkage analysis, the genomic clone was mapped on chromosome 4 between the markers TG 182 and TG 65 in a position corresponding to the Adh1 locus. To further confirm the Adh1 origin of the genomic clone, tobacco plants were transformed with the 6.2 kb Xba I/Hinb III genomic subfragment. Isozyme analysis demonstrated that in transgenic tobacco plants functional tomato specific ADH-1 homodimers were synthesized as well as heterodimers composed of tobacco and tomato subunits.     

In vivo detection of regulatory factor binding sites of Arabidopsis thaliana Adh

In vivo footprinting experiments have been used to analyze the binding of trans-acting regulatory factors in the 5 flanking region upstream of the alcohol dehydrogenase (Adh) gene from Arabidopsis thaliana. Protein-DNA interactions were detected by dimethyl sulfate footprinting and genomic sequencing, using an A. thaliana cell suspension culture that constitutively expressed the Adh gene. Several distinct footprinting domains have been characterized, and the potential effects of the corresponding trans-acting factors have been inferred from a comparison with data from the maize alcohol dehydrogenase-1 (Adh1) gene. One binding site is similar in sequence to one of the anaerobic response elements (ARE) of the maize gene, which has also been shown to bind to a trans-acting factor. Several of the remaining binding sites apparently represent a class of elements sharing the sequence 5-GTGG-3 within their footprint.Comparisons with maize Adh1 in vivo protein interactions reveal that the elements of Adh promoter structure are highly conserved, but the relative and absolute positions of the elements are variable.     

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST 2 and RAT 1 genes of Saccharomyces cerevisiae

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.     

Cloning and analysis of two genes for chalcone synthase from Petunia hybrida

Summary With the help of a cDNA probe for a chalcone synthase gene of Petroselinum a cDNA clone for a chalcone synthase gene of Petunia hybrida could be identified. The homologous cDNA allowed the cloning of two genomic EcoRI fragments from Petunia hybrida containing complete chalcone synthase genes. It could be demonstrated that the genes on the two fragments are different and are not allelic but members of a gene family. The two genes are found in a variety of different Petunia lines including in the two conditional mutants affected in chalcone synthase expression in floral buds, White Joy and Red Star. The structure of the two chs genes from Petunia is compared to the chs gene from Antirrhinum majus.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Identification of <Emphasis Type="Italic">Elymus</Emphasis> (Triticeae,Poaceae) and its related genera genomes by RFLP analysis of PCR-amplified <Emphasis Type="Italic">Adh</Emphasis> genes

Elymus L. is the largest genus in Triticeae, containing about 150 species with four recognized genome donors (St, H, P, and W). Traditionally, the genome compound of this genus is identified based on cytological data. Recently, molecular phylogenetic analysis was used to investigate its genomic combination. Here we describe a restriction fragment length polymorphism (RFLP) assay based on digesting alcohol dehydrogenase (Adh) amplicons with two restriction enzyme combinations, EcoRI–HindIII and EcoRI–PstI, which easily can be used to distinguish Elymus and its closely related genera genomes. The method includes only four steps: (1) amplifying nuclear Adh genes with universal primers; (2) purifying and cloning PCR products; (3) digesting plasmids with restriction enzymes that identify a given genome; (4) running the digested products on an agarose gel and identify the sample based on the restriction profiles. Results showed that: (1) PCR products ranged from 1,200 to 2,000 bp; (2) Adh2 gene was amplified from all the tested genomes; Adh1 gene was amplified from almost all of the tested genomes except the W genome; Adh3 gene was amplified only from the St genome; (3) the EcoRI–HindIII combination was effective to distinguish different Adh gene types (Adh1, Adh2, and Adh3); (4) the Adh2–EcoRI–PstI fragments could be used to distinguish Elymus and its closely related genera genomes. Therefore, This RFLP assay provides an inexpensive and simple means of identifying Elymus genomes.     

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.     

Isolation of silencer-containing sequences causing a tissue-specific position effect on alcohol dehydrogenase expression in Drosophila melanogaster

A transient expression assay has been used to investigate the cause of a tissuespecific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4-kb genomic clone containing the 3.2-kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue-specific pattern resembling the abnormal expression pattern of the position effect. The 3.2-kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2-kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2-kb fragment indicates that it is a noncoding region. A 17-bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2-kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an α₁-tubulin promoter-LacZ fusion construct or an hsp70 promoter-Ach fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5′ silencer and the 3′ element act together to create the tissue specific pcsition effect characteristic of the GC-1 line. © 1994 Wiley-Liss, Inc.     

Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression

In order to scan the 5 flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5 end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from –67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from –142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (–59 and –52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.     

Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots

Alcohol dehydrogenase (Adh) is the key enzyme in alcohol fermentation. We analyzed Adh expression in order to clarify the role of Adh of soybeans (Glycine max) to flooding stress. Proteome analysis confirmed that expression of Adh is significantly upregulated in 4-day-old soybean seedlings subjected to 2 days of flooding. Southern hybridization analysis and soybean genome database search revealed that soybean has at least 6 Adh genes. The GmAdh2 gene that responded to flooding was isolated from soybean cultivar Enrei. Adh2 expression was markedly increased 6 h after flooding and decreased 24 h after floodwater drainage. In situ hybridization and Western blot indicated that flooding strongly induces Adh2 expression in RNA and protein levels in the root apical meristem. Osmotic, cold, or drought stress did not induce expression of Adh2. These results indicate that Adh2 is a flooding-response specific soybean gene expressed in root tissue.     

Organization and expression of mouse Hox3 cluster genes

Summary The three yolk protein genes (yp) of Drosophila melanogaster are transcribed in a sex- and tissue-limited fashion. We have searched for cis-regulatory sequences in regions flanking yp1 and yp2 to identify the elements that confer female-specific expression in the fat body. One such 127 by element has previously been identified in this region. We show here the existence of two additional regions which confer female fat body-specific expression on an Adh reporter gene and on the native yp2 gene, respectively. This suggests some redundancy in the regulation of expression of the yp genes. Computer searches for putative binding sites for the DSX protein, which regulates sex-specific expression of the yp genes, revealed several such sites in our constructs. However, the significance of these is unclear since many such sites also occur in genes which one would not expect to be regulated in a sex-specific manner (e.g. Adh, Actin 5C). We suggest that DSX acts in concert with other proteins to mediate sex- and tissue-specific expression of the yp genes.     

Linkage of the alcohol dehydrogenase structural genes in pearl millet (Pennisetum typhoides)

Pearl millet produces three ADH isozymes, Sets I, II, and III. Naturally occurring ADH electrophoretic variants affecting Sets I and II isozymes but not III have been previously described. Analysis of such variants led to the identification of the Adh1 structural gene. The existence of a second Adh structural gene was inferred from dissociation-reassociation studies of Set II. In the present report, a naturally occurring variant affecting the electrophoretic mobility of Sets III and II but not Set I is described. Analysis of this variant confirms the existence of a second structural gene, Adh2. Crosses utilizing this Adh2 marker reveal a dissimilarity with maize and other plants such as sunflower and narrow-leafed lupins. Adh1 and Adh2 of pearl millet do not segregate independently; indeed, no recombinants have been observed. This is the first major difference encountered in an otherwise remarkably similar genetic and environmental control of the ADH isozymes in maize and millet. The organization of the Adh genes of pearl millet may reflect a more primitive arrangement than that of maize.This work was supported by a PHS National Research Service Award Training Grant in Genetics to the Biology Department of the University of Oregon.     

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.     

Analysis of nucleotide substitutions and amino acid conservation in theDrosophila Adh genomic region

The homologous genomic region that contains two paralogous genes,Adh andAdh-dup, was compared in severalDrosophila species. Sequences were analyzed as follows: a) At the nucleotide level, Ka and Ks values were determined for each pair of species. Ka-Adh and Ka-Adh-dup are not significantly different. However, Ks-Adh values are significantly lower than Ks-Adh-dup, which are more variable. In agreement with other reports, lower Ks values forAdh correlate with a high level of gene expression and relatively high percentage of G+C content in the third codon position, while the opposite applies toAdh-dup. b) At the protein level, amino acid comparisons reveal conserved regions shared by ADH and ADH-DUP, which have been assigned to known functional domains. Key residues for dehydrogenasic function are also found in ADH-DUP, thus pointing to a dehydrogenase activity for ADH-DUP, albeit very different from that of ADH.