Effects of light and acetate on the liberation of zoospores by a mutant strain ofChlamydomonas reinhardtii

In light-dark-synchronized cultures of the unicellular green algaChlamydomonas reinhardtii, release of zoospores from the wall of the mother cell normally takes place during the second half of the dark period. The recently isolated mutant ls, however, needs light for the liberation of zoospores when grown photoautotrophically under a 12 h light-12 h dark regime. The light-induced release of zoospores was found to be prevented by addition of the photosystem-II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Furthermore, light dependence of this process was shown to be abolished when the mutant ls was grown either photoautotrophically under a 14 h light-10 h dark regime or in the presence of acetate. Our findings indicate that the light-dependency of zoospore liberation observed in cultures of this particular mutant during photoautotrophic growth under a 12 h light-12 h dark regime might be attributed to an altered energy metabolism. The light-induced release of zoospores was found to be prevented by addition of cycloheximide or chloramphenicol, antibiotics which inhibit protein biosynthesis by cytoplasmic and organellar ribosomes, respectively. Actinomycin D, an inhibitor of RNA synthesis, however, did not affect the light-induced liberation of zoospores.Sporangia accumulate in stationary cultures of the mutant ls. Release of zoospores was observed when these sporangia were collected by centrifugation and incubated in the light after resuspension in fresh culture medium. Since liberation of zoospores was not observed after dilution of the stationary cultures with fresh culture medium, we suppose that components which interfere with the action of the sporangial autolysin are accumulated in the culture medium of the mutant ls.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Glycerol-, inositol-, and reducing end hexose-containing oligosaccharides in human urine

Ten previously unreported oligosaccharides have been purified from the urines of human subjects using a combination of gel filtration, ion exchange, and thin-layer chromatographies. Their structures were determined by direct probe mass spectrometry, methylation analysis, and proton NMR spectroscopy of the permethylated oligosaccharide alditols.On the basis of composition, the oligosaccharides could be divided into three groups. Five oligosaccharides containing glycerol were characterized as glucosyl1-1glycerol; glucosyl1-1glycerol; galactosyl1-1glycerol; glucosyl-1-1(fucosyl-1-2)glycerol and/or fucosyl-1-1(glucosyl-1-2)glycerol; and glucosyl-1-1(galactosyl-1-2)glycerol or galactosyl-1-1(glucosyl-1-2)glycerol. Four inositol-containing oligosaccharides were characterized as galactosyl1 (fucosyl1)inositol,N-acetylgalactosaminyl1 (fucosyl1)inositol, fucosyl1-2galactosyl1 (N-acetylgalactosaminyl1)inositol and fucosyl1-2galactosyl1-4-N-acetylglucosaminyl1(N-acetylgalactosaminyl1)inositol. Finally, galactosyl1-3(fucosyl1-2)galactosyl1-6galactosyl1-4(fucosyl1-3)glucose, an oligosaccharide with glucose at its reducing end, was tentatively identified. The significance and possible origins of the carbohydrate structures are discussed.     

Biodegradation of seven polychlorinated biphenyls by a newly isolated aerobic bacterium (<Emphasis Type="Italic">Rhodococcus</Emphasis> sp. R04)

An aerobic bacterial strain, designated R04, belonging to the genus Rhodococcus has been isolated and characerized by 16S rDNA analysis. The capability of this strain to degrade seven different polychlorinated biphenyls (CBs), 500 ppm 3-CB, 3,4-CB, 4,4-CB, 2,4,6-CB, 2,4,5-CB, 2,3,4,5-CB and 3,4,3,4-CB in liquid medium, was evaluated. After 5 days of incubation, the concentration of chloride increased to 0.35 mM in cultures containing 3-CB and R04, whereas in cultures with 3,4-CB, 2,3,4,5-CB or 3,4,3,4-CB plus R04 the chloride content increased to 0.1 mM. However, non-stoichiometric amounts of chloride were produced in cultures with R04 and 4,4-CB, 2,4,6-CB and 2,4,5-CB. The spectrum of supernatants from R04 grown on seven PCBs had a UV-visible (UV-VIS) absorption at 200–500 nm, characteristic of biphenyl-derived cleavage products. Gas-chromatographic (GC) analysis showed that R04 was able to transform 100% of 3-CB and 3,4-CB after 1 day of incubation, and 95% of 4,4-CB, 2,4,6-CB, 2,4,5-CB, 2,3,4,5-CB and 3,4,3,4-CB after 5 days of incubation. The position of the chlorine substituents on the rings strongly influenced the degradation of polychlorinated biphenyls (PCBs) and their intermediate metabolites by Rhodococcus sp. R04. The degradation of PCBs was further evaluated by monitoring intermediate metabolites of PCBs.     

Carotenoid pigments in a red strain of Rhizobium from Lotononis bainesii Baker

The carotenoid pigments of a Rhizobium strain isolated from Lotononis bainesii were found to be diglucosyl-4,4-diapocarotene-4,4-dioate and glucosyl-4,4-diapocarotene-4-oate-4-oic acid.5th publication in the series Carotenoids of Rhizobia [4th publication: Helv. chim. Acta 62: 2551–2557 (1979)]     

Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5 regions were translationally fused with the -D-glucuronidase reporter gene (GUS). The GNT1 5 region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5 regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.Department of Plant Molecular Biology, Leiden University     

Effects of cyclic nucleotides on morphogenesis inNostoc muscorum

The filamentous cyanophyteNostoc muscorum A grew aseriately in light in a mineral salts (sugar-free) culture medium supplemented with adenosine 3:5-cyclic-monophosphate or N⁶, O²-dibutyryl adenosine 3:5-cyclic-monophosphate (1 mM). The aseriate morphology thus formed in the light on the 10th day following inoculation was similar to that formed in the dark after 20–30 days growth in cAMP-free medium containing glucose or sucrose. Inoculum previously grown in sucrose- or glucose-containing medium displayed aseriate morphology with lesser proliferation of coccoid cells as compared to inoculum grown in the absence of glucose or sucrose. cGMP, ADP, AMP and inhibitors of phosphodiesterase (theophylline and caffeine) did not have any effect on the persistence of aseriate morphology. However they stimulated cell division at the aseriate stage and delayed the release of hormogonia.Abbreviations cAMP adenosine 3:5-cyclic-monophosphate - db cAMP N⁶, O²-dibutyryl adenosine 3:5-cyclic-monophosphate - cGMP guanosine 3:5-cyclic-monophosphate - ATP adenosine 5-triphosphate - ADP adenosine5-diphosphate - AMP adenosine 5-monophosphate     

Identification and quantitation of adenosine-3′:5′-cyclic monophosphate in plants using gas chromatography-mass spectrometry and high-performance liquid chromatography

Direct evidence has been obtained for the presence of adenosine-3:5-cyclic monophosphate (cAMP) in tobacco (Nicotiana tabacum L.) callus tissue cultures, bean (Phaseolus vulgaris L.) seedlings and immature kernels of sweet corn (Zea mays L.) through the use of a highly specific and sensitive gas chromatography-mass spectrometric assay. Levels of endogenous cAMP ranged from 70 to 126 pmol/g fresh weight. Corresponding levels of cAMP determined for the same samples using radioimmunoassay were consistently three to four times higher. Contrary to previous reports for citrus plants, measurable levels of cAMP could not be detected in young lemon leaves within the limits of detection of the mass-spectrometric assay method. In the case of tobacco callus tissue, the coumarin glucoside, scopolin, which was present in large amounts and showed similar chromatographic behaviour to cAMP, interferred strongly with the mass-spectrometric measurements of cAMP in inadequately purified extracts. The use of high-performance liquid chromatography, in addition to standard chromatographic purification methods, produced highly purified plant extracts for quantitation of cAMP and also provided a method for the separation of cAMP from its 2:3-isomer.Abbreviations cAMP adenosine-3:5-cyclic monophosphate - 2:3-cAMP adenosine-2:3-cyclic monophosphate - GC-MS-MID combined gas chromatography-mass spectrometry with selected multiple-ion-detection - HPLC high-performance liquid chromatography - RIA radioimmunoassay - TLC thin-layer chromatography     

Random coil proton chemical shifts of deoxyribonucleic acids

Sixteen 17-nucleotide DNA sequences have been used to determine the sequence effect on random coil DNA proton chemical shifts. Based on the proton chemical shifts measured for the central nucleotides in 64 triplets and the correction factors determined for the next nearest neighbor effects, a parameter set has been derived for predicting random coil DNA proton chemical shifts. The root-mean-square deviation (RMSD) between the predicted and the observed aromatic H6/H8 proton chemical shifts of 200 data from 22 random coil DNA sequences was determined to be 0.02 ppm with a correlation coefficient of 0.998. For the H1, H2, H2 and H3 sugar protons, the RMSD values between the predicted and the experimental shifts were found to be 0.02, 0.03, 0.03 and 0.02 ppm, respectively.     

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, -acetyldigitoxin, and -acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.Abbreviation DGT UDP-glucose:digitoxin 16-C-glucosyltransferase     

Transfer of germ plasm from the secondary to the primary gene pool in pennisetum

Summary Germ plasm from the A-genome of Pennisetum purpureum Schum. (AABB) of the secondary gene pool was transferred to cultivated pearl millet (AA) [P. glaucum (L.) R. Br.] by pollinating cytoplasmicnuclear male-sterile (cms) pearl millet with fertile allohexaploid pearl millet x P. purpureum hybrids (AAAABB). Certain allohexaploids used as pollinators on cms pearl millet resulted in 14-chromosome diploid pearl millet progenies. Three types of diploid pearl millet plants were produced in addition to the expected 28-chromosome AAAB-genome plants: (1) cms plants with only the A-genome, (2) cms plants with the A- and A-genomes, and (3) fertile plants with the A- and A-genomes. The latter group has allowed the utilization of genes for fertility restoration, stiff stalk, maturity, height, and morphological characteristics from the A-genome of P. purpureum in the pearl millet breeding program. Production of monoploid gametes by the allohexaploids appeared to be genetically controlled.     

Involvement of cyclic adenosine-3′, 5′-monophosphate in chloronema differentiation in protonema cultures of Funaria hygrometrica

The role of purine and pyrimidine ribosides, nucleotides and substituted xanthines in the differentiation of chloronema filaments in suspension cultures of protonema of the moss Funaria hygrometrica Hedw. has been examined. Cyclic adenosine-3,5-monophosphate (cAMP) and mono-and dibutyryl cAMP evoked the maximum response in wild-type protonema. ADP and ATP also enhanced chloronema differentiation but were less active than cAMP; pyrimidine derivatives were completely inactive. Inhibitors of cyclic-nucleotide phosphodiesterase aminophylline, theophylline and ICI 58, 301 (3-acetamido-6-methyl-8-n-propyl-s-triazolo-(4,3a)-pyrazine)-mimicked the effect of cAMP. A leaky, chloronema-repressed mutant was isolated and in this mutant cAMP was much more active than cyclic guanosine monophosphate and ADP in enhancing chloronema differentiation. These results strongly indicate that cAMP is involved in chloronema differentiation in Funaria, and a hypothesis on growth regulation in protonema cell cultures is proposed.Abbreviations cAMP, cyclic AMP cyclic adenosine-3, 5-monophosphate - cCMP, cGMP, cIMP cyclic cytosine-, guanosine-and inosine-3, 5-monophosphates, respectively - IAA indole-3-acetic acid - ICI 58,301 3-acetamido-6-methyl-8-n-propyl-s-triazolo-(4,3a)-pyrazine     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

The sequence and secondary structure of the 3′-UTR affect 3′-end maturation,RNA accumulation,and translation in tobacco chloroplasts

RNA maturation and modulation of RNA stability play important roles in chloroplast gene expression. In vitro and in vivo studies have shown that both the 5- and 3-untranslated regions (UTRs) contain sequence and structural elements that guide these processes, and interact with specific proteins. We have previously characterized the spinach chloroplast petD 3-UTR in detail by in vitro approaches. This stem-loop forming sequence is a weak terminator but is required for RNA maturation and also exhibits sequence-specific protein binding. To test petD 3-UTR function in vivo, tobacco chloroplast transformants were generated containing uidA reporter genes flanked by variants of the petD 3-UTR, including one which does not form an RNA-protein complex in vitro, and one which lacks a stem-loop structure. Analysis of uidA mRNA indicated that a stable secondary structure is required to accumulate a discrete mRNA, and that changes in the 3-UTR sequence which affect protein binding in vitro can also affect RNA metabolism in vivo. The 3-UTR also influenced -glucuronidase protein accumulation, but not in proportion to RNA levels. These results raise the possibility that in tobacco chloroplasts, the 3-UTR may influence translational yield.     

Effects of abscisic acid (ABA) and ABA analogs on freezing tolerance,low-temperature growth,and flowering in rapeseed

Brassica napus and B. campestris are grown in Western Canada in areas subject to unseasonable frosts. At the seedling stage, cultivars of Brassica are very sensitive to frosts of -2° to-5°C, which are either lethal or delay the development of the plant. Seedlings of B. napus and B. campestris, germinated and grown at 10°C (16-h photoperiod), were treated with a foliar spray of either 100 M racemic abscisic acid (ABA), 100 M of various ABA analogs, 0.1% acetone, or were untreated. Freeze tests indicated 2°C of frost tolerance could be gained in B. napus following an application of three ABA analogs. In B. campestris, three analogs also increased freezing tolerance approximately 1.5°C. The analogs 2,3 dihydro ABA and acetylenic divinyl methyl-ABA were effective in both species. Plant fresh weight and dry weight increased in treated plants relative to control or acetone-treated plants after 3 weeks at 10°C. The effect of frost and/or analog treatment on flowering was determined in both species. In B. campestris and B. napus, a mild frost advanced flowering by approximately 2 days compared with nonfrozen control plants. The promotive effect of frost on flowering decreased with increasing severity of the frost. Several of the analog treatments, particularly 2,3 dihydro ABA and acetylenic divinyl ABA, advanced flowering by 2–3 days in both species. The benefit of these ABA analog treatments on flowering was enhanced additionally by a mild frost. Plants treated with either ABA, 2,3 dihydro ABA, 2,3 acetylenic dihydro ABA, or acetylenic divinyl ABA flowered up to 5 days earlier than control plants.     

Regioselective alkylation of benzylβ-d-lactoside and its derivatives by stannylation

The preparation of benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside, which is a key intermediate for chemical synthesis of oligosaccharide components of glycosphingolipids, was achieved by an improved method. The 3-O-p-methoxybenzyl and 3-O-methyl derivatives were prepared from benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside through stannylation. By using benzyl -d-lactoside as starting material, benzyl 3-O-methyl-, 3-O-benzyl- and 3-O-p-methoxybenzyl--d-lactoside were regioselectively synthesized using the same procedure.     

Rapid plant regeneration of pea using thidiazuron

A simple and rapid pea regeneration procedure was developed. An average of up to 20 shoots formed from hypocotyl explants of cvs. Sugar Ann and Patriot cultured on Murashige and Skoog basal medium supplemented with 0.5 or 1.0 M thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea). Hypocotyls of Puget and Sugar Daddy did not respond. Regenerated shoots rooted rapidly when cultured on Murashige and Skoog basal medium containing either 2.0 M -naphthaleneacetic acid or 1.0–2.0 M indole-3-butyric acid. Seeds were harvested from regenerated plants after only 9–11 weeks.Abbreviations BAP 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy acetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)     

ζ-Carotene<Emphasis Type="Italic"> cis</Emphasis> isomers as products and substrates in the plant poly-<Emphasis Type="Italic">cis</Emphasis> carotenoid biosynthetic pathway to lycopene

The plant carotenoid biosynthetic pathway to cyclic carotenes proceeds via carotene precursors in cis configuration. Involvement of individual isomers was elucidated by genetic complementation of desaturations and in vitro reactions of the corresponding enzyme. Determination of substrate and product specificity of phytoene and -carotene desaturase revealed that 15-cis-phytoene is converted to 9,15,9-tricis--carotene with 15,9-dicis-phytofluene as intermediate by the first desaturase. Prior to a subsequent conversion by -carotene desaturase, the 15-cis double bond of 9,15,9-tricis--carotene has to be (photo)isomerized to all-trans. Then, the resulting 9,9-dicis--carotene is utilized by -carotene desaturase via 7,9,9-tricis-neurosporene to 7,9,7,9-tetracis-lycopene. Other -carotene isomers that are assumed to be spontaneous isomerization products were not converted, except for the asymmetric 9-cis--carotene. This isomer is desaturated only to 7,9-dicis-neurosporene resembling a dead-end of the pathway. Prolycopene, the product of the desaturation reactions, is finally isomerized by a specific isomerase to all-trans-lycopene, which is a prerequisite for cyclization to -carotene. The 5-cis-lycopene and the 9-cis-and 13-cis--carotene isomers detected in leaves are thought to originate independently from cis precursors by non-enzymatic isomerization of their all-trans forms.     

Isolation and structural determination of a novel coenzyme from a methane-oxidizing bacterium

The respiratory quinone composition of the obligate methane-utilizing bacterium Methylomonas rubra was examined. A single lipoquinone was isolated which on examination by thin-layer chromatography cochromatographed with coenzyme Q. Reverse-phase partition and argentation high performance liquid chromatography demonstrated the lipoquinone did not correspond to any known coenzyme Q prenologue. On the basis of mass spectrometry and proton nuclear magnetic resonance spectrometry the novel lipoquinone was shown to correspond to 2,3-dimethoxy-5-methyl-6-(11-methylene-3,7,15, 18, 18, 19, 23-heptamethyltetracosa-2, 6, 14, 19, 22-pentaenyl-)-1,4-benzoquinone.     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.