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.     

Physiological and biochemical changes associated with cotton fiber development. VIII. Wall components

Fibers of three cotton cultivars (Gossypium hirsutum L.) H-4, H-8 and (G. arboreum) G. Cot-15, which shows variation in staple length were analyzed for growth in terms of fiber length and fresh and dry mass. From the growth analysis cotton fiber development is divided in four distinct phases i.e. (i) initiation (ii) elongation (iii) secondary thickening and (iv) maturation. Rate of fiber elongation and rate of water content shows close parallelism. Highly esterified and less esterified pectic fraction along with high and low molecular weight xyloglucan fractions were estimated from fiber walls of all the three cotton genotypes. Xyloglucans were fractioned in to high and low molecular weight by alkali treatment, 1 M and 4 M KOH respectively. Xyloglucan content shows inverse correlation with fiber elongation. Role of water content and wall components in determination of staple length in cotton genotypes is discussed.     

Inter-Specific Differences in Cotton for Nutrient Partitioning from Subtending Leaves to Reproductive Parts at Various Developmental Stages: Consequences for Fruit Growth and Yield

A field study was carried out to unravel the inter-specific differences in cotton for the partitioning of N, P, K, S, Ca, Mg, Na and Cl from the subtending leaves to the reproductive parts of Gossypium hirsutum, G. barbadense and G. arboreum at various developmental stages. Results revealed significant differences among the species for the various parameters studied. Overall there was a greater fresh and dry matter yield of various reproductive parts and subtending leaves of G. hirsutum and G. barbadense than G. arboreum, although the leaf photosynthetic rate was similar. Age-dependent increase in leaf area/leaf mass ratio indicated a greater partitioning of earlier acquired assimilates to the growth of reproductive parts. Results indicated greater partitioning of N, P, S and Ca during later reproductive growth (from boll production to its opening) in G. hirsutum and G. barbadense but during earlier reproductive growth in G. arboreum (from bud up to flower formation) as was evident by decreased subtending leaf/reproductive parts ratio. It is concluded that better N, P, S and Ca partitioning ability of G. hirsutum and G. barbadense at the onset of boll development played a major role in the better yield and good quality fiber characteristics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chemotaxonomical investigations of theSymphytum officinale polyploid complex andS. asperum (Boraginaceae): The pyrrolizidine alkaloids

By means of thin layer chromatography in conjunction with mass spectrometry the pyrrolizidine alkaloid patterns derived fromSymphytum asperum, several cytotypes ofS. officinale agg. and the artificial hybrids of the former taxa, were compared. The obtained patterns were not essentially affected by variation in cytotype, harvesting times and -location of plants. Lycopsamine, acetyl-lycopsamine and symphytine or their isomers were generally found in theS. officinale cytotypes, echimidine and symphytine inS. asperum. The interspecific hybrids contained all alkaloids mentioned. The definite lack of echimidine in the 2 n=40 cytotype proves that it is conspecific withS. officinale and does not belong to a hybrid-swarmS. asperum × S. officinale with 2 n=48. Part I of this series of contributions.     

Regeneration of Gossypium hirsutum and G. barbadense from shoot apex tissues for transformation

A method of regenerating cotton plants from the shoot apical meristem of seedlings was developed for use with particle gun and Agrobacterium-mediated transformation. This method was developed to circumvent the problems of genotype restriction and chromosomal damage frequently encountered in cotton regeneration in tissue culture through somatic embryogenesis. In this procedure, the cells of the shoot meristem are targeted for transformation. Normal and fertile plants of Gossypium barbadense Pima S-6, and 19 cultivars of G. hirsutum were regenerated using this method. Shoot regeneration from these tissues was direct and relatively rapid. A MS based, hormone-free medium could be used with all the varieties tested.This project was funded by grants from Cotton Incorporated, Nisshinbo Industries, and a grant from the Texas Agricultural Experiment Station to RHS. Texas Agricultural Experiment Station Technical Article TA-25667.     

The effects of NaCl on antioxidant enzyme activities in callus tissue of salt-tolerant and salt-sensitive cotton cultivars (Gossypium hirsutum L.)

Summary To determine NaCl effects on callus growth and antioxidant activity, callus of a salt-tolerant and a salt-sensitive cultivar of cotton was grown on media amended with 0, 75, and 150 mM NaCl. Callus of the salt-tolerant cultivar, Acala 1517-8 8, grown at 150 mM NaCl, showed significant increases in superoxide dismutase, catalase, ascorbate peroxidase, peroxidase and glutathione reductase activities compared to callus tissue grown at 0 mM NaCl. In contrast, callus tissue of the salt-sensitive cultivar, Deltapine 50, grown at 0, 75, and 150 mM NaCl, showed no difference in the activities of these enzymes. At the 150 mM NaCl treatment, peroxidase was the only antioxidant enzyme from Deltapine 50 with an activity as high as that observed in Acala 1517-88. The NaCl-induced increase in the activity of these enzymes in Acala 1517-88 indicates that callus tissue from the more salt-tolerant cultivar has a higher capacity for scavenging and dismutating superoxide, an increased ability to decompose H₂O₂, and a more active ascorbate-glutathione cycle when grown on media amended with NaCl.     

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

Two unlinked genes, Adh₁ and Adh₂, 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 Adh₂ isozymes were more than twice as active as those of Adh₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh₁^F/Adh₁^F, Adh₂^S/Adh₂^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 Adh₁, 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.     

Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): Evidence for gene duplication

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red mombim) was also higher than that of the host fruit infested with A. fraterculus (guava).This research was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq-PIG 40.2486/82).     

Systematics ofNama (Hydrophyllaceae): Flavonoids and phyletic position of sect.Arachnoidea and sect.Cinerascentia

Eight flavonoids, four 6-oxygenated flavones, two methyl ethers of luteolin, apigenin 6,8-C-diglucoside and quercetin 3-O-glucoside, were isolated fromNama lobbii andN. rothrockii, sole members of sects.Arachnoidea andCinerascentia, respectively. Both taxa diverge markedly from other namas in morphology and chromosome number and their placement inNama has been questioned. The occurrence of 6-oxygenated flavones in these taxa adds to their already distinctive nature. Flavonoid evidence argues that both are more closely allied toEriodictyon than either is toNama.     

A biochemical genetic study of alcohol dehydrogenase isozymes of the medfly,Ceratitis capitata wied

A concerted effort is under way to analyze, at the genetic, biochemical, and molecular level, theAdh gene system in the medflyCeratitis capitata, an important agricultural pest. The isoelectric focusing (IEF) pattern of alcohol dehydrogenase (ADH) of the medfly demonstrates the presence of two well-differentiated, genetically independent dimeric proteins, called ADH-1 and ADH-2. These proteins do not exhibit interlocus heterodimeric isozymes, and the genes are not controlled coordinately during development,Adh ₁ andAdh ₂ being expressed mainly in muscle or in fat body and ovary, respectively. From the intensity of the IEF isozyme patterns, primary alcohols are judged to be better substrates than secondary alcohols, in contrast withDrosophila melanogaster ADH, and ethanol is probably the most efficient substrate for both sets of isozymes. The isoelectric points of ADH-1 (pI=5.4) and ADH-2 (pI=8.6) are different fromD. melanogaster ADH (pI=7.6), but the medfly ADH-1 has a native molecular weight (approx. 58 kD) close to that ofD. melanogaster. A population survey of samples both from laboratory strains and from wild geographically different populations showed that theAdh ₁ locus is more polymorphic thanAdh ₂. The most variable populations are from Africa, the supposed source area of the species. Further, a case of selection at theAdh ₁ locus under laboratory conditions is reported. The hypothesis ofAdh gene duplication and the degree of similarity between medfly andDrosophila ADH are also discussed. This research was supported mainly by National Research Council of Italy, Special Project RAISA, Sub-project No. 2, Paper No. 342. Grants from the International Atomic Energy Agency, Vienna, Austria, from European Communities Commission, Second R & D Programme, “Science and Technology for Development,” and from the Italian Ministry of University and Scientific Research and Technology (“Funds 40%”) also supported this work. This paper was written when the senior author was on leave of absence at the IMBB, Crete, Greece; he was financially supported by an ECC Senior Fellowship.     

Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase null mutants ofArabidopsis thaliana (L.) Heynh

Several mutants have been isolated at theArabidopsis thaliana (L.) Heynh. alcohol dehydrogenase (ADH) gene locus using allyl alcohol selection on ethyl methanesulfonate (EMS)-mutagenized seeds. Eleven mutants were isolated in theADH1-A electrophoretic allele, and 21 in theADH1-S allele. These null mutants are characterized by the absence of measurable ADH activity and genetic data showed that the mutations were confined to theADH1 gene locus ofArabidopsis. Eleven mutants in theADH1-A background were further characterized at the protein and mRNA level. These experiments revealed striking differences in the ADH protein and mRNA content. Some of the mutants did not synthesize any mRNA or ADH-like protein, whereas some of them had a nearly normal level of ADH protein and mRNA. Others had a very low level of both protein and mRNA. ADH null mutants differed physiologically from the wild type by their higher sensitivity to anaerobic treatment in plants and significantly reduced resistance to acetaldehyde in suspension cultures.This research was supported by the Geconcerteerde Onderzoeksactie, Grant 86/91–103, and the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL), Grant 4972A.     

Effect of allopolyploidy on the activity of selected enzymes inHibiscus

Mature seeds of diploid and tetraploidHibiscus species were analyzed for enzyme activity (alcohol dehydrogenase, malate dehydrogenase, leucine aminopeptidase), total protein content, DNA amount and dry weight. The recently formed tetraploid,H. radiatus, generally had enzyme and protein levels very similar to the sum of its progenitors, while the more ancient speciesH. acetosella had several lower levels. This difference may reflect the greater amount of timeH. acetosella has had to evolve dosage compensations.Michigan Agricultural Experiment Station Journal Article 9665.A part of this research was used to satisfy the requirements ofA. Hoisington for a M.S. degree at the University of South Carolina.     

Effect of triacontanol on the lipid composition of cotton (Gossypium hirsutum L.) leaves and its interaction with indole-3-acetic acid and benzyladenine

Application of triacontanol (TRIA), a long chain aliphatic alcohol (C-30), to cotton (Gossypium hirsutum L.) leaves resulted in an increase in dry weight and an alteration in lipid composition. A significant increase in monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) was attained 24 h after TRIA treatment. However, no significant change in any of the individual phospholipids was observed. Benzyladenine (BA) treatment increased only phosphatidylcholine (PC) levels without having any effect on either glycolipids or other phospholipids. Indole-3-acetic acid (IAA) initiated no significant change in the lipid composition. Combined treatment with TRIA and BA resulted in an increase of MGDG, DGDG and PC, indicating that the individual effects of these two growth regulators were not altered.The combined treatment of IAA and TRIA did not bring about any change in the levels of MGDG and DGDG indicating that the effect of TRIA was nullified by IAA. MDGD is known to be involved in the packaging of photosystem I proteins. Whether TRIA-induced increase in dry weight which is due to the enhanced photosynthetic rate, is related to increased MGDG levels is not yet discernible.Abbreviations BA benzyladenine - DGDG digalactosyldiacylglycerol - IAA indole-3-acetic acid - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SQDG sulfoquinovosyldiacylglycerol - TRIA triacontanol     

Interspecific differences in differentiation and dedifferentiation patterns in the genusNicotiana

Differentiation on hormoneless media, habituation ability and crown gall induction inNicotiana tissue cultures have been used as physiological parameters of evolutionary differentiation between species. Some of them on hormone free media differentiated whole plantlets, others produced only shoots or roots or showed undifferentiated growth (habituation), some eventually died. Moreover, the same genotypes showed a differential behaviour as far as tumor formation byAgrobacterium tumefaciens was concerned. Particularly, the competence for crown gall transformation inNicotiana species seems negatively correlated with differentiation capacity and may be ascribed to differences in the plants capacity to synthesize growth regulators. The correlation between the results obtained and the phylogenetic position of the genotypes tested is finally discussed.     

Alcohol dehydrogenase of Biomphalaria glabrata (Mollusca: Pulmonata): Polymorphism,genetic analysis,and interspecific variations

Alcohol dehydrogenase of Biomphalaria glabrata has been characterized by electrophoresis, substrate specificities, and other physicochemical means. It exists as a multiple molecular form possessing a minimum number of three bands in ovotestis, five in digestive gland, and six in albumen gland. Each organ shows characteristic electrophoretic forms which differ in substrate specificities and the response to the organomercurial inhibitor p-hydroxymercuribenzoate. Mercaptoethanol treatment has no effect on any electrophoretic form. Genetic analyses of the electrophoretic variants show that three different loci are responsible for the synthesis of the various electrophoretic forms observed in this species. Different species vary in their electrophoretic patterns. A possible role of alcohol dehydrogenase isozymes in the phylogenetic relationship among three species, B. glabrata, B. tenagophila, and B. straminea, has been discussed.This work was supported by a grant from the Conselho Nacional de Pesquisas, Brazil.     

Comparison of the karyotypes ofPsathyrostachys juncea andP. huashanica (Poaceae) studied by banding techniques

The karyotypes ofP. juncea (Elymus junceus) andP. huashanica (both outbreeders) were investigated by Feulgen-staining and by C-, N-, and Agbanding, based on a single plant in cach case. Both species have 2n=2x=14 and large chromosomes, possibly a generic character. The karyotype ofP. juncea has 8 metacentrics and 6 SAT-chromosomes with minute, heterochromatic satellites while that ofP. huashanica has 9 metacentrics and 5 SAT-chromosomes only, 2 of which with small, heterochromatic satellites. The C-banding patterns ofP. juncea chromosomes comprise from one to five, mostly small, bands at distal, and terminal positions, while those ofP. huashanica chromosomes are characterized by large telomeric bands in most arms. Banding patterns and chromosome morphology allow identification of the homologues of the seven chromosome pairs inP. juncea, but of two pairs inP. huashanica only. The patterns of both taxa are polymorphic, supporting that both taxa are outbreeders. The karyotypic characters suggest thatP. juncea is more closely related toP. fragilis than either is toP. huashanica. N-banding stains weakly. Silver nitrate staining demonstrates that nucleolus organizers of both species have different nucleolus forming capacities. The presence of micronucleoli suggests that both species have an extra unidentified chromosome with nucleolus forming capacity.     

Selection at the alcohol dehydrogenase locus of Drosophila melanogaster: Effects of ethanol and temperature

Drosophila melanogaster larvae were subjected to 10 generations of selection on 6% ethanol at 17, 25, and 30°C. For each temperature there was a significant (P<0.01) increase in the frequency of the Adh isoallele. Controls with no ethanol showed no change in the frequency of the Adh^F isoallele. Larvae subjected to stronger selection on 8% ethanol confirmed the results. When adults of various ages were subjected to 16 and 32°C, the ADH^F isoenzyme retained its twofold advantage in activity over ADH^S regardless of the temperature. The same result was obtained with larvae at 16 and 35°C. Although some effect of temperature was demonstrated, it was concluded that the effect was not strong enough for temperature to be a selective factor under the conditions studied. However, ethanol is a strong selective factor for laboratory populations.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.